The glucagon-like peptide-1 receptor agonist exenatide restores impaired pro-islet amyloid polypeptide processing in cultured human islets: implications in type 2 diabetes and islet transplantation.

AIMS/HYPOTHESIS: Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), is associated with beta cell death in type 2 diabetes as well as in cultured and transplanted human islets. Impaired prohIAPP processing due to beta cell dysfunction is implicated in hIAPP aggregation. We examined whether the glucagon-like peptide-1 receptor (GLP-1R) agonist exenatide can restore impaired prohIAPP processing and reduce hIAPP aggregation in cultured human islets and preserve beta cell function/mass during culture conditions used in clinical islet transplantation. METHODS: Isolated human islets (n = 10 donors) were cultured with or without exenatide in normal or elevated glucose for 2 or 7 days. Beta cell apoptosis, proliferation, mass, function, cJUN N-terminal kinase (JNK) and protein kinase B (PKB) activation and amyloid formation were assessed. ProhIAPP, its intermediates and mature hIAPP were detected. RESULTS: Exenatide-treated islets had markedly lower JNK and caspase-3 activation and beta cell apoptosis, resulting in higher beta/alpha cell ratio and beta cell area than non-treated cultured islets. Exenatide improved beta cell function, manifested as higher insulin response to glucose and insulin content, compared with non-treated cultured islets. Phospho-PKB immunoreactivity was detectable in exenatide-treated but not untreated cultured islets. Islet culture caused impaired prohIAPP processing with decreased mature hIAPP and increased NH(2)-terminally unprocessed prohIAPP levels resulting in higher release of immature hIAPP. Exenatide restored prohIAPP processing and reduced hIAPP aggregation in cultured islets. CONCLUSIONS/INTERPRETATION: Exenatide treatment enhances survival and function of cultured human islets and restores impaired prohIAPP processing in normal and elevated glucose conditions thereby reducing hIAPP aggregation. GLP-1R agonists may preserve beta cells in conditions associated with islet amyloid formation.

Menin and GIP are inversely regulated by food intake and diet via PI3/AKT signaling in the proximal duodenum.

BACKGROUND AND AIMS: Ingestion of food stimulates the secretion of incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 to ensure the proper absorption and storage of nutrients. Menin is the 67 kDa protein product of the MEN1 gene recently reported to have a role in metabolism. In this study, we will determine the regulation of menin in the proximal duodenum by food intake and diet in correlation with GIP levels in the proximal duodenum of mice after an 18 h fast followed by 4 and 7 h refeeding and 3 months of high-fat diet. METHODS: A dual luciferase assay was used to determine GIP promoter activity and ELISA was used to measure the levels of GIP after inhibition of menin through small interfering RNA (siRNA) and exposure to MAPK and AKT inhibitors. Colocalization of menin and GIP were determined by immunofluorescence. RESULTS: Menin and GIP expression are regulated by fasting, refeeding and diet in the proximal duodenum. Overexpression of menin in STC-1 cells significantly inhibited GIP mRNA and promoter activity, whereas menin siRNA upregulated GIP levels. Inhibition of GIP expression by the PI3/AKT inhibitor, LY294002, was abrogated in STC-1 cells with reduced menin levels, whereas the MAPK inhibitor, UO126, inhibited the expression of GIP independent of menin. Exposure of STC-1 cells to GIP reduced menin expression in a dose-dependent manner via PI3K-AKT signaling. CONCLUSION: Feeding and diet regulates the expression of menin, which inversely correlates with GIP levels in the proximal duodenum. In vitro assays indicate that menin is a negative regulator of GIP via inhibition of PI3K-AKT signaling. We show menin colocalizing with GIP in K cells of the proximal gut and hypothesize that downregulation of menin may serve as a mechanism by which GIP is regulated in response to food intake and diet.

Deletion of Fas protects islet beta cells from cytotoxic effects of human islet amyloid polypeptide.

