Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

Abdelhak Mansouri,Veronique Lenoir,Jean Girard,Carole Henique,Frederic Bouillaud,Carina Prip-Buus,Isabelle Cohen,Gwladys Fumey

doi:10.1074/jbc.m110.170431

Abstract

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate's protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.

Highlights

Quantification was done with a Chemigenius apparatus (Syngene), and values are expressed relative to tubulin expression
long-chain fatty acid (LCFA) Metabolism—Consistent with previous studies [18], the endogenous CPT1 protein expressed in mouse C2C12 cells was CPT1A, the liver isoform
Our findings indicate that preincubation with oleate increases mitochondrial LCFA oxidation through a second mechanism, namely activation of AMPK, which leads to acetyl-CoA carboxylase (ACC) inhibition and, to increased CPT1 activity

Summary

Introduction

Lipids were extracted from homogenates G5), CPT1mt expression increased [1-14C]oleate oxidation to according to the method of Bligh and Dyer [29] and analyzed CO2, ASP, and their sum (Total) by 2.5-, 1.5-, and 1.7-fold, by gas-liquid chromatography for determination of neutral respectively, in comparison with control or Ad-LacZ-infected lipid molecular species, ceramides, sphingomyelins (SM) cells (Fig. 1C), despite a 3-fold increase in ACC protein level In control and Ad-LacZ-infected cells (Fig. 4A), only 44% of the metabolized palmitate was oxidized, in contrast to 74% for oleate, with no difference in the CO2/ASP ratio (palmitate, 0.28 Ϯ 0.02; oleate, 0.27 Ϯ 0.02). CPT1mt expression and OA increased [1-14C]palmitate oxidation by 1.6- and 1.4-fold, respectively (Fig. 4C).

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Conclusion