Murine Model Of NASH Research Articles

WED-456 - Tyrosol reduces steatosis, fibrosis and inflammation in a murine model of NASH by modulating the immune hepatic phenotype

WED-456 - Tyrosol reduces steatosis, fibrosis and inflammation in a murine model of NASH by modulating the immune hepatic phenotype

Inhibition of Na/K-ATPase signaling Attenuates Steatohepatitis and Atherosclerosis in Mice Fed a Western Diet.

We have previously reported that the α1 subunit of sodium-potassium adenosine triphosphatase (Na/K-ATPase), acts as a receptor and an amplifier for reactive oxygen species, in addition to its distinct pumping function. On this background, we speculated that the blockade of Na/K-ATPase-induced ROS amplification with a specific peptide, pNaKtide, might attenuate the development of steatohepatitis. To test this hypothesis, pNaKtide was administered to a murine model of NASH: the C57Bl6 mouse fed a "western" diet containing high amounts of fat and fructose. The administration of pNaKtide reduced obesity as well as hepatic steatosis, inflammation and fibrosis. Of interest, we also noted a marked improvement in mitochondrial fatty acid oxidation, insulin sensitivity, dyslipidemia and aortic streaking in this mouse model. To further elucidate the effects of pNaKtide on atherosclerosis, similar studies were performed in ApoE knockout mice also exposed to the western diet. In these mice, pNaKtide not only improved steatohepatitis, dyslipidemia, and insulin sensitivity but also ameliorated significant aortic atherosclerosis. Collectively, this study demonstrates that the Na/K-ATPase/ROS amplification loop contributes significantly to the development and progression of steatohepatitis and atherosclerosis. Furthermore, this study presents a potential treatment, the pNaKtide, for the metabolic syndrome phenotype.

Hepatocyte mARC1 promotes fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with invitro and invivo studies of mARC1 knockdown. Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an invitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)-siRNA to understand the invivo impacts of MTARC1. We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. Invitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and invivo GalNAc-siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown invitro and invivo. Collectively, our findings from human genetics, and invitro and invivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD. We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 forNAFLD.

The administration of pirfenidone modifies the expression of JMJD2B in a murine model of NASH

The administration of pirfenidone modifies the expression of JMJD2B in a murine model of NASH

Moringa oleifera decreases biomarkers of oxidative stress in a murine NASH model

This study aimed to evaluate the effect of Moringa oleifera aqueous extract on biomarkers of oxidative stress in a murine model of NASH. The characterization of the extract was performed by DPPH and ABTS spectrophotometric assays. Male C57BL/6J mice were randomly separated into two groups. 1) Standard diet (ND) (n = 5) (18% lipid) and 2) High fat (HF) diet (n = 10) (60% lipid and 42 g/L sugar in water of use), for 16 weeks. At the end of eight weeks, five HF group mice were divided into a subgroup, 3) Moringa Oleifera (HF + MO), 290 mg/kg/day p.o. for eight weeks. Malondialdehyde (MDA) levels were determined in liver homogenates and the transcriptome by microarray. Differences between groups were determined by ANOVA/Kruskal-Wallis test. The trial was approved by the research ethics committee. Moringa aqueous extract showed antioxidant capacity; DPPH values were 10081.4 0.3 and 22960.4 0.3 for ABTS. Hepatic MDA levels were increased in the HF group compared to the ND group (p<0.05) and decreased in the moringa-treated group (p<0.05). In the transcriptome, mRNAs involved in endoplasmic reticulum stress were underexpressed. An increase in MDA has been demonstrated in a murine model of NAFLD induced by a high-fat diet. In our study, Moringa administration reduces MDA production and gene expression of molecules involved in oxidative stress. MO treatment is a therapeutic alternative for the NASH spectrum of liver disorders. The resources used in this study were from the hospital without any additional financing The authors declare no potential conflicts of interest.

