Effect of berenil on polyamine metabolism in primary cultured rat hepatocytes

• 1.1. Putrescine and spermidine content increased in hepatocytes during culture. In the presence of 10 μM Berenil, putrescine content was further increased, while the increase of spermidine was prevented.
• 2.2. Ornithine decarboxylase activity was markedly reduced, and to a lesser extent also S-adenosyl-methionine decarboxylase activity.
• 3.3. Berenil appears to promote an increase in the transformation of spermidine into putrescine, and to inhibit the polyamine efflux.

     

Study of carbohydrate metabolism in glycogen storing cell lines derived from cultured rat hepatocytes

Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. An increase of glucose-6-phosphate dehydrogenase activity is also found. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.     

Enhancement of glycogen concentrations in primary cultures of rat hepatocytes exposed to glucose and fructose.

Glycogen synthesis in isolated hepatocytes can occur from glucose both by a direct mechanism and by an indirect process in which glucose is first metabolized to C3 intermediates before use for glycogenesis via gluconeogenesis. We studied the incorporation into glycogen of glucose and the gluconeogenic substrate, fructose, in primary cultures of hepatocytes from fasted rats. In the presence of insulin, both glucose and fructose promoted net deposition of glycogen; however, fructose carbon was incorporated into glycogen to a greater extent than that from glucose. When glucose and fructose were administered simultaneously, the glycogenic utilization of glucose was stimulated 2-3-fold, and that of fructose was increased by about 50%. At constant hexose concentrations, the total incorporation of carbon, and the total accumulation of glycogen mass, from glucose and fructose when present together exceeded that from either substrate alone. Fructose did not change the relative proportion of glucose carbon incorporated into glycogen via the indirect (gluconeogenic) mechanism. The synergism of glucose and fructose in glycogen synthesis in isolated rat hepatocytes in primary culture appears to result from a decrease in the rate of degradation of newly deposited glycogen, owing to (i) decreased amount of phosphorylase a mediated by glucose and (ii) noncovalent inhibition of residual phosphorylase activity by some intermediate arising from the metabolism of fructose, presumably fructose 1-phosphate.     

Modulation of glucose metabolism by sulfonylureas in primary cultures of adult rat hepatocytes

Addition of tolbutamide (0.1-5 microM) or glipizide (0.05-5 microM) to primary cultures of adult rat hepatocytes caused a dose-dependent increase of fructose 2,6-bisphosphate concentration. This effect was accompanied by a stimulation of the rate of L-lactate production and by an acceleration of the metabolic flux through the reaction catalysed by 6-phosphofructo 1-kinase. Moreover, the continuous presence of tolbutamide during the first 26 hours of culture mimicked long-term insulin effects by raising fructose 2,6-bisphosphate levels and the rate of L-lactate formation. Glucokinase, 6-phosphofructo 1-kinase and total 6-phosphofructo 2-kinase activities were not found to be significantly different in hepatocytes cultured either in the presence or in the absence of sulfonylurea.     

Dexamethasone and dimethylsulfoxide as distinct regulators of growth and differentiation of cultured suckling rat hepatocytes

Dexamethasone can promote the differentiation of different tissues in vivo while dimethylsulfoxide is a commonly used inducer of differentiation in various tumor cell types in culture. In the present study, the effects of dexamethasone and dimethylsulfoxide on growth and functional activities of cultured differentiating suckling rat hepatocytes stimulated with various combinations of EGF, insulin, and glucagon were evaluated. Hepatocytes stimulated with EGF and either insulin or glucagon entered S phase and mitosis after a lag period of 24 h. These hormonal factors thus provide simple combinations of hepatocyte-growth regulators. Dexamethasone in the presence of EGF and glucagon inhibited the initiation of DNA synthesis and mitosis, but it had no effect on EGF-insulin stimulated cultures. Such a differential effect of dexamethasone was observed at concentrations ranging from 4 nM to 200 microM. alpha-Fetoprotein, albumin, and tyrosine aminotransferase were used as typical markers of hepatocyte differentiation status. Irrespective of the combinations of growth-promoting factors used, dexamethasone inhibited alpha 1-fetoprotein production and maintained albumin production and tyrosine aminotransferase inducibility. In contrast, dimethylsulfoxide at 2% inhibited hepatocyte growth and supported the maintenance of the production of both alpha 1-fetoprotein and albumin, independent of the hormonal growth regulators used. On this basis, dexamethasone and dimethylsulfoxide act as distinct modulators of growth and maturation of cultured differentiating suckling rat hepatocytes.     

