Hepatocytes work in highly structured, repetitive hepatic lobules. Blood flow across the radial axis of the lobule generates oxygen, nutrient, and hormone gradients, which result in zoned spatial variability and functional diversity. This large heterogeneity suggests that hepatocytes in different lobule zones may have distinct gene expression profiles, metabolic features, regenerative capacity, and susceptibility to damage. Here, we describe the principles of liver zonation, introduce metabolomic approaches to study the spatial heterogeneity of the liver, and highlight the possibility of exploring the spatial metabolic profile, leading to a deeper understanding of the tissue metabolic organization. Spatial metabolomics can also reveal intercellular heterogeneity and its contribution to liver disease. These approaches facilitate the global characterization of liver metabolic function with high spatial resolution along physiological and pathological time scales. This review summarizes the state of the art for spatially resolved metabolomic analysis and the challenges that hinder the achievement of metabolome coverage at the single-cell level. We also discuss several major contributions to the understanding of liver spatial metabolism and conclude with our opinion on the future developments and applications of these exciting new technologies.

Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the "gold standard" for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.

Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced β-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and β-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

Bone morphogenetic protein-9 (BMP-9) is a member of the transforming growth factor beta (TGF-β) superfamily of cytokines, which regulate cell growth and differentiation during embryogenesis. Apart of that, the hypoglycemic potential of BMP-9 is of great interest. It has been confirmed that BMP-9, like insulin, improves glycemia in diabetic mice and regulates directional glucose metabolism in hepatocytes; therefore it is proposed to be a candidate hepatic insulin-sensitizing substance (HISS). In liver fibrosis, due to the portocaval shunt, insulin bypasses the organ and the liver undergoes atrophy. Parenteral administration of insulin reverses atrophy by stimulating mitogenic activity of the hepatocytes. Because BMP-9 has a signaling pathway similar to other BMPs and insulin, it is to be expected that BMP-9 has a certain regenerative role in the liver, supporting the above-mentioned is evidence of BMP-9 expression in Dissè's spaces and BMP-7's mitogenic activity in mucosal cells. However, further studies are needed to confirm the possible regenerative role of BMP-9.

Hypoxia and hypoxia-reoxygenation (H-R) regulate human hepatocyte cell death by mediating the accumulation of reactive oxygen species (ROS). Hepatocytes within the liver are organised into peri-portal (PP) and peri-venous (PV) subpopulations. PP and PV hepatocytes differ in size and function. We investigated whether PP and PV human hepatocytes exhibit differential susceptibility to hypoxic stress. Isolated hepatocytes were used in an in vitro model of hypoxia and H-R. ROS production and cell death were assessed using flow cytometry. PV, and not PP hepatocytes, accumulate intracellular ROS in a mitochondrial dependent manner during hypoxia and H-R. This increased ROS regulates hepatocyte apoptosis and necrosis via a mitochondrial pathway. These findings have implications on the understanding of liver injury and application of potential therapeutic strategies.

To study the regulatory mechanisms of sinusoidal regeneration after partial hepatectomy.

We investigated the expression of angiopoietin (Ang)-1, Ang-2, Tie-2, and vascular endothelial growth factor (VEGF) in regenerating liver tissue by quantitative reverse-transcription polymerase chain reaction (RT-PCR) using a LightCycler (Roche Diagnostics) and also immunohistochemical staining after 70% hepatectomy in rats. In the next step, we isolated liver cells (hepatocytes, sinusoidal endothelial cell (SEC), Kupffer cell, and hepatic stellate cells (HSC)) from regenerating liver tissue by in situ collagenase perfusion and counterflow elutriation, to determine potential cellular sources of these angiogenic factors after hepatectomy. Proliferation and apoptosis of SECs were also evaluated by proliferating cell nuclear antigen (PCNA) staining and the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay, respectively.

VEGF mRNA expression increased with a peak at 72 h after hepatectomy, decreasing thereafter. The expression of Ang-1 mRNA was present at detectable levels before hepatectomy and increased slowly with a peak at 96 h. Meanwhile, Ang-2 mRNA was hardly detected before hepatectomy, but was remarkably induced at 120 and 144 h. In isolated cells, VEGF mRNA expression was found mainly in the hepatocyte fraction. Meanwhile, mRNA for Ang-1 and Ang-2 was found in the SEC and HSC fractions, but was more prominent in the latter. The PCNA labeling index of SECs increased slowly, reaching a peak at 72 h, whereas apoptotic SECs were detected between 120 h and 144 h.

