Inducible nitric oxide synthase (iNOS) is induced by inflammatory cytokines in skeletal muscle and fat. It has been proposed that chronic iNOS induction may cause muscle insulin resistance. Here we show that iNOS expression is increased in muscle and fat of genetic and dietary models of obesity. Moreover, mice in which the gene encoding iNOS was disrupted (Nos2-/- mice) are protected from high-fat-induced insulin resistance. Whereas both wild-type and Nos2-/- mice developed obesity on the high-fat diet, obese Nos2-/- mice exhibited improved glucose tolerance, normal insulin sensitivity in vivo and normal insulin-stimulated glucose uptake in muscles. iNOS induction in obese wild-type mice was associated with impairments in phosphatidylinositol 3-kinase and Akt activation by insulin in muscle. These defects were fully prevented in obese Nos2-/- mice. These findings provide genetic evidence that iNOS is involved in the development of muscle insulin resistance in diet-induced obesity.

The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase–deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.

The purpose of this study was to investigate whether in vivo nitric oxide synthase (NOS) inhibition influences insulin-mediated glucose disposal in rat peripheral tissues. The NOS inhibitor N G-nitro-l-arginine methyl ester (l-NAME) or saline was infused constantly during a hyperinsulinemic-euglycemic clamp in normal rats. Glucose utilization rates of insulin-sensitive tissues (individual muscles, heart, and adipose tissues) were simultaneously determined using tracer infusion of 2-deoxy-d-[3H]glucose (2-[3H]DG). NOS blockade with l-NAME resulted in significant ( P < 0.05) reduction in both whole body glucose disposal (−16%, P < 0.01) and plasma 2-[3H]DG disappearance rate (−30%, P < 0.05) during hyperinsulinemic-euglycemic clamp.l-NAME significantly decreased insulin-stimulated glucose uptake in heart (−62%, P = 0.01), soleus (−42%, P = 0.05), red (−53%, P < 0.001) and white (−62%, P < 0.001) gastrocnemius, tibialis (−57%, P < 0.01), and quadriceps (−33%, P < 0.05) muscles. The NOS inhibitor also decreased insulin action in brown interscapular (−47%, P < 0.01), retroperitoneal (−52%, P = 0.07), and gonadal (−66%, P = 0.06) adipose tissues. In contrast to in vivo NOS blockade,l-NAME failed to affect basal or insulin-stimulated 2-[3H]DG transport in isolated soleus or extensor digitorum longus muscles in vitro. These results support the hypothesis that the action of insulin to augment glucose uptake by skeletal muscles and other peripheral insulin-sensitive tissues in vivo is NO dependent.

The stress-activated p38 mitogen-activated protein kinase (MAPK) was recently shown to be activated by insulin in muscle and adipose cells in culture. Here, we explore whether such stimulation is observed in rat skeletal muscle and whether muscle contraction can also affect the enzyme. Insulin injection (2 U over 3.5 min) resulted in increases in p38 MAPK phosphorylation measured in soleus (3.2-fold) and quadriceps (2.2-fold) muscles. Increased phosphorylation (3.5-fold) of an endogenous substrate of p38 MAPK, cAMP response element binder (CREB), was also observed. After in vivo insulin treatment, p38 MAPK␣ and p38 MAPK␤ isoforms were found to be activated (2.1-and 2.4-fold, respectively), using an in vitro kinase assay, in immunoprecipitates from quadriceps muscle extracts. In vitro insulin treatment (1 nmol/l over 4 min) and electrically-induced contraction of isolated extensor digitorum longus (EDL) muscle also doubled the kinase activity of p38 MAPK␣ and p38 MAPK␤. The activity of both isoforms was inhibited in vitro by 10 µmol/l SB203580 in all muscles. To explore the possible participation of p38 MAPK in the stimulation of glucose uptake, EDL and soleus muscles were exposed to increasing doses of SB203580 before and during stimulation by insulin or contraction. SB203580 caused a significant reduction in the insulin-or contractionstimulated 2-deoxyglucose uptake. Maximal inhibition (50-60%) occurred with 10 µmol/l SB203580. These results show that p38 MAPK␣ and -␤ isoforms are activated by insulin and contraction in skeletal muscle. The data further suggest that activation of p38 MAPK may participate in the stimulation of glucose uptake by both stimuli in rat skeletal muscle.

The protein tyrosine phosphatase SHP-1 is a well-known inhibitor of activation-promoting signaling cascades in hematopoietic cells but its potential role in insulin target tissues is unknown. Here we show that Ptpn6(me-v/me-v) (also known as viable motheaten) mice bearing a functionally deficient SHP-1 protein are markedly glucose tolerant and insulin sensitive as compared to wild-type littermates, as a result of enhanced insulin receptor signaling to IRS-PI3K-Akt in liver and muscle. Downregulation of SHP-1 activity in liver of normal mice by adenoviral expression of a catalytically inert mutant of SHP-1, or after small hairpin RNA-mediated SHP-1 silencing, further confirmed this phenotype. Tyrosine phosphorylation of CEACAM1, a modulator of hepatic insulin clearance, and clearance of serum [125I]-insulin were markedly increased in SHP-1-deficient mice or SHP-1-deficient hepatic cells in vitro. These findings show a novel role for SHP-1 in the regulation of glucose homeostasis through modulation of insulin signaling in liver and muscle as well as hepatic insulin clearance.

Background: DGAT1 is a triglyceride biosynthetic enzyme with a possible role in metabolic disorders. Results: T-863, a potent DGAT1 inhibitor acting on the acyl-CoA binding site of DGAT1, decreased body weight, improved insulin sensitivity, and alleviated hepatic steatosis in diet-induced obese mice. Conclusion: These data support further exploration of DGAT1 inhibitors for metabolic disorders. Significance: Our study reveals mechanisms of action for DGAT1 inhibitors.

No abstract

It has been recently proposed that obestatin, a peptide encoded by the ghrelin gene, reduces food intake by activating the orphan G protein-coupled receptor GPR39. To gain further insights into the role of GPR39 in body weight homeostasis, we characterized the phenotype of mice with targeted disruption of the GPR39 gene. Body weight, adiposity, and food intake were found to be similar between GPR39(+/+) and GPR39(-/-) mice. Furthermore, fasting glucose and insulin levels were similar between both genotypes. Injection of obestatin peptide (1 micromol/kg, ip) obtained from multiple sources did not consistently inhibit food intake in wild-type mice after an overnight fast, and no difference in food intake was observed between wild-type and GPR39 knockout mice after injection of the peptide. Finally, ectopic expression of GPR39 in HEK293T cells revealed a constitutive activation of the receptor that was unaffected by stimulation with obestatin. Our phenotypic characterization suggests that GPR39 is not a major modulator of food intake in mice, although a more subtle role cannot be excluded. The role of GPR39 in normal physiology requires further study and should be conducted independently of the function of obestatin.