Diabetes Journal current issue

• Comment on: Robertson (2010) Islet Transplantation a Decade Later and Strategies for Filling a Half-Full Glass. Diabetes;59:1285-1291
• Response to Comment on: Robertson (2010) Islet Transplantation a Decade Later and Strategies for Filling a Half-Full Glass. Diabetes;59:1285-1291
• Response to Comment on: Yang et al. (2010) Associations of Hyperglycemia and Insulin Usage With the Risk of Cancer in Type 2 Diabetes: The Hong Kong Diabetes Registry. Diabetes;59:1254-1260
• Comment on: Chauhan et al. (2010) Impact of Common Variants of PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, CDKN2A, IGF2BP2, and CDKAL1 on the Risk of Type 2 Diabetes in 5,164 Indians. Diabetes;59:2068-2074
• Comment on: Yang et al. (2010) Associations of Hyperglycemia and Insulin Usage With the Risk of Cancer in Type 2 Diabetes: The Hong Kong Diabetes Registry. Diabetes;59:1254-1260
• Response to Comment on: Chauhan et al. (2010) Impact of Common Variants of PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, CDKN2A, IGF2BP2, and CDKAL1 on the Risk of Type 2 Diabetes in 5,164 Indians. Diabetes;59:2068-2074
• Comment on: Matsuoka et al. (2010) Regulation of MafA Expression in Pancreatic {beta}-Cells in db/db Mice With Diabetes. Diabetes;59:1709-1720
• Homozygous Mutations in NEUROD1 Are Responsible for a Novel Syndrome of Permanent Neonatal Diabetes and Neurological Abnormalities
  OBJECTIVE

  NEUROD1 is expressed in both developing and mature β-cells. Studies in mice suggest that this basic helix-loop-helix transcription factor is critical in the development of endocrine cell lineage. Heterozygous mutations have previously been identified as a rare cause of maturity-onset diabetes of the young (MODY). We aimed to explore the potential contribution of NEUROD1 mutations in patients with permanent neonatal diabetes.

  RESEARCH DESIGN AND METHODS

  We sequenced the NEUROD1 gene in 44 unrelated patients with permanent neonatal diabetes of unknown genetic etiology.

  RESULTS

  Two homozygous mutations in NEUROD1 (c.427_ 428del and c.364dupG) were identified in two patients. Both mutations introduced a frameshift that would be predicted to generate a truncated protein completely lacking the activating domain. Both patients had permanent diabetes diagnosed in the first 2 months of life with no evidence of exocrine pancreatic dysfunction and a morphologically normal pancreas on abdominal imaging. In addition to diabetes, they had learning difficulties, severe cerebellar hypoplasia, profound sensorineural deafness, and visual impairment due to severe myopia and retinal dystrophy.

  CONCLUSIONS

  We describe a novel clinical syndrome that results from homozygous loss of function mutations in NEUROD1. It is characterized by permanent neonatal diabetes and a consistent pattern of neurological abnormalities including cerebellar hypoplasia, learning difficulties, sensorineural deafness, and visual impairment. This syndrome highlights the critical role of NEUROD1 in both the development of the endocrine pancreas and the central nervous system in humans.

  
  
  
• Elimination of Negative Feedback Control Mechanisms Along the Insulin Signaling Pathway Improves {beta}-Cell Function Under Stress
  OBJECTIVE

  Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-1 signaling. Here, we examined the role of Ser phosphorylation of IRS-2 in mediating the inhibitory effects of proinflammatory cytokines and cellular stress on β-cell function.

  RESEARCH DESIGN AND METHODS

  Five potential inhibitory Ser sites located proximally to the P-Tyr binding domain of IRS-2 were mutated to Ala. These IRS-2 mutants, denoted IRS-2^5A, and their wild-type controls (IRS-2^WT) were introduced into adenoviral constructs that were infected into Min6 cells or into cultured murine islets.

  RESULTS

  When expressed in cultured mouse islets, IRS-2^5A was better than IRS-2^WT in protecting β-cells from apoptosis induced by a combination of IL-1β, IFN-, TNF-, and Fas ligand. Cytokine-treated islets expressing IRS2^5A secreted significantly more insulin in response to glucose than did islets expressing IRS-2^WT. This could be attributed to the higher transcription of Pdx1 in cytokine-treated islets that expressed IRS-2^5A. Accordingly, transplantation of 200 islets expressing IRS2^5A into STZ-induced diabetic mice restored their ability to respond to a glucose load similar to naïve mice. In contrast, mice transplanted with islets expressing IRS2^WT maintained sustained hyperglycemia 3 days after transplantation.

