Activity In Non-alcoholic Fatty Liver Disease Research Articles

Inducible nitric oxide synthase accelerates nonalcoholic fatty liver disease progression by regulating macrophage autophagy.

Cells and tissues, such as macrophages, express inducible nitric oxide synthase (INOS) after stimulation by certain factors. INOS helps mediate the macrophage inflammatory reaction, but few studies have explored how INOS affects macrophage function in nonalcoholic fatty liver disease (NAFLD). This study investigated the role of INOS-mediated macrophage activity in NAFLD. A high-fat diet was used to establish an NAFLD mouse model. After 12 weeks, blood was collected for immune cell and lipid analyses, and liver tissues were collected for pathological analyses with hematoxylin and eosin and Oil Red O staining. Peritoneal macrophages were extracted in situ, cultured in Dulbecco's modified Eagle's medium, and stimulated with palmitic acid to mimic in vivo conditions for further assays. Real-time polymerase chain reaction, western blot analysis, and immunofluorescence were used to verify the expression of target genes or proteins. In the NAFLD model, INOS expression in macrophages increased, and INOS knockdown significantly decreased the number of macrophages. Pathological examinations confirmed that INOS knockdown slowed NAFLD progression and macrophage infiltration during inflammation. INOS knockdown also enhanced phagocytosis and lipid transport by macrophages, and increased the expression of autophagy-related molecules in macrophages, which improved the autophagy level, promoted apoptotic cell degradation, and maintained intracellular environment homeostasis. These results indicate a correlation between INOS expression and macrophage function in NAFLD.

CT Texture Analysis in Nonalcoholic Fatty Liver Disease (NAFLD)

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease worldwide. There are limited biomarkers that can detect progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The purpose of our study was to utilize CT texture analysis to distinguish steatosis from NASH. 16 patients with NAFLD (38% male, median (interquartile range): age 57 (48-64) years, BMI 37.5 (35.0-46.8) kg/m2) underwent liver biopsy and abdominal non-contrast CT. CT texture analysis was performed to quantify gray-level tissue summaries (e.g., entropy, kurtosis, skewness, and attenuation) using commercially available software (TexRad, Cambridge England). Logistic regression analyses were performed to quantify the association between steatosis/NASH status and CT texture. ROC curve analysis was performed to determine sensitivity, specificity, AUC, 95% CIs, and cutoff values of texture parameters to differentiate steatosis from NASH. By histology, 6/16 (37%) of patients had simple steatosis and 10/16 (63%) had NASH. Patients with NASH had lower entropy (median, interquartile range (IQR): 4.3 (4.1, 4.8) vs. 5.0 (4.9, 5.2), P= 0.013) and lower mean value of positive pixels (MPP) (34.4 (21.8, 52.2) vs. 66.5 (57.0, 70.7), P= 0.009) than those with simple steatosis. Entropy values below 4.73 predict NASH with 100% (95%CI: 67-100%) specificity and 80% (50-100%) sensitivity, AUC: 0.88. MPP values below 54.0 predict NASH with 100% (67-100%) specificity and 100% (50-100%) sensitivity, AUC 0.90. Our study provides preliminary evidence that CT texture analysis may serve as a novel imaging biomarker for disease activity in NAFLD and the discrimination of steatosis and NASH.

Glutathione: Pharmacological aspects and implications for clinical use in non-alcoholic fatty liver disease.

Glutathione is a tripeptide synthesized at cytosolic level, that exists in cells in a reduced form (thiol-reduced-GSH-) and in an oxidized form (disulfide-oxidized). The antioxidant function of GSH has led to speculation about its therapeutic role in numerous chronic diseases characterized by altered redox balance and reduced GSH levels, including, for instance, neurodegenerative disorders, cancer, and chronic liver diseases. Among these latter, non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in hepatocytes, in the absence of alcohol abuse or other steatogenic factors, is one of the most prevalent. The umbrella term NAFLD includes the pure liver fat accumulation, the so-called hepatic steatosis or non-alcoholic fatty liver, and the progressive form with inflammation, also known as non-alcoholic steatohepatitis, which is related to the increase in oxidative stress and reactive oxygen species, eventually leading to liver fibrosis. Although the pathogenetic role of oxidative stress in these diseases is well established, there is still limited evidence on the therapeutic role of GSH in such conditions. Hence, the aim of this review is to depict the current molecular and pharmacological knowledge on glutathione, focusing on the available studies related to its therapeutic activity in NAFLD.

