**Role of Incretin, Incretin Analogues and Dipeptidyl Peptidase 4 Inhibitors in the Pathogenesis and Treatment of Diabetes Mellitus**

Athanasia K. Papazafiropoulou, Marina S. Kardara and Stavros I. Pappas *3rdDepartment of Internal Medicine and Center of Diabetes, General Hospital of Nikaia "Ag. Panteleimon" – Piraeus, Greece* 

### **1. Introduction**

Type 2 diabetes mellitus (T2DM) is increasing in prevalence worldwide, and is expected to affect 440 million people by 2030 (IDF, 2009). Despite the development and use of several medications to control patients' blood glucose levels, the effective management of T2DM continues to be a challenge to physicians. In order to achieve HbA1c targets (<7.0%), patients must reach desirable fasting (90 mg/dL - 130 mg/dL) and postprandial glucose levels (<180 mg/dL) (American Diabetes Association, 2006). However, two thirds of patients with T2DM remain unable to reach the HbA1c targets (Koro, 1988; Fan, 2006).

Blood glucose levels are dependent on the dynamic processes of hepatic production of glucose and skeletal muscle use of glucose. Treatment strategies designed to improve these processes have as a result the improvement in patient's glycemic status. Different agents are currently available, providing physicians with several options for the management of T2DM. These clinical therapies include insulin and oral drugs that are classified as insulin sensitizers (e.g., biguanides and thiazolidinediones), insulin secretagogues (e.g., sulfonylureas and meglitinides), and alpha-glucosidase inhibitors. Newer treatment agents, incretin mimetics and dipeptidyl peptidase 4 (DPP-4) inhibitors, have been recently added to clinicians' therapeutic choices (Drucker, 2003; Drucker, 2006a).

### **2. The incretin effect**

The concept that gut factors stimulate pancreatic endocrine secretion was hypothesized soon after secretin was discovered in 1902 (Kieffer, 1999). In 1906, this notion was tested by giving gut extracts to patients with diabetes, which reduced their glycosuria (Moore, 1906). In the 1920s, based on studies in dogs, the term incretins was introduced for the gastrointestinal hormones released in response to food ingestion (Zunz, 1929). These hormones are responsible for approximately 60% of the insulin secretion following a meal and for the socalled incretin effect. The incretin effect describes the phenomenon that oral glucose leads to

Role of Incretin, Incretin Analogues and Dipeptidyl

**5. T2DM and incretin-based therapies** 

T2DM, two options are presently available:

**6. GLP-1-receptor agonists** 

2. DPP-4 inhibitors as orally active substances

(Zander, 2002).

**6.1 Exenatide** 

(Kolterman, 2005).

Barnett, 2007).

Peptidase 4 Inhibitors in the Pathogenesis and Treatment of Diabetes Mellitus 291

Several studies in T2DM patients have shown that synthetic GLP-1 administration induces insulin secretion, (Nathan, 1992; Nauck, 1993a) slows gastric emptying, and decreases inappropriately elevated glucagons secretion (Nauck, 1993a; Kolterman, 2003). Acute GLP-1 infusion studies showed that GLP-1 improved fasting and post prandial plasma glucose concentrations (Nathan, 1992; Nauck, 1993b). Long-term studies showed that this hormone exerts euglycemic effects, leading to improvements in HbA1c, and induces weight loss

The incretin-based therapies offer a good alternative choice to the established antidiabetic compounds due to their satisfying antihyperglycaemic efficacy, their lack of risk of hypoglycaemia and their positive effects on body weight. In order to utilise GLP-1 action for

Exenatide is the synthetic form of exendin-4, a peptide first discovered in the saliva of the gila monster (heloderma suspectum) in 1992. It has a 53% amino acid sequence homology to human GLP-1 and is a GLP-1 receptor agonist (Eng, 1992). It is administered subcutaneously twice daily. A slow release formulation for once-weekly administration (Exenatide LAR [long-acting release]) is presently in clinical phase III studies (Drucker, 2008). Exenatide has a prolonged half-life in comparison to native GLP-1 of approximately 3.5 h. After subcutaneous injection sufficient plasma concentrations are reached for 4–6 hours

In clinical studies exenatide lowered the HbA1c by 0.8–1.1% (Buse, 2004; DeFronzo, 2005). Exenatide in combination with metformin (Kendall, 2005), sulfonylurea (DeFronzo, 2005), or both (Buse, 2004) resulted in significant mean HbA1c reductions from baseline ranging from –0.77% to –0.86%. Patients also had statistically significant reductions in mean body weight from baseline (–1.6 kg to -2.8 kg). Comparative studies with insulin showed that effects of exenatide on glycaemic parameters are comparable to the improvement seen with insulin therapy (Heine 2005; Gallwitz, 2006; Barnett, 2007; Nauck, 2007). The comparative studies with insulin showed a difference in weight development of 4–5 kg in 30 weeks between the

An improvement of β-cell function [measured with HOMA-β (homeostatic modelling assessment of beta cell function) and the proinsulin: insulin ratio] was also observed in the clinical studies. First phase of insulin secretion was restored after an intravenous glucose

Severe hypoglycaemic events were only observed in exenatide-treated patients who had received combination therapy with sulfonylurea. For this reason a reduction in the dosage of sulfonylurea should be considered when initiating exenatide therapy. In the comparative studies comparing exenatide with insulin treatment, the incidence of nocturnal hypoglycaemic events was lower in the exenatide-treated patients (Gallwitz, 2006;

insulin and exenatide treated groups (Heine 2005; Barnett, 2007a; Nauck, 2007a).

bolus under treatment with exenatide (Gallwitz, 2006; Barnett, 2007b).

1. GLP-1-receptor agonists (or GLP-1 mimetics) as injectable compounds

a greater insulin response than an isoglycaemic intravenous glucose load (McIntyre, 1964; Nauck, 1986).

There are two major incretins: glucosedependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1). In this chapter we will focus on GLP-1 actions, since this molecule is preserved in patients with T2DM.

### **3. Physiological actions of GLP-1**

GLP-1 is a product of the glucagons gene, which is expressed in pancreatic α-cells and in Lcells, located mostly in the lower small intestine and colon. GLP-1 concentrations increase as early as 5 to 10 minutes following ingestion of carbohydrates and lipids, well before the nutrients pass into the lower gut where most L-cells are located (Eissele, 1992; Deacon, 1995). Once released from L- cells, GLP-1 is rapidly metabolized by a widely distributed serine protease, DPP-4, resulting in a half-life of 1 to 2 minutes in the circulation. DPP-4, which is located on endothelial cells as well as in soluble form in plasma, cleaves the two Nterminal amino acids from GLP-1, causing a substantial loss of insulinotropic activity (Deacon, 1995; Vahl, 2003).

GLP-1 stimulates insulin secretion of the β-cells and inhibits glucagon secretion from the αcells. Both actions occur in a glucose-dependent manner and lead to a normalisation of postprandial and fasting hyperglycaemia (Drucker, 2006b). In the gastrointestinal tract, GLP-1 has a direct effect on motility and slows gastric emptying. This effect contributes to a normalisation of postprandial hyperglycaemia and explains why long-term treatment with GLP-1 receptor agonists leads to weight loss (Drucker, 2006b). Under hypoglycaemic conditions the counter-regulation by glucagon is not affected and insulin secretion is not stimulated and, therefore, GLP-1 does not elicit hypoglycaemia (Drucker, 2006b).

Except for its antidiabetic actions, recent findings have shown that application of GLP-1 receptor agonists led to an improvement in cardiovascular parameters (reduction of systolic blood pressure, beneficial effects on myocardial ischaemia in animal models, positive effects on left ventricular function in heart failure) (Papazafiropoulou, 2011). In addition, animal studies in rodents and isolated human islets showed beneficial long-term actions of GLP-1 to β-cell mass (Fehmann, 1992; Brubaker, 2004). Whether these findings will have a positive effect on preventing T2DM progression is not known yet.

### **4. Incretins and the pathogenesis of T2DM**

In T2DM patients the incretin effect is diminished. Incretins does not act as an insulinotropic hormones under chronic hyperglycaemia in T2DM. However, GLP-1 is still able to stimulate insulin secretion under hyperglycaemia in T2DM (Drucker, 2006). In addition, the effects of GLP-1 on gastric emptying and glucagon secretion are maintained in patients with T2DM (Nauck, 1993a).

A study confirmed that the incretin effect is reduced in patients with T2DM (Knop, 2007). Another study showed a significant reduction in the incretin effect and the GLP-1 response to oral glucose in T2DM patients compared with individuals with normal or impaired glucose tolerance (Muscelli, 2008). Notably, impaired actions of GLP-1 may be partially restored by improved glycemic control (Knop, 2007). The findings from a study of obese diabetic mice suggest that the effect of GLP-1 therapy may be caused by improvements in βcell function and insulin sensitivity, as well as by a reduction in gluconeogenesis in the liver (Lee, 2007).

Several studies in T2DM patients have shown that synthetic GLP-1 administration induces insulin secretion, (Nathan, 1992; Nauck, 1993a) slows gastric emptying, and decreases inappropriately elevated glucagons secretion (Nauck, 1993a; Kolterman, 2003). Acute GLP-1 infusion studies showed that GLP-1 improved fasting and post prandial plasma glucose concentrations (Nathan, 1992; Nauck, 1993b). Long-term studies showed that this hormone exerts euglycemic effects, leading to improvements in HbA1c, and induces weight loss (Zander, 2002).

## **5. T2DM and incretin-based therapies**

The incretin-based therapies offer a good alternative choice to the established antidiabetic compounds due to their satisfying antihyperglycaemic efficacy, their lack of risk of hypoglycaemia and their positive effects on body weight. In order to utilise GLP-1 action for T2DM, two options are presently available:


## **6. GLP-1-receptor agonists**

### **6.1 Exenatide**

290 Diabetes – Damages and Treatments

a greater insulin response than an isoglycaemic intravenous glucose load (McIntyre, 1964;

There are two major incretins: glucosedependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1). In this chapter we will focus on GLP-1 actions, since this

GLP-1 is a product of the glucagons gene, which is expressed in pancreatic α-cells and in Lcells, located mostly in the lower small intestine and colon. GLP-1 concentrations increase as early as 5 to 10 minutes following ingestion of carbohydrates and lipids, well before the nutrients pass into the lower gut where most L-cells are located (Eissele, 1992; Deacon, 1995). Once released from L- cells, GLP-1 is rapidly metabolized by a widely distributed serine protease, DPP-4, resulting in a half-life of 1 to 2 minutes in the circulation. DPP-4, which is located on endothelial cells as well as in soluble form in plasma, cleaves the two Nterminal amino acids from GLP-1, causing a substantial loss of insulinotropic activity

GLP-1 stimulates insulin secretion of the β-cells and inhibits glucagon secretion from the αcells. Both actions occur in a glucose-dependent manner and lead to a normalisation of postprandial and fasting hyperglycaemia (Drucker, 2006b). In the gastrointestinal tract, GLP-1 has a direct effect on motility and slows gastric emptying. This effect contributes to a normalisation of postprandial hyperglycaemia and explains why long-term treatment with GLP-1 receptor agonists leads to weight loss (Drucker, 2006b). Under hypoglycaemic conditions the counter-regulation by glucagon is not affected and insulin secretion is not

Except for its antidiabetic actions, recent findings have shown that application of GLP-1 receptor agonists led to an improvement in cardiovascular parameters (reduction of systolic blood pressure, beneficial effects on myocardial ischaemia in animal models, positive effects on left ventricular function in heart failure) (Papazafiropoulou, 2011). In addition, animal studies in rodents and isolated human islets showed beneficial long-term actions of GLP-1 to β-cell mass (Fehmann, 1992; Brubaker, 2004). Whether these findings will have a positive

In T2DM patients the incretin effect is diminished. Incretins does not act as an insulinotropic hormones under chronic hyperglycaemia in T2DM. However, GLP-1 is still able to stimulate insulin secretion under hyperglycaemia in T2DM (Drucker, 2006). In addition, the effects of GLP-1 on gastric emptying and glucagon secretion are maintained in patients with T2DM

A study confirmed that the incretin effect is reduced in patients with T2DM (Knop, 2007). Another study showed a significant reduction in the incretin effect and the GLP-1 response to oral glucose in T2DM patients compared with individuals with normal or impaired glucose tolerance (Muscelli, 2008). Notably, impaired actions of GLP-1 may be partially restored by improved glycemic control (Knop, 2007). The findings from a study of obese diabetic mice suggest that the effect of GLP-1 therapy may be caused by improvements in βcell function and insulin sensitivity, as well as by a reduction in gluconeogenesis in the liver

stimulated and, therefore, GLP-1 does not elicit hypoglycaemia (Drucker, 2006b).

effect on preventing T2DM progression is not known yet.

**4. Incretins and the pathogenesis of T2DM** 

Nauck, 1986).

molecule is preserved in patients with T2DM.

**3. Physiological actions of GLP-1** 

(Deacon, 1995; Vahl, 2003).

(Nauck, 1993a).

(Lee, 2007).

Exenatide is the synthetic form of exendin-4, a peptide first discovered in the saliva of the gila monster (heloderma suspectum) in 1992. It has a 53% amino acid sequence homology to human GLP-1 and is a GLP-1 receptor agonist (Eng, 1992). It is administered subcutaneously twice daily. A slow release formulation for once-weekly administration (Exenatide LAR [long-acting release]) is presently in clinical phase III studies (Drucker, 2008). Exenatide has a prolonged half-life in comparison to native GLP-1 of approximately 3.5 h. After subcutaneous injection sufficient plasma concentrations are reached for 4–6 hours (Kolterman, 2005).

In clinical studies exenatide lowered the HbA1c by 0.8–1.1% (Buse, 2004; DeFronzo, 2005). Exenatide in combination with metformin (Kendall, 2005), sulfonylurea (DeFronzo, 2005), or both (Buse, 2004) resulted in significant mean HbA1c reductions from baseline ranging from –0.77% to –0.86%. Patients also had statistically significant reductions in mean body weight from baseline (–1.6 kg to -2.8 kg). Comparative studies with insulin showed that effects of exenatide on glycaemic parameters are comparable to the improvement seen with insulin therapy (Heine 2005; Gallwitz, 2006; Barnett, 2007; Nauck, 2007). The comparative studies with insulin showed a difference in weight development of 4–5 kg in 30 weeks between the insulin and exenatide treated groups (Heine 2005; Barnett, 2007a; Nauck, 2007a).

An improvement of β-cell function [measured with HOMA-β (homeostatic modelling assessment of beta cell function) and the proinsulin: insulin ratio] was also observed in the clinical studies. First phase of insulin secretion was restored after an intravenous glucose bolus under treatment with exenatide (Gallwitz, 2006; Barnett, 2007b).

Severe hypoglycaemic events were only observed in exenatide-treated patients who had received combination therapy with sulfonylurea. For this reason a reduction in the dosage of sulfonylurea should be considered when initiating exenatide therapy. In the comparative studies comparing exenatide with insulin treatment, the incidence of nocturnal hypoglycaemic events was lower in the exenatide-treated patients (Gallwitz, 2006; Barnett, 2007).

Role of Incretin, Incretin Analogues and Dipeptidyl

**7. DPP-4 Inhibitors** 

renal disease (Bergman, 2007).

**7.2 Vildagliptin** 

(Ristic, 2005).

**7.1 Sitagliptin** 

Peptidase 4 Inhibitors in the Pathogenesis and Treatment of Diabetes Mellitus 293

with a personal or family history of medullary thyroid cancer and in patients with multiple

Data on the pharmacokinetic profile of liraglutide in mild to moderate renal impairment

Sitagliptin was the first DPP-4 inhibitor approved for the T2DM treatment. The recommended dose of once-daily oral sitagliptin is 100 mg. At this dose, sitagliptin can inhibit ~80% of endogenous DPP-4 activity over a 24-hour period (Herman, 2005). Increases

In the monotherapy trials, sitagliptin compared to placebo, resulted in statistically significant improvements in HbA1c and fasting glucose (Aschner, 2006; Raz, 2006; Scott, 2007). Sitagliptin given as add-on therapy to metformin (Charbonnel, 2006) resulted in similar HbA1c and fasting glucose reductions as in the monotherapy trials. Τhe same result was observed, in a 24 week trial, when sitagliptin was added to pioglitazone vs pioglitazone and placebo (Rosenstock, 2006). In another study, reductions from baseline in HbA1c and fasting glucose were similar when sitagliptin was compared to glipizide, (Nauck, 2007). Increases in HOMA-β

Sitagliptin therapy has been shown to be weight neutral in all clinical trials except in one study in which sitagliptin given with metformin resulted in weight reduction of 1.5 kg after 52 weeks of treatment (Nauck, 2007b). The most common side effects of sitagliptin were headache, arthritis, nasopharyngitis, respiratory or urinary tract infections and rarely skin reactions (Aschner, 2006; Raz, 2006; Rosenstock, 2006). The incidence of hypoglycemia was low in these trials (<2%) and was similar to the placebo arms. Dose reduction of sitagliptin has been recommended for patients with moderate or severe renal insufficiency or end stage

Vildagliptin also acts by inhibiting circulating DPP-4 activity. It is available as a 50 mg twice-daily in combination with metformin, sulfonylurea or pioglitazone. Vildagliptin has been studied as monotherapy (Ristic, 2005; Pratley, 2006; Dejager, 2007), in combination with other oral antidiabetic agents (Ahren, 2004; Fonseca, 2007; Rosenstock, 2007), and against active comparator therapies including glitazones (Rosenstock, 2007) and metformin (Schweizer, 2007) Vildagliptin therapy was associated with an increase in HOMA-β (11%

In placebo-controlled trials, vildagliptin monotherapy reduced HbA1c (range 0.5% to 0.9%) and fasting glucose (14.4 mg/dL to 19.8 mg/dL) from baseline. The HbA1c reductions observed with monotherapy were statistically significantly greater than placebo in all trials. In clinical studies testing vildaglitpin in monotherapy or combination therapy with metformin, glimepiride, pioglitazone or insulin, vildagliptin was able to decrease the HbA1c by approximately 0.5–1.0% (Ahren, 2008; Pratley, 2008; Barnett, 2009). Vildagliptin therapy was associated with an increase HOMA-β (11% and 23%) in two monotherapy trials (Ristic, 2005; Pratley, 2006), but improvement relative to placebo was only observed in one trial

endocrine neoplasia syndrome type 2 (US Food and Drug Administration, 2010).

in HOMA-β ranging from 4% to 20% have been shown in the sitagliptin trials.

showed no alteration of the profile (Deacon, 2009a; Vilsboll, 2009).

ranging from 4% to 20% have been shown in the sitagliptin trials.

and 23%) in two monotherapy trials (Ristic, 2005; Pratley, 2006).

