Metformin and Diabetes Mellitus

**3**

**Chapter 1**

**Abstract**

metformin action.

acidosis, diabetes

**1. Introduction**

Metformin Indications,

*Roxana Adriana Stoica, Diana Simona Ștefan,* 

*Adrian Paul Suceveanu, Cristian Serafinceanu*

*Manfredi Rizzo, Andra Iulia Suceveanu,* 

Contraindications

*and Anca Pantea-Stoian*

Dosage, Adverse Reactions, and

Metformin or dimethyl biguanide is the oral antidiabetic drug with the most extensive experience of prescribing in the clinical practice of type 2 diabetes mellitus. In this chapter, we reviewed the indications, contraindications, and adverse drug reactions (ADR) of metformin. The most significant adverse drug reactions of metformin are lactic acidosis, allergies, hypoglycemia, vitamin B12 deficiency, altered taste, and gastrointestinal intolerance. Metformin is contraindicated in severe chronic diseases (hepatic, renal, and cardiac failure) or acute complications of diabetes (ketoacidosis and hyperosmolar state). Metformin is considered by all international guidelines the first-line treatment in type 2 diabetes mellitus (T2DM) together with medical, nutritional therapy. It is one of the most prescribed molecules worldwide. Furthermore, metformin can also be prescribed for other diseases like polycystic ovary syndrome or prediabetes (impaired glucose tolerance/fasting hyperglycemia). Recent studies have shown positive results concerning the use of metformin for cardiovascular or neuroprotective effects; also, several scientific papers are suggesting an antitumor or antiaging effect of metformin. Having such an excellent efficiency in practice, thus predicting its sustainability on the pharmaceutical market, research is directed toward characterizing metformin action on bacteria genera in the gut. Modifying the microbiota composition by pre- and probiotics could improve

**Keywords:** metformin, indication, adverse reaction, gastric intolerance, lactic

Metformin or dimethyl biguanide has its origin in traditional herbal medicine (*Galega officinalis* or goat's rue) that is rich in guanidine. Guanidine was proven to have the capacity to lower blood glucose and was used as an antidiabetic treatment from the 1920s to 1930s. Its administration was interrupted prematurely due to

### **Chapter 1**

## Metformin Indications, Dosage, Adverse Reactions, and Contraindications

*Roxana Adriana Stoica, Diana Simona Ștefan, Manfredi Rizzo, Andra Iulia Suceveanu, Adrian Paul Suceveanu, Cristian Serafinceanu and Anca Pantea-Stoian*

### **Abstract**

Metformin or dimethyl biguanide is the oral antidiabetic drug with the most extensive experience of prescribing in the clinical practice of type 2 diabetes mellitus. In this chapter, we reviewed the indications, contraindications, and adverse drug reactions (ADR) of metformin. The most significant adverse drug reactions of metformin are lactic acidosis, allergies, hypoglycemia, vitamin B12 deficiency, altered taste, and gastrointestinal intolerance. Metformin is contraindicated in severe chronic diseases (hepatic, renal, and cardiac failure) or acute complications of diabetes (ketoacidosis and hyperosmolar state). Metformin is considered by all international guidelines the first-line treatment in type 2 diabetes mellitus (T2DM) together with medical, nutritional therapy. It is one of the most prescribed molecules worldwide. Furthermore, metformin can also be prescribed for other diseases like polycystic ovary syndrome or prediabetes (impaired glucose tolerance/fasting hyperglycemia). Recent studies have shown positive results concerning the use of metformin for cardiovascular or neuroprotective effects; also, several scientific papers are suggesting an antitumor or antiaging effect of metformin. Having such an excellent efficiency in practice, thus predicting its sustainability on the pharmaceutical market, research is directed toward characterizing metformin action on bacteria genera in the gut. Modifying the microbiota composition by pre- and probiotics could improve metformin action.

**Keywords:** metformin, indication, adverse reaction, gastric intolerance, lactic acidosis, diabetes

### **1. Introduction**

Metformin or dimethyl biguanide has its origin in traditional herbal medicine (*Galega officinalis* or goat's rue) that is rich in guanidine. Guanidine was proven to have the capacity to lower blood glucose and was used as an antidiabetic treatment from the 1920s to 1930s. Its administration was interrupted prematurely due to

toxicity. The medicine was valued again between the 1940s and 1950s when Jean Sterne observed the low blood glucose values of patients that were treated with metformin for influenza. Since then, the drug class of biguanides has received much consideration, especially buformin and phenformin in the 1970s and metformin after the 1990s [1].

The 60-year history of biguanides' use is filled with victories and defeats, being the oral antidiabetic drug with the most extensive experience of prescribing in the clinical practice.

We will review in the following pages the indications, contraindications, and adverse drug reactions (ADR) of metformin and the single biguanide approved globally for use nowadays.

An ADR according to the World Health Organization is "a response to a drug which is noxious and unintended, and which occurs at doses normally used in man for the prophylaxis, diagnosis, or therapy of a disease, or the modification of physiological function." A side effect is "an unintended effect occurring at normal dose related to the pharmacological properties" [2].

A contraindication represents "something (such as a symptom or condition) that makes a particular treatment or procedure inadvisable" [3].

### **2. Indications**

### **2.1 Type 2 diabetes mellitus (T2DM)**

All international guidelines consider metformin and lifestyle intervention as the first-line treatment in adults with T2DM in order to improve glycemic control [4]. It can be used either as monotherapy or combination therapy with glucagon-like peptide-1 receptor agonist (GLP-1 RA), sodium-glucose co-transporter inhibitor (SGLT2i), dipeptidyl peptidase-4 inhibitor (DPP4-I), thiazolidinedione (TZD), sulfonylurea (SU), and insulin. Metformin therapy should be continued as long as it is well tolerated and not contraindicated. All other agents, including insulin, should be added to metformin treatment [4].

### **2.2 Prediabetes**

Metformin can be used in order to prevent or delay the onset of T2DM [5]. Although other pharmacological agents have been used in clinical trials (acarbose [6–8], orlistat [9], and rosiglitazone [10]), it appears that metformin has the most reliable evidence base [11–16]. The vast majority of international guidelines recommend metformin use in prediabetes. It can be used together with a combination of a lifestyle intervention for patients with prediabetes: impaired glucose tolerance (2-h post-load glucose 140–199 mg/dL), fasting hyperglycemia (100–125 mg/dl), or A1C 5.7–6.4% [17–23]. Metformin appears to have a more significant advantage when used in patients who are <60 years old and have a BMI >35 kg/m<sup>2</sup> or women with prior gestational diabetes mellitus [16].

### **2.3 Type 1 diabetes mellitus (T1DM)**

Metformin is sometimes used in T1DM to limit insulin dose requirement [24, 25]. The American Diabetes Association states that adding metformin leads to the reduction in body weight and can improve lipid levels, but not HbA1c [4, 26]. The REMOVAL study suggests that metformin might also reduce atherosclerosis progression, thus suggesting to improve CVD risk management in type 1 diabetes [27, 28].

**5**

function [54].

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications*

Lifestyle modification is the first-line therapy for GDM. If glycemic targets are not achieved, then insulin treatment is required for lowering blood glucose; metformin can also be considered if the patient cannot take or declines insulin [29]. Some controlled randomized trials are proving limited efficacy of metformin during pregnancy [30, 31]. Metformin therapy is associated with a lower risk of neonatal hypoglycemia and less maternal weight gain than insulin in systematic reviews [32–34]; metformin may slightly increase the risk of prematurity, and it crosses the placenta [35]. Thus, the ADA considers that metformin should not be used as first-line agents [36].

PCOS patients suffer from insulin resistance and hyperinsulinemia [37]. Metformin has been used for PCOS treatment [38] for treating the metabolic abnormalities of PCOS. A recent meta-analysis [39] demonstrated that metformin

Several studies showed an increased life-span when using metformin (4–6% in different mouse breeds or a mean life-span increased by 14% and maximum life-span increased by 1 month of treatment with metformin is started early in life) [40, 41]. In the United Kingdom Prospective Diabetes Study (UKPDS), the use of metformin decreased the risk of cardiovascular disease, cancer incidence, and

Epidemiological studies reported a positive result of metformin concerning ovarian [43, 44], breast, prostate, or colorectal tumors [45–48] enhancing the antitumor effect of metformin. Furthermore, studies are demonstrating a reduced incidence of several gastroenterological cancers and a reduction in cancer mortality

UKPDS was the first study that demonstrated the cardiovascular benefit of metformin; the risk of all-cause mortality and acute myocardial infarction was significantly reduced in overweight patients with T2DM [42]. The 10-year postinterventional follow-up of the UKPDS survivor cohort revealed that metformin

The cardiovascular protective effects of metformin could be explained by the reduced level of LDL cholesterol [52], the limitation of weight gain, [53] and the improvement of oxidative stress, inflammatory response, and the endothelial cell

It has been reported that patients treated with metformin have lower risk of dementia than those with other diabetes medications [55]. Metformin has a better protective effect on the domain of verbal learning, working memory, and executive

Results of meta-analyses of RCTs (primarily in patients with schizophrenia and schizoaffective disorder) support the use of metformin for weight loss, preventing weight gain associated with second-generation antipsychotics in adult patients [57].

could decrease testosterone and insulin level in women with PCOS.

overall mortality, compared with other antidiabetic drugs [42].

treatment had a long-term benefit on cardiovascular risk [51].

*DOI: http://dx.doi.org/10.5772/intechopen.88675*

**2.4 Gestational diabetes mellitus (GDM)**

**2.5 Polycystic ovary syndrome (PCOS)**

when using metformin [49, 50].

**2.6 Antitumor or antiaging effect of metformin**

**2.7 Cardiovascular or neuroprotective effects**

function than other diabetic treatments [56].

**2.8 Antipsychotic-induced weight gain**

### **2.4 Gestational diabetes mellitus (GDM)**

*Metformin*

after the 1990s [1].

clinical practice.

**2. Indications**

**2.2 Prediabetes**

globally for use nowadays.

related to the pharmacological properties" [2].

**2.1 Type 2 diabetes mellitus (T2DM)**

be added to metformin treatment [4].

with prior gestational diabetes mellitus [16].

**2.3 Type 1 diabetes mellitus (T1DM)**

makes a particular treatment or procedure inadvisable" [3].

toxicity. The medicine was valued again between the 1940s and 1950s when Jean Sterne observed the low blood glucose values of patients that were treated with metformin for influenza. Since then, the drug class of biguanides has received much consideration, especially buformin and phenformin in the 1970s and metformin

The 60-year history of biguanides' use is filled with victories and defeats, being the oral antidiabetic drug with the most extensive experience of prescribing in the

We will review in the following pages the indications, contraindications, and adverse drug reactions (ADR) of metformin and the single biguanide approved

An ADR according to the World Health Organization is "a response to a drug which is noxious and unintended, and which occurs at doses normally used in man for the prophylaxis, diagnosis, or therapy of a disease, or the modification of physiological function." A side effect is "an unintended effect occurring at normal dose

A contraindication represents "something (such as a symptom or condition) that

All international guidelines consider metformin and lifestyle intervention as the first-line treatment in adults with T2DM in order to improve glycemic control [4]. It can be used either as monotherapy or combination therapy with glucagon-like peptide-1 receptor agonist (GLP-1 RA), sodium-glucose co-transporter inhibitor (SGLT2i), dipeptidyl peptidase-4 inhibitor (DPP4-I), thiazolidinedione (TZD), sulfonylurea (SU), and insulin. Metformin therapy should be continued as long as it is well tolerated and not contraindicated. All other agents, including insulin, should

Metformin can be used in order to prevent or delay the onset of T2DM [5]. Although other pharmacological agents have been used in clinical trials (acarbose [6–8], orlistat [9], and rosiglitazone [10]), it appears that metformin has the most reliable evidence base [11–16]. The vast majority of international guidelines recommend metformin use in prediabetes. It can be used together with a combination of a lifestyle intervention for patients with prediabetes: impaired glucose tolerance (2-h post-load glucose 140–199 mg/dL), fasting hyperglycemia (100–125 mg/dl), or A1C 5.7–6.4% [17–23]. Metformin appears to have a more significant advantage

Metformin is sometimes used in T1DM to limit insulin dose requirement [24, 25].

The American Diabetes Association states that adding metformin leads to the reduction in body weight and can improve lipid levels, but not HbA1c [4, 26]. The REMOVAL study suggests that metformin might also reduce atherosclerosis progression, thus suggesting to improve CVD risk management in type 1 diabetes [27, 28].

or women

when used in patients who are <60 years old and have a BMI >35 kg/m<sup>2</sup>

**4**

Lifestyle modification is the first-line therapy for GDM. If glycemic targets are not achieved, then insulin treatment is required for lowering blood glucose; metformin can also be considered if the patient cannot take or declines insulin [29]. Some controlled randomized trials are proving limited efficacy of metformin during pregnancy [30, 31]. Metformin therapy is associated with a lower risk of neonatal hypoglycemia and less maternal weight gain than insulin in systematic reviews [32–34]; metformin may slightly increase the risk of prematurity, and it crosses the placenta [35]. Thus, the ADA considers that metformin should not be used as first-line agents [36].

### **2.5 Polycystic ovary syndrome (PCOS)**

PCOS patients suffer from insulin resistance and hyperinsulinemia [37]. Metformin has been used for PCOS treatment [38] for treating the metabolic abnormalities of PCOS. A recent meta-analysis [39] demonstrated that metformin could decrease testosterone and insulin level in women with PCOS.

### **2.6 Antitumor or antiaging effect of metformin**

Several studies showed an increased life-span when using metformin (4–6% in different mouse breeds or a mean life-span increased by 14% and maximum life-span increased by 1 month of treatment with metformin is started early in life) [40, 41]. In the United Kingdom Prospective Diabetes Study (UKPDS), the use of metformin decreased the risk of cardiovascular disease, cancer incidence, and overall mortality, compared with other antidiabetic drugs [42].

Epidemiological studies reported a positive result of metformin concerning ovarian [43, 44], breast, prostate, or colorectal tumors [45–48] enhancing the antitumor effect of metformin. Furthermore, studies are demonstrating a reduced incidence of several gastroenterological cancers and a reduction in cancer mortality when using metformin [49, 50].

### **2.7 Cardiovascular or neuroprotective effects**

UKPDS was the first study that demonstrated the cardiovascular benefit of metformin; the risk of all-cause mortality and acute myocardial infarction was significantly reduced in overweight patients with T2DM [42]. The 10-year postinterventional follow-up of the UKPDS survivor cohort revealed that metformin treatment had a long-term benefit on cardiovascular risk [51].

The cardiovascular protective effects of metformin could be explained by the reduced level of LDL cholesterol [52], the limitation of weight gain, [53] and the improvement of oxidative stress, inflammatory response, and the endothelial cell function [54].

It has been reported that patients treated with metformin have lower risk of dementia than those with other diabetes medications [55]. Metformin has a better protective effect on the domain of verbal learning, working memory, and executive function than other diabetic treatments [56].

### **2.8 Antipsychotic-induced weight gain**

Results of meta-analyses of RCTs (primarily in patients with schizophrenia and schizoaffective disorder) support the use of metformin for weight loss, preventing weight gain associated with second-generation antipsychotics in adult patients [57]. Metformin can be recommended as a second-line option after nonpharmacologic strategies for managing weight gain in patients with mood disorders and is recognized as often being used as a secondary prevention strategy for antipsychotic-related weight gain [58].

### **3. Dosage**

The dose for glucose-lowering efficacy is usually in the range of 500–2000 mg/ day. There is no standard dosage regimen for the management of hyperglycemia in patients with type 2 diabetes. On the other side, clinically significant responses are not seen at doses below 1500–2000 mg per day.

The dosage of metformin must be individualized for every patient considering effectiveness and tolerance while not exceeding the maximum recommended daily doses (2550 mg in adults and 2000 mg in pediatric patients >10 years of age) (**Table 1**).

Patients that are receiving immediate-release metformin treatment may be switched to extended form once daily with the same total daily dose (up to 2000 mg daily).

In the case of renal impairment, the dosage of metformin must be adjusted (**Table 2**).


### **Table 1.**

*Dosage of metformin.*


### **Table 2.**

*Dosage of metformin for renal impairment.*

### **4. Adverse drug reactions of metformin**

### **4.1 Lactic acidosis (very rare)**

Phenformin and buformin were two potent biguanides that were used in the 1970s for type 2 diabetes treatment. The Swedish Adverse Drug Reaction Committee

**7**

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications*

patients treated with this biguanide and phenformin [61].

analyzed the reports from 1965 to 1977 that involved biguanides (0.6% of the total). The fact that attracted attention was that in 6% of the cases in which the patient died (the majority with lactic acidosis), phenformin was administered [59]. After this committee report analysis, the class was used with precaution, and metformin was favored over phenformin because there was an early study that showed that type 2 diabetic patients admitted in the hospital had a higher mean lactate level when they were treated with other medicine instead of the first-mentioned earlier [59, 60]. A Cochrane meta-analysis that was published in 2006 that analyzed data from 206 trials and cohort studies did not find any case of lactic acidosis in metformintreated patients or the control group. Also, the lactate level was not significantly raised in the metformin group, although there was a small difference between

A case-control study with 10.652 Danish type 2 diabetic patients showed that the lactic acidosis incidence in patients treated with metformin was 391/100.000 person-years, but the use of the drug itself did not elevate the risk; associated

Systemic allergic reactions to metformin are infrequent [63, 64]. It can be used in patients with asthma that have hypersensitivity, without increasing the risk of related outcomes, meaning hospitalizations, asthma-related emergency room visits,

Cutaneous allergic reactions have been described scarcely ever, but clinicians

In monotherapy as a first-line agent, metformin was proven to be safe and beneficial in a recent meta-analysis. The hypoglycemic risk was lower than for

Rare cases in elderly patients, with comorbidities and polypharmacy (angiotensin-converting enzyme inhibitors or nonsteroidal anti-inflammatory drugs) or

The American Diabetes Association Guidelines recommend that potential vitamin B12 deficiency should be taken into consideration and screened in type 2 diabetes patients long-treated with high-dose metformin (more than 2 g/day) [59]. A meta-analysis of 29 studies showed that the metformin-treated group had a

• It generates bacterial overgrowth because it alters bowel movement [70].

Taste disturbance is an adverse effect that can be caused by the accumulation and secretion of metformin in saliva. Lee N et al. demonstrated that the salivary

significantly lower level of this vitamin [69]. The implied mechanisms are:

• The drug acts as a competitor for vitamin B12 absorption.

*DOI: http://dx.doi.org/10.5772/intechopen.88675*

diseases had greater importance [62].

**4.2 Allergic reactions (infrequent)**

should be aware of their existence [66].

monotherapy with sulfonylurea [67].

**4.4 Vitamin B12 deficiency (rare)**

• It affects the intrinsic factor action.

**4.5 Altered taste (frequent)**

combined with malnutrition, have been described [68].

**4.3 Hypoglycemia (very rare)**

or exacerbations [65].

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications DOI: http://dx.doi.org/10.5772/intechopen.88675*

analyzed the reports from 1965 to 1977 that involved biguanides (0.6% of the total). The fact that attracted attention was that in 6% of the cases in which the patient died (the majority with lactic acidosis), phenformin was administered [59]. After this committee report analysis, the class was used with precaution, and metformin was favored over phenformin because there was an early study that showed that type 2 diabetic patients admitted in the hospital had a higher mean lactate level when they were treated with other medicine instead of the first-mentioned earlier [59, 60].

A Cochrane meta-analysis that was published in 2006 that analyzed data from 206 trials and cohort studies did not find any case of lactic acidosis in metformintreated patients or the control group. Also, the lactate level was not significantly raised in the metformin group, although there was a small difference between patients treated with this biguanide and phenformin [61].

A case-control study with 10.652 Danish type 2 diabetic patients showed that the lactic acidosis incidence in patients treated with metformin was 391/100.000 person-years, but the use of the drug itself did not elevate the risk; associated diseases had greater importance [62].

### **4.2 Allergic reactions (infrequent)**

*Metformin*

**3. Dosage**

(**Table 2**).

Geriatric use

Pediatric use >10 years old

*Dosage of metformin.*

**Table 1.**

Adults Immediate-

Metformin can be recommended as a second-line option after nonpharmacologic strategies for managing weight gain in patients with mood disorders and is recognized as often being used as a secondary prevention strategy for antipsychotic-related weight gain [58].

The dose for glucose-lowering efficacy is usually in the range of 500–2000 mg/ day. There is no standard dosage regimen for the management of hyperglycemia in patients with type 2 diabetes. On the other side, clinically significant responses are

The dosage of metformin must be individualized for every patient considering effectiveness and tolerance while not exceeding the maximum recommended daily doses (2550 mg in adults and 2000 mg in pediatric patients >10 years of age) (**Table 1**). Patients that are receiving immediate-release metformin treatment may be switched

to extended form once daily with the same total daily dose (up to 2000 mg daily). In the case of renal impairment, the dosage of metformin must be adjusted

> 500 mg/daily or 850 mg/daily

> 500 mg/daily or 1000 mg/daily

Extended release Not yet established

not seen at doses below 1500–2000 mg per day.

release metformin

Extended-release metformin

> Immediate release

**4. Adverse drug reactions of metformin**

**Renal impairment eGFR**

Phenformin and buformin were two potent biguanides that were used in the 1970s for type 2 diabetes treatment. The Swedish Adverse Drug Reaction Committee

Initiation Contraindicated Not recommended No dose adjustment

Stop Assess the benefit-risk of

**<30 30–45 >45**

With caution; to start at the low end of the dosing range, assess renal function more frequently

**Initial dose Titration dose Maximum dose**

500 mg/weekly or 850 mg/2 weeks

500 mg/daily 500 mg/weekly 2000 mg/daily

500 mg/weekly 2000 mg/daily

continuing therapy

needed

2550 mg/daily

No dose adjustment needed

**4.1 Lactic acidosis (very rare)**

*Dosage of metformin for renal impairment.*

If eGFR falls during treatment

**6**

**Table 2.**

Systemic allergic reactions to metformin are infrequent [63, 64]. It can be used in patients with asthma that have hypersensitivity, without increasing the risk of related outcomes, meaning hospitalizations, asthma-related emergency room visits, or exacerbations [65].

Cutaneous allergic reactions have been described scarcely ever, but clinicians should be aware of their existence [66].

### **4.3 Hypoglycemia (very rare)**

In monotherapy as a first-line agent, metformin was proven to be safe and beneficial in a recent meta-analysis. The hypoglycemic risk was lower than for monotherapy with sulfonylurea [67].

Rare cases in elderly patients, with comorbidities and polypharmacy (angiotensin-converting enzyme inhibitors or nonsteroidal anti-inflammatory drugs) or combined with malnutrition, have been described [68].

### **4.4 Vitamin B12 deficiency (rare)**

The American Diabetes Association Guidelines recommend that potential vitamin B12 deficiency should be taken into consideration and screened in type 2 diabetes patients long-treated with high-dose metformin (more than 2 g/day) [59]. A meta-analysis of 29 studies showed that the metformin-treated group had a significantly lower level of this vitamin [69]. The implied mechanisms are:


### **4.5 Altered taste (frequent)**

Taste disturbance is an adverse effect that can be caused by the accumulation and secretion of metformin in saliva. Lee N et al. demonstrated that the salivary

glands express the organic cation transporter-3 (OCT3) in high amounts that is responsible for metformin carriage and could be involved in the mechanism of this side effect. In animal studies, the OCT3(−/−) mice, the uptake of metformin in the saliva was downregulated [71].

### **4.6 Gastrointestinal intolerance (widespread)**

Gastrointestinal side effects include diarrhea, nausea, meteorism, and constipation and affect approximately 20% of the patients [71, 72].

The hydrochloride salt of metformin is usually administered orally and is absorbed mostly by the small intestine. The concentration inside the enterocyte can reach up to 300 times the level in the circulation and depends on drug transport by organic cation transporter 1 (OCT1) [67]. Also, metformin increases glucose use in the anaerobic cycle and lactate production inside the enterocyte. Local higher production of lactate could be associated with adverse reactions [73].

Scarpello et al. demonstrated that metformin slows the absorption of bile acids, consequently leading to osmotic diarrhea [74]. On the contrary, the serum measures of lactate, serotonin, or bile acids were similar in normal and intolerant volunteers after a 500-mg dose of metformin, making the authors conclude that the intolerance is probably related to local factors within the lumen or enterocyte [73].

Some authors suggested that a reduced function of OCT1 could have an effect on the tolerability of metformin in the digestive system. The population with a reduced-function OCT1 alleles also had a higher increase of metformin intolerance. If this population was additionally treated with an OCT1 inhibitor, the risk increased even more [75]. Thus, patients that are under treatment with other medications that interact with OCT1 could have a higher risk for gastrointestinal ADR [75].

There are several formulations like the immediate-release (IR) tablets that result in high local concentration, extended-release tablets (XR) that have a prolonged discharge of the active molecule due to a dual polymer matrix, and delayed-release tablets (DR). The XR and the DR forms help in uniformly spreading out molecules along the intestinal membrane and prevent intolerance [75].

### **4.7 Hypothyroidism (controversial)**

Metformin acts by activating adenosine monophosphate-activated protein kinase (AMPK), an enzyme that also activates thyroid iodine in vitro models. Thus, it was assumed that metformin could alter thyroid function [76]. In healthy volunteers, only the level of T3 was decreased by metformin administration, but not the iodine uptake, TSH, or fT4 [76].

Following this idea, observational studies proved that metformin treatment could reduce thyroid-stimulating hormone (TSH) level, but randomized control trials performed afterward failed to certify this hypothesis [77].

### **5. Contraindications**

The indications and efficiency of metformin in type 2 diabetes are clearly stated in current guidelines [4] and continue to extend to other branches of medicine. For example, the UKPDS study revealed that metformin is associated with a lower risk of mortality [37], and some researchers tried to use metformin as an antiaging drug. Besides its broad indications, metformin remains contraindicated in many conditions associated with hypoxemia because it can lead to lactic acidosis [78].

**9**

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications*

decision depending on patient particularities and response [79].

heart failure and type 2 diabetes treated with metformin [82].

In type 2 diabetes patients with severe hyperglycemia and ketoacidosis or type 1 diabetes, insulin treatment should be initiated [4]. When the glycemic values are balanced, and if the patient does not have other contraindications, metformin treat-

In type 1 diabetes, metformin is solely administered as an adjuvant because it can reduce the insulin requirements [25]. A randomized controlled trial found that metformin increases the risk for gastrointestinal adverse events in overweight type 1 diabetes patients, with no benefit for glycemic control, so a clinician should reach a

After the warning regarding lactic acidosis, cardiac failure was put on the list with contraindications. Afterward, observational studies [80] and systematic reviews [81, 82] showed that metformin could be used in stable heart failure. If patients develop congestive heart failure or concomitantly have other contraindications or acute diseases, metformin should be stopped. The studies realized and included in the meta-analysis are very heterogeneous, most of them comparing different medications, but with no specifications regarding the mean dose of metformin or other classes. Overall, the mortality rate was 22% lower in patients with

Metformin is restricted in patients with eGFR less than 30 ml/min/1.73 m2 (stage IV CKD), and dose must be adjusted beginning with an eGFR below 45 ml/ min/1.73 m2 (stage IIIb) [4]. In a cohort study of a national registry, metformin was associated with a lower rate of mortality and serious adverse events at an eGFR between 45 and 60 ml/min/1.73 m2 and had neutral effects on the same variables at eGFR between 30 and 45 ml/min/1.73 m2. Although its effect is less evident in stage IV chronic kidney disease, the benefit of biguanide treatment outweighs the ADR

Impaired hepatic function is another warning from the FDA [64]. This term includes a broad spectrum of liver pathology, and metformin treatment should be tailored. In a retrospective study that included patients with cirrhosis, metformin had a protective effect for encephalopathy development [84]. Likewise, in another retrospective study, biguanide treatment was continued after cirrhosis diagnosis and was associated with improved survival [85]. In patients with cirrhosis secondary to hepatitis C virus infection, the risk of hepatocellular carcinoma was reduced

Because the risk of lactic acidosis is higher in patients with altered blood gas exchange like in chronic obstructive pulmonary disease (COPD), asthma, restrictive

A randomized clinical trial used metformin in a rapidly escalated dose after a COPD exacerbation and showed no amelioration in glycemic profile. This could be since

pulmonary pathologies, the FDA and EMA recommend precaution [63, 64].

*DOI: http://dx.doi.org/10.5772/intechopen.88675*

ment can be started in type 2 diabetes [4].

**5.3 Chronic kidney disease (CKD)**

risk in a 4-year follow-up [82, 83].

**5.4 Hepatic failure and cirrhosis**

during a 5-year follow-up [86].

**5.5 Respiratory insufficiency**

**5.1 Ketoacidosis**

**5.2 Cardiac failure**

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications DOI: http://dx.doi.org/10.5772/intechopen.88675*

### **5.1 Ketoacidosis**

*Metformin*

ADR [75].

saliva was downregulated [71].

**4.6 Gastrointestinal intolerance (widespread)**

tion and affect approximately 20% of the patients [71, 72].

along the intestinal membrane and prevent intolerance [75].

trials performed afterward failed to certify this hypothesis [77].

**4.7 Hypothyroidism (controversial)**

iodine uptake, TSH, or fT4 [76].

**5. Contraindications**

glands express the organic cation transporter-3 (OCT3) in high amounts that is responsible for metformin carriage and could be involved in the mechanism of this side effect. In animal studies, the OCT3(−/−) mice, the uptake of metformin in the

Gastrointestinal side effects include diarrhea, nausea, meteorism, and constipa-

Scarpello et al. demonstrated that metformin slows the absorption of bile acids, consequently leading to osmotic diarrhea [74]. On the contrary, the serum measures of lactate, serotonin, or bile acids were similar in normal and intolerant volunteers after a 500-mg dose of metformin, making the authors conclude that the intolerance is probably related to local factors within the lumen or enterocyte [73].

Some authors suggested that a reduced function of OCT1 could have an effect on the tolerability of metformin in the digestive system. The population with a reduced-function OCT1 alleles also had a higher increase of metformin intolerance. If this population was additionally treated with an OCT1 inhibitor, the risk increased even more [75]. Thus, patients that are under treatment with other medications that interact with OCT1 could have a higher risk for gastrointestinal

There are several formulations like the immediate-release (IR) tablets that result in high local concentration, extended-release tablets (XR) that have a prolonged discharge of the active molecule due to a dual polymer matrix, and delayed-release tablets (DR). The XR and the DR forms help in uniformly spreading out molecules

Metformin acts by activating adenosine monophosphate-activated protein kinase (AMPK), an enzyme that also activates thyroid iodine in vitro models. Thus, it was assumed that metformin could alter thyroid function [76]. In healthy volunteers, only the level of T3 was decreased by metformin administration, but not the

Following this idea, observational studies proved that metformin treatment could reduce thyroid-stimulating hormone (TSH) level, but randomized control

The indications and efficiency of metformin in type 2 diabetes are clearly stated in current guidelines [4] and continue to extend to other branches of medicine. For example, the UKPDS study revealed that metformin is associated with a lower risk of mortality [37], and some researchers tried to use metformin as an antiaging drug. Besides its broad indications, metformin remains contraindicated in many condi-

tions associated with hypoxemia because it can lead to lactic acidosis [78].

The hydrochloride salt of metformin is usually administered orally and is absorbed mostly by the small intestine. The concentration inside the enterocyte can reach up to 300 times the level in the circulation and depends on drug transport by organic cation transporter 1 (OCT1) [67]. Also, metformin increases glucose use in the anaerobic cycle and lactate production inside the enterocyte. Local higher

production of lactate could be associated with adverse reactions [73].

**8**

In type 2 diabetes patients with severe hyperglycemia and ketoacidosis or type 1 diabetes, insulin treatment should be initiated [4]. When the glycemic values are balanced, and if the patient does not have other contraindications, metformin treatment can be started in type 2 diabetes [4].

In type 1 diabetes, metformin is solely administered as an adjuvant because it can reduce the insulin requirements [25]. A randomized controlled trial found that metformin increases the risk for gastrointestinal adverse events in overweight type 1 diabetes patients, with no benefit for glycemic control, so a clinician should reach a decision depending on patient particularities and response [79].

### **5.2 Cardiac failure**

After the warning regarding lactic acidosis, cardiac failure was put on the list with contraindications. Afterward, observational studies [80] and systematic reviews [81, 82] showed that metformin could be used in stable heart failure. If patients develop congestive heart failure or concomitantly have other contraindications or acute diseases, metformin should be stopped. The studies realized and included in the meta-analysis are very heterogeneous, most of them comparing different medications, but with no specifications regarding the mean dose of metformin or other classes. Overall, the mortality rate was 22% lower in patients with heart failure and type 2 diabetes treated with metformin [82].

### **5.3 Chronic kidney disease (CKD)**

Metformin is restricted in patients with eGFR less than 30 ml/min/1.73 m2 (stage IV CKD), and dose must be adjusted beginning with an eGFR below 45 ml/ min/1.73 m2 (stage IIIb) [4]. In a cohort study of a national registry, metformin was associated with a lower rate of mortality and serious adverse events at an eGFR between 45 and 60 ml/min/1.73 m2 and had neutral effects on the same variables at eGFR between 30 and 45 ml/min/1.73 m2. Although its effect is less evident in stage IV chronic kidney disease, the benefit of biguanide treatment outweighs the ADR risk in a 4-year follow-up [82, 83].

### **5.4 Hepatic failure and cirrhosis**

Impaired hepatic function is another warning from the FDA [64]. This term includes a broad spectrum of liver pathology, and metformin treatment should be tailored. In a retrospective study that included patients with cirrhosis, metformin had a protective effect for encephalopathy development [84]. Likewise, in another retrospective study, biguanide treatment was continued after cirrhosis diagnosis and was associated with improved survival [85]. In patients with cirrhosis secondary to hepatitis C virus infection, the risk of hepatocellular carcinoma was reduced during a 5-year follow-up [86].

### **5.5 Respiratory insufficiency**

Because the risk of lactic acidosis is higher in patients with altered blood gas exchange like in chronic obstructive pulmonary disease (COPD), asthma, restrictive pulmonary pathologies, the FDA and EMA recommend precaution [63, 64]. A randomized clinical trial used metformin in a rapidly escalated dose after a COPD exacerbation and showed no amelioration in glycemic profile. This could be since

mean in-hospital glycemia was assessed and it usually takes 1–2 weeks for metformin to reach its maximum hypoglycemic potential; there were no cases of lactic acidosis, and mean serum lactate was similar in the intervention and placebo group [87].

### **6. Special populations**

### **6.1 Children**

Metformin is indicated now in children above 10 years [63, 64], although there were studies that included obese participants above 7 years without side effects [88].

### **6.2 Pregnancy**

There are limited data that could not identify a drug-associated risk of miscarriage or congenital disabilities. Metformin use was not associated with any of these maternal or fetal outcomes in post-marketing studies with small sample size or in meta-analyses of the randomized clinical trials that included pregnant women. The risk of stillbirth, congenital disabilities, and macrosomia can be increased if the patients do not have reasonable control under this oral treatment. Thus, the risk is falsely attributed to metformin [89].

### **6.3 Lactation**

Metformin is present in the human milk in insignificant concentration. The potential adverse effect on the child or milk production has not been described [89].

### **6.4 Elderly**

There is a study which compared pharmacokinetics and pharmacodynamics of metformin in the older population (65–85 years) versus young controls. Results showed that the glucose-lowering effect was similar in both groups, although the maximum concentration and exposure were two times higher in the advanced age population. Usually, it is not recommended in patients above 85 years old because they have a reduced eGFR [90].

### **7. Overdosage**

A retrospective cohort study performed in the emergency department analyzed 56 of self-reported metformin overdose from a total of 2872 cases (1.9%). The incidence of hyperlactatemia was 56.4%, and that of metformin-associated lactic acidosis (MALA) was 17.9%. When the patient is co-ingested with acetaminophen, the risk of MALA was higher. No case resulted in death [91].

Treatment in metformin overdose includes supportive care, gastrointestinal decontamination (gastric lavage), alkalinization, and even emergency hemodialysis in severe cases [92].

### **8. Future directions: metformin and metagenome**

There were some studies on human microbiota, which suggested that metformin induces dysbiosis and promotes nutritional imbalances for specific bacterial types

**11**

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications*

in healthy volunteers [93, 94]. *Escherichia* sp. has a selective advantage over other

Twelve bacterial species that were present at baseline predicted the appearance of gastrointestinal adverse events (self-reported) [94]. Characterizing these bacteria genera and modifying the microbiota composition by pre- and probiotics could improve metformin action. Also, these bacteria could be set as new targets for

Besides its controversial history, metformin remains the most used medicine in type 2 diabetes treatment. Progressive dose increases should be encouraged in order to prevent gastrointestinal adverse effects. Lactic acidosis is obsolete if the patient does not have other severe comorbidities. The indications of metformin currently extend to other areas like oncology, endocrinology, and gastroenterology and should offer the scientific world more information about its adverse effects.

Anca Pantea Stoian, MD, PhD; Cristian Serafinceanu MD, PhD; and Manfredi Rizzo, MD, PhD, were advisory boards for AstraZeneca, Eli Lilly, Merck, Novo Nordisk, Sanofi. Anca Pantea Stoian, MD, PhD, is the Vicepresident of Romanian National Committee of Diabetes, Nutrition and Metabolic Diseases, and speaker for Astra Zeneca, Eli-Lilly, Coca-Cola, NovoNordisk, Sanofi. Manfredi Rizzo, MD, PhD, is the Director, Clinical Medical & Regulatory Affairs, Novo Nordisk Europe East and South. Simona Diana Stefan, MD, received speaker fees from Merck, Novo Nordisk, Sanofi. Andra Iulia Suceveanu, MD, PhD; Adrian Paul Suceveanu, MD,

PhD; and Roxana Adriana Stoica, MD, declare no conflict of interest.

*DOI: http://dx.doi.org/10.5772/intechopen.88675*

organisms [95].

diabetes treatment.

**9. Conclusions**

**Conflict of interest**

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications DOI: http://dx.doi.org/10.5772/intechopen.88675*

in healthy volunteers [93, 94]. *Escherichia* sp. has a selective advantage over other organisms [95].

Twelve bacterial species that were present at baseline predicted the appearance of gastrointestinal adverse events (self-reported) [94]. Characterizing these bacteria genera and modifying the microbiota composition by pre- and probiotics could improve metformin action. Also, these bacteria could be set as new targets for diabetes treatment.

### **9. Conclusions**

*Metformin*

**6. Special populations**

falsely attributed to metformin [89].

they have a reduced eGFR [90].

**6.1 Children**

**6.2 Pregnancy**

**6.3 Lactation**

**6.4 Elderly**

**7. Overdosage**

in severe cases [92].

mean in-hospital glycemia was assessed and it usually takes 1–2 weeks for metformin to reach its maximum hypoglycemic potential; there were no cases of lactic acidosis, and mean serum lactate was similar in the intervention and placebo group [87].

Metformin is indicated now in children above 10 years [63, 64], although there were studies that included obese participants above 7 years without side effects [88].

There are limited data that could not identify a drug-associated risk of miscarriage or congenital disabilities. Metformin use was not associated with any of these maternal or fetal outcomes in post-marketing studies with small sample size or in meta-analyses of the randomized clinical trials that included pregnant women. The risk of stillbirth, congenital disabilities, and macrosomia can be increased if the patients do not have reasonable control under this oral treatment. Thus, the risk is

Metformin is present in the human milk in insignificant concentration. The potential adverse effect on the child or milk production has not been described [89].

