BACKGROUND:Alcohol-associated liver disease (ALD) is a major global health condition characterized by inflammation, hepatocellular injury, fibrosis, and impaired liver regeneration. To capture key features of severe ALD, we employed a murine model combining ethanol (EtOH) exposure with galactosamine (GalN), a hepatotoxic agent that sensitizes the liver to endotoxin-induced injury. Using this model, we evaluated the therapeutic efficacy of resolvin D1 (RvD1), a lipid mediator with potent anti-inflammatory and tissue-protective actions. METHODS:C57BL/6J male mice were fed 5% EtOH-containing liquid diet for 10 days and administered GalN (500 mg/kg, i.p.) on days 9, 10, and 11, followed by a single EtOH binge (5 g/kg) on day 11. A separate cohort also received RvD1 (500 ng/mouse, days 7-12) as a therapy. Markers of endotoxemia, liver injury, inflammation, fibrosis, and regeneration were evaluated at 9 and 48 hours after the binge. RESULTS:The EtOH+GalN model reproduced hallmark features of advanced ALD, including acute liver injury, sustained inflammation, induction of pro-fibrotic gene markers, reduced hepatic synthetic function, and markedly impaired regenerative capacity. RvD1 treatment significantly mitigated EtOH+GalN-induced pathology, lowering plasma endotoxin and ALT levels, reducing hepatic necrosis, and limiting neutrophil infiltration. RvD1 also decreased hepatic IL-6 levels at 9 hours and reduced IFN-γ at 48 hours. In addition, RvD1 attenuated the expression of pro-fibrotic genes and improved the diminished regenerative response in EtOH+GalN-treated mice, increasing hepatocyte proliferation at 48 hours and partially restoring plasma albumin concentrations. CONCLUSION:This study established an EtOH+GalN exposure mouse model of severe ALD and demonstrated that RvD1 exerts significant hepatoprotective effects. The findings support RvD1 as a promising therapeutic candidate for advanced ALD and provide the rationale for further mechanistic studies and translational evaluation.

Metabolic and alcohol-related liver disease (MetALD) is a newly defined entity within the spectrum of steatotic liver disease, characterized by the interplay of cardiometabolic risk factors and alcohol consumption. The evolving epidemiology and complex pathophysiology of MetALD present unique challenges and opportunities for clinical trial design. Inclusion criteria should require simultaneous evidence of metabolic dysfunction (at least two cardiometabolic features) and verified quantifiable alcohol exposure recorded over the preceding 3–6 months. Traditional histological end points are limited by invasiveness, sampling error and interpretative variability. Thus, imaging modalities, serum-based fibrosis biomarkers and quantitative measures of alcohol intake are gaining relevance as non-invasive, reproducible and patient-centric end points aiming to improve trial feasibility. Furthermore, incorporating alcohol biomarkers, stratifying patients by metabolic risk factor burden, and using adaptive designs of trials might enhance the precision and generalizability of MetALD clinical trials. Although uncertainties remain regarding optimal patient selection criteria, event rates and the dynamic interplay between metabolic dysfunction and alcohol intake, ongoing research efforts aim to refine diagnostic criteria, standardize methodologies and validate novel end points. These advances will ultimately accelerate drug development, improve trial efficiency and foster interventions to treat MetALD. Metabolic and alcohol-related liver disease presents challenges in clinical trials due to complex pathophysiology. This Review discusses noninvasive imaging, serum biomarkers and adaptive designs as modalities to enhance patient-centric end points, aiming to refine diagnostics and improve drug development.

