Kentaro Toyoda

GLP-1 receptor antagonist as a potential probe for pancreatic β-cell imaging

We examined exendin(9-39), an antagonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), as a potential probe for imaging of pancreatic beta-cells. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Binding assay showed competitive inhibition of [(125)I]BH-exendin(9-39) binding by non-radioactive exendin(9-39). To assess in vivo selectivity, the biodistribution was evaluated by intravenous administration of [(125)I]BH-exendin(9-39) to mice. Radioactivity of harvested pancreas reached highest levels at 60 and 120min among organs examined except lung. Pre-administration of excess non-radioactive exendin(9-39) remarkably and specifically blocked the radioactivity of pancreas. After [(125)I]BH-exendin(9-39) injection into transgenic mice with pancreatic beta-cells expressing GFP, fluorescent and radioactive signals of sections of pancreas were evaluated with an image analyzer. Imaging analysis showed that the fluorescent GFP signals and the radioactive signals were correspondingly located. Thus, the GLP-1R antagonist exendin(9-39) may serve as a useful probe for pancreatic beta-cell imaging.

Curcumin inhibits glucose production in isolated mice hepatocytes

Curcumin is a compound derived from the spice turmeric, and is a potent anti-oxidant, anti-carcinogenic, and anti-hepatotoxic agent. We have investigated the acute effects of curcumin on hepatic glucose production. Gluconeogenesis and glycogenolysis in isolated hepatocytes, and gluconeogenetic enzyme activity after 120 min exposure to curcumin were measured. Hepatic gluconeogenesis from 1 mM pyruvate was inhibited in a concentration-dependent manner, with a maximal decrease of 45% at the concentration of 25 microM. After 120 min exposure to 25 microM curcumin, hepatic gluconeogenesis from 2mM dihydroxyacetone phosphate and hepatic glycogenolysis were inhibited by 35% and 20%, respectively. Insulin also inhibited hepatic gluconeogenesis from 1mM pyruvate and inhibited hepatic glycogenolysis in a concentration-dependent manner. Curcumin (25 microM) showed an additive inhibitory effect with insulin on both hepatic gluconeogenesis and glycogenolysis, indicating that curcumin inhibits hepatic glucose production in an insulin-independent manner. After 120 min exposure to 25 microM curcumin, hepatic glucose-6-phosphatase (G6Pase) activity and phosphoenolpyruvate carboxykinase (PEPCK) activity both were inhibited by 30%, but fructose-1,6-bisphosphatase (FBPase) was not reduced. After 120 min exposure to 25 microM curcumin, phosphorylation of AMP kinase alpha-Thr(172) was increased. Thus, the anti-diabetic effects of curcumin are partly due to a reduction in hepatic glucose production caused by activation of AMP kinase and inhibition of G6Pase activity and PEPCK activity.

Transcriptional Regulatory Factor X6 (Rfx6) Increases Gastric Inhibitory Polypeptide (GIP) Expression in Enteroendocrine K-cells and Is Involved in GIP Hypersecretion in High Fat Diet-induced Obesity

Background: Gastric inhibitory polypeptide (GIP) secreted from enteroendocrine K-cells potentiates insulin secretion and induces energy accumulation into adipose tissue. Results: Transcriptional Rfx6 is expressed in K-cells and increases GIP expression. Rfx6 expression is up-regulated in K-cells of obese mice. Conclusion: Rfx6 plays critical roles in GIP expression and hypersecretion in obesity. Significance: Gene analysis of K-cells isolated from GIP-GFP knock-in mice enabled identification of Rfx6. Gastric inhibitory polypeptide (GIP) is an incretin released from enteroendocrine K-cells in response to nutrient ingestion. GIP potentiates glucose-stimulated insulin secretion and induces energy accumulation into adipose tissue, resulting in obesity. Plasma GIP levels are reported to be increased in the obese state. However, the molecular mechanisms of GIP secretion and high fat diet (HFD)-induced GIP hypersecretion remain unclear, primarily due to difficulties in separating K-cells from other intestinal epithelial cells in vivo. In this study, GIP-GFP knock-in mice that enable us to visualize K-cells by enhanced GFP were established. Microarray analysis of isolated K-cells from these mice revealed that transcriptional regulatory factor X6 (Rfx6) is expressed exclusively in K-cells. In vitro experiments using the mouse intestinal cell line STC-1 showed that knockdown of Rfx6 decreased mRNA expression, cellular content, and secretion of GIP. Rfx6 bound to the region in the gip promoter that regulates gip promoter activity, and overexpression of Rfx6 increased GIP mRNA expression. HFD induced obesity and GIP hypersecretion in GIP-GFP heterozygous mice in vivo. Immunohistochemical and flow cytometry analysis showed no significant difference in K-cell number between control fat diet-fed (CFD) and HFD-fed mice. However, GIP content in the upper small intestine and GIP mRNA expression in K-cells were significantly increased in HFD-fed mice compared with those in CFD-fed mice. Furthermore, expression levels of Rfx6 mRNA were increased in K-cells of HFD-fed mice. These results suggest that Rfx6 increases GIP expression and content in K-cells and is involved in GIP hypersecretion in HFD-induced obesity.

