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

Western 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.