Characterisation of hepatocyte sub-populations generated by centrifugal elutriation

We describe protocols for the fractionation of isolated hepatocytes into eight sub-populations using centrifugal elutriation. The distribution of fluorescein isothiocyanate and acridine orange in hepatocytes prepared from livers pre-perfused with one of these dyes is described and used as an indicator of acinar zone derivation for each population. The cytochrome P-450 content and response to induction by 3-methylcholanthrene and phenobarbitone; the distribution of lactate dehydrogenase, glucose-6-phosphatase, pyruvate kinase and tyrosine aminotransferase activities in the sub-populations is also reported. A marked asymmetry of distribution in all these activities was observed. On the basis of putative zone derivations (based on data of fluorescent dye distribution) of eight factors studied, the distributions of six were consistent with the sub-populations being derived from different acinar zones. Two major discrepancies were noted however, the distribution of pyruvate kinase activity and the response of the sub-populations to phenobarbitone. We conclude from this study that while a metabolic heterogeneity was revealed in the sub-populations generated, further characterisation is required to determine whether acinar zone separation has occurred and if so to what extent.     

Fatty acid-binding protein expression in the liver: its regulation and relationship to the zonation of fatty acid metabolism

Summary Liver fatty acid-binding protein (L-FABP) is expressed in a declining gradient between the portal and central zones of the liver acinus. This paper discusses the results of experimental studies which address the questions: (a) What factors regulate L-FABP expression in liver and produce its acinar gradient? (b) What is the relationship between the acinar gradient of L-FABP and acinar gradients in the transport and metabolism of long-chain fatty acids? Both high-fat diets and clofibrate-treatment increase L-FABP proportionally at both extremes of the liver acinus and the small intestine, with preservation of the L-FABP gradient in both tissues. Female rats differ from males, however, in showing a greater hepatic abundance of L-FABP which is expressed almost equally throughout the acinus. Dietary studies show that L-FABP is induced with increased fatty acid flux derived from dietary fat but not from de novo hepatic fatty acid synthesis. Studies of the synthesis and utilization of fatty acids by hepatocytes isolated from the periportal and pericentral zones of the liver acinus suggest that the acinar gradient of L-FABP is not associated with differences in the instrinsic capacity of zone 1 and zone 3 hepatocytes to utilize or synthesize fatty acids. In addition, studies of the acinar uptake pattern of a fluorescent fatty acid derivative by isolated perfused livers indicate that the acinar distribution of L-FABP does not determine the pattern of fatty acid uptake in the intact acinus. Rather, the acinar gradient of L-FABP is most likely to represent a response to physiological conditions existing in the intact acinus which may include gradients in the flux of fatty acids, fatty acid metabolites and hormones.Abbreviations ALT Alanine Aminotransferase - FABP Fatty Acid Binding Protein - I-FABP Intestinal-type Fatty Acid Binding Protein - L-FABP Liver-type Fatty Acid Binding Protein - 12-NBD-stearate 12-(N-methyl)-N-(7-nitrobenzo-2-oxa-1, 3,-diazol-4-yl)amino)-octadecanoic acid     

Expression of apoptosis on rat liver by hepatic vagus hyperactivity after ventromedial hypothalamic lesioning

We examined whether the Fas (APO-1/CD95)/Fas ligand system mediates apoptosis in rats with ventromedial hypothalamus (VMH) lesions. Northern and Western blotting indicated that VMH lesions lead to a significant increase in Fas mRNA and protein expression from day 1 to day 7 and in Fas ligand mRNA and protein expression from day 2 to day 7. Immunohistochemistry indicated that the region of strongest Fas expression shifted from acinar zone 1 to zones 2 and 3 by day 7 after VMH lesioning and that at days 2-7 Fas-ligand-positive hepatocyte cell membranes and cytoplasm were randomly distributed in acinar zones 1-3. We also analyzed activation of caspase 3-like proteases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Spectrofluorometric assay demonstrated that caspase 3-like activity significantly increased only in hepatocytes after VMH lesioning. Moreover, electron microscopy and TUNEL assay showed that VMH lesions induced apoptosis. All of these effects were completely inhibited by hepatic vagotomy and administration of atropine. Vagal firing after VMH lesioning may stimulate Fas/Fas ligand system-mediated apoptosis through the cholinergic system in the rat liver.     

