Insulin resistance is a major hallmark in the development of type 2 diabetes, which is characterized by an impaired ability of insulin to inhibit glucose output from the liver and to promote glucose uptake in muscle. The nuclear hormone receptor coactivator PGC-1 (peroxisome proliferator-activated (PPAR)-gamma coactivator-1) has been implicated in the onset of type 2 diabetes. Hepatic PGC-1 expression is elevated in mouse models of this disease, where it promotes constitutive activation of gluconeogenesis and fatty acid oxidation through its association with the nuclear hormone receptors HNF-4 and PPAR-alpha, respectively. Here we show that PGC-1-deficient mice, generated by adenoviral delivery of PGC-1 RNA interference (RNAi) to the liver, experience fasting hypoglycemia. Hepatic insulin sensitivity was enhanced in PGC-1-deficient mice, reflecting in part the reduced expression of the mammalian tribbles homolog TRB-3, a fasting-inducible inhibitor of the serine-threonine kinase Akt/PKB (ref. 6). We show here that, in the liver, TRB-3 is a target for PPAR-alpha. Knockdown of hepatic TRB-3 expression improved glucose tolerance, whereas hepatic overexpression of TRB-3 reversed the insulin-sensitive phenotype of PGC-1-deficient mice. These results indicate a link between nuclear hormone receptor and insulin signaling pathways, and suggest a potential role for TRB-3 inhibitors in the treatment of type 2 diabetes.

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Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKK␤, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-␣, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-␣ signaling with neutralizing antibodies to TNF-␣ or its receptors or with a dominant negative TNF-␣ peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-␣ signaling, whereas the FFA-induced increase in TNF-␣ secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-␣-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-␣ is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.

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We investigated the chronic in vivo effect of resistin on insulin sensitivity and glucose metabolism by overexpressing resistin protein in male Wistar rats using intravenous administration of an adenovirus encoding mouse resistin. After 7 days of elevated resistin levels at a supraphysiological concentration, the animals displayed glucose intolerance and hyperinsulinemia during glucose tolerance tests, and insulin tolerance tests demonstrated an impaired glucose-lowering effect of insulin. The glucose clamp studies were performed at submaximal (4 mU/kg/min) and maximal (25 mU/kg/min) insulin infusion rates and demonstrated the presence of insulin resistance induced by elevated resistin levels. Indeed, the insulin-stimulated glucose infusion rate was decreased by 12-31%; suppression of hepatic glucose output was attenuated by 28-55%; and insulin suppression of circulating FFA levels was inhibited by 7%. Insulin receptor substrate-1 and -2 phosphorylation and Akt activation were impaired in muscle and adipose tissue. Interestingly, activation of AMP-activated protein kinase in skeletal muscle, liver, and adipose tissue was also significantly downregulated. Together, these results indicate that chronic "hyper-resistinemia" leads to whole-body insulin resistance involving impaired insulin signaling in skeletal muscle, liver, and adipose tissue, resulting in glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. Thus elevated resistin levels in normal rats fed a regular chow diet produce many of the features of human syndrome X.

Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [␥-32 P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-(PKC-). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulininduced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.Stimulation of glucose transport is a major action of insulin and occurs in the insulin target tissues, muscle and fat, by a process involving translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane (34). GLUT4 proteins are contained in intracellular vesicles which are predominantly localized to a perinuclear compartment in the basal state. After insulin stimulation, the GLUT4-containing vesicles are translocated to the plasma membrane (31). Numerous studies have examined the insulin signaling mechanisms leading to translocation of GLUT4 vesicles to the plasma membrane, and it is understood that this process involves multiple steps (34). These steps include release of vesicles from storage pools, transport to the plasma membrane, proper docking, and fusion with the membrane, and these events are regulated by multiple insulin signaling components (5).It has been shown that different Rab proteins are present in trafficking vesicles (26,43) and that GLUT4 vesicles can contain a number of associated molecules, such as Rab4, Rab5, Rab11, insulin-responsive amino peptidase, and transferrin receptors (27). In previous reports (6,35,...