There is a study which compared pharmacokinetics and pharmacodynamics of metformin in the older population (65–85 years) versus young controls. Results showed that the glucose-lowering effect was similar in both groups, although the maximum concentration and exposure were two times higher in the advanced age population. Usually, it is not recommended in patients above 85 years old because

A retrospective cohort study performed in the emergency department analyzed

56 of self-reported metformin overdose from a total of 2872 cases (1.9%). The incidence of hyperlactatemia was 56.4%, and that of metformin-associated lactic acidosis (MALA) was 17.9%. When the patient is co-ingested with acetaminophen,

Treatment in metformin overdose includes supportive care, gastrointestinal decontamination (gastric lavage), alkalinization, and even emergency hemodialysis

There were some studies on human microbiota, which suggested that metformin induces dysbiosis and promotes nutritional imbalances for specific bacterial types

the risk of MALA was higher. No case resulted in death [91].

**8. Future directions: metformin and metagenome**

**10**

Besides its controversial history, metformin remains the most used medicine in type 2 diabetes treatment. Progressive dose increases should be encouraged in order to prevent gastrointestinal adverse effects. Lactic acidosis is obsolete if the patient does not have other severe comorbidities. The indications of metformin currently extend to other areas like oncology, endocrinology, and gastroenterology and should offer the scientific world more information about its adverse effects.

### **Conflict of interest**

Anca Pantea Stoian, MD, PhD; Cristian Serafinceanu MD, PhD; and Manfredi Rizzo, MD, PhD, were advisory boards for AstraZeneca, Eli Lilly, Merck, Novo Nordisk, Sanofi. Anca Pantea Stoian, MD, PhD, is the Vicepresident of Romanian National Committee of Diabetes, Nutrition and Metabolic Diseases, and speaker for Astra Zeneca, Eli-Lilly, Coca-Cola, NovoNordisk, Sanofi. Manfredi Rizzo, MD, PhD, is the Director, Clinical Medical & Regulatory Affairs, Novo Nordisk Europe East and South. Simona Diana Stefan, MD, received speaker fees from Merck, Novo Nordisk, Sanofi. Andra Iulia Suceveanu, MD, PhD; Adrian Paul Suceveanu, MD, PhD; and Roxana Adriana Stoica, MD, declare no conflict of interest.

### **Author details**

Roxana Adriana Stoica1†, Diana Simona Ștefan1,2†\*, Manfredi Rizzo3,4†, Andra Iulia Suceveanu5†, Adrian Paul Suceveanu5†, Cristian Serafinceanu1,2† and Anca Pantea-Stoian1†

1 University of Medicine and Pharmacy "Carol Davila" Bucharest, Romania

2 National Institute of Diabetes, Nutrition and Metabolic Diseases "Prof. Paulescu N.C." Bucharest, Romania

3 Biomedical Department of Internal Medicine and Medical Specialties School of Medicine, University of Palermo, Palermo, Italy

4 Division of Endocrinology, Diabetes and Metabolism University of South Carolina School of Medicine Columbia, South Carolina, USA

5 University of Medicine "Ovidius" Constanta, Romania

\*Address all correspondence to: simona\_ds2002@yahoo.com

† All authors are with an equal scientific contribution.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**13**

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications*

Association. 2003;**290**:486-494. DOI:

10.1001/jama.290.4.486

diacare.27.1.155

[9] Torgerson JS, Hauptman J, Boldrin MN, Sjöstrom L. XENical in the prevention of diabetes in obese subjects (XENDOS) study. Diabetes Care. 2004;**27**:155-161. DOI: 10.2337/

[10] Gerstein HC, Yusuf S, Bosch J, Pogue J, Sheridan P, Dinccag N, et al. Effect of rosiglitazone on the frequency of diabetes in patients with impaired glucose tolerance or impaired fasting glucose: A randomised controlled trial. Lancet. 2006;**368**:1096-1105. DOI: 10.1016/S0140-6736(06)69420-8

[11] Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. The New England Journal of Medicine. 2002;**346**:393-403. PMID: 11832527

[12] Knowler WC, Fowler SE, Hamman RF, Christophi CA, Hoffman HJ, Brenneman AT, et al. 10-year follow-up of diabetes incidence

and weight loss in the diabetes prevention program outcomes study. Lancet. 2009;**374**:1677-1686. DOI: 10.1016/S0140-6736(09)61457-4

S0140-6736(10)60746-5

[13] Zinman B, Harris SB, Neuman J, Gerstein HC, Retnakaran RR, Raboud J, et al. Low-dose combination therapy with rosiglitazone and metformin to prevent type 2 diabetes mellitus (CANOE trial): A double-blind randomised controlled study. Lancet. 2010;**376**:103-111. DOI: 10.1016/

[14] Ramachandran A, Snehalatha C, Mary S, Mukesh B, Bhaskar AD, Vijay V. The Indian diabetes prevention

programme shows that lifestyle modification and metformin prevent type 2 diabetes in Asian Indian subjects

*DOI: http://dx.doi.org/10.5772/intechopen.88675*

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S102. DOI: 10.2337/dc19-S009

10.2337/dci18-0062

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randomised trial. Lancet.

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s00125-017-4318-z

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[2] Available at: https://www.who.int/ medicines/areas/quality\_safety/safety\_ efficacy/trainingcourses/definitions.pd

[4] American Diabetes Association et al. Diabetes Care. 2019;**42**(Suppl. 1):S90-

[5] Cefalu WT, Riddle M. More evidence for a prevention-related indication for metformin: Let the arguments resume! Diabetes Care. 2019;**42**:499-501. DOI:

[6] Wenying Y, Lixiang L, Jinwu Q, Guangwei L, Zhiqing Y, Xiaoren P. The preventive effect of acarbose and metformin on the IGT population from becoming diabetes mellitus: A 3-year multicentral prospective study. Chinese Journal of Endocrinology and Metabolism. 2001;**17**:131-134. http:// en.cnki.com.cn/Article\_en/CJFDTotal-

[7] Chiasson JL, Josse RG, Gomis R, Hanefeld M, Karasik A, Laakso M. Acarbose for prevention of type 2 diabetes mellitus: The STOP-NIDDM

2002;**359**:2072-2077. DOI: 10.1016/

[8] Chiasson JL, Josse RG, Gomis R, Hanefeld M, Karasik A, Laakso M. Acarbose treatment and the risk of cardiovascular disease and hypertension in patients with impaired glucose tolerance: The STOP-NIDDM trial. Journal of the American Medical

*Metformin Indications, Dosage, Adverse Reactions, and Contraindications DOI: http://dx.doi.org/10.5772/intechopen.88675*

### **References**

*Metformin*

**Author details**

and Anca Pantea-Stoian1†

N.C." Bucharest, Romania

Medicine, University of Palermo, Palermo, Italy

School of Medicine Columbia, South Carolina, USA

5 University of Medicine "Ovidius" Constanta, Romania

† All authors are with an equal scientific contribution.

provided the original work is properly cited.

\*Address all correspondence to: simona\_ds2002@yahoo.com

Roxana Adriana Stoica1†, Diana Simona Ștefan1,2†\*, Manfredi Rizzo3,4†, Andra Iulia Suceveanu5†, Adrian Paul Suceveanu5†, Cristian Serafinceanu1,2†

1 University of Medicine and Pharmacy "Carol Davila" Bucharest, Romania

2 National Institute of Diabetes, Nutrition and Metabolic Diseases "Prof. Paulescu

3 Biomedical Department of Internal Medicine and Medical Specialties School of

4 Division of Endocrinology, Diabetes and Metabolism University of South Carolina

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**12**

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[2] Available at: https://www.who.int/ medicines/areas/quality\_safety/safety\_ efficacy/trainingcourses/definitions.pd [Accessed at 29.06.2019]

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[5] Cefalu WT, Riddle M. More evidence for a prevention-related indication for metformin: Let the arguments resume! Diabetes Care. 2019;**42**:499-501. DOI: 10.2337/dci18-0062

[6] Wenying Y, Lixiang L, Jinwu Q, Guangwei L, Zhiqing Y, Xiaoren P. The preventive effect of acarbose and metformin on the IGT population from becoming diabetes mellitus: A 3-year multicentral prospective study. Chinese Journal of Endocrinology and Metabolism. 2001;**17**:131-134. http:// en.cnki.com.cn/Article\_en/CJFDTotal-ZHNF200103001.htm

[7] Chiasson JL, Josse RG, Gomis R, Hanefeld M, Karasik A, Laakso M. Acarbose for prevention of type 2 diabetes mellitus: The STOP-NIDDM randomised trial. Lancet. 2002;**359**:2072-2077. DOI: 10.1016/ S0140-6736(02)08905-5

[8] Chiasson JL, Josse RG, Gomis R, Hanefeld M, Karasik A, Laakso M. Acarbose treatment and the risk of cardiovascular disease and hypertension in patients with impaired glucose tolerance: The STOP-NIDDM trial. Journal of the American Medical

Association. 2003;**290**:486-494. DOI: 10.1001/jama.290.4.486

[9] Torgerson JS, Hauptman J, Boldrin MN, Sjöstrom L. XENical in the prevention of diabetes in obese subjects (XENDOS) study. Diabetes Care. 2004;**27**:155-161. DOI: 10.2337/ diacare.27.1.155

[10] Gerstein HC, Yusuf S, Bosch J, Pogue J, Sheridan P, Dinccag N, et al. Effect of rosiglitazone on the frequency of diabetes in patients with impaired glucose tolerance or impaired fasting glucose: A randomised controlled trial. Lancet. 2006;**368**:1096-1105. DOI: 10.1016/S0140-6736(06)69420-8

[11] Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. The New England Journal of Medicine. 2002;**346**:393-403. PMID: 11832527

[12] Knowler WC, Fowler SE, Hamman RF, Christophi CA, Hoffman HJ, Brenneman AT, et al. 10-year follow-up of diabetes incidence and weight loss in the diabetes prevention program outcomes study. Lancet. 2009;**374**:1677-1686. DOI: 10.1016/S0140-6736(09)61457-4

[13] Zinman B, Harris SB, Neuman J, Gerstein HC, Retnakaran RR, Raboud J, et al. Low-dose combination therapy with rosiglitazone and metformin to prevent type 2 diabetes mellitus (CANOE trial): A double-blind randomised controlled study. Lancet. 2010;**376**:103-111. DOI: 10.1016/ S0140-6736(10)60746-5

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[15] Andreadis EA, Katsanou PM, Georgiopoulos DX, Tsourous GI, Yfanti GK, Gouveri ET, et al. The effect of metformin on the incidence of type 2 diabetes mellitus and cardiovascular disease risk factors in overweight and obese subjects– the Carmos study. Experimental and Clinical Endocrinology & Diabetes. 2009;**117**:175-180. DOI: 10.1055/s-0028-1087177

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*Metformin*

with impaired glucose tolerance (IDPP1). Diabetologia. 2006;**49**:289- 297. DOI: 10.1007/s00125-005-0097-z of Australia. 2007;**186**:461-465.

[21] Eastern Mediterranean Regional Office (WHO). Guidelines for the Prevention, Management and Care of Diabetes Mellitus. EMRO Technical Publications Series 32. [Internet] Available at: www.emro.who.int/dsaf/

[22] Committee Canadian Diabetes Association Clinical Practice Guidelines Expert. Reducing the risk of developing diabetes. Canadian Journal of Diabetes.

2013;**37**:S16-S19. DOI: 10.1016/j.

Hildemann S. Therapeutic use of metformin in prediabetes and diabetes prevention. Drugs. 2015;**75**(10): 1071-1094. DOI: 10.1007/ s40265-015-0416-8

[24] What role for metformin in type 1 diabetes? Drug and Therapeutics Bulletin. 2018;**56**(7):78-80. DOI:

[25] Vella S, Buetow L, Royle P, et al. The use of metformin in type 1 diabetes:

[26] Meng H, Zhang A, Liang Y, Hao J, Zhang X, Lu J. Effect of metformin on glycaemic control in patients with type 1 diabetes: A meta-analysis of randomized controlled trials. Diabetes/ Metabolism Research and Reviews. 2018;**34**:e2983r. DOI: 10.1002/dmrr.2983

Ford I, REMOVAL Study Group, et al. Cardiovascular and metabolic effects of metformin in patients with type 1 diabetes (REMOVAL): A double-blind, randomised, placebo-controlled trial. The Lancet Diabetes and Endocrinology.

A systematic review of efficacy. Diabetologia. 2010;**53**:809-820. DOI:

10.1007/s00125-009-1636-9

[27] Petrie JR, Chaturvedi N,

2017;**5**:597-609. DOI: 10.1016/ S2213-8587(17)30194-8

[23] Hostalek U, Gwilt M,

10.1136/dtb.2018.7.0645

PMID:17484708

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[70] Andrès E, Noel E, Goichot B. Metformin-associated vitamin B12 deficiency. Archives of Internal Medicine. 2002;**162**(19):2251-2252.

[71] Lee N, Duan H, Hebert MF, Liang CJ, Rice KM, Wang J. Taste of a pill: Organic cation transporter-3 (OCT3) mediates metformin

DOI: 10.1074/jbc.M114.570564

DOI: 10.1111/dom.13264

DIA628>3.0.CO;2-A

[74] Scarpello JH, Hodgson E,

accumulation and secretion in salivary glands. The Journal of Biological Chemistry. 2014;**289**(39):27055-27064.

[72] McCreight LJ, Bailey CJ, Pearson ER. Metformin and the gastrointestinal tract. Diabetologia. 2016;**59**:426-435. DOI: 10.1007/s00125-015-3844-9

[73] McCreight LJ, Stage TB, Connelly P, et al. Pharmacokinetics of metformin in patients with gastrointestinal intolerance. Diabetes, Obesity & Metabolism. 2018;**20**(7):1593-1601.

Howlett HC. Effect of metformin on bile salt circulation and intestinal motility in type 2 diabetes mellitus. Diabetic Medicine. 1998;**15**:651-656. DOI: 10.1002/ (SICI)1096-9136(199808)15:8<651::AID-

[75] Dujic T, Zhou K, Donnelly LA, Tavendale R, Palmer CN, Pearson ER.

Association of organic cation transporter 1 with intolerance to metformin in type 2 diabetes: A GoDARTS study. Diabetes. 2015;**64**:1786-1793. DOI: 10.2337/

[76] Sloot YJE, Janssen MJR, van Herwaarden AE, et al. The influence of energy depletion by metformin or hypocaloric diet on thyroid iodine uptake in healthy volunteers: A randomized Trial. Scientific Reports.

10.1007/s11739-014-1157-5

PMID: 12390080

**18**

db14-1388

[84] Ampuero J, Ranchal I, Nunez D, Diaz-Herrero Mdel M, Maraver M, del Campo JA, et al. Metformin inhibits glutaminase activity and protects against hepatic encephalopathy. PLoS One. 2012;**7**:e49279. DOI: 10.1371/ journal.pone.0049279

[85] Zhang X, Harmsen WS, Mettler TA, Kim WR, Roberts RO, Therneau TM, et al. Continuation of metformin use after a diagnosis of cirrhosis significantly improves survival of patients with diabetes. Hepatology. 2014;**60**:2008-2016. DOI: 10.1002/ hep.27199

[86] Nkontchou G, Cosson E, Aout M, Mahmoudi A, Bourcier V, Charif I, et al. Impact of metformin on the prognosis of cirrhosis induced by viral hepatitis C in diabetic patients. The Journal of Clinical Endocrinology and Metabolism. 2011;**96**:2601-2608. DOI: 10.1210/ jc.2010-2415

[87] Hitchings AW, Lai D, Jones PW, Baker EH, Metformin in COPD Trial Team. Metformin in severe exacerbations of chronic obstructive pulmonary disease: A randomised controlled trial. Thorax. 2016 Jul;**71**(7):587-593. DOI: 10.1136/ thoraxjnl-2015-208035

[88] Pastor-Villaescusa B, Cañete MD, Caballero-Villarraso J, et al. Metformin for obesity in Prepubertal and pubertal children: A randomized controlled Trial. Pediatrics. 2017;**140**(1):e20164285. DOI: 10.1542/peds.2016-4285

[89] Priya G, Kalra S. Metformin in the management of diabetes during pregnancy and lactation. Drugs Context. 2018;**7**:212523. DOI: 10.7573/ dic.212523

[90] Jang K, Chung H, Yoon JS, Moon SJ, Yoon SH, Yu KS, et al. Pharmacokinetics, safety, and tolerability of metformin in healthy elderly subjects. Journal of Clinical Pharmacology. 2016;**6**(9):1104-1110. DOI: 10.1002/jcph.699

[91] Taub ES, Hoffman RS, Manini AF. Incidence and risk factors for hyperlactatemia in ED patients with acute metformin overdose. The American Journal of Emergency Medicine. 2019;(19):30184-30186. DOI: 10.1016/j.ajem.2019.03.033

[92] Wang GS, Hoyte C. Review of Biguanide (metformin) toxicity. Journal of Intensive Care Medicine. 2018;**21**:885066618793385. DOI: 10.1177/0885066618793385

[93] Elbere I, Kalnina I, Silamikelis I, et al. Association of metformin administration with gut microbiome dysbiosis in healthy volunteers. PLoS One. 2018;**13**(9):e0204317. DOI: 10.1371/ journal.pone.0204317

[94] Bryrup T, Thomsen CW, Kern T, et al. Metformin-induced changes of the gut microbiota in healthy young men: Results of a non-blinded, onearmed intervention study. Diabetologia. 2019;**62**(6):1024-1035. DOI: 10.1007/ s00125-019-4848-7

[95] Rosario D, Benfeitas R, Bidkhori G, et al. Understanding the representative gut microbiota dysbiosis in metformintreated type 2 diabetes patients using genome-scale metabolic modeling. Frontiers in Physiology. 2018;**9**:775. DOI: 10.3389/fphys.2018.00775

**21**

**Chapter 2**

**Abstract**

diseases.

**1. Introduction**

cardiovascular, neurodegenerative

prospect for clinical application.

Application

New Insight into Metformin

*Yun Yan, Karen L. Kover and Wayne V. Moore*

Mechanism of Action and Clinical

Metformin is the first-line medication for Type 2 diabetes (T2D) treatment, and it is the only US FDA approved oral antidiabetic medication for pediatric patients with T2D 10 years and older. Metformin is also used to treat polycystic ovary syndrome (PCOS), another condition with underlying insulin resistance. The clinical applications of metformin are continuing to expand into other fields including cancer, aging, cardiovascular diseases, and neurodegenerative diseases. Metformin modulates multiple biological pathways. Its novel properties and effects continue to evolve; however, its molecular mechanism of action remains incompletely understood. In this chapter, we focus on the recent translational research and clinical data on the molecular action of metformin and the evidence linking the effects of metformin on insulin resistance, prediabetes, diabetes, aging, cancer, PCOS, cardiovascular diseases, and neurodegenerative

**Keywords:** metformin, insulin, insulin resistance, diabetes, aging, PCOS, cancer,

Synthesis of metformin was reported in 1922 and its effect of lowering glucose was reported soon after. Metformin was first reported to be used for the treatment of diabetes by French physician Jean Steme in 1957. The effect of metformin on improvement of morbidity and mortality in type 2 diabetes (T2D) was confirmed in the United Kingdom Prospective Diabetes Study (UKPDS), a large clinical trial performed in 1980–1990s [1]. It was approved for T2D treatment in adults by US FDA in 1994 and for pediatric patients 10 years and older in 2000. Metformin is prescribed world-wide as the first-line oral drug for adults and children with T2D. Its physiological effects related to T2D include increase in insulin sensitivity, reduction of gluconeogenesis in the liver, enhanced glucose uptake by muscle, and reduced intestinal glucose absorption. Several molecular mechanisms of action have been proposed but more remain to be discovered. In this chapter, we will review molecular mechanisms of action of metformin and its

### **Chapter 2**

## New Insight into Metformin Mechanism of Action and Clinical Application

*Yun Yan, Karen L. Kover and Wayne V. Moore*

### **Abstract**

Metformin is the first-line medication for Type 2 diabetes (T2D) treatment, and it is the only US FDA approved oral antidiabetic medication for pediatric patients with T2D 10 years and older. Metformin is also used to treat polycystic ovary syndrome (PCOS), another condition with underlying insulin resistance. The clinical applications of metformin are continuing to expand into other fields including cancer, aging, cardiovascular diseases, and neurodegenerative diseases. Metformin modulates multiple biological pathways. Its novel properties and effects continue to evolve; however, its molecular mechanism of action remains incompletely understood. In this chapter, we focus on the recent translational research and clinical data on the molecular action of metformin and the evidence linking the effects of metformin on insulin resistance, prediabetes, diabetes, aging, cancer, PCOS, cardiovascular diseases, and neurodegenerative diseases.

**Keywords:** metformin, insulin, insulin resistance, diabetes, aging, PCOS, cancer, cardiovascular, neurodegenerative

### **1. Introduction**

Synthesis of metformin was reported in 1922 and its effect of lowering glucose was reported soon after. Metformin was first reported to be used for the treatment of diabetes by French physician Jean Steme in 1957. The effect of metformin on improvement of morbidity and mortality in type 2 diabetes (T2D) was confirmed in the United Kingdom Prospective Diabetes Study (UKPDS), a large clinical trial performed in 1980–1990s [1]. It was approved for T2D treatment in adults by US FDA in 1994 and for pediatric patients 10 years and older in 2000. Metformin is prescribed world-wide as the first-line oral drug for adults and children with T2D. Its physiological effects related to T2D include increase in insulin sensitivity, reduction of gluconeogenesis in the liver, enhanced glucose uptake by muscle, and reduced intestinal glucose absorption. Several molecular mechanisms of action have been proposed but more remain to be discovered. In this chapter, we will review molecular mechanisms of action of metformin and its prospect for clinical application.

### **2. Mechanisms of action**

The potential mechanisms of metformin action involve several pathways. The AMPK-pathway plays an important role in metformin actions [2, 3]. Metformin inhibits the mitochondrial respiratory chain (complex I), which increases the AMP to ATP ratio, leading to the phosphorylation of AMP-activated protein kinase (AMPK) at Thr-172. We have demonstrated that metformin treatment increases protein level of phosphorylated AMPK in high-glucose-treated endothelial cells [4]. The phosphorylated AMPK subsequently phosphorylates multiple downstream effectors to regulate cellular metabolism and energy homeostasis [5]. These downstream effectors include thioredoxin interacting protein (TXNIP) and TBC1D1, a RAB-GTPase activating protein and a member of the tre-2/BUB2/cdc1 domain family. Phosphorylated TXNIP and TBC1D1 increase the plasma membrane localization of glucose transporter 1 (GLUT1) and GLUT4, respectively [6, 7], and regulate glycogen synthases (GYS1 and GYS2) to prevent the storage of glycogen [8]. Some actions of metformin have been found to be AMPK-independent [9].

In diabetic mice, metformin has an effect on gut microbiota by inducing a profound shift in the gut microbial community profile, resulting in an increase in the Akkermansia spp. population [10] and cAMP-induced agmatine production [11], which may decrease absorption of glucose from the gastrointestinal tract and increase lipid metabolism respectively. In addition, metformin decreases insulin-induced suppression of fatty acid oxidation and lowers lipid content of hepatic cells [12].

### **3. Insulin resistance**

Insulin resistance (IR) is a condition in which the cellular response to insulin is decreased resulting in elevated insulin levels (hyperinsulinism). When the beta cells are not able to overcome the resistance by producing more insulin, hyperglycemia develops. Insulin resistance is more prevalent in certain racial populations suggesting a genetic basis for the resistance. The major "environmental" risk factors for insulin resistance are obesity and sedentary lifestyle. Exercise and weight loss are established approaches to improve insulin sensitivity and decrease insulin resistance [13]. Insulin resistance may also be the basis for polycystic ovary syndrome (PCOS) in women. Some studies have suggested that metabolic syndrome (insulin resistance, type 2 diabetes, obesity, hyperlipidemia, and hypertension) and PCOS (insulin resistance, hyperandrogenism, amenorrhea, non-obese) are the ends of a spectrum of insulin resistance. The loss of microvascular insulin response and reduction of muscle glucose uptake are early events in the pathogenesis of insulin resistance [14, 15].

Metformin can increase insulin receptor tyrosine kinase activity, enhance glycogen synthesis, and increase the recruitment and activity of GLUT4 glucose transporters. In high-fat-diet-fed insulin resistant rats, metformin improved the insulin sensitivity of vascular and skeletal muscle and restored glucose uptake in insulin resistant skeletal muscle [16]. In adipose tissue, metformin promoted the reesterification of free fatty acids and inhibited lipolysis, which indirectly improved insulin sensitivity through reduced lipotoxicity [17].

Insulin resistance is a risk factor for the development of T2D [18] and occurs earlier than hyperglycemia. Blood-based biomarker that identify insulin resistance earlier than current glycemia-based approaches, including fasting glucose and HbA1C [19] might identify individual's at risk for developing diabetes, and provide a novel tool to monitor metformin treatment in the high risk population. Several blood-based biomarkers of insulin resistance have been identified [19]. Branchedchain amino acids [20] and asymmetric dimethylarginine (ADMA) [21] show an

**23**

**Figure 1.**

*New Insight into Metformin Mechanism of Action and Clinical Application*

Akt1 deficient mice do not exhibit diabetes phenotypes [32, 33].

the cell membrane surface and thereby increases glucose uptake [35].

association with insulin resistance. Metformin decreases the level of circulating branched-chain amino acids and reduces insulin resistance in a high-fat diet mouse model [22]. Metformin treatment lowers plasma ADMA which is associated with

Recent studies indicate that phosphatidylinositol-3-kinase/protein kinase B protein (PI3K/PKB, also known as Akt) signaling pathway is associated with insulin resistance, and plays a critical role in insulin stimulation of glucose transport into cells [24–30]. The key molecules involved in this pathway are PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), and phosphoinositide 3.4.5

Akt has three isoforms Akt1, Akt2 and Akt3 (also referred to as protein kinase B (PKB) α, −β and –γ, respectively). Their domain structures are similar, including a pleckstrin homology (PH) kinase domain at the amino-terminal and a hydrophobic motif (HM) domain at the carboxyl-terminal [31]. Three isoforms share many substrates, but each isoform also has specific substrate. Akt2 is specific for the insulin signaling pathway and plays a critical role in glucose homeostasis. Akt2 deficient mice have insulin resistance, hyperglycemia, and loss of pancreatic β cells while

PIP3 binds to PDK1 and Akt protein and recruits Akt protein to the plasma membrane. PDK1 phosphorylates Akt at Thr308/309 of Akt1/Akt2, respectively of the kinase domain leading to partial Akt activation. PI3K might directly phosphorylate Akt1 at Thr308 [34]. Full Akt activation is associated with a second PI3K phosphorylation of Akt at Ser473/474 of Akt1/Akt2, respectively in the carboxylterminal hydrophobic motif [34]. Subsequently, the phosphorylated Akt2 recruits insulin-regulated GLUT1 and GLUT4 glucose transporters from the cytoplasm onto

GLUT1 is an insulin independent transporter whereas GLUT4 is an insulin dependent transporter. Insulin increases GLUT4 in the cell membrane and promotes the glucose transport into muscle and fatty cells (**Figure 1**). Any defect in Akt pathway along with the downstream molecules could result in insulin resistance [29]. Clinical data indicate that acute myocardial insulin resistance that occurs after cardiac surgery with cardiopulmonary bypass is attributed to Akt inactivation.

*Insulin binds to insulin receptor and induces its dimerization and auto phosphorylation of tyrosine residues in two transmembrane β subunits, which further lead to the phosphorylation of tyrosine residues on the IRS protein. These molecules can further activate PI3K, resulting in activation of PDK1/2. AKT is recruited and gets phosphorylated by PDK1/2. Once activated, AKT promotes GLUT4 translocation to plasma membrane and* 

*facilitates glucose into cell. TXNIP inhibits glucose transporter by promoting GLUT4 endocytosis.*

*DOI: http://dx.doi.org/10.5772/intechopen.91148*

trisphosphate (PIP3).

improved glycemic control in patients with T2D [23].

### *New Insight into Metformin Mechanism of Action and Clinical Application DOI: http://dx.doi.org/10.5772/intechopen.91148*

*Metformin*

**2. Mechanisms of action**

found to be AMPK-independent [9].

insulin sensitivity through reduced lipotoxicity [17].

**3. Insulin resistance**

The potential mechanisms of metformin action involve several pathways. The AMPK-pathway plays an important role in metformin actions [2, 3]. Metformin inhibits the mitochondrial respiratory chain (complex I), which increases the AMP to ATP ratio, leading to the phosphorylation of AMP-activated protein kinase (AMPK) at Thr-172. We have demonstrated that metformin treatment increases protein level of phosphorylated AMPK in high-glucose-treated endothelial cells [4]. The phosphorylated AMPK subsequently phosphorylates multiple downstream effectors to regulate cellular metabolism and energy homeostasis [5]. These downstream effectors include thioredoxin interacting protein (TXNIP) and TBC1D1, a RAB-GTPase activating protein and a member of the tre-2/BUB2/cdc1 domain family. Phosphorylated TXNIP and TBC1D1 increase the plasma membrane localization of glucose transporter 1 (GLUT1) and GLUT4, respectively [6, 7], and regulate glycogen synthases (GYS1 and GYS2) to prevent the storage of glycogen [8]. Some actions of metformin have been

In diabetic mice, metformin has an effect on gut microbiota by inducing a profound shift in the gut microbial community profile, resulting in an increase in the Akkermansia spp. population [10] and cAMP-induced agmatine production [11], which may decrease absorption of glucose from the gastrointestinal tract and increase lipid metabolism respectively. In addition, metformin decreases insulin-induced suppression of fatty acid oxidation and lowers lipid content of hepatic cells [12].

Insulin resistance (IR) is a condition in which the cellular response to insulin is decreased resulting in elevated insulin levels (hyperinsulinism). When the beta cells are not able to overcome the resistance by producing more insulin, hyperglycemia develops. Insulin resistance is more prevalent in certain racial populations suggesting a genetic basis for the resistance. The major "environmental" risk factors for insulin resistance are obesity and sedentary lifestyle. Exercise and weight loss are established approaches to improve insulin sensitivity and decrease insulin resistance [13]. Insulin resistance may also be the basis for polycystic ovary syndrome (PCOS) in women. Some studies have suggested that metabolic syndrome (insulin resistance, type 2 diabetes, obesity, hyperlipidemia, and hypertension) and PCOS (insulin resistance, hyperandrogenism, amenorrhea, non-obese) are the ends of a spectrum of insulin resistance. The loss of microvascular insulin response and reduction of muscle glucose uptake are early events in the pathogenesis of insulin resistance [14, 15]. Metformin can increase insulin receptor tyrosine kinase activity, enhance glycogen synthesis, and increase the recruitment and activity of GLUT4 glucose transporters. In high-fat-diet-fed insulin resistant rats, metformin improved the insulin sensitivity of vascular and skeletal muscle and restored glucose uptake in insulin resistant skeletal muscle [16]. In adipose tissue, metformin promoted the reesterification of free fatty acids and inhibited lipolysis, which indirectly improved

Insulin resistance is a risk factor for the development of T2D [18] and occurs earlier than hyperglycemia. Blood-based biomarker that identify insulin resistance earlier than current glycemia-based approaches, including fasting glucose and HbA1C [19] might identify individual's at risk for developing diabetes, and provide a novel tool to monitor metformin treatment in the high risk population. Several blood-based biomarkers of insulin resistance have been identified [19]. Branchedchain amino acids [20] and asymmetric dimethylarginine (ADMA) [21] show an

**22**

association with insulin resistance. Metformin decreases the level of circulating branched-chain amino acids and reduces insulin resistance in a high-fat diet mouse model [22]. Metformin treatment lowers plasma ADMA which is associated with improved glycemic control in patients with T2D [23].

Recent studies indicate that phosphatidylinositol-3-kinase/protein kinase B protein (PI3K/PKB, also known as Akt) signaling pathway is associated with insulin resistance, and plays a critical role in insulin stimulation of glucose transport into cells [24–30]. The key molecules involved in this pathway are PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), and phosphoinositide 3.4.5 trisphosphate (PIP3).

Akt has three isoforms Akt1, Akt2 and Akt3 (also referred to as protein kinase B (PKB) α, −β and –γ, respectively). Their domain structures are similar, including a pleckstrin homology (PH) kinase domain at the amino-terminal and a hydrophobic motif (HM) domain at the carboxyl-terminal [31]. Three isoforms share many substrates, but each isoform also has specific substrate. Akt2 is specific for the insulin signaling pathway and plays a critical role in glucose homeostasis. Akt2 deficient mice have insulin resistance, hyperglycemia, and loss of pancreatic β cells while Akt1 deficient mice do not exhibit diabetes phenotypes [32, 33].

PIP3 binds to PDK1 and Akt protein and recruits Akt protein to the plasma membrane. PDK1 phosphorylates Akt at Thr308/309 of Akt1/Akt2, respectively of the kinase domain leading to partial Akt activation. PI3K might directly phosphorylate Akt1 at Thr308 [34]. Full Akt activation is associated with a second PI3K phosphorylation of Akt at Ser473/474 of Akt1/Akt2, respectively in the carboxylterminal hydrophobic motif [34]. Subsequently, the phosphorylated Akt2 recruits insulin-regulated GLUT1 and GLUT4 glucose transporters from the cytoplasm onto the cell membrane surface and thereby increases glucose uptake [35].

GLUT1 is an insulin independent transporter whereas GLUT4 is an insulin dependent transporter. Insulin increases GLUT4 in the cell membrane and promotes the glucose transport into muscle and fatty cells (**Figure 1**). Any defect in Akt pathway along with the downstream molecules could result in insulin resistance [29]. Clinical data indicate that acute myocardial insulin resistance that occurs after cardiac surgery with cardiopulmonary bypass is attributed to Akt inactivation.

### **Figure 1.**

*Insulin binds to insulin receptor and induces its dimerization and auto phosphorylation of tyrosine residues in two transmembrane β subunits, which further lead to the phosphorylation of tyrosine residues on the IRS protein. These molecules can further activate PI3K, resulting in activation of PDK1/2. AKT is recruited and gets phosphorylated by PDK1/2. Once activated, AKT promotes GLUT4 translocation to plasma membrane and facilitates glucose into cell. TXNIP inhibits glucose transporter by promoting GLUT4 endocytosis.*

Inactivated Akt impairs the membrane transposition of GLUT4, which results in insulin resistance accompanied with hyperinsulinemia, hyperglycemia and cardiac dysfunction [36]. It has been reported that metformin attenuates insulin resistance by restoring PI3K/Akt/GLUT4 signaling in the hepatocytes of T2D rats [37]. Metformin combined with phloretin, a dihydrochalcone found in fruits, promoted glucose consumption and suppressed gluconeogenesis in skeletal muscle via PI3K/ Akt/GlUT4 signaling pathway in T2D rat models [38].

TXNIP is being considered as a novel mediator of insulin resistance [39, 40]. TXNIP induced by high-glucose concentration is a key intracellular regulator of glucose and lipid metabolism [6]. We have demonstrated that metformin improves endothelial cell function via down-regulation of high-glucose-induced TXNIP transcription [4].

Over expression of TXNIP induces apoptosis of pancreatic β cells and endothelial cells, decreases muscle and adipose insulin sensitivity, promotes GLUT4 endocytosis and reduces glucose uptake in myocytes and adipocytes [4, 41–43]. Reduction of TXNIP expression by RNA interference gene-silencing significantly improves insulin induced glucose uptake in cultured human skeletal muscle cells [41]. TXNIP knockout mice had improved insulin sensitivity and increased glucose uptake in both adipose and skeletal muscle [39]. In PCOS, metformin improved insulin resistance in a PCOS rat model via an AMPK alpha-SIRT1 pathway [44].

### **4. Prediabetes**

New criteria defining prediabetes includes the presence of one or more of the following, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and HbA1C of 5.7–6.4% [45]. The progression from prediabetes to diabetes is related to insulin resistance and β-cell dysfunction. Prediabetes is a serious health condition which increases the risk of developing T2D, heart disease and stroke. In the US, approximately 84 million American adults (more than 1 out of 3) have prediabetes but 90% patients with prediabetes are not aware of their condition [46]. Metformin improves insulin sensitivity and provides an attractive pharmacological intervention for prediabetes [47, 48]. Results from several clinical trials in the prediabetes population, including children, adolescents and adults, have indicated that metformin can delay or halt the progression from prediabetes to diabetes [49–51]. Metformin is generally well tolerated and has no significant safety issues with long-term use for diabetes prevention [48]. In the long-term "Diabetes Prevention Program Outcomes Study (DPPOS)", either lifestyle intervention or metformin significantly reduced diabetes development over 15 years. Lifestyle intervention has been shown similar or greater effectiveness than metformin in clinical trials [52] and remains the cornerstone of care for patients with prediabetes. However, lifestyle interventions are difficult for patients to maintain and often fail to control weight over the long term. Metformin therapy was shown to be just as effective as lifestyle intervention in individual with prediabetes <60 years of age, BMI ≥ 35 kg/m2, and in women with a history of gestational diabetes mellitus [51, 53]. A study showed that metformin was underused in patients with prediabetes and only 3.7% of adult patients with prediabetes were prescribed metformin [54]. Currently metformin is not approved by FDA for prediabetes. Overweight patients with comorbidities may be at increased risk of diabetes. New guidelines recommended that metformin therapy for T2D prevention should be considered in those with prediabetes, especially those with BMI ≥ 35 kg/m2 , those aged <60 years, and women with prior

**25**

*New Insight into Metformin Mechanism of Action and Clinical Application*

tolerability, and the characteristics of each patient [58].

vitamin B12 level is recommended [65].

gestational diabetes mellitus [55]. The combinations of metformin with lifestyle or other treatments have shown more beneficial effects in diabetes prevention [48, 49].

Metformin is approved for use in patients with T2D. It is still under debated whether metformin can be an adjunct therapy for T1D though many overweight T1D patients have been prescribed metformin due to its beneficial effects on

Metformin is considered first-line therapy to treat T2D due to its blood glucoselowering effects, safety and relatively low cost. Metformin lowers blood glucose level by decreasing glucose production in liver, reducing intestinal glucose absorption, increasing insulin sensitivity and promoting muscle glucose uptake in muscle. Metformin treatment can be combined with lifestyle modification and other antidiabetic drugs, such as dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 (GLP-1) receptor agonists or sodium-glucose cotransporter-2 (SGLT2) [56, 57]. Combined therapy is individualized depending on effectiveness, safety,

Metformin is safe and tolerable with the exception of the risk of lactic acidosis in patients with risk factors for lactic acidosis [59], including impairment of renal, cardiac, and hepatic function [60–62]. Another concern is metformin-induced vitamin B12 deficiency; patients who receive long-term metformin treatment (>6 months) at large doses have developed B12 deficiency [63, 64], so that annual screening of

Insulin resistance in T1D patients may contribute to poor glycemic control and is associated with increased insulin dose requirement [66]. Metformin treatment has been shown to increase insulin sensitivity, improve glycemic control, and reduce cardiovascular risk in patients with T1D [67]. The studies reported that metformin used as an adjunct therapy in T1D reduced insulin dose and body weight with no improvement in HbA1c and glycemic control [68, 69]. Another short term adjunct therapy with metformin demonstrated improved glycemic control, insulin sensitivity, and quality of life without weight gain, while long-term (2 years) metformin treatment was associated with decreased BMI [70]. A 1 year retrospective investigation reported an association between metformin as adjunct therapy and decreased glucose levels, decreased prevalence metabolic syndrome traits, and decreased

Metformin was shown to be safe and effective for treatment of pediatric patients

with T2D age 10 to 16 years old [72]. Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) recruited 699 youth and adolescents over a 4-year period. In this cohort study, metformin was used alone or in combination with life style modification or other antidiabetics drugs [73]. Metformin treatment was associated with decreased HbA1c and improved glycemic control in more than

*DOI: http://dx.doi.org/10.5772/intechopen.91148*

**5. Diabetes**

**5.1 Adult T2D**

**5.2 Adult T1D**

insulin dose [71].

**5.3 Pediatric T2D**

improving insulin resistance.

gestational diabetes mellitus [55]. The combinations of metformin with lifestyle or other treatments have shown more beneficial effects in diabetes prevention [48, 49].