Intermediate cells in the adult human pancreas. Contribution to the transformation of differentiated cells in vertebrates

Problems associated with the transformation of differentiated cells in vertebrate organisms are discussed based on electron microscopical results of intermediate cells (i.e. cells with morphological characteristics of exocrine acinar cells and endocrine cells of Langerhans' islets) in the pancreas of human adults with chronic insulin-dependent diabetes mellitus. In this context, reference is made to experimental results of Scarpelli, Rao, and coworkers relating to the occurrence of hepatocyte-like cells in the pancreas of Syrian golden hamsters (Rao and Scarpelli 1980; Scarpelli and Rao 1981; Rao et al. 1983). These observations show that exocrine acinar cells of the pancreas may, even beyond the neonatal period, become transformed, depending upon different triggering stimuli, into different endocrine islet cells, or into hepatocytes, this being accomplished either directly or by new formation of cells (regeneration) with abnormal differentiation (metaplasia). Obviously, transformation is effected through a change in the activation of gene loci: the normally stably blocked genes are partially or completely deblocked for the functions of different endocrine islets cells or hepatocytes, and the original genetic expression of exocrine pancreatic functions is blocked either partially or completely. The results presented and quoted in this paper suggest that in all differentiated cells derived from the endoderm of the foregut, such as duct cells, exocrine and endocrine pancreatic cells, and hepatocytes, functional programs are retained which can be modified in the manner quoted to enable partial or complete transformation into one or another of these differentiated cells in the adult organism.     

Hepatic transdifferentiation in the pancreas

The differentiated state of specialized cells appears to be dependent on interactions between the extracellular microenvironment, cytoplasmic signals and DNA. Perturbations in these interactions lead to phenotypic alterations of the cell—referred to as transdifferentiation. Copper deficiency in rats leads to global acinar cell loss due to apoptosis possibly leading to perturbations in cell–cell interactions and the microenvironment. Acinar cell loss is associated with the proliferation of ductular epithelial and oval cells. Massive depletion of the acinar cell pool creates severe expansion pressure on oval and ductular cells to fill the vacuity. This probably causes a change in the commitment of these cells resulting in transdifferentiation into hepatocytes. Pancreatic hepatocytes exhibit all the morphological and functional properties of liver parenchymal cells.     

A 3-dimensional presentation of the functional liver unit]

The reconstruction of the liver parenchyma of a golden hamster after poisoning with allyl formate is described. Allyl formate primarily destroys the periportal areas and leads, following the microvascularisation of the liver parenchyma, to a necrosis of the hepatocytes progressing towards the terminal hepatic venule. The still intact parenchymal zones can be characterized by the positive PAS reaction. In this study the preterminal and the terminal portal branches as well as zone 3, situated in the vasculatory periphery, were reconstructed. By this method, a three-dimensional presentation of the acinar functional zones was possible for the first time.     

Immunocytochemical localization of exocrine enzymes in rat pancreatic acinar carcinoma

Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.     

Induction and origin of hepatocytes in rat pancreas

2-[4(2,2- Dichlorocyclopropyl )phenoxy]2-methyl propionic acid (ciprofibrate), a peroxisome proliferator , induced hepatocytes in the pancreas of adult male F-344 rats when added to their diet at a dosage of 10 mg/kg body weight for 60-72 wk. These cells are morphologically indistinguishable from hepatic hepatocytes and were usually localized adjacent to islets of Langerhans with extensions into surrounding acinar tissue. A significant increase in the volume density of peroxisomes, together with immunochemically detectable amounts of two peroxisome-associated enzymes, was observed in pancreas with hepatocytes of rats maintained on ciprofibrate. Uricase-containing crystalloid nucleoids, specific for rat hepatocyte peroxisomes, were present in pancreatic hepatocytes. These structures facilitated the identification of cells with hybrid cytoplasmic features characteristic of pancreatic acinar and endocrine cells and hepatocytes. Such cells are presumed to represent a transitional state in which pancreas specific genes are being repressed while liver specific ones are simultaneously expressed. The presence of exocrine and/or endocrine secretory granules in transitional cells indicates that acinar/intermediate cells represent the precursor cell from which pancreatic hepatocytes are derived.     

Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.     

Different proliferative responses of periportal and perivenous hepatocytes to EGF.

Stimulation of DNA synthesis by EGF was compared in cultured periportal and perivenous hepatocyte populations. Periportal hepatocytes responded to EGF more sensitive (IC50-values 20 vs 75 ng/ml) and with a higher maximal stimulation (420 vs 290%) than perivenous hepatocytes with respect to both [3H]thymidine incorporation and labeling index. The glutamine synthetase-positive hepatocytes responded much less to EGF than did the perivenous cells in general. The simultaneous presence of insulin increased the sensitivity for EGF predominantly in the periportal hepatocytes. These inherent differences in the growth potential of hepatocytes from different acinar localizations may contribute to different growth patterns across the lobules in normal and regenerating liver.     

Functional hepatocellular heterogeneity for the production of plasma proteins.

It is now well established that hepatocytes are the main liver cells responsible for the synthesis of plasma proteins produced by the liver. That these cells are not specialized in the production of the different plasma proteins is also well established. Presently the point still debated is whether a functional hepatocellular heterogeneity exists for plasma protein synthesis as for many other hepatocyte functions. Several physiological and pathological situations suggest that this heterogeneity takes place in the hepatocytes of two opposite hepatic lobular zones, the periportal and centrilobular zones. However, this zonal difference, which supposes different regulatory mechanisms, must be confirmed by techniques other than the now classical immunocytochemistry or the in situ hybridization technique recently proposed for the demonstration of mRNAs in hepatocytes. Another hepatocellular heterogeneity, the intercellular heterogeneity, which can be observed in the same lobular zone, is more difficult to analyze, but shows that from hepatocyte to hepatocyte a variation exists in the synthesis of a given plasma protein.     

Ultrastructure and complex carbohydrate ultracytochemistry of the cat parotid gland

The structure and glycoconjugate content of the cat parotid gland were analyzed at electron microscopic level by applying morphological techniques and three ultrastructural histochemical methods - HID-TCH-SP, LID-TCH-SP and PA-TCH-SP. This gland appeared as a typical salivary gland composed of acinar secretory cells, intercalated ducts, striated ducts and excretory ducts. The most common configuration of secretory granules consisted of a dense core surrounded by a variable electron-lucent halo. All ductal segments were characterized by the presence of different cell populations and small apical granules greatly different from those localized in the acinar cells. By using HID-TCH-SP we were able to demonstrate that in a few acinar cells there are sulphated sites, whereas PA-TCH-SP staining revealed the presence of vic-glycol radicals in all acinar cells preferentially located on the halo of secretory granules.     

Almost total conversion of pancreas to liver in the adult rat: a reliable model to study transdifferentiation

Study of transdifferentiation provides an excellent opportunity to investigate various factors and mechanisms involved in repression of activated genes and derepression of inactivated genes. Here we describe a highly reproducible in vivo model, in which hepatocytes are induced in the pancreas of adult rats that were maintained on copper-deficient diet containing a relatively non-toxic copper-chelating agent, triethylenetetramine tetrahydrochloride (0.6% w/w) for 7-9 weeks and then returned to normal rat chow. This dietary manipulation resulted in almost complete loss of pancreatic acinar cells at the end of copper-depletion regimen, and in the development of multiple foci of hepatocytes during recovery phase. In some animals, liver cells occupied more than 60% of pancreatic volume within 6-8 weeks of recovery. Northern blot analysis of total RNA obtained from the pancreas of these rats revealed the expression of albumin mRNA. Albumin was demonstrated in these pancreatic hepatocytes by immunofluorescence. The advantages of this model over the previously described models are: a) low mortality (10%), b) depletion of acinar cells, and c) development of multiple foci of hepatocytes in 100% of rats.     

Hepatocyte heterogeneity in bile formation and hepatobiliary transport of drugs.

