Nonalcoholic Fatty Liver Patients Research Articles

Lipoprotein A, combined with alanine aminotransferase and aspartate aminotransferase, contributes to predicting the occurrence of NASH: a cross-sectional study

BackgroundNonalcoholic steatohepatitis (NASH) progresses from simple nonalcoholic fatty liver (NAFL) and has a poor prognosis. Abnormal lipid metabolism is closely related to the occurrence and development of nonalcoholic fatty liver disease (NAFLD). This study aimed to study the relationships between serum lipid metabolites and NASH, and to improve the early diagnosis of NASH.MethodsThis study included 86 NAFLD patients (23 NASH and 63 NAFL), and 81 unaffected individuals as controls from West China Hospital between October 2018 and May 2019. With lipid metabolites as the focus of the study, the differences in lipid metabolites were compared between the control group, NAFL patients, and NASH patients. Logistic regression analysis was used to examine the risk factors of NASH. Finally, receiver operating characteristic curve (ROC curve) was used to analyze the efficacy of the metabolites in NASH prediction.ResultsThe levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lipoprotein A (LPA) increased with the severity of NAFLD. In NAFLD patients, LPA (OR:1.61; 95%CI: 1.03–2.52) was a potential risk factor for NASH, and ROC analysis showed that the combination of LPA, ALT, and AST had a greater predictive efficiency for NASH.ConclusionsAbnormal apolipoprotein/lipoprotein is closely related to lipid metabolism disorder in patients with NAFLD. In NAFL, the combination of LPA, ALT, and AST contributes to predicting the occurrence of NASH. LPA may be a potential biomarker and therapeutic target for diagnosing and treating NASH.

Activation of Sphingosine Kinase 1/Sphingosine-1-Phosphate Pathway Protects Nonalcoholic Fatty Liver from Ischemia/Reperfusion Injury by Alleviating Oxidative Stress in Hepatocytes

Background: Nonalcoholic fatty liver (NAFL) is emerging as a leading risk factor of hepatic ischemia/reperfusion (I/R) injury lacking of effective therapy. Lipid dyshomeostasis has been implicated in the hepatopathy of NAFL. Herein, we investigate the bioactive lipids that critically regulate I/R injury in NAFL. Methods: Lipidomics were performed to identify dysregulated lipids in mouse and human NAFL with I/R injury. The alteration of corresponding lipid-metabolizing genes was examined. The effects of the dysregulated lipid metabolism on I/R injury in NAFL were evaluated in mice and human cultured hepatocytes. Findings: Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) were uncovered to be substantially activated by I/R in mouse NAFL. Sphingosine kinase 1 (Sphk1) was found to be essential for hepatic S1P generation in response to I/R in mouse NAFL. Sphk1 knockdown inhibited the hepatic S1P rise while accumulating ceramides, leading to aggressive hepatic I/R injury with increased oxidative stress in NAFL mice. In contrast, administration of exogenous S1P protected NAFL mice from hepatic I/R injury. Clinical study revealed a significant activation of SPHK1/S1P pathway by I/R in liver specimens of NAFL patients. In vitro studies on cultured human hepatocytes consolidated that inhibiting the SPHK1/S1P pathway exaggerated I/R-induced damage and oxidative stress in human hepatocytes of NAFL. Interpretations: Activation of SPHK1/S1P pathway is important for protecting NAFL from I/R injury, which may serve as therapeutic targets for hepatic I/R injury in NAFL. Funding Statement: This work was supported by Dean Foundation of Nanfang Hospital, Southern Medical University (2018C029) to JPQ, Guangdong Province Science and Technology Program (2014A020212174) to CJL, Guangdong Province Science and Technology Program (2017A030313684) to JZ, National Natural Science Foundation of China (81803063) to YYL, National Natural Science Foundation of China (81600462) and the Outstanding Youth Development Scheme of Nanfang Hospital, Southern Medical University (2016006) to KW. Declaration of Interests: The authors declare no competing of interest. Ethics Approval Statement: All experiments using human samples were carried out following the protocols approved by the Medical Ethics Committee of Nanfang Hospital Southern Medical University (NFEC-2019-029).

