Dietary Restriction Suppresses Steatosis-Associated Hepatic Tumorigenesis in Hepatitis C Virus Core Gene Transgenic Mice.

BACKGROUND AND AIMS: Dietary restriction (DR) is a preventive strategy for obesity, metabolic syndrome, cardiovascular disease, and diabetes. Although an interconnection between obesity, metabolic syndrome, fatty liver, and hepatocellular carcinoma has been documented, the mechanism and impact of DR on steatosis-derived hepatocarcinogenesis are not fully understood. This study aimed to evaluate whether DR can prevent hepatic tumorigenesis. METHODS: Male hepatitis C virus core gene transgenic (HCVcpTg) mice that develop spontaneous age-dependent insulin resistance, hepatic steatosis, and ensuing liver tumor development without apparent hepatic fibrosis, were fed with either a control diet ad libitum (control group) or 70% of the same control diet (DR group) for 15 months, and liver phenotypes were investigated. RESULTS: DR significantly reduced the number and volume of liver tumors. DR attenuated hepatic oxidative and endoplasmic reticulum stress and markedly suppressed nuclear factor-κB, signal transducer and activator of transcription 3 (STAT3) and STAT5, and phosphorylation of extracellular signal-regulated kinase, leading to downregulation of several pro-oncogenic mediators, such as cyclin D1. Serum insulin and insulin-like growth factor 1 levels, as well as hepatic expression of insulin receptor substrate 1/2, phosphatidylinositol-3 kinase, and serine/threonine-protein kinase AKT, were downregulated by DR. A transcriptome analysis revealed that STAT3 signaling and lipogenesis were the most suppressed hepatocarcinogenic pathways affected by DR. Additionally, DR stimulated autophagy and p62/sequestosome 1 degradation, enhanced phosphorylation of AMP-activated protein kinase α, increased fibroblast growth factor 21 expression, and attenuated expression of senescence-associated secretory phenotypes. CONCLUSION: DR suppressed steatosis-associated hepatic tumorigenesis in HCVcpTg mice, mainly due to attenuation of pathways involved in inflammation, cellular stress, cell proliferation, insulin signaling, and senescence. These findings support the notion that persistent 30% reduction of daily food intake is beneficial for preventing steatosis-associated hepatocarcinogenesis caused by HCV core protein.

A saturated fatty acid-rich diet enhances hepatic lipogenesis and tumorigenesis in HCV core gene transgenic mice.

Previous studies suggested that high consumption of saturated fatty acid (SFA) is a risk factor for liver cancer. However, it remains unclear how dietary SFA affects liver tumorigenesis. This study aimed to investigate the impact of a SFA-rich diet on hepatic tumorigenesis using hepatitis C virus core gene transgenic (HCVcpTg) mice that spontaneously developed hepatic steatosis and tumors with aging. Male HCVcpTg mice were treated for 15 months with a purified control diet or SFA-rich diet prepared by replacing soybean oil in the control diet with hydrogenated coconut oil, and phenotypic changes were assessed. In this special diet, almost all dietary fatty acids were SFA. Long-term feeding of SFA-rich diet to HCVcpTg mice increased hepatic steatosis, liver dysfunction, and the prevalence of liver tumors, likely due to stimulation of de novo lipogenesis, activation of the pro-inflammatory and pro-oncogenic transcription factor nuclear factor-kappa B (NF-κB), enhanced c-Jun N-terminal kinase/activator protein 1 (JNK/AP-1) signaling and induction of the oncogenes cyclin D1 and p62/sequestosome 1. The SFA-rich diet did not affect liver fibrosis or autophagy. Collectively, long-term SFA-rich diet consumption promoted hepatic tumorigenesis mainly through activation of lipogenesis, NF-κB, and JNK/AP-1 signaling. We therefore propose that HCV-infected patients should avoid excessive intake of SFA-rich foods to prevent liver cancer.

A trans-fatty acid-rich diet promotes liver tumorigenesis in HCV core gene transgenic mice.

