Elevated phospholipid hydroperoxide glutathione peroxidase (GPX4) expression modulates oxylipin formation and inhibits age-related skeletal muscle atrophy and weakness.

Our previous studies support a key role for mitochondrial lipid hydroperoxides as important contributors to denervation-related muscle atrophy, including muscle atrophy associated with aging. Phospholipid hydroperoxide glutathione peroxidase 4 (GPX4) is an essential antioxidant enzyme that directly reduces phospholipid hydroperoxides and we previously reported that denervation-induced muscle atrophy is blunted in a mouse model of GPX4 overexpression. Therefore, the goal of the present study was to determine whether GPX4 overexpression can reduce the age-related increase in mitochondrial hydroperoxides in skeletal muscle and ameliorate age-related muscle atrophy and weakness (sarcopenia). Male C57Bl6 WT and GPX4 transgenic (GPX4Tg) mice were studied at 3 to 5 and 23-29 months of age. Basal mitochondrial peroxide generation was reduced by 34% in muscle fibers from aged GPX4Tg compared to old WT mice. GPX4 overexpression also reduced levels of lipid peroxidation products: 4-HNE, MDA, and LOOHs by 38%, 32%, and 84% respectively in aged GPX4Tg mice compared to aged WT mice. Muscle mass was preserved in old GPX4 Tg mice by 11% and specific force generation was 21% higher in old GPX4Tg versus age matched male WT mice. Oxylipins from lipoxygenases (LOX) and cyclooxygenase (COX), as well as less abundant non-enzymatically generated isomers, were significantly reduced by GPX4 overexpression. The expression of cPLA2, 12/15-LOX and COX-2 were 1.9-, 10.5- and 3.4-fold greater in old versus young WT muscle respectively, and 12/15-LOX and COX-2 levels were reduced by 37% and 35%, respectively in muscle from old GPX4Tg mice. Our study suggests that lipid peroxidation products may play an important role in the development of sarcopenia, and their detoxification might be an effective intervention in preventing muscle atrophy.

High glucose-stimulated enhancer of zeste homolog-2 (EZH2) forces suppression of deptor to cause glomerular mesangial cell pathology.

Function of mTORC1 and mTORC2 has emerged as a driver of mesangial cell pathologies in diabetic nephropathy. The mechanism of mTOR activation is poorly understood in this disease. Deptor is a constitutive subunit and a negative regulator of both mTOR complexes. Mechanistic investigation in mesangial cells revealed that high glucose decreased the expression of deptor concomitant with increased mTORC1 and mTORC2 activities, induction of hypertrophy and, expression of fibronectin and PAI-1. shRNAs against deptor mimicked these pathologic outcomes of high glucose. Conversely, overexpression of deptor significantly inhibited all effects of high glucose. To determine the mechanism of deptor suppression, we found that high glucose significantly increased the expression of EZH2, resulting in lysine-27 tri-methylation of histone H3 (H3K27Me3). Employing approaches including pharmacological inhibition, shRNA-mediated downregulation and overexpression of EZH2, we found that EZH2 regulates high glucose-induced deptor suppression along with activation of mTOR, mesangial cell hypertrophy and fibronectin/PAI-1 expression. Moreover, expression of hyperactive mTORC1 reversed shEZH2-mediated inhibition of hypertrophy and expression of fibronectin and PAI-1 by high glucose. Finally, in renal cortex of diabetic mice, we found that enhanced expression of EZH2 is associated with decreased deptor levels and increased mTOR activity and, expression of fibronectin and PAI-1. Together, our findings provide a novel mechanism for mTOR activation via EZH2 to induce mesangial cell hypertrophy and matrix expansion during early progression of diabetic nephropathy. These results suggest a strategy for leveraging the intrinsic effect of deptor to suppress mTOR activity via reducing EZH2 as a novel therapy for diabetic nephropathy.