Contribution of endotoxin to Th17 bias in patients with non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a form of non-alcoholic fatty liver disease (NAFLD) with more severe inflammation-induced liver damage. Microbial products, such as endotoxin, may contribute to the pathogenesis of NASH. In this study, we investigated the effect of serum endotoxin on CD4 T cell inflammation. Age and sex-matched non-obese healthy subjects, subjects with non-alcoholic fatty liver (NAFL) but not steatohepatitis, and NASH patients were recruited for this study. The latter two groups were additionally matched in BMI and diabetes status. We first showed that compared to healthy subjects and NAFL patients, NASH patients presented significantly higher levels of serum endotoxin. Concurrently, NASH patients presented a Th17 bias that was associated with high endotoxin levels. To examine whether endotoxin could directly mediate IL-17 expression from CD4 T cells, naive CD4 T cells were stimulated with varying levels of endotoxin. In healthy subjects and NAFL patients, endotoxin did not act directly on naive CD4 T cells but required the presence of antigen-presenting cells to upregulate IL-17. Inhibition of TLR4 in macrophages, but not in CD4 T cells, could impair endotoxin-mediated IL-17 upregulation. In NASH patients, however, endotoxin at high levels directly, but minimally, increased IL-17 production. We further found that naive CD4 T cells from NASH patients presented significantly higher TLR4 than naive CD4 T cells from healthy subjects and NAFL patients, and CD3/CD28 stimulation could significantly elevate TLR4 expression by naive CD4 T cells. Overall, these data demonstrate that endotoxin promote Th17 bias in NASH patients.

Circulating Levels of Pro-inflammatory Cytokines in Patients with Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis.

Pro-inflammatory cytokines are associated with systemic inflammatory responses. To investigate the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) compared to healthy individuals. This case-control study was conducted on 30 patients with NAFL, 30 patients with NASH, and 30 healthy volunteers. The plasma level of IL-1β, IL-6, and TNF-α were determined by ELISA, and biochemical parameters were measured using colorimetric methods. IL-1β and IL-6 levels were significantly higher in patients with NASH compared with NAFL and control group. However, TNF-α levels had no significant variations in NAFL and NASH patients compared to the control group (p=0.903 and p=0.960, respectively). Results showed that the levels of ALT activity and pro-inflammatory cytokines were higher in patients with NASH compared to control and NAFL subjects; Therefore, steatosis and inflammation develop as a result of excessive pro-inflammatory factors in NASH.

Noninvasive diagnostic criteria for nonalcoholic steatohepatitis based on gene expression levels in peripheral blood mononuclear cells.

Nonalcoholic fatty liver disease (NAFLD) consists of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH); the latter progresses to liver cirrhosis and hepatocellular carcinoma. Discriminating NASH from NAFL typically involves liver biopsy. The mechanism of NASH progression is unclear but may involve immunological pathways. In this study, we examined expression levels of cytokine- and chemokine-encoding genes in peripheral blood mononuclear cells (PBMCs) from NAFLD patients and established immunological criteria for discriminating NASH from NAFL. PBMCs were obtained from 54 patients diagnosed histologically with NAFLD (NAFL, 18; NASH, 36). mRNA was extracted from PBMCs, and expression levels of cytokine- and chemokine-encoding genes were determined by quantitative real-time PCR. Statistical analysis was performed by nonparametric test. Expression levels of interferon (IFN)γ, interleukin (IL)2, IL15, C-C-motif chemokine ligand (CCL)2, IL10, and C-X-C-motif chemokine ligand (CXCL)11 were significantly upregulated in NASH patients compared with NAFL patients. Moreover, their expression levels were positively correlated with the degree of ballooning of hepatocytes but not of steatosis or lobular inflammation. We focused on those encoding IL10, IFNγ, and CCL2, and developed a scoring system to discriminate NASH from NAFL. The discriminatory power of the criteria was validated in an independent cohort. Expression levels of the cytokine- and chemokine-encoding genes in PBMCs were positively correlated with ballooning, suggesting their utility for the diagnosis of NASH. The data indicate that peripheral as well as intrahepatic immunity is involved in the progression of NASH. Our findings afford new insight into immunological mechanisms of NASH and will facilitate its noninvasive diagnosis.

Angiopoietin-Like Protein 8 Is a Novel Vitamin D Receptor Target Gene Involved in Nonalcoholic Fatty Liver Pathogenesis

