Stress In Nonalcoholic Fatty Liver Disease Research Articles

Azoramide ameliorates fructose-induced nonalcoholic fatty liver disease in mice

Nonalcoholic fatty liver disease (NAFLD) is a frequent health problem. The insulin resistance and endoplasmic reticulum (ER) stress have been suggested to play important roles in the development and progression of NAFLD. However these processes and correlations have not fully been understood yet. Azoramide, an antidiabetic drug, has the potential for reducing insulin resistance and ER stress in obese mice. To date, there is no study on the effects of azoramide in NAFLD. The aim of this study was to investigate the potential role of azoramide on insulin resistance and ER stress in NAFLD induced mice.Forty Swiss Albino mice were assigned into control, azoramide, fructose and fructose + azoramide groups. Azoramide group received a single dose of azoramide at 150 mg/kg/day by gavage between 71–77th days. Fructose group was treated with 30% fructose solution for 70 days to generate NAFLD. Fructose + azoramide group was treated with 30% fructose for 70 days and then with a single dose of 150 mg/kg/day azoramide for 7 days. At the end of experiment, blood of mice was taken via cardiac puncture, and the livers were excised and weighted. GRP78 and XBP-1 levels were examined with immunohistochemistry in liver tissues. Liver steatosis was evaluated with H&E, Oil-Red O and Sudan-Black staining. ALT, AST, triglyceride, total cholesterol, VLDL, LDL, HDL, fasting glucose and insulin levels were measured in serum.The body and liver weights, insulin resistance, ER-stress markers, lipid profile, AST, ALT and histopathological changes increased by fructose treatment. Azoramide treatment was generally reversed all these changes.These data offer the first evidence to show that azoramide may serve as a potential treatment agent in NAFLD through decreasing the ER-stress and insulin resistance.

Resveratrol protects against nonalcoholic fatty liver disease by improving lipid metabolism and redox homeostasis via the PPARα pathway.

Resveratrol (RSV), a well-known bioactive compound, has been reported to exert a broad range of health benefits. Accumulating evidence suggests that RSV is beneficial for many metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). This study investigated the preventive and therapeutic effects of RSV on high-fat diet (HFD)-induced NAFLD in rats and palmitate acid (PA)-induced hepatocyte steatosis in HepG2 cells. Hepatocytes were incubated with inhibitors of peroxisome proliferator-activated receptor α (PPARα) or short interfering RNAs (siRNAs) targeting PPARα, AMP-activated protein kinase (AMPK), and protein kinase A (PKA) to determine the underlying mechanisms. We found that RSV noticeably ameliorated HFD-induced hepatic steatosis in rats and inhibited PA-induced lipid accumulation in HepG2 cells. Moreover, RSV improved lipid metabolism, enhanced antioxidant capacity, and restored mitochondrial respiratory chain activities. Incubation with inhibitors of PPARα or PPARα siRNA abolished the protective effects of RSV on lipid metabolism and redox homeostasis. Furthermore, RSV activated the PKA/AMPK/PPARα signaling pathway. Our results provided direct evidence for a novel, PPARα-mediated mechanism responsible for the beneficial effects of RSV on hepatic steatosis. These findings may have important theoretical and application prospects for the prevention and treatment of NAFLD. Novelty RSV improved lipid metabolism and redox homeostasis and oxidative stress in NAFLD via the PKA/AMPK/PPARα signaling pathway. RSV may have a greater beneficial effect in the early prevention of hepatic steatosis.

