Explore the vast range of titles available.

Glucagon secretion by pancreatic α-cells is triggered by hypoglycemia and suppressed by high glucose levels; impaired suppression of glucagon secretion is a hallmark of both type 1 and type 2 diabetes. Here, we show that α-cell glucokinase ( Gck ) plays a role in the control of glucagon secretion. Using mice with α-cell-specific inactivation of Gck ( αGckKO mice), we find that glucokinase is required for the glucose-dependent increase in intracellular ATP/ADP ratio and the closure of K ATP channels in α-cells and the suppression of glucagon secretion at euglycemic and hyperglycemic levels. αGckKO mice display hyperglucagonemia in the fed state, which is associated with increased hepatic gluconeogenic gene expression and hepatic glucose output capacity. In adult mice, fed hyperglucagonemia is further increased and glucose intolerance develops. Thus, glucokinase governs an α-cell metabolic pathway that suppresses secretion at or above normoglycemic levels; abnormal suppression of glucagon secretion deregulates hepatic glucose metabolism and, over time, induces a pre-diabetic phenotype. Glucagon secretion is promoted during hypoglycemia and inhibited by increased glucose levels. Here, Basco et al. show that glucokinase suppresses glucose-regulated glucagon secretion by modulating the intracellular ATP/ADP ratio and the closure of K ATP channels in α-cells.

Increasing GLP-1–Induced β-Cell Proliferation by Silencing the Negative Regulators of Signaling cAMP Response Element Modulator-α and DUSP14 Sonia Klinger 1 , 2 , Carine Poussin 1 , 2 , Marie-Bernard Debril 1 , 2 , Wanda Dolci 1 , 2 , Philippe A. Halban 3 and Bernard Thorens 1 , 2 1 Institute of Physiology, University of Lausanne, Lausanne, Switzerland 2 Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland 3 Department of Genetic Medicine and Development, University Medical Center, University Hospital, Geneva, Switzerland Address correspondence and reprint requests to Bernard Thorens, University of Lausanne, Center for Integrative Genomics, Génopode Building, 1015 Lausanne, Switzerland. E-mail: bernard.thorens{at}unil.ch Abstract OBJECTIVE —Glucagon-like peptide-1 (GLP-1) is a growth and differentiation factor for mature β-cells and their precursors. However, the overall effect of GLP-1 on increasing β-cell mass in both in vivo and in vitro conditions is relatively small, and augmenting this effect would be beneficial for the treatment or prevention of type 1 and type 2 diabetes. Here, we searched for cellular mechanisms that may limit the proliferative effect of GLP-1 and tested whether blocking them could increase β-cell proliferation. RESEARCH DESIGN AND METHODS —We examined GLP-1–regulated genes in βTC-Tet cells by cDNA microarrays. To assess the effect of some of these gene on cell proliferation, we reduced their expression using small heterogenous RNA in β-cell lines and primary mouse islets and measured [ 3 H]thymidine or 5′-bromo-2′-deoxyuridine incorporation. RESULTS —We identified four negative regulators of intracellular signaling that were rapidly and strongly activated by GLP-1: the regulator of G-protein–signaling RGS2; the cAMP response element-binding protein (CREB) antagonists cAMP response element modulator (CREM)-α and ICERI; and the dual specificity phosphatase DUSP14, a negative regulator of the mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase 1/2 (ERK1/2) pathway. We show that knockdown of CREMα or DUSP14 or expression of a dominant-negative form of DUSP14 increased β-cell line proliferation and enhanced the GLP-1–induced proliferation of primary β-cells. CONCLUSIONS —Together, our data show that 1 ) the cAMP/protein kinase A/CREB and MAPK/ERK1/2 pathways can additively control β-cell proliferation, 2 ) β-cells have evolved several mechanisms limiting GLP-1–induced cellular proliferation, and 3 ) blocking these mechanisms increases the positive effect of GLP-1 on β-cell mass. CREB, cAMP response element-binding protein CREM, cAMP response element modulator DMEM, Dulbecco's modified Eagle's medium ERK1/2, extracellular signal-regulated kinase 1/2 FACS, fluorescence-activated cell sorter FBS, fetal bovine serum GAPDH, glyceraldehyde-3-phosphate dehydrogenase GFP, green fluorescent protein GLP-1, glucagon-like peptide-1 MAPK, mitogen-activated protein kinase PI 3-kinase, phosphatidylinositol 3-kinase PKA, protein kinase A Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 19 November 2007. DOI: 10.2337/db07-1414. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-1414 . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received October 4, 2007. Accepted November 13, 2007. DIABETES

