MitoTracker Red for Isolation of Zone-specific Hepatocytes and Characterization of Hepatic Sublobular Metabolism

Overview

Journal Biochem Biophys Res Commun

Publisher Elsevier

Specialty Biochemistry

Date 2024 Aug 15

PMID 39146811

Authors

Matthew Savoca

Kenji Takemoto

Jiangting Hu

Li Li

B Jacob Kendrick

Zhi Zhong

John J Lemasters

Affiliations

Soon will be listed here.

Abstract

Background: The liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics.

Aim: Our goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones.

Methods: Mice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed.

Results: Multiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes.

Conclusions: MTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

References

Samuvel D, Li L, Krishnasamy Y, Gooz M, Takemoto K, Woster P . Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice. Autophagy. 2022; 18(11):2671-2685. PMC: 9629059. DOI: 10.1080/15548627.2022.2046457. View

Lemasters J, Chacon E, Ohata H, Harper I, Nieminen A, Tesfai S . Measurement of electrical potential, pH, and free calcium ion concentration in mitochondria of living cells by laser scanning confocal microscopy. Methods Enzymol. 1995; 260:428-44. DOI: 10.1016/0076-6879(95)60156-2. View

Rappaport A, BOROWY Z, LOUGHEED W, LOTTO W . Subdivision of hexagonal liver lobules into a structural and functional unit; role in hepatic physiology and pathology. Anat Rec. 1954; 119(1):11-33. DOI: 10.1002/ar.1091190103. View

Dall M, Trammell S, Asping M, Hassing A, Agerholm M, Vienberg S . Mitochondrial function in liver cells is resistant to perturbations in NAD salvage capacity. J Biol Chem. 2019; 294(36):13304-13326. PMC: 6737221. DOI: 10.1074/jbc.RA118.006756. View

Abdelmegeed M, Ha S, Choi Y, Akbar M, Song B . Role of CYP2E1 in Mitochondrial Dysfunction and Hepatic Injury by Alcohol and Non-Alcoholic Substances. Curr Mol Pharmacol. 2015; 10(3):207-225. PMC: 4757513. DOI: 10.2174/1874467208666150817111114. View

McCarthy W, Sherlock L, Grayck M, Zheng L, Lacayo O, Solar M . Innate Immune Zonation in the Liver: NF-κB (p50) Activation and C-Reactive Protein Expression in Response to Endotoxemia Are Zone Specific. J Immunol. 2023; 210(9):1372-1385. PMC: 10121917. DOI: 10.4049/jimmunol.2200900. View

Nieminen A, Saylor A, Herman B, Lemasters J . ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition. Am J Physiol. 1994; 267(1 Pt 1):C67-74. DOI: 10.1152/ajpcell.1994.267.1.C67. View

Kang S, Cunningham R, Miller C, Brown L, Cultraro C, Harned A . A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling. Nat Commun. 2024; 15(1):1799. PMC: 10902380. DOI: 10.1038/s41467-024-45751-9. View

Lemasters J . Metabolic implications of non-electrogenic ATP/ADP exchange in cancer cells: A mechanistic basis for the Warburg effect. Biochim Biophys Acta Bioenerg. 2021; 1862(7):148410. PMC: 8096716. DOI: 10.1016/j.bbabio.2021.148410. View

10.

Cunningham R, Porat-Shliom N . Liver Zonation - Revisiting Old Questions With New Technologies. Front Physiol. 2021; 12:732929. PMC: 8458816. DOI: 10.3389/fphys.2021.732929. View

11.

Neve E, Ingelman-Sundberg M . Molecular basis for the transport of cytochrome P450 2E1 to the plasma membrane. J Biol Chem. 2000; 275(22):17130-5. DOI: 10.1074/jbc.M000957200. View

12.

Kasahara D, Sumiyoshi H, Endo H, Yanagawa T, Nakano Y, Matsuki Y . Visualization and isolation of zone-specific murine hepatocytes that maintain distinct cytochrome P450 oxidase expression in primary culture. Biochem Biophys Res Commun. 2020; 528(3):420-425. DOI: 10.1016/j.bbrc.2020.05.202. View

13.

Wei Y, Wang Y, Jia Y, Li L, Yoon J, Zhang S . Liver homeostasis is maintained by midlobular zone 2 hepatocytes. Science. 2021; 371(6532). PMC: 8496420. DOI: 10.1126/science.abb1625. View

14.

Rosenberger F, Thielert M, Strauss M, Schweizer L, Ammar C, Madler S . Spatial single-cell mass spectrometry defines zonation of the hepatocyte proteome. Nat Methods. 2023; 20(10):1530-1536. PMC: 10555842. DOI: 10.1038/s41592-023-02007-6. View

15.

Rodriguez-Enriquez S, Kim I, Currin R, Lemasters J . Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes. Autophagy. 2006; 2(1):39-46. PMC: 4007489. DOI: 10.4161/auto.2229. View

16.

Gebhardt R . Metabolic zonation of the liver: regulation and implications for liver function. Pharmacol Ther. 1992; 53(3):275-354. DOI: 10.1016/0163-7258(92)90055-5. View

17.

Zhong Z, Ramshesh V, Rehman H, Liu Q, Theruvath T, Krishnasamy Y . Acute ethanol causes hepatic mitochondrial depolarization in mice: role of ethanol metabolism. PLoS One. 2014; 9(3):e91308. PMC: 3950152. DOI: 10.1371/journal.pone.0091308. View

18.

Uchiyama A, Kim J, Kon K, Jaeschke H, Ikejima K, Watanabe S . Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury. Hepatology. 2008; 48(5):1644-54. PMC: 2579320. DOI: 10.1002/hep.22498. View

19.

Jarrett S, Rohrer B, Perron N, Beeson C, Boulton M . Assessment of mitochondrial damage in retinal cells and tissues using quantitative polymerase chain reaction for mitochondrial DNA damage and extracellular flux assay for mitochondrial respiration activity. Methods Mol Biol. 2012; 935:227-43. DOI: 10.1007/978-1-62703-080-9_16. View

20.

Nault R, Saha S, Bhattacharya S, Sinha S, Maiti T, Zacharewski T . Single-cell transcriptomics shows dose-dependent disruption of hepatic zonation by TCDD in mice. Toxicol Sci. 2022; 191(1):135-148. PMC: 9887712. DOI: 10.1093/toxsci/kfac109. View