SAT-109 tiRNA-5 Reflects the Severity of Mitochondrial Oxidative Stress in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disease with complicated mechanisms among which disturbance of mitochondria function weighs a lot. Overexpression of CypD, the initial factor in mitochondrial permeability transition pore, led to mitochondrial stress triggered hepatic TG accumulation. tRNA fragments (tRFs) are non-coding RNAs produced by precise cleavage of tRNA and divided into subtypes including tiRNA-5, tiRNA-3, tRF-5, tRF-3 and tRF-1. It is reported that tRFs, especially tiRNA-5 associated with stress. However, whether tRFs connect with NAFLD is unclear. To solve the problem, CypD overexpression models were constructed by injecting mouse AdPPIF virus to 8-weeks male C57BL/6 mice as study group (CH, n=6). Control group (WT, n=6) was established by injecting empty vector virus (AdEGFP). Then tRFs in liver were detected by high-throughput sequencing. Proportion of tRFs in CH was 40.8% higher vs WT. A connection might exist between CypD and tRFs. To validate this speculation, CypD knockdown mice were established as study group and 8-weeks male C57BL/6 as control. Then both groups were fed with 60 kcal% fat diet for 4 weeks or 8 weeks and named as CKO4, CWT4, CKO8 or CWT8 (n=6). Sequencing showed that proportion of tRFs in CKO4 and CKO8 was 42.7% and 31.2% lower vs control respectively, consistent with the speculation. Therefore, bioinformatics analysis was used to explore further. Pie plot based on proportion of each tRFs subtype showed that over 90% tRFs were tRF-5 and tiRNA-5. tiRNA-5 was 38.5% higher in CH vs WT, 27.4% lower in CKO4 vs CWT4 and 36.0% lower in CKO8 vs CWT8, indicating a connection between tiRNA-5 and CypD. Integration of pathological sections, metabolic index and more analysis were done for futher study. PCA and Venn diagram described the difference on the whole while absolute value of Pearson correlation coefficient (CH vs WT=0.84, CKO4 vs CWT4=0.93, CKO8 vs CWT8=0.73) and Scatter plot (CH vs WT =0.69, CKO4 vs CWT4=0.85, CKO8 vs CWT8=0.66) reflected the similarity between study and control group. These data indicated a connection between tRF and the severity of NAFLD assessed by pathology and metabolism index. Finally, a cutoff of fold change ≥ 1.5 was employed to identify the differentially expressed tRFs and most of those were tiRNA-5 as speculated. qRT-PCR was conducted for validation, among which fold change of AS-tDR-000666 (GlyGCC derived tiRNA-5) was significative (CH vs WT=4.29, CKO4 vs CWT4=0.22, CKO8 vs CWT8=0.05). It might be a potential biomarker related with severity of NAFLD. Because the CypD-change-induced mitochondrial oxidative stress was the direct mechanism triggering NAFLD in mouse models above, we could conclude that mitochondrial oxidative stress in NAFLD changed the profile of tRFs and tiRNA-5 had the potential to reflect the severity of mitochondrial oxidative stress in NAFLD. This study provided a new understanding of NAFLD.

Protection of hepatocyte mitochondrial function by blueberry juice and probiotics via SIRT1 regulation in non-alcoholic fatty liver disease.

Mitochondrial dysfunction has been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Our previous study has firstly reported that blueberry juice and probiotics (BP) effectively protect liver function in NAFLD. However, the role of BP in hepatic mitochondria is unknown. Here, we aimed to investigate the effects and mechanisms of BP on the mitochondrial function and oxidative stress of rats with NAFLD. The NAFLD rat models were established and treated with BP and SIRT1 siRNA. The mitochondrial ultrastructure was analyzed by electron microscopy, reactive oxygen species (ROS) was detected by immunofluorescence, and biomarkers of mitochondrial function and oxidative stress were examined via quantitative reverse transcription-PCR, western blot, and immunohistochemistry. Results revealed that BP significantly reversed the NAFLD-induced hepatic mitochondrial damage, mitochondrial swelling, and hepatic necrosis. In particular, BP significantly restored the mitochondrial respiratory function of NAFLD rats by decreasing state 4 and 3 respiration rates, by increasing the respiration control ratio (RCR) and the ADP/O ratio, and by enhancing ATP, ADP, AMP, and EC syntheses. Moreover, BP reduced mitochondrial oxidative stress in NAFLD by decreasing the MDA level, elevating the GSH and SOD levels, and suppressing the ROS activity. Importantly, SIRT1 deficiency significantly abolished the effects of BP on the mitochondria and oxidative stress. Furthermore, BP reversed the decline of PGC-1α expression induced by NAFLD, while SIRT1 silencing significantly suppressed the effects of BP on PGC-1α. In conclusion, BP might effectively protect rats against mitochondrial dysfunction during NAFLD as potential ingredients of functional food, by modulating the SIRT1 expression. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide novel tools for therapeutic intervention.

