**9. Probiotic interventions**

**8. The microbiota in type 2 diabetes**

80 Pathophysiology - Altered Physiological States

were resistant to diet-induced obesity [83].

microbiota change.

in prediabetes.

abundance of *Intestinibacter* sp. (**Figure 2**) [89].

Genetics, lifestyle and increased bodyweight all contribute to the development of type 2 diabetes. Around 80% of individuals with T2D are overweight thus suggesting an important role of diet and microbiota in the pathophysiology of this disease. The link between microbiota and T2D first became evident in studies on germ-free mice. Thus, colonization of germfree animals with microbiota harvested from conventionally raised mice lead to a significant increase in body fat and insulin resistance [82]. A following study showed that germ-free mice

Subsequently, several studies have documented the microbiota shifts associated with T2D. After analysing a cohort of Chinese patients with T2D, Qin et al. showed that the diabetic microbiome is low in butyrate-producing bacteria such as Clostridiales sp., *F. prausnitzii, Roseburia intestinalis* and *E. rectal* [84]. Moreover, the T2D intestinal niche contained opportunistic pathogens including the sulfate-reducing *Desulfovibrio*, *Bacteroides caccae* and *E. coli*. In line with these findings, a study in Scandinavian post-menopausal women revealed decreased levels of *F. praunitzii* and *R. intestinalis* in T2D compared with individuals having impaired glucose tolerance. In addition, both Chinese and Scandinavian T2D cohorts exhibited elevated *Lactobacillus* levels. Obesity and impaired glucose metabolism were reported to have an altered ratio between Bacteroidetes and Firmicutes [85]; however, neither the Chinese nor the Scandinavian study found this

The Chinese study revealed an increase of *E. coli* in T2D patients and another Danish study showed that Proteobacteria levels were elevated in T2D [86]. These Gram-negative bacteria could potentially be involved in the pathophysiology of T2D. Specifically, the lipopolysaccharides (LPS) released by these bacteria could promote a subclinical proinflammation, which is typical to both diabetes and obesity. Recent studies revealed that T2D is characterized by elevated endotoxemia. Indeed, mice receiving high fat (HF) diet until they developed diabetes had endotoxemia, increased intestinal permeability and a distinct microbiots [87]. In addition, the term of metabolic infection has emerged in order to describe the role of the microbiome in endotoxemia-associated inflammation together with insulin resistance in T2D. Endotoxin of microbial origin could play a role in the insulin resistance associated with T2D since blood levels of bacterial DNA (mostly Proteobacteria) were shown to be increased

