Gavini, C. K. | bioRxiv Science

bioRxiv2025

Organelle-specific lipid profiles influence/underlie metabolic health in a nutrition-dependent manner

AbstractWestern diet (WD), characterized by high energy density, saturated fat and sucrose, is a major driver of obesity and insulin resistance (IR). Although dietary fat composition influences systemic lipid metabolism and insulin sensitivity, its impact on subcellular lipid classes distribution and fatty acid (FA) incorporation in skeletal muscle remains poorly defined. We hypothesized that (1) modulating dietary FA intake remodels mitochondrial and lipid droplet (LD) lipid profiles, including phospholipids (PL) and diacylglycerol (DAG) stereoisomers implicated in lipotoxicity; and (2) organelle-specific lipid profiles relate to metabolic health. C57BL/6J mice were fed WD or control chow for 12 weeks. Whole-body metabolism, insulin sensitivity and substrate use were assessed by indirect calorimetry, glucose and insulin tolerance tests, and fasting biomarkers. Mitochondria and LDs were isolated from soleus muscle for organelle-resolved lipidomics. DAG isomers and PL classes were quantified, and FA chain length and saturation patterns were analyzed in total lysate (TL), mitochondria and LDs. Correlations were performed between lipid class abundance and metabolic parameters. WD-fed mice developed obesity, dyslipidemia and early IR. Intramyocellular lipids increased, whereas mitochondrial abundance was unchanged. Organelle-resolved lipidomics revealed distinct subcellular signatures not detectable in TL. DAG FA composition mirrored dietary FA supply across compartments, with WD increasing saturated FA (SFA) and reducing di-unsaturated FA (DiUFA) species. In contrast, PL remodeling was class- and compartment-specific, with coordinated changes in mitochondria and LDs that were masked in whole-muscle TL. Several PL classes, including ether-linked phosphatidylethanolamine (ePE), phosphatidylinositol, PE and phosphatidylcholine in TL and LD, and phosphatidylserine and phosphatidylglycerol (PG) in TL and mitochondria, were strongly associated with insulin sensitivity and substrate utilization in healthy mice, but these relationships were lost under WD. LD-associated sn1,3-DAG content was a strong predictor of metabolic health in lean mice, which related to ATGL abundance. PE and PG in LD were related to obesity markers. Dietary lipid overload induces distinct and compartment-specific remodeling of the skeletal muscle lipidome. DAG and PL classes exhibited divergent FA incorporation across mitochondria, LDs and TL, with coordinated remodeling between LDs and mitochondria that remained undetectable at the whole-muscle level. Several lipid pools, particularly LD-localized 1,3-DAG, PE and PG, were consistently related to metabolic flexibility and markers of metabolic health in healthy muscle and were disrupted in WD-fed mice. These findings identify lipid class identity, FA composition and subcellular localization as key determinants of muscle adaptation to nutritional excess and point to LD phospholipids and DAG stereoisomers as potential early molecular signatures of emerging IR.

physiology↗

bioRxiv2019

Hypothalamic C2-domain protein involved in MC4R trafficking and control of energy balance

ObjectiveRates of overweight and obesity epidemic have risen significantly in the past few decades, and 34% of adults and 15-20% of children and adolescents in the United States are now obese. Melanocortin receptor 4 (MC4R), contributes to appetite control in hypothalamic neurons and is a target for future anti-obesity treatments (such as setmelanotide) or novel drug development effort. Proper MC4R trafficking regulation in hypothalamic neurons is crucial for normal neural control of homeostasis and is altered in obesity and in presence of lipids. The mechanisms underlying altered MC4R trafficking in the context of obesity is still unclear. Here, we discovered that C2CD5 expressed in the hypothalamus is involved in the regulation of MC4R endocytosis. This study unmasked a novel trafficking protein nutritionally regulated in the hypothalamus providing a novel target for MC4R dependent pathways involved in bodyweight homeostasis and Obesity.\n\nMethodsTo evaluate the expression of C2cd5, we first used in situ hybridization and RNAscope technology in combination with electronic microscopy. For in vivo, we characterized the energy balance of wild type (WT) and C2CD5 whole-body knockout (C2CD5KO) mice fed normal chow (NC) and/or western-diet (high-fat/high-sucrose/cholesterol) (WD). To this end, we performed comprehensive longitudinal assessment of bodyweight, energy balance (food intake, energy expenditure, locomotor activity using TSE metabolic cages), and glucose homeostasis. In addition, we evaluated the consequence of loss of C2CD5 on feeding behavior changes normally induced by MC4R agonist (Melanotan, MTII) injection in the paraventricular hypothalamus (PVH). For in vitro approach, we tease out the role of C2CD5 and its calcium sensing domain C2 in MC4R trafficking. We focused on endocytosis of MC4R using an antibody feeding experiment (in a neuronal cell line - Neuro2A (N2A) stably expressing HA-MC4R-GFP; against HA-tag and analyzed by flux cytometry).\n\nResultsWe found that 1) the expression of hypothalamic C2CD5 is decreased in diet-induced obesity models compared to controls, 2) mice lacking C2CD5 exhibit an increase in food intake compared to WT mice, 3) C2CD5 interacts with endocytosis machinery in hypothalamus, 4) loss of functional C2CD5 (lacking C2 domain) blunts MC4R endocytosis in vitro and increases MC4R at the surface that fails to respond to MC4R ligand, and, 5) C2CD5KO mice exhibit decreased acute responses to MTII injection into the PVH.\n\nConclusionsBased on these, we conclude that hypothalamic C2CD5 is involved in MC4R endocytosis and regulate bodyweight homeostasis. These studies suggest that C2CD5 represents a new protein regulated by metabolic cues and involved in metabolic receptor endocytosis. C2CD5 represent a new target and pathway that could be targeted in Obesity.

physiology↗