The most frequent adverse events with exenatide were fullness and nausea. Nausea was the most common reason to stop therapy; with 2–6.4% drop-outs in the clinical studies with exenatide (Gallwitz, 2006; Barnett, 2007). Escalating the dose of exenatide from 5 μg to 10 μg after 4 weeks led to a transient increase in nausea which diminished with continued exposure to the higher dose (Gallwitz, 2006; Barnett, 2007).

In approximately 40% of exenatide-treated patients, anti-exenatide antibodies can be detected. However, over a time period of at least 3 years, these antibody titres did not have any obvious effect on glycaemic control (Drucker, 2008). Cases of acute pancreatitis have been reported since exenatide has been used (Ahmad, 2008; Cure, 2008). In total, the incidence of pancreatitis was low and similar to the elevated risk of pancreatitis that was observed in obese T2DM patients (Dore, 2009).

Exenatide is predominantly eliminated by glomerular filtration followed by proteolytic degradation (Yoo, 2006). Exenatide should not be used in patients with severe renal impairment (creatinine clearance <30 ml/min) or end stage renal disease. Additionally, caution should be applied when initiating or increasing doses of exenatide in patients with moderate renal impairment (creatinine clearance 30–50 ml/min) (Gallwitz, 2006; Barnett, 2007).

### **6.2 Liraglutide**

Liraglutide is the first human GLP-1 analogue. It has two modifications in the amino acid sequence of native GLP-1 and an attachment of a fatty acid side chain to the peptide. It is injected subcutaneously once daily (Agerso, 2002). Liraglutide lowers blood glucose, body weight and food intake in animal models (Sturis, 2003). In clinical studies in approximately 4,200 T2DM patients liraglutide was efficacious and safe (Marre, 2009; Nauck, 2009; Zinman, 2009). In animal studies with diabetic rodents, liraglutide has been shown to increase β-cell mass.

Liraglutide in monotherapy in newly diagnosed T2DM patients led to HbA1c reduction of 0.9–1.1% in a dose of 1.2 or 1.8 mg once daily respectively, over a period of up to 2 years (Garber, 2008). In other studies, the same doses of liraglutide effectively lowered glycaemic parameters in various combinations with oral antidiabetic agents by approximately 1.0–1.5% (Garber, 2008; Garber, 2009).

Liraglutide treatment led to a significant weight loss (Deacon, 2009a; Vilsboll, 2009). The weight loss was accompanied by a more pronounced loss in visceral fat than subcutaneous fat (Deacon, 2009a; Vilsboll, 2009). Furthermore, systolic blood pressure was lowered by 2–6 mmHg in the liraglutide-treated patients. This effect was independent of the weight loss, as the reduction of blood pressure was already observed early on in therapy, when weight loss had not yet occurred (Garber, 2008; Garber, 2009; Zinman, 2009).

The incidence of hypoglycaemic episodes was comparable to placebo in all studies, where no sulfonylurea was used in the combination with liraglutide (Deacon, 2009a; Vilsboll, 2009). Gastrointestinal symptoms were also common, but nausea and vomiting were reported for a short period at the beginning of therapy (Buse, 2009). In the liraglutide clinical trials, there was no evidence of neutralizing antibodies (Garber, 2008; Garber, 2009; Zinman, 2009).

Animal studies showed that a rare type of thyroid cancer known as medullary thyroid cancer was associated with liraglutide in mice and rats, although the relevance of this finding to humans remains unknown. FDA has stipulated that liraglutide be contraindicated in patients with a personal or family history of medullary thyroid cancer and in patients with multiple endocrine neoplasia syndrome type 2 (US Food and Drug Administration, 2010).

Data on the pharmacokinetic profile of liraglutide in mild to moderate renal impairment showed no alteration of the profile (Deacon, 2009a; Vilsboll, 2009).

## **7. DPP-4 Inhibitors**

### **7.1 Sitagliptin**

292 Diabetes – Damages and Treatments

The most frequent adverse events with exenatide were fullness and nausea. Nausea was the most common reason to stop therapy; with 2–6.4% drop-outs in the clinical studies with exenatide (Gallwitz, 2006; Barnett, 2007). Escalating the dose of exenatide from 5 μg to 10 μg after 4 weeks led to a transient increase in nausea which diminished with continued

In approximately 40% of exenatide-treated patients, anti-exenatide antibodies can be detected. However, over a time period of at least 3 years, these antibody titres did not have any obvious effect on glycaemic control (Drucker, 2008). Cases of acute pancreatitis have been reported since exenatide has been used (Ahmad, 2008; Cure, 2008). In total, the incidence of pancreatitis was low and similar to the elevated risk of pancreatitis that was

Exenatide is predominantly eliminated by glomerular filtration followed by proteolytic degradation (Yoo, 2006). Exenatide should not be used in patients with severe renal impairment (creatinine clearance <30 ml/min) or end stage renal disease. Additionally, caution should be applied when initiating or increasing doses of exenatide in patients with moderate renal impairment (creatinine clearance 30–50 ml/min) (Gallwitz, 2006; Barnett,

Liraglutide is the first human GLP-1 analogue. It has two modifications in the amino acid sequence of native GLP-1 and an attachment of a fatty acid side chain to the peptide. It is injected subcutaneously once daily (Agerso, 2002). Liraglutide lowers blood glucose, body weight and food intake in animal models (Sturis, 2003). In clinical studies in approximately 4,200 T2DM patients liraglutide was efficacious and safe (Marre, 2009; Nauck, 2009; Zinman, 2009). In animal studies with diabetic rodents, liraglutide has been shown to increase

Liraglutide in monotherapy in newly diagnosed T2DM patients led to HbA1c reduction of 0.9–1.1% in a dose of 1.2 or 1.8 mg once daily respectively, over a period of up to 2 years (Garber, 2008). In other studies, the same doses of liraglutide effectively lowered glycaemic parameters in various combinations with oral antidiabetic agents by approximately 1.0–1.5%

Liraglutide treatment led to a significant weight loss (Deacon, 2009a; Vilsboll, 2009). The weight loss was accompanied by a more pronounced loss in visceral fat than subcutaneous fat (Deacon, 2009a; Vilsboll, 2009). Furthermore, systolic blood pressure was lowered by 2–6 mmHg in the liraglutide-treated patients. This effect was independent of the weight loss, as the reduction of blood pressure was already observed early on in therapy, when weight loss

The incidence of hypoglycaemic episodes was comparable to placebo in all studies, where no sulfonylurea was used in the combination with liraglutide (Deacon, 2009a; Vilsboll, 2009). Gastrointestinal symptoms were also common, but nausea and vomiting were reported for a short period at the beginning of therapy (Buse, 2009). In the liraglutide clinical trials, there was no evidence of neutralizing antibodies (Garber, 2008; Garber, 2009;

Animal studies showed that a rare type of thyroid cancer known as medullary thyroid cancer was associated with liraglutide in mice and rats, although the relevance of this finding to humans remains unknown. FDA has stipulated that liraglutide be contraindicated in patients

had not yet occurred (Garber, 2008; Garber, 2009; Zinman, 2009).

exposure to the higher dose (Gallwitz, 2006; Barnett, 2007).

observed in obese T2DM patients (Dore, 2009).

2007).

**6.2 Liraglutide** 

β-cell mass.

Zinman, 2009).

(Garber, 2008; Garber, 2009).

Sitagliptin was the first DPP-4 inhibitor approved for the T2DM treatment. The recommended dose of once-daily oral sitagliptin is 100 mg. At this dose, sitagliptin can inhibit ~80% of endogenous DPP-4 activity over a 24-hour period (Herman, 2005). Increases in HOMA-β ranging from 4% to 20% have been shown in the sitagliptin trials.

In the monotherapy trials, sitagliptin compared to placebo, resulted in statistically significant improvements in HbA1c and fasting glucose (Aschner, 2006; Raz, 2006; Scott, 2007). Sitagliptin given as add-on therapy to metformin (Charbonnel, 2006) resulted in similar HbA1c and fasting glucose reductions as in the monotherapy trials. Τhe same result was observed, in a 24 week trial, when sitagliptin was added to pioglitazone vs pioglitazone and placebo (Rosenstock, 2006). In another study, reductions from baseline in HbA1c and fasting glucose were similar when sitagliptin was compared to glipizide, (Nauck, 2007). Increases in HOMA-β ranging from 4% to 20% have been shown in the sitagliptin trials.

Sitagliptin therapy has been shown to be weight neutral in all clinical trials except in one study in which sitagliptin given with metformin resulted in weight reduction of 1.5 kg after 52 weeks of treatment (Nauck, 2007b). The most common side effects of sitagliptin were headache, arthritis, nasopharyngitis, respiratory or urinary tract infections and rarely skin reactions (Aschner, 2006; Raz, 2006; Rosenstock, 2006). The incidence of hypoglycemia was low in these trials (<2%) and was similar to the placebo arms. Dose reduction of sitagliptin has been recommended for patients with moderate or severe renal insufficiency or end stage renal disease (Bergman, 2007).

### **7.2 Vildagliptin**

Vildagliptin also acts by inhibiting circulating DPP-4 activity. It is available as a 50 mg twice-daily in combination with metformin, sulfonylurea or pioglitazone. Vildagliptin has been studied as monotherapy (Ristic, 2005; Pratley, 2006; Dejager, 2007), in combination with other oral antidiabetic agents (Ahren, 2004; Fonseca, 2007; Rosenstock, 2007), and against active comparator therapies including glitazones (Rosenstock, 2007) and metformin (Schweizer, 2007) Vildagliptin therapy was associated with an increase in HOMA-β (11% and 23%) in two monotherapy trials (Ristic, 2005; Pratley, 2006).

In placebo-controlled trials, vildagliptin monotherapy reduced HbA1c (range 0.5% to 0.9%) and fasting glucose (14.4 mg/dL to 19.8 mg/dL) from baseline. The HbA1c reductions observed with monotherapy were statistically significantly greater than placebo in all trials. In clinical studies testing vildaglitpin in monotherapy or combination therapy with metformin, glimepiride, pioglitazone or insulin, vildagliptin was able to decrease the HbA1c by approximately 0.5–1.0% (Ahren, 2008; Pratley, 2008; Barnett, 2009). Vildagliptin therapy was associated with an increase HOMA-β (11% and 23%) in two monotherapy trials (Ristic, 2005; Pratley, 2006), but improvement relative to placebo was only observed in one trial (Ristic, 2005).

Role of Incretin, Incretin Analogues and Dipeptidyl

*Clin Ther*, 29, 2333–2348

*Endocrinology,* 145, 2653–2659

*Diabetes Care*, 29, 2638-2643

*Vasc Health Risk Manag,* 5, 199–211

treatment of type 2 diabetes. *Adv Ther*, 26, 488–499

374, 39–47

1969–1970

*Diabetes Care,* 29(Suppl 1), S43-S48

b. Barnett A (2007) Exenatide. *Expert Opin Pharmacother,* 8, 2593–2608

therapies. *Clin Endocrinol (Oxf),* 70, 343–353

type 2 diabetes. *Diabetes Care*, 27, 2628–2635

Peptidase 4 Inhibitors in the Pathogenesis and Treatment of Diabetes Mellitus 295

American Diabetes Association (2006). Diagnosis and classification of diabetes mellitus.

Aschner P, Kipnes MS, Lunceford JK, Sanchez M, Mickel C, Williams-Herman DE (2006).

Barnett AH (2009). New treatments in type 2 diabetes: a focus on the incretin-based

a. Barnett AH, Burger J, Johns D, Brodows R, Kendall DM, Roberts A, Trautmann ME (2007).

Bergman AJ, Cote J, Yi B, Marbury T, Swan SK, Smith W, Gottesdiener K, Wagner J, Herman

Brubaker PL, Drucker DJ (2004). Minireview: glucagon-like peptides regulate cell

Buse JB, Henry RR, Han J, Kim DD, Fineman MS, Baron AD (2004). Effects of exenatide

Buse JB, Rosenstock J, Sesti G, Schmidt WE, Montanya E, Brett JH, Zychma M, Blonde L

Chacra AR, Tan GH, Apanovitch A, Ravichandran S, List J, Chen R (2009). Saxagliptin

Charbonnel B, Karasik A, Liu J, Wu M, Meininger G (2006). Efficacy and safety of the

Cure P, Pileggi A, Alejandro R (2008). Exenatide and rare adverse events. *N Engl J Med*, 358,

a. Deacon CF (2009). Potential of liraglutide in the treatment of patients with type 2 diabetes.

b**.** Deacon CF, Holst JJ (2009). Saxagliptin: a new dipeptidyl peptidase-4 inhibitor for the

Deacon CF, Johnsen AH, Holst JJ (1995). Degradation of glucagon-like peptide-1 by human

DeFronzo RA, Hissa MN, Garber AJ, Luiz Gross J, Yuyan Duan R, Ravichandran S, Chen RS

endogenous metabolite in vivo. *J Clin Endocrinol Metab*, 80, 952–957

plasma in vitro yields an N-terminally truncated peptide that is a major

(2009). The efficacy and safety of saxagliptin when added to metformin therapy in

dipeptidyl peptidase-4 inhibitor. *Diabetes Care*, 30, 1862-1864

randomised controlled trial. *Int J Clin Pract,* 63, 1395–1406

Effect of the dipeptidyl peptidase-4 inhibitor sitagliptin as monotherapy on glycemic control in patients with type 2 diabetes. *Diabetes Care*, 29, 2632-2637

Tolerability and efficacy of exenatide and titrated insulin glargine in adult patients with type2 diabetes previously uncontrolled with metformin or a sulfonylurea: a multinational, randomized, open-label, two-period, crossover noninferiority trial.

GA (2007). Effect of renal insufficiency on the pharmacokinetics of sitagliptin,a

proliferation and apoptosis in the pancreas, gut, and central nervous system.

(exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with

(2009). Liraglutide once a day versus exenatide twice a day for type 2 diabetes: a 26 week randomised, parallel-group, multinational, open-label trial (LEAD-6). *Lancet*,

added to a submaximal dose of sulphonylurea improves glycaemic control compared with uptitration of sulphonylurea in patients with type 2 diabetes: a

dipeptidyl peptidase-4 inhibitor sitagliptin added to ongoing metformin therapy in patients with type 2 diabetes inadequately controlled with metformin alone.

Vildagliptin has a good safety and tolerability profile and the most common adverse events are flu-like symptoms, headache, dizziness, and rarely liver enzyme elevations. Vildagliptin, like the other DPP-4 inhibitors, is weight-neutral. The incidence of hypoglycemia was low in trials with vildagliptin and similar to the placebo (Fonseca, 2007; Rosenstock, 2007). No dose adjustment is required in patients with mild renal impairment (creatinine clearance 50 ml/min). Vildagliptin should not be used in patients with hepatic impairment, including patients with pre-treatment alanine aminotransferase or aspartate aminotransferase >3x the upper limit of normal.

### **7.3 Saxagliptin**

Saxagliptin also acts by inhibiting circulating DPP-4 activity and is available as a 5 mg oncedaily in combination with metformin, sulfonylurea or pioglitazone. Saxagliptin causes a reduction in HbA1c by 0.7–0.9%. Fasting plasma glucose is also lowered dose dependently lowered by saxagliptin (Rosenstock, 2008). In a study with drug-naïve patients, saxagliptin lowered all glycaemic parameters significantly (Rosenstock, 2009). As an add-on medication to a therapy with either metformin or glitazone, saxagliptin also led to significant metabolic improvements (Chacra, 2009; Deacon, 2009b; DeFronzo, 2009).

Saxagliptin did not cause hypoglycaemia, was well-tolerated and was weight-neutral. A meta-analysis of clinical phase III studies with saxagliptin showed favourable data on the development of cardiovascular events (Wolf, 2009).

### **8. In conclusion**

Incretin-based therapies offer an alternative treatment option for T2DM patients by targeting pancreatic β-cell dysfunction. Both GLP-1 receptor agonists and DPP-4 inhibitors have been shown to be effective in improving glycemic control in patients with T2DM. They appear to be well tolerated, have a low risk of hypoglycaemia, lead to weight reduction or have a neutral effect on weight.

Choice of therapy should be based on a patient's profile and preference, with consideration given to the unique characteristics of the GLP-1 receptor agonists and DPP-4 inhibitors. The most patient-relevant and striking difference between the incretin-based therapies is that GLP-1 receptor agonists are injectable agents, while DPP-4 inhibitors are effective orally. GLP-1 receptor agonists offer more robust HbA1c level reductions and the potential for weight loss. Nausea, the most common adverse event observed with GLP-1 receptor agonist therapy is not observed in treatment with DPP-4 inhibitors. Advances in the investigation of incretin therapies will further improve treatment outcomes for patients with T2DM and help them reach target goals.

