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.

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.

Proposal of Stroop test cut-off values as screening for neuropsychological impairments in cirrhosis: A Japanese multicenter study.

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.

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.

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.

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.

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.

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.

[Case of computed tomography (CT) gastrography for the detection of multiple gastric submucosal tumors: a case report].

A 66-year-old female underwent esophagogastroduodenoscopy, which revealed a 17-mm gastric submucosal tumor (SMT) located in the posterior wall of the upper area of the stomach. She was referred to our hospital for further investigation because of accumulation of radioactive substance in the gastric wall by PET-CT. CT gastrography (CTG) revealed three gastric SMTs in total. Partial gastrectomy was performed; histopathologically, they were diagnosed as gastrointestinal stromal tumor (GIST), benign schwannoma, and necrotic tissue. Gastric schwannoma is very rare, and we could find no cases with both GIST and schwannoma. Additionally, the three gastric SMTs were only detected clearly via CTG. We report on the details of this case and the usefulness of CTG for the detection of gastric SMT.

Innate immune responses involving natural killer and natural killer T cells promote liver regeneration after partial hepatectomy in mice.

To clarify the roles of innate immune cells in liver regeneration, here, we investigated the alteration in regenerative responses after partial hepatectomy (PH) under selective depletion of natural killer (NK) and/or NKT cells. Male, wild-type (WT; C57Bl/6), and CD1d-knockout (KO) mice were injected with anti-NK1.1 or anti-asialo ganglio-N-tetraosylceramide (GM1) antibody and then underwent the 70% PH. Regenerative responses after PH were evaluated, and hepatic expression levels of cytokines and growth factors were measured by real-time RT-PCR and ELISA. Phosphorylation of STAT3 was detected by Western blotting. Depletion of both NK and NKT cells with an anti-NK1.1 antibody in WT mice caused drastic decreases in bromodeoxyuridine uptake, expression of proliferating cell nuclear antigen, and cyclin D1, 48 h after PH. In mice given NK1.1 antibody, increases in hepatic TNF-α, IL-6/phospho-STAT3, and hepatocyte growth factor (HGF) levels following PH were also blunted significantly, whereas IFN-γ mRNA levels were not different. CD1d-KO mice per se showed normal liver regeneration; however, pretreatment with an antiasialo GM1 antibody to CD1d-KO mice, resulting in depletion of both NK and NKT cells, also blunted regenerative responses. Collectively, these observations clearly indicated that depletion of both NK and NKT cells by two different ways results in impaired liver regeneration. NK and NKT cells most likely upregulate TNF-α, IL-6/STAT3, and HGF in a coordinate fashion, thus promoting normal regenerative responses in the liver.

Altered expression and function of hepatic natural killer T cells in obese and diabetic KK-A(y) mice.

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-A(y) mice, an animal model of metabolic syndrome. METHODS: Male, 8-week-old KK-A(y) 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-A(y) 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-A(y) 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-A(y) 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-A(y) 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-A(y) mice even after HF diet feeding. CONCLUSION: KK-A(y) 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.

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.