### **5. Diabetes**

*Metformin*

transcription [4].

pathway [44].

**4. Prediabetes**

Inactivated Akt impairs the membrane transposition of GLUT4, which results in insulin resistance accompanied with hyperinsulinemia, hyperglycemia and cardiac dysfunction [36]. It has been reported that metformin attenuates insulin resistance by restoring PI3K/Akt/GLUT4 signaling in the hepatocytes of T2D rats [37]. Metformin combined with phloretin, a dihydrochalcone found in fruits, promoted glucose consumption and suppressed gluconeogenesis in skeletal muscle via PI3K/

TXNIP is being considered as a novel mediator of insulin resistance [39, 40]. TXNIP induced by high-glucose concentration is a key intracellular regulator of glucose and lipid metabolism [6]. We have demonstrated that metformin improves endothelial cell function via down-regulation of high-glucose-induced TXNIP

Over expression of TXNIP induces apoptosis of pancreatic β cells and endothelial cells, decreases muscle and adipose insulin sensitivity, promotes GLUT4 endocytosis and reduces glucose uptake in myocytes and adipocytes [4, 41–43]. Reduction of TXNIP expression by RNA interference gene-silencing significantly improves insulin induced glucose uptake in cultured human skeletal muscle cells [41]. TXNIP knockout mice had improved insulin sensitivity and increased glucose uptake in both adipose and skeletal muscle [39]. In PCOS, metformin improved insulin resistance in a PCOS rat model via an AMPK alpha-SIRT1

New criteria defining prediabetes includes the presence of one or more of the following, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and HbA1C of 5.7–6.4% [45]. The progression from prediabetes to diabetes is related to insulin resistance and β-cell dysfunction. Prediabetes is a serious health condition which increases the risk of developing T2D, heart disease and stroke. In the US, approximately 84 million American adults (more than 1 out of 3) have prediabetes but 90% patients with prediabetes are not aware of their condition [46]. Metformin improves insulin sensitivity and provides an attractive pharmacological intervention for prediabetes [47, 48]. Results from several clinical trials in the prediabetes population, including children, adolescents and adults, have indicated that metformin can delay or halt the progression from prediabetes to diabetes [49–51]. Metformin is generally well tolerated and has no significant safety issues with long-term use for diabetes prevention [48]. In the long-term "Diabetes Prevention Program Outcomes Study (DPPOS)", either lifestyle intervention or metformin significantly reduced diabetes development over 15 years. Lifestyle intervention has been shown similar or greater effectiveness than metformin in clinical trials [52] and remains the cornerstone of care for patients with prediabetes. However, lifestyle interventions are difficult for patients to maintain and often fail to control weight over the long term. Metformin therapy was shown to be just as effective as lifestyle intervention in individual with prediabetes <60 years of age, BMI ≥ 35 kg/m2, and in women with a history of gestational diabetes mellitus [51, 53]. A study showed that metformin was underused in patients with prediabetes and only 3.7% of adult patients with prediabetes were prescribed metformin [54]. Currently metformin is not approved by FDA for prediabetes. Overweight patients with comorbidities may be at increased risk of diabetes. New guidelines recommended that metformin therapy for T2D prevention should be considered in those with prediabetes,

, those aged <60 years, and women with prior

Akt/GlUT4 signaling pathway in T2D rat models [38].

**24**

especially those with BMI ≥ 35 kg/m2

Metformin is approved for use in patients with T2D. It is still under debated whether metformin can be an adjunct therapy for T1D though many overweight T1D patients have been prescribed metformin due to its beneficial effects on improving insulin resistance.

### **5.1 Adult T2D**

Metformin is considered first-line therapy to treat T2D due to its blood glucoselowering effects, safety and relatively low cost. Metformin lowers blood glucose level by decreasing glucose production in liver, reducing intestinal glucose absorption, increasing insulin sensitivity and promoting muscle glucose uptake in muscle. Metformin treatment can be combined with lifestyle modification and other antidiabetic drugs, such as dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 (GLP-1) receptor agonists or sodium-glucose cotransporter-2 (SGLT2) [56, 57]. Combined therapy is individualized depending on effectiveness, safety, tolerability, and the characteristics of each patient [58].

Metformin is safe and tolerable with the exception of the risk of lactic acidosis in patients with risk factors for lactic acidosis [59], including impairment of renal, cardiac, and hepatic function [60–62]. Another concern is metformin-induced vitamin B12 deficiency; patients who receive long-term metformin treatment (>6 months) at large doses have developed B12 deficiency [63, 64], so that annual screening of vitamin B12 level is recommended [65].

### **5.2 Adult T1D**

Insulin resistance in T1D patients may contribute to poor glycemic control and is associated with increased insulin dose requirement [66]. Metformin treatment has been shown to increase insulin sensitivity, improve glycemic control, and reduce cardiovascular risk in patients with T1D [67]. The studies reported that metformin used as an adjunct therapy in T1D reduced insulin dose and body weight with no improvement in HbA1c and glycemic control [68, 69]. Another short term adjunct therapy with metformin demonstrated improved glycemic control, insulin sensitivity, and quality of life without weight gain, while long-term (2 years) metformin treatment was associated with decreased BMI [70]. A 1 year retrospective investigation reported an association between metformin as adjunct therapy and decreased glucose levels, decreased prevalence metabolic syndrome traits, and decreased insulin dose [71].

### **5.3 Pediatric T2D**

Metformin was shown to be safe and effective for treatment of pediatric patients with T2D age 10 to 16 years old [72]. Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) recruited 699 youth and adolescents over a 4-year period. In this cohort study, metformin was used alone or in combination with life style modification or other antidiabetics drugs [73]. Metformin treatment was associated with decreased HbA1c and improved glycemic control in more than

half of the participants. Metformin plus rosiglitazone was significantly better than metformin monotherapy [74].

### **5.4 Pediatric T1D**

Using metformin to improve glycemic control and insulin sensitivity in youth and adolescents with T1D has been reported in several clinical trials. Studies that report a positive association of metformin have reported: 1. Decreased insulin dose, BMI and waist circumference in adolescents with T1D [75]. 2. Lower daily insulin dose improved whole-body and peripheral insulin resistance in adolescents with T1D who were overweight/obese [76]. 3. Lower insulin dose and improved vascular smooth muscle function and HbA1c children with T1D [77]. 4. Decreased cardiovascular disease risk factors in youth with T1D [78]. 5. Improvement in HbA1c level in adolescents with T1D [79, 80]. In contrast, some trials did not observe improvement in HbA1c [76, 81], or glycemic control. As expected, there was an increased gastrointestinal adverse event in overweight adolescents with T1D [81].

### **6. Aging**

Metformin has attracted interest for its potential effects on aging [82]. Metformin treatment has a positive association with reduction in the incidence of mortality from age-related diseases including diabetes, cancer, cardiovascular diseases, and neurodegenerative diseases. Metformin is reported to increase lifespan in several animal models. Cohort clinical trials, Metformin in Longevity Study (MILES) and Targeting Aging with Metformin (TAME), have been initiated to investigate metformin's anti-aging effects in human.

In several animal models, including nematodes and rodents, metformin has been shown to delay aging. Metformin treated female outbred mice (100 mg/kg in drinking water) showed an increased mean lifespan 37.8% [83]. The effects of metformin treatment were shown to be age dependent in mice. When treatment was started at the early stage of life, middle-age and late stages of life, the mean lifespan was increased by 21%, 7% and 13% respectively compared to the controls [84]. In a mouse breast cancer model, metformin delayed the onset of mammary adenocarcinoma and increased lifespan by a mean of 8% compared to the control group [85]. Metformin prolonged the survival time of male mice with Huntington's disease by 21.1%, but had no effects in female [86]. A recent study found that metformin reduced oxidative stress and inflammation, extended both lifespan and healthspan by 4–6% in different strains of mice, and attenuated the deleterious effects of aging in male mice [87].

Gut microbiota has been shown to affect health status and longevity and play a role in resistance to infection, inflammation, autoimmunity, and cancer, and the regulation of the brain-gut axis [88, 89]. Metformin acts directly on gut bacteria to decrease absorption of glucose, improve lipid metabolism and elevate agmatine production to extend host lifespan [10, 90].

The reported effects of metformin on microbiota and animals have promoted interest in evaluating its effects on human longevity. In 2014, Metformin in Longevity Study (MILES, NCT02432287) clinical trial was initiated to examine the effects of metformin treatment on the biology of aging in humans, and to determine if treatment with metformin (1700 mg/day) could restore more youthful gene expression in elderly people with impaired glucose tolerance. Results from MILES showed that 6-weeks of metformin treatment in older adults (~70-year-old participants) improved age-associated gene expression, and significantly influenced metabolic and non-metabolic pathways in skeletal muscle and subcutaneous

**27**

*New Insight into Metformin Mechanism of Action and Clinical Application*

adipose tissue [91]. Currently, MILES has progressed to a phase 4 trial. Targeting Aging with Metformin (TAME) is managed by America Federation for Aging Research (AFAR) to investigate metformin's ability to delay the onset of comorbidities related to aging. The plan is to recruit 3000 older adults (aged 65–79 years old) without diabetes who will be randomly assigned to 1500 mg metformin daily or placebo for 6 years, with a mean follow-up time of more than 3–5 years (https:// www.afar.org/research/TAME). These ongoing trials are expected to further evalu-

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting about 5–15% of reproductive age women [92, 93]. PCOS is associated with insulin resistance and hyperinsulinemia, even in lean women. The condition puts women at risk for infertility, obesity, diabetes, as well as cardiovascular disease [94]. Metformin has been used to treat PCOS for 25 years and is currently recommended

Clinically, metformin was first reported as a treatment for PCOS in 1994 [95]. A 6-month trial of metformin or placebo in women with PCOS found that metformin improved menstruation and insulin sensitivity, and reduced hyperinsulinemia and hyperandrogenemia [96]. In addition, metformin has been found to inhibit androgen production by repressing the steroidogenic enzymatic activities of 17α-hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) in the theca cells taken from the ovaries of women with PCOS [97].

Women treated with metformin had increased rates of ovulation and pregnancy [93], reduced rates of early pregnancy loss, preterm delivery, preeclampsia, and fetal growth restriction [98, 99], and improved live birth rates [93]. There were no serious adverse effects in pregnant women with PCOS treated with metformin or their offspring [98–100]. These results indicate that the roles of metformin are not only in glucose metabolism, but also in regulating ovarian hormonal activities and

There is not enough evidence to recommend metformin as first-line therapy for women with PCOS but adding metformin to other PCOS treatment seems an optimal option. Gastrointestinal side effects were more common in metformin combined with clomiphene citrate than clomiphene citrate alone, but the combined therapy may have beneficial effects in the rates of ovulation and pregnancy [93, 101]. Combination of metformin with clomiphene citrate can be considered as the first line therapy in anovulatory PCOS women without other infertility factors [102]. Metformin was less effective than clomiphene citrate in obese women with PCOS [93, 102]. Combined therapy of metformin and spironolactone showed greater improvement in menstrual cycles and hyperinsulinemia. Adding metformin to ethinyl estradiol-cyproterone acetate treatment in non-obese women with PCOS resulted in significant decreases in androgen levels and increases sex hormone-binding globulin level, which confirmed that metformin also, has some beneficial effects in non-obese women with PCOS [103]. In a DHEA-induced PCOS rat animal model, metformin

treatment restored ovarian angiogenesis and follicular development [104].

Cardiovascular diseases (CVD) are the leading cause of death and disability in the world. Metformin might have sustained beneficial role on reducing CVD risk

*DOI: http://dx.doi.org/10.5772/intechopen.91148*

ate and update the roles of metformin in antiaging.

in combination with other therapy.

functions in women with PCOS.

**8. Cardiovascular diseases**

**7. PCOS**

### *New Insight into Metformin Mechanism of Action and Clinical Application DOI: http://dx.doi.org/10.5772/intechopen.91148*

adipose tissue [91]. Currently, MILES has progressed to a phase 4 trial. Targeting Aging with Metformin (TAME) is managed by America Federation for Aging Research (AFAR) to investigate metformin's ability to delay the onset of comorbidities related to aging. The plan is to recruit 3000 older adults (aged 65–79 years old) without diabetes who will be randomly assigned to 1500 mg metformin daily or placebo for 6 years, with a mean follow-up time of more than 3–5 years (https:// www.afar.org/research/TAME). These ongoing trials are expected to further evaluate and update the roles of metformin in antiaging.

### **7. PCOS**

*Metformin*

**6. Aging**

metformin monotherapy [74].

**5.4 Pediatric T1D**

half of the participants. Metformin plus rosiglitazone was significantly better than

Using metformin to improve glycemic control and insulin sensitivity in youth and adolescents with T1D has been reported in several clinical trials. Studies that report a positive association of metformin have reported: 1. Decreased insulin dose, BMI and waist circumference in adolescents with T1D [75]. 2. Lower daily insulin dose improved whole-body and peripheral insulin resistance in adolescents with T1D who were overweight/obese [76]. 3. Lower insulin dose and improved vascular smooth muscle function and HbA1c children with T1D [77]. 4. Decreased cardiovascular disease risk factors in youth with T1D [78]. 5. Improvement in HbA1c level in adolescents with T1D [79, 80]. In contrast, some trials did not observe improvement in HbA1c [76, 81], or glycemic control. As expected, there was an increased

gastrointestinal adverse event in overweight adolescents with T1D [81].

investigate metformin's anti-aging effects in human.

production to extend host lifespan [10, 90].

Metformin has attracted interest for its potential effects on aging [82]. Metformin treatment has a positive association with reduction in the incidence of mortality from age-related diseases including diabetes, cancer, cardiovascular diseases, and neurodegenerative diseases. Metformin is reported to increase lifespan in several animal models. Cohort clinical trials, Metformin in Longevity Study (MILES) and Targeting Aging with Metformin (TAME), have been initiated to

In several animal models, including nematodes and rodents, metformin has been shown to delay aging. Metformin treated female outbred mice (100 mg/kg in drinking water) showed an increased mean lifespan 37.8% [83]. The effects of metformin treatment were shown to be age dependent in mice. When treatment was started at the early stage of life, middle-age and late stages of life, the mean lifespan was increased by 21%, 7% and 13% respectively compared to the controls [84]. In a mouse breast cancer model, metformin delayed the onset of mammary adenocarcinoma and increased lifespan by a mean of 8% compared to the control group [85]. Metformin prolonged the survival time of male mice with Huntington's disease by 21.1%, but had no effects in female [86]. A recent study found that metformin reduced oxidative stress and inflammation, extended both lifespan and healthspan by 4–6% in different strains of mice, and attenuated the deleterious effects of aging in male mice [87]. Gut microbiota has been shown to affect health status and longevity and play a role in resistance to infection, inflammation, autoimmunity, and cancer, and the regulation of the brain-gut axis [88, 89]. Metformin acts directly on gut bacteria to decrease absorption of glucose, improve lipid metabolism and elevate agmatine

The reported effects of metformin on microbiota and animals have promoted

interest in evaluating its effects on human longevity. In 2014, Metformin in Longevity Study (MILES, NCT02432287) clinical trial was initiated to examine the effects of metformin treatment on the biology of aging in humans, and to determine if treatment with metformin (1700 mg/day) could restore more youthful gene expression in elderly people with impaired glucose tolerance. Results from MILES showed that 6-weeks of metformin treatment in older adults (~70-year-old participants) improved age-associated gene expression, and significantly influenced metabolic and non-metabolic pathways in skeletal muscle and subcutaneous

**26**

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting about 5–15% of reproductive age women [92, 93]. PCOS is associated with insulin resistance and hyperinsulinemia, even in lean women. The condition puts women at risk for infertility, obesity, diabetes, as well as cardiovascular disease [94]. Metformin has been used to treat PCOS for 25 years and is currently recommended in combination with other therapy.

Clinically, metformin was first reported as a treatment for PCOS in 1994 [95]. A 6-month trial of metformin or placebo in women with PCOS found that metformin improved menstruation and insulin sensitivity, and reduced hyperinsulinemia and hyperandrogenemia [96]. In addition, metformin has been found to inhibit androgen production by repressing the steroidogenic enzymatic activities of 17α-hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) in the theca cells taken from the ovaries of women with PCOS [97].

Women treated with metformin had increased rates of ovulation and pregnancy [93], reduced rates of early pregnancy loss, preterm delivery, preeclampsia, and fetal growth restriction [98, 99], and improved live birth rates [93]. There were no serious adverse effects in pregnant women with PCOS treated with metformin or their offspring [98–100]. These results indicate that the roles of metformin are not only in glucose metabolism, but also in regulating ovarian hormonal activities and functions in women with PCOS.

There is not enough evidence to recommend metformin as first-line therapy for women with PCOS but adding metformin to other PCOS treatment seems an optimal option. Gastrointestinal side effects were more common in metformin combined with clomiphene citrate than clomiphene citrate alone, but the combined therapy may have beneficial effects in the rates of ovulation and pregnancy [93, 101]. Combination of metformin with clomiphene citrate can be considered as the first line therapy in anovulatory PCOS women without other infertility factors [102]. Metformin was less effective than clomiphene citrate in obese women with PCOS [93, 102]. Combined therapy of metformin and spironolactone showed greater improvement in menstrual cycles and hyperinsulinemia. Adding metformin to ethinyl estradiol-cyproterone acetate treatment in non-obese women with PCOS resulted in significant decreases in androgen levels and increases sex hormone-binding globulin level, which confirmed that metformin also, has some beneficial effects in non-obese women with PCOS [103]. In a DHEA-induced PCOS rat animal model, metformin treatment restored ovarian angiogenesis and follicular development [104].

### **8. Cardiovascular diseases**

Cardiovascular diseases (CVD) are the leading cause of death and disability in the world. Metformin might have sustained beneficial role on reducing CVD risk

and mortality [105, 106]. The cardioprotective effects include reduction of weight gain and hyperinsulinemia, improvement of endothelial function and fibrinolysis, and reduction of low-grade inflammation, oxidative stress, and glycation.

Recent clinical studies have shown that metformin has protective effects on vascular endothelial function and angiogenesis in patients with T2D [107]. Several clinical trials have reported that metformin treatment reduced CVD risk in T2D [1, 108]. Recently the efficacy of metformin in modifying CVD outcomes has been challenged [109–111] but updated evidence support that metformin is cardiovascular protective [112]. A meta-analysis that included 40 clinical trials comprising 1,066,408 patients has shown that metformin reduced cardiovascular mortality, all-cause mortality and cardiovascular events in coronary artery disease [105].

Diabetes increases CVD risk and mortality. More than 75% of male and more than 57% female T2D patients died from cardiovascular disease. The mortality of CVD with T2D patients is twice those without T2D [113]. Patients with chronic cardiovascular disease (CVD) comorbidity are likely to benefit from metformin treatment [1, 105, 108]. Metformin is recommended to be used alone or in combination with other drugs as the first line therapy in T2D patients with high risk of CVD, including atherosclerotic cardiovascular disease [114, 115].

Several clinical trials for metformin on participants with or without T1D diabetes have been completed [106]. Trials Metformin in Insulin Resistant Left Ventricular Dysfunction (TAYSIDE, NCT00473876) and Reducing with Metformin Vascular Adverse Lesions of Type 1 Diabetes (REMOVAL, NCT01483560) have promising data. TAYSIDE found that metformin had a beneficial effect in participants with nondiabetic chronic heart failure and insulin resistance, significantly improved the secondary endpoint of the slope of the ratio of minute ventilation to carbon dioxide production, fasting insulin resistance and weight loss [116]. REMOVAL showed that metformin reduced the prespecified tertiary end point of carotid artery intima-media thickness in T1D suggesting a cardiovascular protective effect [117]. In an 8-week period of metformin treatment for nondiabetic participants with cardiac syndrome X, metformin improved endothelium-dependent microvascular response, maximal ST-segment depression, Duke score, and chest pain incidence, which suggested that metformin may improve vascular function and decrease myocardial ischemia [118]. However, several studies reported that metformin was not found to be effective in their participants [106].

Investigation of Metformin in Pre-diabetes on Atherosclerotic Cardiovascular OuTcomes (VA-IMPACT, NCT02915198) and Glucose Lowering in Non-diabetic Hyperglycemia Trial (GLINT, ISRCTN34875079) are current ongoing studies to further evaluate the effects of metformin on CVD [119]. The trials will evaluate the incidence of cardiovascular death and non-fatal myocardial infarction events. Their data will provide more insight on the association of metformin treatment on CVD.

The role of metformin in inhibiting mitochondrial enzymes and activating AMPK pathway are the most likely cellular mechanisms in cardiovascular protection. We have demonstrated that AMPK activated by metformin improved cellular function, decreased apoptosis, and reduced inflammation in vascular endothelial cells [4, 42]. TXNIP is a key regulator of cellular redox state induced by high glucose and promotes high-glucose-induced macrovascular endothelial dysfunction. We have also reported that metformin down-regulated high-glucose-induced TXNIP expression by inactivating ChREBP and Forkhead box O1 (FOXO1) through AMPK pathway (**Figure 2**) [4].

**29**

to cell-cycle arrest [137].

**9. Cancer**

**Figure 2.**

*New Insight into Metformin Mechanism of Action and Clinical Application*

Preexisting diabetes is a risk factor for cancers, including liver, pancreas, endometrium, colon, breast, and bladder cancers [120]. Epidemiological studies show that the incidence of cancer is decreased in patients with T2D treated with metformin [121]. Metformin has shown to inhibit cancer cell growth in clinical trials including cancer patients without diabetes [122–124]. Based on http://ClinicalTrials.gov in January 2020, there are more than 300 clinical trials investigating metformin in cancer treatment, more than 100 of them have been completed. The results were published or posted on http://ClinicalTrials.gov. These trials included patients with or without diabetes with different cancers using metformin treatment or combination of metformin with other anticancer drugs. Accumulating evidence from clinical trials and a national cohort study suggest that metformin treatment may improve therapeutic response and

*Metformin inhibits the nuclear entry of ChREBP and FOXO1 from cytosol and their binding capacity to the TXNIP promoter, thus potently and effectively suppresses TXNIP transcription induced by high glucose at last.* 

*The inhibitory effect of metformin on nuclear translocation is AMPK-phosphorylation-dependent.*

have potential beneficial effects on cancer prevention and therapy [125–127]. The effect of metformin on inhibiting cell proliferation can be classified as AMPK independent and AMPK dependent [128]. Metformin inhibits the electron

production in mitochondrial complex I ATP as well as activation of AMPK [129, 130]. AMPK activated by metformin subsequently regulates cell growth and survival by targeting metabolic enzymes and transporters [131, 132]. AMPK downregulates mTOR activity that plays a central role in the regulation of cell proliferation,

Tumor protein 53 (p53) plays a central role in the cellular responses to repair of DNA damage, cell survival and apoptosis. p53 mutations occur in almost every type of human cancer cells and more than 50% of human cancers have a somatic p53 mutation [136]. AMPK activation induced phosphorylation at Ser15 of p53, leading

Metformin was reported to inhibit melanoma cell invasion and metastasis via an AMPK/p53 dependent manner [138]. In a pre-clinical lymphoma model, metformin

ratio and decrease of ATP

transport chain, resulting in an elevated NADH/NAD+

growth, differentiation, migration, and survival [133–135].

*DOI: http://dx.doi.org/10.5772/intechopen.91148*

*New Insight into Metformin Mechanism of Action and Clinical Application DOI: http://dx.doi.org/10.5772/intechopen.91148*

### **Figure 2.**

*Metformin*

disease [105].

participants [106].

pathway (**Figure 2**) [4].

on CVD.

and mortality [105, 106]. The cardioprotective effects include reduction of weight gain and hyperinsulinemia, improvement of endothelial function and fibrinolysis,

Recent clinical studies have shown that metformin has protective effects on vascular endothelial function and angiogenesis in patients with T2D [107]. Several clinical trials have reported that metformin treatment reduced CVD risk in T2D [1, 108]. Recently the efficacy of metformin in modifying CVD outcomes has been challenged [109–111] but updated evidence support that metformin is cardiovascular protective [112]. A meta-analysis that included 40 clinical trials comprising 1,066,408 patients has shown that metformin reduced cardiovascular mortality, all-cause mortality and cardiovascular events in coronary artery

Diabetes increases CVD risk and mortality. More than 75% of male and more than 57% female T2D patients died from cardiovascular disease. The mortality of CVD with T2D patients is twice those without T2D [113]. Patients with chronic cardiovascular disease (CVD) comorbidity are likely to benefit from metformin treatment [1, 105, 108]. Metformin is recommended to be used alone or in combination with other drugs as the first line therapy in T2D patients with high risk of CVD,

Several clinical trials for metformin on participants with or without T1D diabetes have been completed [106]. Trials Metformin in Insulin Resistant Left Ventricular Dysfunction (TAYSIDE, NCT00473876) and Reducing with Metformin Vascular Adverse Lesions of Type 1 Diabetes (REMOVAL, NCT01483560) have promising data. TAYSIDE found that metformin had a beneficial effect in participants with nondiabetic chronic heart failure and insulin resistance, significantly improved the secondary endpoint of the slope of the ratio of minute ventilation to carbon dioxide production, fasting insulin resistance and weight loss [116]. REMOVAL showed that metformin reduced the prespecified tertiary end point of carotid artery intima-media thickness in T1D suggesting a cardiovascular protective effect [117]. In an 8-week period of metformin treatment for nondiabetic participants with cardiac syndrome X, metformin improved endothelium-dependent microvascular response, maximal ST-segment depression, Duke score, and chest pain incidence, which suggested that metformin may improve vascular function and decrease myocardial ischemia [118]. However, several studies reported that metformin was not found to be effective in their

Investigation of Metformin in Pre-diabetes on Atherosclerotic Cardiovascular OuTcomes (VA-IMPACT, NCT02915198) and Glucose Lowering in Non-diabetic Hyperglycemia Trial (GLINT, ISRCTN34875079) are current ongoing studies to further evaluate the effects of metformin on CVD [119]. The trials will evaluate the incidence of cardiovascular death and non-fatal myocardial infarction events. Their data will provide more insight on the association of metformin treatment

The role of metformin in inhibiting mitochondrial enzymes and activating AMPK pathway are the most likely cellular mechanisms in cardiovascular protection. We have demonstrated that AMPK activated by metformin improved cellular function, decreased apoptosis, and reduced inflammation in vascular endothelial cells [4, 42]. TXNIP is a key regulator of cellular redox state induced by high glucose and promotes high-glucose-induced macrovascular endothelial dysfunction. We have also reported that metformin down-regulated high-glucose-induced TXNIP expression by inactivating ChREBP and Forkhead box O1 (FOXO1) through AMPK

including atherosclerotic cardiovascular disease [114, 115].

and reduction of low-grade inflammation, oxidative stress, and glycation.

**28**

*Metformin inhibits the nuclear entry of ChREBP and FOXO1 from cytosol and their binding capacity to the TXNIP promoter, thus potently and effectively suppresses TXNIP transcription induced by high glucose at last. The inhibitory effect of metformin on nuclear translocation is AMPK-phosphorylation-dependent.*

### **9. Cancer**

Preexisting diabetes is a risk factor for cancers, including liver, pancreas, endometrium, colon, breast, and bladder cancers [120]. Epidemiological studies show that the incidence of cancer is decreased in patients with T2D treated with metformin [121]. Metformin has shown to inhibit cancer cell growth in clinical trials including cancer patients without diabetes [122–124]. Based on http://ClinicalTrials.gov in January 2020, there are more than 300 clinical trials investigating metformin in cancer treatment, more than 100 of them have been completed. The results were published or posted on http://ClinicalTrials.gov. These trials included patients with or without diabetes with different cancers using metformin treatment or combination of metformin with other anticancer drugs. Accumulating evidence from clinical trials and a national cohort study suggest that metformin treatment may improve therapeutic response and have potential beneficial effects on cancer prevention and therapy [125–127].

The effect of metformin on inhibiting cell proliferation can be classified as AMPK independent and AMPK dependent [128]. Metformin inhibits the electron transport chain, resulting in an elevated NADH/NAD+ ratio and decrease of ATP production in mitochondrial complex I ATP as well as activation of AMPK [129, 130]. AMPK activated by metformin subsequently regulates cell growth and survival by targeting metabolic enzymes and transporters [131, 132]. AMPK downregulates mTOR activity that plays a central role in the regulation of cell proliferation, growth, differentiation, migration, and survival [133–135].

Tumor protein 53 (p53) plays a central role in the cellular responses to repair of DNA damage, cell survival and apoptosis. p53 mutations occur in almost every type of human cancer cells and more than 50% of human cancers have a somatic p53 mutation [136]. AMPK activation induced phosphorylation at Ser15 of p53, leading to cell-cycle arrest [137].

Metformin was reported to inhibit melanoma cell invasion and metastasis via an AMPK/p53 dependent manner [138]. In a pre-clinical lymphoma model, metformin treatment resulted in activation of p53, leading to cell apoptosis [139]. In the prostate cancer cells, the combination of metformin and 2-deoxyglucose resulted in p53-dependent cell apoptosis [140]. Metformin has been found to inhibit human cervical cancer cell proliferation and induce apoptosis via modulating p53 and cyclin D1 expression [141].

The effect of metformin on anti-cancer also has a p53-independent mechanism. Metformin has been shown to induce G2M arrest in p53-deficient colorectal cancer cells and tumors. When combined with ionizing radiation metformin therapy enhanced antitumor effects in radioresistant p53-deficient colorectal cancer cells [142]. Treatment with metformin increased apoptosis in p53-deficient human colon cancer cell and reduced tumor growth in xenografts of p53-deficient human colon cancer cells [143].

The p53 homologs, P63 and p73 have overlapping function in tumorigenesis and development [144]. P63 and P73 mutations are rare in human tumors, but they can be overexpressed. P63 plays a critical role in development of squamous epithelium and is overexpressed in squamous cell carcinoma [145]. Metformin inhibited p63 protein expression in squamous carcinoma cell, resulting in decreased cell viability and xenographic tumor growth [146]. P73 overexpression induces apoptosis and cell cycle arrest of tumor cells [147]. AMPK activated by metformin phosphorylated Ser426 of p73 leading to p73 accumulation and cell apoptosis in human colon cancer cells [148].

Metformin may prevent tumorigenesis by inhibiting the insulin like growth factor (IGF)-1 signaling pathway and increasing insulin sensitivity. The proliferation marker Ki-67 was significantly decreased in patients with endometrial cancer cell after metformin treatment [149]. Metformin enhances cytotoxic T lymphocyte (CTL) antitumor activity via activating AMPK to phosphorylate Ser195 of PDL-1 in a murine model of breast cancer which is consistent with the finding that tumor tissues from metformin-treated breast cancer patients exhibited reduced PDL-1 level with AMPK activation [150].

These findings suggest that metformin could be a useful adjuvant agent and has therapeutic benefits in several tumor types, including colorectal, prostate and breast cancers. However, there is limited evidence in other tumor types, and further clinical investigations are needed to evaluate metformin effects in cancer therapy.

### **10. Neurodegenerative diseases**

Metformin is described to have a beneficial effect in neurodegenerative diseases (ND), including dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease and mild cognitive impairment [151, 152].

Population-based studies support an association between the elevated risk of ND in patients with T2D [153–155]. A large population cohort study used Taiwan's National Health Insurance Database to investigate the relationship between dementia, T2D, and metformin treatment. They found that the prevalence of dementia was increased in patients with T2D and that metformin therapy was associated with a 24% decrease in the incidence of dementia in patients with T2D. The combination treatment of metformin with sulfonylureas was associated with a 35% decrease in the risk of dementia in T2D patients over 8 years of observation [156]. In a recent study, long-term (>2 years) metformin therapy was associated with lower incidence of dementia among elderly adults with T2D. Longer term treatment (>4 years) was associated with reduced risk of Alzheimer's and Parkinson's diseases, and none with mild cognitive impairment [157]. A large T2D population cohort study found that sulfonylureas therapy increased the risk of Parkinson's disease, but adding metformin as a co-therapy significantly reduced the risk of Parkinson's disease in T2D

**31**

*New Insight into Metformin Mechanism of Action and Clinical Application*

aggravated neurodegenerative process in ApoE knockout mice [163].

cognitive impairment among older adults with T2D [159].

[158]. Long-term (>6 years) metformin treatment significantly reduced the risk of

In contrast, other studies have shown that the metformin therapy of T2D is associated with: 1. a slightly higher risk of Alzheimer's disease [160], 2. increased risk for cognitive impairment [161], and 3. no beneficial effects on preventing development of Alzheimer's disease after adjusting for underlying risk factors and the duration of diabetes since diagnosis [162]. In addition, metformin treatment

The current evidence suggests that the neuroprotective effects of metformin occur via activation of AMPK/mTOR pathway and inhibition of tau phosphorylation [164, 165]. In addition, it is known that metformin enhances angiogenesis and neurogenesis, induces autophagy, reduces oxidative stress, and improves

Despite the different findings from these studies, a recent meta-analysis suggests

Metformin is currently approved and widely prescribed for patients with T2D and PCOS. The clinical trial data and clinical experience over several decades have demonstrated its safety and efficacy. The interest in metformin therapy has dramatically increased as the population-based cohort studies indicate that metformin can decrease the risk of cancer, cardiovascular and cerebral disease. Current studies indicate that metformin has potential for treatment of T1D, cancer, aging, cardiovascular and neurodegenerative diseases. Translational and clinical trials need to be continued and expanded to determine if there are indications for metformin

Department of Pediatrics, Division of Endocrinology, Children's Mercy Kansas City,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

School of Medicine, University of Missouri Kansas City, Kansas City, USA

that metformin may prevent development of dementia in patients with diabetes indicating that metformin should be continued in patients with T2D patients at risk of the dementia or Alzheimer's disease. Use of metformin to prevent neurodegenerative diseases in people without diabetes is not supported by current evidence [152].

*DOI: http://dx.doi.org/10.5772/intechopen.91148*

neurological deficits [166–170].

therapy in diseases other than T2D.

The authors declare no conflict of interest.

Yun Yan\*, Karen L. Kover and Wayne V. Moore

\*Address all correspondence to: yyan@cmh.edu

provided the original work is properly cited.

**Conflict of interest**

**Author details**

**11. Conclusions**

### *New Insight into Metformin Mechanism of Action and Clinical Application DOI: http://dx.doi.org/10.5772/intechopen.91148*

[158]. Long-term (>6 years) metformin treatment significantly reduced the risk of cognitive impairment among older adults with T2D [159].

In contrast, other studies have shown that the metformin therapy of T2D is associated with: 1. a slightly higher risk of Alzheimer's disease [160], 2. increased risk for cognitive impairment [161], and 3. no beneficial effects on preventing development of Alzheimer's disease after adjusting for underlying risk factors and the duration of diabetes since diagnosis [162]. In addition, metformin treatment aggravated neurodegenerative process in ApoE knockout mice [163].

The current evidence suggests that the neuroprotective effects of metformin occur via activation of AMPK/mTOR pathway and inhibition of tau phosphorylation [164, 165]. In addition, it is known that metformin enhances angiogenesis and neurogenesis, induces autophagy, reduces oxidative stress, and improves neurological deficits [166–170].

Despite the different findings from these studies, a recent meta-analysis suggests that metformin may prevent development of dementia in patients with diabetes indicating that metformin should be continued in patients with T2D patients at risk of the dementia or Alzheimer's disease. Use of metformin to prevent neurodegenerative diseases in people without diabetes is not supported by current evidence [152].

### **11. Conclusions**

*Metformin*

cyclin D1 expression [141].

with AMPK activation [150].

**10. Neurodegenerative diseases**

disease and mild cognitive impairment [151, 152].

cancer cells [143].

treatment resulted in activation of p53, leading to cell apoptosis [139]. In the prostate cancer cells, the combination of metformin and 2-deoxyglucose resulted in p53-dependent cell apoptosis [140]. Metformin has been found to inhibit human cervical cancer cell proliferation and induce apoptosis via modulating p53 and

The effect of metformin on anti-cancer also has a p53-independent mechanism. Metformin has been shown to induce G2M arrest in p53-deficient colorectal cancer cells and tumors. When combined with ionizing radiation metformin therapy enhanced antitumor effects in radioresistant p53-deficient colorectal cancer cells [142]. Treatment with metformin increased apoptosis in p53-deficient human colon cancer cell and reduced tumor growth in xenografts of p53-deficient human colon

The p53 homologs, P63 and p73 have overlapping function in tumorigenesis and development [144]. P63 and P73 mutations are rare in human tumors, but they can be overexpressed. P63 plays a critical role in development of squamous epithelium and is overexpressed in squamous cell carcinoma [145]. Metformin inhibited p63 protein expression in squamous carcinoma cell, resulting in decreased cell viability and xenographic tumor growth [146]. P73 overexpression induces apoptosis and cell cycle arrest of tumor cells [147]. AMPK activated by metformin phosphorylated Ser426 of p73 leading to p73 accumulation and cell apoptosis in human colon cancer cells [148]. Metformin may prevent tumorigenesis by inhibiting the insulin like growth factor (IGF)-1 signaling pathway and increasing insulin sensitivity. The proliferation marker Ki-67 was significantly decreased in patients with endometrial cancer cell after metformin treatment [149]. Metformin enhances cytotoxic T lymphocyte (CTL) antitumor activity via activating AMPK to phosphorylate Ser195 of PDL-1 in a murine model of breast cancer which is consistent with the finding that tumor tissues from metformin-treated breast cancer patients exhibited reduced PDL-1 level

These findings suggest that metformin could be a useful adjuvant agent and has therapeutic benefits in several tumor types, including colorectal, prostate and breast cancers. However, there is limited evidence in other tumor types, and further clinical investigations are needed to evaluate metformin effects in cancer therapy.

Metformin is described to have a beneficial effect in neurodegenerative diseases (ND), including dementia, Alzheimer's disease, Parkinson's disease, Huntington's

Population-based studies support an association between the elevated risk of ND in patients with T2D [153–155]. A large population cohort study used Taiwan's National Health Insurance Database to investigate the relationship between dementia, T2D, and metformin treatment. They found that the prevalence of dementia was increased in patients with T2D and that metformin therapy was associated with a 24% decrease in the incidence of dementia in patients with T2D. The combination treatment of metformin with sulfonylureas was associated with a 35% decrease in the risk of dementia in T2D patients over 8 years of observation [156]. In a recent study, long-term (>2 years) metformin therapy was associated with lower incidence of dementia among elderly adults with T2D. Longer term treatment (>4 years) was associated with reduced risk of Alzheimer's and Parkinson's diseases, and none with mild cognitive impairment [157]. A large T2D population cohort study found that sulfonylureas therapy increased the risk of Parkinson's disease, but adding metformin as a co-therapy significantly reduced the risk of Parkinson's disease in T2D

**30**

Metformin is currently approved and widely prescribed for patients with T2D and PCOS. The clinical trial data and clinical experience over several decades have demonstrated its safety and efficacy. The interest in metformin therapy has dramatically increased as the population-based cohort studies indicate that metformin can decrease the risk of cancer, cardiovascular and cerebral disease. Current studies indicate that metformin has potential for treatment of T1D, cancer, aging, cardiovascular and neurodegenerative diseases. Translational and clinical trials need to be continued and expanded to determine if there are indications for metformin therapy in diseases other than T2D.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Yun Yan\*, Karen L. Kover and Wayne V. Moore Department of Pediatrics, Division of Endocrinology, Children's Mercy Kansas City, School of Medicine, University of Missouri Kansas City, Kansas City, USA

\*Address all correspondence to: yyan@cmh.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[142] Jeong YK, Kim MS, Lee JY, Kim EH, Ha H. Metformin Radiosensitizes p53 deficient colorectal Cancer cells through induction of G2/M arrest and inhibition of DNA repair proteins. PLoS One. 2015;**10**:e0143596. DOI: 10.1371/journal.

