Adiponectin knockout mice on high fat diet develop fibrosing steatohepatitis

Background and Aim:  Low levels of serum adiponectin have been reported to be associated with obesity, diabetes, and non-alcoholic steatohepatitis (NASH), as well as several malignancies. Adiponectin knockout (KO) mice have been reported to cause insulin resistance and neointimal formation of the artery. We used adiponectin KO mice fed a high fat (HF) diet, and investigated the effect of adiponectin on the progression of steatohepatitis and carcinogenesis in vivo .
Methods:  Adiponectin KO mice and wild type (WT) mice were fed a HF diet or normal chow for the periods of 24 and 48 weeks. The HF diet contained 60% of calories from fat.
Results:  The adiponectin KO mice on the HF diet showed obesity, marked elevation of serum transaminase levels, and hyperlipidemia. At 24 weeks, hepatic expression of tumor necrosis factor-α and procollagen α (I) was higher in KO mice as compared with WT mice. At 48 weeks, liver triglyceride contents in KO mice on normal chow were significantly higher than those in WT mice. Hepatocyte ballooning, spotty necrosis, and pericellular fibrosis around central veins were observed in KO mice on the HF diet. The pericellular fibrosis was more severe in KO mice on the HF diet than that in WT mice (1.62% vs 1.16%, P  = 0.033). Liver adenoma and hyperplastic nodules developed in a KO mouse on the HF diet at 48 weeks (12.5%, n  = 1/8), whereas no tumor was detected in WT mice ( n  = 10).
Conclusions:  Adiponectin may play a protective role in the progression of NASH in the early stages by suppressing tumor necrosis factor-α expression and liver fibrosis.     

Hypoadiponectinemia accelerates hepatic tumor formation in a nonalcoholic steatohepatitis mouse model

BACKGROUND/AIMS: Adipose tissue produces a number of adipocytokines, including adiponectin, leptin, and tumor necrosis factor-alpha. Obesity, which is associated with low plasma adiponectin levels, is an independent risk factor for various liver diseases including nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the effects of adiponectin on the progression of NASH to cirrhosis and tumor formation using adiponectin-knockout (KO) mice. METHODS: Using a choline-deficient L-amino acid-defined (CDAA) diet-induced mouse NASH model, liver histology and oxidative stress markers were investigated in KO and wild-type (WT) mice. RESULTS: Hepatic steatosis was enhanced to a greater extent in KO mice, compared to WT mice after a 1-week CDAA diet. After 24 weeks, 6 out of 14 KO mice developed liver cirrhosis and hepatic tumors, whereas the 15 WT mice showed only simple steatosis. In KO mice, hepatic cytochrome P450 2E1 levels were upregulated, and markers of oxidative stress (thiobarbituric acid reactive substances, 8-hydroxydeoxyguanosine-positive cells) were significantly increased compared with WT mice. CONCLUSIONS: Our results indicate that lack of adiponectin enhances the progression of hepatic steatosis, fibrosis, and hepatic tumor formation in an animal model of NASH. Hypoadiponectinemia in obesity could be a risk factor for NASH-related hepatic tumor formation.     

Hepatic vagus nerve regulates Kupffer cell activation via α7 nicotinic acetylcholine receptor in nonalcoholic steatohepatitis

Background

Nonalcoholic fatty liver disease ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). Kupffer cells play a central role in promoting hepatic inflammation, which leads to the development of NASH. We investigated the anti-inflammatory effect of hepatic vagus-mediated stimulation of the α7 nicotinic acetylcholine receptor (α7nAChR) on Kupffer cells in NASH pathogenesis.

Methods

Wild-type (WT) mice undergoing hepatic vagotomy (HV) were fed a methionine- and choline-deficient (MCD) diet for 1 week. α7nAChR knockout (α7KO) chimeric mice were generated by transplanting α7KO bone marrow cells into irradiated and Kupffer cell-deleted WT recipients. Kupffer cells were isolated from WT mice and treated with α7nAChR agonist under stimulation by lipopolysaccharide and/or palmitic acid.