BACKGROUND:How parental alcohol use disorder and liver disease-related mortality influence the risk and the outcomes of alcohol-associated hepatitis (AH) in the offspring is unknown. METHODS:We analyzed data from 2 prospective observational studies of AH cases and heavy drinking controls (HDCs). Family history of parental alcohol use disorder and liver disease mortality was assessed at the study entry. Logistic regression and Cox proportional hazard models were used to assess the influences of family history on AH development and outcome. RESULTS:Data from 1356 participants in two prospective cohorts (926 AH cases and 430 HDC) were combined and analyzed. Parental alcohol use disorder was found in 56.9% of AH cases and 61.1% of HDC; parental death due to liver disease was reported in 7.5% of AH cases and 5.7% of HDC. Multivariable logistic regression showed that parental liver disease-related mortality was associated with more than a doubled risk of AH development in the offspring after controlling for their demographic characteristics and drinking behavior (OR=2.26, 95% CI: [1.22, 4.20]). Moreover, among the AH cases, having a parent die of liver disease significantly increased the 90-day mortality of study participants after adjusting for the effects of other risk factors (HR=2.26, 95% CI: [1.05, 4.86]). CONCLUSIONS:The study highlights the influences of parental death due to liver disease on AH development and mortality. Identifying patients at risk of AH through family history might help facilitate discussions on reducing alcohol consumption.

BACKGROUND AND AIMS:Betaine, 20 g/day for 12 months, reduced liver injury in several trials in non-alcoholic steatohepatitis (NASH). Our aim was to determine the safety and efficacy of lower doses of betaine in clinically diagnosed metabolic dysfunction-associated steatotic liver disease (MASLD) and an elevated ALT. APPROACH AND RESULTS:We performed 3 pilot trials in participants with clinically diagnosed non-cirrhotic MASLD and ALT ≥50 U/L. In the first trial, 44 participants were randomized to 4 or 8 g daily for 12 weeks. In the second trial, 10 participants received 1 g/day for 24 weeks, while 16 participants received 2 g/day for 24 weeks in the third trial. The primary outcome was the percent decline in the abnormal component of ALT (ie, ALT >30 for males or >25 for females). Other outcomes included improvement in absolute ALT and AST, and other serologic tests of liver injury, including metabolomics-advanced steatohepatitis fibrosis (MASEF) score, cytokeratin 18, and pro-C3. Baseline and end-of-treatment data were compared with a paired t test. At baseline, more than 75% of participants had NASH when tested by the MASEF score. ALT, AST, cytokeratin 18, pro-C3, and MASEF score decreased significantly among participants receiving 8, 4​​​​​, and 2 g, but not 1 g. High-density lipoprotein increased in the 4 and 2 g cohorts; low-density lipoprotein did not change. Approximately 35% reported mild, transient gastrointestinal symptoms. CONCLUSIONS:Betaine 8, 4​​​​​​, and 2 g/day for 12-24 weeks significantly reduced ALT and other serologic markers of liver injury among participants with clinically defined MASLD and an elevated ALT.

Background/Objectives: Alcohol-associated hepatitis (AH) is an acute inflammatory condition of alcohol-associated liver disease (ALD) with rapid progression and high mortality. The Age-Bilirubin-INR-Creatinine (ABIC) score is a static algorithm that predicts survivability in AH. The roles of alcohol drinking patterns and nutritional status in AH progression and risk of death are understudied. This study evaluates the impact of alcohol drinking patterns and nutrition on AH progression and mortality. Methods: Sixty-one adult patients diagnosed with AH were stratified by the Model for End-Stage Liver Disease (MELD) as non-severe (MELD < 20, n = 26, Gr.1) and severe (MELD ≥ 20, n = 35, Gr.2). Each group was further subdivided by ABIC: low- (<6.71), intermediate- (6.71–9), and high- (>9) risk categories. We assessed different demographics: nutrition using the Controlling Nutritional Status (CONUT) score; lifetime drinking history (LTDH); recent alcohol use (AUDIT); laboratory measures (complete metabolic panel, complete blood count, and coagulation), and clinical measures (Maddrey DF, Child–Turcotte–Pugh, and Lille). Results: All patients showed a significant and positive correlation between ABIC and LTDH (r = 0.538, p = 0.004), particularly in Gr.2 (r = 0.554, p = 0.011). The low-risk Gr.2 exhibited the highest AST:ALTs. AST:ALTs were significantly associated with LTDH, AUDIT, and CONUT (R2 = 0.539, p = 0.031). In all AH patients with intermediate mortality risk, AST:ALTs were strongly linked to CONUT and LTDH (R2 = 0.657, p = 0.017). Conclusions: Severe AH demonstrates rapid liver injury progression even when the mortality risk is low. Chronic and recent heavy alcohol consumption and poor nutrition adversely impact AH severity and mortality risk. Alcohol intake and nutritional assessments in routine clinicals could identify high-risk patients, thereby improving treatment and a favorable prognosis.

