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232 Mixed Lineage Kinase 3 Mediates the Release of Proinflammatory Extracellular Vesicles in Nonalcoholic Steatohepatitis
AASLD Abstracts
(SIRT3) is an NAD(+)-dependent protein deacetylase that acts as key metabolic sensor and mediates adaptive responses to metabolic stressors such as calorie restriction, fasting and exercise. SIRT3 also has the potential to improve hepatic steatosis and insulin resistance. In the current study, we hypothesized that the hepatic overexpression of SIRT3 might salvage hepatic steatosis and insulin resistance phenotype associated with the MTP deficiency. Methods: To accelerate the phenotype associated with MTP deficiency, 3-4 months old MTPHT mice were fed high-fat diet for 16 weeks. Mice were then subjected to tail vein injection with SIRT3 or β-gal (control) adenovirus. Serum glucose, insulin, triglyceride and β-hydroxybutyrate were evaluated. Liver tissues were used to measure triglyceride content, mitochondrial fatty acid oxidation and oxygen consumption. Hepatic mitochondrial superoxide dismutase2 (SOD2) and basal AKT phosphorylation levels were also assessed. Results: Hepatic overexpression of SIRT3 in MTP-HT mice reduced hepatic steatosis, increased mitochondrial fatty acid oxidation and oxygen consumption. Serum β-hydroxybutyrate levels were increased in response to SIRT3 overexpression consistent with improved mitochondrial fatty acid oxidation. Liver mitochondrial SOD 2 levels increased in response to SIRT 3 expression suggesting increased antioxidant capabilities against oxidative stress. Fasting serum levels of glucose and insulin were reduced. Basal levels of phosphorylated AKT were decreased consistent with improved insulin sensitivity. Conclusions: Hepatic expression of SIRT3 rescues diet-induced hepatic steatosis and may play an important role in regulating oxidative stress and insulin sensitivity. Together, our results suggest that the manipulation of Sirt3 expression may represent a potential novel strategy to manage mitochondrial disorders in humans. Serum and liver changes in response to SIRT3 overexpression
232 Mixed Lineage Kinase 3 Mediates the Release of Proinflammatory Extracellular Vesicles in Nonalcoholic Steatohepatitis Samar H. Ibrahim, Petra Hirsova, Steven F. Bronk, Stephen A. Harrison, Val Goodfellow, Gregory J. Gores Backgrounds and aims: We have recently demonstrated that genetic deletion of mixed lineage kinase 3 (MLK3) reduces inflammation in a murine model of NASH. However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are not fully elucidated. Our hypothesis is that MLK3 mediates the release of chemokine rich extracellular vehicles (EVs), which induce macrophage activation & trafficking to the liver in NASH. Methods: Two MLK3 inhibitors CLFB-1134 and URMC099 were kindly provided by Califia Bio, Inc. Primary mouse hepatocytes (PMH) and Huh7 cells were treated with lysophosphatidylcholine (LPC), a toxic metabolite of saturated fatty acid with and without one of the MlK3 inhibitors. Released EVs were isolated by differential ultracentrifugation, quantified by nanoparticle tracking analysis, and employed for macrophage treatment. EVs protein contents were profiled by mass spectrometry (MSP). Results: LPC treatment of PMH & Huh7 cells induced a 3.6-fold and 140-fold increase in release of EVs, respectively, which was prevented by either genetic knock down or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine CXCL10 in the EVs. CXCL10 was markedly enriched in EVs isolated from LPC treated PMH versus untreated cells, as assessed by immuno-gold electron microscopy and immunoblot analysis. Unexpectedly, either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EV (corrected for the number of EV secreted). Treatment of mouse bone marrow-derived macrophages with physiologically relevant concentrations of lipotoxic hepatocyte-derived EVs induced macrophage activation and chemotaxis, an effect blocked by incubation with CXCL10 neutralizing antiserum. Finally, activating phospho-MLK3 expression was increased in liver biopsies of patients with fatty liver compared to the normal control. In Conclusion: during hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes which are proinflammatory for macrophages. We speculate that these EVs mediate hepatic inflammation in vivo by inducing macrophages trafficking to the liver and that inhibition of MLK3-dependent EV release from hepatocytes could be salutary in human NASH.