Inhibition of GIP signaling modulates adiponectin levels under high-fat diet in mice.

Gastric inhibitory polypeptide (GIP) is an incretin and directly promotes fat accumulation in adipocytes. Inhibition of GIP signaling prevents onset of obesity and increases fat oxidation in peripheral tissues under high-fat diet (HFD), but the mechanism is unknown. In the present study, we investigated the effects of inhibition of GIP signaling on adiponectin levels after 3 weeks of HFD by comparing wild-type (WT) mice and GIP receptor-deficient (Gipr(-/-)) mice. In HFD-fed Gipr(-/-) mice, fat oxidation was significantly increased and adiponectin mRNA levels in white adipose tissue and plasma adiponectin levels were significantly increased compared to those in HFD-fed WT mice. In addition, the PPARalpha mRNA level was increased and the ACC mRNA level was decreased in skeletal muscle of HFD-fed Gipr(-/-) mice compared with those in HFD-fed WT mice. These results indicate that inhibition of GIP signaling increases adiponectin levels, resulting in increased fat oxidation in peripheral tissues under HFD.

GLP-1 receptor signaling protects pancreatic beta cells in intraportal islet transplant by inhibiting apoptosis

To clarify the cytoprotective effect of glucagon-like peptide-1 receptor (GLP-1R) signaling in conditions of glucose toxicity in vivo, we performed murine isogenic islet transplantation with and without exendin-4 treatment. When a suboptimal number of islets (150) were transplanted into streptozotocin-induced diabetic mice, exendin-4 treatment contributed to the restoration of normoglycemia. When 50 islets expressing enhanced green fluorescent protein (EGFP) were transplanted, exendin-4 treatment reversed loss of both the number and mass of islet grafts one and 3 days after transplantation. TUNEL staining revealed that exendin-4 treatment reduced the number of apoptotic beta cells during the early posttransplant phase, indicating that GLP-1R signaling exerts its cytoprotective effect on pancreatic beta cells by inhibiting their apoptosis. This beneficial effect might be used both to ameliorate type 2 diabetes and to improve engraftment rates in clinical islet transplantation.

Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice

Aims/hypothesisMetformin, the major target of which is liver, is commonly used to treat type 2 diabetes. Although metformin activates AMP-activated protein kinase (AMPK) in hepatocytes, the mechanism of activation is still not well known. To investigate AMPK activation by metformin in liver, we examined the role of reactive nitrogen species (RNS) in suppression of hepatic gluconeogenesis.MethodsTo determine RNS, we performed fluorescence examination and immunocytochemical staining in mouse hepatocytes. Since metformin is a mild mitochondrial complex I inhibitor, we compared its effects on suppression of gluconeogenesis, AMPK activation and generation of the RNS peroxynitrite (ONOO−) with those of rotenone, a representative complex I inhibitor. To determine whether endogenous nitric oxide production is required for ONOO− generation and metformin action, we used mice lacking endothelial nitric oxide synthase (eNOS).ResultsMetformin and rotenone significantly decreased gluconeogenesis and increased phosphorylation of AMPK in wild-type mouse hepatocytes. However, unlike rotenone, metformin did not increase the AMP/ATP ratio. It did, however, increase ONOO− generation, whereas rotenone did not. Exposure of eNOS-deficient hepatocytes to metformin did not suppress gluconeogenesis, activate AMPK or increase ONOO− generation. Furthermore, metformin lowered fasting blood glucose levels in wild-type diabetic mice, but not in eNOS-deficient diabetic mice.Conclusions/interpretationActivation of AMPK by metformin is dependent on ONOO−. For metformin action in liver, intra-hepatocellular eNOS is required.

