Organelle-Specific Lipid Profiles Underlie Metabolic Health in a Nutrition-Dependent Manner

Western diet (WD), characterized by high energy density and saturated fat, promotes obesity and insulin resistance (IR), yet how dietary lipid overload remodels skeletal muscle lipids at the subcellular level remains unclear. We investigated whether WD alters lipid class distribution and fatty acid (FA) incorporation within distinct muscle organelles and whether these changes relate to metabolic health. C57BL/6 J mice were fed WD or control chow for 12 weeks. Mitochondria and lipid droplets (LDs) were isolated from soleus for organelle-resolved lipidomics. WD induced obesity, dyslipidemia, early IR, and intramyocellular lipid accumulation without changes in mitochondrial content. Organelle-resolved analyses revealed compartment-specific lipid remodeling, hidden in whole muscle. Diacylglycerol (DAG) FA composition closely reflected dietary FA supply across compartments, whereas phospholipid remodeling was class and organelle dependent, with coordinated changes between mitochondria and LDs. Several phospholipid classes and LD-associated sn-1,3-DAG were associated with insulin sensitivity and substrate use in metabolically healthy mice, but WD disrupted these relationships. These findings demonstrate that lipid class identity, FA composition, and subcellular localization critically shape skeletal muscle responses to nutritional excess. By identifying organelle-specific lipid pools linked to early metabolic dysfunction, this study provides a framework that may inform future translational investigations of IR in human skeletal muscle. Article Highlights Skeletal muscle insulin resistance is linked to lipid metabolism, yet whole-tissue analyses obscure how subcellular lipid remodeling contributes to metabolic dysfunction. We investigated whether Western diet induces compartment-specific changes in skeletal muscle lipid classes and fatty acid (FA) composition and how these relate to metabolic health. Western diet disrupts the relationship between lipids and metabolism. Diacylglycerol FA composition reflected dietary supply. Phospholipid remodeling was class and compartment specific. Lipid droplet-localized 1,3-diacylglycerol, phosphatidylethanolamine, and phosphatidylglycerol reflected muscle health. These results highlight subcellular lipid organization as a key determinant of muscle insulin resistance and provide a framework for identifying early lipid signatures relevant to human diabetes research.