Betaine-homocysteine methyltransferase (BHMT) is an enzyme involved in one-carbon metabolism and plays a crucial role in maintaining liver health. In this study, we investigated the impact of liver-specific deletion of BHMT on liver dysfunction using a mouse model. We generated BHMT floxed mice and bred them with albumin Cre to generate liver-specific BHMT knockout (BHMT LKO) mice. Liver tissues harvested from six-month-old chow-fed BHMT floxed and LKO mice were characterized through histological, biochemical, and molecular analyses. BHMT LKO mice displayed a complete loss of hepatic expression of BHMT mRNA, protein and enzyme activity. Histopathological analysis revealed the development of hepatic steatosis in BHMT LKO mice compared to the floxed mice. These morphological changes were supported by biochemical analysis showing elevated levels of hepatic triglycerides in conjunction with a profound decrease in the methylation potential (i.e., reduced S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) ratio), which was mainly driven by a six- to sevenfold increase in SAH levels. BHMT LKO mice also exhibited increased lipid peroxidation and lysosomal dysfunction compared to floxed mice. Early signs of inflammation were seen in the livers of BHMT LKO mice of both sexes, as evident from significant increase in CD68-positive cells and interleukin 1β levels. Additionally, there was a moderate increase in fibrosis, as evidenced by the upregulated expression of α-smooth muscle actin and collagen II levels and the histological assessment of picrosirius red-stained liver sections of BHMT LKO mice of both sexes compared to their respective counterparts. These findings demonstrate that hepatic BHMT deficiency promotes lipid accumulation, lysosomal/proteasomal dysfunction, and early inflammatory and fibrotic changes in the liver by reducing the methylation potential. Collectively, our results underscore BHMT as a critical regulator of liver homeostasis and a potential therapeutic target in liver-related disorders.

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the transfer of methyl groups to phosphatidylethanolamine to generate phosphatidylcholine (PC). PC produced de novo through this pathway is preferentially used for very-low-density lipoprotein assembly and is essential for its normal hepatic secretion as well as for bile acid detoxification. While human PEMT loss-of-function polymorphisms are linked to increased metabolic dysfunction-associated steatotic liver disease risk, the enzyme's role as a primary driver of progressive liver disease remains underexplored. We utilized PEMT knockout (PEMT KO) mice on a standard chow diet to model this deficiency. Histopathological analysis showed that while 2-month-old PEMT KO mice were protected, 6-month-old KOs of both sexes spontaneously developed extensive micro-and macrovesicular steatosis, parenchymal inflammation, and granulomatous inclusions. This severe, age-dependent pathology was confirmed by elevated hepatic triglyceride levels, bile acids, impaired methylation potential, and a cascade of secondary injuries, including increased oxidative stress, impaired proteasomal function, and the induction of cellular senescence markers. This bile acid toxicity and oxidative stress activated the central innate immune sensor, the NLRP3 inflammasome, driving pronounced macrophage infiltration and chronic inflammation. Picrosirius red staining and protein analysis confirmed the progression to severe pericellular fibrosis. Our study establishes that PEMT deficiency is sufficient to initiate and drive the complete progression from steatosis to advanced liver fibrosis, identifying PEMT as a critical metabolic checkpoint and a potent therapeutic target for mitigating progressive liver disease.

The global incidence of steatotic liver disease (SLD), driven by metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), and the synergistic metabolic dysfunction and alcohol-associated liver disease (MetALD), is fueling a rapid rise in hepatocellular carcinoma (HCC). Regardless of the initial etiology (metabolic, alcoholic, or viral), progression to advanced SLD and HCC is governed by critical shared pathways, which are systemic metabolic dysregulation and inflammation. Identifying a single, targeted pharmacological agent to address this unified pathophysiology is an urgent unmet need. This review addresses the epidemiological links between SLD etiologies and HCC, dissecting their shared metabolic pathophysiology. We evaluate the emerging potential of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) as a multifunctional therapeutic strategy to target this metabolic hepatic nexus. GLP-1 RAs offer a dual central and peripheral mechanism against SLD progression. Centrally, these GLP-1 RAs modulate appetite and reduce the intake and cravings for high-calorie food and alcohol. Peripherally, these agents induce significant weight loss, enhance insulin sensitivity, and reduce hepatic de novo lipogenesis. While their efficacy in resolving metabolic dysfunction-associated steatohepatitis (MASH) is increasingly well documented, their ability to target the metabolic hepatic nexus also suggests a promising therapeutic role in ALD and MetALD. Furthermore, GLP-1 RAs appear to exert direct anti-inflammatory and anticancer effects by activating metabolic sensors, such as the AMPK pathway, to inhibit proliferative signaling. Clinical and preclinical data support the efficacy of GLP-1 RAs in resolving steatosis and SLD progression in metabolic contexts. By targeting shared metabolic dysregulation, GLP-1 RAs emerge as potential candidates for HCC risk mitigation and may serve as future therapeutic adjuvants. Future large-scale, prospective clinical trials are warranted to confirm these benefits, particularly in ALD and MetALD, where underlying metabolic dysfunction remains a significant driver of disease progression.

