三木 隆司

Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. Due to the rarity of FBPase deficiency, the mechanism by which the mutations cause enzyme activity loss still remains unclear. Here we identify compound heterozygous missense mutations of FBP1, c.491G>A (p.G164D) and c.581T>C (p.F194S), in an adult patient with hypoglycemic lactic acidosis. The G164D and F194S FBP1 mutants exhibit decreased FBP1 protein expression and a loss of FBPase enzyme activity. The biochemical phenotypes of all previously reported FBP1 missense mutations in addition to G164D and F194S are classified into three functional categories. Type 1 mutations are located at pivotal residues in enzyme activity motifs and have no effects on protein expression. Type 2 mutations structurally cluster around the substrate binding pocket and are associated with decreased protein expression due to protein misfolding. Type 3 mutations are likely nonpathogenic. These findings demonstrate a key role of protein misfolding in mediating the pathogenesis of FBPase deficiency, particularly for Type 2 mutations. This study provides important insights that certain patients with Type 2 mutations may respond to chaperone molecules.

Glutaminase 2 (GLS2), a master regulator of glutaminolysis that is induced by p53 and converts glutamine to glutamate, is abundant in the liver but also exists in pancreatic β-cells. However, the roles of GLS2 in islets associated with glucose metabolism are unknown, presenting a critical issue. To investigate the roles of GLS2 in pancreatic β-cells in vivo, we generated β-cell-specific Gls2 conditional knockout mice (Gls2 CKO), examined their glucose homeostasis, and validated the findings using a human islet single-cell analysis database. GLS2 expression markedly increased along with p53 in β-cells from control (RIP-Cre) mice fed a high-fat diet. Furthermore, Gls2 CKO exhibited significant diabetes mellitus with gluconeogenesis and insulin resistance when fed a high-fat diet. Despite marked hyperglycaemia, impaired insulin secretion and paradoxical glucagon elevation were observed in high-fat diet-fed Gls2 CKO mice. GLS2 silencing in the pancreatic β-cell line MIN6 revealed downregulation of insulin secretion and intracellular ATP levels, which were closely related to glucose-stimulated insulin secretion. Additionally, analysis of single-cell RNA-sequencing data from human pancreatic islet cells also revealed that GLS2 expression was elevated in β-cells from diabetic donors compared to nondiabetic donors. Consistent with the results of Gls2 CKO, downregulated GLS2 expression in human pancreatic β-cells from diabetic donors was associated with significantly lower insulin gene expression as well as lower expression of members of the insulin secretion pathway, including ATPase and several molecules that signal to insulin secretory granules, in β-cells but higher glucagon gene expression in α-cells. Although the exact mechanism by which β-cell-specific GLS2 regulates insulin and glucagon requires further study, our data indicate that GLS2 in pancreatic β-cells maintains glucose homeostasis under the condition of hyperglycaemia.

The liver stores glycogen and releases glucose into the blood upon increased energy demand. Group 2 innate lymphoid cells (ILC2) in adipose and pancreatic tissues are known for their involvement in glucose homeostasis, but the metabolic contribution of liver ILC2s has not been studied in detail. Here we show that liver ILC2s are directly involved in the regulation of blood glucose levels. Mechanistically, interleukin (IL)-33 treatment induces IL-13 production in liver ILC2s, while directly suppressing gluconeogenesis in a specific Hnf4a/G6pc-high primary hepatocyte cluster via Stat3. These hepatocytes significantly interact with liver ILC2s via IL-13/IL-13 receptor signaling. The results of transcriptional complex analysis and GATA3-ChIP-seq, ATAC-seq, and scRNA-seq trajectory analyses establish a positive regulatory role for the transcription factor GATA3 in IL-13 production by liver ILC2s, while AP-1 family members are shown to suppress IL-13 release. Thus, we identify a regulatory role and molecular mechanism by which liver ILC2s contribute to glucose homeostasis.

Glutamine synthase 2 (GLS2) is a key regulator of glutaminolysis and has been previously implicated in activities consistent with tumor suppression. Here we generated Gls2 knockout (KO) mice that develop late-occurring B cell lymphomas and hepatocellular carcinomas (HCC). Further, Gls2 KO mice subjected to the hepatocarcinogenic Stelic Animal Model (STAM) protocol produce larger HCC tumors than seen in wild-type mice. GLS2 has been shown to promote ferroptosis, a form of cell death characterized by iron-dependent accumulation of lipid peroxides. In line with this, GLS2 deficiency, either in cells derived from Gls2 KO mice or in human cancer cells depleted of GLS2, conferred significant resistance to ferroptosis. Mechanistically, GLS2, but not GLS1, increased lipid ROS production by facilitating the conversion of glutamate to α-ketoglutarate, thereby promoting ferroptosis. Ectopic expression of wild-type GLS2 in a human hepatic adenocarcinoma xenograft model significantly reduced tumor size; this effect was nullified by either expressing a catalytically inactive form of GLS2 or by blocking ferroptosis. Furthermore, analysis of cancer patient datasets supported a role for GLS2-mediated regulation of ferroptosis in human tumor suppression. These data suggest that GLS2 is a bona fide tumor suppressor and that its ability to favor ferroptosis by regulating glutaminolysis contributes to its tumor suppressive function.

