Hepatic Crown-Like Structure: A Unique Histological Feature in Non-Alcoholic Steatohepatitis in Mice and Humans

Although macrophages are thought to be crucial for the pathogenesis of chronic inflammatory diseases, how they are involved in disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is poorly understood. Here we report the unique histological structure termed “hepatic crown-like structures (hCLS)” in the mouse model of human NASH; melanocortin-4 receptor deficient mice fed a Western diet. In hCLS, CD11c-positive macrophages aggregate to surround hepatocytes with large lipid droplets, which is similar to those described in obese adipose tissue. Histological analysis revealed that hCLS is closely associated with activated fibroblasts and collagen deposition. When treatment with clodronate liposomes effectively depletes macrophages scattered in the liver, with those in hCLS intact, hepatic expression of inflammatory and fibrogenic genes is unaffected, suggesting that hCLS is an important source of inflammation and fibrosis during the progression of NASH. Notably, the number of hCLS is positively correlated with the extent of liver fibrosis. We also observed increased number of hCLS in the liver of non-alcoholic fatty liver disease/NASH patients. Collectively, our data provide evidence that hCLS is involved in the development of hepatic inflammation and fibrosis, thereby suggesting its pathophysiologic role in disease progression from simple steatosis to NASH.     

Enzymatically-epoxidized docosahexaenoic acid, 19,20-EpDPE,suppresses hepatic crown-like structure formation and nonalcoholic steatohepatitis fibrosis through GPR120

A hepatic crown-like structure (hCLS) formed by macrophages accumulating around lipid droplets and dead cells in the liver is a unique feature of nonalcoholic steatohepatitis (NASH) that triggers progression of liver fibrosis. As hCLS plays a key role in the progression of NASH fibrosis, hCLS formation has emerged as a potential therapeutic target. n-3 polyunsaturated fatty acids (n-3 PUFAs) have potential suppressive effects on NASH fibrosis; however, the mechanisms underlying this effect are poorly understood. Here, we report that n-3 PUFA-enriched Fat-1 transgenic mice are resistant to hCLS formation and liver fibrosis in a NASH model induced by a combination of high-fat diet, CCl4 and a Liver X receptor (LXR) agonist. Liquid chromatography–tandem mass spectrometry-based mediator lipidomics revealed that the amount of endogenous n-3 PUFA-derived metabolites, such as 17,18-dihydroxyeicosatetraenoic acid (17,18-diHETE), and 19,20-epoxy docosapentaenoic acid (19,20-EpDPE), was significantly elevated in Fat-1 mice, along with hCLS formation. In particular, DHA-derived 19,20-EpDPE produced by Cyp4f18 attenuated the hCLS formation and liver fibrosis in a G protein-coupled receptor 120 (GPR120)-dependent manner. These results indicated that 19,20-EpDPE is an endogenous active metabolite that mediates the preventive effect of n-3 PUFAs against NASH fibrosis.     

The role of AMPKα2 in the HFD-induced nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic steatosis, inflammation and liver fibrosis and has become one of the leading causes of hepatocellular carcinoma and liver failure. However, the underlying molecular mechanism of hepatic steatosis and the progression to nonalcoholic steatohepatitis (NASH) are not fully understood. Herein, we discovered that AMPKα2 catalytic subunit showed reduced expression in the liver following high fat diet (HFD) feeding to mice. Importantly, knockout of AMPKα2 in mice aggravated NAFLD, hepatic steatosis, inflammation and fibrosis. On the other hand, hepatocyte-targeted overexpression of AMPKα2 prevented or reversed NAFLD indications. In vivo mechanistic studies revealed that increased phosphorylation of IKKα/β and NF-κB in HFD-fed AMPKα2^−/− mice compared to WT mice, and treatment of these mouse cohorts with an inhibitor of NF-κB signaling for 4 weeks, effectively attenuated the progression of steatohepatitis and metabolic disorder features. In summary, AMPKα2 provides a protective role in the process of hepatic steatosis to NASH progression through suppression of liver NF-κB signaling.     