AIMS/HYPOTHESIS: Islet amyloid, which is mainly composed of human islet amyloid polypeptide (hIAPP), is a pathological characteristic of type 2 diabetes and also forms in cultured and transplanted islets. We used islet beta cells as well as two ex vivo models of islet amyloid formation, cultured human islets and hIAPP-expressing transgenic mouse islets with or without beta cell Fas deletion, to test whether: (1) the aggregation of endogenous hIAPP induces Fas upregulation in beta cells; and (2) deletion or blocking of Fas protects beta cells from amyloid toxicity. METHODS: INS-1, mouse or human islet cells were cultured with hIAPP alone, or with amyloid inhibitor or Fas antagonist. Non-transduced islets, and human islets or hIAPP-expressing mouse islets transduced with an adenovirus that delivers a human proIAPP-specific small interfering RNA (siRNA) (Ad-ProhIAPP-siRNA) were cultured to form amyloid. Mouse islets expressing hIAPP with or without Fas were similarly cultured. Beta cell Fas upregulation, caspase-3 activation, apoptosis and function, and islet IL-1ß levels were assessed. RESULTS: hIAPP treatment induced Fas upregulation, caspase-3 activation and apoptosis in INS-1 and islet cells. The amyloid inhibitor or Fas antagonist reduced apoptosis in hIAPP-treated beta cells. Islet cells with Fas deletion had lower hIAPP-induced beta cell apoptosis than those expressing Fas. Ad-ProhIAPP-siRNA-mediated amyloid inhibition reduced Fas upregulation and IL-1ß immunoreactivity in human and hIAPP-expressing mouse islets. Cultured hIAPP-expressing mouse islets with Fas deletion had similar amyloid levels, but lower caspase-3 activation and beta cell apoptosis, and a higher islet beta:alpha cell ratio and insulin response to glucose, compared with islets expressing Fas and hIAPP. CONCLUSIONS/INTERPRETATION: The aggregation of biosynthetic hIAPP produced in islets induces beta cell apoptosis, at least partially, via Fas upregulation and the Fas-mediated apoptotic pathway. Deletion of Fas protects islet beta cells from the cytotoxic effects of endogenously secreted (and exogenously applied) hIAPP.

dsAAV8-mediated gene transfer and ß-cell expression of IL-4 and ß-cell growth factors are capable of reversing early-onset diabetes in NOD mice.

Type-I diabetes is a chronic disease mediated by autoimmune destruction of insulin-producing ß-cells. Although progress has been made towards improving diabetes-associated pathologies and the quality of life for those living with diabetes, no therapy has been effective at eliminating disease manifestations or reversing disease progression. Here, we examined whether double-stranded adeno-associated virus serotype 8 (dsAAV8)-mediated gene delivery to endogenous ß-cells of interleukin (IL)-4 in combination with ß-cell growth factors can reverse early-onset diabetes in NOD mice. Our results demonstrate that a single treatment with dsAAV8 vectors expressing IL-4 in combination with glucagon-like peptide-1 or hepatocyte growth factor/NK1 under the regulation of the insulin promoter enhanced ß-cell proliferation and survival in vivo, significantly delaying diabetes progression in NOD mice, and reversing disease in ∼10% of treated NOD mice. These results demonstrate the ability to reverse hyperglycemia in NOD mice with established diabetes by in vivo gene transfer to ß-cells of immunomodulatory factors and ß-cell growth factors.

Reduction of both beta cell death and alpha cell proliferation by dipeptidyl peptidase-4 inhibition in a streptozotocin-induced model of diabetes in mice.

