Antiatherogenic effects of S-nitroso-N-acetylcysteine in hypercholesterolemic LDL receptor knockout mice.

BACKGROUND: The pathophysiology of the NO/NO synthase system and dysfunctional changes in the endothelium in the early phases of the atherogenic process are incompletely understood. In this study, we investigated the effects of the nitrosothiol NO donor S-nitroso-N-acetylcysteine (SNAC) in the early prevention of plaque development in the hypercholesterolemic LDLr-/- mice as well as the changes in endothelium-dependent relaxation and NO synthase expression. METHODS AND RESULTS: LDLr-/- mice were fed a 1.25% cholesterol-enriched diet for 15 days. Plasma cholesterol/triglyceride levels increased and this increase was accompanied by the development of aortic root lesions. Aortic vasorelaxation to acetylcholine was increased, although endothelium-independent relaxation in response to sodium nitroprusside did not change, which suggest stimulated NO release enhanced. This dysfunction was associated with enhanced aortic superoxide production and with increased levels of constitutive NOS isoform expression, particularly neuronal NOS. SNAC (S-nitroso-N-acetylcysteine) administration (0.51 micromol/kg/day i.p. for 15 days) decreased the extent of the plaque by 55% in hypercholesterolemic mice, but had no effects on vasomotor changes. It did, however, lead to a decrease in constitutive NOS expression. The SNAC induced only minor changes in plasma lipid profile. CONCLUSION: The present study has shown that, in early stages of plaque development in LDLr-/- mice, specific changes in NO/NO synthase system develop, that are characterized by increased endothelium-dependent vasorelaxation and increased constitutive NOS expression. Since the development of plaque and the indicator of endothelial cell dysfunction were prevented by SNAC, such treatment may constitute a novel strategy for the halting of progression of early plaque.     

Upregulation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice

We determined the effects of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on parameters of plasma lipoprotein and hepatic lipid metabolism in LDL receptor (LDLr) knockout mice. Dietary n-3 PUFA decreased the rate of appearance and increased the hepatic clearance of IDL/LDL resulting in a marked decrease in the plasma concentration of these particles. Dietary n-3 PUFA increased the hepatic clearance of IDL/LDL through a mechanism that appears to involve apolipoprotein (apo)E but is independent of the LDLr, the LDLr related protein (LRP), the scavenger receptor B1, and the VLDLr. The decreased rate of appearance of IDL/VLDL in the plasma of animals fed n-3 PUFA could be attributed to a marked decrease in the plasma concentration of precursor VLDL. Decreased plasma VLDL concentrations were due in part to decreased hepatic secretion of VLDL triglyceride and cholesteryl esters, which in turn was associated with decreased concentrations of these lipids in liver. Decreased hepatic triglyceride concentrations in animals fed n-3 PUFA were due in part to suppression of fatty acid synthesis as a result of a decrease in sterol regulatory element binding protein-1 (SREBP-1) expression and processing. In conclusion, these studies indicate that n-3 PUFA can markedly decrease the plasma concentration of apoB-containing lipoproteins and enhance hepatic LDL clearance through a mechanism that does not involve the LDLr pathway or LRP.     

Echium oil reduces plasma lipids and hepatic lipogenic gene expression in apoB100-only LDL receptor knockout mice

We tested the hypothesis that dietary supplementation with echium oil (EO), which is enriched in stearidonic acid (SDA; 18:4 n-3), the product of Delta-6 desaturation of 18:3 n-3, will decrease plasma triglyceride (TG) concentrations and result in conversion of SDA to eicosapentaenoic acid (EPA) in the liver. Mildly hypertriglyceridemic mice (apoB100-only LDLrKO) were fed a basal diet containing 10% calories as palm oil (PO) and 0.2% cholesterol for 4 weeks, after which they were randomly assigned to experimental diets consisting of the basal diet plus supplementation of 10% of calories as PO, EO or fish oil (FO) for 8 weeks. The EO and FO experimental diets decreased plasma TG and VLDL lipid concentration, and hepatic TG content compared to PO, and there was a significant correlation between hepatic TG content and plasma TG concentration among diet groups. EO fed mice had plasma and liver lipid EPA enrichment that was greater than PO-fed mice but less than FO-fed mice. Down-regulation of several genes involved in hepatic TG biosynthesis was similar for mice fed EO and FO and significantly lower compared to those fed PO. In conclusion, EO may provide a botanical alternative to FO for reduction of plasma TG concentrations.     

LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice

The very low density lipoprotein receptor (VLDLR) has been proposed to play a role in the delivery of fatty acids to peripheral tissues. However, despite reduced adipose tissue mass in VLDLR-deficient (VLDLR(-)(/-)) mice, this has been difficult to substantiate. In the present study, VLDLR-deficient and VLDLR-overexpressing (PVL) mice were cross-bred onto a low density lipoprotein receptor knockout (LDLR(-)(/-)) background to study the VLDLR under conditions of relatively high serum VLDL and triglyceride levels. Absence of the VLDLR resulted in a significant increase in serum triglyceride levels (1.9-fold) when mice were fed a high fat diet. In contrast, overexpression of the VLDLR resulted in a significant decrease in serum triglyceride levels (2.0-fold) under similar conditions. When kept on a chow diet, a period of prolonged fasting revealed a significant increase in serum triglyceride levels in VLDLR(-)(/-); LDLR(-)(/-) mice (2.3-fold) as compared with LDLR(-)(/-) controls. This could not be attributed to altered apolipoprotein B and VLDL triglyceride production rates. Furthermore, no major differences in nascent VLDL triglyceride content were found between VLDLR(-)(/-); LDLR(-)(/-) mice and LDLR(-)(/-) controls. However, the triglyceride content of circulating VLDL of VLDLR(-)(/-); LDLR(-)(/-) mice (63%) was relatively high as compared with LDLR(-)(/-) controls (49%). These observations suggest that the VLDLR affects peripheral uptake of VLDL triglycerides.In conclusion, under conditions of LDLR deficiency in combination with high fat feeding or prolonged fasting, the effect of the VLDLR on VLDL triglyceride metabolism was revealed.     

Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice

The adult brain contains small populations of neural precursor cells (NPC) that can give rise to new neurons and glia, and may play important roles in learning and memory, and recovery from injury. Growth factors can influence the proliferation, differentiation and survival of NPC, and may mediate responses of NPC to injury and environmental stimuli such as enriched environments and physical activity. We now report that neurotrophin expression and neurogenesis can be modified by a change in diet. When adult mice are maintained on a dietary restriction (DR) feeding regimen, numbers of newly generated cells in the dentate gyrus of the hippocampus are increased, apparently as the result of increased cell survival. The new cells exhibit phenotypes of neurons and astrocytes. Levels of expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are increased by DR, while levels of expression of high-affinity receptors for these neurotrophins (trkB and trkC) are unchanged. In addition, DR increases the ratio of full-length trkB to truncated trkB in the hippocampus. The ability of a change in diet to stimulate neurotrophin expression and enhance neurogenesis has important implications for dietary modification of neuroplasticity and responses of the brain to injury and disease.     

VLDL best predicts aortic root atherosclerosis in LDL receptor deficient mice

Hyperlipidemia is a major risk factor for developing atherosclerosis in humans, and epidemiological studies have correlated specific lipoprotein levels with cardiovascular disease risk. Murine models of atherosclerosis rely on the induction of hyperlipidemia for vascular lesions to form, but the pathogenic contributions attributed to different lipoprotein populations are not well defined. To address this issue, we analyzed over 300 LDL receptor (LDLR) deficient mice that have been fed a high-fat diet and for which a full lipoprotein profile and aortic root atherosclerosis values were assessed. Overall, aortic root atherosclerosis is best predicted by plasma VLDL cholesterol levels with less predictive value derived from either LDL or HDL cholesterol. Triglyceride levels are more atherogenic in female mice, especially immune competent females, and depletion of the adaptive immune system leads to a global reduction in plasma lipid levels and aortic root lesion size yet does not appear to alter the atherogenic potential of individual lipoprotein subspecies. In contrast, HDL-cholesterol is a better predictor of aortic root atherosclerosis in apoE-deficient mice. In summary, this large scale analysis of high-fat diet fed LDLR deficient mice highlight the relationship between different plasma lipid components, especially VLDL-cholesterol, and aortic root atherosclerosis.     

Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice

Despite an only minor reduction in the glomerular filtration rate, uninephrectomy (UNX) markedly accelerates the rate of growth of atherosclerotic plaques in ApoE-/- mice. It has been suggested that vitamin D receptor (VDR) activation exerts an antiproliferative effect on vascular smooth muscle cells, but the side effects may limit its use. To assess a potentially different spectrum of actions, we compared the effects of paricalcitol and calcitriol on remodeling and calcification of the aortic wall in sham-operated and UNX ApoE-/- mice on a diet with normal cholesterol content. Sham-operated and UNX mice were randomly allotted to treatment with solvent, calcitriol (0.03 μg/kg) or paricalcitol (0.1 μg/kg) 5 times/wk intraperitoneally for 10 wk. Semithin (0.6 μm) sections of the aorta were analyzed by 1) morphometry, 2) immunohistochemistry, and 3) Western blotting of key proteins involved in vascular calcification and growth. Compared with sham-operated animals (5.6 ± 0.24), the wall-to-lumen ratio (x100) of the aorta was significantly higher in solvent- and calcitriol-treated UNX animals (6.64 ± 0.27 and 7.17 ± 0.81, respectively, P < 0.05), but not in paricalcitol-treated UNX (6.1 5 ± 0.32). Similar differences were seen with respect to maximal plaque height. Expression of transforming growth factor (TGF)-β1 in aortic intima/plaque was also significantly higher in UNX solvent and UNX calcitriol compared with sham-operated and UNX paricalcitol animals. Treatment with both paricalcitol and calcitriol caused significant elevation of VDR expression in the aorta. While at the dose employed paricalcitol significantly reduced TGF-β expression in plaques, calcitriol in contrast caused significant vascular calcification and elevated expression of related proteins (BMP2, RANKL, and Runx2).     

Effect of nickel chloride on hepatic lipid peroxidation and glutathione concentration in mice

Intraperitoneal administration of nickel chloride enhanced hepatic lipid peroxidation (HLP) in 6-wk-old and 8–12-wk-old male CBA-mice but not in 3-wk-old mice. nickel chloride administration depleted hepatic GSH in 8–12-wk-old mice but not in the younger age groups. After 300 μmol NiCl₂/kg mortality occurred among 8–12-wk-old mice but not among the younger mice. Stimulation of GSH synthesis by administration ofl-2-oxothiazolidine-4-carboxylate reduced nickel chloride induced mortality and HLP. Reduction of GSH synthesis by administration of buthionine sulfoximine (BSO) did not, however, enhance the toxicity of nickel chloride. This might be owing to chelation of the Ni(II)-ion by BSO. The results demonstrate age dependency and a protective effect of enhanced GSH synthesis in nickel chloride stimulated HLP.     

Addition of dietary fat to cholesterol in the diets of LDL receptor knockout mice: effects on plasma insulin, lipoproteins, and atherosclerosis

The factors underlying cardiovascular risk in patients with diabetes have not been clearly elucidated. Efforts to study this in mice have been hindered because the usual atherogenic diets that contain fat and cholesterol also lead to obesity and insulin resistance. We compared plasma glucose, insulin, and atherosclerotic lesion formation in LDL receptor knockout (Ldlr(-/-)) mice fed diets with varying fat and cholesterol content that induced similar lipoprotein profiles. Ldlr(-/-) mice fed a high-fat diet developed obesity, mild hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. Quantitative and qualitative assessments of atherosclerosis were unchanged in diabetic Ldlr(-/-) mice fed a high-fat diet compared with lean nondiabetic control mice after 20 weeks of diet. Although one group of mice fed diets for 40 weeks had larger lesions at the aortic root, this was associated with a more atherogenic lipoprotein profile. The presence of a human aldose reductase transgene had no effect on atherosclerosis in fat-fed Ldlr(-/-) mice with mild diabetes. Our data suggest that when lipoprotein profiles are similar, addition of fat to a cholesterol-rich diet does not increase atherosclerotic lesion formation in Ldlr(-/-) mice.     

Nonalcoholic fatty liver disease is associated with an altered hepatocyte microRNA profile in LDL receptor knockout mice

MicroRNAs modulate processes associated with cell cycle control and differentiation. Here we explored the potential of microRNAs in the modulation of hepatic lipid metabolism and the development of nonalcoholic fatty liver disease.MicroRNA profiles of hepatocytes from low-density lipoprotein (LDL) receptor knockout mice fed a chow diet or a hypertriglyceridemia/fatty liver-inducing Western-type diet (WTD) were determined using quantitative real-time polymerase chain reaction. Ninety-seven of 103 microRNAs measured were expressed by hepatocytes and low variability between hepatocyte pools was observed. Feeding WTD coincided with a marked fivefold decrease in the relative expression level of miR-216 (P<.05) and miR-302a (P<.01). Interestingly, an increased hepatic miR-216 expression was detected in response to fasting. MicroRNA/biological function linkage analysis suggested that the change in hepatocyte microRNA profiles in response to high dietary lipid levels is associated with changes in cell cycle control and proliferation. In accordance with a diminished miR-302a expression on the WTD, hepatocyte mRNA expression levels of miR-302a target genes ABCA1 and in particular ELOVL6 were increased in response to WTD (twofold to ninefold). This suggests a role for miR-302a in hepatic cholesterol, fatty acid and glucose metabolism.In conclusion, we have shown that fatty liver development in LDL receptor knockout mice is associated with a significant change in the hepatocyte microRNA profile, i.e., a fivefold decrease in miR-216 and miR-302a expression. Based upon our comparative gene and microRNA expression studies it is anticipated that miR-302a may prove to be a valuable therapeutic target in the regulation of hepatic fatty acid utilization and insulin resistance.     