BACKGROUND:Ghrelin, a stomach-derived orexigenic peptide, typically rises during fasting to stimulate food intake. Chronic alcohol consumption elevates circulating ghrelin, which induces alcohol craving and increases intake in clinical and preclinical models. By increasing alcohol drive, ghrelin contributes to alcohol use disorder (AUD) and accelerates alcohol-associated liver disease (ALD) by inhibiting insulin secretion and promoting adipose lipolysis. However, the cellular mechanisms by which ethanol dysregulates ghrelin secretion remain unclear. This study investigated how ethanol modulates ghrelin production and disrupts nutrient sensing in ghrelin-secreting cells. METHODS:Stomach (SG-1) and pancreatic (PG-1) ghrelinoma cells were treated with 25 mM or 50 mM ethanol for 48 h, after which ghrelin synthesis and secretion were measured. Further, the interaction between ethanol and other physiological regulators (glucose [10 mM], insulin [20 mM], and palmitic acid [400 μM]) of ghrelin secretion was evaluated. Intracellular glycolytic rate was characterized using Seahorse-XFe analysis. RESULTS:Chronic ethanol exposure (48 h) significantly increased ghrelin secretion and the mRNA encoding for ghrelin and ghrelin O-acyltransferase (GOAT), an enzyme that activates ghrelin. Under normal conditions, high glucose effectively suppressed ghrelin secretion; however, ethanol pretreatment blunted this inhibitory effect. While palmitic acid alone had no effect, its combination with ethanol synergistically enhanced ghrelin secretion. Mechanistically, ethanol-pretreated cells exhibited a metabolic impairment characterized by increased glucose uptake but significantly reduced basal glycolysis, proton efflux rate (PER), and compensatory glycolytic capacity. This metabolic failure was linked to the profound downregulation of the rate-limiting enzymes glucokinase, hexokinase, and pyruvate kinase. CONCLUSIONS:Ethanol drives increased ghrelin by disrupting the glucose-sensing machinery within ghrelin-secreting cells. By suppressing key glycolytic enzymes, ethanol uncouples the cell from glucose, mimicking a state of starvation despite nutrient availability. Targeting these metabolic pathways may provide a novel therapeutic strategy for the interconnected pathologies of AUD and ALD.

This study investigated the complex interplay between two gut hormones, glucagon-like peptide-1 (GLP-1) and ghrelin and their role in the development of alcohol-associated liver disease (ALD). Previous studies conducted in our laboratory and others have shown that chronic alcohol exposure leads to increased serum ghrelin and GLP-1 levels. Paradoxically, despite increased GLP-1, insulin resistance and disrupted hepatic lipid metabolism was noted in chronic ethanol fed animals. These results suggested impaired GLP-1-mediated protective function in the presence of high ghrelin. Our recent studies also revealed that growth hormone secretagogue receptor (GHSR, which is the ghrelin receptor) knockout (GHSR-KO) rats fed ethanol were more insulin sensitive and were resistant to develop ALD despite reduced serum GLP-1 compared to ethanol-fed wildtypes. Based on these considerations, we hypothesized that alcohol-induced increases in ghrelin-GHSR interaction impairs GLP-1-mediated functions. We employed both in vivo and in vitro approaches, including chronic ethanol feeding of wild-type and GHSR-KO rats, ghrelin administration to chow-fed rats, GSHR and GLP-1 receptor (GLP-1R)-transfected hepatocytes, murine enteroendocrine GLUTag and HEK293T cells utilizing several techniques to test our hypothesis. Chronic ethanol feeding in wildtype rats increased GLP-1 and GLP-1R levels, while ethanol-fed GHSR-KO rats did not show this increase. Ghrelin promoted GHSR and GLP-1R interaction/dimerization, thereby reducing GLP-1 effects. Furthermore, in-silico molecular docking analysis identified specific amino acid residues in the transmembrane regions of both receptors that are predicted to mediate this interaction. Alcohol-induced increases in ghrelin modulate GLP-1-mediated functions through GHSR-GLP-1R interactions. Targeting this interaction could offer a potential therapeutic strategy for ALD.

