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.

Research in academic medical centers: two threats to sustainable support.

Reductions in federal support and clinical revenue jeopardize biomedical research and, in turn, clinical medicine.

ß-cell failure in type 2 diabetes: postulated mechanisms and prospects for prevention and treatment.

OBJECTIVE: This article examines the foundation of ß-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS: A group of experts participated in a conference on 14-16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS: The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of ß-cell failure, natural history of ß-cell failure, and impact of therapeutic interventions. CONCLUSIONS: ß-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased ß-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to (1) impact the natural history of ß-cell failure; (2) identify and characterize genetic loci for T2D; (3) target ß-cell signaling, metabolic, and genetic pathways to improve function/mass; (4) develop alternative sources of ß-cells for cell-based therapy; (5) focus on metabolic environment to provide indirect benefit to ß-cells; (6) improve understanding of the physiology of responses to bypass surgery; and (7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and ß-cells.

BH3-only molecule Bim mediates ß-cell death in IRS2 deficiency.

Irs2-deficient mice develop type 2-like diabetes due to a reduction in ß-cell mass and a failure of pancreatic islets to undergo compensatory hyperplasia in response to insulin resistance. In order to define the molecular mechanisms, we knocked down Irs2 gene expression in mouse MIN6 insulinoma cells. Insulin receptor substrate 2 (IRS2) suppression induced apoptotic cell death, which was associated with an increase in expression of the BH3-only molecule Bim. Knockdown (KD) of Bim reduced apoptotic ß-cell death induced by IRS2 suppression. In Irs2-deficient mice, Bim ablation restored ß-cell mass, decreased the number of TUNEL-positive cells, and restored normal glucose tolerance after glucose challenge. FoxO1 mediates Bim upregulation induced by IRS2 suppression, and FoxO1 KD partially inhibits ß-cell death induced by IRS2 suppression. These results suggest that Bim plays an important role in mediating the increase in ß-cell apoptosis and the reduction in ß-cell mass that occurs in IRS2-deficient diabetes.

ß-cell failure in type 2 diabetes: postulated mechanisms and prospects for prevention and treatment.

OBJECTIVE: This article examines the foundation of ß-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS: A group of experts participated in a conference on 14-16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS: The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of ß-cell failure, natural history of ß-cell failure, and impact of therapeutic interventions. CONCLUSIONS: ß-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased ß-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to 1) impact the natural history of ß-cell failure; 2) identify and characterize genetic loci for T2D; 3) target ß-cell signaling, metabolic, and genetic pathways to improve function/mass; 4) develop alternative sources of ß-cells for cell-based therapy; 5) focus on metabolic environment to provide indirect benefit to ß-cells; 6) improve understanding of the physiology of responses to bypass surgery; and 7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and ß-cells.

Role of BH3-only molecules Bim and Puma in ß-cell death in Pdx1 deficiency.

Mutations in pancreatic duodenal homeobox-1 (PDX1) are associated with diabetes in humans. Pdx1-haploinsufficient mice develop diabetes due to an increase in ß-cell death leading to reduced ß-cell mass. For definition of the molecular link between Pdx1 deficiency and ß-cell death, Pdx1-haploinsufficient mice in which the genes for the BH3-only molecules Bim and Puma had been ablated were studied on a high-fat diet. Compared with Pdx1(+/-) mice, animals haploinsufficient for both Pdx1 and Bim or Puma genes showed improved glucose tolerance, enhanced ß-cell mass, and reduction in the number of TUNEL-positive cells in islets. These results suggest that Bim and Puma ablation improves ß-cell survival in Pdx1(+/-) mice. For exploration of the mechanisms responsible for these findings, Pdx1 gene expression was knocked down in mouse MIN6 insulinoma cells resulting in apoptotic cell death that was found to be associated with increased expression of BH3-only molecules Bim and Puma. If the upregulation of Bim and Puma that occurs during Pdx1 suppression was prevented, apoptotic ß-cell death was reduced in vitro. These results suggest that Bim and Puma play an important role in 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.

Ginsenoside Re rapidly reverses insulin resistance in muscles of high-fat diet fed rats.

