Manabu Ishiki

Overexpression of SH2-Containing Inositol Phosphatase 2 Results in Negative Regulation of Insulin-Induced Metabolic Actions in 3T3-L1 Adipocytes via Its 5′-Phosphatase Catalytic Activity

ABSTRACT Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5′-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5′-phosphatase-defective SHIP2 (ΔIP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor β subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or ΔIP-SHIP2. Because WT-SHIP2 possesses the 5′-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of ΔIP-SHIP2, indicating that ΔIP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase Cλ in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase Cλ, whereas these activations were increased by expression of ΔIP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of ΔIP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3β and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of ΔIP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5′-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

Treatment with SRT1720, a SIRT1 activator, ameliorates fatty liver with reduced expression of lipogenic enzymes in MSG mice

Nonalcoholic fatty liver disease (NAFLD) is an abnormal liver metabolism often observed with insulin resistance and metabolic syndrome. Calorie restriction is a useful treatment for NAFLD and reportedly prolongs the life spans of several species in which sirtuin plays an important role. In this study, we examined whether the activation of SIRT1, a mammalian ortholog of sirtuin, may ameliorate the development of NAFLD. Monosodium glutamate (MSG) mice, which exhibited obesity and insulin resistance, were treated with SRT1720, a specific SIRT1 activator from the age of 6-16 wk. Sixteen-week-old MSG mice exhibited increased liver triglyceride content and elevated levels of aminotransferase. SRT1720 treatment significantly reduced these levels without affecting body weight or food intake. These results suggested that the administration of SRT1720 ameliorated the development of NAFLD in MSG mice. The expressions of lipogenic genes, such as sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, and the serum lipid profiles, including free fatty acids, were elevated in MSG mice and were reduced by SRT1720 treatment. SRT1720 treatment also reduced the expressions of lipogenic genes in cultured HepG2 cells. Furthermore, SRT1720 treatment decreased the expressions of marker genes for oxidative stress and inflammatory cytokines in the liver of MSG mice. Taken together, SRT1720 treatment may reduce liver lipid accumulation, at least in part, by directly reducing the expressions of lipogenic genes. The reduction of oxidative stress and inflammation may also be involved in the amelioration of NAFLD.

Association of the polymorphisms in the 5′-untranslated region of PTEN gene with type 2 diabetes in a Japanese population

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P3 to PI(4,5)P2. Since the PI3‐kinase product, PI(3,4,5)P3, is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non‐insulin‐dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non‐diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position −9 (−9C→G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild‐type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin‐induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild‐type PTEN gene. These findings suggest that the change of C to G at position −9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3‐kinase product.

Impact of divergent effects of astaxanthin on insulin signaling in L6 cells.

Because oxidative stress promotes insulin resistance in obesity and type 2 diabetes, it is crucial to find effective antioxidant for the purpose of decreasing this threat. In this study, we explored the effect of astaxanthin, a carotenoid antioxidant, on insulin signaling and investigated whether astaxanthin improves cytokine- and free fatty acid-induced insulin resistance in vitro. We examined the effect of astaxanthin on insulin-stimulated glucose transporter 4 (GLUT4) translocation, glucose uptake, and insulin signaling in cultured rat L6 muscle cells using plasma membrane lawn assay, 2-deoxyglucose uptake, and Western blot analysis. Next, we examined the effect of astaxanthin on TNFα- and palmitate-induced insulin resistance. The amount of reactive oxygen species generated by TNFα or palmitate with or without astaxanthin was evaluated by dichlorofluorescein staining. We also compared the effect of astaxanthin on insulin signaling with that of other antioxidants, α-lipoic acid and α-tocopherol. We observed astaxanthin enhanced insulin-stimulated GLUT4 translocation and glucose uptake, which was associated with an increase in insulin receptor substrate-1 tyrosine and Akt phosphorylation and a decrease in c-Jun N-terminal kinase (JNK) and insulin receptor substrate-1 serine 307 phosphorylation. Furthermore, astaxanthin restored TNFα- and palmitate-induced decreases in insulin-stimulated GLUT4 translocation or glucose uptake with a concomitant decrease in reactive oxygen species generation. α-Lipoic acid enhanced Akt phosphorylation and decreased ERK and JNK phosphorylation, whereas α-tocopherol enhanced ERK and JNK phosphorylation but had little effect on Akt phosphorylation. Collectively these findings indicate astaxanthin is a very effective antioxidant for ameliorating insulin resistance by protecting cells from oxidative stress generated by various stimuli including TNFα and palmitate.

