Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis.

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.

Metabolic control analysis of hepatic glycogen synthesis in vivo.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Characteristics of patients aged over 75 years with hepatitis C virus infection treated with direct-acting antivirals in Japan: Evidence based on the nationwide, real-world database in Japan.

AIM: Direct-acting antivirals (DAAs) have dramatically changed the treatment of chronic hepatitis C. Their high efficacy helps in eradicating hepatitis C virus with few adverse events. Information on real-world use of DAAs therapy in patients aged 75 years and older is inadequate. METHODS: The Japanese DAAs database was constructed in 2014 as a cooperative system between 18 prefectures. The medical reports filled in by doctors and anonymized at the local government office were collected. The patients' demographic features, viral factors, and treatment characteristics were compared among three groups stratified by age when therapy was initiated: Group A (<60 years old), Group B (60-74 years old), and Group C (≥75 years old). RESULTS: Out of the 22,454 patients whose age upon starting therapy could be identified, 24.8% (n = 5597) belonged to Group C, which was ten times the number in the Japanese Interferon Database. Female patients, advanced stages of liver fibrosis, and past history of hepatocellular carcinoma treatment were significantly higher in the older age groups (Group A < B < C), whereas sustained virologic response (SVR) rates were not different (91%-93%). In Group C, multivariate logistic regression analysis revealed that predicting factors for virologic response varied among DAAs regimens. However, the completion of DAAs therapy commonly contributed to SVR, regardless of DAAs regimen. CONCLUSIONS: DAAs therapy is associated with high SVR rates, even in the oldest age group, and therapy should not be withheld on the basis of old age.

Antidiabetic Therapy in the Treatment of Nonalcoholic Steatohepatitis.

Liver-related diseases are the third-leading causes (9.3%) of mortality in type 2 diabetes (T2D) in Japan. T2D is closely associated with nonalcoholic fatty liver disease (NAFLD), which is the most prevalent chronic liver disease worldwide. Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD, can lead to hepatocellular carcinoma (HCC) and hepatic failure. No pharmacotherapies are established for NASH patients with T2D. Though vitamin E is established as a first-line agent for NASH without T2D, its efficacy for NASH with T2D recently failed to be proven. The effects of pioglitazone on NASH histology with T2D have extensively been established, but several concerns exist, such as body weight gain, fluid retention, cancer incidence, and bone fracture. Glucagon-like peptide 1 (GLP-1) receptor agonists and sodium-glucose cotransporter 2 (SGLT2) inhibitors are expected to ameliorate NASH and NAFLD (LEAN study, LEAD trial, and E-LIFT study). Among a variety of SGLT2 inhibitors, dapagliflozin has already entered the phase 3 trial (DEAN study). A key clinical need is to determine the kinds of antidiabetic drugs that are the most appropriate for the treatment of NASH to prevent the progression of hepatic fibrosis, resulting in HCC or liver-related mortality without increasing the risk of cardiovascular or renal events. Combination therapies, such as glucagon receptor agonist/GLP-1 or gastrointestinal peptide/GLP-1, are under development. This review focused on antidiabetic agents and future perspectives on the view of the treatment of NAFLD with T2D.

Clinical features of hepatocellular carcinoma in nonalcoholic fatty liver disease patients without advanced fibrosis.

BACKGROUND AND AIM: The prevalence of hepatocellular carcinoma (HCC) associated with nonalcoholic fatty liver disease (NAFLD-HCC) is increasing. Unfortunately, NAFLD frequently develops into HCC without liver cirrhosis. Therefore, we investigated the clinical features of HCC in NAFLD patients without advanced fibrosis. METHODS: We compared clinical characteristics, survival rates, and recurrence rates between 104 NAFLD-HCC patients diagnosed between January 2000 and December 2016, including 35 without (F0-2) and 69 with advanced fibrosis (F3-F4). Risk factors associated with survival and recurrence were evaluated. RESULTS: In total, 66.3% of those diagnosed had advanced fibrosis, 58.8% in men and 80.5% in women (men vs women, P = 0.03). In NAFLD-HCC without advanced fibrosis, tumor size was significantly larger and liver histological activity was lower than those in patients with advanced fibrosis. Survival rates between the two groups did not differ. Among those achieving curative treatment, the recurrence rate was significantly lower in NAFLD-HCC without advanced fibrosis (P < 0.01). Risk factors of recurrence were male gender, lower serum albumin, and advanced fibrosis. CONCLUSIONS: In men, HCC tended to develop from NAFLD without advanced fibrosis. Although tumor size in NAFLD-HCC without advanced fibrosis is significantly larger, the recurrence rate is significantly lower. Surgical therapy should be strongly considered in these cases.

