Combined weight loss and exercise training alters skeletal muscle subcellular lipid localization and intermuscular adipose tissue cellular composition

Subcellular lipid accumulation and intermuscular adipose tissue (IMAT) accumulation are associated with insulin resistance, but the impact of combined weight loss and exercise training on localization of lipids and IMAT cellular composition is not known. Twenty-one adults with obesity (18F/3M; 46+2y; 35.0+0.9 kg/m²) completed a 3-month supervised weight loss and exercise training intervention. Insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp and basal and insulin-stimulated vastus lateralis biopsies were collected pre- and post-intervention. After the intervention, body weight and body fat decreased (11±1% and 9±1%), while VO₂ peak and insulin sensitivity increased (14±3% and 68±14%). Lipidomics revealed reduced sarcolemmal and nuclear triglycerides, with unchanged whole-muscle triglycerides. Whole muscle 1,2-diacylglycerols increased due to increased nuclear 1,2-diacylglycerols without PKCε, θ, or δ activation. Whole muscle sphingolipid levels increased due to cytosolic accumulation. Single nuclei RNA-seq showed altered IMAT cellular composition, including increased fibro-adipogenic progenitors, vascular cells, and macrophages, and decreased preadipocytes. Bulk muscle RNA-seq indicated upregulation of genes related to muscle remodeling and cellular respiration and there were changes in the relationship between nuclear diacylglycerols and gene expression post-intervention. These findings dissociate improvements in insulin sensitivity from total muscle DAG and sphingolipid levels and highlight roles for subcellular lipid redistribution and IMAT remodeling in insulin sensitization.