Modeling dysbiosis of human NASH in mice: Loss of gut microbiome diversity and overgrowth of Erysipelotrichales.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD) that is responsible for a growing fraction of cirrhosis and liver cancer cases worldwide. Changes in the gut microbiome have been implicated in NASH pathogenesis, but the lack of suitable murine models has been a barrier to progress. We have therefore characterized the microbiome in a well-validated murine NASH model to establish its value in modeling human disease. The composition of intestinal microbiota was monitored in mice on a 12- or 24-week NASH protocol consisting of high fat, high sugar Western Diet (WD) plus once weekly i.p injection of low-dose CCl4. Additional mice were subjected to WD-only or CCl4-only conditions to assess the independent effect of these variables on the microbiome. There was substantial remodeling of the intestinal microbiome in NASH mice, characterized by declines in both species diversity and bacterial abundance. Based on changes to beta diversity, microbiota from NASH mice clustered separately from controls in principal coordinate analyses. A comparison between WD-only and CCl4-only controls with the NASH model identified WD as the primary driver of early changes to the microbiome, resulting in loss of diversity within the 1st week. A NASH signature emerged progressively at weeks 6 and 12, including, most notably, a reproducible bloom of the Firmicute order Erysipelotrichales. We have established a valuable model to study the role of gut microbes in NASH, enabling us to identify a new NASH gut microbiome signature.

Circulating pro fibrotic protein promotes fibrosis in liver

Abstract Background/Introduction Non-alcoholic steatohepatitis (NASH), driven by the obesity epidemic, has become the most common form of liver disease. Inflamed visceral adipose tissue secretes pro-inflammatory adipokines that are causal for systemic metabolic disorders. Role of adipokines in NASH, especially those from brown adipose tissues (BATokine) remain unclear. Purpose To show the pathogenic role of BATokine in NASH. Methods To identify and characterize the pathological roles of pro-fibrotic BATokine, we generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice, and murine systemic or BAT specific knockout (KO) models. We also conducted functional in-vitro studies with differentiated brown adipocytes. Results Analyzing two sets of DNA micro array data with bioinformatics, we identified a secreted form pro-fibrotic protein (sPFP) expressed in dysfunctional brown adipose tissues (BAT) in mice. Testing our biobank samples, we found this protein increased in plasma of NASH patients. We generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice for 9–10 months since 4 weeks of age, and found that sPFP is produced predominantly by BAT. In this model, we also found that sPFP increased in plasma. We generated a murine systemic or BAT specific sPFP knockout (KO) models and found that liver fibrosis ameliorated in these models. We also suppressed circulating sPFP with a peptide vaccine targeting this molecule, and found that sPFP vaccination therapy inhibited liver fibrosis. Next, we generated sPFP gain of function (GOF) model by the administration of plasmid encoding sPFP into skeletal muscle. Liver fibrosis augmented in sPFP-GOF model, and these results suggested that sPFP has causal role for the progression of fibrotic response in liver. In vitro studies with differentiated brown adipocytes showed that metabolic stress increased c-Fos in nuclear, and this was causal for an increase in sPFP level. Conclusions Our results suggest that one of the BATokines, sPFP, contributes for the progression of fibrotic responses in obese-NASH model. Inhibition of sPFP may become a therapy for NASH or obesity related fibrotic disorders. Funding Acknowledgement Type of funding source: None

Lipocalin 2 induces neuroinflammation and blood-brain barrier dysfunction through liver-brain axis in murine model of nonalcoholic steatohepatitis

