Prostate cancer (PCa) is the second most common and a leading cause of cancer-related deaths among men. Current screening methods lack precision in distinguishing aggressive cases, emphasizing a need for tissue-based biomarkers. Although Golgi disorganization, ER stress, and elevated high-mannose (Man) glycoproteins (e.g., Integrin αv, key metastatic player) are recognized features of metastatic prostate tumors, their interrelationships remain unexplored. It is observed that the growth of primary prostate tumors is linked to an increase in endoplasmic reticulum (ER)-plasma membrane (PM) junctions signaling, mediated by STIM1 and ORP5. However, transition to lymph node and tissue metastasis is associated with their downregulation, loss of ER-PM communications, significant Golgi dispersal, and rapid conversion of high-Man glycans in the Golgi to atypical MGAT5-modified sugars that facilitate Integrin αv clustering at the PM via Galectin-3 binding. Golgi dispersal is associated with increased organelle volume and surface area to accommodate heightened trafficking and processing. These findings position STIM1 and ORP5 as biomarkers of aggressive PCa and show that high-Man enrichment is not due to defective maturation but reflects a glycan pool that cancer cells actively utilize, suggesting that the concept of ER stress response in PCa should be redefined to include Golgi reorganization and altered ER-PM junctions.

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.

Alcohol-associated liver disease (ALD) in its earliest form is evidenced as hepatic steatosis which may progress to liver cirrhosis. The mechanisms behind this are poorly understood and therapeutics limited. Liver is a specialized organ exhibiting heterogeneity along the porto-central axis. Periportal preponderance of lipid droplet accumulation was noted in human ALD livers compared to other causes of hepatic steatosis. Using single cell multiomics, we studied transcriptional mechanisms across the hepatic lobule that could account for zonation of lipid droplets in a murine ALD model. Alcohol led to periportal zonation of lipogenesis-associated genes in mice, including Hsd17b13 and Fasn. Chromatin landscape studies demonstrated zonation of master transcription factors that led to these changes in the transcriptome. We utilized these data to provide novel insight into zone-specific HNF4α and PPARα regulation of HSD17B13. We conclude novel mechanisms underlying ALD leading to spatially distinct establishment of hepatic steatosis and provide insight into disease pathogenesis.

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.

The liver stores substantial numbers of neutral lipid organelles termed lipid droplets (LDs) that accumulate within hepatocytes in response to chronic ethanol (EtOH) consumption leading to hepatic steatosis. Mass spectrometry analysis of LDs isolated from EtOH-damaged rat livers revealed a substantial reduction in the valosin-containing protein ATPase (VCP/p97) that acts to remove targeted proteins from cellular membranes for degradation. Experimental disruption of VCP function resulted in an increase in LD content in hepatocytes and mouse livers along with a marked increase in LD-associated hydroxysteroid dehydrogenase (HSD17β13) known to contribute to hepatic steatosis. Surprisingly, treatment of hepatocytes with the proteasome inhibitor MG132 had no effect on HSD17β13 levels, while a disruption of lysosome function and chaperone-mediated autophagy increased cellular HSD17β13 levels substantially. These findings provide new insights into the cellular mechanisms by which the liver regulates its lipid stores and how this is disrupted by chronic EtOH exposure.

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.