Ang-Tie system, together with VEGF, plays a critical role in regulating balance between SEC proliferation and apoptosis during sinusoidal regeneration after hepatectomy. However, the VEGF system plays a more important role in the early phase of sinusoidal regeneration than angiopoietin/Tie system.

The aim of this study was to investigate regulatory mechanisms of sinusoidal endothelial cell (SEC) proliferation after hepatectomy in rats.

We investigated expressions of vascular endothelial cell growth factor (VEGF) and its receptors, flt-1 and KDR/flk-1, in regenerating liver after 70% hepatectomy. Proliferation of both hepatocytes and SECs was also monitored by evaluating the proliferating cell nuclear antigen (PCNA) labeling index. Furthermore, VEGF production by cultured hepatocytes isolated at different times after hepatectomy was measured in vitro.

The expression of VEGF mRNA was increased markedly between 48 and 72 h after hepatectomy, and thereafter decreasing gradually. The immunohistochemical staining revealed that expression of VEGF started to increase 24 h after hepatectomy, with a peak at 72 h, and the majority of the VEGF-positive cells were hepatocytes located in periportal areas. Meanwhile, expression of flt-1 and KDR/flk-1 was observed along the sinusoids even before hepatectomy, but was increased between 72 and 120 h. Furthermore, VEGF production by cultured hepatocytes isolated 72 h after hepatectomy was significantly increased. The PCNA labeling index of the SECs exhibited a delayed and slower regenerative response in comparison to the hepatocytes, reaching a peak at 72 h.

These data strongly suggest that VEGF secreted by proliferating hepatocytes may represent an important stimulator of SEC proliferation.

Cancer influences hepatic amino acid metabolism in the host. To further investigate this relationship, the effects of an implanted fibrosarcoma on specific amino acid transport activities were measured in periportal (PP)- and perivenous (PV)-enriched rat hepatocyte populations. Na(+)-dependent glutamate transport rates were eightfold higher in PV than in PP preparations but were relatively unaffected during tumor growth. System N-mediated glutamine uptake was 75% higher in PV than in PP preparations and was stimulated up to twofold in both regions by tumor burdens of 9 +/- 4% of carcass weight compared with hepatocytes from pair-fed control animals. Excessive tumor burdens (26 +/- 7%) resulted in hypophagia, loss of PV-enriched system N activities, and reduced transporter stimulation. Conversely, saturable arginine uptake was enhanced fourfold in PP preparations and was induced twofold only after excessive tumor burden. These data suggest that hepatic amino acid transporters are differentially influenced by cancer in a spatial and temporal manner, and they represent the first report of reciprocal zonal enrichment of system N and saturable arginine uptake in the mammalian liver.

Cytochromes P-450 (P-450) are members of a multigene superfamily of hemoproteins consisting the microsomal monooxygenase system with NADPH P-450 reductase (reductase) and/or reducing equivalents. Expression of many P-450 isoforms in hepatocytes is shown to be regulated at the level of transcription through interaction between cis-acting elements in the genes and DNA-binding (transacting) factors. Some isoforms of the CYP1A, 2B, 2E, and 3A subfamilies are regulated at the posttranscriptional level. For the topology of P-450 and reductase molecules in ER membrane of hepatocytes, models from stopped flow analysis and electron spin resonance are proposed. The densities of total P-450 and reductase molecules are revealed to be high enough to support the cluster model, suggesting that about ten P-450 molecules form an aggregate and surround one reductase molecule, and therefore the two enzymes form large micelles. ER proliferation after PB administration, which had been correlated with increase in P-450 level, is shown to be probably independent of the increase in P-450 level. There are considerable discrepancies among results reported on sublobular expression of various P-450 isoforms. Causes of the discrepancies are likely to be differences in experimental conditions of histochemical detection carried out and/or in species, strain, and/or sex.

Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from the blood circulation.

