High fasting insulin concentrations may be a pivotal predictor for the severity of hepatic fibrosis beyond the glycemic status in non-alcoholic fatty liver disease patients before development of diabetes mellitus.

BACKGROUND: Insulin resistance and type 2 diabetes mellitus (T2DM) contribute to the progression of non-alcoholic fatty liver disease (NAFLD). However, the relationship between glucose metabolic factors and the histological severity in NAFLD patients before development of T2DM is not well known. METHODS: In 103 biopsy-proven NAFLD patients (68 men and 35 women) with hemoglobin A1c of <6.5% and fasting blood glucose of <126 mg/dL, we investigated whether glucose metabolic factors influenced the severity of hepatic fibrosis without prior known T2DM. RESULTS: Female gender, age, serum aspartate aminotransferase, the aspartate aminotransferase/alanine aminotransferase ratio, fasting immunoreactive insulin (f-IRI), homeostasis model assessment - insulin resistance, hemoglobin A1c, hyaluronic acid, and type IV collagen 7 s were significantly higher, and 1,5-anhydroglucitol was significantly lower, in the fibrosis stage F3 group than in the F0-2 group. Multiple logistic regression analysis showed that only f-IRI (P = 0.006; odds ratio, 1.15151; 95% confidence interval, 1.04198-1.27254) was significantly indicated as a predictive factor for F3. As determined by both forward and backward stepwise selection analyses to optimize the model, f-IRI (P = 0001; odds ratio, 1.16788) remained an independent predictive factor for F3. To discriminate the F3 group from the F0-2 group, the area under the receiver operating characteristic curves showed that fasting insulin was 0.7219, and the best cut-off value of f-IRI was 13.2 µU/mL in the receiver operating characteristic curve analysis. CONCLUSIONS: High fasting insulin concentrations may be a pivotal glucose metabolic predictor for the severity of hepatic fibrosis beyond the glycemic status in NAFLD patients before development of T2DM.

Patatin-like phospholipase domain-containing protein 3 is involved in hepatic fatty acid and triglyceride metabolism through X-box binding protein 1 and modulation of endoplasmic reticulum stress in mice.

AIM: Non-alcoholic steatohepatitis (NASH) is the major cause of chronic liver disease worldwide. Endoplasmic reticulum (ER) stress is considered to be an important pathological characteristic in NASH. A sequence variation (I148M) in the patatin-like phospholipase domain-containing protein 3/adiponutrin (PNPLA3) gene is known to be associated with the development of NASH. However, PNPLA3 deficiency has been considered to not be associated with fatty liver disease. To clarify, therefore, the role of PNPLA3 in liver, we established PNPLA3 knockout (KO) mice and investigated the phenotypes and involved factors under ER stress. METHODS: ER stress was induced by i.p. injection with tunicamycin or with saline at 0 and 24 h in KO and C57BL/6 (wild-type [WT]) mice. At 48 h after the starting of treatment, blood and liver samples were studied. RESULTS: Hepatic steatosis and triglyceride content were remarkably increased in WT mice than in KO mice under ER stress. The hepatic palmitate/oleate ratio was significantly higher originally in KO mice than in WT mice. Moreover, the expression of stearoyl-coenzyme A desaturase-1 (SCD1) in KO mice under ER stress was decreased further than that in WT mice. Expression of ER stress markers X-box binding protein 1 (XBP1) and ERdj4 was increased in WT mice but not in KO mice under ER stress. CONCLUSION: We first demonstrated the hepatic phenotype of PNPLA3 deficiency under ER stress. Our observations would indicate that PNPLA3 has an important role in hepatic fatty acid metabolism and triglyceride accumulation through XBP1 under ER stress.

The Clinical Features of Late Postoperative Cholangitis After Hepaticojejunostomy Brought on by Conditions other than Cancer Recurrence.

