SXRF for Studying the Distribution of Trace Metals in the Pancreas and Liver.

Transition metals such as iron, copper and zinc are required for the normal functioning of biological tissues, whereas others, such as cadmium, are potentially highly toxic. Any disturbances in homeostasis caused by lack of micronutrients in the diet, pollution or genetic heredity result in malfunction and/or diseases. Here, we used synchrotron X-ray fluorescence, SXRF, microscopy and mice with altered functions of major antioxidant enzymes to show that SXRF may become a powerful tool to study biologically relevant metal balance in the pancreas and liver of mice models with disturbed glucose homeostasis.

Interdependencies of Gene Expression and Function between Two Redox Enzymes and REG Family Proteins in Murine Pancreatic Islets and Human Pancreatic Cells.

Our laboratory previously revealed that regenerating islets-derived protein 2 (REG2) was diminished in pancreatic islets of glutathione peroxidase-1-overexpressing mice (Gpx1-OE). It remained unknown if there is an inverse relationship between the expression and function of all Reg family genes and antioxidant enzymes in the pancreatic islets or human pancreatic cells. This research was to determine how altering the Gpx1 and superoxide dismutase-1 (Sod1) genes alone or together (dKO) affected the expression of all seven murine Reg genes in murine pancreatic islets. In Experiment 1, Gpx1-/-, Gpx1-OE, their wild-type (WT), Sod1-/-, dKO, and their WT (male, 8-wk old, n = 4-6) were fed a Se-adequate diet and their islets were collected to assay the mRNA levels of Reg family genes. In Experiment 2, islets from the six groups of mice were treated with phosphate-buffered saline (PBS), REG2, or REG2 mutant protein (1 µg/mL), and/or GPX mimic (ebselen, 50 µM) and SOD mimic (copper [II] diisopropyl salicylate, CuDIPS, 10 µM) for 48 h before the proliferation assay using bromodeoxyuridine (BrdU). In Experiment 3, human pancreatic cells (PANC1) were treated with REG2 (1 µg/mL) and assayed for REG gene expression, GPX1 and SOD1 activities, viability, and responses to Ca2+. Compared with the WT, knockouts of Gpx1 and/or Sod1 up-regulated (p < 0.05) the mRNA levels of most of the murine Reg genes in islets whereas the Gpx1 overexpression down-regulated (p < 0.05) Reg mRNA levels. REG2, but not the REG2 mutant, inhibited islet proliferation in Gpx1 or Sod1-altered mice. Such inhibition was abolished by co-incubation the Gpx1-/- islets with ebselen and the Sod1-/- islets with CuDIPS. Treating PANC1 cells with murine REG2 protein induced expression of its human orthologue REG1B and three other REG genes, but decreased SOD1 and GPX1 activities and cell viability. In conclusion, our results revealed an interdependence of REG family gene expression and/or function on intracellular GPX1 and SOD1 activities in murine islets and human pancreatic cells.

Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

We previously reported a depletion of murine regenerating islet-derived protein 2 (REG2) in pancreatic islets of glutathione peroxidase-1 (Gpx1) overexpressing (OE) mice. The present study was to explore if and how the REG2 depletion contributed to an augmented glucose stimulated insulin secretion (GSIS) in OE islets. After we verified a consistent depletion (90%, p < 0.05) of REG2 mRNA, transcript, and protein in OE islets compared with wild-type (WT) controls, we treated cultured and perifused OE islets (70 islets/sample) with REG2 (1 µg/ml or ml · min) and observed 30-40% (p < 0.05) inhibitions of GSIS by REG2. Subsequently, we obtained evidences of co-immunoprecipitation, cell surface ligand binding, and co-immunofluorescence for a ligand-receptor binding between REG2 and transmembrane, L-type voltage-dependent Ca2+ channel (CaV1.2) in beta TC3 cells. Mutating the C-type lectin binding domain of REG2 or deglycosylating CaV1.2 removed the inhibition of REG2 on GSIS and(or) the putative binding between the two proteins. Treating cultured OE and perifused WT islets with REG2 (1 µg/ml or ml · min) decreased (p < 0.05) Ca2+ influx triggered by glucose or KCl. An intraperitoneal (ip) injection of REG2 (2 µg/g) to OE mice (6-month old, n = 10) decreased their plasma insulin concentration (46%, p < 0.05) and elevated their plasma glucose concentration (25%, p < 0.05) over a 60 min period after glucose challenge (ip, 1 g/kg). In conclusion, our study identifies REG2 as a novel regulator of Ca2+ influx and insulin secretion, and reveals a new cascade of GPX1/REG2/CaV1.2 to explain how REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca2+ influx and augmented GSIS. These findings create new links to bridge redox biology, tissue regeneration, and insulin secretion.

