Norio Harada

Beneficial Effects of Exendin-4 on Experimental Polyneuropathy in Diabetic Mice

OBJECTIVE The therapeutic potential of exendin-4, an agonist of the glucagon-like peptide-1 receptor (GLP-1R), on diabetic polyneuropathy (DPN) in streptozotocin (STZ)-induced diabetic mice was investigated. RESEARCH DESIGN AND METHODS The presence of the GLP-1R in lumbar dorsal root ganglion (DRG) was evaluated by immunohistochemical analyses. DRG neurons were dissected from C57BL6/J mice and cultured with or without Schwann cell–conditioned media in the presence or absence of GLP-1 (7–37) or exendin-4. Then neurite outgrowth was determined. In animal-model experiments, mice were made diabetic by STZ administration, and after 12 weeks of diabetes, exendin-4 (10 nmol/kg) was intraperitoneally administered once daily for 4 weeks. Peripheral nerve function was determined by the current perception threshold and motor and sensory nerve conduction velocity (MNCV and SNCV, respectively). Sciatic nerve blood flow (SNBF) and intraepidermal nerve fiber densities (IENFDs) also were evaluated. RESULTS The expression of the GLP-1R in DRG neurons was confirmed. GLP-1 (7–37) and exendin-4 significantly promoted neurite outgrowth of DRG neurons. Both GLP-1R agonists accelerated the impaired neurite outgrowth of DRG neurons cultured with Schwann cell–conditioned media that mimicked the diabetic condition. At the doses used, exendin-4 had no effect on blood glucose or HbA1c levels. Hypoalgesia and delayed MNCV and SNCV in diabetic mice were improved by exendin-4 without affecting the reduced SNBF. The decreased IENFDs in sole skins of diabetic mice were ameliorated by exendin-4. CONCLUSIONS Our findings indicate that exendin-4 ameliorates the severity of DPN, which may be achieved by its direct actions on DRG neurons and their axons.

Chronic Reduction of GIP Secretion Alleviates Obesity and Insulin Resistance Under High-Fat Diet Conditions.

Gastric inhibitory polypeptide (GIP) exhibits potent insulinotropic effects on β-cells and anabolic effects on bone formation and fat accumulation. We explored the impact of reduced GIP levels in vivo on glucose homeostasis, bone formation, and fat accumulation in a novel GIP-GFP knock-in (KI) mouse. We generated GIP-GFP KI mice with a truncated prepro-GIP gene. The phenotype was assessed in heterozygous and homozygous states in mice on a control fat diet and a high-fat diet (HFD) in vivo and in vitro. Heterozygous GIP-GFP KI mice (GIP-reduced mice [GIPgfp/+]) exhibited reduced GIP secretion; in the homozygous state (GIP-lacking mice [GIPgfp/gfp]), GIP secretion was undetectable. When fed standard chow, GIPgfp/+ and GIPgfp/gfp mice showed mild glucose intolerance with decreased insulin levels; bone volume was decreased in GIPgfp/gfp mice and preserved in GIPgfp/+ mice. Under an HFD, glucose levels during an oral glucose tolerance test were similar in wild-type, GIPgfp/+, and GIPgfp/gfp mice, while insulin secretion remained lower. GIPgfp/+ and GIPgfp/gfp mice showed reduced obesity and reduced insulin resistance, accompanied by higher fat oxidation and energy expenditure. GIP-reduced mice demonstrate that partial reduction of GIP does not extensively alter glucose tolerance, but it alleviates obesity and lessens the degree of insulin resistance under HFD conditions, suggesting a potential therapeutic value.

Pancreatic and Extrapancreatic Effects of Gastric Inhibitory Polypeptide

The hormonal factor(s) implicated as transmitters of signals from the gut to pancreatic β-cells is referred to as incretin, and gastric inhibitory polypeptide (GIP) is identified as one of the incretins. GIP is a gastrointestinal peptide hormone of 42 amino acids that is released from duodenal endocrine K-cells after absorption of glucose or fat and exerts its effects by binding to its specific receptor, the GIP receptor. By generating and characterizing mice with a targeted mutation of the GIP receptor gene, we have shown that GIP has not only an insulinotropic role, but also physiological roles on fat accumulation into adipose tissues and calcium accumulation into bone. We here propose a new acronym, GIP, for gut-derived nutrient-intake polypeptide.