Excess consumption of trans-fatty acid (TFA), an unsaturated fatty acid containing trans double bonds, is a major risk factor for cardiovascular disease and metabolic syndrome. However, little is known about the link between TFA and hepatocellular carcinoma (HCC) despite it being a frequent form of cancer in humans. In this study, the impact of excessive dietary TFA on hepatic tumorigenesis was assessed using hepatitis C virus (HCV) core gene transgenic mice that spontaneously developed HCC. Male transgenic mice were treated for 5 months with either a control diet or an isocaloric TFA-rich diet that replaced the majority of soybean oil with shortening. The prevalence of liver tumors was significantly higher in TFA-rich diet-fed transgenic mice compared with control diet-fed transgenic mice. The TFA-rich diet significantly increased the expression of pro-inflammatory cytokines, as well as oxidative and endoplasmic reticulum stress, and activated nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (NRF2), leading to high p62/sequestosome 1 (SQSTM1) expression. Furthermore, the TFA diet activated extracellular signal-regulated kinase (ERK) and stimulated the Wnt/ß-catenin signaling pathway, synergistically upregulating cyclin D1 and c-Myc, driving cell proliferation. Excess TFA intake also promoted fibrogenesis and ductular reaction, presumably contributing to accelerated liver tumorigenesis. In conclusion, these results demonstrate that a TFA-rich diet promotes hepatic tumorigenesis, mainly due to persistent activation of NF-κB and NRF2-p62/SQSTM1 signaling, ERK and Wnt/ß-catenin pathways and fibrogenesis. Therefore, HCV-infected patients should avoid a TFA-rich diet to prevent liver tumor development.

Correction to: A high-cholesterol diet promotes steatohepatitis and liver tumorigenesis in HCV core gene transgenic mice.

In the original publication of the article.

A high-cholesterol diet promotes steatohepatitis and liver tumorigenesis in HCV core gene transgenic mice.

Previous epidemiological studies have suggested a link between high-cholesterol intake and liver disease progression, including hepatocellular carcinoma (HCC). However, the precise mechanism of hepatotoxicity and hepatocarcinogenesis caused by excessive cholesterol consumption remains unclear. We aimed to investigate the impact of dietary cholesterol using hepatitis C virus core gene transgenic (HCVcpTg) mice, which spontaneously developed HCC with age. Male HCVcpTg mice were treated for 15 months with either a control diet or an isocaloric diet containing 1.5% cholesterol, and liver phenotypes and tumor-associated signaling pathways were evaluated. The high-cholesterol diet-fed HCVcpTg mice exhibited a significantly higher incidence of liver tumors compared with the control diet mice (100% vs. 41%, P < 0.001). The diet induced steatohepatitis with pericellular fibrosis and evoked higher mRNA expression of pro-inflammatory and pro-fibrotic mediators along with enhanced hepatocyte proliferation and greater oxidative and endoplasmic reticulum stress in the liver. Moreover, long-term consumption of cholesterol-rich diet activated nuclear factor-kappa B (NF-κB) and p62/sequestosome 1 (Sqstm1)-nuclear factor erythroid 2 (NRF2) axis, enhanced fibrogenesis, and consequently accelerated hepatic tumorigenesis. In conclusion, these results demonstrate that a high-cholesterol diet facilitates liver tumorigenesis by inducing steatohepatitis, promoting hepatocyte division, and up-regulating cellular stress and pro-inflammatory NF-κB and detoxifying p62/Sqstm1-NRF2 signals. Therefore, high dietary cholesterol should be avoided in HCV-infected patients to prevent development of steatohepatitis, liver fibrosis, and HCC.

Impact of chronic kidney dysfunction on serum Sulfatides and its metabolic pathway in mice.

Serum sulfatides are critical glycosphingolipids that are present in lipoproteins and exert anticoagulant effects. A previous study reported decreased levels of serum sulfatides in hemodialysis patients and suggested an association with cardiovascular disease. However, the mechanism of changes in serum sulfatides in chronic kidney dysfunction has not been well investigated. The current study examined whether a chronic kidney disease (CKD) state could decrease serum sulfatide levels using 5/6 nephrectomy (5/6NCKD) mice, an established CKD murine model, and studied the mechanisms contributing to diminished sulfatides. 5/6NCKD mice and sham operation control mice were sacrificed at the 4th or 12th postoperative week (POW) for measurement of serum sulfatide levels. Hepatic sulfatide content, which is the origin of serum sulfatides, and the expression of sulfatide metabolic enzymes in liver tissue were assessed as well. The 5/6NCKD mice developed CKD and showed increased serum creatinine and indoxyl sulfate. The serum levels and hepatic amounts of sulfatides were significantly decreased in 5/6NCKD mice at both 4 and 12 POW, while the degradative enzymes of sulfatides arylsulfatase A and galactosylceramidase were significantly increased. In a Hepa1-6 murine liver cell line, indoxyl sulfate addition caused intracellular levels of sulfatides to decrease and degradative enzymes of sulfatides to increase in a manner comparable to the changes in 5/6NCKD mice liver tissue. In conclusion, chronic kidney dysfunction causes degradation of sulfatides in the liver to decrease serum sulfatide levels. One explanation of these results is that indoxyl sulfate, a uremic toxin, accelerates the degradation of sulfatides in liver tissue.

