Mitochondrial dysfunction is a characteristic trait of human and rodent obesity, insulin resistance and fatty liver disease. Here we show that high-fat diet (HFD) feeding causes mitochondrial fragmentation in inguinal white adipocytes from male mice, leading to reduced oxidative capacity by a process dependent on the small GTPase RalA. RalA expression and activity are increased in white adipocytes after HFD. Targeted deletion of RalA in white adipocytes prevents fragmentation of mitochondria and diminishes HFD-induced weight gain by increasing fatty acid oxidation. Mechanistically, RalA increases fission in adipocytes by reversing the inhibitory Ser637 phosphorylation of the fission protein Drp1, leading to more mitochondrial fragmentation. Adipose tissue expression of the human homolog of Drp1, DNM1L, is positively correlated with obesity and insulin resistance. Thus, chronic activation of RalA plays a key role in repressing energy expenditure in obese adipose tissue by shifting the balance of mitochondrial dynamics toward excessive fission, contributing to weight gain and metabolic dysfunction.

Mitochondrial dysfunction is a characteristic trait of human and rodent obesity, insulin resistance, and fatty liver disease. Here we report that mitochondria undergo fragmentation and reduced oxidative capacity specifically in inguinal white adipose tissue after feeding mice high fat diet (HFD) by a process dependent on the small GTPase RalA. RalA expression and activity are increased in white adipocytes from mice fed HFD. Targeted deletion of Rala in white adipocytes prevents the obesity-induced fragmentation of mitochondria and produces mice resistant to HFD-induced weight gain via increased fatty acid oxidation. As a result, these mice also exhibit improved glucose tolerance and liver function. In vitro mechanistic studies revealed that RalA suppresses mitochondrial oxidative function in adipocytes by increasing fission through reversing the protein kinase A-catalyzed inhibitory Ser⁶³⁷phosphorylation of the mitochondrial fission protein Drp1. Active RalA recruits protein phosphatase 2A (PP2Aa) to specifically dephosphorylate this inhibitory site on Drp1, activating the protein, thus increasing mitochondrial fission. Adipose tissue expression of the human homolog of Drp1, DNML1, is positively correlated with obesity and insulin resistance in patients. Thus, chronic activation of RalA plays a key role in repressing energy expenditure in obese adipose tissue by shifting the balance of mitochondrial dynamics towards excessive fission, contributing to weight gain and related metabolic dysfunction.