Pancreatic ß-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak.

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in ß-cell death leading to reduced ß-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic ß-cell death. In Pdx1 haploinsufficient mice, Bax ablation in ß-cells increased ß-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates ß-cell apoptosis in Pdx1-deficient diabetes.

Xenin-25 delays gastric emptying and reduces postprandial glucose levels in humans with and without type 2 diabetes.

Xenin-25 (Xen) is a neurotensin-related peptide secreted by a subset of glucose-dependent insulinotropic polypeptide (GIP)-producing enteroendocrine cells. In animals, Xen regulates gastrointestinal function and glucose homeostasis, typically by initiating neural relays. However, little is known about Xen action in humans. This study determines whether exogenously administered Xen modulates gastric emptying and/or insulin secretion rates (ISRs) following meal ingestion. Fasted subjects with normal (NGT) or impaired (IGT) glucose tolerance and Type 2 diabetes mellitus (T2DM; n = 10-14 per group) ingested a liquid mixed meal plus acetaminophen (ACM; to assess gastric emptying) at time zero. On separate occasions, a primed-constant intravenous infusion of vehicle or Xen at 4 (Lo-Xen) or 12 (Hi-Xen) pmol · kg(-1) · min(-1) was administered from zero until 300 min. Some subjects with NGT received 30- and 90-min Hi-Xen infusions. Plasma ACM, glucose, insulin, C-peptide, glucagon, Xen, GIP, and glucagon-like peptide-1 (GLP-1) levels were measured and ISRs calculated. Areas under the curves were compared for treatment effects. Infusion with Hi-Xen, but not Lo-Xen, similarly delayed gastric emptying and reduced postprandial glucose levels in all groups. Infusions for 90 or 300 min, but not 30 min, were equally effective. Hi-Xen reduced plasma GLP-1, but not GIP, levels without altering the insulin secretory response to glucose. Intense staining for Xen receptors was detected on PGP9.5-positive nerve fibers in the longitudinal muscle of the human stomach. Thus Xen reduces gastric emptying in humans with and without T2DM, probably via a neural relay. Moreover, endogenous GLP-1 may not be a major enhancer of insulin secretion in healthy humans under physiological conditions.

Complex patterns of metabolic and Ca²âº entrainment in pancreatic islets by oscillatory glucose.

Glucose-stimulated insulin secretion is pulsatile and driven by intrinsic oscillations in metabolism, electrical activity, and Ca(2+) in pancreatic islets. Periodic variations in glucose can entrain islet Ca(2+) and insulin secretion, possibly promoting interislet synchronization. Here, we used fluorescence microscopy to demonstrate that glucose oscillations can induce distinct 1:1 and 1:2 entrainment of oscillations (one and two oscillations for each period of exogenous stimulus, respectively) in islet Ca(2+), NAD(P)H, and mitochondrial membrane potential. To our knowledge, this is the first demonstration of metabolic entrainment in islets, and we found that entrainment of metabolic oscillations requires voltage-gated Ca(2+) influx. We identified diverse patterns of 1:2 entrainment and showed that islet synchronization during entrainment involves adjustments of both oscillatory phase and period. All experimental findings could be recapitulated by our recently developed mathematical model, and simulations suggested that interislet variability in 1:2 entrainment patterns reflects differences in their glucose sensitivity. Finally, our simulations and recordings showed that a heterogeneous group of islets synchronized during 1:2 entrainment, resulting in a clear oscillatory response from the collective. In summary, we demonstrate that oscillatory glucose can induce complex modes of entrainment of metabolically driven oscillations in islets, and provide additional support for the notion that entrainment promotes interislet synchrony in the pancreas.

Targeting cyclophilin D and the mitochondrial permeability transition enhances beta-cell survival and prevents diabetes in Pdx1 deficiency.

Mutations of the pancreatic duodenal homeobox gene-1, Pdx1, cause heritable diabetes in humans and mice. A central abnormality with Pdx1 deficiency is increased death of beta-cells, leading to decreased beta-cell mass. We show that lentiviral suppression of Pdx1 increases death of mouse insulinoma MIN6 beta-cells associated with dissipation of the mitochondrial inner membrane electrochemical gradient, Deltapsi(m). Preventing mitochondrial permeability transition pore opening with the cyclophilin D inhibitor cyclosporin A restored Deltapsi(m) and rescued cell viability. Reduced beta-cell mass, markers of beta-cell apoptosis, necrosis, and decreased proliferation are present in Pdx1 haploinsufficient mice. Genetic ablation of the Ppif gene, encoding cyclophilin D, restored beta-cell mass and decreased TUNEL and complement complex labeling without affecting beta-cell proliferation. In adult mice maintained on a high-fat diet, Ppif ablation normalized fasting glucose and glucose and insulin responses to acute glucose challenge. Thus, cyclophilin D and the mitochondrial permeability transition are critical regulators of beta-cell death caused by Pdx1 insufficiency.