  CONCLUSIONS

  Elimination of a physiological negative feedback control mechanism along the insulin-signaling pathway that involves Ser/Thr phosphorylation of IRS-2 affords protection against the adverse effects of proinflammatory cytokines and improves β-cell function under stress. Genetic approaches that promote IRS2^5A expression in pancreatic β-cells, therefore, could be considered a rational treatment against β-cell failure after islet transplantation.

  
  
  
• {beta}-Cell Failure in Diet-Induced Obese Mice Stratified According to Body Weight Gain: Secretory Dysfunction and Altered Islet Lipid Metabolism Without Steatosis or Reduced {beta}-Cell Mass
  OBJECTIVE

  C57Bl/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 METHODS

  DIO 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.

  RESULTS

  HDR 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.

  CONCLUSIONS

  β-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.

  
  
  
• The Redox Enzyme p66Shc Contributes to Diabetes and Ischemia-Induced Delay in Cutaneous Wound Healing
  OBJECTIVE

  The redox enzyme p66Shc produces hydrogen peroxide and triggers proapoptotic signals. Genetic deletion of p66Shc prolongs life span and protects against oxidative stress. In the present study, we evaluated the role of p66Shc in an animal model of diabetic wound healing.

  RESEARCH DESIGN AND METHODS

  Skin wounds were created in wild-type (WT) and p66Shc^–/– control and streptozotocin-induced diabetic mice with or without hind limb ischemia. Wounds were assessed for collagen content, thickness and vascularity of granulation tissue, apoptosis, reepithelialization, and expression of c-myc and β-catenin. Response to hind limb ischemia was also evaluated.

  RESULTS

  Diabetes delayed wound healing in WT mice with reduced granulation tissue thickness and vascularity, increased apoptosis, epithelial expression of c-myc, and nuclear localization of β-catenin. These nonhealing features were worsened by hind limb ischemia. Diabetes induced p66Shc expression and activation; wound healing was significantly faster in p66Shc^–/– than in WT diabetic mice, with or without hind limb ischemia, at 1 and 3 months of diabetes duration and in both SV129 and C57BL/6 genetic backgrounds. Deletion of p66Shc reversed nonhealing features, with increased collagen content and granulation tissue thickness, and reduced apoptosis and expression of c-myc and β-catenin. p66Shc deletion improved response to hind limb ischemia in diabetic mice in terms of tissue damage, capillary density, and perfusion. Migration of p66Shc^–/– dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia.

  CONCLUSIONS

  p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia. Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

  
  
  
• Muller Cell-Derived VEGF Is Essential for Diabetes-Induced Retinal Inflammation and Vascular Leakage
  OBJECTIVE

  Vascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of Müller cell-derived VEGF in DR, we disrupted VEGF in Müller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice.

  RESEARCH DESIGN AND METHODS

  Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-, ICAM-1, and NF-B. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses.

  RESULTS

  Diabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice.

  CONCLUSIONS

  Müller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, Müller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.

  
  
  
• Cellularity and Adipogenic Profile of the Abdominal Subcutaneous Adipose Tissue From Obese Adolescents: Association With Insulin Resistance and Hepatic Steatosis
  OBJECTIVE

  We explored whether the distribution of adipose cell size, the estimated total number of adipose cells, and the expression of adipogenic genes in subcutaneous adipose tissue are linked to the phenotype of high visceral and low subcutaneous fat depots in obese adolescents.

  RESEARCH DESIGN AND METHODS

  A total of 38 adolescents with similar degrees of obesity agreed to have a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test and hyperinsulinemic euglycemic clamp) and imaging studies (MRI, DEXA, ¹H-NMR). Subcutaneous periumbilical adipose cell-size distribution and the estimated total number of subcutaneous adipose cells were obtained from tissue biopsy samples fixed in osmium tetroxide and analyzed by Beckman Coulter Multisizer. The adipogenic capacity was measured by Affymetrix GeneChip and quantitative RT-PCR.

