Liver Zonation - Revisiting Old Questions With New Technologies

Abstract

Despite the ever-increasing prevalence of non-alcoholic fatty liver disease (NAFLD), the etiology and pathogenesis remain poorly understood. This is due, in part, to the liver's complex physiology and architecture. The liver maintains glucose and lipid homeostasis by coordinating numerous metabolic processes with great efficiency. This is made possible by the spatial compartmentalization of metabolic pathways a phenomenon known as liver zonation. Despite the importance of zonation to normal liver function, it is unresolved if and how perturbations to liver zonation can drive hepatic pathophysiology and NAFLD development. While hepatocyte heterogeneity has been identified over a century ago, its examination had been severely hindered due to technological limitations. Recent advances in single cell analysis and imaging technologies now permit further characterization of cells across the liver lobule. This review summarizes the advances in examining liver zonation and elucidating its regulatory role in liver physiology and pathology. Understanding the spatial organization of metabolism is vital to further our knowledge of liver disease and to provide targeted therapeutic avenues.

Keywords: architecture; liver; physiology; technologies; zonation.

FIGURE 1

Liver anatomy and the hepatic lobule. The liver is composed of hexagonal units called lobules. Oxygen and nutrient rich blood flows directionally from the hepatic vessels (red) in the corners of the lobule toward a central vein in the middle (blue). Periportal hepatocytes (zone 1) are at the periphery of the lobule, followed by mid-lobular cells, and finally pericentral hepatocytes surround the central vein (zone 3). The variable microenvironment along the periportal-pericentral axis results in graded gene expression and the spatial separation of certain metabolic processes to periportal (red) and pericentral (blue) regions. Certain liver injuries are also zone-dependent, with some originating or confined to periportal (yellow) regions and some to pericentral regions of the lobule (brown). NAFLD, non-alcoholic fatty liver disease.

FIGURE 2

Technological advances in the study of liver zonation. Over the past century, technological advances have driven discoveries related to hepatocyte organization and function. Early observations describing structural heterogeneity were confined to microscopy techniques (Noël, 1923; Kater, 1933; Shank et al., 1959). The development of hepatocyte separation approaches (1950–1980), including perfusion techniques, fluorescent activated cell sorting (FACS), and immunohistochemistry allowed biochemical classification of hepatocyte activities (Baron et al., 1981; Gumucio et al., 1981; Lindros and Penttilä, 1985). Deep insight into the differential gene expression patterns in the hepatic lobule was accomplished in 2017, where single cell RNA sequencing revealed that fifty percent of liver genes are non-uniformly distributed (Halpern et al., 2017). The first three-dimensional (3D) multiphoton microscopy of the human liver lobule reveled disrupted bile canalicular network in nonalcoholic fatty liver disease patients in 2019 (Segovia-Miranda et al., 2019). Recently (2021), changes in gene expression patterns across the lobule were examined in different times of the day revealing yet another layer of complexity in the regulation of compartmentalization (Droin et al., 2021). Advances in mouse genetics and light microscopy now allow the use of Intravital microscopy for examination of the hepatic lobule in real time. Depicted is a projection of 3D volume of the murine lobule acquired with intravital confocal microscopy (PP, periportal; PC, pericentral; bile canaliculi in green, sinusoids in red).

FIGURE 3

Comparison of mouse and human liver lobules. Confocal microscopy image of immunofluorescence-stained mouse and human liver sections illustrate the conservation and differences between species regarding markers of hepatocyte heterogeneity. (A) Mouse liver section with a pericentral (PC) to periportal (PP) axis highlighted showing; a uniform nuclei distribution across the axis (DAPI in yellow), glutamine synthetase (GS) is selectively expressed in pericentral hepatocytes (cyan), and E-cadherin is enriched in periportal hepatocytes (magenta). (B) Human liver section with a pericentral (PC) to periportal (PP) axis highlighted showing; a uniform nuclei distribution across the axis (DAPI in yellow) and glutamine synthetase selectively expressed in pericentral hepatocytes (cyan), while argininosuccinate synthase 1 (ASS1, green) is expressed predominantly in periportal hepatocytes. Pericentral expression of glutamine synthetase is conserved between murine and human. Scale bar = 50 μm.