Unlike isolated single hepatocytes, hepatocyte couplets retain their apical polarity, and, during short-term culture form an enclosed canalicular space or vacuole between the two adjacent cells into which biliary secretion is initiated. Hepatocyte couplets were prepared after partial collagenase perfusion of rat liver. Centrifugal elutriation was used to fractionate the preparation into six couplet-containing suspensions. Image analysis was used to determine the size of cultured couplets. The size of the couplets ranged from 34.1 +/- 0.76 microns and 684 +/- 24.1 microns 2 (mean length and area respectively +/- S.E.M.) in Fraction 2, to 43.7 +/- 0.57 microns and 1033 +/- 33.8 microns 2 length and area respectively in Fraction 7. Glutamine synthetase activity was assessed in each freshly eluted fraction and was shown to be predominant in Fractions 6 and 7. Pretreatment of rats with CCl4, which selectively destroys perivenous hepatocytes, decreased the proportion of couplets in these fractions by over 67%, and their glutamine synthetase activity by over 97%. It was concluded that Fractions 2 and 3 contained predominantly couplets of Zone 1 (periportal) origin, Fractions 4 and 5 those from Zone 2, and Fractions 6 and 7 predominantly couplets of Zone 3 (perivenous) origin. The development of canalicular secretory activity was assessed in the couplets after a 15 min incubation with a fluorescent bile acid, cholyl-lysyl-fluorescein (CLF). This was sigmoidal in all fractions, but slower in the periportal couplets, taking 5.1 h for 50% to show secretory activity in Fraction 2, compared with 2.7 h for Fraction 7. Incubation of hepatocyte couplets with 1 or 10 microM taurodehydrocholate, a non-toxic bile acid analogue, did not influence the rate of development of accumulation of CLF by the couplets or the area of the canalicular vacuole in any fraction. However, it did decrease the CLF content of couplets incubated with CLF for 15 min to a greater extent in those of perivenous origin. After subjecting the couplets to oxidative stress by incubation with 20 microM menadione (2-methyl-1,4-naphthoquinone), it was evident that periportal couplets were less able to maintain canalicular secretory activity than perivenous couplets.

1. Isolated periportal (PP) and perivenous (PV) hepatocytes from normal and inducer-treated rat livers were used to examine the following: intralobular localization of cytochrome P-450IA, P-450IIB, P-450IIE and P-450IIIA dependent monooxygenase activities and effects of phenobarbital (PB), beta-naphthoflavone (BNF) and pregnenolone-16 alpha-carbonitrile (PCN) on the zonal induction of these monooxygenases. 2. 7-Ethoxyresorufin O-deethylase (7EROD), 7-pentoxyresorufin O-dealkylase (7PROD) and N-nitrosodimethylamine N-demethylase (NAND) activities of PP hepatocytes were not significantly different from those of PV hepatocytes. 3. Ethylmorphine N-demethylase (EMND) activity was significantly higher in PV hepatocytes than in PP hepatocytes of normal rats. 4. EMND activity was induced by PCN and PB treatments. The response of EMND activity to PCN treatment was higher in PP hepatocytes than that in PV hepatocytes, and as a result the PV dominance disappeared following PCN treatment. 5. Extents of the response of this activity to PB treatment were similar in PP and PV hepatocytes, and PV dominance remained unchanged even after induction.

Liver parenchyma shows a remarkable heterogeneity of the hepatocytes along the porto-central axis with respect to ultrastructure and enzyme activities resulting in different cellular functions within different zones of the liver lobuli. According to the concept of metabolic zonation, the spatial organization of the various metabolic pathways and functions forms the basis for the efficient adaptation of liver metabolism to the different nutritional requirements of the whole organism in different metabolic states. The present review summarizes current knowledge about this heterogeneity, its development and determination, as well as about its significance for the understanding of all aspects of liver function and pathology, especially of intermediary metabolism, biotransformation of drugs and zonal toxicity of hepatotoxins.

Somatostatin (SS-14) is known as an antigrowth factor for a variety of cell types, including gastrointestinal mucosa, exocrine pancreas, lymphocytes, and some tumors. We have recently identified and biochemically characterized SS-14-binding protein on rat liver plasma membranes (S. E. Raper, P. C. Kothary, and J. DelValle, Gastroenterology 96: A408, 1989; P. C. Kothary et al., Digestion 46 (Suppl 1): 58, 1990). We hypothesized that SS-14 may affect liver growth as well and investigated cellular mechanisms of this phenomenon focusing on the second messenger cAMP. Freshly isolated rat hepatocytes were plated on tissue culture dishes coated with Matrigel (laminin, heparan sulfate, and type IV collagen). The medium was not supplemented with serum or hormones. Either dibutyryl-cAMP (1 mM) or isobutylmethylxanthine (IBMX, 0.1 mM) was added in the presence or absence of SS-14 (10 nM). DNA synthesis was estimated by the rate of [3H]thymidine incorporation into DNA and by the labeling index (an autoradiographic measurement of the number of labeled nuclei). SS-14 significantly inhibited both [3H]thymidine incorporation and labeling index of rat hepatocytes stimulated by dibutyryl-cAMP or IBMX. SS-14 also inhibited intracellular cAMP accumulation stimulated by IBMX. We conclude that SS-14 exerts at least part of its antiproliferative effects via the adenylate cyclase system. Further study using other signal transduction systems may yield more information about mechanisms of hepatocyte growth.