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.     

Discovery of XEN445: A potent and selective endothelial lipase inhibitor raises plasma HDL-cholesterol concentration in mice

Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardiovascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors. XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice.     

In vivo contribution of LCAT to apolipoprotein B lipoprotein cholesteryl esters in LDL receptor and apolipoprotein E knockout mice

Previous studies have indicated that LCAT may play a role in the generation of cholesteryl esters (CE) in plasma apolipoprotein B (apoB) lipoproteins. The purpose of the present study was to examine the quantitative importance of LCAT on apoB lipoprotein CE fatty acid (CEFA) composition. LCAT(-/-) mice were crossed into the LDL receptor (LDLr)(-/-) and apoE(-/-) background to retard the clearance and increase the concentration of apoB lipoprotein in plasma. Plasma free cholesterol was significantly elevated but total and esterified cholesterol concentrations were not significantly affected by removal of functioning LCAT in either the LDLr(-/-) or apoE(-/-) mice consuming a chow diet. However, when functional LCAT was removed from LDLr(-/-) mice, the CEFA ratio (saturated + monounsaturated/polyunsaturated) of plasma LDL increased 7-fold because of a 2-fold increase in saturated and monounsaturated CE, a 40% reduction in cholesteryl linoleate, and a complete absence of long chain (>18 carbon) polyunsaturated CE (20:4, 20:5n-3, and 22:6n-3), from 29.3% to 0%. Removal of functional LCAT from apoE(-/-) mice resulted in only a 1.6-fold increase in the CEFA ratio, due primarily to a complete elimination of long chain CE (7.7% to 0%).Our results demonstrate that LCAT contributes significantly to the CEFA pool of apoB lipoprotein and is the only source of plasma long chain polyunsaturated CE in these mice.     

Dietary tryptophan alleviates dextran sodium sulfate-induced colitis through aryl hydrocarbon receptor in mice

Ulcerative colitis is the typical progression of chronic inflammatory bowel disease. Amino acids, particularly tryptophan, have been reported to exert a protective effect against colitis induced by dextran sodium sulfate (DSS), but the precise underlying mechanisms remain incompletely clarified. Tryptophan metabolites are recognized to function as endogenous ligands for aryl hydrocarbon receptor (Ahr), which is a critical regulator of inflammation and immunity. Thus, we conducted this study to investigate whether dietary tryptophan supplementation protects against DSS-induced colitis by acting through Ahr. Female wild-type (WT) and Ahr-deficient (knockout; KO) mice (10–12 weeks old) were divided into four groups and fed either a control or 0.5% tryptophan diet. The tryptophan diet ameliorated DSS-induced colitis symptoms and severity in WT mice but not in KO mice, and the diet reduced the mRNA expression of Il-6, Tnfα, Il-1β and the chemokines Ccl2, Cxcl1 and Cxcl2 in the WT groups. Furthermore, Il-22 and Stat3 mRNA expression in the colon was elevated in WT mice fed with the tryptophan diet, which mainly protected epithelial layer integrity, and Ahr also modulated immune homeostasis by regulating Foxp3 and Il-17 mRNA expression. These data suggest that tryptophan-containing diet might ameliorate DSS-induced acute colitis and regulate epithelial homeostasis through Ahr. Thus, tryptophan could serve as a promising preventive agent in the treatment of ulcerative colitis.     