Background: α-cyclodextrin (α-CD) is one of the dietary fibers that may have a beneficial effect on cholesterol and/or glucose metabolism, but its efficacy and mode of action remain unclear. Methods: In the present study, we examined the anti-hyperglycemic effect of α-CD after oral loading of glucose and liquid meal in mice. Results: Administration of 2 g/kg α-CD suppressed hyperglycemia after glucose loading, which was associated with increased glucagon-like peptide 1 (GLP-1) secretion and enhanced hepatic glucose sequestration. By contrast, 1 g/kg α-CD similarly suppressed hyperglycemia, but without increasing secretions of GLP-1 and insulin. Furthermore, oral α-CD administration disrupts lipid micelle formation through its inclusion of lecithin in the gut luminal fluid. Importantly, prior inclusion of α-CD with lecithin in vitro nullified the anti-hyperglycemic effect of α-CD in vivo, which was associated with increased intestinal mRNA expressions of SREBP2-target genes (Ldlr, Hmgcr, Pcsk9, and Srebp2). Conclusions: α-CD elicits its anti-hyperglycemic effect after glucose loading by inducing lecithin inclusion in the gut lumen and activating SREBP2, which is known to induce cholecystokinin secretion to suppress hepatic glucose production via a gut/brain/liver axis.

As glucose-dependent insulinotropic polypeptide (GIP) possesses pro-adipogenic action, the suppression of the GIP hypersecretion seen in obesity might represent a novel therapeutic approach to the treatment of obesity. However, the mechanism of GIP hypersecretion remains largely unknown. In the present study, we investigated GIP secretion in two mouse models of obesity: High-fat diet-induced obese (DIO) mice and leptin-deficient Lepob/ob mice. In DIO mice, plasma GIP was increased along with an increase in GIP mRNA expression in the lower small intestine. Despite the robust alteration in the gut microbiome in DIO mice, co-administration of maltose and the α-glucosidase inhibitor (α-GI) miglitol induced the microbiome-mediated suppression of GIP secretion. The plasma GIP levels of Lepob/ob mice were also elevated and were suppressed by fat transplantation. The GIP mRNA expression in fat tissue was not increased in Lepob/ob mice, while the expression of an interleukin-1 receptor antagonist (IL-1Ra) was increased. Fat transplantation suppressed the expression of IL-1Ra. The plasma IL-1Ra levels were positively correlated with the plasma GIP levels. Accordingly, although circulating GIP levels are increased in both DIO and Lepob/ob mice, the underlying mechanisms differ, and the anti-obesity actions of α-GIs and leptin sensitizers may be mediated partly by the suppression of GIP secretion.

Gut microbiota mediates the effects of diet, thereby modifying host metabolism and the incidence of metabolic disorders. Increased consumption of omega-6 polyunsaturated fatty acid (PUFA) that is abundant in Western diet contributes to obesity and related diseases. Although gut-microbiota-related metabolic pathways of dietary PUFAs were recently elucidated, the effects on host physiological function remain unclear. Here, we demonstrate that gut microbiota confers host resistance to high-fat diet (HFD)-induced obesity by modulating dietary PUFAs metabolism. Supplementation of 10-hydroxy-cis-12-octadecenoic acid (HYA), an initial linoleic acid-related gut-microbial metabolite, attenuates HFD-induced obesity in mice without eliciting arachidonic acid-mediated adipose inflammation and by improving metabolic condition via free fatty acid receptors. Moreover, Lactobacillus-colonized mice show similar effects with elevated HYA levels. Our findings illustrate the interplay between gut microbiota and host energy metabolism via the metabolites of dietary omega-6-FAs thereby shedding light on the prevention and treatment of metabolic disorders by targeting gut microbial metabolites.

We recently reported the reduced ATP-sensitive potassium (KATP) channel activities in the transgenic mouse heart overexpressing the vascular type KATP channel pore-forming subunit (Kir6.1). Although dysfunction of cardiac KATP channel has been nominated as a cause of cardiomyopathy in human, these transgenic mice looked normal as wild-type (WT) during the experiment period (~20 weeks). Extended observation period revealed unexpected deaths beginning from 30 weeks and about 50% of the transgenic mice died by 55 weeks. Surface ECG recordings from the transgenic mice at rest demonstrated the normal sinus rhythm and the regular ECG complex as well as the control WT mice except for prolonged QT interval. However, the stress ECG test with noradrenaline revealed abnormal intraventricular conduction delay and arrhythmogeneity in the transgenic mouse. Fibrotic changes in the heart tissue were remarkable in aged transgenic mice, and the cardiac fibrosis developed progressively at least from the age of 30 weeks. Gene expression analyses revealed the differentiation of cardiac fibroblasts to myofibroblasts with elevated cytokine expressions was initiated way in advance before the fibrotic changes and the upregulation of BNP in the ventricle. In sum, Kir6.1TG mice provide an electro-pathological disease concept originated from KATP channel dysfunction.

AIMS/INTRODUCTION: Impaired glucose tolerance (IGT) is a subtype of prediabetes, a condition having high risk for development to diabetes mellitus, but its pathophysiology is not fully understood. In the present study, we examined metabolic changes in IGT by using two types (D-glucose [Glc] and partial hydrolysate of starch [PHS]) of oral glucose tolerance tests (OGTTs), with emphasis on serum incretins and metabolites. MATERIALS AND METHODS: We carried out the two types of OGTT (Glc/OGTT and PHS/OGTT) in 99 young Japanese individuals who had tested either positive (GU+ ; n = 48) or negative (GU- ; n = 51) for glycosuria. After OGTT, they were sub-grouped into five categories: normal glucose tolerance (NGT) in the GU- group (GU- /NGT; n = 49), NGT in the GU+ group (GU+ /NGT; n = 28), IGT (n = 12), diabetes mellitus (n = 1) and renal glycosuria (n = 9). Serum incretin and metabolites of GU- /NGT and IGT were then measured. RESULTS: When the serum insulin level at each time-point during PHS/OGTT was expressed as its ratio relative to Glc/OGTT, it was increased time-dependently in GU- /NGT, but not in IGT. Such an increase in the ratio was also detected of serum incretin levels in GU- /NGT, but not in IGT, suggesting a lack of deceleration of oligosaccharide absorption in IGT. Metabolome analysis showed a difference in the serum levels of two metabolites of unknown function in mammals, methylcysteine and sedoheptulose 1,7-bisphosphate, between GU- /NGT and IGT. CONCLUSIONS: Comparison of PHS/OGTT and Glc/OGTT showed that oligosaccharide absorption was accelerated in IGT. Methylcysteine and sedoheptulose 1,7-bisphosphate could be novel markers for dysregulated glucose metabolism.

