Raman imaging of rat nonalcoholic fatty liver tissues reveals distinct biomolecular states.

An essential challenge in diagnosing states of nonalcoholic fatty liver disease (NAFLD) is the early prediction of progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) before the disease progresses. Histological diagnoses of NAFLD rely on the appearance of anomalous tissue morphologies, and it is difficult to segment the biomolecular environment of the tissue through a conventional histopathological approach. Here, we show that hyperspectral Raman imaging provides diagnostic information on NAFLD in rats, as spectral changes among disease states can be detected before histological characteristics emerge. Our results demonstrate that Raman imaging of NAFLD can be a useful tool for histopathologists, offering biomolecular distinctions among tissue states that cannot be observed through standard histopathological means.

IL-33-ILC2 axis promotes anti-tumor CD8+ T cell responses via OX40 signaling.

Group 2 innate lymphoid cells (ILC2s) are resident cells and participate in innate and adaptive immunity. In the tumor microenvironment (TME), ILC2s contribute to both tumorigenesis and inhibition of tumor growth, but the true role of ILC2s in TME construction remains unclear. We show that IL-33 treatment induces an anti-tumor effect in vivo in a mouse model of melanoma in which ILC2s and CD8+ T cells infiltrate into tumor tissue. This anti-tumor effect is dependent on CD8+ T cells, however, IL-33 does not act directly on CD8+ T cells because the cells lack ST2, the receptor for IL-33. ILC2s and CD8+ T cells in tumors of IL-33-treated mice express OX40 ligand (OX40L) and OX40, respectively, and in vivo blockade of OX40L-OX40 interaction canceled the anti-tumor effect of IL-33. Co-culture of CD8+ T cells expressing OX40 with IL-33-stimulated ILC2 expressing OX40L promoted cell activation and proliferation of CD8+ T cells, which was significantly suppressed by administration of anti-OX40L blocking antibody. Thus, the IL-33-ILC2 axis promotes CD8+ T cell responses via OX40/OX40L interaction and exerts an anti-tumor effect.

Label-free Assessment of the Nascent State of Rat Non-alcoholic Fatty Liver Disease Using Spontaneous Raman Microscopy.

Spontaneous Raman microscopy, which can detect molecular vibrations in cells and tissues, could be a useful tool for the label-free assessment of non-alcoholic fatty liver disease (NAFLD). However, it is unclear whether it can be used to evaluate the nascent state of NAFLD. To address this, we analyzed the Raman spectra of rat liver tissues in the nascent state of NAFLD upon excitation at 532 nm. Raman and histochemical analyses were performed of liver tissues from rats fed a high-fat, high-cholesterol diet (HFHCD). Raman microscopic imaging analysis of formalin-fixed thin tissue slices showed hepatic steatosis, as revealed by the Raman band at 2,854 cm-1, whereas lipid droplets were not detectable by hematoxylin-eosin staining of images until 3 days after feeding a HFHCD. Raman signals of retinol at 1,588 cm-1 emitted from hepatic stellate cells were distributed alongside hepatic cords; the retinol content rapidly decreased after feeding a HFHCD, whereas hepatic lipid content increased inversely. Raman microscopic analysis of the surface of fresh ex vivo livers enabled early detection of lipid accumulation after a 1-day feeding a HFHCD. In conclusion, spontaneous Raman microscopy can be applied to the label-free evaluation of the nascent state of NAFLD liver tissues.

Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.

OBJECTIVE: Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM-VCTE), Fibrosis-4 Index (FIB-4) and NAFLD (non-alcoholic fatty liver disease) Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies. DESIGN: Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individually and in sequential combinations. RESULTS: Data were included from 37 primary studies (n=5735; 45% women; median age: 54 years; median body mass index: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs (<1.3; ≥2.67) followed by LSM-VCTE cut-offs (<8.0; ≥10.0 kPa) to rule-in or rule-out advanced fibrosis had sensitivity and specificity (95% CI) of 66% (63-68) and 86% (84-87) with 33% needing a biopsy to establish a final diagnosis. FIB-4 cut-offs (<1.3; ≥3.48) followed by LSM cut-offs (<8.0; ≥20.0 kPa) to rule out advanced fibrosis or rule in cirrhosis had a sensitivity of 38% (37-39) and specificity of 90% (89-91) with 19% needing biopsy. CONCLUSION: Sequential combinations of markers with a lower cut-off to rule-out advanced fibrosis and a higher cut-off to rule-in cirrhosis can reduce the need for liver biopsies.

