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Aim: Following the coronavirus disease outbreak, a state of public emergency was declared worldwide, which enforced lifestyle changes. This study therefore aimed to investigate the changes in lifestyle, body composition, hepatic steatosis, and fibrosis in patients with chronic liver disease (CLD) under lockdown. Methods: During the lockdown period, 1344 patients with CLD answered a lifestyle questionnaire. In 298 patients, body composition and liver stiffness measure (LSM)/controlled attenuation parameter (CAP) were analyzed by InBody and FibroScan, respectively, and serial data were obtained in 137 patients. Results: More than half of the CLD patients answered decreases in physical activity and frequency of outings during lockdown, while diet was less affected. Overall, 58% of patients showed elevations in CAP values, which were not different statistically over time. Women, but not men, were more likely to increase CAP values during lockdown. Neither LSM nor serum fibrosis markers were elevated chronologically during lockdown. In men, body mass index (BMI), body fat percentage, and visceral fat area (VFA) were significantly increased, whereas in women, lower‐limb muscle mass was significantly decreased. Patients with decreased SMI showed elevations in CAP and VFA values, and patients who exercised less showed increases in BMI. Conclusion: In response to lockdown, men tended to increase body fat but the degree of hepatic steatosis was less affected, while women were more likely to exacerbate hepatic steatosis with skeletal muscle loss among CLD patients. Gender‐specific approaches need to be established for management of CLD patients to avoid exacerbation or comorbidity of steatotic liver disease. [ABSTRACT FROM AUTHOR]

Aim: Despite its relevant clinical impact and high prevalence, covert hepatic encephalopathy (HE) still remains underdiagnosed. As patients with liver cirrhosis tend to be older in Japan, more suitable tests for the elderly and cut‐off values based on this attribute are needed. Recently, a Stroop test has been developed and validated for the screening and diagnosis of covert HE in the United States. The present study aims to establish the cut‐off values of the Stroop test to screen covert HE. Methods: This study was a prospective multicenter cross‐sectional endeavor. We undertook a survey of 311 cirrhotic patients, administering the number connection test (NCT)‐A and ‐B and the Stroop‐off and ‐on test. Results: We determined the cut‐off values of Stroop test results for cirrhotic patients in a variety of age ranges. The cut‐off value of the Stroop test was strongly correlated with age. There was a significant correlation between the results of NCT‐B and age, and Stroop‐on test results showed a correlation with serum albumin (Alb) levels. Serum Alb ≤3.2 g/dl could have the potential to be an objective biomarker of covert HE. In addition, stepwise logistic regression analysis revealed a relationship between the results of the Stroop‐on test and plasma ammonia levels. Conclusions: We established the cut‐off values of Stroop test results and confirmed the efficacy of the Stroop test as a simple tool for assessing cognitive alterations. The Stroop test could be suitable as a necessary minimum for the diagnosis of covert HE. [ABSTRACT FROM AUTHOR]

Background and Aim: Aging is an independent risk factor for the progression of non‐alcoholic steatohepatitis. Here, we investigated the role of age‐related alterations in fatty acid metabolism in dietary steatohepatitis using lipidomics analysis. Methods: Male 8‐week and 55‐week‐old C57BL/6 J mice were fed a high‐fat diet (HFD) for 8 weeks. The quality and quantity of lipid molecular species in the liver were evaluated using the lipidomics approach. Results: Elder mice fed an HFD developed more severe steatohepatitis than young mice. Oxidative stress and inflammatory cytokines in the liver were exacerbated following HFD feeding in elder mice compared with young mice. In elder mice, de novo fatty acid synthesis was promoted, whereas β oxidation was blunted following HFD feeding, and lipid secretion from the liver was reduced. The expression of sirtuin 1 was not only reduced with age as expected but also significantly decreased due to intake of HFD. In the lipidomics analysis, the concentrations of diacylglycerol and TAG molecular species containing monounsaturated fatty acids were markedly increased following HFD feeding in elder mice compared with young mice. In contrast, the concentration of phosphatidylethanolamine and phosphatidylcholine molecular species containing polyunsaturated fatty acids were remarkably decreased following HFD feeding in elder mice compared with young mice, and the expression of fatty acid desaturase was blunted. Conclusions: Aging‐dependent alterations in lipid metabolism under excessive lipid supply most likely enhance hepatic lipotoxicity, thereby exacerbating metabolic steatohepatitis in elderly. [ABSTRACT FROM AUTHOR]

