The long-term objective of this project is to understand the mechanisms that distinguish human thermogenic adipocyte metabolism and contribute to metabolic health. During the past funding periods, we have been successful in three significant lines of research, as follows: a, We have developed a model to interrogate human adipose tissue physiological function in vivo in mice. b, We have discovered at least 4 distinct adipocyte subtypes differentiating from human mesenchymal progenitors, associated with specific adipose tissue functions and differentially enriched in human adipose tissue depots, and c, We have discovered LINC00473, a primate-specific long non-coding RNA specifically expressed in thermogenic adipocytes and involved in lipolysis and mitochondrial oxidative metabolism. LINC00473 levels are decreased in obesity and Type 2 Diabetes, consistent
with an important physiological role. We will now further elucidate molecular mechanisms by which LINC00473 regulates these pathways and its contribution to systemic energy homeostasis. Aim 1. To elucidate the mechanism by which LINC00473 interacts with lipid droplet and mitochondrial proteins and the effect of these interactions on fatty acid and mitochondrial oxidative metabolism. Using affinity isolation and mass spectrometry, we have identified lipid droplet and mitochondrial proteins that interact with LINC00473. We will now: 1A. Define direct and indirect interactions between LINC00473 and candidate proteins, using molecular approaches including ChIRP and APEX2 proximity labeling. 1B. Define the kinetics of assembly of LINC00473-protein complexes and their relationship with stimulated lipolysis and respiration, and 1C. Define the role of specific interactions though expression of LINC00473 mutant constructs. Aim 2. To test the hypothesis that LINC00473 mediates a feedback mechanism to control lipolysis. We shall: 2A. Determine whether inhibition of LINC00473 expression upon PLIN1 depletion is due to enhanced basal lipolysis. 2B. Determine whether impaired stimulation of lipolysis in PLIN1 depleted cells is due LINC00473 supprssion. 2C. Determine whether PLIN1 depletion suppresses LINC00473 through decreased transcription, enhanced degradation or both. Aim 3. We will test the role of LINC00473 on thermogenic adipose tissue development and systemic glucose metabolism. Using techniques to generate human adipose tissue in mice we shall: 3A. Assess the effects of overexpression or knockdown of LINC00473 on dynamics of adipose tissue growth, vascularization, and innervation. 3B. Determine how adipose tissue developed from adipocytes expressing high or low levels of LINC00473 responds to physiological stimuli such as cold. 3C. Determine whether adipose tissue developed from adipocytes expressing high or low levels of LINC00473 differentially affects systemic glucose metabolism. This work will reveal molecular mechanisms that define human thermogenic adipocyte function, and how these mechanisms operate systemically and could impact metabolic disease.