234 GDNF Regulates Hepatic Steatosis Through Modulation of miRNA-122 and miRNA-107 Ross B. Deppe, Simon M. Mwangi, Alton B. Farris, Shanthi Srinivasan Aims: Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming a global health problem that demands quick action to stem its spread. We have previously reported that glial cell line derived neurotrophic factor (GDNF) is protective against high fat diet-induced obesity and hepatic steatosis however the mechanisms involved have not yet been determined. Here we examined the effects of GDNF on the expression in the liver of genes known to be involved in NAFLD and of microRNAs (miRNA s) that regulate these genes. Methods: Six weeks old wild type (WT) mice and GDNF transgenic (GDNF Tg) littermates that overexpressed GDNF in glia were fed a high fat diet (HFD; 60% cal from fat) or a regular diet (RD; 18% cal from fat) for 12 weeks. Histological scoring of NAFLD was performed on H& E and Sirius Red-stained liver sections. In vitro the effects of GDNF were assessed using Hep G2 cells cultured for 66 h in medium supplemented with or without GDNF, oleic acid and palmitate. Gene expression and miRNA levels were assessed by real-time PCR. Results: Liver from WT mice fed the HFD had significant mixed macrovesicular and microvesicular steatosis (10 + 1.58%, P<0.001, n=5), but no evidence of inflammation or fibrosis. They also had significantly increased expression of mRNA for the fatty acid transporter CD36 and the enzyme stearoyl-Coenzyme A desaturase 1 (Scd1) which catalyzes the synthesis of monosaturated fatty acids. In contrast, livers from GDNF Tg mice fed the HFD showed no evidence of steatosis, inflammation or fibrosis. They also had significantly decreased expression of mRNA for the de novo lipogenesis enzymes fatty acid synthase (Fasn), Scd1, and diacylglycerol O-acyltransferase 2 (Dgat2), and of mRNA for PPAR-gamma and PPAR-α, that are associated with increased hepatic steatosis. The levels of miR-122 and miR-107, that down-regulate liver Fasn expression, were significantly increased (respectively, 1.365 + 0.05 fold, P<0.001 and 1.312 + 0.087 fold, P<0.05) in liver from GDNF Tg mice fed the HFD, but unchanged in liver from WT mice fed the HFD relative to those in liver from WT mice fed RD. In vitro, GDNF enhanced the levels of miR-122 in Hep G2 cells (1.455 + 0.1, P<0.01, n=6, relative to vehicle) and blunted oleic acid and palmitate-induced miR122 suppression. GDNF also significantly enhanced the levels of miR-107 in Hep G2 cells cultured in GDNF alone (1.461 + 0.115 fold, P<0.05, n=6) or in the presence of oleic acid and palmitate (1.667 + 0.055, P<0.001, n=6). Conclusions: We demonstrate a novel role for GDNF in protecting against hepatic steatosis through miR-122 and miR-107 induced modulation of pro-steatotic genes. These miRNAs could be potential therapeutic targets for hepatic steatosis.