Hepatic vitamin D receptor (VDR) expression is increased in patients with nonalcoholic fatty liver (NAFL) and is required for liver steatosis in an NAFL mouse model. However, how hepatocyte VDR is involved in setting up steatosis remains unclear. The authors transduced human hepatocyte-derived cells with an adenoviral vector encoding human VDR and found that angiopoietin-like protein 8 (ANGPTL8) expression was increased upon VDR activation by vitamin D or lithocholic acid. The mRNA levels of hepatic VDR- and vitamin D-related genes [cytochrome P450 (CYP) 2R1, CYP27A1, and CYP3A4] were higher in NAFL patients compared with normal liver subjects. Noteworthy, hepatic ANGPTL8 mRNA and protein levels were elevated in NAFL patients, and its mRNA correlated with VDR mRNA and with the steatosis grade. Moreover, increases in serum conjugated bile acids, including the VDR agonist glycine-lithocholic acid, were observed in NAFL patients. Additionally, free fatty acids and insulin were able to up-regulate both VDR and ANGPTL8 mRNA in human hepatocytes, whereas ANGPTL8 gene knockdown attenuated free fatty acids-induced triglyceride accumulation in these cells. In conclusion, activated VDR up-regulates ANGPTL8 expression, contributing to triglyceride accumulation in human hepatocytes. Moreover, hepatic ANGPTL8 mRNA positively correlates with VDR mRNA content and the grade of steatosis in NAFL patients, suggesting that this novel pathway may play a key role in the pathogenesis of hepatosteatosis.

Circulating Phospholipid Patterns in NAFLD Patients Associated with a Combination of Metabolic Risk Factors.

Background: Non-alcoholic fatty liver disease (NAFLD) is associated with inefficient macro- and micronutrient metabolism, and alteration of circulating phospholipid compositions defines the signature of NAFLD. This current study aimed to assess the pattern of serum phospholipids in the spectrum of NAFLD, and its related comorbidities and genetic modifications. Methods: 97 patients with diagnosed NAFLD were recruited at a single center during 2013–2016. Based on histological and transient elastography assessment, 69 patients were divided into non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL) subgroups. 28 patients served as healthy controls. Serum phospholipids were determined by liquid-chromatography mass spectrometry (LC-MS/MS). Results: The total content of phosphatidylcholine (PC) and sphingomyelin in the serum was significantly increased in NAFL and NASH patients, compared to healthy controls. In addition, serum lysophospatidylethanolamine levels were significantly decreased in NAFL and NASH individuals. Circulating PC species, containing linoleic and α-linolenic acids, were markedly increased in NAFLD patients with hypertension, compared to NAFLD patients without hypertension. The pattern of phospholipids did not differ between NAFLD patients with diabetes and those without diabetes. However, NAFLD patients with hyperglycemia (blood glucose level (BGL) >100 mg/dL) exhibited significantly a higher amount of monounsaturated phosphatidylethanolamine than those with low blood glucose levels. In addition, NAFLD patients with proven GG-genotype of PNPLA3, who were at higher risk for the development of progressive disease with fibrosis, showed lower levels of circulating plasmalogens, especially 16:0, compared to those with CC- and CG-allele. Conclusions: Our extended lipidomic study presents a unique metabolic profile of circulating phospholipids associated with the presence of metabolic risk factors or the genetic background of NAFLD patients.

Association of serum ferritin with non-alcoholic fatty liver disease: a meta-analysis

BackgroundA growing number of studies reported the connection between the level of serum ferritin (SFL) and non-alcoholic fatty liver disease (NAFLD). However, such connection was still disputable. The aim of our meta-analysis was to estimate SFL between the groups as below: patients with NAFLD against control group; non-alcoholic steatohepatitis (NASH) patients against control group; non-alcoholic fatty liver (NAFL) patients against a control group and NASH patients vs NAFL patients.MethodsWe screened the studies in PubMed, EMBASE, the Cochrane Database and the Cochrane Central register controlled trials from the beginning to July 10, 2016 to find the studies indicated the connection between SFL and NAFLD (NAFL and/or NASH). Fourteen published studies which evaluate the SFL in NAFLD patients were selected.ResultsHigher SFL was noticed in NAFLD patients against control group (standardized mean difference [SMD] 1.01; 95% CI 0.89, 1.13), NASH patients against control group (SMD 1.21; 95% CI 1.00, 1.42), NAFL patients against control group (SMD 0.51; 95% CI 0.24, 0.79) and NASH patients against NAFL patients (SMD 0.63; 95% CI 0.52, 0.75). These results remained unaltered actually after the elimination of studies which were focused on paediatric or adolescent populations. Higher SFL was presented in NAFLD patients against the control group (SMD 1.08; 95% CI 0.95, 1.20) in adults and NASH patients against NAFL patients in adults (SMD 0.74; 95% CI 0.62, 0.87). The connection between SFL and NASH against NAFL group in paediatric or adolescent populations was observed inconsistently (SMD 0.10; 95% CI -0.18, 0.38).ConclusionsThe level of SFL was elevated in patients with NAFLD (NAFL and/or NASH) compared with the controls. Compared with NAFL, The level of SFL was increased in NASH. The result remained unaltered actually after the elimination of studies focused on paediatric or adolescent populations.

