Swelling of rat hepatocytes stimulates glycogen synthesis

In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following observations. 1) The extent of stimulation of glycogen synthesis by both metabolizable and nonmetabolizable amino acids was directly proportional to their ability to increase cell volume, except for proline, which stimulated glycogen synthesis more than could be accounted for by the increase in cell volume. 2) Both cell swelling and stimulation of glycogen synthesis by amino acids were prevented when hepatocytes were incubated in hyperosmotic media containing sucrose or raffinose. 3) Increasing the cell volume by incubating hepatocytes in Na(+)-depleted media in the absence of amino acids also stimulated glycogen synthesis. 4) Stimulation of glycogen synthesis by Na+ depletion was prevented by restoring the normal osmolarity with sucrose, but not with choline chloride which, by itself, stimulated glycogen synthesis and increased the cell volume. It is concluded that stimulation of glycogen synthesis by amino acids is due, at least in part, to an increase in hepatocyte volume resulting from amino acid uptake, and that hepatocyte swelling per se stimulates glycogen synthesis.     

Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes.

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.     

Zonal distribution of glycogen synthesis in isolated rat hepatocytes

Incubation of hepatocytes isolated from fasted rats with [14C]glucose for short periods of time showed that the initial stages of glycogen synthesis occur near the plasma membrane. Incubation with [14C]glucose followed by cold glucose demonstrated that glycogen synthesis is always active at the hepatocyte periphery and that previously synthesised glycogen moves towards the centre of the cell, while its place is filled by newly synthesised molecules. However, the reverse experiment, incubation with cold glucose before addition of [14C]glucose, showed that, as glycogen synthesis progresses, it also becomes gradually active in more internal sites of the hepatocyte. These results indicate a spatial order in the synthesis of hepatic glycogen.     

Transforming growth factor alpha inhibits glycogen synthesis and counteracts the stimulation by insulin in hepatocytes.

Rat transforming growth factor alpha (TGF alpha) inhibits glycogen synthesis in rat and guinea pig hepatocyte cultures and counteracts the stimulation of glycogen deposition and activation of glycogen synthase caused by insulin. The EC50 for inhibition of glycogen deposition was 0.2nM. The inhibition of glycogen synthesis was also observed in the absence of extracellular Ca2+ and was not blocked by indomethacin, suggesting that it is not mediated by production of prostaglandins. Since TGF alpha is produced by hepatocytes during liver regeneration and by macrophages during endotoxin stimulation, it may have an autocrine/paracrine effect on hepatic carbohydrate metabolism in these states, and may account for the low hepatic glycogen levels during liver regeneration and the impaired glucose tolerance associated with sepsis.     

Inhibition of glycogen synthesis in rat hepatocytes by medium Zn2+

In hepatocytes from fasted rats, Zn2+ in the range from 0 to 500 microM has relatively minor effects on gluconeogenesis from most substrates, or on ureagenesis from NH3. In hepatocytes from fed rats, Zn2+ does not affect glycogenolysis. In hepatocytes from fasted rats, in which glycogen is being actively synthesized using the substrate combination (Katz et al. (1976) Proc. Natl.Acad.Sci.USA 73,3433-3437) of glucose, lactate and glutamine (all 10mM), Zn2+ markedly inhibits glycogen synthesis, with total inhibition at 500 microM, and a half maximal effect in the range from 50 to 100 microM. Dipicolinate (pyridine 2,6-dicarboxylate), a zinc chelator, is about as effective as L-glutamine in activating glycogen synthesis with the substrate combination of dihydroxyacetone, lactate and glucose (all 10mM). This suggests the possible hypothesis that endogenous Zn2+ might control the rate of glycogen synthesis in vivo. However, alternate explanations such as metabolite accumulation are also possible, since dipicolinate causes inhibition of gluconeogenesis from L-lactate.     

Effect of mercaptopicolinic acid and of transaminase inhibitors on glycogen synthesis by rat hepatocytes.

To explore the mechanism of the stimulation of glycogen synthesis by amino acids (1) we have studied the effects of transaminase inhibitors and of mercaptopicolinic acid, (MPA) an inhibitor of phosphoenol pyruvate carboxykinase. Mercaptopicolinic acid enhanced glycogen synthesis from fructose, dihydroxyacetone and xylitol. Stimulation of glycogen synthesis with hepatocytes from fasted rats by 0.5 mM mercaptopicolinic acid was 50–70% as effective as 10 mM glutamine. With hepatocytes from fed rats, the stimulation of glycogen synthesis by mercaptopicolinic acid was more pronounced, and stimulation by mercaptopicolinic acid and amino acids was additive. Glycogen synthesis as high as 1% in wet weight per hour was attained in hepatocytes with a high initial glycogen content. Over 80% of glycogen synthase was in the active (a) form. Amino oxyacetic acid greatly depressed or abolished the stimulatory effect of glutamine and asparagine and of mercatopicolinic acid, and induced extensive glycogen breakdown in hepatocytes of fed rats.     

