Hepatic metabolic adaptation and adipose tissue expansion are altered in mice with steatohepatitis induced by high-fat high sucrose diet

Background: Obesity is a chronic progressive disease with several metabolic alterations. Nonalcoholic fatty liver disease (NAFLD) is an important comorbidity of obesity that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis or hepatocarcinoma. This study aimed at clarifying the molecular mechanisms underlying the metabolic alterations in hepatic and adipose tissue during high-fat high-sucrose diet-induced NAFLD development in mice. Methods: Twenty-four male mice (C57BL/6J) were randomly allocated into 3 groups (n = 8 mice per group) to receive a chow diet, a high-fat diet (HFD), or a high-fat high-sucrose diet (HF-HSD) for 20 weeks. At sacrifice, liver and adipose tissue were obtained for histopathological, metabolomic, and protein expression analyses. Results: HF-HSD (but not HFD) was associated with NASH and increased oxidative stress. These animals presented an inhibition of hepatic autophagy and alterations in AMP-activated protein kinase/mammalian target of rapamycin activity. We also observed that the ability of metabolic adaptation was adversely affected by the increase of damaged mitochondria. NASH development was associated with changes in adipose tissue dynamics and increased amounts of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids in visceral adipose tissue. Conclusion: HF-HSD led to a metabolic blockage and impaired hepatic mitochondria turnover. In addition, the continuous accumulation of fatty acids produced adipose tissue dysfunction and hepatic fat accumulation that favored the progression to NASH.     

Oxidative Stress Induced Inflammation Initiates Functional Decline of Tear Production

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b₅₆₀ large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease.     

Comprehensive characterization of metabolic,inflammatory and fibrotic changes in a mouse model of diet-derived nonalcoholic steatohepatitis

The objective of this study was to develop a well-characterized mouse model of nonalcoholic steatohepatitis (NASH) with a strong manifestation of liver fibrosis. The progression of metabolic, inflammatory and fibrotic features of this mouse model was monitored by performing in vivo time-course study. Male C57BL/6J mice were fed a high-fat/high-sucrose/high-cholesterol diet (34% fat, 34% sucrose and 2.0% cholesterol, by weight) for 2, 4, 6, 8, 10, 12, 14 or 16 weeks to induce obesity-associated metabolic dysfunctions, inflammation and fibrosis in the liver and white adipose tissue (WAT). Body and liver weights were gradually increased with significant hepatic triglyceride accumulation, i.e., liver steatosis, and marked elevation of serum alanine transaminase levels at week 10. While hepatic inflammation was displayed with the highest expression of macrophage markers and M1 markers at week 6, liver fibrosis determined by collagen accumulation was continuously increased to week 16. In epididymal WAT, weights and adipocyte size peaked at week 6–8. The increased expression of fibrogenic genes preceded inflammatory features (week 2 to 6 vs. week 6 to 16), suggesting that early fibrosis may trigger inflammatory events in the WAT. This study established a mouse model of diet-induced NASH with a strong manifestation of liver fibrosis. This mouse model will be a valuable in vivo tool in studying the pathophysiology of NASH and also in testing preventive and therapeutic potentials of dietary components and drugs against NASH with liver fibrosis.     

Mitochondrial reactive oxygen species generation by the SDHC V69E mutation causes low birth weight and neonatal growth retardation

We have previously demonstrated that excessive mitochondrial reactive oxygen species caused by mutations in the SDHC subunit of Complex II resulted in premature death in C. elegans and Drosophila, tumors in mouse cells and infertility in transgenic mice. We now report the generation and initial characterization of conditional transgenic mice (Tet-mev-1) using our uniquely developed Tet-On/Off system, which equilibrates transgene expression to endogenous levels. The mice experienced mitochondrial respiratory chain dysfunction that induced reactive oxygen species overproduction. The mitochondrial oxidative stress resulted in excessive apoptosis leading to low birth weight and growth retardation in the neonatal developmental phase in Tet-mev-1 mice.     

The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H₂O₂, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm^−/− mice had significantly less hepatic fibrosis than Atm^+/+ or Atm^+/− mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression.     