Early Tumor Shrinkage as a Predictive Factor for Outcomes in Hepatocellular Carcinoma Patients Treated with Lenvatinib: A Multicenter Analysis.

We investigated the association between early tumor shrinkage (ETS) and treatment outcome in patients with hepatocellular carcinoma treated with lenvatinib (LEN). A retrospective analysis was performed in 104 patients. ETS was defined as tumor shrinkage at the first evaluation in the sum of target lesions' longest diameters from baseline according to the Response Evaluation Criteria in Solid Tumors (RECIST). The median overall survival (OS) was not reached, whereas the median progression-free survival (PFS) was 5.0 months. The receiver operating characteristic curve analysis in differentiating long-term responders (PFS ≥ 5.0 months) from short-term responders (PFS < 5.0 months) revealed an ETS cut-off value of 10%. ETS ≥ 10% was significantly correlated with better PFS and OS compared with ETS < 10%. Additionally, ETS ≥ 10% showed a better discrimination ability on prognosis compared with modified RECIST-based objective response at the first evaluation. Multivariate analysis confirmed ETS ≥ 10% as an independent predictor of better OS, as well as a Child-Pugh score of 5 and macrovascular invasion. In conclusion, ETS ≥ 10% was strongly associated with outcome in patients treated with LEN. This biomarker could allow earlier assessment of the treatment response and guide treatment decision-making for HCC.

Impact of Relative Dose Intensity of Early-phase Lenvatinib Treatment on Therapeutic Response in Hepatocellular Carcinoma.

BACKGROUND: Factors associated with response to lenvatinib have not been clarified in patients with hepatocellular carcinoma (HCC). PATIENTS AND METHODS: This study retrospectively analyzed 50 patients treated with lenvatinib as first-line therapy between March 2018 and March 2019. Patients were divided into two groups by the Modified Response Evaluation Criteria in Solid Tumours (mRECIST) (responders and non-responders, whose best overall responses were complete (CR)/partial response (PR) and stable (SD)/progressive disease (PD), respectively). Factors associated with response were assessed, including the relative dose intensity 8 weeks after lenvatinib induction (8W-RDI). RESULTS: The best overall responses were 0/22/14/14 of CR/PR/SD/PD. Multivariate analysis revealed that only 8W-RDI was significantly associated with response. The receiver operating characteristic curve for 8W-RDI in differentiating responders from non-responders revealed a cut-off value of 75%. Patients with 8W-RDI ≥75% experienced a higher response rate and longer progression-free survival than patients with 8W-RDI <75%. CONCLUSION: Our results suggest that maintaining an RDI ≥75% during the initial 8 weeks of lenvatinib treatment has a favorable impact on response.

Raman spectroscopic histology using machine learning for nonalcoholic fatty liver disease.

Histopathology requires the expertise of specialists to diagnose morphological features of cells and tissues. Raman imaging can provide additional biochemical information to benefit histological disease diagnosis. Using a dietary model of nonalcoholic fatty liver disease in rats, we combine Raman imaging with machine learning and information theory to evaluate cellular-level information in liver tissue samples. After increasing signal-to-noise ratio in the Raman images through superpixel segmentation, we extract biochemically distinct regions within liver tissues, allowing for quantification of characteristic biochemical components such as vitamin A and lipids. Armed with microscopic information about the biochemical composition of the liver tissues, we group tissues having similar composition, providing a descriptor enabling inference of tissue states, contributing valuable information to histological inspection.

Insulin resistance increases the risk of incident type 2 diabetes mellitus in patients with non-alcoholic fatty liver disease.