Genome-wide DNA methylation analysis during non-alcoholic steatohepatitis-related multistage hepatocarcinogenesis: comparison with hepatitis virus-related carcinogenesis.

The aim of this study was to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Single-CpG-resolution genome-wide DNA methylation analysis was performed on 264 liver tissue samples using the Illumina Infinium HumanMethylation450 BeadChip. After Bonferroni correction, 3331 probes showed significant DNA methylation alterations in 113 samples of non-cancerous liver tissue showing NASH (NASH-N) as compared with 55 samples of normal liver tissue (NLT). Principal component analysis using the 3331 probes revealed distinct DNA methylation profiles of NASH-N samples that were different from those of NLT samples and 37 samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection (viral-N). Receiver operating characteristic curve analysis identified 194 probes that were able to discriminate NASH-N samples from viral-N samples with area under the curve values of more than 0.95. Jonckheere-Terptsra trend test revealed that DNA methylation alterations in NASH-N samples from patients without hepatocellular carcinoma (HCC) were inherited by or strengthened in NASH-N samples from patients with HCC, and then inherited by or further strengthened in 22 samples of NASH-related HCC (NASH-T) themselves. NASH- and NASH-related HCC-specific DNA methylation alterations, which were not evident in viral-N samples and 37 samples of HCC associated with HBV or HCV infection, were observed in tumor-related genes, such as WHSC1, and were frequently associated with mRNA expression abnormalities. These data suggested that NASH-specific DNA methylation alterations may participate in NASH-related multistage hepatocarcinogenesis.

Accuracy analysis of intrahepatic fat density measurements using dual-energy computed tomography: Validation using a test phantom.

BACKGROUND: Currently, no standardized method for measuring intrahepatic fat density via conventional computed tomography (CT) exists. OBJECTIVE: We aim to quantify intrahepatic fat density via material decomposition analysis using rapid kilovolt peak-switching dual-energy (RSDE) CT. METHODS: Homogenized porcine liver and fat (lard) were mixed in various ratios to produce phantoms for fat density verification. The actual fat density was measured on the basis of the phantom volume and weight, and these measurements were used as reference densities. The fat and liver mass attenuation coefficients, which were used as the material basis pairs, were employed in the material decomposition analysis. Then, the measured fat density of each phantom was compared with the reference densities. RESULTS: For fat content differences exceeding 2%, the measured fat density for the phantoms became statistically significant (p < 0.01). The correlation between the reference densities and RSDE-measured fat densities was reasonably high (R > 0.9997); this indicates the validity of this analysis method. CONCLUSIONS: Intrahepatic fat density can be measured using the mass attenuation coefficients of fat and liver in a material decomposition analysis. Given the knowledge of the accuracy and the limitations found in this study, our method can quantitatively evaluate fat density.

Hepatitis B virus vaccination-related seroprevalence among health-care personnel in a Japanese tertiary medical center.

AIM: Few studies concerning the protective management of hepatitis B virus (HBV) infection among health-care personnel (HCP), excluding occult HBV or carriers, have been reported. Therefore, we undertook a cross-sectional study of the updated status of HBV vaccine management by measuring the antibody to hepatitis B surface antigen (anti-HBs) along with the antibody to hepatitis B core antigen (anti-HBc). METHODS: Both anti-HBs and anti-HBc were assessed in 1085 HCP employed by our institute. Hepatitis B virus vaccination-related histories were recorded using self-administered questionnaires. RESULTS: Of 1085 HCP, 27 (2.5%) were positive for anti-HBc, and its positive rate increased with age. Of the 1058 subjects with negative anti-HBc, 879 (83.1%) were positive for anti-HBs. The median titer of anti-HBs was 71.1 mIU/mL, which was higher in female subjects (P = 0.037). By age group, the positive rate of anti-HBs were 77.5%, 89.3%, 90.8%, and 81.6% in the groups aged ≤29, 30-39, 40-49, and ≥50 years, respectively (P < 0.001). Of the 908 subjects who reported receiving HBV vaccination, 6 (0.7%) were positive for anti-HBc. Among them, one subject was suspected to have a possible subclinical HBV infection after the HBV vaccination. CONCLUSION: We report the current HBV vaccination-related seroprevalence of anti-HBs along with anti-HBc in a Japanese tertiary medical institution consisting of more than 1000 HCP, which was an level comparable to similar sized hospitals in developed countries. Anti-HBc would be important for understanding HBV status, but not necessary for general HBV vaccine management for HCP.