### **9. References**

Agerso H, Jensen LB, Elbrond B, Rolan P, Zdravkovic M (2002). The pharmacokinetics, pharmacodynamics, safety and tolerability of NN2211, a new long-acting GLP-1 derivative, in healthy men. *Diabetologia,* 45,195–202

Ahmad SR, Swann J (2008). Exenatide and rare adverse events. *N Engl J Med,* 358,1970–1971

Ahren B (2008). Emerging dipeptidyl peptidase-4 inhibitors for the treatment of diabetes. *Expert Opin Emerg Drugs,* 13, 593–607

Vildagliptin has a good safety and tolerability profile and the most common adverse events are flu-like symptoms, headache, dizziness, and rarely liver enzyme elevations. Vildagliptin, like the other DPP-4 inhibitors, is weight-neutral. The incidence of hypoglycemia was low in trials with vildagliptin and similar to the placebo (Fonseca, 2007; Rosenstock, 2007). No dose adjustment is required in patients with mild renal impairment (creatinine clearance 50 ml/min). Vildagliptin should not be used in patients with hepatic impairment, including patients with pre-treatment alanine aminotransferase or aspartate aminotransferase >3x the

Saxagliptin also acts by inhibiting circulating DPP-4 activity and is available as a 5 mg oncedaily in combination with metformin, sulfonylurea or pioglitazone. Saxagliptin causes a reduction in HbA1c by 0.7–0.9%. Fasting plasma glucose is also lowered dose dependently lowered by saxagliptin (Rosenstock, 2008). In a study with drug-naïve patients, saxagliptin lowered all glycaemic parameters significantly (Rosenstock, 2009). As an add-on medication to a therapy with either metformin or glitazone, saxagliptin also led to significant metabolic

Saxagliptin did not cause hypoglycaemia, was well-tolerated and was weight-neutral. A meta-analysis of clinical phase III studies with saxagliptin showed favourable data on the

Incretin-based therapies offer an alternative treatment option for T2DM patients by targeting pancreatic β-cell dysfunction. Both GLP-1 receptor agonists and DPP-4 inhibitors have been shown to be effective in improving glycemic control in patients with T2DM. They appear to be well tolerated, have a low risk of hypoglycaemia, lead to weight reduction or

Choice of therapy should be based on a patient's profile and preference, with consideration given to the unique characteristics of the GLP-1 receptor agonists and DPP-4 inhibitors. The most patient-relevant and striking difference between the incretin-based therapies is that GLP-1 receptor agonists are injectable agents, while DPP-4 inhibitors are effective orally. GLP-1 receptor agonists offer more robust HbA1c level reductions and the potential for weight loss. Nausea, the most common adverse event observed with GLP-1 receptor agonist therapy is not observed in treatment with DPP-4 inhibitors. Advances in the investigation of incretin therapies will further improve treatment outcomes for patients with T2DM and help

Agerso H, Jensen LB, Elbrond B, Rolan P, Zdravkovic M (2002). The pharmacokinetics,

Ahmad SR, Swann J (2008). Exenatide and rare adverse events. *N Engl J Med,* 358,1970–1971 Ahren B (2008). Emerging dipeptidyl peptidase-4 inhibitors for the treatment of diabetes.

derivative, in healthy men. *Diabetologia,* 45,195–202

*Expert Opin Emerg Drugs,* 13, 593–607

pharmacodynamics, safety and tolerability of NN2211, a new long-acting GLP-1

improvements (Chacra, 2009; Deacon, 2009b; DeFronzo, 2009).

development of cardiovascular events (Wolf, 2009).

upper limit of normal.

**7.3 Saxagliptin** 

**8. In conclusion** 

have a neutral effect on weight.

them reach target goals.

**9. References** 


Role of Incretin, Incretin Analogues and Dipeptidyl

http://www.diabetesatlas.org

state? *Diabetes,* 56, 1951–1959

1091

*Care*, 27,17-20

56, 1671–1679

tolerance. *Lancet*, 41, 20–21

Peptidase 4 Inhibitors in the Pathogenesis and Treatment of Diabetes Mellitus 297

Garber A, Henry RR, Ratner RE, Hale P, Chang CT, Bode B (2009). Monotherapy with

Heine RJ, van Gaal LF, Johns D, Mihm MJ, Widel MH, Brodows RG (2005). Exenatide versus

Herman GA, Stevens C, Van Dyck K, Bergman A, Yi B, De Smet M, Snyder K, Hilliard D,

International Diabetes Federation (IDF) (2009). Diabetes Atlas. Available at

Kendall DM, Riddle MC, Rosenstock J, Zhuang D, Kim DD, Fineman MS, Baron AD (2005).

Knop FK, Vilsbøll T, Højberg PV, Larsen S, Madsbad S, Vølund A, Holst JJ, Krarup T (2007).

Kolterman OG, Buse JB, Fineman MS, Gaines E, Heintz S, Bicsak TA, Taylor K, Kim D,

Kolterman OG, Kim DD, Shen L, Ruggles JA, Nielsen LL, Fineman MS, Baron AD (2005).

Koro CE, Bowlin SJ, Bourgeois N, Fedder DO (2004). Glycemic control from 1988 to 2000

Lee YS, Shin S, Shigihara T, Hahm E, Liu MJ, Han J, Yoon JW Jun HS (2007). Glucagon-like

Marre M, Shaw J, Brandle M, Bebakar WM, Kamaruddin NA, Strand J, Zdravkovic M, Le

McIntyre N, Holdsworth CD, Turner DS (1964). New interpretation of oral glucose

Moore B, Edie E, Abram J (1906). On the treatment of diabetes mellitus by acid extract of

studies with single oral doses. *Clin Pharmacol Ther*, 78, 675-688

mono 2-year results. *Diabetes,* 58(Suppl 1), 162, OR

randomized trial. *Ann Intern Med,* 143, 559–569

diabetes. *J Clin Endocrinol Metab,* 88, 3082–3089

type 2 diabetes mellitus. *Am J Health Syst Pharm*, 62, 173–181

Type 2 diabetes (LEAD-1 SU). *Diabet Med,* 26, 268–278

duodenal mucous membrane. *Biochem J*, 1, 28–38

liraglutide, a once-daily human GLP-1 analog, provides sustained reductions in A1C, FPG, and weight compared with glimepiride in type 2 diabetes: LEAD-3

insulin glargine in patients with suboptimally controlled type 2 diabetes: a

Tanen M, Tanaka W, Wang AQ, Zeng W, Musson D, Winchell G, Davies MJ, Ramael S, Gottesdiener KM, Wagner JA (2005). Pharmacokinetics and pharmacodynamics of sitagliptin, an inhibitor of dipeptidyl peptidase IV, in healthy subjects: results from two randomized, double-blind, placebo-controlled

Effects of exenatide (exendin-4) on glycemic control over 30 weeks in patients with type 2 diabetes treated with metformin and a sulfonylurea. *Diabetes Care*, 28,1083–

Reduced incretin effect in type 2 diabetes: cause or consequence of the diabetic

Aisporna M, Wang Y Baron AD (2003). Synthetic exendin-4 (exenatide) signifi cantly reduces postprandial and fasting plasma glucose in subjects with type 2

Pharmacokinetics, pharmacodynamics, and safety of exenatide in patients with

among US adults diagnosed with type 2 diabetes: a preliminary report. *Diabetes* 

peptide-1 gene therapy in obese diabetic mice results in long-term cure of diabetes by improving insulin sensitivity and reducing hepatic gluconeogenesis. *Diabetes,*

Thi TD, Colagiuri S (2009). Liraglutide, a once-daily human GLP-1 analogue, added to a sulphonylurea over 26 weeks produces greater improvements in glycaemic and weight control compared with adding rosiglitazone or placebo in subjects with

patients with inadequately controlled type 2 diabetes with metformin alone. *Diabetes Care*, 32, 1649–1655


DeFronzo RA, Ratner RE, Han J, Kim DD, Fineman MS, Baron AD (2005). Effects of

Dejager S, Razac S, Foley JE, Schweizer A (2007). Vildagliptin in drug-naive patients with

Dore DD, Seeger JD, Arnold Chan K (2009). Use of a claimsbased active drug safety

b. Drucker DJ, Nauck MA (2006). The incretin system: glucagon-like peptide-1 receptor

Drucker DJ (2003). Enhancing incretin action for the treatment of type 2 diabetes. *Diabetes* 

a. Drucker DJ (2006). Incretin-based therapies: a clinical need filled by unique metabolic

Eissele R, Göke R, Willemer S, Harthus HP, Vermeer H, Arnold R, Göke B (1992). Glucagon-

Eng J, Kleinman WA, Singh L, Singh G, Raufman JP (1992). Isolation and characterization of

Fan T, Koro CE, Fedder DO, Bowlin SJ (2006). Ethnic disparitiesand trends in glycemic

Fehmann HC, Habener JF (1992). Insulinotropic hormone glucagon-like peptide-I(7–37)

Fonseca V, Schweizer A, Albrecht D, Baron MA, Chang I, Dejager S (2007). Addition of

Gallwitz B (2006). Exenatide in type 2 diabetes: treatment effects in clinical studies and

Garber A, Henry R, Ratner R, Garcia-Hernandez PA, Rodriguez-Pattzi H, Olvera-Alvarez I,

III, double-blind, parallel-treatment trial. *Lancet*, 373, 473–481

insulinoma beta TC-1 cells. *Endocrinology*, 130,159–166

animal study data. *Int J Clin Pract,* 60,1654–1661

randomised, openlabel, non-inferiority study. *Lancet,* 372, 1240–1250 Drucker DJ, Nauck MA (2006). The incretin system: glucagonlike peptide-1 receptor

treated patients with type 2 diabetes. *Diabetes Care,* 28, 1092–1100

*Diabetes Care*, 32, 1649–1655

1705

1705

*Care*, 26, 2929-2940

*Eur J Clin Invest*, 22, 283–291

*Chem,* 267, 7402–7405

*Care*, 29, 1924-1925

50, 1148-1155.

dose study. *Horm Metab Res,* 39, 218-223

effects. *Diabetes Educ*, 32(Suppl 2), 65S-71S

patients with inadequately controlled type 2 diabetes with metformin alone.

exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin-

type 2 diabetes: a 24-week, doubleblind, randomized, placebo-controlled, multiple-

surveillance system to assess the risk of acute pancreatitis with exenatide or sitagliptin compared to metformin or glyburide. *Curr Med Res Opin,* 25,1019–1027 Drucker DJ, Buse JB, Taylor K, Kendall DM, Trautmann M, Zhuang D, Porter L (2008).

Exenatide once weekly versus twice daily for the treatment of type 2 diabetes: a

agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. *Lancet*, 368, 1696–

agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. *Lancet*, 368,1696–

like peptide-1 cells in the gastrointestinal tract and pancreas of rat, pig and man.

exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. *J Biol* 

control among adults with type 2 diabetes in the U.S. from 1988 to 2002. *Diabetes* 

stimulation of proinsulin gene expression and proinsulin biosynthesis in

vildagliptin to insulin improves glycaemiccontrol in type 2 diabetes. *Diabetologia,*

Hale PM, Zdravkovic M, Bode B (2008). Liraglutide versus glimepiride monotherapy for type 2 diabetes (LEAD-3 Mono): a randomised, 52-week, phase


Role of Incretin, Incretin Analogues and Dipeptidyl

25, 2401–2411

*Obes Metab*, 9, 175-185

*Metab,* 10, 376–386

*Pharmacol*, 140:123–132

Victoza (liraglutide).

101–113

*Med,* 25,152–156

*Ann Endocrinol (Paris),* 69, 164–165

diabetes. *Ann Pharmacother*, 40, 1777–1784

type 2 diabetes. *Int J Clin Pract*, 61, 171-180

Peptidase 4 Inhibitors in the Pathogenesis and Treatment of Diabetes Mellitus 299

Rosenstock J, Aguilar-Salinas C, Klein E, Nepal S, List J, Chen R (2009). Effect of saxagliptin

Rosenstock J, Baron MA, Camisasca RP, Cressier F, Couturier A, Dejager S (2007). Efficacy

Rosenstock J, Brazg R, Andryuk PJ, Lu K, Stein P (2006). Efficacy and safety of the

Rosenstock J, Reusch J, Bush M, Yang F, Stewart M (2009). The potential of albiglutide, a

Rosenstock J, Sankoh S, List JF (2008). Glucose-lowering activity of the dipeptidyl peptidase-

Scott R, Wu M, Sanchez M, Stein P (2007). Efficacy and tolerability of the dipeptidyl

Sturis J, Gotfredsen CF, Romer J, Rolin B, Ribel U, Brand CL, Wilken M, Wassermann K,

US Food and Drug Administration**.** Questions and answers—safety requirements for

http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPa

Vahl TP, Paty BW, Fuller BD, Prigeon RL, D'Alessio DA (2003). Effects of GLP-1-(7-36) NH2,

Vilsboll T (2009). Liraglutide: a new treatment for type 2 diabetes. *Drugs Today (Barc)*, 45,

Vilsboll T, Brock B, Perrild H, Levin K, Lervang HH, Kolendorf K, Krarup T, Schmitz O,

Werner U (2008). Preclinical pharmacology of the new GLP-1 receptor agonist AVE0010.

Wolf R, Frederich R, Fiedorek FT, Donovan M, Xu Z, Harris S, Chen R (2009). Evaluation of

Yoo BK, Triller DM, Yoo DJ (2006). Exenatide: a new option for the treatment of type 2

CV risk in the saxagliptin clinical trials. *Diabetes,* 59(Suppl 1), 8

GLP-1-(7-37), and GLP-1- (9-36)NH2 on intravenous glucose tolerance and glucoseinduced insulin secretion in healthy humans. *J Clin Endocrinol Metab,* 88, 1772–1779

Zdravkovic M, Le-Thi T, Madsbad S (2008). Liraglutide, a once-daily human GLP-1 analogue, improves pancreatic B-cell function and arginine-stimulated insulin secretion during hyperglycaemia in patients with Type 2 diabetes mellitus. *Diabet* 

tients andProviders/ucm198543.htm. Accessed August 9, 2010.

placebocontrolled, parallel-group study. *Clin Ther*, 28, 1556-1568

monotherapy in treatment-naivepatients with type 2 diabetes. *Curr Med Res Opin*,

and tolerability of initial combination therapy with vildagliptin and pioglitazone compared with component monotherapy in patients with type 2 diabetes. *Diabetes* 

dipeptidyl peptidase-4 inhibitor sitagliptin added to ongoing pioglitazone therapy in patients with type 2 diabetes: a 24-week, multicenter, randomized, double-blind,

long-acting GLP-1 receptor agonist, in type 2 diabetes: a randomized controlled trial exploring weekly, biweekly, and monthly dosing. *Diabetes Care,* 32, 1880–1886

4 inhibitor saxagliptin indrug-naive patients with type 2 diabetes. *Diabetes Obes* 

peptidase-4 inhibitor sitagliptin as monotherapy over 12 weeks in patients with

Deacon CF, Carr RD, Knudsen LB (2003). GLP-1 derivative liraglutide in rats with beta-cell deficiencies: influence of metabolic state on beta-cell mass dynamics. *Br J* 


Muscelli E, Mari A, Casolaro A, Camastra S, Seghieri G, Gastaldelli A, Holst JJ, Ferrannini E

Nauck M, Frid A, Hermansen K, Shah NS, Tankova T, Mitha IH, Zdravkovic M, During M,

(liraglutide effect and action in diabetes)-2 study. *Diabetes Care*, 32, 84–90 A**.** Nauck MA, Duran S, Kim D, Johns D, Northrup J, Festa A, Brodows R, Trautmann M

A. Nauck MA, Heimesaat MM, Orskov C, Holst JJ, Ebert R, Creutzfeldt W (1993). Preserved

Nauck MA, Homberger E, Siegel EG, Allen RC, Eaton RP, Ebert R, Creutzfeldt W (1986).

