ABSTRACT
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.
Subject(s)
Alzheimer Disease , Bacteria , Butyrates , Disease Models, Animal , Dysbiosis , Gastrointestinal Microbiome , Memory Disorders , Animals , Butyrates/metabolism , Mice , Alzheimer Disease/microbiology , Alzheimer Disease/pathology , Alzheimer Disease/metabolism , Memory Disorders/microbiology , Memory Disorders/metabolism , Memory Disorders/pathology , Bacteria/classification , Bacteria/metabolism , Bacteria/genetics , Bacteria/isolation & purification , Dysbiosis/microbiology , Hippocampus/metabolism , Hippocampus/pathology , Mice, Transgenic , Male , Disease Progression , Brain-Gut Axis/physiology , Brain/metabolism , Brain/pathologyABSTRACT
The interactions of different dietary doses of copper with fructose contribute to the development of metabolic dysfunction-associated steatotic liver disease (MASLD) via the gut-liver axis. The underlying mechanisms remain elusive. The aim of this study was to identify the specific pathways leading to gut barrier dysfunction in the ileum using a proteomics approach in a rat model. Male weanling Sprague Dawley rats were fed diets with adequate copper (CuA), marginal copper (CuM), or supplemented copper (CuS) in the absence or presence of fructose supplementation (CuAF, CuMF, and CuSF) for 4 weeks. Ileum protein was extracted and analyzed with an LC-MS. A total of 2847 differentially expressed proteins (DEPs) were identified and submitted to functional enrichment analysis. As a result, the ileum proteome and signaling pathways that were differentially altered were revealed. Of note, the CuAF is characterized by the enrichment of oxidative phosphorylation and ribosome as analyzed with the KEGG; the CuMF is characterized by an enriched arachidonic acid metabolism pathway; and focal adhesion, the regulation of the actin cytoskeleton, and tight junction were significantly enriched by the CuSF. In conclusion, our proteomics analysis identified the specific pathways in the ileum related to the different dietary doses of copper-fructose interactions, suggesting that distinct mechanisms in the gut are involved in the development of MASLD.
Subject(s)
Copper , Fructose , Ileum , Liver , Proteomics , Rats, Sprague-Dawley , Animals , Fructose/administration & dosage , Fructose/adverse effects , Male , Copper/metabolism , Proteomics/methods , Ileum/metabolism , Ileum/drug effects , Liver/metabolism , Liver/drug effects , Rats , Diet , Proteome/metabolism , Signal Transduction/drug effects , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/etiology , Dietary SupplementsABSTRACT
BACKGROUND: Alcohol-associated hepatitis (AH) is plagued with high mortality and difficulty in identifying at-risk patients. The extracellular matrix undergoes significant remodeling during inflammatory liver injury and could potentially be used for mortality prediction. METHODS: EDTA plasma samples were collected from patients with AH (n = 62); Model for End-Stage Liver Disease score defined AH severity as moderate (12-20; n = 28) and severe (>20; n = 34). The peptidome data were collected by high resolution, high mass accuracy UPLC-MS. Univariate and multivariate analyses identified differentially abundant peptides, which were used for Gene Ontology, parent protein matrisomal composition, and protease involvement. Machine-learning methods were used to develop mortality predictors. RESULTS: Analysis of plasma peptides from patients with AH and healthy controls identified over 1600 significant peptide features corresponding to 130 proteins. These were enriched for extracellular matrix fragments in AH samples, likely related to the turnover of hepatic-derived proteins. Analysis of moderate versus severe AH peptidomes was dominated by changes in peptides from collagen 1A1 and fibrinogen A proteins. The dominant proteases for the AH peptidome spectrum appear to be CAPN1 and MMP12. Causal graphical modeling identified 3 peptides directly linked to 90-day mortality in >90% of the learned graphs. These peptides improved the accuracy of mortality prediction over the Model for End-Stage Liver Disease score and were used to create a clinically applicable mortality prediction assay. CONCLUSIONS: A signature based on plasma peptidome is a novel, noninvasive method for prognosis stratification in patients with AH. Our results could also lead to new mechanistic and/or surrogate biomarkers to identify new AH mechanisms.