AIMS/HYPOTHESIS: Incretins stimulate insulin secretion in a glucose-dependent manner but also promote pancreatic beta cell protection. Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new glucose-lowering treatment that blocks incretin degradation by DPP-4. We assessed whether DPP-4 inhibition suppresses the progression to hyperglycaemia in a low-dose streptozotocin (STZ)-induced diabetic mouse model, and then investigated how DPP-4 inhibition affects islet function and morphology. METHODS: The DPP-4 inhibitor, des-fluoro-sitagliptin (SITA), was administered to mice during and after STZ injections, and in some mice also before STZ. RESULTS: In control mice, STZ resulted in hyperglycaemia associated with impaired insulin secretion and excess glucagon secretion. In SITA-treated STZ mice, these metabolic abnormalities were improved, particularly when SITA administration was initiated before STZ injections. We observed beta cell loss and dramatic alpha cell expansion associated with decreased insulin content and increased glucagon content after STZ administration. In SITA-treated mice, islet architecture and insulin content were preserved, and no significant increase in glucagon content was observed. After STZ exposure, beta cell apoptosis increased before hyperglycaemia, and SITA treatment reduced the number of apoptotic beta cells. Interestingly, alpha cell proliferation was observed in non-treated mice after STZ injection, but the proliferation was not observed in SITA-treated mice. CONCLUSIONS/INTERPRETATION: Our results suggest that the ability of DPP-4 inhibition to suppress the progression to STZ-induced hyperglycaemia involves both alleviation of beta cell death and alpha cell proliferation.

Immunohistochemical characterisation of cells co-producing insulin and glucagon in the developing human pancreas.

AIMS/HYPOTHESIS: In adult human islets, insulin and glucagon production is largely restricted to individual cell populations. The production of these hormones is less segregated during development and during the differentiation of human pluripotent stem cells towards pancreatic lineages. We therefore sought to characterise the transcription factor profile of these cells that co-produce insulin and glucagon in the developing human pancreas, and thus to gain insight into their potential fate during normal pancreas development. METHODS: An immunohistochemical analysis was performed on human pancreas sections from fetal donors aged 9 to 21 weeks and from adult donors between the ages of 17 and 55 years. RESULTS: Endocrine cells were observed within the pancreas at all ages examined, with cells co-producing insulin and glucagon observed as early as 9 weeks of fetal age. The population of cells that co-produce insulin and glucagon generally decreased in prevalence with age, with negligible numbers in adult pancreas. From 9 to 16 weeks, the population of glucagon-only cells increased, while the insulin-only cells decreased in abundance. Cells that co-produced insulin and glucagon also produced the alpha cell transcription factor, aristaless related homeobox (ARX), and lacked the beta cell transcription factors pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1) and v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA). CONCLUSIONS/INTERPRETATION: Our results indicate that cells co-producing insulin and glucagon in the developing human pancreas share a transcription factor profile that is similar to that of mature alpha cells and suggest that some maturing alpha cells briefly exhibit ectopic insulin expression. Thus cells that co-produce insulin and glucagon may represent a transient cell population, which gives rise to mature alpha cells.

Hepatic leptin signalling and subdiaphragmatic vagal efferents are not required for leptin-induced increases of plasma IGF binding protein-2 (IGFBP-2) in ob/ob mice.

AIMS/HYPOTHESIS: The fat-derived hormone leptin plays a crucial role in the maintenance of normal body weight and energy expenditure as well as in glucose homeostasis. Recently, it was reported that the liver-derived protein, insulin-like growth factor binding protein-2 (IGFBP-2), is responsible for at least some of the glucose-normalising effects of leptin. However, the exact mechanism by which leptin upregulates IGFBP-2 production is unknown. Since it is believed that circulating IGFBP-2 is predominantly derived from the liver and leptin has been shown to have both direct and indirect actions on the liver, we hypothesised that leptin signalling in hepatocytes or via brain-liver vagal efferents may mediate leptin control of IGFBP-2 production. METHODS: To address our hypothesis, we assessed leptin action on glucose homeostasis and plasma IGFBP-2 levels in both leptin-deficient ob/ob mice with a liver-specific loss of leptin signalling and ob/ob mice with a subdiaphragmatic vagotomy. We also examined whether restoring hepatic leptin signalling in leptin receptor-deficient db/db mice could increase plasma IGFBP-2 levels. RESULTS: Continuous leptin administration increased plasma IGFBP-2 levels in a dose-dependent manner, in association with reduced plasma glucose and insulin levels. Interestingly, leptin was still able to increase plasma IGFBP-2 levels and improve glucose homeostasis in both ob/ob mouse models to the same extent as their littermate controls. Further, restoration of hepatic leptin signalling in db/db mice did not increase either hepatic or plasma IGFBP-2 levels. CONCLUSIONS/INTERPRETATION: Taken together, these data indicate that hepatic leptin signalling and subdiaphragmatic vagal inputs are not required for leptin upregulation of plasma IGFBP-2 nor blood glucose lowering in ob/ob mice.