L-Carnitine Prevents Progression of Non-Alcoholic Steatohepatitis in a Mouse Model with Upregulation of Mitochondrial Pathway

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease characterized by lobular inflammation, hepatocellular ballooning, and fibrosis with an inherent risk for progression to cirrhosis and hepatocellular carcinoma (HCC). Mitochondrial dysfunction appears to play a role in the progression from simple steatosis to NASH. L-carnitine (L-b-hydroxy-g-N-trimethylaminobutyric acid), an essential nutrient that converts fat into energy in mitochondria, has been shown to ameliorate liver damage. The aim of the present study was to explore the preventive and therapeutic effect of L-carnitine in NASH model mice. Eight-week-old male STAM mice, a NASH-cirrhosis-hepatocarcinogenic model, were divided into 3 experimental groups and fed as follows: 1) high-fat diet (HFD) (control group); 2) HFD mixed with 0.28% L-carnitine (L-carnitine group); and 3) HFD mixed with 0.01% α-tocopherol (α-tocopherol group). After 4 or 8 weeks, mice were sacrificed. Blood samples and livers were collected, and hepatic tumors were counted and measured. Livers were subjected to histological study, immunohistochemical staining of 4-hydroxynonenal and ferritin, determination of 8-OHdG levels, mRNA and protein expressions for multiple genes, and metabolomic analysis. The intestinal microbiome was also analyzed. L-carnitine increased hepatic expression of genes related to long-chain fatty acid transport, mitochondrial β-oxidation, and antioxidant enzymes following suppression of hepatic oxidative stress markers and inflammatory cytokines in NASH, and mice treated with L-carnitine developed fewer liver tumors. Although α-tocopherol resulted in NASH improvement in the same manner as L-carnitine, it increased periodontitis-related microbiotic changes and hepatic iron transport-related gene expression and led to less effective for anti-hepatocarcinogenesis.

Conclusion

L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model by upregulating the mitochondrial β-oxidation and redox system.     

Alpha-lipoic acid attenuates methionine choline deficient diet-induced steatohepatitis in C57BL/6 mice

AimsNon-alcoholic steatohepatitis (NASH) is a liver disease that causes fat accumulation, inflammation and fibrosis. Increased oxidative stress contributes to hepatic inflammation and fibrosis by upregulation of Cytochrome P450 2E1 (CYP2E1), endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) activity. This study examined whether alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, prevents steatohepatitis through the inhibition of several pathways involved in hepatic inflammation and fibrosis.Main MethodsC57BL/6 mice were fed an MCD diet with or without ALA for 4 weeks. Liver sections from mice on control or MCD diets with or without ALA were stained with hematoxylin-eosin, oil red O, and anti-4-HNE antibody. The effects of ALA on methionine-choline deficient MCD-diet induced plasma AST and ALT as well as tissue TBARS were measured. The effects of ALA on CYP2E1 expression, ER stress, MAPK levels, and NF-κB activity in MCD diet-fed mice liver were measured by northern and western blot analysis.Key findingsDietary supplementation with ALA reduced MCD diet-induced hepatic lipid accumulation, hepatic inflammation, TBARS, 4-HNE, and plasma ALT and AST levels. These effects were associated with a reduced expression of CYP2E1 and reduced ER stress and MAPK and NF-κB activity.SignificanceTaken together, the results of the present study indicate that ALA attenuates steatohepatitis through inhibition of several pathways, and provide the possibility that ALA can be used to prevent the development and progression of non-alcoholic fatty liver disease in patients who have strong risk factors for NASH.     

Protective effects of azelaic acid against high-fat diet-induced oxidative stress in liver, kidney and heart of C57BL/6J mice

Excess fat intake induces hyperinsulinaemia, increases nutrient uptake and lipid accumulation, amplifies ROS generation, establishes oxidative stress and morphological changes leading to tissue injury in the liver, kidney and heart of high-fat diet (HFD)-fed mice. The effect of azelaic acid (AzA), a C9 α,ω-dicarboxylic acid, against HFD-induced oxidative stress was investigated by assaying the activities and levels of antioxidants and oxidative stress markers in the liver, kidney and heart of C57BL/6J mice. Mice were segregated into two groups, one fed standard diet (NC) and the other fed high-fat diet (HFD) for 15 weeks. HFD-fed mice were subjected to intragastric administration of AzA (80 mg/kg BW)/RSG (10 mg/kg BW) during 11-15 weeks. Glucose, insulin, triglycerides, hepatic and nephritic markers were analysed in the plasma and the activity of enzymatic, non-enzymatic antioxidants and lipid peroxidation markers were examined in the plasma/erythrocytes, liver, kidney and heart of normal and experimental mice. We inferred significant decrease in enzymatic and non-enzymatic antioxidants along with significant increase in glucose, insulin, hepatic and nephritic markers, triglycerides and lipid peroxidation markers in HFD-fed mice. Administration of AzA could positively restore the levels of plasma glucose, insulin, triglycerides, hepatic and nephritic markers to near normal. AzA increased the levels of enzymatic and nonenzymatic antioxidants with significant reduction in the levels of lipid peroxidation markers. Histopathological examination of liver, kidney and heart substantiated these results. Hence, we put forward that AzA could counteract the potential injurious effects of HFD-induced oxidative stress in C57BL/6J mice.     

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.     

Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: Involvement of mitochondrial and NAD(P)H oxidase systems

Mitochondrial and NADPH oxidase systems and oxidative stress were investigated in 12 week high-fat high-sucrose (HFHS) diet-fed rats. A protective effect of wine polyphenol (PP) extract was also examined. In liver, maximal activities of CII and CII+III mitochondrial complexes were decreased but NADPH oxidase expression (p22^phox and p47^phox) and NADPH oxidase-dependent superoxide anion production were not modified, whereas oxidative stress (lipid and protein oxidation products and antioxidant systems) was increased with HFHS diet. In muscle, anion superoxide production was slightly increased while mitochondrial complex activities and lipid and protein oxidation products were not modified with HFHS diet. In heart, NADPH oxidase expression and superoxide anion production were increased, and maximal activity of mitochondrial respiratory chain complexes or oxidative stress parameters were not modified. Wine polyphenol extract had an inhibiting effect on liver oxidative stress and on heart NADPH oxidase expression and superoxide anion production, and on induction of hepatic steatosis with HFHS diet. Induction of mitochondrial dysfunction could be a primary event in the development of oxidative stress in liver, while in skeletal muscle and in heart the NADPH oxidase system seems to be mainly involved in oxidative stress. Wine polyphenol extract was shown to partially prevent oxidative stress in liver and heart tissues and to nearly completely prevent steatosis development in liver.     

Sugar kelp (Saccharina latissima) inhibits hepatic inflammation and fibrosis in a mouse model of diet-induced nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH), closely associated with obesity, is a health concern worldwide. We investigated whether the consumption of U.S.-grown sugar kelp (Saccharina latissima), an edible brown alga, can prevent obesity-associated metabolic disturbances and NASH in a mouse model of diet-induced NASH. Male C57BL/6J mice were fed a low-fat diet, a high-fat/high-sucrose/high-cholesterol diet (HF), or a HF diet containing sugar kelp (HF-Kelp) for 14 weeks. HF-Kelp group showed lower body weight with increased O₂ consumption, CO₂ production, physical activity, and energy expenditure compared with the HF. In the liver, there were significant decreases in weight, triglycerides, total cholesterol, and steatosis with HF-Kelp. The HF-Kelp group decreased hepatic expression of a macrophage marker adhesion G protein-coupled receptor E1 (Adgre1) and an M1 macrophage marker integrin alpha x (Itgax). HF-Kelp group also exhibited decreased liver fibrosis, as evidenced by less expression of fibrogenic genes and collagen accumulation than those of HF group. In epididymal white adipose tissue (eWAT), HF-Kelp group exhibited decreases in eWAT weight and adipocyte size compared with those of the HF. HF-Kelp group showed decreased expression of collagen type VI alpha 1 chain, Adgre1, Itgax, and tumor necrosis factor α in eWAT. We demonstrated, for the first time, that the consumption of U.S-grown sugar kelp prevented the development of obesity and its associated metabolic disturbances, steatosis, inflammation, and fibrosis in the liver and eWAT of a diet-induced NASH mouse model.     

Dietary fat,fatty acid saturation and mitochondrial bioenergetics

Fat intake alters mitochondrial lipid composition which can affect function. We used novel methodology to assess bioenergetics, including simultaneous ATP and reactive oxygen species (ROS) production, in liver and heart mitochondria of C57BL/6 mice fed diets of variant fatty acid content and saturation. Our methodology allowed us to clamp ADP concentration and membrane potential (ΔΨ) at fixed levels. Mice received a control diet for 17–19 weeks, a high-fat (HF) diet (60 % lard) for 17–19 weeks, or HF for 12 weeks followed by 6–7 weeks of HF with 50 % of fat as menhaden oil (MO) which is rich in n-3 fatty acids. ATP production was determined as conversion of 2-deoxyglucose to 2-deoxyglucose phosphate by NMR spectroscopy. Respiration and ATP production were significantly reduced at all levels of ADP and resultant clamped ΔΨ in liver mitochondria from mice fed HF compared to controls. At given ΔΨ, ROS production per mg mitochondrial protein, per unit respiration, or per ATP generated were greater for liver mitochondria of HF-fed mice compared to control or MO-fed mice. Moreover, these ROS metrics began to increase at a lower ΔΨ threshold. Similar, but less marked, changes were observed in heart mitochondria of HF-fed mice compared to controls. No changes in mitochondrial bioenergetics were observed in studies of separate mice fed HF versus control for only 12 weeks. In summary, HF feeding of sufficient duration impairs mitochondrial bioenergetics and is associated with a greater ROS “cost” of ATP production compared to controls. These effects are, in part, mitigated by MO.     

Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis

Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 +/- 0.5 g (means +/- SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system.     

Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H(2)O(2) and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H(2)O(2) production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.     

The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H₂O₂, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm^?/? mice had significantly less hepatic fibrosis than Atm^+/+ or Atm^+/? mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression.     