Subject(s)
Extracellular Matrix , Hepatitis, Alcoholic , Humans , Male , Prognosis , Female , Hepatitis, Alcoholic/blood , Hepatitis, Alcoholic/mortality , Extracellular Matrix/metabolism , Middle Aged , Adult , Peptides/blood , Biomarkers/blood , Severity of Illness Index , Machine Learning , Case-Control Studies , ProteomicsABSTRACT
BACKGROUND AND AIMS: In a recent trial, patients with severe alcohol-associated hepatitis treated with anakinra plus zinc (A+Z) had lower survival and higher acute kidney injury (AKI) rates versus prednisone (PRED). We characterize the clinical factors and potential mechanisms associated with AKI development in that trial. APPROACH AND RESULTS: Data from 147 participants in a multicenter randomized clinical trial (74 A+Z, 73 PRED) were analyzed. AKI, AKI phenotypes, and kidney injury biomarkers were compared between participants who did/did not develop AKI in the 2 treatment arms. Multivariable competing risk analyses were performed to identify baseline risk factors for incident AKI, with death treated as a competing event. Risk factors considered were age, sex, mean arterial pressure, white blood cell count, albumin, MELD, ascites, HE, and treatment arm. At baseline, no participants had AKI; 33% (n=49) developed AKI during follow-up. AKI incidence was higher in A+Z than in PRED (45% [n=33] versus 22% [n=16], p =0.001). AKI phenotypes were similar between the 2 treatment arms ( p =0.361), but peak AKI severity was greater in A+Z than PRED (stage 3 n=21 [63.6%] vs. n=8 [50.0%], p =0.035). At baseline, urine-neutrophil-gelatinase-associated lipocalin levels were similar between participants who developed AKI in both treatment arms ( p =0.319). However, day 7 and 14 urine-neutrophil-gelatinase-associated lipocalin levels were significantly elevated in participants treated with A+Z who developed AKI versus participants treated with PRED who developed AKI ( p =0.002 and 0.032, respectively). On multivariable competing risk analysis, only A+Z was independently associated with incident AKI (subdistribution hazard ratio 2.35, p =0.005). CONCLUSIONS: AKI occurred more frequently and was more severe in participants treated with A+Z. A+Z-treated participants with AKI had higher urine-neutrophil-gelatinase-associated lipocalin, suggesting that A+Z maybe nephrotoxic in patients with severe alcohol-associated hepatitis.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Delta-5 Fatty Acid Desaturase , Diet, Western , Fatty Acid Desaturases , Hepatocytes , Animals , Male , Rats , Delta-5 Fatty Acid Desaturase/metabolism , Dependovirus/genetics , Diet, Western/adverse effects , Disease Models, Animal , Fatty Acid Desaturases/metabolism , Fatty Acid Desaturases/genetics , Fructose/metabolism , Hepatocytes/metabolism , Liver/metabolism , Phenotype , Rats, Sprague-Dawley , Triglycerides/metabolismABSTRACT
Diagnostic challenges continue to impede development of effective therapies for successful management of alcohol-associated hepatitis (AH), creating an unmet need to identify noninvasive biomarkers for AH. In murine models, complement contributes to ethanol-induced liver injury. Therefore, we hypothesized that complement proteins could be rational diagnostic/prognostic biomarkers in AH. Here, we performed a comparative analysis of data derived from human hepatic and serum proteome to identify and characterize complement protein signatures in severe AH (sAH). The quantity of multiple complement proteins was perturbed in liver and serum proteome of patients with sAH. Multiple complement proteins differentiated patients with sAH from those with alcohol cirrhosis (AC) or alcohol use disorder (AUD) and healthy controls (HCs). Serum collectin 11 and C1q binding protein were strongly associated with sAH and exhibited good discriminatory performance among patients with sAH, AC, or AUD and HCs. Furthermore, complement component receptor 1-like protein was negatively associated with pro-inflammatory cytokines. Additionally, lower serum MBL associated serine protease 1 and coagulation factor II independently predicted 90-day mortality. In summary, meta-analysis of proteomic profiles from liver and circulation revealed complement protein signatures of sAH, highlighting a complex perturbation of complement and identifying potential diagnostic and prognostic biomarkers for patients with sAH.