BackgroundRecent clinical and basic research implicated a strong correlation between NAFLD/NASH phenotypes with ectopic manifestations including neuroinflammation and neurodegeneration, but the mediators and critical pathways involved are not well understood. Lipocalin 2 (Lcn2) is one of the important mediators exclusively produced in the liver and circulation during NASH pathology.MethodsUsing murine model of NASH, we studied the role of Lcn2 as a potent mediator of neuroinflammation and neurodegeneration in NASH pathology via the liver-brain axis.ResultsResults showed that high circulatory Lcn2 activated 24p3R (Lipocalin2 receptor) in the brain and induced the release of high mobility group box 1 (HMGB1) preferably from brain cells. Released HMGB1 acted as a preferential ligand to toll-like receptor 4 (TLR4) and induced oxidative stress by activation of NOX-2 signaling involving activated p65 protein of the NF-κB complex. Further, the HMGB1-derived downstream signaling cascade activated NLRP3 inflammasome and release of proinflammatory cytokines IL-6 and IL-1β from brain cells. In addition, to advance our present understanding, in vitro studies were performed in primary brain endothelial cells where results showed high circulatory Lcn2 influenced HMGB1 secretion. Mechanistically, we also showed that elevated Lcn2 level in underlying NASH might be a likely cause for induction of blood-brain barrier dysfunction since the adipokine decreased the expression of tight junction protein Claudin 5 and caused subsequent elevation of pro-inflammatory cytokines IL-6 and IL-1β.ConclusionIn conclusion, the NASH-induced brain pathology might be because of increased Lcn2-induced release of HMGB1 and accompanying neuroinflammation.

FRI-284-A combination of fenofibrate with a liver-targeted acetyl-CoA carboxylase inhibitor enhances efficacy while reversing plasma triglyceride increases in a murine model of NASH

FRI-284-A combination of fenofibrate with a liver-targeted acetyl-CoA carboxylase inhibitor enhances efficacy while reversing plasma triglyceride increases in a murine model of NASH

RETRACTED ARTICLE: pNaKtide Attenuates Steatohepatitis and Atherosclerosis by Blocking Na/K-ATPase/ROS Amplification in C57Bl6 and ApoE Knockout Mice Fed a Western Diet

We have previously reported that the α1 subunit of sodium potassium adenosine triphosphatase (Na/K-ATPase), acts as a receptor and an amplifier for reactive oxygen species, in addition to its distinct pumping function. On this background, we speculated that blockade of Na/K-ATPase-induced ROS amplification with a specific peptide, pNaKtide, might attenuate the development of steatohepatitis. To test this hypothesis, pNaKtide was administered to a murine model of NASH: the C57Bl6 mouse fed a “western” diet containing high amounts of fat and fructose. The administration of pNaKtide reduced obesity as well as hepatic steatosis, inflammation and fibrosis. Of interest, we also noted marked improvement in mitochondrial fatty acid oxidation, insulin sensitivity, dyslipidemia and aortic streaking in this mouse model. To further elucidate the effects of pNaKtide on atherosclerosis, similar studies were performed in ApoE knockout mice also exposed to the western diet. In these mice, pNaKtide not only improved steatohepatitis, dyslipidemia, and insulin sensitivity, but also ameliorated significant aortic atherosclerosis. Collectively, this study demonstrates that the Na/K-ATPase/ROS amplification loop contributes significantly to the development and progression of steatohepatitis and atherosclerosis. And furthermore, this study presents a potential treatment, the pNaKtide, for the metabolic syndrome phenotype.

FRI-352 - A liver-targeted acetyl CoA carboxylase inhibitor reduces hepatic steatosis and liver injury in a murine model of NASH

FRI-352 - A liver-targeted acetyl CoA carboxylase inhibitor reduces hepatic steatosis and liver injury in a murine model of NASH

Calycosin attenuates triglyceride accumulation and hepatic fibrosis in murine model of non-alcoholic steatohepatitis via activating farnesoid X receptor

