Voluntary Exercise Can Ameliorate Insulin Resistance by Reducing iNOS-Mediated S-Nitrosylation of Akt in the Liver in Obese Rats

Voluntary exercise can ameliorate insulin resistance. The underlying mechanism, however, remains to be elucidated. We previously demonstrated that inducible nitric oxide synthase (iNOS) in the liver plays an important role in hepatic insulin resistance in the setting of obesity. In this study, we tried to verify our hypothesis that voluntary exercise improves insulin resistance by reducing the expression of iNOS and subsequent S-nitrosylation of key molecules of glucose metabolism in the liver. Twenty-one Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus, and 18 non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats were randomly assigned to a sedentary group or exercise group subjected to voluntary wheel running for 20 weeks. The voluntary exercise significantly reduced the fasting blood glucose and HOMA-IR in the OLETF rats. In addition, the exercise decreased the amount of iNOS mRNA in the liver in the OLETF rats. Moreover, exercise reduced the levels of S-nitrosylated Akt in the liver, which were increased in the OLETF rats, to those observed in the LETO rats. These findings support our hypothesis that voluntary exercise improves insulin resistance, at least partly, by suppressing the iNOS expression and subsequent S-nitrosylation of Akt, a key molecule of the signal transduction pathways in glucose metabolism in the liver

iNOS Is Required for IL-1β-Induced Suppressed PDX-1 Expression, Inactivation of IRS-2/Akt Pathway and NF-κB Activation in Pancreatic β-Cells

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Protective Roles of S-Nitrosoglutathione Reductase (GSNOR), a Negative Regulator of Protein S-Nitrosylation, in Obesity- and Streptozotocin-Induced Diabetes: iNOS-Dependent and -Independent Mechanisms

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A Deficiency of Herp, an Endoplasmic Reticulum Stress Protein, Suppresses Atherosclerosis in ApoE Knockout Mice by Attenuating Inflammatory Responses

<div><p>Herp was originally identified as an endoplasmic reticulum (ER) stress protein in vascular endothelial cells. ER stress is induced in atherosclerotic lesions, but it is not known whether Herp plays any role in the development of atherosclerosis. To address this question, we generated Herp- and apolipoprotein E (apoE)-deficient mice (Herp^−/−; apoE^−/− mice) by crossbreeding Herp^−/− mice and apoE^−/− mice. Herp was expressed in the endothelial cells and medial smooth muscle cells of the aorta, as well as in a subset of macrophages in the atherosclerotic lesions in apoE^−/− mice, while there was no expression of Herp in the Herp^−/−; apoE^−/− mice. The doubly deficient mice developed significantly fewer atherosclerotic lesions than the apoE^−/− mice at 36 and 72 weeks of age, whereas the plasma levels of cholesterol and triglycerides were not significantly different between the strains. The plasma levels of non-esterified fatty acids were significantly lower in the Herp^−/−; apoE^−/− mice when they were eight and 16 weeks old. The gene expression levels of ER stress response proteins (GRP78 and CHOP) and inflammatory cytokines (IL-1β, IL-6, TNF-α and MCP-1) in the aorta were significantly lower in Herp^−/−; apoE^−/− mice than in apoE^−/− mice, suggesting that Herp mediated ER stress-induced inflammation. In fact, peritoneal macrophages isolated from Herp-deficient mice and RAW264.7 macrophages in which Herp was eliminated with a siRNA expressed lower levels of mRNA for inflammatory cytokines when they were treated with tunicamycin. Herp deficiency affected the major mediators of the unfolded protein response, including IRE1 and PERK, but not ATF6. These findings suggest that a deficiency of Herp suppressed the development of atherosclerosis by attenuating the ER stress-induced inflammatory reactions.</p></div

Herp expression in the artery.

<p>A: The amount for Herp mRNA was determined by a real-time PCR in the aortas of apoE^−/− mice (open columns) and Herp^−/−; apoE^−/−mice (closed columns). Each mRNA expression level was normalized to that of GAPDH. The mRNA levels are shown as relative ratios to the mRNA level of eight-week-old apoE^−/− mice. Each value represents the mean ± SEM of five mice. N.D. indicates non-detectable. ** p<0.01 vs. apoE^−/− mice at eight weeks of age. B: Immunostaining of Herp in the normal (upper panels) and atherosclerotic (lower panels) aortas of apoE^−/− and Herp^−/−; apoE^−/− mice at 72 weeks of age. Blue; hematoxylin, Red; Herp (diaminobenzidine; DAB). The bar shows 50 μm.</p

Herp-deficient RAW264.7 macrophages showed reduced expression of IL-1β and IL-6 in response to ER stress.

<p>RAW264.7 macrophages were treated with a siRNA against Herp or a control siRNA for 48 μM tunicamycin for 6 hrs. The mRNA expression levels of ER stress-related proteins and cytokines were analyzed by a real-time PCR analysis. Open columns show unstimulated cells (DMSO), closed columns show cells stimulated with tunicamycin (TM). The amounts of mRNA were normalized to that of GAPDH. The mRNA levels were shown as a relative ratio to the mRNA level of tunicamycin-treated macrophages, which were transfected with control siRNA. A: IL-1β, B: IL-6, C: Herp, D: GRP78, E: CHOP, F: MCP-1, G: TNF-α, H: SERP1, I: Sec61b, J: SEL1L and K: ATF4. Each column represents the mean ± SEM; n = 3 per group. * p<0.05, ** p<0.01, *** p<0.001, vs unstimulated control macrophages; ¶p<0.05, ¶¶p<0.01, ¶¶¶p<0.001 vs unstimulated siHerp macrophages. †p<0.05, ††p<0.01, †††p<0.001 vs stimulated siHerp macrophages. N.D.; not detected. n.d.; no difference.</p

The expression levels of IL-1β, MCP-1 and VCAM-1 were reduced in the aortas of Herp^−/−; apoE^−/− mice.

<p>Total RNA was prepared from the entire aortas of apoE^−/− and Herp^−/−; apoE^−/− mice. The mRNA expression levels were determined by real-time PCR. Each mRNA level was normalized to that of GAPDH mRNA. The mRNA levels are shown as the relative ratio to the mRNA level of eight or 20-week-old apoE^−/− mice. A: IL-1β, B: GRP78, C: IL-6, D: MCP-1, E: CHOP, F: VCAM-1, G: SERP1, H: Sec61b, I: SEL1L and J: ATF4. Open columns show apoE^−/−, and closed columns show Herp^−/−; apoE^−/− mice. Each column represents the mean ± SEM of five mice. N.D. indicates non-detectable. * p<0.05, ** p<0.01 vs. apoE^−/− mice at the same age. A, B: Immunostaining of IL-1β and GRP78 in the atherosclerotic aortas of apoE^−/− and Herp^−/−; apoE^−/− mice at 72 weeks of age. Blue; hematoxylin, Red; GRP78 or IL-1β (DAB). The bar shows 50 μm.</p

Effect of Herp deficiency on the plasma glucose and lipid levels.

<p>The body weight (A) and the plasma levels of glucose (B), cholesterol (C), triglycerides (D) and NEFA (E) following overnight fasting at eight, 16, 24, 36 and 72 weeks of age, were determined as described in the Methods. Open circles show apoE^−/−, and closed squares show Herp^−/−; apoE^−/− mice. Each value represents the mean ± SEM; n = 5 per group. * p<0.01 vs. Herp^−/−; apoE^−/− mice at the same age.</p