Overexpression of manganese superoxide dismutase ameliorates high-fat diet-induced insulin resistance in rat skeletal muscle

Abstract

Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.

Fig. 1.

Successful overexpression of manganese superoxide dismutase (MnSOD) in TC muscle. A: mRNA expression of endogenous and exogenous mnsod in control and MnSOD in vivo electroporation (IVE) tibialis cranialis (TC) muscle (n = 9/group). B: protein expression in whole muscle lysates of endogenous and exogenous MnSOD; n = 9/group. C: Western blots of whole muscle lysates comparing expression of endogenous and exogenous MnSOD protein. D: protein expression in whole muscle lysates of Cu/Zn-SOD (n = 6/group). E: whole muscle lysate activity assay for MnSOD. Enzyme activity measured by spectrophotometer (n = 6–8/group). F: Western blots of mitochondria-enriched, cytosol-enriched, and total muscle lysates from control and MnSOD IVE TC muscle. Blots with antibodies to MnSOD (22 and 24 kDa for endogenous and exogenous MnSOD, respectively), Myc tag (construct MnSOD only), lactate dehydrogenase (cytosolic marker), and voltage-dependent anion channel (VDAC)/porin (mitochondrial marker) are shown. G: Western blots using less stringent denaturing of control and MnSOD IVE TC muscle. High-molecular-weight tetramer is visualized using antibodies for MnSOD (all samples) and using the anti-Myc antibody (MnSOD IVE samples only). ***P < 0.001; **P < 0.01.

Fig. 2.

Downstream changes due to MnSOD overexpression. A: representative protein expression by Western blot showing expression of subunits of the electron transport chain complex I, complex II, complex III, complex IV, and complex V. Whole muscle lysate activity assay for glutathione peroxidase (B) and catalase (C). Enzyme activity was measured by spectrophotometer (n = 6–8/group). **P < 0.01.

Fig. 3.

MnSOD overexpression ameliorates the reduction in glucose uptake due to high-fat diet. Glucose uptake into muscle (Rg′) of chow- and high-fat-fed rats as determined by 2-[³H]deoxyglucose radiolabeled tracer uptake into control and MnSOD IVE TC muscle under euglycemic hyperinsulinemic clamp conditions (n = 6/group) *P < 0.05; **P < 0.01.

Fig. 4.

Overexpression of MnSOD protects against increased protein carbonylation induced by a high-fat diet. Protein carbonylation levels in MnSOD overexpression and control muscles from chow- and high-fat diet-fed rats, as determined by enzymatic derivatization of the carbonyl groups into 2,4-dinitrophenylhydrazone by 2,4-dinitrophenylhydrazine, followed by resolution using PAGE and detection using a primary antibody to the dinitrophenylhydrazine moiety on the protein (n = 6/group). **P < 0.01.