One caveat of the currently available human studies is the lack of information regarding the role of antidiabetic medication in altering the microbiota. The first-line drug of choice for type 2 diabetes treatment is represented by metformin. In the Swedish study, the diabetic patients received metformin treatment and their microbiota was enriched in Enterobacteriaceae and had low levels of *Eubacterium* and *Clostridium*. In mice-fed a high-fat diet, metformin was shown to affect both the host glucose metabolism as well as the microbiota by increasing the levels of *Akkermansia* [88]. Recently, metformin treatment has also been shown to alter the microbiota composition in T2D patients by increasing *Escherichia* sp. and decreasing the Due to their anti-inflammatory, hypoglycaemic, insulinotropic, antioxidative and satietogenis properties, probiotics can be employed as a treatment for T2D. The insulinotropic effect of genetically engineered *Escherichia coli Nissle 1917* for GLP-1 was investigated in Caco-2 cells; it was observed that the probiotic strain stimulated the epithelial cells leading to the secretion of insulin corresponding to blood insulin concentration of 164 pmol/ml to 164 nmol/ml [90]. In addition, Paszti-Gere et al. reported that oxidative stress causing damage to insulin-secreting ß-cells was counteracted by metabolites of *Lactobacillus plantarum* 2142. Specifically, the spent culture supernatant of *L. plantarum 2142* decreased the oxidative stress-induced overexpression of proinflammatory cytokines IL-8 and TNF-α in IPEC-J2 cell line [91]. The multiple mechanisms of probiotics in T2D treatment have emerged from studies by using animal models. Oral administration (0.05%) or diet supplementation (0.1%) of heat-killed *L. casei* in different mouse models including KK-Ay mice, NOD mice and Alloxan-induced diabetic mice reduced the plasma glucose level and diabetes development [92, 93]. Feeding neonatal STZ-induced diabetic (n-STZ) rats with a diet containing *Lactobacillus rhamnosus GG* for a period of 9 weeks determined a lower blood haemoglobin level and an improved glucose tolerance in comparison to the control group receiving a conventional diet. The *L. rhamnosus GG* treatment group had a serum insulin level significantly higher than the control group at 30 min after glucose loading [94]. Furthermore, feeding VSL#3 lowered β-cell destruction and inflammation in NOD mice, and this effect was accompanied by increased IL-10 secretion in pancreas, Peyer's patches and spleen. In a separate study, the feeding of a probiotic containing *Lactobacillus acidophilus NCDC14* and *L. casei NCDC19* significantly lowered free fatty acids, the blood glucose and glycosylated haemoglobin, and trigycerides in fructose-induced diabetic rats [95]. The feeding of the same probiotic to STZ-induced rats suppressed the STZ-induced oxidative damage in pancreatic tissues by inhibiting lipid peroxidation, generation of nitric oxide and improved the antioxidant potential of glutathione, superoxide dismutase, and catalase and glutathione peroxidase. These data suggest that oral administration of the probiotic significantly ameliorated the risk factors such as dyslipidemia, hyperglycemia and oxidative stress in diabetic rats [96]. Probiotic pre-treatment with a mixture containing *Bifidobacterium lactis, L. acidophilus* and *L. rhamnosus* lowered the blood glucose and improved the bioavailability of gliclazide, a second-generation sulphonylurea used for treating non-insulin dependent diabetes mellitus T2D in alloxan induced diabetic rats [97]. The antidiabetic effects against insulin resistance of different probiotics can also be due to increased liver natural killer T (NKT) cells. NKT cells are involved in regulating the inflammatory process in the liver which is the main organ responsible for inflammation-mediated insulin resistance. Depletion of liver NKT enhanced the production of pro-inflammatory cytokines, and HFD was known to induce depletion of hepatic NKT cells leading to insulin resistance. HFD-induced depletion of NKT cells in male C57BL-6 mice was significantly improved by administration of the VSL#3 probiotic. This probiotic treatment also leads to weight loss, and improved insulin resistance and inflammation by modulating TNF-α expression and reducing NF-kB binding activity [98]. Treatment with *L. plantarum* DSM 15313 and *L. reuteri* GMNL-263 was reported to lower the blood glucose and glycosylated haemoglobin, in HFD-fed C57BL/6 J mice and STZ-induced diabetic rats [99, 100]. DCs from NOD mice were stimulated with three different strains of lactobacilli including *L. casei*, *L. reuteri* and *L. plantarum* for a period of 24 h. Out of the strains tested, *L. casei* was found to induce DCs to generate the highest level of IL-10 and the lowest level of IL-12 expression. When the *L. casei*-stimulated DCs were transferred to NOD mice, they showed a significant delay in diabetes incidence [101]. *Bifidobacterium longum* CGMCC NO. 2107 added as a supplemed in HFD was shown to reduce the metabolic endotoxin (LPS) plasma concentrations and to improve intestinal inflammation [102]. Amar et al. analysed the effect probiotic treatment has on mucosal dysbiosis, bacterial translocation and glucose metabolism [103]. The results obtained revealed that the bacterial translocation was prevented in mice lacking the microbial pattern recognition receptors Nod1 or CD14. Nevertheless, it was increased in Myd88 deficient mice and ob/ob mouse under the same conditions. In addition, the administration of *Bifidobacterium animalis* subsp*. lactis 420* reduced the bacterial translocation to mesenteric adipose tissue, decreasing the expression of major pro-inflammatory cytokines TNF-a, IL-1b and IL-6 in mesenteric adipose tissue, liver and muscle. In addition, *B. animalis* subsp*. lactis 420* also improved the insulin sensitivity and fasting hyperinsulinaemia in HFD fed mice [103].

