Hepatic steatosis and skeletal muscle alterations during the COVID-19 lockdown in a cohort of patients with chronic liver disease in Japan.

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.

Benefit of an action camera in endoscopy education for medical students under COVID-19.

BACKGROUND: Endoscopy is an important form of clinical gastroenterology education because it gives students the opportunity to learn about diagnosis procedures and even treatment. During the COVID-19 pandemic, medical students were observed from outside the endoscopy room due to the risk of airborne infection. In this study, we investigated the efficacy of combining endoscopy education with doctor's-eye-view videos of the procedure obtained using live-action cameras (GoPro®). METHODS: From February to May 2021, endoscopists wore GoPro Hero8 cameras on their heads to display a doctor's-eye view video outside the room. The efficacy of the GoPro videos in combination with endoscopic monitoring was evaluated by 15 participating medical students. The participants rated the efficacy on a 5-point scale and commented on the positive and negative points. RESULTS: A total of 78.6% of participants evaluated the GoPro as good; 57.2% answered that it increased their understanding, with 71.4% stating that it increased their understanding of procedures in particular. A total of 85.7% of the students answered that their interest in endoscopy had increased, and 85.7% evaluated the benefit of the GoPro videos as good. In addition, 64.3% answered that the method was effective in preventing COVID-19 infection. Education using GoPro videos enabled students to feel as if they were conducting the endoscopy themselves and enabled them to concentrate on learning. CONCLUSIONS: Practical endoscopic education using a GoPro is an effective educational tool that not only increases understanding of endoscopic practice but also stimulates students' interest and awareness of their future as doctors.

Autophagic dysfunction in the liver enhances the expression of insoluble nuclear proteins 14-3-3ζ and importin α4.

AIMS: Autophagic dysfunction is associated with the progression of various liver diseases, including nonalcoholic fatty liver disease (NAFLD). However, serum markers for evaluating autophagic function have not been reported. Highly insoluble nuclear proteins participate in many cellular functions and are potential diagnostic markers for cancer. We performed a proteomic analysis of the hepatic nuclear insoluble fraction to identify novel autophagy-related diagnostic biomarkers. MAIN METHODS: The insoluble nuclear protein fraction was extracted from the livers of Atg7F/F, Atg7F/F:alb-Cre (hepatocyte-specific autophagy-deficient mice), C57BL/6 J, and KKAy (NAFLD model) mice. Proteins were separated by two-dimensional electrophoresis and visualized by silver staining. Protein spots were identified using mass spectrometry. The localization of proteins in hepatocytes was verified by immunofluorescence using a confocal microscope. KEY FINDINGS: The levels of insoluble nuclear proteins 14-3-3ζ and importin α4 were upregulated following hepatic autophagy dysfunction and were detectable in serum. Under normal conditions, these proteins are mainly distributed in the cytoplasm, whereas autophagic dysfunction induces their translocation to the nucleus. Incubation with an autophagy inhibitor up-regulated these proteins expression in the insoluble nuclear fraction of primary hepatocytes. Treatment with EGF or insulin enhanced 14-3-3ζ expression in the nuclear insoluble fraction; in contrast, the addition of rapamycin downregulated 14-3-3ζ expression. Importin α4 expression was increased in the nuclear insoluble fraction after incubation with tunicamycin or hydrogen peroxide. SIGNIFICANCE: Accumulation of 14-3-3ζ and importin α4 as nuclear-insoluble proteins may be associated with autophagic dysfunction. Our findings indicate that these proteins might be useful diagnostic biomarkers for liver diseases with autophagic disorders.

Carbohydrate-deficient transferrin is a sensitive marker of alcohol consumption in fatty liver disease.