AIM: Type 2 diabetes mellitus (T2DM) is a major complication of patients with non-alcoholic fatty liver disease (NAFLD). The aim of this retrospective study is to determine the risk factors for development of T2DM in patients with biopsy-proven NAFLD. METHODS: One hundred and sixty two consecutive patients with biopsy-proven NAFLD who received a 75-g oral glucose tolerance test were enrolled as the total cohort. Among them, we analyzed 89 patients without T2DM diagnosed by oral glucose tolerance test to estimate the cumulative rate for development of T2DM as the follow-up cohort. RESULTS: Of 162 patients, the glucose tolerance pattern were DM in 45 patients (27.8%), impaired glucose tolerance in 68 (42.0%), and normal glucose tolerance in 49 (30.2%). Patients with NAFL tended to be more likely to have normal glucose tolerance than those with non-alcoholic steatohepatitis (NASH). The serum levels of pre- and post-load insulin were significantly higher in the NASH group. Of 89 patients without T2DM, 13 patients newly developed T2DM during a follow-up period of 5.2 years. The cumulative rate of T2DM incidence was 8.8% at the end of the 5th year and 23.4% at the end of the 10th year. Multivariate analysis identified homeostasis model of assessment - insulin resistance (≥3.85, hazard ratio 40.1, P = 0.033) as an independent risk factor for development of T2DM. CONCLUSIONS: Patients with NASH have an underlying potential of glucose intolerance. In NAFLD patients, insulin resistance is the most important risk factor for the incidence of T2DM. Appropriate therapy against insulin resistance could be needed for patients with NAFLD to prevent development of T2DM.

Hepatic nucleotide binding oligomerization domain-like receptors pyrin domain-containing 3 inflammasomes are associated with the histologic severity of non-alcoholic fatty liver disease.

AIM: To examine the role of nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes in the development of non-alcoholic fatty liver disease (NAFLD). METHODS: Levels of mRNAs encoding NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, procaspase-1, interleukin (IL)-1ß, and IL-18 were quantified by real-time polymerase chain reaction in 91 liver samples and 37 blood samples from biopsy-proven patients with NAFLD. Adiponutrin (also called PNPLA3) polymorphisms (rs738409, C > G) were determined in 74 samples by genotyping assays. Serum IL-1ß and IL-18 levels were measured by enzyme-linked immunosorbent assay and liver tissue caspase-1 expression by immunostaining. RESULTS: Hepatic NLRP3, procaspase-1, IL-1ß, and IL-18 mRNA levels were significantly higher in NAFLD patients than in controls and were significantly associated with adiponutrin G alleles. Blood procaspase-1 mRNA was significantly higher in NAFLD patients than in healthy controls. Hepatic procaspase-1 and IL-1ß mRNA levels correlated significantly with lobular inflammation, hepatocyte ballooning, and NAFLD activity score. Serum IL-18 levels were significantly higher in NAFLD patients than in controls, while IL-1ß levels were non-significantly higher. Serum IL-1ß and IL-18 concentrations correlated significantly with steatosis, NAFLD activity score, and transaminase levels. Serum IL-1ß levels were significantly associated with adiponutrin G alleles. Scattered caspase-1-positive cells were present in portal tracts and inflammatory foci and around ballooning hepatocytes. Immunofluorescence staining showed that caspase-1 colocalized with the macrophage marker CD68. CONCLUSIONS: The NLRP3 inflammasomes are primed in the liver, influenced by adiponutrin genotypes, and activated in Kupffer cells and/or macrophages in NAFLD, leading to histological progression through IL-1ß and IL-18 production.

Liver stiffness measurement to platelet ratio index predicts the stage of liver fibrosis in non-alcoholic fatty liver disease.