  RESULTS

  Subjects were divided into two groups: high versus low ratio of visceral to visceral + subcutaneous fat (VAT/[VAT+SAT]). The cell-size distribution curves were significantly different between the high and low VAT/(VAT+SAT) groups, even after adjusting for age, sex, and ethnicity (MANOVA P = 0.035). Surprisingly, the fraction of large adipocytes was significantly lower (P < 0.01) in the group with high VAT/(VAT+SAT), along with the estimated total number of large adipose cells (P < 0.05), while the mean diameter was increased (P < 0.01). From the microarray analyses emerged a lower expression of lipogenesis/adipogenesis markers (sterol regulatory element binding protein-1, acetyl-CoA carboxylase, fatty acid synthase) in the group with high VAT/(VAT+SAT), which was confirmed by RT-PCR.

  CONCLUSIONS

  A reduced lipo-/adipogenic capacity, fraction, and estimated number of large subcutaneous adipocytes may contribute to the abnormal distribution of abdominal fat and hepatic steatosis, as well as to insulin resistance in obese adolescents.

  
  
  
• Age-Period-Cohort Analysis of 1990-2003 Incidence Time Trends of Childhood Diabetes in Italy: The RIDI Study
  OBJECTIVE

  To investigate age-period-cohort effects on the temporal trend of type 1 diabetes in children age 0–14 years in Italian registries.

  RESEARCH DESIGN AND METHODS

  This report is based on 5,180 incident cases in the period 1990–2003 from the Registry for Type 1 Diabetes Mellitus in Italy (RIDI). Multilevel (random intercept) Poisson regression models were used to model the effects of sex, age, calendar time, and birth cohorts on temporal trends, taking into account the registry-level variance component.

  RESULTS

  The incidence rate was 12.26 per 100,000 person-years and significantly higher in boys (13.13 [95% CI 12.66–13.62]) than in girls (11.35 [10.90–11.82]). Large geographical variations in incidence within Italy were evident; incidence was highest in Sardinia, intermediate in Central-Southern Italy, and high in Northern Italy, particularly in the Trento Province, where the incidence rate was 18.67 per 100,000 person-years. An increasing temporal trend was evident (2.94% per year [95% CI 2.22–3.67]). With respect to the calendar period 1990–1992, the incidence rates increased linearly by 15, 27, 35, and 40% in the following time periods (P for trend < 0.001). With respect to the 1987–1993 birth cohort, the incidence rate ratio increased approximately linearly from 0.63 (95% CI 0.54–0.73) in the 1975–1981 cohort to 1.38 (1.06–1.80) in the 1999–2003 cohort. The best model, however, included sex, age, and a linear time trend (drift).

  CONCLUSIONS

  Large geographical variations and an increasing temporal trend in diabetes incidence are evident among type 1 diabetic children in Italy. Age-period-cohort analysis shows that the variation over time has a linear component that cannot be ascribed to either the calendar period or the birth cohort.

  
  
  
• Brain Insulin Action Regulates Hypothalamic Glucose Sensing and the Counterregulatory Response to Hypoglycemia
  OBJECTIVE

  An impaired ability to sense and appropriately respond to insulin-induced hypoglycemia is a common and serious complication faced by insulin-treated diabetic patients. This study tests the hypothesis that insulin acts directly in the brain to regulate critical glucose-sensing neurons in the hypothalamus to mediate the counterregulatory response to hypoglycemia.

  RESEARCH DESIGN AND METHODS

  To delineate insulin actions in the brain, neuron-specific insulin receptor knockout (NIRKO) mice and littermate controls were subjected to graded hypoglycemic (100, 70, 50, and 30 mg/dl) hyperinsulinemic (20 mU/kg/min) clamps and nonhypoglycemic stressors (e.g., restraint, heat). Subsequently, counterregulatory responses, hypothalamic neuronal activation (with transcriptional marker c-fos), and regional brain glucose uptake (via ¹⁴C-2deoxyglucose autoradiography) were measured. Additionally, electrophysiological activity of individual glucose-inhibited neurons and hypothalamic glucose sensing protein expression (GLUTs, glucokinase) were measured.

  RESULTS

  NIRKO mice revealed a glycemia-dependent impairment in the sympathoadrenal response to hypoglycemia and demonstrated markedly reduced (3-fold) hypothalamic c-fos activation in response to hypoglycemia but not other stressors. Glucose-inhibited neurons in the ventromedial hypothalamus of NIRKO mice displayed significantly blunted glucose responsiveness (membrane potential and input resistance responses were blunted 66 and 80%, respectively). Further, hypothalamic expression of the insulin-responsive GLUT 4, but not glucokinase, was reduced by 30% in NIRKO mice while regional brain glucose uptake remained unaltered.

