Aging is a risk factor for the development of chronic metabolic diseases such as type 2 diabetes, cardiovascular disease, neurodegenerative disease, and cancer. These aging-related diseases are known to be clustered together; aging-associated neurodegenerative disorders frequently coincide with type 2 diabetes, insulin resistance, obesity, and metabolic syndrome. This suggests that neurodegenerative disorders and type 2 diabetes share common genetic and metabolic features. Therefore, studying the role of the gene that contributes to neurodegenerative diseases in obesity and diabetes is of particular importance, as it may provide a clue for the pathological mechanism of the development of aging-related diseases. Intensive investigation on the underlying mechanisms of cellular aging has suggested that autophagy-lysosome and mitochondrial function plays an essential role in the regulation of cellular homeostasis and health (1). The autophagy-lysosome system is one of the main intracellular proteolytic systems that is responsible for the clearance of unwanted intracellular macromolecules and organelles (2). Cellular aging is associated with the disruption of this homeostatic mechanism, leading to the accumulation of oxidized/misfolded proteins, lipids, and damaged organelles (3). However, the genes and proteins that regulate autophagy-lysosomal activation and contribute to cellular aging remain largely unidentified.Autophagy is a highly regulated process that is inducible by nutrient deprivation and cellular stress. During nutrient deprivation, multiple signaling pathways cooperate to Abstract In this study, we investigated the role and mechanism of Niemann-Pick type C (NPC)2 in regulating lysosomal activity, mitophagy, and mitochondrial function in adipocytes. We found that knocking down NPC2 impaired lysosomal activity, as evidenced by the reduced mature cathepsin B, the increased accumulation of light chain 3 (LC3) and p62, and the decreased autophagic flux. In NPC2-knockdown (kd) adipocytes, the starvation-induced conversion of LC3-I to LC3-II was abolished. More interestingly, the majority of NPC2 was found in the mitochondrial fraction, and NPC2 deficiency led to impaired autophagic flux and decreased induction of LC3-II in the mitochondrial fraction during mitochondrial stress. Moreover, cellular respiration profiling revealed that NPC2-kd adipocytes had significantly decreased basal/maximal respiration and mitochondrial gene expression compared with scrambled cells, suggesting mitochondrial dysfunction. Additionally, we found that the mitochondrial recruitment of LC3-II induced by lipopolysaccharide (LPS), but not TNF, was blunted in NPC2-kd adipocytes. Most intriguingly, NPC2-kd selectively diminished LPS-induced NFB and ERK1/2 phosphorylation and the expression of pro-inflammatory genes, indicating that tolllike receptor signaling activation is impaired in the absence of NPC2. Our results suggest that NPC2 is in a mitochondrially associated autophagosome and plays an important role in regulating mitophag...