BackgroundNon-alcoholic steatohepatitis (NASH) represents the more severe end of hepatic steatosis and is associated with progressive liver disease. Calycosin, derived from the root of Radix Astragali, has been demonstrated to have favorable efficacy on acute liver injury. PurposeThe present study was to investigate the hepatoprotective effect of calycosin on attenuating triglyceride accumulation and hepatic fibrosis, as well as explore the potential mechanism in murine model of NASH. Study designThe C57BL/6 male mice were fed with methionine choline deficient (MCD) diet for 4 weeks to induce NASH and treated with or without calycosin by oral gavage for 4 weeks. MethodsThe body weight, liver weight and the liver to body weight ratios were measured. Serum ALT, AST, TG, TC, FFA, MCP-1 and mKC levels were accessed by biochemical methods. H&E staining and Oil red O staining were used to identify the amelioration of liver histopathology. Immunohistochemistry of a-SMA, Masson trichrome staining and Sirius red staining were used to identify the amelioration of hepatic fibrosis. The quantitative real-time-PCR and Western blot were applied to observe the expression changes of key factors involved in triglyceride synthesis, free fatty acid β-oxidation and hepatic fibrosis. ResultsCalycosin significantly inhibited body weight loss induced by MCD diet, decreased the ALT and AST activities, MCP-1 and mKC in a dose-dependent manner. The H&E and Oil red O staining indicated calycosin effectively improved hepatic steatosis, improved the degree of triglyceride accumulation. Masson trichrome and Sirius red staining indicated that calycosin treatment remarkably attenuated the degree of hepatic fibrosis. Immunohistochemistry of a-SMA demonstrated that calycosin attenuated hepatic fibrosis by inhibiting hepatic stellate cell activation. Further, calycosin inhibited the expression of SREBP-1c, FASN, ACC and SCD1 involved in triglyceride synthesis, promoted the expression of PPARa, CPT1, Syndecan-1 and LPL involved in free fatty acid β-oxidation. The above effects of calycosin were attributed to FXR activation. ConclusionCalycosin attenuates triglyceride accumulation and hepatic fibrosis to protect against NASH via FXR activation.

Seven weeks of Western diet in apolipoprotein-E-deficient mice induce metabolic syndrome and non-alcoholic steatohepatitis with liver fibrosis.

Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis, inflammation and fibrosis, which might progress to cirrhosis. Human NASH is associated with metabolic syndrome (MS). Currently, rodent NASH models either lack significant fibrosis or MS. ApoE−/− mice are a MS model used in cardiovascular research. The aim of this work was to establish and characterise a novel mouse NASH model with significant fibrosis and MS. ApoE−/− and wild-type mice (wt) were fed either a western-diet (WD), methionine-choline-deficient-diet (MCD) or normal chow. Liver histology, RT-PCR, hepatic hydroxyproline content, triglycerides and cholesterol levels, and fasting glucose levels assessed hepatic steatosis, inflammation and fibrosis. Further, portal pressure was measured invasively, and kidney pathology was assessed by histology. ApoE−/− mice receiving WD showed abnormal glucose tolerance, hepatomegaly, weight gain and full spectrum of NASH including hepatic steatosis, fibrosis and inflammation, with no sign of renal damage. MCD-animals showed less severe liver fibrosis, but detectable renal pathological changes, besides weight loss and unchanged glucose tolerance. This study describes a murine NASH model with distinct hepatic steatosis, inflammation and fibrosis, without renal pathology. ApoE−/− mice receiving WD represent a novel and fast model with all characteristic features of NASH and MS well suitable for NASH research.

232 Mixed Lineage Kinase 3 Mediates the Release of Proinflammatory Extracellular Vesicles in Nonalcoholic Steatohepatitis

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.

233 Hepatic Overexpression of SIRT3 in Mice Heterozygous for Mitochondrial Trifunctional Protein Rescues Hepatic Steatosis and Improves Insulin Sensitivity

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.