Growth arrest and DNA damage-inducible 45α protects against nonalcoholic steatohepatitis induced by methionine- and choline-deficient diet.

Growth arrest and DNA damage-inducible 45 α (Gadd45α) is a stress-inducible protein that plays an important role in cell survival/death and DNA repair, but its contribution to the development of nonalcoholic steatohepatitis (NASH) has not been investigated. C57BL/6 Gadd45a-null and wild-type (WT) mice were treated with a methionine and choline-deficient diet (MCD) for eight weeks and phenotypic changes examined. Gadd45a-null mice had more severe hepatic inflammation and fibrosis, higher levels of mRNAs encoding pro-inflammatory, pro-fibrotic, and pro-apoptotic proteins, and greater oxidative and endoplasmic reticulum (ER) stress compared with WT mice. Indeed, Gadd45a mRNA was induced in response to ER stress in primary hepatocytes. Lipidomic analysis of NASH livers demonstrated decreased triacylglycerol (TG) in MCD-treated Gadd45a-null mice, which was associated with increased mRNAs encoding phospholipase D (Pld1/2), phosphatidic acid phosphatase type 2A, and choline/ethanolamine phosphotransferase 1 (Cept1), involved in the phosphatidylcholine-phosphatidic acid-diacylglycerol cycle, and decreased mRNAs encoding fatty acid (FA)-binding protein 1 (Fabp1) and FA transport protein 5. Treatment of cultured primary hepatocytes with tumor necrosis factor α, transforming growth factor ß, and hydrogen peroxide led to the corresponding induction of Fabp1, Pld1/2, and Cept1 mRNAs. Collectively, Gadd45α plays protective roles against MCD-induced NASH likely due to attenuating cellular stress and ensuing inflammatory signaling. These results also suggest an interconnection between hepatocyte injury, inflammation and disrupted glycerophospholipid/FA metabolism that yields a possible mechanism for decreased TG accumulation with NASH progression (i.e., "burned-out" NASH).

Targeting nuclear receptors for the treatment of fatty liver disease.

Ligand-activated nuclear receptors, including peroxisome proliferator-activated receptor alpha (PPARα), pregnane X receptor, and constitutive androstane receptor, were first identified as key regulators of the responses against chemical toxicants. However, numerous studies using mouse disease models and human samples have revealed critical roles for these receptors and others, such as PPARß/δ, PPARγ, farnesoid X receptor (FXR), and liver X receptor (LXR), in maintaining nutrient/energy homeostasis in part through modulation of the gut-liver-adipose axis. Recently, disorders associated with disrupted nutrient/energy homeostasis, e.g., obesity, metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD), are increasing worldwide. Notably, in NAFLD, a progressive subtype exists, designated as non-alcoholic steatohepatitis (NASH) that is characterized by typical histological features resembling alcoholic steatohepatitis (ASH), and NASH/ASH are recognized as major causes of hepatitis virus-unrelated liver cirrhosis and hepatocellular carcinoma. Since hepatic steatosis is basically caused by an imbalance between fat/energy influx and utilization, abnormal signaling of these nuclear receptors contribute to the pathogenesis of fatty liver disease. Standard therapeutic interventions have not been fully established for fatty liver disease, but some new agents that activate or inhibit nuclear receptor signaling have shown promise as possible therapeutic targets. In this review, we summarize recent findings on the roles of nuclear receptors in fatty liver disease and discuss future perspectives to develop promising pharmacological strategies targeting nuclear receptors for NAFLD/NASH.

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

AIM: To assess whether surrogate biomarkers of endotoxemia were correlated with the histological features of nonalcoholic fatty liver disease (NAFLD). METHODS: One 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. RESULTS: Serum 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. CONCLUSION: LBP/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.