B. Nauck MA, Kleine N, Orskov C, Holst JJ, Willms B, Creutzfeldt W (1993). Normalization

type 2 (non-insulin-dependent) diabetic patients. *Diabetologia*, 36, 741–744 B. Nauck MA, Meininger G, Sheng D, Terranella L, Stein PP (2007). Efficacy and safety of the

Papazafiropoulou A, Pappas S, Papadogiannis D, Tentolouris N (2011). Cardiovascular

Pratley RE (2008). Overview of glucagon-like peptide-1 analogs and dipeptidyl peptidase-4

Pratley RE, Jauffret-Kamel S, Galbreath E, Holmes D (2006). Twelveweek monotherapy with

Raz I, Hanefeld M, Xu L, Caria C, Williams-Herman D, Khatami H (2006). Efficacy and

Retterstol K (2009). Taspoglutide: a long acting human glucagonlike polypeptide-1

Ristic S, Byiers S, Foley J, Holmes D (2005). Improved glycaemic control with dipeptidyl

the DPP-4 inhibitor vildagliptin improves glycemic control in subjects with type 2

safety of the dipeptidyl peptidase-4 inhibitorsitagliptin as monotherapy in patients

peptidase-4 inhibition in patients with type2 diabetes: vildagliptin (LAF237) dose

Effects of Glucagon-like Peptide 1. *Mini Rev Med Chem,* 11, 97-105

inhibitors for type 2 diabetes. *Medscape J Med*, 10, 171

with type 2 diabetes mellitus. *Diabetologia*, 49, 2564-2571.

analogue. *Expert Opin Investig Drugs* 18, 1405–1411

diabetes. *Horm Metab Res*, 38, 423-428

response. *Diabetes Obes Metab*, 7, 692-698

metformin: a non-inferiority study. *Diabetologia*, 50, 259–267

and C-peptide responses. *J Clin Endocrinol Metab*, 63, 492–498

normal subjects and type 2 diabetic patients. *Diabetes*, 57, 1340–1348 Nathan DM, Schreiber E, Fogel H, Mojsov S, Habener JF (1992). Insulinotropic action of

270–276

*Invest,* 91, 301–307

194-205

(2008). Separate impact of obesity and glucose tolerance on the incretin effect in

glucagonlike peptide-I-(7-37) in diabetic and nondiabetic subjects. *Diabetes Care*, 15,

Matthews DR (2009). Efficacy and safety comparison of liraglutide, glimepiride, and placebo, all in combination with metformin, in type 2 diabetes: the LEAD

(2007). A comparison of twicedailyexenatide and biphasic insulin aspart in patients with type 2 diabetes who were suboptimally controlled with sulfonylurea and

incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus. *J Clin* 

Incretin effects of increasing glucose loads in man calculated from venous insulin

of fasting hyperglycaemia by exogenous glucagon-like peptide 1 (7-36 amide) in

dipeptidyl peptidase-4 inhibitor, sitagliptin, compared with the sulfonylurea, glipizide, in patients with type 2 diabetes inadequately controlled on metformin alone: a randomized, double-blind, non-inferiority trial. *Diabetes Obes Metab*, 9,


**16** 

*Korea* 

Sang Won Suh

**Zinc Translocation Causes** 

*Department of Physiology, Hallym University,* 

 *College of Medicine, Chuncheon,* 

**Hypoglycemia-Induced Neuron Death** 

Hypoglycemia is a common but serious problem among type1 and type 2 diabetic patients receiving intensive treatment with glucose-lowering drugs such as insulin or sulfonylurea. Moderate hypoglycemia is occurring 0.1-0.3 episode/patient per day and is usually corrected by patients themselves or just ignored. However, severe hypoglycemia causes unconsciousness and it may lead to neuronal injury in the cerebral cortex and hippocampus. Hypoglycemic neuronal death is resulted from a cascade of several events after prolonged period of lack of glucose since brain exclusively use glucose (Auer et al., 1984a; Auer and Siesjo, 1993; Auer et al., 1984b). Sustained release of glutamate from presynaptic terminals into the extracellular space and activation of glutamate receptors has been suggested as a necessary upstream event in this neuron death cascade (Auer and Siesjo, 1993; Wieloch, 1985). Also mitochondrial membrane permeability (Friberg et al., 1998), calpain activation (Ferrand-Drake et al., 2003), PARP-1 activation (Suh et al., 2003) and NADPH oxidase activation-induced ROS production (Suh et al., 2007; Suh et al., 2008) have been shown to be possible downstream events. Our lab has undertaken studies to establish whether vesicular zinc release and subsequent zinc translocation into postsynaptic neurons is an important upstream step in this hypoglycemia-induced neuron death process. Using an animal model of insulin-induced hypoglycemia we have shown that: (I) vesicular zinc is released from hippocampal mossy fiber terminals; (II) intracellular zinc accumulation is induced in the hippocampal neurons; (III) neuronal death is reduced by zinc chelation or zinc transporter gene deletion; (IV) PARP-1 activation is reduced by zinc chelation; (V) ROS production is reduced by zinc chelation after hypoglycemia and glucose reperfusion (HG/GR); and (VI) hypothermia prevented hypoglycemia-induced zinc release and neuron death. Together, these results suggest that zinc translocation is an upstream step linking HG/GR to PARP-1 activation, to NADPH oxidase activation and neuronal death in brain regions containing high concentrations of vesicular zinc. Zinc translocation into postsynaptic neurons was also demonstrated in the hippocampal slice model with combined oxygen and glucose deprivation (OGD) where neuronal zinc accumulation into the hippocampal CA1 neurons is blocked by extracellular zinc chelator, CaEDTA (Yin et al., 2002). In addition, hippocampal slices prepared from zinc transporter 3 (ZnT3) knockout mouse, which have little or no vesicular zinc in neuronal terminals, showed no zinc accumulation in post-synaptic neurons following OGD and hypoglycemia (Suh et al., 2008). These hippocampal slice experiments

**1. Introduction** 


## **Zinc Translocation Causes Hypoglycemia-Induced Neuron Death**

### Sang Won Suh

*Department of Physiology, Hallym University, College of Medicine, Chuncheon, Korea* 

### **1. Introduction**

300 Diabetes – Damages and Treatments

Zander M, Madsbad S, Madsen JL, Holst JJ (2002). Effect of 6-week course of glucagon-like

Zinman B, Gerich J, Buse JB, Lewin A, Schwartz S, Raskin P, Hale PM, Zdravkovic M,

Zunz E, La Barre J (1929). Contributions a l'étude des variations physiologiques de la

type 2 diabetes (LEAD-4 Met+TZD). *Diabetes Care*, 32,1224–1230

diabetes: a parallel-group study. *Lancet,* 359, 824–830

pancréas. *Arch Int Physiol Biochim,* 31, 20–44

peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2

Blonde L (2009). Efficacy and safety of the human glucagon-like peptide-1 analog liraglutide in combination with metformin and thiazolidinedione in patients with

sécrétion interne du pancréas: relations entre les sécrétions externe et interne du

Hypoglycemia is a common but serious problem among type1 and type 2 diabetic patients receiving intensive treatment with glucose-lowering drugs such as insulin or sulfonylurea. Moderate hypoglycemia is occurring 0.1-0.3 episode/patient per day and is usually corrected by patients themselves or just ignored. However, severe hypoglycemia causes unconsciousness and it may lead to neuronal injury in the cerebral cortex and hippocampus. Hypoglycemic neuronal death is resulted from a cascade of several events after prolonged period of lack of glucose since brain exclusively use glucose (Auer et al., 1984a; Auer and Siesjo, 1993; Auer et al., 1984b). Sustained release of glutamate from presynaptic terminals into the extracellular space and activation of glutamate receptors has been suggested as a necessary upstream event in this neuron death cascade (Auer and Siesjo, 1993; Wieloch, 1985). Also mitochondrial membrane permeability (Friberg et al., 1998), calpain activation (Ferrand-Drake et al., 2003), PARP-1 activation (Suh et al., 2003) and NADPH oxidase activation-induced ROS production (Suh et al., 2007; Suh et al., 2008) have been shown to be possible downstream events. Our lab has undertaken studies to establish whether vesicular zinc release and subsequent zinc translocation into postsynaptic neurons is an important upstream step in this hypoglycemia-induced neuron death process. Using an animal model of insulin-induced hypoglycemia we have shown that: (I) vesicular zinc is released from hippocampal mossy fiber terminals; (II) intracellular zinc accumulation is induced in the hippocampal neurons; (III) neuronal death is reduced by zinc chelation or zinc transporter gene deletion; (IV) PARP-1 activation is reduced by zinc chelation; (V) ROS production is reduced by zinc chelation after hypoglycemia and glucose reperfusion (HG/GR); and (VI) hypothermia prevented hypoglycemia-induced zinc release and neuron death. Together, these results suggest that zinc translocation is an upstream step linking HG/GR to PARP-1 activation, to NADPH oxidase activation and neuronal death in brain regions containing high concentrations of vesicular zinc. Zinc translocation into postsynaptic neurons was also demonstrated in the hippocampal slice model with combined oxygen and glucose deprivation (OGD) where neuronal zinc accumulation into the hippocampal CA1 neurons is blocked by extracellular zinc chelator, CaEDTA (Yin et al., 2002). In addition, hippocampal slices prepared from zinc transporter 3 (ZnT3) knockout mouse, which have little or no vesicular zinc in neuronal terminals, showed no zinc accumulation in post-synaptic neurons following OGD and hypoglycemia (Suh et al., 2008). These hippocampal slice experiments

Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 303

Chelatable zinc (free or weakly bound to proteins) is present in a subset of glutamatergic axon terminals throughout the mammalian forebrain, especially in the hippocampus and in the cerebral cortex (Danscher et al., 1985) (Frederickson, 1989). The chelatable zinc is mainly localized in synaptic vesicles of excitatory presynaptic neuron terminals (Perez-Clausell and Danscher, 1985) and is released into the extracellular space during paroxysmal neuronal activity or membrane depolarization (Assaf and Chung, 1984; Howell et al., 1984). This zinc release has been suggested to contribute to neuronal death in several disease conditions, such as seizure (Frederickson et al., 1988; Suh et al., 2001), ischemia (Koh et al., 1996; Tonder et al., 1990) and traumatic brain injury (Suh et al., 2000). Zinc can induce the production of reactive oxygen species (ROS) and PARP-1 activation in cell cultures (Kim et al., 1999; Sensi et al., 1999a; Sheline et al., 2000), suggesting a possible role of zinc in hypoglycemia-induced neuronal death. Our previous study showed that hypoglycemia induces vesicular zinc release from the synaptic terminals. We also found that hypoglycemia increases neuronal zinc accumulation in postsynaptic neurons, which is prevented by intracerebroventricular injection of the Zn2+ chelator CaEDTA (Suh et al., 2004; Suh et al., 2008) or intraperitoneal

Oxidative stress and zinc release are both known to contribute to neuronal death after hypoglycemia; however, the temporal relationships between these events are not well established. Our study demonstrated that the vesicular zinc release from hippocampal mossy fiber and subsequent translocation into postsynaptic neurons occurs immediately after HG/GR. We used the fluorescent dye TSQ, which binds free zinc (Frederickson et al., 1987). The vesicular zinc signal detected by TSQ showed a partial decrease (release from mossy fiber terminal) after 60 minutes of hypoglycemia alone (HG alone), but was almost completely absent after 30 minutes of hypoglycemia followed by 30 minutes of glucose reperfusion (HG/GR) (Figure 1A) (Suh et al., 2004; Suh et al., 2007). This result suggests that vesicular zinc release from hippocampal mossy fiber is not caused by hypoglycemia itself but caused by a combination of hypoglycemia and subsequent glucose reperfusion. Conversely, TSQ staining in the postsynaptic pyramidal neuron bodies was absent under sham operated conditions or hypoglycemia alone, but TSQ intensity in the cytoplasm of CA1 neurons was increased 3 hours after 30 minutes of hypoglycemia and 30 minutes glucose reperfusion (HG/GR) (Figure 1B). This represents translocation of presynaptic zinc to postsynaptic neuron of CA1 pyramidal neurons. This initial cytoplasmic zinc increase was prevented by intracerebroventricular (i.c.v) injection of the zinc chelator, CaEDTA. Without zinc chelation, this intraneuronal zinc accumulation continued to increase until 24 hours after hypoglycemia and glucose reperfusion (Suh et al., 2008). However, CaEDTA treatment also prevented this continuous intracellular zinc accumulation when evaluated at 24 hours later, suggesting that released zinc from the synaptic vesicles translocated into the post-synaptic neurons during several hours after hypoglycemia and glucose reperfusion conditions (Figure 1C). From these findings, we speculate that zinc release/translocation is a key upstream step in the sequence of events leading to neuronal death after HG/GR (Suh et al., 2004; Suh et al., 2007). However, the identity of the factor(s) involved in the intermediating step(s) for HG/GR-induced vesicular zinc release and translocation process

**2. Role of zinc in hypoglycemic neuronal death** 

injection of clioquinol (CQ) (Shin et al., 2010).

**2.1 Vesicular zinc release and translocation after hypoglycemia** 

with zinc chelator or with ZnT3 KO mice suggest that the zinc signal observed in postsynaptic hippocampal neurons as shown in our previous study (Suh et al., 2003) was a result of zinc translocation from the presynaptic terminals.

Fig. 1. Vesicular zinc release and translocation after hypoglycemia.

A) TSQ fluorescent images show vesicular zinc release from presynaptic terminals of hippocampal mossy fibers after hypoglycemia/ glucose reperfusion (HG/GR). Intense TSQ fluorescent signal (white color in the figure) in the mossy fiber of sham operated rats indicates high vesicular zinc contents in the vesicle. However, the diminished TSQ fluorescent intensity in the HG/GR rats indicates that bulk of vesicular zinc has been released and therefore presynaptic vesicular zinc contents are reduced at the time when the brain section was evaluated. TSQ fluorescent intensity in mossy fiber is decreased after 60 minutes of hypoglycemia (HG alone). TSQ fluorescent intensity is further decreased after 30 minutes hypoglycemia and 30 minutes glucose reperfusion (HG/GR), which represents mossy fiber vesicular zinc release from presynaptic terminals. A schematic drawing represents vesicular zinc release from presynaptic terminals after HG/GR. B) TSQ fluorescent images show zinc translocation into postsynaptic neurons of hippocampal CA1 pyramidal neurons 3 hours after hypoglycemia. Zinc accumulation in the intracellular space can be detected in this early time point. A schematic drawing represents intracellular zinc accumulation 3 hours after HG/GR. C) TSQ fluorescent images show zinc accumulation into postsynaptic neurons 24 hours after HG/GR. Intense zinc accumulation in the intracellular space is detected in this time point. A schematic drawing represents intracellular zinc accumulation 24 hours after HG/GR. Scale bar in (A) is 200 μm and in (B) and (C) are 20 μm.

with zinc chelator or with ZnT3 KO mice suggest that the zinc signal observed in postsynaptic hippocampal neurons as shown in our previous study (Suh et al., 2003) was a result

of zinc translocation from the presynaptic terminals.

Fig. 1. Vesicular zinc release and translocation after hypoglycemia.

A) TSQ fluorescent images show vesicular zinc release from presynaptic terminals of hippocampal mossy fibers after hypoglycemia/ glucose reperfusion (HG/GR). Intense TSQ fluorescent signal (white color in the figure) in the mossy fiber of sham operated rats indicates high vesicular zinc contents in the vesicle. However, the diminished TSQ fluorescent intensity in the HG/GR rats indicates that bulk of vesicular zinc has been released and therefore presynaptic vesicular zinc contents are reduced at the time when the brain section was evaluated. TSQ fluorescent intensity in mossy fiber is decreased after 60 minutes of hypoglycemia (HG alone). TSQ fluorescent intensity is further decreased after 30 minutes hypoglycemia and 30 minutes glucose reperfusion (HG/GR), which represents mossy fiber vesicular zinc release from presynaptic terminals. A schematic drawing represents vesicular zinc release from presynaptic terminals after HG/GR. B) TSQ

fluorescent images show zinc translocation into postsynaptic neurons of hippocampal CA1 pyramidal neurons 3 hours after hypoglycemia. Zinc accumulation in the intracellular space can be detected in this early time point. A schematic drawing represents intracellular zinc accumulation 3 hours after HG/GR. C) TSQ fluorescent images show zinc accumulation into postsynaptic neurons 24 hours after HG/GR. Intense zinc accumulation in the intracellular space is detected in this time point. A schematic drawing represents intracellular zinc accumulation 24 hours after HG/GR. Scale bar in (A) is 200 μm and in (B) and (C) are 20 μm.

### **2. Role of zinc in hypoglycemic neuronal death**

Chelatable zinc (free or weakly bound to proteins) is present in a subset of glutamatergic axon terminals throughout the mammalian forebrain, especially in the hippocampus and in the cerebral cortex (Danscher et al., 1985) (Frederickson, 1989). The chelatable zinc is mainly localized in synaptic vesicles of excitatory presynaptic neuron terminals (Perez-Clausell and Danscher, 1985) and is released into the extracellular space during paroxysmal neuronal activity or membrane depolarization (Assaf and Chung, 1984; Howell et al., 1984). This zinc release has been suggested to contribute to neuronal death in several disease conditions, such as seizure (Frederickson et al., 1988; Suh et al., 2001), ischemia (Koh et al., 1996; Tonder et al., 1990) and traumatic brain injury (Suh et al., 2000). Zinc can induce the production of reactive oxygen species (ROS) and PARP-1 activation in cell cultures (Kim et al., 1999; Sensi et al., 1999a; Sheline et al., 2000), suggesting a possible role of zinc in hypoglycemia-induced neuronal death. Our previous study showed that hypoglycemia induces vesicular zinc release from the synaptic terminals. We also found that hypoglycemia increases neuronal zinc accumulation in postsynaptic neurons, which is prevented by intracerebroventricular injection of the Zn2+ chelator CaEDTA (Suh et al., 2004; Suh et al., 2008) or intraperitoneal injection of clioquinol (CQ) (Shin et al., 2010).

