Chronic Binge Alcohol Administration Research Articles

Characterizing MetALD in a Nonhuman Primate Model using High Fat and Sucrose Diet and Chronic Binge Alcohol Administration

Mechanisms leading to metabolic and alcohol associated liver disease (MetALD) are not clearly delineated. This study examined the differential regulation of markers of hepatic mitochondrial function in nonhuman primates fed a high fat/high sucrose (HFSD) and administered chronic binge alcohol (CBA) or vehicle as compared to control-fed animals. We hypothesized that markers of hepatic dysregulation due to HFSD will be further altered by the combination of CBA/HFSD. Eight rhesus macaques were fed HFSD (27% sucrose and 42% fat, by kcal) with n=4 receiving vehicle and n=4 receiving 13-14g/kg/wk of ethanol through a gastric catheter. Rhesus macaques fed standard primate chow were used as reference controls (n=4-6). Liver RNA was isolated for determining expression of genes regulating mitochondrial biogenesis and function using RT-qPCR. Results were analyzed using one-way ANOVA and post-hoc pairwise multiple comparisons were performed using Tukey’s method, with alpha set to 0.05. Expression of inflammatory genes was significantly altered across the groups (p<0.05). HFSD increased tumor necrosis factor alpha (p=0.01), interleukin 1-beta (p=0.04), cluster of differentiation 38 (p=0.01), and transforming growth factor beta (p=0.01) expression compared to control. Both HFSD and CBA/HFSD increased interleukin-10 compared to control (p=0.02, p=0.03). Gene expression of mitochondrial function and oxidative stress regulators were significantly altered across the groups (p<0.05). CBA/HFSD decreased expression of peroxisome proliferator-activated receptor-gamma coactivator-1 beta (p=0.04) and nuclear respiratory factor 2 (p=0.04) compared to control. Both HFSD and CBA/HFSD decreased peroxisome proliferator-activated receptor alpha expression compared to control (p=0.01, 0.01). HFSD decreased superoxide dismutase 2 (SOD2) expression compared to control (p=0.01) and increased uncoupling protein 2 (p=0.01). SOD2 expression was increased only in CBA/HFSD compared to HFSD (p=0.01). These results reveal marked HFSD-mediated increases in markers of inflammation and CBA/HFSD-mediated changes in markers of mitochondrial function regulation and oxidative stress regulation. These findings suggest a greater impact of calorie-dense diets than CBA on hepatic inflammation and a combined impact of CBA and HFSD on mitochondrial regulation. Ongoing studies will examine oxidative phosphorylation complex protein activities and expression to elucidate and integrate HFSD or CBA mediated changes in hepatic bioenergetic capacity. Supported by F30AA030910, P60AA009803, and T32AA007577. 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.

