Dr. Kohli is a consultant for and received grants from Johnson & Johnson. Dr. Seeley is a consultant for and is on the speakers' bureau of Amylin Pharmaceuticals. He is also on the speakers' bureau of Eli Lilly and Johnson & Johnson. He is on the speakers' bureau of Novo Nordick and Merck. He owns stock in Zafgen Inc.
Diets high in saturated fat and fructose have been implicated in the development of obesity and nonalcoholic steatohepatitis (NASH) in humans. We hypothesized that mice exposed to a similar diet would develop NASH with fibrosis associated with increased hepatic oxidative stress that would be further reflected by increased plasma levels of the respiratory chain component, oxidized coenzyme Q9 (oxCoQ9). Adult male C57Bl/6 mice were randomly assigned to chow, high-fat (HF), or high-fat high-carbohydrate (HFHC) diets for 16 weeks. The chow and HF mice had free access to pure water, whereas the HFHC group received water with 55% fructose and 45% sucrose (wt/vol). The HFHC and HF groups had increased body weight, body fat mass, fasting glucose, and were insulin-resistant compared with chow mice. HF and HFHC consumed similar calories. Hepatic triglyceride content, plasma alanine aminotransferase, and liver weight were significantly increased in HF and HFHC mice compared with chow mice. Plasma cholesterol (P < 0.001), histological hepatic fibrosis, liver hydroxyproline content (P = 0.006), collagen 1 messenger RNA (P = 0.003), CD11b-F4/80+Gr1+ monocytes (P < 0.0001), transforming growth factor β1 mRNA (P = 0.04), and α-smooth muscle actin messenger RNA (P = 0.001) levels were significantly increased in HFHC mice. Hepatic oxidative stress, as indicated by liver superoxide expression (P = 0.002), 4-hydroxynonenal, and plasma oxCoQ9 (P < 0.001) levels, was highest in HFHC mice. Conclusion: These findings demonstrate that nongenetically modified mice maintained on an HFHC diet in addition to developing obesity have increased hepatic ROS and a NASH-like phenotype with significant fibrosis. Plasma oxCoQ9 correlated with fibrosis progression. The mechanism of fibrosis may involve fructose inducing increased ROS associated with CD11b+F4/80+Gr1+ hepatic macrophage aggregation, resulting in transforming growth factor β1–signaled collagen deposition and histologically visible hepatic fibrosis. (HEPATOLOGY 2010)
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Epidemiologic data suggest that there has been a significant rise in calories consumed from saturated fat and fructose-rich foods.1 This has been paralleled by an increasing prevalence of obesity and its associated hepatic comorbidity, nonalcoholic fatty liver disease (NAFLD).2 Natural history studies of NAFLD indicate that the presence of fibrosis within the more severe phenotype, nonalcoholic steatohepatitis (NASH), is an important predictor of adverse long-term outcomes, including diabetes and progression to cirrhosis.3, 4 Fibrosis progression to cirrhosis is thought to be modulated through hepatic reactive oxygen species (ROS) generation, macrophage activation, and transforming growth factor β (TGF-β)-mediated collagen deposition.5-7 Although recent data have highlighted potential biomarkers for distinguishing NAFLD from NASH, liver biopsy continues to be the gold standard for monitoring fibrosis progression in NASH.8 The role of saturated fat and fructose in triggering the mechanisms of fibrosis progression in NASH remain to be clearly elucidated.9
Our understanding of this process has been hampered by the lack of a comprehensive and physiologic small animal model of NASH with fibrosis. To date, small animal models of NASH with fibrosis involve genetic manipulation,10-12 forced overfeeding,13 or contrived diets deficient in methionine and choline (MCD).14-17 Although each of these models has been valuable, they fail to address key aspects of the process in humans. For example, few humans have diets that are deficient in methionine and choline. Moreover, rodents exposed to methionine- and choline-deficient diets are not obese; rather, they lose weight and become more insulin-sensitive.17
Recent studies, particularly the ALIOS diet using ad libitum high-fructose and high–trans fat diets in small animals, have had some success in generating steatosis with inflammation but failed to produce significant fibrosis.18, 19 Lieber et al.20 fed a high-fat-liquid diet (71% kcal from fat) to rats ad libitum, but these animals only developed steatosis without any fibrosis or collagen deposition. Genetically modified mice (such as liver-specific phosphotase and tensin homolog–suppressed10 or carcinoembryonic antigen-related cell adhesion molecule–inactivated21) do produce fibrosis when metabolically challenged with high-fat diets, but nongenetically modified animals either take very long periods or require large animal models to generate NASH with fibrosis.22, 23 Thus, a key goal of the present study was to develop a model of NASH that produces significant fibrosis in the context of diet-induced obesity in nongenetically modified mice.