#### **2.1 Vesicular zinc release and translocation after hypoglycemia**

Oxidative stress and zinc release are both known to contribute to neuronal death after hypoglycemia; however, the temporal relationships between these events are not well established. Our study demonstrated that the vesicular zinc release from hippocampal mossy fiber and subsequent translocation into postsynaptic neurons occurs immediately after HG/GR. We used the fluorescent dye TSQ, which binds free zinc (Frederickson et al., 1987). The vesicular zinc signal detected by TSQ showed a partial decrease (release from mossy fiber terminal) after 60 minutes of hypoglycemia alone (HG alone), but was almost completely absent after 30 minutes of hypoglycemia followed by 30 minutes of glucose reperfusion (HG/GR) (Figure 1A) (Suh et al., 2004; Suh et al., 2007). This result suggests that vesicular zinc release from hippocampal mossy fiber is not caused by hypoglycemia itself but caused by a combination of hypoglycemia and subsequent glucose reperfusion. Conversely, TSQ staining in the postsynaptic pyramidal neuron bodies was absent under sham operated conditions or hypoglycemia alone, but TSQ intensity in the cytoplasm of CA1 neurons was increased 3 hours after 30 minutes of hypoglycemia and 30 minutes glucose reperfusion (HG/GR) (Figure 1B). This represents translocation of presynaptic zinc to postsynaptic neuron of CA1 pyramidal neurons. This initial cytoplasmic zinc increase was prevented by intracerebroventricular (i.c.v) injection of the zinc chelator, CaEDTA. Without zinc chelation, this intraneuronal zinc accumulation continued to increase until 24 hours after hypoglycemia and glucose reperfusion (Suh et al., 2008). However, CaEDTA treatment also prevented this continuous intracellular zinc accumulation when evaluated at 24 hours later, suggesting that released zinc from the synaptic vesicles translocated into the post-synaptic neurons during several hours after hypoglycemia and glucose reperfusion conditions (Figure 1C). From these findings, we speculate that zinc release/translocation is a key upstream step in the sequence of events leading to neuronal death after HG/GR (Suh et al., 2004; Suh et al., 2007). However, the identity of the factor(s) involved in the intermediating step(s) for HG/GR-induced vesicular zinc release and translocation process

Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 305

Fig. 3. Key aspects of hypoglycemia-induced neuronal death by zinc.

nucleus. 6) Neuron death.

1) Nitric oxide (NO) production after hypoglycemia/ glucose reperfusion leads to release of

Since peroxynitrite (highly neurotoxic) is formed by reaction of nitric oxide (NO) with superoxide (Beckman and Koppenol, 1996), our previous study also sought to clarify the role of superoxide formation on presynaptic zinc release from hippocampal mossy fiber and postsynaptic zinc accumulation in the hippocampal CA1 neurons after hypoglycemic insult. This study showed that over-expression of SOD-1 significantly reduced hypoglycemiainduced neuronal death (Suh et al., 2007). To determine whether the neuroprotective role of SOD-1 over-expression was due to reduced release of vesicular zinc, SOD-1 transgenic rats were subjected to hypoglycemia. From this study, we concluded that SOD-1 overexpression had no effect on hypoglycemia-induced vesicular zinc release or on the initial zinc translocation into hippocampal postsynaptic neurons when evaluated at 3 hours after hypoglycemia, but that SOD-1 overexpression did reduce neuronal death and neuronal zinc accumulation when evaluated at 24 hours after hypoglycemia. These results suggest that vesicular zinc release occurs upstream of ROS production, but that ROS production continues to promote to zinc accumulation in post-synaptic neurons at later time points

zinc together with glutamate from presynaptic terminals. 2) Zinc translocates into intracellular space. 3) Translocated zinc activates NADPH oxidase. 4) NADPH oxidase activation induces ROS production. 5) Production of superoxide from NADPH oxidase induces DNA damage and activation of poly(ADP-ribose) polymerase-1 (PARP-1) in the

is unknown. In our prior study, nitrotyrosine formation was detected shortly after glucose reperfusion, but not during hypoglycemia per se (Suh et al., 2003). Subsequently we found that a neuron specific NOS inhibitor, 7-NI, significantly inhibited hypoglycemia-induced vesicular zinc release from hippocampal mossy fiber (Fig 2A). 7-NI also prevented intracellular zinc accumulation and neuronal death at 24 hour post-HG/GR time point (Fig. 2B) (Suh et al., 2003). These findings suggest that nitric oxide production is an event upstream of vesicular zinc release and postsynaptic zinc accumulation. This observation is consistent with previous studies in which intra-hippocampal injection of nitric oxide donor (Spermino-NONOate) induced vesicular zinc release and intracellular zinc accumulation (Cuajungco and Lees, 1998; Frederickson et al., 2002).

is unknown. In our prior study, nitrotyrosine formation was detected shortly after glucose reperfusion, but not during hypoglycemia per se (Suh et al., 2003). Subsequently we found that a neuron specific NOS inhibitor, 7-NI, significantly inhibited hypoglycemia-induced vesicular zinc release from hippocampal mossy fiber (Fig 2A). 7-NI also prevented intracellular zinc accumulation and neuronal death at 24 hour post-HG/GR time point (Fig. 2B) (Suh et al., 2003). These findings suggest that nitric oxide production is an event upstream of vesicular zinc release and postsynaptic zinc accumulation. This observation is consistent with previous studies in which intra-hippocampal injection of nitric oxide donor (Spermino-NONOate) induced vesicular zinc release and intracellular zinc accumulation

Fig. 2. A) Vesicular zinc release after hypoglycemia/glucose reperfusion is prevented by NOS inhibitor. Vesicular zinc release was evaluated at hippocampal hilus by TSQ

(HG+GR+SOD), whereas the NOS inhibitor 7-NI almost completely prevented vesicular zinc release from the hilus mossy fiber area. Graph shows TSQ fluorescence intensity. Data are mean + s.e.m; n = 10; \* P < 0.05. # P < 0.05. B) Intracellular zinc accumulation and neuronal death after hypoglycemia/ glucose reperfusion (HG/GR). Images show neuronal zinc accumulation at 3 or 24 hours after HG/GR and neuronal death at 24 hours after HG/GR. TSQ intensity in CA1 pyramidal neurons is increased compared to sham operated rats by 3 hours after HG, and further increased at 24 hours. CA1 pyramidal neurons show Fluoro-Jade B staining (green) at 24 hours after HG/GR. Scale bar = 50 μm. n = 3-4. This

SOD-1 over-expressing rats (SOD-1 Tg) show similar zinc release after HG/GR

figure is modified from our previous published paper (Suh et al., JCBFM, 2008).

fluorescence. The TSQ signal loss is apparent after 30 minutes of HG and 30 minutes of GR.

(Cuajungco and Lees, 1998; Frederickson et al., 2002).

Since peroxynitrite (highly neurotoxic) is formed by reaction of nitric oxide (NO) with superoxide (Beckman and Koppenol, 1996), our previous study also sought to clarify the role of superoxide formation on presynaptic zinc release from hippocampal mossy fiber and postsynaptic zinc accumulation in the hippocampal CA1 neurons after hypoglycemic insult. This study showed that over-expression of SOD-1 significantly reduced hypoglycemiainduced neuronal death (Suh et al., 2007). To determine whether the neuroprotective role of SOD-1 over-expression was due to reduced release of vesicular zinc, SOD-1 transgenic rats were subjected to hypoglycemia. From this study, we concluded that SOD-1 overexpression had no effect on hypoglycemia-induced vesicular zinc release or on the initial zinc translocation into hippocampal postsynaptic neurons when evaluated at 3 hours after hypoglycemia, but that SOD-1 overexpression did reduce neuronal death and neuronal zinc accumulation when evaluated at 24 hours after hypoglycemia. These results suggest that vesicular zinc release occurs upstream of ROS production, but that ROS production continues to promote to zinc accumulation in post-synaptic neurons at later time points

Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 307

by glucose reperfusion through a process requiring extracellular zinc signaling. To further confirm that vesicular zinc release is involved in HG/GR-induced ROS production and neuron death, we used the ZnT3-/- mouse, which has no vesicular zinc in the presynaptic terminals (Suh et al., 2007). The ZnT3-/- mice showed diminished ROS production at 3 hours after HG/GR and reduced neuronal death 7 days after HG/GR (Figure 5). This result confirms prior reports that zinc chelation prevents ROS production and neuron death after HG/GR (Suh et al., 2004; Suh et al., 2007) and strongly suggests that it is the vesicular zinc

Fig. 4. Hypoglycemia/ glucose reperfusion-induced ROS production is mediated by zincinduced NADPH oxidase activation. ROS production in neurons detected by ethidium (Et)

A) The zinc chelator, CaEDTA, reduces HG/GR-induced Et production in the CA1 neurons.

ZnEDTA is the control. Rats were treated with saline, 100 mM CaEDTA, or 100 mM ZnEDTA. Scale bar is 50 μm. B) Schematic drawing of p47phox and p67phox translocation to

plasma membrane by zinc translocation into neuron.

fluorescence.

pool that contributes to neuronal demise in this setting.

(Figure 2, 3). This suggests that protein-bound zinc can be liberated by reactive oxygen species (ROS) such as superoxide. Thus, if neuronal SOD concentrations are adequate for clearance of superoxide, further intracellular free zinc release can be prevented even though initial zinc translocation event has occurred. Conversely, if superoxide production is not cleared or stabilized, intracellular free zinc will continue to increase to the point of neuronal demise. This result suggests that in addition to presynaptically-released Zn2+ , hippocampal neurons also have a pool of intracellularly releasable Zn2+. Intracellularly derived zinc may arise from metallothionein (MTs) or other zinc binding proteins. MTs play a major role in modulating neuron death after seizure or ischemia as these proteins release a substantial amount of Zn2+ under conditions of oxidative stress. This notion is supported by prior studies suggesting that non-vesicular zinc may be also important in promoting brain injury (Lee et al., 2000).

#### **2.2 The role of zinc on hypoglycemia-induced ROS production**

The mechanism by which ROS production is aggravated by intracellular zinc influx has not been firmly established. Several lines of evidence suggest that zinc induces increased mitochondrial ROS production (Sensi et al., 1999b). However, in cell culture models, zinc has been identified as an activator of NADPH oxidase, an enzyme that produces superoxide. NADPH oxidase is present in many cell types including neurons (Kim and Koh, 2002; Noh and Koh, 2000). NADPH oxidase is a multi-component enzyme comprising a plasma membrane-bound subunit, gp91; a membrane-associated flavocytochrome, cytochrome b558; and at least three cytosolic subunits, p47phox, p67phox and the small G protein Rac2 (Groemping and Rittinger, 2005). During activation, the p47phox component is phosphorylated and translocates to the plasma membrane, where it associates with the other subunits to form the active enzyme complex. The methoxy-substituted catechol, apocynin, blocks this assembly but does not inhibit mitochondrial dehydrogenases (Dodd and Pearse, 2000; Stolk et al., 1994). Interestingly, our previous studies examining the production of ROS in the brain during hypoglycemic insult suggest that superoxide is formed primarily during the glucose reperfusion period. The mechanism by which NADPH oxidase is activated in non-phagocytic cells is not well understood, but zinc has been identified as both an inducer of neuronal NADPH oxidase activity (Kim and Koh, 2002; Noh and Koh, 2000) and a contributor to hypoglycemic neuronal death (Suh et al., 2008). High concentrations of presynaptic zinc are present in the brain regions most vulnerable to hypoglycemic injury (Frederickson et al., 2005; Suh et al., 2004). Recently, we published that vesicular zinc release is required for NADPH oxidase activation in HG/GR (Suh et al., 2007). Rats pre-treated with an intracerebroventricular injection of the zinc chelator CaEDTA showed reduced neuronal ROS formation, suggesting that vesicular zinc release is an upstream event of NADPH oxidase activation. ZnEDTA, used as a control, showed no effect on ROS production. The translocation of NADPH oxidase subunits, p47phox or p61phox, to the plasma membrane in cortical neuronal cultures subjected to glucose deprivation followed by glucose reperfusion was blocked by CaEDTA, but not by ZnEDTA (Figure 4). Moreover we demonstrated that zinc-induced ROS production in neuron cultures was almost completely absent in cultures from mice deficient in the p47phox subunit of NADPH oxidase and in wt neurons treated with the NADPH oxidase assembly inhibitor apocynin (Stolk et al., 1994; Suh et al., 2008). These results suggest that NADPH oxidase subunit assembly is triggered

(Figure 2, 3). This suggests that protein-bound zinc can be liberated by reactive oxygen species (ROS) such as superoxide. Thus, if neuronal SOD concentrations are adequate for clearance of superoxide, further intracellular free zinc release can be prevented even though initial zinc translocation event has occurred. Conversely, if superoxide production is not cleared or stabilized, intracellular free zinc will continue to increase to the point of neuronal demise. This result suggests that in addition to presynaptically-released Zn2+ , hippocampal neurons also have a pool of intracellularly releasable Zn2+. Intracellularly derived zinc may arise from metallothionein (MTs) or other zinc binding proteins. MTs play a major role in modulating neuron death after seizure or ischemia as these proteins release a substantial amount of Zn2+ under conditions of oxidative stress. This notion is supported by prior studies suggesting that non-vesicular zinc may be also important in promoting brain injury

The mechanism by which ROS production is aggravated by intracellular zinc influx has not been firmly established. Several lines of evidence suggest that zinc induces increased mitochondrial ROS production (Sensi et al., 1999b). However, in cell culture models, zinc has been identified as an activator of NADPH oxidase, an enzyme that produces superoxide. NADPH oxidase is present in many cell types including neurons (Kim and Koh, 2002; Noh and Koh, 2000). NADPH oxidase is a multi-component enzyme comprising a plasma membrane-bound subunit, gp91; a membrane-associated flavocytochrome, cytochrome b558; and at least three cytosolic subunits, p47phox, p67phox and the small G protein Rac2 (Groemping and Rittinger, 2005). During activation, the p47phox component is phosphorylated and translocates to the plasma membrane, where it associates with the other subunits to form the active enzyme complex. The methoxy-substituted catechol, apocynin, blocks this assembly but does not inhibit mitochondrial dehydrogenases (Dodd and Pearse, 2000; Stolk et al., 1994). Interestingly, our previous studies examining the production of ROS in the brain during hypoglycemic insult suggest that superoxide is formed primarily during the glucose reperfusion period. The mechanism by which NADPH oxidase is activated in non-phagocytic cells is not well understood, but zinc has been identified as both an inducer of neuronal NADPH oxidase activity (Kim and Koh, 2002; Noh and Koh, 2000) and a contributor to hypoglycemic neuronal death (Suh et al., 2008). High concentrations of presynaptic zinc are present in the brain regions most vulnerable to hypoglycemic injury (Frederickson et al., 2005; Suh et al., 2004). Recently, we published that vesicular zinc release is required for NADPH oxidase activation in HG/GR (Suh et al., 2007). Rats pre-treated with an intracerebroventricular injection of the zinc chelator CaEDTA showed reduced neuronal ROS formation, suggesting that vesicular zinc release is an upstream event of NADPH oxidase activation. ZnEDTA, used as a control, showed no effect on ROS production. The translocation of NADPH oxidase subunits, p47phox or p61phox, to the plasma membrane in cortical neuronal cultures subjected to glucose deprivation followed by glucose reperfusion was blocked by CaEDTA, but not by ZnEDTA (Figure 4). Moreover we demonstrated that zinc-induced ROS production in neuron cultures was almost completely absent in cultures from mice deficient in the p47phox subunit of NADPH oxidase and in wt neurons treated with the NADPH oxidase assembly inhibitor apocynin (Stolk et al., 1994; Suh et al., 2008). These results suggest that NADPH oxidase subunit assembly is triggered

**2.2 The role of zinc on hypoglycemia-induced ROS production** 

(Lee et al., 2000).

by glucose reperfusion through a process requiring extracellular zinc signaling. To further confirm that vesicular zinc release is involved in HG/GR-induced ROS production and neuron death, we used the ZnT3-/- mouse, which has no vesicular zinc in the presynaptic terminals (Suh et al., 2007). The ZnT3-/- mice showed diminished ROS production at 3 hours after HG/GR and reduced neuronal death 7 days after HG/GR (Figure 5). This result confirms prior reports that zinc chelation prevents ROS production and neuron death after HG/GR (Suh et al., 2004; Suh et al., 2007) and strongly suggests that it is the vesicular zinc pool that contributes to neuronal demise in this setting.

Fig. 4. Hypoglycemia/ glucose reperfusion-induced ROS production is mediated by zincinduced NADPH oxidase activation. ROS production in neurons detected by ethidium (Et) fluorescence.

A) The zinc chelator, CaEDTA, reduces HG/GR-induced Et production in the CA1 neurons. ZnEDTA is the control. Rats were treated with saline, 100 mM CaEDTA, or 100 mM ZnEDTA. Scale bar is 50 μm. B) Schematic drawing of p47phox and p67phox translocation to plasma membrane by zinc translocation into neuron.

Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 309

and memory (Suh et al., 2003). Administration of PARP-1 inhibitors at time points up to 3 hours after HG/GR was effective in reducing neuronal death, suggesting both that PARP-1 is a downstream event in the HG/GR cell death pathway and that PARP-1 inhibitors might

A link between zinc release and PARP-1 activation has been suggested by studies showing PARP-1 activation and PARP-1 mediated neuronal death after neuronal exposure to zinc in cell culture, and the ability of PARP-1 inhibitors to abrogate zinc-induced cell death (Kim and Koh, 2002; Sheline et al., 2000; Sheline et al., 2003; Virag and Szabo, 2002). How zinc leads to PARP-1 activation has not been firmly established, but zinc has been shown to induce formation of reactive oxygen species through actions on mitochondria (Ichord et al., 1999) and through up-regulation of NADPH oxidase and neuronal nitric oxide synthase (Kim et al., 2002). Our previous study showed that the zinc chelator CaEDTA attenuated poly(ADPribose) formation in the post-synaptic pyramidal cells after HG/GR, suggesting that zinc translocation may be an upstream event in hypoglycemia-induced PARP-1 activation. This result, coupled with the marked reduction in neuronal death observed with CaEDTA, and the prior observation that PARP-1 inhibitors reduce hypoglycemic neuronal death (Frederickson et al., 2002), suggests a sequential process of zinc entry, PARP-1 activation, and cell death triggered by HG/GR. These results do not, however, exclude other mechanisms by which

be useful in the clinical treatment of hypoglycemic brain injury (Figure 6).

vesicular zinc release could contribute to hypoglycemic neuronal death.