cFLIP Protein Prevents Tumor Necrosis Factor-α–Mediated Induction of Caspase-8–Dependent Apoptosis in Insulin-Secreting βTc-Tet Cells Sandra Cottet , Philippe Dupraz , Fabienne Hamburger , Wanda Dolci , Muriel Jaquet and Bernard Thorens Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland Abstract Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to β-cell dysfunction and destruction. The exact role played by interferon-γ, tumor necrosis factor (TNF)-α, and interleukin-1β in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, βTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-α, in combination with interleukin-1β and interferon-γ. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in βTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1β–mediated transcriptional activity of nuclear factor (NF)-κB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-α–induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-γ alone could induce these β-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse β-cells against TNF-α–induced caspase-8 activation and apoptosis and is correlated with enhanced NF-κB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor–triggered responses by directing the intracellular signals from β-cell death to β-cell survival. Footnotes Address correspondence and reprint requests to Bernard Thorens, Institute of Pharmacology and Toxicology, University of Lausanne, 27 rue du Bugnon, 1005 Lausanne, Switzerland. E-mail: bthorens{at}ipharm.unil.ch . S.C. and P.D. contributed equally to this work. Received for publication 3 July 2001 and accepted in revised form 27 February 2002. cFLIP, cellular FLICE-inhibitory protein; CHX, cycloheximide; DD, death domain; DED, death effector domain; DISC, death-inducing signaling complex; ELISA, enzyme-linked immunosorbent assay; FADD: Fas-associated death domain protein; FLAG, octapeptide tag; FLICE: FADD-like interleukin-1β–converting enzyme; GSIS, glucose-stimulated insulin secretion; IAP, inhibitor of apoptosis; IBMX, isobutylmethylxanthine; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; JAK, Janus kinase; KRBH, HEPES-buffered Krebs-Ringer bicarbonate buffer; NF, nuclear factor; PGK, phosphoglycerate kinase; p NA, p -nitroanilide; RIP, receptor-interacting protein; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; TNFR1, TNF-α receptor 1; TRADD, TNF receptor–associated death-domain protein; TRAF, TNF receptor–associated factor; DIABETES

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca ²⁺ homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo–exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.

Glucose sensing by the hepatoportal sensor is GLUT2-dependent: in vivo analysis in GLUT2-null mice. R Burcelin , W Dolci and B Thorens Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland. Abstract In the preceding article, we demonstrated that activation of the hepatoportal glucose sensor led to a paradoxical development of hypoglycemia that was associated with increased glucose utilization by a subset of tissues. In this study, we tested whether GLUT2 plays a role in the portal glucose-sensing system that is similar to its involvement in pancreatic beta-cells. Awake RIPGLUT1 x GLUT2-/- and control mice were infused with glucose through the portal (Po-) or the femoral (Fe-) vein for 3 h at a rate equivalent to the endogenous glucose production rate. Blood glucose and plasma insulin concentrations were continuously monitored. Glucose turnover, glycolysis, and glycogen synthesis rates were determined by the 3H-glucose infusion technique. We showed that portal glucose infusion in RIPGLUT1 x GLUT24-/- mice did not induce the hypoglycemia observed in control mice but, in contrast, led to a transient hyperglycemic state followed by a return to normoglycemia; this glycemic pattern was similar to that observed in control Fe-mice and RIPGLUT1 x GLUT2-/- Fe-mice. Plasma insulin profiles during the infusion period were similar in control and RIPGLUT1 x GLUT2-/- Po- and Fe-mice. The lack of hypoglycemia development in RIPGLUT1 x GLUT2-/- mice was not due to the absence of GLUT2 in the liver. Indeed, reexpression by transgenesis of this transporter in hepatocytes did not restore the development of hypoglycemia after initiating portal vein glucose infusion. In the absence of GLUT2, glucose turnover increased in Po-mice to the same extent as that in RIPGLUT1 x GLUT2-/- or control Fe-mice. Finally, co-infusion of somatostatin with glucose prevented development of hypoglycemia in control Po-mice, but it did not affect the glycemia or insulinemia of RIPGLUT1 x GLUT2-/- Po-mice. Together, our data demonstrate that GLUT2 is required for the function of the hepatoportal glucose sensor and that somatostatin could inhibit the glucose signal by interfering with GLUT2-expressing sensing units.