Reduced lysosomal acid lipase (LAL) activity in NAFLD is related to the severity of liver disease ‘‘First study from the Asia-Pacific’’

Background and Aim: Non-alcoholic fatty liver disease (NAFLD) is estimated to be the most common liver disorder and presents as a major health burden globally. Pathogenesis of NAFLD is multi-factorial. Lysosomal Acid Lipase impairment is suggested as one of the involved pathogenic mechanisms in NAFLD. This study aimed to explore LAL activity in patients with NAFLD and its relationship with the disease severity.

Paraoxonase 1 and Non-Alcoholic Fatty Liver Disease: A Meta-Analysis.

Oxidative stress is involved in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). However, reliable biomarkers of NAFLD in relation to oxidative stress are not available. While paraoxonase 1 (PON1) is an antioxidant biomarker, there appears to be mixed data on PON-1 in patients with NAFLD. The aim of this meta-analysis was to assess the current data on PON1 activity (i.e., paraoxonase and arylesterase) in patients with NAFLD. A PubMed, CENTRAL, and Embase search identified 12 eligible articles. In the meta-analysis, the paraoxonase activity was low in patients with NAFLD (mean difference (MD) −27.17 U/L; 95% confidence interval (CI) −37.31 to −17.03). No difference was noted in the arylesterase activity (MD 2.45 U/L; 95% CI −39.83 to 44.74). In a subgroup analysis, the paraoxonase activity was low in biopsy-proven nonalcoholic steatohepatitis (MD −92.11 U/L; 95% CI −115.11 to −69.11), while the activity in NAFLD as diagnosed by ultrasonography or laboratory data was similar (MD −2.91 U/L; 95% CI −11.63 to 5.80) to that of non-NAFLD. In summary, the PON1, especially paraoxonase, activity could be a useful biomarker of NAFLD. Further studies are warranted to ascertain the relevance of PON1 measurements in patients with NAFLD.

Insulin resistance is positively associated with plasma cathepsin D activity in NAFLD patients.

Previous studies associated plasma cathepsin D (CTSD) activity with hepatic insulin resistance in overweight and obese humans. Insulin resistance is a major feature of non-alcoholic fatty liver disease (NAFLD) and is one of the multiple hits determining the progression towards non-alcoholic steatohepatitis (NASH). In line, we have previously demonstrated that plasma CTSD levels are increased in NASH patients. However, it is not known whether insulin resistance associates with plasma CTSD activity in NAFLD. To increase our understanding regarding the mechanisms by which insulin resistance mediates NAFLD, fifty-five liver biopsy or MRI-proven NAFLD patients (BMI>25kg/m2) were included to investigate the link between plasma CTSD activity to insulin resistance in NAFLD. We concluded that HOMA-IR and plasma insulin levels are independently associated with plasma CTSD activity in NAFLD patients (standardized coefficient β: 0.412, 95% Cl: 0.142~0.679, p=0.004 and standardized coefficient β: 0.495, 95% Cl: 0.236~0.758, p=0.000, respectively). Together with previous studies, these data suggest that insulin resistance may link to NAFLD via elevation of CTSD activity in plasma. As such, these data pave the way for testing CTSD inhibitors as a pharmacological treatment of NAFLD.

Butyrate Priming Improves Gut Health by Modulating Host Protease Activity in Murine Non‐Alcoholic Fatty Liver Disease

