Date of Award

Spring 4-15-2025

Embargo Period

4-15-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

John Lemasters

Second Advisor

Zhi Zhong

Third Advisor

Danyelle Townsend

Fourth Advisor

Don Rockey

Fifth Advisor

Stephen Duncan

Abstract

Background: Ethanol (EtOH) consumption causes hepatocellular mitochondrial depolarization (mtDepo) and mitophagy. Here, the Aims were to develop techniques to characterize sublobular hepatocellular metabolism and the distribution of mtDepo and mitophagy after acute EtOH. Methods: Wildtype and GFP-LC3 transgenic mice were treated with EtOH or vehicle and administered the covalently labeling mitochondrial membrane potential (ΔΨ) reporter, MitoTracker Red (MTR), and/or the non-covalent ΔΨ reporter, rhodamine 123 (Rh123), prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted. Enrichment in E-cadherin (ECAD) and cytochrome P4502E1 in periportal (PP) and pericentral (PC) hepatocytes, respectively, validated the sort. Oxygen consumption rates (OCR) were assessed by Seahorse respirometry. Results: Multiphoton microscopy showed that Rh123 and MTR fluorescence distributed zonally, decreasing from periportal (PP) to pericentral (PC) zones in a flow-dependent fashion. After vehicle, intracellular MTR fluorescence was mitochondrial in all liver zones, signifying mitochondrial polarization. OCRs (ATP-linked, proton leak-linked, and maximal) of sorted PP hepatocytes were ~4 times that of PC. After EtOH, intracellular MTR fluorescence became non-mitochondrial in the central half but not the portal half of liver lobules, signifying mtDepo. Mitophagy indicated by GFP-LC3 puncta was also only increased in the central half. After EtOH, OCRs of both PP and PC hepatocytes were ~2-times that of vehicle, but PC hepatocytes displayed greater proportional increases. In PC hepatocytes, basal OCR was inhibited by oligomycin and stimulated by uncoupler, suggesting reversal of mtDepo, which Rh123 uptake confirmed. Conclusions: After EtOH, mtDepo is restricted to the central half of liver lobules with a sharp midzonal demarcation from the portal half. In sorted hepatocytes without EtOH treatment, OCRs of PP hepatocytes are substantially greater than PC, but both PP and PC OCRs increase after EtOH. However, mtDepo in PC hepatocytes after in vivo EtOH treatment reverses during isolation. Increased mitochondrial respiratory capacity in all sublobular zones likely adaptively promotes detoxifying EtOH metabolism. These findings document sharp zonal differences in hepatic metabolic responses to EtOH exposure that may help shed light on the progression to alcoholic liver disease.

Rights

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