Age-dependent PPARα activation induces hepatic sulfatide accumulation in transgenic mice carrying the hepatitis C virus core gene.

Sulfatides, a type of glycosphingolipid, are associated with carcinogenesis. Peroxisome proliferator-activated receptor α (PPARα) is involved in the regulation of sulfatide metabolism as well as in cancer development. We previously reported that transgenic (Tg) mice expressing hepatitis C virus core protein (HCVcp) exhibited age-dependent PPARα activation and carcinogenesis in liver. However, the metabolism of sulfatides in hepatocellular carcinoma is unknown. To examine the relationship between sulfatide metabolism, carcinogenesis, HCVcp, and PPARα, age-dependent changes of these factors were examined in HCVcpTg, PPARα inhibitor-treated HCVcpTg, and non-Tg mice. The sulfatide content in liver, the hepatic expression of two key enzymes catalyzing the initial and last reactions in sulfatide synthesis, the hepatic expression of known sulfatide-transferring protein, oxidative stress, and hepatic PPARα expression and its activation were age-dependently increased in HCVcpTg mice. The increased synthesis and accumulation of sulfatides and PPARα activation were significantly enhanced in liver cancer lesions. These changes were attenuated by PPARα inhibitor treatment and not observed in non-Tg mice. These results suggest that HCVcp-induced age-dependent PPARα activation increases synthesis of sulfatides and the resulting sulfatide accumulation affects HCV-related liver cancer. The monitoring of hepatic sulfatide content and the modulation of sulfatide generation by intervention using a PPARα inhibitor might be useful for the prediction and prevention of HCV-related hepatocarcinogenesis, respectively.

Mechanism of the development of nonalcoholic steatohepatitis after pancreaticoduodenectomy.

BACKGROUND AND AIM: It is recognized that nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), may develop after pancreaticoduodenectomy (PD). However, the mechanism of NASH development remains unclear. This study aimed to examine the changes in gene expression associated with NASH occurrence following PD. METHODS: The expression of genes related to fatty acid/triglyceride (FA/TG) metabolism and inflammatory signaling was examined using liver samples obtained from 7 post-PD NASH patients and compared with 6 healthy individuals and 32 conventional NASH patients. RESULTS: The livers of post-PD NASH patients demonstrated significant up-regulation of the genes encoding CD36, FA-binding proteins 1 and 4, acetyl-coenzyme A carboxylase α, diacylglycerol acyltransferase 2, and peroxisome proliferator-activated receptor (PPAR) γ compared with normal and conventional NASH livers. Although serum apolipoprotein B (ApoB) and TG were decreased in post-PD NASH patients, the mRNAs of ApoB and microsomal TG transfer protein were robustly increased, indicating impaired TG export from the liver as very-low-density lipoprotein (VLDL). Additionally, elevated mRNA levels of myeloid differentiation primary response 88 and superoxide dismutases in post-PD NASH livers suggested significant activation of innate immune response and augmentation of oxidative stress generation. CONCLUSIONS: Enhanced FA uptake into hepatocytes and lipogenesis, up-regulation of PPARγ, and disruption of VLDL excretion into the circulation are possible mechanisms of steatogenesis after PD. GENERAL SIGNIFICANCE: These results provide a basis for understanding the pathogenesis of NAFLD/NASH following PD.

Purification and identification of antihypertensive peptides from fermented buckwheat sprouts.

Buckwheat (Fagopyrum esculentum) is rich in antihypertensive compounds. This study investigated the effect of lactic-fermented buckwheat sprouts (neo-FBS) on level, identification, and potency of blood pressure-lowering (BPL) compounds. A single oral dose of 1.0 mg/kg body weight buckwheat sprouts (BS) in spontaneously hypertensive rats did not show significant BPL activity, whereas neo-FBS significantly decreased blood pressure. HPLC of neo-FBS identified two peaks absent in the profile of BS. The peak exhibiting potent BPL activity was fractionated, and six peptides (DVWY, FDART, FQ, VAE, VVG, and WTFR) and tyrosine were identified by LC-MS/MS and Edman degradation. Single oral dose administration of the peptides revealed significant BPL effect of all the peptides, with the most potent being DVWY, FQ, and VVG. DVWY, VAE, and WTFR are novel. This study demonstrates that lactic fermentation of BS produces new, highly potent antihypertensive peptides and increases active compounds GABA and tyrosine already present in BS.