Structural mitochondrial damage is a significant pathophysiologic feature of human NASH with fibrosis.24 The generation of ROS by the damaged mitochondrial respiratory chain and concomitant release of lipid peroxidation products produce detrimental effects.25 Plasma levels of antioxidants such as reduced coenzyme Q (redCoQ) correlate negatively with increasing fibrosis in NAFLD.26 Furthermore, fructose has been shown in mice to activate macrophages27 and induce fibrogenesis through ROS-dependent mechanisms.28
Based on these data, we tested the hypothesis that mice given ad libitum access to a high-calorie diet with predominantly medium chain hydrogenated saturated trans fatty acids (contrasting with the ALIOS diet, which had long chain saturated trans fats18) and fructose would induce increased hepatic ROS and generate significant fibrosis. Our data represent a significant advance to the study of NAFLD in that within 16 weeks, an ad libitum access to this diet yields obesity, insulin resistance, and NASH with fibrosis in nongenetically modified mice. This phenotype develops in the background of increased hepatic ROS and proinflammatory macrophages, driving TGF-β and α-smooth muscle actin (α-SMA)–driven collagen deposition.
Six- to eight-week-old male C57Bl/6 mice (Jackson Laboratory, Bar Harbor, ME) were group-housed in cages in a temperature-controlled vivarium (22 ± 2°C) on a 12-hour light/dark schedule at the University of Cincinnati. Animals were randomly assigned to a chow diet (Teklad; Harlan, Madison, WI), a high-fat (HF) diet (Surwit diet [58 kcal % fat]; Research Diets, New Brunswick, NJ), or a high-fat, high-carbohydrate (HFHC) diet (Surwit diet) and drinking water enriched with high-fructose corn syrup equivalent. A total of 42 g/L of carbohydrates was mixed in drinking water at a ratio of 55% fructose (Acros Organics, Morris Plains, NJ) and 45% sucrose (Sigma-Aldrich, St. Louis, MO) by weight. Animals were provided ad libitum access to these diets for 16 weeks. Body weights were measured weekly, and percent body fat was measured at 12 weeks using Echo MRI (Echo Medical Systems, Houston, TX). All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and Cincinnati Children's Hospital Medical Center.
Blood Glucose, Plasma Insulin, and Insulin Resistance Measurements
Glucose was measured at 12 weeks after a 4-hour fast using Accu-Check glucose meter (Roche Diagnostics, Indianapolis, IN). Plasma insulin was measured using a mouse insulin enzyme-linked immunosorbent assay kit (Crystal Chem, Downers Grove, IL). Insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR).29
Liver sections for histology were obtained at sacrifice after 16 weeks, fixed in 10% formalin, and stained with hematoxylin-eosin or trichrome by the Cincinnati Digestive Health Center Histopathology Core. Histology was read by a single independent pathologist blinded to experimental design and treatment groups. Briefly, steatosis was graded (0-3), lobular inflammation was scored (0-3), and ballooning was rated (0-2).30 Fibrosis was staged separately on a scale of 0-4. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling was performed as described.31
Hepatic Triglyceride and Plasma Alanine Aminotransferase, Triglyceride, and Cholesterol Quantification
Liver triglyceride (TG) content was determined at 16 weeks as described.32 Briefly, 100 mg of wet liver tissue was homogenized and the enzymatic assay was performed using a Triglycerides Reagent Set (Pointe Scientific, Inc., Canton, MI). Photometric absorbance was read at 500 nm. Blood was collected at 16 weeks and was used to measure alanine aminotransferase (ALT), TG, and cholesterol using a DiscretPak ALT Reagent Kit (Catachem, Bridgeport, CT), Triglycerides Reagent Set (Pointe Scientific, Inc.), and Infinity Cholesterol Liquid Stable Reagent (Thermo Electron, Waltham, MA), respectively.