BACKGROUND: The prevalence of nonalcoholic fatty liver disease (NAFLD) and alcohol-associated/related liver disease (ALD) with metabolic syndrome is increasing globally. Metabolic syndrome and excessive alcohol consumption synergically exacerbate liver pathologies; therefore, drinking-specific serum markers unaffected by liver injury or metabolic syndrome are essential for assessing alcohol consumption. We evaluated the ratio of carbohydrate-deficient transferrin to total transferrin (%CDT) in patients with fatty liver disease, particularly focusing on its correlation with metabolic factors (UMIN000033550). METHODS: A total of 120 patients with fatty liver disease, including ALD and NAFLD, were screened for alcohol misuse using the Alcohol Use Disorders Identification Test. Associations of metabolic syndrome-related factors and hepatic steatosis/liver stiffness with drinking markers, such as %CDT, gamma-glutamyl transferase (GGT), and mean corpuscular volume (MCV), were assessed using multiple linear regression analyses. RESULTS: %CDT significantly increased with 3-4 drinks/day. The optimal cutoff value for identifying non- to light drinkers was 1.78% (sensitivity, 71.8%; specificity, 83.7%; and area under the receiver operating characteristic curve [AUROC], 0.851), which was significantly higher than that for GGT. The cutoff value for identifying heavy drinkers was 2.08% (sensitivity, 65.5%; specificity, 86.8%; and AUROC, 0.815). Multiple regression analysis revealed that this proportion was negatively correlated with body mass index, whereas GGT and MCV were influenced by multiple factors involved in liver injury and dyslipidemia. CONCLUSIONS: %CDT showed a strong correlation with alcohol consumption, independent of liver damage, steatosis/stiffness, or metabolic syndrome-related factors, indicating that it is a useful drinking marker for the accurate diagnosis of NAFLD and ALD.

Gender-specific development of experimental autoimmune cholangitis induced by double-stranded RNA.

Here we investigated the gender difference in murine cholangitis resembling human primary biliary cholangitis (PBC) caused by synthetic double-stranded RNA, and underlying hepatic innate immune responses. Female C57Bl/6 mice given repeated injections of polyinosinic-polycytidylic acid (poly I:C) for 24 weeks developed overt cholangitis with positive serum anti-mitochondria-M2 antibody, whereas male mice showed minimal pathological changes without induction in autoantibody. Poly I:C induced hepatic inflammatory cytokines and type-I interferons predominantly in females. Hepatic expression levels of toll-like receptor (TLR) 3 and melanoma differentiation-associated protein (MDA) 5 were equivalent in both genders; however, both mRNA and protein levels of retinoic acid-inducible gene (RIG)-I were nearly doubled in female livers. Following 4-week injections of poly I:C, not only hepatic RIG-I, but also TLR3 and MDA5 showed female-predominance. Moreover, hepatic RIG-I levels were 25% lower in ovariectomized mice, whereas supplementation of 17 ß-estradiol enhanced hepatic RIG-I expression, as well as cytokine induction. These results clearly indicate that hepatic RIG-I expression is potentiated by estrogen, and triggers gender-dependent hepatic innate immune response against double-stranded RNA, which most likely play a pivotal role in the pathogenesis of autoimmune cholangiopathies including PBC.

Nonalcoholic fatty liver disease and alcohol-related liver disease: From clinical aspects to pathophysiological insights.

Two major causes of steatohepatitis are alcohol and metabolic syndrome. Although the underlying causes of alcoholrelated liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) differ, there are certain similarities in terms of the mode of disease progression and underlying pathophysiological mechanisms. Further, excessive alcohol consumption is often seen in patients with metabolic syndrome, and alcoholic hepatitis exacerbation by comorbidity with metabolic syndrome is an emerging clinical problem. There are certain ethnic differences in the development of both NAFLD and ALD. Especially, Asian populations tend to be more susceptible to NAFLD, and genetic polymorphisms in patatin-like phospholipase domain-containing 3 (PNPLA3) play a key role in both NAFLD and ALD. From the viewpoint of pathophysiology, cellular stress responses, including autophagy and endoplasmic reticulum (ER) stress, are involved in the development of cellular injury in steatohepatitis. Further, gutderived bacterial products and innate immune responses in the liver most likely play a profound role in the pathogenesis of both ALD and NASH. Though the recent progress in the treatment of viral hepatitis has reduced the prevalence of viral-related development of hepatocellular carcinoma (HCC), non-viral HCC is increasing. Alcohol and metabolic syndrome synergistically exacerbate progression of steatohepatitis, resulting in carcinogenesis. The gut-liver axis is a potential therapeutic and prophylactic target for steatohepatitis and subsequent carcinogenesis.

Aging exacerbates high-fat diet-induced steatohepatitis through alteration in hepatic lipid metabolism in mice.

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 ß 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.

Rifaximin prevents ethanol-induced liver injury in obese KK-Ay mice through modulation of small intestinal microbiota signature.

