Lack of TRPM2 impaired insulin secretion and glucose metabolisms in mice.
ABSTRACT: OBJECTIVE: TRPM2 is a Ca²(+)-permeable nonselective cation channel activated by adenosine dinucleotides. We previously demonstrated that TRPM2 is activated by coapplication of heat and intracellular cyclic adenosine 5'-diphosphoribose, which has been suggested to be involved in intracellular Ca²(+) increase in immunocytes and pancreatic ?-cells. To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice. RESEARCH DESIGN AND METHODS: Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed in TRPM2-KO and wild-type mice. We also measured cytosolic free Ca²(+) in single pancreatic cells using fura-2 microfluorometry and insulin secretion from pancreatic islets. RESULTS: Basal blood glucose levels were higher in TRPM2-KO mice than in wild-type mice without any difference in plasma insulin levels. The OGTT and IPGTT demonstrated that blood glucose levels in TRPM2-KO mice were higher than those in wild-type mice, which was associated with an impairment in insulin secretion. In isolated ?-cells, smaller intracellular Ca²(+) increase was observed in response to high concentrations of glucose and incretin hormone in TRPM2-KO cells than in wild-type cells. Moreover, insulin secretion from the islets of TRPM2-KO mice in response to glucose and incretin hormone treatment was impaired, whereas the response to tolbutamide, an ATP-sensitive potassium channel inhibitor, was not different between the two groups. CONCLUSIONS: These results indicate that TRPM2 is involved in insulin secretion stimulated by glucose and that further potentiated by incretins. Thus, TRPM2 may be a new target for diabetes therapy.
Project description:AIMS/HYPOTHESIS:The aim of this study was to elucidate the impact of 3'-phosphoinositide-dependent protein kinase-1 (PDPK1) in vascular endothelial cells on the maintenance of pancreatic beta cell mass and function. METHODS:Male vascular endothelial cell-specific Pdpk1-knockout mice (Tie2+/-/Pdpk1flox/flox mice) and their wild-type littermates (Tie2-/-/Pdpk1flox/flox mice; control) were used for this study. At 12 weeks of age, an IPGTT and OGTT were conducted. Pancreatic blood flow was measured under anaesthesia. Thereafter, islet blood flow was measured by the microsphere method. Mice were killed for islet isolation and further functional study and mRNA was extracted from islets. Pancreases were sampled for immunohistochemical analyses. RESULTS:During the IPGTT, the blood glucose level was comparable between knockout mice and control flox mice, although serum insulin level was significantly lower in knockout mice. During the OGTT, glucose tolerance deteriorated slightly in knockout mice, accompanied by a decreased serum insulin level. During an IPGTT after pre-treatment with exendin-4 (Ex-4), glucose tolerance was significantly impaired in knockout mice. In fact, glucose-stimulated insulin secretion of isolated islets from knockout mice was significantly reduced compared with control flox mice, and addition of Ex-4 revealed impaired sensitivity to incretin hormones in islets of knockout mice. In immunohistochemical analyses, both alpha and beta cell masses were significantly reduced in knockout mice. In addition, the CD31-positive area was significantly decreased in islets of knockout mice. The proportion of pimonidazole-positive islets was significantly increased in knockout mice. mRNA expression levels related to insulin biosynthesis (Ins1, Ins2, Mafa, Pdx1 and Neurod [also known as Neurod1]) and beta cell function (such as Gck and Slc2a2) were significantly decreased in islets of knockout mice. Microsphere experiments revealed remarkably reduced islet blood flow. In addition, mRNA expression levels of Hif1? (also known as Hif1a) and its downstream factors such as Adm, Eno1, Tpi1 (also known as Ets1), Hmox1 and Vegfa, were significantly increased in islets of knockout mice, indicating that islets of knockout mice were in a more hypoxic state than those of control flox mice. As a result, mRNA expression levels related to adaptive unfolded protein response and endoplasmic reticulum stress-related apoptotic genes were significantly elevated in islets of knockout mice. In addition, inflammatory cytokine levels were increased in islets of knockout mice. Electron microscopy revealed reduced endothelial fenestration and thickening of basal membrane of vascular endothelium in islets of knockout mice. CONCLUSIONS/INTERPRETATION:Vascular endothelial PDPK1 plays an important role in the maintenance of pancreatic beta cell mass and function by maintaining vascularity of pancreas and islets and protecting them from hypoxia, hypoxia-related endoplasmic reticulum stress, inflammation and distortion of capillary structure.