Fig. 6. Hypoglycemia/ glucose reperfusion-induced poly(ADP-ribose) formation in CA1

A) Poly(ADP-ribose) immunoreactivity was only modestly increased at termination of immediately after HG/GR (0 hr), but was markedly increased at 3 hr after insult, and then slowly declined after that point in the hippocampal CA1 and DG area. Scale bar is 50 μm. B) Poly(ADP-ribose) formation was reduced by administration of zinc chelator, CaEDTA, at

hippocampus in rats.

the time of glucose correction. Scale bar is 50 μm.

Fig. 5. Hypoglycemia/ glucose reperfusion-induced ROS production and neuronal injury is prevented by ZnT3 gene deletion in mice.

A) Vesicular zinc in the mouse hippocampus imaged with TSQ fluorescence (white) from wild-type mice and from ZnT3-/- mice. Scale bar is 500 μm. B) To characterize the source of ROS production in hypoglycemic neuronal injury, we used a rat model of insulin-induced hypoglycemia and evaluated the production of reactive oxygen species with dihydroethidium. Dihydroethidium is oxidized by superoxide and superoxide reaction products to form fluorescent ethidium (Et) species, which are then trapped within cells by DNA binding.In the ZnT3-/- mice, hypoglycemia-induced ROS production is almost completely prevented. Scale bar is 50 μm. C) Neuronal death (FJB (+) neurons) in ZnT3-/ mice was significantly less than wild type mice. Scale bar is 100 μm. Part of this figure is modified from our previous published paper (Suh et al., JCBFM, 2008).

### **2.3 The role of zinc on hypoglycemia-induced PARP-1 activation**

PARP-1 activation has been shown to mediate neuronal death in a variety of disorders including ischemia, trauma, and inflammation (Virag and Szabo, 2002). PARP-1 uses the ADP-ribose group of NAD+ to form branched ADP-ribose polymers on specific acceptor proteins in the vicinity of DNA strand breaks or kinks (Burzio et al., 1979; D'Amours et al., 1999). Formation of these polymers facilitates DNA repair and prevents chromatid exchange, but extensive PARP-1 activation can promote cell death through a processes involving mitochondrial permeability transition and release of apoptosis inducing factor (Alano et al., 2004; Ha and Snyder, 1999; Yu et al., 2002). Our previous study showed that PARP-1 activation was substantially increased in hippocampal neurons after HG/GR. Rats treated with PARP-1 inhibitors after HG/GR showed a striking reduction in neuronal death, coupled with improved performance on the Morris water maze, a test of spatial learning

Fig. 5. Hypoglycemia/ glucose reperfusion-induced ROS production and neuronal injury is

A) Vesicular zinc in the mouse hippocampus imaged with TSQ fluorescence (white) from wild-type mice and from ZnT3-/- mice. Scale bar is 500 μm. B) To characterize the source of ROS production in hypoglycemic neuronal injury, we used a rat model of insulin-induced

dihydroethidium. Dihydroethidium is oxidized by superoxide and superoxide reaction products to form fluorescent ethidium (Et) species, which are then trapped within cells by DNA binding.In the ZnT3-/- mice, hypoglycemia-induced ROS production is almost completely prevented. Scale bar is 50 μm. C) Neuronal death (FJB (+) neurons) in ZnT3-/ mice was significantly less than wild type mice. Scale bar is 100 μm. Part of this figure is

PARP-1 activation has been shown to mediate neuronal death in a variety of disorders including ischemia, trauma, and inflammation (Virag and Szabo, 2002). PARP-1 uses the ADP-ribose group of NAD+ to form branched ADP-ribose polymers on specific acceptor proteins in the vicinity of DNA strand breaks or kinks (Burzio et al., 1979; D'Amours et al., 1999). Formation of these polymers facilitates DNA repair and prevents chromatid exchange, but extensive PARP-1 activation can promote cell death through a processes involving mitochondrial permeability transition and release of apoptosis inducing factor (Alano et al., 2004; Ha and Snyder, 1999; Yu et al., 2002). Our previous study showed that PARP-1 activation was substantially increased in hippocampal neurons after HG/GR. Rats treated with PARP-1 inhibitors after HG/GR showed a striking reduction in neuronal death, coupled with improved performance on the Morris water maze, a test of spatial learning

hypoglycemia and evaluated the production of reactive oxygen species with

modified from our previous published paper (Suh et al., JCBFM, 2008).

**2.3 The role of zinc on hypoglycemia-induced PARP-1 activation** 

prevented by ZnT3 gene deletion in mice.

and memory (Suh et al., 2003). Administration of PARP-1 inhibitors at time points up to 3 hours after HG/GR was effective in reducing neuronal death, suggesting both that PARP-1 is a downstream event in the HG/GR cell death pathway and that PARP-1 inhibitors might be useful in the clinical treatment of hypoglycemic brain injury (Figure 6).

A link between zinc release and PARP-1 activation has been suggested by studies showing PARP-1 activation and PARP-1 mediated neuronal death after neuronal exposure to zinc in cell culture, and the ability of PARP-1 inhibitors to abrogate zinc-induced cell death (Kim and Koh, 2002; Sheline et al., 2000; Sheline et al., 2003; Virag and Szabo, 2002). How zinc leads to PARP-1 activation has not been firmly established, but zinc has been shown to induce formation of reactive oxygen species through actions on mitochondria (Ichord et al., 1999) and through up-regulation of NADPH oxidase and neuronal nitric oxide synthase (Kim et al., 2002). Our previous study showed that the zinc chelator CaEDTA attenuated poly(ADPribose) formation in the post-synaptic pyramidal cells after HG/GR, suggesting that zinc translocation may be an upstream event in hypoglycemia-induced PARP-1 activation. This result, coupled with the marked reduction in neuronal death observed with CaEDTA, and the prior observation that PARP-1 inhibitors reduce hypoglycemic neuronal death (Frederickson et al., 2002), suggests a sequential process of zinc entry, PARP-1 activation, and cell death triggered by HG/GR. These results do not, however, exclude other mechanisms by which vesicular zinc release could contribute to hypoglycemic neuronal death.

Fig. 6. Hypoglycemia/ glucose reperfusion-induced poly(ADP-ribose) formation in CA1 hippocampus in rats.

A) Poly(ADP-ribose) immunoreactivity was only modestly increased at termination of immediately after HG/GR (0 hr), but was markedly increased at 3 hr after insult, and then slowly declined after that point in the hippocampal CA1 and DG area. Scale bar is 50 μm. B) Poly(ADP-ribose) formation was reduced by administration of zinc chelator, CaEDTA, at the time of glucose correction. Scale bar is 50 μm.

Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 311

Fig. 7. Hypoglycemia-induced microglia activation is prevented by zinc chelation.

activation in the hippocampal CA1 region. However, zinc chelation by CaEDTA

saline treated group.

(A) Morphological change and intensity of immunostaining of microglia after hypoglycemia is affected by zinc chelation. Hypoglycemia (HG+saline) substantially increased microglia

mild hypothermia also can prevent hypoglycemia-induced neuronal death. Neuronal death evaluated in hippocampal area shows that hypothermia significantly reduced neuronal death while hyperthermia applied after hypoglycemic events aggravated the neuronal death (Shin et al., 2010). The neuroprotective effects of hypothermia after hypoglycemia in our previous study, however, differ from those reported in previous studies (Agardh et al., 1992). Agardh et al. reported that mild hypothermia applied before and during of hypoglycemia (before and entire period of iso-EEG period) produced a similar degree of neuronal death compared to normothermic animals. No neuroprotective effect of hypothermia was seen in the hypoglycemic animals. The differences between our study and Agardh et al.'s may be explained by the onset of hypothermia application. Agardh et al. applied hypothermia before and during the iso-EEG period. However, in our study,

(HG+CaEDTA) or clioquinol (HG+CQ) significantly reduced microglia activation in the above areas. Scale bar=100 *μ*m. (B) Quantification of microglia activation was performed in the hippocampal CA1 area. As shown in the images, microglia activation is strongly prevented by zinc chelation. Data are mean±s.e.m. (*n*=3 to 6); \**P*<0.05 compared with the

#### **2.4 The role of zinc on hypoglycemia-induced microglia activation**

Microglia is thought to be the resident immune cells of the central nervous system (CNS). Under physical conditions, resting microglia adopts the characteristic ramified morphological appearance and scatter throughout mature CNS to play role in the immune surveillance and host defense. The resting microglia transform into an activated states including amoeboid morphology, up-regulation of proliferation and release of proinflammatory mediators, when the cells bind to pathogen-derived molecules or other microglial activating agents. The pro-inflammatory cytokines such as interleukin-1 and tumor necrosis factor alpha, released from activated microglia following ischemia, brain trauma and the other brain damages (Clausen et al., 2005; Sairanen et al., 1997; Saito et al., 1996; Taupin et al., 1993), are thought to be associated with neuronal death (Loddick and Rothwell, 1996; Lu et al., 2005; Yamasaki et al., 1995). On the other hand, these cytokines have been reported to induce nerve growth factor expression or cell survival signaling (DeKosky et al., 1994), (Fontaine et al., 2002) (Herx et al., 2000). Moreover activated microglia have been reported to release neurotrophic factors such as brain-derived neurotrophic factor (Lee et al., 2002b). These reports are implying that microglia activation is not only neurotoxic but neurotrophic. However, the factors that trigger microglial activation have not been completely understood. Recently, poly (ADP-ribose) polymerase (PARP)-1 has been known to act as a coactivator of nuclear factor kappa B (NF-kB), which leads to microglial migration on excitotoxically damaged organotypic hippocampal slice culture, and neuronal cell death (Chiarugi and Moskowitz, 2003) and (Ullrich et al., 2001). Furthermore, in zinc-induced cell death of neuron cultures, PARP-1 has been reported to be activated by zinc through NADPH oxidase pathway (Sheline et al., 2003), (Kim and Koh, 2002). In our previous study, we sought to examine whether zinc induces microglial activation and how microglia is activated by zinc. We found that zinc can induce microglial activation which mediated by PARP-1 activation though NADPH oxidase pathway and that microglial activation in mice ischemic brain are blocked by zinc chelator (Kauppinen et al., 2008). During severe hypoglycemia, glucose reperfusion and its neurotoxic cascade may not only damage neurons directly, but may also promote neuronal injury indirectly via microglia activation. Microglia activation is a gradual process including change of morphology from highly ramified into an amoeboid shape, proliferation, migration to injury site, increased expression of surface molecules, increased secretion of cytokines, chemokines, free radicals and proteases, and assumption of phagocytotic activity (Kreutzberg, 1996). We tested whether zinc chelation prevents microglia activation after hypoglycemia. Both CaEDTA and CQ substantially decreased hypoglycemia-induced microglia activation in the hippocampal CA1 pyramidal area (Figure 7).

#### **2.5 Prevention of hypoglycemia-induced neuronal death by hypothermia**

Our previous study presented that mild hypothermia reduces hypoglycemia-induced neuronal death in the hippocampus, whereas hyperthermia aggravates those brain injuries. We suggested that hypothermia (lowering brain temperature) prevents hypoglycemiainduced neuronal death by reduction of vesicular zinc release, superoxide production and microglia activation, where temperature dependent vesicular zinc release was a key event upstream of hypoglycemia-induced superoxide production and microglia activation.

Mild hypothermia has been known as the most effective approach to prevent neuronal death after cerebral ischemia (Busto et al., 1987; Maier et al., 2002), traumatic brain injury (Clifton et al., 1991; Suh et al., 2006) and prolonged seizure (Liu et al., 1993). We found that

Microglia is thought to be the resident immune cells of the central nervous system (CNS). Under physical conditions, resting microglia adopts the characteristic ramified morphological appearance and scatter throughout mature CNS to play role in the immune surveillance and host defense. The resting microglia transform into an activated states including amoeboid morphology, up-regulation of proliferation and release of proinflammatory mediators, when the cells bind to pathogen-derived molecules or other microglial activating agents. The pro-inflammatory cytokines such as interleukin-1 and tumor necrosis factor alpha, released from activated microglia following ischemia, brain trauma and the other brain damages (Clausen et al., 2005; Sairanen et al., 1997; Saito et al., 1996; Taupin et al., 1993), are thought to be associated with neuronal death (Loddick and Rothwell, 1996; Lu et al., 2005; Yamasaki et al., 1995). On the other hand, these cytokines have been reported to induce nerve growth factor expression or cell survival signaling (DeKosky et al., 1994), (Fontaine et al., 2002) (Herx et al., 2000). Moreover activated microglia have been reported to release neurotrophic factors such as brain-derived neurotrophic factor (Lee et al., 2002b). These reports are implying that microglia activation is not only neurotoxic but neurotrophic. However, the factors that trigger microglial activation have not been completely understood. Recently, poly (ADP-ribose) polymerase (PARP)-1 has been known to act as a coactivator of nuclear factor kappa B (NF-kB), which leads to microglial migration on excitotoxically damaged organotypic hippocampal slice culture, and neuronal cell death (Chiarugi and Moskowitz, 2003) and (Ullrich et al., 2001). Furthermore, in zinc-induced cell death of neuron cultures, PARP-1 has been reported to be activated by zinc through NADPH oxidase pathway (Sheline et al., 2003), (Kim and Koh, 2002). In our previous study, we sought to examine whether zinc induces microglial activation and how microglia is activated by zinc. We found that zinc can induce microglial activation which mediated by PARP-1 activation though NADPH oxidase pathway and that microglial activation in mice ischemic brain are blocked by zinc chelator (Kauppinen et al., 2008). During severe hypoglycemia, glucose reperfusion and its neurotoxic cascade may not only damage neurons directly, but may also promote neuronal injury indirectly via microglia activation. Microglia activation is a gradual process including change of morphology from highly ramified into an amoeboid shape, proliferation, migration to injury site, increased expression of surface molecules, increased secretion of cytokines, chemokines, free radicals and proteases, and assumption of phagocytotic activity (Kreutzberg, 1996). We tested whether zinc chelation prevents microglia activation after hypoglycemia. Both CaEDTA and CQ substantially decreased hypoglycemia-induced microglia activation in the hippocampal

**2.4 The role of zinc on hypoglycemia-induced microglia activation** 

CA1 pyramidal area (Figure 7).

**2.5 Prevention of hypoglycemia-induced neuronal death by hypothermia** 

Our previous study presented that mild hypothermia reduces hypoglycemia-induced neuronal death in the hippocampus, whereas hyperthermia aggravates those brain injuries. We suggested that hypothermia (lowering brain temperature) prevents hypoglycemiainduced neuronal death by reduction of vesicular zinc release, superoxide production and microglia activation, where temperature dependent vesicular zinc release was a key event

Mild hypothermia has been known as the most effective approach to prevent neuronal death after cerebral ischemia (Busto et al., 1987; Maier et al., 2002), traumatic brain injury (Clifton et al., 1991; Suh et al., 2006) and prolonged seizure (Liu et al., 1993). We found that

upstream of hypoglycemia-induced superoxide production and microglia activation.

Fig. 7. Hypoglycemia-induced microglia activation is prevented by zinc chelation. (A) Morphological change and intensity of immunostaining of microglia after hypoglycemia is affected by zinc chelation. Hypoglycemia (HG+saline) substantially increased microglia activation in the hippocampal CA1 region. However, zinc chelation by CaEDTA (HG+CaEDTA) or clioquinol (HG+CQ) significantly reduced microglia activation in the above areas. Scale bar=100 *μ*m. (B) Quantification of microglia activation was performed in the hippocampal CA1 area. As shown in the images, microglia activation is strongly prevented by zinc chelation. Data are mean±s.e.m. (*n*=3 to 6); \**P*<0.05 compared with the saline treated group.

mild hypothermia also can prevent hypoglycemia-induced neuronal death. Neuronal death evaluated in hippocampal area shows that hypothermia significantly reduced neuronal death while hyperthermia applied after hypoglycemic events aggravated the neuronal death (Shin et al., 2010). The neuroprotective effects of hypothermia after hypoglycemia in our previous study, however, differ from those reported in previous studies (Agardh et al., 1992). Agardh et al. reported that mild hypothermia applied before and during of hypoglycemia (before and entire period of iso-EEG period) produced a similar degree of neuronal death compared to normothermic animals. No neuroprotective effect of hypothermia was seen in the hypoglycemic animals. The differences between our study and Agardh et al.'s may be explained by the onset of hypothermia application. Agardh et al. applied hypothermia before and during the iso-EEG period. However, in our study,

Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 313

Fig. 8. Temperature dependent hypoglycemic neuronal death is mediated by zinc release

(Sham) and hypoglycemia (HG) experienced rats. Hypothermia group (Hypo) almost completely prevented synaptic zinc release. Scale bar = 500 μm. (B) Bar graph shows

0.05 compared with normothermic reperfusion group. (C) Photomicrographs of TSQ fluorescence staining shows zinc accumulation in the hippocampal CA1 neurons after hypoglycemia. Scale bar = 100 μm. (D) Bar graph shows quantitated TSQ (+) neurons in the CA1 area. Data are mean ± s.e.m. (n = 5-7). \**p* < 0.05 compared with normothermic glucose reperfusion group. (E-H) Zinc chelators, CaEDTA or clioquinol (CQ), prevents hypoglycemiainduced neuronal death. (E and G). FJB (+) neurons were reduced by CaEDTA or CQ injection

even after hyperthermic reperfusion. Scale bar = 100 μm. (F and G) graphs represent

figure is modified from our previous published paper (Suh et al., JCBFM, 2010).