BACKGROUND:It is unclear whether physical activity (PA) and advanced fibrosis are associated in alcohol-associated liver disease (ALD) and metabolic dysfunction and alcohol-associated liver disease (MetALD). We examined the association between work-related PA (WPA) or leisure-time PA (LTPA) and advanced fibrosis across the steatotic liver disease (SLD) spectrum. METHODS:Data obtained from the National Health and Nutrition Examination Survey (2017-2020) in adults with hepatic steatosis were included and categorized into MASLD (n=2236), MetALD (n=1355), and ALD (n=457) based on alcohol use. PA was quantified as metabolic equivalent (MET)-minutes per week, and ≥600 MET-min/wk was used as the threshold for high activity. Adjusted logistic regression models for confounders (sex, race/ethnicity, education, income, metabolic risk factors, and etiology of SLD) were used to analyze the relationship of PA types with at-risk advanced fibrosis, according to an Agile 3+ score ≥0.45. RESULTS:Among 4342 SLD participants, LTPA was strongly linked to a lower risk of at-risk advanced fibrosis in MASLD (OR=0.56, 95% CI: 0.34-0.93), as well as MetALD and ALD combined (OR=0.55, 95% CI: 0.34-0.89). WPA was not associated with improved advanced fibrosis. There were no interactions between WPA and SLD subtypes (p=0.98), and between LTPA and SLD subtypes (p=0.55). CONCLUSIONS:LTPA but not WPA is protective for at-risk advanced fibrosis in patients with MASLD and MetALD. These findings showcase the importance of structured LTPA interventions to mitigate fibrosis risk in SLD. Larger studies are needed to examine the benefits of PA in patients with ALD and to delineate exercise prescriptions for patients with SLD.

Exposure to pollutants, including the ubiquitous "forever chemical," perfluorooctane sulfonate (PFOS) has increasingly been associated with metabolic dysfunction-associated steatotic liver disease. Recent epidemiological evidence has identified associations between per- and polyfluoroalkyl substances (PFAS) exposure and increased liver injury in alcohol consumers, suggesting potential interactions between these exposures. However, the intersection of pollutant exposures and alcohol-associated liver disease (ALD) is not well studied. We hypothesize that pollutants may disrupt hepatic metabolism to modify ALD severity. Recently, we developed a two-hit (ethanol [EtOH] plus pollutant) mouse model, enabling testing of this hypothesis. Here, we elucidate the metabolic and disease-modifying effects of PFOS in this model. Male C57BL/6J mice were fed isocaloric control or 5% EtOH Lieber-DeCarli diet for 15 days. From day 6 of feeding, mice were concurrently gavaged with 1 mg/kg PFOS or 2% tween-80 vehicle for 10 days, followed by a 5 g/kg EtOH binge dose and euthanized 5 to 6 h later. Approximately 60% of the administered PFOS dose accumulated in the liver. PFOS exacerbated EtOH-induced hepatic steatosis and was associated by higher levels of plasma very low-density lipoprotein and alanine aminotransferase. PFOS upregulated hepatic EtOH-metabolizing enzymes and lowered blood alcohol levels. Ingenuity Pathway Analysis (IPA) Top Toxicity Functions/Lists associated with hepatic gene expression following PFOS co-exposure in EtOH-fed mice included: Fatty acid metabolism and liver steatosis; nuclear receptor activation, cytochrome P450, and reactive oxygen species; apoptosis; liver fibrosis; and hepatocellular carcinoma (HCC). Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses similarly revealed enrichment in fatty acid, xenobiotic, alcohol, or glutathione metabolic processes; and peroxisome proliferator-activated receptor (PPAR) signaling. PFOS upregulated hepatic expression of several nuclear receptors (e.g. Pparα, Car, and Pxr) and their P450 target genes (e.g. Cyp4a10, Cyp2b10, and Cyp3a11) by real-time-PCR or Western blot, confirming key IPA predictions. PFOS is a metabolism-disrupting chemical that worsens ALD severity. PFOS activated hepatic nuclear receptors and enriched hepatic transcriptional pathways associated with steatosis, xenobiotic metabolism, oxidative stress, cell death, fibrosis, and HCC. These data demonstrate a novel mechanism whereby PFOS exacerbates ALD through coordinated dysregulation of lipid homeostasis and liver injury, potentially mediated by nuclear receptor activation. The identification of PFOS as an ALD risk modifier highlights the critical need to evaluate environmental pollutants as potential contributors to liver disease progression. More data are required on environmental pollution as a disease-modifying factor in ALD. Impact Statement: The present study demonstrates that PFOS exacerbates alcohol-induced liver injury through nuclear receptor activation and metabolic disruption. These findings provide novel insights into how environmental pollutants can act as significant risk modifiers in ALD, how PFAS exposures may contribute to the growing public health burden of liver disease, and underscore the importance of considering combined risk factors in developing targeted interventions.