  CONCLUSIONS

  Chronically, insulin acts in the brain to regulate the counterregulatory response to hypoglycemia by directly altering glucose sensing in hypothalamic neurons and shifting the glycemic levels necessary to elicit a normal sympathoadrenal response.

  
  
  
• Human Immune System Development and Rejection of Human Islet Allografts in Spontaneously Diabetic NOD-Rag1null IL2r{gamma}null Ins2Akita Mice
  OBJECTIVE

  To create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell–derived β-cells in the absence or presence of a functional human immune system.

  RESEARCH DESIGN AND METHODS

  We backcrossed the Ins2^Akita mutation onto the NOD-Rag1^null IL2r^null strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts.

  RESULTS

  We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2r^null, Rag1^null, and Ins2^Akita genes in NOD-Rag1^null IL2r^null Ins2^Akita (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2r^null and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts.

  CONCLUSIONS

  NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell–derived β-cells in the absence or presence of an alloreactive human immune system.

  
  
  
• Improvement of Retinal Vascular Injury in Diabetic Rats by Statins Is Associated With the Inhibition of Mitochondrial Reactive Oxygen Species Pathway Mediated by Peroxisome Proliferator-Activated Receptor {gamma} Coactivator 1{alpha}
  OBJECTIVE

  Mitochondrial reactive oxygen species (ROS) plays a key role in diabetic retinopathy (DR) pathogenesis. However, whether simvastatin decreases diabetes-induced mitochondrial ROS production remains uncertain. The aim of this study was to clarify the beneficial effects and mechanism of action of simvastatin against diabetes-induced retinal vascular damage.

  RESEARCH DESIGN AND METHODS

  Diabetic rats and control animals were randomly assigned to receive simvastatin or vehicle for 24 weeks, and bovine retinal capillary endothelial cells (BRECs) were incubated with normal or high glucose with or without simvastatin. Vascular endothelial growth factor (VEGF) and peroxisome proliferator–activated receptor coactivator 1 (PGC-1) in the rat retinas or BRECs were examined by Western blotting and real-time RT-PCR, and poly (ADP-ribose) polymerase (PARP), and p38 MAPK were examined by Western blotting. Mitochondrial membrane potential (m) and ROS production were assayed using the potentiometric dye 5,5',6,6'- Tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) or CM-H₂DCFDA fluorescent probes.

  RESULTS

  Simvastatin significantly upregulated PGC-1 (P < 0.01), subsequently decreased m (P < 0.05) and ROS generation (P < 0.01), inhibited PARP activation (P < 0.01), and further reduced VEGF expression (P < 0.01) and p38 MAPK activity (P < 0.01). Those changes were associated with the decrease of retinal vascular permeability, retinal capillary cells apoptosis, and formation of acellular capillaries.

  CONCLUSIONS

  Simvastatin decreases diabetes-induced mitochondrial ROS production and exerts protective effects against early retinal vascular damage in diabetic rats in association with the inhibition of mitochondrial ROS/PARP pathway mediated by PGC-1. The understanding of the mechanisms of action of statins has important implications in the prevention and treatment of mitochondrial oxidative stress-related illness such as DR.

  
  
  
• A Novel Clinically Relevant Strategy to Abrogate Autoimmunity and Regulate Alloimmunity in NOD Mice
  OBJECTIVE

  To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal.

  RESEARCH DESIGN AND METHODS

  Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.

  RESULTS

  BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice).

  CONCLUSIONS

  The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

  
  
  
• Immune Cell-Derived C3 Is Required for Autoimmune Diabetes Induced by Multiple Low Doses of Streptozotocin
  OBJECTIVE

  The complement system contributes to autoimmune injury, but its involvement in promoting the development of autoimmune diabetes is unknown. In this study, our goal was to ascertain the role of complement C3 in autoimmune diabetes.

  RESEARCH DESIGN AND METHODS

  Susceptibility to diabetes development after multiple low-dose streptozotocin treatment in wild-type (WT) and C3-deficient mice was analyzed. Bone marrow chimeras, luminex, and quantitative reverse transcription PCR assays were performed to evaluate the phenotypic and immunologic impact of C3 in the development of this diabetes model.