Benzo[a]pyrene enhances lipid peroxidation induced DNA damage in aorta of apolipoprotein E knockout mice

The genotoxic compound benzo[a]pyrene (B[a]P) enhances atherosclerotic plaque progression, possibly by inducing oxidative stress and subsequent lipid peroxidation (LPO). Since LPO plays a key role in atherosclerosis, stable LPO derived DNA modifications such as 1,N6-ethenodeoxy-adenosine (epsilondA) and 3,N4-ethenodeoxy-cytidine (epsilondC) may be useful biomarkers for in vivo oxidative stress. In this study, benzo[a]pyrene-diol-epoxide (BPDE)-DNA, epsilondA and epsilondC were determined by 32P-postlabelling in apolipoprotein E knockout (ApoE-KO) mice treated with 5mg/kg B[a]P by gavage. After 4 days, BPDE-DNA adduct levels were higher in aorta (10.8 +/- 1.4 adducts/10(8) nucleotides) than in lung (3.3 +/- 0.7, P < 0.05), which is a known target organ for B[a]P. Levels of epsilondA were higher in aorta of B[a]P-exposed animals than in unexposed controls (8.1 +/- 4.4 vs 3.4 +/- 2.1 adducts per 10(8) parent nucleotides, P < 0.05). On the other hand, epsilondC levels were not affected by B[a]P exposure. Serum low density lipoprotein (LDL) levels were lower in B[a]P-exposed mice than in controls (9.3 +/- 3.7 and 13.3 +/- 4.0mmol/l, respectively), whereas high density lipoprotein (HDL) levels were higher (1.4 +/- 1.6 and 0.4 +/- 0.3mmol/l, respectively). Consequently, a three-fold difference in the LDL/HDL ratio was observed (P = 0.001). epsilondA levels were positively related with plasma HDL concentrations (R = 0.68, P = 0.02), suggesting that the HDL mediated protection of the vessel wall against reactive lipid peroxides was reduced in B[a]P-exposed apoE-KO mice. Our observations show that direct as well as lipid peroxidation induced DNA damage is formed by B[a]P in aorta of apoE-KO mice, which may be involved in atherosclerotic plaque progression. This study further indicates that etheno-DNA adducts are useful biomarkers for in vivo oxidative stress in atherosclerosis.     

Fenofibrate improves lipid metabolism and obesity in ovariectomized LDL receptor-null mice

We investigated whether fenofibrate improves lipid metabolism and obesity in female ovariectomized (OVX) or sham-operated (SO) low density lipoprotein receptor-null (LDLR-null) mice. All mice fed a high-fat diet exhibited increases in serum triglycerides and cholesterol as well as in body weight and white adipose tissue (WAT) mass compared to mice fed a low fat control diet. However, fenofibrate prevented high-fat diet-induced increases in body weight and WAT mass in female OVX LDLR-null mice, but not in SO mice. In addition, administration of fenofibrate reduced serum lipids and hepatic apolipoprotein C-III mRNA while increasing the mRNA of acyl-CoA oxidase in both groups of mice, however, these effects were more pronounced in OVX LDLR-null mice. The results of this study provide first evidence that fenofibrate improves both lipid metabolism and obesity, in part through PPARalpha activation, in female OVX LDLR-null mice.     

Influence of lipoprotein-lipase activity on plasma triacylglycerol concentration and lipid storage in three genotypes of ducks

The lipoprotein-lipase (LPL) hydrolyses the triacylglycerols (TG) secreted by the liver and, thus, allows the storage of lipids onto the extrahepatic tissues. The LPL activity has been studied by injection of LPL antibodies in three genotypes of ducks (Muscovy (Cairina moschat), Pekin (Anas plathyrhynchos) and Mule (hybrids of male Muscovy ducks and female Pekin ducks)) under overfeeding condition. The results show a similar weight gain between injected and control animals. A higher liver steatosis is observed in Mule ducks (616 ± 18 g; 8.79% of body mass (BW)) and Muscovy ducks (514 ± 13 g; 7.05% BW) compared to Pekin ducks (353 ± 21 g; 5.89% BW, p < 0.05). Pekin ducks showed a much marked extrahepatic fattening of abdominal and subcutaneous adipose tissues. The LPL activity was evaluated by comparing the evolution of the plasma TG concentrations after injections of saline (control animals) or injections of specific LPL-antibodies. Inhibition of LPL activity performed by intravenous injections of LPL-antibodies showed a spectacular increase in the plasma TG concentrations in the three genotypes. That increase was considerably higher in Pekin ducks (98 ± 10 g/L) compared to Muscovy ducks (35 ± 2 g/L, p < 0.01) and Mule ducks (30 ± 4 g/L, p < 0.01). Those data suggest that a high export of lipids synthesized in liver and a high LPL activity occur in overfed Pekin ducks, which can favour the extrahepatic fattening to the detriment of the liver steatosis, and conversely in overfed Muscovy and Mule ducks.     

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

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