was shown to improve the antioxidant status and lipid profile in T2D patients. Several randomized, double blind, placebo-controlled clinical trials evaluated the effects of probiotic administration on antioxidant status, blood glucose and lipid profile in T2D. The patients with T2D mellitus enrolled in these studies were divided into two groups: the probiotic interven-

 cfu/mL *B. lactis Bb12*, whereas the control group consumed 300 g/d of conventional yoghurt during a period of 6 weeks. The probiotic treatment cohort exhibited a significant decrease in fasting blood glucose as well as an increase in the activities of the erythrocyte superoxide dismutase and glutathione peroxidase. In addition, the total cholesterol: high-density lipoprotein (HDL)-C and LDL-C: HDL-C ratios were decreased in the probiotic-treated patients compared to the control [104, 105]. Another randomized, double-blind, placebo-controlled study was performed on 20 elderly diabetic volunteers aged 50–60 years, over for a period of 30 days to study the effect of a symbiotic drink (a preparation with a combination of both probiotics and pre-

cfu/mL of *L. acidophilus*, and 2 g oligofructose harboured a signifi-

The Intricate Relationship between Diabetes, Diet and the Gut Microbiota

biotics) on glycaemia and cholesterol levels. The symbiotic group that consumed 108

function recovery time and length of hospitalization were also recorded [107].

cantly increased HDL cholesterol, and a decrease in fasting glycemia but, importantly, no significant changes were observed in the placebo group [106]. Recently, in a study by Shao et al., 67 diabetic patients with gastrointestinal cancer were randomized into the probiotic treatment group (33 patients receiving enteral nutrition with probiotics, glutamine and fish oil) and the control group (34 patients receiving regular enteral nutrition). Fasting blood glucose and insulin were recorded on the day before surgery and post-operative days 3 and 7. Insulin resistance index (HOMA-IR) was calculated as well by using the homeostasis model assessment (HOMA) for both groups, and the supplementary data on incidence of nosocomial infections, intestinal

The enteral nutrition with probiotics, glutathione and fish oil was associated with a low fasting insulin and insulin resistance index compared to the control group. The length of hospital stay was significantly decreased from 21 to 17 days in the treatment group. Nevertheless, no significant differences in nosocomial infection and intestinal function recovery were observed between the two groups. The role of maternal probiotic-supplemented dietary counseling during pregnancy on colostrum adiponectin concentration in neonatal nutrition, metabolism and immunity was analysed in a randomized, placebo-controlled study by Luoto et al. [108]. Specifically, 256 pregnant women were randomized into three groups: dietary intervention with probiotics (diet/ *L. rhamnosus GG* and *B. lactis*), with placebo (diet/placebo) and a control cohort (control/placebo). Dietary intake was analysed by food records at each pregnancy trimester, and subsequently colostrum samples were collected after birth for the analysis of adiponectin concentration. An improved adiponectin concentration is a parameter of neonatal metabolic homeostasis and is also an indicator of reduced chances of gestational diabetes. Probiotic treatment increased the colostrum adiponectin concentration compared to the control (12.7 ng/ml vs. 10.2 ng/ml). Nevertheless, other studies state that probiotic use does not provide a benefit for the diabetic host. For instance, a randomized, double-blinded clinical trial using the commercial probiotic *L. acidophilus NCFM* in a group of 45 men for a timeframe of 4 weeks revealed that there were no changes in the expression of baseline inflammatory markers and in the systemic inflammatory response following probiotic treatment [109].