OBJECTIVE: In a previous study, it was found that a ginseng berry extract with a high content of the ginsenoside Re normalized blood glucose in ob/ob mice. The objective of this study was to evaluate the effect of the ginsenoside Re on insulin resistance of glucose transport in muscles of rats made insulin resistant with a high-fat diet. MATERIAL/METHOD: Rats were fed either rat chow or a high-fat diet for 5 weeks. The rats were then euthanized, and insulin stimulated glucose transport activity was measured in epitrochlearis and soleus muscle strips in vitro. RESULTS: Treatment of muscles with Re alone had no effect on glucose transport. The high-fat diet resulted in ~50% decreases in insulin responsiveness of GLUT4 translocation to the cell surface and glucose transport in epitrochlearis and soleus muscles. Treatment of muscles with Re in vitro for 90 min completely reversed the high-fat diet-induced insulin resistance of glucose transport and GLUT4 translocation. This effect of Re is specific for insulin stimulated glucose transport, as Re treatment did not reverse the high-fat diet-induced resistance of skeletal muscle glucose transport to stimulation by contractions or hypoxia. CONCLUSIONS: Our results show that the ginsenoside Re induces a remarkably rapid reversal of high-fat diet-induced insulin resistance of muscle glucose transport by reversing the impairment of insulin-stimulated GLUT4 translocation to the cell surface.

Xenin-25 amplifies GIP-mediated insulin secretion in humans with normal and impaired glucose tolerance but not type 2 diabetes.

Glucose-dependent insulinotropic polypeptide (GIP) potentiates glucose-stimulated insulin secretion (GSIS). This response is blunted in type 2 diabetes (T2DM). Xenin-25 is a 25-amino acid neurotensin-related peptide that amplifies GIP-mediated GSIS in hyperglycemic mice. This study determines if xenin-25 amplifies GIP-mediated GSIS in humans with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or T2DM. Each fasting subject received graded glucose infusions to progressively raise plasma glucose concentrations, along with vehicle alone, GIP, xenin-25, or GIP plus xenin-25. Plasma glucose, insulin, C-peptide, and glucagon levels and insulin secretion rates (ISRs) were determined. GIP amplified GSIS in all groups. Initially, this response was rapid, profound, transient, and essentially glucose independent. Thereafter, ISRs increased as a function of plasma glucose. Although magnitudes of insulin secretory responses to GIP were similar in all groups, ISRs were not restored to normal in subjects with IGT and T2DM. Xenin-25 alone had no effect on ISRs or plasma glucagon levels, but the combination of GIP plus xenin-25 transiently increased ISR and plasma glucagon levels in subjects with NGT and IGT but not T2DM. Since xenin-25 signaling to islets is mediated by a cholinergic relay, impaired islet responses in T2DM may reflect defective neuronal, rather than GIP, signaling.

Moderate effect of duodenal-jejunal bypass surgery on glucose homeostasis in patients with type 2 diabetes.

Gastric bypass surgery causes resolution of type 2 diabetes (T2DM), which has led to the hypothesis that upper gastrointestinal (UGI) tract diversion, itself, improves glycemic control. The purpose of this study was to determine whether UGI tract bypass without gastric exclusion has therapeutic effects in patients with T2DM. We performed a prospective trial to assess glucose and ß-cell response to an oral glucose load before and at 6, 9, and 12 months after duodenal-jejunal bypass (DJB) surgery. Thirty-five overweight or obese adults (BMI: 27.0 ± 4.0 kg/m(2)) with T2DM and 35 sex-, age-, race-, and BMI-matched subjects with normal glucose tolerance (NGT) were studied. Subjects lost weight after surgery, which was greatest at 3 months (6.9 ± 4.9%) with subsequent regain to 4.2 ± 5.3% weight loss at 12 months after surgery. Glycated hemoglobin (HbA(1c)) decreased from 9.3 ± 1.6% before to 7.7 ± 2.0% at 12 months after surgery (P < 0.001), in conjunction with a 20% decrease in the use of diabetes medications (P < 0.05); 7 (20%) subjects achieved remission of diabetes (no medications and HbA(1c) <6.5%). The area under the curve after glucose ingestion was ~20% lower for glucose but doubled for insulin and C-peptide at 12 months, compared with pre-surgery values (all P < 0.01). However, the ß-cell response was still 70% lower than subjects with NGT (P < 0.001). DJB surgery improves glycemic control and increases, but does not normalize the ß-cell response to glucose ingestion. These findings suggest that altering the intestinal site of delivery of ingested nutrients has moderate therapeutic effects by improving ß-cell function and glycemic control.