Exacerbation of alcoholic hepatitis (AH) with comorbid metabolic syndrome is an emerging clinical problem, where microbiota plays a profound role in the pathogenesis. Here, we investigated the effect of rifaximin (RFX) on liver injury following chronic-binge ethanol (EtOH) administration in KK-Ay mice, a rodent model of metabolic syndrome. Female, 8-wk-old KK-Ay mice were fed Lieber-DeCarli diet (5% EtOH) for 10 days, following a single EtOH gavage (4 g/kg body wt). Some mice were given RFX (0.1 g/L, in liquid diet) orally. Small intestinal contents were collected from mice without binge. Intestinal microbiota was quantified using aerobic and anaerobic culturing techniques and further analyzed by 16S rRNA sequencing in detail. EtOH feeding/binge caused hepatic steatosis, oxidative stress, and induction of inflammatory cytokines in KK-Ay mice, which were markedly prevented by RFX treatment. Hepatic mRNA levels for cluster of differentiation 14, Toll-like receptor (TLR) 4, TLR2, and NADPH oxidase 2 were increased following EtOH feeding/binge, and administration of RFX completely suppressed their increase. The net amount of small intestinal bacteria was increased over threefold after chronic EtOH feeding as expected; however, RFX did not prevent this net increase. Intriguingly, the profile of small intestinal microbiota was dramatically changed following EtOH feeding in the order level, where the Erysipelotrichales predominated in the relative abundance. In sharp contrast, RFX drastically blunted the EtOH-induced increases in the Erysipelotrichales almost completely, with increased proportion of the Bacteroidales. In conclusion, RFX prevents AH through modulation of small intestinal microbiota/innate immune responses in obese KK-Ay mice.NEW & NOTEWORTHY Here we demonstrated that rifaximin (RFX) prevents chronic-binge ethanol (EtOH)-induced steatohepatitis in KK-Ay mice. Chronic EtOH feeding caused small intestinal bacterial overgrowth, with drastic alteration in the microbiota profile predominating the order Erysipelotrichales. RFX minimized this EtOH induction in Erysipelotrichales with substitutive increases in Bacteroidales. RFX also prevented EtOH-induced increases in portal lipopolysaccharide, and hepatic cluster of differentiation 14, toll-like receptor (TLR) 2, and TLR4 mRNA levels, suggesting the potential involvement of microbiota-related innate immune responses.

The Chemical Chaperone 4-Phenylbutyric Acid Prevents Alcohol-Induced Liver Injury in Obese KK-Ay Mice.

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.

Cathepsin L-deficiency enhances liver regeneration after partial hepatectomy.

AIM: Cathepsin L (Ctsl) plays a pivotal role in lysosomal and autophagic proteolysis. Previous investigations revealed that partial hepatectomy (PH) decreases biosynthesis of cathepsins in liver, followed by suppression of lysosomal and autophagic proteolysis during liver regeneration. Conversely, it was reported that autophagy-deficiency suppressed liver regeneration. Thus, the purpose of this study is to determine if Ctsl deficiency affects liver regeneration after PH. METHODS: 70% of PH was performed in male Ctsl-deficient mice (Ctsl-/-) and wild-type littermates (Ctsl +/+) after PH. Mice were sacrificed and wet weight of the whole remaining liver was measured. Bromodeoxyuridine (BrdU)-immunostaining of liver sections was performed. Expression of cyclin D1, p62, LC-3, Nrf2, cleaved-Notch1, Hes1 was evaluated by western blot analysis. NQO1 mRNA expression was measured by realtime-PCR. RESULTS: After a 70% of PH, the liver mass was significantly restored within 5 days in Ctsl-/- mice compared to wild-type. Ctsl-deficiency enhanced the increases in both the rate of BrdU-positive cells and cyclin D1 expression after PH more than wild-type mice. On the other hand, Ctsl-deficiency upregulated p62, cleaved-Notch1 and Hes1 expression after PH. Moreover, the protein level of Nrf2 in the nucleus and mRNA expression of NQO1 in the liver after PH was also up-regulated in Ctsl-/- mice. CONCLUSIONS: These findings suggest that accumulation of p62 due to loss of Ctsl plays an important role in liver regeneration through activation of Nrf2-Notch1 signaling. Taken together, Ctsl might be a new therapeutic target on disorder of liver regeneration.

Formation of p62-positive inclusion body is associated with macrophage polarization in non-alcoholic fatty liver disease.

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.

A Customized Online Nutrition Guidance System Is Effective for Treating Patients with Nonalcoholic Fatty Liver Disease by Supporting Continuity of Diet Therapy at Home: A Pilot Study.