cfu/mL *L. acidophilus La5* and

http://dx.doi.org/10.5772/intechopen.70602

cfu/mL

83

tion group consumed 300 g/d of probiotic yoghurt containing 106

106

*Bifidobacterium bifidum*, 108

Since there are a few reports in this area, the knowledge regarding the efficacy of probiotic administration in diabetic human subjects is quite limited. Consumption of probiotic yoghurt was shown to improve the antioxidant status and lipid profile in T2D patients. Several randomized, double blind, placebo-controlled clinical trials evaluated the effects of probiotic administration on antioxidant status, blood glucose and lipid profile in T2D. The patients with T2D mellitus enrolled in these studies were divided into two groups: the probiotic intervention group consumed 300 g/d of probiotic yoghurt containing 106 cfu/mL *L. acidophilus La5* and 106 cfu/mL *B. lactis Bb12*, whereas the control group consumed 300 g/d of conventional yoghurt during a period of 6 weeks. The probiotic treatment cohort exhibited a significant decrease in fasting blood glucose as well as an increase in the activities of the erythrocyte superoxide dismutase and glutathione peroxidase. In addition, the total cholesterol: high-density lipoprotein (HDL)-C and LDL-C: HDL-C ratios were decreased in the probiotic-treated patients compared to the control [104, 105]. Another randomized, double-blind, placebo-controlled study was performed on 20 elderly diabetic volunteers aged 50–60 years, over for a period of 30 days to study the effect of a symbiotic drink (a preparation with a combination of both probiotics and prebiotics) on glycaemia and cholesterol levels. The symbiotic group that consumed 108 cfu/mL *Bifidobacterium bifidum*, 108 cfu/mL of *L. acidophilus*, and 2 g oligofructose harboured a significantly increased HDL cholesterol, and a decrease in fasting glycemia but, importantly, no significant changes were observed in the placebo group [106]. Recently, in a study by Shao et al., 67 diabetic patients with gastrointestinal cancer were randomized into the probiotic treatment group (33 patients receiving enteral nutrition with probiotics, glutamine and fish oil) and the control group (34 patients receiving regular enteral nutrition). Fasting blood glucose and insulin were recorded on the day before surgery and post-operative days 3 and 7. Insulin resistance index (HOMA-IR) was calculated as well by using the homeostasis model assessment (HOMA) for both groups, and the supplementary data on incidence of nosocomial infections, intestinal function recovery time and length of hospitalization were also recorded [107].

receiving a conventional diet. The *L. rhamnosus GG* treatment group had a serum insulin level significantly higher than the control group at 30 min after glucose loading [94]. Furthermore, feeding VSL#3 lowered β-cell destruction and inflammation in NOD mice, and this effect was accompanied by increased IL-10 secretion in pancreas, Peyer's patches and spleen. In a separate study, the feeding of a probiotic containing *Lactobacillus acidophilus NCDC14* and *L. casei NCDC19* significantly lowered free fatty acids, the blood glucose and glycosylated haemoglobin, and trigycerides in fructose-induced diabetic rats [95]. The feeding of the same probiotic to STZ-induced rats suppressed the STZ-induced oxidative damage in pancreatic tissues by inhibiting lipid peroxidation, generation of nitric oxide and improved the antioxidant potential of glutathione, superoxide dismutase, and catalase and glutathione peroxidase. These data suggest that oral administration of the probiotic significantly ameliorated the risk factors such as dyslipidemia, hyperglycemia and oxidative stress in diabetic rats [96]. Probiotic pre-treatment with a mixture containing *Bifidobacterium lactis, L. acidophilus* and *L. rhamnosus* lowered the blood glucose and improved the bioavailability of gliclazide, a second-generation sulphonylurea used for treating non-insulin dependent diabetes mellitus T2D in alloxan induced diabetic rats [97]. The antidiabetic effects against insulin resistance of different probiotics can also be due to increased liver natural killer T (NKT) cells. NKT cells are involved in regulating the inflammatory process in the liver which is the main organ responsible for inflammation-mediated insulin resistance. Depletion of liver NKT enhanced the production of pro-inflammatory cytokines, and HFD was known to induce depletion of hepatic NKT cells leading to insulin resistance. HFD-induced depletion of NKT cells in male C57BL-6 mice was significantly improved by administration of the VSL#3 probiotic. This probiotic treatment also leads to weight loss, and improved insulin resistance and inflammation by modulating TNF-α expression and reducing NF-kB binding activity [98]. Treatment with *L. plantarum* DSM 15313 and *L. reuteri* GMNL-263 was reported to lower the blood glucose and glycosylated haemoglobin, in HFD-fed C57BL/6 J mice and STZ-induced diabetic rats [99, 100]. DCs from NOD mice were stimulated with three different strains of lactobacilli including *L. casei*, *L. reuteri* and *L. plantarum* for a period of 24 h. Out of the strains tested, *L. casei* was found to induce DCs to generate the highest level of IL-10 and the lowest level of IL-12 expression. When the *L. casei*-stimulated DCs were transferred to NOD mice, they showed a significant delay in diabetes incidence [101]. *Bifidobacterium longum* CGMCC NO. 2107 added as a supplemed in HFD was shown to reduce the metabolic endotoxin (LPS) plasma concentrations and to improve intestinal inflammation [102]. Amar et al. analysed the effect probiotic treatment has on mucosal dysbiosis, bacterial translocation and glucose metabolism [103]. The results obtained revealed that the bacterial translocation was prevented in mice lacking the microbial pattern recognition receptors Nod1 or CD14. Nevertheless, it was increased in Myd88 deficient mice and ob/ob mouse under the same conditions. In addition, the administration of *Bifidobacterium animalis* subsp*. lactis 420* reduced the bacterial translocation to mesenteric adipose tissue, decreasing the expression of major pro-inflammatory cytokines TNF-a, IL-1b and IL-6 in mesenteric adipose tissue, liver and muscle. In addition, *B. animalis* subsp*. lactis 420* also improved the insulin sensitivity and fasting hyperinsulinaemia

