Oxidative phosphorylation and fatty acid oxidation are two major metabolic pathways in mitochondria. Acyl-CoA dehydrogenase 9 (ACAD9), an enzyme assumed to play a role in fatty acid oxidation, was recently identified as a factor involved in complex I biogenesis. Here we further investigated the role of ACAD9's enzymatic activity in fatty acid oxidation and complex I biogenesis. We provide evidence indicating that ACAD9 displays enzyme activity in vivo. Knockdown experiments in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts revealed that ACAD9 is responsible for the production of C14:1-carnitine from oleate and C12-carnitine from palmitate. These results explain the origin of these obscure acylcarnitines that are used to diagnose VLCAD deficiency in humans. Knockdown of ACAD9 in control fibroblasts did not reveal changes in the acylcarnitine profiles upon fatty acid loading. Next, we investigated whether catalytic activity of ACAD9 was necessary for complex I biogenesis. Catalytically inactive ACAD9 gave partial-to-complete rescue of complex I biogenesis in ACAD9-deficient cells and was incorporated in high-molecular-weight assembly intermediates. Our results underscore the importance of the ACAD9 protein in complex I assembly and suggest that the enzymatic activity is a rudiment of the duplication event.

In adipocytes, mitochondrial uncoupling is known to trigger a triglyceride loss comparable with the one induced by TNFα, a proinflammatory cytokine. However, the impact of a mitochondrial uncoupling on the abundance/composition of mitochondria and its connection with triglyceride content in adipocytes is largely unknown. In this work, the effects of a mild mitochondrial uncoupling triggered by FCCP were investigated on the mitochondrial population of 3T3-L1 adipocytes by both quantitative and qualitative approaches. We found that mild mitochondrial uncoupling does not stimulate mitochondrial biogenesis in adipocytes but induces an adaptive cell response characterized by quantitative modifications of mitochondrial protein content. Superoxide anion radical level was increased in mitochondria of both TNFα- and FCCP-treated adipocytes, whereas mitochondrial DNA copy number was significantly higher only in TNFα-treated cells. Subproteomic analysis revealed that the abundance of pyruvate carboxylase was reduced significantly in mitochondria of TNFα- and FCCP-treated adipocytes. Functional study showed that overexpression of this major enzyme of lipid metabolism is able to prevent the triglyceride content reduction in adipocytes exposed to mitochondrial uncoupling or TNFα. These results suggest a new mechanism by which the effects of mitochondrial uncoupling might limit triglyceride accumulation in adipocytes.