AIM: Platelet count and liver stiffness measurement (LSM) using transient elastography could identify significant fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). We constructed a novel index combining LSM with platelet count for staging fibrosis in Japanese patients with NAFLD. METHODS: We recruited 173 Japanese patients with liver biopsy-proven NAFLD. The areas under the receiver operating characteristic curves were calculated and compared with established parameters and scoring systems for staging liver fibrosis. RESULTS: After excluding 10 patients in whom LSM failed, 163 patients with NAFLD were enrolled. The areas under the receiver operating characteristic curves of the LSM/platelet ratio (LPR) index for detecting fibrosis ≥stage 1, ≥stage 2, and ≥stage 3 were the greatest (0.835, 0.913, and 0.936, respectively) compared with those for various other parameters and established scoring systems, such as LSM, type IV collagen 7 s domain, platelet count, NAFIC score, fibrosis-4 index, NAFLD fibrosis score, aspartate aminotransferase/alanine aminotransferase ratio, and aspartate aminotransferase to platelet ratio index. The optimal cut-off, positive predictive, and negative predictive values of the LPR index for detecting ≥stage 3 fibrosis were 0.60, 48.9%, and 99.2%, whereas those of LSM were 10.0 kPa, 35.0%, and 99.0%, respectively. The novel LPR index helps avoid biopsies in a larger percentage of patients with NAFLD compared with that LSM alone. CONCLUSIONS: The LPR index was the best predictor for staging fibrosis in patients with NAFLD. It represents a simple and non-invasive alternative to liver biopsy in clinical practice.

Development of hepatocellular carcinoma in Japanese patients with biopsy-proven non-alcoholic fatty liver disease: Association between PNPLA3 genotype and hepatocarcinogenesis/fibrosis progression.

AIM: Some patients with non-alcoholic fatty liver disease (NAFLD) develop hepatocellular carcinoma (HCC). Patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 (encoding the I148M variant) has been associated with advanced fibrosis and HCC. We determined the risk factors for HCC, including the PNPLA3 rs738409 polymorphism, in Japanese patients with biopsy-proven NAFLD. METHODS: In this retrospective cohort study, we analyzed hepatocarcinogenesis in 238 patients. PNPLA3 rs738409 genotype was determined by allelic discrimination in 130 patients. Among them, 86 patients who were followed up for >5 years and without liver cirrhosis were analyzed to clarify the relationship between PNPLA3 genotype and long-term changes in biomarkers. RESULTS: Of 238 patients, PNPLA3 genotype frequencies were: CC, 0.14; CG, 0.46; and GG, 0.40. During a follow-up period of 6.1 years, 10 patients (4.2%) with non-alcoholic steatohepatitis developed HCC. The cumulative rate of HCC was 1.9% at the end of the 5th year and 8.3% at the end of the 10th year. Multivariate analysis identified PNPLA3 genotype GG (hazard ratio, 6.36; P = 0.019) and fibrosis stage (fibrosis stage 3/4; hazard ratio, 24.4; P = 0.011) as predictors of HCC development. In the long follow-up cohort, a larger reduction in platelet count was found in the GG group (P = 0.032) despite a larger reduction in alanine aminotransferase (P = 0.023) compared to that in the CC/CG group. CONCLUSIONS: In Japanese patients with NAFLD, severe fibrosis and PNPLA3 GG genotype were predictors of HCC development, independent of other known risk factors. Patients with the PNPLA3 GG genotype have the potential for a decreased platelet count, even when alanine aminotransferase levels are well controlled.

Effect of sodium glucose cotransporter 2 inhibitor on liver function tests in Japanese patients with non-alcoholic fatty liver disease and type 2 diabetes mellitus.

AIM: No pharmacological therapies have been established for non-alcoholic fatty liver disease (NAFLD). Sodium glucose cotransporter 2 inhibitor (SGLT2I) was developed for the treatment of adults with type 2 diabetes mellitus (T2DM). The aim of this retrospective study is to evaluate the efficacy of SGLT2I in NAFLD patients with T2DM. METHODS: Twenty-four biopsy-proven NAFLD patients with T2DM who received SGLT2I for 24 weeks were retrospectively enrolled as the SGLT2I group. Another 21 NAFLD patients with T2DM treated with dipeptidyl peptidase-4 inhibitor (DPP4I) for 24 weeks were selected as the DPP4I group. Clinical data were evaluated at baseline and at 4, 12, and 24 weeks. Seventeen patients in the SGLT2I group were evaluated by body composition before and after therapy. RESULTS: Not only body weight and hemoglobin A1c but also transaminase activities were significantly decreased in the SGLT2I group. Reductions in transaminase activities were similar between SGLT2I and DPP4I groups. In the SGLT2I group, body mass index and fasting plasma glucose also decreased after the treatment. CONCLUSION: Sodium glucose cotransporter 2 inhibitor can be a novel promising agent for the treatment for NAFLD patients with T2DM. Prospective randomized controlled trials are warranted to confirm this efficacy of SGLT2I on NAFLD with T2DM.

