Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3−/− livers at 3 months of age. Livers of Sirt3−/− mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Re-introduction of wild-type, but not deacetylation null Sirt3, into Sirt3−/− MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSODK122-R), increased MnSOD activity when expressed in MnSOD−/− MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3−/− MEFs with lenti-MnSODK122-R inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3−/− livers and these irradiated livers displayed significant IR-induced cell damage and micro-vacuolization in their hepatocytes.

OBJECTIVEInsulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here.RESEARCH DESIGN AND METHODSMuscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry–based quantification using normalized spectral abundance factors.RESULTSOf 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased).CONCLUSIONSThe results confirm the reduction in mitochondrial proteins in insulin-resistant muscle and suggest that changes in muscle structure, protein degradation, and folding also characterize insulin resistance.

Rationale Clinical studies have shown that Sirt3 expression declines by 40% by age 65 paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 due to increased NADH and acetyl-CoA levels. Sirt3 impairment reduces the activity of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), due to hyperacetylation. Objective In this study we examined if loss of Sirt3 activity increases vascular oxidative stress due to SOD2 hyperacetylation and promotes endothelial dysfunction and hypertension. Methods and Results Hypertension was markedly increased in Sirt3 knockout (Sirt3−/−) and SOD2 depleted (SOD2+/−) mice in response to low dose of angiotensin II (0.3 mg/kg/day) compared with wild-type C57Bl/6J mice. Sirt3 depletion increased SOD2 acetylation, elevated mitochondrial O2•, and diminished endothelial nitric oxide. Angiotensin II induced hypertension was associated with Sirt3 S-glutathionylation, acetylation of vascular SOD2 and reduced SOD2 activity. Scavenging of mitochondrial H2O2 in mCAT mice prevented Sirt3 and SOD2 impairment and attenuated hypertension. Treatment of mice after onset of hypertension with a mitochondria-targeted H2O2 scavenger, mitoEbselen, reduced Sirt3 S-glutathionylation, diminished SOD2 acetylation and reduced blood pressure in wild-type but not in Sirt3−/− mice while an SOD2 mimetic, mitoTEMPO, reduced blood pressure and improved vasorelaxation both in Sirt3−/− and wild type mice. SOD2 acetylation had an inverse correlation with SOD2 activity and a direct correlation with the severity of hypertension. Analysis of human subjects with essential hypertension showed 2.6-fold increase in SOD2 acetylation and 1.4-fold decrease in Sirt3 levels while SOD2 expression was not affected. Conclusions Our data suggest that diminished Sirt3 expression and redox inactivation of Sirt3 lead to SOD2 inactivation and contributes to the pathogenesis of hypertension.

fatty liver disease occurs frequently in the setting of metabolic syndrome, but the factors leading to nonalcoholic steatohepatitis (NASH) are not fully understood. This study investigated Toll-like receptor 4 (TLR4) signaling in human liver with the goal of delineating whether activation of this pathway segregates those with nonalcoholic fatty liver from those with NASH. Experiments were performed using liver biopsy tissue obtained from class III obese subjects undergoing bariatric surgery, and extended to an immortalized human hepatocyte HepaRG cell line and primary human hepatocytes. The bacterial endotoxin lipopolysaccharide (LPS) and total free fatty acid levels were significantly increased in plasma of NASH patients. TLR4 mRNA levels were significantly increased in subjects with NASH compared with NAFL as was interferon regulatory factor (IRF) 3 in the myeloid differentiation factor 88-independent signaling pathway. In HepaRG cells, nuclear factor-B (NF-B) nuclear translocation and functional activity increased following treatment with the fatty acid, palmitate, and following exposure to LPS compared with hepatocytes stimulated with a lipogenic treatment that induced de novo lipogenesis. Palmitate and LPS induction of NF-B activity was partially attenuated by chemical-or small-interfering RNA-mediated inhibition of TLR4. Expression of TLR4 and its downstream mediators was upregulated with palmitate and LPS. Similar results were observed using primary human hepatocytes from a lean donor. Interestingly, NF-B activity assays showed obese donor hepatocytes were resistant to chemical TLR4 inhibition. In conclusion, TLR4 expression is upregulated in a large cohort of NASH patients, compared with those with NAFL, and this occurs within the setting of increased LPS and fatty acids. nonalcoholic steatohepatitis; palmitate; Toll-like receptor 4; nuclear factor-

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.