Pathophysiological analysis of primary biliary cirrhosis focusing on choline/phospholipid metabolism.

BACKGROUND & AIMS: Injury to biliary epithelial cells caused by disorders in bile composition may be the initial step in the pathogenesis of primary biliary cirrhosis (PBC). We therefore examined choline/phospholipid metabolism in livers of patients with PBC. METHODS: Hepatic levels of mRNA encoded by choline metabolism-related genes in early stage PBC patients were quantified by real-time RT-PCR. Serum cholesterol and triglyceride concentrations in each lipoprotein compartment and serum/tissue choline levels were also measured. OCT1 expression was quantified by genotype (rs683369 and rs622342). RESULTS: Serum choline concentrations were significantly higher in PBC patients than in normal individuals, with the concentrations in the former lowered by treatment with fibrates. Hepatic choline levels were markedly lower in PBC patients than in controls. The levels of expression of genes associated with choline uptake (OCT1 and CTL1), phosphatidylcholine synthesis (PEMT and BHMT), and phosphatidylcholine transport (MDR3) were significantly upregulated in PBC compared with control livers. Serum cholesterol concentrations and the cholesterol/triglyceride ratio in serum very low density lipoprotein were markedly higher in PBC patients than in controls. In PBC liver, OCT1 protein levels were lower in patients with minor (CG/GG at rs683369 and/or CC at rs622342) than major (CC at rs683369 and AA at rs622342) genotypes of the OCT1 gene. CONCLUSION: During early stage PBC, hepatocellular choline uptake and PC synthesis become dysregulated. OCT1 genotypes may influence the pathogenesis of PBC.

Deficiency of eNOS exacerbates early-stage NAFLD pathogenesis by changing the fat distribution.

BACKGROUND: Although many factors and molecules that are closely associated with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) have been reported, the role of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) in the pathogenesis of NAFLD/NASH remains unclear. We therefore investigated the role of eNOS-derived NO in NAFLD pathogenesis using systemic eNOS-knockout mice fed a high-fat diet. METHODS: eNOS-knockout and wild-type mice were fed a basal diet or a high-fat diet for 12 weeks. Lipid accumulation and inflammation were evaluated in the liver, and various factors that are closely associated with NAFLD/NASH and hepatic tissue blood flow were analyzed. RESULTS: Lipid accumulation and inflammation were more extensive in the liver and lipid accumulation was less extensive in the visceral fat tissue in eNOS-knockout mice, compared with wild-type mice, after 12 weeks of being fed a high-fat diet. While systemic insulin resistance was comparable between the eNOS-knockout and wild-type mice fed a high-fat diet, hepatic tissue blood flow was significantly suppressed in the eNOS-knockout mice, compared with the wild-type mice, in mice fed a high-fat diet. The microsomal triglyceride transfer protein activity was down-regulated in eNOS-knockout mice, compared with wild-type mice, in mice fed a high-fat diet. CONCLUSIONS: A deficiency of eNOS-derived NO may exacerbate the early-stage of NASH pathogenesis by changing the fat distribution in a mouse model via the regulation of hepatic tissue blood flow.

Deficiency of iNOS-derived NO accelerates lipid accumulation-independent liver fibrosis in non-alcoholic steatohepatitis mouse model.

BACKGROUND: Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. METHODS: iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. RESULTS: Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. CONCLUSIONS: iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation.

Abdominal visceral fat accumulation measured by computed tomography associated with an increased risk of gallstone disease.

BACKGROUND AND AIM: Visceral adiposity is a strong determinant of insulin resistance, which decreases cholecystokinin response sensitivity, and increases cholesterol saturation in the gallbladder bile; thus, it potentially relates to gallstone disease development. We aimed to investigate whether visceral fat measured by computed tomography (CT) is a risk factor for gallstone disease. METHODS: A cohort of 717 participants undergoing CT and ultrasonography was analyzed. The associations between body mass index (BMI), visceral adipose tissue (VAT) area, subcutaneous adipose tissue (SAT) area, and gallstone disease were analyzed adjusted for age, sex, hypertension, diabetes, and dyslipidemia. RESULTS: In multivariate analysis, gallstone disease was significantly associated with VAT and SAT areas for both categorical data and trend (P for trend < 0.001, 0.009), but not body mass index (BMI). Among patients with BMI < 25, gallstone disease remained significantly associated with VAT area (P for trend 0.021) and SAT area (P for trend 0.005). Interactions between the obesity indices and being elderly on the risk of gallstone disease were found; specifically BMI (P = 0.005), SAT (P < 0.001), and VAT (P = 0.154). A significant association between all obesity indices and gallstone disease was seen in patients aged < 65 but not among those aged ≥ 65. However, no significant association was noted between the obesity indices and sex. CONCLUSIONS: CT-measured adipose tissue, rather than BMI, was a better predictor for risk of gallstone disease. This finding applies to younger people or even those with normal body weight, suggesting the importance of abdominal visceral fat accumulation in the development of gallstone disease.

