Expression of differentiated function by hepatocytes in primary culture: variable effects of glucagon and insulin on gluconeogenesis during cell growth

Hormonal effects on gluconeogenesis from lactate were studied during the growth cycle of adult rat parenchyma liver cells using a primary monolayer culture system previously described [25]. Basal and glucagon-stimulated gluconeogenic ability were found to decline rapidly during log phase, insulin-stimulated growth. A progressive recovery of gluconeogenesis activity was observed after cell division subsided. Rates of lactate-gluconeogenesis were found also to decline in the absence of prior insulin exposure. This decline was not as rapid as the loss observed in cells cultured with insulin. However, in insulin-deficient cultures gluconeogenesis was completely abolished after 12 days and did not reelevate with further incubation unless cells were washed and exposed to glucagon. Decreasing growth rates of insulin-supplemented cultures by decreasing serum concentrations resulted in comparatively higher gluconeogenic activity. The results presented here are consistent with previous observations of hepatic parenchymal expression of 'differentiated function' during cellular growth phases in culture (i.e., differentiated functions are generally lost during rapid growth and regained as cells become quiescent). The present study, however, presents unexpected effects of insulin on the apparent growth-state dependent gluconeogenic recovery. Our data imply that although insulin has long been known to inhibit gluconeogenesis, its presence in culture may facilitate long-term basal maintenance of gluconeogenic enzyme activity. Insulin also functions as a growth factor whose initial mitogenic effect correlates with decreased gluconeogenic function. These changes show no simple or predictive correlation with cyclic nucleotide metabolism.     

Control of growth and expression of differentiated functions of mature hepatocytes in primary culture

Since methods to disperse and culture hepatocytes were developed 15 years ago, numerous investigations have shown that primary cultures of mature hepatocytes retain most liver functions and respond as well to various hormones as those in vivo. Thus they are the most suitable system in vitro for studies on the liver. Moreover, recently it was found that differentiated hepatocytes in culture can grow under certain conditions and that this growth is regulated not only by several hormones, such as insulin, epidermal growth factor and serum growth factor, but also by a cell membrane factor and proteins in the environmental matrix through cell contact. This article describes the biochemical characterization of regulatory factors for hepatocyte growth and functions and their reciprocal expression. The mechanisms of liver regeneration, differentiation and carcinogenesis and the importance of the tissue architecture for these events are discussed mainly on the basis of our findings.     

Reciprocal changes of insulin and glucagon receptors in primary cultured hepatocytes

The specific [125I]insulin binding to primary cultured hepatocytes was significantly greater than that to freshly isolated hepatocytes. Low affinity insulin binding sites in cultured cells were 6-fold greater in number than those of freshly isolated cells without a significant change in high affinity sites. However, both sensitivity (insulin concentration for half maximum stimulation) and responsiveness (% of increase above the basal level) to insulin for the stimulation of ODC activity were similar for isolated and cultured cells indicating an important role of high affinity sites in the insulin action. On the other hand, the specific [125I]glucagon binding to cultured cells was significantly decreased. Low affinity glucagon binding sites in cultured cells decreased by about 50% in cultured cells without a significant change in high affinity sites. Both sensitivity and responsiveness to glucagon for the stimulation of ketogenesis from palmitate also decreased as compared with those of isolated cells, indicating an important role of low affinity sites in the glucagon action. These results indicate that insulin and glucagon receptors were reciprocally changed in cultured cells, as compared with isolated cells.     

Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats.

In hepatocytes from 48 h-starved rats identical glucagon dose-response curves were obtained for the stimulation of gluconeogenesis from lactate, for ketogenesis and for the decreasing of the C5-dicarboxylate pool. Glucagon (20 nM) caused a 5-fold increase in 3-hydroxybutyrate formation, but decreased acetoacetate production 50% of that of the control. In hepatocytes from biotin-deficient rats glucagon no longer stimulated gluconeogenesis from lactate, but still produced its effects on the mitochondrial redox state and the C5-dicarboxylate pool. The results suggest that the primary site of the hormone action on gluconeogenesis is located within the mitochondria rather than in the cytosol.     

