Regulation of Lipolysis by 14-3-3 Proteins on Human Adipocyte Lipid Droplets

Abstract

Lipid droplets (LDs) in adipocytes are pivotal for systemic lipid metabolism, serving as storage centers during nutritional surplus and as sources of fatty acids when energy is needed. These LDs react to hormonal stimuli like catecholamines and insulin, and their impaired response can lead to dysregulated lipolysis, lipotoxicity, and an increased risk of metabolic diseases. The specific mechanisms behind lipid release in human adipocytes remain largely unexplored. This study aims to elucidate the control of lipid mobilization in human adipocytes. We utilized advanced techniques to generate and differentiate primary progenitor cells on a large scale. Employing proximity labeling with enhanced ascorbate peroxidase (APEX2), we identified the interactome of perilipin 1 (PLIN1), a key LD component protein, under various lipolytic states. Through LC-MS/MS, we discovered 70 proteins interacting specifically with PLIN1. This includes PNPLA2 and LIPE, vital for regulated triglyceride hydrolysis, and four 14-3-3 protein family members (YWHAB, YWHAE, YWHAZ, YWHAG), which are known to regulate diverse signaling pathways. Our functional studies revealed that YWHAB is essential for maximal cyclic adenosine monophosphate (cAMP)-stimulated lipolysis, as its CRISPR-Cas9-mediated knockout mitigates lipolysis through a mechanism independent of insulin signaling. In summary, our use of proximity labeling not only comprehensively mapped the LD proteome in human adipocytes but also unveiled new regulatory mechanisms in adipocyte lipolysis control, specifically involving 14-3-3 proteins.