The Loss of Drp1 Improves Skeletal Muscle Insulin Action in Primary Myotubes Derived from Humans with Severe Obesity.

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. Excessive Drp1-mediated mitochondrial fission in skeletal muscle from humans with severe obesity is associated with impaired insulin action. However, it remains unclear whether specific inhibition of Drp1 in skeletal muscle cells alleviates insulin resistance in obesity. Therefore, this study aims to determine the direct role of Drp1 on regulating insulin action in human skeletal muscle cells derived from humans with severe obesity. Human skeletal muscle cells from six lean, insulin-sensitive (LN, BMI = 22.7 ± 1.2 kg/m ,HOMA-IR = 1.9 ± 0.4) and six severely obese, insulin-resistant (OB, BMI = 47.3 ± 2.8 kg/m , HOMA-IR = 3.4 ± 0.4) subjects were pooled together, respectively. At 90% confluency, myoblasts were transfected using polyethyleneimine with a Drp1 shRNA (shDrp1) or scramble shRNA constructs (shCtrl). After 48 h, the medium was replaced with differentiation media with puromycin. On day 7 of differentiation, the mitochondrial network, reactive oxygen species (ROS), insulin signaling, glucose uptake, and protein markers of mitochondrial dynamics and mitochondrial content were assessed. RNA sequencing was also performed on OB-shCtrl and OB-shDrp1 myotubes. Differentially regulated genes were identified, and a gene set enrichment was used to determine pathway modulations. Drp1 protein expression was reduced in OB-shDrp1 myotubes compared to LN-shCtrl and OB-shCtrl (72% and 78%, respectively, P<0.05). OB-shCtrl myotubes exhibited fragmented mitochondrial networks with an increase in the number of non-networked individual mitochondria compared to the LN-shCtrl (P<0.05). The loss of Drp1 in OB-shDrp1 myotubes restored the mitochondrial network structure with the reduced number of non-networked mitochondria compared to OB-shCtrl (P<0.05), and is not different from LN-shCtrl. There were no differences in protein expression of markers of mitochondrial dynamics and content. Regarding insulin action, insulin-stimulated Akt Ser phosphorylation and glucose uptake (over basal condition) were both reduced in OB-shCtrl myotubes compared to LN-shCtrl (P<0.05). Importantly, OB-shDrp1 myotubes significantly increased insulin-stimulated Akt Ser phosphorylation and glucose uptake compared to OB-shCtrl (P<0.05). In addition, ROS was elevated in OB-shCtrl myotubes when compared to LN-shCtrl (P<0.05) but was reduced in OB-shDrp1 myotubes (P<0.05). Lastly, the loss of Drp1 revealed an upregulation in genes responsible for fatty acid oxidation and downregulation in genes for glycolysis in OB-shDrp1 myotubes compared to OB-shCtrl. These results demonstrate that the loss of Drp1 improves mitochondrial morphology and enhances insulin action, which may, at least partially, be due to reduced ROS production and improved fat oxidation in skeletal muscle cells from humans with severe obesity. Our data suggest that Drp1 may serve as an important regulator of skeletal muscle insulin action.