The role of oxidative stress in the development of obesity and obesity-related metabolic disorders

Obesity is a serious medical condition, defined as excessive accumulation of fat. Abdominal fat is recognized as the major risk for obesity related diseases such as: hypertension, dyslipidemia, type 2 diabetes mellitus, coronary heart disease, stroke, non-alcoholic fatty liver disease etc. Fat accumulation is also related to pro-oxidant and pro-inflammatory states. Recently published articles suggest that oxidative stress may be a link between obesity and related complications. Adiposity leads to increased oxidative stress via several multiple biochemical processes such as superoxide generation through the action of NADPH oxidase, glyceraldehyde auto-oxidation, oxidative phosphorylation, protein kinase C (PKC) activation, and polyol and hexosamine pathways. On the other hand, oxidative stress plays a causative role in the development of obesity, by stimulating the deposition of adipose tissue, including preadipocyte proliferation, adipocyte differentiation and growth. Exercise-induced weight loss can improve the redox state by modulating both oxidative stress and antioxidant promoters, which reduce endothelial dysfunction and inflammation.     

α-亚麻酸对糖尿病大鼠炎症介质和氧化应激的影响

目的:观察α-亚麻酸(ALA)对糖尿病大鼠体内炎症介质和氧化应激的影响,探讨ALA在糖尿病防治中的作用。方法:雄性SD大鼠高脂饮食喂养4周后,腹腔注射链脲佐菌素(STZ)30 mg/kg建立2型糖尿病(T2DM)模型。将大鼠随机分为3组(n=10):正常对照组、糖尿病模型组和ALA治疗组(500μg/kg.d)。4周后测定大鼠血清中肿瘤坏死因子(TNF-α)、可溶性P-选择素(sP-selectin)、可溶性细胞间黏附分子(sICAM-1)、一氧化氮(NO)、丙二醛(MDA)的含量以及超氧化物岐化酶(SOD)和过氧化氢酶(CAT)的活性。结果:与正常对照组相比,糖尿病大鼠血清中炎症介质TNF-α、sP-selectin和sICAM-1的含量增加,血清NO含量下降而MDA升高,同时抗氧化酶SOD和CAT的活性降低;ALA治疗可显著降低糖尿病大鼠血清中TNF-α、sP-selectin和sICAM-1的含量(与STZ+vehicle组相比,P<0.01),增加血清NO水平并减少MDA含量,升高抗氧化酶SOD和CAT的活性(与STZ+vehicle组相比,均P<0.05)。结论:ALA可显著降低糖尿病大鼠血清炎症介质的生成,减轻氧化应激水平,具有抗炎和抗氧化作用。提示ALA对糖尿病及糖尿病并发症的发生发展可能具有一定的防治作用。     

Functions and oxidative stress status of leukocytes in patients with nephrotic syndrome

This study was conducted to establish the functions and oxidative stress status in leukocytes of adult patients with nephrotic syndrome. Thirty adult patients with nephrotic syndrome and 32 controls were included. Phagocytosis ability, the killing ability of the micro-organism phagosited of polymorphonuclear leukocytes (PMNL) and monocytes, along with oxidative stress parameters of PMNLs were assessed. There was no statistically significant difference in phagocytosis function of PMNLs and monocytes of patients when compared to those of controls. PMNL burst activities of the patient and control groups also showed no difference; however, the monocyte burst activities of patients were significant (p = 0.012). The glutathione peroxidase (GSH-Px) activities in PMNLs of the patients with nephrotic syndrome were significantly higher (p = 0.026) when compared to those of controls. In comparison with those of the control subjects, the patients had also higher selenium levels in their PMNLs (p < 0.001). Although PMNL malonyldialdehyde (MDA) levels of the patients seem to be higher than those of controls, the difference had no statistical significance (p = 0.071). Conclusively, in the patients with nephrotic syndrome, PMNLs appear to be exposed to an oxidative stress as indicated by their increased GSH-Px activities and selenium content. However, PMNLs in nephrotic syndrome patients seem to be coping with the insulting oxidative stress, as suggested by their near-normal MDA productions. Furthermore, these data suggest that nephrotic syndrome appears not to have an influence on phagocytosis and killing abilities of granulocytes and monocytes as long as these cells can overcome the oxidative stress to which they are exposed in this disease.     

