椎葉 一心

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.

Age-associated declines in skeletal muscle function are linked to cellular senescence and mitochondrial alterations, yet mitochondrial phenotypes in aged human myoblasts remain insufficiently characterized. Here, we examined primary skeletal muscle myoblasts from young and elderly donors to assess mitochondrial function, morphology, and mitochondria-endoplasmic reticulum (ER) contact sites (MERCS). Myoblasts from older donors exhibited senescence features, including elevated SA-β-gal activity and reduced Lamin B1 expression, accompanied by increased mitochondrial oxidative stress. Despite marked mitochondrial hyperfusion and increased mitochondrial DNA content, mitochondrial oxygen consumption rate and membrane potential per mitochondrial area were comparable between young and old cells. MERCS were significantly elevated in aged myoblasts and were reduced by scavenging mitochondrial reactive oxygen species (mtROS), indicating an association between oxidative stress and MERCS formation. These findings suggest that mitochondrial hyperfusion and enhanced MERCS accompany cellular aging in human myoblasts and may contribute to maintaining mitochondrial function under elevated oxidative stress.

Abstract Mitochondria play a central role in cellular energy metabolism and homeostasis, and their dysfunction is closely linked to the progression of age-related diseases. The mitochondrial ubiquitin ligase MITOL (also known as MARCHF5) is a key regulator of mitochondrial dynamics and function, and reduced MITOL expression in the mouse heart has been implicated in mitochondrial dysfunction and cardiac aging. In this study, we identified berberrubine as a compound that promotes MITOL expression and activates mitochondria. We further assembled a group of berberrubine-based compounds, including its quinoid form and a newly developed water-soluble derivative, and collectively named them “Mitorubin” as mitochondria-activating compounds with therapeutic potential. While conventional berberrubine has poor water solubility, the addition of acetic acid significantly improved its solubility, enabling formulation as a solution. Mitorubin enhanced MITOL expression in cultured cells, increased mitochondrial DNA content and expression of mitochondrial proteins, and promoted mitochondrial respiration. In a model of age-related cardiac dysfunction, oral administration of Mitorubin restored mitochondrial function, improved cardiac performance, suppressed myocardial hypertrophy, and alleviated pulmonary congestion. Moreover, Mitorubin did not shorten lifespan in aged mice and significantly extended lifespan in high-fat diet-fed mice, suggesting both safety and efficacy under chronic administration. These findings suggest that Mitorubin is a promising mitochondrial activator and may represent a novel therapeutic strategy for age-related diseases.