Malnutrition, defined as deficiency, excess, or imbalance of nutrients, is a common complication in patients with liver disease, especially those with cirrhosis. Malnutrition may present as an isolated micronutrient deficiency, such as zinc deficiency, and it commonly presents as frailty and/or sarcopenia in patients with advanced liver disease. Patients with cirrhosis and/or alcohol-associated hepatitis should be assessed for malnutrition because it adversely affects patient outcomes including mortality, as well as waitlist and posttransplant outcomes among liver transplant candidates. The prevalence of malnutrition varies based on the method of assessment and disease severity, being higher in those with advanced liver disease. Among stable outpatients with cirrhosis, counseling should be done to eat small frequent meals, a night-time snack between 7 PM and 10 PM, and 2 or more cups of coffee daily. In selected patients with metabolic dysfunction-associated steatohepatitis, vitamin E 800 IU/d should be provided. Among hospitalized patients with cirrhosis, nutritional supplementation preferably by enteral route should be implemented in those with poor oral intake of daily requirements of proteins and/or calories. Protein intake should not be restricted including patients with decompensated cirrhosis and hepatic encephalopathy. A vegetable source of protein seems to be better tolerated than an animal source of protein in patients with hepatic encephalopathy. Branched chain amino acids augment the efficacy of lactulose and rifaximin in the treatment of hepatic encephalopathy. Level of evidence and strength of recommendations were evaluated using the Grading of Recommendations, Assessment, Development, and Evaluations system. This guideline was developed under the auspices of the American College of Gastroenterology Practice Parameters Committee.

BACKGROUND:During the coronavirus disease 2019 (COVID-19) pandemic, there was a marked increase in alcohol consumption. COVID-19 superimposed on underlying liver disease notably worsens the outcome of many forms of liver injury. The goal of a current pilot study was to test the dual exposure of alcohol and COVID-19 infection in an experimental animal model of alcohol-associated liver disease (ALD). METHODS:After 4 weeks of ethanol (EtOH) feeding, C57BL/6 male mice received SARS-CoV-2 (SARS2-N501YMA30) intranasally at 3 × 102, 1 × 103, 3 × 103, and 1 × 104 plaque-forming units (PFU). Mice were then weighed/monitored daily for morbidity/mortality for 10 days while continuing EtOH consumption. Markers of liver inflammation, injury, and intestinal barrier integrity were evaluated. RESULTS:A similar gradual weight loss was observed in all inoculated mice (slightly less in the 3 × 102 group) up to post-infection day 4. Greater mortality was observed in mice receiving the highest viral dose at days 3 and 4 post-infection. The majority of the surviving mice subjected to EtOH and inoculated with 3 × 103 or 1 × 104 PFU rapidly lost 25% of their body weight and were euthanized on post-infection day 4. Analysis of liver health in animals that survived to the end of the experiment exhibited no significant changes in hepatic steatosis but had a limited increase in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at all viral doses versus EtOH alone. However, the 1 × 104 PFU viral dose exacerbated EtOH-induced hepatic inflammation characterized by elevated levels of several pro-inflammatory cytokines, including Il-6 and Tnf-α. There was limited effect of viral infection on the intestine. CONCLUSIONS:SARS-CoV-2 infection caused a dose-dependent negative impact on body weight and survival in mice fed EtOH. This pilot study suggests that early mortality observed after high-dose SARS-CoV-2 challenge could be due, in part, to hepatic dysfunction following chronic EtOH feeding.