Reports from clinical investigations implicate a strong association of non‐alcoholic fatty liver disease (NAFLD) and inflammatory bowel diseases (IBD). Recently we reported a rapid alteration of gut microbiome in murine NAFLD model that resulted in gut leaching, tight junction protein alteration and intestinal inflammation. Advancing our present understanding, we show that altered gut microbiome in murine NAFLD significantly decreased microbial proteases content in the host gut micro‐environment. Dysbiosis significantly increased expression of host proteases derived from gut immune cells which were associated with intestinal inflammation and gut leaching. Results also showed significant upregulated expression of a disintegrin and metalloproteinase 17 (ADAM17) and serine proteases Granzyme M, Cathepsin G and Proteinase 3 in intestines of NAFLD mice. Increased expression of these proteases were associated with activation of protease activated receptor 1 (PAR1), significant over production of pro inflammatory cytokines TNFα and IL‐1β, decreased expression of Occludin, and increased expression of Claudin2. Most interestingly administration of butyrate to NAFLD mouse group significantly increased microbial protease content in gut, decreased expression of ADAM17, Granzyme M and Cathepsin G, and increased expression of protective matrix metalloproteinases 7 and 2 (MMP7 and MMP2). Decreased expression of ADAM17, Granzyme M and Cathepsin G improved gut leaching by upregulation of Occludin and down regulation of Claudin 2 expression, decreased PAR1 expression, and decreased TNFα and IL‐1β secretion. Furthermore, increased expression of MMP 2 and MMP 7 showed elevated expression of anti‐inflammatory cytokines IL‐4 and IL‐10 in small intestine. Thus, in conclusion our present study demonstrates that butyrate plays a crucial role in gut homeostasis by a previously unknown function of modulating host protease activity in NAFLD and can be of significant therapeutic value.Support or Funding InformationThis project has been supported by NIH grants # 2P01AT003961 (Project 4) and #P20GM103641‐07(Project3) to Dr. Saurabh Chatterjee

Clinical validation of the FLIP algorithm and the SAF score in patients with non-alcoholic fatty liver disease

Histological classifications used to diagnose/stage non-alcoholic fatty liver disease (NAFLD) are based on morphology, with undetermined clinical correlates and relevance. We assessed the clinical relevance of the fatty liver inhibition of progression (FLIP) algorithm and the steatosis, activity, and fibrosis (SAF) scoring system. One hundred and forty consecutive patients with suspected NAFLD and a separate validation cohort of 78 patients enrolled in a therapeutic trial, all with central reading of liver biopsy, were included. FLIP and SAF were used to categorize patients with non-alcoholic steatohepatitis (NASH), non-NASH NAFLD (NAFL), or non-NAFLD. The SAF activity score assessed hepatocyte ballooning and lobular inflammation; a histologically severe disease was defined as a SAF activity score of ≥3 and/or bridging fibrosis or cirrhosis. Clinical, biochemical, and metabolic data were analyzed in relation to histology. Patients with NASH according to the FLIP algorithm had a clinical profile distinct from those with NAFL, with a higher prevalence of metabolic risk factors (increased body mass index [BMI], central obesity, serum glucose, and glycated hemoglobin), more severe insulin resistance (fasting insulin and homeostasis model assessment for insulin resistance [HOMA-IR] values), and higher levels of aminotransferases. Similar findings were documented for patients with severe disease vs. those without. Positive linear trends existed between NASH or severe disease and increasing BMI and HOMA-IR. There was a strong association between liver fibrosis and NASH or SAF-defined scores of activity. Patients with either significant or bridging fibrosis overwhelmingly had NASH, and bridging fibrosis most often coexisted with severe activity. The FLIP algorithm/SAF score, although based on purely morphological grounds, are clinically relevant, as they identify patients with distinct clinical and biological profiles of disease severity. Disease activity in NAFLD is associated with fibrosis severity. The examination of liver tissue under the microscope (histology) serves to define the type and severity of non-alcoholic fatty liver disease morphologically, and is also used to determine improvement in therapeutic or natural history clinical trials. The FLIP algorithm/SAF classification is a new histological classification well validated on morphological but not clinical grounds. Here, we demonstrate that different disease categories defined by the FLIP/SAF classification correspond to entities of different clinical and biological severity. We also show a strong association between the activity of steatohepatitis (defined histologically) and the amount of fibrotic scar.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease

In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlklin fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

The Serum Oxidative/Anti-oxidative Stress Balance Becomes Dysregulated in Patients with Non-alcoholic Steatohepatitis Associated with Hepatocellular Carcinoma.