Continuity is required for diet therapy, but it depends on patients. We examined the utility of a new tool, the customized online nutrition guidance system, in patients with nonalcoholic fatty liver disease (NAFLD). Seven patients plotted their body weight (BW) and marked a customized task card on completion for 90 days on a website. The instructors encouraged them by e-mail. BW, serum transaminase levels, and system usage were evaluated. The results showed that BW and serum alanine aminotransferase levels were significantly lower than at baseline. BW and task visualization as well as encouragement by e-mails were effective in ensuring continuity. Thus, this system is effective in keeping NAFLD patients motivated to continue their diet therapy.

Spleen-derived lipocalin-2 in the portal vein regulates Kupffer cells activation and attenuates the development of liver fibrosis in mice.

The liver has an immune tolerance against gut-derived products from the portal vein (PV). A disruption of the gut-liver axis leads to liver injury and fibrosis. The spleen is connected to the PV and regulates immune functions. However, possible splenic effects on liver fibrosis development are unclear. Lipocalin-2 (Lcn2) is an antimicrobial protein that regulates macrophage activation. To clarify the role of the spleen in liver fibrosis development, we induced liver fibrosis in mice after splenectomy, and investigated liver fibrosis development. Liver fibrosis resulted in significantly increased splenic Lcn2 levels, but all other measured cytokine levels were unchanged. Splenectomized mice showed enhanced liver fibrosis and inflammation accompanied by significantly decreased Lcn2 levels in PV. Lipopolysaccharide-stimulated primary Kupffer cells, resident liver macrophages, which were treated with recombinant Lcn2 (rLcn2) produced less tumor necrosis factor-α and Ccl2 and the activation of hepatic stellate cells, the effector cells for collagen production in the liver, was suppressed by co-culture with rLcn2-treated Kupffer cells. In addition, the involvement of gut-derived products in splenectomized mice was evaluated by gut sterilization. Interestingly, gut sterilization blocked the effect of splenectomy on liver fibrosis development. In conclusion, spleen deficiency accelerated liver fibrosis development and decreased PV Lcn2 levels. The mechanism of splenic protection against liver fibrosis development may involve the splenic Lcn2, triggered by gut-derived products that enter the liver through the PV, regulates Kupffer cells activated by the gut-liver axis. Thus, the splenic Lcn2 may have an important role in regulating the immune tolerance of the liver in liver fibrosis development.

Sodium 4-phenylbutyric acid prevents murine acetaminophen hepatotoxicity by minimizing endoplasmic reticulum stress.

BACKGROUND: Acetaminophen (APAP) overdose induces severe oxidative stress followed by hepatocyte apoptosis/necrosis. Previous studies have indicated that endoplasmic reticulum (ER) stress is involved in the cell death process. Therefore, we investigated the effect of the chemical chaperone 4-phenyl butyric acid (PBA) on APAP-induced liver injury in mice. METHODS: Eight-week-old male C57Bl6/J mice were given a single intraperitoneal (i.p.) injection of APAP (450 mg/kg body weight), following which some were repeatedly injected with PBA (120 mg/kg body weight, i.p.) every 3 h starting at 0.5 h after the APAP challenge. All mice were then serially euthanized up to 12 h later. RESULTS: PBA treatment dramatically ameliorated the massive hepatocyte apoptosis/necrosis that was observed 6 h after APAP administration. PBA also significantly prevented the APAP-induced increases in cleaved activating transcription factor 6 and phosphorylation of c-Jun N-terminal protein kinase and significantly blunted the increases in mRNA levels for binding immunoglobulin protein, spliced X-box binding protein-1, and C/EBP homologous protein. Moreover, PBA significantly prevented APAP-induced Bax translocation to the mitochondria, and the expression of heme oxygenase-1 mRNA and 4-hydroxynonenal. By contrast, PBA did not affect hepatic glutathione depletion following APAP administration, reflecting APAP metabolism. CONCLUSIONS: PBA prevents APAP-induced liver injury even when an APAP challenge precedes its administration. The underlying mechanism of action most likely involves the prevention of ER stress-induced apoptosis/necrosis in the hepatocytes during APAP intoxication.

L-carnitine prevents metabolic steatohepatitis in obese diabetic KK-Ay mice.

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 ß-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 ß-hydroxybutyrate in the liver was not affected by HFD alone. In contrast, L-carnitine treatment significantly increased hepatic ß-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 ß-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.

Glycine prevents metabolic steatohepatitis in diabetic KK-Ay mice through modulation of hepatic innate immunity.