Xenin-25 potentiates glucose-dependent insulinotropic polypeptide action via a novel cholinergic relay mechanism.

The intestinal peptides GLP-1 and GIP potentiate glucose-mediated insulin release. Agents that increase GLP-1 action are effective therapies in type 2 diabetes mellitus (T2DM). However, GIP action is blunted in T2DM, and GIP-based therapies have not been developed. Thus, it is important to increase our understanding of the mechanisms of GIP action. We developed mice lacking GIP-producing K cells. Like humans with T2DM, "GIP/DT" animals exhibited a normal insulin secretory response to exogenous GLP-1 but a blunted response to GIP. Pharmacologic doses of xenin-25, another peptide produced by K cells, restored the GIP-mediated insulin secretory response and reduced hyperglycemia in GIP/DT mice. Xenin-25 alone had no effect. Studies with islets, insulin-producing cell lines, and perfused pancreata indicated xenin-25 does not enhance GIP-mediated insulin release by acting directly on the beta-cell. The in vivo effects of xenin-25 to potentiate insulin release were inhibited by atropine sulfate and atropine methyl bromide but not by hexamethonium. Consistent with this, carbachol potentiated GIP-mediated insulin release from in situ perfused pancreata of GIP/DT mice. In vivo, xenin-25 did not activate c-fos expression in the hind brain or paraventricular nucleus of the hypothalamus indicating that central nervous system activation is not required. These data suggest that xenin-25 potentiates GIP-mediated insulin release by activating non-ganglionic cholinergic neurons that innervate the islets, presumably part of an enteric-neuronal-pancreatic pathway. Xenin-25, or molecules that increase acetylcholine receptor signaling in beta-cells, may represent a novel approach to overcome GIP resistance and therefore treat humans with T2DM.

Carboxypeptidase E mediates palmitate-induced beta-cell ER stress and apoptosis.

Obesity is a principal risk factor for type 2 diabetes, and elevated fatty acids reduce beta-cell function and survival. An unbiased proteomic screen was used to identify targets of palmitate in beta-cell death. The most significantly altered protein in both human islets and MIN6 beta-cells treated with palmitate was carboxypeptidase E (CPE). Palmitate reduced CPE protein levels within 2 h, preceding endoplasmic reticulum (ER) stress and cell death, by a mechanism involving CPE translocation to Golgi and lysosomal degradation. Palmitate metabolism and Ca(2+) flux were also required for CPE proteolysis and beta-cell death. Chronic palmitate exposure increased the ratio of proinsulin to insulin. CPE null islets had increased apoptosis in vivo and in vitro. Reducing CPE by approximately 30% using shRNA also increased ER stress and apoptosis. Conversely, overexpression of CPE partially rescued beta-cells from palmitate-induced ER stress and apoptosis. Thus, carboxypeptidase E degradation contributes to palmitate-induced beta-cell ER stress and apoptosis. CPE is a major link between hyperlipidemia and beta-cell death pathways in diabetes.

Diabetic pancreatic beta cells ARNT all they should be.

A complex network of interacting transcription factors plays a critical role in normal pancreatic beta cell function, with mutations in certain transcription factor genes known to cause diabetes. In a recent issue of Cell, Gunton et al.(2005) demonstrate a role for the transcription factor ARNT/HIF1beta (hydrocarbon nuclear receptor translocator/hypoxia-inducible factor 1 beta) in normal beta cell function. ARNT expression is reduced in diabetic human islets and beta cell-specific ARNT knockout mice show the impaired glucose tolerance and abnormal insulin secretion that are characteristic of type 2 diabetes.

Calpain-10 is a component of the obesity-related quantitative trait locus Adip1.