Ginseng and ginsenoside Re do not improve ß-cell function or insulin sensitivity in overweight and obese subjects with impaired glucose tolerance or diabetes.

OBJECTIVE: Ginseng and its active component, ginsenoside Re, are popular herbal products that are advocated for treatment of diabetes. The purpose of this study was to determine whether ginseng or ginsenoside Re improves ß-cell function and insulin sensitivity (IS) in insulin-resistant subjects. RESEARCH DESIGN AND METHODS: Overweight or obese subjects (BMI = 34 ± 1 kg/m²) with impaired glucose tolerance or newly diagnosed type 2 diabetes were randomized to 30 days of treatment with ginseng root extract (8 g/day), ginsenoside Re (250-500 mg/day), or placebo. ß-Cell function was assessed as the disposition index (DI) and measured by a frequently sampled oral glucose tolerance test, and IS was assessed as the relative increase in glucose disposal during a hyperinsulinemic-euglycemic clamp procedure plus stable isotope tracer infusion. RESULTS: Values for DI and IS after therapy (Post) were not different from values before therapy (Pre) in the placebo (DI: Pre, 5.8 ± 0.9 × 10⁻³ and Post, 5.8 ± 0.8 × 10⁻³, P = 0.99; IS: Pre,165 ± 29% and Post, 185 ± 24%, P = 0.34), ginseng (DI: Pre, 7.7 ± 2.0 × 10⁻³ and Post, 6.0 ± 0.8 × 10⁻³, P = 0.29; IS: Pre, 171 ± 72% and Post,137 ± 59%, P = 0.88), and ginsenoside Re (DI: Pre, 7.4 ± 3.0 × 10⁻³ and Post, 5.9 ± 1.1 × 10⁻³, P = 0.50; IS: Pre, 117 ± 31% and Post, 134 ± 34%, P = 0.44) groups. Ginsenosides Re, Rb1, and Rb2 were not detectable in plasma after treatment with ginseng root extract or ginsenoside Re. CONCLUSIONS: Oral ginseng or ginsenoside Re therapy does not improve ß-cell function or IS in overweight/obese subjects with impaired glucose tolerance or newly diagnosed diabetes. Poor systemic bioavailability might be responsible for the absence of a therapeutic effect.

Loss of Nix in Pdx1-deficient mice prevents apoptotic and necrotic ß cell death and diabetes.

Mutations in pancreatic duodenal homeobox (PDX1) are linked to human type 2 diabetes and maturity-onset diabetes of the young type 4. Consistent with this, Pdx1-haploinsufficient mice develop diabetes. Both apoptosis and necrosis of ß cells are mechanistically implicated in diabetes in these mice, but a molecular link between Pdx1 and these 2 forms of cell death has not been defined. In this study, we introduced an shRNA into mouse insulinoma MIN6 cells to deplete Pdx1 and found that expression of proapoptotic genes, including NIP3-like protein X (Nix), was increased. Forced Nix expression in MIN6 and pancreatic islet ß cells induced programmed cell death by simultaneously activating apoptotic and mitochondrial permeability transition-dependent necrotic pathways. Preventing Nix upregulation during Pdx1 suppression abrogated apoptotic and necrotic ß cell death in vitro. In Pdx1-haploinsufficient mice, Nix ablation normalized pancreatic islet architecture, ß cell mass, and insulin secretion and eliminated reactive hyperglycemia after glucose challenge. These results establish Nix as a critical mediator of ß cell apoptosis and programmed necrosis in Pdx1-deficient diabetes.

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.

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.

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.