Subject(s)
Biomarkers , Complement System Proteins , Hepatitis, Alcoholic , Proteomics , Humans , Hepatitis, Alcoholic/blood , Hepatitis, Alcoholic/mortality , Hepatitis, Alcoholic/diagnosis , Proteomics/methods , Male , Female , Complement System Proteins/metabolism , Biomarkers/blood , Middle Aged , Adult , Liver/metabolism , Liver/pathology , Alcoholism/blood , Alcoholism/complications , Proteome/metabolism , Prognosis , AgedABSTRACT
Lipids play a significant role in life activities and participate in the biological system through different pathways. Although comprehensive two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) has been developed to profile lipid abundance changes, lipid identification and quantification from 2DLC-MS data remain a challenge. We created Lipid Wizard, open-source software for lipid assignment and isotopic peak stripping of the 2DLC-MS data. Lipid Wizard takes the peak list deconvoluted from the 2DLC-MS data as input and assigns each isotopic peak to the lipids recorded in the LIPID MAPS database by precursor ion m/z matching. The matched lipids are then filtered by the first-dimension retention time (1D RT), followed by the second-dimension retention time (2D RT), where the 2D RT of each lipid is predicted using an equivalent carbon number (ECN) model. The remaining assigned lipids are used for isotopic peak stripping via an iterative linear regression. The performance of Lipid Wizard was tested using a set of lipid standards and then applied to study the lipid changes in the livers of mice (fat-1) fed with alcohol.
Subject(s)
Lipids , Liquid Chromatography-Mass Spectrometry , Mice , Animals , Lipids/analysis , Software , Liver/chemistry , Databases, FactualABSTRACT
BACKGROUND & AIMS: Severe alcohol-associated hepatitis (SAH) is associated with high 90-day mortality. Glucocorticoid therapy for 28 days improves 30- but not 90-day survival. We assessed the efficacy and safety of a combination of anakinra, an IL-1 antagonist, plus zinc (A+Z) compared to prednisone using the Day-7 Lille score as a stopping rule in patients with SAH. METHODS: In this phase IIb double-blind randomized trial in adults with SAH and MELD scores of 20-35, participants were randomized to receive either daily anakinra 100 mg subcutaneously for 14 days plus daily zinc sulfate 220 mg orally for 90 days, or daily prednisone 40 mg orally for 30 days. Prednisone or prednisone placebo was stopped if Day-7 Lille score was >0.45. All study drugs were stopped for uncontrolled infection or ≥5 point increase in MELD score. The primary endpoint was overall survival at 90 days. RESULTS: Seventy-three participants were randomized to prednisone and 74 to A+Z. The trial was stopped early after a prespecified interim analysis showed prednisone was associated with higher 90-day overall survival (90% vs. 70%; hazard ratio for death = 0.34, 95% CI 0.14-0.83, p = 0.018) and transplant-free survival (88% vs. 64%; hazard ratio for transplant or death = 0.30, 95% CI 0.13-0.69, p = 0.004) than A+Z. Acute kidney injury was more frequent with A+Z (45%) than prednisone (22%) (p = 0.001), but rates of infection were similar (31% in A+Z vs. 27% in prednisone, p = 0.389). CONCLUSIONS: Participants with SAH treated with prednisone using the Day-7 Lille score as a stopping rule had significantly higher overall and transplant-free 90-day survival and lower incidence of acute kidney injury than those treated with A+Z. IMPACT AND IMPLICATIONS: There is no approved treatment for severe alcohol-associated hepatitis (SAH). In this double-blind randomized trial, patients with SAH treated with prednisone using the Lille stopping rule on Day 7 had higher 90-day overall and transplant-free survival and lower rates of acute kidney injury compared to patients treated with a combination of anakinra and zinc. The data support continued use of glucocorticoids for patients with SAH, with treatment discontinuation for those with a Lille score >0.45 on Day 7. TRIAL REGISTRATION: NCT04072822.