233 Hepatic Overexpression of SIRT3 in Mice Heterozygous for Mitochondrial Trifunctional Protein Rescues Hepatic Steatosis and Improves Insulin Sensitivity Fatiha Nassir, Justin J. Arndt, Jamal A. Ibdah Background: Mitochondrial trifunctional protein (MTP) is an enzyme complex responsible for catalyzing the last three steps in long-chain fatty acid β-oxidation. Studies using heterozygous mice for MTP (HT-MTP), generated by our group, have linked defect in mitochondrial dysfunction with hepatic steatosis and insulin resistance in 15-18 months mice. Sirtuin 3
S-973
AASLD Abstracts
(SIRT3) is an NAD(+)-dependent protein deacetylase that acts as key metabolic sensor and mediates adaptive responses to metabolic stressors such as calorie restriction, fasting and exercise. SIRT3 also has the potential to improve hepatic steatosis and insulin resistance. In the current study, we hypothesized that the hepatic overexpression of SIRT3 might salvage hepatic steatosis and insulin resistance phenotype associated with the MTP deficiency. Methods: To accelerate the phenotype associated with MTP deficiency, 3-4 months old MTPHT mice were fed high-fat diet for 16 weeks. Mice were then subjected to tail vein injection with SIRT3 or β-gal (control) adenovirus. Serum glucose, insulin, triglyceride and β-hydroxybutyrate were evaluated. Liver tissues were used to measure triglyceride content, mitochondrial fatty acid oxidation and oxygen consumption. Hepatic mitochondrial superoxide dismutase2 (SOD2) and basal AKT phosphorylation levels were also assessed. Results: Hepatic overexpression of SIRT3 in MTP-HT mice reduced hepatic steatosis, increased mitochondrial fatty acid oxidation and oxygen consumption. Serum β-hydroxybutyrate levels were increased in response to SIRT3 overexpression consistent with improved mitochondrial fatty acid oxidation. Liver mitochondrial SOD 2 levels increased in response to SIRT 3 expression suggesting increased antioxidant capabilities against oxidative stress. Fasting serum levels of glucose and insulin were reduced. Basal levels of phosphorylated AKT were decreased consistent with improved insulin sensitivity. Conclusions: Hepatic expression of SIRT3 rescues diet-induced hepatic steatosis and may play an important role in regulating oxidative stress and insulin sensitivity. Together, our results suggest that the manipulation of Sirt3 expression may represent a potential novel strategy to manage mitochondrial disorders in humans. Serum and liver changes in response to SIRT3 overexpression
232 Mixed Lineage Kinase 3 Mediates the Release of Proinflammatory Extracellular Vesicles in Nonalcoholic Steatohepatitis Samar H. Ibrahim, Petra Hirsova, Steven F. Bronk, Stephen A. Harrison, Val Goodfellow, Gregory J. Gores Backgrounds and aims: We have recently demonstrated that genetic deletion of mixed lineage kinase 3 (MLK3) reduces inflammation in a murine model of NASH. However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are not fully elucidated. Our hypothesis is that MLK3 mediates the release of chemokine rich extracellular vehicles (EVs), which induce macrophage activation & trafficking to the liver in NASH. Methods: Two MLK3 inhibitors CLFB-1134 and URMC099 were kindly provided by Califia Bio, Inc. Primary mouse hepatocytes (PMH) and Huh7 cells were treated with lysophosphatidylcholine (LPC), a toxic metabolite of saturated fatty acid with and without one of the MlK3 inhibitors. Released EVs were isolated by differential ultracentrifugation, quantified by nanoparticle tracking analysis, and employed for macrophage treatment. EVs protein contents were profiled by mass spectrometry (MSP). Results: LPC treatment of PMH & Huh7 cells induced a 3.6-fold and 140-fold increase in release of EVs, respectively, which was prevented by either genetic knock down or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine CXCL10 in the EVs. CXCL10 was markedly enriched in EVs isolated from LPC treated PMH versus untreated cells, as assessed by immuno-gold electron microscopy and immunoblot analysis. Unexpectedly, either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EV (corrected for the number of EV secreted). Treatment of mouse bone marrow-derived macrophages with physiologically relevant concentrations of lipotoxic hepatocyte-derived EVs induced macrophage activation and chemotaxis, an effect blocked by incubation with CXCL10 neutralizing antiserum. Finally, activating phospho-MLK3 expression was increased in liver biopsies of patients with fatty liver compared to the normal control. In Conclusion: during hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes which are proinflammatory for macrophages. We speculate that these EVs mediate hepatic inflammation in vivo by inducing macrophages trafficking to the liver and that inhibition of MLK3-dependent EV release from hepatocytes could be salutary in human NASH.