PURPOSE: Postoperative cholangitis and anastomotic strictures (AS) are long-term complications of biliary-enteric anastomosis (BEA). METHODS: We retrospectively reviewed data of patients who underwent bile duct resection with or without hepatectomy and investigated the risk factors for postoperative cholangitis, benign AS, and incidence of Clavien-Dindo (C-D) >Grade III complications. RESULTS: Overall, data of 189 patients (115 men and 74 women) were retrospectively analyzed. The median patient age was 73 years. Thirty-five patients (18.5%) developed postoperative cholangitis, and 16 (8.4%) developed postoperative AS. Male sex and serious postoperative complications (C-D ≥ Grade III) were independent risk factors for cholangitis. The incidence of serious postoperative complications was 32.3%. Hypertension, preoperative biliary drainage, C-reactive protein-albumin ratio ≥.22, and bile duct resection with hepatectomy were potential risk factors for serious postoperative complications. CONCLUSIONS: The incidence rates of postoperative cholangitis and AS after BEA were 18.5% and 8.4%, respectively. Male sex and serious postoperative complications (C-D ≥ Grade III) were independent risk factors for postoperative cholangitis.

Possibility of Neoadjuvant Treatment for Radiologically Judged Resectable Pancreatic Cancer.

Survival remains poor even after resection of pancreatic cancer and the postoperative recurrence rate is extremely high. Thus, neoadjuvant treatment may improve outcomes for resectable pancreatic cancer (RPC). This study evaluated the efficacy of neoadjuvant therapy for radiologically judged RPC. A prospectively maintained institutional database was reviewed to identify patients who underwent potentially curative resection of radiologically judged RPC. Patient characteristics and intermediate-term outcomes were compared between groups that received neoadjuvant treatment or upfront surgery (UFS). We identified 353 eligible patients, including 55 patients who received neoadjuvant chemoradiotherapy (CRT group), 53 patients who received neoadjuvant gemcitabine plus nab-paclitaxel (GnP group), and 245 patients who underwent UFS (UFS group). The cumulative rates of pancreatic cancer recurrence at 2 years after pancreatic surgery were 49.5% in the UFS, 48.1% in the CRT group, and 52.7% in the GnP group. The recurrence rate tended to be improved after neoadjuvant treatment, although the difference was not significant at this follow-up point. While the clinical TNM classifications were noticeably different from the final pathological findings, the clinical and pathological TNM classifications were more similar in the groups that underwent neoadjuvant treatment. Neoadjuvant treatment can help identify good surgical candidates and avoid unnecessary laparotomy. Our results also suggest that neoadjuvant therapy might help improve the preoperative diagnostic accuracy for patients with RPC.

A novel and comprehensive mouse model of human non-alcoholic steatohepatitis with the full range of dysmetabolic and histological abnormalities induced by gold thioglucose and a high-fat diet.

BACKGROUND: The search for effective treatments of non-alcoholic steatohepatitis (NASH), now the most common chronic liver disease in affluent countries, is hindered by a lack of animal models having the range of anthropometric and pathophysiological features as human NASH. AIMS: To examine if mice treated with gold thioglucose (GTG) - known to induce lesions in the ventromedial hypothalamus, leading to hyperphagia and obesity - and then fed a high-fat diet (HF) had a comprehensive histological and dysmetabolic phenotype resembling human NASH. METHODS: C57BL/6 mice were injected intraperitoneally with GTG and then fed HF for 12 weeks (GTG+HF). The extent of abdominal adiposity was assayed by CT scanning. A glucose tolerance test and an insulin tolerance test were performed to evaluate insulin resistance (IR). Histological, molecular and biochemical analyses were also performed. RESULTS: Gold thioglucose+HF induced dysmetabolism, with hyperphagia, obesity with increased abdominal adiposity, IR and consequent steatohepatitis, with hepatocyte ballooning, Mallory-Denk bodies, perivenular and pericellular fibrosis as seen in adult NASH, paralleled by an increased expression of the profibrogenic factors, transforming growth factor-ß1 and TIMP-1. Plasma adiponectin and the expression of adiponectin receptor 1 and receptor 2 were decreased, while PPAR-γ and FAS were increased in the livers of GTG+HF mice. In addition, GTG+HF mice showed glucose intolerance and severe IR. CONCLUSIONS: Treatment with GTG and HF diet induce, in mice, a comprehensive model of human NASH, with the full range of dysmetabolic and histological abnormalities.