Background: Co‐occurrence of metabolic syndrome and chronic alcohol consumption is increasing worldwide. The present study investigated the effect of the chemical chaperone 4‐phenylbutyric acid (PBA)—which has been shown to alleviate dietary steatohepatitis caused by endoplasmic reticulum (ER) stress—on chronic‐plus‐binge ethanol (EtOH)–induced liver injury in a mouse model of obesity. Methods: Male KK‐Ay mice (8 weeks old) were fed a Lieber–DeCarli diet (5% EtOH) for 10 days. Some mice were given PBA intraperitoneally (120 mg/kg body weight, daily) during the experimental period. On day 11, mice were gavaged with a single dose of EtOH (4 g/kg body weight). Control mice were given a dextrin gavage after being pair‐fed a control diet. All mice were then serially euthanized before or at 9 hours after gavage. Results: Chronic‐plus‐binge EtOH intake induced massive hepatic steatosis along with hepatocyte apoptosis and inflammation, which was reversed by PBA treatment. Administration of PBA also suppressed chronic‐plus‐binge EtOH–induced up‐regulation of ER stress‐related genes including binding immunoglobulin protein (Bip), unspliced and spliced forms of X‐box‐binding protein‐1 (uXBP1 and sXBP1, respectively), inositol trisphosphate receptor (IP3R), and C/EBP homologous protein (CHOP). Further, it blocked chronic‐plus‐binge EtOH–induced expression of the oxidative stress marker heme oxygenase‐1 (HO‐1) and 4‐hydroxynonenal. Chronic EtOH alone (without binge) increased Bip and uXBP1, but it did not affect those of sXBP1, IP3R, CHOP, or HO‐1. PBA reversed the prebinge expression of these genes to control levels, but it did not affect chronic EtOH‐induced hepatic activity of cytochrome P450 2E1. Conclusions: Binge EtOH intake after chronic consumption induces massive ER stress‐related oxidative stress and liver injury in a mouse model of obesity through dysregulation of the unfolded protein response. PBA ameliorated chronic‐plus‐binge EtOH–induced liver injury by reducing ER and oxidative stress after an EtOH binge. Chronic‐plus‐binge ethanol (EtOH) feeding induced severe hepatic steatosis with hepatocyte apoptosis in obese KK‐Ay mice through dysregulation of the unfolded protein response. The treatment with 4‐phenylbutyrate (PBA) ameliorated chronic‐plus‐binge EtOH‐induced liver injury by reducing ER and oxidative stress after an EtOH binge (A: immunohistological staining for ccCK18, scale bar = 100 μm, B‐D: RT‐PCR for sXBP1, CHOP, or HO‐1). The components of ER stress pathway are potential therapeutic targets for the management and prevention of alcoholic liver disease. [ABSTRACT FROM AUTHOR]

