The Circadian Clock Component NPAS2 is modulated by AMPK and Regulates Substrate Utilization and Mitochondrial...

The molecular circadian clock coordinates cellular and systemic metabolism across the day, yet how it integrates with energy-sensing pathways in human skeletal muscle remains incompletely understood. AMP-activated protein kinase (AMPK) functions as a cellular energy sensor and has been implicated in circadian regulation; however, its role in coupling clock components to metabolic programming in human muscle has not been established. Here, we investigated the interaction between AMPK signaling and the circadian machinery in primary human myotubes, focusing on the downstream clock components NPAS2 and DBP. Silencing of the AMPKα subunit altered core clock gene expression, increasing CRY1 and CRY2 while reducing NPAS2 and DBP expression, with rhythmicity preserved. To determine the functional relevance of these changes, we examined metabolic phenotypes following siRNA-mediated silencing of NPAS2. NPAS2 knockdown reduced basal oxidative metabolism, decreased abundance of electron transport chain subunits, and increased β-oxidation flux, while maximal mitochondrial respiratory capacity remained intact. These alterations occurred independently of canonical AMPK-ACC signaling but were associated with reduced HIF1A expression. In contrast, DBP silencing reduced glucose oxidation but did not recapitulate the broader metabolic remodeling observed with NPAS2 knockdown. Together, these findings identify NPAS2 as a downstream mediator linking AMPK-dependent circadian signaling to skeletal muscle metabolic regulation. Disruption of this axis alters substrate utilization and impairs mitochondrial function, revealing a mechanism by which circadian clock components contribute to metabolic regulation in human skeletal muscle.