Project Summary Obesity is a leading risk factor for type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular diseases. A central driver of pathogenesis in obesity-associated disorders is the insufficient lipid-storing capacity of adipocytes and subsequent lipid deposition in extra-adipose organs. The lipid droplet (LD) is the organelle responsible for lipid storage and mobilization in adipocytes. It remains to be elucidated whether proteins and pathways regulating LD structure and function constitute limiting factors governing the lipid-storing capacity of adipocytes, and thus play an essential role in determining one’s susceptibility to obesity-associated disorders. We observed that mice deficient in CLSTN3B, a mammalian adipocyte-specific protein, are more prone to high-fat diet-induced metabolic disorders compared with body weight-matched wild-type mice, whereas the adipose-specific clstn3b transgenic mice display the opposite phenotype. Preliminary evidence shows that CLSTN3B localizes to endoplasmic reticulum (ER)/LD contact sites and ablation of CLSTN3B results in an impaired coating of LDs by phospholipids and proteins. Our overall objectives are to (i) establish the significance of CLSTN3B expressed in white adipocytes to the metabolic phenotype; (ii) reveal the molecular mechanism of CLSTN3B action at the ER/LD contact sites. The central hypothesis is that CLSTN3B enhances the structural and functional integrity of LDs, improves white adipocyte lipid-storing capacity, and contributes to the maintenance of metabolic health under obese conditions; mechanistically, this is achieved by replenishing LD surface phospholipids and promoting the binding of LD-targeting proteins. We will test this hypothesis by pursuing three specific aims: 1) Show that CLSTN3B expressed in white adipocytes is the main contributor to the metabolic benefits upon high-fat diet feeding; 2) Show that CLSTN3B promotes phospholipids transfer between ER and LD; 3) Probe the role of the C-terminal ER luminal segment of CLSTN3B in the formation of ER/LD contacts. For the first aim, we will construct genetic models allowing specific assessment of white adipocyte-derived CLSTN3B. For the second aim, we will design in vitro reconstituted phospholipid transfer assays and examine the functional significance of LD surface phospholipid density. For the third aim, we will use biochemical approaches to identify potential binding partners of the ER luminal C-terminal fragment of CLSTN3B, followed by assessing the significance of such interactions using cellular and animal models. The proposed research is innovative because it dissects the molecular mechanism of a novel protein and explains susceptibility to obesity-associated disorders from a novel perspective. The proposed research is significant because it aims to establish an integrated understanding encompassing interorganelle communication and metabolic physiology at the organismal level. Our long-term goal is to ...