Aim: Hepatic inclusion composed of autophagy‐specific substrate p62 is one of the histological features of non‐alcoholic fatty liver disease (NAFLD) and can be a precursor to hepatic carcinogenesis. The expression of p62 was enhanced by not only autophagic dysfunction but also oxidative stress and inflammation. M1/M2 phenotypic balance of macrophages plays a pivotal role in the progression of NAFLD. We evaluated the correlation between macrophage polarization and the formation of p62 aggregation in NAFLD. Methods: Liver biopsy specimens from NAFLD patients were analyzed by immunohistochemical staining for M1 macrophage marker CD11c, M2 macrophage marker CD163, and p62/SQSTM1 (p62). The histological severity of NAFLD is assessed by a NAFLD activity score (NAS). The number of autophagic vesicles in hepatocytes was visualized and counted by using transmission electron microscopy. Results: The aggregation of p62 was undetectable in control, whereas hepatocytes with p62 aggregation were observed in approximately 88% of NAFLD specimens. The number of hepatocytes with p62 aggregation was positively correlated with the number of autophagic vesicles, serum alanine aminotransferase, NAS, fibrosis, and the number of CD11c‐positive cells, but not CD163‐positive cells. Assembly of CD11c‐positive cells was observed around hepatocytes with p62 aggregation. The ratio of CD11c/CD163‐positive macrophages was significantly associated with the formation of p62 aggregation. Conclusions: These findings indicate that chronic inflammation by M1‐polarization of macrophages contributes to the disease progression from simple steatosis to non‐alcoholic steatohepatitis in concert with autophagic dysfunction. [ABSTRACT FROM AUTHOR]

Aim Pharmacological treatment for metabolic syndrome-related non-alcoholic steatohepatitis has not been established. We investigated the effect of L-carnitine, an essential substance for β-oxidation, on metabolic steatohepatitis in mice. Methods Male KK-Ay mice were fed a high-fat diet (HFD) for 8 weeks, with supplementation of L-carnitine (1.25 mg/mL) in drinking water for the latter 4 weeks. Results Serum total carnitine levels were decreased following HFD feeding, whereas the levels were reversed almost completely by L-carnitine supplementation. In mice given L-carnitine, exacerbation of hepatic steatosis and hepatocyte apoptosis was markedly prevented even though HFD feeding was continued. Body weight gain, as well as hyperlipidemia, hyperglycemia, and hyperinsulinemia, following HFD feeding were also significantly prevented in mice given L-carnitine. High-fat diet feeding elevated hepatic expression levels of carnitine palmitoyltransferase 1A mRNA; however, production of β-hydroxybutyrate in the liver was not affected by HFD alone. In contrast, L-carnitine treatment significantly increased hepatic β-hydroxybutyrate contents in HFD-fed mice. L-carnitine also blunted HFD induction in sterol regulatory element binding protein-1c mRNA in the liver. Furthermore, L-carnitine inhibited HFD-induced serine phosphorylation of insulin receptor substrate-1 in the liver. L-carnitine decreased hepatic free fatty acid content in 1 week, with morphological improvement of swollen mitochondria in hepatocytes, and increases in hepatic adenosine 5'-triphosphate content. Conclusions L-carnitine ameliorates steatohepatitis in KK-Ay mice fed an HFD, most likely through facilitating mitochondrial β-oxidation, normalizing insulin signals, and inhibiting de novo lipogenesis in the liver. It is therefore postulated that supplementation of L-carnitine is a promising approach for prevention and treatment of metabolic syndrome-related non-alcoholic steatohepatitis. [ABSTRACT FROM AUTHOR]