Association of renal damage and oxidative stress in a rat model of metabolic syndrome. Influence of gender

This study investigated the association between nephropathy and oxidative stress, by measurement of systolic blood pressure, lipid peroxidation, activities of catalase, manganese- and copper-zinc-superoxide dismutase and endothelial nitric oxide synthase expression and concentrations of nitrates/nitrites in kidneys from rats with Metabolic Syndrome. Weaning female or male rats had 30% sucrose to drink for 24 weeks (Metabolic Syndrome). Modulation by sex hormones was investigated by gonadectomy and hormone replacement. In Metabolic Syndrome, Castrated Metabolic Syndrome + Testosterone males and Ovariectomized Metabolic Syndrome females had increased blood pressure, proteinuria and lipid peroxidation. Nitrates/nitrites and activities of catalase, manganese and copper-zinc-superoxide dismutase decreased vs intact Control, Castrated Metabolic Syndrome males, intact Metabolic Syndrome and Ovariectomized Metabolic Syndrome + Estradiol females. The results suggest that sex hormones modulate the activity of superoxide-dismutase, catalase and endothelial nitric oxide-synthase. Ovariectomy decreased the protection against oxidative stress in females; the opposite occurred in castrated males.     

Hydroxytyrosol prevents dermal papilla cells inflammation under oxidative stress by inducing autophagy

Hydroxytyrosol (HT), a primary phenolic antioxidant in olive oil, can afford protection from oxidative stress (OS) in different cells, including skin cells. In particular, it regulates several inflammation‐associated processes as well as in improving the antioxidant defense system. However, there is no information about HT used in the treatment of hair loss. This work aimed at exploring the potential protective actions of HT against OS in rat dermal papilla cells. After treatment, the related expression of protein and messenger RNA were detected using morphological and molecular analyses. The results showed that HT significantly reduced intracellular reactive oxygen species level, apoptotic markers and inflammation induced by OS and enhanced cell survival by regulating autophagy. Furthermore, HT enhanced the secretion of hair growth factors in the anti‐inflammation process. These results suggest that HT has a significant protective ability against OS and encourage the use of this biological ingredient as a possible tool to prevent alopecia.     

Discovery of biomarkers for oxidative stress based on cellular metabolomics

Oxidative stress has a close relationship with various pathologic physiology phenomena and the potential biomarkers of oxidative stress may provide evidence for clinical diagnosis or disease prevention. Metabolomics was employed to identify the potential biomarkers of oxidative stress. High-performance liquid chromatography–diode array detector, mass spectrometry and partial least squares discriminate analysis were used in this study. The 10, 15 and 13 metabolites were considered to discriminate the model group, vitamin E-treated group and l-glutathione-treated group, respectively. Some of them have been identified, namely, malic acid, vitamin C, reduced glutathione and tryptophan. Identification of other potential biomarkers should be conducted and their physiological significance also needs to be elaborated.     

Inhibitory effects of hydrogen sulphide on pulmonary fibrosis in smoking rats via attenuation of oxidative stress and inflammation

Accumulating evidence has demonstrated that hydrogen sulphide (H₂S) is involved in the pathogenesis of various respiratory diseases. In the present study, we established a rat model of passive smoking and investigated whether or not H₂S has protective effects against pulmonary fibrosis induced by chronic cigarette smoke exposure. Rat lung tissues were stained with haematoxylin‐eosin and Masson's trichrome. The expression of type I collagen was detected by immunohistochemistry. Oxidative stress was evaluated by detecting serum levels of malondialdehyde, superoxide dismutase and glutathione peroxidase and measuring reactive oxygen species generation in lung tissue. Inflammation was assessed by measuring serum levels of inflammatory cytokines, including high‐sensitivity C‐reactive protein, tumour necrosis factor‐α, interleukin (IL)‐1β and IL‐6. The protein expression of Nrf2, NF‐κB and phosphorylated mitogen‐activated protein kinases (MAPKs) in the pulmonary tissue was determined by Western blotting. Our findings indicated that administration of NaHS (a donor of H₂S) could protect against pulmonary fibrosis in the smoking rats. H₂S was found to induce the nuclear accumulation of Nrf2 in lung tissue and consequently up‐regulate the expression of antioxidant genes HO‐1 and Trx‐1 in the smoking rats. Moreover, H₂S could also reduce cigarette smoking‐induced inflammation by inhibiting the phosphorylation of ERK 1/2, JNK and p38 MAPKs and negatively regulating NF‐κB activation. In conclusion, our study suggests that H₂S has protective effects against pulmonary fibrosis in the smoking rats by attenuating oxidative stress and inflammation.     