  RESULTS

  Coincident with the induced elevations in blood glucose levels, we documented alternative pathway complement component gene expression within the islets of the diabetic WT mice. When we repeated the experiments with C3-deficient mice, we observed complete resistance to disease, as assessed by the absence of histologic insulitis and the absence of T-cell reactivity to islet antigens. Studies of WT chimeras bearing C3-deficient bone marrow cells showed that bone marrow cell–derived C3, and not serum C3, is involved in the induction of diabetes in this model.

  CONCLUSIONS

  The data reveal a key role for immune cell–derived C3 in the pathogenesis of murine multiple low-dose streptozotocin-induced diabetes and support the concept that immune cell mediated diabetes is in part complement-dependent.

  
  
  
• Increased Brain Fatty Acid Uptake in Metabolic Syndrome
  OBJECTIVE

  To test whether brain fatty acid uptake is enhanced in obese subjects with metabolic syndrome (MS) and whether weight reduction modifies it.

  RESEARCH DESIGN AND METHODS

  We measured brain fatty acid uptake in a group of 23 patients with MS and 7 age-matched healthy control subjects during fasting conditions using positron emission tomography (PET) with [¹¹C]-palmitate and [¹⁸F]fluoro-6-thia-heptadecanoic acid ([¹⁸F]-FTHA). Sixteen MS subjects were restudied after 6 weeks of very low calorie diet intervention.

  RESULTS

  At baseline, brain global fatty acid uptake derived from [¹⁸F]-FTHA was 50% higher in patients with MS compared with control subjects. The mean percentage increment was 130% in the white matter, 47% in the gray matter, and uniform across brain regions. In the MS group, the nonoxidized fraction measured using [¹¹C]-palmitate was 86% higher. Brain fatty acid uptake measured with [¹⁸F]-FTHA-PET was associated with age, fasting serum insulin, and homeostasis model assessment (HOMA) index. Both total and nonoxidized fractions of fatty acid uptake were associated with BMI. Rapid weight reduction decreased brain fatty acid uptake by 17%.

  CONCLUSIONS

  To our knowledge, this is the first study on humans to observe enhanced brain fatty acid uptake in patients with MS. Both fatty acid uptake and accumulation appear to be increased in MS patients and reversed by weight reduction.

  
  
  
• Membrane Potential-Dependent Inactivation of Voltage-Gated Ion Channels in {alpha}-Cells Inhibits Glucagon Secretion From Human Islets
  OBJECTIVE

  To document the properties of the voltage-gated ion channels in human pancreatic -cells and their role in glucagon release.

  RESEARCH DESIGN AND METHODS

  Glucagon release was measured from intact islets. [Ca²⁺]_i was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated -cells identified by immunocytochemistry.

  RESULTS

  Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl₂. Glucose remained inhibitory in the presence of ZnCl₂ and after blockade of type-2 somatostatin receptors. Human -cells are electrically active at 1 mmol/l glucose. Inhibition of K_ATP-channels with tolbutamide depolarized -cells by 10 mV and reduced the action potential amplitude. Human -cells contain heteropodatoxin-sensitive A-type K⁺-channels, stromatoxin-sensitive delayed rectifying K⁺-channels, tetrodotoxin-sensitive Na⁺-currents, and low-threshold T-type, isradipine-sensitive L-type, and -agatoxin-sensitive P/Q-type Ca²⁺-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40–70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, -agatoxin, and isradipine. The [Ca²⁺]_i oscillations depend principally on Ca²⁺-influx via L-type Ca²⁺-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below –20 mV and peaked at 0 mV. Blocking P/Q-type Ca²⁺-currents abolished depolarization-evoked exocytosis.

  CONCLUSIONS

  Human -cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

  
  
  
• Targeted Disruption of Pancreatic-Derived Factor (PANDER, FAM3B) Impairs Pancreatic {beta}-Cell Function
  OBJECTIVE

  Pancreatic-derived factor (PANDER, FAM3B) is a pancreatic islet-specific cytokine-like protein that is secreted from β-cells upon glucose stimulation. The biological function of PANDER is unknown, and to address this we generated and characterized a PANDER knockout mouse.

  RESEARCH DESIGN AND METHODS

  To generate the PANDER knockout mouse, the PANDER gene was disrupted and its expression was inhibited by homologous recombination via replacement of the first two exons, secretion signal peptide and transcriptional start site, with the neomycin gene. PANDER^–/– mice were then phenotyped by a number of in vitro and in vivo tests to evaluate potential effects on glucose regulation, insulin sensitivity, and β-cell morphology and function.