(A-D) Vesicular zinc release and translocation is aggravated by hyperthermia but is prevented by hypothermia. (A) represents TSQ fluorescence images of hippocampus from sham operated

quantitated TSQ fluorescence intensity from hilus area. Data are mean + s.e.m. (n=7-12). \* *P* <

quantitated neuronal death in the hippocampal CA1 and subiculum area after hypoglycemia. Data are the mean ± s.e.m (n=5-7) \**p* < 0.05 compared with saline treated rats. Part of this

cannot exclude the possibility that intracellularly originated free zinc also contributes to hippocampal neuron cell death after hypoglycemia as previously suggested (Aizenman et al., 2000). Anatomical and physiological studies have shown that DG neurons contain a high concentration of vesicular zinc in their synaptic terminals which is released with neuronal activity. Intraneuronal accumulation of zinc may arise from cytoplasmic organelles or

and translocation.

hypothermia applied after the iso-EEG period was terminated, i.e. brain temperature was decreased during the glucose reperfusion period after hypoglycemia. Since we have previously shown that hypoglycemia-induced neuronal death is not initiated during the period of glucose deprivation but instead during glucose reperfusion period, it may be that the hypothermic application before and during the isoelectric period was not sufficient to prevent neuronal death after hypoglycemic events. In our experimental setting we also found that hypothermia application before and during the iso-EEG period had no statistically significant neuroprotective effects as seen in the previous study (Agardh et al., 1992), strengthening our hypothesis that brain temperature is a critical factor during glucose reperfusion period after hypoglycemia.

Suggested neuroprotective mechanisms of mild hypothermia on several brain injuries are based on decreases in cerebral metabolic requirement (Erecinska et al., 2003), intracranial pressure (Soukup et al., 2002), glutamate release from presynaptic vesicles (Arai et al., 1993; Ichord et al., 1999), free radical generation (Globus et al., 1995; Horiguchi et al., 2003) and inflammatory reaction (Kumar and Evans, 1997; Wang et al., 2002). Previously, we have shown that hypothermia reduced vesicular zinc release and subsequent neuronal death after traumatic brain injury (Suh et al., 2006). We also have shown that hypoglycemia-induced neuronal death is mediated by vesicular zinc release and translocation (Suh et al., 2004; Suh et al., 2008). Therefore, we hypothesized that mild hypothermia has neuroprotective effects by reduction of the vesicular zinc release after hypoglycemia. Although zinc is released from presynaptic terminals as a component of normal physiologic signaling at zinc-modulated synapses (Li et al., 2001), a large amount of vesicular zinc released together with glutamate may enter postsynaptic neurons through glutamate receptors (Weiss and Sensi, 2000; Weiss et al., 2000) or voltage-sensitive calcium channels (Sensi et al., 1999b). Zinc translocation into post-synaptic neurons after hypoglycemia has been demonstrated by our lab (Suh et al., 2004; Suh et al., 2007; Suh et al., 2008). Many brain areas with high vesicular zinc level exhibit high vulnerability to hypoglycemia, but this correlation is not always true. Some brain areas with high vesicular zinc concentration are not correspondingly sensitive to hypoglycemia, and conversely some brain areas that are highly sensitive to hypoglycemia are not rich in vesicular zinc (Frederickson et al., 2000). Thus vesicular zinc is not the sole determinant of neuronal vulnerability to hypoglycemia, but may be a contributory factor in areas where vesicular concentrations are high. The zinc chelator CaEDTA was used to evaluate a causal role for extracellular zinc elevations in subsequent post-synaptic neuronal zinc accumulation and death after hypoglycemia. The utility of CaEDTA as a zinc chelator has been established in ischemia, brain trauma and epilepsy studies (Frederickson et al., 2002; Koh et al., 1996; Lee et al., 2002a). Interestingly, Aizenmann et al. suggested that the large fraction of zinc existing in the form of thiol-zinc-metalloproteins can be released from oxidation of intracellular zinc binding proteins (e.g. metallothionein) by oxidative stress. Zinc liberated in such a manner may then become cytotoxic (Aizenman et al., 2000). Our study showed that application of mild hypothermia significantly reduced hypoglycemiainduce neuronal death by reducing presynaptic zinc release and translocation into postsynaptic neurons (Figure 8) (Shin et al., 2010). Hyperthermia applied after hypoglycemia aggravates this zinc release and translocation compared to normothermia applied animals. From these results, we conclude that neuroprotective effects of mild hypothermia after hypoglycemia can be achieved by reduction of synaptic zinc release and subsequent zinc translocation. However, our study also found that zinc dependent DG neuron degeneration was prevented by the cell permeable zinc chelator, CQ. We therefore

hypothermia applied after the iso-EEG period was terminated, i.e. brain temperature was decreased during the glucose reperfusion period after hypoglycemia. Since we have previously shown that hypoglycemia-induced neuronal death is not initiated during the period of glucose deprivation but instead during glucose reperfusion period, it may be that the hypothermic application before and during the isoelectric period was not sufficient to prevent neuronal death after hypoglycemic events. In our experimental setting we also found that hypothermia application before and during the iso-EEG period had no statistically significant neuroprotective effects as seen in the previous study (Agardh et al., 1992), strengthening our hypothesis that brain temperature is a critical factor during glucose

Suggested neuroprotective mechanisms of mild hypothermia on several brain injuries are based on decreases in cerebral metabolic requirement (Erecinska et al., 2003), intracranial pressure (Soukup et al., 2002), glutamate release from presynaptic vesicles (Arai et al., 1993; Ichord et al., 1999), free radical generation (Globus et al., 1995; Horiguchi et al., 2003) and inflammatory reaction (Kumar and Evans, 1997; Wang et al., 2002). Previously, we have shown that hypothermia reduced vesicular zinc release and subsequent neuronal death after traumatic brain injury (Suh et al., 2006). We also have shown that hypoglycemia-induced neuronal death is mediated by vesicular zinc release and translocation (Suh et al., 2004; Suh et al., 2008). Therefore, we hypothesized that mild hypothermia has neuroprotective effects by reduction of the vesicular zinc release after hypoglycemia. Although zinc is released from presynaptic terminals as a component of normal physiologic signaling at zinc-modulated synapses (Li et al., 2001), a large amount of vesicular zinc released together with glutamate may enter postsynaptic neurons through glutamate receptors (Weiss and Sensi, 2000; Weiss et al., 2000) or voltage-sensitive calcium channels (Sensi et al., 1999b). Zinc translocation into post-synaptic neurons after hypoglycemia has been demonstrated by our lab (Suh et al., 2004; Suh et al., 2007; Suh et al., 2008). Many brain areas with high vesicular zinc level exhibit high vulnerability to hypoglycemia, but this correlation is not always true. Some brain areas with high vesicular zinc concentration are not correspondingly sensitive to hypoglycemia, and conversely some brain areas that are highly sensitive to hypoglycemia are not rich in vesicular zinc (Frederickson et al., 2000). Thus vesicular zinc is not the sole determinant of neuronal vulnerability to hypoglycemia, but may be a contributory factor in areas where vesicular concentrations are high. The zinc chelator CaEDTA was used to evaluate a causal role for extracellular zinc elevations in subsequent post-synaptic neuronal zinc accumulation and death after hypoglycemia. The utility of CaEDTA as a zinc chelator has been established in ischemia, brain trauma and epilepsy studies (Frederickson et al., 2002; Koh et al., 1996; Lee et al., 2002a). Interestingly, Aizenmann et al. suggested that the large fraction of zinc existing in the form of thiol-zinc-metalloproteins can be released from oxidation of intracellular zinc binding proteins (e.g. metallothionein) by oxidative stress. Zinc liberated in such a manner may then become cytotoxic (Aizenman et al., 2000). Our study showed that application of mild hypothermia significantly reduced hypoglycemiainduce neuronal death by reducing presynaptic zinc release and translocation into postsynaptic neurons (Figure 8) (Shin et al., 2010). Hyperthermia applied after hypoglycemia aggravates this zinc release and translocation compared to normothermia applied animals. From these results, we conclude that neuroprotective effects of mild hypothermia after hypoglycemia can be achieved by reduction of synaptic zinc release and subsequent zinc translocation. However, our study also found that zinc dependent DG neuron degeneration was prevented by the cell permeable zinc chelator, CQ. We therefore

reperfusion period after hypoglycemia.

Fig. 8. Temperature dependent hypoglycemic neuronal death is mediated by zinc release and translocation.

(A-D) Vesicular zinc release and translocation is aggravated by hyperthermia but is prevented by hypothermia. (A) represents TSQ fluorescence images of hippocampus from sham operated (Sham) and hypoglycemia (HG) experienced rats. Hypothermia group (Hypo) almost completely prevented synaptic zinc release. Scale bar = 500 μm. (B) Bar graph shows quantitated TSQ fluorescence intensity from hilus area. Data are mean + s.e.m. (n=7-12). \* *P* < 0.05 compared with normothermic reperfusion group. (C) Photomicrographs of TSQ fluorescence staining shows zinc accumulation in the hippocampal CA1 neurons after hypoglycemia. Scale bar = 100 μm. (D) Bar graph shows quantitated TSQ (+) neurons in the CA1 area. Data are mean ± s.e.m. (n = 5-7). \**p* < 0.05 compared with normothermic glucose reperfusion group. (E-H) Zinc chelators, CaEDTA or clioquinol (CQ), prevents hypoglycemiainduced neuronal death. (E and G). FJB (+) neurons were reduced by CaEDTA or CQ injection even after hyperthermic reperfusion. Scale bar = 100 μm. (F and G) graphs represent quantitated neuronal death in the hippocampal CA1 and subiculum area after hypoglycemia. Data are the mean ± s.e.m (n=5-7) \**p* < 0.05 compared with saline treated rats. Part of this figure is modified from our previous published paper (Suh et al., JCBFM, 2010).

cannot exclude the possibility that intracellularly originated free zinc also contributes to hippocampal neuron cell death after hypoglycemia as previously suggested (Aizenman et al., 2000). Anatomical and physiological studies have shown that DG neurons contain a high concentration of vesicular zinc in their synaptic terminals which is released with neuronal activity. Intraneuronal accumulation of zinc may arise from cytoplasmic organelles or

Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 315

1) Modulation of vesicular zinc release by gene manipulation; 2) Prevention of vesicular zinc

intervention strategy requires a highly zinc specific chelator, which also can permeate blood brain barrier and has no side effects. No such agent is currently available and further investigation will be necessary to identify and develop candidate drugs for this purpose.

Vesicular zinc release and subsequent translocation of this ion into postsynaptic neurons has been known as a key upstream event of hypoglycemia-induced neuron death. Thus, zinc chelation is a promising target for the treatment of severe hypoglycemia-induced neuron

This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A100687) and Korea Science and

Agardh, C.D., Smith, M.L., and Siesjo, B.K. (1992). The influence of hypothermia on hypoglycemia-induced brain damage in the rat. Acta Neuropathol *83*, 379-385. Aizenman, E., Stout, A.K., Hartnett, K.A., Dineley, K.E., McLaughlin, B., and Reynolds, I.J.

Alano, C.C., Ying, W., and Swanson, R.A. (2004). Poly(ADP-ribose) polymerase-1-mediated

Arai, H., Uto, A., Ogawa, Y., and Sato, K. (1993). Effect of low temperature on glutamate-

Assaf, S.Y., and Chung, S.H. (1984). Release of endogenous Zn2+ from brain tissue during

Auer, R.N., Olsson, Y., and Siesjo, B.K. (1984a). Hypoglycemic brain injury in the rat.

Auer, R.N., and Siesjo, B.K. (1993). Hypoglycaemia: brain neurochemistry and

Auer, R.N., Wieloch, T., Olsson, Y., and Siesjo, B.K. (1984b). The distribution of

Beckman, J.S., and Koppenol, W.H. (1996). Nitric oxide, superoxide, and peroxynitrite: the

neuropathology. Baillieres Clin Endocrinol Metab *7*, 611-625.

hypoglycemic brain damage. Acta Neuropathol *64*, 177-191.

good, the bad, and ugly. Am J Physiol *271*, C1424-1437.

intracellular zinc release. J Neurochem *75*, 1878-1888.

transition. J Biol Chem *279*, 18895-18902.

neurons. Neurosci Lett *163*, 132-134.

activity. Nature *308*, 734-736.

study. Diabetes *33*, 1090-1098.

(2000). Induction of neuronal apoptosis by thiol oxidation: putative role of

cell death in astrocytes requires NAD+ depletion and mitochondrial permeability

induced intracellular calcium accumulation and cell death in cultured hippocampal

Correlation of density of brain damage with the EEG isoelectric time: a quantitative

death. However, still further studies will be needed to apply this concept to human.

release by NOS inhibition; 3) hypothermia; 4) Chelation of extracellular zinc by zinc chelators; 5) Inhibition of NADPH oxidase activation; 6) Increase of SOD function; 7) PARP-1 inhibition. Among them, we speculate that prevention of vesicular zinc release and translocation would be the most promising intervention strategies. However, this

**4. Conclusion** 

**6. References** 

**5. Acknowledgement** 

Engineering Foundation (KOSEF- 2009-0078399).

proteins rather than from presynaptic terminals of stratum moleculare. However, the source of intraneuronal accumulation of zinc in DG neurons still requires further study. An additional unsolved question arises regarding how the extracellular zinc chelator, CaEDTA also prevented DG neuron death if intraneuronal zinc accumulation originates from cytoplasmic sources.

Taken together, the present study shows that post-hypoglycemic (glucose reperfusion period) brain temperature can modulate the outcome of brain injury, i.e. hypothermia significantly reduces, while hyperthermia aggravates, neuronal death after hypoglycemia through inhibition of vesicular zinc release, reduction of ROS production and prevention of microglia activation. Therefore, cautious brain temperature monitoring and maintaining lower brain temperature during glucose reperfusion period may predict a better clinical outcome after a severe hypoglycemic episode.

## **3. Proposed intervention strategies for hypoglycemia-induced neuron death**

Taken together the present book chapter suggests a sequence of events that lead to neuronal death after HG/GR. Glucose reperfusion initiates nitric oxide production, which leads to vesicular zinc release, which in turn activates neuronal NADPH oxidase. ROS produced by NADPH oxidase leads to increased zinc accumulation, PARP-1 activation, and resultant cell death. Therefore, based on these studies, the present review suggests that following intervention strategies for preventing hypoglycemia-induced neuron death. As we described in schematic drawing (Figure 9), there are at least 6 different possible approaches.

Fig. 9. Proposed intervention strategies for preventing hypoglycemia/ glucose reperfusioninduced neuron death. This schematic drawing indicates that hypoglycemia/ glucose reperfusion-induced neuron death can be prevented by several intervention methods. 1) Vesicular zinc content modulation by gene or chemical manipulation. 2) Vesicular zinc release inhibition by NO inhibitor. 3) Vesicular zinc release inhibition by hypothermia. 4) Zinc chelation in the extracellular space. 5) Inhibition of NADPH oxidase activation. 6) Scavenging or dismutating of reactive oxygen species. 7) Inhibition of PARP-1 activation. Round red colored dot represents ionic zinc. Symbol X represents intervention.

1) Modulation of vesicular zinc release by gene manipulation; 2) Prevention of vesicular zinc release by NOS inhibition; 3) hypothermia; 4) Chelation of extracellular zinc by zinc chelators; 5) Inhibition of NADPH oxidase activation; 6) Increase of SOD function; 7) PARP-1 inhibition. Among them, we speculate that prevention of vesicular zinc release and translocation would be the most promising intervention strategies. However, this intervention strategy requires a highly zinc specific chelator, which also can permeate blood brain barrier and has no side effects. No such agent is currently available and further investigation will be necessary to identify and develop candidate drugs for this purpose.

### **4. Conclusion**

314 Diabetes – Damages and Treatments

proteins rather than from presynaptic terminals of stratum moleculare. However, the source of intraneuronal accumulation of zinc in DG neurons still requires further study. An additional unsolved question arises regarding how the extracellular zinc chelator, CaEDTA also prevented DG neuron death if intraneuronal zinc accumulation originates from

Taken together, the present study shows that post-hypoglycemic (glucose reperfusion period) brain temperature can modulate the outcome of brain injury, i.e. hypothermia significantly reduces, while hyperthermia aggravates, neuronal death after hypoglycemia through inhibition of vesicular zinc release, reduction of ROS production and prevention of microglia activation. Therefore, cautious brain temperature monitoring and maintaining lower brain temperature during glucose reperfusion period may predict a better clinical

**3. Proposed intervention strategies for hypoglycemia-induced neuron death**  Taken together the present book chapter suggests a sequence of events that lead to neuronal death after HG/GR. Glucose reperfusion initiates nitric oxide production, which leads to vesicular zinc release, which in turn activates neuronal NADPH oxidase. ROS produced by NADPH oxidase leads to increased zinc accumulation, PARP-1 activation, and resultant cell death. Therefore, based on these studies, the present review suggests that following intervention strategies for preventing hypoglycemia-induced neuron death. As we described in schematic drawing (Figure 9), there are at least 6 different possible approaches.

Fig. 9. Proposed intervention strategies for preventing hypoglycemia/ glucose reperfusioninduced neuron death. This schematic drawing indicates that hypoglycemia/ glucose reperfusion-induced neuron death can be prevented by several intervention methods. 1) Vesicular zinc content modulation by gene or chemical manipulation. 2) Vesicular zinc release inhibition by NO inhibitor. 3) Vesicular zinc release inhibition by hypothermia. 4) Zinc chelation in the extracellular space. 5) Inhibition of NADPH oxidase activation. 6) Scavenging or dismutating of reactive oxygen species. 7) Inhibition of PARP-1 activation.

Round red colored dot represents ionic zinc. Symbol X represents intervention.

cytoplasmic sources.

outcome after a severe hypoglycemic episode.

