Preferential suppression of insulin-stimulated proliferation of cultured hepatocytes by somatostatin: Evidence for receptor-mediated growth regulation

The role of somatostatin (SS-14) in the regulation of rat liver regeneration was examined by using thymidine incorporation into hepatocyte DNA labeled with tritiated thymidine, a nuclear-labeling index, and the binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes isolated at various times after partial hepatectomy. The data demonstrated no suppressive effect of SS-14 on insulin and glucagon-stimulated thymidine incorporation into hepatocyte DNA as early as 2 h after partial hepatectomy. These data were substantiated by a nuclear labeling index studies. At 2 h, ¹²⁵I-tyr¹¹-SS-14 binding to its specific sites on isolated hepatocytes was undetectable. There was a time-dependent increase in binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes obtained at various times after partial hepatectomy. There was a significant decrease in the number of binding sites after partial hepatectomy as determined by Scatchard analysis. The data were supported by autoradiography analysis of affinity labeled ¹²⁵I-tyr¹¹-SS-14-binding protein complex followed by SDS-PAGE. SS-14 also inhibited intracellular cAMP in hepatocytes obtained at 18 h after hepatectomy. The data are consistent with the hypothesis that SS-14 participates via its own receptor in the regulation of the liver regeneration. © 1995 Wiley-Liss, Inc.     

Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.     

Inhibitors of Protein Biosynthesis Can Stimulate Proliferation of Mouse Hepatocytes in Vitro

Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1–4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor of protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation.     

Similar induction of the hepatic EGF receptor in vivo by EGF and partial hepatectomy

A 2-fold increase in the level of epidermal growth factor (EGF) receptor mRNA accompanied by a similar increase in newly synthesized ligand-binding EGF-receptors was observed 2-4 h after intraportal EGF-injections and 2-4 h after partial hepatectomy. After this initial increase, the EGF-receptor levels decreased back to control levels 6-8 h after EGF-injections and below control levels 6-8 h after partial hepatectomy. EGF was also found to influence the degradation of endocytosed [125I]-EGF 3 h after injection to a similar extent as partial hepatectomy. The similar effects of EGF and partial hepatectomy suggests that EGF or EGF-like factors may be mechanistically involved in the early "promotion" stage during the pre-replicative phase of liver regeneration. The EGF-induced effects are, however, at least not alone responsible for the replicative process.     

Changes in lipoprotein binding and uptake by hepatocytes during rat liver regeneration

The binding and uptake of cholesterol enriched lipoproteins by isolated hepatocytes was decreased at 16 hours after partial hepatectomy, with a tendency to return to control values as the regeneration proceeds. The number of lipoprotein binding sites of total cellular membranes remained similar to control at 16 and 24 hours. The plasma lipoprotein pattern, determined by electrophoretic analysis, showed a lower per cent of very low density lipoproteins (VLDL) and a higher per cent of low density lipoproteins (LDL) at 16 and 24 hours post-partial hepatectomy. At these times, plasma lecithin: cholesterol acyltransferase (LCAT) activity was decreased. It is intriguing to suggest that the regenerating liver could regulated the blood lipoprotein pattern and the uptake of lipoproteins by modulating the surface expression of the receptors.     

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Liver regenerative potential of the lake frog Rana ridibunda after partial hepatectomy

A histomorphological study of the regenerating liver of Rana ridibunda, within 2 months after partial hepatectomy, shows that regenerative processes on the wound surface are slowly proceeding. Processes of reticular fiber reconstruction occurred in the composition of the basal membrane of liver sinusoids. A cytophotometric study shows that glandular cells in R. ridibunda liver are commonly tetraploid. The post-traumatic regeneration of the liver after partial hepatectomy involves activation of DNA synthesis in hepatocytes, leading to increase in their ploidy. Within the 1st month of regeneration, the mitotic index of hepatocytes substantially increased. Regeneration of glandular parenchyma of the liver is accompanied by a quantitative increase in binucleate hepatocytes, which is most highly expressed within 5-20 days after partial hepatectomy.     