We hypothesized that somatostatin-14 (SS-14) might inhibit insulin-stimulated hepatic growth. Rat hepatocytes were isolated by a two-step collagenase perfusion technique and cultured on Matrigel. Differentiated hepatocyte function was documented by albumin synthesis. Hepatocytes were incubated with insulin in the presence or absence of SS-14. Hepatocyte proliferation was assessed by tritiated thymidine ([3H]thy) incorporation into DNA. [3H]thy incorporation was increased by 230% in the presence of insulin and was essentially abolished by the addition of SS-14. Insulin-stimulated cyclic-AMP accumulation was also decreased from 190 to 108% of control levels (P less than 0.05) by the addition of SS-14. Pretreatment with pertussis toxin, which inactivates the inhibitory G-protein, Gi, blocked the effect of SS-14.

(i) In the rat, SS-14 effectively blocks insulin-stimulated [3H]thy incorporation into DNA, possibly by blocking intracellular cAMP accumulation. (ii) Pertussis toxin blocks the growth inhibitory effects of SS-14, suggesting that inhibitory G proteins are involved in the mechanism of SS-14 action. Somatostatin may be useful in studying the role of second messengers in cell growth.

Technological advances in the separation and culture of mammalian hepatocytes have facilitated the use of these cells as the foundation for either hepatocyte transplantation or hepatocyte-seeded hollow fiber liver assist devices (LAD). To fully appreciate the practical applications of these tissue engineering solutions, it is necessary to understand the types of human liver failure as well as the corresponding animal models. The most immediate application of this type of technology is the treatment of hepatic encephalopathy (HE), an acute and highly fatal complication of fulminant hepatic failure. Although the pathogenesis of HE is unknown, failure of the detoxification function of the liver is accepted as playing an important role in this disorder. Consequently, the assaying and preservation of P450 activity in the grafted cells or in the LAD must be among the main targets of this research. This review explores the problems in hepatocyte transplantation and culture that deserve special consideration and emphasizes the conditions contributing to the in vitro maintenance of phenotypic expression of these cells.

Periportal (pp) or perivenous (pv) liver parenchymal cells from female adult Uje: WIST rats were isolated after retro- or antegrade digitonin infusion followed by collagenase perfusion in the opposite direction. The morphological results revealed a distinct acinar-related destruction of the pv- or pp-zone by digitonin. The remaining cells of the respective other zone showed a good structural maintenance. After subsequent conventional collagenase perfusion the yield, viability and structural integrity of the isolated hepatocytes were high. The zonal cell separation was indicated by significant differences in the pp marker glucose-6-phosphatase and the pv marker glutamine synthetase found in the isolated pp or pv cell populations. Under our experimental conditions including the use of female rats, the alanine aminotransferase and glutamate dehydrogenase as well as ethylmorphine N-demethylase and ethoxycoumarin O-deethylase activities were evenly distributed in both preparations. Under stimulating conditions the capacity for urea synthesis was similar in both pv and pp cells.

Approximately 20 to 24 samples (0.1-0.2 g each) were obtained from each of six isolated rat livers following steady-state infusion of quinidine. The concentrations of quinidine, analyzed by an HPLC method, were found to vary markedly within each lobe (up to approximately 52-fold) or between lobes (up to approximately 25-fold) from the same liver. The maximum intrahepatic concentration difference in the six livers studied was 208-fold. Implications of the present study in the determination of liver drug concentration, and of the partition coefficient between liver and venous drug concentration in physiological pharmacokinetic modeling, as well as in hepatic modeling, are discussed.