Anabolic regulation of gluconeogenesis by insulin in isolated rat hepatocytes

The role of substrate availability in the regulation of gluconeogenesis in isolated rat hepatocytes was studied using [U-14C]alanine as a tracer in the presence of different concentrations of L-alanine in the incubation medium. At low alanine concentrations (0.5 mM) insulin decreased the 14C incorporation into the glucose pool and increased the incorporation of tracer carbons into the protein and lipid pools and into CO2. The net radioactivity lost from the glucose pool was only a small percentage of the total increase in the activity of the protein, lipid, CO2, or glycogen pools, supporting the notion that the effect of insulin in diminishing gluconeogenesis is secondary to its effects on pathways using pyruvate. At higher concentrations of alanine (2.5, 5.0, and 10.0 mM) in the incubation medium insulin increased the movement of alanine carbons into protein and glucose. This suggests that at higher substrate concentrations the ability of the liver to synthesize proteins is overwhelmed and the pyruvate carbons are forced into the gluconeogenesis pathway. These results were further confirmed by using [U-14C]lactate. The increases in observed specific activity of glucose following insulin administration would not be possible if insulin acted by affecting the activity of any enzyme directly involved in the formation or utilization of pyruvate, most of which have been proposed as sites of insulin action. Data presented show that insulin "inhibits" gluconeogenesis by affecting a change in substrate availability.     

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.     

Expression of cell adhesion molecule and albumin genes in primary culture of rat hepatocytes

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.     

DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP

Epidermal growth factor (EGF) especially in combination with insulin and glucagon, has been shown to stimulate DNA synthesis in liver cells, both in the whole animal and in cell cultures. As a further development we have found that in primary monolayer cultures of freshly isolated adult rat liver parenchymal cells, in which contamination with nonparenchymal cells was negligible, DNA synthesis was substantially stimulated by these substances. In control cultures, incorporation of [3H]thymidine into DNA and labeling of nuclei in autoradiographs was low. The stimulation by EGF was enhanced by insulin and glucagon, whereas these hormones by themselves exhibited only limited activity. These observations were made in cultures of hepatocytes that were never exposed to serum, even during cell isolation and plating. Hence for stimulation of DNA synthesis under these conditions neither serum factors nor interactions with other types of cells or their products were required. The effects of glucagon were reproduced by substances that elevate intracellular concentration of cyclic-AMP, including cholera toxin, isoproterenol, and methylisobutylxanthine. These various substances, especially EGF, glucagon, or cyclic-AMP, altered the morphological characteristics of the cultures during early stages, promoting cellular spreading and aggregation.     

Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum-free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three-carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8- to 12-fold increase over basal glucose production). Half-maximum stimulation of gluconeogenesis was seen with 3.6 × 10^?10 M glucagon and 3.6 × 10^?8 M dexamethasone. Maximum stimulation was oberved with 10^?7 M glucagon and 10^?6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon-stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2-fold increase) or 15 hours (1.7-fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.     

Effects of glucagon and alpha- and beta-agonists on glycogenolysis and gluconeogenesis in isolated ovine hepatocytes

(1) The effects of glucagon, dibutyryl cyclic AMP, vasopressin, phenylephrine, and isoproterenol on glycogenolysis and gluconeogenesis were investigated using isolated ovine hepatocytes. (2) Glycogenolysis was stimulated by all effectors except vasopressin. The response to alpha-agonists was greater than that of beta-agonists in older animals. Stimulation by beta-agonists increased after 30 h primary culture. (3) Gluconeogenesis from propionate or L-lactate plus pyruvate was stimulated to a small extent by dibutyryl cyclic AMP, glucagon and isoproterenol but not by vasopressin or phenylephrine. (4) No effects of lactation were observed. (5) Data are compared to results obtained in other species and the physiological significance of the results in relation to the ruminant is discussed.     

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.     

Diazepam: in vitro effects on glucagon and insulin release

Diazepam suppressed arginine-induced glucagon release from the isolated perfused rat pancreas in a dose-dependent manner, with an IC50 of approximately 65 microM. In contrast, insulin release was enhanced by 10-50 microM diazepam, but inhibited by higher concentrations of drug. Thus, 50 microM diazepam simultaneously suppressed glucagon and increased insulin release in this model. The potentiation of insulin release may result from phosphodiesterase inhibition. The inhibitory effects on hormone release are discussed in terms of diazepam's molecular conformation, which is similar to that of diphenylhydantoin, an inhibitor of both glucagon and insulin release in the isolated perfused rat pancreas. The possibility is also considered that the conformation of both compounds might be similar to the apparent active site of the hormone release inhibitor somatostatin.