Safety and effectiveness of low-dose propofol sedation during and after esophagogastroduodenoscopy in child A and B cirrhotic patients.

BACKGROUND: Effective and safe sedation for patients with liver cirrhosis is problematic. AIM: To examine the safety and effectiveness of low-dose propofol sedation during and after esophagogastroduodenoscopy (EGD) in cirrhotic patients. METHODS: Study 1 was a prospective study in cirrhotic patients who underwent diagnostic EGD under propofol sedation. Propofol was given by bolus injection with an age-adjusted standard protocol consisting of 40 mg for patients <70 years, 30 mg for patients aged 70-89 years; additional injections of 20 mg propofol were given up to a maximum of 120 mg. The principal parameter was the occurrence of adverse events within 24 h after EGD. Secondary parameters included successful procedures, complications, and full recovery within 60 min. In Study 2, the residual effects of propofol were evaluated using a driving simulator and blood propofol concentrations in a subset of cirrhotic patients undergoing EGD and compared with healthy individuals. The principal parameter was driving ability. RESULTS: Study 1: Consecutive cirrhotic patients were entered and all 163 successfully completed EGD. The mean dose of propofol was 46 mg (range 30-120 mg). No complications occurred. Full recovery had occurred in 100 % 60 min after the procedure. No adverse events occurred within 24 h after EGD. Study 2: There were no significant differences in blood propofol levels between cirrhotic patients (n = 21) and healthy individuals (n = 20) after sedation. In cirrhotic patients, there was no deterioration in driving ability as compared with healthy individuals. CONCLUSION: Low-dose propofol sedation provided safe and effective sedation for EGD in cirrhotic patients with rapid recovery.

Long-term improvement of oxidative stress via kidney transplantation ameliorates serum sulfatide levels.

BACKGROUND: Oxidative stress (OS) is a strong risk factor for cardiovascular disease (CVD). The incidence of CVD is lower among kidney transplantation (KT) recipients than hemodialysis patients, and the reduction in OS may be one reason for this difference. Recently, serum sulfatides were recognized as a candidate inhibitory factor of CVD affected by OS. However, the long-term changes in OS and serum sulfatide levels in KT recipients are unknown. METHODS: We investigated the long-term changes in a serum OS marker, malondialdehyde (MDA), and the serum sulfatide levels in 17 KT recipients. Multiple regression analysis was used to analyze the factors correlated with serum sulfatide levels. RESULTS: The high serum levels of MDA in the KT recipients decreased dramatically but were still high 1 year after KT surgery. MDA levels decreased further and reached near-normal levels more than 3 years after the surgery. Similarly, over the same 3 years, the low serum sulfatide levels increased to near-normal levels, reaching saturation. Multiple regression analysis showed that the most significant factors influencing serum sulfatide levels were MDA and total cholesterol content. CONCLUSIONS: The current results show that over the long term, the internal improvement brought about by successful KT can normalize OS. Oxidative normalization was significantly correlated with the restoration of serum sulfatide levels, which were also influenced by lipoprotein metabolism. The amelioration of serum sulfatide levels might contribute to the low incidence of CVD in KT recipients.

PPARα is down-regulated following liver transplantation in mice.

BACKGROUND & AIMS: Graft dysfunction is one of the major complications after liver transplantation, but its precise mechanism remains unclear. Since steatotic liver grafts are susceptible to post-transplant dysfunction, and peroxisome proliferator-activated receptor (PPAR) α plays an important role in the maintenance of hepatic lipid homeostasis, we examined the role of PPARα in liver transplantation. METHODS: Livers were harvested from Sv/129 wild-type (Ppara(+/+)) mice and PPARα-null (Ppara(-/-)) mice and transplanted orthotopically into syngeneic Ppara(+/+) mice. RESULTS: Hepatocellular damage was unexpectedly milder in transplanted Ppara(-/-) livers compared with Ppara(+/+) ones. This was likely due to decreased lipid peroxides in the Ppara(-/-) livers, as revealed by the lower levels of fatty acid oxidation (FAO) enzymes, which are major sources of reactive oxygen species. Hepatic PPARα and its target genes, such as FAO enzymes and pyruvate dehydrogenase kinase 4, were strongly down-regulated after transplantation, which was associated with increases in hepatic tumor necrosis factor-α expression and nuclear factor-κB activity. Inhibiting post-transplant PPARα down-regulation by clofibrate treatment markedly augmented oxidative stress and hepatocellular injury. CONCLUSIONS: Down-regulation of PPARα seemed to be an adaptive response to metabolic alterations following liver transplantation. These results provide novel information to the understanding of the pathogenesis of early post-transplant events.