Subject(s)
Acute Kidney Injury , Hepatitis, Alcoholic , Adult , Humans , Prednisone/adverse effects , Interleukin 1 Receptor Antagonist Protein/adverse effects , Zinc/therapeutic use , Hepatitis, Alcoholic/drug therapy , Double-Blind Method , Acute Kidney Injury/chemically induced , Acute Kidney Injury/drug therapy , Treatment OutcomeABSTRACT
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.
Subject(s)
Elasticity Imaging Techniques , Non-alcoholic Fatty Liver Disease , Adult , Humans , United States/epidemiology , Non-alcoholic Fatty Liver Disease/diagnostic imaging , Elasticity Imaging Techniques/adverse effects , Nutrition Surveys , Vibration , Prospective Studies , Liver Cirrhosis/diagnostic imaging , Liver Cirrhosis/etiology , Liver/diagnostic imaging , FibrosisABSTRACT
Gut microbiota function has numerous effects on humans and the diet humans consume has emerged as a pivotal determinant of gut microbiota function. Here, a new concept that gut microbiota can be trained by diet-derived exosome-like nanoparticles (ELNs) to release healthy outer membrane vesicles (OMVs) is introduced. Specifically, OMVs released from garlic ELN (GaELNs) trained human gut Akkermansia muciniphila (A. muciniphila) can reverse high-fat diet-induced type 2 diabetes (T2DM) in mice. Oral administration of OMVs released from GaELNs trained A. muciniphila can traffick to the brain where they are taken up by microglial cells, resulting in inhibition of high-fat diet-induced brain inflammation. GaELNs treatment increases the levels of OMV Amuc-1100, P9, and phosphatidylcholines. Increasing the levels of Amuc-1100 and P9 leads to increasing the GLP-1 plasma level. Increasing the levels of phosphatidylcholines is required for inhibition of cGas and STING-mediated inflammation and GLP-1R crosstalk with the insulin pathway that leads to increasing expression of Insulin Receptor Substrate (IRS1 and IRS2) on OMV targeted cells. These findings reveal a molecular mechanism whereby OMVs from plant nanoparticle-trained gut bacteria regulate genes expressed in the brain, and have implications for the treatment of brain dysfunction caused by a metabolic syndrome.
Subject(s)
Brain-Gut Axis , Diabetes Mellitus, Type 2 , Exosomes , Garlic , Gastrointestinal Microbiome , Nanoparticles , Diabetes Mellitus, Type 2/metabolism , Garlic/chemistry , Animals , Nanoparticles/chemistry , Exosomes/metabolism , Mice , Akkermansia , Humans , Male , Diet, High-Fat , Mice, Inbred C57BL , Brain/metabolism , Brain/pathologyABSTRACT
PURPOSE OF REVIEW: To delineate common and uncommon dietary and nutritional deficiencies in individuals with chronic heavy alcohol use and alcohol use disorder and to highlight important advances in the nutrition field in patients ranging from those with alcohol use disorder (AUD) and no liver disease to those with decompensated alcohol-associated liver disease (ALD). RECENT FINDINGS: Patients with AUD may have nutritional deficiencies, especially isolated nutrient deficiencies, such as thiamine or zinc deficiencies. This should not be surprising, as alcohol is a major source of "empty calories." It is devoid of critical macronutrients, such as protein, and micronutrients including important vitamins and minerals. Patients with AUD frequently drink much more than often appreciated (10-20 drinks a day). Patients with AUD and early ALD often begin to develop more apparent nutritional deficiencies. Healthcare providers need to be aware of the presenting features of individual nutrient deficiencies, such as thiamine deficiency, and to provide prompt treatment. In patients with more advanced liver disease, malnutrition correlates with severity of liver disease. It is important to understand the value of nutritional support throughout the spectrum of AUD. SUMMARY: We review nutritional deficiencies in the spectrum of patients with AUD and ALD and highlight new information and recommendations.