Evaluation of postprandial hypoglycemia in patients with nonalcoholic fatty liver disease by oral glucose tolerance testing and continuous glucose monitoring.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is often associated with insulin resistance and glucose intolerance. Postprandial hypoglycemia frequently occurs in NAFLD patients; however, the details remain unclear. PATIENTS AND METHODS: The 75-g oral glucose tolerance test (75gOGTT) in 502 patients with biopsy-proven NAFLD and continuous glucose monitoring (CGM) in 20 patients were performed, and the characteristics and causes of postprandial hypoglycemia were investigated. RESULTS: The proportion of patients in the Hypo subgroup [plasma glucose (PG) at 180 min

Differences in characteristics of glucose intolerance between patients with NAFLD and chronic hepatitis C as determined by CGMS.

Glucose intolerance frequently develops in accordance with the progression of chronic liver disease. However, differences in the characteristics of glucose intolerance between patients with nonalcoholic fatty liver disease (NAFLD) and those with chronic hepatitis C (C-CH) remain incompletely understood. To clarify these differences, patients with NAFLD (n = 37) and C-CH (n = 40) were evaluated with a continuous glucose monitoring system (CGMS). In the patients with NAFLD, Maximum blood glucose concentration and blood glucose swings were significantly correlated with hepatic fibrosis markers. In the patients with C-CH, however, those two CGMS parameters were negatively correlated with the serum albumin (ALB) concentration. Furthermore, in the patients with C-CH with an ALB concentration of ≤4.0 g/dl, those two CGMS parameters were negatively correlated with the ALB concentration with greater statistical significance. In conclusion, obvious differences in the characteristics of glucose intolerance between patients with NAFLD and those with C-CH were clarified. In patients with NAFLD, glucose intolerance gradually progressed in accordance with the progression of hepatic fibrosis. In those with C-CH, glucose intolerance suddenly developed upon the appearance of hypoalbuminaemia.

Overexpression of carbonic anhydrase-related protein XI promotes proliferation and invasion of gastrointestinal stromal tumors.

Three isoforms of carbonic anhydrase-related protein (CA-RP) are evolutionally well conserved among the CA gene family but lack classical CA activity. Although the biological function of CA-RPs is unknown, overexpression of CA-RP VIII has been reported in certain tumor types. Based on the finding that CA-RPs are commonly expressed in the neuronal cells, we investigated expression of all three CA-RPs in gastrointestinal stromal tumor (GIST). In contrast to no detectable signal of any of the three CA-RPs in intestinal cells of Cajal, immunohistochemical analysis showed distinct cytoplasmic expressions of CA-RPs VIII and XI in 13 (59%) and 20 (91%) of 22 GIST tissue specimens, respectively. The positive signals for both CA-RPs VIII and XI were more intense in the periphery than in the central part of GISTs, whereas no significant signal for CA-RP X expression was observed in any of the GISTs. These expression patterns of CA-RPs were consistently observed by reverse transcription-polymerase chain reaction-Southern blot and immunocytochemistry in the cultured GIST cell line GIST-T1. Ectopic expression of CA-RP XI in GIST-T1 cells induced cell proliferation and invasion in vitro. These findings indicate that CA-RP XI plays a role in the development of GIST.

Expression of carbonic anhydrase-related protein CA-RP VIII in non-small cell lung cancer.

Carbonic anhydrase-related protein (CA-RP VIII) lacks a Zn-binding motif which is essential for carbonic anhydrase activity. Therefore, CA-RP VIII is believed to have a no catalytic activity and a new biological property. In the present study, CA-RP VIII expression in non-tumorous lung and non-small cell lung carcinomas was investigated. Little or no expression of CA-RP VIII was observed in human lungs by Northern-blot analysis. Reverse-transcription polymerase chain reaction analysis demonstrated CA-RP VIII mRNA in developing human lungs and, to a lesser extent, in normal lungs. Subsequent immunohistochemical staining revealed that CA-RP VIII was expressed in the pulmonary epithelium in developing lungs; however, CA-RP VIII expression was restricted in bronchial ciliated cells in adult lungs. Neither bronchial gland nor squamous metaplasia of interstitial pneumonia expressed CA-RP VIII. In contrast, CA-RP VIII was strongly expressed in almost all archival lung cancer specimens, which included 24 squamous cell carcinomas, 25 adenocarcinomas, and 6 adenosquamous cell carcinomas. Cancer cells at the front of tumor progression expressed CA-RP VIII in particular abundance. The present findings suggest that CA-RP VIII may play a role in non-small lung cell carcinomas.