FGF-21 enhances islet engraftment in mouse syngeneic islet transplantation model.

To clarify the effect of fibroblast growth factor-21 (FGF-21) on islet transplantation, a suboptimal number of islets were transplanted into streptozotocin (STZ)-induced diabetic mice with or without FGF-21 treatment. Three-day treatment with FGF-21 contributed to restoration of normoglycemia by suppressing islet graft loss. The FGF-21-treated mice showed lower glycemic levels despite similar insulin content in the graft than that in untreated mice on day 3, indicating that FGF-21 not only has a cytoprotective effect but also decreases β-cell load by increasing insulin sensitivity. These results suggest that FGF-21 may be useful as a treatment to improve islet engraftment rates in clinical islet transplantation.

Effect of high dietary fat on insulin secretion in genetically diabetic Goto-Kakizaki rats

Introduction and Aim To clarify the effects of a high fat-diet on insulin secretion from genetically diabetic beta cells, Goto-Kakizaki rats and Wistar rats were subjected to oral glucose tolerance test (OGTT) after 12-week high-fat feeding. Methodology We compared Wistar and Goto-Kakizaki (GK) rats fed a high-fat diet (45% fat content) for 12 weeks, measuring insulin secretion and insulin release. Results Insulin secretion during oral glucose tolerance test (OGTT) was enhanced in high-fat diet–fed Wistar rats (WF) with normal glucose tolerance. Insulin secretion in high-fat diet–fed GK rats (GF) during OGTT also was enhanced together with deteriorated glucose tolerance. Basal insulin release from the isolated perfused pancreas at 3.3 m M glucose in WF was comparable to that in normal chow-fed Wistar rats (WN), but basal insulin release in GF was remarkably higher than in normal chow–fed GK rats (GN). Stimulated insulin release induced by 16.7 m M glucose was remarkably increased in WF compared with WN. Total insulin release at 16.7 m M glucose in both GK rat groups was similar and minimal. Conclusion These results indicate that normal pancreatic &bgr;-cells have the ability to secrete sufficient insulin to compensate for the insulin resistance induced by a high-fat diet. In contrast, glucose metabolism in diabetic rats after high-fat diet deteriorated partly because of insufficient insulin secretion caused by genetic defects and lipotoxicity due to chronically high FFA levels.

The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice.

Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic β cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucose absorption in vivo was measured by single-pass perfusion method. Incorporation of [(14)C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [(14)C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin-mediated pathway rather than through a GLP-1-mediated pathway.

Exendin-4 protects pancreatic beta cells from the cytotoxic effect of rapamycin by inhibiting JNK and p38 phosphorylation.

It has been reported that the immunosuppressant rapamycin decreases the viability of pancreatic beta cells. In contrast, exendin-4, an analogue of glucagon-like peptide-1, has been found to inhibit beta cell death and to increase beta cell mass. We investigated the effects of exendin-4 on the cytotoxic effect of rapamycin in beta cells. Incubation with 10 nM rapamycin induced cell death in 12 h in murine beta cell line MIN6 cells and Wistar rat islets, but not when coincubated with 10 nM exendin-4. Rapamycin was found to increase phosphorylation of c-Jun amino-terminal kinase (JNK) and p38 in 30 minutes in MIN6 cells and Wistar rat islets while exendin-4 decreased their phosphorylation. Akt and extracellular signal-regulated kinase (ERK) were not involved in the cytoprotective effect of exendin-4. These results indicate that exendin-4 may exert its protective effect against rapamycin-induced cell death in pancreatic beta cells by inhibiting JNK and p38 signaling.