Subject(s)
Alcoholism , Liver Diseases, Alcoholic , Malnutrition , Humans , Alcoholism/complications , Malnutrition/therapy , Vitamins , Nutritional Status , MineralsABSTRACT
INTRODUCTION: This study is to evaluate the safety and pharmacokinetics (PK) of larsucosterol (DUR-928 or 25HC3S) in subjects with alcohol-associated hepatitis (AH), a devastating acute illness without US Food and Drug Administration-approved therapies. METHODS: This phase 2a, multicenter, open-label, dose escalation study evaluated the safety, PK, and efficacy signals of larsucosterol in 19 clinically diagnosed subjects with AH. Based on the model for end-stage liver disease (MELD) score, 7 subjects were considered to have moderate AH and 12 to have severe AH. All subjects received 1 or 2 intravenous infusions (72 hours apart) of larsucosterol at a dose of 30, 90, or 150 mg and were followed up for 28 days. Efficacy signals from a subgroup of subjects with severe AH were compared with those from 2 matched arms of those with severe AH treated with standard of care (SOC), including corticosteroids, from a contemporaneous study. RESULTS: All 19 larsucosterol-treated subjects survived the 28-day study. Fourteen (74%) of all subjects including 8 (67%) of the subjects with severe AH were discharged ≤72 hours after receiving a single infusion. There were no drug-related serious adverse events nor early terminations due to the treatment. PK profiles were not affected by disease severity. Biochemical parameters improved in most subjects. Serum bilirubin levels declined notably from baseline to day 7 and day 28, and MELD scores were reduced at day 28. The efficacy signals compared favorably with those from 2 matched groups treated with SOC. Lille scores at day 7 were <0.45 in 16 of the 18 (89%) subjects with day 7 samples. Lille scores from 8 subjects with severe AH who received 30 or 90 mg larsucosterol (doses used in phase 2b trial) were statistically significantly lower ( P < 0.01) than those from subjects with severe AH treated with SOC from the contemporaneous study. DISCUSSION: Larsucosterol was well tolerated at all 3 doses in subjects with AH without safety concerns. Data from this pilot study showed promising efficacy signals in subjects with AH. Larsucosterol is being evaluated in a phase 2b multicenter, randomized, double-blinded, placebo-controlled (AHFIRM) trial.
Subject(s)
End Stage Liver Disease , Hepatitis, Alcoholic , Humans , Pilot Projects , Severity of Illness Index , Hepatitis, Alcoholic/drug therapy , Hepatitis, Alcoholic/diagnosisABSTRACT
Alcohol-associated liver disease (ALD) is a serious public health problem with limited pharmacologic options. The goal of the current study was to investigate the efficacy of pharmacologic inhibition of soluble epoxide hydrolase (sEH), an enzyme involved in lipid metabolism, in experimental ALD, and to examine the underlying mechanisms. C57BL/6J male mice were subjected to acute-on-chronic ethanol (EtOH) feeding with or without the sEH inhibitor 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB). Liver injury was assessed by multiple end points. Liver epoxy fatty acids and dihydroxy fatty acids were measured by targeted metabolomics. Whole-liver RNA sequencing was performed, and free modified RNA bases were measured by mass spectrometry. EtOH-induced liver injury was ameliorated by TUCB treatment as evidenced by reduced plasma alanine aminotransferase levels and was associated with attenuated alcohol-induced endoplasmic reticulum stress, reduced neutrophil infiltration, and increased numbers of hepatic M2 macrophages. TUCB altered liver epoxy and dihydroxy fatty acids and led to a unique hepatic transcriptional profile characterized by decreased expression of genes involved in apoptosis, inflammation, fibrosis, and carcinogenesis. Several modified RNA bases were robustly changed by TUCB, including N6-methyladenosine and 2-methylthio-N6-threonylcarbamoyladenosine. These findings show the beneficial effects of sEH inhibition by TUCB in experimental EtOH-induced liver injury, warranting further mechanistic studies to explore the underlying mechanisms, and highlighting the translational potential of sEH as a drug target for this disease.
Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Liver Diseases, Alcoholic , Mice , Animals , Male , Epoxide Hydrolases/genetics , Epoxide Hydrolases/metabolism , Transcriptome , Mice, Inbred C57BL , Liver Diseases, Alcoholic/genetics , Fatty Acids , Ethanol , RNAABSTRACT
BACKGROUND: Severe alcoholic hepatitis (AH) has a high short-term mortality rate. The MELD assesses disease severity and mortality; however, it is not specific for AH. We screened plasma samples from patients with severe AH for biomarkers of multiple pathological processes and identified predictors of short-term mortality. METHODS: Plasma was collected at baseline from 85 patients with severe AH (MELD≥20, Maddrey's discriminant function≥32) enrolled in the Defeat Alcoholic Steatohepatitis clinical trial (investigating IL-1 receptor antagonist+pentoxifylline+zinc vs. methylprednisolone+placebo). Samples were analyzed for 43 biomarkers and the markers' association with 28- and 90-day mortalities was assessed. RESULTS: Thirty-one (36.5%) patients died during the 90-day follow-up with similar ratios in the treatment groups. Eight biomarkers showed an association with mortality. IL-6, IL-22, interferon-α2, soluble TNF receptor 1, lipocalin-2, and α-fetoprotein levels were associated with 28-day mortality, while IL-6, IL-13, and endotoxin levels with 90-day mortality. In multivariable Cox regression, encephalopathy, lipocalin-2, and α-fetoprotein levels were independent predictors of 28-day mortality, and IL-6, IL-13, international normalized ratio levels, and age were independent predictors of 90-day mortality. The combination of IL-13 and age had superior performance in predicting 90-day mortality compared with MELD in the total cohort and the individual treatment groups. CONCLUSIONS: We identified predictors of short-term mortality in a cohort exclusively involving patients with severe AH. We created a composite score of IL-13 and age that predicts 90-day mortality regardless of the treatment type with a performance superior to MELD in severe AH.
Subject(s)
Age Factors , Hepatitis, Alcoholic , Interleukin-13 , Humans , alpha-Fetoproteins , Biomarkers/blood , Hepatitis, Alcoholic/mortality , Interleukin-13/blood , Interleukin-6 , Lipocalin-2ABSTRACT
BACKGROUND: Cyclic nucleotides are second messengers, which play significant roles in numerous biological processes. Previous work has shown that cAMP and cGMP signaling regulates various pathways in liver cells, including Kupffer cells, hepatocytes, hepatic stellate cells, and cellular components of hepatic sinusoids. Importantly, it has been shown that cAMP levels and enzymes involved in cAMP homeostasis are affected by alcohol. Although the role of cyclic nucleotide signaling is strongly implicated in several pathological pathways in liver diseases, studies describing the changes in genes regulating cyclic nucleotide metabolism in ALD are lacking. METHODS: Male C57B/6 mice were used in an intragastric model of alcohol-associated steatohepatitis (ASH). Liver injury, inflammation, and fibrogenesis were evaluated by measuring plasma levels of injury markers, liver tissue cytokines, and gene expression analyses. Liver transcriptome analysis was performed to examine the effects of alcohol on regulators of cyclic AMP and GMP levels and signaling. cAMP and cGMP levels were measured in mouse livers as well as in livers from healthy human donors and patients with alcohol-associated hepatitis (AH). RESULTS: Our results show significant changes in several phosphodiesterases (PDEs) with specificity to degrade cAMP (Pde4a, Pde4d, and Pde8a) and cGMP (Pde5a, Pde6d, and Pde9a), as well as dual-specificity PDEs (Pde1a and