Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K<Superscript>+</Superscript> channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H₂O₂ release using different substrates and ATP-sensitive K⁺ transport activities are increased in mitochondria from animals on high fat diets. The increase in H₂O₂ release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K⁺ channels, indicating it was not related to an observed increase in K⁺ transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K⁺ transport in mitochondria can be modulated by diet.     

Effect of prolonged ethanol ingestion on hepatic lipogenesis and related enzyme activities

1. Hepatic lipogenesis in vivo and the activities of enzymes associated with fatty acid synthesis in the liver were studied in rats fed for 21 days on liquid diets containing ethanol. 2. The ethanol-fed rats developed a moderate hepatic triacylglycerol accumulation during this period. When carbohydrate was replaced by ethanol in the diet, the rate of fatty acid synthesis was slower in the ethanol-fed rats on low-, medium- and high-fat diets than in the appropriate controls. However, when the fat/carbohydrate ratio was kept the same in the ethanol-fed and control rats, ethanol had no influence on the rate of fatty acid synthesis. 3. Glucose 6-phosphate dehydrogenase activity was lower in the ethanol-fed group. ;Malic' enzyme activity did not change during the ethanol treatment when the fat/carbohydrate ratio was kept unchanged. 4. The ATP citrate lyase activity was lower in the ethanol-fed rats on all diets, whereas acetyl-CoA synthetase activity was independent of the composition of the control diet, but was lower in the ethanol-fed rats, in which the concentration of the active form of pyruvate dehydrogenase was also lower. 5. It is concluded that hepatic fatty acid synthesis does not play any major role in ethanol-induced triacylglycerol accumulation. Careful design of the diets is necessary to reveal the specific effects of ethanol on the enzymes associated with lipogenesis.     

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.     

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.     

High Fat Feeding Induces Hepatic Fatty Acid Elongation in Mice

Background

High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated.

Methodology/Principal Findings

To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-¹³C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 µg/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 µg/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8±0.4% vs. 8.1±0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized.

Conclusions/Significance

High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice.     

Overexpression of miR-155 in the Liver of Transgenic Mice Alters the Expression Profiling of Hepatic Genes Associated with Lipid Metabolism

Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice) for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL), as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into the metabolic state of the liver in Rm155LG/Alb-Cre mice.     

EXTL2 controls liver regeneration and aortic calcification through xylose kinase-dependent regulation of glycosaminoglycan biosynthesis

The gene products of two members of the EXT gene family, EXT1 and EXT2, function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of the three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes an N-acetylhexosaminyltransferase. However, both the role of EXTL2 in glycosaminoglycan (GAG) biosynthesis and the biological significance of EXTL2 remain unclear. Interestingly, EXTL2 can transfer a GlcNAc residue to the tetrasaccharide linkage region when this region is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminate chain elongation. Production of GAGs was significantly higher in EXTL2-knockout mice than in wild-type mice. EXTL2-knockout mice are viable and apparently healthy during development and after birth. Therefore, EXTL2-knockout mice were analyzed following the experimental induction of two separate pathological conditions. Carbon tetrachloride (CCl₄) was used to induce liver failure, and 5/6th nephrectomy in combination with a high-phosphate diet was used to induce chronic kidney disease (CKD). Under conditions of CCl₄-induced liver failure, hepatocyte proliferation following CCl₄ treatment was lower in EXTL2-knockout mice than in wild-type mice; consequently, liver regeneration was impaired in EXTL2-knockout mice. This reduction in hepatocyte proliferation resulted partially because EXTL2-knockout mice experienced less hepatocyte-growth-factor-mediated signaling than did wild-type mice. Under conditions of induced CKD, matrix mineralization in vascular smooth muscle cells (VSMCs) in aortic rings of EXTL2-knockout mice was enhanced relative to that in wild-type mice. Altered biosynthesis of GAGs in EXTL2-knockout mice affected bone-morphogenetic-protein signaling, and consequently enhanced the differentiation of VSMCs into osteoblasts. Taken together, these results indicated that the EXTL2-dependent mechanism that regulates GAG biosynthesis is important for the maintenance of tissue homeostasis under pathological conditions, that is, lack of EXTL2 causes GAG overproduction and structural changes of GAGs associated with pathological processes.     