Genetic analysis of KCNJ8 has pointed a mutation (S422L) as a susceptible link to J wave syndrome (JWS). In vitro expression study indicated that the ATP-sensitive K+ (K-ATP) channel with the S422L mutation has the gain-of-function with reduced sensitivity to ATP. However, the electrophysiological impact of KCNJ8 has not been elucidated in vivo. Transgenic mouse strains overexpressing KCNJ8 S422L variant (TGmt) or WT (TGWT) in cardiomyocytes have been created to investigate the influence of KCNJ8 in cardiomyocytes and the JWS-related feature of the S422L variant on the cardiac electrophysiology. These TG strains demonstrated distinct changes in the J-ST segment of ECG with marked QT prolongation, which might be ascribed to the action potential prolongation resulting from the reduction of voltage-dependent K+ currents in ventricular cells. The pinacidil-induced K-ATP current was decreased in these TG myocytes and no obvious difference between TG and non-TG (WT) myocytes in the ATP sensitivity of the KATP channel was observed although the open probability of the KATP channels was significantly lower in TG myocytes than WT. These transgenic mouse strains with distinct ECG changes suggested that the S422L mutation in KCNJ8 gene is not a direct cause of JWS. (C) 2017 The Authors. Production and hosting by Elsevier B.V.

Although the two anti-diabetic drugs, dipeptidyl peptidase-4 inhibitors (DPP4is) and glucagon-like peptide-1 (GLP-1) receptor agonists (GLP1RAs), have distinct effects on the dynamics of circulating incretins, little is known of the difference in their consequences on morphology and function of pancreatic islets. We examined these in a mouse model of β cell injury/regeneration. The model mice were generated so as to express diphtheria toxin (DT) receptor and a fluorescent protein (Tomato) specifically in β cells. The mice were treated with a DPP4i (MK-0626) and a GLP1RA (liraglutide), singly or doubly, and the morphology and function of the islets were compared. Prior administration of MK-0626 and/or liraglutide similarly protected β cells from DT-induced cell death, indicating that enhanced GLP-1 signaling can account for the cytoprotection. However, 2-week intervention of MK-0626 and/or liraglutide in DT-injected mice resulted in different islet morphology and function: β cell proliferation and glucose-stimulated insulin secretion (GSIS) were increased by MK-0626 but not by liraglutide; α cell mass was decreased by liraglutide but not by MK-0626. Although liraglutide administration nullified MK-0626-induced β cell proliferation, their co-administration resulted in increased GSIS, decreased α cell mass, and improved glucose tolerance. The pro-proliferative effect of MK-0626 was lost by co-administration of the GLP-1 receptor antagonist exendin-(9-39), indicating that GLP-1 signaling is required for this effect. Comparison of the effects of DPP4is and/or GLP1RAs treatment in a single mouse model shows that the two anti-diabetic drugs have distinct consequences on islet morphology and function.

<title>Abstract</title> Dmbx1 is a brain-specific homeodomain transcription factor expressed primarily during embryogenesis, and its systemic disruption (Dmbx1−/−) in the ICR mouse strain resulted in leanness associated with impaired long-lasting orexigenic effect of agouti-related peptide (AgRP). Because spatial and temporal expression patterns of Dmbx1 change dramatically during embryogenesis, it remains unknown when and where Dmbx1 plays a critical role in energy homeostasis. In the present study, the physiological roles of Dmbx1 were examined by its conditional disruption (Dmbx1loxP/loxP) in the C57BL/6 mouse strain. Although Dmbx1 disruption in fetal brain resulted in neonatal lethality, its disruption by synapsin promoter-driven Cre recombinase, which eliminated Dmbx1 expression postnatally, exempted the mice (Syn-Cre;Dmbx1loxP/loxP mice) from lethality. Syn-Cre;Dmbx1loxP/loxP mice show mild leanness and impaired long-lasting orexigenic action of AgRP, demonstrating the physiological relevance of Dmbx1 in the adult. Visualization of Dmbx1-expressing neurons in adult brain using the mice harboring tamoxifen-inducible Cre recombinase in the Dmbx1 locus (Dmbx1CreERT2/+ mice) revealed Dmbx1 expression in small numbers of neurons in restricted regions, including the lateral parabrachial nucleus (LPB). Notably, c-Fos expression in LPB was increased at 48 hours after AgRP administration in Dmbx1loxP/loxP mice but not in Syn-Cre;Dmbx1loxP/loxP mice. These c-Fos-positive neurons in LPB did not coincide with neurons expressing Dmbx1 or melanocortin 4 receptor but did coincide with those expressing calcitonin gene-related peptide. Accordingly, Dmbx1 in the adult LPB is required for the long-lasting orexigenic effect of AgRP via the neural circuitry involving calcitonin gene-related peptide neurons.