Project description:BACKGROUND:Previously, we found a significant relationship in a rat study between energy intake and bile acid (BA) metabolism especially 12?-hydroxylated (12?OH) BAs. The present study was designed to reveal relationships among BA metabolism, glucose tolerance, and cecal organic acids in rats fed a high-fat and high-sucrose diet (HFS) by using multivariate and multiple regression analyses in two types of glucose tolerance tests (GTTs). METHODS:Male WKAH/HkmSlc rats were fed with a control or a HFS for 13?weeks. Oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT) were performed at week 9 and 11, respectively. BAs were analyzed by using ultra high-performance liquid chromatography-mass spectrometry. Organic acid concentrations in cecal contents were analyzed by using ultra high-performance liquid chromatography with post-column pH buffered electric conductivity method. RESULTS:A positive correlation of aortic 12?OH BA concentration was observed with energy intake and visceral adipose tissue weight. We found that an increase of 12?OH BAs in enterohepatic circulation, intestinal contents and feces in the HFS-fed rats compared to those in control rats regardless of no significant increase of total BA concentration in the feces in the test period. Fecal 12?OH BA concentration was positively correlated with maximal insulin level in OGTT and area under curve of insulin in IPGTT. There was a positive correlation between aortic 12?OH BAs concentration and changes in plasma glucose level in both OGTT and IPGTT. In contrast, a decrease in the concentration of organic acids was observed in the cecal contents of the HFS-fed rats. Multiple linear regression analysis in the IPGTT revealed that the concentrations of aortic 12?OH BA and cecal acetic acid were the predictors of insulin secretion. Moreover, there was a positive correlation between concentration of portal 12?OH BAs and change in insulin concentration of peripheral blood in the IPGTT. CONCLUSION:The distribution analysis of BA compositions accompanied by GTTs revealed a close relationship between 12?OH BA metabolism and insulin secretion in GTTs in rats.
Project description:OBJECTIVE:Islets secrete neurotransmitters including glutamate which participate in fine regulation of islet function. The excitatory ionotropic glutamate receptor GluK2 of the kainate receptor family is widely expressed in brain and also found in islets, mainly in ? and ? cells. ? cells co-release glucagon and glutamate and the latter increases glucagon release via ionotropic glutamate receptors. However, neither the precise nature of the ionotropic glutamate receptor involved nor its role in glucose homeostasis is known. As isoform specific pharmacology is not available, we investigated this question in constitutive GluK2 knock-out mice (GluK2-/-) using adult and middle-aged animals to also gain insight in a potential role during aging. METHODS:We compared wild-type GluK2+/+ and knock-out GluK2-/- mice using adult (14-20 weeks) and middle-aged animals (40-52 weeks). Glucose (oral OGTT and intraperitoneal IPGTT) and insulin tolerance as well as pyruvate challenge tests were performed according to standard procedures. Parasympathetic activity, which stimulates hormones secretion, was measured by electrophysiology in vivo. Isolated islets were used in vitro to determine islet ?-cell electrical activity on multi-electrode arrays and dynamic secretion of insulin as well as glucagon was determined by ELISA. RESULTS:Adult GluK2-/- mice exhibit an improved glucose tolerance (OGTT and IPGTT), and this was also apparent in middle-aged mice, whereas the outcome of pyruvate challenge was slightly improved only in middle-aged GluK2-/- mice. Similarly, insulin sensitivity was markedly enhanced in middle-aged GluK2-/- animals. Basal and glucose-induced insulin secretion in vivo was slightly lower in GluK2-/- mice, whereas fasting glucagonemia was strongly reduced. In vivo recordings of parasympathetic activity showed an increase in basal activity in GluK2-/- mice which represents most likely an adaptive mechanism to counteract hypoglucagonemia rather than altered neuronal mechanism. In vitro recording demonstrated an improvement of glucose-induced electrical activity of ?-cells in islets obtained from GluK2-/- mice at both ages. Finally, glucose-induced insulin secretion in vitro was increased in GluK2-/- islets, whereas glucagon secretion at 2 mmol/l of glucose was considerably reduced. CONCLUSIONS:These observations indicate a general role for kainate receptors in glucose homeostasis and specifically suggest a negative effect of GluK2 on glucose homeostasis and preservation of islet function during aging. Our observations raise the possibility that blockade of GluK2 may provide benefits in glucose homeostasis especially during aging.