Abstract-Nitric oxide (NO) derived from endothelial cells is profoundly related to the maintenance of physiological vascular tone. Impairment of endothelial NO generation brought about by gene polymorphism is considered the major deterioration factor for progressive renal disease, including diabetic nephropathy. The present study aimed to elucidate the Glu298Asp polymorphism of endothelial NO synthase (eNOS) in patients with end-stage renal disease (ESRD) and its role as a predisposing factor for cardiovascular complications. Glu298Asp in exon 7 of the eNOS gene was determined by polymerase chain reaction, followed by restriction fragment length polymorphism analysis, in ESRD patients (nϭ185) and compared with that of unrelated healthy individuals (nϭ304). Key Words: polymorphism Ⅲ polymerase chain reaction Ⅲ nitric oxide synthase Ⅲ diabetes mellitus T he endothelial isoform of nitric oxide (NO) synthase (eNOS, NOSIII) is a constitutively expressed 135 kDa protein predominantly associated with the particulated specific structures in the plasmalemmal membrane, caveolae, of vascular endothelial cells. 1,2 NO is produced from L-arginine and diffuses to vascular smooth muscle cells, where it increases the concentration of cGMP by stimulating soluble guanylate cyclase, leading to vascular relaxation. It also inhibits platelet and/or leukocyte adhesion to vascular endothelium. Therefore, the impairment of eNOS expression has been considered a primary factor for diseases such as hypertension, coronary artery disease, thromboembolic diseases, and atherosclerosis. Indeed, knocking out the gene encoding eNOS in mice resulted in significant hypertension, and aortic rings from these animals studied ex vivo displayed no relaxation in response to acetylcholine. 3 Patients with endstage renal disease (ESRD) derived from diabetes mellitus (DM) nephropathy have a higher prevalence of cardiovascular complications than those with non-DM ESRD, and this limits their 5-year survival rate to less than 50%. Therefore, polymorphism in eNOS is considered one of the major predisposing factors for endothelial dysfunction.The gene polymorphisms of eNOS have been detected at 4b/4a variable number of tandem repeats in intron 4, Glu298Asp in exon 7, CA repeat in intron 13, A27 to C (A to C nucleotide conversion) in intron 18, and G10 to T in intron 23. A part of candidate variations (4b/4a tandem repeats in intron 4) has been investigated in glomerulonephritis and/or ESRD patients, but no conclusive results have been obtained so far. Recently, the G/T polymorphism in exon 7 coding for Glu298Asp was detected and reported to have a remarkable association with coronary spasm, acute myocardial infarction, 4 and hypertension. 5 To date, this is the only known eNOS polymorphism associated with an altered protein sequence, though recent expression studies have demonstrated no functional difference between 298Glu and 298Asp despite the accumulating clinical evidence. 6,7

Background The number of patients suffering from diabetic nephropathy resulting in end-stage kidney disease is increasing worldwide. In clinical settings, there are limited data regarding the impact of the urinary albumin-to-creatinine ratio (UACR) and reduced estimated glomerular filtration rate (eGFR) on renal and cardiovascular outcomes and all-cause mortality. Methods We performed a historical cohort study of 4328 Japanese participants with type 2 diabetes from 10 centers. Risks for renal events (requirement for dialysis or transplantation, or half reduction in eGFR), cardiovascular events (cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke), and all-cause mortality were assessed according to UACR and eGFR levels. Results During follow-up (median 7.0 years, interquartile range 3.0-8.0 years), 419 renal events, 605 cardiovascular events and 236 deaths occurred. The UACR levels increased the risk and the adjusted hazard ratios for these three events. In addition to the effects of UACR levels, eGFR stages significantly increased the adjusted hazard ratios for renal events and all-cause mortality, especially in 123Clin Exp Nephrol DOI 10.1007/s10157-013-0879-4 patients with macroalbuminuria. Diabetic nephropathy score, based on the prognostic factors, well predicted incidence rates per 1000 patient/year for each event.Conclusions Increased UACR levels were closely related to the increase in risks for renal, cardiovascular events and all-cause mortality in Japanese patients with type 2 diabetes, whereas the association between high levels of UACR and reduced eGFR was a strong predictor for renal events.