Sirtuins in mitochondrial physiology and energy homeostasis.

Mitochondrial sirtuins integrate cellular metabolic and energetic state with post-translational regulation of mitochondrial proteins, thereby influencing bioenergetics, redox homeostasis, and metabolic flexibility. Despite extensive study of individual sirtuins, a comprehensive understanding of their function remains limited by methodological and conceptual challenges. These arise from the complex metabolic environment in which mitochondrial sirtuins act, including compartmentalized NAD+ pools, metabolite-driven non-enzymatic acylation, overlapping substrate specificities, and limited tools to accurately characterize enzyme-specific activity, particularly for SIRT4. This review aims to highlight current experimental approaches used to study mitochondrial sirtuins, and the need to integrate enzymatic measurements with metabolic and physiological readouts. We discuss the importance of considering mitochondrial spatial heterogeneity within cells, tissue-specific metabolic context, and temporal dynamics of metabolic state when interpreting sirtuin activity. Recent advances in quantitative proteomics, metabolite profiling, and mitochondria-specific analyses provide new opportunities to resolve these complexities. Moving forward, integrating these approaches with a systems-level and quantitative framework will be critical to fully understand how mitochondrial sirtuins orchestrate metabolic regulation across cellular and organismal scales.