Priyanka Voori Giri

• Starvation, a global crisis affecting millions, stems from various factors like poverty, conflicts, and climate change, particularly impacting low-income regions. In the medical realm, treating malnourished patients poses complex challenges including addressing their unique nutritional needs, managing complications such as electrolyte imbalances and organ dysfunction, and mitigating the risk of refeeding syndrome a potentially life-threatening condition characterized by metabolic disturbances following the reintroduction of nutrition after a period of starvation. In the pharmaceutical arena, starvation serves as a critical research model for investigating metabolic adaptations, nutrient sensing pathways, and therapeutic targets implicated in metabolic diseases, cancer, and aging. By studying the physiological responses to starvation, researchers gain insights into the molecular mechanisms governing energy metabolism, substrate utilization, and cellular homeostasis, informing the development of novel pharmacological agents and therapeutic strategies. During starvation, the skeletal muscles undergo metabolic changes to adapt to nutrient deprivation. Initially, glycogen stores are depleted, leading to increased fatty acid oxidation. Muscle protein breakdown provides amino acids for gluconeogenesis, resulting in muscle wasting. Autophagy is upregulated to maintain cellular integrity and energy production. These adaptations ensure the survival of vital organs and energy homeostasis during starvation. One regulator of metabolic processes is the nutrient-sensitive transcription factor EB (TFEB). TFEB promotes the expression of genes involved in fatty acid oxidation (FAO) (PGC-1α/PPARGC1A, PPARα/Ppara), glucose oxidation (GO) (HKII/HK2, GLUT1/Slc2a1, GLUT4/Slc2a4, and IRS2/Irs2) and energy production by oxidative phosphorylation (OXPHOS, complex I-V). TFEB may therefore play a role in stress-induced metabolic remodelling of the skeletal muscle. Previous research has emphasized TFEB's critical role in orchestrating cellular responses to nutrient stress, particularly in liver tissue. However, the specific influence of TFEB on skeletal muscle metabolism during starvation is poorly understood. This study aims to elucidate TFEB's role in skeletal muscle metabolism during starvation for this we employed both in vitro and in vivo approaches. In in vitro studies C2C12 mouse skeletal muscle myocytes were used to validate TFEB nuclear translocation during starvation. TFEB overexpression and silencing experiments were conducted in C2C12 cells. Seahorse assays were used to analyse changes in cellular respiration and ATP production. Our research began with in vitro studies to establish a foundation for understanding TFEB's role in skeletal muscle metabolism during starvation. Using C2C12 mouse skeletal muscle myocytes, we first validated TFEB's nuclear translocation under starvation conditions, observing an upregulation of genes associated with mitochondrial biogenesis and autophagy. This initial finding prompted us to explore the differential impacts of TFEB overexpression versus deletion on metabolic remodelling and energy homeostasis in skeletal myocytes. Employing seahorse assays, we analysed cellular respiration, ATP production rates, and metabolic changes in C2C12 skeletal myoblasts overexpressing TFEB. The results revealed a striking metabolic shift from oxidative phosphorylation (OXPHOS) to glycolytic respiration, accompanied by increased overall metabolic activity, higher ATP production, and enhanced maximal and reserve respiratory capacities. This metabolic reprogramming suggested an improved ability of skeletal myoblasts to respond to increased energy demands and adapt to stressful conditions like starvation. Conversely, when we silenced Tfeb using siRNA under starvation conditions, we observed a trend towards decreased maximal mitochondrial respiration, indicating potential mitochondrial dysfunction in the absence of TFEB. These compelling in vitro findings led us to extend our investigation to in vivo models, where we examined the consequences of skeletal muscle-specific TFEB deletion on mitochondrial biogenesis and bioenergetics. This comprehensive approach allowed us to bridge the gap between cellular and organismal levels, providing a more holistic understanding of TFEB's critical role in skeletal muscle metabolism during nutrient deprivation. The second phase of this study addressed a critical knowledge gap by employing a starvation-induced mouse model to compare the metabolic profiles of skeletal muscle-specific TFEB knockout (cKO) mice with those of wild-type (WT) counterparts under physiological, fed, and starved conditions. Initially, the skeletal muscle-specific deletion of Tfeb (TfebloxP/loxP; MCK-CRE; cKO) showed no significant impact on muscle weight under normal conditions compared to controls (TfebloxP/loxP; +/+; WT). However, when subjected to a 48-h starvation period, cKO mice exhibited marked physiological changes. Comprehensive analyses, including morphological, histological, and qRT-PCR assessments, revealed that cKO mice experienced greater body weight loss and downregulation of genes involved in key metabolic processes such as fatty acid oxidation, glucose metabolism, and autophagy compared to their WT counterparts. Proteomic and transcriptomic analyses further elucidated the differential responses of metabolic pathways across various skeletal muscle types following TFEB deletion. Notably, TFEB deficiency led to disruptions in both glucose and lipid oxidation pathways, reduced autophagic and mitophagic genes, and ubiquitin ligase genes within skeletal muscles under starvation conditions. In summary, this study elucidates the multifaceted role of TFEB in regulating metabolic pathways within skeletal muscle during starvation. By integrating proteomic and transcriptomic analyses with functional assessments, we provide compelling evidence that TFEB is crucial for maintaining energy homeostasis, mitochondrial function, and metabolic flexibility under nutrient-deprived conditions. These findings contribute to our understanding of how transcription factors like TFEB orchestrate cellular responses to metabolic stressors. Further investigations are warranted to explore the precise molecular mechanisms through which TFEB regulates these critical processes in skeletal muscle physiology.