Hepatic Hydroxyproline Quantification
One hundred milligrams of liver was homogenized, to which HCl was added and samples were baked at 110°C for 18 hours. Aliquots were evaporated and pH was neutralized. Chloramine-T solution was added and samples were incubated at room temperature. Ehrich's reagent was then added, after which samples were incubated at 50°C and absorbance was measured at 550 nm.
Fecal Fat Quantification
Total fatty acid-based compounds in the feces were quantified by saponifying a sample of feces, to which a known mass of heptadecanoic acid was added. The total fatty acids in a known mass of feces was calculated by way of gas chromatograpy as described.33
RNA was isolated from flash frozen liver tissues. Total RNA was isolated using TRIzol reagent protocol (Molecular Research Center, Cincinnati, OH). Isolated RNA was treated with RNase-Free DNase (Fisher Scientific, Pittsburgh, PA) and purified on an RNeasy Mini Spin Column (Qiagen, Valencia, CA). Complementary DNA was made using the TaqMan reverse transcription protocol and an Eppendorf Mastercycler polymerase chain reaction (PCR) machine (Eppendorf North America, Westbury, NY). A predesigned, validated, gene-specific TaqMan probe was used for collagen 1 and α-SMA. The primer sequence for TGF-β1 was: CGT AGT AGA CGA TGG GCA GTG G (reverse), TAT TTG GAG CCT GGA CAC ACA G (forward). Messenger RNA (mRNA) expression was obtained using Stratagene SYBR green real-time kinetic PCR on a Stratagene Mx3005 multiplex quantitative PCR machine (Stratagene, Agilent Technologies, La Jolla, CA). Relative expression was determined by comparison of dT values relative to glyceraldehyde 3-phosphate dehydrogenase expression using the 2-ΔΔCT method.
Identification of Macrophages by Flow Cytometry
Single liver cell suspensions were prepared by mincing and passing over 40 μm cell strainers (Fisher Scientific, Pittsburgh, PA). After centrifugation at 2,000 rpm, a cell pellet was mixed with 33% Percoll (Sigma-Aldrich, St. Louis, MO) in RPMI 1640 solution (Invitrogen, Carlsbad, CA). Cell suspension was centrifuged at 2,000 rpm for 20 minutes at room temperature, the cell pellet was removed and washed, and red blood cells were lysed with 1× lysis buffer (eBioscience, San Diego, CA). Cells were suspended in 50 μL fluorescence-activated cell sorting buffer and Fc receptor was blocked with anti-mouse CD16/32 (clone 93, eBioscience). Cells were stained with CD11b-PerCP-Cy5.5 (clone M1/70), F4/80-PE (clone BM8), and Gr1-FITC (clone 1A8) (eBioscience). Cells were acquired on a FacsCanto FlowCytometer (BD Biosciences, San Jose, CA) and data were analyzed using FlowJo software version 7.5 (TreeStar, Ashland, OR).
ROS Staining of Liver Sections
Frozen liver sections were rehydrated in phosphate-buffered saline (PBS). Stock dihydroethidium (DHE) (Sigma-Aldrich) solution was diluted in dimethyl sulfoxide (Sigma-Aldrich). Slides were incubated in DHE solution and washed with 1× phosphate-buffered saline and placed on coverslips using 80% glycerol in phosphate-buffered saline. Fluorescence was recorded and quantified using Texas red filter on an upright Olympus BX51 microscope using DPControler software (Olympus, Hamburg, Germany) and IMAGE J software (National Institutes of Health, Bethesda, MD).34
Liver sections were incubated in 10% normal horse serum after blocking. Sections were incubated with the 4-hydroxynonenal primary antibody (Alpha Diagnostic International, San Antonio, TX) overnight and then incubated with secondary biotin conjugated antibody (Alpha Diagnostic International). Avidin–biotin peroxidase complex (Vector Laboratories, Burlingame, CA) staining was performed with diaminobenzidine (Vector Laboratories). The sections were counterstained with Mayer's hematoxylin.