BACKGROUND:There is a robust link between chronic alcohol intake and the development of alcohol-associated liver disease (ALD). Over 90% of excessive alcohol drinkers develop hepatic steatosis that can progress to an advanced liver injury state. However, this progression depends on many extrahepatic factors including age, which is also a predictor of ALD-related mortality. This study aimed to identify selected pathological changes in rats of different ages with chronic ethanol administration for the same duration to gain insights into the effects of aging in the development and progression of ALD. METHODS:Male Wistar rats of young (4 months), middle (8-12 months), and older (24 months) age were pair-fed for 6 weeks with Lieber-DeCarli control or ethanol diet. At the end of the experimental period, rats were euthanized and serum and tissues (liver, gut, and adipose) were collected for analyses. RESULTS:Chronic ethanol feeding increased serum hepatic injury markers, circulating nonesterified free fatty acids, and hepatic triglycerides across the different age groups compared to their respective controls, with the higher levels seen in the middle-aged and old ethanol-fed rats compared to young ethanol-fed rats. Further, histopathological evaluation and quantitative analysis of inflammatory and fibrotic markers revealed more progressive liver injury in older ethanol-fed rats compared to young and middle-aged counterparts. We also observed increased intestinal permeability, as indicated by lower ileal expression of tight junction proteins and higher serum endotoxin levels in older ethanol-fed rats. Aging alone adversely affected several of these injury markers in older control-fed rats compared to middle-aged and young control-fed rats. CONCLUSION:Our findings indicate that aging significantly influences the development of liver injury after chronic alcohol intake.

Aging is a critical factor influencing susceptibility to hepatic injury. In this study, the spontaneous development of liver injury with advancing age and potential sex-related differences in these processes are examined. This study focuses on key mechanisms such as fatty acid metabolism, immune response, and cellular stress in male and female C57BL/6 mice. Aged male and female mice (20 to 22 months old) exhibited higher body weight and an altered metabolic profile and fatty acid metabolism compared to their younger counterparts (8 to 10 weeks old). In addition, increased oxidative stress, cellular senescence, expression of inflammatory markers, and cytokines/chemokines levels were also observed in aged male and female mice compared to younger mice. Furthermore, the aged mice exhibited increased indices of hepatic fibrosis, evident from the upregulation of smooth muscle actin-α, collagen, and transforming growth factor-β. In conclusion, aging promotes spontaneous liver injury by increasing indices of oxidative stress, steatosis, inflammation, and fibrosis. These results highlight the impact of chronological age on the liver that can increase its susceptibility to secondary hepatic stressors such as alcohol, high-calorie diet, or hepatotropic infections. Understanding how metabolic and inflammatory pathways change with aging in males and females is essential for elucidating the mechanisms that drive chronic liver disease progression. These insights are particularly important for developing targeted, sex-specific prevention and therapeutic strategies for the aging population.

Quorum sensing (QS) is the ability of bacteria to monitor their population density and adjust gene expression accordingly. QS-regulated processes include host-microbe interactions, horizontal gene transfer, and multicellular behaviours (such as the growth and development of biofilm). The creation, transfer, and perception of bacterial chemicals known as autoinducers or QS signals are necessary for QS signalling (e.g. N-acylhomoserine lactones). Quorum quenching (QQ), another name for the disruption of QS signalling, comprises a wide range of events and mechanisms that are described and analysed in this study. In order to better comprehend the targets of the QQ phenomena that organisms have naturally developed and are currently being actively researched from practical perspectives, we first surveyed the diversity of QS-signals and QS-associated responses. Next, the mechanisms, molecular players, and targets related to QS interference are discussed, with a focus on natural QQ enzymes and compounds that function as QS inhibitors. To illustrate the processes and biological functions of QS inhibition in microbe -microbe and host-microbe interactions, a few QQ paradigms are described in detail. Finally, certain QQ techniques are offered as potential instruments in a variety of industries, including agriculture, medical, aquaculture, crop production, and anti-biofouling areas. Published by Elsevier Masson SAS on behalf of Institut Pasteur.