Glucose-6-phosphatase (G6Pase) is a key regulator of gluconeogenesis. We previously found that administration of glycerol, a substrate for gluconeogenesis, transactivates G6Pase in the mouse liver. To clarify its cell-autonomous transcriptional activation in hepatocytes, we examined the mechanism of expression of the gene G6pc, which encodes G6Pase, in rat hepatoma cell line FAO cells. Endogenous G6pc expression in FAO cells was increased by glycerol administration as well as by the fatty acid oleate. Luciferase reporter assay revealed that the ~2.0 kb mouse G6pc promoter contains the element(s) responsible for glycerol-stimulated G6pc transactivation. Using several deletion- or chimeric-constructs of G6pc promoter, we found that the DNA response element for hepatocyte nuclear factor 4α (HNF4α) (−77/−65) in the G6pc promoter is essential for transactivation by glycerol. Similarly to glycerol, oleate also increased G6pc expression through its action on the HNF4α element (−77/−65). Furthermore, the reporter activities were higher in the cells co-treated with glycerol plus oleate than in those singly treated with glycerol or oleate. In addition, the temporal profiles of G6pc expression differed between glycerol and oleate administration. Our present results suggest that glycerol and oleate induce G6pc expression both via the HNF4αelement (−77/−65) and also through other regulatory mechanisms.

BACKGROUND/OBJECTIVES: Brain-derived neurotrophic factor (BDNF) and its receptor (tropomyosin-related kinase B: TrkB, also known as Ntrk2) have a key role in central regulation of the energy balance. BDNF and TrkB are also expressed in the peripheral tissues, including adipose tissue, but their peripheral role has been unclear. Here we report on the functional significance of the adipose tissue BDNF/TrkB axis in metabolic homeostasis. MATERIALS AND METHODS: To examine the role of the BDNF/TrkB axis in the central nervous system and in adipose tissue, we generated adipocyte-specific or neuron-specific BDNF/TrkB conditional knockout (CKO) mice. Then we compared the feeding behavior and metabolic profile between each type of CKO mouse and their littermates. RESULTS: Bdnf expression was significantly increased in the adipose tissue of mice receiving a high-calorie diet, whereas Ntrk2 expression was decreased. The Bdnf/Ntrk2 expression ratio of adipose tissue was higher in female mice than male mice. Fabp4-Cre mice are widely used to establish adipocyte-specific CKO mice. However, we found that Fabp4-Cre-induced deletion of Bdnf or Ntrk2 led to hyperphagia, obesity, and aggressiveness, presumably due to ectopic Fabp4-Cre mediated gene recombination in the brain. Next, we attempted to more specifically delete Bdnf or Ntrk2 in adipocytes using Adipoq-Cre mice. Expression of Ntrk2, but not Bdnf, in the adipose tissue was reduced by Adipoq-Cre mediated gene recombination, indicating that adipocytes only expressed TrkB. No phenotypic changes were detected when Adipoq-Cre TrkB CKO mice were fed a normal diet, whereas female CKO mice receiving a high-calorie diet showed a decrease in food intake and resistance to obesity. CONCLUSIONS: The adipose tissue BDNF/TrkB axis has a substantial influence on the feeding behavior and obesity in female mice.

High-fat diet (HFD) triggers insulin resistance and diabetes mellitus, but their link remains unclear. Characterization of overt hyperglycemia in insulin receptor mutant (Insr(P1195L/+)) mice exposed to HFD (Insr(P1195L/+)/HFD mice) revealed increased glucose-6-phosphatase (G6pc) expression in liver and increased gluconeogenesis from glycerol. Lipolysis in white adipose tissues (WAT) and lipolysis-induced blood glucose rise were increased in Insr(P1195L/+)/HFD mice, while wild-type WAT transplantation ameliorated the hyperglycemia and the increased G6pc expression. We found that the expressions of genes involved in bile acid (BA) metabolism were altered in Insr(P1195L/+)/HFD liver. Among these, the expression of Cyp7a1, a BA synthesis enzyme, was insulin-dependent and was markedly decreased in Insr(P1195L/+)/HFD liver. Reduced Cyp7a1 expression in Insr(P1195L/+)/HFD liver was rescued by WAT transplantation, and the expression of Cyp7a1 was suppressed by glycerol administration in wild-type liver. These findings suggest that unsuppressed lipolysis in adipocytes elicited by HFD feeding is linked with enhanced gluconeogenesis from glycerol and with alterations in BA physiology in Insr(P1195L/+)/HFD liver.

Adenosine triphosphate-sensitive K(+) (KATP) channels play an essential role in glucose-induced insulin secretion from pancreatic β-cells. It was recently reported that the KATP channel is also found in the enteroendocrine K-cells and L-cells that secrete glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), respectively. In the present study, we investigated the involvement of the KATP channel in fructose-induced GIP, GLP-1 and insulin secretion in mice. Fructose stimulated GIP secretion, but pretreatment with diazoxide, a KATP channel activator, did not affect fructose-induced GIP secretion under streptozotocin-induced hyperglycemic conditions. Fructose significantly stimulated insulin secretion in Kir6.2 (+/+) mice, but not in mice lacking KATP channels (Kir6.2 (-/-) ), and fructose stimulated GLP-1 secretion in both Kir6.2 (+/+) mice and Kir6.2 (-/-) mice under the normoglycemic condition. In addition, diazoxide completely blocked fructose-induced insulin secretion in Kir6.2 (+/+) mice and in MIN6-K8 β-cells. These results show that fructose-induced GIP and GLP-1 secretion is KATP channel-independent and that fructose-induced insulin secretion is KATP channel-dependent.

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.