Inhibitory effect of IL-8 on insulin action in human adipocytes via MAP kinase pathway

BackgroundVarious cytokines and other compounds are produced in human adipose tissue and might have functions in the adipose tissue. They might be involved in complications associated with obesity and diabetes. Recently, interleukin-8 (IL-8) has been shown to be produced and released from human adipose tissue and/or adipocytes, suggesting IL-8 involvement in some obesity-related health complications. Therefore, we found it of interest to investigate whether IL-8 is involved in the insulin action in human adipocytes.MethodsThe IL-8 levels in the medium were measured using ELISA. The IL-8 mRNA expression was analyzed using Northern blot analysis. The phosphorylation of Akt was analyzed using Western blot analysis. Furthermore, we examined the effect of IL-8 on the phosphorylation of Akt induced by insulin.ResultsThe level of IL-8 in the medium and the IL-8 mRNA expression after stimulation with either TNF-α, IL-1β, or CRP was significantly enhanced in human adipocytes. It is particularly interesting that IL-8 per se also enhanced IL-8 mRNA expression. The IL-8 induced-IL-8 mRNA expression was inhibited by PD98059 (a MEK inhibitor) or SB203580 (a p38 MAPK inhibitor). The IL-8 inhibited insulin-induced Akt phosphorylation. The inhibitory effect of IL-8 was eliminated by either PD 98059 or SB203580.ConclusionThese data suggest that IL-8 is a main adipocytokine producing insulin resistance via the inhibition of insulin-induced Akt phosphorylation in adipocytes. The attenuation of IL-8 action might be a target for prevention of diabetes and its complications.

Effect of glimepiride (HOE 490) on insulin receptors of skeletal muscles from genetically diabetic KK-Ay mouse

A new sulfonylurea, glimepiride (HOE 490), has been developed for the glycemic control in non-insulin-dependent diabetes mellitus. We examined the effect of glimepiride on glucose and insulin levels in KK-Ay mice, an animal model of non-insulin-dependent diabetes mellitus, which is characterized by hyperglycemia and hyperinsulinemia. Administration of glimepiride (0.5 mg/kg/day) for 8 weeks to KK-Ay mice resulted in decrease in glucose (297 +/- 36 to 250 +/- 51 mg/dl) and insulin (76 +/- 14 to 41 +/- 14 microU/ml) levels. To clarify the mechanism of the agent, we examined the effect of this new drug on insulin receptors in the skeletal muscles. There was no difference in insulin binding to the receptors from both glimepiride-treated and -untreated KK-Ay mice muscles. The insulin-stimulated autophosphorylation of insulin receptors from KK-Ay mice was decreased compared to that from normal mice (5 +/- 1 vs. 39 +/- 13% over basal). Glimepiride did not ameliorate impaired insulin-stimulated insulin receptor autophosphorylation. To determine the effect of glimepiride on post-insulin receptor signaling pathway, we measured 2-[3H]glycerol incorporation into diacylglycerol in the cultured rat fibroblast cell line overexpressing human insulin receptors. Glimepiride (100 microM) as well as insulin (10 nM) significantly stimulated diacylglycerol production. These results suggest that glimepiride has a potent extrapancreatic effect on glucose metabolism and may directly stimulate glucose transport activity through phospholipid signaling pathway, but not through insulin receptor kinase signaling pathway.

FTO Gene Polymorphism Is Associated with Type 2 Diabetes through Its Effect on Increasing the Maximum BMI in Japanese Men

Aim Several studies have demonstrated that polymorphisms within the fat-mass and obesity-associated gene (FTO) are associated with type 2 diabetes (T2D). However, whether the effects of the FTO locus on T2D susceptibility are independent of fat-mass increases remains controversial. To investigate this issue, we examined the association of FTO variants with T2D and various aspects of BMI history during adult life in a Japanese population. Methods We genotyped SNPs within FTO (rs1121980 and rs1558902) in 760 Japanese patients with T2D who had reached a lifetime maximum BMI (BMImax) before or at the time of diagnosis and 693 control individuals with information regarding their BMImax. Results The BMImax showed the strongest association with T2D risk among the BMIs evaluated in this study. In the sex-combined analysis, FTO SNPs were not associated with any of the BMI variables or with T2D, but in sex-stratified analyses, both SNPs were significantly associated with the BMImax and rs1558902 was associated with T2D in men. The association of the SNPs with T2D remained significant after adjustments for the current BMI and age, whereas the T2D association of the SNP was no longer significant after adjustments for BMImax and age. Conclusions These results suggest that the effects of FTO polymorphisms on T2D susceptibility in Japanese men are mediated through their effect on increasing the BMImax before or at the time of diagnosis.

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts☆

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.