Increased expression of c-jun in nonalcoholic fatty liver disease

The lack of animal models which closely mimic the human situation is one main reason why the molecular mechanisms causing progression from nonalcoholic fatty liver to (NASH) still are elusive. In addition to the quantity of consumed calories, certain dietary components, namely saturated fatty acids, fructose, and cholesterol are increasingly recognized as dietary factors affecting NAFLD progression. The aim of this study was to developed and characterize a NASH inducing diet (ND) containing these nutrients in a composition which mimics Western food. Methods and Results: Feeding ND to male C57Bl6/N mice for 12 weeks induced significant body weight gain, an impaired glucose tolerance and elevated fasting blood glucose levels as well as decreased adiponectin and increased leptin serum levels. Furthermore, ND-feeding led to marked hepatic steatosis and distinct signs of hepatic oxidative stress and lipid peroxidation. In line with this, ND-fed mice exhibited more apoptotic hepatocytes and elevated transaminase serum levels as well as hepatic inflammation, activation of hepatic stellate cells, and beginning fibrosis, as assessed by histology and hydroxyprolin analysis. Transcriptome-wide gene expression analysis and classification in Gene Ontology categories revealed main alterations in the categories „lipid metabolic process”, “innate immune response” and “inflammatory response“ in the liver of HD-fed mice, which closely resembled NASH patients. Search for over-represented transcription factor target sites among the differenzially expressed genes identified AP-1 as the most likely factor to cause the transcriptional changes. Interestingly, protein-protein interaction network analysis identified c-JUN (a component if the AP-1 complex) as hub. Westernblot and immunohistochemical analysis confirmed c-jun-phosphorylation and nuclear translocation in murine ND-livers as well as NASH patients. Unexpectedly, also expression of (unphosphorylated) c-jun was significantly enhanced not only in NASH, but also in fatty liver without inflammation. Immunohistochemical analysis of a tissue micro array containing 80 human NAFLD tissues revealed a significant correlation between c-jun expression and the extent of hepatic steatosis. Conclusions: Our novel murine NASH-model induces important pathomechanisms also found in human NASH and led to the identification of increased c-jun already in simple steatosis. It is likely that higher abundance of this crucial pro-inflammatory regulator facilitates NASH upon additional stimuli. The identification of the latter as well as c-jun itself appear as attractive prognostic and therapeutic targets of NAFLD progression.

691 Cell-Specific Roles of miR-155 and miR-122 in Inflammation, Immunity and Fibrosis in Non-Alcoholic Steatohepatitis

ligand. IL-1β and inflammasome components were measured from the cells and supernatant. TLR2 deficient mice were fed a CDAA diet to assess the requirement of TLR2 in NASH. (Results) The CDAA diet feeding for 22 weeks induced steatohepatitis, obesity and insulin resistance. Hepatic and blood FFAs were increased in WT mice fed the CDAA diet compared with those fed a standard diet. Hepatic and blood IL-1β levels were also increased in WT mice fed the CDAA diet. CDAA diet feeding increased in expression of inflammasome components including nalp3, nalp1, ASC, caspase-1, and AIM2. InWTKupffer cells, palmitic acid did not increase expression of IL-1β and inflammasome components. In contrast, a synthetic TLR2 ligand increased gene expression of IL-1β as well as an inflammasome component nalp3. Although a synthetic TLR2 ligand increased mRNA expressions of IL-1β and nalp3, active form of IL-1β was not detected from the supernatant. Interestingly, palmitic acids treatment induced secretion of active IL-1β form in Kupffer cells primed with TLR2 ligand. Finally, we tested the role of TLR2 in murine NASH model. TLR2 deficient mice fed the CDAA showed less steatohepatitis and lower expression of inflammasome components than WT mice. (Conclusion) TLR2 signaling induces expression of inflammasome components and the subsequent treatment with palmitic acid induces activation of IL-1β. Thus, activation of inflammasome and IL-1β require both FFA stimulation and TLR2 signaling in NASH.

The Hepatic Inflammsome Has an Increased NK-T and CD8+ T- Cell Signature in a Murine Model of Fibrotic Nonalcoholic Steatohepatitis (NASH)