The Annexin A2-Notch regulatory loop in hepatocytes promotes liver fibrosis in NAFLD by increasing osteopontin expression

BackgroundThe mechanisms underlying the progression of liver disease from simple hepatic steatosis to advanced nonalcoholic steatohepatitis (NASH) and liver fibrosis warrant further investigation. Increased mRNA levels of Annexin A2 protein (Anxa2) have been observed in patients with NASH. However, the role of Anxa2 in NASH remains unclear.MethodsThe protein levels of Anxa2 were analyzed in the livers of mice and patients with NASH. Anxa2-knockout and -knockdown mice were generated, and NASH was induced through a high fructose, palmitate, and cholesterol (FPC) diet or methionine- and choline-deficient (MCD) diet.FindingsWe found elevated expression of Anxa2 in the livers of patients and mice with NASH. Anxa2 knockdown but not knockout ameliorated liver fibrosis in both FPC and MCD diet–fed mice. Liver-specific Anxa2 overexpression increased collagen deposition in mice fed a normal diet. Mechanistically, Anxa2 overexpression in hepatocytes promoted hepatic stellate cell activation in a paracrine manner by increasing osteopontin expression. Notch inhibition suppressed the exogenous overexpression of Anxa2-induced osteopontin and endogenous Anxa2 expression. Additionally, Anxa2 overexpression accelerated the progression of nonalcoholic fatty liver disease (NAFLD) in mice fed a high-fat diet. Moreover, Anxa2 levels were higher in NAFLD patients with advanced liver fibrosis than in those with mild liver fibrosis, as determined using the Gene Expression Omnibus database.InterpretationIn conclusion, we found increased Anxa2 expression in hepatocytes promoted liver fibrosis in NASH mice by increasing osteopontin expression. The Anxa2-Notch positive regulatory loop contributes to this process and represents a novel target for the treatment of NASH-related liver fibrosis.     

Role of Pin1 Protein in the Pathogenesis of Nonalcoholic Steatohepatitis in a Rodent Model

Nonalcoholic steatohepatitis (NASH) is a disorder characterized by simultaneous fat accumulation and chronic inflammation in the liver. In this study, Pin1 expression was revealed to be markedly increased in the livers of mice with methionine choline-deficient (MCD) diet-induced NASH, a rodent model of NASH. In addition, Pin1 KO mice were highly resistant to MCD-induced NASH, based on a series of data showing simultaneous fat accumulation, chronic inflammation, and fibrosis in the liver. In terms of Pin1-induced fat accumulation, it was revealed that the expression levels of peroxisome proliferator-activated receptor α and its target genes were higher in the livers of Pin1 KO mice than in controls. Thus, resistance of Pin1 KO mice to hepatic steatosis is partially attributable to the lack of Pin1-induced down-regulation of peroxisome proliferator-activated receptor α, although multiple other mechanisms are apparently involved. Another mechanism involves the enhancing effect of hematopoietic Pin1 on the expressions of inflammatory cytokines such as tumor necrosis factor and monocyte chemoattractant protein 1 through NF-κB activation, eventually leading to hepatic fibrosis. Finally, to distinguish the roles of hematopoietic or nonhematopoietic Pin1 in NASH development, mice lacking Pin1 in either nonhematopoietic or hematopoietic cells were produced by bone marrow transplantation between wild-type and Pin1 KO mice. The mice having nonhematopoietic Pin1 exhibited fat accumulation without liver fibrosis on the MCD diet. Thus, hepatic Pin1 appears to be directly involved in the fat accumulation in hepatocytes, whereas Pin1 in hematopoietic cells contributes to inflammation and fibrosis. In summary, this is the first study to demonstrate that Pin1 plays critical roles in NASH development. This report also raises the possibility that hepatic Pin1 inhibition to the appropriate level might provide a novel therapeutic strategy for NASH.     

iPla2β deficiency in mice fed with MCD diet does not correct the defect of phospholipid remodeling but attenuates hepatocellular injury via an inhibition of lipid uptake genes