[143] Buzzai M, Jones RG, Amaravadi RK, Lum JJ, DeBerardinis RJ, Zhao F, et al. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Cancer Research. 2007;**67**:6745-6752. DOI: 10.1158/0008-5472.CAN-06-4447

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2010;**2**:a004887. DOI: 10.1101/

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Ponnamperuma RM, King KE, et al. Molecular mechanisms of p63-mediated

cshperspect.a004887

Coutandin D, Candi E, Melino G. p63 and p73, the ancestors of p53. Cold Spring Harbor Perspectives in Biology.

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[132] Herzig S, Shaw RJ. AMPK: Guardian of metabolism and mitochondrial homeostasis. Nature Reviews. Molecular Cell Biology. 2018;**19**:121-135. DOI:

[133] Huang S, Houghton PJ. Targeting mTOR signaling for cancer therapy. Current Opinion in Pharmacology.

[134] Vogt PK. PI 3-kinase, mTOR, protein synthesis and cancer. Trends in Molecular Medicine. 2001;**7**:482-484. DOI: 10.1616/s1471-4914(01)0216-x

[135] Schmelzle T, Hall MN. TOR, a central controller of cell growth. Cell. 2000;**103**:253-262. DOI: 10.1016/

[136] Muller PA, Vousden KH. p53 mutations in cancer. Nature Cell Biology. 2013;**15**:2-8. DOI: 10.1038/

[137] Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, et al. AMPactivated protein kinase induces a p53-dependent metabolic checkpoint. Molecular Cell. 2005;**18**:283-293. DOI:

10.1016/j.molcel.2005.03.027

[138] Cerezo M, Tichet M, Abbe P, Ohanna M, Lehraiki A, Rouaud F, et al. Metformin blocks melanoma invasion and metastasis development in AMPK/ p53-dependent manner. Molecular Cancer Therapeutics. 2013;**12**:1605-1615. DOI: 10.1158/1535-7163.MCT-12-1226-T

[139] Juan J, Gu QZ, Mavis C, Czuczman MS, Hernandezollizaliturri FJ. Metformin

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[147] Yoon MK, Ha JH, Lee MS, Chi SW. Structure and apoptotic function of p73. BMB Reports. 2015;**48**:81-90. DOI: 10.5483/ bmbrep.2015.48.2.255

[148] Adamovich Y, Adler J, Meltser V, Reuven N, Shaul Y. AMPK couples p73 with p53 in cell fate decision. Cell Death and Differentiation. 2014;**21**:1451-1459. DOI: 10.1038/cdd.2014.60

[149] Schuler KM, Rambally BS, DiFurio MJ, Sampey BP, Gehrig PA, Makowski L, et al. Antiproliferative and metabolic effects of metformin in a preoperative window clinical trial for endometrial cancer. Cancer Medicine. 2015;**4**:161-173. DOI: 10.1002/cam4.353

[150] Cha JH, Yang WH, Xia W, Wei Y, Chan LC, Lim SO, et al. Metformin promotes antitumor immunity via endoplasmic-reticulum-associated degradation of PD-L1. Molecular Cell. 2018;**71**:606-620 .e7. DOI: 10.1016/j. molcel.2018.07.030

[151] Wang YW, He SJ, Feng X, Cheng J, Luo YT, Tian L, et al. Metformin: A review of its potential indications. Drug Design, Development and Therapy. 2017;**11**:2421-2429. DOI: 10.2147/DDDT. S141675

[152] Campbell JM, Stephenson MD, de Courten B, Chapman I, Bellman SM, Aromataris E. Metformin use associated with reduced risk of dementia in

patients with Diabetes: A systematic review and meta-analysis. Journal of Alzheimer's Disease. 2018;**65**:1225-1236. DOI: 10.3233/JAD-180263

[153] Moreira RO, Campos SC, Soldera AL. Type 2 Diabetes mellitus and Alzheimer's disease: From physiopathology to treatment implications. Diabetes/Metabolism Research and Reviews. 2013;**71**:365-376. DOI: 10.1002/dmrr.2442

[154] Gudala K, Bansal D, Schifano F, Bhansali A. Diabetes mellitus and risk of dementia: A meta-analysis of prospective observational studies. Journal of Diabetes Investigation. 2013;**4**:640-650. DOI: 10.1111/jdi.12087

[155] Xu W, Caracciolo B, Wang HX, Winblad B, Backman L, Qiu C, et al. Accelerated progression from mild cognitive impairment to dementia in people with diabetes. Diabetes. 2010;**59**:2928-2935. DOI: 10.2337/ db10-0539

[156] Hsu CC, Wahlqvist ML, Lee MS, Tsai HN. Incidence of dementia is increased in type 2 diabetes and reduced by the use of sulfonylureas and metformin. Journal of Alzheimer's Disease. 2011;**24**:485-493. DOI: 10.3233/ JAD-2011-101524

[157] Shi Q, Liu S, Fonseca VA, Thethi TK, Shi L. Effect of metformin on neurodegenerative disease among elderly adult US veterans with type 2 diabetes mellitus. BMJ Open. 2019;**9**:e024954. DOI: 10.1136/ bmjopen-2018-024954

[158] Wahlqvist ML, Lee MS, Hsu CC, Chuang SY, Lee JT, Tsai HN. Metformininclusive sulfonylurea therapy reduces the risk of Parkinson's disease occurring with type 2 diabetes in a Taiwanese population cohort. Parkinsonism & Related Disorders. 2012;**18**:753-758. DOI: 10.1016/j.parkreldis.2012.03.010

[159] Ng TP, Feng L, Yap KB, Lee TS, Tan CH, Winblad B. Longterm metformin usage and cognitive function among older adults with diabetes. Journal of Alzheimer's Disease. 2014;**41**:61-68. DOI: 10.3233/JAD-131901

[160] Imfeld P, Bodmer M, Jick SS, Meier CR. Metformin, other antidiabetic drugs, and risk of Alzheimer's disease: A population-based case-control study. Journal of the American Geriatrics Society. 2012;**60**:916-921. DOI: 10.1111/j.1532-5415.2012.03916.x

[161] Moore EM, Mander AG, Ames D, Kotowicz MA, Carne RP, Brodaty H, et al. Increased risk of cognitive impairment in patients with diabetes is associated with metformin. Diabetes Care. 2013;**36**:2981-2987. DOI: 10.2337/dc13-0229

[162] Huang CC, Chung CM, Leu HB, Lin LY, Chiu CC, Hsu CY, et al. Diabetes mellitus and the risk of Alzheimer's disease: A nationwide population-based study. PLoS One. 2014;**9**:e87095. DOI: 10.1371/journal.pone.0087095

[163] Kuhla A, Brichmann E, Ruhlmann C, Thiele R, Meuth L, Vollmar B. Metformin therapy aggravates neurodegenerative processes in ApoE−/− mice. Journal of Alzheimer's Disease. 2019;**68**:1415-1427. DOI: 10.3233/JAD-181017

[164] Curry DW, Stutz B, Andrews ZB, Elsworth JD. Targeting AMPK signaling as a Neuroprotective strategy in Parkinson's disease. Journal of Parkinson's Disease. 2018;**8**:161-181. DOI: 10.3233/JPD-171296

[165] Kickstein E, Krauss S, Thornhill P, Rutschow D, Zeller R, Sharkey J, et al. Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**:21830-21835. DOI: 10.1073/pnas.0912793107

[166] Paseban M, Mohebbati R, Niazmand S, Sathyapalan T, Sahebkar A. Comparison of the neuroprotective effects of aspirin, atorvastatin, captopril and metformin in diabetes mellitus. Biomolecules. 2019;**9**:1-12. DOI: 10.3390/biom9040118

[167] Alzoubi KH, Khabour OF, Al-Azzam SI, Tashtoush MH, Mhaidat NM. Metformin eased cognitive impairment induced by chronic L-methionine administration: Potential role of oxidative stress. Current Neuropharmacology. 2014;**12**:186-192. DOI: 10.2174/1570159X1166613112022 3201

[168] Jiang T, Yu JT, Zhu XC, Wang HF, Tan MS, Cao L, et al. Acute metformin preconditioning confers neuroprotection against focal cerebral ischaemia by pre-activation of AMPK-dependent autophagy. British Journal of Pharmacology. 2014;**171**:3146-3157. DOI: 10.1111/bph.12655

[169] Venna VR, Li J, Hammond MD, Mancini NS, McCullough LD. Chronic metformin treatment improves poststroke angiogenesis and recovery after experimental stroke. The European Journal of Neuroscience. 2014;**39**:2129- 2138. DOI: 10.1111/ejn.12556

[170] Jin Q, Cheng J, Liu Y, Wu J, Wang X, Wei S, et al. Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain, Behavior, and Immunity. 2014;**40**: 131-142. DOI: 10.1016/j.bbi.2014.03.003

**45**

**1. Introduction**

**Chapter 3**

**Abstract**

Metformin and Its Benefits

*and Adrian-Paul Suceveanu*

thus provide a better outcome for this illness.

in Improving Gut Microbiota

Disturbances in Diabetes Patients

*Andra Iulia-Suceveanu, Sergiu Ioan Micu, Claudia Voinea,* 

*Madalina Elena Manea, Doina Catrinoiu, Laura Mazilu,* 

*Anca Pantea Stoian, Irinel Parepa, Roxana Adriana Stoica* 

The human gastrointestinal tract presents a vastly population of microorganisms, called the microbiota. The presence of these microorganisms offers many benefits to the host, through a range of physiological functions. However, there is a potential for these mechanisms to be disrupted condition, known as dysbiosis. Recent results are showing important associations between diabetes and the gut microbiota and how the intestinal flora can influence the prognosis of this illness. Microbial intestinal imbalance has been linked to alterations in insulin sensitivity and in glucose metabolism and may play an important role in the development of diabetes. Metformin is one of the most important and widely used first-line medications for the management of type 2 diabetes (T2D). It is a complex drug with multiple sites of action and multiple molecular mechanisms. In recent years, attention has been directed to other modes of action, other than the classic ones, with increasing evidence of a major key role of the intestine. By analysing the effects of metformin on the homeostasis of the microbiota of diabetes patients, our present topic becomes one of the major importance in understanding how metformin therapy can improve gut microbiota dysbiosis and

**Keywords:** metformin, diabetes mellitus, gut dysbiosis, improvement, microbiota

The human gastrointestinal tract hosts a complex population of microorganisms. The function and composition of the gut microbiota vary from an individual to another, factors contributing to its differences being various. The mode of birth, the type of diet, exercise, body mass index, different diseases and therapies are factors that influence the gut microbiota composition and function. Type 2 diabetes (T2D), a highly prevalent metabolic disease, is lately characterized as a disease with significant alteration of the composition and function of the gut microbiota. New therapeutic targets are revealed, and researchers are thoroughly exploring these possible pathways and hypotheses to understand the pathogeny of

### **Chapter 3**

*Metformin*

[159] Ng TP, Feng L, Yap KB, Lee TS, Tan CH, Winblad B. Longterm metformin usage and cognitive function among older adults with diabetes. Journal of Alzheimer's Disease. 2014;**41**:61-68. DOI: 10.3233/JAD-131901 [166] Paseban M, Mohebbati R,

DOI: 10.3390/biom9040118

[167] Alzoubi KH, Khabour OF, Al-Azzam SI, Tashtoush MH,

impairment induced by chronic

role of oxidative stress. Current Neuropharmacology. 2014;**12**:186-192. DOI: 10.2174/1570159X1166613112022

3201

10.1111/bph.12655

Niazmand S, Sathyapalan T, Sahebkar A. Comparison of the neuroprotective effects of aspirin, atorvastatin, captopril and metformin in diabetes mellitus. Biomolecules. 2019;**9**:1-12.

Mhaidat NM. Metformin eased cognitive

L-methionine administration: Potential

[168] Jiang T, Yu JT, Zhu XC, Wang HF, Tan MS, Cao L, et al. Acute metformin preconditioning confers neuroprotection against focal cerebral ischaemia by pre-activation of AMPK-dependent autophagy. British Journal of

Pharmacology. 2014;**171**:3146-3157. DOI:

[169] Venna VR, Li J, Hammond MD, Mancini NS, McCullough LD. Chronic metformin treatment improves poststroke angiogenesis and recovery after experimental stroke. The European Journal of Neuroscience. 2014;**39**:2129-

2138. DOI: 10.1111/ejn.12556

[170] Jin Q, Cheng J, Liu Y, Wu J, Wang X, Wei S, et al. Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke. Brain, Behavior, and Immunity. 2014;**40**: 131-142. DOI: 10.1016/j.bbi.2014.03.003

[160] Imfeld P, Bodmer M, Jick SS, Meier CR. Metformin, other antidiabetic drugs, and risk of Alzheimer's disease: A population-based case-control study. Journal of the American Geriatrics Society. 2012;**60**:916-921. DOI: 10.1111/j.1532-5415.2012.03916.x

[161] Moore EM, Mander AG, Ames D, Kotowicz MA, Carne RP, Brodaty H, et al. Increased risk of cognitive impairment in patients with diabetes is associated with metformin. Diabetes Care. 2013;**36**:2981-2987. DOI:

[162] Huang CC, Chung CM, Leu HB, Lin LY, Chiu CC, Hsu CY, et al. Diabetes mellitus and the risk of Alzheimer's disease: A nationwide population-based study. PLoS One. 2014;**9**:e87095. DOI:

processes in ApoE−/− mice. Journal of Alzheimer's Disease. 2019;**68**:1415-1427.

[164] Curry DW, Stutz B, Andrews ZB, Elsworth JD. Targeting AMPK signaling

[165] Kickstein E, Krauss S, Thornhill P, Rutschow D, Zeller R, Sharkey J, et al. Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**:21830-21835. DOI:

as a Neuroprotective strategy in Parkinson's disease. Journal of Parkinson's Disease. 2018;**8**:161-181.

10.1371/journal.pone.0087095

[163] Kuhla A, Brichmann E, Ruhlmann C, Thiele R, Meuth L, Vollmar B. Metformin therapy aggravates neurodegenerative

DOI: 10.3233/JAD-181017

DOI: 10.3233/JPD-171296

10.1073/pnas.0912793107

10.2337/dc13-0229

**44**

## Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients

*Andra Iulia-Suceveanu, Sergiu Ioan Micu, Claudia Voinea, Madalina Elena Manea, Doina Catrinoiu, Laura Mazilu, Anca Pantea Stoian, Irinel Parepa, Roxana Adriana Stoica and Adrian-Paul Suceveanu*

### **Abstract**

The human gastrointestinal tract presents a vastly population of microorganisms, called the microbiota. The presence of these microorganisms offers many benefits to the host, through a range of physiological functions. However, there is a potential for these mechanisms to be disrupted condition, known as dysbiosis. Recent results are showing important associations between diabetes and the gut microbiota and how the intestinal flora can influence the prognosis of this illness. Microbial intestinal imbalance has been linked to alterations in insulin sensitivity and in glucose metabolism and may play an important role in the development of diabetes. Metformin is one of the most important and widely used first-line medications for the management of type 2 diabetes (T2D). It is a complex drug with multiple sites of action and multiple molecular mechanisms. In recent years, attention has been directed to other modes of action, other than the classic ones, with increasing evidence of a major key role of the intestine. By analysing the effects of metformin on the homeostasis of the microbiota of diabetes patients, our present topic becomes one of the major importance in understanding how metformin therapy can improve gut microbiota dysbiosis and thus provide a better outcome for this illness.

**Keywords:** metformin, diabetes mellitus, gut dysbiosis, improvement, microbiota

### **1. Introduction**

The human gastrointestinal tract hosts a complex population of microorganisms. The function and composition of the gut microbiota vary from an individual to another, factors contributing to its differences being various. The mode of birth, the type of diet, exercise, body mass index, different diseases and therapies are factors that influence the gut microbiota composition and function. Type 2 diabetes (T2D), a highly prevalent metabolic disease, is lately characterized as a disease with significant alteration of the composition and function of the gut microbiota. New therapeutic targets are revealed, and researchers are thoroughly exploring these possible pathways and hypotheses to understand the pathogeny of the disease better and also to better manage the treatment options. Metformin, one of the most widely used first-line medication for the management of type 2 diabetes, looks to present other modes of action than the classic ones involving liver metabolism. Studies proved that metformin could modulate the gut microbiota disturbances encountered in type 2 diabetes, in this way improving the outcome of the disease.

### **1.1 The gut microbiota: definition, development and structure**

Among other things, the cohabitation of the man with the environment is at the root of the human evolution, an extraordinary example in this sense being the relationship between humans and microorganisms.

The digestive tract hosts a complex, vast and dynamic community of microorganisms, called the microbiota. Together they form a mutualist relationship, with profound implications for the host both during homeostasis and disease [1].

It is worth mentioning that the gut is not the only place where there is a population of microorganisms with which the human organism is in such a connection (e.g., the skin also harbouring a plethora of bacteria) [2].

The composition of the microbiota varies from individual to another but also from segment to segment of the digestive tract and includes species from all three domains of life: bacteria, Archaea and Eukarya. All of the species are classified into 12 different phyla, of which more than 90% belong to *Actinobacteria*, *Bacteroidetes*, *Firmicutes* and *Proteobacteria* [3].

The process of colonizing the digestive tube with microorganisms is classically believed to begin at birth by "seeding" the newborn with microorganisms originating from the mother's genital area (vaginal passage, mother's areola), the skin, and the microbiota of the contacts in the surrounding environment, and from then the development continues throughout life. In recent years, however, this theory is challenged by a series of studies that have shown the presence of microorganisms in uterine tissues (e.g., the placenta, suggesting that colonization could be initiated before birth, by haematogenous sowing) [4]. At the age of 3–4 years, the core of the microbiota is relatively defined, and its structure is similar to that of the adult but is continuously subject to change depending on various external and internal factors.

Even if the core of the microbiota is established from an early age, several factors contribute to carve its form, explaining its variations from an individual to another [5]:


**47**

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients*

As stated before, the composition also depends on the digestive tract region (biogeography), this being explained by the physiological properties of the digestive segment. For instance, in the small intestine, the pH is lower and the transit time is shorter, which is why only rapidly growing bacteria, with the ability to adhere to the surface, are thought to survive. On the other hand, the colon shows a favourable environment for the development of microorganisms. It is worth mentioning that there are differences in the composition between faecal/luminal and mucosal bacteria [6]. In its final form, this whole microsystem consists of over 2000 species, which make up altogether more than 100 trillion cells, about 10 times more than the cells

The microbiota exerts a significant influence on the host during homeostasis and disease, with profound implications for the proper body's physiological functions,

• Mechanic barrier—strengthening the gut integrity, shaping and regenerating

• Biologic active barrier—consuming the feeding substrates for pathogens [7]

• Key regulators of digestion—involvement in the metabolism of biliary salts,

• Synthesis of vitamins—principal reservoir for B complex vitamins [11]

Any perturbation of the healthy gut microbiota that disrupts the mutualist relationship between the organism and the associated microbes is called dysbiosis.

• Drug therapy: antibiotics, chemotherapy, antiviral drugs and hormone therapy

• Synthesis of dopamine, serotonin and other neurotransmitters [12]

The underlying cause of a gut dysbiosis may be the following [13]:

• Diseases: cancers, hepatopancreatic diseases and diabetes

*DOI: http://dx.doi.org/10.5772/intechopen.88749*

**1.2 Functions**

of the human body, hence the name of "superorganism".

considering the microbiota as a "forgotten organ".

• Harvesting energy [9]

• Unbalanced diet

• Regulating host immunity [10]

**1.3 Dysbiosis: definition, causes and consequences**

The antagonist term of dysbiosis is eubiosis.

• Chronic and acute infections

• Presence of intestinal parasites

• Local inflammation

• Frequent enemas

The leading roles of the gut flora are the following:

the intestinal epithelium, protecting against pathogens [7]

short-chain fatty acids (SCFAs), lipids and glucides [8]


*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients DOI: http://dx.doi.org/10.5772/intechopen.88749*

As stated before, the composition also depends on the digestive tract region (biogeography), this being explained by the physiological properties of the digestive segment. For instance, in the small intestine, the pH is lower and the transit time is shorter, which is why only rapidly growing bacteria, with the ability to adhere to the surface, are thought to survive. On the other hand, the colon shows a favourable environment for the development of microorganisms. It is worth mentioning that there are differences in the composition between faecal/luminal and mucosal bacteria [6].

In its final form, this whole microsystem consists of over 2000 species, which make up altogether more than 100 trillion cells, about 10 times more than the cells of the human body, hence the name of "superorganism".

### **1.2 Functions**

*Metformin*

the disease.

the disease better and also to better manage the treatment options. Metformin, one of the most widely used first-line medication for the management of type 2 diabetes, looks to present other modes of action than the classic ones involving liver metabolism. Studies proved that metformin could modulate the gut microbiota disturbances encountered in type 2 diabetes, in this way improving the outcome of

Among other things, the cohabitation of the man with the environment is at the root of the human evolution, an extraordinary example in this sense being the

The digestive tract hosts a complex, vast and dynamic community of microorganisms, called the microbiota. Together they form a mutualist relationship, with profound implications for the host both during homeostasis and disease [1].

It is worth mentioning that the gut is not the only place where there is a population of microorganisms with which the human organism is in such a connection

The composition of the microbiota varies from individual to another but also from segment to segment of the digestive tract and includes species from all three domains of life: bacteria, Archaea and Eukarya. All of the species are classified into 12 different phyla, of which more than 90% belong to *Actinobacteria*, *Bacteroidetes*,

The process of colonizing the digestive tube with microorganisms is classically believed to begin at birth by "seeding" the newborn with microorganisms originating from the mother's genital area (vaginal passage, mother's areola), the skin, and the microbiota of the contacts in the surrounding environment, and from then the development continues throughout life. In recent years, however, this theory is challenged by a series of studies that have shown the presence of microorganisms in uterine tissues (e.g., the placenta, suggesting that colonization could be initiated before birth, by haematogenous sowing) [4]. At the age of 3–4 years, the core of the microbiota is relatively defined, and its structure is similar to that of the adult but is continuously subject to change depending on various external and internal

Even if the core of the microbiota is established from an early age, several factors contribute to carve its form, explaining its variations from an individual to another [5]:

• Diet (starting with breast milk that plays an essential role in the development

**1.1 The gut microbiota: definition, development and structure**

relationship between humans and microorganisms.

(e.g., the skin also harbouring a plethora of bacteria) [2].

*Firmicutes* and *Proteobacteria* [3].

**46**

factors.

• Type of birth

• Gestational age

of the flora)

• Physical activity

• Geographic region and cultural habits

• Drugs (especially antibiotic therapy)

• Ageing

• Diseases

The microbiota exerts a significant influence on the host during homeostasis and disease, with profound implications for the proper body's physiological functions, considering the microbiota as a "forgotten organ".

The leading roles of the gut flora are the following:


### **1.3 Dysbiosis: definition, causes and consequences**

Any perturbation of the healthy gut microbiota that disrupts the mutualist relationship between the organism and the associated microbes is called dysbiosis. The antagonist term of dysbiosis is eubiosis.

The underlying cause of a gut dysbiosis may be the following [13]:


### *Metformin*

The effects of dysbiosis are reflected in the processes of the internal environment, contributing to the emergence of numerous pathological conditions such as [14]:


### **2. Dysbiosis: microbiota in type 2 diabetes**

The relationship between gut microbiota and diabetes is not fully understood, but changes in its composition and function can contribute to the onset and maintenance of insulin resistance, thus influencing the prognosis of this illness. Both T2D patients and those that are at high risk of developing this disease seem to have an imbalance in the composition and function of the microbiota, just like a "metabolic dysbiosis".

Analysing the literature, the main changes observed in the microbiota composition of diabetic patients are [20–23]:


The Gram-negative bacteria (*E. coli*, *Bacteroidetes* and *Proteobacteria*) present lipopolysaccharides (LPS) at the surface of the membrane. Lipopolysaccharides are also known as endotoxins. They are large molecules consisting of a lipid and a polysaccharide composed of O-antigen with an outer core and an inner core joined by a covalent bond [24]. LPS are found to be elevated in the plasma of diabetic and obese patients by crossing an altered intestinal barrier (leaky gut). Accumulating, they trigger an inflammatory reaction called endotoxinemia. This systemic inflammatory response is associated with dyslipidaemia, increased blood pressure, but

**49**

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients*

also, with insulin resistance and earlier onset of diabetes through a variety of

• Activation of pro-inflammatory kinases: mitogen-activated protein kinases

• Increased expression of inflammatory proteins: tumour necrosis factor-α

The passage of LPS through the intestinal mucosa is due to increased intestinal permeability (so-called leaky gut) that can be explained by the diminishing of butyrate and mucin-degrading bacteria such as *Roseburia*, *Butyrivibrio* and *Akkermansia muciniphila*. Furthermore, this epithelial dysfunction can determine an important translocation of intestinal bacteria into the adipose tissue, which maintains a low-grade inflammation and insulin resistance, process called "meta-

At the moment, the amount of information regarding an alleged link between gut microbiota and T1D is modest. Several studies showed similarities between the disturbances of the microbiota found in T2D and T1D patients: reduced population of *Firmicutes* and increased the population of *Bacteroidetes* and increased in intestinal permeability. Increased gut permeability might contribute to pancreatic β-cell damage due to the increased absorption of exogenous antigens such as Streptomyces toxin—streptozotocin—that has tropism for pancreatic tissue and can cause lesions at its level [28].

There are also mechanisms mediated by the gut microbiota such as the production of short-chain fatty acids and secondary bile acids (SBA) that counteract those pro-inflammatory- and insulin-resistant effects. These mechanisms can be affected

SCFAs are produced from dietary fibres that are fermented by the intestinal bacteria. Acetate, butyrate and propionate are the three most common SCFAs. They exert an essential role in the metabolism of carbohydrates, lipids, in maintaining the integrity of the intestinal barrier and in modulating inflammatory reactions

• Maintaining the integrity of the colon epithelium: Butyric acid is the primary energy source of the colon's epithelial cells. It stimulates the proliferation but also the differentiation and apoptosis of the colonocyte, thus participating in the coordination of its life cycle. It also participates in the regulation of tight

• Improves carbohydrate metabolism: Propionate lowers the accumulation of lipids in the adipose tissue and reduces hepatic lipogenesis thus decreases the

*2.1.1 Dysbiosis: protective anti-inflammatory- and anti-insulin-resistant* 

junction proteins (claudin 1 and zonula occludens).

• Impaired insulin signalling at the level of insulin receptor substrate 1

(TNFα), monocyte chemotactic protein and interleukin 6

*DOI: http://dx.doi.org/10.5772/intechopen.88749*

and I kappa B kinase complex

• Inhibition of glucose transport

**2.1 Dysbiosis: microbiota in type 1 diabetes (T1D)**

mechanisms such as [25]:

bolic infection" [26, 27].

*mechanisms*

in the case of dysbiosis.

through a variety of functions [29]:

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients DOI: http://dx.doi.org/10.5772/intechopen.88749*

also, with insulin resistance and earlier onset of diabetes through a variety of mechanisms such as [25]:


*Metformin*

• Autoimmune diseases [15]

• Neurological disorders [19]

tion of diabetic patients are [20–23]:

**2. Dysbiosis: microbiota in type 2 diabetes**

• Prematurely ageing

• Allergies [16]

• Obesity

• Diabetes

dysbiosis".

*muciniphila*

*Proteobacteria*

*Eggerthella lenta*

• Cancers [18]

• Atherosclerosis [17]

The effects of dysbiosis are reflected in the processes of the internal environment,

The relationship between gut microbiota and diabetes is not fully understood, but changes in its composition and function can contribute to the onset and maintenance of insulin resistance, thus influencing the prognosis of this illness. Both T2D patients and those that are at high risk of developing this disease seem to have an imbalance in the composition and function of the microbiota, just like a "metabolic

Analysing the literature, the main changes observed in the microbiota composi-

• Decrease in bacteria that regulate intestinal permeability, such as *Akkermansia* 

• Increase in various opportunistic pathogens such as *Clostridium symbiosum* and

The Gram-negative bacteria (*E. coli*, *Bacteroidetes* and *Proteobacteria*) present lipopolysaccharides (LPS) at the surface of the membrane. Lipopolysaccharides are also known as endotoxins. They are large molecules consisting of a lipid and a polysaccharide composed of O-antigen with an outer core and an inner core joined by a covalent bond [24]. LPS are found to be elevated in the plasma of diabetic and obese patients by crossing an altered intestinal barrier (leaky gut). Accumulating, they trigger an inflammatory reaction called endotoxinemia. This systemic inflammatory response is associated with dyslipidaemia, increased blood pressure, but

• Reduced Gram-positive bacteria such as bacteria from phyla *Firmicutes*

• Reduced butyrate-producing bacteria, such as *Roseburia* and *Butyrivibrio*

• Increased Gram-negative bacteria, such as *Bacteroides*, *E. coli* and

contributing to the emergence of numerous pathological conditions such as [14]:

**48**

The passage of LPS through the intestinal mucosa is due to increased intestinal permeability (so-called leaky gut) that can be explained by the diminishing of butyrate and mucin-degrading bacteria such as *Roseburia*, *Butyrivibrio* and *Akkermansia muciniphila*. Furthermore, this epithelial dysfunction can determine an important translocation of intestinal bacteria into the adipose tissue, which maintains a low-grade inflammation and insulin resistance, process called "metabolic infection" [26, 27].

### **2.1 Dysbiosis: microbiota in type 1 diabetes (T1D)**

At the moment, the amount of information regarding an alleged link between gut microbiota and T1D is modest. Several studies showed similarities between the disturbances of the microbiota found in T2D and T1D patients: reduced population of *Firmicutes* and increased the population of *Bacteroidetes* and increased in intestinal permeability. Increased gut permeability might contribute to pancreatic β-cell damage due to the increased absorption of exogenous antigens such as Streptomyces toxin—streptozotocin—that has tropism for pancreatic tissue and can cause lesions at its level [28].

*2.1.1 Dysbiosis: protective anti-inflammatory- and anti-insulin-resistant mechanisms*

There are also mechanisms mediated by the gut microbiota such as the production of short-chain fatty acids and secondary bile acids (SBA) that counteract those pro-inflammatory- and insulin-resistant effects. These mechanisms can be affected in the case of dysbiosis.

SCFAs are produced from dietary fibres that are fermented by the intestinal bacteria. Acetate, butyrate and propionate are the three most common SCFAs. They exert an essential role in the metabolism of carbohydrates, lipids, in maintaining the integrity of the intestinal barrier and in modulating inflammatory reactions through a variety of functions [29]:


insulin resistance. Propionate and acetate also stimulate the production of glucagon-like peptide-1.

• Anti-inflammatory role: Butyric acid plays an essential role in maintaining the integrity of the intestinal mucosa, preventing endotoxemia and metabolic infection. Butyric acid also inhibits the nuclear factor kappa-beta from the macrophages that cause a suppression of TNF-alpha, IL-6 and myeloperoxidase activity.

At the intestinal level, bacteria metabolize primary bile acids (cholic and chenodeoxycholic acids) to secondary bile acids (deoxycholic and lithocholic acids). Bile acids are involved in multiple metabolic pathways, research over the last decades, demonstrating an essential role against inflammation and insulin resistance. Secondary bile acids contribute to a decrease in insulin resistance through:


In terms of their anti-inflammatory role, lithocholic acid inhibits the release of pro-inflammatory cytokines TNF-alpha, IL1 and IL6 from colon epithelium [32].

### **3. Metformin and the gut**

Metformin presents as a sophisticated drug having multiple sites of action and various molecular mechanisms. Lately, attention has been directed to other modes of action, different than the classic ones. Its action at the intestinal level was suggested by the results of several studies that showed the following:


**51**

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients*

• Increase microbes from *Verrucomicrobiaceae*, *Porphyromonadaceae*,

Regarding the effects of metformin on the gut microbiota, studies have shown that administration of metformin produced several changes in the composition of

*Rikenellaceae*, *Akkermansia muciniphila*, and *Prevotellaceae* spp. moreover,

• Decrease of the *Lachnospiraceae*, *Rhodobacteraceae* spp., *Peptostreptococcaceae*

Furthermore by comparing the modified microbiome profile by metformin treatment, with the microbiome profiles under various disease situations, these changes have been negatively correlated with multiple diseases that have an inflammatory pathogenic substrate such as colitis, chronic diarrhoea and irritable bowel syndrome, suggesting that its anti-inflammatory proprieties can be determined

The main side effects of metformin are gastrointestinal: nausea, vomiting, diarrhoea and abdominal pain. These side effects occur most frequently at the beginning of treatment, and in most cases, they disappear spontaneously. The cause of these side effects is not fully understood and may be due to the growth of opportunistic pathogenic bacteria from *Escherichia* to *Shigella* spp. which are shown to increase at the beginning of treatment. If we relate to the increase of these opportunistic pathogens, the further reduction of side effects can be caused by a reduction of the substrate to which these microorganisms are dependent (substrates provided by polysaccharide-degrading anaerobes) through diet and an increase of anaerobic

As stated before, the gut microbiota profile is profoundly modified in T2D patients in terms of its structure and composition. Administration of metformin results in improved glucose metabolism, but the way this is achieved is not fully understood, and its implications upon the intestinal flora are incompletely discovered. Analysing data from the literature, administration of metformin causes the composition to change and, therefore, the physiology of the microbiota as well.

Administration of metformin is associated with an essential decrease in

*Bacteroides fragilis* is an obligately anaerobic, Gram-negative, rod-shaped bacteria, whose essential feature in metabolic pathology is the presence of capsular lipopolysaccharides. LPS are found to be elevated in the plasma of diabetic and obese patients and are associated with dyslipidaemia and increased blood pressure but also with insulin resistance and earlier onset of diabetes through a plenty of mechanisms that have been described previously. Colonizing mice with *Bacteroides fragilis* by transferring stool samples enriched with these bacteria determines an increase in body weight, impaired glucose tolerance and a decrease

*DOI: http://dx.doi.org/10.5772/intechopen.88749*

the intestinal flora such as the following [36]:

species from *Escherichia-Shigella* sp.

through regulation of the microbiota homeostasis.

mucus-associated bacteria such as *Akkermansia muciniphila* [37].

**4. Metformin and the microbiota of type 2 diabetes**

**5. Metformin and** *Bacteroides fragilis*

*Bacteroides fragilis* [38].

in insulin sensitivity.

and *Clostridiaceae*

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients DOI: http://dx.doi.org/10.5772/intechopen.88749*

Regarding the effects of metformin on the gut microbiota, studies have shown that administration of metformin produced several changes in the composition of the intestinal flora such as the following [36]:


Furthermore by comparing the modified microbiome profile by metformin treatment, with the microbiome profiles under various disease situations, these changes have been negatively correlated with multiple diseases that have an inflammatory pathogenic substrate such as colitis, chronic diarrhoea and irritable bowel syndrome, suggesting that its anti-inflammatory proprieties can be determined through regulation of the microbiota homeostasis.

The main side effects of metformin are gastrointestinal: nausea, vomiting, diarrhoea and abdominal pain. These side effects occur most frequently at the beginning of treatment, and in most cases, they disappear spontaneously. The cause of these side effects is not fully understood and may be due to the growth of opportunistic pathogenic bacteria from *Escherichia* to *Shigella* spp. which are shown to increase at the beginning of treatment. If we relate to the increase of these opportunistic pathogens, the further reduction of side effects can be caused by a reduction of the substrate to which these microorganisms are dependent (substrates provided by polysaccharide-degrading anaerobes) through diet and an increase of anaerobic mucus-associated bacteria such as *Akkermansia muciniphila* [37].

### **4. Metformin and the microbiota of type 2 diabetes**

As stated before, the gut microbiota profile is profoundly modified in T2D patients in terms of its structure and composition. Administration of metformin results in improved glucose metabolism, but the way this is achieved is not fully understood, and its implications upon the intestinal flora are incompletely discovered. Analysing data from the literature, administration of metformin causes the composition to change and, therefore, the physiology of the microbiota as well.

### **5. Metformin and** *Bacteroides fragilis*

Administration of metformin is associated with an essential decrease in *Bacteroides fragilis* [38].

*Bacteroides fragilis* is an obligately anaerobic, Gram-negative, rod-shaped bacteria, whose essential feature in metabolic pathology is the presence of capsular lipopolysaccharides. LPS are found to be elevated in the plasma of diabetic and obese patients and are associated with dyslipidaemia and increased blood pressure but also with insulin resistance and earlier onset of diabetes through a plenty of mechanisms that have been described previously. Colonizing mice with *Bacteroides fragilis* by transferring stool samples enriched with these bacteria determines an increase in body weight, impaired glucose tolerance and a decrease in insulin sensitivity.

*Metformin*

glucagon-like peptide-1.

G-protein-coupled receptor 1 [30]

• Increasing triglyceride clearance

the metabolism of lipids

**3. Metformin and the gut**

diabetes [35].

dase activity.

(FXR)

insulin resistance. Propionate and acetate also stimulate the production of

• Anti-inflammatory role: Butyric acid plays an essential role in maintaining the integrity of the intestinal mucosa, preventing endotoxemia and metabolic infection. Butyric acid also inhibits the nuclear factor kappa-beta from the macrophages that cause a suppression of TNF-alpha, IL-6 and myeloperoxi-

At the intestinal level, bacteria metabolize primary bile acids (cholic and chenodeoxycholic acids) to secondary bile acids (deoxycholic and lithocholic acids). Bile acids are involved in multiple metabolic pathways, research over the last decades, demonstrating an essential role against inflammation and insulin resistance. Secondary bile acids contribute to a decrease in insulin resistance through:

• Modulating glucose absorption through interaction with farnesoid X receptor

• Modulating energy expenditure: increase energy expenditure in brown adipose tissue by activating enzyme type 2 iodothyronine deiodinase and oxygen

• Bile acids are the major pathway for catabolism of cholesterol, thus regulating

In terms of their anti-inflammatory role, lithocholic acid inhibits the release of pro-inflammatory cytokines TNF-alpha, IL1 and IL6 from colon epithelium [32].

Metformin presents as a sophisticated drug having multiple sites of action and various molecular mechanisms. Lately, attention has been directed to other modes of action, different than the classic ones. Its action at the intestinal level was sug-

• A delayed-release formula is retained almost entirely in the gut, with minimal systemic absorption. It is effective at lowering blood glucose as the standard immediate-release formulation in individuals with type 2 diabetes [33].

• In diabetic rats, intravenous administration of metformin is less effective than intra-duodenal administration for lowering blood glucose levels [34].

• Human genetic studies proved that variants in SLC22A1 gene (the gene encoding OCT1), which reduce hepatic uptake of metformin, do not impact upon the efficacy of metformin to lower HbA1c in individuals with type 2

gested by the results of several studies that showed the following:

• Stimulating the production of glucagon-like peptide-1 by binding to

consumption, thus contributing to the prevention of obesity [31]

**50**

Mechanisms by which metformin has determined the decrease of this species have not been elucidated but have been assumed since *Bacteroides fragilis* were reduced in mice that received stool samples from patients who had been given metformin.

Besides reducing *Bacteroides fragilis*, the bile acid glycoursodeoxycholic (GUDCA) is increased through decreasing the bacteria's bile salt hydrolase activity. GUDCA is a glycine-conjugated form of the secondary bile acid deoxycholic acid, which has been known to have anti-inflammatory proprieties by reducing the levels of pro-inflammatory cytokines. Another biological function of GUDCA is to antagonize the farnesoid X receptor.

The FXR is predominantly found at the intestinal and hepatic tissue. Bile acids are the major ligands (activators) of this receptor. It is mainly involved in the metabolism of bile acids but also of carbohydrates and lipids.

The primary functions of FXR activation is the suppression of cholesterol 7 alpha-hydroxylase (CYP7A1), which reduces the synthesis of bile acids (via the feedback mechanism, FXR is activated by bile acids and further determines the suppression of this enzyme, thus reducing the synthesis of bile acids). FXR inhibition produces an increase in bile acids improving metabolic endpoints due to their anti-inflammatory and insulin sensitivity effects [39].