Musashi expression in ß-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes.

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic ß-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates ß-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of ß-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and ß-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and ß-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory ß-cell proliferation and the loss of ß-cell gene expression seen in specific phases of the progression to type 2 diabetes.

Reprogramming gut and pancreas endocrine cells to treat diabetes.

Multiple approaches have been investigated with the ultimate goal of providing insulin independence to patients with either type 1 or type 2 diabetes. Approaches to produce insulin-secreting cells in culture, convert non-ß-cells into functional ß-cells or engineer autologous cells to express and secrete insulin in a meal-responsive manner have all been described. This research has been facilitated by significant improvements in both viral and non-viral gene delivery approaches that have enabled new experimental strategies. Many studies have examined possible avenues to confer islet cytoprotection against immune rejection, inflammation and apoptosis by genetic manipulation of islet cells prior to islet transplantation. Here we review several reports based on the reprogramming of pancreas and gut endocrine cells to treat diabetes.

New aspects of an old drug: metformin as a glucagon-like peptide 1 (GLP-1) enhancer and sensitiser.

The two major deficits in type 2 diabetes, insulin resistance and impaired beta cell function, are often treated with metformin and incretin-based drugs, respectively. However, there may be unappreciated benefits of this combination of therapies. In this issue of Diabetologia, Maida et al. (doi: 10.1007/s00125-010-1937-z) report that metformin acutely increases plasma levels of glucagon-like peptide 1 (GLP-1) in mice. Moreover, they show that metformin enhances the expression of the genes encoding the receptors for both GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) in mouse islets and also increases the effects of GIP and GLP-1 on insulin secretion from beta cells. Interestingly, these incretin-sensitising effects of metformin appear to be mediated by a peroxisome proliferator-activated receptor α-dependent pathway, as opposed to the more commonly ascribed pathway of metformin action involving AMP-activated protein kinase. These provocative findings by Maida et al. extend our understanding of the mechanism of action of metformin and provide further insights into the benefits of combining metformin with incretin-based drugs to combat diabetes.

Differences between amyloid toxicity in alpha and beta cells in human and mouse islets and the role of caspase-3.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by decreased beta cell mass and islet amyloid formation. Islet amyloid formed by aggregation of human islet amyloid polypeptide (hIAPP) is associated with beta cell apoptosis. We used human and transgenic mouse islets in culture to examine whether deletion of caspase-3 protects islets from apoptosis induced by endogenously produced and exogenously applied hIAPP and compared hIAPP toxicity in islet alpha and beta cells. METHODS: Human and wild-type or caspase-3 knockout mouse islet cells were treated with hIAPP. Rat insulinoma INS-1 cells were similarly cultured with hIAPP and the amyloid inhibitor Congo Red or caspase-3 inhibitor. Human and hIAPP-expressing caspase-3 knockout mouse islets were cultured to form amyloid fibrils and assessed for beta and alpha cell apoptosis, beta cell function and caspase-3 activation. RESULTS: hIAPP-treated INS-1 cells had increased caspase-3 activation and apoptosis, both of which were reduced by inhibitors of amyloid or caspase-3. Similarly, hIAPP-treated human and mouse islet beta cells had elevated active caspase-3- and TUNEL-positive cells, whereas mouse islet cells lacking caspase-3 had markedly lower beta cell but comparable alpha cell apoptosis. During culture, human islets that formed amyloid had higher active caspase-3- and TUNEL-positive beta cells than those without detectable amyloid. Finally, cultured hIAPP-expressing mouse islets lacking caspase-3 had markedly lower beta cell apoptosis than those expressing caspase-3, associated with an increase in islet beta cell/alpha cell ratio, insulin content and glucose response. CONCLUSIONS/INTERPRETATION: Prevention of caspase-3 activation protects islet beta cells from apoptosis induced by fibrillogenesis of endogenously secreted and exogenously applied hIAPP. Islet beta cells are more susceptible to hIAPP toxicity than alpha cells cultured under the same conditions.