Quantification of CoQ9 was performed as described.35 Plasma with internal standard CoQ11 was injected into an automated high-performance liquid chromatographic system equipped with a coulometer detector. Quantification of oxCoQ9 was obtained using ChromQuest software (Fisher Scientific, Pittsburgh, PA). After injection, the extract was mixed with 1,4-benzoquinone, incubated, and then injected into the high-performance liquid chromatographic system for measuring total CoQ9. Concentration of reduced coenzyme Q9 was achieved by subtracting oxCoQ9 from total CoQ9.
Statistical comparison between groups and treatments was performed using one-way analysis of variance (ANOVA) and post hoc Tukey's test. Student t tests were used when comparing two groups. A P value of <0.05 was considered statistically significant. Data are presented as the mean ± SEM.
Food Intake, Body Weight, Body Composition, and Insulin Resistance
The HFHC diet–fed and HF diet–fed mice consumed more total calories per day (12.54 ± 0.6 and 11.76 ± 1.5 kcal/day, respectively) than their chow-fed controls (8.67 ± 1.6). There was no difference between HF and HFHC in terms of total calories consumed or stool output per day. Percent fat content in fecal material was also similar between the HF (2.46 ± 0.6%) and HFHC (2.08 ± 0.7%) groups. Mice fed HFHC and HF diets gained more weight than mice fed the chow diet. HFHC and HF mice had mean body weights of 50.5 ± 0.8 g and 53.18 ± 1.8 g, respectively (Fig. 1A), compared with a mean body weight of 31.94 ± 0.2 g for chow-fed mice at 16 weeks. Total body fat mass estimation by way of magnetic resonance imaging at 12 weeks demonstrated that HFHC mice (18.66 ± 0.7 g) and HF mice (18.40 ± 0.9 g) had significantly greater body fat compared with chow-fed mice (2.82 ± 0.6 g; P < 0.0001) (Fig. 1B). Fasting plasma glucose levels were higher in HFHC (223.6 ± 7 mg/dL) and HF (235.4 ± 10 mg/dL) mice than in chow-fed mice (160.4 ± 7.3 mg/dL; P < 0.0001) (Fig. 1C). Similarly, fasting insulin was higher in HFHC mice (7.7 ± 1 ng/mL) and HF mice (10.3 ± 0.9 ng/mL) compared with chow-fed mice (1.9 ± 0.1 ng/mL; P < 0.0001) (Fig. 1D). Glucose and insulin values were used to estimate insulin resistance as HOMA-IR calculations, and both HFHC (4.2 ± 0.6) and HF (5.9 ± 0.5) mice were significantly insulin-resistant compared with chow-fed mice (1.1 ± 0.4; P < 0.0001) (Fig. 1E). Thus, both HFHC and HF mice were significantly obese and insulin-resistant compared with chow mice.
Hepatic Steatosis, Inflammation, and Apoptosis
Histologic examination of livers from HFHC and HF mice demonstrated substantial steatosis with inflammatory changes. Microvesicular and macrovesicular steatosis were clearly visible on routine histology staining with hematoxylin-eosin after 16 weeks (Fig. 2A). For sections with a steatosis score of 3, the distribution of steatosis in both the HF group and the HFHC group was panlobular. Nearly all hepatocytes have microvesicular steatosis, and many had both microvesicular and macrovesicular steatosis with a random distribution. For sections with a steatosis score ≤2, there was a panlobular distribution of steatosis in the HF group, but there was evidence of zone II sparing in the HFHC group. Lobular inflammation was prominent in HFHC sections similar to human NASH descriptions (Fig. 2B). Confirming the histological impressions, the weights of the livers of HFHC and HF mice were significantly higher compared with chow-fed mice (P < 0.0001) (Fig. 2E). Similarly, TG content at 16 weeks was higher in HFHC mice (1,955 ± 430 mg/dL per 100 mg wet liver) and HF mice (1,096 ± 115) compared with chow mice (276 ± 34; P < 0.0001 [one-way ANOVA]) (Fig. 2C). Plasma ALT levels were also greater in both HFHC (217.3 ± 40.2 IU/L) and HF mice (187 ± 47 IU/L) at 16 weeks compared with chow-fed mice (70.9 ± 5.4 IU/L; P < 0.0001) (Fig. 2D). Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling was increased in both HFHC and HF mice compared with chow-fed mice (data not shown). Thus, both HFHC and HF mice had significantly more hepatic steatosis, inflammation, and apoptosis than chow-fed mice.