The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis

The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.     

Adipokines and adipocyte function in Clock mutant mice that retain melatonin rhythmicity

Clock(δ19)+MEL mutant mice, which retain melatonin rhythmicity, but lack peripheral tissue rhythmicity have impaired glucose tolerance, but reduced plasma free fatty acids, increased plasma adiponectin, and improved insulin sensitivity. Here, we report their response to a high-fat diet and adipocyte rhythmicity and function. The diet increased epigonadal fat weight similarly (twofold) in both wild-type and Clock(δ19)+MEL mice. The Clock(δ19) mutation abolished rhythmicity of Per2, Rev erbα and peroxisome proliferator-activated receptor-γ (Pparγ ) mRNA in epigonadal fat, but not Bmal1 mRNA, and reduced Rev erbα mRNA by 59 and 70% compared to the wild-type mice on the control and high-fat diets, respectively. The mutants had increased Adipoq mRNA expression in epigonadal fat (22%; P < 0.05) on a control diet, but showed no further change on a high-fat diet, and no change in Lep, Nampt or Retn mRNA on either diet. The Clock(δ19) mutation abolished rhythmicity of genes in epigonadal fat that contribute to plasma free fatty acids for mice on both diets, and increased Lipe mRNA expression in those on the high-fat diet. The persistent melatonin rhythm and reduced plasma free fatty acids in Clock(δ19)+MEL mutants may contribute to their enhanced insulin sensitivity, ameliorate the extent of impaired glucose homeostasis, and protect against the adverse effects of a high-fat diet.     

Hepatic mitochondrial energetics during catch-up fat with high-fat diets rich in lard or safflower oil

We have investigated whether altered hepatic mitochondrial energetics could explain the differential effects of high-fat diets with low or high ω6 polyunsaturated fatty acid content (lard vs. safflower oil) on the efficiency of body fat recovery (catch-up fat) during refeeding after caloric restriction. After 2 weeks of caloric restriction, rats were isocalorically refed with a low-fat diet (LF) or high-fat diets made from either lard or safflower oil for 1 week, and energy balance and body composition changes were assessed. Hepatic mitochondrial energetics were determined from measurements of liver mitochondrial mass, respiratory capacities, and proton leak. Compared to rats refed the LF, the groups refed high-fat diets showed lower energy expenditure and increased efficiency of fat gain; these differences were less marked with high-safflower oil than with high-lard diet. The increase in efficiency of catch-up fat by the high-fat diets could not be attributed to differences in liver mitochondrial activity. By contrast, the lower fat gain with high-safflower oil than with high-lard diet is accompanied by higher mitochondrial proton leak and increased proportion of arachidonic acid in mitochondrial membranes. In conclusion, the higher efficiency for catch-up fat on high-lard diet than on LF cannot be explained by altered hepatic mitochondrial energetics. By contrast, the ability of the high-safflower oil diet to produce a less pronounced increase in the efficiency of catch-up fat may partly reside in increased incorporation of arachidonic acid in hepatic mitochondrial membranes, leading to enhanced proton leak and mitochondrial uncoupling.     

Kefir prevented excess fat accumulation in diet-induced obese mice

Excessive body fat accumulation can result in obesity, which is a serious health concern. Kefir, a probiotic, has recently shown possible health benefits in fighting obesity. This study investigated the inhibitory effects of 0.1 and 0.2% kefir powder on fat accumulation in adipose and liver tissues of high-fat diet (HFD)-induced obese mice. Kefir reduced body weight and epididymal fat pad weight and decreased adipocyte diameters in HFD-induced obese mice. This was supported by decreased expression of genes related to adipogenesis and lipogenesis as well as reduced proinflammatory marker levels in epididymal fat. Along with reduced hepatic triacylglycerol concentrations and serum alanine transaminase and aspartate transaminase activities, genes related to lipogenesis and fatty acid oxidation were downregulated and upregulated, respectively, in liver tissue. Kefir also decreased serum triacylglycerol, total cholesterol, and low-density lipoprotein–cholesterol concentrations. Overall, kefir has the potential to prevent obesity.     

