Project Summary: Adipose tissue is a dynamic energy depot that supports metabolic function and survival during fasting. When the ability of adipocytes to dynamically release energy in the form of fatty acids is impaired, fat cells become hypertrophied and develop ER stress, insulin resistance, express hypoxic signaling programs and inflammatory mediators, all of which contribute to the pathobiology of cardiometabolic disease. The canonical view of adipocyte energy balance is that insulin drives energy storage in lipids with feeding, while counterregulatory hormones drive release of three fatty acid chains from each triglyceride molecule with fasting through the sequential action of three lipases, including the rate-limiting adipose triglyceride lipase (ATGL). In preliminary data contained in this grant proposal, our laboratory discovered evidence that fasting-lipolysis is largely preserved when the canonical ATGL-dependent lipolytic pathway is targeted during prolonged fasting. Instead, our preliminary data points to a critical role for a complementary, alternative pathway involving lysosomes and the lysosomal lipase (lysosomal acid lipase-LIPA). Our preliminary data further demonstrate that members of the MiT/TFE transcription factor family, including the master regulator of lysosomal biogenesis, TFEB, are required for lipolysis with fasting. These preliminary data support the central hypothesis of this project: lysosomal digestion of lipid in adipocytes is a critical mechanism of fatty acid release during fasting. In this proposal we seek to leverage innovative methods, including inducible adipocyte targeted loss of function studies in vivo, state of the art multi-omics studies of MiT/TFE transcription factor regulatory circuitry, and high-resolution imaging mass spectrometry—which enables quantitative tracking of stable isotope lipid labels at the sub-organelle level— to examine the underlying mechanisms and the physiological implications of this novel lipolytic pathway in adipocytes. In Aim 1, we will define the molecular and cell biological mechanisms that govern mobilization of fatty acids from adipocytes with fasting. In Aim 2, we will define the role of the lysosomal lipolysis pathway in systemic lipid homeostasis and energy balance in the context of fasting and disease-relevant stressors of aging and diet-induced obesity. Decades of data suggests that lipolysis in adipocytes, which is a major determinant of circulating fatty acids and systemic lipid metabolism, is important to cardiometabolic diseases such as diabetes mellitus. If our central hypothesis is correct, that an alternative lysosomal pathway is operative with fasting, then our understanding of systemic fatty acid metabolism is incomplete. Completion of this project holds promise to reorient our understanding of how adipocytes regulate systemic lipid metabolism and to determine whether this core function is a determinant of metabolic disease.