Effect of 12-week dulaglutide therapy in Japanese patients with biopsy-proven non-alcoholic fatty liver disease and type 2 diabetes mellitus.

AIMS: No pharmacological therapies have been established for non-alcoholic fatty liver disease (NAFLD) with type 2 diabetes mellitus (T2DM). The aim of this retrospective study is to evaluate the efficacy and safety of dulaglutide, a novel glucagon-like peptidase-1 receptor agonist, in Japanese NAFLD patients with T2DM. METHODS: Fifteen biopsy-proven NAFLD patients with T2DM refractory to diet intervention who received once weekly dulaglutide 0.75 mg for 12 weeks were retrospectively enrolled after exclusion of two patients by 12 weeks. In five patients, transient elastography and body composition were also evaluated before and after the treatment. RESULTS: Not only body weight and hemoglobin A1c but also transaminase activities were significantly decreased after the 12-week therapy with dulaglutide. Total body fat mass and liver stiffness measurement also decreased after the treatment. CONCLUSION: Dulaglutide, a new glucagon-like peptidase-1 receptor agonist, could be a novel promising agent for the treatment for NAFLD patients with T2DM due to its efficacy in body weight reduction, the nature of weekly injection, and patient preference. Prospective randomized controlled trials are warranted to confirm this impact of dulaglutide on NAFLD with T2DM.

Association of coronary artery calcification with liver fibrosis in Japanese patients with non-alcoholic fatty liver disease.

AIMS: Cardiovascular events are the leading cause of death among patients with non-alcoholic fatty liver disease (NAFLD), but their relationship remains unclear. This study examined the association between coronary atherosclerosis and liver fibrosis, represented by the coronary artery calcification (CAC) score and non-invasive fibrosis markers, respectively. METHODS: Among 698 patients with chest pain or electrocardiographic abnormalities who underwent coronary computed tomography (CT) between April 2006 and March 2010, those with known liver disorders or history of emergency coronary angioplasty were excluded, leaving 366 patients for this study. Diagnosis of NAFLD was based on abdominal CT and history of alcohol consumption. Subjects with CAC of 100 AU or more were categorized into the high-risk group for cardiovascular events. Patient records were examined for clinical parameters including CAC score and non-invasive fibrosis marker FIB-4 index. RESULTS: Ninety-four patients (25.7%) had NAFLD. In this group, univariate analysis identified old age, high diastolic blood pressure, high liver to spleen ratio and high FIB-4 index as risk factors for cardiovascular events and multivariate analysis identified age of 66 years or older and FIB-4 index of 2.09 or more as the significant risk factors. For the observation period until August 2014, the cumulative proportion of PCI performance was significantly higher in patients with FIB-4 of 2.09 or more than those with FIB-4 of less than 2.09. CONCLUSION: The progression of arteriosclerosis and that of liver fibrosis may be associated in NAFLD patients. The FIB-4 index can be easily determined and thus can be a useful marker for predicting cardiovascular events in NAFLD patients.

Drug-induced liver injury in a chronic hepatitis C patient treated by peginterferon, ribavirin and simeprevir.