Group VIA calcium-independent phospholipase A2 (iPla2β) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2β deletion protects hepatic steatosis and obesity in genetic ob/ob and obese mice chronically fed with high-fat diet by replenishing the loss of hepatic phospholipids (PL). As mouse feeding with methionine- and choline-deficient (MCD) diet is a model of lean NASH, we tested whether iPla2β-null mice could still be protected since PL syntheses are disturbed. MCD-diet feeding of female wild-type for 5 weeks induced hepatic steatosis with a severe reduction of body and visceral fat weights concomitant with a decrease of hepatic phosphatidylcholine. These parameters were not altered in MCD-fed iPla2β-null mice. However, iPla2β deficiency attenuated MCD-induced elevation of serum transaminase activities and hepatic expression of fatty-acid translocase Cd36, fatty-acid binding protein-4, peroxisome-proliferator activated receptorγ, and HDL-uptake scavenger receptor B type 1. The reduction of lipid uptake genes was consistent with a decrease of hepatic esterified and unesterified fatty acids and cholesterol esters. On the contrary, iPla2β deficiency under MCD did not have any effects on inflammasomes and pro-inflammatory markers but exacerbated hepatic expression of myofibroblast α-smooth muscle actin and vimentin. Thus, without any rescue of PL loss, iPla2β inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2β mediates hepatic steatosis and lipotoxicity in hepatocytes in both obese and lean NASH, but elicits exacerbated liver fibrosis in lean NASH likely by affecting other cell types.     

Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive disease and poses a high risk of severe liver damage. However, the pathogenesis of NASH is still unclear. Accumulation of lipid droplets and insulin resistance is the hallmark of NASH. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in glucose metabolism via regulating the activity of pyruvate dehydrogenase complex (PDC). Here, we demonstrated a novel of PDK4 in NASH by regulating hepatic steatosis and insulin signaling pathway in methionine and choline deficient (MCD) diet induced NASH model. Hepatic PDK4 levels were highly induced in human patients with NASH and MCD diet fed mice, as well as in hepatocytes treated with oleic acid. The glucose and lipid metabolism were impaired in Pdk4^?/? mice. Pdk4 deficiency ameliorated the hepatic steatosis significantly in NASH mice. Pdk4^?/?-MCD mice had reduced liver weights and triglyceride (TG) levels. And Pdk4 deficiency dramatically reduced the expression of genes related to fatty acid uptake, synthesis and gluconeogenesis. In addition, elevated phosphorylated AMPK (p-AMPK), p-SAPK/JNK and diminished p-ERK, p-P38, p-Akt and p-mTOR/p-4EBP1 proteins were observed. In conclusion, our data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK4 may be a new therapeutic strategy against NAFLD.     

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background

Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model.

Methodology/Principal Findings

Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH.

Conclusions/Significance

These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.     

Vismodegib Suppresses TRAIL-mediated Liver Injury in a Mouse Model of Nonalcoholic Steatohepatitis

Hedgehog signaling pathway activation has been implicated in the pathogenesis of NASH. Despite this concept, hedgehog pathway inhibitors have not been explored. Thus, we examined the effect of vismodegib, a hedgehog signaling pathway inhibitor, in a diet-induced model of NASH. C57BL/6 mice were placed on 3-month chow or FFC (high saturated fats, fructose, and cholesterol) diet. One week prior to sacrifice, mice were treated with vismodegib or vehicle. Mice fed the FFC diet developed significant steatosis, which was unchanged by vismodegib therapy. In contrast, vismodegib significantly attenuated FFC-induced liver injury as manifested by reduced serum ALT and hepatic TUNEL-positive cells. In line with the decreased apoptosis, vismodegib prevented FFC-induced strong upregulation of death receptor DR5 and its ligand TRAIL. In addition, FFC-fed mice, but not chow-fed animals, underwent significant liver injury and apoptosis following treatment with a DR5 agonist; however, this injury was prevented by pre-treatment with vismodegib. Consistent with a reduction in liver injury, vismodegib normalized FFC-induced markers of inflammation including mRNA for TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 and a variety of macrophage markers. Furthermore, vismodegib in FFC-fed mice abrogated indices of hepatic fibrogenesis. In conclusion, inhibition of hedgehog signaling with vismodegib appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition may be salutary in human NASH.     

