We previously reported that prenatal and suckling exposure to a maternal diet rich in animal fat leads to cardiovascular dysfunction in young adult rat offspring with subsequent development of dyslipidemia and hyperglycemia. We have further investigated glucose homeostasis in adult female offspring by euglycemic-hyperinsulinemic clamp and by dynamic assessment of glucose-stimulated insulin secretion in isolated, perifused pancreatic islet cells. Additionally, given the link between reduced mitochondrial DNA (mtDNA) content and the development of type 2 diabetes mellitus, we have measured mtDNA in organs from young adult animals. Sprague-Dawley rats were fed a diet rich in animal fat or normal chow throughout pregnancy and weaning. Infusion of insulin (5 mU.kg(-1).min(-1)) resulted in a higher steady-state plasma insulin concentration in 1-year-old offspring of fat-fed dams (OHF, n = 4) vs. offspring of control dams (OC, n = 4, P < 0.01). Glucose-stimulated insulin secretion in isolated islets from 9-mo-old OHF was significantly reduced compared with OC (n = 4, P < 0.05). Transmission electron micrography showed altered insulin secretory granule morphology in OHF pancreatic beta-cells. Kidney mtDNA was reduced in 3-mo-old OHF [16S-to-18S gene ratio: OC (n = 10) 1.05 +/- 0.19 vs. OHF (n = 10) 0.66 +/- 0.06, P < 0.05]. At 6 mo, gene chip microarray of OHF aorta showed reduced expression of the mitochondrial genome. Prenatal and suckling exposure to a diet rich in animal fat leads to whole body insulin resistance and pancreatic beta-cell dysfunction in adulthood, which is preceded by reduced tissue mtDNA content and altered mitochondrial gene expression.

Defective insulin secretion in response to glucose is an important component of the β cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS), oxygen-sensing pathways may modulate insulin release. The von Hippel-Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation. To determine the role of this pathway in controlling glucose-stimulated insulin release from pancreatic β cells, we generated mice lacking Vhl in pancreatic β cells (βVhlKO mice) and mice lacking Vhl in the pancreas (PVhlKO mice). Both mouse strains developed glucose intolerance with impaired insulin secretion. Furthermore, deletion of Vhl in β cells or the pancreas altered expression of genes involved in β cell function, including those involved in glucose transport and glycolysis, and isolated βVhlKO and PVhlKO islets displayed impaired glucose uptake and defective glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of Hif-1α expression, and deletion of Hif1a in Vhl-deficient β cells restored GSIS. Consistent with this, expression of activated Hif-1α in a mouse β cell line impaired GSIS. These data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose homeostasis and that activation of this pathway in response to decreased islet oxygenation may contribute to β cell dysfunction. IntroductionBlood glucose levels are normally tightly controlled by the regulation of insulin release from the pancreatic β cells. Glucose-stimulated insulin secretion (GSIS) is a complex metabolic process involving the uptake and phosphorylation of glucose via GLUT2 transporters and glucokinase (Gck), respectively, metabolism of glucose-6-phosphate via the glycolytic pathway, and subsequent activation of mitochondrial metabolism to produce coupling factors such as ATP (1). A rise in the cytoplasmic ATP/ADP ratio leads to closure of K ATP channels, depolarization of the plasma membrane, opening of voltage-sensitive Ca 2+ channels, and activation of Ca 2+ -dependent exocytotic mechanisms, resulting in insulin secretion (1). This metabolic sensing mechanism requires molecular oxygen for the quantitative generation of ATP from glucose. Understanding the complex physi-

