Akihiro Hamasaki

Overexpression of SIRT5 confirms its involvement in deacetylation and activation of carbamoyl phosphate synthetase 1

SIR2 protein, an NAD-dependent deacetylase, is localized to nucleus and is involved in life span extension by calorie restriction in yeast. In mammals, among the seven SIR2 homologues (SIRT1-7), SIRT3, 4, and 5 are localized to mitochondria. As SIRT5 mRNA levels in liver are increased by fasting, the physiological role of SIRT5 was investigated in liver of SIRT5-overexpressing transgenic (SIRT5 Tg) mice. We identified carbamoyl phosphate synthetase 1 (CPS1), a key enzyme of the urea cycle that catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate, as a target of SIRT5 by two-dimensional electrophoresis comparing mitochondrial proteins in livers of SIRT5 Tg and wild-type mice. CPS1 protein was more deacetylated and activated in liver of SIRT5 Tg mice than in wild-type. In addition, urea production was upregulated in hepatocytes of SIRT5 Tg mice. These results agree with those of a previous study using SIRT5 knockout (KO) mice. Because ammonia generated during fasting is toxic, SIRT5 protein might play a protective role by converting ammonia to non-toxic urea through deacetylation and activation of CPS1.

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.

Analysis of factors influencing pancreatic β-cell function in Japanese patients with type 2 diabetes: Association with body mass index and duration of diabetic exposure

AIMS To elucidate the clinical factors affecting beta-cell function, serum C-peptide immunoreactivity (CPR) levels of patients with type 2 diabetes were analyzed. METHODS Seven hundred Japanese patients with type 2 diabetes were enrolled. beta-Cell function was evaluated by fasting CPR (FCPR), 6 min after intravenous injection of 1mg glucagon (CPR-6 min), and the increment of CPR (DeltaCPR). Simple regression analysis between FCPR, CPR-6 min, and DeltaCPR and measures of variables and stepwise multiple regression analysis were carried out. RESULTS Years from diagnosis and BMI were the major independent variables predicting beta-cell function. Years from diagnosis was negatively correlated with CPR-6 min (P<0.0001, r=-0.271), and decrease in CPR-6 min was 0.050 ng/(ml year). BMI was positively correlated with CPR-6 min (P<0.0001, r=0.369). When subjects were divided according to BMI, the decrease in CPR-6 min per year in the high-BMI group (0.068 ng/(ml year)) was greater than that in the low-BMI group (0.035 ng/(ml year)). CONCLUSION A linear decline in endogenous insulin secretion over more than several decades of diabetes was confirmed by this cross-sectional study. The duration of diabetes exposure and BMI are thus major factors in beta-cell function in Japanese patients with type 2 diabetes.

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.

Utility of indices using C‐peptide levels for indication of insulin therapy to achieve good glycemic control in Japanese patients with type 2 diabetes

Aims/Introduction:  Type 2 diabetes is progressive in that therapy must be altered over time, which is partly as a result of the progressive loss of pancreatic β‐cell function. To elucidate the relationship between residual endogenous insulin secretion and the necessity of insulin therapy to achieve good glycemic control, indices using serum C‐peptide immunoreactivity (CPR) were analyzed in patients with type 2 diabetes.

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.

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.

Plasma gastric inhibitory polypeptide and glucagon‐like peptide‐1 levels after glucose loading are associated with different factors in Japanese subjects

Aims/Introduction:  Gastric inhibitory polypeptide (GIP) and glucagon‐like peptide‐1 (GLP‐1) are major incretins that potentiate insulin secretion from pancreatic β‐cells. The factors responsible for incretin secretion have been reported in Caucasian subjects, but have not been thoroughly evaluated in Japanese subjects. We evaluated the factors associated with incretin secretion during oral glucose tolerance test (OGTT) in Japanese subjects with normal glucose tolerance (NGT).