The modulation of hepatic adenosine triphosphate and inflammation by eicosapentaenoic acid during severe fibrotic progression in the SHRSP5/Dmcr rat model

AimsEicosapentaenoic acid (EPA) can ameliorate certain liver lesions involved in non-alcoholic steatohepatitis (NASH). A previous study has found that stroke-prone spontaneously hypertensive 5/Dmcr (SHRSP5/Dmcr) rats fed a high fat-cholesterol (HFC) diet developed fibrotic steatohepatitis with histological similarities to NASH. This study evaluated the potential effects and mechanisms of action of EPA supplementation using this rodent model.Main methodsMale rats were randomly assigned to groups that were fed with either the stroke-prone (SP) diet or HFC diet with or without EPA for 2, 8 and 14 weeks, respectively. The liver histopathology, biochemical features, mRNA and protein levels, and nuclear factor-κB (NF-κB) DNA binding activity were determined.Key findingsThe SP diet-fed rats presented normal livers. Conversely, the HFC diet-fed rats developed microvesicular/macrovesicular steatosis, inflammation, ballooning degeneration and severe fibrosis. At 2 weeks, the administration of EPA inhibited hepatic inflammatory recruitment by blocking the phosphorylation of inhibitor of κB-α (IκBα), which antagonizes the NF-κB activation pathway. The dietary supplementation of EPA for 8 weeks ameliorated hepatic triglyceride accumulation and macrovesicular steatosis by inhibiting the HFC diet-induced decrease in the protein levels of enzymes involved in fatty acid β-oxidation including carnitine palmitoyltransferase 1, very long chain acyl-CoA dehydrogenase and peroxisomal bifunctional protein. Although the administration of EPA elicited no histologically detectable effects on severe fibrosis at 14 weeks, it restored an HFC diet-induced decline in hepatic adenosine triphosphate (ATP) levels and suppressed ballooning degeneration, suggesting that EPA may inhibit HFC diet-induced ATP loss and cell death.SignificanceInitial amelioration of the inflammation and steatosis in the rats after EPA supplementation indicates a possibility to treat steatohepatitis. Additionally, this study provides new insights into the roles of EPA in hepatic ATP depletion and subsequent hepatocellular injury during severe fibrosis.     

The establishment of a novel non-alcoholic steatohepatitis model accompanied with obesity and insulin resistance in mice

Non-alcoholic steatohepatitis (NASH) is a hepatic manifestation of the metabolic syndrome that can progress to liver cirrhosis. The major aim of this study was to establish a novel NASH mouse model accompanied by obesity and insulin resistance, then explore the molecular mechanisms of NASH and evaluate the effects of both the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist fenofibrate and the PPARgamma agonist rosiglitazone in this established NASH model. The novel model was induced in C57BL/6 mice by 23 weeks of ad libitum feeding of a modified high-fat diet (mHFD), with lower methinione and choline and higher fat content. In comparison to the controls, the model animals developed pronounced obesity, dyslipidemia and insulin resistance. Marked liver lesions characterized by severe steatosis, inflammation, fibrosis, increased hepatic triglyceride content, and elevated serum alanine aminotransferase (ALT) levels were observed in the models. In this novel model, treatment with fenofibrate or rosiglitazone significantly improved insulin sensitivity and corrected dyslipidemia; however, fenofibrate was more effective than rosiglitazone in improving hepatic morphology and ALT levels. Further study showed that long-term feeding of mHFD significantly increased expression of mRNA for hepatic PPARgamma, adipose fatty acid binding protein (ap2) and CD36 and suppressed expression of mRNA for hepatic PPARalpha and carnitine palmitoyl transferase-1a (CPT-1a). These results showed the successful establishment of the combined NASH and obese-insulin resistance mouse model. Additionally, aberrant expressions of hepatic PPARalpha and PPARgamma may play a major role in the pathogenesis of NASH by affecting hepatic lipogenesis and fatty acid oxidation in this novel model.     

Hyperresponsivity to low-dose endotoxin during progression to nonalcoholic steatohepatitis is regulated by leptin-mediated signaling

Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH.     

Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model

The pathogenesis of nonalcoholic steatohepatitis (NASH) is poorly defined. Feeding mice a diet deficient in methionine and choline (MCD diet) induces experimental NASH. Osteopontin (OPN) is a Th1 cytokine that plays an important role in several fibroinflammatory diseases. We examined the role of OPN in the development of experimental NASH. A/J mice were fed MCD or control diet for up to 12 wk, and serum alanine aminotransferase (ALT), liver histology, oxidative stress, and the expressions of OPN, TNF-alpha, and collagen I were assessed at various time points. MCD diet-fed mice developed hepatic steatosis starting after 1 wk and inflammation by 2 wk; serum ALT increased from day 3. Hepatic collagen I mRNA expression increased during 1-4 wk, and fibrosis appeared at 8 wk. OPN protein expression was markedly increased on day 1 of MCD diet and persisted up to 8 wk, whereas OPN mRNA expression was increased at week 4. TNF-alpha expression was increased from day 3 to 2 wk, and evidence of oxidative stress did not appear until 8 wk. Increased expression of OPN was predominantly localized in hepatocytes. Hepatocytes in culture also produced OPN, which was stimulated by transforming growth factor-beta and TNF-alpha. Moreover, MCD diet-induced increases in serum ALT levels, hepatic inflammation, and fibrosis were markedly reduced in OPN(-/-) mice when compared with OPN(+/+) mice. In conclusion, our results demonstrate an upregulation of OPN expression early in the development of steatohepatitis and suggest an important role for OPN in signaling the onset of liver injury and fibrosis in experimental NASH.     

Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2

Inflammatory cell infiltration in the liver is a hallmark of nonalcoholic steatohepatitis (NASH). The chemokine-chemokine receptor interaction induces inflammatory cell recruitment. CC-chemokine receptor (CCR)2 is expressed on hepatic macrophages and hepatic stellate cells. This study aims to investigate the therapeutic potential of CCR2 to NASH. Twenty-two weeks on a choline-deficient amino acid-defined (CDAA) diet induced steatosis, inflammatory cell infiltration, and liver fibrosis with increased CCR2 and monocyte chemoattractant protein (MCP)-1 expression in the wild-type livers. The infiltrated macrophages expressed CD68, CCR2, and a marker of bone marrow-derived monocytes, Ly6C. CCR2(-/-) mice had less steatosis, inflammatory cell infiltration, and fibrosis, and hepatic macrophages expressing CD68 and Ly6C were decreased. Toll-like receptor (TLR)4(-/-), TLR9(-/-), and MyD88(-/-) mice had reduced hepatic macrophage infiltration with decreased MCP-1 and CCR2 expression because TLR signaling is a potent inducer of MCP-1. To assess the role of Kupffer cells at the onset of NASH, Kupffer cells were depleted by liposomal clodronate. The Kupffer cell depletion ameliorated steatohepatitis with a decrease in the MCP-1 expression and recruitment of Ly6C-expressing macrophages at the onset of NASH. Finally, to test the therapeutic potential of targeting CCR2, a CCR2 inhibitor was administered to mice on a CDAA diet. The pharmaceutical inhibition of CCR2 prevented infiltration of the Ly6C-positive macrophages, resulting in an inhibition of liver inflammation and fibrosis. We concluded that CCR2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes.     

Fibroblast Growth Factor-5 Participates in the Progression of Hepatic Fibrosis

Non-alcoholic steatohepatitis (NASH) is characterized by the presence of steatosis, inflammation, and fibrosis and is believed to develop via a “two-hit process”; however, its pathophysiology remains unclear. Fibroblast growth factors (FGFs) are heparin-binding polypeptides with diverse biological activities in many developmental and metabolic processes. In particular, FGF5 is associated with high blood pressure. We investigated the function of FGF5 in vivo using spontaneously Fgf5 null mice and explored the role of diet in the development of NASH. Mice fed a high-fat diet gained little weight and had higher serum alanine transaminase, aspartate amino transferase, and non–high-density lipoprotein-cholesterol levels. Liver histology indicated marked inflammation, focal necrosis, fat deposition, and fibrosis, similar to the characteristics of NASH. FGF5 and a high-fat diet play significant roles in the pathophysiology of hepatic fibrosis and Fgf5 null mice may provide a suitable model for liver fibrosis or NASH.     

