Skeletal muscle is responsible for nearly 70 % of insulin-stimulated glucose processing, making it the body's largest energy reservoir. Severe obesity and type 2 diabetes mellitus reduce glucose uptake and processing in skeletal muscle, impairing muscle performance. Muscle glucose, fatty acids, and lipids processing is closely linked to its fiber type composition, which includes slow (type I) or fast (type IIa, IIx, IIb) fibers, each with distinct metabolic characteristics. Shifts in fiber type composition significantly impact whole-body energy homeostasis. Mitochondria, the most abundant organelles in both fiber types, have their activity regulated by fusion and fission processes responsive to nutritional and metabolic signals. Understanding how obesity and pre-diabetic stages affect individual muscle cells is vital for comprehending metabolic changes in muscle tissue.

To investigate metabolic changes between fiber types in response to high-fat diet (HFD), we isolated 50 single soleus muscle fibers of wild-type mice subjected to a control diet (CD) or HFD (n=4) for 16-weeks. Single myofibers were transferred to 96-well-plates and subjected to tryptic digestion according to the SP3 protocol. Desalted peptides were analysed with short 21-min LC-MS gradients in data-independent acquisition mode with an ion mobility mass spectrometer.

In total we identified ~2,600 proteins and ~2,000 proteins per single muscle cell. Marker proteins of metabolic pathways, including the citric acid cycle, ß-oxidation and fatty acid metabolism, were significantly altered, confirming the pre-diabetic phenotype in response to the HFD. The significant decrease in the fibre type-specific protein MYH7, consequently type I fibres, complicated comparative analysis between CD and HFD conditions. To address this, we used over 50 known fiber type proteins for cluster analysis, resulting in four clusters representing type I, type IIa fibers, mixed type I/IIa, and mixed type IIa/IIx fibers under normal and HFD conditions.

Our analysis indicated an increase in mitochondrial content in pre-diabetic mice, with an increased abundance of the mitochondrial fission protein 1 (MTFP1) exclusively in type IIa fibers. Immunostaining against MTPF1 and TOM20 substantiated our proteomics findings and revealed an elevated mitochondrial fission in type IIa fibers compared to slow type I fibers in the soleus. Our analysis revealed significant metabolic changes in type IIa compared to type I fibers, suggesting greater sensitivity of type IIa fibers to stress conditions such as increased fatty acid intake. This study also introduced an alternative bioinformatic approach for classifying muscle fiber types independently of MYH, enabling more accurate comparability between fiber types. We are convinced that analysing individual muscle fibers will provide a more precise view of metabolic changes during aging, in metabolic syndromes and neuromuscular diseases such as amyotrophic lateral sclerosis.