Oral ingestion of carbohydrate triggers glucagon-like peptide 1 (GLP1) secretion, but the molecular mechanism remains elusive. By measuring GLP1 concentrations in murine portal vein, we found that the ATP-sensitive K⁺(K_ATP) channel is not essential for glucose-induced GLP1 secretion from enteroendocrine L cells, while the sodium-glucose co-transporter 1 (SGLT1) is required, at least in the early phase (5 min) of secretion. By contrast, co-administration of the α-glucosidase inhibitor (α-GI) miglitol plus maltose evoked late-phase secretion in a glucose transporter 2-dependent manner. We found that GLP1 secretion induced by miglitol plus maltose was significantly higher than that by another α-GI, acarbose, plus maltose, despite the fact that acarbose inhibits maltase more potently than miglitol. As miglitol activates SGLT3, we compared the effects of miglitol on GLP1 secretion with those of acarbose, which failed to depolarize the<italic>Xenopus laevis</italic>oocytes expressing human SGLT3. Oral administration of miglitol activated duodenal enterochromaffin (EC) cells as assessed by immunostaining of phosphorylated calcium–calmodulin kinase 2 (phospho-CaMK2). In contrast, acarbose activated much fewer enteroendocrine cells, having only modest phospho-CaMK2 immunoreactivity. Single administration of miglitol triggered no GLP1 secretion, and GLP1 secretion by miglitol plus maltose was significantly attenuated by atropine pretreatment, suggesting regulation via vagal nerve. Thus, while α-GIs generally delay carbohydrate absorption and potentiate GLP1 secretion, miglitol also activates duodenal EC cells, possibly via SGLT3, and potentiates GLP1 secretion through the parasympathetic nervous system.

AIMS: Spontaneously diabetic Torii (SDT) rats exhibit vascular abnormalities in pancreatic islets as the initial changes at pre-diabetes stage (8 weeks old), which is followed by β cell deterioration. In the present study, we investigated pathophysiological interactions between β cells and intra-islet microvasculature of SDT rats at pre- and peri-onset of diabetes. METHODS: SDT rats were treated with Habu snake venom (HSV) to assess its hemorrhagic effects in glomeruli and pancreatic islets. SDT rats were treated with streptozotocin (STZ) to assess acute β cell fragility toward cytotoxic insult and the late-stage consequence of β cell ablation in neighboring structures. The receptor tyrosine kinase inhibitor sunitinib was administered to SDT rats to examine its therapeutic effect. RESULTS: HSV administration at 5 weeks old induced severe hemorrhage in and around islets in SDT rats. By contrast, precedent β cell depletion using STZ ameliorated hemorrhage, inflammation, and fibrosis around the islets at 13 weeks old, which is normally seen in SDT rats of this age. Blockade of vascular endothelial growth factor (VEGF)-like activity attenuated HSV-induced hemorrhage in SDT islets. VEGF release from SDT islets was increased at 13 weeks old but not at 5 weeks old, while interleukin-1β release was increased as early as 5 weeks old. Sunitinib treatment started at 5 weeks of age inhibited the onset of intra-islet hemorrhage, β cell loss, and hyperglycemia in SDT rats. CONCLUSIONS: Enhanced VEGF signaling in islets contributes to β cell injury, microvascular failure, and consequential diabetes in SDT rats.

Glucose-dependent insulinotropic polypeptide (GIP), a gut hormone secreted from intestinal K-cells, potentiates insulin secretion. Both K-cells and pancreatic β-cells are glucose-responsive and equipped with a similar glucose-sensing apparatus that includes glucokinase and an ATP-sensitive K⁺(K_ATP) channel comprising KIR6.2 and sulfonylurea receptor 1. In absorptive epithelial cells and enteroendocrine cells, sodium glucose co-transporter 1 (SGLT1) is also known to play an important role in glucose absorption and glucose-induced incretin secretion. However, the glucose-sensing mechanism in K-cells is not fully understood. In this study, we examined the involvement of SGLT1 (SLC5A1) and the K_ATPchannels in glucose sensing in GIP secretion in both normal and streptozotocin-induced diabetic mice. Glimepiride, a sulfonylurea, did not induce GIP secretion and pretreatment with diazoxide, a K_ATPchannel activator, did not affect glucose-induced GIP secretion in the normal state. In mice lacking K_ATPchannels (<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice), glucose-induced GIP secretion was enhanced compared with control (<italic>Kir6.2</italic>^{<italic>+</italic><italic>/</italic><italic>+</italic>}) mice, but was completely blocked by the SGLT1 inhibitor phlorizin. In<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice, intestinal glucose absorption through SGLT1 was enhanced compared with that in<italic>Kir6.2</italic>^{<italic>+</italic><italic>/</italic><italic>+</italic>}mice. On the other hand, glucose-induced GIP secretion was enhanced in the diabetic state in<italic>Kir6.2</italic>^{<italic>+</italic><italic>/</italic><italic>+</italic>}mice. This GIP secretion was partially blocked by phlorizin, but was completely blocked by pretreatment with diazoxide in addition to phlorizin administration. These results demonstrate that glucose-induced GIP secretion depends primarily on SGLT1 in the normal state, whereas the K_ATPchannel as well as SGLT1 is involved in GIP secretion in the diabetic state<italic>in vivo</italic>.