Alcohol-associated liver disease (ALD) is a chronic condition caused by excessive alcohol consumption, with limited effective pharmacological treatments currently available. The inhibition of soluble epoxide hydrolase (s-EH) has emerged as a promising therapeutic strategy in experimental ALD. In this study, we developed a novel liver-targeted formulation of the s-EH inhibitor t-TUCB, encapsulated in fusogenic lipid vesicles (FLVs), and tested its therapeutic efficacy in a mouse model of ALD. This formulation achieved high encapsulation efficiency and targeted primarily hepatocytes. Male C57BL/6J mice were fed an ethanol-containing liquid diet for 10 days, followed by a single ethanol binge on day 11. A subset of mice received a single intraperitoneal dose of t-TUCB-FLVs two hours before the binge. t-TUCB-FLVs significantly attenuated liver injury as assessed by multiple endpoints, including decreased plasma ALT levels and hepatocyte cell death. Spatial transcriptomic analysis revealed that t-TUCB-FLV treatment modulated gene expression in hepatocytes across multiple key pathways, including xenobiotic metabolism, carbohydrate and lipid metabolism, bile acid homeostasis, inflammation, energy balance, and circadian rhythm. Collectively, these findings support liver-specific s-EH inhibition as a potentially effective therapeutic approach for ALD.

Alterations in the gut-microbiome-brain axis are increasingly being recognized to be involved in Alzheimer’s disease (AD) pathogenesis. However, the functional consequences of enteric dysbiosis linking gut microbiota and brain pathology in AD progression remain largely undetermined. The present work investigated the causal role of age-associated temporal decline in butyrate-producing bacteria and butyrate in the etiopathogenesis of AD. Longitudinal metagenomics, neuropathological, and memory analyses were performed in the 3×Tg-AD mouse model. Metataxonomic analyses showed a significant temporal decline in the alpha diversity marked by a decrease in butyrate-producing bacterial communities and a concurrent reduction in cecal butyrate production. Inferred metagenomics analysis identified the bacterial acetyl-CoA pathway as the main butyrate synthesis pathway impacted. Concomitantly, there was an age-associated decline in the transcriptionally permissive acetylation of histone 3 at lysines 9 and 14 (H3K9/K14-Ac) in hippocampal neurons. Importantly, these microbiome-gut-brain changes preceded AD-related neuropathology, including oxidative stress, tau hyperphosphorylation, memory deficits, and neuromuscular dysfunction, which manifest by 17–18 months. Initiation of oral administration of tributyrin, a butyrate prodrug, at 6 months of age mitigated the age-related decline in butyrate-producing bacteria, protected the H3K9/K14-Ac status, and attenuated the development of neuropathological and cognitive changes associated with AD pathogenesis. These data causally implicate age-associated decline in butyrate-producing bacteria as a key pathogenic feature of the microbiome-gut-brain axis affecting the onset and progression of AD. Importantly, the regulation of butyrate-producing bacteria and consequent butyrate synthesis could be a significant therapeutic strategy in the prevention and treatment of AD.

BACKGROUND:Non-alcoholic fatty liver disease is a growing problem in the United States, contributing to a range of liver disease as well as cardiovascular disease. ALT is the most widely used liver chemistry for NAFLD evaluation. We hypothesized that the normal range many laboratories use was too high, missing many patients with clinically important steatosis and/or fibrosis. METHODS:This study utilized 2017-2018 NHANES data including 9254 participants. We compared four different upper limits of normal for ALT with specific measurements of steatosis and liver stiffness as determined by liver elastography with FibroScan®. Liver stiffness was further characterized as showing any fibrosis or advanced fibrosis. After exclusions, our final pool was 4184 for liver stiffness measurement and 4183 for steatosis grade as measured by Controlled Attenuation Parameter (CAP). Using these variables, we performed logistic regression between ALT and CAP, and ALT and fibrosis/advanced fibrosis, and did a Receiver Operating Characteristic curve. RESULTS:Based on three of the most widely used cut off values for ALT, we found that ALT does not reliably rule out NAFLD in over 50% of cases. It also missed 45.9-64.2% of patients with liver fibrosis. CONCLUSIONS:Our study revealed that ALT is an inaccurate marker for NAFLD as measured by FibroScan® with CAP greater than or equal to 300 dB/m. Accuracy improved specific risk factors were considered. These data also showed that ALT was a poor marker for liver fibrosis. We conclude that there is no single ALT level that accurately predicts hepatic steatosis or fibrosis.