Pde10a) in ASH mouse livers. Adenylyl cyclases (ACs) 7 and 9, which are responsible for cAMP generation, were also affected by alcohol. Importantly, adenosine receptor 1, which has been implicated in the pathogenesis of liver diseases, was significantly increased by alcohol. Adrenoceptors 1 and 3 (Adrb), which couple with stimulatory G protein to regulate cAMP and cGMP signaling, were significantly decreased. Additionally, beta arrestin 2, which interacts with cAMP-specific PDE4D to desensitize G-protein-coupled receptor to generate cAMP, was significantly increased by alcohol. Notably, we observed that cAMP levels are much higher than cGMP levels in the livers of humans and mice; however, alcohol affected them differently. Specifically, cGMP levels were higher in patients with AH and ASH mice livers compared with controls. As expected, these changes in liver cyclic nucleotide signaling were associated with increased inflammation, steatosis, apoptosis, and fibrogenesis. CONCLUSIONS: These data strongly implicate dysregulated cAMP and cGMP signaling in the pathogenesis of ASH. Future studies to identify changes in these regulators in a cell-specific manner could lead to the development of novel targeted therapies for ASH.
ABSTRACT
Introduction: Patients with alcohol use disorder (AUD) exhibit symptoms such as alcohol withdrawal, depression, and cravings. The gut-immune response may play a significant role in manifesting these specific symptoms associated with AUD. This study examined the role of gut dysfunction, proinflammatory cytokines, and hormones in characterizing AUD symptoms. Methods: Forty-eight AUD patients [men (n = 34) and women (n = 14)] aged 23-63 years were grouped using the Clinical Institute Withdrawal Assessment of Alcohol Scale (CIWA) as clinically significant (CS-CIWA [score > 10] [n = 22]) and a clinically not-significant group (NCS-CIWA [score ≤ 10] [n = 26]). Clinical data (CIWA, 90-day timeline followback [TLFB90], and lifetime drinking history [LTDH]) and blood samples (for testing proinflammatory cytokines, hormones, and markers of intestinal permeability) were analyzed. A subset of 16 AUD patients was assessed upon admission for their craving tendencies related to drug-seeking behavior using the Penn-Alcohol Craving Score (PACS). Results: CS-CIWA group patients exhibited unique and significantly higher levels of adiponectin and interleukin (IL)-6 compared to NCS-CIWA. In the CS group, there were significant and high effects of association for the withdrawal score with gut-immune markers (lipopolysaccharide [LPS], adiponectin, IL-6, and IL-8) and for withdrawal-associated depression with gut-immune markers (scored using MADRS with LPS, soluble cells of differentiation type 14 [sCD14], IL-6, and IL-8). Craving (assessed by PACS, the Penn-Alcohol Craving Scale) was significantly characterized by what could be described as gut dysregulation (LBP [lipopolysaccharide binding protein] and leptin) and candidate proinflammatory (IL-1ß and TNF-α) markers. Such a pathway model describes the heavy drinking phenotype, HDD90 (heavy drinking days past 90 days), with even higher effects (R2 = 0.955, p = 0.006) in the AUD patients, who had higher ratings for cravings (PACS > 5). Discussion: The interaction of gut dysfunction cytokines involved in both inflammation and mediating activity constitutes a novel pathophysiological gut-brain axis for withdrawal symptoms and withdrawal-associated depression and craving symptoms in AUD. AUD patients with reported cravings show a significant characterization of the gut-brain axis response to heavy drinking. Trial registration: ClinicalTrials.gov, identifier: NCT# 00106106.