Since there are a few reports in this area, the knowledge regarding the efficacy of probiotic administration in diabetic human subjects is quite limited. Consumption of probiotic yoghurt

in HFD fed mice [103].

82 Pathophysiology - Altered Physiological States

The enteral nutrition with probiotics, glutathione and fish oil was associated with a low fasting insulin and insulin resistance index compared to the control group. The length of hospital stay was significantly decreased from 21 to 17 days in the treatment group. Nevertheless, no significant differences in nosocomial infection and intestinal function recovery were observed between the two groups. The role of maternal probiotic-supplemented dietary counseling during pregnancy on colostrum adiponectin concentration in neonatal nutrition, metabolism and immunity was analysed in a randomized, placebo-controlled study by Luoto et al. [108]. Specifically, 256 pregnant women were randomized into three groups: dietary intervention with probiotics (diet/ *L. rhamnosus GG* and *B. lactis*), with placebo (diet/placebo) and a control cohort (control/placebo). Dietary intake was analysed by food records at each pregnancy trimester, and subsequently colostrum samples were collected after birth for the analysis of adiponectin concentration. An improved adiponectin concentration is a parameter of neonatal metabolic homeostasis and is also an indicator of reduced chances of gestational diabetes. Probiotic treatment increased the colostrum adiponectin concentration compared to the control (12.7 ng/ml vs. 10.2 ng/ml). Nevertheless, other studies state that probiotic use does not provide a benefit for the diabetic host. For instance, a randomized, double-blinded clinical trial using the commercial probiotic *L. acidophilus NCFM* in a group of 45 men for a timeframe of 4 weeks revealed that there were no changes in the expression of baseline inflammatory markers and in the systemic inflammatory response following probiotic treatment [109].

#### **10. Prebiotics: a useful tool for the management of diabetes**

Prebiotics were initially defined as "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health" [110]. Later, prebiotics were designated as selectively fermented ingredients that allow certain changes in the composition and/or activity of the gastrointestinal microbiota that confer benefits upon host well-being and health [111]. Prebiotic substances need to meet certain criteria such as: (i) fermentation by the commensal microbiota; (ii) selective stimulation of the growth and/or activity of probiotic bacteria; and (iii) resistance to gastric pH, hydrolysis by the host enzymes and gastrointestinal absorption [112]. The currently known prebiotics which achieve the aforementioned criteria include non-digestible carbohydrates, fructooligosaccharides, galactooligosaccharides and lactulose. Prebiotics, such as fructooligosaccharides and inulin, undergo digestion by probiotics such as bifidobacteria and stimulate their growth [113, 114]. Besides their involvement in stimulating the expansion of probiotics, prebiotics also stimulate immunity, inhibit pathogen growth and produce vitamins. In addition, prebiotics were suggested to promote cell differentiation, cell-cycle arrest and apoptosis of transformed colonocytes by epigenetic modifications and by decreasing the transformation of bile acids [110]. Prebiotics administration may have a regulatory role in modulating endogenous metabolism since the SCFAs obtained as an end product of the carbohydrate metabolism improve glucose tolerance. SCFAs also decrease glucagon levels and activate glucagon-like peptide1 (GLP-1), which can stimulate the elevation of insulin production and elevate insulin sensitivity [115, 116]. SCFAs were shown to have an important role in T2DM patients because they promote secretion of GLP-1, a hormone that inhibits glucagon secretion, decreases hepatic gluconeogenesis and improves insulin sensitivity [117].

was no control drink for comparison [124]. Meta-analyses of acute or short-term, randomized controlled trials revealed that chocolate or cocoa-reduced insulin and fasting insulin after glucose challenge and improved insulin resistance with no effect on fasting glucose and glycated