Components of metabolic syndrome predicting diabetes: no role of inflammation or dyslipidemia

Objective: The diagnostic criteria and the clinical usefulness of the metabolic syndrome (MetSy) are currently questioned. The objective was to describe the structure of MetSy and to evaluate its components for prediction of diabetes type 2 (T2DM). Research Methods and Procedures: This was a case‐referent study nested within a population‐based health survey. Among 33,336 participants, we identified 177 initially non‐diabetic individuals who developed T2DM after 0.1 to 10.5 years (mean, 5.4 years), and, for each diabetes case, two referents matched for sex, age, and year of health survey. Baseline variables included oral glucose tolerance test, BMI, blood pressure, blood lipids, adipokines, inflammatory markers, insulin resistance, and β‐cell function. Exploratory and confirmative factor analyses were applied to hypothesize the structure of the MetSy. The prediction of T2DM by the different factors was evaluated by multivariate logistic regression analysis. Results: A hypothetical five‐factor model of intercorrelated composite factors was generated. The inflammation, dyslipidemia, and blood pressure factors were predicitive only in univariate analysis. In multivariable analyses, two factors independently and significantly predicted T2DM: an obesity/insulin resistance factor and a glycemia factor. The composite factors did not improve the prediction of T2DM compared with single variables. Among the original variables, fasting glucose, proinsulin, BMI, and blood pressure values were predictive of T2DM. Discussion: Our data support the concept of a MetSy, and we propose five separate clusters of components. The inflammation and dyslipidemia factors were not independently associated with diabetes risk. In contrast, obesity and accompanying insulin resistance and β‐cell decompensation seem to be two core perturbations promoting and predicting progression to T2DM.     

Neferine suppresses autophagy-induced inflammation,oxidative stress and adipocyte differentiation in Graves' orbitopathy

Previous studies in Graves’ orbitopathy (GO) patient-derived fibroblasts showed that inhibition of autophagy suppresses adipogenic differentiation. Autophagy activation is associated with inflammation, production of reactive oxygen species and fibrosis. Neferine is an alkaloid extracted from Nelumbo nucifera, which induces Nrf2 expression and inhibits autophagy. Here, we have elucidated the role of neferine on interleukin (IL)-13-induced autophagy using patient-derived orbital fibroblasts as an in vitro model of GO. GO patient-derived orbital fibroblasts were isolated and cultured to generate an in vitro model of GO. Autophagy was determined by Western blot detection of the markers such as Beclin-1, Atg-5 and LC3 and by immunofluorescence detection of autophagosome formation. Analysis of differentiation towards an adipogenic lineage was performed by Oil red O staining. The expression of inflammatory factors was detected by ELISA and semiquantitative RT-PCR. Neferine inhibited autophagy in GO orbital fibroblasts, as indicated by the suppression of IL-13-induced autophagosome formation, overexpression of autophagy markers, increased LC3-II/LC3-I levels and finally down-regulation of p62. Neferine suppressed IL-13-induced inflammation, ROS generation, fibrosis and adipogenic differentiation in GO patient-derived orbital fibroblasts. The anti-inflammatory, antioxidant and antiadipogenic effects of neferine were accompanied by the up-regulation of Nrf2. These results indicated that orbital tissue remodelling and inflammation in GO may be mediated by autophagy, and neferine suppressed autophagy-related inflammation and adipogenesis through a mechanism involving Nrf2.     

Perilipin 5 deficiency promotes atherosclerosis progression through accelerating inflammation,apoptosis, and oxidative stress

Excessive plasma triglyceride (TG) and cholesterol levels promote the progression of several prevalent cardiovascular risk factors, including atherosclerosis, which is a leading death cause. Perilipin 5 (Plin5), an important perilipin protein, is abundant in tissues with very active lipid catabolism and is involved in the regulation of oxidative stress. Although inflammation and oxidative stress play a critical role in atherosclerosis development, the underlying mechanisms are complex and not completely understood. In the present study, we demonstrated the role of Plin5 in high-fat-diet-induced atherosclerosis in apolipoprotein E null (ApoE^−/−) mice. Our results suggested that Plin5 expressions increased in the artery tissues of ApoE^−/− mice. ApoE/Plin5 double knockout (ApoE^−/−Plin5^−/−) exacerbated severer atherogenesis, accompanied with significantly disturbed plasma metabolic profiles, such as elevated TG, total cholesterol, and low-density lipoprotein cholesterol levels and reduced high-density lipoprotein cholesterol contents. ApoE^−/−Plin5^−/− exhibited a higher number of inflammatory monocytes and neutrophils, as well as overexpression of cytokines and chemokines linked with an inflammatory response. Consistently, the IκBα/nuclear factor kappa B pathway was strongly activated in ApoE^−/−Plin5^−/−. Notably, apoptosis was dramatically induced by ApoE^−/−Plin5^−/−, as evidenced by increased cleavage of Caspase-3 and Poly (ADP-ribose) polymerase-2. In addition, ApoE^−/−Plin5^−/− contributed to oxidative stress generation in the aortic tissues, which was linked with the activation of phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinases pathways. In vitro, oxidized low-density lipoprotein (ox-LDL) increased Plin5 expression in RAW264.7 cells. Its knockdown enhanced inflammation, apoptosis, oxidative stress, and lipid accumulation, while promotion of Plin5 markedly reduced all the effects induced by ox-LDL in cells. These studies strongly supported that Plin5 could be a new regulator against atherosclerosis, providing new insights on therapeutic solutions.     