Studies on satellite nucleoli in rat hepatocytes and Novikoff hepatoma cells

Summary The present study was undertaken to provide information on the presence and frequency of satellite nucleoli in cells with increased nucleolar biosynthetic activity. The number of hepatocytes containing satellite nucleoli was analyzed in rat liver, regenerating liver 18 h after partial hepatectomy and in Novikoff hepatoma ascites cells. In comparison with hepatocytes of normal liver, the number of both stimulated hepatocytes and malignant hepatoma cells containing satellite nucleoli was significantly reduced. The results also indicated that whereas most satellite nucleoli contain protein C23, a smaller percentage contain protein B23.     

Ultrastructural zonal heterogeneity of hepatocytes and mitochondria within the hepatic acinus during liver regeneration after partial hepatectomy

BACKGROUND INFORMATION: Partial hepatectomy (70%) induces cell proliferation until the original mass of the liver is restored. In the first 24 h after partial hepatectomy, drastic changes in the metabolism of the remaining liver have been shown to occur. To evaluate changes in hepatocyte ultrastructure within the hepatic acinus during the liver regenerative process, we investigated, by light and electron microscopy observations on specimens taken 0 h, 24 h and 96 h after partial hepatectomy, the hepatocyte structure and ultrastructure in the periportal and pericentral area of the hepatic acinus, with a particular emphasis on mitochondria ultrastructure. Moreover, some biochemical events that could affect the mitochondria ultrastructure and function were investigated. RESULTS: We found that, 24 h after partial hepatectomy, mitochondria with altered ultrastructure were preferentially localized in the periportal area. Periportal hepatocytes showed also an increase in the number of peroxisomes, free ribosomes, lysosomes and autophagosomes. Altered mitochondria showed swelling, an ultrastructural index of increased membrane permeability, a reduction in the number of cristae, and a rarefied, often vacuoled, matrix. Consistently, an increase in the mitochondrial oxidized/reduced glutathione ratio was found as well as calcium release from mitochondria in a manner inhibited by cyclosporin A. Interestingly, light and electron microscopy analysis showed that the hepatocytes in the periportal area were the cells with the major structural attributes to proliferate. At 96 h after partial hepatectomy, the preferential zonation of altered mitochondria was lost and the normal mitochondrial membrane permeability properties were restored. CONCLUSIONS: We suggest that 24 h after partial hepatectomy, a preferential zonation of altered mitochondria in the periportal hepatocytes could be involved in the changes of metabolic and functional heterogeneity of the hepatocytes within the hepatic acinus during the regenerative process.     

Insulin receptors in developing rat liver: Acquisition of down regulation in the immediate postnatal period

Alterations in the high and low affinity insulin receptor concentrations in developing rat liver were investigated. The number of high affinity receptors in partially purified plasma membranes from fetal rats increased from Days 19 through 22 of gestation, with no further increase in binding during the postnatal period. Fetuses of diabetic rats had approximately three times as many high affinity insulin receptors as age-matched fetuses of normal rats; however, by 1 day after birth the receptor number decreased to the normal level. Neither the number of low affinity receptors nor the affinity of insulin binding to high or low affinity receptors changed during development or between offspring of normal and diabetic rats. These changes in the number of high affinity hepatic insulin receptors from prenatal animals did not correlate with the concentration of plasma insulin. When suckling pups were rendered diabetic the changes in the number of high affinity insulin receptors correlated with alterations in plasma insulin concentrations. The number of high affinity sites/microgram DNA in hepatocytes from Day 18 fetal rats was not altered when cells were cultured for 48 h in medium containing 0, 250, or 5000 μU/ml of added insulin. When cultured hepatocytes derived from 1-day-old and adult rats were maintained in medium with added insulin concentrations of 250 or 5000 μU/ml the number of high affinity receptors/microgram DNA decreased as compared to the number of high affinity receptors in hepatocytes cultured in medium with no added insulin. This decrease in receptor number was accompanied by an increase in the affinity of insulin binding to its high affinity receptors. The data show that (i) only the high affinity insulin receptor number increases in rat liver during the prenatal period, (ii) fetuses of diabetic rats show a greater increase in high affinity receptors than do fetuses of normal animals, and (iii) the phenomenon of down regulation for high affinity insulin receptors is not observed in fetal rat liver, but is acquired in the immediate postnatal period.     

Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats.

Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis, which is essential for both healing of injured tissue and proliferation of carcinoma cells. In this study we elucidated the expression and role of VEGF in rat liver regeneration after partial hepatectomy. VEGF expression was mainly detected in periportal hepatocytes and reached a maximal level 48-72 hr after partial hepatectomy by both immunohistochemistry and in situ hybridization. Similarly, immunohistochemistry for Ki-67 showed that the proliferative activity of sinusoidal endothelial cells was highest in the periportal area and reached a maximal level 72 hr after partial hepatectomy. Moreover, neutralization of VEGF significantly inhibited proliferative activity of hepatocytes (p<0. 0001), as well as sinusoidal endothelial cells (p<0.001), at 48 and 96 hr after partial hepatectomy. Conversely, injection of VEGF significantly promoted proliferative activity of hepatocytes (p<0. 0001) as well as sinusoidal endothelial cells (p<0.0005) at 48 hr after partial hepatectomy. These results suggest that VEGF promotes proliferation of hepatocytes through reconstruction of liver sinusoids by proliferation of sinusoidal endothelial cells. Furthermore, these data point to a new therapeutic strategy, the use of VEGF and other hepatocyte growth factors in fulminant or severe acute hepatitis.     

Dynamic changes in protein components of the tight junction during liver regeneration

The construction of the hepatocyte tight junction is one of the most important events during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division. To understand this event at the molecular level, we examined the expression of tight junction proteins by Western blot analysis and their cellular localization by immunofluorescence microscopy in regenerating rat liver after two-thirds hepatectomy. The levels of tight junction components such as claudin-3, ZO-1 and atypical protein kinase C (PKC)-specific interacting protein (ASIP) increased two- to three-fold over control levels in coordination with a peak 2-3 days after partial hepatectomy, whereas occludin levels remained unchanged. The bile canaliculi outlined by tight junction components and actin filaments reveal significant morphological changes from 2-3 days after partial hepatectomy. During this period, claudin-3/ZO-1 and ASIP/ZO-1 were nearly co-localized, whereas occludin was locally reduced or almost absent on the bile canaliculi outlined by ZO-1 staining. The uncoupled localization of F-actin and tight junction components was often observed. The function of hepatocytes, as revealed by the serum bile acids level, was distorted temporally at an early stage of regeneration but mostly restored 3 days after partial hepatectomy. These observations suggest that the de novo construction of tight junctions proceeds mainly 2-3 days after partial hepatectomy in parallel with the cell polarization required for hepatocyte function. However, the complete normalization of the composition of the tight junction components, such as occludin and the association with F-actin, requires additional time, which may support the regeneration of fully polarized normal hepatocytes.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.     

Acid phosphatase distribution in the liver in cirrhosis

The distribution of acid phosphatase in liver cirrhosis, as well as in its reverse development, was investigated in mice using histochemistry and electron histochemistry methods. Histochemistry demonstrated a sharp activity increase of acid phosphatase (as compared with the same in the material of partial hepatectomy) in liver cells (especially hepatocytes) during liver cirrhosis regression 10 days after a partial hepatectomy. Electron histochemistry has shown the enzyme withdraw out of hepatocytes and connective tissue cells of fibrotic stratum in the extra-cell medium. The reaction product localized on the neighbouring collagen fibres giving evidence that during reverse development of liver cirrhosis the lisosomal enzyme release from specified cells by means of exocytosis and they are involved in the lysis of collagen.     