Alcohol-associated liver disease (ALD) is a major health problem of global importance, caused by chronic alcohol consumption, leading to the accumulation of reactive oxygen species (ROS) and subsequent oxidative stress—a central mechanism in liver injury. Superoxide dismutase 1 (SOD1), a Cu-Zn containing antioxidant enzyme, plays a crucial role in attenuating ALD-induced oxidative stress triggered by ethanol metabolism. However, alcohol exposure, whether chronic, acute or binge, differentially affects SOD1 levels, either diminishing its expression or temporarily compensating for alcohol-induced oxidative damage. Regardless, overexpression of SOD1 reverses early stages of ethanol-induced liver inflammation and injury in animal models, highlighting the protective role of SOD1. Current therapies, including alcohol abstinence, corticosteroids, and pentoxifylline, have limited long-term efficacy. Antioxidant-based treatments, such as N-acetylcysteine (NAC) and S-adenosyl-L-methionine (SAM), have demonstrated moderate benefits. While combination therapies like NAC with prednisolone yield more promising outcomes, these benefits are often limited in duration. The use of natural compounds including nutraceuticals and probiotics provide liver protection by enhancing antioxidant defenses, reducing inflammation, and mitigating alcohol-induced liver damage. In particular, these compounds upregulate antioxidant enzymes like SOD1. Recent research suggests that enhancing the activity of SOD1, particularly through nanoformulated SOD1 (NanoSOD1), which had direct effect on the oxidative stress at the cellular level, could offer a promising therapeutic option for ALD. NanoSOD1 aims to improve the bioavailability and stability of SOD1, offering a targeted approach to reduce oxidative stress and protect against liver damage. The effectiveness of NanoSOD1 to improve antioxidant defenses suggests a valuable therapeutic arsenal in ALD treatment. Taken together, given the limited treatment options for ALD, increasing SOD1 activity is essential for managing the progression of the disease.

Arachidonic acid (AA), an omega-6 polyunsaturated fatty acid, is abundant in animal-derived food and is widely present in phospholipids of plasma membrane. Recent studies reported that ethanol exposure leads to the activation of prostaglandin signaling via increasing the levels of AA and its metabolites in cardiomyocytes. To test the hypothesis that AA contributes at least in part, to ethanol-induced cardiomyocyte injury, a chronic ethanol feeding model was used, in which male Wistar rats were fed Lieber-Decarli ethanol diet 6.7% (v/v) or isocaloric control diet for 6 weeks. Gas chromatography analysis indicated that ethanol exposure increased the AA content in rat myocardial phospholipids along with increased protein levels of endoplasmic reticulum (ER) stress markers and a decrease in the level of NADH: ubiquinone oxidoreductase subunit B8, a mitochondrial complex I subunit. In addition, an in vitro model was used in which H9c2 cells, a rat cardiomyoblast cell line, were exposed to AA and/or ethanol (ET), and markers of steatosis and endoplasmic reticulum stress, and mitochondrial respiration were assessed. Of note, AA supplementation potentiated ethanol-induced steatosis. H9c2 cells receiving ET + AA showed an increase in the expression of ER stress markers, including glucose-regulated protein 78 and activating transcription factor 4, compared with controls. Interestingly, compared to ET treatment, ET + AA treatment led to a significant decrease in basal respiration and ATP-linked respiration, indicating an impaired mitochondrial respiration in H9c2 cardiomyoblasts. Finally, inhibiting long-chain acyl CoA synthases by Triacsin C attenuated ET + AA treatment-induced steatosis but increased mitochondrial respiration in H9c2 cells. Collectively, these data suggested that AA supplementation promotes ethanol-induced steatosis and endoplasmic reticulum stress with a concomitant impairment in mitochondrial respiration in H9c2 cardiomyoblasts, and Triacsin C treatment inhibits steatosis but enhances mitochondrial respiration possibly via altered fatty acid partitioning between synthetic and oxidative processes.