CI-981, a novel synthetic inhibitor of HMG-CoA reductase, was previously reported to be highly liver-selective using an ex vivo approach. In order to determine liver-selectivity at the cellular level, CI-981 was evaluated in cell culture and compared to lovastatin, pravastatin, fluvastatin and BMY-21950. Using human cell lines, none of the compounds tested showed liver-selectivity, i.e. strong inhibition of cholesterol synthesis in Hep-G2 cells (liver model) but weak inhibition in human fibroblasts (peripheral cell model). In contrast, all drugs tested produced equal and potent inhibition of sterol synthesis in primary cultures of rat hepatocytes, and CI-981, pravastatin and BMY-21950 were more than 100-fold more potent in rat hepatocytes compared to human fibroblasts. Since all compounds were also equally potent at inhibiting sterol synthesis in a rat subcellular system and in vivo, the data suggest that the use of Hep-G2 cells may not be the cell system of choice in which to study inhibition of hepatic cholesterogenesis or to demonstrate liver selectivity of inhibitors of HMG-CoA reductase.

1. Rats were exposed to hypobaric hypoxia (equivalent altitude 4500 m), 2 x 2 hr per day, for 5 days. Isolated hepatocytes were prepared on day 6 after 18 hr of fast and also from control normoxic animals. The hepatocytes were incubated (120 min) with various substrates. 2. ATP contents were lower in hepatocytes from exposed as compared to control animals whether at the beginning (14%) or at the end (-6 to -33%) of incubation depending on the substrate. 3. Gluconeogenesis from all precursors (lactate, alanine, pyruvate, glutamine) was significantly reduced (40-50%) in exposed as compared to control animals. 4. Ureogenesis from alanine and from pyruvate + NH4Cl was also markedly depressed in exposed animals but no differences were noticed with glutamine or lactate + NH4Cl and alanine + NH4Cl. 5. Results are discussed in relation to known effects of acute and chronic hypoxia, interrelationship between gluconeogenesis and ureogenesis, taking into account the inhomogeneity of liver and the metabolic properties of periportal and perivenous hepatocytes.

Hepatocytes in the proximal (zone 1) and distal (zone 3) regions of the liver acinus are selectively stained by perfusion of the isolated rat liver with 0.2-20 microM acridine orange (AO). After 10-60 min of anterograde perfusion, AO fluorescence is visible in zone 1 cells, whereas retrograde perfusion stains cells of zone 3. In this paper, we describe a technique to isolate a mixed population of fluorescent and nonfluorescent hepatocytes (cells from all acinar zones, which do not loose the zone specific AO labeling) and to separate these cells according to their zonal origin by fluorescence activated cell sorting. The zonal populations obtained were either fluorescent or nonfluorescent (purity greater than 95%). Separated cell fractions differed in their enzyme content (5' nucleotidase, succinate-dehydrogenase, beta-glucuronidase). An unidentified AO metabolite, which is not found in bile after retrograde perfusion (not formed in zone 3 cells), is also absent after retrograde perfusion in sorted fluorescent cells (zone 3 cells), indicating zonal purity of sorted cells.

The liver is described as a composite system consisting of a set of operative creodic microunits open to a continuous flow of matter, energy and informations. Its dynamics depend on two interactive and interrelated subsystems with actions described as homopoiesis and homeorhesis, making it an autoisodiasostic system. The system's emergent (equifinal) or emergence states, operative potential, diffusion and reaction phenomena and compensation states are also formally described. For readers not familiar with the language of general system theory, of system dynamics and of categorical analysis, a glossary of some terms is provided.

A large number of histological, histochemical and biochemical techniques are available for studying liver cell heterogeneity. Structural differences are recognized by morphometric analyses of electron micrographs. The zonal heterogeneity of enzyme activities can be demonstrated by histochemistry and more precisely by ultramicrobiochemical assays in microdissected periportal and perivenous tissue. Immunohistochemistry is useful for quantifying and localizing proteins, especially isoenzymes, without depending on their biological activity. The zonal quantification of specific mRNA can be achieved by in situ hybridization. The different structural and enzymic equipment of periportal and perivenous tissue found by these techniques has led to the concept of metabolic zonation. This hypothesis can be confirmed by determination of metabolic rates in perfused liver after selective zonal damage, in separated periportal and perivenous hepatocytes as well as in periportal and perivenous tissue of perfused liver by non-invasive techniques.

