Hijiri Oshio

In mitochondria, the pyruvate dehydrogenase complex (PDHC) serves as a key metabolic regulator by converting glycolysis-derived pyruvate into acetyl-CoA, thereby controlling carbon flux into the tricarboxylic acid (TCA) cycle. PDHC activity is tightly regulated by two post-translational modifications: phosphorylation of the E1 subunit and lipoylation of the E2 subunit. While phosphorylation of E1 reversibly suppresses pyruvate dehydrogenase (PDH) activity, lipoylation of E2 is essential for intracomplex electron transfer reactions, and together these modifications define PDHC enzymatic activity. Mitochondrial respiratory supercomplexes (SCs) play a critical role in efficient electron transfer during mitochondrial respiration, and PDH has been reported to regulate SC organization. However, it remains unclear whether this regulatory mechanism, including subunit phosphorylation, is linked to protein lipoylation. In this study, we examined the impact of protein lipoylation on the phosphorylation status of the PDHC E1 subunit and on mitochondrial respiratory supercomplex formation during C2C12 differentiation. To this end, suppression of lipoic acid synthase (LIAS), a key enzyme responsible for mitochondrial protein lipoylation, in C2C12 cells resulted in dephosphorylation of the PDHC E1 subunit and formation of specific mitochondrial respiratory supercomplexes. These findings suggest that PDHC E1 dephosphorylation and specific mitochondrial respiratory supercomplex assembly can occur under conditions of impaired E2 lipoylation.

Brown adipose tissue (BAT) is a major site of non-shivering thermogenesis, where mitochondria generate heat instead of adenosine triphosphate (ATP). The thermogenesis occurs through the activity of uncoupling protein 1 (UCP1) which specifically resides in the mitochondrial inner membrane and dissipates the mitochondrial proton gradient upon activation by long-chain free fatty acids (FFA). Although UCP1-independent proton leak has been reported, the mechanism underlying UCP1-independent mitochondrial membrane depolarization remains largely unknown. Here, using primary brown adipocytes, we found that cold-mimicking stimulation induces mitochondrial membrane depolarization even under UCP1 knockout and knockdown conditions. Furthermore, during cold-mimicking stimulation, palmitic acid shows the most prominent increase in a lipolysis-dependent manner. Notably, palmitic acid directly decreases mitochondrial membrane potential specifically in mitochondria isolated from BAT, but not in those isolated from liver or brain. These findings suggest that palmitic acid contributes to mitochondrial depolarization in BAT, thereby contributing to UCP1-independent depolarization.