Dietary Selenium Across Species.

This review traces the discoveries that led to the recognition of selenium (Se) as an essential nutrient and discusses Se-responsive diseases in animals and humans in the context of current understanding of the molecular mechanisms of their pathogeneses. The article includes a comprehensive analysis of dietary sources, nutritional utilization, metabolic functions, and dietary requirements of Se across various species. We also compare the function and regulation of selenogenomes and selenoproteomes among rodents, food animals, and humans. The review addresses the metabolic impacts of high dietary Se intakes in different species and recent revelations of Se metabolites, means of increasing Se status, and the recycling of Se in food systems and ecosystems. Finally, research needs are identified for supporting basic science and practical applications of dietary Se in food, nutrition, and health across species.

Glutathione peroxidase-1 inhibits transcription of regenerating islet-derived protein-2 in pancreatic islets.

Our group previously demonstrated that overexpression of selenium-dependent glutathione peroxidase-1 (GPX1) in mice (OE) led to escalated glucose-stimulated insulin secretion and hyperinsulinemia. Because we found a strong correlation of this phenotype with a diminished expression of regenerating islet-derived protein 2 (REG2) in the OE pancreatic islets, the present study was to reveal underlying mechanisms for that down-regulation of REG2 by GPX1 as a major scavenger of reactive oxygen species. We first treated the OE and wild-type (WT) mice and their islets with ROS-generating diquat, streptozotocin, and H2O2 and ROS-scavenging ebselen and N-acetylcysteine (NAC). Their effects on pancreatic and islet REG2 protein and(or) secretion were opposite (P < 0.05). Thereafter, we identified 13 transcriptional factors with putative binding sites in the Reg2 proximate promoter, and found that only activator protein-1 (AP-1) and albumin D box-binding protein (DBP) mRNA and protein levels were affected (elevated) (P < 0.05) by the GPX1 overproduction in the OE pancreatic islets compared with the WT islets. Contrary to that of Reg2 expression, their mRNA abundances in the cultured islets were elevated (P < 0.05) by ebselen and NAC, but decreased (P < 0.05) by H2O2. Both AP-1 and DBP could bind to the Reg2 promoter at the location of -168 to 0 base pair (bp) in the OE islets. Deleting the AP-1 (-143/-137 and -60/-57 bp) and(or) DBP (-35/-29 bp) binding domains in the Reg2 promoter attenuated and(or) abolished the inhibition of Reg2 promoter activation by ebselen as the GPX1 mimic in ßTC-3 cells. In conclusion, the down-regulation of Reg2 expression in the GPX1-overproducing pancreatic islets was mediated by a transcriptional inhibition of the gene through two ROS responsive transcription factors AP-1 and DBP. Our findings reveal GPX1 as a novel regulator of Reg2 expression, and linking these two previously-unrelated proteins will have broad biomedical implications.

Dietary selenium deficiency partially rescues type 2 diabetes-like phenotypes of glutathione peroxidase-1-overexpressing male mice.

This study was conducted to determine whether dietary Se deficiency precluded overproduction of glutathione peroxidase-1 (GPX1) activity in mice overexpressing (OE) this gene and thus rescued their type 2 diabetes-like phenotypes. A total of 20 male OE and wild-type (WT) mice were fed an Se-deficient (<0.02 mg/kg) diet or an Se-supplemented (0.3 mg/kg as sodium selenite) diet from 1 to 5 mo of age. Dietary Se deficiency eliminated or attenuated (P < 0.05) genotype differences in concentrations of blood glucose, plasma insulin, and/or hepatic lipids, insulin sensitivity, and glucose-stimulated insulin secretion at the end of the study. Dietary Se deficiency decreased (P < 0.05) OE islet mRNA levels of 2 key transcriptional activators (Beta2 and Foxa2) and removed genotype differences in islet mRNA levels of 7 genes (Beta2, Cfos, Foxa2, Pregluc, Ins1, p53, and Sur1) related to insulin synthesis and secretion. Compared with those of the Se-adequate OE mice, the Se-deficient OE mice had lower (P < 0.05) hepatic mRNA levels of 2 key rate-limiting enzymes for lipogenesis (Acc1) and glycolysis (Gk1), along with lower (P < 0.05) activities of hepatic glucokinase and muscle phosphoenolpyruvate carboxykinase. Dietary Se deficiency also decreased (P < 0.05) blood glucose and hepatic lipid concentrations in the WT mice. In conclusion, dietary Se deficiency precluded the overproduction of GPX1 in full-fed OE mice and partially rescued their metabolic syndromes. This alleviation resulted from modulating the expression and/or function of proinsulin genes, lipogenesis rate-limiting enzyme genes, and key glycolysis and gluconeogenesis enzymes in islets, liver, and muscle.

