The Dynamin-related GTPase, Drp1 (encoded by Dnm1l) plays a central role in mitochondrial fission and is requisite for numerous cellular processes however its role in muscle metabolism remains unclear. Herein, we show that among human tissues, the highest number of gene correlations with DNM1L  are in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced Complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized Complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial Complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

RNAseq: A homogenous portion of frozen skeletal muscle samples was homogenized using a tissue homogenizer in Trizol. RNA was isolated using the Qiagen RNeasy Mini QIAcube Kit following the manufacturer’s instructions. Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit and RiboZero Gold following the manufacturer’s instructions. The resulting libraries were combined into two pools and sequenced on an Illumina HiSeq 4000 within the UCLA Neuroscience Genomics core facility following in-house established protocols. For metabolomics, muscle samples were processed and analyzed by Metabolon Inc.