Compared roles of glucose, galactose and fructose as glycogen precursors during the acute response to insulin in cultured rat foetal hepatocytes

1. The efficiency of the contribution of hexoses to basal- and stimulated-glycogenesis, when studied in cultured 18 day-old rat foetal hepatocytes in the presence of glucose, was as follows: galactose greater than glucose greater than fructose. 2. Glucose deprivation had opposite effects on the contributions of [14C]galactose (decreased) and [14C]fructose (increased) to glycogenesis, which occurred independently of insulin and were reversed by glucose concentrations as low as 30-100 microM. 3. The stimulation of glycogenesis by insulin measured with [14C]glucose (3.2-fold) was superior to that obtained with either [14C]galactose or [14C]fructose (2.7-fold in both cases), which revealed a specific beneficial effect of insulin on glucose contribution.     

Glucagon and regulation of glucose metabolism

As a counterregulatory hormone for insulin, glucagon plays a critical role in maintaining glucose homeostasis in vivo in both animals and humans. To increase blood glucose, glucagon promotes hepatic glucose output by increasing glycogenolysis and gluconeogenesis and by decreasing glycogenesis and glycolysis in a concerted fashion via multiple mechanisms. Compared with healthy subjects, diabetic patients and animals have abnormal secretion of not only insulin but also glucagon. Hyperglucagonemia and altered insulin-to-glucagon ratios play important roles in initiating and maintaining pathological hyperglycemic states. Not surprisingly, glucagon and glucagon receptor have been pursued extensively in recent years as potential targets for the therapeutic treatment of diabetes.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

Pathway of glycogen synthesis from glucose in hepatocytes maintained in primary culture

Glycogen synthesis was examined in primary cultures of adult rat hepatocytes that had been isolated from rats following a 24-h fast. Glycogen synthesis was dependent on the concentration of glucose in the culture medium and also required the presence of insulin. The addition of dexamethasone to the culture medium also increased the amount of glycogen synthesis. When the culture medium was supplemented with [U-14C,3-3H]glucose, it was found that approximately 60% of the glucose incorporated into glycogen was not derived from the pool of labeled glucose. In addition, the relative ratio of 3H/14C in the newly synthesized glycogen was approximately 50% of the ratio of the two isotopes in glucose in the culture medium, indicating that the glucose had undergone metabolism prior to its incorporation into glycogen. However, when hepatocytes were isolated from rats that had been fed ad libitum and the synthesis of glycogen from [U-14C,3-3H]glucose was followed, the relative ratio of the two isotopes in glycogen was similar to that measured for glucose in the culture medium, indicating that the glucose was directly incorporated into glycogen without any apparent metabolism. These results indicate that the synthesis of glycogen from glucose may, at least in part, follow an indirect pathway whereby glucose is metabolized prior to incorporation of the carbon into glycogen, but that the pathway followed for the synthesis of glycogen is dependent on the prior metabolic state of the animal.     

Contribution of glucose and gluconeogenic substrates to insulin-stimulated glycogen synthesis in cultured fetal hepatocytes