### **6. Metformin and** *Akkermansia muciniphila*

As stated before, the epithelial barrier of T2D patients is affected by an increase in its permeability (so-called leaky gut) followed by a migration of different toxins such as LPS in the systemic circulation causing inflammatory responses, insulin resistance and impaired glucose tolerance. In addition to these changes, a decrease in the *Akkermansia muciniphila* population was observed.

*Akkermansia muciniphila* is a mucin-degrading bacterium of the phylum *Verrucomicrobia* that resides predominantly in the mucus layer of the colon, where it is involved in maintaining intestinal integrity by promoting mucus secretion and making the barrier mechanism more stable and therefore decreasing its epithelial permeability. Oral supplementation with this bacterial population was shown to reduce intestinal permeability and improve glucose metabolism [40, 41].

A significant change in the composition of the microbiota under metformin treatment regarding intestinal permeability is represented by an increase in the population of *Akkermansia muciniphila*. The mechanism by which this process is accomplished is not fully understood, but it seems that these bacteria metabolise unabsorbable carbohydrates and mucin in short-chain fatty acids, which in turn will be used as fuel for goblet cells. Stimulated goblet cells will further produce mucin, in this way leading to the thickening of the mucus layer and thus to a decrease in the epithelial permeability. Besides increasing the population of *A. muciniphila*, administration of metformin is associated with an increase in the density of mucin-producing goblet cells probably through the indirect mechanism stated above [42, 43] **Figure 1**.

### **6.1 Metformin and SCAF-producing bacteria**

One of the main features of the dysbiosis found in T2D patients is the decrease in butyrate-producing bacteria such as *Roseburia* and *Butyrivibrio*.

**53**

carbohydrates.

**Figure 1.**

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients*

*Roseburia* is a Gram-positive anaerobic bacteria member of the *Firmicutes* phyla

As stated above, short-chain fatty acids such as butyrate, propionate, and acetate are the product of gut microbiota activity, resulting from the fermentation of the carbohydrates that escapes the absorption process, playing an essential role in the process of enhancing intestinal integrity, reducing inflammation and improv-

Significant increase of butyrate-producing bacteria, especially *Butyrivibrio* and

The genus *Bifidobacterium* is a Gram-positive microorganism, member of the *Bifidobacteriaceae* family, belonging to the great *Actinobacteria* phylum, one of the

Oral supplementation of *L. casei* and *B. bifidum*, which are frequently used as a probiotic treatment option, alone and in combination, has been shown to improve insulin resistance (decreased fasting blood glucose, decrease HbA1C) and lower the serum lipid levels by enhancing short-chain fatty acids production, and thus

Administration of metformin has been shown to increase the population of *Bifidobacterium adolescentis*, *Bifidobacterium bifidum*, and also *Lactobacillus* [47].

*Lactobacillus* is a Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria that produces lactic acid from converting

named in honour of distinguished microbiologist Theodor Rosebury [45].

*Roseburia*, is observed in T2D patients treated with metformin [43].

ing the metabolism of glucose and lipids.

Akkermansia muciniphila *mode of action [42, 43].*

most abundant species of the gut microbiota.

improving the outcome of T2D patients [46].

**6.2 Metformin and probiotics**

*DOI: http://dx.doi.org/10.5772/intechopen.88749*

*Butyrivibrio* is a Gram-negative, anaerobic bacteria belonging to the *Clostridia* class, which was first described in the mid-twentieth century [44].

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients DOI: http://dx.doi.org/10.5772/intechopen.88749*

**Figure 1.** Akkermansia muciniphila *mode of action [42, 43].*

*Roseburia* is a Gram-positive anaerobic bacteria member of the *Firmicutes* phyla named in honour of distinguished microbiologist Theodor Rosebury [45].

As stated above, short-chain fatty acids such as butyrate, propionate, and acetate are the product of gut microbiota activity, resulting from the fermentation of the carbohydrates that escapes the absorption process, playing an essential role in the process of enhancing intestinal integrity, reducing inflammation and improving the metabolism of glucose and lipids.

Significant increase of butyrate-producing bacteria, especially *Butyrivibrio* and *Roseburia*, is observed in T2D patients treated with metformin [43].

### **6.2 Metformin and probiotics**

The genus *Bifidobacterium* is a Gram-positive microorganism, member of the *Bifidobacteriaceae* family, belonging to the great *Actinobacteria* phylum, one of the most abundant species of the gut microbiota.

*Lactobacillus* is a Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria that produces lactic acid from converting carbohydrates.

Oral supplementation of *L. casei* and *B. bifidum*, which are frequently used as a probiotic treatment option, alone and in combination, has been shown to improve insulin resistance (decreased fasting blood glucose, decrease HbA1C) and lower the serum lipid levels by enhancing short-chain fatty acids production, and thus improving the outcome of T2D patients [46].

Administration of metformin has been shown to increase the population of *Bifidobacterium adolescentis*, *Bifidobacterium bifidum*, and also *Lactobacillus* [47].

*Metformin*

metformin.

antagonize the farnesoid X receptor.

Mechanisms by which metformin has determined the decrease of this species have not been elucidated but have been assumed since *Bacteroides fragilis* were reduced in mice that received stool samples from patients who had been given

The FXR is predominantly found at the intestinal and hepatic tissue. Bile acids

The primary functions of FXR activation is the suppression of cholesterol 7 alpha-hydroxylase (CYP7A1), which reduces the synthesis of bile acids (via the feedback mechanism, FXR is activated by bile acids and further determines the suppression of this enzyme, thus reducing the synthesis of bile acids). FXR inhibition produces an increase in bile acids improving metabolic endpoints due to their

As stated before, the epithelial barrier of T2D patients is affected by an increase in its permeability (so-called leaky gut) followed by a migration of different toxins such as LPS in the systemic circulation causing inflammatory responses, insulin resistance and impaired glucose tolerance. In addition to these changes, a decrease

*Akkermansia muciniphila* is a mucin-degrading bacterium of the phylum *Verrucomicrobia* that resides predominantly in the mucus layer of the colon, where it is involved in maintaining intestinal integrity by promoting mucus secretion and making the barrier mechanism more stable and therefore

decreasing its epithelial permeability. Oral supplementation with this bacterial population was shown to reduce intestinal permeability and improve glucose

A significant change in the composition of the microbiota under metformin treatment regarding intestinal permeability is represented by an increase in the population of *Akkermansia muciniphila*. The mechanism by which this process is accomplished is not fully understood, but it seems that these bacteria metabolise unabsorbable carbohydrates and mucin in short-chain fatty acids, which in turn will be used as fuel for goblet cells. Stimulated goblet cells will further produce mucin, in this way leading to the thickening of the mucus layer and thus to a decrease in the epithelial permeability. Besides increasing the population of *A. muciniphila*, administration of metformin is associated with an increase in the density of mucin-producing goblet cells probably through the indirect mechanism

One of the main features of the dysbiosis found in T2D patients is the decrease in

*Butyrivibrio* is a Gram-negative, anaerobic bacteria belonging to the *Clostridia*

Besides reducing *Bacteroides fragilis*, the bile acid glycoursodeoxycholic (GUDCA) is increased through decreasing the bacteria's bile salt hydrolase activity. GUDCA is a glycine-conjugated form of the secondary bile acid deoxycholic acid, which has been known to have anti-inflammatory proprieties by reducing the levels of pro-inflammatory cytokines. Another biological function of GUDCA is to

are the major ligands (activators) of this receptor. It is mainly involved in the

metabolism of bile acids but also of carbohydrates and lipids.

anti-inflammatory and insulin sensitivity effects [39].

**6. Metformin and** *Akkermansia muciniphila*

in the *Akkermansia muciniphila* population was observed.

**52**

metabolism [40, 41].

stated above [42, 43] **Figure 1**.

**6.1 Metformin and SCAF-producing bacteria**

butyrate-producing bacteria such as *Roseburia* and *Butyrivibrio*.

class, which was first described in the mid-twentieth century [44].

### **6.3 Metformin and** *Adlercreutzia*

The soybean, a legume species native from East Asia, is widely grown for its edible bean, which has numerous uses. It has been assumed that soy foods contribute to reducing the risk of T2D and the progression of this disease in diabetic patients although opinions are divided by the results of studies which inform this theory or rather confirm it [48].

At the gut level, the main species that metabolizes soybean isoflavonoids to equol are the ones from *Adlercreutzia*. It is worth mentioning that not in all people isoflavonoids are metabolized to equol (so-called equol producers). It was speculated that the health benefits of soy-based diets might be higher in equol producers than in equol nonproducers [49].

It seems that metformin treatment increases the population of *Adlercreutzia* in diabetic patients and therefore stimulating the production of equol, thus enhancing soy-based diet health benefits [50].

### **6.4 Summary of changes found after and before metformin treatment**

These tables help summarise the changes found in the gut microbiota both before and after metformin treatment in T2D patients **Tables 1** and **2**.


### **Table 1.**

*Summary of changes in microbiota composition before and after metformin treatment of T2D.*


**55**

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients*

Alterations of the intestinal microbiota are a key element in understanding the pathophysiology of diabetes and maybe to explain the variability in terms of its therapeutic response and complications occurrence in different patients.

Metformin exerts a significant influence on the bacterial constellation found in

With changes in both composition and function, modulation of the intestinal flora of patients with type 2 diabetes mellitus, obtained by various methods, can bring a better outcome of diabetes patients and can improve the morbidity and

the gut, bringing a significant contribution to restoring its balance.

\*, Sergiu Ioan Micu1

1 Internal Medicine—Gastroenterology Department, Faculty of Medicine,

2 Endocrinology Department, Faculty of Medicine, "Ovidius" University,

3 Diabetes Mellitus and Nutritional Diseases Department, Faculty of Medicine,

4 Oncology Department, Faculty of Medicine, "Ovidius" University, Constanta,

5 Diabetes Mellitus and Nutritional Diseases Department, University of Medicine

6 Cardiology Department, Faculty of Medicine, "Ovidius" University, Constanta,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Doina Catrinoiu3

, Roxana Adriana Stoica5

"Ovidius" University, Constanta, Romania

"Ovidius" University, Constanta, Romania

provided the original work is properly cited.

and Pharmacy "Carol Davila", Bucharest, Romania

\*Address all correspondence to: andrasuceveanu@yahoo.com

, Claudia Voinea<sup>2</sup>

and Adrian-Paul Suceveanu1

, Laura Mazilu4

,

, Anca Pantea Stoian<sup>5</sup>

,

mortality rates of this widely present metabolic disease.

*DOI: http://dx.doi.org/10.5772/intechopen.88749*

**7. Conclusions**

**Author details**

Irinel Parepa6

Romania

Romania

Andra Iulia-Suceveanu1

Madalina Elena Manea3

Constanta, Romania

### **Table 2.**

*Summary of changes in the functions of microbiota before and after metformin treatment.*

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients DOI: http://dx.doi.org/10.5772/intechopen.88749*

### **7. Conclusions**

*Metformin*

**6.3 Metformin and** *Adlercreutzia*

theory or rather confirm it [48].

than in equol nonproducers [49].

soy-based diet health benefits [50].

The soybean, a legume species native from East Asia, is widely grown for its edible bean, which has numerous uses. It has been assumed that soy foods contribute to reducing the risk of T2D and the progression of this disease in diabetic patients although opinions are divided by the results of studies which inform this

At the gut level, the main species that metabolizes soybean isoflavonoids to equol are the ones from *Adlercreutzia*. It is worth mentioning that not in all people isoflavonoids are metabolized to equol (so-called equol producers). It was speculated that the health benefits of soy-based diets might be higher in equol producers

It seems that metformin treatment increases the population of *Adlercreutzia* in diabetic patients and therefore stimulating the production of equol, thus enhancing

**6.4 Summary of changes found after and before metformin treatment**

before and after metformin treatment in T2D patients **Tables 1** and **2**.

Reduced Gram-positive bacteria, such as bacteria from phyla

Reduced butyrate-producing bacteria, such as *Roseburia* and

Decrease in bacteria that regulate intestinal permeability, such

Increased Gram-negative bacteria, such as *Bacteroides*, *E. coli*

Increase in various opportunistic pathogens, such as *Clostridium symbiosum* and *Eggerthella lenta*

**Mechanisms before metformin Mechanisms after metformin** Decrease production of SCFAs Increased production of SCFAs

*Summary of changes in microbiota composition before and after metformin treatment of T2D.*

Endotoxemia and metabolic infection Reduced endotoxemia Inflammation Decreased inflammation Insulin resistance Increased insulin sensitivity

*Summary of changes in the functions of microbiota before and after metformin treatment.*

Epithelial dysfunction and increased intestinal

permeability

*Firmicutes*

*Butyrivibrio*

as *Akkermansia muciniphila*

and *Proteobacteria*

Decrease production of bile acids Increased bile acid production, especially GUDCA

Increased systemic LPS Decreased in LPS migration, reduced systemic LPS

Inhibition of farnesoid X receptor

Increased production of equol

permeability

Enhancing the intestinal barrier, decreasing its

Increased *Firmicutes*

*Bifidobacterium* and Increase *Adlercreutzia*

*muciniphila*

Increased *Roseburia* and *Butyrivibrio*

Significant increase of *Akkermansia* 

Increased probiotic bacteria, such as

Significant decrease of *Bacteroides fragilis*

These tables help summarise the changes found in the gut microbiota both

**Structure before metformin treatment Structure after metformin treatment**

**54**

**Table 2.**

**Table 1.**

Alterations of the intestinal microbiota are a key element in understanding the pathophysiology of diabetes and maybe to explain the variability in terms of its therapeutic response and complications occurrence in different patients.

Metformin exerts a significant influence on the bacterial constellation found in the gut, bringing a significant contribution to restoring its balance.

With changes in both composition and function, modulation of the intestinal flora of patients with type 2 diabetes mellitus, obtained by various methods, can bring a better outcome of diabetes patients and can improve the morbidity and mortality rates of this widely present metabolic disease.

### **Author details**

Andra Iulia-Suceveanu1 \*, Sergiu Ioan Micu1 , Claudia Voinea<sup>2</sup> , Madalina Elena Manea3 , Doina Catrinoiu3 , Laura Mazilu4 , Anca Pantea Stoian<sup>5</sup> , Irinel Parepa6 , Roxana Adriana Stoica<sup>5</sup> and Adrian-Paul Suceveanu1

1 Internal Medicine—Gastroenterology Department, Faculty of Medicine, "Ovidius" University, Constanta, Romania

2 Endocrinology Department, Faculty of Medicine, "Ovidius" University, Constanta, Romania

3 Diabetes Mellitus and Nutritional Diseases Department, Faculty of Medicine, "Ovidius" University, Constanta, Romania

4 Oncology Department, Faculty of Medicine, "Ovidius" University, Constanta, Romania

5 Diabetes Mellitus and Nutritional Diseases Department, University of Medicine and Pharmacy "Carol Davila", Bucharest, Romania

6 Cardiology Department, Faculty of Medicine, "Ovidius" University, Constanta, Romania

\*Address all correspondence to: andrasuceveanu@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[22] Harsch IA, Konturek PC. The role of gut microbiota in obesity and type 2 and type 1 diabetes mellitus: New insights into "old" diseases. Medical Science. 2018;**6**(2):32. DOI: 10.3390/ medsci6020032

[23] Aw W, Fukuda S. Understanding the role of the gut ecosystem in diabetes mellitus. Journal of Diabetes Investigation. 2018;**9**(1):5-12. DOI: 10.1111/jdi.12673

[24] https://en.wikipedia.org/wiki/ Lipopolysaccharide

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[27] Burcelin R, Serino M, Chabo C, Garidou L, Pomié C, Courtney M, et al. Metagenome and metabolism: The tissue microbiota hypothesis. Diabetes, Obesity & Metabolism. 2013;**15**(Suppl. 3):61-70. DOI: 10.1111/ dom.12157

[28] Zheng P, Li Z, Zhou Z. Gut microbiome in type 1 diabetes: A comprehensive review. Diabetes/ Metabolism Research and Reviews. 2018;**34**(7):e3043. DOI: 10.1002/ dmrr.3043

[29] Tan J, McKenzie C, Potamitis M, Thorburn AN, Mackay CR, Macia L. The role of short-chain fatty acids in health and disease. Advances in Immunology. 2014;**121**:91-119. DOI: 10.1016/B978-0-12-800100-4.00003-9

[30] Albaugh VL, Banan B, Antoun J, Xiong Y, Guo Y, Ping J, et al. Role of bile acids and GLP-1 in mediating the metabolic improvements of bariatric surgery. Gastroenterology Journal. 2019;**156**(4):1041-1051.e4. DOI: 10.1053/j.gastro.2018.11.017

[31] Watanabe M et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature. 2006;**439**(7075):484-489

[32] Ward BJ, Lajczak-Mc GJ, Kelly N, O'Dwyer OM, Giddam AK, et al. Ursodeoxycholic acid and lithocholic acid exert anti-inflammatory actions in the colon. AJP Gastrointestinal

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[42] Shin NR, Lee JC, Lee HY, Kim MS, Whon TW, Lee MS, et al. An increase in the *Akkermansia* spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice. Gut. 2014;**63**(5):727-735. DOI: 10.1136/ gutjnl-2012-303839

[43] de la Cuesta-Zuluaga MNT, Corrales-Agudelo V, Velásquez-Mejía EP, Carmona JA, Abad JM, Escobar JS. Metformin is associated with higher relative abundance of mucindegrading *Akkermansia muciniphila* and several short-chain fatty acid-producing microbiota in the gut. Diabetes Care. 2017;**40**(1):54-62. DOI: 10.2337/ dc16-1324

[44] https://en.wikipedia.org/wiki/ Butyrivibrio

[45] https://en.wikipedia.org/wiki/ Roseburia

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[47] Rodriguez J, Hiel S, Delzenne NM. Metformin: Old friend, new ways of action-implication of the gut microbiome? Current Opinion in Clinical Nutrition and Metabolic Care. 2018;**21**(4):294-301. DOI: 10.1097/ MCO.0000000000000468

[48] Mueller NT, Odegaard AO, Gross MD, Koh W-P, Yu MC, Yuan J-M, et al. Soy intake and risk of type 2 diabetes mellitus in Chinese Singaporeans. European Journal of Nutrition. 2012;**51**(8):1033-1040. DOI:

10.1007/s00394-011-0276-2

10.1007/s12263-012-0292-8

10.1155/2018/1890978

[49] Hong K-W et al. Epidemiological profiles between equol producers and nonproducers: A genome wide association study of the equolproducing phenotype. Genes & Nutrition. 2012;**7**(4):567-574. DOI:

[50] Lv Y, Zhao X, Guo W, Gao Y, Yang S, Li Z, et al. The relationship between frequently used glucose-lowering agents and gut microbiota in type 2 diabetes mellitus. Journal Diabetes Research. 2018;**2018**:1890978. DOI:

*Metformin and Its Benefits in Improving Gut Microbiota Disturbances in Diabetes Patients DOI: http://dx.doi.org/10.5772/intechopen.88749*

[47] Rodriguez J, Hiel S, Delzenne NM. Metformin: Old friend, new ways of action-implication of the gut microbiome? Current Opinion in Clinical Nutrition and Metabolic Care. 2018;**21**(4):294-301. DOI: 10.1097/ MCO.0000000000000468

*Metformin*

and Liver Physiology. 2017;**312**. DOI:

unconjugated bilirubin. Journal of Neuropathology and Experimental Neurology. 2007;**66**(9):789-798

[40] Geerlings SY, Kostopoulos I, de Vos WM, Belzer C. *Akkermansia muciniphila* in the human

gastrointestinal tract: When, where, and how?. Microorganisms 2018;**6**(3):

[41] Naito Y, Uchiyama K, Takagi T. A next-generation beneficial microbe: *Akkermansia muciniphila*. Journal of Clinical Biochemistry and Nutrition. 2018;**63**(1):33-35. DOI: 10.3164/

[42] Shin NR, Lee JC, Lee HY, Kim MS, Whon TW, Lee MS, et al. An increase in the *Akkermansia* spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice. Gut. 2014;**63**(5):727-735. DOI: 10.1136/

pii E75. DOI: 10.3390/ microorganisms6030075.

jcbn.18-57

gutjnl-2012-303839

dc16-1324

Butyrivibrio

Roseburia

[43] de la Cuesta-Zuluaga MNT, Corrales-Agudelo V, Velásquez-Mejía EP, Carmona JA, Abad JM,

[44] https://en.wikipedia.org/wiki/

[45] https://en.wikipedia.org/wiki/

*casei* and *Bifidobacterium bifidum* ameliorated hyperglycemia,

dyslipidemia, and oxidative stress in diabetic rats. International Journal of Preventive Medicine. 2016;**7**:102. DOI:

Singh R. Administration of *Lactobacillus* 

[46] Sharma P, Bhardwaj P,

10.4103/2008-7802.188870

Escobar JS. Metformin is associated with higher relative abundance of mucindegrading *Akkermansia muciniphila* and several short-chain fatty acid-producing microbiota in the gut. Diabetes Care. 2017;**40**(1):54-62. DOI: 10.2337/

[33] DeFronzo RA, Buse JB, Kim T, et al. Once-daily delayed-release metformin lowers plasma glucose and enhances fasting and postprandial GLP-1 and PYY: Results from two randomised trials. Diabetologia. 2016;**59**:1645. DOI:

10.1152/ajpgi.00256.2016

10.1007/s00125-016-3992-6

Opinion. 1984;**9**(1):47-51

[34] Bonora E, Cigolini M, Bosello O, Zancanaro C, Capretti L, Zavaroni I, et al. Lack of effect of intravenous metformin on plasma concentrations of glucose, insulin, C-peptide, glucagon and growth hormone in non-diabetic subjects. Current Medical Research and

[35] Sundelin E, Gormsen LC, Jensen JB, Vendelbo MH, Jakobsen S, Munk OL, et al. Genetic polymorphisms in organic cation transporter 1 attenuates hepatic metformin exposure in humans. Clinical Pharmacology and Therapeutics.

2017;**102**(5):841-848. DOI: 10.1002/cpt.701

[36] Ma W, Chen J, Meng Y, Yang J, Cui Q, Zhou Y. Metformin alters gut microbiota of healthy mice: Implication for its potential role in gut microbiota homeostasis. Frontiers in Microbiology. 2018;**9**:1336. DOI: 10.3389/fmicb.

[37] Elbere I et al. Association of metformin administration with gut microbiome dysbiosis in healthy volunteers. PLoS ONE. 2018;**13**(9):e0204317. DOI: 10.1371/

[38] Sun L et al. Gut microbiota and intestinal FXR mediate the clinical benefits of metformin. Nature

Medicine. 2018;**24**(12):1919-1929. DOI:

[39] Fernandes A, Vaz AR, Falcao AS, et al. Glycoursodeoxycholic acid and interleukin-10 modulate the reactivity

journal.pone.0204317

10.1038/s41591-018-0222-4

of rat cortical astrocytes to

2018.01336

**58**

[48] Mueller NT, Odegaard AO, Gross MD, Koh W-P, Yu MC, Yuan J-M, et al. Soy intake and risk of type 2 diabetes mellitus in Chinese Singaporeans. European Journal of Nutrition. 2012;**51**(8):1033-1040. DOI: 10.1007/s00394-011-0276-2

[49] Hong K-W et al. Epidemiological profiles between equol producers and nonproducers: A genome wide association study of the equolproducing phenotype. Genes & Nutrition. 2012;**7**(4):567-574. DOI: 10.1007/s12263-012-0292-8

[50] Lv Y, Zhao X, Guo W, Gao Y, Yang S, Li Z, et al. The relationship between frequently used glucose-lowering agents and gut microbiota in type 2 diabetes mellitus. Journal Diabetes Research. 2018;**2018**:1890978. DOI: 10.1155/2018/1890978

**61**

**Chapter 4**

**Abstract**

**1. Introduction**

Potential Protective Effects

Complications in Patients with

*Jasna Kusturica, Aida Kulo, Maida Rakanović-Todić,* 

Diabetes mellitus (DM) as a chronic condition is a growing global problem. Its numerous complications, including ocular diseases, affect patients' quality and length of life. Metformin is an effective, safe, and inexpensive first-line pharmacotherapy for type 2 diabetes (T2D). The current evidence indicates metformin's multiple sites of action and multiple molecular mechanisms leading to its beneficial impact on metabolism, inflammation, oxidative stress, aging, as well as to its cardiovascular, neurological, bone, and antiproliferative properties. These impacts are the result of its acting on adenosine monophosphate-activated protein kinase (AMPK) dependent and AMPK-independent pathways. Limited data suggest the protective role of metformin on microvascular ocular complications, including retinopathy, glaucoma, and age-related macular degeneration in patients with T2D. However, to confirm its mentioned protective and therapeutic effects, more large, randomized,

**Keywords:** type 2 diabetes, metformin, molecular mechanisms, ocular complications

T1D is commonly diagnosed in childhood and early adolescence, affects men and women equally, and shows the highest prevalence in the white race. T2D occurs in older life, while an increase in incidence is associated with poorer socioeconomic

Diabetes mellitus (DM) is a chronic systemic disease accompanied by impaired metabolism of carbohydrates, proteins, and fats. The American Diabetes Association (ADA) [1] distinguishes two basic types of diabetes mellitus, type 1 (T1D) and type 2 (T2D), while, in addition, gestational diabetes and specific forms of the disease are also recognized. The main pathophysiologic events in DM are insulin deficiency and insulin resistance. The most significant event is insulin resistance that develops in target tissues of action of insulin (muscle, fat tissues, and liver). In T1D, autoimmune destruction of β cells of the pancreatic islets (Langerhans islets) leads to deficient production and absolute insulin deficiency, while in T2D, insulin secretion is considered insufficient to overcome insulin

of Metformin on Ocular

*Lejla Burnazović-Ristić and Sanita Maleškić*

double-blind, and placebo-controlled clinical studies are needed.

resistance in peripheral tissues (relative insulin deficiency).

Type 2 Diabetes

### **Chapter 4**

## Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2 Diabetes

*Jasna Kusturica, Aida Kulo, Maida Rakanović-Todić, Lejla Burnazović-Ristić and Sanita Maleškić*

### **Abstract**

Diabetes mellitus (DM) as a chronic condition is a growing global problem. Its numerous complications, including ocular diseases, affect patients' quality and length of life. Metformin is an effective, safe, and inexpensive first-line pharmacotherapy for type 2 diabetes (T2D). The current evidence indicates metformin's multiple sites of action and multiple molecular mechanisms leading to its beneficial impact on metabolism, inflammation, oxidative stress, aging, as well as to its cardiovascular, neurological, bone, and antiproliferative properties. These impacts are the result of its acting on adenosine monophosphate-activated protein kinase (AMPK) dependent and AMPK-independent pathways. Limited data suggest the protective role of metformin on microvascular ocular complications, including retinopathy, glaucoma, and age-related macular degeneration in patients with T2D. However, to confirm its mentioned protective and therapeutic effects, more large, randomized, double-blind, and placebo-controlled clinical studies are needed.

**Keywords:** type 2 diabetes, metformin, molecular mechanisms, ocular complications

### **1. Introduction**

Diabetes mellitus (DM) is a chronic systemic disease accompanied by impaired metabolism of carbohydrates, proteins, and fats. The American Diabetes Association (ADA) [1] distinguishes two basic types of diabetes mellitus, type 1 (T1D) and type 2 (T2D), while, in addition, gestational diabetes and specific forms of the disease are also recognized. The main pathophysiologic events in DM are insulin deficiency and insulin resistance. The most significant event is insulin resistance that develops in target tissues of action of insulin (muscle, fat tissues, and liver). In T1D, autoimmune destruction of β cells of the pancreatic islets (Langerhans islets) leads to deficient production and absolute insulin deficiency, while in T2D, insulin secretion is considered insufficient to overcome insulin resistance in peripheral tissues (relative insulin deficiency).

T1D is commonly diagnosed in childhood and early adolescence, affects men and women equally, and shows the highest prevalence in the white race. T2D occurs in older life, while an increase in incidence is associated with poorer socioeconomic

status, and an increase in risks is associated with lower economic income, education levels, and unemployment. Overall, DM prevalence is expected to increase to 10.1% in the coming decades [2]. The global trend of the increasing prevalence of both types of DM implies a significant influence of environmental factors on the development of the disease.

The polygenic inheritance of DM has been suggested, with different gene variants that contribute to the overall risk of disease [3, 4]. The risk of developing the disease in the offspring is higher if one parent has T2D (~40%) and T1D (~5%). Gene variants that associate with type 1 and type 2 diseases have a different genetic basis. A limited number of specific gene variants characterize a small subset of patients with Maturity-onset diabetes of the young, a monogenic disease with autosomal dominant transmission [4].

A fundamental pathogenic event in the etiology of T1D is an aberrant immune response and production of autoantibodies to β cells. In children and adolescents with T1D, the polyendocrine autoimmune syndrome has also been described, which involves the expression of autoimmune activity against more than one endocrine organ. T1D is associated with the incidence of autoimmune thyroiditis, celiac and autoimmune gastric disease, and other rare autoimmune conditions [5, 6]. Molecular mimicry and viral infections have been investigated the longest, while recently the focus of research is covering deficiencies in immunoregulation that have been identified in patients with T1D [4]. The interaction of genetic and environmental factors may be important for triggering autoimmune events and the onset of T1D [3]. Association was established between the occurrence of T1D and the consumption of foods rich in nitrates or nitrites, low serum vitamin D levels, or early exposure to enteroviral and other infections. The timing of the introduction of cereals and gluten into the diet and alterations of the gut microbiome were suggested to affect the β-cell autoimmune response with autoantibody production [7]. Consistently, a pattern of assimilation of the local incidence rate of T1D has been observed in persons who migrated from lower geographical areas to a higher incidence area [3].

The increase in T2D prevalence has been particularly linked to obesity, sedentary lifestyles, and unhealthy diets. One of the major risk factors for T2D is obesity. Insulin resistance is thought to develop with increasing fat deposition in the liver and muscle. Visceral obesity contributes to the development of insulin resistance and possibly independently contributes to the development of T2D [8]. In prediabetes and early-stage T2D, partial reversibility of insulin secretion disorders has been observed after the restriction in the high-calorie intake and weight loss [9].

Three symptoms characterize the early onset of DM, i.e., hyperglycemia, polyuria, and increased thirst. The recommended diagnostic criteria and therapeutic monitoring of DM are based on impaired fasting glucose levels, impaired glucose tolerance test, and measuring glycosylated hemoglobin Type A1C (HbA1C). HbA1C is an indicator of long-term glycemic control (over the period of past 2–3 months), as it reflects the average level of glucose to which the erythrocytes were exposed to. In the treatment of DM, special attention is given to a balanced diet and physical activity. Administrations of exogenous insulin and insulin analogs are the first-line treatments for T1D. Insulin therapy requires an individualized approach and involves maintaining blood glucose levels as close as possible to reference levels while avoiding hypoglycemia, which is the most significant side effect of this treatment. Glycemia regulation in T2D is being attempted by oral antidiabetic agents, and if adequate control of the disease cannot be established, insulin therapy is initiated. Antidiabetics usually work by increasing the secretion of insulin from the pancreatic β cells or by reducing the insulin resistance. Also, drugs have been developed both to reduce the postprandial glycemia by slowing and reducing the

**63**

*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2…*

absorption of food from the gut and to reduce the production and release of glucose

Complications of the disease significantly influence the quality of life of patients with DM. Acute complications of diabetes are metabolic and, in their extreme form, include diabetic ketoacidosis and nonketotic hyperosmolar coma. While those acute complications can directly endanger the patient's life, late chronic complications are significant due to the impact on the quality of life and morbidity and mortality associated with the disease itself. Both, acute and chronic complications are in inverse onset with the degree of metabolic control of the disease [4]. HbA1C level showed association with risks of cardiovascular disease [10] and is considered to be

Chronic DM complications can be a cause of cardiovascular events, renal failure,

The impact of glycemic control on the development of microvascular complications of T2D has been documented in large prospective studies [12, 14–16]. The DISCOVER study was conducted in 38 countries and included 16,000 patients with T2D, with an average disease duration of 4.1 years [12]. The results of this study indicated that the prevalences of microvascular and macrovascular complications were 18.8 and 12.7%, respectively. The most common microvascular complications included peripheral neuropathy (7.7%), chronic kidney disease (5.0%), and albuminuria (4.3%). Coronary artery disease (8.2%), heart failure (3.3%), and stroke (2.2%) were the most commonly reported macrovascular complications. An association was observed for the following factors of risk: age, male gender, diabetes

In the development of diabetic neuropathy, the changes in cellular metabolism that result from hyperglycemia and dyslipidemia are leading to oxidative stress as a leading causative factor [17]. Hyperglycemia also exerts a negative effect on the β cells themselves, due to the increased formation of reactive oxygen species (ROS). β cells have reduced amounts of catalase enzyme and superoxide dismutase that metabolize ROS under normal conditions, and an increased amount of ROS acti-

Several mechanisms underlie the onset of microvascular complications, and their common feature is the formation of excess oxygen radicals that cause DNA damage. In hyperglycemia, an accumulation of advanced glycation end (AGE) product and increases in the activity of the hexosamine biosynthesis pathway, polyol pathway, and protein kinase C (PKC) are described [13, 17, 18]. High plasma glucose concentrations cause glycation of amine groups in proteins, and consequently, AGE is formed. AGE causes changes in the signaling pathway of macrophages or vascular endothelial cells with the release of various cytokines and increases the expression of vascular endothelial growth factor (VEGF), which causes increased vascular permeability and retinal angiogenesis [19]. Also, AGE-

mediated ROS generation is considered as a pathogenesis factor [17].

blindness, or lower limb amputation. They are classified as macrovascular and microvascular. Coronary disease and myocardial infarction arise as macrovascular complications of DM. It is estimated that 80% of patients with T2D develop cardiovascular complications [12]. Microvascular complications of DM include diabetic retinopathy (DR), nephropathy, and neuropathy. Retinal capillary endothelial cells, mesangial cells of the renal glomeruli, glial cells, and Schwann cells of the peripheral nerves are particularly exposed as they lack the ability to inhibit glucose

*DOI: http://dx.doi.org/10.5772/intechopen.91263*

associated with microvascular disease [11].

**2. Chronic complications of the disease**

duration, and history of hypoglycemia.

vates proapoptotic nuclear factor kappa B (NF-κB).

transport to the cell under hyperglycemia conditions [13].

from the liver.

*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2… DOI: http://dx.doi.org/10.5772/intechopen.91263*

absorption of food from the gut and to reduce the production and release of glucose from the liver.

Complications of the disease significantly influence the quality of life of patients with DM. Acute complications of diabetes are metabolic and, in their extreme form, include diabetic ketoacidosis and nonketotic hyperosmolar coma. While those acute complications can directly endanger the patient's life, late chronic complications are significant due to the impact on the quality of life and morbidity and mortality associated with the disease itself. Both, acute and chronic complications are in inverse onset with the degree of metabolic control of the disease [4]. HbA1C level showed association with risks of cardiovascular disease [10] and is considered to be associated with microvascular disease [11].

### **2. Chronic complications of the disease**

*Metformin*

opment of the disease.

incidence area [3].

autosomal dominant transmission [4].

status, and an increase in risks is associated with lower economic income, education levels, and unemployment. Overall, DM prevalence is expected to increase to 10.1% in the coming decades [2]. The global trend of the increasing prevalence of both types of DM implies a significant influence of environmental factors on the devel-

The polygenic inheritance of DM has been suggested, with different gene variants that contribute to the overall risk of disease [3, 4]. The risk of developing the disease in the offspring is higher if one parent has T2D (~40%) and T1D (~5%). Gene variants that associate with type 1 and type 2 diseases have a different genetic basis. A limited number of specific gene variants characterize a small subset of patients with Maturity-onset diabetes of the young, a monogenic disease with

A fundamental pathogenic event in the etiology of T1D is an aberrant immune response and production of autoantibodies to β cells. In children and adolescents with T1D, the polyendocrine autoimmune syndrome has also been described, which involves the expression of autoimmune activity against more than one endocrine organ. T1D is associated with the incidence of autoimmune thyroiditis, celiac and autoimmune gastric disease, and other rare autoimmune conditions [5, 6]. Molecular mimicry and viral infections have been investigated the longest, while recently the focus of research is covering deficiencies in immunoregulation that have been identified in patients with T1D [4]. The interaction of genetic and environmental factors may be important for triggering autoimmune events and the onset of T1D [3]. Association was established between the occurrence of T1D and the consumption of foods rich in nitrates or nitrites, low serum vitamin D levels, or early exposure to enteroviral and other infections. The timing of the introduction of cereals and gluten into the diet and alterations of the gut microbiome were suggested to affect the β-cell autoimmune response with autoantibody production [7]. Consistently, a pattern of assimilation of the local incidence rate of T1D has been observed in persons who migrated from lower geographical areas to a higher

The increase in T2D prevalence has been particularly linked to obesity, sedentary lifestyles, and unhealthy diets. One of the major risk factors for T2D is obesity. Insulin resistance is thought to develop with increasing fat deposition in the liver and muscle. Visceral obesity contributes to the development of insulin resistance and possibly independently contributes to the development of T2D [8]. In prediabetes and early-stage T2D, partial reversibility of insulin secretion disorders has been

Three symptoms characterize the early onset of DM, i.e., hyperglycemia, polyuria, and increased thirst. The recommended diagnostic criteria and therapeutic monitoring of DM are based on impaired fasting glucose levels, impaired glucose tolerance test, and measuring glycosylated hemoglobin Type A1C (HbA1C). HbA1C is an indicator of long-term glycemic control (over the period of past 2–3 months), as it reflects the average level of glucose to which the erythrocytes were exposed to. In the treatment of DM, special attention is given to a balanced diet and physical activity. Administrations of exogenous insulin and insulin analogs are the first-line treatments for T1D. Insulin therapy requires an individualized approach and involves maintaining blood glucose levels as close as possible to reference levels while avoiding hypoglycemia, which is the most significant side effect of this treatment. Glycemia regulation in T2D is being attempted by oral antidiabetic agents, and if adequate control of the disease cannot be established, insulin therapy is initiated. Antidiabetics usually work by increasing the secretion of insulin from the pancreatic β cells or by reducing the insulin resistance. Also, drugs have been developed both to reduce the postprandial glycemia by slowing and reducing the

observed after the restriction in the high-calorie intake and weight loss [9].

**62**

Chronic DM complications can be a cause of cardiovascular events, renal failure, blindness, or lower limb amputation. They are classified as macrovascular and microvascular. Coronary disease and myocardial infarction arise as macrovascular complications of DM. It is estimated that 80% of patients with T2D develop cardiovascular complications [12]. Microvascular complications of DM include diabetic retinopathy (DR), nephropathy, and neuropathy. Retinal capillary endothelial cells, mesangial cells of the renal glomeruli, glial cells, and Schwann cells of the peripheral nerves are particularly exposed as they lack the ability to inhibit glucose transport to the cell under hyperglycemia conditions [13].

The impact of glycemic control on the development of microvascular complications of T2D has been documented in large prospective studies [12, 14–16]. The DISCOVER study was conducted in 38 countries and included 16,000 patients with T2D, with an average disease duration of 4.1 years [12]. The results of this study indicated that the prevalences of microvascular and macrovascular complications were 18.8 and 12.7%, respectively. The most common microvascular complications included peripheral neuropathy (7.7%), chronic kidney disease (5.0%), and albuminuria (4.3%). Coronary artery disease (8.2%), heart failure (3.3%), and stroke (2.2%) were the most commonly reported macrovascular complications. An association was observed for the following factors of risk: age, male gender, diabetes duration, and history of hypoglycemia.

In the development of diabetic neuropathy, the changes in cellular metabolism that result from hyperglycemia and dyslipidemia are leading to oxidative stress as a leading causative factor [17]. Hyperglycemia also exerts a negative effect on the β cells themselves, due to the increased formation of reactive oxygen species (ROS). β cells have reduced amounts of catalase enzyme and superoxide dismutase that metabolize ROS under normal conditions, and an increased amount of ROS activates proapoptotic nuclear factor kappa B (NF-κB).