Glucose-induced insulin secretion from pancreatic β-cells critically depends on the activity of ATP-sensitive K⁺channels (K_ATPchannel). We previously generated mice lacking<italic>Kir6.2</italic>, the pore subunit of the β-cell K_ATPchannel (<italic>Kir6.2</italic>^{<italic>−/−</italic>}), that show almost no insulin secretion in response to glucose<italic>in vitro</italic>. In this study, we compared insulin secretion by voluntary feeding (self-motivated, oral nutrient ingestion) and by forced feeding (intra-gastric nutrient injection via gavage) in wild-type (<italic>Kir6.2</italic>^{<italic>+</italic><italic>/</italic><italic>+</italic>}) and<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice. Under<italic>ad libitum</italic>feeding or during voluntary feeding of standard chow, blood glucose levels and plasma insulin levels were similar in<italic>Kir6.2</italic>^{<italic>+</italic><italic>/</italic><italic>+</italic>}and<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice. By voluntary feeding of carbohydrate alone, insulin secretion was induced significantly in<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice but was markedly attenuated compared with that in<italic>Kir6.2</italic>^{<italic>+</italic><italic>/</italic><italic>+</italic>}mice. On forced feeding of standard chow or carbohydrate alone, the insulin secretory response was markedly impaired or completely absent in<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice. Pretreatment with a muscarine receptor antagonist, atropine methyl nitrate, which does not cross the blood–brain barrier, almost completely blocked insulin secretion induced by voluntary feeding of standard chow or carbohydrate in<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice. Substantial glucose-induced insulin secretion was induced in the pancreas perfusion study of<italic>Kir6.2</italic>^{<italic>−/−</italic>}mice only in the presence of carbamylcholine. These results suggest that a K_ATPchannel-independent mechanism mediated by the vagal nerve plays a critical role in insulin secretion in response to nutrients<italic>in vivo</italic>.

UNLABELLED: Aims/Introduction:  Oral ingestion of carbohydrate triggers secretion of glucagon-like peptide (GLP)-1, which inhibits the postprandial rise in blood glucose levels. However, the mechanism of carbohydrate-induced GLP-1 secretion from enteroendocrine L cells remains unclear. In the present study, GLP-1 secretion was examined by meal tolerance tests of healthy Japanese volunteers. MATERIALS AND METHODS:   Twenty-one healthy Japanese men participated in the study. The meal tolerance test was performed with modified nutrient compositions, with or without pretreatment with the α-glucosidase inhibitor acarbose, or with substitution of sucrose with an equivalent dose of sweeteners in the meal. Blood concentrations of glucose, insulin, GLP-1, and apolipoprotein (Apo) B-48 were measured. RESULTS:   GLP-1 secretion started concomitant with the increase in blood glucose levels 10 min after meal ingestion. Insulin secretion started at 5 min, before the increase in blood glucose levels, reflecting the contribution of direct nutrient stimulation on the former parameter and neural regulation in the latter. Carbohydrate retention in the gut lumen induced by acarbose pretreatment extended postprandial GLP-1 secretion and negated the increase in serum ApoB-48 levels. GLP-1 secretion was markedly decreased by a reduction in the amount of sucrose in the meal and was not restored by an equivalent dose of sweeteners used to compensate for the sweet taste. CONCLUSIONS:   The results indicate that direct stimulation of L cells with sugar, but not sweetener, is required for carbohydrate-induced GLP-1 secretion. In addition, inhibition of digestion of dietary carbohydrate by α-glucosidase inhibitors may prevent postprandial hyperglycemia by increasing GLP-1 secretion and by inhibiting glucose absorption. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00163.x, 2011).

Some class I antiarrhythmic drugs induce a sporadic hypoglycemia by producing insulin secretion via inhibition of ATP-sensitive K+ (K-ATP) channels of pancreatic beta-cells. It remains undetermined whether amiodarone produces insulin secretion by inhibiting K-ATP channels. In this study, effects of amiodarone on K-ATP channels, L-type Ca2+ channel, membrane potential, and insulin secretion were examined and compared with those of quinidine in a beta-cell line (MIN6). Amiodarone as well as quinidine inhibited the openings of the K-ATP channel in a concentration-dependent manner without affecting its unitary amplitude in inside-out membrane patches of single MIN6 cells, and the IC50 values were 0.24 and 4.9 mu M, respectively. The L-type Ca2+ current was also inhibited by amiodarone as well as quinidine in a concentration-dependent manner. Although glibenclamide (0.1 mu M) or quinidine (10 mu M) significantly potentiated the insulin secretion from MIN6 cells, amiodarone (1 - 30 mu M) failed to increase insulin secretion. Amiodarone (30 mu M) and nifedipine (10 mu M) significantly inhibited the increase in insulin secretion produced by 0.1 mu M glibenclamide. Amiodarone (30 mu M) produced a gradual decrease of the membrane potential, but did not produce repetitive electrical activity in MIN6 cells. Glibenclamide (1 mu M) produced a slow depolarization, followed by spiking activity which was inhibited by 30 mu M amiodarone. Thus, amiodarone is unlikely to produce hypoglycemia in spite of potent inhibitory action on K-ATP channels in insulin-secreting cells, possibly due to its Ca2+ channel-blocking action.

The primary cilium is now considered to function as a fundamental, not rudimentary, structure for mechanical and chemical sensing by individual cells. Primary cilia in neurons express type III adenylyl cyclase (ACIII) and GPCRs for somatostatin (somatostatin receptor 3, SSTR3), serotonin, and melanin-concentrating hormone. The present immunohistochemical and electron microscopic study revealed an abundant occurrence of SSTR3-expressing solitary cilia in insulin- and growth hormone-secreting cells of the mouse. The SSTR3 immunoreactivity was restricted to the plasma membrane of cilia in both cell types, differing from previously reported immunohistochemical localization of SSTRs to cell bodies. The primary cilia in the islet cells were longer than those in the pituitary cells and extended for a long distance in the intercellular canalicules endowed with microvilli. No other endocrine organs were provided with the SSTR3-expressing primary cilia, while the primary cilia in these organs were frequently immunolabeled with ACIII antibody. Since the somatostatin inhibition of both insulin and GH release is regulated mainly by SSTR1 and SSTR5, the primary cilia expressing SSTR3 may be involved in a signaling which differs from that via other SSTR subtypes expressing in cell bodies.