A 56-year-old male patient with chronic hepatitis C was treated with pegylated interferon (PEG IFN)-α-2b and ribavirin (RBV) for 72 weeks in 2006. The patient achieved an early virological response (EVR); however, hepatitis C relapsed 12 weeks after discontinuation of PEG IFN and RBV. In 2012, the patient was treated with a PEG IFN/RBV/telaprevir combination therapy. After 5 days of treatment, he suffered from a telaprevir-associated skin rash on his body and four limbs. He chose to be treated with PEG IFN and RBV until 60 weeks. He again achieved EVR but no sustained virological response. In 2014, he was treated with PEG IFN/RBV/simeprevir combination therapy. He achieved rapid virological response, but after 6 weeks of therapy, a striking elevation of serum aminotransferase level was recorded with no accompanying skin rash; he was admitted to our hospital. PEG IFN/RBV/simeprevir was stopped, but sodium valproate (400 mg/day), which had been administrated for more than 10 years to prevent epilepsy was continued. Liver biopsy revealed typical features of drug-induced liver injury. After stopping PEG IFN/RBV/simeprevir, serum aminotransferase levels soon returned to the normal range. We diagnosed this case to be simeprevir-induced hepatitis clinically and histologically. Physicians need to stay alert to the possibility of drug-induced liver injury in using simeprevir.

Helicobacter pylori infection might have a potential role in hepatocyte ballooning in nonalcoholic fatty liver disease.

BACKGROUND: Clinical data regarding Helicobacter pylori (H. pylori) infection in nonalcoholic fatty liver disease (NAFLD) are limited. The aim was to evaluate H. pylori infection in patients with NAFLD and its association with disease severity. METHODS: One hundred and thirty patients with biopsy-proven NAFLD [43 with nonalcoholic fatty liver (NAFL) and 87 with nonalcoholic steatohepatitis (NASH)] were recruited for blood samples for anti-H. pylori immunoglobulin G (IgG) and standard biochemical tests were obtained after overnight fasting. Glucose tolerance was evaluated by 75-g oral glucose tolerance test. Liver biopsies were scored for NAFLD activity score (NAS), fibrosis and iron deposits. RESULTS: H. pylori IgG seropositivity was found in 40 % of patients overall. The prevalence of NASH was significantly higher in the patients with H. pylori IgG seropositivity (81 %) than in those without (58 %, p = 0.008). Glucose intolerance was similar between the two groups. The total NAS and the grade of hepatocyte ballooning were higher in the patients with H. pylori IgG seropositivity than in those without, while the hepatic iron grade was lower in the patients with H. pylori IgG seropositivity than in those without. H. pylori infection (p = 0.030), female gender (p = 0.029), and NAFIC score ≥ 2 points (p < 0.001) could independently predict NASH in logistic regression analysis, independent of age, obesity and glucose tolerance. CONCLUSION: The association of H. pylori seropositivity with hepatocyte ballooning suggests that H. pylori infection may represent another contributing factor in the progression from NAFL to NASH. Eradicating H. pylori infection may have therapeutic prospects in NASH treatment.

Predictors of malignancies and overall mortality in Japanese patients with biopsy-proven non-alcoholic fatty liver disease.

AIM: Some patients with non-alcoholic fatty liver disease (NAFLD) develop hepatocellular carcinoma (HCC) and have higher mortality than others. The evidence causally linking NAFLD to extrahepatic malignancies is scarce. Our aim was to determine the incidence of and risk factors for HCC, extrahepatic cancer and mortality in Japanese patients with biopsy-proven NAFLD. METHODS: This retrospective cohort study analyzed outcomes including onset of malignant tumors and death in 312 patients with NAFLD diagnosed by liver biopsy. RESULTS: Of 312 patients, 176 (56.4%) were diagnosed with non-alcoholic steatohepatitis. During a median follow-up period of 4.8 years (range, 0.3-15.8), six patients (1.9%) developed HCC, and 20 (6.4%) developed extrahepatic cancer. Multivariate analysis identified fibrosis stage (≥3; hazard ratio [HR], 12.3; 95% confidence interval [CI], 1.11-136.0; P = 0.041) as a predictor for HCC and type IV collagen 7s (>5 ng/mL; HR, 1.74; 95% CI, 1.08-2.79; P = 0.022) as a predictor for extrahepatic cancer. Eight patients (2.6%) died during the follow-up period. The most common cause of death was extrahepatic malignancy. None died of cardiovascular disease. Multivariate analysis identified type IV collagen 7s (>5 ng/mL; HR, 3.38; 95% CI, 1.17-9.76; P = 0.024) as a predictor for mortality. CONCLUSION: The incidence of extrahepatic cancer was higher than that of HCC. Severe fibrosis was a predictor for HCC. Patients with NAFLD and elevated type IV collagen 7s levels are at increased risk for extrahepatic cancer and overall mortality.