In rat hepatocytes, we examined the relationship between cell volume, bleb formation, and loss of cell viability during chemical hypoxia with KCN plus iodoacetic acid. In hypotonic media (150-200 mosmol/kgH2O), cells swelled to a greater extent during chemical hypoxia than in isotonic media, but rates of cell killing were identical. Sucrose (300 mM) added to isotonic media prevented early cell swelling but actually accelerated cell killing. In contrast, mannitol (300 mM) improved cell survival but did not prevent cell swelling. Bleb formation occurred regardless of buffer tonicity. The antioxidants desferrioxamine and cyanidanol but not superoxide dismutase +/- catalase delayed lethal cell injury. Cell killing was greater during aerobic compared with anaerobic chemical hypoxia. Hydroperoxide formation was measured using a dichlorofluorescin assay and was accelerated during aerobic but not anaerobic chemical hypoxia. The results indicate that cell swelling is not the driving force for bleb formation or lethal cell injury. We conclude that "reductive stress" caused by respiratory inhibition favors formation of toxic oxygen species and may contribute to lethal cell injury during intermittent or incomplete oxygen deprivation.

The zonal distribution of enzyme activities was measured by quantitative cytochemistry in cryosections of liver from three normal children and five infants with idiopathic hepatitis of infancy. Optimal conditions for cytochemical reactions were first validated in rat liver and subsequently used in human livers to quantify zonal activities of acid phosphatase (AP), succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), glucose-6-phosphatase (G6P) and NADPH-dehydrogenase (ND). In normal rat and human livers, activities were greater for SDH and G6P in periportal and for GDH and ND in perivenular hepatocytes, while AP was evenly distributed along the sinusoids. In five infants with idiopathic hepatitis of infancy (IHI), a similar trend of distribution was observed for the two mitochondrial (SDH and GDH) and the two microsomal (G6P and ND) enzymes, although the distribution gradient was less pronounced than, in normal livers. AP showed a mildly greater periportal than perivenular activity. This preliminary study shows that a similar metabolic zonation exists for these enzymes in human livers as is observed in rats.

Liver parenchymal cells from the periportal and centrilobular zones differ in their morphological, biochemical and functional characteristics. In an effort to obtain fractions enriched in either periportal or centrilobular cells, isolated rat liver parenchymal cells were separated into five subpopulations by centrifugal elutriation. The mean diameters of the cells present in fractions I-V were 19.6, 21.1, 21.8, 22.7 and 23.5 micron, respectively. The content of cytochrome P-450 as well as benzphetamine N-demethylase and 7-ethoxyresorufin O-deethylase activities were higher in the larger parenchymal cells than in the smaller ones. After administration of phenobarbital the content of cytochrome P-450 was approximately two-fold greater in the cells present in fractions 3-5, when compared to the same subpopulations isolated from untreated rats; the activity of benzphetamine N-demethylase was enhanced to a similar extent in all five fractions. 3-Methylcholanthrene treatment resulted in a significant increase of cytochrome P-450 content and 7-ethoxyresorufin O-deethylase activity in all five fractions: both parameters were slightly higher in fractions 4 and 5 than in fractions 1 and 2. In conclusion, the elutriated liver parenchymal cells seem to preserve the biochemical heterogeneity observed in the intact liver; the potential enrichment of periportal and centrilobular cells in the different fractions by centrifugal elutriation is discussed.

Periportal and perivenous hepatocytes were isolated from rat liver by digitonin/collagenase perfusion for investigating the acinar heterogeneity of amino acid transport activities related to glutamine and ammonia metabolism. Immunocytochemical staining of the respective subpopulations for glutamine synthetase demonstrated that periportal subpopulations were essentially free of glutamine synthetase-positive cells, whereas perivenous subpopulations showed a 2- to 3-fold enrichment of glutamine synthetase-positive hepatocytes. The high perivenous/periportal ratio of 59 found for glutamine synthetase activity as well as the perivenous/periportal ratios of other marker enzymes further indicated the good separation of periportal and perivenous cells. alpha-Aminoisobutyric acid, histidine and glutamate were used to determine the distribution pattern of amino acid transport systems A, N and G-, as well as of the sodium-independent uptake of these compounds 1 hr after isolation and after maximal hormonal stimulation during primary culture. The strong heterogeneity of the sodium-independent transport of histidine, characterized by higher perivenous transport rates [perivenous/periportal ratio: 1.5 (1 hr) to 3.5 (48 hr)], suggests a significant role of facilitated diffusion, presumably in glutamine export. Conversely, the strong heterogeneity of the sodium-dependent glutamate transport (System G-) characterized by higher uptake rates in nonstimulated [perivenous/periportal ratio: 6.6 (1 hr)] and in hormonally treated perivenous hepatocytes (perivenous/periportal ratio: 2.2) reflects its possible significance with respect to the substrate availability for glutamine synthesis. The observed heterogeneities provide a basis for understanding how substrate fluxes related to glutamine metabolism might be established and regulated.(ABSTRACT TRUNCATED AT 250 WORDS)