Several mechanisms underlie the onset of microvascular complications, and their common feature is the formation of excess oxygen radicals that cause DNA damage. In hyperglycemia, an accumulation of advanced glycation end (AGE) product and increases in the activity of the hexosamine biosynthesis pathway, polyol pathway, and protein kinase C (PKC) are described [13, 17, 18]. High plasma glucose concentrations cause glycation of amine groups in proteins, and consequently, AGE is formed. AGE causes changes in the signaling pathway of macrophages or vascular endothelial cells with the release of various cytokines and increases the expression of vascular endothelial growth factor (VEGF), which causes increased vascular permeability and retinal angiogenesis [19]. Also, AGEmediated ROS generation is considered as a pathogenesis factor [17].

In addition, hyperglycemia increases the activity of the hexosamine pathway, the synthesis of diacylglycerol (DAG), and the activity of aldose reductase within the polyol pathway. Fructose-6-phosphate synthesis of glucosamine-6-phosphate is the first step in the hexosamine biosynthesis pathway. Activation of the hexosamine pathway increases the formation of uridine diphosphate N-acetylglucosamine, which is a substrate donor and catalyzes the binding of monosaccharide GlcNAc to serine and threonine residues of cytosolic and nuclear proteins, including the transcription factor NF-κB. DAG activates PKC isoforms, while basal membrane thickening, increased permeability, coagulation and contractility abnormalities, increased angiogenesis, and cardiomyopathy are all considered to be related to PKC activation. Increased activity of the polyol pathway leads to increased sorbitol formation. When converting glucose to sorbitol, nicotinamide adenine dinucleotide phosphate is consumed, and the production of reduced glutathione as a key antioxidant in the cell is reduced. All these cause the cell to be more susceptible to oxidative stress. Finally, the interaction of metabolic and vascular disorders leads to impaired cellular function and, over the long term, can mediate cell damage and apoptosis.

### **2.1 Ocular complications of DM**

Ocular complications of DM include DR, glaucoma, and cataracts.

The most common ocular complication is DR. Its occurrence is associated with patient age, duration of DM, and hyperglycemia [20]. The contribution of inflammation-mediated pathways and angiogenesis to the progression of DR has been documented [21, 22]. One of the first clinical features of DR is proliferation of endothelial cells and forming of the microaneurysms in retinal capillaries [23]. Capillary damage of ischemia gradually leads to neovascularization. Newly formed capillaries are prone to microhemorrhages. The VEGF signaling is considered to have a significant role in the regulation of neovascularization in retina and pathogenesis of DR [23–25]. Recent advances in treatment of DR include developments in anti-VEGF therapy, which is associated with significant reductions in vision loss due to DR [23].

VEGF levels could be influenced by oxidative stress and formation of ROS, and it has been suggested that exposition of retinal cells to H2O2 might be important in stimulation of VEGF-dependent angiogenesis. Imbalance of VEGF isoforms in retinal cells has been observed *in vivo* [24]. Nevertheless, altered expression of VEGF in retinal pigment epithelial (RPE) cells of normoglycemic and diabetic mice was not observed, whereas expression of antiangiogenic VEGF165b isoform was significantly reduced in diabetic retina. Authors suggested that both hyperglycemia and oxidative stress contribute to the changes in balance of pro- and antiangiogenic factors in the retina.

Along with DR, ocular complications of DM include glaucoma and cataracts. Although age is the most significant risk factor in glaucoma development, DM has been confirmed as an etiological factor for neovascular glaucoma, while there are controversial opinions regarding open-angle glaucoma (OAG) and angle-closure glaucoma (ACG) [26]. The association of T2D and cataract has been demonstrated [26, 27], and assumed underlying mechanisms are compiled of increased oxidative stress, activation of the polyol pathway leading to an increase in the osmotic stress, and glycation of lens proteins [26, 28].

### **3. Method**

We performed a short review to assess and discuss potential protective effects of metformin on ocular complications in patients with T2D.

**65**

*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2…*

Changed levels of not only VEGF-A, one of the most potent members of angiogenic factor family, but also of its isoforms such as VEGF120, VEGF164, and VEGF188 are results of hyperglycemia and oxidative stress in mice [24, 25]. Previous studies have shown that angiogenesis and neovascularization in the eyes of diabetic patients, including DR, are result of increased level of VEGFs [29, 30]. Metformin was shown to mediate the reduction of the VEGF-A expression and angiogenic inhibitors in CD34+ cells under the state of hyperglycemia-hypoxia [31]. Other preliminary study reports that compared to significantly increased plasma VEGF levels in patients treated with pioglitazone, no change in VEGF levels was detected in patients treated with metformin [32]. It is interesting that change of VEGF-A during metformin therapy is independent of metformin-associated effects regarding BMI, HbA1C levels, and waist circumference of fat percentage. Even when the blood glucose and HbA1C levels were not in the recommended range, patients treated with metformin had a lower incidence of ocular complications than

The beneficial effects of metformin were detected in patients with DR [25, 33]. It was documented that 45.5% of patients from the nonmetformin group developed DR compared to 27.3% of patients from the group treated with metformin [34]. However, metformin protective effects on DR are not purely clear. Several studies investigated its effects on vascular endothelium of retina, mainly focusing on pathological background and features of angiogenesis and inflammation. There is evidence that metformin could potently protect endothelial cells via antiangiogenic,

Han et al. [37] in their *in vitro* study found that metformin directly inhibits angiogenesis of human retinal vascular endothelial cells (hRVECs) and has prevented tumor necrosis factor alpha (TNFα)-induced upregulation of multiple

Retinal degenerations are characterized by a progressive loss of photoreceptors or their support cells, the retinal pigmented epithelium (RPE). Xu et al. [38] used metformin to determine whether stimulation of the adenosine monophosphateactivated protein kinase (AMPK) pathway protects the photoreceptors and the RPE from retinal degeneration (**Table 1**). Metformin was able to protect the photoreceptors from light damage, delay rod, and cone degeneration in the Rd10 model and to increase the resistance of the RPE to the injury. Also, authors concluded that metformin's mechanism of protection was associated with increased mitochondrial

The long-term oral metformin was associated with significantly reduced severity of DR in patients with T2D [39]. It could be explained by metformin-induced restoration of energy balance in the retina through activation of AMPK [25]. AMPK

Apart from glycemic control, metformin has shown to have antiinflammatory, antiangiogenic, and calorie restriction-related antiaging activity. Limited data suggest the protective role of metformin on microvascular ocular complications in patients with T2D. The list of studies regarding the link between metformin and

**4. Metformin: protective effects on ocular complications**

ocular involvements in diabetes is presented in **Table 1**.

**4.1 Link between metformin and VEGF-A**

patients in the nonmetformin group [33].

inflammatory cytokines in hRVECs.

biogenesis and reduced oxidative stress.

**4.2 Protective effect on diabetic retinopathy**

antiinflammatory, and antioxidant mechanisms [35, 36].

*DOI: http://dx.doi.org/10.5772/intechopen.91263*

*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2… DOI: http://dx.doi.org/10.5772/intechopen.91263*

### **4. Metformin: protective effects on ocular complications**

Apart from glycemic control, metformin has shown to have antiinflammatory, antiangiogenic, and calorie restriction-related antiaging activity. Limited data suggest the protective role of metformin on microvascular ocular complications in patients with T2D. The list of studies regarding the link between metformin and ocular involvements in diabetes is presented in **Table 1**.

### **4.1 Link between metformin and VEGF-A**

*Metformin*

In addition, hyperglycemia increases the activity of the hexosamine pathway, the synthesis of diacylglycerol (DAG), and the activity of aldose reductase within the polyol pathway. Fructose-6-phosphate synthesis of glucosamine-6-phosphate is the first step in the hexosamine biosynthesis pathway. Activation of the hexosamine pathway increases the formation of uridine diphosphate N-acetylglucosamine, which is a substrate donor and catalyzes the binding of monosaccharide GlcNAc to serine and threonine residues of cytosolic and nuclear proteins, including the transcription factor NF-κB. DAG activates PKC isoforms, while basal membrane thickening, increased permeability, coagulation and contractility abnormalities, increased angiogenesis, and cardiomyopathy are all considered to be related to PKC activation. Increased activity of the polyol pathway leads to increased sorbitol formation. When converting glucose to sorbitol, nicotinamide adenine dinucleotide phosphate is consumed, and the production of reduced glutathione as a key antioxidant in the cell is reduced. All these cause the cell to be more susceptible to oxidative stress. Finally, the interaction of metabolic and vascular disorders leads to impaired cellular func-

tion and, over the long term, can mediate cell damage and apoptosis.

Ocular complications of DM include DR, glaucoma, and cataracts.

The most common ocular complication is DR. Its occurrence is associated with patient age, duration of DM, and hyperglycemia [20]. The contribution of inflammation-mediated pathways and angiogenesis to the progression of DR has been documented [21, 22]. One of the first clinical features of DR is proliferation of endothelial cells and forming of the microaneurysms in retinal capillaries [23]. Capillary damage of ischemia gradually leads to neovascularization. Newly formed capillaries are prone to microhemorrhages. The VEGF signaling is considered to have a significant role in the regulation of neovascularization in retina and pathogenesis of DR [23–25]. Recent advances in treatment of DR include developments in anti-VEGF therapy, which is associated with significant reductions in vision loss due

VEGF levels could be influenced by oxidative stress and formation of ROS, and it has been suggested that exposition of retinal cells to H2O2 might be important in stimulation of VEGF-dependent angiogenesis. Imbalance of VEGF isoforms in retinal cells has been observed *in vivo* [24]. Nevertheless, altered expression of VEGF in retinal pigment epithelial (RPE) cells of normoglycemic and diabetic mice was not observed, whereas expression of antiangiogenic VEGF165b isoform was significantly reduced in diabetic retina. Authors suggested that both hyperglycemia and oxidative stress contribute to the changes in balance of pro- and antiangiogenic factors in the retina. Along with DR, ocular complications of DM include glaucoma and cataracts. Although age is the most significant risk factor in glaucoma development, DM has been confirmed as an etiological factor for neovascular glaucoma, while there are controversial opinions regarding open-angle glaucoma (OAG) and angle-closure glaucoma (ACG) [26]. The association of T2D and cataract has been demonstrated [26, 27], and assumed underlying mechanisms are compiled of increased oxidative stress, activation of the polyol pathway leading to an increase in the osmotic stress,

We performed a short review to assess and discuss potential protective effects of

**2.1 Ocular complications of DM**

and glycation of lens proteins [26, 28].

metformin on ocular complications in patients with T2D.

**64**

**3. Method**

to DR [23].

Changed levels of not only VEGF-A, one of the most potent members of angiogenic factor family, but also of its isoforms such as VEGF120, VEGF164, and VEGF188 are results of hyperglycemia and oxidative stress in mice [24, 25]. Previous studies have shown that angiogenesis and neovascularization in the eyes of diabetic patients, including DR, are result of increased level of VEGFs [29, 30]. Metformin was shown to mediate the reduction of the VEGF-A expression and angiogenic inhibitors in CD34+ cells under the state of hyperglycemia-hypoxia [31]. Other preliminary study reports that compared to significantly increased plasma VEGF levels in patients treated with pioglitazone, no change in VEGF levels was detected in patients treated with metformin [32]. It is interesting that change of VEGF-A during metformin therapy is independent of metformin-associated effects regarding BMI, HbA1C levels, and waist circumference of fat percentage. Even when the blood glucose and HbA1C levels were not in the recommended range, patients treated with metformin had a lower incidence of ocular complications than patients in the nonmetformin group [33].

### **4.2 Protective effect on diabetic retinopathy**

The beneficial effects of metformin were detected in patients with DR [25, 33]. It was documented that 45.5% of patients from the nonmetformin group developed DR compared to 27.3% of patients from the group treated with metformin [34]. However, metformin protective effects on DR are not purely clear. Several studies investigated its effects on vascular endothelium of retina, mainly focusing on pathological background and features of angiogenesis and inflammation. There is evidence that metformin could potently protect endothelial cells via antiangiogenic, antiinflammatory, and antioxidant mechanisms [35, 36].

Han et al. [37] in their *in vitro* study found that metformin directly inhibits angiogenesis of human retinal vascular endothelial cells (hRVECs) and has prevented tumor necrosis factor alpha (TNFα)-induced upregulation of multiple inflammatory cytokines in hRVECs.

Retinal degenerations are characterized by a progressive loss of photoreceptors or their support cells, the retinal pigmented epithelium (RPE). Xu et al. [38] used metformin to determine whether stimulation of the adenosine monophosphateactivated protein kinase (AMPK) pathway protects the photoreceptors and the RPE from retinal degeneration (**Table 1**). Metformin was able to protect the photoreceptors from light damage, delay rod, and cone degeneration in the Rd10 model and to increase the resistance of the RPE to the injury. Also, authors concluded that metformin's mechanism of protection was associated with increased mitochondrial biogenesis and reduced oxidative stress.

The long-term oral metformin was associated with significantly reduced severity of DR in patients with T2D [39]. It could be explained by metformin-induced restoration of energy balance in the retina through activation of AMPK [25]. AMPK


**67**

*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2…*

Metformin effects on the development of DR were tested in STZ-induced diabetic model in mice.

Retinal tissue and D407 RPE cells from wild-type and Ins2Akita mouse model of diabetes were used as experimental models.

Retrospective cohort study with patients with T2D aged ≥40 years and with no preexisting record of OAG.

Longitudinal data from a large database were used, and patients with diabetes, aged ≥40 with no preexisting OAG, were monitored for incident

*AMD: Age-Related Macular Degeneration; DR: Diabetic Retinopathy; hRVEC: human retinal vascular endothelial cell; vldlr-/-mice: very-low-density lipoprotein receptor knockout mutant mouse; STZ: streptozotocin; AMPK: adenosine monophosphate-activated protein kinase; RPE: retinal pigmented epithelium; VEGF-A: vascular endothelial cell growth factor A; OAG: Open-Angle Glaucoma; POAG: primary open-angle glaucoma.*

OAG.

*List of studies regarding the link between metformin and ocular involvements in diabetes.*

**Study title Study design Study outcome Ref.**

Metformin inhibited VEGF signaling by inducing VEGF-A mRNA splicing to VEGF120 isoform, creating a potential for new treatment option for DR.

Both hyperglycemia and oxidative stress disrupted the equilibrium between pro- and antiangiogenic factors in the retina. Hyperglycemia

contributed to deregulation of the expression of VEGF proteins and the production of ROS in RPE cells. Pathological H2O2 levels downregulated the

VEGF165b.

Metformin use was associated with reduction in risk of developing OAG. Proposed mechanisms involved improved glycemic control or effects involving neurogenesis, inflammatory systems, or longevity pathways.

Metformin use was associated with reduced risk of OAG, on a dose-dependent manner. Proposed mechanisms involved neurogenesis, longevity pathways, and/or reduced inflammation.

[25]

[24]

[43]

[46]

activation was suggested to be protective for the tissues that are undergoing metabolic stress. However, the regulation on endothelial inflammatory and angiogenic responses by metformin also has been shown through both AMPK-dependent and

According to a retrospective study [41], there is a correlation between the longterm metformin treatment and reduced severity of DR in patients with T2D regardless of their HbA1c level, gender, race or treatment with sulfonylurea or insulin. In summary, metformin might be used for the purpose of reducing DR progres-

AMPK-independent mechanisms [37, 40].

sion in patients with long history of T2D.

*DOI: http://dx.doi.org/10.5772/intechopen.91263*

Metformin Inhibits the Development of Diabetic Retinopathy through Inducing Alternative Splicing of VEGF-A

Oxidative Stress Modulates the Expression of VEGF Isoforms in the Diabetic Retina

Association of Geroprotective Effects of Metformin and Risk of Open-Angle Glaucoma in Persons with Diabetes Mellitus

Targeting aging: Geroprotective Medication Metformin Reduces Risk of Adultonset Open-angle Glaucoma

**Authors, Year**

Yi QY et al., 2016

Simão S et al., 2016

Lin H-C et al., 2015

Richards JE et al., 2014

**Table 1.**


*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2… DOI: http://dx.doi.org/10.5772/intechopen.91263*

*AMD: Age-Related Macular Degeneration; DR: Diabetic Retinopathy; hRVEC: human retinal vascular endothelial cell; vldlr-/-mice: very-low-density lipoprotein receptor knockout mutant mouse; STZ: streptozotocin; AMPK: adenosine monophosphate-activated protein kinase; RPE: retinal pigmented epithelium; VEGF-A: vascular endothelial cell growth factor A; OAG: Open-Angle Glaucoma; POAG: primary open-angle glaucoma.*

### **Table 1.**

*Metformin*

**Authors, Year**

Brown EE et al., 2019

Chen YY et al., 2019

Li Y et al., 2018

Han J et al., 2018

Xu L et al., 2018

Maleskic S et al., 2017

The Common Antidiabetic Drug Metformin Reduces Odds of Developing Age-Related Macular Degeneration

Association Between Metformin and a Lower Risk of Age-Related Macular Degeneration in Patients with Type 2

Diabetes

Association of Metformin Treatment with Reduced Severity of Diabetic Retinopathy in Type 2 Diabetic Patients

Metformin Suppresses Retinal Angiogenesis and Inflammation In Vitro and In Vivo

Stimulation of AMPK Prevents Degeneration of Photoreceptors and the Retinal Pigment Epithelium

Metformin Use Associated with Protective Effects for Ocular Complications in Patients with Type 2 Diabetes – Observational Study

**Study title Study design Study outcome Ref.**

Patients treated with metformin had decreased odds of developing AMD suggesting its therapeutic role in development or progression of AMD in patients at risk.

Metformin use, especially in higher doses, was associated with significantly lower risk of development of AMD.

Long-term use of metformin was independently associated with significant lower rate of severe nonproliferative DR or proliferative DR in patients with T2D ≥15 years.

Metformin showed potent antiangiogenic and antiinflammatory effects on hRVECs, reduced retinal neovascularization in vldlr−/− mice, and suppressed leukostasis in STZ-induced diabetic mice, suggesting its potential to target key pathogenic components

in DR.

By stimulation of AMPK metformin protected photoreceptors and the RPE in three different mouse models of retinal degeneration, including acute bright light damage, Pde6brd10 inherited retinitis pigmentosa, and sodium iodate-induced RPE injury. Local expression of AMPK catalytic subunit α2 was required for those effects.

Metformin use was associated with fewer ocular complications with decreased odds of both glaucoma and DR compared to other oral antihyperglycemic agents. [47]

[48]

[41]

[37]

[38]

[33]

Retrospective case-control study with medical records from patients ˃55 years. Three controls were matched for every AMD case, defined by Int. Class. of Diseases, 9th Revision code, based on Charlson Comorbidity Index.

Population-based retrospective cohort study with 68,205 patients with

Retrospective chart review study with 335 patients with DR and with T2D ≥15 years. The severity of DR was determined by Early Treatment Diabetic Retinopathy Study scale.

Metformin effects and mechanism were tested in vitro in hRVEC culture and in vivo in vldlr−/− mice.

In vivo study with metformin tested in three different mouse models of retinal degeneration: a light-induced degenerative model, the Pde6brd10 inherited retinal degeneration model, and a model of sodium iodateinduced RPE and retinal injury, as well as in AMPK retinal knockout mice.

Observational study with medical records from 234 patients with T2D (190 patients using metformin and 44 using other oral antihyperglycemic agents).

T2D.

**66**

*List of studies regarding the link between metformin and ocular involvements in diabetes.*

activation was suggested to be protective for the tissues that are undergoing metabolic stress. However, the regulation on endothelial inflammatory and angiogenic responses by metformin also has been shown through both AMPK-dependent and AMPK-independent mechanisms [37, 40].

According to a retrospective study [41], there is a correlation between the longterm metformin treatment and reduced severity of DR in patients with T2D regardless of their HbA1c level, gender, race or treatment with sulfonylurea or insulin.

In summary, metformin might be used for the purpose of reducing DR progression in patients with long history of T2D.

### **4.3 Protective effect on glaucoma**

Glaucoma is a type of neuropathy, and association with DM was identified – it could cause optic neuropathy [42]. The thicker central cornea in patients with DM than in healthy subjects could be a cause of higher intraocular pressure in those patients [26]. A retrospective cohort study showed that metformin use is associated with reduced risk of developing open-angle glaucoma and suggested that metformin could have an impact on glaucoma risk on multiple levels including glycemic control and calorie restriction (CR) [43]. As previous studies suggested that agerelated tissue changes significantly contribute to glaucoma development [44], the antiaging effect of metformin as a CR mimetic drug could delay the progression of tissue damage [45].

Risk reduction of glaucoma was shown to be dose-dependent for metformin and independent of glycemic control in the population with DM [46]. In the observational study, patients treated with metformin had a lower prevalence of glaucoma than patients treated with other oral antidiabetic medications, 3.2 vs. 11.4%, respectively [33].

### **4.4 Protective effect on age-related macular degeneration**

Recently, the first studies on this topic indicated an association between metformin use and the reduction of age-related macular degeneration (AMD) development [47, 48]. Those authors assumed metformin's protective role in development or progression of AMD based on both its antiinflammatory and antioxidative properties and on AMD pathogenesis. Namely, besides environmental and genetic factors, AMD pathogenesis involves inflammation and oxidative stress, which can lead to choroidal neovascularization and geographic atrophy with potential loss of vision [47–50].

In study Chen et al., both the incidence of AMD (3.4 vs. 6.6%) and cumulative hazard for AMD were significantly lower among metformin users than nonusers. Lower hazard ratios for AMD were shown to be associated with higher dose of metformin and longer duration of therapy, and they remained even after adjustment for the patients' age, gender, and comorbidities [48].

Similar results were found in the study by Brown et al., where decreased odds of developing AMD, except for metformin, were not associated with dipeptidyl peptidase 4 inhibitors, selective serotonin reuptake inhibitors, tetracyclic antidepressants, and statins [47].

Almost 8.4 million people worldwide are affected by AMD [51]. It is the most common cause of vision impairment in the developed countries, and the third one, after uncorrected refractive errors and cataract, globally [52–54]. Estimated blindness prevalence related to AMD is 8.7% [55]. However, it is projected that due to the extended life expectancy, the number of people with AMD will increase [52–54]. Current AMD therapy with anti-VEGF drugs is costly, i.e., the cost of an injection of anti-VEGF is up to £800, and usually eight injections per year are recommended [51]. Therefore, as metformin is well-known cheap drug, its potentially protective effect on AMD is promising, especially for countries with limited health care resources.

### **5. Conclusion**

Metformin is effective, well-tolerated, and inexpensive first-line pharmacotherapy for T2D. Its additional potential protective effects on ocular complications

**69**

**Author details**

and Sanita Maleškić

*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2…*

in patients with T2D may have a major beneficial impact on the disease course and quality and length of their life. Well-designed randomized controlled clinical trials should be conducted to evaluate the effects of metformin either on the prevention of ocular complication or on the therapy of already developed ocular complications

Jasna Kusturica\*, Aida Kulo, Maida Rakanović-Todić, Lejla Burnazović-Ristić

University of Sarajevo, Sarajevo, Bosnia and Herzegovina

provided the original work is properly cited.

\*Address all correspondence to: jasna.kusturica@mf.unsa.ba

Institute of Pharmacology, Clinical Pharmacology and Toxicology, Medical Faculty,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*DOI: http://dx.doi.org/10.5772/intechopen.91263*

The authors declare no conflict of interest.

in patients with T2D.

**Conflict of interest**

*Potential Protective Effects of Metformin on Ocular Complications in Patients with Type 2… DOI: http://dx.doi.org/10.5772/intechopen.91263*

in patients with T2D may have a major beneficial impact on the disease course and quality and length of their life. Well-designed randomized controlled clinical trials should be conducted to evaluate the effects of metformin either on the prevention of ocular complication or on the therapy of already developed ocular complications in patients with T2D.

### **Conflict of interest**

*Metformin*

tissue damage [45].

respectively [33].

vision [47–50].

pressants, and statins [47].

**4.3 Protective effect on glaucoma**

Glaucoma is a type of neuropathy, and association with DM was identified – it could cause optic neuropathy [42]. The thicker central cornea in patients with DM than in healthy subjects could be a cause of higher intraocular pressure in those patients [26]. A retrospective cohort study showed that metformin use is associated with reduced risk of developing open-angle glaucoma and suggested that metformin could have an impact on glaucoma risk on multiple levels including glycemic control and calorie restriction (CR) [43]. As previous studies suggested that agerelated tissue changes significantly contribute to glaucoma development [44], the antiaging effect of metformin as a CR mimetic drug could delay the progression of

Risk reduction of glaucoma was shown to be dose-dependent for metformin and independent of glycemic control in the population with DM [46]. In the observational study, patients treated with metformin had a lower prevalence of glaucoma than patients treated with other oral antidiabetic medications, 3.2 vs. 11.4%,

Recently, the first studies on this topic indicated an association between metformin use and the reduction of age-related macular degeneration (AMD) development [47, 48]. Those authors assumed metformin's protective role in development or progression of AMD based on both its antiinflammatory and antioxidative properties and on AMD pathogenesis. Namely, besides environmental and genetic factors, AMD pathogenesis involves inflammation and oxidative stress, which can lead to choroidal neovascularization and geographic atrophy with potential loss of

In study Chen et al., both the incidence of AMD (3.4 vs. 6.6%) and cumulative hazard for AMD were significantly lower among metformin users than nonusers. Lower hazard ratios for AMD were shown to be associated with higher dose of metformin and longer duration of therapy, and they remained even after adjustment for

Similar results were found in the study by Brown et al., where decreased odds of developing AMD, except for metformin, were not associated with dipeptidyl peptidase 4 inhibitors, selective serotonin reuptake inhibitors, tetracyclic antide-

Almost 8.4 million people worldwide are affected by AMD [51]. It is the most common cause of vision impairment in the developed countries, and the third one, after uncorrected refractive errors and cataract, globally [52–54]. Estimated blindness prevalence related to AMD is 8.7% [55]. However, it is projected that due to the extended life expectancy, the number of people with AMD will increase [52–54]. Current AMD therapy with anti-VEGF drugs is costly, i.e., the cost of an injection of anti-VEGF is up to £800, and usually eight injections per year are recommended [51]. Therefore, as metformin is well-known cheap drug, its potentially protective effect on AMD is promising, especially for countries with limited health care

Metformin is effective, well-tolerated, and inexpensive first-line pharmacotherapy for T2D. Its additional potential protective effects on ocular complications

**4.4 Protective effect on age-related macular degeneration**

the patients' age, gender, and comorbidities [48].

**68**

resources.

**5. Conclusion**

The authors declare no conflict of interest.

### **Author details**

Jasna Kusturica\*, Aida Kulo, Maida Rakanović-Todić, Lejla Burnazović-Ristić and Sanita Maleškić Institute of Pharmacology, Clinical Pharmacology and Toxicology, Medical Faculty, University of Sarajevo, Sarajevo, Bosnia and Herzegovina

\*Address all correspondence to: jasna.kusturica@mf.unsa.ba

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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in vitro and in vivo. PLoS One. 2018;**13**(3):e0193031. DOI: 10.1371/

[38] Xu L, Kong L, Wang J, Ash JD. Stimulation of AMPK prevents

degeneration of photoreceptors and the retinal pigment epithelium. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(41):10475-10480. DOI:

journal.pone.0193031

10.1073/pnas.1802724115

2014;**55**:1069

[39] Munie M, Ryu C, Noorulla S, Rana S, Malach D, Qiao X, et al. Effect of metformin on the development and severity of diabetic retinopathy. ARVO Annual Meeting Abstract. Investigative Ophthalmology & Visual Science.

Farhatullah S, Heutling D, Mitchell D, et al. Metformin decreases angiogenesis

ama2006-124.196

2013;**54**:2449

[28] Jeganathan VS, Wang JJ, Wong TY. Ocular association of diabetes other than diabetic retinopathy. Diabetes Care. 2008;**31**(9):1905-1912. DOI:

**72**

[41] Li Y, Ryu C, Munie M, Noorulla S, Rana S, Edwards P, et al. Association of metformin treatment with reduced severity of diabetic retinopathy in type 2 diabetic patients. Journal of Diabetes Research. 2018;**2018**:2801450. DOI: 10.1155/2018/2801450

[42] Zhou M, Wang W, Huang W, Zhang X. Diabetes mellitus as a risk factor for open-angle glaucoma: A systematic review and meta-analysis. PLoS One. 2014;**9**(8):e102972. DOI: 10.1371/journal.pone.0102972

[43] Lin H-C, Stein JD, Nan B, Childers D, Newman-Casey PA, Thompson DA, et al. Association of Geroprotective effects of metformin and risk of open-angle glaucoma in persons with diabetes mellitus. JAMA Ophthalmology. 2015;**133**(8):915-923. DOI: 10.1001/ jamaophthalmol.2015.1440

[44] Guedes G, Tsai JC, Loewen N. Glaucoma and aging. Current Aging Science. 2011;**4**(2):110-117. DOI: 10.2174/1874609811104020110

[45] Anisimov VN. Metformin: Do we finally have an anti-aging medication? Cell Cycle. 2013;**12**(22):3483-3489. DOI: 10.4161/cc.26928

[46] Richards JE, Lin HC, Nan B, Talwar N, Childers D, Newman-Casey PA, et al. Targeting aging: Geroprotective medication metformin reduces risk of adult-onset open-angle glaucoma. Investigative Ophthalmology & Visual Science. 2014;**55**:1668

[47] Brown EE, Ball JD, Chen Z, Khurshid GS, Prosperi M, Ash JD. The common antidiabetic drug metformin

reduces odds of developing age-related macular degeneration. Investigative Ophthalmology & Visual Science. 2019;**60**(5):1470-1477. DOI: 10.1167/ iovs.18-26422

[48] Chen Y-Y, Shen Y-C, Lai Y-J, Wang C-Y, Lin K-H, Feng S-C, et al. Association between metformin and a lower risk of age-related macular degeneration in patients with type 2 diabetes. Journal of Ophthalmology. 2019:1649156. DOI: 10.1155/2019/1649156

[49] Lambert NG, ElShelmani H, Singh MK, Mansergh FC, Wride MA, Padilla M, et al. Risk factors and biomarkers of age-related macular degeneration. Progress in Retinal and Eye Research. 2016;**54, 54**:64-102. DOI: 10.1016/j.preteyeres.2016.04.003

[50] Moschos MM, Nitoda E, Chatziralli IP, Demopoulos CA. Agerelated macular degeneration: Pathogenesis, genetic background, and the role of nutritional supplements. Journal of Chemistry. 2014;**9**:317536. DOI: 10.1155/2014/317536

[51] The Lancet Editorial. Agerelated macular degeneration: Treatment at what cost? The Lancet. 2018;**392**(10153):1090. DOI: 10.1016/ S0140-6736(18)32291

[52] Friedman DS, O'Colmain BJ, Muñoz B, Tomany SC, McCarty C, de Jong PT, et al. Prevalence of age-related macular degeneration in the United States. Archives of Ophthalmology. 2004;**122**(4):564-572. DOI: 10.1001/ archopht.122.4.564

[53] Colijn JM, Buitendijk GHS, Prokofyeva E, Alves D, Cachulo ML, Khawaja AP, et al. Prevalence of age-related macular degeneration in Europe: The past and the future. Journal of Ophthalmology. 2017;**124**(12): 1753-1763. DOI: 10.1016/j. ophtha.2017.05.035

*Metformin*

[54] Bourne RRA, Flaxman SR, Braithwaite T, Cicinelli MV, Das A, Jonas JB, et al. Magnitude, temporal trends, and projections of the global prevalence of blindness and distance and near vision impairment: a systematic review and metaanalysis. The Lancet Global Health. 2017;**5**(9):e888-e897. DOI: 10.1016/ S2214-109X(17)30293-0

[55] WHO. Blindness and vision impairment. Available from: https:// www.who.int/news-room/fact-sheets/ detail/blindness-and-visual-impairment [Accessed: 8 October 2019]

**75**

stress and inflammation.

biologically active substances [4].

**Chapter 5**

**Abstract**

**1. Introduction**

Mellitus

*Galega officinalis* L. and

Immunological Status in Diabetes

*Mariia Nagalievska, Halyna Hachkova and Nataliia Sybirna*

Under diabetes mellitus, the administration of *Galega officinalis* promotes restoration of leukocyte precursors' bone marrow pool and normalizes their proliferative activity. This plant protects the functional state of leukocytes by modulating actin cytoskeleton formation and through quantitative redistribution of leukocyte membrane glycoconjugates. *Galega officinalis* prevents the development of diabetesassociated oxidative stress which results in antiapoptotic activity. The normalization of leukocytes' proliferative and functional capacity by *Galega officinalis*, along with its antiapoptotic and hypoglycemic effects, can improve the course of the disease

and may prevent the development of complications of diabetes.

in their morphology and functional state [1, 3].

**Keywords:** *Galega officinalis*, diabetes mellitus, leukocytes, immune system

Diabetes mellitus belongs to a group of metabolic diseases accompanied by chronic inflammation and attenuation of the immune response, which subsequently contributes to the development of a number of complications [1]. Cells that are most affected by glycemic status and insulin level are leukocytes, which play major roles in inflammation and immune responses [2]. Constant high glucose levels result in the formation of cytotoxic compounds, leading to lower viability of peripheral blood leukocytes. This is mediated by enhanced reactive species production, activation of mitogen-activated protein kinase (MAPK) pathway, high levels of proinflammatory and poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) transcription factors, as well as inactivation of pro-survival pathways which altogether leads to increased apoptosis rate. The alterations in these molecular pathways are usually associated with increased leukocyte mobilization, which causes changes

The multitude of diabetes mellitus complications creates the need for drugs with

In many cases medicinal plants can be safe and effective alternatives to synthetic

a wide spectrum of action, which would not only provide effective reduction of blood glucose but would also exhibit cytoprotective properties. The most commonly used anti-diabetes drug globally is metformin. Metformin shows a pleiotropic effect mediated by its hypoglycemic function, as well as inhibitory effect on oxidative

compounds in disease management, since they possess a unique composition of

### **Chapter 5**

*Metformin*

[54] Bourne RRA, Flaxman SR, Braithwaite T, Cicinelli MV, Das A, Jonas JB, et al. Magnitude, temporal trends, and projections of the global prevalence of blindness and distance and near vision impairment:

a systematic review and metaanalysis. The Lancet Global Health. 2017;**5**(9):e888-e897. DOI: 10.1016/

[55] WHO. Blindness and vision impairment. Available from: https:// www.who.int/news-room/fact-sheets/ detail/blindness-and-visual-impairment

[Accessed: 8 October 2019]

S2214-109X(17)30293-0

**74**

## *Galega officinalis* L. and Immunological Status in Diabetes Mellitus

*Mariia Nagalievska, Halyna Hachkova and Nataliia Sybirna*

### **Abstract**

Under diabetes mellitus, the administration of *Galega officinalis* promotes restoration of leukocyte precursors' bone marrow pool and normalizes their proliferative activity. This plant protects the functional state of leukocytes by modulating actin cytoskeleton formation and through quantitative redistribution of leukocyte membrane glycoconjugates. *Galega officinalis* prevents the development of diabetesassociated oxidative stress which results in antiapoptotic activity. The normalization of leukocytes' proliferative and functional capacity by *Galega officinalis*, along with its antiapoptotic and hypoglycemic effects, can improve the course of the disease and may prevent the development of complications of diabetes.

**Keywords:** *Galega officinalis*, diabetes mellitus, leukocytes, immune system

### **1. Introduction**

Diabetes mellitus belongs to a group of metabolic diseases accompanied by chronic inflammation and attenuation of the immune response, which subsequently contributes to the development of a number of complications [1]. Cells that are most affected by glycemic status and insulin level are leukocytes, which play major roles in inflammation and immune responses [2]. Constant high glucose levels result in the formation of cytotoxic compounds, leading to lower viability of peripheral blood leukocytes. This is mediated by enhanced reactive species production, activation of mitogen-activated protein kinase (MAPK) pathway, high levels of proinflammatory and poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) transcription factors, as well as inactivation of pro-survival pathways which altogether leads to increased apoptosis rate. The alterations in these molecular pathways are usually associated with increased leukocyte mobilization, which causes changes in their morphology and functional state [1, 3].

The multitude of diabetes mellitus complications creates the need for drugs with a wide spectrum of action, which would not only provide effective reduction of blood glucose but would also exhibit cytoprotective properties. The most commonly used anti-diabetes drug globally is metformin. Metformin shows a pleiotropic effect mediated by its hypoglycemic function, as well as inhibitory effect on oxidative stress and inflammation.

In many cases medicinal plants can be safe and effective alternatives to synthetic compounds in disease management, since they possess a unique composition of biologically active substances [4].

*Galega officinalis* (*Galega*, goat's rue, French lilac) is a promising plant that can be used for treatment of a wide range of inflammatory diseases, including diabetes mellitus. *G. officinalis* is well-known for its hypoglycemic action, and it has been long used as part of a plant mixture for treatment of diabetes mellitus [5]. For a long time, the antidiabetic effect of *G. officinalis* was associated with high content of alkaloid galegine, which is one of the main components of this plant's leaves. In fact, metformin, discussed above, is a synthetic form of galegine, which was originally used to treat diabetes mellitus type 2 [5]. The toxicity of *G. officinalis'* alkaloids decreased its attractiveness as a hypoglycemic drug. However, it was found that even the non-alkaloid extract has a hypoglycemic effect and is potentially nontoxic [6, 7]. Based on such historical use and a large number of recent scientific studies, *G. officinalis* is a source of potent biologically active substances for the prevention and treatment of diabetes mellitus [8].

### **2. Effects of metformin on the immune system**

Metformin (N,N-dimethylbiguanide) is an oral antihyperglycemic agent, which from a chemical point of view is a synthetic derivative of guanidine. The hypoglycemic effect of this drug is realized through the inhibition of hepatic glucose production, reducing intestinal glucose absorption and improving glucose uptake and utilization by peripheral tissues. Recent research has shed light on the pleiotropic effect of metformin, ranging from hypoglycemic function to cardio- and nephroprotection, as well as inhibitory effects on oxidative stress and inflammation [9–11].

The scientific data concerning the influence of metformin on the immune system is controversial, and its effect strongly depends on the pathology in which it is used. For example, metformin enhances antitumor immunity, but in other contexts, it can act as an anti-inflammatory or immunosuppressive agent [8]. Metformin can suppress senescence- and cancer-related inflammation. The majority of experimental data indicates that metformin modulates leukocytes' functional activity by activating 5′ adenosine monophosphate-activated protein kinase (AMPK). Metformin can activate AMPK in multiple cell populations, including macrophages and neutrophils [12, 13]. It has also been demonstrated that metformin inhibits innate immune response to fungal infection in an AMPK-dependent manner and lessens central nervous system inflammation [14].

Considering the significant modulating effect of metformin on the immune system, it is unsurprising that it has a strong effect on immunocompetent blood cells, which we discuss below.

### **2.1 Metformin influence on defective hematopoiesis**

Studies conducted on Fanconi anemia mice showed the unique property of metformin to improve hematopoiesis by restoring hematopoietic stem cell (HSC) numbers. It also delays tumor formation, presumably via reduction of DNA damage induced by aldehydes [15]. An important part of metformin protective effect may be conferred by aldehyde detoxification. Other mechanisms by which metformin may act to protect the cell's DNA are reducing the activity of mitochondrial complex 1 activity, thus potentially reducing oxidative DNA damage. It is also possible that metformin can switch the metabolic balance between oxidative phosphorylation and anaerobic glycolysis and downregulate inflammatory pathways which are thought to contribute to bone marrow failure [15]. Another study demonstrates that metformin treatment significantly inhibited the total-body irradiation-induced increase in the levels of DNA double-strand breaks and reactive oxygen species

**77**

Galega officinalis *L. and Immunological Status in Diabetes Mellitus*

modulates the expression of antioxidant enzymes in HSCs [16].