Novel findings for the development of drug therapy for various liver diseases: Liver microsomal triglyceride transfer protein activator may be a possible therapeutic agent in non-alcoholic steatohepatitis.

The factors involved in the progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH) are not fully understood and thus it is urgently needed to elucidate these factors. Steatosis is not causal in the development of NASH, but rather it sensitizes the liver to the damaging effects of second hits such that stressors innocuous to a healthy liver lead to the development of NASH in the steatotic liver. In the previous study, most of the hepatic lipid metabolite profiles were similar in the NAFL and NASH groups. However, very-low-density lipoprotein (VLDL) synthesis, especially hepatic microsomal triglyceride transfer protein (MTP) mRNA expression, was impaired in the NASH group. Moreover, NASH showed significantly higher incidence of minor alley appearance compared with NAFL, indicating the possibility of association between NASH pathogenesis and decreased congenital MTP activity. MTP is one of the enzymes that transfer triglycerides to nascent apolipoprotein B, producing VLDL and removing lipid from the hepatocyte. A growing body of literature suggests that the measurement of hepatic MTP expression may be helpful for diagnosis; and moreover, hepatic MTP activator may be a possible therapeutic agent for the treatment of NASH.

Serum nitric oxide metabolite as a biomarker of visceral fat accumulation: clinical significance of measurement for nitrate/nitrite.

BACKGROUND: A visceral fat area of more than 100 cm2 as measured by computed tomography (CT) at the umbilical level has been included as a criterion for obesity in all the proposed criteria for metabolic syndrome. However, CT cannot be used frequently because of radiation exposure. We evaluated the usefulness of measurement of the serum levels of nitric oxide (NO), instead of CT and the waist circumference, as a marker of abdominal visceral fat accumulation. MATERIAL/METHODS: The study was carried out in 80 subjects. The serum levels of NO metabolites (nitrate/nitrite) were measured using the Griess reagent. RESULTS: Simple and multiple regression analysis revealed that the serum levels of NO metabolites showed the greatest degree of correlation with the visceral fat area (r = 0.743, p<0.0001), and corresponded to a visceral fat area of 100 cm2, as determined using the ROC curve, was 21.0 µmol/ml (sensitivity 88%, specificity 82%); this method was more sensitive than the waist circumference for evaluation of the visceral fat accumulation. CONCLUSIONS: Measurement of the serum levels of NO metabolites may be a simple, safe, convenient and reliable method for the evaluation of visceral fat accumulation in clinical diagnostic screening.

FIB-4 First in the Diagnostic Algorithm of Metabolic-Dysfunction-Associated Fatty Liver Disease in the Era of the Global Metabodemic.

The prevalence of obesity or metabolic syndrome is increasing worldwide (globally metabodemic). Approximately 25% of the adult general population is suffering from nonalcoholic fatty liver disease (NAFLD), which has become a serious health problem. In 2020, global experts suggested that the nomenclature of NAFLD should be updated to metabolic-dysfunction-associated fatty liver disease (MAFLD). Hepatic fibrosis is the most significant determinant of all cause- and liver -related mortality in MAFLD. The non-invasive test (NIT) is urgently required to evaluate hepatic fibrosis in MAFLD. The fibrosis-4 (FIB-4) index is the first triaging tool for excluding advanced fibrosis because of its accuracy, simplicity, and cheapness, especially for general physicians or endocrinologists, although the FIB-4 index has several drawbacks. Accumulating evidence has suggested that vibration-controlled transient elastography (VCTE) and the enhanced liver fibrosis (ELF) test may become useful as the second step after triaging by the FIB-4 index. The leading cause of mortality in MAFLD is cardiovascular disease (CVD), extrahepatic malignancy, and liver-related diseases. MAFLD often complicates chronic kidney disease (CKD), resulting in increased simultaneous liver kidney transplantation. The FIB-4 index could be a predictor of not only liver-related mortality and incident hepatocellular carcinoma, but also prevalent and incident CKD, CVD, and extrahepatic malignancy. Although NITs as milestones for evaluating treatment efficacy have never been established, the FIB-4 index is expected to reflect histological hepatic fibrosis after treatment in several longitudinal studies. We here review the role of the FIB-4 index in the management of MAFLD.