Glycogen synthesis by rat hepatocytes.

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.     

Amiloride inhibits protein synthesis in isolated rat hepatocytes

The effects of amiloride on Na⁺ ion influx, amino acid transport, protein synthesis and RNA synthesis have been studied in isolated rat hepatocytes. The initial rate of ²²Na⁺ uptake and the amount of ²²Na⁺ taken up at later time points were decreased in hepatocytes incubated in the presence of amiloride. Amiloride inhibited by about 25% the influx of α-methylamino[1?¹⁴C]isobutyric acid, a specific substrate for the A (Alanine preferring) system of neutral amino acid transport. By contrast, the activity of system L (Leucine preferring) was not affected by amiloride. Rates of protein synthesis were determined by using high extracellular concentrations of [¹⁴C]valine in order to maintain a constant amino acid precursor pool. Amiloride inhibited protein synthesis by 85% and had no effect on RNA synthesis. Half-maximal inhibition of protein synthesis occurred with amiloride at about 150 μM. In the absence of Na⁺ in the incubation medium, the rate of protein synthesis was reduced by about 35% and no further inhibition was observed with amiloride. These results suggest that in isolated rat hepatocytes protein synthesis is partially dependent on Na⁺, and that amiloride is an efficient inhibitor of protein synthesis.     

Eicosapentaenoic acid inhibits synthesis and secretion of triacylglycerols by cultured rat hepatocytes

Primary cultures of rat hepatocytes were used to study the effects of eicosapentaenoic and oleic acid on synthesis and secretion of triacylglycerols associated with very low density lipoproteins. From the experiments the following was observed. Oleic acid markedly stimulates secretion as well as synthesis of triacylglycerols, whereas eicosapentaenoic acid causes very little or no increase in secretion or synthesis as compared to a fatty-acid-free medium. The effects could already be observed after 15 min incubation. The inhibitory effect of eicosapentaenoic acid is reversible within 1-2 h. Eicosapentaenoic acid inhibits much of the stimulatory effect of oleic acid on synthesis and secretion of triacylglycerols. The cellular uptake of eicosapentaenoic acid is somewhat higher than that of oleic acid and the metabolism of these fatty acids to acid-soluble materials is similar. Eicosapentaenoic acid does not affect the secretory pathway of triacylglycerols per se. From these results it may be concluded that the mechanism for the inhibitory effect of eicosapentaenoic acid on triacylglycerol secretion is probably via reduced triacylglycerol synthesis.     

The molecular mechanism by which adrenalin inhibits glycogen synthesis.

Improved methodology was used to establish that the phosphorylation of a serine located 10 residues from the N-terminus of glycogen synthase (N10) increases from 0.12 mol.mol-1 to 0.54 mol.mol-1 in vivo in response to adrenalin. The only 'N10 kinase' detected in muscle extracts was casein kinase-1 (CK1), although its activity was unaffected by injection of adrenalin in vivo or by incubation with cyclic-AMP-dependent protein kinase and MgATP in vitro. Prior phosphorylation of the serine residue N7 by phosphorylase kinase increased sixfold the rate of phosphorylation of glycogen synthase by CK1, and altered the specificity of CK1 so that it phosphorylated the serine residue N10 specifically. Stoichiometric phosphorylation of N7 decreased the activity ratio (+/- glucose 6-phosphate) of glycogen synthase from 0.80 to 0.45, and subsequent phosphorylation of N10 to 0.8 mol.mol-1 produced a further decrease to 0.17, demonstrating that N10 phosphorylation inhibits glycogen synthase. The major 'N10 phosphatase' in skeletal muscle extracts was identified as the glycogen-associated form of protein phosphatase-1 (PP1G), accounting for approximately 75% of the N10 phosphatase activity in the extracts and about 90% of the activity in isolated glycogen particles. Phosphorylation of N10, after prior phosphorylation of N7, decreased the rate of dephosphorylation of N7. These results, in conjunction with previous findings, establish that adrenalin inhibits glycogen synthase by increasing the phosphorylation of N7, N10 and three further serines located 30, 34 and 38 residues from the start of the C-terminal CNBr peptide (termed the region C30-C38). They also indicate that increased phosphorylation of N10, the region C30-C38, and perhaps N7, is initiated through the inhibition of PP1G by adrenalin, which results from phosphorylation of its glycogen-targetting subunit by cyclic-AMP-dependent protein kinase [Hubbard, M.J. & Cohen, P. (1989) Eur. J. Biochem. 186, 711-716]. The conclusion that direct phosphorylation of glycogen synthase by cyclic-AMP-dependent protein kinase makes little contribution to inhibition by adrenalin, is at variance with the teachings of the major textbooks of biochemistry.     