Hepatic Fibrosis and Profibrogenic Gene Signatures
Trichrome-stained liver sections from HFHC mice demonstrated significant fibrosis (Fig. 3A). Fibrosis was first observed in mice after 14 weeks. After 16 weeks, fibrosis was clearly visible in half of the mice (Table 1). When seen in a section, fibrosis was extensive and was seen in most portal areas. At 16 weeks, 33% of mice had stage 1a or 1c fibrosis with perisinusoidal or portal/periportal fibrosis, whereas 16% had stage 2 fibrosis with perisinusoidal and portal/periportal fibrosis. Perisinusoidal fibrosis was seen in both zones 1 and 2. Periportal fibrosis was seen in all portal triads and there was extension of fibers between portal tracts as well. Thus, the distribution of fibrosis seen in HFHC liver sections was akin to human NASH biopsies, with the fibrosis being either predominantly zone 1 (as seen in pediatric patients) or perisinusoidal (seen more often in adult patients) (Fig. 3A). HF and chow-fed mice had no evidence of significant fibrosis on histology. Reverse-transcription PCR (RT-PCR) for hepatic collagen 1 mRNA expression was significantly higher in HFHC mice (7.36 ± 2.1 fold) compared with HF mice (0.92 ± 0.6 fold) and when normalized to chow-fed mice (1.0 ± 0.1) at 16 weeks (P = 0.0031) (Fig. 3B). Similarly, mRNA expression of TGF-β1 was significantly higher in HFHC mice (3.72 ± 1.3 fold) when normalized to chow-fed mice (1.0 ± 0.2) at 16 weeks (P = 0.04) (Fig. 3C). Hepatic levels of hydroxyproline were higher in the HFHC mice (0.94 ± 0.05 mg per 100 mg liver) compared with both HF mice (0.63 ± 0.04; P < 0.01) and chow-fed mice (0.61 ± 0.01; P < 0.01) (Fig. 3D). Thus, HFHC mice had significantly more hepatic fibrosis and profibrogenic gene signatures than HF and chow-fed mice.
Table 1. Histologic Characteristics After 16 Weeks on Diet
HFHC and HF mice had a higher steatosis grade than chow-fed mice (one-way ANOVA; P < 0.0001 in all three groups). ***P < 0.001, *P < 0.05 (Tukey's post test). HFHC liver histology was significantly different from both HF and chow-fed mice after 16 weeks for fibrosis and lobular inflammation score (one-way ANOVA; P < 0.0001 [fibrosis] and P = 0.0195 [inflammation]). †††P < 0.001, †P < 0.05 (Tukey's post test). Data are expressed as the mean ± SEM (HFHC, n = 6; HF, n = 8; chow-fed, n = 8).
Hepatic Macrophage Population and Stellate Cell Activation
The macrophage inflammatory Gr1+ subset is massively recruited into the liver upon toxic injury and may differentiate into fibrocytes.7, 36 We found that HFHC mice (2.03 ± 0.3%) had an approximately 10-fold increase in CD11b+F4/80+ cells compared with HF mice (0.03 ± 0.0%) and chow-fed mice (0.35 ± 0.1%; P < 0.0001) (Fig. 4A,B). Upon gating on CD11b+F4/80+ cells, the Gr1+ subset of cells were 10-fold higher in HFHC mice (1.12 ± 0.2%) compared with either HF (0.08 ± 0.0%) or chow-fed mice (0.1 ± 0.0%; P < 0.0001) (Fig. 4C). Further mRNA gene expression for α-SMA was three-fold higher in HFHC mice compared with HF mice and was undetectable in the livers of chow-fed mice (Fig. 4D). Thus, HFHC mice had a significantly more proinflammatory monocyte population compared with HF and chow-fed mice, which may signal stellate cell activation.