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Consumption of dark chocolate containing 500 mg polyphenols for a period of 4 weeks reduced blood pressure (BP), fasting glucose and insulin resistance in lean and overweight females compared to 20 g of placebo dark chocolate with negligible polyphenol content [126]. Drinking cocoa flavanols (902 mg) for 12 weeks also improved insulin sensitivity in overweight and obese individuals compared to a low-flavanol cocoa drink [127]. In contrast, daily consumption of 25 g dark chocolate for 8 weeks did not ameliorate fasting glucose, insulin and HbA1c levels in hypertensive diabetic subjects compared to those consuming 25 g of white chocolate [128]. Given the conflicting results obtained, current data are insufficient to

Cinnamon contains several polyphenols such as procyanidin, cinnamtannin trans-cinnamic acid and flavones (cinnamaldehyde and trans-cinnamaldehyde) and catechin, and several studies have shown the positive effects of cinnamon on glycaemic control [123]. Two clinical studies reported positive effects of cinnamon on fasting blood glucose levels, but no significant changes of HbA1c, LDL, HDL, total cholesterol or TG [129, 130]. Other studies reported no significant changes in fasting glucose, lipids, HbA1c, or insulin levels in 43 subjects with T2D receiving 1 g of cinnamon daily for 3 months [131], 25 postmenopausal women with T2D taking 1.5 g of cinnamon daily for 6 weeks [132], in 11 healthy subjects taking cinnamon (3 g) daily for 4 weeks [133], and in 72 adolescents with T1D taking 1 g of cinnamon daily [134]. A randomized, placebo-controlled, double-blind clinical trial of 58 subjects with T2D found that intake of 2 g daily of cinnamon for 12 weeks significantly reduced HbA1c, systolic blood and diastolic blood pressure [135]. Whole grains including wheat, soy, rye and flaxseed and nuts such as almonds, pecans and hazelnuts are an important source of polyphenols [136]. Whole grain intake is associated with a reduced risk of T2D, but the mechanism of the protection is not well understood [137]. Extra virgin olive oil and olive leafs are another source of polyphenols such as oleuropein and hydroxytyrosol, and they are suggested to have beneficial effects in T2D [138]. The Mediterranean diet supplemented with virgin olive oil or nuts harboured anti-inflammatory effects by decreasing chemokines, interleukin-6 (IL-6) and adhesion molecules, and T-lymphocytes and monocytes [139]. A study of 3541 patients with high cardiovascular risk revealed that a Mediterranean diet rich in extra virgin oil leads to a 40%

Supplementation with olive leaf polyphenols improved insulin sensitivity and pancreatic β-cells secretory capacity after oral glucose challenge in overweight, middle-aged men at the risk of developing metabolic syndrome [141, 142]. Supplementation with a 500 mg olive leaf extract tablet for 14 weeks in subjects with T2D significantly lowered HbA1c and fasting insu-

Red wine, berries, grape skins, rhubarb roots, red wine and peanuts, and the roots of rhubarb are sources of resveratrol, a polyphenol naturally synthesized by plants in response to infection and injury. Resveratrol supplementation in obese men for a period of 30 days reduced

haemoglobin (HbA1c) [125].

use cocoa polyphenols for glycaemic control.

reduction in the risk of T2D compared with the control group [140].

lin but had no effects on postprandial insulin levels [142, 143].

Prebiotics were also suggested to lead to hypercholesterolemia by lowering cholesterol absorption and by the generation of SCFAs upon selective fermentation by commensal microbiota [118]. A daily intake of 20 g of the prebiotic inulin significantly lowered serum triglycerides compared to the control group. Inulin treatment also decreased serum LDL-cholesterol and increased serum HDL-cholesterol [119]. Moreover, normolipidemic individuals consuming 18% of inulin on a daily basis without any other dietary restrictions exhibited a decrease in total plasma cholesterol and triacylglycerols as well as an increased fecal concentration of Lactobacilluslactate [120]. The inclusion of inulin in the diet of rats increased the excretions of fecal lipids and cholesterol compared to that of the control group due to a reduced cholesterol absorption [121]. Other prebiotics including resistant starches and their derivatives, oligodextrans, lactose, lactoferrin-derived peptides and N-acetylchitooligosaccharides were also shown to have hypocholesterolaemic effects in T2DM patients who are at high risk of developing cardiovascular complications [112]. A diet enriched with arabinoxylan and resistant starch consumed by adults with metabolic syndrome leads to a reduction in the total species diversity of the faecal associated intestinal microbiota and an increase in *Bifidobacterium* and butyrate levels [122].