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2 alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2 alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2 alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2 alpha primes insulin granules for fusion. Thus, Rim2 alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2 alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2 alpha plays a critical role in exocytosis of these dense-core granules.

Pancreatic beta cells, organized in the islets of Langerhans, sense glucose and secrete appropriate amounts of insulin. We have studied the roles of LKB1, a conserved kinase implicated in the control of cell polarity and energy metabolism, in adult beta cells. LKB1-deficient beta cells show a dramatic increase in insulin secretion in vivo. Histologically, LKB1-deficient beta cells have striking alterations in the localization of the nucleus and cilia relative to blood vessels, suggesting a shift from hepatocyte-like to columnar polarity. Additionally, LKB1 deficiency causes a 65% increase in beta cell volume. We show that distinct targets of LKB1 mediate these effects. LKB1 controls beta cell size, but not polarity, via the mTOR pathway. Conversely, the precise position of the beta cell nucleus, but not cell size, is controlled by the LKB1 target Par1b. Insulin secretion and content are restricted by LKB1, at least in part, via AMPK. These results expose a molecular mechanism, orchestrated by LKB1, for the coordinated maintenance of beta cell size, form, and function.

Epac2, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rap1, is activated by adenosine 3&apos;,5&apos;-monophosphate. Fluorescence resonance energy transfer and binding experiments revealed that sulfonylureas, widely used antidiabetic drugs, interact directly with Epac2. Sulfonylureas activated Rap1 specifically through Epac2. Sulfonylurea-stimulated insulin secretion was reduced both in vitro and in vivo in mice lacking Epac2, and the glucose-lowering effect of the sulfonylurea tolbutamide was decreased in these mice. Epac2 thus contributes to the effect of sulfonylureas to promote insulin secretion. Because Epac2 is also required for the action of incretins, gut hormones crucial for potentiating insulin secretion, it may be a promising target for antidiabetic drug development.

cAMP is a well-known regulator of exocytosis, and cAMP-GEFII (Epac2) is involved in the potentiation of cAMP-dependent, PKA-independent regulated exocytosis in secretory cells. However, the mechanisms of its action are not fully understood. In the course of our study of Epac2 knockout mice, we identified a novel splicing variant of Epac2, which we designate Epac2B, while renaming the previously identified Epac2 Epac2A. Epac2B, which lacks the first cAMP-binding domain A in the N-terminus but has the second cAMP-binding domain B of Epac2A, possesses GEF activity towards Rap 1, as was found for Epac2A. Immunocytochemical analysis revealed that exogenously introduced Epac2A into insulin-secreting MIN6 cells was localized near the plasma membrane, while Epac2B was found primarily in the cytoplasm. Interestingly, cAMP-binding domain A alone introduced into MIN6 cells was also localized near the plasma membrane. In MIN6 cells, Epac2A was involved in triggering hormone secretion by stimulation with 5.6 mM glucose plus I mM 8-Bromo-cAMP, but Epac2B was not. The addition of a membrane-targeting signal to the N-terminus of Epac2B was able to mimic the effect of Epac2A on hormone secretion. Thus, the present study indicates that the N-terminal cAMP-binding domain A of Epac2A plays a critical role in determining its subcellular localization and potentiating insulin secretion by cAMP.

We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K(+) (K(ATP)) channels (Kir6.2 (-/-) mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. Pancreas perfusion experiments were performed using wild-type (Kir6.2 (+/+) ) or Kir6.2 (-/-) mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2 (-/-) mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K(ATP) channels with glibenclamide. The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2 (-/-) pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K(ATP) channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2 (+/+) , as in Kir6.2 (-/-) pancreases. Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2 (-/-) beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.

KCNJ11 null mutants, lacking Kir6.2 ATP-sensitive K+ (KATp) channels, exhibit a marked susceptibility towards hypertension (HTN)-induced heart failure. To gain insight into the molecular alterations induced by knockout of this metabolic sensor under hemodynamic stress, wild-type (WT) and Kir6.2 knockout (Kir6.2-KO) cardiac proteomes were profiled by comparative 2-DE and Orbitrap MS. Despite equivalent systemic HTN produced by chronic hyperaldosteronism, 114 unique proteins were altered in Kir6.2-KO compared to WT hearts. Bioinformatic analysis linked the primary biological function of the KATp channel-dependent protein cohort to energetic metabolism (64% of proteins), followed by signaling infrastructure (36%) including oxidoreductases, stress-related chaperones, processes supporting protein degradation, transcription and translation, and cytostructure. Mapped protein-protein relationships authenticated the primary impact on metabolic pathways, delineating the KATp channel-dependent subproteome within a nonstochastic network. Iterative systems interrogation of the proteomic web prioritized heart-specific adverse effects, i.e., "Cardiac Damage", "Cardiac Enlargement", and "Cardiac Fibrosis", exposing a predisposition for the development of cardiomyopathic traits in the hypertensive Kir6.2-KO. Validating this maladaptive forecast, phenotyping documented an aggravated myocardial contractile performance, a massive interstitial fibrosis and an exaggerated left ventricular size, all prognostic indices of poor outcome. Thus, Kir6.2 ablation engenders unfavorable proteomic remodeling in hypertensive hearts, providing a composite molecular substrate for pathologic stress-associated cardiovascular disease.