MicroRNA-206 improves alcohol-mediated decreased myoblast differentiation

Background: With recent advances in antiretroviral therapy (ART), people living with HIV (PLWH) have a near-normal life expectancy but have higher risk for aging-related comorbidities, such as metabolic disorders and frailty earlier in life than the general population. At-risk alcohol use is twice as likely in PLWH compared to the general population and alcohol-related myopathy occurs in 40- 60% of people with alcohol use disorder. Previous studies demonstrated that in vivo chronic binge alcohol (CBA) administration in simian immunodeficiency virus (SIV)-infected male rhesus macaques decreased myoblast differentiation and downregulated microRNA-206 (miR-206) expression in skeletal muscle. We hypothesized that increasing miR-206 expression would increase differentiation in CBA-derived primary macaque myoblasts. Methods: Eight SIV-infected female rhesus macaques received either water (VEH) or CBA (13- 14 g/kg/week) for 14.5 months. Three months into either VEH or CBA administration, animals were vaginally infected with SIVmac251, and ART was started 2.5 months later. Animals were sacrificed after 9 months, and primary myoblasts isolated from vastus lateralis muscle 24 hours after the last dose of alcohol (blood alcohol=0 mM). Myoblasts at passage 4 (VEH & CBA) were proliferated for three days. Cells were then transfected with either miR-206 mimic or control (miR scramble). Four experimental groups were studied at day 5 of differentiation: VEH+scramble, CBA+scramble, VEH+miR-206, and CBA+miR-206. Expression of miR-206 was determined by qPCR. Myoblast differentiation was determined by myotube density, calculated as the average number of myotubes per frame, and fusion index calculated as the percentage of myonuclei that fused into myotubes. Results: In non-transfected CBA-derived cells, myotube density and miR-206 expression was lower compared to VEH-derived myotubes (p<0.001 and p<0.05, respectively). miR-206 transfection resulted in over 150-fold higher miR-206 expression compared to VEH-scramble (p<0.05) in CBA- and VEH-derived cells. miR-206 overexpression significantly increased myotube fusion index (p<0.05) compared to myoblasts transfected with miR scramble in CBA-derived cells. Conclusions: Data show significant improvement of alcohol-mediated decreased myoblast differentiation with miR-206 overexpression. These findings suggest that increasing muscle miR-206 can potentially be used as a therapeutic strategy to improve regenerative capacity in response to muscle injury or atrophy. (Supported by: T35AA021097, F30AA029358, P60AA009803, K01AA024494) This is the full abstract presented at the American Physiology Summit 2023 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.

High‐Fat High‐Sucrose Diet Increases ACE2 Receptor Expression in Lung and Pancreatic Islets in SIV‐Infected Rhesus Macaques: Implications for Increased Risk for SARS‐CoV‐2 Infection

People with pre‐existing conditions including obesity, diabetes, and kidney disease are at greater risk for complications of COVID‐19, the disease caused by SARS‐CoV‐2 infection. Predisposing risk factors could include poor dietary quality and at‐risk alcohol use. Increased survival and aging of people living with HIV (PLWH) on antiretroviral therapy (ART) is complicated by comorbidities including at‐risk alcohol use, metabolic dysregulation, diabetes, and chronic renal disease, with obesity and metabolic syndrome highest in southern states. We hypothesize that poor dietary quality in combination with at‐risk alcohol use may increase risk factors for SARS‐CoV‐2 infection in PLWH. To test this hypothesis, biospecimen obtained from a longitudinal study in female rhesus macaques (macaca mulatta) were used to determine whether high‐fat high‐sucrose diet (HFD; protein/fat/carbohydrates 16/42/42% of total kcal and 27% sucrose by weight) and chronic binge alcohol administration (CBA) increased expression of the machinery required for SARS‐CoV‐2 cell entry. Female macaques (n=10) were assigned to HFD or standard chow diet (SD; protein/fat/carbohydrates 29/14/57% of total kcal) and CBA (50‐60 mM peak blood alcohol, 5 days/week) or isovolumetric water (VEH) beginning 3 months before SIVmac251 infection. ART was initiated at 2.5 months of SIV infection. Tissue samples including lung, pancreas, and kidney were collected at study endpoint (~12 months post‐SIV infection). Immunohistochemistry was performed on formalin‐fixed, paraffin embedded samples to determine protein expression of angiotensin converting enzyme 2 (ACE2) receptor and transmembrane serine protease 2 (TMPRSS2). Images were obtained at 20x magnification and fluorescence intensity was used for quantification. RNA was extracted from frozen samples and mRNA expression of ACE2 and TMPRSS2 was measured by qPCR and quantified relative to ribosomal protein S13. Normality of data was verified and outliers identified using Grubbs test. Data were then analyzed using a 2 (alcohol) × 2 (diet) ANOVA. Protein expression of ACE2 in the lung (p<0.01, η2=0.59), whole pancreas (p<0.05, η2=0.64), and pancreatic islets (p<0.05, η2=0.44) was greater in HFD‐ than SD‐fed macaques and was not significantly altered by CBA. No alcohol‐ or diet‐mediated differences in ACE2 protein expression were observed for whole kidney or proximal tubules. No alcohol‐ or diet‐mediated differences in protein expression of TMPRSS2 or relative mRNA expression of ACE2 or TMPRSS2 were observed. Results indicate that a high‐fat, high‐sucrose diet increases expression of the ACE2 receptor in SIV‐infected female macaques. Because the ACE2 receptor is required for SARS‐CoV‐2 cell entry, this diet‐mediated increase in expression may increase risk for greater vulnerability to COVID‐19 disease and associated complications. These data provide direct evidence for a link between dietary quality and risk for SARS‐CoV‐2 infection in the context of SIV/HIV infection, urging diet counseling and increased access to higher‐quality foods in this population.