Knockouts of SOD1 and GPX1 exert different impacts on murine islet function and pancreatic integrity.

Metabolic subtlety and clinical relevance of different forms of reactive oxygen species in diabetes remain unclear. Using single knockout of Cu,Zn-superoxide dismutase (SOD1(-/-)) or Se-glutathione peroxidase-1 (GPX1(-/-)) and their double-knockout (DKO) mouse models, we determined if elevating endogenously-derived superoxide and hydroperoxide exerted distinct impacts and mechanisms on body glucose homeostasis. Whereas the three knockout groups displayed decreased plasma insulin concentrations and islet ß-cells mass, only SOD1(-/-) showed decreased body weight, increased blood glucose, and blocked glucose-stimulated insulin secretion. Null of SOD1 and GPX1 elevated respective islet superoxide and hydroperoxide production, and upregulated p53 phosphorylation. Knockout of SOD1 downregulated the foxhead box A2/pancreatic and duodenal homeobox 1 pathway in a superoxide-dependent fashion at epigenetic, mRNA, and protein levels in islets, but improved insulin signaling in liver and muscle. The SOD1(-/-) mice showed more apparent pancreatitis than the GPX1(-/-) mice that were more susceptible to the cerulein-induced amylase increase. Knockout of SOD1 impaired islet function, pancreas integrity, and body glucose homeostasis more than that of GPX1. Simultaneous ablation of both enzymes did not result in additive or aggravated metabolic outcomes.

Two tales of antioxidant enzymes on ß cells and diabetes.

Pancreatic islets contain low activities of catalase, selenium-dependent glutathione peroxidase 1 (GPX1), and Cu,Zn-superoxide dismutase 1 (SOD1). Thus, enhancing expression of these enzymes in islets has been unquestionably favored. However, such an attempt has produced variable metabolic outcomes. While ß cell-specific overexpression of Sod1 enhanced mouse resistance to streptozotocin-induced diabetes, the same manipulation of catalase aggravated onset of type 1 diabetes in nonobese diabetic mice. Global overexpression of Gpx1 in mice induced type 2 diabetes-like phenotypes. Although knockouts of Gpx1 and Sod1 each alone or together decreased pancreatic ß cell mass and plasma insulin concentrations, these knockouts improved body insulin sensitivity to different extents. Pancreatic duodenal homeobox 1, forkhead box A2, and uncoupling protein 2 are three key regulators of ß cell mass, insulin synthesis, and glucose-stimulated insulin secretion. Phenotypes resulted from altering GPX1 and/or SOD1 were partly mediated through these factors, along with protein kinase B and c-jun terminal kinase. A shifted reactive oxygen species inhibition of protein tyrosine phosphatases in insulin signaling might be attributed to altered insulin sensitivity. Overall, metabolic roles of antioxidant enzymes in ß cells and diabetes depend on body oxidative status and target functions. Revealing regulatory mechanisms for this type of dual role will help prevent potential pro-diabetic risk of antioxidant over-supplementation to humans.

Impacts of dietary selenium deficiency on metabolic phenotypes of diet-restricted GPX1-overexpressing mice.

We previously reported a spontaneous development of type 2 diabetes-like phenotypes in glutathione peroxidase-1 (GPX1)-overexpressing (OE) mice. Diet restriction of these mice rescued all their phenotypes, except for hyperinsulinemia and hypersecretion of insulin. This study was to determine whether dietary Se deficiency eliminated these two primary effects of GPX1 overproduction. Forty-seven male OE and wild-type (WT) mice were fed an Se-adequate (0.4 mg Se/kg) or deficient (<0.02 mg Se/kg) diet at 2 to 3 g (full-fed = 5 g) per day from 4 to 12 weeks of age. Although dietary Se deficiency did not rescue the primary phenotypes of the diet-restricted OE mice, it exerted a strong effect (p < 0.05) on mRNA or protein levels (or both) of 14 molecules involved in islet insulin synthesis and secretion and hepatic lipogenesis. Dietary Se deficiency exhibited a hypoinsulinemic trend in OE mice and a strong hypolipidemic effect (p < 0.05) in the liver of WT mice. Hepatic lipogenesis was attenuated in OE compared with WT mice. In conclusion, diet restriction might be too overwhelming to allow a demonstration of a dietary Se-depletion effect on the OE phenotypes. Full-fed animals could offer a better chance to illustrate such effects and the underlying mechanisms.

Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development.

The onset of pancreas development in the foregut endoderm is marked by activation of the homeobox gene Pdx1 (IPF1). Pdx1 is essential for the expansion of the pancreatic primordium and the development of endocrine islets. The control of Pdx1 expression has been only partially elucidated. We demonstrate here that the winged-helix transcription factors Foxa1 and Foxa2 co-occupy multiple regulatory domains in the Pdx1 gene. Compound conditional ablation of both Foxa1 and Foxa2 in the pancreatic primordium results in complete loss of Pdx1 expression and severe pancreatic hypoplasia. Mutant mice exhibit hyperglycemia with severely disrupted acinar and islet development, and die shortly after birth. Assessment of developmental markers in the mutant pancreas revealed a failure in the expansion of the pancreatic anlage, a blockage of exocrine and endocrine cell differentiation, and an arrest at the primitive duct stage. Comparing their relative developmental activity, we find that Foxa2 is the major regulator in promoting pancreas development and cell differentiation. Using chromatin immunoprecipitations (ChIP) and ChIP sequencing (ChIPSeq) of fetal pancreas and islet chromatin, we demonstrate that Foxa1 and Foxa2 predominantly occupy a distal enhancer at -6.4 kb relative to the transcriptional start site in the Pdx1 gene. In addition, occupancy of the well-characterized proximal Pdx1 enhancer by Foxa1 and Foxa2 is developmental stage-dependent. Thus, the regulation of Pdx1 expression by Foxa1 and Foxa2 is a key early event controlling the expansion and differentiation of the pancreatic primordia.

Metabolic and ionic coupling factors in amino acid-stimulated insulin release in pancreatic beta-HC9 cells.

Fuel stimulation of insulin secretion from pancreatic beta-cells is thought to be mediated by metabolic coupling factors that are generated by energized mitochondria, including protons, adenine nucleotides, and perhaps certain amino acids (AA), as for instance aspartate, glutamate, or glutamine (Q). The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or AA, alone or combined, using (31)P, (23)Na and (1)H NMR technology, respirometry, and biochemical analysis to study the metabolic events that occur in continuously superfused mouse beta-HC9 cells contained in agarose beads and enhanced by the phosphodiesterase inhibitor IBMX. Exposing beta-HC9 cells to high glucose or 3.5 mM of a physiological mixture of 18 AA (AAM) plus 2 mM glutamine caused a marked stimulation of insulin secretion associated with increased oxygen consumption, cAMP release, and phosphorylation potential as evidenced by higher phosphocreatine and lower P(i) peak areas of (31)P NMR spectra. Diazoxide blocked stimulation of IR completely, suggesting involvement of ATP-dependent potassium (K(ATP)) channels in this process. However, levels of MgATP and MgADP concentrations, which regulate channel activity, changed only slowly and little, whereas the rate of insulin release increased fast and very markedly. The involvement of other candidate coupling factors was therefore considered. High glucose or AAM + Q increased pH(i). The availability of temporal pH profiles allowed the precise computation of the phosphate potential (ATP/P(i) x ADP) in fuel-stimulated IR. Intracellular Na+ levels were greatly elevated by AAM + Q. However, glutamine alone or together with 2-amino-2-norbornanecarboxylic acid (which activates glutamate dehydrogenase) decreased beta-cell Na levels. Stimulation of beta-cells by glucose in the presence of AAM + Q (0.5 mM) was associated with rising cellular concentrations of glutamate and glutamine and strikingly lower aspartate levels. Methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the glucose enhancement of AMM + Q-induced IR and associated changes in glutamine and aspartate but did not prevent the accumulation of glutamate. The results of this study demonstrate again that an increased phosphate potential and a functional K(ATP) channel are essential for metabolic coupling during fuel-stimulated insulin release but illustrate that determining the identity and relative importance of all participating coupling factors and second messengers remains a challenge largely unmet.

Foxa1-deficient mice exhibit impaired insulin secretion due to uncoupled oxidative phosphorylation.