High fat diet induced models of simple hepatic steatosis have been shown to result in a decreased hepatic natural killerT (NKT) cell population. Interestingly, NKT cells are known to correlate positively with the development of adipose tissue inflammation and glucose intolerance in diet-induced obesity. Recent data implicates refined carbohydrates (Fructose) in the progression of hepatic steatosis to advanced fibrotic NASH in humans. This has also been replicated in a novel model of obesity and NASH with fibrosis in mice. We therefore hypothesized that NKT cells populate the livers in a murine model of advanced NASH with fibrosis. Methods: Adult male C57BL6 mice were randomly assigned to control (C) or high fat high carbohydrate diet (HFHC). The C group received regular chow and water while the HFHC group received diet with Medium Chain Triglycerides (MCT; 58% kcal) and water supplemented with 55% fructose & 45% sucrose. Mice were sacrificed at 16 wks of age and underwent hepatic mononuclear cell phenotyping by flow cytometry. Results: Mice receiving HFHC diet gained significantly more weight than C fed mice starting at 4 wk (p<0.01), with a Mean ± SE weight of 45.84±1.4g at 16 wk compared to 31.33±1.0g for C fed mice (p<0.0001). Fasting glucose was higher in HFHC mice at 16 wks (176.3±9.2 mg/dl; p=0.001). Liver weight at 16 weeks was higher in the HFHC group (2.44±0.2g) compared to the C fed mice (1.47±0.03g; p<0.01). Steatosis as measured by liver triacylglycerol (TG) content was also higher in HFHC mice at 16 wks (2354±487.8 mg/dl/100 mg wet liver) compared to the C fed mice (p= 0.001). Inflammation as measured by serum alanine aminotransferase (ALT) levels was higher in HFHC mice (135.8±24.28 IU/L) at 16 wks compared to the C fed mice (p=0.019). Flow cytometric analysis identified increased NK-T cell (CD3+NK1.1+) signature in mice receiving HFHC (3.7±2.3x104 cells) compared to C fed mice (1.1±0.6x104 cells; p=0.02). While hepatic CD1d-restricted invariant NK-T CD4+ and CD8+ cell populations remained unchanged, total CD3+CD8+ T-cells significantly increased in the HFHC group (1.93±0.79x105) vs. C fed (1.0±0.7x105; p=0.04). A corresponding increase in CD8+ T-cells expressing the activation marker CD69 was also seen [(2.02±1.01x104 (C) vs 6.85±2.6x104 (HFHC); p<0.01]. Liver fibrosis was visible at sacrifice in majority of the HFHC fed mice (Stage 1c and 2) while none was seen in C diet fed mice. Conclusion: HFHC diet produces a relatively rapid and progressive liver injury resulting in NASH with fibrosis in a background of diet-induced obesity. In this system, which closely resembles the human phenotype of advanced NASH with fibrosis, we observed a significant increase in hepatic NK-T and CD8+ T-cells. These cell types may potentially typify the hepatic inflammasome in the progression of human disease to NASH with fibrosis.

Effect of disease progression on liver apparent diffusion coefficient values in a murine model of NASH at 11.7 tesla MRI

To evaluate the apparent diffusion coefficient (ADC) values of liver in a murine model of non-alcoholic steatohepatitis using 11.7 Tesla (T) MRI. This animal study was IACUC approved. Seventeen male C57BL/6 mice were divided into control (n = 3) and experimental groups (n = 14) fed a methionine-deficient choline-deficient (MCD) diet to induce steatohepatitis. Livers underwent ex vivo diffusion-weighted MR imaging and ADC maps were calculated. A pathologist determined subjective scores of steatosis, classified from 0 to 3. Digital image analysis was used to determine percentage areas of steatosis. Graphs comparing ADC to subjective and digital image analysis (DIA) determinations of steatosis were plotted. Subjective assessments of steatosis ranged up to values of 3 and DIA determined areas of steatosis to range up to approximately 16%. ADC values approximated 800 × 10(-6) mm(2) /s (range, 749-811 × 10(-6) mm(2) /s, mean 786 × 10(-6) mm(2) /s) in controls and 500 × 10(-6) mm(2) /s (range, 478-733 × 10(-6) mm(2) /s, mean 625 × 10(-6) mm(2) /s) in experimental mice. Moderate correlation between ADC and subjective scores of steatosis (R = -0.56) was observed. Strong correlation between ADC values and percentage areas of steatosis was between ADC values and percentage areas of steatosis was observed greater (R = -0.81) and very strong correlation was observed with the exclusion of a single outlying data point (R = -0.91). Based on the comparison of ADC values and steatosis determinations by DIA, increasing degrees of steatosis are seen to result in decreased hepatic ADC values.