Correction of Liver Steatosis by a Hydrophobic Iminosugar Modulating Glycosphingolipids Metabolism

The iminosugar N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynoijirimycin (AMP-DNM), an inhibitor of glycosphingolipid (GSL) biosynthesis is known to ameliorate diabetes, insulin sensitivity and to prevent liver steatosis in ob/ob mice. Thus far the effect of GSL synthesis inhibition on pre-existing NASH has not yet been assessed. To investigate it, LDLR(−/−) mice were kept on a western-type diet for 12 weeks to induce NASH. Next, the diet was continued for 6 weeks in presence or not of AMP-DNM in the diet. AMP-DNM treated mice showed less liver steatosis, inflammation and fibrosis. Induction of fatty acid beta-oxydation was observed, as well as a reduction of plasma lipids. Our study demonstrates that AMP-DNM treatment is able to significantly correct pre-existing NASH, suggesting that inhibiting GSL synthesis may represent a novel strategy for the treatment of this pathology.     

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.     

Kuppfer Cells Trigger Nonalcoholic Steatohepatitis Development in Diet-induced Mouse Model through Tumor Necrosis Factor-α Production

Nonalcoholic steatohepatitis (NASH), characterized by lipid deposits within hepatocytes (steatosis), is associated with hepatic injury and inflammation and leads to the development of fibrosis, cirrhosis, and hepatocarcinoma. However, the pathogenic mechanism of NASH is not well understood. To determine the role of distinct innate myeloid subsets in the development of NASH, we examined the contribution of liver resident macrophages (i.e. Kupffer cells) and blood-derived monocytes in triggering liver inflammation and hepatic damage. Employing a murine model of NASH, we discovered a previously unappreciated role for TNFα and Kupffer cells in the initiation and progression of NASH. Sequential depletion of Kupffer cells reduced the incidence of liver injury, steatosis, and proinflammatory monocyte infiltration. Furthermore, our data show a differential contribution of Kupffer cells and blood monocytes during the development of NASH; Kupffer cells increased their production of TNFα, followed by infiltration of CD11b^intLy6C^hi monocytes, 2 and 10 days, respectively, after starting the methionine/choline-deficient (MCD) diet. Importantly, targeted knockdown of TNFα expression in myeloid cells decreased the incidence of NASH development by decreasing steatosis, liver damage, monocyte infiltration, and the production of inflammatory chemokines. Our findings suggest that the increase of TNFα-producing Kupffer cells in the liver is crucial for the early phase of NASH development by promoting blood monocyte infiltration through the production of IP-10 and MCP-1.     

Liver Injury and Fibrosis Induced by Dietary Challenge in the Ossabaw Miniature Swine

BackgroundOssabaw miniature swine when fed a diet high in fructose, saturated fat and cholesterol (NASH diet) develop metabolic syndrome and nonalcoholic steatohepatitis (NASH) characterized by liver injury and fibrosis. This study was conducted to further characterize the development of NASH in this large animal model.MethodsOssabaw swine were fed standard chow (control group; n = 6) or NASH diet (n = 6) for 24 weeks. Blood and liver tissue were collected and liver histology were characterized at 0, 8, 16 and 24 weeks of dietary intervention. Hepatic apoptosis and lipid levels were assessed at week 24.ResultsThe NASH diet group developed metabolic syndrome and progressive histologic features of NASH including: (a) hepatocyte ballooning at 8 weeks which progressed to extensive ballooning (>90% hepatocytes), (b) hepatic fibrosis at week 16, which progressed to moderate fibrosis, and (c) Kupffer cell accumulation with vacuolization at 8 weeks which progressed through week 24. The NASH diet group showed increased hepatocyte apoptosis that correlated with hepatic total and free cholesterol and free fatty acids, but not esterified cholesterol or triglycerides.ConclusionsThis report further characterizes the progression of diet-induced NASH in the Ossabaw swine model. In Ossabaw swine fed the NASH diet: (a) hepatocyte injury and fibrosis can occur without macrovesicular steatosis or excess triglyceride accumulation; (b) hepatocyte ballooning generally precedes the development of fibrosis; (c) there is increased hepatocyte apoptosis, and it is correlated more significantly with hepatic free cholesterol than hepatic free fatty acids and had no correlation with hepatic triglycerides.