Beneficial effects of hydro-alcoholic extract of Caralluma fimbriata against high-fat diet-induced insulin resistance and oxidative stress in Wistar male rats

The main aim of this study was to investigate the beneficial effects of hydro-alcoholic extract of Caralluma fimbriata (CFE) on the effects of high-fat diet feeding on insulin resistance and oxidative stress in Wistar rats. High-fat diet (60 % of fat) and CFE (200 mg/kg body weight/day) were given concurrently to the rats for a period of 90 days. Feeding with high-fat diet resulted in the development of hyperglycemia, hyperinsulinemia, hyperleptinemia, and hypertriglyceridemia and impaired insulin sensitivity (P?<?0.05). Administration of CFE to high-fat diet-fed rats for 90 days resulted in a significant improvement in plasma glucose, insulin, leptin, and triglycerides. Regarding liver antioxidant status, high-fat fed rats showed higher levels of lipid peroxidation, protein oxidation and lower GSH levels and lower activities of enzymatic antioxidants, while CFE treatment prevented all these observed abnormalities. In conclusion, intake of CFE may be beneficial for the suppression of high-fat diet-induced insulin resistance and oxidative stress.     

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt^?/? mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt^?/? mice. Treatment with vitamin E (0.5?g/kg) for 3?weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt^?/? mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.     

Adaptive failure to high-fat diet characterizes steatohepatitis in Alms1 mutant mice

The biochemical differences between simple steatosis, a benign liver disease, and non-alcoholic steatohepatitis, which leads to cirrhosis, are unclear. Fat aussie is an obese mouse strain with a truncating mutation (foz) in the Alms1 gene. Chow-fed female foz/foz mice develop obesity, diabetes, and simple steatosis. We fed foz/foz and wildtype mice a high-fat diet. Foz/foz mice developed serum ALT elevation and severe steatohepatitis with hepatocyte ballooning, inflammation, and fibrosis; wildtype mice showed simple steatosis. Biochemical pathways favoring hepatocellular lipid accumulation (fatty acid uptake; lipogenesis) and lipid disposal (fatty acid beta-oxidation; triglyceride egress) were both induced by high-fat feeding in wildtype but not foz/foz mice. The resulting extremely high hepatic triglyceride levels were associated with induction of mitochondrial uncoupling protein-2 and adipocyte-specific fatty acid binding protein-2, but not cytochrome P4502e1 or lipid peroxidation. In this model of metabolic syndrome, transition of steatosis to steatohepatitis was associated with hypoadiponectinemia, a mediator of hepatic fatty acid disposal pathways.     

High-fat diet decreases activity of the oxidative phosphorylation complexes and causes nonalcoholic steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is the most frequent histological finding in individuals with abnormal liver-function tests in the Western countries. In previous studies, we have shown that oxidative phosphorylation (OXPHOS) is decreased in individuals with NAFLD, but the cause of this mitochondrial dysfunction remains uncertain. The aims of this study were to determine whether feeding mice a high-fat diet (HFD) induces any change in the activity of OXPHOS, and to investigate the mechanisms involved in the pathogenesis of this defect. To that end, 30 mice were distributed between five groups: control mice fed a standard diet, and mice on a HFD and treated with saline solution, melatonin (an antioxidant), MnTBAP (a superoxide dismutase analog) or uric acid (a scavenger of peroxynitrite) for 28 weeks intraperitoneously. In the liver of these mice, we studied histology, activity and assembly of OXPHOS complexes, levels of subunits of these complexes, gene expression of these subunits, oxidative and nitrosative stress, and oxidative DNA damage. In HFD-fed mice, we found nonalcoholic steatohepatitis, increased gene expression of TNFα, IFNγ, MCP-1, caspase-3, TGFβ1 and collagen α1(I), and increased levels of 3-tyrosine nitrated proteins. The activity and assembly of all OXPHOS complexes was decreased to about 50–60%. The amount of all studied OXPHOS subunits was markedly decreased, particularly the mitochondrial-DNA-encoded subunits. Gene expression of mitochondrial-DNA-encoded subunits was decreased to about 60% of control. There was oxidative damage to mitochondrial DNA but not to genomic DNA. Treatment of HFD-fed mice with melatonin, MnTBAP or uric acid prevented all changes observed in untreated HFD-fed mice. We conclude that a HFD decreased OXPHOS enzymatic activity owing to a decreased amount of fully assembled complexes caused by a reduced synthesis of their subunits. Antioxidants and antiperoxynitrites prevented all of these changes, suggesting that nitro-oxidative stress played a key role in the pathogenesis of these alterations. Treatment with these agents might prevent the development of NAFLD in humans.KEY WORDS: Mitochondrial respiratory chain, Nonalcoholic steatohepatitis, NADPH oxidase, Oxidative phosphorylation, Proteomic, Nitro-oxidative stress