Portal glucose infusion in the mouse induces hypoglycemia: evidence that the hepatoportal glucose sensor stimulates glucose utilization. R Burcelin , W Dolci and B Thorens Institute of Pharmacology, University of Lausanne, Switzerland. Abstract To analyze the role of the murine hepatoportal glucose sensor in the control of whole-body glucose metabolism, we infused glucose at a rate corresponding to the endogenous glucose production rate through the portal vein of conscious mice (Po-mice) that were fasted for 6 h. Mice infused with glucose at the same rate through the femoral vein (Fe-mice) and mice infused with a saline solution (Sal-mice) were used as controls. In Po-mice, hypoglycemia progressively developed until glucose levels dropped to a nadir of 2.3 +/- 0.1 mmol/l, whereas in Fe-mice, glycemia rapidly and transiently developed, and glucose levels increased to 7.7 +/- 0.6 mmol/l before progressively returning to fasting glycemic levels. Plasma insulin levels were similar in both Po- and Fe-mice during and at the end of the infusion periods (21.2 +/- 2.2 vs. 25.7 +/- 0.9 microU/ml, respectively, at 180 min of infusion). The whole-body glucose turnover rate was significantly higher in Po-mice than in Fe-mice (45.9 +/- 3.8 vs. 37.7 +/- 2.0 mg x kg(-1) x min)-1), respectively) and in Sal-mice (24.4 +/- 1.8 mg x kg(-1) x min(-1)). Somatostatin co-infusion with glucose in Po-mice prevented hypoglycemia without modifying the plasma insulin profile. Finally, tissue glucose clearance, which was determined after injecting 14C-2-deoxyglucose, increased to a higher level in Po-mice versus Fe-mice in the heart, brown adipose tissue, and the soleus muscle. Our data show that stimulation of the hepatoportal glucose sensor induced hypoglycemia and increased glucose utilization by a combination of insulin-dependent and insulin-independent or -sensitizing mechanisms. Furthermore, activation of the glucose sensor and/or transmission of its signal to target tissues can be blocked by somatostatin.

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular ... homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo-exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters. (ProQuest: ... denotes formulae/symbols omitted.)

Phosphoinositides, synthesized from myo ‐inositol, play a critical role in the development of growth cones and in synaptic activity. As neurons cannot synthesize inositol, they take it up from the extracellular milieu. Here, we demonstrate that, in brain and PC12 cells, the recently identified H + / myo ‐inositol symporter HMIT is present in intracellular vesicles that are distinct from synaptic and dense‐core vesicles. We further show that HMIT can be triggered to appear on the cell surface following cell depolarization, activation of protein kinase C or increased intracellular calcium concentrations. HMIT cell surface expression takes place preferentially in regions of nerve growth and at varicosities and leads to increased myo ‐inositol uptake. The symporter is then endocytosed in a dynamin‐dependent manner and becomes available for a subsequent cycle of stimulated exocytosis. HMIT is thus expressed in a vesicular compartment involved in activity‐dependent regulation of myo ‐inositol uptake in neurons. This may be essential for sustained signaling and vesicular traffic activities in growth cones and at synapses.

Glucagon-like peptide-1 (GLP-1) is a growth and differentiation factor for mature beta-cells and their precursors. However, the overall effect of GLP-1 on increasing beta-cell mass in both in vivo and in vitro conditions is relatively small, and augmenting this effect would be beneficial for the treatment or prevention of type 1 and type 2 diabetes. Here, we searched for cellular mechanisms that may limit the proliferative effect of GLP-1 and tested whether blocking them could increase beta-cell proliferation. We examined GLP-1-regulated genes in beta TC-Tet cells by cDNA microarrays. To assess the effect of some of these gene on cell proliferation, we reduced their expression using small heterogenous RNA in beta-cell lines and primary mouse islets and measured [(3)H]thymidine or 5'-bromo-2'-deoxyuridine incorporation. We identified four negative regulators of intracellular signaling that were rapidly and strongly activated by GLP-1: the regulator of G-protein-signaling RGS2; the cAMP response element-binding protein (CREB) antagonists cAMP response element modulator (CREM)-alpha and ICERI; and the dual specificity phosphatase DUSP14, a negative regulator of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. We show that knockdown of CREMalpha or DUSP14 or expression of a dominant-negative form of DUSP14 increased beta-cell line proliferation and enhanced the GLP-1-induced proliferation of primary beta-cells. Together, our data show that 1) the cAMP/protein kinase A/CREB and MAPK/ERK1/2 pathways can additively control beta-cell proliferation, 2) beta-cells have evolved several mechanisms limiting GLP-1-induced cellular proliferation, and 3) blocking these mechanisms increases the positive effect of GLP-1 on beta-cell mass.