**2.2 Influence of metformin on functional state of leukocytes**

enhanced uptake of bacteria by phagocytic cells [12, 13].

IκB-α degradation and thus prevention of NF-κB activation [18].

transduction via the phosphoinositide 3 kinase pathway [19].

**2.3 Effects of metformin on oxidative stress**

from glycemic control [14].

vascular cells [20, 21].

(ROS) by attenuation of NOX4 expression in HSCs. Furthermore, metformin

Many diabetic patients who receive metformin show significantly reduced neutrophil-to-lymphocyte ratio [9]. Metformin is able to reduce hyperneutrophilia in girls with hyperinsulinemic hyperandrogenism and improves white blood cell count in women with polycystic ovary syndrome, two conditions characterized by a pronounced systemic inflammatory state [17]. Metformin increased the number of CD8-positive tumor-infiltrating lymphocytes. Normalizing effect of metformin on the number of immunocompetent cells is associated with its ability to upregulate

AMPK and as a consequence of altering energy metabolism in the cell [14].

Apart from metformin influence on immunocompetent cell number, this drug also can modulate their functional activity. As expected for an AMPK activator, metformin enhances cell mobility and phagocytosis, in particular in macrophages that show enhanced uptake of bacteria, synthetic beads, or apoptotic cells. The effects of AMPK activation may be due to its ability to increase availability of cell surface receptors, including αM integrin or Fc receptors or due to mechanisms that involve suppression of TLR4-associated signaling pathways. Metformin by activating AMPK regulates the process of inflammation resolution—efferocytosis and

Additionally, in patients with prediabetes, metformin treatment reduces the concentration of neutrophil extracellular trap (NET) components independently

The normalization of phagocytosis processes and NETosis under metformin administration could suggest an effect of this drug on neutrophil activation. Indeed, metformin attenuates neutrophil activation via inhibition of mitochondrial respiratory complex I, potentially through intracellular H2O2-mediated inhibition of

Immune system modulation by metformin can be realized not only by its direct influence on the immunocompetent cells but also by its ability to regulate chemokine level. Metformin causes a decrease in inflammatory markers in plasma, including soluble intercellular adhesion molecule, vascular cell adhesion molecule-1, macrophage migration inhibitory factor, C-reactive protein, IL-6, and IL-8. The anti-inflammatory action of metformin is realized by suppressing Akt, Erk1/2, and NF-B translocation. Such changes lead to blocking of pro-inflammatory signal

Immunosuppressive effect of metformin can be mediated by its ability to inhibit the expression of pro-inflammatory mediators (IFN-, TNF-, IL-1, IL-6, IL-17, iNOS, MMP9, and RANTES) and infiltration of immune cells, which was blocked by reducing the expression of CAMs (ICAM, VCAM, and E-selectin) on

Oxidative stress is the leading cause of microvascular and cardiovascular diabetes complications [22]. Disruption of glucose metabolism causes mitochondrial superoxide overproduction in cells. An increased amount of superoxide leads to overactivity of polyol and hexosamine pathways, increased formation of AGEs (advanced glycation end products) and its receptors, and activation of protein kinase C isoforms. Altogether, this leads to the development of complications of diabetes. Simultaneously endothelial nitric oxide synthase is inactivated. Changes

*DOI: http://dx.doi.org/10.5772/intechopen.88802*

*Metformin*

and treatment of diabetes mellitus [8].

**2. Effects of metformin on the immune system**

lessens central nervous system inflammation [14].

**2.1 Metformin influence on defective hematopoiesis**

cells, which we discuss below.

*Galega officinalis* (*Galega*, goat's rue, French lilac) is a promising plant that can be used for treatment of a wide range of inflammatory diseases, including diabetes mellitus. *G. officinalis* is well-known for its hypoglycemic action, and it has been long used as part of a plant mixture for treatment of diabetes mellitus [5]. For a long time, the antidiabetic effect of *G. officinalis* was associated with high content of alkaloid galegine, which is one of the main components of this plant's leaves. In fact, metformin, discussed above, is a synthetic form of galegine, which was originally used to treat diabetes mellitus type 2 [5]. The toxicity of *G. officinalis'* alkaloids decreased its attractiveness as a hypoglycemic drug. However, it was found that even the non-alkaloid extract has a hypoglycemic effect and is potentially nontoxic [6, 7]. Based on such historical use and a large number of recent scientific studies, *G. officinalis* is a source of potent biologically active substances for the prevention

Metformin (N,N-dimethylbiguanide) is an oral antihyperglycemic agent, which from a chemical point of view is a synthetic derivative of guanidine. The hypoglycemic effect of this drug is realized through the inhibition of hepatic glucose production, reducing intestinal glucose absorption and improving glucose uptake and utilization by peripheral tissues. Recent research has shed light on the pleiotropic effect of metformin, ranging from hypoglycemic function to cardio- and nephroprotection, as well as inhibitory effects on oxidative stress and inflammation [9–11]. The scientific data concerning the influence of metformin on the immune system is controversial, and its effect strongly depends on the pathology in which it is used. For example, metformin enhances antitumor immunity, but in other contexts, it can act as an anti-inflammatory or immunosuppressive agent [8]. Metformin can suppress senescence- and cancer-related inflammation. The majority of experimental data indicates that metformin modulates leukocytes' functional activity by activating 5′ adenosine monophosphate-activated protein kinase (AMPK). Metformin can activate AMPK in multiple cell populations, including macrophages and neutrophils [12, 13]. It has also been demonstrated that metformin inhibits innate immune response to fungal infection in an AMPK-dependent manner and

Considering the significant modulating effect of metformin on the immune system, it is unsurprising that it has a strong effect on immunocompetent blood

Studies conducted on Fanconi anemia mice showed the unique property of metformin to improve hematopoiesis by restoring hematopoietic stem cell (HSC) numbers. It also delays tumor formation, presumably via reduction of DNA damage induced by aldehydes [15]. An important part of metformin protective effect may be conferred by aldehyde detoxification. Other mechanisms by which metformin may act to protect the cell's DNA are reducing the activity of mitochondrial complex 1 activity, thus potentially reducing oxidative DNA damage. It is also possible that metformin can switch the metabolic balance between oxidative phosphorylation and anaerobic glycolysis and downregulate inflammatory pathways which are thought to contribute to bone marrow failure [15]. Another study demonstrates that metformin treatment significantly inhibited the total-body irradiation-induced increase in the levels of DNA double-strand breaks and reactive oxygen species

**76**

(ROS) by attenuation of NOX4 expression in HSCs. Furthermore, metformin modulates the expression of antioxidant enzymes in HSCs [16].

### **2.2 Influence of metformin on functional state of leukocytes**

Many diabetic patients who receive metformin show significantly reduced neutrophil-to-lymphocyte ratio [9]. Metformin is able to reduce hyperneutrophilia in girls with hyperinsulinemic hyperandrogenism and improves white blood cell count in women with polycystic ovary syndrome, two conditions characterized by a pronounced systemic inflammatory state [17]. Metformin increased the number of CD8-positive tumor-infiltrating lymphocytes. Normalizing effect of metformin on the number of immunocompetent cells is associated with its ability to upregulate AMPK and as a consequence of altering energy metabolism in the cell [14].

Apart from metformin influence on immunocompetent cell number, this drug also can modulate their functional activity. As expected for an AMPK activator, metformin enhances cell mobility and phagocytosis, in particular in macrophages that show enhanced uptake of bacteria, synthetic beads, or apoptotic cells. The effects of AMPK activation may be due to its ability to increase availability of cell surface receptors, including αM integrin or Fc receptors or due to mechanisms that involve suppression of TLR4-associated signaling pathways. Metformin by activating AMPK regulates the process of inflammation resolution—efferocytosis and enhanced uptake of bacteria by phagocytic cells [12, 13].

Additionally, in patients with prediabetes, metformin treatment reduces the concentration of neutrophil extracellular trap (NET) components independently from glycemic control [14].

The normalization of phagocytosis processes and NETosis under metformin administration could suggest an effect of this drug on neutrophil activation. Indeed, metformin attenuates neutrophil activation via inhibition of mitochondrial respiratory complex I, potentially through intracellular H2O2-mediated inhibition of IκB-α degradation and thus prevention of NF-κB activation [18].

Immune system modulation by metformin can be realized not only by its direct influence on the immunocompetent cells but also by its ability to regulate chemokine level. Metformin causes a decrease in inflammatory markers in plasma, including soluble intercellular adhesion molecule, vascular cell adhesion molecule-1, macrophage migration inhibitory factor, C-reactive protein, IL-6, and IL-8. The anti-inflammatory action of metformin is realized by suppressing Akt, Erk1/2, and NF-B translocation. Such changes lead to blocking of pro-inflammatory signal transduction via the phosphoinositide 3 kinase pathway [19].

Immunosuppressive effect of metformin can be mediated by its ability to inhibit the expression of pro-inflammatory mediators (IFN-, TNF-, IL-1, IL-6, IL-17, iNOS, MMP9, and RANTES) and infiltration of immune cells, which was blocked by reducing the expression of CAMs (ICAM, VCAM, and E-selectin) on vascular cells [20, 21].

### **2.3 Effects of metformin on oxidative stress**

Oxidative stress is the leading cause of microvascular and cardiovascular diabetes complications [22]. Disruption of glucose metabolism causes mitochondrial superoxide overproduction in cells. An increased amount of superoxide leads to overactivity of polyol and hexosamine pathways, increased formation of AGEs (advanced glycation end products) and its receptors, and activation of protein kinase C isoforms. Altogether, this leads to the development of complications of diabetes. Simultaneously endothelial nitric oxide synthase is inactivated. Changes

in the activity of these signaling pathways result in increased intracellular ROS and activation of pro-inflammatory pathways [22].

Considering such intimate link between diabetes and oxidative stress, antidiabetes treatments should not only reduce blood sugar but should also possess strong antioxidant properties. Metformin satisfies both criteria; as in addition to a hypoglycemic effect, it improves the immunological parameters of patients, presumably through its antioxidant properties [23]. In aortic endothelial cells, metformin has been shown to inhibit high glucose-dependent ROS overproduction, which was mediated by a reduction in NADPH oxidase activity and an inhibition of the respiratory chain complex 1. Another possible mechanism of metformin antioxidant properties is its ability to activate AMPK with the ensuing induction of manganese superoxide dismutase and expression of the antioxidant thioredoxin and endothelial NO synthase (eNOS). Additionally, metformin is able to reduce AGEs synthesis and the expression of their specific cell receptor called RAGE in endothelial cells [16, 23]. In addition to the abovementioned indirect mechanisms of modulation of superoxide anion intracellular production, it was found that metformin can directly scavenge ROS, in particular • OH but not O2 • [16].

While leukocytes actively participate in ROS generation, they are highly sensitive to ROS-mediated oxidative damage. Metformin was demonstrated to have a protective effect against oxidative stress in immunocompetent cells [24].

Furthermore, metformin modulates the function of fMLP-activated polymorphonuclear neutrophils that quench the products of oxidative burst. Researchers hypothesized that metformin may recognize specific cell membrane sites, thereby inducing intracellular signal transduction resulting in changes in NADPH oxidase activity or in other sources of intracellular ROS [25]. Furthermore, metformin-induced decrease in ROS levels led to a partial inhibition of lipid peroxidation in lymphocytes [26].

### **2.4 A protective role of metformin against apoptosis**

Most chronic diseases, including diabetes mellitus, are accompanied by oxidative stress, which may result in apoptosis of different types of cells [27]. Metformin has been shown to have protective role on apoptosis. The inhibition of apoptosis by metformin has been described in many cell types and under various conditions. There may be several mechanisms of apoptosis prevention. Firstly, metformin possesses good radical scavenging activity. Secondly, metformin can regulate caspase levels and induce xenobiotic phase II enzymes [28].

A number of authors have concluded that metformin exerts a neuroprotective effect by decreasing mitochondria-dependent apoptosis. This is achieved through the inhibition of permeability transition pore opening, blocking the release of cytochrome c and preventing subsequent cell death [29]. A protective role of metformin against programmed cell death is likely mediated by maintaining mitochondria integrity and reducing Ca2+. This drug also lowers the expression of caspase-3, cytochrome c, and cleaved caspase-9 and reduces fragmentation of PARP-1 while increasing the expression of Bcl-2 [29]. A similar protective effect of metformin has been described for primary rat hepatocytes. Metformin may protect against apoptosis by induction of menadione-induced heme oxygenase-1 and bcl-xl expression and the reduction of c-Jun N-terminal kinase activation [30, 31].

Given the ability of metformin to inhibit apoptosis of different cells in a variety of pathologies, it is possible to assume that it has a similar effect on immunocompetent blood cells. Indeed, it was shown that metformin markedly decreased the percentage of apoptotic cells in bone marrow cells of rats [32]. It also reduces the activation of macrophages and inhibits the expression of COX-2 and caspase-3, thereby attenuating inflammatory responses and apoptosis [33].

**79**

diabetes [6].

Galega officinalis *L. and Immunological Status in Diabetes Mellitus*

Treatment with metformin reduces the amount of oxidant-induced DNA damage in lymphocytes. It was shown that pharmacological concentration (50 μM) of metformin could protect against prooxidant stimulus-induced DNA damage at early but not late stages. Thus, metformin likely exerts an antiapoptotic effect by reduc-

*Galega officinalis* (goat's rue) is a toxic leguminous plant originated in the Eastern Mediterranean and Black Sea regions but now has been spread in southeastern parts of Europe and the Middle East. In the medieval period, this plant was traditionally used for the treatment of diabetes [5, 34]. *G. officinalis* contains a large number of secondary metabolites with pronounced biological properties, among which are alkaloids, saponins, flavonoids, tannins, fatty acids, and phytoestrogens [35].

The non-alkaloid extract of *G. officinalis* can be obtained by a two-step extraction [6, 7]. In the first stage, the biologically active substances are obtained by plant material infusion in 96 % ethanol. After alcohol evaporation, equal volumes of water and chloroform are added to the residue. The obtained chloroform fraction should be evaporated to obtain the solid residue, which is then dissolved in water to form an emulsion. The latter is not stable and eventually forms a precipitate. The stability of emulsions is very important; their stratification affects the accuracy of active substance content measurement. To solve this problem, the biocomplex PS (surface-active products of *Pseudomonas* sp. PS-17 biosynthesis) can be used [7]. Using gas chromatography/mass spectrometry method, it was established that the biocomplex PS consists of methyl ester of decenoic acid and dodecenoic acid. These surfactants were added to the initial mixture obtained by the addition of water to non-alkaloid fraction of *G. officinalis*. Such extraction and stabilization yield a

Crucially, such non-alkaloid fraction of *G. officinalis* extract exhibited a hypoglycemic effect in streptozotocin-induced diabetes mellitus if administered for 14 days at 600 mg/kg per day. Notably, blood glucose concentration decreased to

Blood glucose measurement evaluates current glucose concentration, which may depend on many factors (the intake and composition of food, physical activity and their intensity, the emotional state of the patient, and even the time of the day) [37]. Thus, blood glucose concentration may not reflect the actual degree of diabetes compensation, potentially resulting in medication under- or overdosing. Therefore, today, the key indicator for treatment quality and risk of diabetes complications is the level of glycosylated hemoglobin (HbA1c) [37]. Notably, the non-alkaloid fraction of *Galega officinalis* extract normalizes HbA1c content under

Sugar-reducing effect of non-alkaloid extract may be due to its complex composition [6, 36, 38]. Gas chromatography/mass spectrometry detected phytol as a component of non-alkaloid fraction of *Galega officinalis* extract. Phytol might contribute to the extract's sugar-lowering effect, as it is known to lower insulin resistance and sensitivity of muscles to insulin and to reduce gluconeogenesis [39]. It has been shown that phytol can increase the expression of *GLUT2* and *glucokinase*

**3. Effects of** *Galega officinalis* **L. on immunocompetent cells under** 

**3.1 Component composition and hypoglycemic effect of non-alkaloid** 

*DOI: http://dx.doi.org/10.5772/intechopen.88802*

ing caspase-3 and caspase-8 activities [28].

**diabetes mellitus**

**extract of** *Galega officinalis*

physiological values [6, 7].

stable water emulsion without toxic alkaloids [6, 36].

Galega officinalis *L. and Immunological Status in Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.88802*

*Metformin*

in the activity of these signaling pathways result in increased intracellular ROS and

Considering such intimate link between diabetes and oxidative stress, antidiabetes treatments should not only reduce blood sugar but should also possess strong antioxidant properties. Metformin satisfies both criteria; as in addition to a hypoglycemic effect, it improves the immunological parameters of patients, presumably through its antioxidant properties [23]. In aortic endothelial cells, metformin has been shown to inhibit high glucose-dependent ROS overproduction, which was mediated by a reduction in NADPH oxidase activity and an inhibition of the respiratory chain complex 1. Another possible mechanism of metformin antioxidant properties is its ability to activate AMPK with the ensuing induction of manganese superoxide dismutase and expression of the antioxidant thioredoxin and endothelial NO synthase (eNOS). Additionally, metformin is able to reduce AGEs synthesis and the expression of their specific cell receptor called RAGE in endothelial cells [16, 23]. In addition to the abovementioned indirect mechanisms of modulation of superoxide anion intracellular production, it was found that metformin can directly

OH but not O2

protective effect against oxidative stress in immunocompetent cells [24].

**2.4 A protective role of metformin against apoptosis**

levels and induce xenobiotic phase II enzymes [28].

and the reduction of c-Jun N-terminal kinase activation [30, 31].

thereby attenuating inflammatory responses and apoptosis [33].

• [16]. While leukocytes actively participate in ROS generation, they are highly sensitive to ROS-mediated oxidative damage. Metformin was demonstrated to have a

Furthermore, metformin modulates the function of fMLP-activated polymorphonuclear neutrophils that quench the products of oxidative burst. Researchers hypothesized that metformin may recognize specific cell membrane sites, thereby inducing intracellular signal transduction resulting in changes in NADPH oxidase activity or in other sources of intracellular ROS [25]. Furthermore, metformin-induced decrease in ROS levels led to a partial inhibition of lipid peroxidation in lymphocytes [26].

Most chronic diseases, including diabetes mellitus, are accompanied by oxidative stress, which may result in apoptosis of different types of cells [27]. Metformin has been shown to have protective role on apoptosis. The inhibition of apoptosis by metformin has been described in many cell types and under various conditions. There may be several mechanisms of apoptosis prevention. Firstly, metformin possesses good radical scavenging activity. Secondly, metformin can regulate caspase

A number of authors have concluded that metformin exerts a neuroprotective effect by decreasing mitochondria-dependent apoptosis. This is achieved through the inhibition of permeability transition pore opening, blocking the release of cytochrome c and preventing subsequent cell death [29]. A protective role of metformin against programmed cell death is likely mediated by maintaining mitochondria integrity and reducing Ca2+. This drug also lowers the expression of caspase-3, cytochrome c, and cleaved caspase-9 and reduces fragmentation of PARP-1 while increasing the expression of Bcl-2 [29]. A similar protective effect of metformin has been described for primary rat hepatocytes. Metformin may protect against apoptosis by induction of menadione-induced heme oxygenase-1 and bcl-xl expression

Given the ability of metformin to inhibit apoptosis of different cells in a variety of pathologies, it is possible to assume that it has a similar effect on immunocompetent blood cells. Indeed, it was shown that metformin markedly decreased the percentage of apoptotic cells in bone marrow cells of rats [32]. It also reduces the activation of macrophages and inhibits the expression of COX-2 and caspase-3,

activation of pro-inflammatory pathways [22].

scavenge ROS, in particular •

**78**

Treatment with metformin reduces the amount of oxidant-induced DNA damage in lymphocytes. It was shown that pharmacological concentration (50 μM) of metformin could protect against prooxidant stimulus-induced DNA damage at early but not late stages. Thus, metformin likely exerts an antiapoptotic effect by reducing caspase-3 and caspase-8 activities [28].

### **3. Effects of** *Galega officinalis* **L. on immunocompetent cells under diabetes mellitus**

*Galega officinalis* (goat's rue) is a toxic leguminous plant originated in the Eastern Mediterranean and Black Sea regions but now has been spread in southeastern parts of Europe and the Middle East. In the medieval period, this plant was traditionally used for the treatment of diabetes [5, 34]. *G. officinalis* contains a large number of secondary metabolites with pronounced biological properties, among which are alkaloids, saponins, flavonoids, tannins, fatty acids, and phytoestrogens [35].

### **3.1 Component composition and hypoglycemic effect of non-alkaloid extract of** *Galega officinalis*

The non-alkaloid extract of *G. officinalis* can be obtained by a two-step extraction [6, 7]. In the first stage, the biologically active substances are obtained by plant material infusion in 96 % ethanol. After alcohol evaporation, equal volumes of water and chloroform are added to the residue. The obtained chloroform fraction should be evaporated to obtain the solid residue, which is then dissolved in water to form an emulsion. The latter is not stable and eventually forms a precipitate. The stability of emulsions is very important; their stratification affects the accuracy of active substance content measurement. To solve this problem, the biocomplex PS (surface-active products of *Pseudomonas* sp. PS-17 biosynthesis) can be used [7]. Using gas chromatography/mass spectrometry method, it was established that the biocomplex PS consists of methyl ester of decenoic acid and dodecenoic acid. These surfactants were added to the initial mixture obtained by the addition of water to non-alkaloid fraction of *G. officinalis*. Such extraction and stabilization yield a stable water emulsion without toxic alkaloids [6, 36].

Crucially, such non-alkaloid fraction of *G. officinalis* extract exhibited a hypoglycemic effect in streptozotocin-induced diabetes mellitus if administered for 14 days at 600 mg/kg per day. Notably, blood glucose concentration decreased to physiological values [6, 7].

Blood glucose measurement evaluates current glucose concentration, which may depend on many factors (the intake and composition of food, physical activity and their intensity, the emotional state of the patient, and even the time of the day) [37]. Thus, blood glucose concentration may not reflect the actual degree of diabetes compensation, potentially resulting in medication under- or overdosing. Therefore, today, the key indicator for treatment quality and risk of diabetes complications is the level of glycosylated hemoglobin (HbA1c) [37]. Notably, the non-alkaloid fraction of *Galega officinalis* extract normalizes HbA1c content under diabetes [6].

Sugar-reducing effect of non-alkaloid extract may be due to its complex composition [6, 36, 38]. Gas chromatography/mass spectrometry detected phytol as a component of non-alkaloid fraction of *Galega officinalis* extract. Phytol might contribute to the extract's sugar-lowering effect, as it is known to lower insulin resistance and sensitivity of muscles to insulin and to reduce gluconeogenesis [39]. It has been shown that phytol can increase the expression of *GLUT2* and *glucokinase* genes through activation of RXR (retinoid X receptor) [39], which are otherwise downregulated under diabetes mellitus. Palmitic acid esters in the extract could also cause a dose-dependent decrease in blood plasma glucose in animals with experimental diabetes mellitus [40]. Furthermore, non-alkaloid fraction of *Galega officinalis* extract contains high levels of phytosterols (campesterol and stigmasterol) that, in addition to the ability to inhibit cholesterol adsorption, can reduce the level of glycosylated hemoglobin [41, 42].

Another notable biologically active substance from *Galega officinalis* is α-amyrin. It has a hypoglycemic action and can influence endocannabinoid system. Some ligands for cannabinoid CB1 receptors can directly bind and allosterically regulate Kir6.2/SUR1 K (ATP) channels, thereby controlling glucose-stimulated insulin release. In addition, α- and β-amyrin, due to their anti-inflammatory and antioxidant properties, have a positive effect on the state of animals with streptozotocin diabetes [43].

It has been shown that quinazoline derivatives are capable to lower blood glucose level and body weight in obese animals [44]. Notably, the non-alkaloid fraction of *Galega officinalis* contains such substances (2-methyl-1,2,3a,4,5-hexahydropyrrolo[1,2-a]quinazoline). These derivatives can increase the activity of AMPK, which results in increased glucose adsorption by muscle cells. It has been found that AMPK, in addition to regulating insulin release by pancreatic cells, inhibits the activity of acetyl-CoA-carboxylase and hydroxymethylglutaryl-CoA-reductase in fat cells, thereby inhibiting the biosynthesis of fatty acids and cholesterol [45].

High content of alpha-linolenic acid in *Galega* extract is also noteworthy. Omega-3 polyunsaturated fatty acids increase cell membrane fluidity, as well as the number of insulin receptors, the affinity of insulin to these receptors, and the number of type 4 glucose transporters; they also regulate the balance between proand antioxidants [46].

Based on the above statement, the sugar-lowering effect of the non-alkaloid fraction of *Galega officinalis* extract is likely due to the presence of phytol, ethyl ester of palmitic acid, phytosterols (campesterol and stigmasterol), and quinazoline derivatives, acting separately or synergistically [6].

### **3.2 Regulation of bone marrow cells proliferation by** *Galega officinalis*

Many of diabetes complications are induced by the intensification of chronic inflammation and attenuation of the immune response. Leukocytes play major roles in inflammation and immune responses. Diabetes mellitus is accompanied by infectious and inflammatory processes, of which the most frequent are bacterial infections, which are accompanied by relapses and are difficult to treat. Changes in the proliferative activity and ratio of leukocytes and changes in their functional properties and activation of free radical oxidation are among probable causes of the propensity of patients with diabetes mellitus to infectious processes and their compromised immunological status [2].

Therefore, the measurement of the hypoglycemic effect is insufficient when testing the effectiveness of new antidiabetic agents. It is also necessary to evaluate the effect of potential hypoglycemic drugs on cells that are susceptible to metabolic changes in diabetes mellitus. Cells whose function is very significantly affected in the course of diabetes mellitus are white blood cells. High levels of glucose in the bloodstream cause inflammation, which primarily affects blood cells, in particular, leukocytes [47, 48].

In addition to a broad spectrum of substances with a hypoglycemic effect, the non-alkaloid fraction of *Galega officinalis* extract contains compounds with potential immunomodulatory effect. *Galega officinalis* normalizes differential count of

**81**

Galega officinalis *L. and Immunological Status in Diabetes Mellitus*

leukocytes in conditions of diabetes mellitus. In particular, it leads to an increase in the number of segmented and band neutrophils while overall lowering the number of lymphocytes to almost control values [49]. This indicates a normalization of the cell-mediated immune response, as one of the most important factors determining the activity of the immune system of an organism [49]. The normalization of the content of immunocompetent cells in blood after treatment of diabetic rats with *Galega* extract may be due to the influence of its biologically active substances on

The non-alkaloid fraction of *Galega officinalis* extract, as a source of biologically active substances with wide range of actions, significantly affects the proliferative activity of bone marrow cells in conditions of diabetes. In particular, in rats with streptozotocin-induced diabetes mellitus, the administration of *Galega officinalis* extract caused a significant decrease in leukocyte proliferation, which is otherwise very high under diabetes. However, a more detailed analysis showed that despite the overall growth of leukocyte proliferation under diabetes mellitus, the abundance of not all leukocyte types increases in the bone marrow [38]. In particular, under diabetes a reduction in the number of myeloblasts was shown, with the following decrease of juvenile and staff neutrophils. By contrast, lymphoblast numbers increased. Interestingly, the number of lymphocytes in the bone marrow does not undergo significant changes, potentially because immature lymphocytes leave the bone marrow towards the bloodstream. Since the non-alkaloid fraction of *Galega officinalis* extract can regulate the proliferative activity of leukocyte precursors, it is able to influence on the content of different types of leukocytes. *Galega officinalis* extract administration causes a decline in lymphoblasts and segmented granulocytes number, as well as an increase in numbers of lymphocytes and juvenile and staff granulocytes in the bone marrow of animals with diabetes mellitus. It has been proposed that this effect is due to the extract's ability to regulate the tumor necrosis factor α (TNF-α) content, the amount of which significantly increases in diabetes

Furthermore, the revealed influence of *Galega officinalis* extract on the proliferative activity of leukocytes may relate to the presence of inositol [50], fatty acids [51, 52], especially α-linolenic acid [53–55], flavonoids [56–59], phytol [60], squalene [61], campesterol, and stigmasterol [62] as well as α-amyrin [38, 63].

**3.3 Influence of** *Galega officinalis* **on functional state of leukocytes and their** 

In diabetes, abnormal immune response manifests itself not only in the imbalance in the process of leukocytes proliferation but also in the disruption of these cells' functional activity. The main effectors of the inflammatory process are phagocytes [64]. The effectiveness of phagocytic response is largely determined by the nature and intensity of its initial stage—chemotaxis. However, because of its complexity, chemotaxis is one of the most vulnerable forms of neutrophil reactivity [65]. Therefore, the impairment of the functional capacity of phagocytes and other immunocytes is associated with the pathology of movement of these cells. The main mechanism that allows cell motility is actin polymerization, as it underlies in the

In animals with diabetes, the non-alkaloid fraction of *Galega officinalis* extract causes a decrease in filamentous actin (F-actin) content; this can testify about the reduction in the formation of short pseudopodia on the leukocytes surface. These data indicate that the use of this extract reduces the change in the structural and functional properties of leukocytes, as well as decrease of leukocyte pre-activated state [67]. It is possible that the extract-induced decrease in actin polymerization

*DOI: http://dx.doi.org/10.5772/intechopen.88802*

the proliferation of these cells.

mellitus [38].

**antioxidant-prooxidant balance**

formation of stress fibrils, lamellipodia, and filopodia [66].

### Galega officinalis *L. and Immunological Status in Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.88802*

*Metformin*

diabetes [43].

and antioxidants [46].

derivatives, acting separately or synergistically [6].

compromised immunological status [2].

level of glycosylated hemoglobin [41, 42].

genes through activation of RXR (retinoid X receptor) [39], which are otherwise downregulated under diabetes mellitus. Palmitic acid esters in the extract could also cause a dose-dependent decrease in blood plasma glucose in animals with experimental diabetes mellitus [40]. Furthermore, non-alkaloid fraction of *Galega officinalis* extract contains high levels of phytosterols (campesterol and stigmasterol) that, in addition to the ability to inhibit cholesterol adsorption, can reduce the

Another notable biologically active substance from *Galega officinalis* is α-amyrin.

It has been shown that quinazoline derivatives are capable to lower blood glucose

It has a hypoglycemic action and can influence endocannabinoid system. Some ligands for cannabinoid CB1 receptors can directly bind and allosterically regulate Kir6.2/SUR1 K (ATP) channels, thereby controlling glucose-stimulated insulin release. In addition, α- and β-amyrin, due to their anti-inflammatory and antioxidant properties, have a positive effect on the state of animals with streptozotocin

level and body weight in obese animals [44]. Notably, the non-alkaloid fraction of *Galega officinalis* contains such substances (2-methyl-1,2,3a,4,5-hexahydropyrrolo[1,2-a]quinazoline). These derivatives can increase the activity of AMPK, which results in increased glucose adsorption by muscle cells. It has been found that AMPK, in addition to regulating insulin release by pancreatic cells, inhibits the activity of acetyl-CoA-carboxylase and hydroxymethylglutaryl-CoA-reductase in fat cells, thereby inhibiting the biosynthesis of fatty acids and cholesterol [45]. High content of alpha-linolenic acid in *Galega* extract is also noteworthy. Omega-3 polyunsaturated fatty acids increase cell membrane fluidity, as well as the number of insulin receptors, the affinity of insulin to these receptors, and the number of type 4 glucose transporters; they also regulate the balance between pro-

Based on the above statement, the sugar-lowering effect of the non-alkaloid fraction of *Galega officinalis* extract is likely due to the presence of phytol, ethyl ester of palmitic acid, phytosterols (campesterol and stigmasterol), and quinazoline

Many of diabetes complications are induced by the intensification of chronic inflammation and attenuation of the immune response. Leukocytes play major roles in inflammation and immune responses. Diabetes mellitus is accompanied by infectious and inflammatory processes, of which the most frequent are bacterial infections, which are accompanied by relapses and are difficult to treat. Changes in the proliferative activity and ratio of leukocytes and changes in their functional properties and activation of free radical oxidation are among probable causes of the propensity of patients with diabetes mellitus to infectious processes and their

Therefore, the measurement of the hypoglycemic effect is insufficient when testing the effectiveness of new antidiabetic agents. It is also necessary to evaluate the effect of potential hypoglycemic drugs on cells that are susceptible to metabolic changes in diabetes mellitus. Cells whose function is very significantly affected in the course of diabetes mellitus are white blood cells. High levels of glucose in the bloodstream cause inflammation, which primarily affects blood cells, in particular,

In addition to a broad spectrum of substances with a hypoglycemic effect, the non-alkaloid fraction of *Galega officinalis* extract contains compounds with potential immunomodulatory effect. *Galega officinalis* normalizes differential count of

**3.2 Regulation of bone marrow cells proliferation by** *Galega officinalis*

**80**

leukocytes [47, 48].

leukocytes in conditions of diabetes mellitus. In particular, it leads to an increase in the number of segmented and band neutrophils while overall lowering the number of lymphocytes to almost control values [49]. This indicates a normalization of the cell-mediated immune response, as one of the most important factors determining the activity of the immune system of an organism [49]. The normalization of the content of immunocompetent cells in blood after treatment of diabetic rats with *Galega* extract may be due to the influence of its biologically active substances on the proliferation of these cells.

The non-alkaloid fraction of *Galega officinalis* extract, as a source of biologically active substances with wide range of actions, significantly affects the proliferative activity of bone marrow cells in conditions of diabetes. In particular, in rats with streptozotocin-induced diabetes mellitus, the administration of *Galega officinalis* extract caused a significant decrease in leukocyte proliferation, which is otherwise very high under diabetes. However, a more detailed analysis showed that despite the overall growth of leukocyte proliferation under diabetes mellitus, the abundance of not all leukocyte types increases in the bone marrow [38]. In particular, under diabetes a reduction in the number of myeloblasts was shown, with the following decrease of juvenile and staff neutrophils. By contrast, lymphoblast numbers increased. Interestingly, the number of lymphocytes in the bone marrow does not undergo significant changes, potentially because immature lymphocytes leave the bone marrow towards the bloodstream. Since the non-alkaloid fraction of *Galega officinalis* extract can regulate the proliferative activity of leukocyte precursors, it is able to influence on the content of different types of leukocytes. *Galega officinalis* extract administration causes a decline in lymphoblasts and segmented granulocytes number, as well as an increase in numbers of lymphocytes and juvenile and staff granulocytes in the bone marrow of animals with diabetes mellitus. It has been proposed that this effect is due to the extract's ability to regulate the tumor necrosis factor α (TNF-α) content, the amount of which significantly increases in diabetes mellitus [38].

Furthermore, the revealed influence of *Galega officinalis* extract on the proliferative activity of leukocytes may relate to the presence of inositol [50], fatty acids [51, 52], especially α-linolenic acid [53–55], flavonoids [56–59], phytol [60], squalene [61], campesterol, and stigmasterol [62] as well as α-amyrin [38, 63].

### **3.3 Influence of** *Galega officinalis* **on functional state of leukocytes and their antioxidant-prooxidant balance**

In diabetes, abnormal immune response manifests itself not only in the imbalance in the process of leukocytes proliferation but also in the disruption of these cells' functional activity. The main effectors of the inflammatory process are phagocytes [64]. The effectiveness of phagocytic response is largely determined by the nature and intensity of its initial stage—chemotaxis. However, because of its complexity, chemotaxis is one of the most vulnerable forms of neutrophil reactivity [65]. Therefore, the impairment of the functional capacity of phagocytes and other immunocytes is associated with the pathology of movement of these cells. The main mechanism that allows cell motility is actin polymerization, as it underlies in the formation of stress fibrils, lamellipodia, and filopodia [66].

In animals with diabetes, the non-alkaloid fraction of *Galega officinalis* extract causes a decrease in filamentous actin (F-actin) content; this can testify about the reduction in the formation of short pseudopodia on the leukocytes surface. These data indicate that the use of this extract reduces the change in the structural and functional properties of leukocytes, as well as decrease of leukocyte pre-activated state [67]. It is possible that the extract-induced decrease in actin polymerization

might regulate integrin-dependent interaction with vascular endothelium necessary for leukocytes penetration through the blood vessel wall during inflammatory processes [68].

F-actin is represented by two pools: (1) long microfilaments (the constitutive fraction of cytoskeleton) located near the cell membrane and reaching towards the center of the cell and (2) short microfilaments located in the submembrane cortical network. Short filaments form a very dynamic fraction, since they are the first ones to initiate polymerization of actin membrane filaments at the time of leukocytes activation [69]. Along with F-actin high content in blood leukocytes in diabetes mellitus condition, the process of its polymerization is intensified with the formation of fraction of short actin filaments. The source of monomers for this polymerization is, to a large extent, products of cytoskeleton filaments depolymerization and, to a lesser extent, the cellular pool of monomeric actin. The increase in actin polymerization may be due to an increase in the phosphatidylinositol amount observed in diabetes mellitus [70]. These cellular messengers may act as inhibitors of phosphorylation of actin regulatory proteins that affect the redistribution of actin filaments and reduce the content of cytoskeleton actin filaments and proportionally increase the level of actin in the short filaments and monomers fractions [71].

The administration of the non-alkaloid fraction of *Galega officinalis* extract in leukocytes of animals with diabetes causes a pronounced depolymerization of short actin filaments. It is accompanied by the formation of actin monomers and their polymerization to a fraction of cytoskeleton filaments. *Galega*-induced changes in actin cytoskeleton organization of leukocytes under prolonged hyperglycemia are probably due to a decrease in the pre-activated state of leukocytes. This effect is mainly achieved by a decrease in the intensity of activation and translocation of the phosphatidylinositol-3′-kinase regulatory subunit in the cytoskeleton sites [68, 72]. Reduced amount of phosphatidylinositol-3′-kinase reaction products (phosphatidylinositol-3,4-diphosphate and phosphatidylinositol-1,3,4-triphosphate) in the cell results in association of the CAP protein with actin filaments, resulting in inhibition of actin polymerization [71].

As mentioned above, diabetes mellitus type 1 is characterized by pre-activated state of leukocytes. This state is associated with the structural and functional rearrangement of the receptor apparatus of these cells. Often, such alterations are realized through changes in the structure of surface glycoproteins that contain sialic acid [73]. In diabetes, N-acetyl-β,D-glucosamine residues are exposed to a greater degree compared to healthy subjects, while the exposure of sialic acids linked by α2→3 and α2→6-glycoside bonds to subterminal residues (β, D-galactose, or N-acetylgalactosamine) decreases. Quantitative redistribution of glycoconjugates in leukocyte membranes leads to the modification of signaling networks involved in intercellular interactions, as well as, to the disruption of the aggregation and adhesiveness of these cells [67]. Activation of membrane-bound neuraminidases in diabetes mellitus leads to a decrease in the total level of sialic acids on the cell membrane. Desialylation is accompanied by increased content of subterminal monosaccharide—β, D-galactose. Galactose-containing glycoproteins regulate leukocyte migration during the inflammatory process, accompanied by a dynamic rearrangement of actin cytoskeleton [74].

The non-alkaloid fraction of *Galega officinalis* extract normalizes the content and structures of the glycoproteins' carbohydrate determinants that form leukocytes' glycocalyx.

Reduction in N-acetyl-β,D-glucosamine residue content upon *Galega officinalis* administration is important to restore normal leukocyte function. Normalized content of such receptors indicates completion of leukocytes pre-activation. It is known that N-acetyl-β,D-glucosamine-containing glycoproteins include a receptor

**83**

phytosterols, and amyrin [38].