Clinical trials reported that dietary polyphenols increase the population of *Bifidobacterium* sp. in the gut [123]. Daily consumption of red wine polyphenols for a period of 4 weeks significantly increased the levels of *Bifidobacterium, Prevotella, Bacteroides, Bacteroides uniformis, Eggerthella lenta, Enterococcus,* and *Blautia coccoides-E. rectale* groups compared with baseline, but there was no control drink for comparison [124]. Meta-analyses of acute or short-term, randomized controlled trials revealed that chocolate or cocoa-reduced insulin and fasting insulin after glucose challenge and improved insulin resistance with no effect on fasting glucose and glycated haemoglobin (HbA1c) [125].

**10. Prebiotics: a useful tool for the management of diabetes**

84 Pathophysiology - Altered Physiological States

Prebiotics were initially defined as "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health" [110]. Later, prebiotics were designated as selectively fermented ingredients that allow certain changes in the composition and/or activity of the gastrointestinal microbiota that confer benefits upon host well-being and health [111]. Prebiotic substances need to meet certain criteria such as: (i) fermentation by the commensal microbiota; (ii) selective stimulation of the growth and/or activity of probiotic bacteria; and (iii) resistance to gastric pH, hydrolysis by the host enzymes and gastrointestinal absorption [112]. The currently known prebiotics which achieve the aforementioned criteria include non-digestible carbohydrates, fructooligosaccharides, galactooligosaccharides and lactulose. Prebiotics, such as fructooligosaccharides and inulin, undergo digestion by probiotics such as bifidobacteria and stimulate their growth [113, 114]. Besides their involvement in stimulating the expansion of probiotics, prebiotics also stimulate immunity, inhibit pathogen growth and produce vitamins. In addition, prebiotics were suggested to promote cell differentiation, cell-cycle arrest and apoptosis of transformed colonocytes by epigenetic modifications and by decreasing the transformation of bile acids [110]. Prebiotics administration may have a regulatory role in modulating endogenous metabolism since the SCFAs obtained as an end product of the carbohydrate metabolism improve glucose tolerance. SCFAs also decrease glucagon levels and activate glucagon-like peptide1 (GLP-1), which can stimulate the elevation of insulin production and elevate insulin sensitivity [115, 116]. SCFAs were shown to have an important role in T2DM patients because they promote secretion of GLP-1, a hormone that inhibits glucagon secretion, decreases hepatic gluconeogenesis and improves insulin sensitivity [117].

Prebiotics were also suggested to lead to hypercholesterolemia by lowering cholesterol absorption and by the generation of SCFAs upon selective fermentation by commensal microbiota [118]. A daily intake of 20 g of the prebiotic inulin significantly lowered serum triglycerides compared to the control group. Inulin treatment also decreased serum LDL-cholesterol and increased serum HDL-cholesterol [119]. Moreover, normolipidemic individuals consuming 18% of inulin on a daily basis without any other dietary restrictions exhibited a decrease in total plasma cholesterol and triacylglycerols as well as an increased fecal concentration of Lactobacilluslactate [120]. The inclusion of inulin in the diet of rats increased the excretions of fecal lipids and cholesterol compared to that of the control group due to a reduced cholesterol absorption [121]. Other prebiotics including resistant starches and their derivatives, oligodextrans, lactose, lactoferrin-derived peptides and N-acetylchitooligosaccharides were also shown to have hypocholesterolaemic effects in T2DM patients who are at high risk of developing cardiovascular complications [112]. A diet enriched with arabinoxylan and resistant starch consumed by adults with metabolic syndrome leads to a reduction in the total species diversity of the faecal associ-

ated intestinal microbiota and an increase in *Bifidobacterium* and butyrate levels [122].

