Liver Zonation – Revisiting Old Questions With New Technologies

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.

Temporal Analyses of Postnatal Liver Development and Maturation by Single Cell Transcriptomics

Liver is the major metabolic organ, although its postnatal development and maturation are inadequately understood. We analyzed 52,834 single cell transcriptomes and identified 31 cell types or states in mouse livers at postnatal day 1, 3, 7, 21 and 56. We observed unexpectedly high levels of hepatocyte heterogeneity in the developing liver and progressive construction of the zonated metabolic functions from pericentral to periportal hepatocytes, which was orchestrated with development of sinusoid endothelial, stellate and Kupffer cells. Trajectory and gene regulatory analyses captured 36 transcription factors, including a circadian regulator Bhlhe40, in programming liver development. Remarkably, we identified a special group of macrophages enriched at day 7 with a hybrid phenotype of macrophages and endothelial cells, which may regulate sinusoidal construction and Treg cell function. This study provides a comprehensive atlas that covers all hepatic cell types instrumental for further dissection of liver development, metabolic functions and diseases.

Intracellular lipid content is a key intrinsic determinant for hepatocyte viability and metabolic and inflammatory states in mice

The liver is an essential metabolic organ. In addition to metabolizing glucose and lipids, hepatocytes also secrete various cytokines that modulate both hepatocyte metabolism and liver inflammation. Hepatocyte injury and death and liver inflammation are the major contributors to liver diseases, including nonalcoholic steatohepatitis (NASH). Anatomic locations have a profound effect on hepatocyte metabolism, and liver zonation describes the metabolic heterogeneity of hepatocytes along the portovenous axis. However, it is unclear whether hepatocyte heterogeneity is affected by intrinsic factors and whether dietary fat, a risk factor for NASH, has distinct detrimental effects on different hepatocyte subpopulations. Here, we showed that mouse livers contained both high-lipid and low-lipid subpopulations of hepatocytes. The high-lipid subpopulation was more susceptible to injury and apoptosis and produced more proinflamatrory cytokines after treatment with endotoxin and saturated fatty acids. Dietary fat consumption further increased fatty acid uptake, intracellular lipid levels, hepatocyte injury and death, and the expression of proinflammatory cytokines in the high-lipid subpopulation. In contrast, dietary fat slightly increased lipid levels, cell death, and expression of proinflammatory cytokines in the low-lipid subpopulation. The low-lipid subpopulation produced more glucose. Fat consumption further activated the gluconeogenic program in the low-lipid, but not the high-lipid, subpopulations. These data suggest that intracellular lipid content is a key intrinsic determinant for hepatocyte heterogeneity of metabolic, inflammatory, and survival states.

Dynamic Network Reconstruction from Gene Expression Data Describing the Effect of LiCl Stimulation on Hepatocytes

Summary Wnt/β-catenin signalling plays an important role in zonation of liver parenchyma and in patterning of hepatocyte heterogeneity. A characteristic marker of this heterogeneity is glutamine synthetase, which is expressed only in a subset of pericentrally located hepatocytes. To investigate, whether and how the Wnt/β-catenin signalling pathway is involved a culture of hepatocytes was stimulated by LiCl. This resulted in an increase in the specific GS activity, indicating that the Wnt/β-catenin pathway may participate in regulating GS levels. Affymetrix GeneChip oligonucleotide arrays were used to monitor the gene expression changes during a period from 2 to 24 hours after stimulation by LiCl. Samples from a cultivation without stimulation were used as controls. Based on the gene expression profiles a hypothetic signal transduction network was constructed by a reverse engineering algorithm. The network robustness was tested and the most stable structure was identified.

Hepatocyte heterogeneity in response to extracellular adenosine

Metabolic and haemodynamic effects of adenosine were studied in antegrade and retrograde rat liver perfusions with influent nucleoside concentrations either below (i.e. 20 microM) or exceeding (i.e. 200-300 microM) the single-pass clearance capacity of the liver. Adenosine (20 microM) increased in antegrade perfusions the perfusion pressure and markedly stimulated prostaglandin D2, thromboxane B2 and glucose output, whereas in retrograde perfusions no pressure and eicosanoid response occurred and glucose output was stimulated only slightly. The perfusion-direction-dependent differences in the glucose and pressure response to adenosine (20 microM) were fully abolished in presence of ibuprofen (50 microM). When the adenosine concentration in influent was raised to 200-300 microM, i.e. to a concentration exceeding single-pass clearance of the nucleoside, the adenosine-induced prostaglandin D2 release was about 10-fold higher in retrograde perfusions than in antegrade perfusions. On the other hand, both adenosine (20-300 microM)-induced cyclic AMP (cAMP) and K+ release from the liver were not affected by the direction of perfusion, and maximal effects on cAMP release were observed at influent adenosine concentrations of 100 microM. The basal rate (adenosine absent) of prostaglandin D2 and thromboxane B2 release was about 10-fold higher in retrograde than in antegrade perfusion experiments, whereas the basal cAMP release from the liver was not affected by the direction of perfusion. Maximal adenosine-stimulated glucose output was significantly higher in antegrade than in retrograde perfusions at all adenosine concentrations tested (range 10-300 microM). Ibuprofen abolished this difference, indicating that eicosanoids liberated under the influence of adenosine contribute to the glycogenolytic response in antegrade, but not in retrograde, perfusion. Desensitization occurred following repetitive adenosine infusion; this was more pronounced for adenosine-induced prostaglandin release than for cAMP or K+ efflux. The data suggest the following. (i) Both cAMP and eicosanoids are involved in the stimulation of glycogenolysis by adenosine. (ii) Eicosanoids are probably liberated under the influence of extracellular adenosine from a portal pre-sinusoidal compartment and accordingly stimulate glycogenolysis only in antegrade perfusions. Thus signals derived from portal vein structures can modulate hepatocellular function. (iii) Contractile elements are probably located also inside the liver acinus. (iv) Eicosanoids released into the hepatic vein reflect less than 10% of hepatic eicosanoid formation, because of marked clearance by perivenous hepatocytes.