Exercise Training Attenuates Non-Alcoholic Fatty Liver Disease in rats with Diabetes via Endoplasmic Reticulum Stress

Non-alcoholic fatty liver disease (NAFLD) is highly prevalent in type 2 diabetes (T2D). Recent evidence supports that endoplasmic reticulum (ER) stress plays a critical role in the development of NAFLD. Although exercise training has been commonly prescribed for the treatment of NAFLD, the effects of different types of exercise on ER stress in NAFLD under type 2 diabetic condition are largely unknown. PURPOSE: The purpose of this study was to determine the effects of aerobic and resistance exercises on hepatic ER stress response and NAFLD in rats with T2D. METHODS: Male Sprague-Dawley rats were randomly assigned to four groups (n=10/group): Control (CON), T2D, T2D with aerobic exercise (T2D+AE; treadmill walking at 30 m/min, 0° incline, 60 mins/day, 5 day/week, for 8 weeks) and T2D with resistance exercise (T2D+RE; climbing a 80° incline vertical ladder with weights progressively increased from 50% to 100% of maximal carrying capacity on the tail, 3 times/day, 5 days/ week, for 8 weeks). Liver tissue samples were collected for histopathological analysis of the density of lipid droplets, and immunoblot analysis of expression levels of ER stress proteins, including glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), caspase 12, and c-Jun N-terminal kinase (JNK). One-way ANOVAs and Tukey’s -test were used for data analysis. RESULTS: The density of lipid droplets in the liver was significantly higher in the T2D group than in the CON group (p<0.01), but was significantly lower in the T2D+AE and T2D+RE groups when compared to the T2D group (both p<0.01). In addition, the T2D group had significantly higher levels of protein expression of GRP78, CHOP, caspase 12 and JNK when compared to the CON group (all p<0.01). Both exercise groups had significantly lower levels of protein expression of GRP 78, CHOP, Caspase 12 and JNK when compared to the T2D group (p<0.05 to p<0.01). CONCLUSION: Our findings suggest that both aerobic and resistance exercises are protective against NAFLD in rats with T2D by potentially regulating proteins involved in ER stress response.

Vitamin E as a Treatment for Nonalcoholic Fatty Liver Disease: Reality or Myth?

Obesity is one of the major epidemics of this millennium, and its incidence is growing worldwide. Following the epidemics of obesity, nonalcoholic fatty liver disease (NAFLD) has become a disease of increasing prevalence and a leading cause of morbidity and mortality closely related to cardiovascular disease, malignancies, and cirrhosis. It is believed that oxidative stress is a main player in the development and progression of NAFLD. Currently, a pharmacological approach has become necessary in NAFLD because of a failure to modify lifestyle and dietary habits in most patients. Vitamin E is a potent antioxidant that has been shown to reduce oxidative stress in NAFLD. This review summarizes the biological activities of vitamin E, with a primary focus on its therapeutic efficacy in NAFLD.