Biliopancreatic fistula caused by an intraductal papillary-mucinous tumor of the pancreas confirmed by biochemical analysis of mucin.

Intraductal papillary-mucinous tumor of the pancreas is occasionally accompanied by biliopancreatic fistula. However, it is difficult to show the inflow of mucin produced by the tumor into the common bile duct. To confirm the biliopancreatic fistula, the mucin-rich fraction was purified from the bile and stained with antimucin antibodies. Western blot analysis showed characteristic smear staining patterns for mucin molecules with three types of antimucin antibodies. Immunohistochemical analysis with the antibody showed significant signals of the cancer cells and the luminal content of the dilated pancreatic duct. These results showed that the bile contained an abundance of mucin, which was produced by the primary pancreatic tumor. In cases with intraductal papillary-mucinous tumor of the pancreas, biochemical analysis of mucin molecules in the bile can be of clinical use in consideration of pathological process of tumor progression.

Diminished cellular immune response to carbonic anhydrase II in patients with Sjögren's syndrome and idiopathic chronic pancreatitis.

CONTEXT: A serum antibody to carbonic anhydrase II has been reported in patients with Sjögren's syndrome and idiopathic chronic pancreatitis. OBJECTIVE: To evaluate cellular immune response to carbonic anhydrase II in patients with Sjögren's syndrome and idiopathic chronic pancreatitis. PATIENTS: Idiopathic chronic pancreatitis (n=23), Sjögren's syndrome (n=12), alcoholic chronic pancreatitis (n=3) and normal controls (n=13). MAIN OUTCOME MEASURES: Proliferation assay of peripheral blood mononuclear cells. RESULTS: Notable increased proliferation of the mononuclear cells upon stimulation with carbonic anhydrase II was observed in 2 patients with idiopathic chronic pancreatitis (9%) and 2 patients with Sjögren's syndrome (17%) but not in patients with alcoholic chronic pancreatitis nor in normal controls. Among the four study groups, there was no significant difference in the prevalence rate of the positive proliferative responses (P=0.444). CONCLUSION: Carbonic anhydrase II may not be a major target antigen for the immunological process in the pathogenesis of Sjögren's syndrome and idiopathic chronic pancreatitis. Serum antibody to carbonic anhydrase II may be detected in these patients as a consequence of the immune reaction against other antigens which mimic carbonic anhydrase II.

Bofutsushosan, a Japanese herbal (Kampo) medicine, attenuates progression of nonalcoholic steatohepatitis in mice.

BACKGROUND: Obesity-induced liver disease (nonalcoholic fatty liver disease, NAFLD) is now the commonest cause of chronic liver disease in affluent nations. There are presently no proven treatments for NAFLD or its more severe stage, nonalcoholic steatohepatitis (NASH). Bofutsushosan (BTS), a Japanese herbal (Kampo) medicine, long used as an anti-obesity medicine in Japan and other Asian countries, has been shown to reduce body weight and improve insulin resistance (IR) and hepatic steatosis. The precise mechanism of action of BTS, however, remains unclear. To evaluate the ability of BTS to prevent the development of NASH, and determine the mediators and pathways involved. METHODS: C57BL/6 mice were injected intra-peritoneally with gold-thioglucose and fed a high-fat diet (HF) or HF diet admixed with either 2 or 5 % BTS for 12 weeks. The effectiveness of BTS in attenuating features of NASH and the mechanisms through which BTS attenuated NASH were then assayed through an assessment of the anthropometric, radiological, biochemical and histological parameters. RESULTS: BTS attenuated the progression of NASH through induction of adiponectin and its receptors along with an induction of PPAR-α and PPAR-γ, decreased expression of SREBP-1c, increased hepatic fatty acid oxidation and increased hepatic export of triglycerides. BTS moreover, reduced IR through phosphorylation of the protein kinase, Akt. CONCLUSIONS: BTS through induction of adiponectin signaling and Akt attenuated development of NASH. Identification of the active entity in BTS should allow development of novel treatments for NASH.

