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Elevated Drp1-mediated mitochondrial fission was proposed to promote NAFLD, as inhibition of hepatic Drp1 in early life prevented high-fat diet induced liver steatosis in mice. However, whether Drp1-knockdown (Drp1si) can reverse established NASH in adulthood is unknown. Hepatocyte-restricted delivery of siRNA conjugated to N-acetyl-galactosamine (GalNac) is a safe and FDA-approved approach to selectively manipulate hepatic gene expression and treat human liver disease. Here, we show that hepatic Drp1si in adult healthy mice decreased body weight by 12% and induced liver damage, as shown by the elevation in circulating transaminases, liver inflammation, fibrosis and necrosis (p In conclusion, our study supports that increased Drp1 activity is an adaptative mechanism counteracting NASH that prevents ISR overactivation and mitigates ER stress to limit fibrosis, inflammation, and necrosis. Our study argues against blocking Drp1 in hepatocytes to combat NASH. Disclosure J. Steffen: Employee; Janssen Research & Development, LLC. J. Ngo: None. S. Wang: None. H. F. Kramer: Employee; Janssen Research & Development, LLC. L. D. Norquay: Employee; Janssen Pharmaceuticals, Inc. A. Pocai: Employee; Johnson & Johnson. O. Shirihai: None. M. Liesa: None.

OBJECTIVE—Insulin and contraction increase skeletal muscle glucose uptake through distinct and additive mechanisms. However, recent reports have demonstrated that both signals converge on the Akt substrate of 160 kDa (AS160), a protein that regulates GLUT4 translocation. Although AS160 phosphorylation is believed to be the primary factor affecting its activity, AS160 also possesses a calmodulin-binding domain (CBD). This raises the possibility that contraction-stimulated increases in Ca2+/calmodulin could also modulate AS160 function. RESEARCH DESIGN AND METHODS—To evaluate the AS160 CBD in skeletal muscle, empty-vector, wild-type, or CBD-mutant AS160 cDNAs were injected into mouse muscles followed by in vivo electroporation. One week later, AS160 was overexpressed by ∼14-fold over endogenous protein. RESULTS—Immunoprecipitates of wild-type and CBD-mutant AS160 were incubated with biotinylated calmodulin in the presence of Ca2+. Wild-type AS160, but not the CBD-mutant AS160, associated with calmodulin. Next, we measured insulin- and contraction-stimulated glucose uptake in vivo. Compared with empty-vector and wild-type AS160, insulin-stimulated glucose uptake was not altered in muscles expressing CBD-mutant AS160. In contrast, contraction-stimulated glucose uptake was significantly decreased in CBD-mutant–expressing muscles. This inhibitory effect on glucose uptake was not associated with aberrant contraction-stimulated AS160 phosphorylation. Interestingly, AS160 expressing both calmodulin-binding and Rab-GAP (GTPase-activating protein) domain point mutations (CBD + R/K) fully restored contraction-stimulated glucose uptake. CONCLUSIONS—Our results suggest that the AS160 CBD directly regulates contraction-induced glucose uptake in mouse muscle and that calmodulin provides an additional means of modulating AS160 Rab-GAP function independent of phosphorylation. These findings define a novel AS160 signaling component, unique to contraction and not insulin, leading to glucose uptake in skeletal muscle.

Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK alpha2-inactive (alpha2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK alpha2i transgenic mice. Combined AMPK alpha2 and Akt inhibition by wortmannin treatment of AMPK alpha2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK alpha2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.