In previous studies we have shown that hepatocytes stream from the portal tract toward the terminal hepatic vein. The present study provides evidence that littoral cells participate in the same cell stream and that the liver actually streams en masse. Littoral cells stand for sinusoidal endothelia and Kupffer cells. Thirty male adult rats, random bred, were injected with 0.5 microCi [3H]-thymidine, specific activity 5 Ci/mmol/g body weight. The rats were killed in groups of five, at the following times: 1 h, 14, 30, 60, 90 and 120 days. The livers were processed histologically and dipped into liquid emulsion for autoradiography. In each animal 50 labelled hepatocytes and 50 littoral cells were randomly selected and their distance from the nearest terminal portal tract rim was measured. Both cell populations renew their cells continuously. Each consists of two cell types, progenitors, residing around the portal tract up to the distance of 200 microns, and functional cells, which inhabit the rest of the acinus. The two regions where the different cell types reside are known respectively as progenitor (P) and functional (Q) compartments. Both cells are formed in the P-compartment and advance jointly along a trajectory, the tissue radius, toward the terminal hepatic vein where they die. They progress at a daily velocity of 2 micron. Since both advance at the same speed, as long as they exist they remain neighbours. Liver parenchyma and stroma thus stream en masse.(ABSTRACT TRUNCATED AT 250 WORDS)

Thirty male adult rats, were injected with [3H]-thymidine, [3H]TdR. The animals were killed in groups of five, at the following times: 1 h, 14, 30, 60, 90 and 120 days. The livers were fixed in formalin, embedded in paraffin and cut into 5-micron-thick sections, which were then dipped into liquid emulsion and exposed for 3 weeks. The grain content of each sampled cell was counted and its distance from the nearest portal tract rim measured. Since the cells were labelled once and the label was available only for a short period, cell displacement could be followed with time. One hour after labelling, most labelled cells were confined within the 300 micron distance from the portal space rim. From then on, labelled cells advanced at a daily velocity of 2.5 micron toward the terminal hepatic vein. The liver acinus is composed of two compartments: a progenitor compartment extending up to 300 micron from the portal tract and a functional compartment covering the remaining acinus portion. Such an arrangement is known as a two-compartment cell renewal system, and is found in renewing tissues like crypt-villus and epidermis. The hepatocyte moves from the portal space toward the terminal hepatic vein, proceeding along a trajectory, denominated as tissue radius. Since cells move in one direction, the further a cell is from the portal tract, the older it is. One may therefore estimate cell age by measuring its distance from the origin. Since the tissue radius portrays cells of all ages, it describes the entire life history of one cell. The advancing hepatocytes traverse the three acinus zones.(ABSTRACT TRUNCATED AT 250 WORDS)

Within the hepatic acinus, the functional unit of liver parenchyma, the induction of cytochrome P-450 protein by phenobarbital is manifested primarily in hepatocytes located closer to the hepatic venule, i.e., distal hepatocytes. The objective of this study was to determine the levels of cytochromes P-450b and P-450e mRNAs in populations of hepatocytes originating in the proximal or distal half of the liver acinus in the rat, as an approach to the elucidation of the mechanisms responsible for the heterogeneous zonal expression of cytochrome P-450 protein. The development of a new method to isolate hepatocytes originating from the proximal or distal half of the liver acinus enabled the measurement of total cytochrome P-450 content and of cytochromes P-450b and P-450e mRNAs in these hepatocytes. Levels of cytochromes P-450b and P-450e mRNAs were assessed in proximal and distal hepatocytes by Northern blot hybridization of poly(A+)RNA with a cDNA recognizing sequences of these two cytochromes. The kinetics of induction were defined by measuring these parameters after a single phenobarbital injection. Cytochrome P-450 mRNA levels reached maximum induction at 16 hr, returning to basal values by 48 hr. In contrast, total cytochrome P-450 microsomal protein content reached maximum induction after 33 hr. Hepatocytes of the distal half of the hepatic acinus responded to phenobarbital with higher levels of cytochromes P-450b and P-450e mRNAs than proximal hepatocytes. These results indicated that there is modulation of the expression of the cytochromes P-450b and P-450e genes within the hepatic acinus.