  RESULTS

  Glucose tolerance tests demonstrated significantly higher blood glucose levels in PANDER^–/– versus wild-type male mice. To identify the mechanism of the glucose intolerance, insulin sensitivity and pancreatic β-cell function were examined. Hyperinsulinemic-euglycemic clamps and insulin tolerance testing showed similar insulin sensitivity for both the PANDER^–/– and wild-type mice. The in vivo insulin response following intraperitoneal glucose injection surprisingly produced significantly higher insulin levels in the PANDER^–/– mice, whereas insulin release was blunted with arginine administration. Islet perifusion and calcium imaging studies showed abnormal responses of the PANDER^–/– islets to glucose stimulation. In contrast, neither islet architecture nor insulin content was impacted by the loss of PANDER. Interestingly, the elevated insulin levels identified in vivo were attributed to decreased hepatic insulin clearance in the PANDER^–/– islets. Taken together, these results demonstrated decreased pancreatic β-cell function in the PANDER^–/– mouse.

  CONCLUSIONS

  These results support a potential role of PANDER in the pancreatic β-cell for regulation or facilitation of insulin secretion.

  
  
  
• Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft
  OBJECTIVE

  The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets.

  RESEARCH DESIGN AND METHODS

  Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO–expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice.

  RESULTS

  IDO-expressing grafts survived significantly longer than controls (41.2 ± 1.64 vs. 12.9 ± 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming.

  CONCLUSIONS

  Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts.

  
  
  
• Induction of Chimerism Permits Low-Dose Islet Grafts in the Liver or Pancreas to Reverse Refractory Autoimmune Diabetes
  OBJECTIVE

  To test whether induction of chimerism lowers the amount of donor islets required for reversal of diabetes and renders the pancreas a suitable site for islet grafts in autoimmune diabetic mice.

  RESEARCH DESIGN AND METHODS

  The required donor islet dose for reversal of diabetes in late-stage diabetic NOD mice after transplantation into the liver or pancreas was compared under immunosuppression or after induction of chimerism. Recipient mice were monitored for blood glucose levels and measured for insulin-secretion capacity. Islet grafts were evaluated for β-cell proliferation, β-cell functional gene expression, and revascularization.

  RESULTS

  With immunosuppression, transplantation of 1,000, but not 600, donor islets was able to reverse diabetes when transplanted into the liver, but transplantation of 1,000 islets was not able to reverse diabetes when transplanted into the pancreas. In contrast, after induction of chimerism, transplantation of as few as 100 donor islets was able to reverse diabetes when transplanted into either the liver or pancreas. Interestingly, when lower doses (50 or 25) of islets were transplanted, donor islets in the pancreas were much more effective in reversal of diabetes than in the liver, which was associated with higher β-cell replication rate, better β-cell functional gene expression, and higher vascular density of graft islets in the pancreas.

  CONCLUSIONS

  Induction of chimerism not only provides immune tolerance to donor islets, but also markedly reduces the required amount of donor islets for reversal of diabetes. In addition, this process renders the pancreas a more superior site than the liver for donor islets in autoimmune mice.

  
  
  
• Inflammatory Tendencies and Overproduction of IL-17 in the Colon of Young NOD Mice Are Counteracted With Diet Change
  OBJECTIVE

  Dietary factors influence diabetes development in the NOD mouse. Diet affects the composition of microbiota in the distal intestine, which may subsequently influence intestinal immune homeostasis. However, the specific effects of antidiabetogenic diets on gut immunity and the explicit associations between intestinal immune disruption and type 1 diabetes onset remain unclear.

  RESEARCH DESIGN AND METHODS

  Gut microbiota of NOD mice fed a conventional diet or ProSobee formula were compared using gas chromatography. Colonic lamina propria immune cells were characterized in terms of activation markers, cytokine mRNA and Th17 and Foxp3⁺ T-cell numbers, using real-time PCR and flow cytometry. Activation of diabetogenic CD4 T-cells by purified B-cells was assessed in both groups. Immune tolerance to autologous commensal bacteria was evaluated in vitro using thymidine-incorporation tests.

  RESULTS

  Young NOD mice showed a disturbed tolerance to autologous commensal bacteria. Increased numbers of activated CD4 T-cells and (CD11b⁺CD11c⁺) dendritic cells and elevated levels of Th17 cells and IL23 mRNA were moreover observed in colon lamina propria. These phenomena were abolished when mice were fed an antidiabetogenic diet. The antidiabetogenic diet also altered the expression levels of costimulatory molecules and the capacity of peritoneal B-cells to induce insulin-specific CD4 T-cell proliferation.