Hepatic ROS, Plasma Lipid, and CoQ Studies
At 16 weeks, HFHC mouse livers had more DHE staining (40.3 ± 2.9 FU/HPF) compared with those of HF mice (28.3 ± 2.9 FU/HPF) or chow-fed mice (17 ± 1.0 FU/HPF; P = 0.002) (Fig. 5A,C). We also observed increased 4-hydroxynonenal staining in HFHC mouse hepatocytes compared with HF and chow-fed mice (Fig. 5B). The plasma TG levels were not significantly different between the three groups, but serum cholesterol was significantly higher in HFHC mice (372.3 ± 21.9 mg/dL) compared with both HF mice (277.3 ± 50.5 mg/dL; P < 0.001) and chow-fed mice (127.5 ± 7.1 mg/dL; P < 0.001) (Fig. 5E). Plasma oxCoQ 9 levels in mice at 16 weeks were significantly higher in HFHC mice (0.06 ± 0.004 μg/mL) compared with HF mice (0.03 ± 0.004 μg/mL) and chow-fed mice (0.02 ± 0.004 μg/mL; P < 0.0001) (Table 2 and Fig. 5D). The correlation of liver tissue collagen 1 mRNA relative expression and absolute plasma oxCoQ 9 levels had an R2 value of 0.51. Thus, the fructose-containing HFHC diet had the most hepatic ROS, hypercholesterolemia, and hepatic fibrosis. This was mirrored by the levels of plasma oxCoQ9, which differed significantly among all three groups and correlated with the presence of fibrosis in this model.
Table 2. Plasma CoQ9 Profile After 16 Weeks on Diet
HFHC and HF mice had higher oxCoQ9 plasma levels (one-way ANOVA; P < 0.0001). ***P < 0.001, *P < 0.05 (Tukey's post test). HFHC mice also had significantly increased total plasma CoQ9 levels compared with both HF and chow-fed mice after 16 weeks (one-way ANOVA; P = 0.0141). ††P < 0.05 (Tukey's post test). Data are expressed as the mean ± SEM (HFHC, n = 5; HF, n = 5; chow-fed, n = 5).
The rising rates of NASH make addressing the underlying causes of this serious condition more pressing. Hepatic steatosis is common in obese patients, but only a subset of these patients develop NASH, emphasizing the contribution of genetic and potential environmental risk factors. Human NASH histopathology has been associated with steatosis, lobular and portal inflammation, hepatocyte ballooning, and fibrosis. Specifically, zone 3 predominant macrovesicular steatosis, ballooning, and perisinusoidal fibrosis is deemed consistent with adult or type 1 NASH. Type 2 or pediatric NASH histopathology has been reported to have panacinar or periportal (zone 1) steatosis, rare ballooning and portal tract expansion by chronic inflammation or fibrosis.37 Individuals who have NASH with fibrosis have progressive disease and greater morbidity and mortality including the potential for cirrhosis, liver failure, and liver transplantation.3 However, the specific biological determinants that lead to development of NASH with fibrosis are not well-defined.
Fructose consumption accounts for approximately 10.2% of all calories in our average diet in the United States.38 In comparison with other simple sugars such as glucose, use of fructose for hepatic metabolism is not restricted by the rate-limiting step of phosphofructokinase, thus avoiding the regulating action of insulin.39 Fructose intake is two- to three-fold higher in patients with NASH compared with body mass index–matched controls, and daily fructose ingestion has been associated with increased hepatic fibrosis.40, 41 These epidemiologic data prompted us to investigate the potential mechanistic role that fructose and other simple sugars may play in the development of NASH.