URB597 ameliorates the deleterious effects induced by binge alcohol consumption in adolescent rats

Heavy episodic drinking or binge drinking during adolescence may elicit serious neurotoxic consequences in cerebral areas (e.g., the prefrontal cortex, i.e., PFC) and the hippocampus, delay the maturation of the brain and increase the probability of drug abuse and dependence. The endocannabinoid system plays an important role in neuroprotection by reducing oxidative stress and neuroinflammation. In the present study, we aimed to investigate whether URB597, an inhibitor of the metabolic enzyme of the endocannabinoid anandamide (AEA), altered the effects of acute and chronic alcohol administration beginning during rat adolescence on recognition memory, neuroinflammation and brain-derived neurotrophic factor (BDNF) levels. The animals received intraperitoneal injections of URB597 (0.3 mg/Kg) or vehicle followed by the oral administration of ethanol (3 or 6 g/Kg) or distilled water for 3 consecutive days in one week (acute binging) or over 4 weeks (chronic binging). The groups were submitted to the novel object recognition task, and their PFCs and hippocampi were removed for analyses of the cytokine and BDNF levels. URB597 potentiated long-term memory after the 3 mg/Kg acute alcohol administration. The chronic binge alcohol administration increased the interferon (IFN)-γ and tumor necrosis factor (TNF)-α levels in the PFC and hippocampus and the interleukin (IL)-10 and BDNF levels in the PFC, and these effects were prevented by URB597. Our results indicate that the neuromodulation facilitated by AEA can reduce the neuroimmune response induced by the chronic administration of alcohol beginning in adolescence in rats.

The New Orleans Alcohol Use in HIV Study: Launching a Translational Investigation of the Interaction of Alcohol Use with Biological and Socioenvironmental Risk Factors for Multimorbidity in People Living with HIV.

Alcohol use disorders (AUDs) are highly prevalent in people living with HIV (PLWH) and are associated with increased HIV risk behaviors, suboptimal treatment adherence, potential interaction with medication pharmacodynamics, and greater risk for disease progression. Preclinical studies show that chronic binge alcohol administration accelerates disease progression and aggravates pathogenesis in the simian immunodeficiency virus (SIV)-infected rhesus macaque model despite viral suppression by antiretroviral therapy. To translate preclinical findings in the rhesus macaque model of chronic binge alcohol administration and SIV infection and to address areas of uncertainty surrounding the biological mechanisms and socioenvironmental modifiers that contribute to the relationship between alcohol use and HIV-associated comorbidities, precocious aging, and disease progression, we designed a translational multiproject, longitudinal, cohort study, and the New Orleans Alcohol Use in HIV (NOAH) Study. The NOAH Study is led by a multidisciplinary team of scientists, with a research focus on the interaction of AUD and HIV. The overarching hypothesis is that alcohol use will lead to adverse health outcomes in PLWH. In this report, we describe the study design and baseline descriptive characteristics of our cohort. Three-hundred and sixty-five participants completed the baseline testing. The cohort is predominantly male (69%) and African American (83.5%). The majority of participants report incomes below 200% of the federal poverty level. CD4 counts<200cells/μl were found in 12.8% and viral loads<50copies/ml were found in 73.6%. These HIV status variables did not differ based upon alcohol use. The NOAH Study facilitates bidirectional translational investigation of alcohol's impact on PLWH. Translation of preclinical findings to PLWH permits confirmation of basic biological mechanisms in humans and also allows incorporation of sociobehavioral factors that may affect biology but are challenging to replicate in preclinical models.