  CONCLUSIONS

  Young NOD mice show signs of subclinical colitis, but the symptoms are alleviated by a diet change to an antidiabetogenic diet. Disrupted immune tolerance in the distal intestine may influence peritoneal cell pools and B-cell–mediated activation of diabetogenic T-cells.

  
  
  
• Nitric Oxide Synthesis Is Reduced in Subjects With Type 2 Diabetes and Nephropathy
  OBJECTIVE

  Nitric oxide (NO) is a key metabolic and vascular regulator. Its production is stimulated by insulin. A reduced urinary excretion of NO products (NOx) is frequently found in type 2 diabetes, particularly in association with nephropathy. However, whether the decreased NOx excretion in type 2 diabetes is caused by a defective NOx production from arginine in response to hyperinsulinemia has never been studied.

  RESEARCH DESIGN AND METHODS

  We measured NOx fractional (FSR) and absolute (ASR) synthesis rates in type 2 diabetic patients with diabetic nephropathy and in control subjects, after l-[¹⁵N₂-guanidino]-arginine infusion, and use of precursor–product relationships. The study was conducted both before and after an euglycemic hyperinsulinemic (~1,000–1,200 pmol/l) clamp.

  RESULTS

  In type 2 diabetes, NOx FSR was reduced both under basal (19.3 ± 3.9% per day, vs. 22.9 ± 4.5% per day in control subjects) and hyperinsulinemic states (24.0 ± 5.6% per day, vs. 37.9 ± 6.4% per day in control subjects; P < 0.03 by ANOVA). Similarly, in type 2 diabetes, NOx ASR was lower than in control subjects under both conditions (basal, 0.32 ± 0.06 vs. 0.89 ± 0.34 mol per day; hyperinsulinemia, 0.35 ± 0.07 vs. 1.15 ± 0.38 mol per day; P = 0.01 by ANOVA). In type 2 diabetes, the ability of insulin to stimulate both the FSR (4.7 ± 3.2% per day) and the ASR (0.03 ± 0.04 mol per day) of NOx was several-fold lower than that in control subjects (15.0 ± 2.9% per day and 0.25 ± 0.07 mol per day, P < 0.03 and P < 0.02, respectively). Also the fraction of arginine flux converted to NOx (basal, 0.22 ± 0.05% vs. 0.65 ± 0.25%; hyperinsulinemia, 0.32 ± 0.06% vs. 1.03 ± 0.33%) was sharply reduced in the patients (P < 0.01 by ANOVA).

  CONCLUSIONS

  In type 2 diabetic patients with nephropathy, intravascular NOx synthesis from arginine is decreased under both basal and hyperinsulinemic states. This defect extends the concept of insulin resistance to NO metabolism.

  
  
  
• Antigen-Specific Immunotherapy for Type 1 Diabetes: Maximizing the Potential
• Ghrelin Suppresses Glucose-Stimulated Insulin Secretion and Deteriorates Glucose Tolerance in Healthy Humans
  OBJECTIVE

  The orexigenic gut hormone ghrelin and its receptor are present in pancreatic islets. Although ghrelin reduces insulin secretion in rodents, its effect on insulin secretion in humans has not been established. The goal of this study was to test the hypothesis that circulating ghrelin suppresses glucose-stimulated insulin secretion in healthy subjects.

  RESEARCH DESIGN AND METHODS

  Ghrelin (0.3, 0.9 and 1.5 nmol/kg/h) or saline was infused for more than 65 min in 12 healthy patients (8 male/4 female) on 4 separate occasions in a counterbalanced fashion. An intravenous glucose tolerance test was performed during steady state plasma ghrelin levels. The acute insulin response to intravenous glucose (AIRg) was calculated from plasma insulin concentrations between 2 and 10 min after the glucose bolus. Intravenous glucose tolerance was measured as the glucose disappearance constant (Kg) from 10 to 30 min.

  RESULTS

  The three ghrelin infusions raised plasma total ghrelin concentrations to 4-, 15-, and 23-fold above the fasting level, respectively. Ghrelin infusion did not alter fasting plasma insulin or glucose, but compared with saline, the 0.3, 0.9, and 1.5 nmol/kg/h doses decreased AIRg (2,152 ± 448 vs. 1,478 ± 2,889, 1,419 ± 275, and 1,120 ± 174 pmol/l) and Kg (0.3 and 1.5 nmol/kg/h doses only) significantly (P < 0.05 for all). Ghrelin infusion raised plasma growth hormone and serum cortisol concentrations significantly (P < 0.001 for both), but had no effect on glucagon, epinephrine, or norepinephrine levels (P = 0.44, 0.74, and 0.48, respectively).