Regulation of SREBP-2 intracellular trafficking improves impaired autophagic flux and alleviates endoplasmic reticulum stress in NAFLD

Sterol regulatory element-binding protein 2 (SREBP-2), is a well-known transcriptional regulator of cholesterol metabolism. SREBP-2 is activated unconventionally to promote excessive cholesterol accumulation in non-alcoholic fatty liver disease (NAFLD). In addition, recent studies suggested that excessive lipid and cholesterol accumulation can weaken cellular autophagy function and promote endoplasmic reticulum stress (ERS). However, it remains unknown whether regulation of SREBP-2 processing modulates autophagy and ERS. In this study, we demonstrated that inhibition of SREBP-2 intracellular trafficking by site-1 protease (S1P) and site-2 protease (S2P) specific inhibitors, or shRNAs targeting S1P and S2P, upregulated gene and protein expression of autophagy markers, and improved the impaired autophagic flux induced in both cell and mouse models of NAFLD. Furthermore, increased lipid degradation by autophagy could repress PERK-P-EIF2α signaling. Taken together, these findings suggest that regulating the nuclear transport of SREBP-2 reduces lipid deposition and ERS via an autophagy-dependent pathway.

ROS correlates intimately with the progression of non-alcoholic fatty liver disease to hepatocarcinoma

The prevalence of non-alcoholic fatty liver disease (NAFLD) is rapidly increasing. NAFLD affects the health of one-third of the world’s population in forms that range from simple steatosis to hepatocellular injury, inflammation, fibrosis, liver cirrhosis and hepatocarcinoma. Oxidative stress in the progression of NAFLD to hepatocarcinoma is gaining increasing attention. Sustained and excessive reactive oxygen species (ROS), which lead to oxidative stress, are involved in all pathophysiological stages of NAFLD and contribute to the occurrence of hepatocarcinoma. Antioxidants in natural plant extracts that activate nuclear factor related to E2 factor 2 (Nrf2) can effectively suppress the progression. The cellular anti-oxidative system has great importance in the prevention and reversal of NAFLD. This review summarizes the current knowledge of the involvement of oxidative stress in NAFLD to hepatocarcinoma.

NKT cell modulates NAFLD potentiation of metabolic oxidative stress-induced mesangial cell activation and proximal tubular toxicity.

Obesity and nonalcoholic fatty liver disease (NAFLD) are associated with the development and progression of chronic kidney disease. We recently showed that NAFLD induces liver-specific cytochrome P-450 (CYP)2E1-mediated metabolic oxidative stress after administration of the CYP2E1 substrate bromodichloromethane (BDCM) (Seth RK, Das S, Kumar A, Chanda A, Kadiiska MB, Michelotti G, Manautou J, Diehl AM, Chatterjee S. Toxicol Appl Pharmacol 274: 42-54, 2014; Seth RK, Kumar A, Das S, Kadiiska MB, Michelotti G, Diehl AM, Chatterjee S. Toxicol Sci 134:291-303, 2013). The present study examined the effects of CYP2E1-mediated oxidative stress in NAFLD leading to kidney toxicity. Mice were fed a high-fat diet for 12 wk to induce NAFLD. NAFLD mice were exposed to BDCM, a CYP2E1 substrate, for 4 wk. NAFLD + BDCM increased CYP2E1-mediated lipid peroxidation in proximal tubular cells compared with mice with NAFLD alone or BDCM-treated lean mice, thus ruling out the exclusive role of BDCM. Lipid peroxidation increased IL-1β, TNF-α, and interferon-γ. In parallel, mesangial cell activation was observed by increased α-smooth muscle actin and transforming growth factor-β, which was blocked by the CYP2E1 inhibitor diallyl sulphide both in vivo and in vitro. Mice lacking natural killer T cells (CD1d knockout mice) showed elevated (>4-fold) proinflammatory mediator release, increased Toll-like receptor (TLR)4 and PDGF2 mRNA, and mesangial cell activation in the kidney. Finally, NAFLD CD1D knockout mice treated with BDCM exhibited increased high mobility group box 1 and Fas ligand levels and TUNEL-positive nuclei, indicating that higher cell death was attenuated in TLR4 knockout mice. Tubular cells showed increased cell death and cytokine release when incubated with activated mesangial cells. In summary, an underlying condition of progressive NAFLD causes renal immunotoxicity and aberrant glomerular function possibly through high mobility group box 1-dependent TLR4 signaling and mesangial cell activation, which, in turn, is modulated by intrinsic CD1D-dependent natural killer T cells.