Previous research highlighted the involvement of the cannabinoid CB1 receptor in regulating the physiology of hepatocytes and hepatic stellate cells. The inhibition of the CB1 receptor via peripherally restricted CB1 receptor inverse agonist JD5037 has shown promise in inhibiting liver fibrosis in mice treated with CCl4. However, its efficacy in phospholipid transporter-deficiency-induced liver fibrosis remains uncertain. In this study, we investigated the effectiveness of JD5037 in Mdr2−/− mice. Mdr2 (Abcb4) is a mouse ortholog of the human MDR3 (ABCB4) gene encoding for the canalicular phospholipid transporter. Genetic disruption of the Mdr2 gene in mice causes a complete absence of phosphatidylcholine from bile, leading to liver injury and fibrosis. Mdr2−/− mice develop spontaneous fibrosis during growth. JD5037 was orally administered to the mice for four weeks starting at eight weeks of age. Liver fibrosis, bile acid levels, inflammation, and injury were assessed. Additionally, JD5037 was administered to three-week-old mice to evaluate its preventive effects on fibrosis development. Our findings corroborate previous observations regarding global CB1 receptor inverse agonists. Four weeks of JD5037 treatment in eight-week-old Mdr2−/− mice with established fibrosis led to reduced body weight gains. However, contrary to expectations, JD5037 significantly exacerbated liver injury, evidenced by elevated serum ALT and ALP levels and exacerbated liver histology. Notably, JD5037-treated Mdr2−/− mice exhibited significantly heightened serum bile acid levels. Furthermore, JD5037 treatment intensified liver fibrosis, increased fibrogenic gene expression, stimulated ductular reaction, and upregulated hepatic proinflammatory cytokines. Importantly, JD5037 failed to prevent liver fibrosis formation in three-week-old Mdr2−/− mice. In summary, our study reveals the exacerbating effect of JD5037 on liver fibrosis in genetically MDR2-deficient mice. These findings underscore the need for caution in the use of peripherally restricted CB1R inverse agonists for liver fibrosis treatment, particularly in cases of dysfunctional hepatic phospholipid transporter.

Background: Virtually the entire spectrum of liver disease is observed in association with type 2 diabetes mellitus (T2DM); indeed, T2DM is now the most common cause of liver disease in the U.S. We conducted a pilot study to investigate the relevance of increased microbial translocation and systemic inflammation in the development of liver injury in patients with T2DM. Methods: Patients with T2DM (n = 17) and non-diabetic controls (NDC; n = 11) aged 25–80 yrs. participated in this study. Serum levels of endotoxin, calprotectin, soluble CD14 and CD163, and several inflammatory cytokines were measured. In addition to standard liver injury markers, ALT and AST, novel serum markers of liver injury, keratin 18 (K-18) M30 (apoptosis-associated caspase-cleaved keratin 18), and M65 (soluble keratin 18) were evaluated. Statistical analyses were performed using the Mann–Whitney test to assess differences between study groups. Pearson’s correlation analysis was performed to determine the strength of association between two variables using GraphPad Prism 9.5.0 software. Results: Patients with T2DM had significantly higher levels of sCD14 in comparison to NDC, suggesting an increase in gut permeability, microbial translocation, and monocyte/macrophage activation. Importantly, relevant to the ensuing inflammatory responses, the increase in sCD14 in patients with T2DM was accompanied by a significant increase in sCD163, a marker of hepatic Kupffer cell activation and inflammation. Further, a positive correlation was observed between sCD163 and endotoxin and sCD14 in T2DM patients but not in NDC. In association with these changes, keratin 18 (K-18)-based serum markers (M65 and M30) that reflect hepatocyte death were significantly higher in the T2DM group indicating ongoing liver injury. Notably, both M65 and M30 levels correlated with sCD14 and sCD163, suggesting that immune cell activation and hepatic inflammation may be linked to the development of liver injury in T2DM. Conclusions: These findings suggest that the pathogenic changes in the gut–liver axis, marked by increased microbial translocation, may be a major component in the etiology of hepatocyte inflammation and injury in patients with T2DM. However, larger longitudinal studies, including histological evidence, are needed to confirm these observations.