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (KATP) channel-dependent Ca(2+) influx with a subsequent increase of the cytosolic free Ca(2+) concentration ([Ca(2+)](c)) (triggering pathway) and a KATP channel-independent augmentation of secretion without further increase of [Ca(2)](c) (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking KATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca(2+)](c) and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca(2+)](c) and insulin secretion were marginally elevated, and high glucose increased [Ca(2+)](c) only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mM KCl). Culture in 10 mM glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca(2+)](c) with modulation of [Ca(2+)](c) oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca(2+)](c) and insulin secretion, by inducing KATP channel-independent depolarization and Ca(2+) influx via voltage-dependent Ca(2+) channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca(2+)](c), but culture reveals a glucose-responsive phenotype mainly by increasing [Ca(2+)](c). The results support models implicating a KATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that KATP channels are not the only possible transducers of metabolic effects on the triggering Ca(2+) signal. (Endocrinology 150: 33-45, 2009)

Responding for rewarding brain stimulation has been used to track hedonic status in animals. In addition to neurochemical alterations, stimulation of the lateral hypothalamus or ventral tegmentum has been shown to influence immunological processes, including elevation of peripheral natural killer cell activity. In the present study, we examined whether ventral tegmental area (VTA) stimulation or environmental enrichment altered the severity of lipopolysaccharide (LPS)-induced sickness behaviors and the provocation of cytokine expression induced by the endotoxin. Accordingly, rats received either trials of brain stimulation reward or exposure to an enriched environment and subsequently challenged with 150 ug/kg i.p. of LPS. Groups receiving LPS and saline injections without further manipulation were also included. Using the real-time RT-PCR and a multiplex bead assay, mRNA and protein levels for several cytokines and their receptors were determined to evaluate how these may vary as a consequence of reward. Both brain stimulation and environmental enrichment similarly diminished sickness behaviors associated with the endotoxin. Receptor gene levels were generally stable across groups. Levels of IL-6 within the VTA were increased in the group receiving LPS challenge alone and environmental enrichment was associated with modestly reduced IL-6 levels within this brain region. Taken together, these data suggest that rewarding brain stimulation and environmental enrichment buffer against malaise provoked by endotoxin challenge. Moreover, IL-6 expression within the VTA may influence the development of sickness behavior following inflammatory stimuli. (C) 2008 Elsevier B.V. All rights reserved.

The role of cardiac sarcolemmal ATP-sensitive K(+) (K(ATP)) channels in the regulation of sinoatrial node (SAN) automaticity is not well defined. Using mice with homozygous knockout (KO) of the Kir6.2 (a pore-forming subunit of cardiac K(ATP) channel) gene, we investigated the pathophysiological role of K(ATP) channels in SAN cells during hypoxia. Langendorff-perfused mouse hearts were exposed to hypoxic and glucose-free conditions (hypoxia). After 5 min of hypoxia, sinus cycle length (CL) was prolonged from 207 +/- 10 to 613 +/- 84 ms (P &lt; 0.001) in wild-type (WT) hearts. In Kir6.2 KO hearts, CL was slightly prolonged from 198 +/- 17 to 265 +/- 32 ms. The CL of spontaneous action potentials of WT SAN cells, recorded in the current-clamp mode, was markedly prolonged from 410 +/- 56 to 605 +/- 108 ms (n = 6, P &lt; 0.05) with a decrease of the slope of the diastolic depolarization (SDD) after the application of the K(+) channel opener pinacidil (100 mu M). Pinacidil induced a glibenclamide (1 mu M)-sensitive outward current, which was recorded in the voltage-clamp mode, only in WT SAN cells. During metabolic inhibition by 2,4-dinitrophenol, CL was prolonged from 292 +/- 38 to 585 +/- 91 ms (P &lt; 0.05) with a decrease of SDD in WT SAN cells but not in Kir6.2 KO SAN cells. Diastolic Ca(2+) concentration, measured by fluo-3 fluorescence, was decreased in WT SAN cells but increased in Kir6.2 KO SAN cells after short-term metabolic inhibition. In conclusion, the present study using Kir6.2 KO mice indicates that, during hypoxia, activation of sarcolemmal K(ATP) channels in SAN cells inhibits SAN automaticity, which is important for the protection of SAN cells.

In wild-type mice, a single injection of streptozotocin (STZ, 200 mg/kg body wt) caused within 4 days severe hyperglycemia, hypoinsulinemia, significant glucose intolerance, loss of body weight, and the disappearance of pancreatic beta-cells. However, in ATP-sensitive K+ channel (K-ATP channel)-deficient mice (Kir6.2(-/-) mice), STZ had none of these effects. Exposing isolated pancreatic islets to STZ caused severe damage in wild-type but not in Kir6.2(-/-) islets. Following a single injection, plasma STZ levels were slightly less in Kir6.2(-/-) mice than in wild-type mice. Despite the difference in plasma STZ, wild-type and Kir6.2(-/-) liver accumulated the same amount of STZ, whereas Kir6.2(-/-) pancreas accumulated 4.1-fold less STZ than wild-type pancreas. Kir6.2(-/-) isolated pancreatic islets also transported less glucose than wild- type ones. Quantification of glucose transporter 2 (GLUT2) protein content by Western blot using an antibody with an epitope in the extracellular loop showed no significant difference in GLUT2 content between wild- type and Kir6.2(-/-) pancreatic islets. However, visualization by immunofluorescence with the same antibody gave rise to 32% less fluorescence in Kir6.2(-/-) pancreatic islets. The fluorescence intensity using another antibody, with an epitope in the COOH terminus, was 5.6 times less in Kir6.2(-/-) than in wild-type pancreatic islets. We conclude that 1) Kir6.2(-/-) mice are STZ resistant because of a decrease in STZ transport by GLUT2 in pancreatic beta-cells and 2) the decreased transport is due to a downregulation of GLUT2 activity involving an effect at the COOH terminus.