Growth differentiation factor 15 predicts advanced fibrosis in biopsy-proven non-alcoholic fatty liver disease.

We explored whether growth differentiation factor 15 (GDF15) affects the histological severity of non-alcoholic fatty liver disease (NAFLD) independent of insulin resistance. In a biopsy-proven NAFLD cohort, we measured serum GDF15 levels using enzyme-linked immunosorbent assays. Among 190 subjects (mean age, 53±14years; men, 52.1%), 72 (men, 65.3%) and 78 (men, 44.9%) were diagnosed with biopsy-proven non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) respectively. GDF15 levels were significantly higher in NASH patients than in controls (P=.010) or NAFL patients (P=.001). Subjects with advanced fibrosis (≥F3) also showed higher GDF15 levels compared to the others (F0-2; P<.001). Among NAFLD patients, the highest quartile of GDF15 levels was significantly associated with a risk of advanced fibrosis even after adjustment for age, gender, body mass index, smoking status, hypertension, diabetes, aspartate aminotransferase, platelet, albumin, insulin resistance and low skeletal muscle mass (odds ratio, 4.27; 95% confidence interval, 1.04-17.63), but not with NASH risk. GDF15 levels showed a significant positive correlation with liver stiffness (Spearman's ρ, .525; P<.001). Palmitate treatment increased the GDF15 mRNA expression level significantly in Kupffer cells, but not in hepatocytes. In LX-2 cells, GDF15 treatment resulted in enhanced expression of α-smooth muscle actin and collagen I, as well as phosphorylation of SMAD2 and SMAD3. Our findings suggest that GDF15 may serve as a novel biomarker of advanced fibrosis in NAFLD, thereby indicating the need for urgent anti-fibrotic pharmacotherapy.

Serum thymosin beta4 as a noninvasive biomarker in patients with nonalcoholic steatohepatitis

The aim of the study was to determine whether serum thymosin beta4 (Tβ4) can be a useful noninvasive biomarker to differentiate between nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver (NAFL). The study included 24 NAFL patients and 21 NASH patients. The levels of Tβ4, 8-hydroxydeoxyguanosine acid (8-OhdG), liver function parameters, blood lipid, and glucose were detected in the venous blood of all patients. The NAFLD histological activity score (NAS) was examined in biopsy specimens from all patients. Statistical analysis was performed in order to find differences between the two abovementioned groups. In addition, receiver operator characteristic (ROC) analyses for alanine aminotransferase (ALT) and Tβ4 levels were performed in NAFL and NASH patients and the cut-off value was determined. Associations between the variables were tested using correlation coefficient calculations. Statistical significance was set at a p value of < 0.05. Serum Tβ4 content was 5.12 ± 1.87 mg/l in the NAFL group and 2.98 ± 1.35 mg/l in the NASH group (p < 0.001). Serum Tβ4 content and NAS, histological features of hepatic steatosis, lobular inflammation and ballooning, ALT, glucose and 8-OhdG levels were negatively correlated (p < 0.05 for all) in the NASH group. The correlation coefficient values were -0.530, -0.562, -0.574, -0.438, -0.446, -0.426 and -0.563, respectively. On the basis of ROC analysis, the best predictive Tβ4 cut-off value for detecting NASH was 3.94 mg/l (85.7% sensitivity and 79.2% specificity, which were higher than those of ALT). Serum Tβ4 level can be used as a biomarker for the diagnosis of NASH and was negatively correlated with the oxidation state of the liver.

Association between endotoxemia and histological features of nonalcoholic fatty liver disease.

AIMTo assess whether surrogate biomarkers of endotoxemia were correlated with the histological features of nonalcoholic fatty liver disease (NAFLD).METHODSOne hundred twenty-six NAFLD patients who had undergone percutaneous liver biopsy were enrolled. Serum lipopolysaccharide (LPS)-binding protein (LBP) and anti-endotoxin core immunoglobulin G (EndoCab IgG) antibody concentrations at the time of liver biopsy were measured using the enzyme-linked immunosorbent assays to examine for relationships between biomarker levels and histological scores.RESULTSSerum LBP concentration was significantly increased in nonalcoholic steatohepatitis (NASH) patients as compared with nonalcoholic fatty liver (NAFL) subjects and was correlated with steatosis (r = 0.38, P < 0.0001) and ballooning scores (r = 0.23, P = 0.01), but not with the severity of lobular inflammation or fibrosis. Multivariate linear regression analysis revealed that LBP was associated with steatosis score and circulating C-reactive protein, aspartate aminotransferase, and fibrinogen levels. Serum EndoCab IgG concentration was comparable between NASH and NAFL patients. No meaningful correlations were detected between EndoCab IgG and histological findings.CONCLUSIONLBP/EndoCab IgG were not correlated with lobular inflammation or fibrosis. More accurate LPS biomarkers are required to stringently assess the contribution of endotoxemia to conventional NASH.