Olive oil-derived nitro-fatty acids: protection of mitochondrial function in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive liver fat deposition in the absence of significant alcohol intake. Since extra virgin olive oil (EVOO) reduces fat accumulation, we analyzed the involvement of nitro-fatty acids (NO₂-FA) on the beneficial effects of EVOO consumption on NAFLD. Nitro-fatty acids formation was observed during digestion in mice supplemented with EVOO and nitrite. Mice fed with a high-fat diet (HF) presented lower plasma NO₂-FA levels than normal chow, and circulating concentrations recovered when the HF diet was supplemented with 10% EVOO plus nitrite. Under NO₂-FA formation conditions, liver hemoxygenase-1 expression significantly increased while decreased body weight and fat liver accumulation. Mitochondrial dysfunction plays a central role in the pathogenesis of NAFLD while NO₂-FA has been shown to protect from mitochondrial oxidative damage. Accordingly, an improvement of respiratory indexes was observed when mice were supplemented with both EVOO plus nitrite. Liver mitochondrial complexes II and V activities were greater in mice with EVOO supplementation and further improved in the presence of nitrite. Overall, our results strongly suggest a positive correlation between NO₂-OA formation from EVOO and the observed improvement of mitochondrial function in NAFLD. The formation of NO₂-FA can account for the health benefits associated with EVOO consumption.     

Effect of dietary lipid content on lipid transport and storage during larval development of Manduca sexta

Manduca sexta larvae were raised on diets containing either 1.2% fat (control diet), 5.9% fat (high-fat diet) or on a fat-free diet. Insects raised on the control and high-fat diets did not differ significantly in body weight, whereas animals raised on the fat-free diet were significantly smaller. The fat content of the diet had no effect on the hemolymph concentration of lipophorin. During the larval period, lipophorin isolated from animals on the high-fat diet contained more lipid, and lipophorin isolated from animals on the fat-free diet contained less lipid than lipophorin isolated from control animals. However, lipophorin isolated from animals during the prepupal period had the same composition regardless of diet. Compared to controls, animals on the high-fat diet had a larger mass of fat body which contained more stored triacylglycerol, while animals on the fat-free diet had a smaller mass of fat body which contained less stored triacylglycerol. As the fat content of the diet was increased, the fatty acid composition of fat body triacylglycerols reflected more closely that of the dietary lipid.     

Hepatic steatosis and increased adiposity in mice consuming rapidly vs. slowly absorbed carbohydrate

Objective: Non‐alcoholic fatty liver disease (NAFLD) is fast becoming a major public health concern, coincident with the increasing prevalence of obesity. Although lifestyle greatly influences development of NAFLD, the specific dietary causes remain largely unknown. The purpose of this study was to determine whether a diet high in rapidly absorbed carbohydrate (RAC) vs. slowly absorbed carbohydrate (SAC), controlled for confounding dietary factors, causes NAFLD in mice with similar body weight. An animal model was chosen because of logistical and ethical challenges to conducting this study in humans. Research Methods and Procedures: Male 129SvPas mice were fed diets high in either RAC (amylopectin; high glycemic index) or SAC (amylose; low glycemic index) for 25 weeks. Diets were controlled for macronutrient and micronutrient content, differing only in starch type. Body weight and composition were measured throughout the study. Hepatic and plasma triacylglycerol concentrations were quantified at the end of the study. Results: Body weight was not significantly different between the two groups. However, total body adiposity increased twice as much, in absolute terms, in the mice fed RAC vs. SAC (12.2 ± 2.9% vs. 6.1 ± 4.2%, p < 0.0001). Hepatic triacylglycerol content was 2‐fold greater in the RAC group (20.7 ± 9.4 vs. 9.6 ± 4.9 mg/g, p = 0.01). In addition, plasma insulin and triacylglycerol concentrations were higher in the RAC group. Discussion: A diet high in RAC causes accumulation of fat in liver, adipose tissue, and plasma in mice. Therefore, a low glycemic index diet may help prevent or treat NAFLD in humans.     

Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high saturated fat diet

Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it may have detrimental effects by inhibiting fatty acid oxidation. Peroxisome proliferator-activated receptor α (PPARα) agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment using a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild-type and PDK4 knockout mice fed a high-fat diet. As expected, treatment of wild-type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, reduced blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid, and a reduction in the capacity for fatty acid synthesis as a result of PDK4 deficiency.     

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.