SAT-249 - Ethanol Rapidly Activates Carbohydrate Responsive Element Binding Protein-Dependent Metabolic Reprogramming In The Liver.

Background and Rationale: Alcohol consumption causes ~180,000 deaths annually in the U.S, primarily due to liver disease, imposing a significant health and economic burden. Despite this, the earliest molecular responses of ethanol remain poorly defined. We have previously identified that ethanol increases glycerol-3-phosphate (G3P) levels, which acts as an activator for the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in the liver. ChREBP is known to regulate glycolytic and lipogenic genes. However, its downstream gene network under acute ethanol exposure remains unclear.

Methods: To define these early transcriptional programs, liver specific ChREBP knockout and control mice were subjected to ethanol or water via oral gavage, and liver tissues were collected at 1 and 3 hours. RNA-seq, followed by DAVID Gene Ontology pathway analyses were performed.

Results: Acute ethanol exposure induced rapid transcriptional changes in the liver, revealing a temporal shift from 1 to 3 hours. Gene Ontology analysis displayed activation of stress-response pathways, including oxidative stress and inflammation within 1 hour. Interestingly, transcription factors Bhlhe40 and Bhlhe41, known players in nutrient metabolism, were significantly upregulated in a ChREBP dependent manner. By 3 hours, the program switched to coordinated metabolic reprogramming, with upregulation of Gpi1 and Gpd1, which is consistent with our previous finding of increased G3P levels upon ethanol exposure, as well as Glut4, Fasn, Acc, Pnpla3. This upregulation was absent in ChREBP knockout mice. Notably, Bhlhe40 and Bhlhe41 were not induced at 3 hours, positioning these regulators as early effectors of ChREBP that drive the downstream metabolic remodeling. To test this hypothesis, we collected livers from hepatocyte-specific BHLHE40 knockout and control mice after 3 hours of ethanol exposure. qRT-PCR analysis revealed a compensatory upregulation of Bhlhe41, with no differences in metabolic gene expression between knockout and control mice. These findings indicate functional redundancy and a combined role for BHLHE40 and BHLHE41 in the ethanol response. We are currently generating BHLHE40/41 double knockout mice for evaluation.

Conclusion: Our findings demonstrate a rapid ChREBP dependent transcriptional program, transitioning from early stress response to structured metabolic remodeling following ethanol exposure. Completion of this work will provide insights into how ethanol primes the liver during early events in alcoholic liver diseases.

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