Glycemic variability is an independent predictive factor for development of hepatic fibrosis in nonalcoholic fatty liver disease.

UNLABELLED: Patients with nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) often have metabolic disorders including insulin resistance and type 2 diabetes mellitus (T2DM). We clarified the predictive factors in glucose metabolism for progression of hepatic fibrosis in patients with NAFLD by the 75-g oral glucose tolerance test (75gOGTT) and a continuous glucose monitoring system (CGMS). One hundred sixty-nine patients (68 female and 101 male patients) with biopsy-proven NAFLD with performance with 75gOGTT were enrolled and divided into four groups according to the stage of hepatic fibrosis (F0-3). The proportion of patients with T2DM significantly gradually increased, HbA1c and the homeostasis model assessment of insulin resistance were significantly elevated, and 1,5-anhydroglucitol (1,5-AG) was remarkably decreased with the progression of fibrosis. In the 75gOGTT, both plasma glucose and insulin secretion were remarkably increased with the progression of fibrosis. The only factor significantly associated with advanced fibrosis was 1,5-AG (P = 0.008) as determined by multivariate logistic regression analysis. We next evaluated the changes in blood glucose during 24 hours by monitoring with the CGMS to confirm the relationship between glycemic variability and progression of fibrosis. Variability of median glucose, standard deviation of median glucose (P = 0.0022), maximum blood glucose (P = 0.0019), and ΔMin-max blood glucose (P = 0.0029) were remarkably higher in severe fibrosis than in mild fibrosis. CONCLUSION: Hyperinsulinemia and hyperglycemia, especially glycemic variability, are important predictive factors in glucose impairment for the progression of hepatic fibrosis in NAFLD.

Usefulness of Technetium-99 m-2-methoxy-isobutyl-isonitrile liver scintigraphy for evaluating disease activity of non-alcoholic fatty liver disease.

AIM: Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD). Therefore, it is important to evaluate disease activity and distinguish NASH from simple steatosis in NAFLD. Technetium-99 m-2-methoxy-isobutyl-isonitrile ((99m) Tc-MIBI) is a lipophilic cation designed for myocardial perfusion scintigraphy in the diagnosis of ischemic heart diseases, and its retention reflects mitochondrial function. It was reported that hepatic mitochondrial abnormalities would be an important predictive factor for NASH disease progression. The aim of this study was to examine the clinical usefulness of (99m) Tc-MIBI liver scintigraphy for evaluating disease activity of NAFLD and distinguishing NASH from simple steatosis in patients with NAFLD. METHODS: Twenty-six patients with biopsy-proven NAFLD were enrolled. Clinicolaboratory tests and (99m) Tc-MIBI liver scintigraphy were performed. To evaluate hepatic uptake, regions of interest were set at the liver and heart, and the uptake ratio of the liver to heart (liver/heart ratio) was calculated. RESULTS: All patients with NAFLD were classified into three groups according to the NAFLD activity score: non-NASH (simple steatosis) (n = 4), borderline NASH (n = 11), and NASH (n = 11). Liver/heart ratios were significantly lower in NASH than in simple steatosis (P < 0.05). Moreover, liver/heart ratios were significantly correlated with NAFLD activity scores among the patients (r = -0.413, P < 0.05). CONCLUSIONS: The present study indicates that (99m) Tc-MIBI liver scintigraphy would be a useful non-invasive functional imaging method with which to evaluate disease activity of NAFLD and distinguish NASH from simple steatosis.

Angiotensinogen gene haplotype is associated with the prevalence of Japanese non-alcoholic steatohepatitis.