Clinical characteristics of de novo nonalcoholic fatty liver disease following pancreaticoduodenectomy.

BACKGROUND: Hepatic steatosis may develop after pancreatic resection, but its clinicopathological features remain unclear. We explored the clinical characteristics of newly appearing nonalcoholic fatty liver disease (NAFLD) after pancreaticoduodenectomy (PD), designated as de novo NAFLD after PD. METHODS: Of 83 patients who underwent PD between 2001 and 2006, the patients with regular alcohol consumption after PD (n = 3), those who were unavailable for regular abdominal computed tomography follow-up (n = 12), and those who died within 6 months of PD (n = 8) were excluded from the study. In the remaining 60 patients, the prevalence and clinical features of de novo NAFLD after PD were examined. RESULTS: NAFLD developed after PD in 14 (23%) patients in our cohort. Liver biopsy was performed in 8 patients and all showed typical steatohepatitis. Compared with the patients who had conventional nonalcoholic steatohepatitis (NASH), patients with post-PD de novo NASH demonstrated significant decreases in body mass index and lower levels of serum albumin, cholesterol, apolipoprotein B, and homeostasis model assessment for insulin resistance. Multivariate logistic regression analysis revealed that pancreatic head cancer was associated with an increased risk of developing NAFLD after PD (odds ratio 12.0, 95% confidence interval 2.0-71.4, P = 0.006). Increased dosage of oral pancreatic enzymes significantly ameliorated the steatosis, as well as leading to the recovery of body weight loss and resolution of the biochemical abnormalities. CONCLUSIONS: De novo NAFLD/NASH after PD is characterized by non-obesity and lack of hyperlipidemia and insulin resistance and is associated with pancreatic exocrine insufficiency. In such patients, intensifying pancreatic enzyme supplementation may be useful.

Down-regulation of SREBP-1c is associated with the development of burned-out NASH.

BACKGROUND & AIMS: It is well-known that hepatic triglycerides (TG) diminish with the progression of non-alcoholic steatohepatitis (NASH), which has been designated as burned-out NASH, but its mechanism remains unclear. We aimed to explore the changes in hepatic fatty acid (FA) and TG metabolism with disease progression. METHODS: Hepatic expression of key genes in healthy individuals (n=6) and patients with simple steatosis (SS, n=10), mild NASH (fibrosis stage 1-2, n=20), and advanced NASH (fibrosis stage 3-4, n=20) were assessed by quantitative polymerase chain reaction. RESULTS: Hepatic expression of genes related to FA uptake and oxidation and very-low-density lipoprotein synthesis/export did not differ among the groups. However, the mRNA levels of sterol regulatory element-binding protein (SREBP)-1c and its downstream genes FA synthase, acetyl-coenzyme A carboxylase 1, and diacylglycerol acyltransferase 1 were inversely correlated with fibrosis stage. Immunoblot analysis revealed a remarkable reduction in mature SREBP-1c levels in advanced NASH. Furthermore, hepatic expression of tumor necrosis factor-alpha increased in accordance with fibrosis progression, which was possibly related to the decrease in hepatic SREBP-1c expression. CONCLUSIONS: Down-regulation of SREBP-1c and lipogenic enzymes may be associated with the development of burned-out NASH.

Nonalcoholic fatty liver disease in Japanese junior high school students: its prevalence and relationship to lifestyle habits.