Abstract 4421: Molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver

Abstract (Background) The incidence of hepatocellular carcinoma (HCC) arising from non-alcoholic fatty liver (NAFL) has been recently increasing according to increase of patients having metabolic syndrome. HCC, which shows steatosis, cellular ballooning, Mallory-Denk bodies, inflammation and fibrosis in cancer tissue, are recognized as steatohepatitic HCC (SH-HCC). This HCC phenotype is more frequently found in NAFL patients than in viral-hepatitic patients. (Purpose) The purpose of this study is to elucidate molecular features of SH-HCC with NAFL background. (Methods) To profile SH-HCC-related genes, mRNA expression patterns in SH-HCCs were compared with those in viral hepatitis HCCs by RNA array. RNA expression of the identified SH-HCC related gene was analyzed in three human liver carcinoma cell lines (HepG2, Huh7, and HLE) in normal conditioned medium. By mimicking the lipid-rich environment, these cell lines were also cultured in the presence of saturated (palmitic or stearic acid, recognized as endoplasmic reticulum stressors) or unsaturated fatty acids (oleic acid: OA). To address the role of SH-HCC-related gene, cell proliferation and apoptosis were assessed in the presence or absence of fatty acids, by knock-down of the objective gene using siRNA. (Results) (1) Caveolin (cav)-1 and -2 were identified as SH-HCC related genes (1.63 and 1.58 fold expression of cav-1 and -2, respectively) by RNA microarray analysis. (2) HLE and Huh7 showed high and low expression of both cav-1 and -2 mRNA, whereas HepG2 did not express either on qRT-PCR. (3) In the presence of OA, HepG2 and Huh7 did not well proliferate, whereas HLE cell proliferation increased with concentration of OA and culture duration. (4) Knock-down of cav-1 and -2 canceled OA-induced increase of cell proliferation. In the same condition, expression of cleaved caspase 3 was increased, suggesting the promotion of apoptosis. (Consideration) Cav-1 and -2, the important protein for endocytosis and cellular signaling, are highly expressed in SH-HCC. These proteins would increase cell proliferation and inhibit apoptosis in the presence of unsaturated fatty acids, presumably by using these fatty acids as energy source or by caveolae-related anti-apoptotic process. Citation Format: Makoto Takeda, Takanori Sakaguchi, Ryouta Kiuchi, Takanori Hiraide, Yasushi Shibasaki, Yoshihumi Morita, Hirotoshi Kikuchi, Mitsutoshi Setou, Hiroyuki Konno. Molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4421.

PNPLA3 as a Genetic Determinant of Risk for and Severity of Non-alcoholic Fatty Liver Disease Spectrum.

Background and Aims: Patatin-like phospholipase domain protein 3 (PNPLA3) polymorphisms (rs738409 C>G) are associated with non-alcoholic fatty liver disease (NAFLD). We performed a systematic review and meta-analysis to examine the association of PNPLA3 polymorphisms with the spectrum and severity of this disease. Methods: Studies evaluating the association between the PNPLA3 polymorphism spectrum (fatty liver, steatohepatitis, cirrhosis, and hepatocellular carcinoma) and NAFLD were included. Pooled data are reported as odds ratios (ORs) with 95% confidence intervals. Results: Of 393 potentially relevant studies, 35 on NAFLD were included in the analysis. Compared to healthy controls, the pooled ORs for rs738409 CG and GG compared to CC among patients with non-alcoholic fatty liver (NAFL) were 1.46 (1.16–1.85) and 2.76 (2.30–3.13), and were 1.75 (1.24–2.46) and 4.44 (2.92–6.76) among patients with non-alcoholic steatohepatitis respectively. The respective ORs for CG and GG compared to the CC genotype were 2.35 (0.90–6.13) and 5.05 (1.47–17.29) when comparing non-alcoholic hepatocellular carcinoma to NAFL patients. Among the NAFLD patients, the ORs for G allele frequency when comparing steatosis grade 2–3 to grade 0–1 NAFL, when comparing the NAFLD activity score of ≥ 4 to score ≤ 3, when comparing NASH to NAFLD, when comparing the presence of lobular inflammation to absence, and when comparing the presence of hepatocyte ballooning to absence were 2.33 (1.43–3.80), 1.80 (1.36–2.37), 1.66 (1.42–1.94), 1.58 (1.19–2.10), and 2.63 (1.87–3.69) respectively. Subgroup analysis based on ethnicity showed similar results. Conclusions: PNPLA3 polymorphisms have strong association with the risk for and severity of NAFLDs. PNPLA3 polymorphism plays an evolving role in diagnosis and treatment decisions in patients with NAFLD.

