Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxP technology in combination with gene targeting to perform global, ␤ cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. ␤ cell-and hepatocytespecific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/ enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a ␤ cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in ␤ cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the ␤ cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both ␤ cell and hepatic GK contribute to the hyperglycemia of MODY-2.

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and ␤-cell mass, mice with ␤-cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of ␤-cell secretagogues to islets, we showed that reduced insulin output is not a result of ␤-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation. Diabetes

Long-chain fatty acids amplify insulin secretion from the pancreatic beta cell. The G protein-coupled receptor GPR40 is specifically expressed in beta cells and is activated by fatty acids. Loss of function of GPR40 was shown to markedly inhibit fatty-acid stimulation of insulin secretion in vitro. However, the role of GPR40 in acute regulation of insulin secretion in vivo remains unclear. To this aim, we generated GPR40 knock-out (KO) mice and examined glucose homeostasis, insulin secretion in response to glucose and Intralipid in vivo, and insulin secretion in vitro after short-and long-term exposure to fatty acids. Our results show that GPR40 KO mice have essentially normal glucose tolerance and insulin secretion in response to glucose. Insulin secretion in response to Intralipid was reduced by approximately 50%. In isolated islets, insulin secretion in response to glucose and other secretagogues was unaltered, but fatty-acid potentiation of insulin release was markedly reduced. Islets from GPR40 KO mice were as sensitive to fatty-acid inhibition of insulin secretion upon prolonged exposure as islets from wild-type animals. We conclude that GPR40 contributes approximately half of the full insulin secretory response to fatty acids in mice, but does not play a role in the mechanisms of lipotoxicity.Long-chain fatty acids are essential regulators of normal pancreatic beta-cell function, and are likely to play a role in the pathogenesis of beta-cell dysfunction in type 2 diabetes (reviewed in (1)). Under normal circumstances, fatty acids do not initiate insulin release, but amplify glucose-stimulated insulin secretion (GSIS) (2-5). Fatty-acid potentiation of insulin secretion has physiological implications, particularly after a period of fasting (6). Until recently, the prevailing model postulated that the effects of fatty acids on the beta cell were mediated by their intracellular metabolism and the generation of lipid derived signals which, in turn, potentiate GSIS (2;7). According to this hypothesis, fatty acids are transported across the plasma membrane and activated into their long-chain coenzyme A esters, which in turn modulate a number of intracellular targets that influence insulin secretion. Moreover, evidence suggests that intracellular fatty-acid metabolism is a key component of both nutrient-and nonnutrient-induced insulin secretion (7). In contrast to their acute, stimulatory effect on GSIS, prolonged exposure to elevated levels of fatty acids impairs beta-cell function, a phenomenon referred to as lipotoxicity (reviewed in (1)). The mechanisms of lipotoxicity remain poorly understood but have been proposed to also involve intracellular metabolism of fatty acids and the generation of lipid-derived metabolites (8).The models described above have been challenged by the observation that fatty acids activate the G-protein coupled receptor (GPCR) GPR40 (9-11), also referred to as the free fatty-acid 1 receptor (FFA 1 R) (12;13). GPR40 belongs to a class of GPCR with high structural conservation, o...

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Excess glucagon levels contribute to the hyperglycemia associated with type 2 diabetes. Reducing glucagon receptor expression may thus ameliorate the consequences of hyperglucagonemia and improve blood glucose control in diabetic patients. This study describes the antidiabetic effects of a specific glucagon receptor antisense oligonucleotide (GR-ASO) in db/db mice. The ability of GR-ASOs to inhibit glucagon receptor mRNA expression was demonstrated in primary mouse hepatocytes by quantitative real-time RT-PCR. Intraperitoneal administration of GR-ASO at a dosage of 25 mg/kg twice a week in db/db mice for 3 weeks resulted in 1) decreased glucagon receptor mRNA expression in liver; 2) decreased glucagon-stimulated cAMP production in hepatocytes isolated from GR-ASO-treated db/db mice; 3) significantly reduced blood levels of glucose, triglyceride, and free fatty acids; 4) improved glucose tolerance; and 5) a diminished hyperglycemic response to glucagon challenge. Neither lean nor db/db mice treated with GR-ASO exhibited hypoglycemia. Suppression of GR expression was also associated with increased ( approximately 10-fold) levels of plasma glucagon. No changes were observed in pancreatic islet cytoarchitecture, islet size, or alpha-cell number. However, alpha-cell glucagon levels were increased significantly. Our studies support the concept that antagonism of glucagon receptors could be an effective approach for controlling blood glucose in diabetes.

Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response.

Nonsteroidal antiinf lammatory drugs reduce the risk of colon cancer, possibly via cyclooxygenase (COX) inhibition. The growth factor-inducible COX-2, which is overexpressed in neoplastic colonic tissue, is an attractive target to mediate this effect. Herein we have exploited the ability of a human colon cancer cell line, HCA-7 Colony 29, to polarize when cultured on Transwell (Costar) filters to study COX-2 production and the vectorial release of prostaglandins (PGs). Administration of type ␣ transforming growth factor to the basolateral compartment, in which the epidermal growth factor receptor (EGFR) resides, results in a marked induction of COX-2 immunoreactivity at the base of the cells and the unexpected appearance of COX-2 in the nucleus. The increase in COX-2 protein is associated with a dose-and time-dependent increase in PG levels in the basolateral, but not apical, medium. Amphiregulin is the most abundantly expressed EGFR ligand in these cells, and the protein is present at the basolateral surface. EGFR blockade reduces baseline COX-2 immunoreactivity, PG levels, and mitogenesis in a concentration-dependent manner. Two specific COX-2 inhibitors, SC-58125 and NS 398, also, in a dose-dependent manner, attenuate baseline and type ␣ transforming growth factor-stimulated mitogenesis, although PG levels are decreased >90% at all concentrations of inhibitor tested. These findings show that activation of the EGFR stimulates COX-2 production and its translocation to the nucleus, vectorial release of PGs, and mitogenesis in polarized HCA-7 Colony 29 cells.In the gastrointestinal tract, prostaglandins (PGs) mediate important functions, including motility, vascular tone, angiogenesis, mucosal protection, and immune responsiveness (1). Inasmuch as epithelial cells are capable of PG synthesis, it is feasible that PGs synthesized in the gastrointestinal epithelium regulate these functions by paracrine pathways in response to luminal or serosal stimuli. Although data exist in support of vectorial release of PGs in the isolated rat colon (2), as well as in other tissues and polarized kidney-derived cells (3-5), regulatory mechanisms have not been defined more precisely.Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid (AA) to PGs and other eicosanoids. Two isoforms of the enzyme have been characterized. COX-1 in most cells is expressed constitutively, and a second inducible form known as COX-2 has been identified (refs. 6-8; for review see ref. 9). Recent observations indicate that many colonic polyps and cancers overexpress COX-2 (10-12) and that inhibition of this enzyme by nonsteroidal antiinflammatory drugs decreases the risk of colonic neoplasia (13-20), emphasizing the importance of defining potential autocrine and paracrine pathways for regulation of gastrointestinal epithelial growth by COX. Signaling through the epidermal growth factor receptor (EGFR) induces COX-2 expression, and unregulated overexpression of COX-2 results in a tumorigenic phenotype in the rat intestinal e...

OBJECTIVEC57Bl/6 mice develop obesity and mild hyperglycemia when fed a high-fat diet (HFD). Although diet-induced obesity (DIO) is a widely studied model of type 2 diabetes, little is known about β-cell failure in these mice.RESEARCH DESIGN AND METHODSDIO mice were separated in two groups according to body weight gain: low- and high-HFD responders (LDR and HDR). We examined whether mild hyperglycemia in HDR mice is due to reduced β-cell mass or function and studied islet metabolism and signaling.RESULTSHDR mice were more obese, hyperinsulinemic, insulin resistant, and hyperglycemic and showed a more altered plasma lipid profile than LDR. LDR mice largely compensated insulin resistance, whereas HDR showed perturbed glucose homeostasis. Neither LDR nor HDR mice showed reduced β-cell mass, altered islet glucose metabolism, and triglyceride deposition. Insulin secretion in response to glucose, KCl, and arginine was impaired in LDR and almost abolished in HDR islets. Palmitate partially restored glucose- and KCl-stimulated secretion. The glucose-induced rise in ATP was reduced in both DIO groups, and the glucose-induced rise in Ca2+ was reduced in HDR islets relatively to LDR. Glucose-stimulated lipolysis was decreased in LDR and HDR islets, whereas fat oxidation was increased in HDR islets only. Fatty acid esterification processes were markedly diminished, and free cholesterol accumulated in HDR islets.CONCLUSIONSβ-Cell failure in HDR mice is not due to reduced β-cell mass and glucose metabolism or steatosis but to a secretory dysfunction that is possibly due to altered ATP/Ca2+ and lipid signaling, as well as free cholesterol deposition.