Project description:Cholecystokinin (CCK) is released in response to lipid intake and stimulates insulin secretion. We hypothesized that CCK deficiency would alter the regulation of insulin secretion and glucose homeostasis.We used quantitative magnetic resonance imaging to determine body composition and studied plasma glucose and insulin secretion of CCK gene knockout (CCK-KO) mice and their wild-type controls using intraperitoneal glucose and arginine infusions. The area of anti-insulin staining in pancreatic islets was measured by immunohistochemistry. Insulin sensitivity was assessed with euglycemic-hyperinsulemic clamps.CCK-KO mice fed a low-fat diet had a reduced acute insulin response to glucose but a normal response to arginine and normal glucose tolerance, associated with a trend toward greater insulin sensitivity. However, when fed a high-fat diet (HFD) for 10 weeks, CCK-KO mice developed glucose intolerance despite increased insulin sensitivity that was associated with low insulin secretion in response to both glucose and arginine. The deficiency of insulin secretion in CCK-KO mice was not associated with changes in ?-cell or islet size.CCK is involved in regulating insulin secretion and glucose tolerance in mice eating an HFD. The impaired insulin response to intraperitoneal stimuli that do not typically elicit CCK release suggests that this hormone has chronic effects on ?-cell adaptation to diet in addition to acute incretin actions.
Project description:OBJECTIVE:KCNQ1 gene polymorphisms are associated with type 2 diabetes. This linkage appears to be mediated by altered beta-cell function. In an attempt to study underlying mechanisms, we examined the effect of four KCNQ1 single nucleotide polymorphisms (SNPs) on insulin secretion upon different stimuli. RESEARCH DESIGN AND METHODS:We genotyped 1,578 nondiabetic subjects at increased risk of type 2 diabetes for rs151290, rs2237892, rs2237895, and rs2237897. All participants underwent an oral glucose tolerance test (OGTT); glucagon-like peptide (GLP)-1 and gastric inhibitory peptide secretion was measured in 170 participants. In 519 participants, a hyperinsulinemic-euglycemic clamp was performed, in 314 participants an intravenous glucose tolerance test (IVGTT), and in 102 subjects a hyperglycemic clamp combined with GLP-1 and arginine stimuli. RESULTS:rs151290 was nominally associated with 30-min C-peptide levels during OGTT, first-phase insulin secretion, and insulinogenic index after adjustment in the dominant model (all P < or = 0.01). rs2237892, rs2237895, and rs2237897 were nominally associated with OGTT-derived insulin secretion indexes (all P < 0.05). No SNPs were associated with beta-cell function during intravenous glucose or GLP-1 administration. However, rs151290 was associated with glucose-stimulated gastric inhibitory polypeptide and GLP-1 increase after adjustment in the dominant model (P = 0.0042 and P = 0.0198, respectively). No associations were detected between the other SNPs and basal or stimulated incretin levels (all P > or = 0.05). CONCLUSIONS:Common genetic variation in KCNQ1 is associated with insulin secretion upon oral glucose load in a German population at increased risk of type 2 diabetes. The discrepancy between orally and intravenously administered glucose seems to be explained not by altered incretin signaling but most likely by changes in incretin secretion.
Project description:This letter provides the first pharmacological proof of principle that the sst3 receptor mediates glucose-stimulated insulin secretion (GSIS) from pancreatic ?-cells. To enable these studies, we identified the selective sst3 antagonist (1R,3R)-3-(5-phenyl-1H-imidazol-2-yl)-1-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-?-carboline (5a), with improved ion channel selectivity and mouse pharmacokinetic properties as compared to previously described tetrahydro-?-carboline imidazole sst3 antagonists. We demonstrated that compound 5a enhances GSIS in pancreatic ?-cells and blocks glucose excursion induced by dextrose challenge in ipGTT and OGTT models in mice. Finally, we provided strong evidence that these effects are mechanism-based in an ipGTT study, showing reduction of glucose excursion in wild-type but not sst3 knockout mice. Thus, we have shown that antagonism of sst3 represents a new mechanism with potential in treating type 2 diabetes mellitus.