Aim Recent evidences indicate that hepatic steatosis suppresses autophagic proteolysis. The present study evaluated the correlation between autophagic function and cathepsin expression in the liver from patients with non-alcoholic fatty liver disease ( NAFLD). Methods Liver biopsy specimens were obtained from patients with chronic liver diseases (chronic hepatitis C [ CHC; n = 20], chronic hepatitis B [ CHB; n = 16], primary biliary cirrhosis [ PBC; n = 23], NAFLD [ n = 22] and control [ n = 14]). The number of autophagic vesicles in hepatocytes was counted by using transmission electron microscopy. Expression of cathepsin B, D, L and p62 in the liver section was analyzed by immunohistochemical staining. The histological severity of NAFLD is assessed by NAFLD activity score ( NAS). Results The number of autophagic vesicles in hepatocytes was significantly increased in both CHC and NAFLD groups, but not CHB and PBC, more than control. Although hepatocytes with aggregation of p62 were observed in less than 15% of CHC, p62 aggregation was detected in approximately 65% of NAFLD. Cathepsin B, D and L expression was significantly suppressed in the liver from NAFLD patients. Suppression of cathepsin B, D and L expression was not observed in CHB, CHC and PBC. In NAFLD patients, p62 aggregation was correlated with serum alanine aminotransferase value and inflammatory activity by NAS. Conclusion These results indicate that a decrease in hepatic cathepsin expression in NAFLD is associated with autophagic dysfunction. Hepatic inflammation correlates with autophagic dysfunction in NAFLD. These findings indicate that the suppression of autophagic proteolysis by hepatic steatosis is involved in the pathogenesis of NAFLD. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the role of natural killer (NK)T cells in the pathogenesis of non-alcoholic steatohepatitis (NASH), here we investigated the expression and function of hepatic NKT cells in KK-Ay mice, an animal model of metabolic syndrome. Methods: Male, 8-week-old KK-Ay and C57Bl/6 mice were fed a high-fat (HF) diet for 4 weeks. Some mice were given daily intragastric injections of pioglitazone for 5 days prior to or after dietary treatment. Results: In untreated KK-Ay mice, the percentages of NKT cells in liver mononucleolar cells were nearly one-third of those in C57Bl/6 controls. Elevations in interleukin (IL)-4 and interferon (IFN)-γ mRNA in the liver after a single injection of α-galactosylceramide (GalCer) were blunted in KK-Ay mice largely. Percentages of NKT cells, as well as GalCer-induced increases in IL-4 mRNA, were blunted significantly in both strains after HF diet feeding for 4 weeks. Interestingly, KK-Ay mice pretreated with pioglitazone showed significant increases in NKT cell proportion, and GalCer-induced increases in IL-4 and IFN-γ mRNA were also enhanced by pioglitazone. In KK-Ay mice, the percentages of annexin V positive NKT cells were nearly 2.5-fold higher than those in C57Bl/6 controls; however, pioglitazone decreased annexin V positive cells significantly. Moreover, pioglitazone increased NKT cell fraction in KK-Ay mice even after HF diet feeding. Conclusion: KK-Ay mice exhibit proportional and functional alterations in hepatic NKT cells in close relation with the development of steatohepatitis, and it is postulated that pioglitazone improves steatohepatitis in part through restoration of hepatic NKT cells. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated a dietary model of steatohepatitis caused by methionine- and choline-deficiency (MCD) in obese, diabetic KK–Ay mice. Male KK–Ay mice and C57Bl/6 mice were fed an MCD diet for up to 8 weeks, and liver pathology was evaluated. Hepatic steatosis and inflammatory infiltration were more prominent in KK–Ay mice than in C57Bl/6 mice 4 weeks after feeding with MCD diet. MCD diet-induced increases in tumor necrosis factor (TNF)-α mRNA levels, as well as lipid peroxidation, in the liver were also potentiated significantly in KK–Ay mice. Extended degree of hepatic fibrosis was observed in KK–Ay mice as compared to C57Bl/6 mice 8 weeks after feeding with MCD diet. Indeed, α1(I)procollagen and transforming growth factor (TGF)-β1 mRNA levels were significantly higher in KK–Ay mice following dietary treatment. Serum adiponectin levels were elevated nearly two-fold when C57Bl/6 mice were given MCD diet for 4 weeks; however, serum adiponectin levels in KK–Ay mice fed both the control- and MCD diet were the same, reaching the values almost 1/2 of those in C57Bl/6 mice. In conclusion, KK–Ay mice exhibit increased susceptibility to MCD diet-induced steatohepatitis, where hypoadiponectinemia most likely plays a key role in exacerbation of both inflammatory and profibrogenic responses. [Copyright &y& Elsevier]