Role of Hepatic Progenitor Cells in Nonalcoholic Fatty Liver Disease Development: Cellular Cross-Talks and Molecular Networks

Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of diseases ranging from simple fatty liver to nonalcoholic steatohepatitis, (NASH) which may progress to cirrhosis and hepatocellular carcinoma. NASH has been independently correlated with atherosclerosis progression and cardiovascular risk. NASH development is characterized by intricate interactions between resident and recruited cells that enable liver damage progression. The increasing general agreement is that the cross-talk between hepatocytes, hepatic stellate cells (HSCs) and macrophages in NAFLD has a main role in the derangement of lipid homeostasis, insulin resistance, danger recognition, immune tolerance response and fibrogenesis. Moreover, several evidences have suggested that hepatic stem/progenitor cell (HPCs) activation is a component of the adaptive response of the liver to oxidative stress in NAFLD. HPC activation determines the appearance of a ductular reaction. In NASH, ductular reaction is independently correlated with progressive portal fibrosis raising the possibility of a periportal fibrogenetic pathway for fibrogenesis that is parallel to the deposition of subsinusoidal collagen in zone 3 by HSCs. Recent evidences indicated that adipokines, a class of circulating factors, have a key role in the cross-talk among HSCs, HPCs and liver macrophages. This review will be focused on cellular cross-talk and the relative molecular networks which are at the base of NASH progression and fibrosis.

Absence of adipose triglyceride lipase protects from hepatic endoplasmic reticulum stress in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by triglyceride (TG) accumulation and endoplasmic reticulum (ER) stress. Because fatty acids (FAs) may trigger ER stress, we hypothesized that the absence of adipose triglyceride lipase (ATGL/PNPLA2)-the main enzyme for intracellular lipolysis, releasing FAs, and closest homolog to adiponutrin (PNPLA3) recently implicated in the pathogenesis of NAFLD-protects against hepatic ER stress. Wild-type (WT) and ATGL knockout (KO) mice were challenged with tunicamycin (TM) to induce ER stress. Serum biochemistry, hepatic TG and FA profiles, liver histology, and gene expression for markers of hepatic lipid metabolism, ER stress, and inflammation were explored. Moreover, cell-culture experiments were performed in Hepa1.6 cells after the knockdown of ATGL before FA and TM treatment. TM increased hepatic TG accumulation in ATGL KO, but not in WT, mice. Lipogenesis and β-oxidation were repressed at the gene-expression level (sterol regulatory element-binding transcription factor 1c, fatty acid synthase, acetyl coenzyme A carboxylase 2, and carnitine palmitoyltransferase 1 alpha) in both WT and ATGL KO mice. Genes for very-low-density lipoprotein (VLDL) synthesis (microsomal triglyceride transfer protein and apolipoprotein B) were down-regulated by TM in WT and even more in ATGL KO mice, which displayed strongly reduced serum VLDL cholesterol levels. Notably, ER stress markers glucose-regulated protein, C/EBP homolog protein, spliced X-box-binding protein, endoplasmic-reticulum-localized DnaJ homolog 4, and inflammatory markers Tnfα and iNos were induced exclusively in TM-treated WT, but not ATGL KO, mice. Total hepatic FA profiling revealed a higher palmitic acid/oleic acid (PA/OA) ratio in WT mice, compared to ATGL KO mice, at baseline. Phosphoinositide-3-kinase inhibitor-known to be involved in FA-derived ER stress and blocked by OA-was increased in TM-treated WT mice only. In line with this, in vitro OA protected hepatocytes from TM-induced ER stress. Lack of ATGL may protect from hepatic ER stress through alterations in FA composition. ATGL could constitute a new therapeutic strategy to target ER stress in NAFLD.