Project description:The incretin effect on insulin secretion was investigated in 8 subjects with type 2 diabetes (T2D) and 8 with normal glucose tolerance (NGT), using 25, 75, and 125 g oral glucose tolerance tests (OGTT) and isoglycemic intravenous glucose infusions (IIGI). The ß-cell response was evaluated using a model embedding a dose-response (slope=glucose sensitivity), an early response (rate sensitivity), and potentiation (time-related secretion increase). The incretin effect, as OGTT/IIGI ratio, was calculated for each parameter. In NGT, the incretin effect on total secretion increased with dose (1.3 ± 0.1, 1.7 ± 0.2, 2.2 ± 0.2 fold of IIGI, P<0.0001), mediated by a dose-dependent increase of the incretin effect on glucose sensitivity (1.9 ± 0.4, 2.4 ± 0.4, 3.1 ± 0.4, P=0.005), and a dose-independent enhancement of the incretin effect on rate sensitivity (894 , 454 , 783  pmol m(-2) L mmol(-1) above IIGI; median [interquartile range], P<0.0001). The incretin effect on potentiation also increased (0.97 ± 0.06, 1.45 ± 0.20, 1.24 ± 0.16, P<0.0001). In T2D, the incretin effect on total secretion (1.0 ± 0.1, 1.1 ± 0.1, 1.3 ± 0.1, P=0.004) and glucose sensitivity (1.2 ± 0.2, 1.3 ± 0.2, 2.0 ± 0.2, P=0.005) were impaired vs NGT; however, the incretin effect on rate sensitivity increased already at 25 g (269 , 284 , 193  pmol m(-2) L mmol(-1) above IIGI; negligible IIGI rate sensitivity in T2D prevented the calculation of the fold increment). OGTT did not stimulate potentiation above IIGI (0.94 ± 0.04, 0.89 ± 0.06, 1.06 ± 0.09; P<0.01 vs NGT). In the whole group, the incretin effect was inversely associated with total secretion during IIGI, although systematically lower in T2D. In conclusion, 1) In NGT, glucose sensitivity and potentiation mediate the dose-dependent incretin effect increase; 2) In T2D, the incretin effect is blunted vs NGT, but rate sensitivity is enhanced at all loads; 3) Relatively lower incretin effect in NGT is associated with higher secretion during IIGI, suggesting that the reduced incretin effect does not result from ß-cell dysfunction.
Project description:Patients admitted to the intensive care unit often develop hyperglycaemia, but the underlying mechanisms have not been fully described. The incretin effect is reduced in patients with type 2 diabetes. Type 2 diabetes and critical illness have phenotypical similarities, such as hyperglycaemia, insulin resistance and systemic inflammation. Previous studies have shown beneficial effects of exogenous glucagon-like peptide (GLP)-1 on glycaemia in critically ill patients, a phenomenon also seen in patients with type 2 diabetes. In this study, we hypothesised that the incretin effect, which is mediated by the incretin hormones GLP-1 and glucose-dependent insulinotropic peptide (GIP), is impaired in critically ill patients.The incretin effect (i.e., the relative difference between the insulin response to oral and intravenous glucose administration) was investigated in a cross-sectional case-control study. Eight critically ill patients without diabetes admitted to a mixed intensive care unit and eight healthy control subjects without diabetes, matched at group level by age, sex and body mass index, were included in the study. All subjects underwent an oral glucose tolerance test (OGTT) followed by an intravenous glucose infusion (IVGI) on the next day to mimic the blood glucose profile from the OGTT. Blood glucose, serum insulin, serum C-peptide and plasma levels of GLP-1, GIP, glucagon and proinflammatory cytokines were measured intermittently. The incretin effect was calculated as the increase in insulin secretion during oral versus intravenous glucose administration in six patients. The groups were compared using either Student's t test or a mixed model of repeated measurements.Blood glucose levels were matched between the OGTT and the IVGI in both groups. Compared with control subjects, proinflammatory cytokines, tumour necrosis factor ? and interleukin 6, were higher in patients than in control subjects. The endogenous response of GIP and glucagon, but not GLP-1, to the OGTT was greater in patients. The insulin response to the OGTT did not differ between groups, whereas the insulin response to the IVGI was higher in patients. Consequently, the calculated incretin effect was lower in patients (23 vs. 57%, p=0.003).In critically ill patients, the incretin effect was reduced. This resembles previous findings in patients with type 2 diabetes.ClinicalTrials.gov identifier: NCT01347801 . Registered on 2 May 2011.