Usefulness of Beta-Cryptoxanthin for Nonalcoholic Fatty Liver Diseases

Usefulness of Beta-Cryptoxanthin for Nonalcoholic Fatty Liver Diseases We previously reported that in nonalcoholic steatohepatitis (NASH) patients, there was excess intake of carbohydrates and low intake of protein, polyunsaturated fatty acids and zinc. We also reported that a two-year modification of these diets was effective for improving anthropometric and biological parameters in NASH patients. There have been a few reports on NASH patients about the effectiveness of antioxidant carotenoids, especially betacryptoxanthin (b-crypt). In this study, we clarified intake and serum levels of b-crypt in NASH patients, and conducted a clinical trial to assess whether b-crypt inhibits the progression of NASH. Intake and serum levels of b-crypt were significantly lower in NASH patients than in nonalcoholic fatty liver (NAFL) patients and healthy controls. We randomly assigned NAFLD patients to receive either a trial beverage containing 3 mg of b-crypt or a placebo beverage for 12 weeks. In both NASH and NAFL patients, serum GGT levels were decreased with b-crypt treatment, as compared with placebo. Serum oxidative LDL and interleukin (IL)-6 levels were decreased and serum superoxide dismutase and IL-10 levels were increased in both NASH and NAFL patients who received b-crypt treatment. We also randomly assigned NAFLD patients to receive either a trial beverage containing 3 mg of b-crypt, 12 mg of Zn and 30 mg of a-tocopherol or a trial beverage containing only 3 mg of b-crypt. Both trial beverages had the same effect. These findings suggest that b-crypt treatment is effective in promoting anti-oxidant and antiinflammation activities in NAFLD patients.

Progression from Nonalcoholic Fatty Liver to Nonalcoholic Steatohepatitis Is Marked by a Higher Frequency of Th17 Cells in the Liver and an Increased Th17/Resting Regulatory T Cell Ratio in Peripheral Blood and in the Liver.

Nonalcoholic fatty liver disease is increasing in prevalence. It can be subdivided into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). Five to twenty percent of cases progress from NAFL to NASH. Increased hepatic Th17 cells and IL-17 expression were observed in NASH mice and patients, respectively. We analyzed CD4(+) effector T cells and regulatory T cells (Tregs) from peripheral blood and livers of NAFL and NASH patients. A total of 51 NAFL patients, 30 NASH patients, 31 nonalcoholic fatty liver disease patients (without histology), and 43 healthy controls were included. FACS analysis was performed on PBMCs and intrahepatic lymphocytes. Compared with healthy controls, a lower frequency of resting Tregs (rTregs; CD4(+)CD45RA(+)CD25(++)) and higher frequencies of IFN-γ(+) and/or IL-4(+) cells were detected among CD4(+) T cells of peripheral blood in NASH, and to a lesser degree in NAFL. In hepatic tissue, NAFL to NASH progression was marked by an increase in IL-17(+) cells among intrahepatic CD4(+) T cells. To define immunological parameters in peripheral blood to distinguish NAFL from NASH, we calculated different ratios. Th17/rTreg and Th2/rTreg ratios were significantly increased in NASH versus NAFL. The relevance of our findings for NASH pathogenesis was highlighted by the normalization of all of the changes 1 y after bariatric surgery. In conclusion, our data indicate that NAFL patients show changes in their immune cell profile compared with healthy controls. NAFL to NASH progression is marked by an increased frequency of IL-17(+) cells among intrahepatic CD4(+) T cells and higher Th17/rTreg and Th2/rTreg ratios in peripheral blood.

Lower levels of insulin-like growth factor-1 standard deviation score are associated with histological severity of non-alcoholic fatty liver disease.