Development and application of oxidative stress biomarkers

Abstract

Oxidative stress may cause a wide variety of free radical reactions to produce deleterious modifications in membranes, proteins, enzymes, and DNA. Reactive Oxygen Species (ROS) generated by myeloperoxidase (MPO) can induce lipid peroxidation and also play an important role in the generation of reactive chlorinating and brominating species. As the universal biomarkers, chemical, and immunochemical approach on oxidatively modified and halogenated tyrosines has been carried out. As amido-type adduct biomarkers, chemical, and immunochemical evaluation of hexanoyl- and propanoyl-lysines, hexanoyl- and propanoyl-dopamines and phospholipids were prepared and developed for application of evaluation of novel antioxidative functional food factors. We have also involved in application of oxidatively modified DNAs such as 8-hydroxy- and 8-halogenated deoxyguanosines as the useful biomarkers for age-related diseases using both in vitro and in vivo systems. Application of these oxidative stress biomarkers for novel type of functional food development and recent approach for development of novel evaluation systems are also discussed.     

Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity

The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and β-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.     

Effects of different intensities of continuous training on vascular inflammation and oxidative stress in spontaneously hypertensive rats

We aimed to study the effects and underlying mechanism of different intensities of continuous training (CT) on vascular inflammation and oxidative stress in spontaneously hypertensive rats (SHR). Rats were divided into five groups (n = 12): Wistar-Kyoto rats sedentary group (WKY-S), sedentary group (SHR-S), low-intensity CT group (SHR-L), medium-intensity CT group (SHR-M) and high-intensity CT group (SHR-H). Changes in body mass, heart rate and blood pressure were recorded. The rats were euthanized after 14 weeks, and blood and vascular tissue samples were collected. Haematoxylin and Eosin staining was used to observe the aortic morphology, and Western blot was used to detect the expression of mesenteric artery proteins. After CT, the mean arterial pressures improved in SHR-L and SHR-M and increased in SHR-H compared with those in SHR-S. Vascular inflammation and oxidative stress levels significantly subsided in SHR-L and SHR-M (p < 0.05), whereas in SHR-H, only vascular inflammation significantly subsided (p < 0.05), and oxidative stress remained unchanged (p > 0.05). AMPK and SIRT1/3 expressions in SHR-L and SHR-M were significantly up-regulated than those in SHR-S (p < 0.05). These results indicated that low- and medium-intensity CT can effectively reduce the inflammatory response and oxidative stress of SHR vascular tissue, and high-intensity CT can improve vascular tissue inflammation but not oxidative stress.     

Effects of chitosan oligosaccharides on intestinal oxidative stress and inflammation response in heat stressed rats

This study was to verify the effects of chitosan oligosaccharides (COS) on intestinal integrity, oxidative status, and inflammatory response in a heat-stressed rat model. A total of 24 male Sprague Dawley rats were randomly divided into 3 treatment: CON, the control group; HS, the heat stress group; HSC, the heat stress group with 200 mg/kg COS. Rats in the HS and HSC group exposed to a cyclical heat stress for 7 consecutive days. The CON and HS group provided basal diet, and the HSC group provided the same diet with 200 mg/kg COS. Compared with the HS group, rats in the HSC group had lower serum diamine oxidase and D-lactate acid level, higher villus height of jejunum and ileum, lower malondialdehyde (MDA) content in duodenum, jejunum, and ileum mucosa, higher glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (T-AOC) activity in duodenum mucosa, higher T-AOC activity in jejunum mucosa, and higher glutathione (GSH) level in ileum mucosa. Compared with the HS group, rats in the HSC group had higher interleukin-10 (IL-10) level, but lower tumor necrosis factor-α (TNF-α) level in duodenum, jejunum, and ileum mucosa. These results indicated that COS may alleviate intestinal damage under heat stress condition, probably by modulating intestinal inflammatory response and oxidative status.     