Backgrounds: Alcohol-associated liver disease (ALD) is one of the leading causes of liver diseases. Thromboxane-prostanoid receptor (TP-R) is widely expressed in the liver and is activated by thromboxane A2 (TXA2). A previous study reported an increase in TP-R mRNA level in liver after ethanol feeding. Preclinical studies have documented that TP-R antagonists exert a protective effect against cardiovascular and liver diseases. In particular, TP-R antagonist is reported to attenuate ALD in rodents. However, the mechanism(s) by which antagonizing TP-R attenuates ALD remains unclear. Methods: Herein, we used the chronic plus binge ethanol feeding model. Briefly, male, C57BL/6 wild-type mice (8-week) were fed a Lieber-Decarli ethanol (5% v/v) diet (ET, n = 10) or isocaloric control diet (CON, n = 6) for ten days followed by a single binge administration of ethanol or maltose-dextrin via oral gavage. A cohort of ethanol-fed mice received SQ 29,548, a TP-R antagonist (ET+SQ, n = 8) by oral administration. Results: Western blot analysis showed that the protein level of thromboxane A2 synthase 1 ( P<0.05), responsible for TXA2 production, was upregulated in the mouse liver upon ethanol exposure. Of note, ethanol feeding significantly increased plasma aspartate transferase (AST) level ( P<0.05), which was abolished by SQ administration ( P<0.05). Moreover, compared with CON mice, ET mice showed an increase in liver protein levels of pro-inflammatory markers, including TNFa ( P<0.05), cluster of differentiation 68 (CD68, P<0.001), and vascular cell adhesion protein 1 ( P<0.05). In addition, the pro-fibrogenic markers including collagen type 1 alpha 1 chain (COL1A1, P<0.05) and matrix metalloproteinase 9 (MMP9, P<0.01) were also increased in ET mice. However, these alterations were not found in ET+SQ mice compared to their controls. In particular, ET+SQ mice exhibited a marked reduction in hepatic protein levels of CD68 ( P<0.001), COL1A1 ( P<0.001), and MMP9 ( P<0.01) versus ET mice. Interestingly, we noted that ethanol consumption decreases the protein level of NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), a component of mitochondrial complex I, in mouse liver ( P<0.05), which was reversed by SQ administration( P<0.05). Conclusion: These findings suggest that blockade of TP-R activity attenuates ethanol-induced liver inflammation and fibrosis possibly through improving the function of mitochondrial complex I. This study was supported by the NIH-NIAAA P50 award-Alcohol Center of Research-Nebraska (P50AA030407-5130). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heavy alcohol use is the leading etiology of non-ischemic dilated cardiomyopathy. Alcohol-Associated Cardiomyopathy (ACM) is a specific cardiac muscle disease which is characterized by inflammation, abnormal fatty acid metabolism, and oxidative stress. TP-R, a G-protein coupled receptor, is widely expressed in the myocardium, and plays a critical role in the pathogenesis of various cardiac diseases including hypertensive heart disease and dilated cardiomyopathy. TP-R is activated by thromboxane A2 (TXA2) and 8-isoprostane. Of note, clinical studies have shown that chronic alcohol use markedly increases the level of thromboxane B2, a stable metabolite of TXA2, in plasma of alcohol use disorder patients. However, the role of TP-R signaling in the pathogenesis of ACM remains unclear. Therefore, we hypothesize that TP-R signaling mediates adverse effects of alcohol on the myocardium. To test this hypothesis, we used the chronic plus binge ethanol feeding model. C57BL/6 wild-type male mice (8-week) were fed with Lieber-Decarli ethanol (5% v/v) diet (ET, n = 10) or isocaloric control diet (CON, n = 6) for ten days followed by single binge of ethanol or maltose-dextrin via oral gavage. A cohort of ethanol-fed mice received SQ 29,548, a TP-R antagonist (ET+SQ, n = 8). Our data indicated that mouse myocardial protein levels of thromboxane A2 synthase (TBXAS1, P <0.001), an enzyme responsible for synthesis of TXA2, was increased in response to ethanol exposure. Concomitantly, ethanol-fed mice displayed upregulated myocardial protein levels of pro-inflammatory mediators including tumor necrosis factor alpha (TNF-α, P <0.001) and secreted interleukin 1 beta (IL1β, P <0.001) compared with their controls. These adverse alterations induced by the ethanol diet were ameliorated by the pharmacological inhibition of TP-R. Interestingly, RNA-sequencing and western blotting analysis showed that expression of thioredoxin-interacting protein (TXNIP) was upregulated in response to ethanol exposure ( P <0.001). Meanwhile, mice fed with ethanol diet had the increased myocardial protein level of NLR family pyrin domain containing 3 (NLRP3, P <0.01) compared with CON mice. These ethanol-induced increases in protein levels of TXNIP ( P <0.001) and NLRP3 ( P <0.01) were attenuated due to SQ 29,548 administration. Accordingly, these findings lead us to suggest that pharmaceutical inhibiting TP-R has protective effects on attenuating ethanol-induced myocardial inflammation by inhibiting TXNIP-NLRP3-IL1β axis.

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.

BACKGROUND:Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. METHODS AND RESULTS:Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. CONCLUSIONS:These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.