Research article for 1 revision to the Chemico-Biological InteractionModulatory roles of capsaicin on thermogenesis in C2C12 myoblasts and the skeletal muscle of mice.

Capsaicin, a polyphenol, is known to regulate energy expenditure and thermogenesis in adipocytes and muscles. However, its role in modulating uncoupling proteins (UCPs) and adenosine triphosphate (ATP)-dependent thermogenesis in muscles remains unclear. This study investigated the mechanisms underlying the role of capsaicin in modulating the UCP- and ATP-dependent thermogenesis in C2C12 myoblasts, as well as the gastrocnemius (GM) and soleus muscles (SM) of mice. We employed molecular dynamics (MD), quantitative real-time polymerase chain reactions (qRT-PCR), immunoblots, staining methods, and assay kits to investigate the role of capsaicin on thermogenesis and its modulatory roles on the transient receptor potential cation channel subfamily V member 1 (TRPV1) and α-/β-adrenergic receptors (ARs) using in vitro and in vivo models. Our findings demonstrate that capsaicin treatment in high-fat diet-induced obese mice reduces weight gain and elevates the expression of UCP- and ATP-dependent thermogenic effectors through ATP-consuming calcium and creatine futile cycles. In in vitro and in vivo models capsaicin treatment elevated the expression of sarcoendoplasmic/endoplasmic reticulum calcium ATPases (SERCA-1 and -2), ryanodine receptors (RYR-1 and -2), uncoupling proteins (UCP-2 and -3), creatine kinase B (CKB), and creatine kinase mitochondrial 2 (CKMT2), through activation of TRPV1, α1-, β2-, and β3-AR as well as the suppressed expression of α2-AR. Furthermore, our results also indicate that capsaicin promotes myotube development and enhances lipid metabolism in C2C12 cells. We found that capsaicin increased intracellular Ca levels and the expression of the voltage-dependent anion channel (VDAC) and mitochondrial calcium uniporter (MCU), suggesting that elevated mitochondrial Ca levels boost the expression of oxidative phosphorylation protein complexes via the activation of the ATP-futile cycle. Mechanistic studies in C2C12 cells revealed that TRPV1 is likely dispensable for capsaicin-induced thermogenesis, and TRPV1 and α1-AR may synergistically induce thermogenesis. Collectively, our findings have uncovered a novel mechanism of UCP- and ATP-dependent thermogenesis and its associated pathways in both cellular and animal models which is crucial for designing therapeutic strategies to address obesity and associated metabolic diseases.