We previously mapped Adip1, an obesity quantitative trait locus (QTL), to the central portion of murine chromosome 1 containing the calpain-10 (Capn10) gene. Human studies have associated calpain-10 (CAPN10) variants with type 2 diabetes and various metabolic traits. We performed a quantitative hybrid complementation test (QHCT) to determine whether differences attributed to Adip1 are the result of variant Capn10 alleles in LG/J and SM/J mice. We crossed LG/J and SM/J to wild-type (C57BL/6J) and Capn10 knockout (Capn10(-/-)) mice to form four F(1) hybrid groups: LG/J by wild-type, LG/J by Capn10(-/-), SM/J by wild-type, and SM/J by Capn10(-/-). We performed a two-way ANOVA with the experimental strain, tester strain, and their interaction as the factors. Significant interaction indicates a quantitative failure to complement. We found failure to complement for fat, organ, and body weights, and leptin, female free fatty acid, and triglyceride levels. Capn10(-/-) resulted in heavier weights and higher serum levels in LG/J crosses but not in SM/J crosses. For glucose tolerance and insulin response tests, the Capn10(-/-) allele resulted in lower glucose levels in crosses with SM/J but had no effect in the LG/J crosses. Differences between the LG/J and SM/J Capn10 alleles are the likely source of some of the QTL effects mapped to Adip1 in the LG/J-by-SM/J cross. Capn10 plays an important role in regulating obesity and diabetes in mice.

Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation.

There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1(+/-) mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1(+/-) mice to Becn1(+/-) mice. After 1 week on a high fat diet, 4-week-old Pdx1(+/-) Becn1(+/-) mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1(+/-) mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.

International trial of the Edmonton protocol for islet transplantation.

BACKGROUND: Islet transplantation offers the potential to improve glycemic control in a subgroup of patients with type 1 diabetes mellitus who are disabled by refractory hypoglycemia. We conducted an international, multicenter trial to explore the feasibility and reproducibility of islet transplantation with the use of a single common protocol (the Edmonton protocol). METHODS: We enrolled 36 subjects with type 1 diabetes mellitus, who underwent islet transplantation at nine international sites. Islets were prepared from pancreases of deceased donors and were transplanted within 2 hours after purification, without culture. The primary end point was defined as insulin independence with adequate glycemic control 1 year after the final transplantation. RESULTS: Of the 36 subjects, 16 (44%) met the primary end point, 10 (28%) had partial function, and 10 (28%) had complete graft loss 1 year after the final transplantation. A total of 21 subjects (58%) attained insulin independence with good glycemic control at any point throughout the trial. Of these subjects, 16 (76%) required insulin again at 2 years; 5 of the 16 subjects who reached the primary end point (31%) remained insulin-independent at 2 years. CONCLUSIONS: Islet transplantation with the use of the Edmonton protocol can successfully restore long-term endogenous insulin production and glycemic stability in subjects with type 1 diabetes mellitus and unstable control, but insulin independence is usually not sustainable. Persistent islet function even without insulin independence provides both protection from severe hypoglycemia and improved levels of glycated hemoglobin. (ClinicalTrials.gov number, NCT00014911 [ClinicalTrials.gov].).

Suppressed insulin signaling and increased apoptosis in CD38-null islets.

CD38 is a multifunctional enzyme capable of generating metabolites that release Ca2+ from intracellular stores, including nicotinic acid adenine dinucleotide phosphate (NAADP). A number of studies have led to the controversial proposal that CD38 mediates an alternate pathway for glucose-stimulated insulin release and contributes to the pathogenesis of diabetes. It has recently been shown that NAADP mediates Ca2+ mobilization by insulin in human pancreatic beta-cells. In the present study, we report altered Ca2+ homeostasis and reduced responsiveness to insulin, but not glucose, in Cd38-/- beta-cells. In keeping with the antiapoptotic role of insulin signaling, Cd38-/- islets were significantly more susceptible to apoptosis compared with islets isolated from littermate controls. This finding correlated with disrupted islet architecture and reduced beta-cell mass in Cd38-/- mice, both in the context of a normal lab diet and a high-fat diet. Nevertheless, we did not find robust differences in glucose homeostasis in vivo or glucose signaling in vitro in Cd38-/- mice on the C57BL/6 genetic background, in contrast to previous studies by others of Cd38 knockout mice on the ICR background. Thus, our results suggest that CD38 plays a role in novel antiapoptotic signaling pathways but does not directly control glucose signaling in pancreatic beta-cells.

Defective insulin secretion and increased susceptibility to experimental diabetes are induced by reduced Akt activity in pancreatic islet beta cells.