Strategies for prevention and treatment of nonalcoholic steatohepatitis remain to be established. We evaluated the effect of glycine on metabolic steatohepatitis in genetically obese, diabetic KK-Ay mice. Male KK-Ay mice were fed a diet containing 5% glycine for 4 wk, and liver pathology was evaluated. Hepatic mRNA levels for lipid-regulating molecules, cytokines/chemokines, and macrophage M1/M2 markers were determined by real-time RT-PCR. Hepatic expression of natural killer (NK) T cells was analyzed by flow cytometry. Body weight gain was significantly blunted and development of hepatic steatosis and inflammatory infiltration were remarkably prevented in mice fed the glycine-containing diet compared with controls. Indeed, hepatic induction levels of molecules related to lipogenesis were largely blunted in the glycine diet-fed mice. Elevations of hepatic mRNA levels for TNFα and chemokine (C-C motif) ligand 2 were also remarkably blunted in the glycine diet-fed mice. Furthermore, suppression of hepatic NK T cells was reversed in glycine diet-fed KK-Ay mice, and basal hepatic expression levels of NK T cell-derived cytokines, such as IL-4 and IL-13, were increased. Moreover, hepatic mRNA levels of arginase-1, a marker of macrophage M2 transformation, were significantly increased in glycine diet-fed mice. In addition, dietary glycine improved glucose tolerance and hyperinsulinemia in KK-Ay mice. These observations clearly indicate that glycine prevents maturity-onset obesity and metabolic steatohepatitis in genetically diabetic KK-Ay mice. The underlying mechanisms most likely include normalization of hepatic innate immune responses involving NK T cells and M2 transformation of Kupffer cells. It is proposed that glycine is a promising immunonutrient for prevention and treatment of metabolic syndrome-related nonalcoholic steatohepatitis.

Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis.

Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis. The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD.

Sodium 4-phenylbutyrate prevents murine dietary steatohepatitis caused by trans-fatty acid plus fructose.

Excess consumption of trans-fatty acid could increase the risk of non-alcoholic steatohepatitis (NASH); however, treatment targeting trans-fatty acid-induced NASH has not been examined. Here we focused on the influence of trans-fatty acid intake on endoplasmic reticulum (ER) stress in hepatocytes, so we investigated the effect of the chemical chaperone 4-phenylbutyric acid (PBA), on trans-fatty acid-caused steatohepatitis using diabetic KK-A(y) mice. Elaidic acid (EA, trans-fatty acid) alone did not cause definitive liver injury. In contrast, EA plus low-dose fructose induced extensive apoptosis in hepatocytes with severe fat accumulation. EA plus fructose significantly increased ER stress markers such as glucose-regulated protein 78 (GRP78), eukaryotic initiation factor 2α (eIF2α) and phosphorylated c-jun N-terminal kinase (JNK), while PBA significantly reduced this response. In vitro, EA promoted expression of GRP78 and phosphorylation of eIF2α in primary-cultured hepatocytes. EA also increased hepatocellular susceptibility to low-dose tert-butyl hydroperoxide. Treatment with PBA significantly reduced these responses. In conclusion, EA potentiates susceptibly to non-hazardous dose of fructose, and increases ER and oxidative stress. PBA improved steatohepatitis induced by EA plus fructose through amelioration of ER stress. Therefore, ER stress-targeted therapy using a chemical chaperone is a promising novel strategy for trans-fatty acid-induced steatohepatitis.

Recurrent severe acute hepatitis caused by hypereosinophilic syndrome associated with elevated serum immunoglobulin G4 levels.

A 46-year-old male was admitted to our hospital with severe acute hepatitis, hypereosinophilia, and serum immunoglobulin G4 (IgG4) elevation. Plasma exchange was performed, and he was treated by prednisolone; however, his hepatitis recurred twice over the following twelve months. Transjuglar liver biopsy was performed at the third onset, which demonstrated extensive hepatocyte necrosis, congestion, and severe eosinophil infiltration. We diagnosed hypereosinophilic syndrome (HES)-related hepatitis. Although no cholangitis was detected by imaging and pathological diagnosis, IgG4-positive cells were detected in the liver and bone marrow. Furthermore, the elevation of serum IgG4 levels was associated with the eosinophil count and his clinical condition. After the addition of azathioprine to his treatment regimen, no reoccurrence was observed. IgG4-positive cells may have contributed to the severity and refractoriness of this recurrent acute HES-related hepatitis.

Abnormality of autophagic function and cathepsin expression in the liver from patients with non-alcoholic fatty liver disease.

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.