Vesicular zinc release and subsequent translocation of this ion into postsynaptic neurons has been known as a key upstream event of hypoglycemia-induced neuron death. Thus, zinc chelation is a promising target for the treatment of severe hypoglycemia-induced neuron death. However, still further studies will be needed to apply this concept to human.

### **5. Acknowledgement**

This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A100687) and Korea Science and Engineering Foundation (KOSEF- 2009-0078399).

### **6. References**


Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 317

Frederickson, C.J., Hernandez, M.D., Goik, S.A., Morton, J.D., and McGinty, J.F. (1988). Loss

Frederickson, C.J., Kasarskis, E.J., Ringo, D., and Frederickson, R.E. (1987). A quinoline

Frederickson, C.J., Koh, J.Y., and Bush, A.I. (2005). The neurobiology of zinc in health and

Frederickson, C.J., Suh, S.W., Silva, D., and Thompson, R.B. (2000). Importance of zinc in the central nervous system: the zinc-containing neuron. J Nutr *130*, 1471S-1483S. Friberg, H., Ferrand-Drake, M., Bengtsson, F., Halestrap, A.P., and Wieloch, T. (1998).

Globus, M.Y., Busto, R., Lin, B., Schnippering, H., and Ginsberg, M.D. (1995). Detection of

intraischemic brain temperature modulation. J Neurochem *65*, 1250-1256. Groemping, Y., and Rittinger, K. (2005). Activation and assembly of the NADPH oxidase: a

Ha, H.C., and Snyder, S.H. (1999). Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc Natl Acad Sci U S A *96*, 13978-13982. Herx, L.M., Rivest, S., and Yong, V.W. (2000). Central nervous system-initiated

Howell, G.A., Welch, M.G., and Frederickson, C.J. (1984). Stimulation-induced uptake and

Ichord, R.N., Northington, F.J., van Wylen, D., Johnston, M.V., Kwon, C., and Traystman,

coma in piglets are temperature sensitive. Am J Physiol *276*, H2053-2062. Kauppinen, T.M., Higashi, Y., Suh, S.W., Escartin, C., Nagasawa, K., and Swanson, R.A.

Kim, T.Y., Hwang, J.J., Yun, S.H., Jung, M.W., and Koh, J.Y. (2002). Augmentation by zinc of

Kim, Y.H., Kim, E.Y., Gwag, B.J., Sohn, S., and Koh, J.Y. (1999). Zinc-induced cortical

Kim, Y.H., and Koh, J.Y. (2002). The role of NADPH oxidase and neuronal nitric oxide

Koh, J.Y., Suh, S.W., Gwag, B.J., He, Y.Y., Hsu, C.Y., and Choi, D.W. (1996). The role of zinc

(2008). Zinc triggers microglial activation. J Neurosci *28*, 5827-5835.

mediation by Src family tyrosine kinases. Synapse *46*, 49-56.

Neuroscience *89*, 175-182.

1013-1016.

cortical culture. Exp Neurol *177*, 407-418.

production of ciliary neurotrophic factor. J Immunol *165*, 2232-2239. Horiguchi, T., Shimizu, K., Ogino, M., Suga, S., Inamasu, J., and Kawase, T. (2003).

transient forebrain ischemia in rats. J Neurotrauma *20*, 511-520.

release of zinc in hippocampal slices. Nature *308*, 736-738.

seizures: a histofluorescence study. Brain Res *446*, 383-386.

(bouton zinc) in the brain. J Neurosci Methods *20*, 91-103.

disease. Nat Rev Neurosci *6*, 449-462.

cell death. J Neurosci *18*, 5151-5159.

structural perspective. Biochem J *386*, 401-416.

of zinc staining from hippocampal mossy fibers during kainic acid induced

fluorescence method for visualizing and assaying the histochemically reactive zinc

Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in

free radical activity during transient global ischemia and recirculation: effects of

inflammation and neurotrophism in trauma: IL-1 beta is required for the

Postischemic hypothermia inhibits the generation of hydroxyl radical following

R.J. (1999). Brain O2 consumption and glutamate release during hypoglycemic

NMDA receptor-mediated synaptic responses in CA1 of rat hippocampal slices:

neuronal death with features of apoptosis and necrosis: mediation by free radicals.

synthase in zinc-induced poly(ADP-ribose) polymerase activation and cell death in

in selective neuronal death after transient global cerebral ischemia. Science *272*,


Burzio, L.O., Riquelme, P.T., and Koide, S.S. (1979). ADP ribosylation of rat liver

Busto, R., Dietrich, W.D., Globus, M.Y., Valdes, I., Scheinberg, P., and Ginsberg, M.D. (1987).

Chiarugi, A., and Moskowitz, M.A. (2003). Poly(ADP-ribose) polymerase-1 activity

Clausen, B.H., Lambertsen, K.L., Meldgaard, M., and Finsen, B. (2005). A quantitative in situ

Clifton, G.L., Jiang, J.Y., Lyeth, B.G., Jenkins, L.W., Hamm, R.J., and Hayes, R.L. (1991).

Cuajungco, M.P., and Lees, G.J. (1998). Nitric oxide generators produce accumulation of chelatable zinc in hippocampal neuronal perikarya. Brain Res *799*, 118-129. D'Amours, D., Desnoyers, S., D'Silva, I., and Poirier, G.G. (1999). Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem J *342*, 249-268. Danscher, G., Howell, G., Perez-Clausell, J., and Hertel, N. (1985). The dithizone, Timm's

DeKosky, S.T., Goss, J.R., Miller, P.D., Styren, S.D., Kochanek, P.M., and Marion, D. (1994).

Dodd, O.J., and Pearse, D.B. (2000). Effect of the NADPH oxidase inhibitor apocynin on ischemia-reperfusion lung injury. Am J Physiol Heart Circ Physiol *279*, H303-312. Erecinska, M., Thoresen, M., and Silver, I.A. (2003). Effects of hypothermia on energy

Ferrand-Drake, M., Zhu, C., Gido, G., Hansen, A.J., Karlsson, J.O., Bahr, B.A., Zamzami, N.,

Fontaine, V., Mohand-Said, S., Hanoteau, N., Fuchs, C., Pfizenmaier, K., and Eisel, U. (2002).

Frederickson, C.J. (1989). Neurobiology of zinc and zinc-containing neurons. Int Rev

Frederickson, C.J., Cuajungco, M.P., LaBuda, C.J., and Suh, S.W. (2002). Nitric oxide causes apparent release of zinc from presynaptic boutons. Neuroscience *115*, 471-474.

of ischemic neuronal injury. J Cereb Blood Flow Metab *7*, 729-738.

for neurodegenerative disorders. J Neurochem *85*, 306-317.

occlusion in mice. Neuroscience *132*, 879-892.

injury. J Cereb Blood Flow Metab *11*, 114-121.

422.

173-177.

513-530.

1442.

*22*, RC216.

Neurobiol *31*, 145-238.

Small differences in intraischemic brain temperature critically determine the extent

promotes NF-kappaB-driven transcription and microglial activation: implication

hybridization and polymerase chain reaction study of microglial-macrophage expression of interleukin-1beta mRNA following permanent middle cerebral artery

Marked protection by moderate hypothermia after experimental traumatic brain

sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS. A proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitally treated with dithizone. Histochemistry *83*, 419-

Upregulation of nerve growth factor following cortical trauma. Exp Neurol *130*,

metabolism in Mammalian central nervous system. J Cereb Blood Flow Metab *23*,

Kroemer, G., Chan, P.H., Wieloch, T., and Blomgren, K. (2003). Cyclosporin A prevents calpain activation despite increased intracellular calcium concentrations, as well as translocation of apoptosis-inducing factor, cytochrome c and caspase-3 activation in neurons exposed to transient hypoglycemia. J Neurochem *85*, 1431-

Neurodegenerative and neuroprotective effects of tumor Necrosis factor (TNF) in retinal ischemia: opposite roles of TNF receptor 1 and TNF receptor 2. J Neurosci

nucleosomal core histones. J Biol Chem *254*, 3029-3037.


Zinc Translocation Causes Hypoglycemia-Induced Neuron Death 319

Sheline, C.T., Behrens, M.M., and Choi, D.W. (2000). Zinc-induced cortical neuronal death:

Sheline, C.T., Wang, H., Cai, A.L., Dawson, V.L., and Choi, D.W. (2003). Involvement of poly

Shin, B.S., Won, S.J., Yoo, B.H., Kauppinen, T.M., and Suh, S.W. (2010). Prevention of

Soukup, J., Zauner, A., Doppenberg, E.M., Menzel, M., Gilman, C., Young, H.F., and

Stolk, J., Hiltermann, T.J., Dijkman, J.H., and Verhoeven, A.J. (1994). Characteristics of the

Suh, S.W., Aoyama, K., Chen, Y., Garnier, P., Matsumori, Y., Gum, E., Liu, J., and Swanson,

Suh, S.W., Chen, J.W., Motamedi, M., Bell, B., Listiak, K., Pons, N.F., Danscher, G., and

Suh, S.W., Frederickson, C.J., and Danscher, G. (2006). Neurotoxic zinc translocation into

Suh, S.W., Garnier, P., Aoyama, K., Chen, Y., and Swanson, R.A. (2004). Zinc release contributes to hypoglycemia-induced neuronal death. Neurobiol Dis *16*, 538-545. Suh, S.W., Gum, E.T., Hamby, A.M., Chan, P.H., and Swanson, R.A. (2007). Hypoglycemic

Suh, S.W., Hamby, A.M., Gum, E.T., Shin, B.S., Won, S.J., Sheline, C.T., Chan, P.H., and

Taupin, V., Toulmond, S., Serrano, A., Benavides, J., and Zavala, F. (1993). Increase in IL-6,

Tonder, N., Johansen, F.F., Frederickson, C.J., Zimmer, J., and Diemer, N.H. (1990). Possible

hypoglycemic neuronal death. J Cereb Blood Flow Metab *28*, 1697-1706. Suh, S.W., Thompson, R.B., and Frederickson, C.J. (2001). Loss of vesicular zinc and

neuronal injury after traumatic brain injury. Brain Res *852*, 268-273.

cerebral blood flow, and outcome. J Neurotrauma *19*, 559-571.

substituted catechol. Am J Respir Cell Mol Biol *11*, 95-102.

glycolysis. J Neurosci *20*, 3139-3146.

1402-1409.

*30*, 390-402.

169.

*12*, 1523-1525.

Neurosci *23*, 10681-10690.

NADPH oxidase. J Clin Invest *117*, 910-918.

ligand. J Neuroimmunol *42*, 177-185.

ischemia in the adult rat. Neurosci Lett *109*, 247-252.

contribution of energy failure attributable to loss of NAD(+) and inhibition of

ADP ribosyl polymerase-1 in acute but not chronic zinc toxicity. Eur J Neurosci *18*,

hypoglycemia-induced neuronal death by hypothermia. J Cereb Blood Flow Metab

Bullock, R. (2002). The importance of brain temperature in patients after severe head injury: relationship to intracranial pressure, cerebral perfusion pressure,

inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-

R.A. (2003). Hypoglycemic neuronal death and cognitive impairment are prevented by poly(ADP-ribose) polymerase inhibitors administered after hypoglycemia. J

Frederickson, C.J. (2000). Evidence that synaptically-released zinc contributes to

hippocampal neurons is inhibited by hypothermia and is aggravated by hyperthermia after traumatic brain injury in rats. J Cereb Blood Flow Metab *26*, 161-

neuronal death is triggered by glucose reperfusion and activation of neuronal

Swanson, R.A. (2008). Sequential release of nitric oxide, zinc, and superoxide in

appearance of perikaryal zinc after seizures induced by pilocarpine. Neuroreport

IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine

role of zinc in the selective degeneration of dentate hilar neurons after cerebral


Kreutzberg, G.W. (1996). Microglia: a sensor for pathological events in the CNS. Trends

Kumar, K., and Evans, A.T. (1997). Effect of hypothermia on microglial reaction in ischemic

Lee, J.Y., Cole, T.B., Palmiter, R.D., and Koh, J.Y. (2000). Accumulation of zinc in

Lee, J.Y., Cole, T.B., Palmiter, R.D., Suh, S.W., and Koh, J.Y. (2002a). Contribution by

Lee, T.H., Kato, H., Chen, S.T., Kogure, K., and Itoyama, Y. (2002b). Expression disparity of

Li, Y., Hough, C.J., Suh, S.W., Sarvey, J.M., and Frederickson, C.J. (2001). Rapid translocation

Liu, Z., Gatt, A., Mikati, M., and Holmes, G.L. (1993). Effect of temperature on kainic acid-

Loddick, S.A., and Rothwell, N.J. (1996). Neuroprotective effects of human recombinant

Lu, K.T., Wang, Y.W., Yang, J.T., Yang, Y.L., and Chen, H.I. (2005). Effect of interleukin-1 on

Maier, C.M., Sun, G.H., Cheng, D., Yenari, M.A., Chan, P.H., and Steinberg, G.K. (2002).

Noh, K.M., and Koh, J.Y. (2000). Induction and activation by zinc of NADPH oxidase in

Perez-Clausell, J., and Danscher, G. (1985). Intravesicular localization of zinc in rat

Sairanen, T.R., Lindsberg, P.J., Brenner, M., and Siren, A.L. (1997). Global forebrain ischemia

Sensi, S.L., Yin, H.Z., Carriedo, S.G., Rao, S.S., and Weiss, J.H. (1999a). Preferential Zn2+

cultured cortical neurons and astrocytes. J Neurosci *20*, RC111.

telencephalic boutons. A histochemical study. Brain Res *337*, 91-98.

degenerating hippocampal neurons of ZnT3-null mice after seizures: evidence

synaptic zinc to the gender-disparate plaque formation in human Swedish mutant

brain-derived neurotrophic factor immunoreactivity and mRNA in ischemic

of Zn(2+) from presynaptic terminals into postsynaptic hippocampal neurons after

interleukin-1 receptor antagonist in focal cerebral ischaemia in the rat. J Cereb

traumatic brain injury-induced damage to hippocampal neurons. J Neurotrauma

Effects of mild hypothermia on superoxide anion production, superoxide dismutase expression, and activity following transient focal cerebral ischemia.

results in differential cellular expression of interleukin-1beta (IL-1beta) and its receptor at mRNA and protein level. J Cereb Blood Flow Metab *17*, 1107-1120. Saito, K., Suyama, K., Nishida, K., Sei, Y., and Basile, A.S. (1996). Early increases in TNF-

alpha, IL-6 and IL-1 beta levels following transient cerebral ischemia in gerbil brain.

influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production. Proc Natl Acad Sci U S A *96*, 2414-2419. Sensi, S.L., Yin, H.Z., and Weiss, J.H. (1999b). Glutamate triggers preferential Zn2+ flux

through Ca2+ permeable AMPA channels and consequent ROS production.

Neurosci *19*, 312-318.

brain. Neuroreport *8*, 947-950.

against synaptic vesicle origin. J Neurosci *20*, RC79.

hippocampal neurons. Neuroreport *13*, 2271-2275.

induced seizures. Brain Res *631*, 51-58.

Blood Flow Metab *16*, 932-940.

Neurobiol Dis *11*, 28-42.

Neurosci Lett *206*, 149-152.

Neuroreport *10*, 1723-1727.

*22*, 885-895.

physiological stimulation. J Neurophysiol *86*, 2597-2604.

APP transgenic mice. Proc Natl Acad Sci U S A *99*, 7705-7710.


**17** 

*China* 

**Congenital Hyperinsulinism** 

*Key Laboratory of Endocrinology, the Ministry of Health,* 

 *Diabetes Institute, Shanghai Sixth Hospital Affiliated to* 

*Department of Endocrinology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College,* 

Congenital hyperinsulinism (CHI) is the most common cause of persistent and recurrent hypoglycaemia in neonates and infants during their first year of livies. CHI may lead to severe mental retardation and epilepsy if not treated properly. Both sporadic and familial variants of CHI are recognized, and of which sporadic forms is relatively uncommon (incidence 1 per 35,000 live births), comparing with the highly consanguinious familial forms high rates of consanguinity; with incidence may be as high as 1 in 2,500 live births in the corresponding communities. The clinical severity of CHI varies mainly with age of onset of hypoglycaemia (severe hypoglycaemia in neonates) and is remarkedly predictive in terms

Hypoglycemia in children is defined by a glucose plasma level below 2.8 or 3 mmol/l, It is a life-threatening condition that requires being diagnosed and treated promptly and appropriately to avoid brain damage and general distress. Congenital hyperinsulinism is due to an inappropriate insulin over-secretion by the β-cells. Insulin is known to be the only hormone to decrease plasma glucose level, and the function of which is realized by inhibiting hepatic glycogenolysis and boosting muscle uptake as well as reducing lipolysis and ketogenesis. Mechanisms above might explain the major characteristic clinical findings of neonatal hyperinsulinism (HI): the increased glucose requirement to correct hypoglycemia, the

Several pathways are involved in the regulation of insulin secretion by the pancreatic β-cell, helping explaining the effectiveness of diazoxide, somatostatin, calcium channel inhibitors and protein restricted diet treatments(Fig. 1). Glucose and other substrates, such as amino acids, stimulate insulin secretion , by raising the intracytosolic ATP/ADP ratio. Glucokinase enzyme initiates the β-cell glucose metabolism. It has a high Km for glucose so that the blood concentration of glucose directly determines the rate in glucose oxidation of β-cell and

responsiveness to exogenous glucagon , and the absence of ketone bodies detected.

**1. Introduction** 

of therapeutic outcome and genetic counseling.

**2. Physiopathology of hypoglycemia** 

Xinhua Xiao and Si Chen

*1 Shuaifuyuan, Wangfujing ST, Beijing ,* 

*Shanghai Jiaotong University, Shanghai,* 