Project description:BACKGROUND: To date, fasting state- and different oral glucose tolerance test (OGTT)-derived measures are used to estimate insulin release with reasonable effort in large human cohorts required, e.g., for genetic studies. Here, we evaluated twelve common (or recently introduced) fasting state-/OGTT-derived indices for their suitability to detect genetically determined ?-cell dysfunction. METHODOLOGY/PRINCIPAL FINDINGS: A cohort of 1364 White European individuals at increased risk for type 2 diabetes was characterized by OGTT with glucose, insulin, and C-peptide measurements and genotyped for single nucleotide polymorphisms (SNPs) known to affect glucose- and incretin-stimulated insulin secretion. One fasting state- and eleven OGTT-derived indices were calculated and statistically evaluated. After adjustment for confounding variables, all tested SNPs were significantly associated with at least two insulin secretion measures (p?0.05). The indices were ranked according to their associations' statistical power, and the ranks an index obtained for its associations with all the tested SNPs (or a subset) were summed up resulting in a final ranking. This approach revealed area under the curve (AUC)(Insulin(0-30))/AUC(Glucose(0-30)) as the best-ranked index to detect SNP-dependent differences in insulin release. Moreover, AUC(Insulin(0-30))/AUC(Glucose(0-30)), corrected insulin response (CIR), AUC(C-Peptide(0-30))/AUC(Glucose(0-30)), AUC(C-Peptide(0-120))/AUC(Glucose(0-120)), two different formulas for the incremental insulin response from 0-30 min, i.e., the insulinogenic indices (IGI)(2) and IGI(1), and insulin 30 min were significantly higher-ranked than homeostasis model assessment of ?-cell function (HOMA-B; p<0.05). AUC(C-Peptide(0-120))/AUC(Glucose(0-120)) was best-ranked for the detection of SNPs involved in incretin-stimulated insulin secretion. In all analyses, HOMA-? displayed the highest rank sums and, thus, scored last. CONCLUSIONS/SIGNIFICANCE: With AUC(Insulin(0-30))/AUC(Glucose(0-30),) CIR, AUC(C-Peptide(0-30))/AUC(Glucose(0-30)), AUC(C-Peptide(0-120))/AUC(Glucose(0-120)), IGI(2), IGI(1), and insulin 30 min, dynamic measures of insulin secretion based on early insulin and C-peptide responses to oral glucose represent measures which are more appropriate to assess genetically determined ?-cell dysfunction than fasting measures, i.e., HOMA-B. Genes predominantly influencing the incretin axis may possibly be best detected by AUC(C-Peptide(0-120))/AUC(Glucose(0-120)).
Project description:OBJECTIVE:Several single nucleotide polymorphisms (SNPs) in diabetes risk genes reduce glucose- and/or incretin-induced insulin secretion. Here, we investigated interactions between glycemia and such diabetes risk polymorphisms. RESEARCH DESIGN AND METHODS:Insulin secretion was assessed by insulinogenic index and areas under the curve of C-peptide/glucose in 1,576 subjects using an oral glucose tolerance test (OGTT). Participants were genotyped for 10 diabetes risk SNPs associated with ?-cell dysfunction: rs5215 (KCNJ11), rs13266634 (SLC30A8), rs7754840 (CDKAL1), rs10811661 (CDKN2A/2B), rs10830963 (MTNR1B), rs7903146 (TCF7L2), rs10010131 (WFS1), rs7923837 (HHEX), rs151290 (KCNQ1), and rs4402960 (IGF2BP2). Furthermore, the impact of the interaction between genetic variation in TCF7L2 and glycemia on changes in insulin secretion was tested in 315 individuals taking part in a lifestyle intervention study. RESULTS:For the SNPs in TCF7L2 and WFS1, we found a significant interaction between glucose control and insulin secretion (all P ? 0.0018 for glucose × genotype). When plotting insulin secretion against glucose at 120 min OGTT, the compromising SNP effects on insulin secretion are most apparent under high glucose. In the longitudinal study, rs7903146 in TCF7L2 showed a significant interaction with baseline glucose tolerance upon change in insulin secretion (P = 0.0027). Increased glucose levels at baseline predicted an increase in insulin secretion upon improvement of glycemia by lifestyle intervention only in carriers of the risk alleles. CONCLUSIONS:For the diabetes risk genes TCF7L2 and WFS1, which are associated with impaired incretin signaling, the level of glycemia determines SNP effects on insulin secretion. This indicates the increasing relevance of these SNPs during the progression of prediabetes stages toward clinically overt type 2 diabetes.