Clinical trials reported that dietary polyphenols increase the population of *Bifidobacterium* sp. in the gut [123]. Daily consumption of red wine polyphenols for a period of 4 weeks significantly increased the levels of *Bifidobacterium, Prevotella, Bacteroides, Bacteroides uniformis, Eggerthella lenta, Enterococcus,* and *Blautia coccoides-E. rectale* groups compared with baseline, but there Consumption of dark chocolate containing 500 mg polyphenols for a period of 4 weeks reduced blood pressure (BP), fasting glucose and insulin resistance in lean and overweight females compared to 20 g of placebo dark chocolate with negligible polyphenol content [126]. Drinking cocoa flavanols (902 mg) for 12 weeks also improved insulin sensitivity in overweight and obese individuals compared to a low-flavanol cocoa drink [127]. In contrast, daily consumption of 25 g dark chocolate for 8 weeks did not ameliorate fasting glucose, insulin and HbA1c levels in hypertensive diabetic subjects compared to those consuming 25 g of white chocolate [128]. Given the conflicting results obtained, current data are insufficient to use cocoa polyphenols for glycaemic control.

Cinnamon contains several polyphenols such as procyanidin, cinnamtannin trans-cinnamic acid and flavones (cinnamaldehyde and trans-cinnamaldehyde) and catechin, and several studies have shown the positive effects of cinnamon on glycaemic control [123]. Two clinical studies reported positive effects of cinnamon on fasting blood glucose levels, but no significant changes of HbA1c, LDL, HDL, total cholesterol or TG [129, 130]. Other studies reported no significant changes in fasting glucose, lipids, HbA1c, or insulin levels in 43 subjects with T2D receiving 1 g of cinnamon daily for 3 months [131], 25 postmenopausal women with T2D taking 1.5 g of cinnamon daily for 6 weeks [132], in 11 healthy subjects taking cinnamon (3 g) daily for 4 weeks [133], and in 72 adolescents with T1D taking 1 g of cinnamon daily [134]. A randomized, placebo-controlled, double-blind clinical trial of 58 subjects with T2D found that intake of 2 g daily of cinnamon for 12 weeks significantly reduced HbA1c, systolic blood and diastolic blood pressure [135]. Whole grains including wheat, soy, rye and flaxseed and nuts such as almonds, pecans and hazelnuts are an important source of polyphenols [136]. Whole grain intake is associated with a reduced risk of T2D, but the mechanism of the protection is not well understood [137]. Extra virgin olive oil and olive leafs are another source of polyphenols such as oleuropein and hydroxytyrosol, and they are suggested to have beneficial effects in T2D [138]. The Mediterranean diet supplemented with virgin olive oil or nuts harboured anti-inflammatory effects by decreasing chemokines, interleukin-6 (IL-6) and adhesion molecules, and T-lymphocytes and monocytes [139]. A study of 3541 patients with high cardiovascular risk revealed that a Mediterranean diet rich in extra virgin oil leads to a 40% reduction in the risk of T2D compared with the control group [140].

Supplementation with olive leaf polyphenols improved insulin sensitivity and pancreatic β-cells secretory capacity after oral glucose challenge in overweight, middle-aged men at the risk of developing metabolic syndrome [141, 142]. Supplementation with a 500 mg olive leaf extract tablet for 14 weeks in subjects with T2D significantly lowered HbA1c and fasting insulin but had no effects on postprandial insulin levels [142, 143].

Red wine, berries, grape skins, rhubarb roots, red wine and peanuts, and the roots of rhubarb are sources of resveratrol, a polyphenol naturally synthesized by plants in response to infection and injury. Resveratrol supplementation in obese men for a period of 30 days reduced glucose, insulin, insulin resistance index and leptin, and decreased inflammatory markers (TNF-α, leukocytes). Even though resveratrol supplementation also decreased adipose tissue lipolysis and plasma fatty acid and glycerol in the postprandial state [144], the study lacked some of the necessary controls therefore more investigations are needed in order to state that resveratrol has antidiabetic effects.

[3] Grigore Mihaescu CC. Immunology and Immunopathology. Editura Medicala; Bucharest,

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[4] Pettitt DJ, Talton J, Dabelea D, Divers J, Imperatore G, Lawrence JM, Liese AD, Linder B, Mayer-Davis EJ, Pihoker C, et al. Prevalence of diabetes in U.S. Youth in 2009: The search

[5] Kantarova D, Buc M. Genetic susceptibility to type 1 diabetes mellitus in humans.

[6] Atkinson MA, Eisenbarth GS. Type 1 diabetes: New perspectives on disease pathogen-

[7] Kasper D et al. Harrison's Principles of Internal Medicine. McGraw-Hill; NewYork,

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