The roles played by arachidonic acid and its cyclooxygenase (COX)-generated and lipoxygenase (LOX)-generated metabolites have been studied using rodent islets and insulin-secreting cell lines, but very little is known about COX and LOX isoform expression and the effects of modulation of arachidonic acid generation and metabolism in human islets. We have used RT-PCR to identify mRNAs for cytosolic phospholipase A 2 (cPLA 2 ), COX-1, COX-2, 5-LOX, and 12-LOX in isolated human islets. COX-3 and 15-LOX were not expressed by human islets. Perifusion experiments with human islets indicated that PLA 2 inhibition inhibited glucose-stimulated insulin secretion, whereas inhibitors of COX-2 and 12-LOX enzymes enhanced basal insulin secretion and also secretory responses induced by 20 mmol/l glucose or by 50 mol/l arachidonic acid. Inhibition of COX-1 with 100 mol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion. These data indicate that the stimulation of insulin secretion from human islets in response to arachidonic acid does not require its metabolism through COX-2 and 5-/12-LOX pathways. The products of COX-2 and LOX activities have been implicated in cytokine-mediated damage of ␤-cells, so selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: they would minimize ␤-cell dysfunction while maintaining insulin secretion through enhancing endogenous arachidonic acid levels. Diabetes 56:197-203, 2007 P hospholipases A 2 (PLA 2 ) constitute a large family of enzymes that hydrolyze the sn-2 position of membrane phospholipids to generate arachidonic acid. Secretory type I (1) and type II (1,2) PLA 2 enzymes have been identified in islets, as have the Ca 2ϩ -dependent type IV cytosolic PLA 2 (cPLA 2 ) (1-3) and the Ca 2ϩ -independent type VI isozymes (4), and it is well-established that insulin secretion from pancreatic ␤-cells is stimulated by arachidonic acid (5-7). Although arachidonic acid is known to exert direct functional effects in vitro, it is also further metabolized by cyclooxygenase (COX) enzymes to produce prostaglandins (rev. in 8) and lipoxygenases (LOX) to produce hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (rev. in 9). Two COX genes have been cloned: the COX-1 gene codes for COX-1 and the COX-3 splice variant (10), and the COX-2 gene codes for the COX-2 isoform. Depending on the oxygenation site in arachidonic acid, the LOX enzymes are termed 5-, 12-, and 15-LOX.The roles played by COX and LOX enzymes in ␤-cells have not been fully established, but there is good evidence that both COX-2 (11-14) and 12-LOX (15,16) play roles in cytokine-mediated damage of ␤-cells. Furthermore, signaling through the 12-LOX pathway is reported to upregulate COX-2 gene expression (17).Although there are some contradictory reports about the effects of COX and LOX products on insulin secretion, the consensus view is that prostaglandins (particularly prostaglandin E 2 [PGE 2 ]) have inhibitory effects, whereas HETEs and leukotrienes are stimulator...

Many plant-based products have been suggested as potential antidiabetic agents, but few have been shown to be effective in treating the symptoms of Type 2 diabetes mellitus (T2DM) in human studies, and little is known of their mechanisms of action. Extracts of Gymnema sylvestre (GS) have been used for the treatment of T2DM in India for centuries. The effects of a novel high molecular weight GS extract, Om Santal Adivasi, (OSA(R)) on plasma insulin, C-peptide and glucose in a small cohort of patients with T2DM are reported here. Oral administration of OSA(R) (1 g/day, 60 days) induced significant increases in circulating insulin and C-peptide, which were associated with significant reductions in fasting and post-prandial blood glucose. In vitro measurements using isolated human islets of Langerhans demonstrated direct stimulatory effects of OSA(R) on insulin secretion from human ß-cells, consistent with an in vivo mode of action through enhancing insulin secretion. These in vivo and in vitro observations suggest that OSA(R) may provide a potential alternative therapy for the hyperglycemia associated with T2DM.

Aims/hypothesis Insulin signalling pathways regulate pancreatic beta cell function. Conditional gene targeting using the Cre/loxP system has demonstrated that mice lacking insulin receptor substrate 2 (IRS2) in the beta cell have reduced beta cell mass. However, these studies have been complicated by hypothalamic deletion when the RIPCre (B6.Cg-tg(Ins2-cre) 25Mgn/J) transgenic mouse (expressing Cre recombinase under the control of the rat insulin II promoter) is used to delete floxed alleles in insulin-expressing cells. These features have led to marked insulin resistance making the beta cellautonomous role of IRS2 difficult to determine. To establish the effect of deleting Irs2 only in the pancreas, we generated PIrs2KO mice in which Cre recombinase expression was driven by the promoter of the pancreatic and duodenal homeobox factor 1 (Pdx1, also known as Ipf1) gene. Materials and methodsIn vivo glucose homeostasis was examined in PIrs2KO mice using glucose tolerance and glucose-stimulated insulin secretion tests. Endocrine cell mass was determined by morphometric analysis. Islet function was examined in static cultures and by performing calcium imaging in Fluo3am-loaded beta cells. Islet gene expression was determined by RT-PCR. Results The PIrs2KO mice displayed glucose intolerance and impaired glucose-stimulated insulin secretion in vivo. Pancreatic insulin and glucagon content and beta and alpha cell mass were reduced. Glucose-stimulated insulin secretion and calcium mobilisation were attenuated in PIrs2KO islets. Expression of the Glut2 gene (also known as Slc2a2) was also reduced in PIrs2KO mice. Conclusions/interpretation These studies suggest that IRS2-dependent signalling in pancreatic islets is required not only for the maintenance of normal beta and alpha cell mass but is also involved in the regulation of insulin secretion.