BACKGROUND: Despite the increase in nonalcoholic fatty liver disease (NAFLD) in Japanese adults, its prevalence in adolescents remains unclear. This prompted us to evaluate the incidence and clinical characteristics of NAFLD among junior high school students. METHODS: A population-based cross-sectional study was conducted among students in a single junior high school in Nagano prefecture. Serum alanine aminotransferase (ALT) and gamma-glutamyltransferase (gammaGT) measurements and abdominal ultrasonography were performed in 249 and 288 students in 2004 and 2007, respectively. In the latter survey, student lifestyle habits were also assessed, using questionnaires. RESULTS: The prevalence of NAFLD was 4.4% and 4.5% in 2004 and 2007, respectively, which was lower than that of obesity (10.0% and 5.9%). Body mass index and ALT and gammaGT levels increased significantly with hepatic steatosis severity. Multivariate logistic regression analysis demonstrated that the presence of obesity and an ALT level of 30 U/L or more were independent predictors of NAFLD (odds ratio 16.9, P<0.001 and odds ratio 16.6, P=0.001, respectively). The ratios of students commuting to and from school by car and not doing sports outside of school were higher in NAFLD students compared with non-NAFLD ones. Such tendencies were observed independently of the presence of obesity. Additionally, one obese student with severe steatosis and liver dysfunction was diagnosed as having nonalcoholic steatohepatitis (NASH). CONCLUSIONS: Approximately 4% of junior high school students had NAFLD that was primarily associated with obesity and reduced daily physical activity. Serum ALT measurement during school check-ups is recommended for the early detection of young adolescent NAFLD/NASH.

Serum fragmented cytokeratin 18 levels reflect the histologic activity score of nonalcoholic fatty liver disease more accurately than serum alanine aminotransferase levels.

BACKGROUND AND GOALS: Reliable noninvasive biomarkers to assess the histologic activity of nonalcoholic fatty liver disease (NAFLD) have not been established. As the frequency of Mallory bodies is known to be closely associated with the disease severity, we hypothesized that serum levels of Mallory body-related proteins were correlated with NAFLD histologic activity and evaluated this possibility. STUDY: Serum levels of total and fragmented cytokeratin (CK) 18, heat shock protein (Hsp) 70, Hsp90alpha, ubiquitin+1, and p38alpha at the time of liver biopsy were measured in 118 NAFLD patients and their association with histologic findings and NAFLD histologic activity score (NAS) was investigated. RESULTS: Serum levels of both forms of CK18 and Hsp90alpha were markedly higher in patients having nonalcoholic steatohepatitis (NASH) compared with non-NASH ones. Both forms of CK18 significantly correlated with degree of steatosis, lobular inflammation, and ballooning, and showed stronger positive correlations with NAS than serum aspartate and alanine aminotransferase (AST and ALT). Multiple regression analysis further revealed that fragmented CK18 and AST were effective predictors of NAS, with the former being the more definitive of the two (P<0.001 vs. 0.005). In 20 NAFLD patients who received a follow-up biopsy, changes in fragmented CK18 levels, but not AST or ALT levels, closely paralleled those in NAS. CONCLUSIONS: These results establish the usefulness of fragmented CK18 measurement for assessing and monitoring the histologic activity of NAFLD.

Efficacy and safety of addition of minor bloodletting (petit phlebotomy) in hepatitis C virus-infected patients receiving regular glycyrrhizin injections.

BACKGROUND: Hepatoprotective therapies that include regular glycyrrhizin injections (GIs) are beneficial for chronic hepatitis C patients, but are sometimes insufficient for normalizing serum alanine aminotransferase (ALT) levels. Here, we evaluated whether the addition of minor bloodletting, named petit phlebotomy (PP), prior to each GI could further reduce serum ALT concentrations in such patients. METHODS: Seventy-six hepatitis C virus (HCV)-infected patients receiving regular GI, with persistently abnormal serum ALT levels, were randomly divided into GI + PP and GI groups and monitored for 12 months. PP was performed before every GI to a total 60 ml of blood a week. The primary PP endpoint was a serum ferritin level of less than 20 ng/ml. PP was suspended upon reaching the endpoint, but was resumed as needed. The efficacy of the addition of PP was evaluated by measuring changes in serum ALT levels. RESULTS: Two patients in each group dropped out because of the appearance of hepatocellular carcinoma. The remainder completed the 12-month treatment with no serious adverse events. Serum ALT and ferritin levels were significantly decreased in the GI + PP group (from 67 +/- 34 to 44 +/- 14 U/l and from 163 +/- 127 to 25 +/- 21 ng/ml, respectively, both P < 0.001), but these changes were not seen in the GI group. Although 20 patients in the GI + PP group had compensated cirrhosis, no significant reductions in serum albumin concentrations were observed. CONCLUSIONS: The addition of PP is effective and safe for improving serum aminotransferase levels in HCV-infected patients receiving regular GI, even in those with compensated cirrhosis.