Galega officinalis *L. and Immunological Status in Diabetes Mellitus*

for N-formyl-methionyl-leucyl-phenylalanine, which stimulates a respiratory burst in neutrophil granulocytes by activating NADPH oxidase [75]. Also, N-acetylβ,D-glucosamine-containing glycoconjugates are involved in the adhesion of leukocytes to the endothelium during inflammation (through cell surface receptor macrophage-1 antigen or complement receptor 3, which mediates the interaction of neutrophil granulocytes with intercellular adhesion molecule-1) [75]. Thus, the normalization of the receptor content, which has N-acetyl-β,D-glucosamine in its structure, improves the cell's response to extracellular stimuli with a corresponding

Under streptozotocin-induced diabetes, the administration of the non-alkaloid fraction of *Galega officinalis* extract increases the content of α(2→3)-bond sialic acids to physiological levels. It is possible that this effect is due to the influence of the extract's biologically active substances on the activity of enzymes involved in the cleavage or transfer of sialic acid residues (neuraminidase and trans-sialidase) [67, 76]. Glycoproteins that contain sialic acids are structural components of the leukocyte co-receptor complex CD3, which is present in all mature T-lymphocytes and is involved in their activation. It can be assumed that the use of *Galega officinalis* may lead to the restoration of the structure of carbohydrate determinants of the glycoprotein subunit CD3-γ or CD3-ε in the CD3 co-receptor. This in turn inhibits the attenuation of T cells maturation and, as a consequence, prevents the develop-

Consequently, receptor apparatus restoration by *Galega officinalis* extract determines the normalization of the cells' response to extracellular signals, which ultimately leads to the reorganization of actin cytoskeleton elements. However, the leukocyte migration, and therefore the state of actin cytoskeleton, depends on the presence of adhesion molecules on leukocyte surface and on the presence of chemokines. One of these chemokines is TNF-α, a pleiotropic pro-inflammatory cytokine. Through the activation of various signaling cascades, it regulates cell proliferation, differentiation, migration, and apoptosis [80, 81]. An increase in cytokine concentrations under diabetes [38, 67] stimulates leukocyte actin polymerization. TNF-α induces a brief increase in polymerized actin content by activating the Rho/ROCK (Rho-related protein kinase) signaling pathway in neutrophils. The activation of the Rho/ROCK signaling pathway leads to the reorganization of the neutrophil cytoskeleton inducing the formation of stress fibers [82–84]. *Galega* extract decreases TNF-α content to physiological levels. This effect is believed to be related to the presence of anti-inflammatory compounds, including flavonoids, methyl ester of linolenic acid, and α-amyrin [67]. Thus, the non-alkaloid fraction of *Galega officinalis* extract reduces leukocyte pre-activation by acting both on cellular receptor apparatus and on chemokine content in the medium. Reducing diabetes-induced leukocytes pre-activated state by *Galega* extract can significantly improve these cells' functional state. One of the most important functional properties of neutrophils is their bactericidal action. It has been discovered that *Galega officinalis* greatly improved the microbe killing properties of cells. In particular, the non-alkaloid fraction of *Galega officinalis* extract causes a decrease in neutrophils myeloperoxidase content, whereas in conditions of diabetes, the content of this enzyme increases [38, 85]. Inhibition of myeloperoxidase production by neutrophils can play an important role in the prevention of vascular damage mediated by leukocytes. It is known that the excessive amount of myeloperoxidase can cause damage of the blood vessel walls by producing strong oxidants (HOCl and HOBr) or by nitration of the tyrosine residues in proteins. Altogether this can eventually result in cardiovascular diseases [86, 87]. It has been proposed that such inhibiting effect of *Galega officinalis* extract may be due to the synergistic action of phytol, flavonoids, squalene,

*DOI: http://dx.doi.org/10.5772/intechopen.88802*

restoration of the functional state of leukocytes.

ment of the immune deficiency [77–79].

### Galega officinalis *L. and Immunological Status in Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.88802*

*Metformin*

processes [68].

might regulate integrin-dependent interaction with vascular endothelium necessary for leukocytes penetration through the blood vessel wall during inflammatory

F-actin is represented by two pools: (1) long microfilaments (the constitutive fraction of cytoskeleton) located near the cell membrane and reaching towards the center of the cell and (2) short microfilaments located in the submembrane cortical network. Short filaments form a very dynamic fraction, since they are the first ones to initiate polymerization of actin membrane filaments at the time of leukocytes activation [69]. Along with F-actin high content in blood leukocytes in diabetes mellitus condition, the process of its polymerization is intensified with the formation of fraction of short actin filaments. The source of monomers for this polymerization is, to a large extent, products of cytoskeleton filaments depolymerization and, to a lesser extent, the cellular pool of monomeric actin. The increase in actin polymerization may be due to an increase in the phosphatidylinositol amount observed in diabetes mellitus [70]. These cellular messengers may act as inhibitors of phosphorylation of actin regulatory proteins that affect the redistribution of actin filaments and reduce the content of cytoskeleton actin filaments and proportionally increase the level of

The administration of the non-alkaloid fraction of *Galega officinalis* extract in leukocytes of animals with diabetes causes a pronounced depolymerization of short actin filaments. It is accompanied by the formation of actin monomers and their polymerization to a fraction of cytoskeleton filaments. *Galega*-induced changes in actin cytoskeleton organization of leukocytes under prolonged hyperglycemia are probably due to a decrease in the pre-activated state of leukocytes. This effect is mainly achieved by a decrease in the intensity of activation and translocation of the phosphatidylinositol-3′-kinase regulatory subunit in the cytoskeleton sites [68, 72]. Reduced amount of phosphatidylinositol-3′-kinase reaction products (phosphatidylinositol-3,4-diphosphate and phosphatidylinositol-1,3,4-triphosphate) in the cell results in association of the CAP protein with actin filaments, resulting in inhibition

As mentioned above, diabetes mellitus type 1 is characterized by pre-activated

The non-alkaloid fraction of *Galega officinalis* extract normalizes the content and structures of the glycoproteins' carbohydrate determinants that form leuko-

Reduction in N-acetyl-β,D-glucosamine residue content upon *Galega officinalis* administration is important to restore normal leukocyte function. Normalized content of such receptors indicates completion of leukocytes pre-activation. It is known that N-acetyl-β,D-glucosamine-containing glycoproteins include a receptor

state of leukocytes. This state is associated with the structural and functional rearrangement of the receptor apparatus of these cells. Often, such alterations are realized through changes in the structure of surface glycoproteins that contain sialic acid [73]. In diabetes, N-acetyl-β,D-glucosamine residues are exposed to a greater degree compared to healthy subjects, while the exposure of sialic acids linked by α2→3 and α2→6-glycoside bonds to subterminal residues (β, D-galactose, or N-acetylgalactosamine) decreases. Quantitative redistribution of glycoconjugates in leukocyte membranes leads to the modification of signaling networks involved in intercellular interactions, as well as, to the disruption of the aggregation and adhesiveness of these cells [67]. Activation of membrane-bound neuraminidases in diabetes mellitus leads to a decrease in the total level of sialic acids on the cell membrane. Desialylation is accompanied by increased content of subterminal monosaccharide—β, D-galactose. Galactose-containing glycoproteins regulate leukocyte migration during the inflammatory process, accompanied by a dynamic

actin in the short filaments and monomers fractions [71].

of actin polymerization [71].

rearrangement of actin cytoskeleton [74].

**82**

cytes' glycocalyx.

for N-formyl-methionyl-leucyl-phenylalanine, which stimulates a respiratory burst in neutrophil granulocytes by activating NADPH oxidase [75]. Also, N-acetylβ,D-glucosamine-containing glycoconjugates are involved in the adhesion of leukocytes to the endothelium during inflammation (through cell surface receptor macrophage-1 antigen or complement receptor 3, which mediates the interaction of neutrophil granulocytes with intercellular adhesion molecule-1) [75]. Thus, the normalization of the receptor content, which has N-acetyl-β,D-glucosamine in its structure, improves the cell's response to extracellular stimuli with a corresponding restoration of the functional state of leukocytes.

Under streptozotocin-induced diabetes, the administration of the non-alkaloid fraction of *Galega officinalis* extract increases the content of α(2→3)-bond sialic acids to physiological levels. It is possible that this effect is due to the influence of the extract's biologically active substances on the activity of enzymes involved in the cleavage or transfer of sialic acid residues (neuraminidase and trans-sialidase) [67, 76]. Glycoproteins that contain sialic acids are structural components of the leukocyte co-receptor complex CD3, which is present in all mature T-lymphocytes and is involved in their activation. It can be assumed that the use of *Galega officinalis* may lead to the restoration of the structure of carbohydrate determinants of the glycoprotein subunit CD3-γ or CD3-ε in the CD3 co-receptor. This in turn inhibits the attenuation of T cells maturation and, as a consequence, prevents the development of the immune deficiency [77–79].

Consequently, receptor apparatus restoration by *Galega officinalis* extract determines the normalization of the cells' response to extracellular signals, which ultimately leads to the reorganization of actin cytoskeleton elements. However, the leukocyte migration, and therefore the state of actin cytoskeleton, depends on the presence of adhesion molecules on leukocyte surface and on the presence of chemokines. One of these chemokines is TNF-α, a pleiotropic pro-inflammatory cytokine. Through the activation of various signaling cascades, it regulates cell proliferation, differentiation, migration, and apoptosis [80, 81]. An increase in cytokine concentrations under diabetes [38, 67] stimulates leukocyte actin polymerization. TNF-α induces a brief increase in polymerized actin content by activating the Rho/ROCK (Rho-related protein kinase) signaling pathway in neutrophils. The activation of the Rho/ROCK signaling pathway leads to the reorganization of the neutrophil cytoskeleton inducing the formation of stress fibers [82–84]. *Galega* extract decreases TNF-α content to physiological levels. This effect is believed to be related to the presence of anti-inflammatory compounds, including flavonoids, methyl ester of linolenic acid, and α-amyrin [67].

Thus, the non-alkaloid fraction of *Galega officinalis* extract reduces leukocyte pre-activation by acting both on cellular receptor apparatus and on chemokine content in the medium. Reducing diabetes-induced leukocytes pre-activated state by *Galega* extract can significantly improve these cells' functional state. One of the most important functional properties of neutrophils is their bactericidal action. It has been discovered that *Galega officinalis* greatly improved the microbe killing properties of cells. In particular, the non-alkaloid fraction of *Galega officinalis* extract causes a decrease in neutrophils myeloperoxidase content, whereas in conditions of diabetes, the content of this enzyme increases [38, 85]. Inhibition of myeloperoxidase production by neutrophils can play an important role in the prevention of vascular damage mediated by leukocytes. It is known that the excessive amount of myeloperoxidase can cause damage of the blood vessel walls by producing strong oxidants (HOCl and HOBr) or by nitration of the tyrosine residues in proteins. Altogether this can eventually result in cardiovascular diseases [86, 87]. It has been proposed that such inhibiting effect of *Galega officinalis* extract may be due to the synergistic action of phytol, flavonoids, squalene, phytosterols, and amyrin [38].

### *Metformin*

Along with the decrease in the content of myeloperoxidase, the non-alkaloid fraction of the *Galega officinalis* extract also reduces the content of cationic proteins [38] that mediate the killing of a variety of microorganisms through ion pore formation in their membranes [88]. The latter effect is associated with the presence of flavonoids in the extract [38], because these compounds are able to inhibit cationic protein secretion [89].

Thus, the use of alkaloid-free *Galega officinalis* extract for the treatment of diabetes leads to the restoration of functional properties of leukocytes, as indicated by the reconstitution of glycoconjugate receptors on leukocyte membranes, normalization of the ratio of polymerized and unpolymerized actin, as well as restoration of bactericidal properties of these cells.

Diabetes is accompanied by neutrophil malfunction caused, to a large extent, by the development of oxidative-nitrative stress [90]. Oxidative stress leads to the activation of immunocompetent blood cells and their aggregation and adhesion. Further, an increase in the synthesis of arachidonic acid and its metabolites, cytokines, oxygen radicals, and secretion of lysosomal enzymes take place in activated leukocytes. Altogether, it ultimately leads to the development of atherosclerosis [91].

Due to the presence of a large number of biologically active substances with a potential antioxidant effect in the non-alkaloid fraction of *Galega officinalis* extract, it is possible to use this extract as a potential source of antioxidants. Indeed, under diabetes mellitus, the non-alkaloid fraction of *Galega officinalis* extract causes a significant reduction in ROS content in leukocytes, which is otherwise elevated in the pathology [92]. Reduction of ROS generation by leukocytes may be due to the influence of *Galega* extract on the activity of the three main enzymatic systems responsible for generation ROS: membrane-bound NADPH oxidase, peroxidase myeloperoxidase in neutrophils and eosinophil peroxidase in eosinophils, as well as NO synthase. Indeed, a decrease in the content of myeloperoxidase in polymorphonuclear leukocytes [38] and reduction of the total activity of NO synthase was confirmed [93]. In addition to decreasing the activity of ROS synthesis enzymatic systems, the non-alkaloid extract of *Galega officinalis* significantly reduces the processes of protein and lipid oxidative modification. This effect is due to a decrease in total ROS content and NO stable metabolites (nitrite and nitrate anions), with the corresponding termination of biosubstrate oxidation by free radicals. Reduction of oxidative modified proteins and lipids stops the chain reaction of oxidative-nitric stress in conditions of diabetes and confirms the antioxidant effect of the *Galega officinalis* extract [38, 93].

The negative action of ROS in the body is counterbalanced by an antioxidant system, whose functioning is aimed at neutralizing free radicals, as well as repairing damages caused by them [94]. However, in conditions of oxidative-nitrative stress, which is largely activated during diabetes, antioxidant system of blood cells cannot fully implement its protective and adaptive mechanisms. The abnormal functioning of the immune system is evident from a decrease in the superoxide dismutase, catalase, and glutathione peroxidase activity in leukocytes. Under diabetes, the non-alkaloid fraction *of Galega officinalis* extract has a protective effect on the key components of the antioxidant defense system, causing a significant increase in superoxide dismutase and catalase activities [92]. Restoration of antioxidant defense enzymes activity by biologically active substances may be caused by inhibition of the glycosylation of these enzymes, mediated by the hypoglycemic effect of the extract. The increased activity of the antioxidant enzymes is in line with the observed suppression of the formation of oxygen and nitrogen reactive forms, as well as protein and lipid oxidation [38, 93].

The protective effect of the non-alkaloid fraction of *Galega officinalis* extract on blood cells can be explained by its ability to regulate the prooxidant-antioxidant

**85**

apoptosis [75, 97].

Galega officinalis *L. and Immunological Status in Diabetes Mellitus*

balance by means of scavenging free radicals and preventing the inhibition of key components of enzymatic antioxidant system. The main active ingredients of the extract that exhibit antioxidant properties are phytol, showing its properties due to its hydroxyl group [95] and, flavonoids, serving as a traps for electrons and free radicals and thus suppressing the chain reactions of free radical biosubstrate oxidation [38, 89, 93]. Also, α-amyrin [43] and α-linoleic acid [46] possess pronounced

**3.4** *Galega officinalis* **prevents leukocytes apoptosis induced by diabetes** 

fragmentation, which is a biochemical marker of apoptosis [97].

The development of diabetes mellitus is accompanied by a significant intensification of oxidative-nitrative stress, resulting in the formation of substances with a strong proapoptotic effect. Especially sensitive to such substances are blood cells, including leukocytes. The response of immune cells to antigenic stimuli, as well as the nature, dynamics, and duration of the immune response and immunological tolerance formation are partially regulated through programmed cell death [96]. The non-alkaloid fraction of *Galega officinalis* extract causes inhibition of DNA

Other studies have shown that the use of the non-alkaloid fraction of *Galega officinalis* extract in animals with diabetes leads to a reduction of lymphocytes with features of apoptosis, in particular to reduction of phosphatidylserine (PS) residue translocation from the inner to the outer side of the membrane [38]. Changes in the intensity of lymphocyte apoptosis may be due to the effect of extract on the content of TNF-α. It is known that TNF-α reacts with the so-called death receptors and activates procaspases that trigger the apoptotic cascade [98]. Thus, a decrease in TNF-α content might suggest that one of the mechanisms by which *Galega officinalis* inhibits apoptosis in immunocompetent cells is by suppressing the extrinsic, or

Another evidence for the activation of the extrinsic apoptosis pathway under diabetes is exposure on leukocytes' immature membrane epitopes with modified sialic acid content. It takes place in response to the loss of surface membrane during cytoplasmic membrane blebbing [99]. The administration of *Galega officinalis* extract to diabetic animals causes an increase in the content of sialic acid residues linked by α(2→3) and α(2→6) glycosidic bonds with the subterminal surface

On the other hand, it has been found that *Galega officinalis* is able to regulate the processes of the intrinsic (mitochondrial) pathway of apoptosis. In particular, it reduces the levels of the apoptosis regulatory proteins p53 and Bcl-2 [75, 97]. It is known that cell damage results in p53 translocation from the cytoplasm into the mitochondria [100]. In the mitochondria this protein undergoes rapid enzymatic de-ubiquitination that yields an active form which interacts with BH4 domain of antiapoptotic proteins Bcl-XL and Bcl-2 [100]. Binding to antiapoptotic proteins induces the release and activation of proapoptotic proteins Bax and Bid. Such interactions lead to the release of cytochrome c and induction of apoptosis [101, 102]. At the same time, *Galega officinalis* in leukocytes regulates the content of Bcl-2, a protein that inhibits both p53-dependent and p53-independent pathways of apoptosis. Reduction of this protein content promotes the formation of ion channels in mitochondria membrane, thus stabilizing the mitochondrial cytochrome c oxidase and regulating the activation of proteins that are involved in

Another significant confirmation of *Galega officinalis* antiapoptotic action is the reduction of the content of PARylated proteins in leukocytes under diabetes [75].

*DOI: http://dx.doi.org/10.5772/intechopen.88802*

death receptor, apoptosis pathway [38].

glycoconjugate residues of rat leukocytes [75].

antioxidant activities.

**mellitus**

Galega officinalis *L. and Immunological Status in Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.88802*

*Metformin*

protein secretion [89].

*officinalis* extract [38, 93].

well as protein and lipid oxidation [38, 93].

bactericidal properties of these cells.

Along with the decrease in the content of myeloperoxidase, the non-alkaloid fraction of the *Galega officinalis* extract also reduces the content of cationic proteins [38] that mediate the killing of a variety of microorganisms through ion pore formation in their membranes [88]. The latter effect is associated with the presence of flavonoids in the extract [38], because these compounds are able to inhibit cationic

Thus, the use of alkaloid-free *Galega officinalis* extract for the treatment of diabetes leads to the restoration of functional properties of leukocytes, as indicated by the reconstitution of glycoconjugate receptors on leukocyte membranes, normalization of the ratio of polymerized and unpolymerized actin, as well as restoration of

Diabetes is accompanied by neutrophil malfunction caused, to a large extent, by the development of oxidative-nitrative stress [90]. Oxidative stress leads to the activation of immunocompetent blood cells and their aggregation and adhesion. Further, an increase in the synthesis of arachidonic acid and its metabolites, cytokines, oxygen radicals, and secretion of lysosomal enzymes take place in activated leukocytes. Altogether, it ultimately leads to the development of atherosclerosis [91]. Due to the presence of a large number of biologically active substances with a potential antioxidant effect in the non-alkaloid fraction of *Galega officinalis* extract, it is possible to use this extract as a potential source of antioxidants. Indeed, under diabetes mellitus, the non-alkaloid fraction of *Galega officinalis* extract causes a significant reduction in ROS content in leukocytes, which is otherwise elevated in the pathology [92]. Reduction of ROS generation by leukocytes may be due to the influence of *Galega* extract on the activity of the three main enzymatic systems responsible for generation ROS: membrane-bound NADPH oxidase, peroxidase myeloperoxidase in neutrophils and eosinophil peroxidase in eosinophils, as well as NO synthase. Indeed, a decrease in the content of myeloperoxidase in polymorphonuclear leukocytes [38] and reduction of the total activity of NO synthase was confirmed [93]. In addition to decreasing the activity of ROS synthesis enzymatic systems, the non-alkaloid extract of *Galega officinalis* significantly reduces the processes of protein and lipid oxidative modification. This effect is due to a decrease in total ROS content and NO stable metabolites (nitrite and nitrate anions), with the corresponding termination of biosubstrate oxidation by free radicals. Reduction of oxidative modified proteins and lipids stops the chain reaction of oxidative-nitric stress in conditions of diabetes and confirms the antioxidant effect of the *Galega* 

The negative action of ROS in the body is counterbalanced by an antioxidant system, whose functioning is aimed at neutralizing free radicals, as well as repairing damages caused by them [94]. However, in conditions of oxidative-nitrative stress, which is largely activated during diabetes, antioxidant system of blood cells cannot fully implement its protective and adaptive mechanisms. The abnormal functioning of the immune system is evident from a decrease in the superoxide dismutase, catalase, and glutathione peroxidase activity in leukocytes. Under diabetes, the non-alkaloid fraction *of Galega officinalis* extract has a protective effect on the key components of the antioxidant defense system, causing a significant increase in superoxide dismutase and catalase activities [92]. Restoration of antioxidant defense enzymes activity by biologically active substances may be caused by inhibition of the glycosylation of these enzymes, mediated by the hypoglycemic effect of the extract. The increased activity of the antioxidant enzymes is in line with the observed suppression of the formation of oxygen and nitrogen reactive forms, as

The protective effect of the non-alkaloid fraction of *Galega officinalis* extract on blood cells can be explained by its ability to regulate the prooxidant-antioxidant

**84**

balance by means of scavenging free radicals and preventing the inhibition of key components of enzymatic antioxidant system. The main active ingredients of the extract that exhibit antioxidant properties are phytol, showing its properties due to its hydroxyl group [95] and, flavonoids, serving as a traps for electrons and free radicals and thus suppressing the chain reactions of free radical biosubstrate oxidation [38, 89, 93]. Also, α-amyrin [43] and α-linoleic acid [46] possess pronounced antioxidant activities.

### **3.4** *Galega officinalis* **prevents leukocytes apoptosis induced by diabetes mellitus**

The development of diabetes mellitus is accompanied by a significant intensification of oxidative-nitrative stress, resulting in the formation of substances with a strong proapoptotic effect. Especially sensitive to such substances are blood cells, including leukocytes. The response of immune cells to antigenic stimuli, as well as the nature, dynamics, and duration of the immune response and immunological tolerance formation are partially regulated through programmed cell death [96]. The non-alkaloid fraction of *Galega officinalis* extract causes inhibition of DNA fragmentation, which is a biochemical marker of apoptosis [97].

Other studies have shown that the use of the non-alkaloid fraction of *Galega officinalis* extract in animals with diabetes leads to a reduction of lymphocytes with features of apoptosis, in particular to reduction of phosphatidylserine (PS) residue translocation from the inner to the outer side of the membrane [38]. Changes in the intensity of lymphocyte apoptosis may be due to the effect of extract on the content of TNF-α. It is known that TNF-α reacts with the so-called death receptors and activates procaspases that trigger the apoptotic cascade [98]. Thus, a decrease in TNF-α content might suggest that one of the mechanisms by which *Galega officinalis* inhibits apoptosis in immunocompetent cells is by suppressing the extrinsic, or death receptor, apoptosis pathway [38].

Another evidence for the activation of the extrinsic apoptosis pathway under diabetes is exposure on leukocytes' immature membrane epitopes with modified sialic acid content. It takes place in response to the loss of surface membrane during cytoplasmic membrane blebbing [99]. The administration of *Galega officinalis* extract to diabetic animals causes an increase in the content of sialic acid residues linked by α(2→3) and α(2→6) glycosidic bonds with the subterminal surface glycoconjugate residues of rat leukocytes [75].

On the other hand, it has been found that *Galega officinalis* is able to regulate the processes of the intrinsic (mitochondrial) pathway of apoptosis. In particular, it reduces the levels of the apoptosis regulatory proteins p53 and Bcl-2 [75, 97]. It is known that cell damage results in p53 translocation from the cytoplasm into the mitochondria [100]. In the mitochondria this protein undergoes rapid enzymatic de-ubiquitination that yields an active form which interacts with BH4 domain of antiapoptotic proteins Bcl-XL and Bcl-2 [100]. Binding to antiapoptotic proteins induces the release and activation of proapoptotic proteins Bax and Bid. Such interactions lead to the release of cytochrome c and induction of apoptosis [101, 102]. At the same time, *Galega officinalis* in leukocytes regulates the content of Bcl-2, a protein that inhibits both p53-dependent and p53-independent pathways of apoptosis. Reduction of this protein content promotes the formation of ion channels in mitochondria membrane, thus stabilizing the mitochondrial cytochrome c oxidase and regulating the activation of proteins that are involved in apoptosis [75, 97].

Another significant confirmation of *Galega officinalis* antiapoptotic action is the reduction of the content of PARylated proteins in leukocytes under diabetes [75].

This indicates a decrease in DNA damage with the corresponding inhibition of DNA repair complex (base excision repair in response to single-stranded DNA breaks and nucleotide excision repair), which includes poly (ADP-ribose) polymerase enzyme [103]. Thus, *Galega*-induced decrease in protein PARylation could stem from inhibition of poly (ADP-ribose) polymerase activity, which can be assumed to prevent ribosylation of a number of proteins, including glyceraldehyde-3-phosphate dehydrogenase. In the presence of excess glucose, this results in inactivation of the polyol and hexosamine pathways, thereby preventing the accumulation of products and precursors of nonenzymatic glycosylation and activation of protein kinase C. As the final result, this leads to the inhibition of oxidative-nitric stress manifestations and prevents the occurrence of chronic diabetic lesions [75].

The established antiapoptotic effect of *Galega officinalis* extract is mediated by sugar-reducing, antioxidant, and anti-inflammatory properties of its components. In particular, the composition of the extract revealed a number of compounds that have potentially hypoglycemic (phytol, ethyl ester of palmitic acid, campesterol, stigmasterol, and quinazoline derivatives), antioxidant (phytol, flavonoids, vitamin E), and anti-inflammatory (flavonoids, methyl ester of linolenic acid, α-amyrin) effects [38].

### **4. Conclusions**

Metformin has become widely used in the treatment of diabetes mellitus type 2 over the last period of time. This is due to the fact that metformin, along with its hypoglycemic effect, has the potential to modulate the functioning of immunocompetent blood cells. Metformin transiently inhibits NADH:ubiquinone oxidoreductase of the mitochondrial electron transport chain. This inhibition leads to the activation of the energy sensor 5′-AMP-activated protein kinase. The activation of this enzyme results in a whole range of metabolic changes in the immunocompetent cells. Metformin is able to regulate the processes of bone marrow cell proliferation, affect the functional activity, and regulate the apoptosis processes of immunocompetent cells.

To date, practically all mechanisms of therapeutic influence of metformin are well described. Instead, the plant from which this biguanide was first obtained somewhat become underestimated. Under diabetes mellitus type 1, the non-alkaloid fraction of *Galega officinalis* possesses pronounced hypoglycemic effect. The non-alkaloid fraction of *Galega officinalis* normalizes the leukocyte proliferation processes by restoring the neutrophils bone marrow pool and reducing the lymphoblasts number. This extract affects the functional state of immunocompetent cells in blood, leading to quantitative redistribution and structural alterations of carbohydrate determinants in leukocyte membranes, reorganization of actin cytoskeleton, as well as affecting the bactericidal function of neutrophils. Furthermore, nonalkaloid fraction of *Galega officinalis* predetermines the suppression of leukocyte to genetically programmed death. The multifactorial effect of *Galega officinalis* extract under diabetes may be, on the one hand, due to its potent hypoglycemic effect, and, on the other hand, due to its ability to regulate the prooxidant-antioxidant balance by scavenging free radicals and preventing the inhibition of key enzymatic components of the antioxidant defense system.

**87**

**Author details**

of Lviv, Lviv, Ukraine

Mariia Nagalievska\*, Halyna Hachkova and Nataliia Sybirna

\*Address all correspondence to: khmarija@gmail.com

provided the original work is properly cited.

Department of Biochemistry, Faculty of Biology, Ivan Franko National University

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Galega officinalis *L. and Immunological Status in Diabetes Mellitus*

*DOI: http://dx.doi.org/10.5772/intechopen.88802*

### **Conflict of interest**

The authors declare no conflict of interest.

Galega officinalis *L. and Immunological Status in Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.88802*

*Metformin*

effects [38].

petent cells.

**4. Conclusions**

This indicates a decrease in DNA damage with the corresponding inhibition of DNA repair complex (base excision repair in response to single-stranded DNA breaks and nucleotide excision repair), which includes poly (ADP-ribose) polymerase enzyme [103]. Thus, *Galega*-induced decrease in protein PARylation could stem from inhibition of poly (ADP-ribose) polymerase activity, which can be assumed to prevent ribosylation of a number of proteins, including glyceraldehyde-3-phosphate dehydrogenase. In the presence of excess glucose, this results in inactivation of the polyol and hexosamine pathways, thereby preventing the accumulation of products and precursors of nonenzymatic glycosylation and activation of protein kinase C. As the final result, this leads to the inhibition of oxidative-nitric stress manifesta-

The established antiapoptotic effect of *Galega officinalis* extract is mediated by sugar-reducing, antioxidant, and anti-inflammatory properties of its components. In particular, the composition of the extract revealed a number of compounds that have potentially hypoglycemic (phytol, ethyl ester of palmitic acid, campesterol, stigmasterol, and quinazoline derivatives), antioxidant (phytol, flavonoids, vitamin E), and anti-inflammatory (flavonoids, methyl ester of linolenic acid, α-amyrin)

Metformin has become widely used in the treatment of diabetes mellitus type 2 over the last period of time. This is due to the fact that metformin, along with its hypoglycemic effect, has the potential to modulate the functioning of immunocompetent blood cells. Metformin transiently inhibits NADH:ubiquinone oxidoreductase of the mitochondrial electron transport chain. This inhibition leads to the activation of the energy sensor 5′-AMP-activated protein kinase. The activation of this enzyme results in a whole range of metabolic changes in the immunocompetent cells. Metformin is able to regulate the processes of bone marrow cell proliferation, affect the functional activity, and regulate the apoptosis processes of immunocom-

To date, practically all mechanisms of therapeutic influence of metformin are well described. Instead, the plant from which this biguanide was first obtained somewhat become underestimated. Under diabetes mellitus type 1, the non-alkaloid fraction of *Galega officinalis* possesses pronounced hypoglycemic effect. The non-alkaloid fraction of *Galega officinalis* normalizes the leukocyte proliferation processes by restoring the neutrophils bone marrow pool and reducing the lymphoblasts number. This extract affects the functional state of immunocompetent cells in blood, leading to quantitative redistribution and structural alterations of carbohydrate determinants in leukocyte membranes, reorganization of actin cytoskeleton, as well as affecting the bactericidal function of neutrophils. Furthermore, nonalkaloid fraction of *Galega officinalis* predetermines the suppression of leukocyte to genetically programmed death. The multifactorial effect of *Galega officinalis* extract under diabetes may be, on the one hand, due to its potent hypoglycemic effect, and, on the other hand, due to its ability to regulate the prooxidant-antioxidant balance by scavenging free radicals and preventing the inhibition of key enzymatic compo-

tions and prevents the occurrence of chronic diabetic lesions [75].

**86**

nents of the antioxidant defense system.

The authors declare no conflict of interest.

**Conflict of interest**

### **Author details**

Mariia Nagalievska\*, Halyna Hachkova and Nataliia Sybirna Department of Biochemistry, Faculty of Biology, Ivan Franko National University of Lviv, Lviv, Ukraine

\*Address all correspondence to: khmarija@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[20] Nath N, Khan M, Paintlia MK, Hoda MN, Giri S. Metformin attenuated the autoimmune disease of the central nervous system in animal models

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[14] Pollak M. The effects of metformin on gut microbiota and the immune system as research frontiers. Diabetologia. 2017;**60**:1662-1667. DOI: 10.1007/s00125-017-4352-x

[15] Zhang Q, Tang W, Deater M, Phan N, Marcogliese AN, Li H, et al. Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice. Blood. 2016;**128**(24):2774-2784. DOI: 10.1182/blood2015-11-683490

[16] Xu G, Wu H, Zhang J, Li D, Wang Y, Wang Y, et al. Metformin ameliorates ionizing irradiation-induced longterm hematopoietic stem cell injury in mice. Free Radical Biology and Medicine. 2015;**87**:15-25. DOI: 10.1016/j. freeradbiomed.2015.05.045

[17] Orio F, Manguso F, Di Biase S, Falbo A, Giallauria F, Labella D, et al. Metformin administration improves leukocyte count in women with polycystic ovary syndrome: A 6-month prospective study. European Journal of Endocrinology. 2007;**157**:69-73. DOI: 10.1530/EJE-07-0133

[18] Kebir DE, Filep JG. Role of neutrophil apoptosis in the resolution of inflammation. The Scientific World Journal. 2010;**10**:1731-1748. DOI: 10.1100/tsw.2010.169

[19] Isoda K, Young JL, Zirlik A, MacFarlane LA, Tsuboi N, Gerdes N, et al. Metformin inhibits proinflammatory responses and nuclear factor-B in human vascular wall cells. Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;**26**:611-617. DOI: 10.1161/01.ATV.0000201938.78044.75

[20] Nath N, Khan M, Paintlia MK, Hoda MN, Giri S. Metformin attenuated the autoimmune disease of the central nervous system in animal models

of multiple sclerosis. The Journal of Immunology. 2009;**182**:8005-8014. DOI: 10.4049/jimmunol.0803563

[21] Han J, Li Y, Liu X, Zhou T, Sun H, Edwards P, et al. Metformin suppresses retinal angiogenesis and inflammation in vitro and in vivo. PLoS One. 2018;**13**(3):e0193031. DOI: 10.1371/ journal.pone.0193031

[22] Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circulation Research. 2010;**107**:1058-1070. DOI: 10.1161/ CIRCRESAHA.110.223545

[23] Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: From mechanisms of action to therapies. Cell Metabolism. 2014;**20**(6):953-966. DOI: 10.1016/j.cmet.2014.09.018

[24] Victor VM, Rovira-Llopis S, Banuls C, Diaz-Morales N, Castello´ R, Falcon1 R, et al. Effects of metformin on mitochondrial function of leukocytes from polycystic ovary syndrome patients with insulin resistance. European Journal of Endocrinology. 2015;**173**:683-691. DOI: 10.1530/ EJE-15-0572

[25] Bonnefont-Rousselot D, Raji B, Walrand S, Garde's-Albert M, Jore D, Legrand A, et al. An intracellular modulation of free radical production could contribute to the beneficial effects of metformin towards oxidative stress. Metabolism. 2003;**52**(5):586-589. DOI: 10.1053/meta.2003.50093

[26] Onaran I, Guven GS, Ozdas SB, Kanigur G, Vehid S. Metformin does not prevent DNA damage in lymphocytes despite its antioxidant properties against cumene hydroperoxideinduced oxidative stress. Mutation Research. 2006;**611**:1-8. DOI: 10.1016/j. mrgentox.2006.06.036

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*Metformin*

ng0193-77

immunodeficiency. Nature Genetics. 1993;**3**(1):77-81. DOI: 10.1038/

Science. 2013;**54**(3):2373-2383. DOI:

Myeloperoxidase selectively binds and selectively kills microbes. Infection and Immunity. 2011;**79**(1):474-485. DOI:

development of oxidative stress general pathology and pathophysiology. Bulletin of Experimental Biology and Medicine. 2012;**154**(1):23-26. DOI: 10.1007/

Myeloperoxidase and cardiovascular

Thrombosis, and Vascular Biology. 2005;**25**:1102-1111. DOI: 10.1161/01.

[88] Cruse JM, Lewis RE. Atlas of Immunology. 2nd ed. CRC Press LLC;

[90] Lee HB, Ha H, King GL. Reactive

Averous G, Faure A, Jesel L, Germain P, et al. Increased levels of procoagulant tissue factor-bearing microparticles within the occluded coronary artery of patients with ST-segment elevation myocardial infarction:

10.1167/iovs.12-10757

10.1128/IAI.00910-09

s10517-012-1865-7

2004. 958 p

[87] Nicholls SJ, Hazen SL.

disease. Arteriosclerosis,

ATV.0000163262.83456.6d

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ASN.0000077403.06195.D2

[91] Morel O, Pereira B,

[85] Allen RC, Stephens JT.

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blood-2007-08-109769

[80] Baud V, Karin M. Signal

S0962-8924(01)02064-5

jimmunol.170.11.5704

s00018-009-0189-x

10.1038/nrm1128.

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[81] Nakao S, Kuwano T, Ishibashi T, Kuwano M, Ono M. Synergistic effect of TNF-alpha in soluble VCAM-1 induced angiogenesis through alpha 4 integrins. Journal of Immunology. 2003;**170**(11):5704-5711. DOI: 10.4049/

[82] Hahmann C, Schroeter T. Rhokinase inhibitors as therapeutics: From pan inhibition to isoform selectivity. Cellular and Molecular Life Sciences. 2010;**67**(2):171-177. DOI: 10.1007/

[83] Riento K, Ridley AJROCKS. Multifunctional kinases in cell

[84] Arita R, Nakao S, Kita T,

behaviours. Nature Reviews. Molecular Cell Biology. 2003;**4**(6):446-456. DOI:

Kawahara S, Asato R, Yoshida S, et al. A key role for ROCK in TNF-a–mediated diabetic microvascular damage. Investigative Ophthalmology & Visual

**94**

[92] Lupak MI, Khokhla MR, Hachkova GY, Kanyuka OP, Klymyshyn NI, Chajka YP, et al. The alkaloid-free fraction from *Galega officinalis* extract prevents oxidative stress under experimental diabetes mellitus. Ukranian Biochemical Journal. 2015;**87**(4):78-86. DOI: 10.15407/ ubj87.04.078

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[94] Atalay V, Laaksonen DE, Niskanen L. Altered antioxidant enzyme defenses in insulin-dependent diabetic men with increased resting and exercise-induced oxidative stress. Acta Physiologica Scandinavica. 1997;**161**:195-201. DOI: 10.1046/j.1365-201X.1997.00200.x

[95] De Menezes Patrício Santos СС, Salvadori MS, Mota VG, Costa LM, De Almeida AAC, Lopes De Oliveira GA, et al. Antinociceptive and antioxidant activities of phytol in vivo and in vitro models. Neuroscience Journal. 2013;**2013**:1-9. DOI: 10.1155/2013/949452

[96] Hetts SW. To die or not to die: An overview of apoptosis and its role in disease. Journal of the American Medical Association. 1998;**279**(4):300- 307. DOI: 10.1001/jama.279.4.300

[97] Khokhla M, Кleveta G, Chajka YA, Skybitska M, Sybirna N. The influence of *Galega officinalis* on rats leukocytes apoptosis under the experimental

diabetes mellitus type 1. Visnyk of Lviv University. Biological Series. 2012;**60**:117-125

[98] Deng Y, Ren X, Yang L, Lin Y, Wu X. A JNK-dependent pathway is required for TNFalpha-induced apoptosis. Cell. 2003;**115**(1):61-70. DOI: 10.1016/S0092-8674(03)00757-8

[99] Meesmann HM, Fehr EM, Kierschke S, Herrmann M, Bilyy R, Heyder P, et al. Decrease of sialic acid residues as an eat-me signal on the surface of apoptotic lymphocytes. Journal of Cell Science. 2010;**123**(Pt19):3347-3356. DOI: 10.1242/jcs.066696

[100] Braithwaite A, Royds J, Jackson P. The p53 story: Layers of complexity. Carcinogenesis. 2005 Jul;**26**(7):1161- 1169. DOI: 10.1093/carcin/bgi091

[101] Erster S, Mihara M, Kim R. In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation. Molecular and Cellular Biology. 2004;**24**(15):6728-6741. DOI: 10.1128/ MCB.24.15.6728-6741.2004

[102] Janicke RU, Sohn D, Schulze-Osthoff K. The dark side of a tumor suppressor: Anti-apoptotic p53. Cell Death and Differentiation. 2008 Jun;**15**(6):959-976. DOI: 10.1038/ cdd.2008.33

[103] Schmitz H. Reversible nuclear translocation of glyceraldehyde-3 phosphate dehydrogenase upon serum depletion. European Journal of Cell Biology. 2001;**80**(6):419-427. DOI: 10.1078/0171-9335-00174

**97**

Section 2

Metformin and

Reproductive System

Section 2
