was also positively associated with greater BMI (r=0.29; p<0.01) and hepatic steatosis score (r=0.33; p<0.01) and inversely with HDL (r=-0.28; p<0.01). Levels of ADMA were not associated with hepatic steatosis. Conclusions: These findings are the first to suggest that iNOS is an important mediator for increased hepatic steatosis in NAFLD. Further, allelic variation of iNOS, as well as circulating concentration may participate in the development of steatosis. iNOS may provide pathogenic clues that connect NAFLD with CVD. Mo1460 APOLIPOPROTEIN (APO)A-V DEFICIENCY ENHANCES HEPATIC SYNTHESIS OF CERAMIDE, A MAJOR FATTY ACID METABOLITE, TRIGGERING A PROGRESSION FROM SIMPLE STEATOSIS TO NONALCOHOLIC STEATOHEPATITIS (NASH) IN CHOW-FED APOA-V KO MICE Helen H. Wang, Guangyong Peng, Ornella de Bari, David A. Ford, Linda S. Zhang, Robert Ryan, Brent Neuschwander-Tetri, Patrick Tso, David Q. Wang Background: Many clinical studies have found that the accumulation of excess triglycerides (TG) in the liver truly is just an "innocent bystander" in the process leading to liver injury and inflammation, and other lipid metabolites may mediate hepatocyte lipotoxicity leading to NASH. We reported at the 2014 DDW that a major feature of the apoA-V KO mouse model that distinguishes it from other animal models of nonalcoholic fatty liver disease is that simple steatosis can spontaneously evolve to NASH, even on a chow diet. We hypothesize that elevated ceramide levels are responsible for triggering NASH due to its lipotoxicity that activates inflammatory responses, leading to hepatocyte apoptosis in chow-fed apoA-V KO mice. Methods: Male apoA-V KO and WT mice were fed standard rodent chow diet for 9 mo. Liver pathology was examined by H&E staining and immunohistochemistry. Lipidomic analyses were performed by ESI-MS/MS. Primary hepatocytes from these mice were treated with 0 or 80 μM of ceramide. Gene expression was studied by qPCR and Western blotting. Results: On chow, liver steatosis developed at the age of 4 mo, gradually advancing to NASH at the age of 7 mo in apoA-V KO, but not WT, mice. Hepatic total TG and cholesteryl ester concentrations were significantly higher in KO mice than in WT mice, with palmitic, palmitoleic, oleic and linoleic acids being the major fatty acids in hepatic TG. ApoA-V deficiency promotes ceramide synthesis by increasing expression of hepatic serine palmi- toyltransferase (SPT). Hepatic ceramide concentrations were higher in apoA-V KO mice than in WT mice (296±25 vs 132±14 nmol/g tissue, P<0.01). In contrast, SPT expression was significantly reduced in apoA-V KO mice transduced with AAV2/8-APOA-V, but not AAV2/ 8-LacZ (as control), thus reducing ceramide synthesis. Due to its cellular lipotoxicity, elevated ceramide induced hepatic lipo-apoptosis through TNFα by enhancing both caspase-induced apoptosis and ROS-mediated mitochondrial dysfunction in apoA-V KO, but not WT, mice. Apoptotic hepatocytes found by TUNEL and caspase-3 activation detected by immunohisto- chemistry were more frequent in the livers of KO mice after they developed NASH compared to aged-matched WT mice. After 12-h treatment with 80 μM of ceramide, numbers of TUNEL positive nuclei (i.e., apoptotic hepatocytes) and caspase-3 activation detected by immunocytochemistry were higher in apoA-V KO hepatocytes than in WT hepatocytes. Moreover, the percentage of early apoptotic cells detected by flow cytometry with annexin V staining was significantly higher in apoA-V KO hepatocytes than in WT hepatocytes. Conclusions: ApoA-V KO mice are an excellent animal model for studying the pathogenesis of NASH. Elevated ceramide levels may play a critical role in triggering hepatocyte apoptosis and liver injury, promoting the development of NASH in chow-fed apoA-V KO mice. Mo1461 DIETARY INTERVENTIONS AND THE ROLE OF THE MICROBIOME IN TREATMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE Fotini Tania Mitsinikos, Denise Chac, Nick Shillingford, William DePaolo Background: Non-alcoholic fatty liver disease (NAFLD) is a major health problem in many countries throughout the world. There is a clinical spectrum in NAFLD, ranging from steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis and alterations within the gut microbiota. Major factors contributing to NAFLD are over-nutrition and obesity. Therefore, dietary modifications are a cornerstone of treatment, despite low adherence and high relapse rates. Studies have examined the effects of diets with different fat and sugar content on the microbiota and NAFLD, yet few have examined the effects of a dietary intervention on a NAFLD-conditioned microbiota. Hypothesis: Replacing a NAFLD-inducing diet with a low fat/low fiber (LFF) or high saturated fat/high fiber (HFF) diet will reverse steatosis, normalize liver enzymes and dampen inflammation in a microbiome-dependent manner. Method: C57BL/6 mice were fed a NAFLD-inducing diet (50.5% of kcal from fat) for 6 weeks