Growth hormone (GH) deficiency may be associated with histological progression of non-alcoholic fatty liver disease (NAFLD) which includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Insulin-like growth factor 1 (IGF-1) is mainly produced by hepatocytes and its secretion is stimulated by GH. Our aim was to determine whether more histologically advanced NAFLD is associated with low circulating levels of IGF-1 in Japanese patients. Serum samples were obtained in 199 Japanese patients with biopsy-proven NAFLD and in 2911 sex- and age-matched healthy people undergoing health checkups. The serum levels of IGF-1 were measured using a commercially available immunoradiometric assay. The standard deviation scores (SDS) of IGF-1 according to age and sex were also calculated in NAFLD patients. The serum IGF-1 levels in NAFLD patients were significantly lower (median, 112 ng/mL) compared with the control population (median, 121 ng/mL, P < 0.0001). IGF-1 SDS less than -2.0 SD from median were found in 11.6% of 199 patients. NASH patients exhibited significantly lower levels of IGF-1 SDS (n = 130; median, -0.7) compared with NAFL patients (n = 69; median, -0.3; P = 0.026). The IGF-1 SDS values decreased significantly with increasing lobular inflammation (P < 0.001) and fibrosis (P < 0.001). In multiple regressions, the association between the IGF-1 SDS values and the severity of NAFLD persisted after adjusting for age, sex and insulin resistance. Low levels of circulating IGF-1 may have a role in the development of advanced NAFLD, independent of insulin resistance. Supplementation with GH/IGF-1 may be a candidate for the treatment of NASH.

Increased frequencies of activated effector CD4+ T cells in the peripheral blood and hepatic tissue of patients with NAFL and NASH

Background: The prevalence of non-alcoholic fatty liver disease (NAFLD) ranges between 20 – 35% in Europe. In 5 to 20% there is a progression from non-alcoholic fatty liver (NAFL) to a non-alcoholic steatohepatitis (NASH) but it is still not understood why some people develop NASH. The current hypothesis about the pathogenesis is a two hit theory. The “first hit” causes a steatosis and the “second hit” in form of bacterial translocation leads to an inflammation and the development of NASH. Hepatic Th17 cell infiltration was observed in a NASH mouse model and higher IL-17 and IL-21 gene expression in human liver of NASH patients. We hypothesized that the phenotype of peripheral CD4+ T cells might be predictive for the degree and quality of hepatic T cell infiltration and histopathology. Aims: Characterization of CD4+ regulatory T cells (Tregs) and conventional CD4+ T cells in peripheral blood and hepatic tissue in patients with NAFL and NASH. Methods: 50 patients with histology-proven NAFL or NASH and 44 healthy controls (HC) were included in this study. PBMCs of peripheral blood and hepatic tissue were characterized by multi-colour FACS analysis. CD4+ T cells were stimulated with PMA and ionomycin for intracellular detection of cytokine production (IL-17, IL-4, INF-g, IL-21). Results: Patients were older and had a higher BMI in comparison to HC. In patients with NAFL and NASH a lower frequency of resting Tregs (CD4+CD45RA+CD25++) was observed in peripheral blood. We found changes in effector CD4+ T cells with higher frequencies of IFN-g+, IL-21+ and IL4+ cells among CD4+ T cells of the peripheral blood of patients. In hepatic tissue, higher frequency of IL17+, IFN-g+, IL21+ and IL4+ cells were measured among CD4+ T cells than in the peripheral blood. CD4+ T cells in peripheral blood and hepatic tissue contained higher frequencies of HLA-DR+, i.e. activated cells. Conclusions: The peripheral blood and hepatic tissue of patients with NAFLD contained higher frequencies of activated effector cells among CD4+ T cells. NAFL patients show a “prehepatitic” immune cell profile very similar to that seen in NASH. Our data suggest that interfering with the effector CD4+ T cell response might prevent progression from NAFL to NASH.

Application of localized 31P MRS saturation transfer at 7 T for measurement of ATP metabolism in the liver: reproducibility and initial clinical application in patients with non-alcoholic fatty liver disease

Saturation transfer (ST) phosphorus MR spectroscopy ((31)P MRS) enables in vivo insight into energy metabolism and thus could identify liver conditions currently diagnosed only by biopsy. This study assesses the reproducibility of the localized (31)P MRS ST in liver at 7 T and tests its potential for noninvasive differentiation of non-alcoholic fatty liver (NAFL) and steatohepatitis (NASH). After the ethics committee approval, reproducibility of the localized (31)P MRS ST at 7 T and the biological variation of acquired hepato-metabolic parameters were assessed in healthy volunteers. Subsequently, 16 suspected NAFL/NASH patients underwent MRS measurements and diagnostic liver biopsy. The Pi-to-ATP exchange parameters were compared between the groups by a Mann-Whitney U test and related to the liver fat content estimated by a single-voxel proton ((1)H) MRS, measured at 3 T. The mean exchange rate constant (k) in healthy volunteers was 0.31 ± 0.03 s(-1) with a coefficient of variation of 9.0 %. Significantly lower exchange rates (p < 0.01) were found in NASH patients (k = 0.17 ± 0.04 s(-1)) when compared to healthy volunteers, and NAFL patients (k = 0.30 ± 0.05 s(-1)). Significant correlation was found between the k value and the liver fat content (r = 0.824, p < 0.01). Our data suggest that the (31)P MRS ST technique provides a tool for gaining insight into hepatic ATP metabolism and could contribute to the differentiation of NAFL and NASH. • 1D localized (31) P MRS saturation transfer in the liver is reproducible at 7 T • NASH patients have decreased hepatic Pi-to-ATP exchange rate • In this study, hepatic metabolic activity correlates with liver fat content.