ABSTRACT
PURPOSE: Chemokine-driven leukocyte infiltration and sustained inflammation contribute to alcohol-associated liver disease (ALD). Elevated hepatic CCL2 expression, seen in ALD, is associated with disease severity. However, mechanisms of CCL2 regulation are not completely elucidated. Post-translational modifications (PTMs) of proteins, particularly acetylation, modulate gene expression. This study examined the acetylation changes of promoter-associated histone-H3 and key transcription factor-NFκB in regulating hepatic CCL2 expression and subsequent inflammation and injury. Further, the effect of therapeutic modulation of the acetylation state by tributyrin (TB), a butyrate prodrug, was assessed. METHODS: Hepatic CCL2 expression was assessed in mice fed control (PF) or an ethanol-containing Lieber-DeCarli (5% v/v, EF) diet for 7 weeks with or without oral administration of tributyrin (TB, 2 g/kg, 5 days/week). A chromatin immunoprecipitation (ChIP) assay evaluated promoter-associated modifications. Nuclear association between SIRT1, p300, and NFκB-p65 and acetylation changes of p65 were determined using immunoprecipitation and Western blot analyses. A Student's t-test and one-way ANOVA determined the significance. RESULTS: Ethanol significantly increased promoter-associated histone-H3-lysine-9 acetylation (H3K9Ac), reflecting a transcriptionally permissive state with a resultant increase in hepatic CCL2 mRNA and protein expression. Moreover, increased lysine-310-acetylation of nuclear RelA/p65 decreased its association with SIRT1, a class III HDAC, but concomitantly increased with p300, a histone acetyltransferase. This further led to enhanced recruitment of NF-κB/p65 and RNA polymerase-II to the CCL2 promoter. Oral TB administration prevented ethanol-associated acetylation changes, thus downregulating CCL2 expression, hepatic neutrophil infiltration, and inflammation/ injury. CONCLUSION: The modulation of a protein acetylation state via ethanol or TB mechanistically regulates hepatic CCL2 upregulation in ALD.
Subject(s)
Hepatitis , Histones , Mice , Animals , Histones/metabolism , NF-kappa B/metabolism , Ethanol , Lysine/metabolism , Sirtuin 1/metabolism , Acetylation , Protein Processing, Post-Translational , InflammationABSTRACT
BACKGROUND: Chronic alcohol consumption impairs gut barrier function and perturbs the gut microbiome. Although shifts in bacterial communities in patients with alcohol-associated liver disease (ALD) have been characterized, less is known about the interactions between host metabolism and circulating microbe-derived metabolites during the progression of ALD. METHODS: A large panel of gut microbiome-derived metabolites of aromatic amino acids was quantified by stable isotope dilution liquid chromatography with online tandem mass spectrometry in plasma from healthy controls (n = 29), heavy drinkers (n = 10), patients with moderate (n = 16) or severe alcohol-associated hepatitis (n = 40), and alcohol-associated cirrhosis (n = 10). RESULTS: The tryptophan metabolites, serotonin and indole-3-propionic acid, and tyrosine metabolites, p-cresol sulfate, and p-cresol glucuronide, were decreased in patients with ALD. Patients with severe alcohol-associated hepatitis and alcohol-associated cirrhosis had the largest decrease in concentrations of tryptophan and tyrosine-derived metabolites compared to healthy control. Western blot analysis and interrogation of bulk RNA sequencing data from patients with various liver pathologies revealed perturbations in hepatic expression of phase II metabolism enzymes involved in sulfonation and glucuronidation in patients with severe forms of ALD. CONCLUSIONS: We identified several metabolites decreased in ALD and disruptions of hepatic phase II metabolism. These results indicate that patients with more advanced stages of ALD, including severe alcohol-associated hepatitis and alcohol-associated cirrhosis, had complex perturbations in metabolite concentrations that likely reflect both changes in the composition of the gut microbiome community and the ability of the host to enzymatically modify the gut-derived metabolites.