Free Fatty Acid Receptor GPR120 Is Highly Expressed in Enteroendocrine K Cells of the Upper Small Intestine and Has a Critical Role in GIP Secretion After Fat Ingestion

Gastric inhibitory polypeptide (GIP) is an incretin secreted from enteroendocrine K cells in response to meal ingestion. Recently free fatty acid receptor G protein-coupled receptor (GPR) 120 was identified as a lipid sensor involved in glucagon-like peptide-1 secretion. However, Gpr 120 gene expression and its role in K cells remain unclear, partly due to difficulties in separation of K cells from other intestinal epithelial cells. In this study, we purified K cells using GIP-green fluorescent protein (GFP) knock-in mice, in which K cells can be visualized by GFP fluorescence. GFP-positive cells (K cells) were observed in the small intestine but not in the stomach and colon. K cell number and GIP content in K cells were significantly higher in the upper small intestine than those in the lower small intestine. We also examined the expression levels of several free fatty acid receptors in K cells. Among free fatty acid receptors, GPR120 was highly expressed in the K cells of the upper small intestine compared with the lower small intestine. To clarify the role of GPR120 on K cells in vivo, we used GPR120-deficient mice (GPR120(-/-)). GPR120(-/-) exhibited significantly lower GIP secretion (75% reduction, P < .01) after lard oil ingestion compared with that in wild-type mice. Consistently, pharmacological inhibition of GPR120 with grifolic acid methyl ether in wild-type mice significantly attenuated lard oil-induced GIP secretion. In conclusion, GPR120 is expressed abundantly in K cells of the upper small intestine and plays a critical role in lipid-induced GIP secretion.

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.

Effect of corosolic acid on gluconeogenesis in rat liver

Corosolic acid (CRA), an active component of Banaba leaves (Lagerstroemia speciosa L.), decreases blood glucose in diabetic animals and humans. In this study, we investigated the mechanism of action of CRA on gluconeogenesis in rat liver. CRA (20-100 microM) dose-dependently decreased gluconeogenesis in perfused liver and in isolated hepatocytes. Fructose-2,6-bisphosphate (F-2,6-BP), a gluconeogenic intermediate, plays a critical role in hepatic glucose output by regulating gluconeogenesis and glycolysis in the liver. CRA increased the production of F-2,6-BP along with a decrease in intracellular levels of cAMP both in the presence and in the absence of forskolin in isolated hepatocytes. While a cAMP-dependent protein kinase (PKA) inhibitor inhibited hepatic gluconeogenesis, the drug did not intensify the inhibitory effect of CRA on hepatic gluconeogenesis in isolated hepatocytes. These results indicate that CRA inhibits gluconeogenesis by increasing the production of F-2,6-BP by lowering the cAMP level and inhibiting PKA activity in isolated hepatocytes. Furthermore, CRA increased glucokinase activity in isolated hepatocytes without affecting glucose-6-phosphatase activity, suggesting the promotion of glycolysis. These effects on hepatic glucose metabolism may underlie the various anti-diabetic actions of CRA.

Early phase glucagon and insulin secretory abnormalities, but not incretin secretion, are similarly responsible for hyperglycemia after ingestion of nutrients

AIMS Hypersecretion of glucagon and reduced insulin secretion both contribute to hyperglycemia in type 2 diabetes (T2DM). However, the relative contributions of impaired glucagon and insulin secretions in glucose excursions at the various stages of T2DM development remain to be determined. METHODS The responses of glucagon and insulin as well as those of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) were examined before and after ingestion of glucose or mixed meal in Japanese subjects with normal or impaired glucose tolerance (NGT and IGT) and in non-obese, untreated T2DM of short duration. RESULTS In OGTT, T2DM showed a rise in glucagon at 0-30 min, unlike NGT and IGT, along with reduced insulin. In MTT, all three groups showed a rise in glucagon at 0-30 min, with that in T2DM being highest, while T2DM showed a significant reduction in insulin. Linear regression analyses revealed that glucose area under the curve (AUC)0-120 min was associated with glucagon-AUC0-30 min and insulin-AUC0-30 min in both OGTT and MTT. Total and biologically intact GIP and GLP-1 levels were similar among the three groups. CONCLUSIONS Disordered early phase insulin and glucagon secretions but not incretin secretion are involved in hyperglycemia after ingestion of nutrients in T2DM of even a short duration.

Effects of glucose and meal ingestion on incretin secretion in Japanese subjects with normal glucose tolerance

Aims/Introduction:  Gastric inhibitory polypeptide (GIP) and glucagon‐like peptide‐1 (GLP‐1) are the major incretins; their secretion after various nutrient loads are well‐evaluated in Caucasians. However, little is known of the relationship between incretin secretion and differing nutritional loading in Japanese subjects. In the present study, we evaluated GIP and GLP‐1 secretion in Japanese subjects with normal glucose tolerance (NGT) after glucose loading (75 g glucose and 17 g glucose) and meal ingestion.