The insulin and IGF signaling pathways are critical for development and maintenance of pancreatic beta cell mass and function. The serine-threonine kinase Akt is one of several mediators regulated by these pathways. We have studied the role of Akt in pancreatic beta cell physiology by generating transgenic mice expressing a kinase-dead mutant of this enzyme in beta cells. Reduction of Akt activity in transgenic animals resulted in impaired glucose tolerance due to defective insulin secretion. The mechanisms involved in dysregulation of secretion in these mice lie at the level of insulin exocytosis and are not the result of abnormalities in glucose signaling or function of voltage-gated Ca2+ channels. Therefore, transgenic mice showed increased susceptibility to developing glucose intolerance and diabetes following fat feeding. These observations suggest that Akt plays a novel and important role in the regulation of distal components of the secretory pathway and that this enzyme represents a therapeutic target for improvement of beta cell function in diabetes.

Increased islet apoptosis in Pdx1+/- mice.

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency.

We have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia, and elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small-intestinal absorptive dysfunction, which was not previously clinically suspected. We postulate that PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject A's negligible PC1 activity, some mature ACTH and glucagon-like peptide 17-36(amide) were detectable in her plasma, suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with the phenotype of mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species.

Two-hour seven-sample oral glucose tolerance test and meal protocol: minimal model assessment of beta-cell responsivity and insulin sensitivity in nondiabetic individuals.

Highly informative yet simple protocols to assess insulin secretion and action would considerably enhance the quality of epidemiological and large-scale clinical trials. In an effort to develop such protocols, a 5-h, 11-sample oral glucose tolerance test (OGTT) was performed in 100 individuals and a 7-h, 21-sample meal in another 100. Plasma glucose, insulin, and C-peptide concentrations were measured. We show that virtually the same minimal model assessment of beta-cell responsivity (dynamic [Phi(d)] and static [Phi(s)]), insulin sensitivity (Si), and disposition index (DI) can be obtained with a reduced seven-sample 2-h protocol: Phi(d), reduced versus full: 871.50 vs. 873.32, r = 0.98 in OGTT and 494.88 vs. 477.99 10(-9), r = 0.91 in meal; Phi(s): 42.36 vs. 44.35, r = 0.88 in OGTT and 35.31 vs. 35.37 10(-9) min(-1), r = 0.90 in meal; Si: 24.33 vs. 22.77 10(-5) dl x kg(-1) x min(-1) per pmol/l, r = 0.89 in OGTT and 19.03 vs. 19.77 10(-5) dl x kg(-1) x min(-1) per pmol/l, r = 0.85 in meal; and DI: 1,282.26 vs. 1,273.23, r = 0.84 in OGTT and 726.92 vs. 776.97 10(-14) dl . kg(-1) x min(-2) per pmol/l, r = 0.84 in meal. This reduced protocol will facilitate the study of insulin secretion and action under physiological conditions in nondiabetic humans.

Rosiglitazone reduces glucose-stimulated insulin secretion rate and increases insulin clearance in nondiabetic, insulin-resistant individuals.

Compensatory hyperinsulinemia permitting insulin-resistant individuals to maintain normal glucose tolerance is associated with a left shift in the glucose-stimulated insulin secretion rate (GS-ISR) dose-response curve and decrease in the insulin metabolic clearance rate (I-MCR). To see whether these changes would reverse with improvement in insulin sensitivity, 14 nondiabetic insulin-resistant subjects received rosiglitazone for 12 weeks (4 mg daily for 4 weeks and then 8 mg daily for 8 weeks). Insulin-mediated glucose uptake was quantified by measuring the steady-state plasma glucose concentration during the insulin suppression test. GS-ISR and I-MCR were determined during a 240-min graded intravenous glucose infusion. I-MCR was also calculated during the insulin suppression test. After rosiglitazone treatment, insulin sensitivity improved with significant fall in steady-state plasma glucose (means +/- SE from 13.5 +/- 0.62 to 9.8 +/- 1.02 mmol/l, P < 0.001). In response, the integrated GS-ISR decreased by 21% (P < 0.001), with a right shift in the dose-response curve. Calculated I-MCR increased by 34% (P = 0.008) during the insulin suppression test and by 21% (P = 0.03) during the graded glucose infusion. In conclusion, enhanced insulin sensitivity in rosiglitazone-treated nondiabetic insulin-resistant individuals was associated with a shift to the right in the GS-ISR dose-response curve and an increase in I-MCR.