DsAAV8-mediated expression of glucagon-like peptide-1 in pancreatic beta-cells ameliorates streptozotocin-induced diabetes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that performs a wide array of well-characterized antidiabetic actions, including stimulation of glucose-dependent insulin secretion, upregulation of insulin gene expression and improvements in beta-cell survival. GLP-1-receptor agonists have been developed for treatment of diabetes; however, the short biological half-lives of these peptide-based therapeutics requires that frequent injections be administered to maintain sufficient circulating levels. Thus, novel methods of delivering GLP-1 remain an important avenue of active research. It has recently been demonstrated that self-complimentary, double-stranded, adeno-associated virus serotype-8 (DsAAV8) can efficiently transduce pancreatic beta-cells in vivo, resulting in long-term transgene expression. In this study, we engineered a DsAAV8 vector containing a GLP-1 transgene driven by the mouse insulin-II promoter (MIP). Biological activity of the GLP-1 produced from this transgene was assessed using a luciferase-based bioassay. DsAAV8-MIP-GLP-1 was delivered via intraperitoneal injection and beta-cell damage induced by multiple low dose streptozotocin (STZ) administration. Glucose tolerance was assessed following intraperitoneal glucose injections and beta-cell proliferation measured by PCNA expression. Expression of GLP-1 in Min6 beta-cells resulted in glucose-dependent secretion of biologically active GLP-1. Intraperitoneal delivery of DsAAV8-MIP-GLP-1 to mice led to localized GLP-1 expression in beta-cells and protection against development of diabetes induced by multiple low-dose STZ administration. This protection was associated with significant increase in beta-cell proliferation. Results from this study indicate that expression and secretion of GLP-1 from beta-cells in vivo via DsAAV8 represents a novel therapeutic strategy for treatment of diabetes.

Exercise restores endothelial function independently of weight loss or hyperglycaemic status in db/db mice.

AIMS/HYPOTHESIS: Exercise ameliorates oxidative stress-mediated diabetic vascular endothelial dysfunction through poorly defined mechanisms. We hypothesised that, in addition to improving metabolic parameters, upregulation of antioxidants such as superoxide dismutase (SOD) mediates exercise-induced reductions of oxidative stress and increased nitric oxide (NO) bioavailability, and also restores vasodilatation. METHODS: Type 2 diabetic db/db and normoglycaemic wild-type mice were exercised at moderate intensity for 1 h a day for 7 weeks, leading to a 10% body weight loss. Sedentary animals or those undergoing a low-intensity exercise regimen causing non-significant weight loss were also used. We examined aortic endothelial cell function, NO bioavailability and various biomarkers of oxidative stress. RESULTS: Moderate-intensity exercise lowered body weight, increased mitochondrial manganese SOD (MnSOD) and both total and phosphorylated (Ser1177) endothelial nitric oxide synthase (eNOS) protein production; it also reduced whole-body (plasma 8-isoprostane) and tissue oxidative stress (nitrotyrosine immunostaining or protein carbonyl levels in the aorta). Low-intensity exercise did not alter body weight; however, it upregulated cytosolic Cu/Zn-SOD instead of MnSOD, and still demonstrated all the above benefits in the db/db aorta. Importantly, both exercise protocols improved endothelial-dependent vasodilatation and NO bioavailability without altering hyperglycaemic status in db/db mice. CONCLUSIONS/INTERPRETATION: Exercise reverses diabetic vascular endothelial dysfunction independently of improvements in body weight or hyperglycaemia. Our data suggest that upregulation of eNOS and specific SOD isoforms could play important roles in improving NO bioavailability, as well as in reversing endothelial dysfunction in type 2 diabetes patients through lifestyle modifications in the management of diabetes.

Effect of exercise on augmented aortic vasoconstriction in the db/db mouse model of type-II diabetes.