Regulation of glycogen synthesis from glucose and gluconeogenic precursors by insulin in periportal and perivenous rat hepatocytes.

Glycogen synthesis in hepatocyte cultures is dependent on: (1) the nutritional state of the donor rat, (2) the acinar origin of the hepatocytes, (3) the concentrations of glucose and gluconeogenic precursors, and (4) insulin. High concentrations of glucose (15-25 mM) and gluconeogenic precursors (10 mM-lactate and 1 mM-pyruvate) had a synergistic effect on glycogen deposition in both periportal and perivenous hepatocytes. When hepatocytes were challenged with glucose, lactate and pyruvate in the absence of insulin, glycogen was deposited at a linear rate for 2 h and then reached a plateau. However, in the presence of insulin, the initial rate of glycogen deposition was increased (20-40%) and glycogen deposition continued for more than 4 h. Consequently, insulin had a more marked effect on the glycogen accumulated in the cell after 4 h (100-200% increase) than on the initial rate of glycogen deposition. Glycogen accumulation in hepatocyte cultures prepared from rats that were fasted for 24 h and then re-fed for 3 h before liver perfusion was 2-fold higher than in hepatocytes from rats fed ad libitum and 4-fold higher than in hepatocytes from fasted rats. The incorporation of [14C]lactate into glycogen was 2-4-fold higher in periportal than in perivenous hepatocytes in both the absence and the presence of insulin, whereas the incorporation of [14C]glucose into glycogen was similar in periportal and perivenous hepatocytes in the absence of insulin, but higher in perivenous hepatocytes in the presence of insulin. Rates of glycogen deposition in the combined presence of glucose and gluconeogenic precursors were similar in periportal and perivenous hepatocytes, whereas in the presence of glucose alone, rates of glycogen deposition paralleled the incorporation of [14C]glucose into glycogen and were higher in perivenous hepatocytes in the presence of insulin. It is concluded that periportal and perivenous hepatocytes utilize different substrates for glycogen synthesis, but differences between the two cell populations in the relative utilization of glucose and gluconeogenic precursors are dependent on the presence of insulin and on the nutritional state of the rat.     

Stabilization of glycogen stores and stimulation of glycogen synthesis in hepatocytes by phenacyl imidazolium compounds

LY177507 is representative of a series of phenacyl imidazolium compounds that cause marked lowering of blood glucose levels in animal models of noninsulin-dependent diabetes mellitus. In studies conducted with isolated rat hepatocytes, LY177507 inhibited net glucose production from a variety of substrates, inhibited glycolysis from exogenous glucose and endogenous glycogen, inhibited glycogenolysis, and stimulated glycogenesis. These effects of LY177507 appear to be the consequence of activation of glycogen synthase and inactivation of glycogen phosphorylase. In vivo studies with normal fed rats demonstrated a decrease in blood glucose, an increase in hepatic glycogen stores, and an inactivation of glycogen phosphorylase. Phenacyl imidazolium compounds appear to lower blood glucose levels and affect hepatic carbohydrate metabolism by a mechanism unlike other known hypoglycemic compounds.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits DNA synthesis in rat primary hepatocytes

Treatment of Sprague-Dawley (SD) rats with a dosing regimen of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) maintaining a steady-state liver concentration of 150 ng/g results in enhanced hepatocyte proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule (Fox et al. (1993) Cancer Res., 53, 2265–2271). Here, we report an initial characterization of the actions of TCDD on hepatocyte proliferation by monitoring DNA synthesis in primary hepatocytes isolated from SD rats. TCDD caused a dose-dependent inhibition (EC₅₀ = 10 pM) of DNA synthesis in primary hepatocytes isolated from either male or female SD rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor, hepatocyte growth factor, and transforming growth factor ). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with transforming growth factor-β1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is non-responsive to estradiol, independent of TGF-β, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.     