AIM: Non-alcoholic steatohepatitis (NASH) patients frequently have hypertension, which is considered to be an important predictive factor for the subsequent development of hepatic fibrosis. The renin-angiotensin system is also known to contribute to the progression of NASH. Various types of functional single-nucleotide polymorphisms (SNPs) involved in the development of NASH have been proposed. Angiotensinogen (AGT) gene SNPs related to cardiovascular diseases have been reported. We aimed to evaluate the involvement of the AGT gene haplotype in Japanese NASH patients. METHODS: Previously described genotypes of SNPs of the AGT gene, rs4762 C/T polymorphism (T207M), rs699 C/T polymorphism (T268M), and rs7079 C/A polymorphism (C11537A), were determined in 124 Japanese biopsy-proven NASH patients and 150 healthy volunteers (controls). RESULTS: The allele and genotype frequencies in rs4762 and rs699 SNPs in NASH patients were similar to those in controls, while the frequency of the A allele and A/- genotype in rs7079 SNPs were much higher in NASH patients than in controls. In addition, the 3-SNP haplotype CTA was significantly over-represented in NASH patients compared with controls. Regarding clinical features of NASH patients, diastolic blood pressures in patients with the CTA/- genotype were much higher than in patients with other genotypes. CONCLUSIONS: We found a 3-SNP haplotype of the AGT gene that is involved in the development of NASH and influences hypertension in NASH patients. These results provide new insight into the therapy of NASH patients with the CTA haplotype using ACE inhibitors or angiotensin II type 1 receptor blockers.

The latest idea in NAFLD/NASH pathogenesis.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by fatty accumulation in the liver without alcohol consumption. NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), which may progress to end-stage liver disease. The prevalence of NAFLD is rising because of an increasing prevalence of obesity and metabolic syndrome. The progression of these diseases is considered to be related to metabolic syndrome, which is characterized by obesity, glucose impairment, dyslipidemia, hypertension, and adipocytokine impairment. In addition, the pathogenesis of NAFLD/NASH is considered to be multifactorial and complex and is influenced by lifestyle habits, nutritional factors, and genetics. In particular, the PNPLA3 gene has been recently recognized as the most important functional gene polymorphism in the progression of NASH. Disruption in hepatic lipid metabolism is closely related to the development of fatty liver. Accumulation of excess triglycerides (TGs) induces hepatic steatosis. However, TG accumulation itself is not harmful to hepatocytes and may instead act as a protective mechanism against free fatty acid (FFA)-induced lipotoxicity. Excess FFAs also contribute to hepatotoxicity in NAFLD/NASH because oxidation of FFAs in hepatic microsomes generates excessive oxidative stress. Oxidative stress is considered one of the most important pathogenic factors in the development of NASH. Mitochondrial abnormalities, which are frequently observed in NASH-affected livers, are associated with impaired electron transport and result in further oxidative stress formation. The aims of this review are to assess the mechanisms of lipid metabolism and hepatic steatosis, the background of the disease, and the potential molecular mechanisms involved.

Clinicopathological features and medical management of intraductal papillary mucinous neoplasms.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) are a clinicopathological entity that is being diagnosed with increasing frequency. However, the best approach to medical management of IPMN needs to be clarified. The aim of the present study was to identify preoperative features that may be predictors of malignant IPMN, and to define the medical management of IPMN of the pancreas. METHODS: A total of 23 patients who underwent surgical resection for IPMN of the pancreas at Kochi Medical School between 1982 and 2004 were examined. Multivariate Cox regression analysis was used to identify factors independently associated with IPM carcinoma. RESULTS: Among the 23 patients, 12 had IPMN adenoma, three had borderline IPMN, four had IPMN with carcinoma in situ, and four had IPMN with invasive carcinoma. In multivariate analysis, elevated serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 levels were found to be preoperative predictors of malignant IPMN. These results suggest that the following IPMN of the pancreas should be resected: (i) IPMN of the pancreas situated in the main duct; (ii) IPMN located in the branch duct if the size of the cystic lesion is >30 mm and the mural nodules are >5 mm in height by endoscopic ultrasound (EUS); and (iii) the diameter of the main pancreatic duct is >10 mm by endoscopic retrograde pancreatography (ERP). Careful observation of patients with branch-type IPMN with small cysts and/or without mural nodules is recommended as a management strategy. CONCLUSION: The present study reinforces the need for immediate surgical resection of malignant IPMN and suggests indicators for IPMN that should assist physicians in making decisions on treatment options.