Objective Oxidative stress is associated with the progression of chronic liver disease. Non-alcoholic fatty liver disease (NAFLD) is also an oxidative stress-related disease. However, the oxidative/anti-oxidative balance has not been fully characterized in NAFLD. The objective of the present study was to investigate the balance between oxidative stress and the anti-oxidative activity in NAFLD, including non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC). Patients We recruited 69 patients with histologically proven NAFLD without HCC (NAFLD; n=58), and with NASH-related HCC (NASH-HCC; n=11). The 58 NAFLD patients included patients with non-alcoholic fatty liver (NAFL; n=14) and NASH (n=44). Methods The serum levels of reactive oxygen metabolites (ROM) and anti-oxidative markers (OXY) were determined and then used to calculate the oxidative index. The correlations among such factors as ROM, OXY, oxidative index, and clinical characteristics were investigated. Results In NAFLD, ROM positively correlated with the body mass index (BMI), hemoglobin A1c (HbA1c), C-reactive protein (CRP), and the histological grade or inflammatory scores, while only high HbA1c and CRP levels were significant factors that correlated with a higher ROM according to a multivariate analysis. OXY positively correlated with the platelet counts, albumin, and creatinine levels, while negatively correlating with age. However, it improved after treatment intervention. The oxidative index positively correlated with BMI, CRP, and HbA1c. The NASH-HCC patients exhibited a lower OXY than the NASH patients, probably due to the effects of aging. Conclusion Oxidative stress correlated with the levels of NASH activity markers, while the anti-oxidative function was preserved in younger patients as well as in patients with a well-preserved liver function. The NASH-HCC patients tended to be older and exhibited a diminished anti-oxidative function.

A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.

Nutrition and physical activity in NAFLD: an overview of the epidemiological evidence.

Nonalcoholic fatty liver disease (NAFLD) has been recognized as a major health burden. The high prevalence of NAFLD is probably due to the contemporary epidemics of obesity, unhealthy dietary pattern, and sedentary lifestyle. The efficacy and safety profile of pharmacotherapy in the treatment of NAFLD remains uncertain and obesity is strongly associated with hepatic steatosis; therefore, the first line of treatment is lifestyle modification. The usual management of NAFLD includes gradual weight reduction and increased physical activity (PA) leading to an improvement in serum liver enzymes, reduced hepatic fatty infiltration, and, in some cases, a reduced degree of hepatic inflammation and fibrosis. Nutrition has been demonstrated to be associated with NAFLD and Non-alcoholic steatohepatitis (NASH) in both animals and humans, and thus serves as a major route of prevention and treatment. However, most human studies are observational and retrospective, allowing limited inference about causal associations. Large prospective studies and clinical trials are now needed to establish a causal relationship. Based on available data, patients should optimally achieve a 5%-10% weight reduction. Setting realistic goals is essential for long-term successful lifestyle modification and more effort must be devoted to informing NAFLD patients of the health benefits of even a modest weight reduction. Furthermore, all NAFLD patients, whether obese or of normal weight, should be informed that a healthy diet has benefits beyond weight reduction. They should be advised to reduce saturated/trans fat and increase polyunsaturated fat, with special emphasize on omega-3 fatty acids. They should reduce added sugar to its minimum, try to avoid soft drinks containing sugar, including fruit juices that contain a lot of fructose, and increase their fiber intake. For the heavy meat eaters, especially those of red and processed meats, less meat and increased fish intake should be recommended. Minimizing fast food intake will also help maintain a healthy diet. PA should be integrated into behavioral therapy in NAFLD, as even small gains in PA and fitness may have significant health benefits. Potentially therapeutic dietary supplements are vitamin E and vitamin D, but both warrant further research. Unbalanced nutrition is not only strongly associated with NAFLD, but is also a risk factor that a large portion of the population is exposed to. Therefore, it is important to identify dietary patterns that will serve as modifiable risk factors for the prevention of NAFLD and its complications.

