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Lipid rafts are subdomains of the cell membrane that are rich in cholesterol and glycolipids, and they are involved in various cellular processes and pathophysiological mechanisms. However, the specific role of lipid rafts in hepatocyte dysfunction during the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) is not fully understood. In this study, we investigated the impact of lipid rafts on insulin sensitivity and hepatocyte injury induced by saturated free fatty acids (sFFAs) using primary-cultured mouse hepatocytes. Treatment of primary hepatocytes with palmitic acid (PA) resulted in significant lipid droplet accumulation in the cytoplasm and a marked increase in the number of cells with accumulation of lipid raft. The addition of cholesterol oxidase (CHOX), which disrupts lipid rafts, did not affect PA-induced lipid droplet formation, but significantly reduced the number of cells with accumulation of lipid raft. In PA-treated hepatocytes, insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-2 and Akt was markedly decreased, but this effect was alleviated by CHOX. Furthermore, PA-pretreated hepatocytes exhibited substantial increases in reactive oxygen species (ROS) production and cell death when exposed to low doses of tert-butyl hydroperoxide (t-BuOOH). In contrast, treatment with CHOX after PA significantly reduced ROS production and cell death following t-BuOOH. These results suggest that PA-induced accumulation of lipid raft not only exacerbates insulin resistance but also enhances responsiveness to oxidative stress stimuli, contributing to MASLD pathogenesis. Modulation of lipid rafts may represent a promising therapeutic target for MASLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Unlabelled: Iron overload exacerbates various liver diseases. In hepatocytes, a portion of non-heme iron is sequestered in lysosomes and endosomes. The precise mechanisms by which lysosomal iron participates in hepatocellular injury remain uncertain. Here, our aim was to determine the role of intracellular movement of chelatable iron in oxidative stress-induced killing to cultured hepatocytes from C3Heb mice and Sprague-Dawley rats. Mitochondrial polarization and chelatable iron were visualized by confocal microscopy of tetramethylrhodamine methylester (TMRM) and quenching of calcein, respectively. Cell viability and hydroperoxide formation (a measure of lipid peroxidation) were measured fluorometrically using propidium iodide and chloromethyl dihydrodichlorofluorescein, respectively. After collapse of lysosomal/endosomal acidic pH gradients with bafilomycin (50 nM), an inhibitor of the vacuolar proton-pumping adenosine triphosphatase, cytosolic calcein fluorescence became quenched. Deferoxamine mesylate and starch-deferoxamine (1 mM) prevented bafilomycin-induced calcein quenching, indicating that bafilomycin induced release of chelatable iron from lysosomes/endosomes. Bafilomycin also quenched calcein fluorescence in mitochondria, which was blocked by 20 microM Ru360, an inhibitor of the mitochondrial calcium uniporter, consistent with mitochondrial iron uptake by the uniporter. Bafilomycin alone was not sufficient to induce mitochondrial depolarization and cell killing, but in the presence of low-dose tert-butylhydroperoxide (25 microM), bafilomycin enhanced hydroperoxide generation, leading to mitochondrial depolarization and subsequent cell death., Conclusion: Taken together, the results are consistent with the conclusion that bafilomycin induces release of chelatable iron from lysosomes/endosomes, which is taken up by mitochondria. Oxidative stress and chelatable iron thus act as two "hits" synergistically promoting toxic radical formation, mitochondrial dysfunction, and cell death. This pathway of intracellular iron translocation is a potential therapeutic target against oxidative stress-mediated hepatotoxicity.

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]

Despite pathophysiological similarities to alcoholic liver disease, susceptibility to acetaminophen hepatotoxicity in metabolic syndrome-related nonalcoholic steatohepatitis (NASH) has not been well elucidated. In this study, therefore, we investigated acetaminophen-induced liver injury in KK-Ay mice, an animal model of metabolic syndrome. Twelve-week-old male KK-Ay and C57B1/6 mice were injected intraperitoneally with 300 or 600 mg/kg acetaminophen, and euthanized 6 h later. Liver histology was assessed, and hepatic expression of 4-hydroxy-2-nonenal was detected by immunohistochemistry. Levels of reduced glutathione were determined spectrophotometrically. Phosphorylation of c-Jun NH2-terminal kinase (JNK) was analyzed by Western blotting. Hepatocytes were isolated from both strains by collagenase perfusion, and cell death and oxidative stress were measured fluorometrically by use of propidium iodide and 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester, respectively. Acetaminophen induced more severe necrosis and apoptosis of hepatocytes in KK-Ay mice than in C57B1/6 mice and significantly increased serum alanine aminotransferase levels in KK-Ay mice. Acetaminophen-induction of 4-hydroxy-2-nonenal in the liver was potentiated, whereas the levels of reduced glutathione in liver were lower in KK-Ay mice. Acetaminophen-induced phosphorylation of JNK in the liver was also enhanced in KK-Ay mice. Exposure to 20 µM tert-butyl hydroperoxide did not kill hepatocytes isolated from C57B1/6 mice but induced cell death and higher oxidative stress in hepatocytes from KK-Ay mice. These results demonstrated that acetaminophen toxicity is increased in diabetic KK-Ay mice mainly due to enhanced oxidative stress in hepatocytes, suggesting that metabolic syndrome-related steatohepatitis is an exacerbating factor for acetaminophen-induced liver injury. [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]

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]