The present study focused on the development of a dietary model of NASH. To this end, we compared HF mice with mice maintained on the same diet but also given ad libitum access to fructose in their drinking water (HFHC). Although weight gain, body fat, insulin resistance, and liver steatosis were similar between the two groups (and elevated relative to mice maintained on chow), mice fed the HFHC diet had increased hepatic oxidative stress, CD11b+F4/80+ Gr1+ macrophages in the liver, TGF-β1–driven fibrogenesis and collagen deposition compared with weight-matched controls in the HF group. Thus, while HF diets produce a range of the components of the metabolic syndrome, fructose consumption would appear necessary to move the process from liver fat deposition alone to fibrogenesis.
ROS has been thought to be an important trigger for hepatic stellate cell activation and for promoting expression of fibrogenic molecules such as α-SMA, TGF-β1, and collagen 1.15, 28, 42, 43 Recently, fructose-fed rats have been reported to develop hepatocyte damage with a decrease in the mitochondrial membrane potential similar to that induced by low noncytotoxic doses of exogenous ROS.44 In vitro studies have also shown that the cytotoxic mechanism involving fructose-driven ROS formation precedes hepatocytotoxicity, and that this cell injury could be prevented by ROS scavengers.44 We therefore investigated this as a potential process in our model and demonstrated that HFHC mice had significantly higher ROS levels compared with both HF and chow-fed mice (Fig. 5).
Previous studies performed with fructose diets have reported insulin resistance and severe necroinflammatory NAFLD but not NASH with fibrosis.18, 19 In contrast to the ALIOS diet, which provided fructose water in gelatin form and long chain–saturated trans fats in their solid diet, our HF diet provided 58% of calories from medium chain–saturated trans fats and fructose and sucrose in their regular drinking water. This diet resulted in 50% of the mice in the HFHC group having fibrosis with a minority having stage 2 fibrosis (Table 2). Karlmark et al.7 highlighted the role of CD11+F4/80+Gr1+ monocytes in perpetuating hepatic stellate cell–driven TGF-β1–dependent fibrosis. More recently, Niedermeier et al.36 reported that Gr1+ monocytes may be essential in the production of murine fibrocytes. In our experiment, intrahepatic CD11+F4/80+Gr1+ monocyte-derived macrophages were 10-fold higher than either HF or chow-fed mice, with 50% of the macrophages in HFHC livers being Gr1+ (Fig. 4). We propose that the conversion of CD11b+F4/80+Gr1+ monocytes into fibrocytes maybe responsible for the increased collagen 1 deposition through ROS-driven TGF-β signaling and stellate cell activation.
In humans, studies have shown extensive mitochondrial damage including paracrystalline inclusion bodies, megamitochondria, damaged respiratory chain and low adenosine triphosphate production with NASH.24 We have previously reported that increased ROS released from damaged mitochondrial respiratory chain is important in NAFLD development.45 CoQ is an important element in the mitochondrial respiratory chain, contributing to the transport of electrons across complex III involving the Qo and Qi sites within the inner mitochondrial membrane to enable generation of adenosine triphosphate. Lower redCoQ plasma levels are present in patients with cirrhosis and redCoQ acts as a lipid soluble antioxidant in hepatocytes in culture.46, 47 Supplementation with CoQ has also been reported to inhibit liver fibrosis through suppression of TGF-β1 expression in mice.48 We demonstrate that plasma levels of oxCoQ9 correlate well with collagen 1 mRNA in liver tissue. We also present data that plasma levels of oxCoQ9 can discriminate between NASH with fibrosis and NASH without fibrosis, with our HFHC (NASH with fibrosis) mice having higher levels compared with HF mice (NASH without fibrosis) or chow-fed mice (normal histology) (Fig. 5).
In conclusion, we believe that our ad libitum dietary model results in NASH with fibrosis in nongenetically modified obese mice. Our data suggest that the mechanism of fibrosis in this model may involve fructose producing an increased ROS signature in the liver associated with CD11b+F4/80+Gr1+ macrophage aggregation resulting in TGF-β1 signaled collagen deposition and histologically visible hepatic fibrosis.