In the past two decades many studies have been devoted to the involvement of the periportal (zone-1) and perivenous (zone-3) hepatocytes in bile formation and hepatobiliary transport of endogenous and exogenous compounds. It became clear that such a heterogeneity in transport function can, in principle, be due to the different localization of the cells in the acinus with respect to the incoming blood, to intrinsic differences between the cells or to both. In this review we first discuss the techniques used to study hepatocyte heterogeneity in hepatobiliary transport function. Combinations of such techniques can be used to discriminate between cellular heterogeneity due to acinar localization as opposed to intrinsic differences. These techniques include: normal and retrograde perfusions of isolated perfused livers; autoradiographic, fluorimetric and histochemical localization of injected substrates; separation of isolated hepatocytes into fractions enriched in periportal and perivenous cells; measurements of fluorescent surface signals with microlight guides; selective zonal toxicity, and pharmacokinetic modelling and analysis. Subsequently, for each of the rate-limiting steps in the hepatobiliary transport of organic compounds, the basic mechanisms are summarized and the available knowledge on the involvement of the cells from the various zones in these transport steps is discussed. The available literature data indicate that heterogeneity in transport function is often due to the localization of the cells in the acinus: the periportal cells are the first to come into contact with the portal blood and are thus exposed to the highest substrate concentration. Consequently they obtain the most prominent task in further disposition of the particular compound. It follows that the extent of involvement of the perivenous cells in drug disposition is implicitly determined by the activity of the periportal cells. Because of the potential saturation of elimination processes in the periportal cells, the involvement of perivenous cells may vary with the input concentration. In addition, real intrinsic differences have been established in the hepatobiliary transport of some substrates. These are probably based on differences in the cellular content of carrier- and receptor-binding and/or metabolizing proteins. In some cases these intrinsic differences may be secondary to existing sinusoidal gradients of endogenous compounds, such as O2, amino acids, bile acids or monosaccharides. Yet, data on the heterogeneity of hepatocytes in the various transport steps are far from complete or are even totally lacking, especially for human liver. A multi-experimental approach and advanced technology will be needed in the future to gain more insight into the acinar organization of bile formation and hepatobiliary transport of drugs in the human.     

Separation of hepatocytes of different acinar zones by flow cytometry

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.     

Copper deprivation in rats induces islet hyperplasia and hepatic metaplasia in the pancreas

BACKGROUND INFORMATION: Prolonged copper deprivation in rats followed by refeeding with a normal diet has previously been used to induce the appearance of hepatocyte-like cells in the pancreas, but the effects on islet size and morphology have not been determined. RESULTS: In the present study we investigated the distribution of pancreatic alpha- and beta-cells and of hepatocytes in adult rats fed a copper-deficient diet followed by refeeding with a normal diet. Immunohistochemical staining for insulin and glucagon showed that the islets of the copper-deficient group were up to 2.4 times larger in mass compared with controls. The islets were disorganized, with alpha-cells found in multiple layers at the periphery of the islet and sometimes deep in the core. Isolated alpha- and beta-cells were also found in increased numbers in the ductular system. Copper deprivation caused almost complete ablation of the acinar cells, and refeeding induced adipogenesis, acinar regeneration and hepatocyte-like cells. Ductular proliferation and nerve hyperplasia were also present. The hepatocytes tended to be associated with islets or with ducts, rather than with residual pancreatic exocrine tissue. CONCLUSIONS: These data show that copper deficiency in rats, as well as inducing the appearance of hepatocytes, is capable of causing islet hyperplasia.     

Spontaneous formation of intercellular bile canaliculi and hybrid biliary-pancreatic canaliculi in co-culture of hepatocytes and exocrine pancreas cells from carp

When cultured together in a primary serum-free hormone-free system, hepatocytes and exocrine pancreas cells from the carp, Cyprinus carpio, spontaneously establish unique morphological structures that do not occur in vivo. These structures include intercellular bile canaliculi between neighbouring hepatocytes and hybrid canaliculi between hepatocytes and pancreas cells. In vivo, carp hepatocytes form only unicellular bile canaliculi; hybrid canaliculi between hepatocytes and exocrine pancreas cells do not exist at all in nature. This study shows that, in an artificial environment, cells are able spontaneously to establish novel morphological structures that are absent in the animal from which the cells have been obtained. Received: 3 January 1996 / Accepted: 17 March 1997