The development of alcohol-associated liver disease (ALD) is associated with disorganized Golgi apparatus and accelerated phagophore formation. While Golgi membranes may contribute to phagophores, association between Golgi alterations and macroautophagy/autophagy remains unclear. GOLGA4/p230 (golgin A4), a dimeric Golgi matrix protein, participates in phagophore formation, but the underlying mechanism is elusive. Our prior research identified ethanol (EtOH)-induced Golgi scattering, disrupting intra-Golgi trafficking and depleting RAB3D GTPase from the trans-Golgi. Employing various techniques, we analyzed diverse cellular and animal models representing chronic and chronic/binge alcohol consumption. In trans-Golgi of non-treated hepatocytes, we found a triple complex formed between RAB3D, GOLGA4, and MYH10/NMIIB (myosin, heavy polypeptide 10, non-muscle). However, EtOH-induced RAB3D downregulation led to MYH10 segregation from the Golgi, accompanied by Golgi fragmentation and tethering of the MYH10 isoform, MYH9/NMIIA, to dispersed Golgi membranes. EtOH-activated autophagic flux is evident through increased WIPI2 recruitment to the Golgi, phagophore formation, enhanced LC3B lipidation, and reduced SQSTM1/p62. Although GOLGA4 dimerization and intra-Golgi localization are unaffected, loss of RAB3D leads to an extension of the cytoplasmic N terminal domain of GOLGA4, forming GOLGA4-positive phagophores. Autophagy inhibition by hydroxychloroquine (HCQ) prevents alcohol-mediated Golgi disorganization, restores distribution of ASGR (asialoglycoprotein receptor), and mitigates COL (collagen) deposition and steatosis. In contrast to short-term exposure to HCQ, extended co-treatment with both EtOH and HCQ results in the depletion of LC3B protein via proteasomal degradation. Thus, (a) RAB3D deficiency and GOLGA4 conformational changes are pivotal in MYH9-driven, EtOH-mediated Golgiphagy, and (b) HCQ treatment holds promise as a therapeutic approach for alcohol-induced liver injury.Abbreviation: ACTB: actin, beta; ALD: alcohol-associated liver disease; ASGR: asialoglycoprotein receptor; AV: autophagic vacuoles; EM: electron microscopy; ER: endoplasmic reticulum; EtOH: ethanol; HCQ: hydroxychloroquine; IP: immunoprecipitation; KD: knockdown; KO: knockout; MYH10/NMIIB: myosin, heavy polypeptide 10, non-muscle; MYH9/NMIIA: myosin, heavy polypeptide 9, non-muscle; PLA: proximity ligation assay; ORO: Oil Red O staining; PM: plasma membrane; TGN: trans-Golgi network; SIM: structured illumination super-resolution microscopy.

Fatty liver is the earliest response of the liver to excessive alcohol consumption. Previously we identified that chronic alcohol administration increases levels of stomach-derived hormone, ghrelin, which by reducing circulating insulin levels, ultimately contributes to the development of alcohol-associated liver disease (ALD). In addition, ghrelin directly promotes fat accumulation in hepatocytes by enhancing de novo lipogenesis. Other than promoting ALD, ghrelin is known to increase alcohol craving and intake. In this study, we used a ghrelin receptor (GHSR) knockout (KO) rat model to characterize the specific contribution of ghrelin in the development of ALD with emphasis on energy homeostasis. Male Wistar wild type (WT) and GHSR-KO rats were pair-fed the Lieber-DeCarli control or ethanol diet for 6 weeks. At the end of the feeding period, glucose tolerance test was conducted, and tissue samples were collected. We observed reduced alcohol intake by GHSR-KOs compared to a previous study where WT rats were fed ethanol diet ad libitum. Further, when the WTs were pair-fed to GHSR-KOs, the KO rats exhibited resistance to develop ALD through improving insulin secretion/sensitivity to reduce adipose lipolysis and hepatic fatty acid uptake/synthesis and increase fatty acid oxidation. Furthermore, proteomic data revealed that ethanol-fed KO exhibit less alcohol-induced mitochondrial dysfunction and oxidative stress than WT rats. Proteomic data also confirmed that the ethanol-fed KOs are insulin sensitive and are resistant to hepatic steatosis development compared to WT rats. Together, these data confirm that inhibiting ghrelin action prevent alcohol-induced liver and adipose dysfunction independent of reducing alcohol intake.

Fatty liver is the earliest response to excessive ethanol consumption, which increases the susceptibility of the liver to develop advanced stage of liver disease. Our previous studies have revealed that chronic alcohol administration alters metabolic hormone levels and their functions. Of current interest to our laboratory is glucagon-like peptide 1 (GLP-1), a widely studied hormone known to reduce insulin resistance and hepatic fat accumulation in patients with metabolic-associated fatty liver disease. In this study, we examined the beneficial effects of exendin-4 (a GLP-1 receptor agonist) in an experimental rat model of ALD. Male Wistar rats were pairfed the Lieber-DeCarli control or ethanol diet. After 4 weeks of this feeding regimen, a subset of rats in each group were intraperitoneally injected every other day with either saline or exendin-4 at a dose of 3 nmol/kg/day (total 13 doses) while still being fed their respective diet. At the end of the treatment, rats were fasted for 6 h and glucose tolerance test was conducted. The following day, the rats were euthanized, and the blood and tissue samples collected for subsequent analysis. We found that exendin-4 treatment had no significant effect on body weight gain among the experimental groups. Exendin-4-treated ethanol rats exhibited improved alcohol-induced alterations in liver/body weight and adipose/body weight ratio, serum ALT, NEFA, insulin, adiponectin and hepatic triglyceride levels. Reduction in indices of hepatic steatosis in exendin-4 treated ethanol-fed rats was attributed to improved insulin signaling and fat metabolism. These results strongly suggest that exendin-4 mitigates alcohol-associated hepatic steatosis by regulating fat metabolism.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ1–42) fibril formation in vitro and suppressed Aβ1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.