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<0.001) . Decreased body weight was largely explained by Gdf15 actions decreasing food intake by 11%, as Gdf15 receptor (Gfral) KO prevented the decrease in food intake and mitigated the weight loss caused by Drp1-KD. An activation of the Atf4- integrated stress response (ISR) in liver explained the 4x fold increase in hepatic Gdf15 expression. Drp1si increased both ER stress and the activity of the mitochondrial protease Oma1, two processes that converge in increasing EIf2alpha phosphorylation and Atf4 expression. While NASH induced by GAN diet did not activate Oma1 by itself, Drp1si in mice with NASH was sufficient to activate Oma1 and exacerbate NASH-induced ER-stress. These led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis. 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:The mitochondrial fission protein Drp1 was proposed to promote NAFLD, as inhibition of hepatocyte Drp1 early in life prevents liver steatosis induced by high-fat diet in mice. However, whether Drp1-knockdown in older mice can reverse established NASH is unknown.METHODS:N-acetylgalactosamine-siRNA conjugates, an FDA approved method to deliver siRNA selectively to hepatocytes, were used to knockdown hepatocyte-Drp1 in mice (NAG-Drp1si). NASH was induced in C57BL/6NTac mice by Gubra-Amylin-NASH diet (D09100310, 40% fat, 22% fructose and 2% cholesterol) and treatment with NAG-Drp1si was started at week 24 of diet. Circulating transaminases, liver histology, gene expression of fibrosis and inflammation markers, and hydroxyproline synthesis determined NASH severity. Liver NEFA and triglycerides were quantified by GC/MS. Mitochondrial function was determined by respirometry. Western blots of Oma1, Opa1, p-eIf2α, as well as transcriptional analyses of Atf4-regulated genes determined ISR engagement.RESULTS:NAG-Drp1si treatment decreased body weight and induced liver inflammation in adult healthy mice. Increased hepatic Gdf15 production was the major contributor to body-weight loss caused by NAG-Drp1si treatment, as Gdf15 receptor deletion (Gfral KO) prevented the decrease in food intake and mitigated weight loss. NAG-Drp1si activated the Atf4-controlled integrated stress response (ISR) to increase hepatic Gdf15 expression. NAG-Drp1si in healthy mice caused ER stress and activated the mitochondrial protease Oma1, which are the ER and mitochondrial triggers that activate the Atf4-controlled ISR. Remarkably, induction of NASH was not sufficient to activate Oma1 in liver. However, NAG-Drp1si treatment was sufficient to activate Oma1 in adult mice with NASH, as well as exacerbating NASH-induced ER stress. Consequently, NAG-Drp1si treatment in mice with NASH led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis.CONCLUSION:Drp1 mitigates NASH by decreasing ER stress, preventing Oma1 activation and ISR exacerbation. The elevation in Gdf15 actions induced by NAG-Drp1si might represent an adaptive response decreasing the nutrient load to liver when mitochondria are misfunctional. Our study argues against blocking Drp1 in hepatocytes to combat NASH.

The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.

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.

AS160 phosphorylation has recently emerged as a critical regulatory step in contraction- and insulin-stimulated glucose uptake in skeletal muscle. Mice express a long and short AS160 splice-variant differing by a single exon. Using PCR we determined that skeletal muscle expresses 17-fold more long form than short form AS160 mRNA. With cell free assays we found that two putative AS160 kinases in skeletal muscle, AMP-activated protein kinase (AMPK) and Akt, directly phosphorylate the AS160 long form. To compare fiber type-specific AS160 regulation, we used soleus (SOL, rich in type I fibers), gastrocnemius (GAS, both type II and I), and tibialis anterior (TA, predominantly type II) muscles. Relative AS160 protein expression was greatest in SOL (2.3 ± 0.08) compared to GAS (1.0 ± 0.03) and TA (0.45 ± 0.04). In contrast, AS160 phosphorylation in response to in vivo treatment with AICAR (AMPK activator) or insulin was greatest in TA [AICAR: TA (1.8 ± 0.06); GAS (1.3 ± 0.03); SOL (1.09 ± 0.17), Insulin: TA (2.1 ± 0.2); GAS (1.9 ± 0.3); SOL (1.8 ± 0.3)]. Thus, type II fibers have an increased ratio of AS160 phosphorylation to AS160 protein expression. We conclude that the AS160 long form is the predominant splice-variant expressed in skeletal muscle, and that AMPK and Akt are AS160 kinases. Fiber-type is a key determinant of AS160 phosphorylation in skeletal muscle. Support: NIH F32 DK07851, R01DK25336, R01AR42238

Mitogen-activated protein kinases (MAPKs) and NF-kappaB are two major regulators of gene transcription and metabolism in response to oxidative, energetic, and mechanical stress in skeletal muscle. Chronic activation of these signaling pathways has been implicated in the development and perpetuation of various pathologies, such as diabetes and cachexia. However, both MAPK and NF-kappaB are also stimulated by exercise, which promotes improvements in fuel homeostasis and can prevent skeletal muscle atrophy. This review will first discuss the major MAPK signaling modules in skeletal muscle, their differential activation by exercise, and speculated functions on acute substrate metabolism and exercise-induced gene expression. Focus will then shift to examination of the NF-kappaB pathway, including its mechanism of activation by cellular stress and its putative mediation of exercise-stimulated adaptations in antioxidant status, tissue regeneration, and metabolism. Although limited, there is additional evidence to suggest cross talk between MAPK and NF-kappaB signals with exercise. The objectives herein are twofold: 1) to determine how and why exercise activates MAPK and NF-kappaB; and 2) to resolve their paradoxical activation during diseased and healthy conditions.

The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr(308) or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser(9) phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr(308) phosphorylation and GSK-3alpha Ser(21) phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3alpha Ser(21) phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.

Insulin and contraction are potent stimulators of GLUT4 translocation and increase skeletal muscle glucose uptake. We recently identified the Rab GTPase-activating protein (GAP) AS160 as a putative point of convergence linking distinct upstream signaling cascades induced by insulin and contraction in mouse skeletal muscle. Here, we studied the functional implications of these AS160 signaling events by using an in vivo electroporation technique to overexpress wild type and three AS160 mutants in mouse tibialis anterior muscles: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate sites (4P); 2) AS160 mutated to abolish Rab GTPase activity (R/K); and 3) double mutant AS160 containing both 4P and R/K mutations (2M). One week following gene injection, protein expression for all AS160 isoforms was elevated over 7-fold. To determine the effects of AS160 on insulin- and contraction-stimulated glucose uptake in transfected muscles, we measured [3H]2-deoxyglucose uptake in vivo following intravenous glucose administration and in situ muscle contraction, respectively. Insulin-stimulated glucose uptake was significantly inhibited in muscles overexpressing 4P mutant AS160. However, this inhibition was completely prevented by concomitant disruption of AS160 Rab GAP activity. Transfection with 4P mutant AS160 also significantly impaired contraction-stimulated glucose uptake, as did overexpression of wild type AS160. In contrast, overexpressing mutant AS160 lacking Rab GAP activity resulted in increases in both sham and contraction-stimulated muscles. These data suggest that AS160 regulates both insulin- and contraction-stimulated glucose metabolism in mouse skeletal muscle in vivo and that the effects of mutant AS160 on the actions of insulin and contraction are not identical. Our findings directly implicate AS160 as a critical convergence factor for independent stimulators of skeletal muscle glucose uptake.

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 alpha 2-inactive (alpha 2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK alpha 2i transgenic mice. Combined AMPK alpha 2 and Akt inhibition by wortmannin treatment of AMPK alpha 2 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 alpha 2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.