We evaluated the effects of exercise on the vascular constrictor responses to alpha-adrenergic stimulation in the db/db mice. Twenty male db/db and their age-matched wild-type (WT) mice were exercised (1 hour/day, five days a week). Mice were anesthetized 7 weeks later, thoracic aortae were mounted in wire myograph and constrictor responses to phenylephrine (PE, 1 nM-10 microM) were obtained. Citrate synthase activity measured in the thigh adductor muscle was significantly increased in db/db mice that were exercise trained. Maximal force generated by PE was markedly greater in db/db aortae and exercise did not attenuate this augmented contractile response. Vessels were incubated with inhibitors of nitric oxide synthase (L-NAME, 200 microM), endothelin receptors (bosentan, 10 microM), protein kinase C (PKC) (calphostin C, 5 microM), cyclooxygenase (indomethacin, 10 microM) or Rho-kinase (Y-27632, 0.1 microM). Only calphostin-C normalized the augmented PE-induced constriction in db/db and db/db- exercised mice to that observed in WT (p<0.05). Cumulative additions of indolactam, a PKC activator, induced significantly greater constrictor responses in aortic rings of db/db mice compared to WT and exercise did not affect this response. Our data suggest that the augmented vasoconstriction observed in the aorta of db/db mice is likely due to increased PKC activity and that exercise do not ameliorate this increased PKC-mediated vasoconstriction.

Effects of dipeptidyl peptidase IV on the satiety actions of peptide YY.

AIMS/HYPOTHESIS: Dipeptidyl peptidase IV (DP IV) inhibitors are currently being developed to prolong the biological activity of insulinotropic peptides as a novel approach in the treatment of diabetes. We hypothesised that DP IV inhibition could attenuate the satiety actions of peptide YY (PYY) by altering the conversion of PYY(1-36) to PYY(3-36). MATERIALS AND METHODS: The effects of PYY delivered by osmotic mini-pumps were assessed in rats treated with a DP IV inhibitor and in a rat model deficient in DP IV. RESULTS: Pharmacological levels of total PYY were found in the circulation after the exogenous administration of PYY(3-36). While both PYY(1-36) and PYY(3-36) reduced food intake in normal rats, PYY(1-36) was ineffective in rats deficient in DP IV. When re-fed after a 24-h fast, DP IV-deficient rats exhibited higher food intake and weight gain than normal rats. Moreover, unlike controls, there was no postprandial increase in PYY levels in DP IV-deficient rats. Despite these findings, administration of a DP IV inhibitor, Pro-boroPro, did not alter the acute anorectic effects of exogenous PYY(1-36) in normal rats. This could be the result of the protection of other appetite regulatory peptides or the generation of PYY(3-36) by remaining DP IV activity or other dipeptidyl peptidases. CONCLUSIONS/INTERPRETATION: Although DP IV inhibition with Pro-boroPro attenuated the generation of PYY(3-36), our results indicate that short-term DP IV inhibition does not eliminate the satiety actions of exogenously administered PYY(1-36) at the doses tested.

Leptin resistance following over-expression of protein tyrosine phosphatase 1B in liver.

Obesity is typically associated with resistance to leptin, yet the mechanism by which leptin signaling becomes impaired is poorly understood. Here we sought to determine if the development of obesity and leptin resistance correlates with increased expression of protein tyrosine phosphatase 1B (PTP1B) in peripheral tissues and whether over-expression of this phosphatase, specifically in liver, could alter the leptin-mediated effects on feeding and glucose metabolism. Obesity was induced in mice through a high-fat diet that resulted in hyperglycemia, hyperinsulinemia and hyperleptinemia. Resistance to leptin was confirmed as exogenous leptin administration reduced food intake in animals on low-fat, but not high-fat diets. Diet-induced resistance to leptin and insulin was associated with increased hepatic levels of PTP1B. Intriguingly, hepatic adenoviral over-expression of PTP1B in ob/ob mice attenuated the ability of exogenous leptin to reduce both plasma glucose levels and food intake. These findings suggest that leptin reduces both plasma glucose and food intake in part through actions on the liver, and hepatic leptin resistance resulting from over-expression of PTP1B may contribute to the development of both diabetes and obesity.