Abstract: In this study, we evaluated whether hepatic steatosis affects the viral response to interferon (IFN) and ribavirin combination therapy in Japanese patients with chronic hepatitis C (CHC). Eighty CHC patients treated with IFN α-2b and ribavirin for 24 weeks were evaluated retrospectively. Liver biopsy specimens were assessed histopathologically, and grade of steatosis was scored as follows: grade 0: <5%; grade 1: 5–33%; grade 2: 33–66%; grade 3: >66%. Sustained viral response (SVR) was defined as negative for HCV-RNA by high-sensitivity qualitative reverse transcription-polymerase chain reaction (RT-PCR) at 24 weeks post-treatment. Hepatic steatosis graded 2 and higher was seen in 28.8% patients, whose average BMI were significantly higher than those in grade 0 patients. Grade of steatosis was well correlated with elevation in serum aminotransferases and gamma-glutamyltranspeptidase (γ-GTP) levels, but not with histological degree of inflammation and fibrosis. The SVR rates were significantly lower in the group with overt steatosis (grade 2/3) as compared to the group with less steatosis (grade 0/1), the values being 30.4% and 57.9%, respectively. Moreover, grade of steatosis was selected as an independent negative factor for SVR in multivariate analysis. In conclusion, hepatic steatosis is an important predictor of poor response to therapy of IFNα-2b and ribavirin in patients with CHC. [Copyright &y& Elsevier]

A letter to the editor is presented regarding non-alcoholic steatohepatitis caused by exposure to a high total dosage of glucocorticoids.

Acetaminophen overdose causes liver injury by mechanisms involving glutathione depletion, oxidative stress and mitochondrial dysfunction. The role of apoptosis in acetaminophen-induced cell killing is still controversial. Here, our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen-induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. Acetaminophen (10 μmol/L) induced necrotic killing in approximately 50% of hepatocytes after 6 h and cyclosporin A (CsA), MPT inhibitor, temporarily decreased necrotic killing after 6 h, but cytoprotection was lost after 16 h. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization at approximately 4.5 h after acetaminophen. CsA delayed these changes indicative of the MPT to about 11 h after acetaminophen. TUNEL labeling and caspase 3 activation also increased after acetaminophen. Fructose (20 mmol/L, an ATP-generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis. In conclusion, acetaminophen induces the MPT and ATP-depletion-dependent necrosis or caspase-dependent apoptosis as determined, in part, by ATP availability from glycolysis. [ABSTRACT FROM AUTHOR]

Obesity and insulin resistance are the key factors for progression of hepatic fibrosis in various chronic liver diseases including non-alcoholic steatohepatitis (NASH). Recently it has been shown that leptin plays a pivotal role in development of hepatic fibrosis. Leptin promotes hepatic fibrogenesis through upregulation of transforming growth factor-β in Kupffer cells and sinusoidal endothelial cells. Further, leptin facilitates proliferation and prevents apoptosis of hepatic stellate cells. There is a paradox, however, in that ob/ob mice and Zucker rats, which are the obese and diabetic strains, had minimal profibrogenic responses in the liver, most likely because they lack leptin and its receptors. To establish a more clinically relevant model to study the mechanism of fibrogenesis under steatohepatitis, fatty changes and profibrogenic responses in the liver caused by methionine–choline deficiency (MCD) were investigated in the KK-Ay mouse, which is an obese and diabetic strain. KK-Ay mice developed more severe hepatic steatosis, inflammation and fibrosis induced by an MCD diet as compared to C57Bl/6 controls. Importantly, KK-Ay mice lack physiological upregulation of adiponectin levels, suggesting that adiponectin plays a pivotal role not only in regulation of insulin sensitivity but also in modulation of inflammatory and profibrogenic responses in dietary steatohepatitis. Collectively, these findings support the hypothesis that the balance of adipocytokine expression is a key regulator for the progression of hepatic fibrosis in the setting of steatohepatitis. [ABSTRACT FROM AUTHOR]