Chronic binge alcohol administration dysregulates global regulatory gene networks associated with skeletal muscle wasting in simian immunodeficiency virus-infected macaques

BackgroundThere are more than 1 million persons living with HIV/AIDS (PLWHA) in the United States and approximately 40 % of them have a history of alcohol use disorders (AUD). Chronic heavy alcohol consumption and HIV/AIDS both result in reduced lean body mass and muscle dysfunction, increasing the incidence of comorbid conditions. Previous studies from our laboratory using rhesus macaques infected with Simian Immunodeficiency Virus (SIV) demonstrated that chronic binge alcohol (CBA) administration in the absence of antiretroviral therapy exacerbates skeletal muscle (SKM) wasting at end-stage SIV disease. The aim of this study was to characterize how CBA alters global gene regulatory networks that lead to SKM wasting at end-stage disease. Administration of intragastric alcohol or sucrose to male rhesus macaques began 3 months prior to SIV infection and continued throughout the duration of study. High-output array analysis was used to determine CBA-dependent changes in mRNA expression, miRNA expression, and promoter methylation status of SKM at end-stage disease (~10 months post-SIV) from healthy control (control), sucrose-administered, SIV-infected (SUC/SIV), and CBA-administered/SIV-infected (CBA/SIV) macaques.ResultsIn addition to previously reported effects on the extracellular matrix and the promotion of a pro-inflammatory environment, we found that CBA adversely affects gene regulatory networks that involve “universal” cellular functions, protein homeostasis, calcium and ion homeostasis, neuronal growth and signaling, and satellite cell growth and survival.ConclusionsThe results from this study provide an overview of the impact of CBA on gene regulatory networks involved in biological functions, including transcriptional and epigenetic processes, illustrating the genetic and molecular mechanisms associated with CBA-dependent SKM wasting at end-stage SIV infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2329-z) contains supplementary material, which is available to authorized users.

Chronic Binge Alcohol Administration in Simian Immunodeficiency Virus‐Infected Rhesus Macaques Modulates Pre‐Frontal Cortex Expression of Brain‐Derived Neurotrophic Factor and Microtubule‐Associated Protein 2

Persons living with HIV/AIDS have a higher incidence of alcohol use and abuse. Previous studies by our group revealed that chronic binge alcohol (CBA) administration unmasks cognitive deficits in SIV-infected rhesus macaques. Chronic alcohol abuse and HIV infection result in loss of neurotrophic factors and neurons. We hypothesized that CBA administration would synergize with SIV to produce greater suppression of neurotrophic factor expression compared to SUC/SIV. Rhesus macaques were fitted with an intra-gastric catheter for delivery of alcohol (CBA, n = 7) or isocaloric sucrose (SUC, n = 7) for the duration of the experiment. Macaques were infected with SIVmac251 three months after initiation of CBA or SUC, one SUC animal was not infected. Macaques were euthanized when end-stage criteria were met (∼4 or 18 months post-inoculation), brains were excised and dissected. Pre-frontal cortex (PFC), caudate (Cd) and hippocampus (Hp) were used for analysis of tissue viral load and gene expression of BDNF and MAP2. Viral load was not significantly different in CBA animals or between brain regions. PFC was the only brain in which viral load was above the limit of detection in all animals. BDNF expression was lower in CBA/SIV only in the PFC (p=NS). MAP2 expression was lower in PFC and Cd (p=NS). Gene expression of MAP2 was positively correlated with BDNF in the PFC (p<0.01), Cd (p<0.01) and Hp (p=0.03). The results suggest that the PFC is most susceptible to combined SIV and CBA insult. Supported by AA07577, AA09803, AA11290.