Dynamic expression of the cell adhesion molecule cell-CAM 105 in fetal and regenerating rat liver

Cell-CAM 105 is an integral cell surface glycoprotein that is involved in cell-cell adhesion of adult rat hepatocytes in vitro. In the present report we used a radio-immunoassay, a quantitative immunoblotting technique and immunofluorescence microscopy to investigate the expression of cell-CAM 105 in fetal and regenerating rat liver. In the fetal liver cell-CAM 105 did not appear until day 16 of the gestation, when it increased rapidly to reach the level found in adult liver, 3 weeks after birth. In liver regenerating after partial hepatectomy a transient decrease in the amount of cell-CAM 105 was observed in the plasma membranes of the hepatocytes. A significant decrease was observed as early as 12 h after partial hepatectomy, reaching a minimum by 3 days after the operation, corresponding to approx. 35% of the amount of cell-CAM 105 in normal liver. The amount then increased slowly and was back to the normal level by about 15 days after partial hepatectomy. The results indicate that cell-CAM 105 exerts its major function in terminally differentiated cells. An excellent correlation was seen between the kinetics of the expression of cell-CAM 105 and of reported changes of both enzymatic and organizational patterns of hepatocytes in regenerating and fetal liver. This suggests that cell-CAM 105 could be important for the development and maintenance of the cell-cell binding and organizational pattern characteristic of terminally differentiated hepatocytes.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Translocation of hepatocyte lysosomes following partial hepatectomy and its inhibition by colchicine

Hepatocyte microtubules were studied during the translocation of lysosomes which follows partial hepatectomy. In hepatocytes of normal rat liver the lysosomes are seen mainly along the bile canaliculi. After partial hepatectomy, these lysosomes lose their pericanalicular arrangement and move towards large inclusions (‘protein droplets’) which result from the marked pinocytosis induced by the removal of about two-thirds of the liver mass. The lysosomes fuse with the inclusions, and by 4 h after partial hepatectomy most of the inclusions demonstrate acid phosphatase activity histochemically. Many microtubules are found in close proximity to the lysosomes. When the rats are treated with colchicine prior to the partial hepatectomy, events are markedly different. The lysosomes change their location but do not hit the cytoplasmic inclusions, and the inclusions remain negative for acid phosphatase activity even 4 h post-hepatectomy. Quantitative ultrastructural study shows that the volume density of microtubules in the hepatocytes decreases to one-third of that in control hepatocytes. This strongly suggests an involvement of microtubules in giving orientation to the translocation of hepatocyte lysosomes under these conditions.     

Comitogenic effects of vasoactive intestinal polypeptide on rat hepatocytes

The primary mitogens such as epidermal growth factor and transforming growth factor-α are known to stimulate DNA synthesis in primary cultures of adult rat hepatocytes. Vasoactive intestinal polypeptide (VIP) was found to amplify DNA synthesis induced by the primary mitogens and thus acted as a comitogen. The comitogenic effect of VIP was specific for the culture medium, suggesting that minor components in the medium were required for hepatocytes to fully respond to VIP. Glutamic acid is probably one of these minor components, although other components present in the nutrient-rich medium were also necessary for the full comitogenic effect. Other comitogens such as insulin, vasopressin, and angiotensin II interacted additively with low concentrations of VIP. The comitogenic effect of VIP was also found in hepatocytes cultured from regenerating rat liver after a partial hepatectomy. In the regenerating hepatocyte cultures, VIP can act as a mitogen even in the absence of the primary mitogen EGF. VIP mRNA was found in several organs including brain, intestine, and liver, and its expression was slightly induced in liver 24 h after a partial hepatectomy. These results suggest that VIP can act as a hepatic comitogen and may play a role in liver cell proliferation. © 1996 Wiley-Liss, Inc.