Oxidant Stress and Antioxidant Status Among Patients With Nonalcoholic Fatty Liver Disease (NAFLD)

One of the major pathogenic mechanisms for progression of nonalcoholic fatty liver disease (NAFLD) is oxidative stress. Recently, many studies have demonstrated the role of oxidative stress in NAFLD however, studies describing the antioxidant status in these patients are lacking. To study the levels of oxidative stress and antioxidant status among patients with NAFLD. It was a prospective study in which 29 patients with NAFLD, 25 diseased controls with chronic viral hepatitis, and 23 healthy controls were enrolled. Apart from standard biochemical parameters, lipid peroxidation products were measured as thiobarbituric acid reactive substances. As measures of antioxidant capacity, superoxide dismutase, vitamin C levels and ferric reducing ability of plasma were measured. Level of thiobarbituric acid reactive substances was significantly higher among NAFLD patients as compared with diseased [4.7 nmol/mL (1.0 to 10.2) vs. 2.4 nmol/mL (0.8 to 10.7); P=0.02] or healthy controls [4.7 nmol/mL (1.0 to 10.2) vs. 1.8 nmol/mL (0.5 to 4.1); P=0.0001]. FRAP was found to be significantly higher in patients with NAFLD as compared with healthy controls [450.3 (197.6 to 733.3) vs. 340.8 (141.6 to 697.5) mumol Fe liberated; P=0.04], even though it was similar between NAFLD and diseased controls. Among NAFLD patients, there was no significant correlation between histological grading or staging and levels of pro and antioxidants. Products of lipid peroxidation are significantly increased among patients with NAFLD as compared with chronic viral hepatitis or healthy controls. Larger studies and newer markers of oxidative stress are required to clarify the association between oxidative stress and histological severity in NAFLD.

Review article: role of oxidative stress in the progression of non‐alcoholic steatosis

The mechanisms responsible for the progression of nonalcoholic fatty liver disease (NAFLD) to more severe liver injury are still poorly understood. Data from animal models suggest that oxidative stress contributes to steatohepatitis and an increase of lipid peroxidation has been documented in human NAFLD. By measuring the titers of circulating antibodies against lipid peroxidation products as markers of oxidative stress we have observed that NAFLD patients have titers of these antibodies significantly higher than in controls. Moreover, the titers of lipid peroxidation-related antibodies are associated with a 3-fold increase in the risk of developing advanced fibrosis/cirrhosis. Although the mechanisms causing oxidative stress in NAFLD have not been elucidated, these results support the involvement of lipid peroxidation in the processes leading to liver fibrosis associated with NAFLD.

Lipid Peroxidation in Bariatric Candidates with Nonalcoholic Fatty Liver Disease (NAFLD) – Preliminary Findings

Pathogenesis of nonalcoholic fatty liver disease (NAFLD) remains incompletely known, and oxidative stress is one of the mechanisms incriminated. The aim of this study was to evaluate the role of liver oxidative stress in NAFLD affecting morbidly obese patients. 39 consecutive patients with BMI >40 kg/m2 submitted to Roux-en-Y gastric bypass were enrolled, and wedge liver biopsy was obtained during operation. Oxidative stress was measured by concentration of hydroperoxides (CEOOH) in liver tissue. Female gender was dominant (89.7%) and median age was 43.6 +/- 11.1 years. Histology showed fatty liver in 92.3%, including 43.6% with nonalcoholic steatohepatitis (NASH), 48.7% with isolated steatosis and just 7.7% with normal liver. Liver cirrhosis was present in 11.7% of those with nonalcoholic steatohepatitis. Concentration of CEOOH was increased in the liver of patients with NASH when compared to isolated steatosis and normal liver (0.26+/- 0.17, 0.20+/- 0.01 and 0.14+/- 0.00 nmol/mg protein, respectively) (P < 0.01). Liver biochemical variables were normal in 92.3% of all cases, and no difference between NASH and isolated steatosis could be demonstrated. 1) Nonalcoholic steatosis, steatohepatitis and cirrhosis were identified in substantial numbers of morbidly obese patients; 2) Concentration of hydroperoxides was increased in steatohepatitis, consistent with a pathogenetic role for oxidative stress in this condition.