Foxa1 (formerly hepatic nuclear factor 3alpha) belongs to the family of Foxa genes that are expressed in early development and takes part in the differentiation of endoderm-derived organs and the regulation of glucose homeostasis. Foxa1-/- pups are growth retarded and hypoglycemic but glucose intolerant in response to an intraperitoneal glucose challenge. However, the mechanism of glucose intolerance in this model has not been investigated. Here, we show that Foxa1-/- islets exhibit decreased glucose-stimulated insulin release in islet perifusion experiments and have significantly reduced pancreatic insulin and glucagon content. Moreover, Foxa1-/- beta-cells exhibit attenuated calcium influx in response to glucose and glyburide, suggesting an insulin secretion defect either at the level or upstream of the ATP-sensitive K+ channel. Intracellular ATP levels after incubation with 10 mmol/l glucose were about 2.5 times lower in Foxa1-/- islets compared with controls. This diminished ATP synthesis could be explained by increased expression of the mitochondrial uncoupling protein uncoupling protein 2 (UCP2) in Foxa1-deficient islets, resulting in partially uncoupled mitochondria. Chromatin immunoprecipitation assays indicate that UCP2 is a direct transcriptional target of Foxa1 in vivo. Thus, we have identified a novel function for Foxa1 in the regulation of oxidative phosphorylation in pancreatic beta-cells.

Cholinergic regulation of fuel-induced hormone secretion and respiration of SUR1-/- mouse islets.

Neural and endocrine factors (i.e., Ach and GLP-1) restore defective glucose-stimulated insulin release in pancreatic islets lacking sulfonylurea type 1 receptors (SUR1(-/-)) (Doliba NM, Qin W, Vatamaniuk MZ, Li C, Zelent D, Najafi H, Buettger CW, Collins HW, Carr RD, Magnuson MA, and Matschinsky FM. Am J Physiol Endocrinol Metab 286: E834-E843, 2004). The goal of the present study was to assess fuel-induced respiration in SUR1(-/-) islets and to correlate it with changes in intracellular Ca(2+), insulin, and glucagon secretion. By use of a method based on O(2) quenching of phosphorescence, the O(2) consumption rate (OCR) of isolated islets was measured online in a perifusion system. Basal insulin release (IR) was 7-10 times higher in SUR1(-/-) compared with control (CON) islets, but the OCR was comparable. The effect of high glucose (16.7 mM) on IR and OCR was markedly reduced in SUR1(-/-) islets compared with CON. Ach (0.5 microM) in the presence of 16.7 mM glucose caused a large burst of IR in CON and SUR1(-/-) islets with minor changes in OCR in both groups of islets. In SUR1(-/-) islets, high glucose failed to inhibit glucagon secretion during stimulation with amino acids or Ach. We conclude that 1) reduced glucose responsiveness of SUR1(-/-) islets may be in part due to impaired energetics, as evidenced by significant decrease in glucose-stimulated OCR; 2) elevated intracellular Ca(2+) levels may contribute to altered insulin and glucagon secretion in SUR1(-/-) islets; and 3) The amplitudes of the changes in OCR during glucose and Ach stimulation do not correlate with IR in normal and SUR1(-/-) islets suggesting that the energy requirements for exocytosis are minor compared with other ATP-consuming reactions.

The MODY1 gene HNF-4alpha regulates selected genes involved in insulin secretion.

Mutations in the gene encoding hepatocyte nuclear factor-4alpha (HNF-4alpha) result in maturity-onset diabetes of the young (MODY). To determine the contribution of HNF-4alpha to the maintenance of glucose homeostasis by the beta cell in vivo, we derived a conditional knockout of HNF-4alpha using the Cre-loxP system. Surprisingly, deletion of HNF-4alpha in beta cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the mutant beta cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60% reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays that the Kir6.2 gene is a transcriptional target of HNF-4alpha. Our data provide genetic evidence that HNF-4alpha is required in the pancreatic beta cell for regulation of the pathway of insulin secretion dependent on the ATP-dependent potassium channel.

Foxa2 regulates multiple pathways of insulin secretion.

