Tripartite ER-Mitochondria-Lipid Droplets contact sites control adipocyte metabolic flexibility

Abstract

Obesity is a major risk factor for cardiometabolic diseases, with adipocyte dysfunction playing a central role. Understanding how lipid storage and mobilization are regulated--and disrupted--in adipocytes is key to addressing obesity-associated complications. The ER-anchored protein Seipin controls lipid droplet (LD) biogenesis and maintenance, and its loss disrupts ER-LD contact sites. In humans, Seipin deficiency causes generalized lipodystrophy, a severe form of adipocyte dysfunction. We previously showed that Seipin also localizes at ER-mitochondria contact sites (MAM), where it regulates calcium exchange and mitochondrial function. Here, we examined whether Seipin targeting to MAM and ER-LD sites overlaps functionally. We analyzed subcutaneous adipose tissue (AT) from inducible Seipin-knockout mice using transmission electron microscopy (TEM) and proximity ligation assays (PLA) to quantify membrane contact sites (MCS) involving the ER, LDs, and mitochondria. In control mice, feeding reduced MAMs while increasing ER-LD and mitochondria-LD contacts, whereas Seipin deficiency abolished this remodeling. Specifically, under lipid loading, MAMs located in proximity to LDs--tripartite contact sites known as MAM-LD--were increased in control but not in Seipin-deficient adipocytes. Fluorescence recovery after photobleaching assays revealed that Seipin depletion impairs triglyceride transfer to LDs, an effect rescued by the MAM-LD-reinforcing synthetic peptide Linker-ER-Mi. Importantly, this rescue was abolished by silencing the mitochondrial calcium uniporter, demonstrating that calcium exchange is critical for triglyceride storage in LDs. We further investigated how MAM-LD remodeling influences adipocyte metabolic flexibility. Using TEM and PLA, we monitored two MAM subtypes: those forming MAM-LD and those engaging cytosolic mitochondria (MAM-CM). During adipogenesis, MAM-LD frequency increased while MAM-CM decreased. Similarly, in mouse AT and 3T3-L1 adipocytes, lipid loading selectively promoted MAM-LD. Notably, this adaptive remodeling of membrane contact sites was blunted in the adipose tissue of diet-induced obese mice. Genetic disruption of MCS in 3T3-L1 adipocytes altered lipid flux, impaired lipolysis, and reduced insulin signaling. Together, our findings identify MAM-LD contacts as key regulators of adipocyte lipid handling and metabolic flexibility, whose disruption may underlie the metabolic inflexibility of obesity.