The Efficacy and Safety of Traditional Chinese Medicine (Jiang Zhi Granule) for Nonalcoholic Fatty Liver: A Multicenter, Randomized, Placebo-Controlled Study

Objective. To evaluate the efficacy and safety of Jiang Zhi Granule (JZG), a Chinese herbal formula, in patients with nonalcoholic fatty liver (NAFL). Methods. A multicenter, randomized, double-blind, placebo-controlled, parallel clinical trial was conducted for 24 weeks in 224 patients with NAFL at 6 university-affiliated hospitals. Patients were randomized 1 : 1 to receive JZG and placebo, respectively. Primary outcome was the change of liver to spleen ratio (L/S ratio) over computed tomography (CT). Secondary outcomes included body mass index (BMI), serum triglyceride (TG), and total cholesterol (TC) levels. Results. Of all the 224 eligible patients, 221 patients were analyzed in the full analysis set (FAS), 205 in the per protocol set (PPS), and 3 patients were withdrawn prematurely. For FAS, JZG significantly increased L/S ratio from 0.74 ± 0.21 to 0.99 ± 0.24 compared to that from 0.79 ± 0.18 to 0.85 ± 0.27 in placebo group (P = 0.0011). For PPS, it showed an increase of 0.26 ± 0.23 of L/S ratio in the patients on JZG versus 0.07 ± 0.22 in those on placebo (P = 0.0003). Superiority of JZG over placebo was also observed with greater reduction in BMI (P < 0.05) in both FAS and PPS. No observable difference in decrease of serum TC and TG was recorded (P > 0.05). There were no serious adverse events (AEs) in the study process and safety indices were normal in both groups. Conclusions. The Chinese herbal formula JZG was found to be superior to placebo in increasing L/S ratio and reducing BMI in NAFL patients. It was also well tolerated in patients and might be a safe and effective medicine for NAFL.

Serum homocysteine levels in patients with nonalcoholic fatty liver disease

Background and rational for the study. Nonalcoholic fatty liver disease (NAFLD) is regarded as the hepatic component of insulin resistance (IR) syndrome, but data on serum homocysteine (HCY) are limited. The aim of the study was the evaluation of serum HCY levels in patients with NAFLD.Material and methods. Thirty-one patients (54 ± 11 years, 8 males) with biopsy-proven NAFLD, 15 with simple nonalcoholic fatty liver (NAFL) and 16 with nonalcoholic steatohepatitis (NASH), and 22 healthy controls (52 ± 9 years, 5 males) matched for gender, age and body mass index (BMI), were recruited. Blood samples for HCY, folate, vitamin B12, insulin and standard biochemical tests were obtained after overnight fasting. Homeostatic model of assessment-insulin resistance (HOMA-IR) was calculated.Results. There was no difference in mean serum HCY levels between controls and NAFLD patients (12.6 ± 4.6 vs. 13.5 ± 2.6 mmol/L, respectively; p = 0.432). Serum folate and vitamin B12 were also similar between the study groups. Mean age, BMI, serum folate and vitamin B12 did not differ between NAFL and NASH patients. However, when compared with NAFL patients, NASH patients had lower mean serum HCY levels (12.3 ± 2.5 vs. 14.7 ± 2.1 mmol/L; p = 0.006). HCY was lower by increasing the grading of fibrosis (p = 0.005), portal inflammation (p = 0.029) and steatosis location (p = 0.021). In logistic regression analysis, HCY independently predicted NASH (p = 0.045) after adjustment for gender, age, BMI, AST, glucose and HOMA-IR.Conclusion. Our data suggest that serum HCY levels are lower in NASH compared with NAFL patients and can independently predict NASH. Serum HCY might represent another non-invasive marker for the assessment of NAFLD.