Chronic binge alcohol administration accentuates expression of pro-fibrotic and inflammatory genes in the skeletal muscle of simian immunodeficiency virus-infected macaques.

Chronic binge alcohol (CBA) administration exacerbates skeletal muscle (SKM) wasting at the terminal stage of simian immunodeficiency virus (SIV) infection in rhesus macaques. This is associated with a pro-inflammatory and oxidative milieu which we have previously shown to be associated with a disrupted balance between anabolic and catabolic mechanisms. In this study, we attempted to characterize the SKM gene expression signature in CBA-administered SIV-infected macaques, using the same animals from the previous study. Administration of intragastric alcohol or sucrose to male rhesus macaques began 3months prior to SIV infection and continued throughout the duration of study. Gene transcriptomes of SKM excised at necropsy (~10months post-SIV) from healthy naïve control (Control), sucrose-administered, SIV-infected (SUC-SIV), and CBA-administered, SIV-infected (CBA-SIV) macaques were evaluated in microarray data sets. The Protein Analysis Through Evolutionary Relationships classification tool was used to filter differentially regulated genes based on their predicted function into select biological processes relevant to SKM wasting which were inflammation, extracellular matrix (ECM) remodeling, and metabolism. In total, 1,124 genes were differentially regulated between SUC-SIV and Controls, 2,022 genes were differentially expressed between the CBA-SIV and Controls, and 836 genes were differentially expressed between CBA-SIV and SUC-SIV animals. The relevance of altered gene expression was reflected in the up-regulation of pro-inflammatory CCL-2, CCL-8, CX3CL1, SELE, HP, and TNFRS10A mRNA expression. In addition, ECM remodeling was reflected in the up-regulation of TIMP-1, MMP 2, and MMP 9 mRNA expression and transforming growth factor-beta 1 protein expression. In addition, hydroxyproline content and picrosirius staining reflected increased collagen deposition in the CBA-SIV muscle tissue. The results of the study demonstrate SKM inflammation as an important underlying mechanism for muscle wasting. In addition, the study provides evidence of SKM fibrotic transformation as a factor in CBA-induced accentuation of SIV-associated muscle wasting.

Transient Receptor Potential Vanilloid 1 Gene Deficiency Ameliorates Hepatic Injury in a Mouse Model of Chronic Binge Alcohol-Induced Alcoholic Liver Disease

Experimental alcohol-induced liver injury is exacerbated by a high polyunsaturated fat diet rich in linoleic acid. We postulated that bioactive oxidized linoleic acid metabolites (OXLAMs) play a critical role in the development/progression of alcohol-mediated hepatic inflammation and injury. OXLAMs are endogenous ligands for transient receptor potential vanilloid 1 (TRPV1). Herein, we evaluated the role of signaling through TRPV1 in an experimental animal model of alcoholic liver disease (ALD). Chronic binge alcohol administration increased plasma OXLAM levels, specifically 9- and 13-hydroxy-octadecadienoic acids. This effect was associated with up-regulation of hepatic TRPV1. Exposure of hepatocytes to these OXLAMs invitro resulted in activation of TRPV1 signal transduction with increased intracellular Ca(2+) levels. Genetic depletion of TRPV1 did not blunt hepatic steatosis caused by ethanol, but prevented hepatic injury. TRPV1 deficiency protected from hepatocyte death and prevented the increase in proinflammatory cytokine and chemokine expression, including tumor necrosis factor-α, IL-6, macrophage inflammatory protein-2, and monocyte chemotactic protein 1. TRPV1 depletion markedly blunted ethanol-mediated induction of plasminogen activator inhibitor-1, an important alcohol-induced hepatic inflammation mediator, via fibrin accumulation. This study indicates, for the first time, that TRPV1 receptor pathway may be involved in hepatic inflammatory response in an experimental animal model of ALD. TRPV1-OXLAM interactions appear to play a significant role in hepatic inflammation/injury, further supporting an important role for dietary lipids in ALD.