Serum Dipeptidyl Peptidase-4 Activity in Insulin Resistant Patients with Non-Alcoholic Fatty Liver Disease: A Novel Liver Disease Biomarker

Background In a cross-sectional study we studied the fasting serum DPP-4 enzymatic activity (sDPP-4) and the insulin resistance index (HOMA2-IR) in gliptin naïve patients with type 2 diabetes and in non-alcoholic fatty liver disease (NAFLD) and in healthy controls (CNTRL).Methods and Findings sDPP-4 was measured by kinetic assay in 39 NAFLD (F/M:19/20, mean age: 47.42 yrs) and 82 type 2 diabetes (F/M:48/34, 62.8 yrs) patients and 26 (F/M:14/12, 35.3 yrs) controls. Definition of T2D group as patients with type 2 diabetes but without clinically obvious liver disease created non-overlapping study groups. Diagnosis of NAFLD was based on ultrasonography and the exclusion of other etiololgy. Patients in T2D and NAFLD groups were similarly obese. 75 g CH OGTT in 39 NAFLD patients: 24-NGT, 4-IGT or IFG (“prediabetes”), 11-type 2 diabetes. HOMA2-IR: CNTRL: 1.44; T2D-group: 2.62 (p = 0.046 vs CNTRL, parametric tests); NAFLD(NGTonly): 3.23 (p = 0.0013 vs CNTRL); NAFLD(IFG/IGT/type 2 diabetes): 3.82 (p<0.001 vs CNTRL, p = 0.049 vs 2TD group). sDPP-4 activity was higher in NAFLD both with NGT (mean:33.08U/L) and abnormal glucose metabolism (30.38U/L) than in CNTRL (25.89U/L, p<0.001 and p = 0.013) or in T2D groups (23.97U/L, p<0.001 and p = 0.004). Correlations in NAFLD among sDPP-4 and ALT: r = 0.4637,p = 0.0038 and γGT: r = 0.4991,p = 0.0017 and HOMA2-IR: r = 0.5295,p = 0.0026 and among HOMA2-IR and ALT: r = 0.4340,p = 0.0147 and γGT: r = 0.4128,p = 0.0210.Conclusions The fasting serum DPP-4 activity was not increased in T2D provided that patients with liver disease were intentionally excluded. The high serum DPP-4 activities in NAFLD were correlated with liver tests but not with the fasting plasma glucose or HbA1C supporting that the excess is of hepatic origin and it might contribute to the speedup of metabolic deterioration. The correlation among γGT, ALT and serum DPP-4 activity and also between serum DPP-4 activity and HOMA2-IR in NAFLD strongly suggests that serum DPP-4 activity should be considered as a novel liver disease biomarker.

The association between increased alanine aminotransferase activity and metabolic factors in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has been associated with metabolic disorders, including central obesity, dyslipidema, hypertension, and hyperglycemia. Metabolic syndrome, obesity, and insulin resistance are major risk factors in the pathogenesis of NAFLD. The aim of this study was to identify the relative contribution of the metabolic syndrome, obesity, and insulin resistance to alanine aminotransferase (ALT) activity in NAFLD. A total of 3091 subjects diagnosed with fatty liver by ultrasonography were enrolled. All components of metabolic syndrome criteria, anthropometric parameters, fasting insulin levels, high-sensitivity C-reactive protein (hs-CRP) as an inflammation marker, and ALT were measured in each subject. Homeostasis model assessment—insulin resistance (HOMA-IR) as a measure of insulin resistance and body mass index (BMI) as a measure of obesity were calculated. The prevalence of increased ALT levels (>40 IU/L) was 26.7%. Increased ALT activity was significantly associated with the following characteristics: male sex, young age, increased triglycerides, fasting glucose, fasting insulin, HOMA-IR, hs-CRP, waist circumference, BMI and diastolic blood pressure, and decreased high-density lipoprotein cholesterol (HDL-C). According to the increase in the number of metabolic syndrome components, BMI, HOMA-IR, and hs-CRP, the prevalence and odds ratio for having increased ALT activity were significantly increased. Central obesity, raised triglycerides, reduced HDL-C, and raised fasting glucose were strongly associated with increased ALT activity. In conclusion, a number of metabolic syndrome components, obesity, insulin resistance, and hs-CRP, are strong predictors of increased ALT activity in NAFLD. Central obesity, raised triglycerides, reduced HDL-C, and raised fasting glucose are metabolic syndrome components that contributed to increased ALT activity.