Results

HV aggravated MCD diet-induced NASH in both steatosis and inflammation. The hepatic inflammatory response, including the upregulation of tumor necrosis factor alpha (TNFα), interleukin (IL)-12, and monocyte chemoattractant protein 1 (MCP-1), was accelerated in HV mice, accompanied by the downregulation of PPARα pathway genes. Kupffer cells were highly activated via the phosphorylation and nuclear translocation of nuclear factor-kappa B (NF-κB) in MCD diet-fed HV mice. The α7nAchR agonist suppressed the inflammatory response of primary Kupffer cells induced by lipopolysaccharide and palmitic acid by attenuating the NF-κB cascade. α7KO chimeric mice fed an MCD diet for 1 week developed advanced NASH with highly activated Kupffer cells. The hepatic expression of TNFα, IL-12, and MCP-1 was upregulated in α7KO chimeric mice, accompanied by abnormal lipid metabolism.

Conclusions

Hepatic vagus activity regulates the inflammatory response of Kupffer cells via α7nAChR in NASH development.

     

Hepatic AdipoR2 signaling plays a protective role against progression of nonalcoholic steatohepatitis in mice

It is unclear how hepatic adiponectin resistance and sensitivity mediated by the adiponectin receptor, AdipoR2, contributes to the progression of nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the roles of hepatic AdipoR2 in NASH, using an animal model. We fed C57BL/6 mice a methionine-deficient and choline-deficient (MCD) diet for up to 8 weeks and analyzed changes in liver pathology caused by either an AdipoR2 short hairpin RNA-expressing adenovirus or an AdipoR2-overexpressing adenovirus. Inhibition of hepatic AdipoR2 expression aggravated the pathological state of NASH at all stages: fatty changes, inflammation, and fibrosis. In contrast, enhancement of AdipoR2 expression in the liver improved NASH at every stage, from the early stage to the progression of fibrosis. Inhibition of AdipoR2 signaling in the liver diminished hepatic peroxisome proliferator activated receptor (PPAR)-alpha signaling, with decreased expression of acyl-CoA oxidase (ACO) and catalase, leading to an increase in lipid peroxidation. Hepatic AdipoR2 overexpression had the opposite effect. Reactive oxygen species (ROS) accumulation in liver increases hepatic production of transforming growth factor (TGF)-beta1 at all stages of NASH; adiponectin/AdipoR2 signaling ameliorated TGF-beta-induced ROS accumulation in primary cultured hepatocytes, by enhancing PPAR-alpha activity and catalase expression. CONCLUSION: The adiponectin resistance and sensitivity mediated by AdipoR2 in hepatocytes regulated steatohepatitis progression by changing PPAR-alpha activity and ROS accumulation, a process in which TGF-beta signaling is implicated. Thus, the liver AdipoR2 signaling pathway could be a promising target in treating NASH.     

Apoptosis in experimental NASH is associated with p53 activation and TRAIL receptor expression

Background and Aims:  We examined extrinsic and intrinsic (endogenous) mitochondrial apoptosis pathways in experimental non-alcoholic steatohepatitis (NASH).
Methods:  To assess extrinsic pathways, we measured hepatic expression of death-inducing cytokine receptors (tumor necrosis factor-α-receptor (TNF-R)1, TNF-R2, Fas, and TNFα-related apoptosis-inducing ligand-receptor (TRAIL-R) mRNA, TUNEL, caspase 3 activation, liver injury and liver pathology in mice fed a methionine and choline deficient (MCD) diet. For endogenous stress pathways, we determined serum insulin-like growth factor-1 (IGF-1), hepatic p53, Bcl-XL, tBid and p21 expression.
Results:  Methionine and choline deficient feeding increased alanine aminotransferase (ALT) and apoptosis from day 10, without increases in TNF-R1, TNF-R2, and Fas. However, murine TRAIL receptors, particularly decoyTRAIL-R1/TNFRSFH23 and Killer/DR5 mRNA increased. MCD feeding enhanced hepatic p53 expression, corresponding to ∼50% fall in serum IGF-1, decreased Bcl-XL, enhanced Bid cleavage to tBid, and up-regulation of p21. Nutritional restitution experiments showed that correcting either methionine or choline deficiency suppressed liver inflammation (extrinsic pathway), but failed to correct apoptosis, IGF-1 or p53.
Conclusions:  Methionine and choline deficiency lower IGF-1 to de-repress p53 during induction of steatohepatitis. The p53 induced by nutritional stress is biologically active in mediating mitochondrial cell death pathways, but may also be responsible for TRAIL receptor expression, thereby linking intrinsic and exogenous apoptosis pathways in NASH.     

Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice

BACKGROUND: While tumour necrosis factor alpha (TNF-alpha) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. METHODS: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. RESULTS: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-alpha, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-alpha administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. CONCLUSIONS: Enhancement of the TNF-alpha/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.     

Expression of adiponectin and its receptors in livers of morbidly obese patients with non-alcoholic fatty liver disease

Background and Aim:  Obesity is one of the risk factors for non-alcoholic fatty liver disease (NAFLD) and a common disease that comprises simple steatosis and non-alcoholic steatohepatitis (NASH), and can eventually lead to liver cirrhosis. Adiponectin is an adipocyte-derived protein that has anti-obesity, antidiabetic and anti-inflammatory properties, and is considered to possess a hepatoprotective function. Its role in the development and progression of NAFLD in morbidly obese patients is unknown. In this study, we examined the expression levels of adiponectin and its receptors in liver biopsies of morbidly obese patients and then determined whether there was an association with the disease severity.
Methods:  Liver biopsies from 30 morbidly obese patients (18 NASH vs 12 steatosis) were analyzed. The needle core biopsies were subjected to routine histological examination and stained immunohistochemically for adiponectin, adiponectin receptor I (adipoRI) and receptor II (adipoRII).
Results:  The two groups were comparable with respect to body mass index, age and gender distribution. The expression of adiponectin decreased in liver biopsies with NASH as compared to those with simple steatosis (1.61 ± 0.70 vs 2.25 ± 0.75, P  = 0.028). Spearman's rank correlation coefficient analysis showed that the staining intensity of adiponectin negatively correlated with the grade of inflammation ( r  = −0.368, P  = 0.045) and stage of fibrosis ( r  = −0.380, P  = 0.038). There was no significant difference in expression of adipoRI and adipoRII between the two groups.
Conclusion:  These findings indicate that decreased liver adiponectin expression may play a role in the development and progression of NAFLD, from simple steatosis to NASH, in morbidly obese patients.     

MCD-induced steatohepatitis is associated with hepatic adiponectin resistance and adipogenic transformation of hepatocytes

BACKGROUND/AIMS: In these studies, we tested the hypothesis that increased lipid intake would exacerbate the severity of nutritional steatohepatitis. METHODS: C57Bl/6J mice were fed methionine-and-choline deficient (MCD) diets containing 20% (high) or 5% (low) fat by weight for 3 weeks and compared to lipid-matched controls. RESULTS: MCD feeding increased serum ALT levels and induced hepatic steatosis, lobular inflammation and ballooning degeneration of hepatocytes, irrespective of dietary fat content. Hepatic triglyceride accumulation was similar between high and low-fat MCD-fed mice, but lipoperoxide levels were approximately 3-fold higher in the high-fat MCD-fed animals. Serum adiponectin levels increased in MCD-fed mice, although to a lesser extent in high-fat fed animals. AMPK phosphorylation was correspondingly increased in muscle of MCD-fed mice, but hepatic AMPK phosphorylation decreased, and there was little evidence of PPARalpha activation, suggesting impaired adiponectin action in the livers of MCD-fed animals. Hepatocyte PPARgamma mRNA levels increased in MCD-fed mice, and were associated with increased aP2 expression, indicating adipogenic transformation of hepatocytes. CONCLUSIONS: Increased dietary lipid intake did not alter steatohepatitis severity in MCD-fed mice despite increased lipoperoxide accumulation. Instead, steatohepatitis was associated with impaired hepatic adiponectin action, and adipogenic transformation of hepatocytes in both low and high-fat MCD-fed mice.     

Isolated idiopathic bile ductular hyperplasia in patients with persistently abnormal liver function tests