Multiple requirements for glycogen synthesis by hepatocytes isolated from fasted rats

Glycogen synthesis from various combinations of substrates by hepatocytes isolated from rats fasted 24 h was studied. As reported by Katz et al. (Katz, J., Golden, S., and Wals, P. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3433-3437), appreciable rates of glycogen synthesis occurred only in the presence of gluconeogenic precursors and one of several amino acids, which includes L-glutamine. L-Leucine had negligible effects on glycogen synthesis from 20 mM dihydroxyacetone and/or 15 mM glucose when L-glutamine was not added to the medium. In the presence of 10 mM L-glutamine, L-leucine greatly increased glycogen synthesis from these substrates. alpha-Ketoisocaproate was ineffective, as was oleate. NH4Cl depressed glycogen synthesis from 10 mM glucose plus 20 mM dihydroxyacetone in the absence of added L-glutamine and enhanced that in its presence, but these effects were weak compared to those of L-leucine. The amino acid analogues L-norvaline and L-norleucine exerted effects that were similar to those exerted by L-leucine. Under all conditions studied, cycloheximide and puromycin inhibited net glycogen synthesis. Cycloheximide did not stimulate gluconeogenesis from dihydroxyacetone, or phosphorylase in hepatocytes from starved rats, or glycogenolysis in hepatocytes from fed rats. Puromycin, however, stimulated glycogenolysis in hepatocytes from fed rats. Glycogen synthesis from 20 mM dihydroxyacetone proceeds with a pronounced initial lag phase that can be shortened by incubation of cells with glutamine plus leucine before addition of dihydroxyacetone. Concurrent measurements of glycogen synthesis, glycogen synthase, and gluconeogenesis under different conditions reveal that in addition to protein synthesis, activation of glycogen synthase, which must occur to allow glycogen synthesis in hepatocytes, requires a second component which can be satisfied by addition of dihydroxyacetone or fructose to the cells.     

Bile acids secretion and synthesis by isolated rat hepatocytes.

Bile acids secretion and their distribution were studied in isolated rat hepatocytes. Bile acids secretion was linearly related with time for first three hours of incubation and the net secretion rate was 23.2 ± 2.74 nmoles per g cells (wet weight) per minute. Isolated hepatocytes synthesized relatively more chenodeoxycholic acid than cholic acid compared to whole animal. These results suggest that isolated hepatocytes synthesize and secrete bile acids and thus provide experimental system to study the effect of drugs on bile acids secretion and synthesis at cellular level.     

Short-term stimulation of net glycogen production by insulin in rat hepatocytes

Isolated liver cells from 24 h starved rats were incubated in Krebs-Ringer buffer containing 4% albumin. In the presence of 10, 20 and 30 mM glucose, addition of insulin stimulated net glycogen production by 52, 39 and 20%, respectively. 2 . 10(-9) M insulin was required for half-maximal stimulation. Increases of glycogen production and of glycogen synthase a activity were observed after 15-30 min of incubation with insulin. The stimulatory effect of insulin was additive to that of lithium. In agreement with the literature, insulin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen deposition whereby a decrease of glucagon-elevated cyclic AMP levels was observed. In addition, we found that insulin also decreased the basal cyclic AMP levels in the absence of added glucagon by 22%. It is concluded that physiological concentrations of insulin stimulate net glycogen deposition in hepatocytes from fasted rats; the decrease of basal cyclic AMP levels upon insulin addition may play a role in the mechanism of the hormone action.     

Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes. Inverse relationship to glycogen content

Cultured rat hepatocytes were used to characterize the relationship between cellular glycogen content and the basal rate, as well as response to insulin of glycogen synthesis. Depending on the concentration of medium glucose, glycogen-depleted monolayers accumulated glycogen between 24 and 48 h of culture up to the fed in vivo level. Insulin at 100 nM stimulated glycogen deposition 20-fold at 1 mM and 1.5-fold at 50 mM glucose. The rate of further glycogen storage decreased with time and increasing glycogen content. In hepatocytes preincubated with 1-50 mM glucose during 24-48 h, short-term basal and insulin-dependent incorporation of 10 mM [14C]glucose into glycogen was inversely related to the actual cellular glycogen content. This was not due to different intracellular dilution of the label, since the specific radioactivity of UDP-glucose was similar in all groups. 125I-Insulin binding indicated that insulin receptors were also not involved in this phenomenon. An inverse relationship was also found between glycogen content and the stimulation of glycogen synthase I activity by insulin, whereas the basal activity of the enzyme was dissociated from the rate of incorporation of [14C]glucose. Basal net glycogen deposition at 10 mM glucose was also inversely related to cellular glycogen; however, no such relation was evident in the presence of insulin due to the overlapping inhibition of glycogenolysis. These studies suggest that the glycogen-mediated inhibition of the activation of glycogen synthase I is operative in the cultured hepatocyte and leads to an apparent inverse relationship between the actual glycogen content and basal as well as insulin-dependent glycogenesis.     