Nonalcoholic fatty liver disease: US-based acoustic radiation force impulse elastography.

PURPOSE: To investigate the clinical usefulness of ultrasonography-based acoustic radiation force impulse (ARFI) elastography (ie, ARFI sonoelastography) in patients with a diagnosis of nonalcoholic fatty liver disease (NAFLD) and compare ARFI sonoelastography results with transient sonoelastography and serum fibrosis marker test results. MATERIALS AND METHODS: Written informed consent was obtained from all subjects, and the local ethics committee approved the study. Fifty-four patients with a liver biopsy-confirmed diagnosis of NAFLD (mean age, 50.6 years +/- 13.7) were examined. All patients with NAFLD and healthy volunteers underwent ARFI sonoelastography, transient sonoelastography, and serum liver fibrosis marker testing (hyaluronic acids, type IV collagen 7 S domain). Ten healthy volunteers underwent ARFI sonoelastography. ARFI sonoelastography results were compared with liver biopsy findings, the reference standard. ARFI sonoelastography findings were compared with liver biopsy, transient sonoelastography, and serum fibrosis marker test results. Student t testing was used for univariate comparisons; Kruskal-Wallis testing, for assessments involving more than two independent groups; and areas under the receiver operating characteristic curve (A(z)), to assess the sensitivity and specificity of ARFI sonoelastography for detection of stage 3 and stage 4 fibrosis. RESULTS: Median velocities in the patients with NAFLD were 1.040 m/sec for those with stage 0 fibrosis, 1.120 m/sec for those with stage 1, 1.130 m/sec for those with stage 2, 1.780 m/sec for those with stage 3, and 2.180 m/sec for those with stage 4. The A(z) for the diagnosis of hepatic fibrosis stages 3 or higher was 0.973 (optimal cutoff value, 1.77 m/sec; sensitivity, 100%; specificity, 91%), while that for the diagnosis of stage 4 fibrosis was 0.976 (optimal cutoff value, 1.90 m/sec; sensitivity, 100%; specificity, 96%). Significant correlations between median velocity measured by using ARFI sonoelastography and the following parameters were observed: liver stiffness measured with transient sonoelastography (r = 0.75, P < .0001), serum level of hyaluronic acid(r = 0.459, P = .0009), and serum level of type IV collagen 7 S domain (r = 0.445, P = .0015). CONCLUSION: There is a significant positive correlation between median velocity measured by using ARFI sonoelastography and severity of liver fibrosis in patients with NAFLD. The results of ARFI sonoelastography were similar to those of transient sonoelastography.

Metformin suppresses azoxymethane-induced colorectal aberrant crypt foci by activating AMP-activated protein kinase.

Metformin is widely used for the treatment of diabetes mellitus. Adenosine monophosphate-activated protein kinase (AMPK) is known to be activated by metformin and to inhibit the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway plays an important role in the protein translational machinery and cell proliferation. We examined the effect of metformin on the suppression of colorectal carcinogenesis in chemical carcinogen-induced models. Seven-wk-old BALB/c mice were intraperitoneally (i.p.) injected with azoxymethane (AOM, 10 mg/kg) and then treated with or without metformin (250 mg/kg/d) for 6 wk (for the investigation of aberrant crypt foci [ACF] formation) or 32 wk (for polyp formation). We next investigated colonic epithelial proliferation using bromodeoxyuridine (BrdU) and the proliferating cell nuclear antigen (PCNA) labeling indices. Furthermore, to examine the indirect effect of metformin, the insulin resistance status and the serum lipid levels were assessed. Treatment with metformin significantly reduced ACF formation. The effect of metformin on colon polyp inhibition was relatively modest. No significant difference in body weight or glucose concentration was observed. The BrdU and PCNA indices decreased in mice treated with metformin. A Western blot analysis revealed that the phosphorylated mTOR, S6 kinase, and S6 protein levels in the colonic mucosa decreased significantly in mice treated with metformin. In conclusion, metformin suppresses colonic epithelial proliferation via the inhibition of the mTOR pathway through the activation of AMPK. As metformin is already used daily as an antidiabetic drug, it might be a safe and promising candidate for the chemoprevention of colorectal cancer.