234 GDNF Regulates Hepatic Steatosis Through Modulation of miRNA-122 and miRNA-107 Ross B. Deppe, Simon M. Mwangi, Alton B. Farris, Shanthi Srinivasan Aims: Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming a global health problem that demands quick action to stem its spread. We have previously reported that glial cell line derived neurotrophic factor (GDNF) is protective against high fat diet-induced obesity and hepatic steatosis however the mechanisms involved have not yet been determined. Here we examined the effects of GDNF on the expression in the liver of genes known to be involved in NAFLD and of microRNAs (miRNA s) that regulate these genes. Methods: Six weeks old wild type (WT) mice and GDNF transgenic (GDNF Tg) littermates that overexpressed GDNF in glia were fed a high fat diet (HFD; 60% cal from fat) or a regular diet (RD; 18% cal from fat) for 12 weeks. Histological scoring of NAFLD was performed on H& E and Sirius Red-stained liver sections. In vitro the effects of GDNF were assessed using Hep G2 cells cultured for 66 h in medium supplemented with or without GDNF, oleic acid and palmitate. Gene expression and miRNA levels were assessed by real-time PCR. Results: Liver from WT mice fed the HFD had significant mixed macrovesicular and microvesicular steatosis (10 + 1.58%, P<0.001, n=5), but no evidence of inflammation or fibrosis. They also had significantly increased expression of mRNA for the fatty acid transporter CD36 and the enzyme stearoyl-Coenzyme A desaturase 1 (Scd1) which catalyzes the synthesis of monosaturated fatty acids. In contrast, livers from GDNF Tg mice fed the HFD showed no evidence of steatosis, inflammation or fibrosis. They also had significantly decreased expression of mRNA for the de novo lipogenesis enzymes fatty acid synthase (Fasn), Scd1, and diacylglycerol O-acyltransferase 2 (Dgat2), and of mRNA for PPAR-gamma and PPAR-α, that are associated with increased hepatic steatosis. The levels of miR-122 and miR-107, that down-regulate liver Fasn expression, were significantly increased (respectively, 1.365 + 0.05 fold, P<0.001 and 1.312 + 0.087 fold, P<0.05) in liver from GDNF Tg mice fed the HFD, but unchanged in liver from WT mice fed the HFD relative to those in liver from WT mice fed RD. In vitro, GDNF enhanced the levels of miR-122 in Hep G2 cells (1.455 + 0.1, P<0.01, n=6, relative to vehicle) and blunted oleic acid and palmitate-induced miR122 suppression. GDNF also significantly enhanced the levels of miR-107 in Hep G2 cells cultured in GDNF alone (1.461 + 0.115 fold, P<0.05, n=6) or in the presence of oleic acid and palmitate (1.667 + 0.055, P<0.001, n=6). Conclusions: We demonstrate a novel role for GDNF in protecting against hepatic steatosis through miR-122 and miR-107 induced modulation of pro-steatotic genes. These miRNAs could be potential therapeutic targets for hepatic steatosis.
233 Hepatic Overexpression of SIRT3 in Mice Heterozygous for Mitochondrial Trifunctional Protein Rescues Hepatic Steatosis and Improves Insulin Sensitivity Fatiha Nassir, Justin J. Arndt, Jamal A. Ibdah Background: Mitochondrial trifunctional protein (MTP) is an enzyme complex responsible for catalyzing the last three steps in long-chain fatty acid β-oxidation. Studies using heterozygous mice for MTP (HT-MTP), generated by our group, have linked defect in mitochondrial dysfunction with hepatic steatosis and insulin resistance in 15-18 months mice. Sirtuin 3
S-973
AASLD Abstracts