Blockade of interleukin 6 signalling ameliorates systemic insulin resistance through upregulation of glucose uptake in skeletal muscle and improves hepatic steatosis in high-fat diet fed mice.

BACKGROUND & AIMS: Mice fed high-fat diet (HFD) demonstrate obesity-related systemic insulin resistance (IR). Aim of this study is to clarify the role of interleukin (IL)-6 in IR in vivo focusing on skeletal muscle, adipose tissue and liver. METHODS: Plasma markers of IR and hepatic IL-6 signalling were examined in eight-week HFD feeding C57/BL6 mice. Furthermore, IR-related molecules in skeletal muscles, adipose tissues and livers were investigated following a single injection of anti- IL-6 receptor neutralizing antibody (MR16-1) in two-week HFD feeding mice. To investigate the role of IL-6 in hepatic steatosis by prolonged HFD, hepatic triglyceride accumulation was assessed in eight-week HFD feeding mice with continuous MR16-1 treatment. RESULTS: High-fat diet for both 2 and 8 weeks elevated plasma IL-6, insulin and leptin, which were decreased by MR16-1 treatment. A single injection of MR16-1 ameliorated IR as assessed by glucose and insulin tolerance test, which may be attributable to upregulation of glucose transporter type 4 via phosphorylation of AMP-activated protein kinase as well as upregulation of peroxisome proliferator-activated receptor alpha in livers and, particularly, in skeletal muscles. MR16-1 also decreased mRNA expression of leptin and tumour necrosis factor-alpha and increased that of adiponectin in adipose tissue. High-fat diet for 8 weeks, not 2 weeks, induced hepatic steatosis and increased hepatic triglyceride content, all of which were ameliorated by MR16-1 treatment. CONCLUSIONS: Blockade of excessive IL-6 stimulus ameliorated HFD-induced IR in a skeletal muscle and modulated the production of adipokines from an early stage of NAFLD, leading to prevention of liver steatosis for a long term.

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

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Serum alanine aminotransferase predicts the histological course of non-alcoholic steatohepatitis in Japanese patients.

AIM: Some cases with non-alcoholic fatty liver disease (NAFLD), particularly non-alcoholic steatohepatitis (NASH), can ultimately progress to liver cirrhosis. However, studies to clarify factors predictive of histological change in patients with NASH remain scarce. Our aim is to determine predictors of histological progression in Japanese patients with biopsy-proven NASH. METHODS: This retrospective cohort study enrolled 52 patients with NASH who underwent serial liver biopsies. Histological evaluation included NAFLD activity score (NAS) and liver fibrosis. The median interval between initial and second liver biopsies was 968 days. An alanine aminotransferase (ALT) response was defined as a decrease of 30% or more from baseline. RESULTS: Of 52 patients, NAS was ameliorated in 30.8%, deteriorated in 30.8% and remained unchanged in 38.4%. Liver fibrosis was improved in 25.0% of patients, progressed in 25.0% and remained stable in 50.0%. Multivariate analysis identified ALT non-response as a predictor of deterioration of NAS (hazard ratio [HR], 5.85; P = 0.031) and progression of liver fibrosis (HR, 4.50; P = 0.029). The mean annual rate of fibrosis was 0.002 stages/year overall, increasing to 0.15 stages/year in ALT non-responders. CONCLUSION: A lack of reduction in serum ALT level by at least 30% from baseline was a predictor for histological progression in patients with NASH. Serum ALT level is a better predictor of histological change than insulin resistance or bodyweight and can be a valid index in treatment. Serum ALT should be strictly controlled to prevent liver histological progression in patients with NASH.