  CONCLUSIONS

  This is a robust proof-of-concept study showing that exogenous ghrelin reduces glucose-stimulated insulin secretion and glucose disappearance in healthy humans. Our findings raise the possibility that endogenous ghrelin has a role in physiologic insulin secretion, and that ghrelin antagonists could improve β-cell function.

  
  
  
• Kinetics of Contraction-Induced GLUT4 Translocation in Skeletal Muscle Fibers From Living Mice
  OBJECTIVE

  Exercise is an important strategy for the treatment of type 2 diabetes. This is due in part to an increase in glucose transport that occurs in the working skeletal muscles. Glucose transport is regulated by GLUT4 translocation in muscle, but the molecular machinery mediating this process is poorly understood. The purpose of this study was to 1) use a novel imaging system to elucidate the kinetics of contraction-induced GLUT4 translocation in skeletal muscle and 2) determine the function of AMP-activated protein kinase 2 (AMPK2) in this process.

  RESEARCH DESIGN AND METHODS

  Confocal imaging was used to visualize GLUT4-enhanced green fluorescent protein (EGFP) in transfected quadriceps muscle fibers in living mice subjected to contractions or the AMPK-activator AICAR.

  RESULTS

  Contraction increased GLUT4-EGFP translocation from intracellular vesicle depots to both the sarcolemma and t-tubules with similar kinetics, although translocation was greater with contractions elicited by higher voltage. Re-internalization of GLUT4 did not begin until 10 min after contractions ceased and was not complete until 130 min after contractions. AICAR increased GLUT4-EGFP translocation to both sarcolemma and t-tubules with similar kinetics. Ablation of AMPK2 activity in AMPK2 inactive transgenic mice did not change GLUT4-EGFP's basal localization, contraction-stimulated intracellular GLUT4-EGFP vesicle depletion, translocation, or re-internalization, but diminished AICAR-induced translocation.

  CONCLUSIONS

  We have developed a novel imaging system to study contraction-stimulated GLUT4 translocation in living mice. Contractions increase GLUT4 translocation to the sarcolemma and t-tubules with similar kinetics and do not require AMPK2 activity.

  
  
  
• Sleep Restriction for 1 Week Reduces Insulin Sensitivity in Healthy Men
  OBJECTIVE

  Short sleep duration is associated with impaired glucose tolerance and an increased risk of diabetes. The effects of sleep restriction on insulin sensitivity have not been established. This study tests the hypothesis that decreasing nighttime sleep duration reduces insulin sensitivity and assesses the effects of a drug, modafinil, that increases alertness during wakefulness.

  RESEARCH DESIGN AND METHODS

  This 12-day inpatient General Clinical Research Center study included 20 healthy men (age 20–35 years and BMI 20–30 kg/m²). Subjects spent 10 h/night in bed for ≥8 nights including three inpatient nights (sleep-replete condition), followed by 5 h/night in bed for 7 nights (sleep-restricted condition). Subjects received 300 mg/day modafinil or placebo during sleep restriction. Diet and activity were controlled. On the last 2 days of each condition, we assessed glucose metabolism by intravenous glucose tolerance test (IVGTT) and euglycemic-hyperinsulinemic clamp. Salivary cortisol, 24-h urinary catecholamines, and neurobehavioral performance were measured.

  RESULTS

  IVGTT-derived insulin sensitivity was reduced by (means ± SD) 20 ± 24% after sleep restriction (P = 0.001), without significant alterations in the insulin secretory response. Similarly, insulin sensitivity assessed by clamp was reduced by 11 ± 5.5% (P < 0.04) after sleep restriction. Glucose tolerance and the disposition index were reduced by sleep restriction. These outcomes were not affected by modafinil treatment. Changes in insulin sensitivity did not correlate with changes in salivary cortisol (increase of 51 ± 8% with sleep restriction, P < 0.02), urinary catecholamines, or slow wave sleep.

  CONCLUSIONS

  Sleep restriction (5 h/night) for 1 week significantly reduces insulin sensitivity, raising concerns about effects of chronic insufficient sleep on disease processes associated with insulin resistance.