Fatty acid desaturase 1 (FADS1) is a rate-limiting enzyme in long-chain polyunsaturated fatty acid (LCPUFA) synthesis. Reduced activity of FADS1 was observed in metabolic dysfunction-associated steatotic liver disease (MASLD). The aim of this study was to determine whether adeno-associated virus serotype 8 (AAV8) mediated hepatocyte-specific overexpression of Fads1 (AAV8-Fads1) attenuates western diet-induced metabolic phenotypes in a rat model. Male weanling Sprague-Dawley rats were fed with a chow diet, or low-fat high-fructose (LFHFr) or high-fat high-fructose diet (HFHFr) ad libitum for 8 weeks. Metabolic phenotypes were evaluated at the endpoint. AAV8-Fads1 injection restored hepatic FADS1 protein levels in both LFHFr and HFHFr-fed rats. While AAV8-Fads1 injection led to improved glucose tolerance and insulin signaling in LFHFr-fed rats, it significantly reduced plasma triglyceride (by ~50%) and hepatic cholesterol levels (by ~25%) in HFHFr-fed rats. Hepatic lipidomics analysis showed that FADS1 activity was rescued by AAV8-FADS1 in HFHFr-fed rats, as shown by the restored arachidonic acid (AA)/dihomo-γ-linolenic acid (DGLA) ratio, and that was associated with reduced monounsaturated fatty acid (MUFA). Our data suggest that the beneficial role of AAV8-Fads1 is likely mediated by the inhibition of fatty acid re-esterification. FADS1 is a promising therapeutic target for MASLD in a diet-dependent manner.

Alcohol-associated liver disease (ALD) is a global healthcare problem with limited treatment options. Recently, Fpr2-/- (formyl peptide receptor 2 knockout) mice were shown to develop exacerbated alcohol-induced liver injury and inflammation, but the associated mechanisms remain elusive. Objective: To identify hepatic proteomic changes associated with exacerbated EtOH-induced liver injury in Fpr2-/- mice. Methods: A proteomic analysis was conducted on liver homogenates from WT and Fpr2-/- mice (n=3/group) that were fed either a control diet (pair-fed mice, PF) or a 5% ethanol (EtOH)-containing diet for 4 weeks followed by a single EtOH binge (5 g/kg). Liver antioxidants including Zinc (Zn) and total glutathione (GSH) were measured via colorimetric assay. Hepatic gene expression was assessed by RT qPCR analysis. Data were compared using a one-way ANOVA with a Tukey multiple comparison test. Results: There were 977 downregulated and 45 upregulated significant protein changes between Fpr2-/-EtOH and WT EtOH mice. Among the downregulated proteins in Fpr2-/-EtOH vs WT EtOH mice were the blood coagulation factors, F2 (mRNA & protein) and F9 (protein). Zn binding proteins, MT1 (protein) and MT2 (mRNA & protein), were significantly upregulated in Fpr2-/- EtOH vs WT EtOH mice. Increased expression of these Zn-binding proteins was associated with decreased free hepatic Zn in Fpr2-/- EtOH vs Fpr2-/- PF mice. Lastly, the rate limiting enzyme for glutathione synthesis, GCLC, and total liver GSH were downregulated in Fpr2-/-EtOH vs WT EtOH mice. Conclusions: These data suggest that loss of Fpr2 in the context of ALD may lead to impaired hepatic Zn and glutathione antioxidant system, contributing to oxidative stress and hepatocellular death. In addition, Fpr2-mediated regulation of F2 and F9 protein expression appears to be compromised which may contribute to ALD- induced coagulopathy. Further investigation is required to understand precisely how FPR2 impacts these processes in ALD.