Oxidative stress-mediated mitochondrial dysfunction contributes to angiotensin II-induced nonalcoholic fatty liver disease in transgenic Ren2 rats.

Emerging evidence indicates that impaired mitochondrial fatty acid beta-oxidation plays a key role in liver steatosis. We have recently demonstrated that increased angiotensin (ANG) II causes progressive hepatic steatosis associated with oxidative stress; however, the underlying mechanisms remain unclear. We hypothesized that ANG II causes hepatic mitochondrial oxidative damage and impairs mitochondrial beta-oxidation, thereby leading to hepatic steatosis. We used the Ren2 rat with elevated endogenous ANG II levels to evaluate mitochondrial ultrastructural changes, gene expression levels, and beta-oxidation. Compared with Sprague-Dawley littermates, Ren2 livers exhibited mitochondrial damage and reduced beta-oxidation, as evidenced by ultrastructural abnormalities, decrease of mitochondrial content, percentage of palmitate oxidation to CO(2), enzymatic activities (beta-HAD and citrate synthase), and the expression levels of cytochrome c, cytochrome c oxidase subunit 1, and mitochondrial transcription factor A. These abnormalities were improved with either ANG II receptor blocker valsartan or superoxide dismutase/catalase mimetic tempol treatment. Both valsartan and tempol substantially attenuated mitochondrial lipid peroxidation in Ren2 livers. Interestingly, there was no difference in the expression of key enzymes (ACC1 and FAS) for fatty acid syntheses and their transcription factors (SREBP-1c and ChREBP) between Sprague-Dawley, untreated Ren2, and valsartan- or tempol-treated Ren2 rats. These results document that ANG II induces mitochondrial oxidative damage and impairs mitochondrial beta-oxidation, contributing to liver steatosis.

Angiotensin II-induced non-alcoholic fatty liver disease is mediated by oxidative stress in transgenic TG(mRen2)27(Ren2) rats.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease (NAFLD) is a common health problem and includes a spectrum of hepatic steatosis, steatohepatitis and fibrosis. The renin-angiotensin system (RAS) plays a vital role in blood pressure regulation and appears to promote hepatic fibrogenesis. We hypothesized that increased RAS activity causes NAFLD due to increased hepatic oxidative stress. METHODS: We employed the transgenic TG(mRen2)27(Ren2) hypertensive rat, harboring the mouse renin gene with elevated tissue Angiotensin II (Ang II). RESULTS: Compared with normotensive Sprague-Dawley (SD) control rats, Ren2 developed significant hepatic steatosis by 9 weeks of age that progressed to marked steatohepatitis and fibrosis by 12 weeks. These changes were associated with increased levels of hepatic reactive oxygen species (ROS) and lipid peroxidation. Accordingly, 9-week-old Ren2 rats were treated for 3 weeks with valsartan, an angiotensin type 1 receptor blocker, or tempol, a superoxide dismutase/catalase mimetic. Hepatic indices for oxidative stress, steatosis, inflammation and fibrosis were markedly attenuated by both valsartan and tempol treatment. CONCLUSIONS: This study suggests that Ang II causes development and progression of NAFLD in the transgenic Ren2 rat model by increasing hepatic ROS. Our findings also support a potential role of RAS in prevention and treatment of NAFLD.

NADPH oxidase contributes to vascular inflammation, insulin resistance, and remodeling in the transgenic (mRen2) rat.

Reduced insulin sensitivity is characteristic of various pathological conditions such as type 2 diabetes mellitus and hypertension. Angiotensin II, acting through its angiotensin type 1 receptor, inhibits the actions of insulin in the vasculature which may lead to deleterious effects such as vascular inflammation, remodeling, endothelial dysfunction, and insulin resistance. In contrast, insulin normally exerts vasodilatory, antiinflammatory, and prosurvival actions. To explore the impact of angiotensin II on insulin signaling, NADPH oxidase-derived reactive oxygen species formation, vascular inflammation, apoptosis, and remodeling, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and exhibits elevated tissue angiotensin II levels. Compared with Sprague-Dawley controls, Ren2 aortas exhibited greater NADPH oxidase activity, reactive oxygen species levels, C-reactive protein, tumor necrosis factor-alpha expression, apoptosis, and wall thickness, which were significantly attenuated by in vivo treatment with angiotensin type 1 receptor blockade (valsartan) or the superoxide dismutase/catalase mimetic (tempol). There was substantially diminished Akt and endothelial NO synthase activation in Ren2 aortas in response to in vivo insulin stimulation, and this was significantly improved by in vivo treatment with valsartan or tempol. In vivo treatment with valsartan, but not tempol, significantly reduced blood pressure in Ren2 rats. Further, there was reduced insulin induced Akt activation and increased tumor necrosis factor-alpha levels in vascular smooth muscle cells from Ren2 and Sprague-Dawley rats treated with angiotensin II, abnormalities that were abrogated by angiotensin type 1 receptor blockade with valsartan or antioxidant N-acetylcysteine. Collectively, these data suggest that increased angiotensin type 1 receptor/NADPH oxidase activation/reactive oxygen species contribute to vascular insulin resistance, endothelial dysfunction, apoptosis, and inflammation.

Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells.

The renin-angiotensin system (RAS) and reactive oxygen species (ROS) have been implicated in the development of insulin resistance and its related complications. There is also evidence that angiotensin II (Ang II)-induced generation of ROS contributes to the development of insulin resistance in skeletal muscle, although the precise mechanisms remain unknown. In the present study, we found that Ang II markedly enhanced NADPH oxidase activity and consequent ROS generation in L6 myotubes. These effects were blocked by the angiotensin II type 1 receptor blocker losartan, and by the NADPH oxidase inhibitor apocynin. Ang II also promoted the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox to the plasma membrane within 15 min. Furthermore, Ang II abolished insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), activation of protein kinase B (Akt), and glucose transporter-4 (GLUT4) translocation to the plasma membrane, which was reversed by pretreating myotubes with losartan or apocynin. Finally, small interfering RNA (siRNA)-specific gene silencing targeted specifically against p47phox (p47siRNA), in both L6 and primary myotubes, reduced the cognate protein expression, decreased NADPH oxidase activity, restored Ang II-impaired IRS1 and Akt activation as well as GLUT4 translocation by insulin. These results suggest a pivotal role for NADPH oxidase activation and ROS generation in Ang II-induced inhibition of insulin signaling in skeletal muscle cells.