BACKGROUND/AIMS: Feeding mice a methionine choline deficient (MCD) diet serves as a nutritional model of non-alcoholic steatohepatitis (NASH). NASH and alcohol-induced steatohepatitis are histologically similar, suggesting a similar pathogenesis. Pentoxifylline (PTX) attenuates TNF-alpha production, acts as an antioxidant and decreases mortality in alcoholic steatohepatitis. The aim of our study is to determine if PTX attenuates MCD diet induced steatohepatitis and determine the mechanism of this effect. METHODS: Mice were placed on an MCD or control diet for 2 weeks and were treated with or without PTX. Serum ALT, liver histology, and inflammatory mechanisms were evaluated. RESULTS: PTX attenuates MCD diet induced steatohepatitis, decreasing both serum ALT levels and hepatic inflammation. Serum ALT levels were reduced approximately 50% in the MCD+PTX group compared to the MCD group. Hepatic glutathione levels were significantly higher in the MCD+PTX group compared to the MCD group. There was also a reduction in TNF-alpha mRNA in female mice treated with PTX. MCD+PTX mice had increased hepatic triglyceride content compared to the MCD mice, but less histologic evidence of inflammation despite the increased steatosis. Serum lipid and bile salt levels also were similar in PTX and vehicle control treated mice. CONCLUSIONS: PTX decreases serum ALT levels and hepatic inflammation in the MCD model of steatohepatitis, likely via increasing glutathione levels or reducing TNF-alpha expression.     

Antioxidants vitamin E and 1-aminobenzotriazole prevent experimental non-alcoholic steatohepatitis in mice

Objective. Hepatic oxidative stress plays a key role in the development of non-alcoholic steatohepatitis (NASH). However, the protective effects of antioxidants on NASH are largely unknown. The aim of this study was to elucidate the effect and mechanism of antioxidants on NASH in mice. Material and methods. C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 10 days or 3 weeks to induce steatohepatitis. Antioxidants (vitamin E, ABT, or vitamin E plus ABT) were supplemented in mice fed a MCD diet, respectively. The effect of antioxidants on oxidative stress and apoptosis was assessed, and activation of adiponectin and expressions of inflammatory factors, apoptosis-related genes, and fibrosis-related genes were assayed. Results. MCD feeding in mice showed increasing serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels, and progressive hepatic injury including hepatic steatosis and inflammatory infiltration. Administration of antioxidants vitamin E and/or ABT significantly lowered serum ALAT and ASAT levels (p<0.001) and ameliorated hepatic steatosis and necroinflammation. These effects were associated with repressed hepatic lipid peroxides through reducing hepatic MDA content and enhancing hepatic superoxide dismutase (SOD) activity; down-regulated inflammatory factor COX-2, lowered activity of NF-κB, up-regulated anti-apoptotic gene Bcl-2, and down-regulated pro-apoptotic gene Bax suppressed expression of the fibrotic genes TGF-β1 and MMP2. Moreover, expression of the anti-inflammatory factor adiponectin was also induced by vitamin E or ABT. A combination of vitamin E and ABT showed an additive effect on preventing liver injury. Conclusions. The present study provides morphological and molecular biological evidence for the protective role of the antioxidant vitamins E and ABT in ameliorating oxidative stress, hepatic apoptosis, and necroinflammation in experimental nutritional steatohepatitis.     

miR-21-5p promotes NASH-related hepatocarcinogenesis

Background and Aims

The mechanisms governing the progression of non-alcoholic fatty liver disease (NAFLD) towards steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain elusive. Here, we evaluated the role of hsa-miRNA-21-5p in NASH-related hepatocarcinogenesis.

Methods

Hepatic hsa-miR-21-5p expression was evaluated in two cohorts of patients with biopsy-proven NAFLD (n = 199) or HCC (n = 366 HCC and n = 11 NAFLD-HCC). Serum/liver metabolomic profiles were correlated with hsa-miR-21-5p in NAFLD obese patients. Wild-type (WT) and Mir21 KO mice were fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks to induce NASH and NASH-HCC, respectively.

Results

In obese individuals, hsa-miR-21-5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile. CDAA-fed WT mice displayed increased hepatic mmu-miR-21-5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre-neoplastic nodules, hyperplastic foci and deregulated cancer-related pathways. Mir21 KO mice exhibited peroxisome-proliferator-activated receptor α (PPARα) activation, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis, with the pro-inflammatory/fibrogenic milieu reversing to baseline levels. In parallel, Mir21 KO mice displayed reduced number of pre-neoplastic nodules, hepatocyte proliferation and activation of oncogenic signalling, being protected from NASH-associated carcinogenesis. The hsa-miRNA-21-5p/PPARα pathway was similarly deregulated in patients with HCC- or NASH-related HCC, correlating with HCC markers and worse prognosis.