The regulation of insulin secretion by pancreatic beta cells is perturbed in several diseases, including adult-onset (type 2) diabetes and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). The first mouse model for PHHI has a conditional deletion of the gene encoding the winged-helix transcription factor Foxa2 (Forkhead box a2, formerly Hepatocyte nuclear factor 3beta) in pancreatic beta cells. Using isolated islets, we found that Foxa2 deficiency resulted in excessive insulin release in response to amino acids and complete loss of glucose-stimulated insulin secretion. Most PHHI cases are associated with mutations in SUR1 (Sulfonylurea receptor 1) or KIR6.2 (Inward rectifier K(+) channel member 6.2), which encode the subunits of the ATP-sensitive K(+) channel, and RNA in situ hybridization of mutant mouse islets revealed that expression of both genes is Foxa2 dependent. We utilized expression profiling to identify additional targets of Foxa2. Strikingly, one of these genes, Hadhsc, encodes short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase, deficiency of which has been shown to cause PHHI in humans. Hadhsc is a direct target of Foxa2, as demonstrated by cotransfection as well as in vivo chromatin immunoprecipitation experiments using isolated islets. Thus, we have established Foxa2 as an essential activator of genes that function in multiple pathways governing insulin secretion.

Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine.

Inhibition of ATP-sensitive K+ (K(ATP)) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic beta-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1-/-) in beta-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1-/- mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-1 (GLP-1) on IR and free intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated or cultured islets of SUR1-/- mice and B6D2F1 controls (SUR1+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1-/-) islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1-/- islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca2+ stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca2+]i at 0.1-1 microM or biphasic changes (an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i) at 1-100 microM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the beta-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]i in turn sensitizes the beta-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca2+]i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia.

Differential effects of glucose and glyburide on energetics and Na+ levels of betaHC9 cells: nuclear magnetic resonance spectroscopy and respirometry studies.

In the present study, noninvasive (31)P and (23)Na(+)-nuclear magnetic resonance (NMR) technology and respirometry were used to compare the effect of high glucose (30 mmol/l) with the effect of the antidiabetic sulfonylurea (SU) compound glyburide (GLY) on energy metabolism, Na(+) flux, insulin, and cAMP release of continuously superfused beta-HC9 cells encapsulated in microscopic agarose beads. Both high glucose and GLY increased oxygen consumption in beta-HC9 cells (15-30%) with a maximal effect at 8 mmol/l for glucose and at 250 nmol/l for GLY. At the same time, insulin release from beta-cells increased by 15- and 25-fold with high glucose or GLY, respectively. The P-creatine (PCr) level was greatly increased and inorganic phosphate (P(i)) was decreased with 30 mmol/l glucose in contrast to the decreased level of PCr and increased P(i) with GLY. ATP levels remained unchanged during both interventions. Studies on isolated mitochondria of beta-HC9 cells showed that GLY added to mitochondria oxidizing glutamine or glutamate abolished the stimulation of respiration by ADP (state 3) meanwhile leaving state 3 respiration unchanged during oxidation of other substrates. Exposure of beta-HC9 cells to 5 mmol/l glucose decreased intracellular Na(+) levels monitored by (23)Na(+)-NMR spectroscopy and 30 mmol/l glucose resulted in a further decrease in cytosolic Na(+). In contrast, Na(+) increased when 1 micro mol/l GLY was added to the perfusate containing 5 mmol/l glucose. These data support the hypothesis that glucose activates the beta-cell through a "push mechanism" due to substrate pressure enhancing fuel flux, energy production, and extrusion of Na(+) from the cells in contrast to SU receptor (SUR)-1 inhibitors, which may modify intermediary and energy metabolism secondarily through a "pull mechanism" due to higher energy demand resulting from increased ion fluxes and the exocytotic work load.

Foxa2 controls Pdx1 gene expression in pancreatic beta-cells in vivo.

Differentiation of early foregut endoderm into pancreatic endocrine and exocrine cells depends on a cascade of gene activation events controlled by various transcription factors. Prior in vitro analysis has suggested that the forkhead/winged helix transcription factor Foxa2 (formerly HNF-3beta) is a major upstream regulator of Pdx1, a homeobox gene essential for pancreatic development. Pdx1 is also essential for the maintenance of glucose homeostasis, as its human orthologue, IPF-1, is mutated in a subset of patients with early-onset type 2 diabetes (MODY4). To analyze the Foxa2/Pdx1 regulatory cascade during pancreatic beta-cell differentiation, we used conditional gene ablation of Foxa2 in mice. We demonstrated that the deletion of Foxa2 in beta-cell-specific knockout mice results in downregulation of Pdx1 mRNA and subsequent reduction of PDX-1 protein levels in islets. These data represent the first in vivo demonstration that Foxa2 acts upstream of Pdx1 in the differentiated beta-cell.