Corosolic acid prevents oxidative stress, inflammation and hypertension in SHR/NDmcr-cp rats, a model of metabolic syndrome

Corosolic acid (CRA), a constituent of banaba leaves, has been reported to have anti-inflammatory and hypoglycemic activities. The aim of this study was to determine the effects of CRA on metabolic risk factors including obesity, hypertension, hyperinsulinemia, hyperglycemia, and hyperlipidemia together with oxidative stress and inflammation, all of which are characteristic of the SHR/NDmcr-cp (cp/cp) (SHR-cp) rat, an animal model of metabolic syndrome. Six-week-old male SHR-cp rats were fed a high fat diet containing 0.072% CRA for 14 weeks. Treatment with CRA lowered blood pressure, which was elevated in control animals, by 10% after 8 weeks, and serum free fatty acids by 21% after 2 weeks. CRA treatment resulted in decreases in the levels of the oxidative stress markers thiobarbituric acid-reactive substances and 8-hydroxydeoxyguanosine by 27% and 59%, respectively, after 2 weeks. CRA treatment also reduced the levels of myeloperoxidase markers, 3-nitrotyrosine and 3-chlorotyrosine by 38% and 39%, respectively, after 10 weeks, and tended to decrease the levels of high sensitivity C-reactive protein, a marker of inflammation, after 6 weeks. However, CRA had no effect on weight gain or hyperglycemia. These results demonstrate that CRA can ameliorate hypertension, abnormal lipid metabolism, and oxidative stress as well as the inflammatory state in SHR-cp rats. This implies that CRA can be beneficial for preventing atherosclerosis-related diseases that are an increasing health care problem worldwide.     

IP3R attenuates oxidative stress and inflammation damage in smoking-induced COPD by promoting autophagy

Tobacco smoking is one of the most important risk factors for chronic obstructive pulmonary disease (COPD). However, the most critical genes and proteins remain poorly understood. Therefore, we aimed to investigate these hub genes and proteins in tobacco smoke-induced COPD, together with the potential mechanism(s). Differentially expressed genes (DEGs) were analysed between smokers and patients with COPD. mRNA expression and protein expression of IP₃R were confirmed in patients with COPD and extracted smoke solution (ESS)-treated human bronchial epithelial (HBE) cells. Moreover, expression of oxidative stress, inflammatory cytokines and/or autophagy-related protein was tested when IP₃R was silenced or overexpressed in ESS-treated and/or 3-MA-treated cells. A total of 30 DEGs were obtained between patients with COPD and smoker samples. IP₃R was identified as one of the key targets in tobacco smoke-induced COPD. In addition, IP₃R was significantly decreased in patients with COPD and ESS-treated cells. Loss of IP₃R statistically increased expression of oxidative stress and inflammatory cytokines in ESS-treated HBE cells, and overexpression of IP₃R reversed the above functions. Furthermore, the autophagy-related proteins (Atg5, LC3 and Beclin1) were statistically decreased, and p62 was increased by silencing of IP₃R cells, while overexpression of IP₃R showed contrary results. Additionally, we detected that administration of 3-MA significantly reversed the protective effects of IP₃R overexpression on ESS-induced oxidative stress and inflammatory injury. Our results suggest that IP₃R might exert a protective role against ESS-induced oxidative stress and inflammation damage in HBE cells. These protective effects might be associated with promoting autophagy.     

Inhibition of inflammation and oxidative stress by an imidazopyridine derivative X22 prevents heart injury from obesity

Inflammation and oxidative stress plays an important role in the development of obesity‐related complications and cardiovascular disease. Benzimidazole and imidazopyridine compounds are a class of compounds with a variety of activities, including anti‐inflammatory, antioxidant and anti‐cancer. X22 is an imidazopyridine derivative we synthesized and evaluated previously for anti‐inflammatory activity in lipopolysaccharide‐stimulated macrophages. However, its ability to alleviate obesity‐induced heart injury via its anti‐inflammatory actions was unclear. This study was designed to evaluate the cardioprotective effects of X22 using cell culture studies and a high‐fat diet rat model. We observed that palmitic acid treatment in cardiac‐derived H9c2 cells induced a significant increase in reactive oxygen species, inflammation, apoptosis, fibrosis and hypertrophy. All of these changes were inhibited by treatment with X22. Furthermore, oral administration of X22 suppressed high‐fat diet‐induced oxidative stress, inflammation, apoptosis, hypertrophy and fibrosis in rat heart tissues and decreased serum lipid concentration. We also found that the anti‐inflammatory and anti‐oxidative actions of X22 were associated with Nrf2 activation and nuclear factor‐kappaB (NF‐κB) inhibition, respectively, both in vitro and in vivo. The results of this study indicate that X22 may be a promising cardioprotective agent and that Nrf2 and NF‐κB may be important therapeutic targets for obesity‐related complications.     

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.