Conclusions

Hsa-miR-21-5p is a key inducer of whole-spectrum NAFLD progression, from simple steatosis to NASH and NASH-associated carcinogenesis. The inhibition of hsa-miR-21-5p, leading to a pro-metabolic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC.     

The Transition from Fatty Liver to NASH Associates with SAMe Depletion in db/db Mice Fed a Methionine Choline-Deficient Diet

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in the Western population. By mechanisms that are not completely understood, this disease may progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). db/db mice spontaneously develop hepatic steatosis, which progresses to NASH when these mice are fed a methionine choline-deficient (MCD) diet. The goal of our studies was to identify lipid and methionine metabolism pathways affected by MCD feeding to determine potential causal events leading to the development of NASH from benign steatosis. db/db mice fed the MCD diet for 2 weeks exhibited signs of incipient NASH development such as upregulated cytokines and chemokines. At this time point, MCD diet feeding caused S-adenosylmethionine (SAMe) depletion in db/db mice, while wild-type mice on the same diet retained hepatic SAMe levels. SAMe depletion exerts pleiotropic effects upon liver homeostasis and is commonly associated with a variety of liver insults such as thioacetamide, CCL(4), and alcohol treatment; thus, SAMe depletion may serve as the second hit in NASH development. It is possible that differences in hepatic lipid and/or methionine metabolism between wild-type and db/db mice underlay the differential maintenance of SAMe levels during methionine and choline restriction. Indeed, db/db mice exhibited inhibited lipid oxidation pathways, which may be a priming factor for NASH development, and db/db mice fed the MCD diet had differential methionine adenosyltransferase (MAT) expression. The occurrence of SAMe depletion at this early, benign stage of NASH development in db/db mice with fatty liver suggests that SAMe supplementation may be (A) targeted to individuals susceptible to NASH (i.e., NAFLD patients) and (B) preventative of NASH before substantial liver injury has occurred.     

Rodent nutritional model of steatohepatitis: Effects of endotoxin (lipopolysaccharide) and tumor necrosis factor alpha deficiency

Background and Aims: Intestinal endotoxin (lipopolysaccharide) is thought to contribute to liver injury in both alcoholic and nonalcoholic steatohepatitis (NASH). Tumor necrosis factor alpha (TNFα) is an important mediator of this process and is considered central to the inflammatory response in NASH. This study aimed to investigate the effects of lipopolysaccharide on liver injury in the methionine choline deficient (MCD) nutritional model of NASH, and to determine if TNFα is required for the development of steatohepatitis in this model. Method: Male C57/BL6 mice received a MCD diet for 4 weeks, whilst a control group received an identical diet supplemented with 0.2% choline bitartrate and 0.3% methionine. At 4 weeks, mice received either an intraperitoneal injection of lipopolysaccharide (0.5 µg/g body mass) or sterile saline, and were killed 24 h thereafter. In a separate study, TNFα knockout and wild type C57BL/6 mice received either MCD or control diets for 4 weeks. Serum transaminase levels, liver histology (steatosis, inflammation and apoptosis), hepatic triglyceride concentration and hepatic lipid peroxidation products (conjugated dienes, lipid hydroperoxides and thiobarbituric reactive substances, free and total) were evaluated. Results: Intraperitoneal administration of lipopolysaccharide augmented serum alanine aminotransferase (ALT) levels (P < 0.02), hepatic inflammation (P < 0.025), apoptosis (P < 0.01) and free thiobarbituric acid reactive substances (P < 0.025) in MCD mice. TNFα knockout mice fed the MCD diet developed steatohepatitis with histological and biochemical changes similar to those seen in wild type counterparts. Conclusions: Lipopolysaccharide augments liver injury in MCD mice, and TNFα is not required for the development of steatohepatitis in MCD mice.     

Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis

In the early stages of nonalcoholic fatty liver disease (NAFLD), triglycerides accumulate in hepatocytes. Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in hepatocyte triglyceride biosynthesis. DGAT2 antisense oligonucleotide (ASO) treatment improved hepatic steatosis dramatically in a previous study of obese mice. According to the 2-hit hypothesis for progression of NAFLD, hepatic steatosis is a risk factor for nonalcoholic steatohepatitis (NASH) and fibrosis. To evaluate this hypothesis, we inhibited DGAT2 in a mouse model of NASH induced by a diet deficient in methionine and choline (MCD). Six-week-old genetically obese and diabetic male db/db mice were fed either the control or the MCD diet for 4 or 8 weeks. The MCD diet group was treated with either 25 mg/kg DGAT2 ASO or saline intraperitoneally twice weekly. Hepatic steatosis, injury, fibrosis, markers of lipid peroxidation/oxidant stress, and systemic insulin sensitivity were evaluated. Hepatic steatosis, necroinflammation, and fibrosis were increased in saline-treated MCD diet-fed mice compared to controls. Treating MCD diet-fed mice with DGAT2 ASO for 4 and 8 weeks decreased hepatic steatosis, but increased hepatic free fatty acids, cytochrome P4502E1, markers of lipid peroxidation/oxidant stress, lobular necroinflammation, and fibrosis. Progression of liver damage occurred despite reduced hepatic expression of tumor necrosis factor alpha, increased serum adiponectin, and striking improvement in systemic insulin sensitivity. CONCLUSION: Results from this mouse model would suggest accumulation of triglycerides may be a protective mechanism to prevent progressive liver damage in NAFLD.     

Sitagliptin attenuates methionine/choline-deficient diet-induced steatohepatitis

Aims

Accumulating evidence suggests that inhibitors of dipeptidyl peptidase-4 (DPP-4), such as sitagliptin, may play an important role in the prevention of non-alcoholic steatohepatitis (NASH). This study was conducted to elucidate whether sitagliptin could prevent steatohepatitis by inhibiting pathways involved in hepatic steatosis, inflammation, and fibrosis.

Methods

C57BL/6 mice were fed a methionine/choline-deficient (MCD) diet with or without supplement with sitagliptin for 5 weeks. Liver and adipose tissue from mice were examined histologically and immunohistochemically to estimate the effect of sitagliptin on the development of NASH.

Results

Supplementation with sitagliptin resulted in significant improvement of MCD diet-induced fat accumulation in the liver. In addition, sitagliptin treatment lowered fatty acid uptake, expression of VLDL receptor and hepatic triglyceride content. Sitagliptin also effectively attenuated MCD diet-induced hepatic inflammation, endoplasmic reticulum (ER) stress, and liver injury, as evidenced by reduced proinflammatory cytokine levels, ER stress markers, and TUNEL staining. Expression of CYP2E1 and 4NHE were strongly increased by the MCD diet, but this effect was successfully prevented by sitagliptin treatment. Furthermore, sitagliptin significantly decreased levels of MCD diet-induced fibrosis-associated proteins such as fibronectin and α-SMA in the liver. Inflammatory and atrophic changes of adipose tissue by MCD diet were restored by sitagliptin treatment.

Conclusions

Sitagliptin attenuated MCD diet-induced hepatic steatosis, inflammation, and fibrosis in mice through amelioration of mechanisms responsible for the development of NASH, including CD36 expression, NF-κB activation, ER stress, CYP2E1 expression, and lipid peroxidation. Treatment with sitagliptin may represent an effective approach for the prevention and treatment of NASH.     

Potential role of chitotriosidase gene in nonalcoholic fatty liver disease evolution

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by steatosis and periportal and lobular inflammation. The molecular mechanisms involved in the anomalous behavior of liver cells have only partially been disclosed. Human Chitotriosidase (Chit) is a member of the chitinase family that it is mainly synthesized by activated macrophages. We investigated chitotriosidase gene expression in Kupffer cells to determine the potential implication of this enzyme in the inflammation and in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis. METHODS: Seventy-five liver biopsies from 40 subjects with NASH, 20 with simple steatosis, and 15 controls were used to detect CHIT expression, tumor necrosis factor-alpha (TNF-alpha), alpha-smooth muscle actin (alpha-SMA), and lipid peroxidation. RESULTS: CHIT was expressed exclusively by Kupffer cells. The levels of CHIT expression were significantly higher in NASH patients than in simple steatosis patients and in the control group. In addition, we found that CHIT over-expression influenced hepatic stellate cells activation, as demonstrated by the significant correlation between CHIT and alpha-SMA expression in NASH patients. A significant correlation was observed also between CHIT, TNF-alpha and lipid peroxidation in both NASH and simple steatosis. CONCLUSION: These results suggest that CHIT over-produced by Kupffer cells may contribute to the progression of hepatic fibrosis.