Plasma protein synthesis by isolated rat hepatocytes

A system of preparation of rat hepatocytes with extended viability has been developed to study the role of hormones and other plasma components upon secretory protein synthesis. Hepatocytes maintained in minimal essential medium reduced the levels of all amino acids in the medium except the slowly catabolized amino acids leucine, isoleucine, and valine, which steadily increase as the result of catabolism of liver protein. Although the liver cells catabolize 10-15% of their own protein during a 20-h incubation, the cells continue to secrete protein in a linear fashion throughout the period. The effects of insulin, cortisol, and epinephrine on general protein synthesis, and specifically on fibrinogen and albumin synthesis, have been tested on cells from both normal rats and adrenalectomized rats. Cells from normal animals show preinduction of tyrosine amino transferase (TAT), having at the time of isolation a high level of enzyme which shows only an increase of approximately 60% upon incubation with cortisol. In contrast, cells from adrenalectomized animals initially have a low level of enzyme which increases fourfold over a period of 9 h. The effects of both epinephrine and cortisol on protein synthesis are also much larger in cells from adrenalectomized animals. After a delay of several hours, cortisol increases fibrinogen synthesis sharply, so that at the end of the 20-h incubation, cells treated with hormone have secreted nearly 2.5 times as much fibrinogen as control cells. The effect is specific; cortisol stimulates neither albumin secretion nor intracellular protein synthesis. The combination of cortisol and epinephrine strongly depresses albumin synthesis in both types of cells. Insulin enhances albumin and general protein synthesis but has little effect on fibrinogen synthesis.     

Dehydroepiandrosterone inhibits DNA synthesis of rat hepatocytes induced by partial hepatectomy or mitogen (ciprofibrate)

In a previous study we have shown that dehydroepiandrosterone (DHEA) inhibits hepatocyte DNA synthesis after short-term administration and induces hepatocellular carcinomas after long-term administration in the rat. It is not known whether DHEA is also capable of inhibiting replicative and mitogen-induced DNA synthesis. In the present study, we have evaluated the effect of DHEA on DNA synthesis in the rat liver after partial hepatectomy and mitogen administration. After partial hepatectomy, DHEA significantly inhibited DNA synthesis at 20, 26, 32 and 38 h. Similarly, combined administration of ciprofibrate, a peroxisome proliferator and mitogen, and DHEA also resulted in significant hepatocyte DNA synthesis. However, DHEA did not affect liver enlargement caused by ciprofibrate. This experimental system will serve as useful tool to evaluate the role of cell proliferation in carcinogenesis.     

Effect of down-regulation and return of insulin receptors on glycogen synthesis in cultured rat hepatocytes

The dependence of the regulation of insulin receptors by insulin on the time hepatocytes were maintained in culture and the relationship between the return of down-regulated receptors and glycogen synthesis from labelled glucose were investigated in primary cultures of adult rat hepatocytes. Insulin receptor numbers, but not ligand affinity, decreased significantly within the first 24 h of culture, even in the absence of insulin, and then returned to the immediate 'post-attachment' level during 24-48 h. Therefore, down-regulation of insulin receptors by 10 nmol/l insulin was only minor during the 1st day in culture, but amounted to 50% of control levels after the 2nd day, whereas the rate of insulin degradation remained unaltered throughout the entire period of culture. When down-regulated monolayers were switched to insulin-free medium, receptors returned to control levels within 5-10 h. The reduced basal rate of glycogenesis as well as insulin-sensitivity and insulin responsiveness of this metabolic pathway also gradually increased to control levels. However, the time-dependent receptor return was dissociated from the increase in insulin-sensitivity, emphasising the importance of postbinding events. Since the changes both in basal rates and in insulin responsiveness of glycogenesis during the period of receptor return were inversely related to differences in the actual glycogen content between control and down-regulated cells, cellular glycogen content might participate in the regulation of glycogenesis as a 'feedback inhibitor'.