Chronic binge alcohol consumption does not diminish effectiveness of continuous antiretroviral suppression of viral load in simian immunodeficiency virus-infected macaques.

Alcohol use disorders (AUDs) are a frequent comorbidity in a large percentage of people living with HIV/AIDS (PLWHA). PLWHA with comorbid AUDs are consistently found to perform poorly at most levels of the HIV treatment cascade, resulting in a higher likelihood of virologic nonsuppression. This has been partly attributed to lower rates of persistence with and adherence to antiretroviral therapies (ART). Focus groups of in-care PLWHA identify the need to suspend ART on drinking days because of the potential for toxicity and/or lack of therapeutic effectiveness. The aim of this study was to examine whether chronic binge alcohol (CBA) consumption decreases the effectiveness of uninterrupted ART, specifically that of nucleoside reverse-transcriptase inhibitors (NRTI) tenofovir and emtricitabine in suppressing viral replication, or results in drug toxicity in simian immunodeficiency virus (SIV)-infected rhesus macaques. Daily CBA or isocaloric sucrose (SUC) administration was initiated 3 months prior to intrarectal SIVmac251 inoculation and continued throughout the study period. ART was initiated 2.5 months after SIV infection and continued through the study period. CBA administration did not prevent or delay the ART-mediated reduction in viral load. Following ART, circulating levels of total protein and creatinine were significantly higher than baseline values in both SUC- and CBA-treated animals, but still within a normal range. No evidence of ART toxicity was observed in either CBA- or SUC-administered macaques. These findings indicate that CBA does not attenuate effectiveness of NRTI suppression of viral load, nor does it appear to interact with NRTI to produce toxicity during the initial 2 months of treatment. We conclude that while efforts to reduce AUD in PLWHA should be a priority, counseling on the importance of adherence to ART even on drinking days should also be promoted.

Reduced Hypothalamic POMC and Anterior Pituitary CRF1 Receptor mRNA Levels After Acute, but Not Chronic, Daily “Binge”Intragastric Alcohol Administration

Endogenous corticotropin-releasing factor (CRF), its pituitary CRF1 receptor, and proopiomelanocortin (POMC) may be involved in the hypothalamic-pituitary-adrenal (HPA) responses to alcohol. Alcohol (1.5 g/kg) or water was administered intragastrically to male Fischer rats after the "binge" pattern regimen, that is, three times daily at 1 hr intervals at the beginning of the light cycle. The levels of CRF, CRF1 receptor, and POMC mRNAs in the hypothalamic-pituitary axis were measured after acute (1 day) or chronic (14 days) binge pattern alcohol administration. Plasma levels of ACTH and corticosterone were measured to examine time-dependent alterations of HPA responses. Plasma ACTH and corticosterone levels were elevated dramatically after 1 day of acute binge pattern alcohol administration. After 14 days of chronic alcohol, however, no elevation in plasma ACTH levels and an attenuated elevation in plasma corticosterone levels were found. CRF mRNA levels in the hypothalamus were not altered after either acute or chronic alcohol administration. CRF1 receptor mRNA levels in the anterior pituitary were decreased significantly after acute administration, with no change after chronic alcohol administration. POMC mRNA levels in the anterior pituitary were not altered by either acute or chronic alcohol administration. In the hypothalamus, POMC mRNA levels were decreased significantly after acute but not chronic binge alcohol administration. These results suggest that (1) rats exposed to chronic binge alcohol develop tolerance in HPA activity, as shown by no elevation of ACTH and an attenuated corticosterone response to chronic alcohol after initial dramatic elevations by acute alcohol administration; (2) a concurrent acute decrease in CRF1 receptor mRNA levels in the anterior pituitary is associated with increased HPA activity, and (3) alterations of POMC gene expression in the hypothalamic region may have implications for a molecular understanding of the neuroendocrine response to alcohol.