and randomly assigned to one of the following groups: 1) No treatment (continue NAFLD- inducing diet), 2) High saturated fat/high fiber (HFF), 3) Low saturated fat/low fiber (LFF). After 6 weeks of the dietary intervention, liver histology, serum AST/ALT, and cytokines were evaluated while 16S rRNA sequencing was performed on fecal and ileal contents. To dissociate the effect of diet from the microbiome, mice received a NAFLD-inducing diet for 6 weeks and randomized into one of the following groups for a fecal microbiota transplant (FMT): 1) NAFLD FMT, 2) LFF FMT, 3) HFF FMT. After the FMT the mice continued the NAFLD-inducing diet for 2 weeks then liver histopathology, AST/ALT and cytokines were evaluated. Results: Dietary intervention with LFF lead to complete reversal of steatosis and a reduction in AST/ALT levels in both LFF and HFF groups. Despite histological improve- ments, the livers of LFF and HFF mice had elevated levels of inflammatory cytokines.16S sequencing analysis revealed a reduction in diversity in mice fed the NAFLD-inducing diet with a complete loss of Bacteroides and an expansion in Akkermansia mucinophila. Mice receiving the LFF intervention diet maintained Akkermansia but also had an increase in Bifidobacteria. In contrast, each mouse receiving the HFF diet responded uniquely. FMT of either the LFF or HFF contents to NAFLD-fed mice lead to a partial reversal of steatosis but had little effect on ALT, AST and promoted liver inflammation. Conclusion: We demon- strate that the effect of dietary interventions is not microbiome-dependent as transfer of a microbiota was not sufficient to transfer protection if the NAFLD-inducing diet was main- tained and instead caused more inflammation. These data also suggest that adherence to a S-685 AGA Abstracts particular diet may be critical in determining the outcomes of treatment and underscores the complicated interplay between the microbiota, diet and disease. Mo1462 MYOKINES AS A MEDIATOR OF ANTI-NAFLD BENEFIT OF EXERCISE: CLINICAL AND BIOLOGICAL IMPLICATIONS Jonathan T. Ohm, Samuel J. Miller, Michelle Zhang, Priyanka Majety, Sanjib Chowdhury, Navneet Momi, Ashish K. Tiwari, Hemant K. Roy Background With obesity pandemic, the prevalence of fatty liver diseases (NAFLD and NASH) is constantly rising. NAFLD/NASH is projected to become the most common etiology of cirrhosis and a major determinant of hepatocellular carcinoma (HCC) burden. Exercise has been shown to improve hepatic steatosis regardless of weight loss but the mechanism remains unclear. Anti-NAFLD benefits of exercise were replicated in animal models which implicated the role of AMP-activated protein kinase (AMPK) signaling. Emerging evidence suggests that some of the health benefits of exercise may be mediated via myokines (cytokines secreted by contracting/exercising skeletal muscles). We, therefore, using a novel cell culture model of exercise aimed to prove the hypothesis that myokines were indeed important in the anti-NAFLD effect of exercise. Methods Concentrated exercise-conditioned media (in vitro exercise model with differentiated mouse skeletal muscle cells,C2C12, stimulated with C-Pace) was compared to similar media from non-contracted cells. We treated human HCC cell line HepG2 with concentrated myokines and analysed results using lipidomic analysis (mass spectroscopy), immunoblot, and Seahorse analysis. Results Higher overall concentra- tion of myokines in the exercise-conditioned media was confirmed with 40-gene myokine array (Figure 1). Treatment of HepG2 cells with myokine-rich supernatant lead to 30% decrease in total fatty acids on lipidomic analysis. Immunoblot analysis showed that treatment with myokine-rich supernatant increased HepG2 expresion of phospho-AMPK, phospho- acetyl CoA carboxylase (pACC, rate limiting in fatty acid oxidation) and pAKT by 2 fold, 2.5 fold and 1.7 fold, respectively (p<0.05); Similarly, myokine treatment resulted a 25% decrease in extracellular acidification rate (ECAR) of HepG2 as determined by Seahorse XF analysis, indicative of decreased glycolysis which translates into lesser production of acetyl CoA, a key substrate for fatty acid synthesis. These effects were abrogated when only supernatants >50kDa were used (which excludes myokines because their molecular weight is typically <50kDa), further implicating myokines as the effector molecules of anti-NAFLD benefit of exercise. Conclusion We demonstrate, for the first time, that myokines released from exercising skeletal muscles can decrease liver fat through both increased oxidation and decreased synthesis of fatty acids. This may have implications for the type/duration of exercise needed for mitigation of NAFLD and provides a platform to elucidate the precise molecular targets. Further studies are ongoigng to identify specific myokine/group of myokines that mediate the anti-NAFLD benefit of exercise. Myokine array Myokine-pAMPK-pAKT pathway AGA Abstracts