Dysfunctional very-low-density lipoprotein synthesis and release is a key factor in nonalcoholic steatohepatitis pathogenesis.

UNLABELLED: The specific mechanisms of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) pathogenesis remain unknown. In the present study we investigated the differences between NAFL and NASH in terms of liver lipid metabolites and serum lipoprotein. In all, 104 Japanese subjects (50 men and 54 postmenopausal women) with histologically verified NAFL disease (NAFLD) (51 with NAFL, 53 with NASH) were evaluated; all diagnoses were based on liver biopsy findings and the proposed diagnostic criteria. To investigate the differences between NAFL and NASH in humans, we carefully examined (1) lipid inflow in the liver, (2) lipid outflow from the liver, (3) very-low-density lipoprotein (VLDL) synthesis in the liver, (4) triglyceride (TG) metabolites in the liver, and (5) lipid changes and oxidative DNA damage. Most of the hepatic lipid metabolite profiles were similar in the NAFL and NASH groups. However, VLDL synthesis and lipid outflow from the liver were impaired, and surplus TGs might have been produced as a result of lipid oxidation and oxidative DNA damage in the NASH group. CONCLUSION: A growing body of literature suggests that a deterioration in fatty acid oxidation and VLDL secretion from the liver, caused by the impediment of VLDL synthesis, might induce serious lipid oxidation and DNA oxidative damage, impacting the degree of liver injury and thereby contributing to the progression of NASH. Therefore, dysfunctional VLDL synthesis and release may be a key factor in progression to NASH.

Nitric oxide plays a crucial role in the development/progression of nonalcoholic steatohepatitis in the choline-deficient, l-amino acid-defined diet-fed rat model.

BACKGROUND: The pathogenesis of nonalcoholic steatohepatitis (NASH) is still unclear. Recently, the 2-hit hypothesis was proposed, in which nitric oxide production, representing oxidative stress, was proposed as a very important candidate for the second hit. METHODS: The total study period was 10 weeks. A total of 20 rats were randomly divided into 2 groups. Group 1 was administered the Choline-Deficient, l-Amino Acid-Defined diet to produce a NASH model, and Group 2 as control received the Choline-Sufficient, l-Amino Acid-defined diet. The blood and tissue concentrations of nitrate + nitrite were measured using the Griess reagent and the expression levels of inducible nitric oxide synthase (iNOS) proteins and mRNA was determined by Western blotting. RESULTS: In regard to nitric oxide (NO) and NO metabolites, there were significant differences in the blood (especially portal venous blood) as well as tissue (liver and visceral fat) concentrations between the 2 animal groups; the amounts of NO metabolites in the tissues were much higher in the NASH models. The level of nitrotyrosine was much markedly higher in the NASH models than in the controls. In regard to the tissue expression of iNOS a significant difference between the 2 groups was found in the visceral fat, especially in the mesenterium. CONCLUSIONS: Based on these results, we hypothesize that the iNOS expression and NO levels in the visceral fat increase, with increased diffusion of NO and its metabolites into the liver, resulting in increased nitrotyrosine formation in the liver; this, in turn, induces inflammation, apoptosis, and fibrosis in the liver, which are one of the characteristic features of NASH.

Serum ferritin is a clinical biomarker in Japanese patients with nonalcoholic steatohepatitis (NASH) independent of HFE gene mutation.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver injury. The spectrum of NAFLD is broad, extending from simple steatosis through nonalcoholic steatohepatitis (NASH). Iron is regarded as a putative element that interacts with oxygen radicals, and high rates of hyperferritinemia and increased hepatic iron stores have been demonstrated in NASH. We investigated serum ferritin concentrations, HFE gene mutations, and insulin resistance in Japanese NASH patients and the diagnostic utility of serum ferritin concentrations as a means of distinguishing NASH. Serum ferritin concentrations were measured in 86 patients with histopathologically verified NAFLD (24 with steatosis and 62 with NASH) and 20 control subjects, they were tested for HFE gene mutations and their insulin resistance was measured. The serum ferritin concentration was significantly higher in the NASH patients than in the patients with simple steatosis (P = 0.006). There was no significant difference between the groups in HFE gene mutation (C282Y, H63D, and S65C), and the serum ferritin level was related with insulin resistance. The area under the ROC curve was 0.732 for distinguishing NASH from simple steatosis (P = 0.005; 95% CI, 0.596-0.856). In conclusion high serum ferritin concentrations are a distinguishing feature of Japanese NASH patients independent of HFE gene mutations.