Leptin reduces glucose transport and cellular ATP levels in INS-1 beta-cells.

Leptin suppresses insulin secretion by opening ATP-sensitive K(+) (K(ATP)) channels and hyperpolarizing beta-cells. We measured the intracellular concentration of ATP ([ATP](i)) in tumor-derived beta-cells, INS-1, and found that leptin reduced [ATP](i) by approximately 30%, suggesting that the opening of K(ATP) channels by leptin is mediated by decreased [ATP](i). A reduction in glucose availability for metabolism may explain the decreased [ATP](i), since leptin (30 min) reduced glucose transport into INS-1 cells by approximately 35%, compared to vehicle-treated cells. The twofold induction of GLUT2 phosphorylation by GLP-1, an insulin secretagogue, was abolished by leptin. Therefore, the acute effect of leptin could involve covalent modification of GLUT2. These findings suggest that leptin may inhibit insulin secretion by reducing [ATP](i) as a result of reduced glucose availability for the metabolic pathway. In addition, leptin reduced glucose transport by 35% in isolated rat hepatocytes that also express GLUT2, suggesting that glucose transport may also be altered by leptin in other glucose-responsive tissues such as the liver.

Glucose-dependent insulinotropic polypeptide as a regulator of beta cell function and fate.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that is secreted in response to food intake and modulates beta cell function. It may also regulate beta cell fate. Released from the nutrient-sensing K-cells of the upper intestine, GIP acts on various tissues, including pancreatic beta cells, via interaction with its G-protein-coupled receptor. Perhaps the most important effect of GIP is its potentiation of insulin secretion. Indeed, pharmacological blockade or genetic knockout of its receptor delays glucose-dependent insulin secretion. Exposure to GIP also enhances the beta cell response to future nutrient stimulation and upregulates transcription of key beta cell genes. There is emerging evidence that like the related hormone glucagon-like peptide-1, GIP may function as a beta cell growth factor and anti-apoptotic agent, further supporting a role for this hormone in balancing beta cell function to changing metabolic conditions. Overproduction of GIP in response to increased nutrient loads may, however, contribute to the pathophysiology of obesity. Interestingly, its insulinotropic effect is lost in type 2 diabetes, perhaps because of hyperglycemia-induced receptor desensitization. A better understanding of GIP's effects on the beta cell under normal and pathological conditions may facilitate the design of GIP derivatives for the treatment of metabolic disorders.

The multifaceted potential of glucagon-like peptide-1 as a therapeutic agent.

Glucagon-like peptide-1 (GLP-1), an intestinal gut hormone, is rapidly emerging as a new therapeutic agent for the treatment of diabetes mellitus. GLP-1, released from intestinal L-cells, is renowned for its potent stimulation of insulin biosynthesis and release from pancreatic b-cells. Exogenous administration of GLP-1 to subjects with type 2 diabetes results in the normalization of plasma glucose concentrations, in part, as a result of augmented glucose-stimulated insulin secretion. However, it is now recognized that GLP-1 has several other anti-diabetic actions that collectively improve the type 2 diabetic phenotype, and may also prove beneficial in the treatment of type 1 diabetes. These effects include the deceleration of gastric emptying and promotion of satiety, thereby reducing the availability of nutrients for absorption and reducing the requirement for insulin secretion. GLP-1 also reduces plasma glucose levels by suppressing glucagon secretion from pancreatic a-cells and potentially by improving insulin sensitivity in peripheral tissues. Further-more, GLP-1 upregulates expression of b-cell genes (GLUT2, glucokinase, insulin, and PDX-1) and promotes b-cell neogenesis and differentiation of ductal cells into insulin secreting cells. Although initial clinical trials indicate GLP-1 has excellent therapeutic potential, its relatively short-lived biological activity and delivery difficulties limit its appeal. Several approaches that are currently being explored to overcome these limitations include mobilizing endogenous GLP-1 release, preserving the biological activity of the native peptide, and developing GLP-1 analogues with extended durations of action.