The influence of medium composition on basal and insulin-stimulated glycogenesis was studied in cultured 17-day-old rat fetal hepatocytes, which contain no glycogen at the time of transplantation. Continuous-labeling ¹⁴C-glucose experiments were used to determine both glycogen content and glycogen labeling. The specific activity of glucose units in the newly formed glycogen (a) was compared to that of the medium glucose (b): the ratio a/b expresses the contribution of medium glucose to glycogen formation. In standard medium (5.5 mM glucose), this ratio averaged 0.60. Variations of glucose concentration in the medium from 1 to 40 mM were accompanied by a progressive increase in both glycogen content and the ratio a/b (up to 0.80). Supplementation of standard medium with fructose, galactose, glycerol, or lactate-pyruvate decreased the hepatocyte glucose uptake from the medium. Galactose (1 to 5 mM) or lactate-pyruvate (5 mM) enhanced the glycogen content whereas glycerol or fructose (1 to 5 mM) had no effect. The ratio a/b, not modified by glycerol or lactate-pyruvate, was decreased to 0.45 by fructose (5 mM). Galactose at concentrations as low as 1 to 2 mM brought the ratio down to 0.30, indicating that it is a superior precursor of glycogen as compared to glucose. When the hepatocytes were grown in the presence of 10 nM insulin, the glycogen content was constantly higher than in the absence of the hormone (2-fold stimulation). Also the amplitude of the glycogenic effect of insulin was similar whatever the modifications of the medium, whereas ratio a/b and glucose uptake were hardly increased by insulin. Thus several substrates can contribute to glycogen formation (especially galactose) in cultured fetal hepatocytes and the essential effect of insulin is a stimulation of the final step of the glycogenosynthetic pathway.     

Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

Addition of platelet-activating factor (PAF) to cells doubly labeled with [14C]glycerol plus [3H]arachidonic acid resulted in a transient decrease of [14C]glycerol-labeled phosphatidylinositol (PI) and a transient increase of [14C]glycerol-labeled lysophosphatidylinositol (LPI). [3H]Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The 3H/14C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of [14C]glycerol-labeled DG paralleled the loss of triacyl [14C]glycerol and the 3H/14C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- [3H]inositol-prelabeled cells, PAF induced a transient decrease of [3H]phosphatidylinositol-4,5-bis-phosphate (TPI) and [3H]phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with 32Pi induced a transient decrease of [32P]polyphosphoinositides at 20 sec to 1 min. [32P]LPI appeared within 10 sec after stimulation and paralleled the loss of [32P]PI. [3H]Inositol triphosphate, [3H]inositol diphosphate, and [3H]inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.     

Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes.

1. Adult rat hepatocytes were isolated by collagenase perfusion and were maintained in monolayer culture for 24h. 2. Choline metabolism and phosphatidylcholine biosynthesis were studied in these cells by performing pulse-chase studies at physiological concentrations (1-40 microM) of (Me-3H)-labelled or unlabelled choline in the culture medium. 3. During the 15 min pulse incubation, choline entering the cells was rapidly phosphorylated to phosphocholine or oxidized to betaine. Low concentrations of choline in the medium decreased the relative amount of choline oxidized. 4. During the 3 h chase period, the radioactivity in the phosphocholine pool was transferred to phosphatidylcholine. Very little radioactivity was associated with CDP-choline. These results provide good evidence that the rate-limiting step for phosphatidylcholine biosynthesis in these cultured hepatocytes is the conversion of phosphocholine into CDP-choline. Similar results were obtained for all concentrations of choline in the culture medium. 5. Cellular concentrations of phosphocholine were unaffected by the concentration of choline (1-40 microM) in the medium. 6. The majority of the label associated with betaine was secreted into the culture medium during the chase incubation. 7. From the pulse-chase studies, and the cellular phosphocholine concentrations, it was possible to estimate the rate of phosphatidylcholine biosynthesis (2.2, 2.8, 3.1 and 3.7 nmol/min per g wet weight of cells cultured in 1, 5, 10 and 40 microM-choline respectively for up to 4.25 h).     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells.

N-Glycosylation, biosynthesis and degradation of dipeptidylpeptidase IV (EC 3.4.14.5) (DPP IV) were comparatively studied in primary cultured rat hepatocytes and Morris hepatoma 7777 cells (MH 7777 cells). DPP IV had a molecular mass of 105 kDa in rat hepatocytes and of 103 kDa in MH 7777 cells as assessed by SDS/PAGE under reducing conditions. This difference in molecular mass was caused by differences in covalently attached N-glycans. DPP IV from hepatoma cells contained a higher proportion of N-glycans of the oligomannosidic or hybrid type and therefore migrated at a slightly lower molecular mass. In both cell types DPP IV was initially synthesized as a 97-kDa precursor which was completely susceptible to digestion with endo-beta-N-acetylglucosaminidase H converting the molecular mass to 84 kDa. The precursor was processed to the mature forms of DPP IV, glycosylated with N-glycans mainly of the complex type with a half-life of 20-25 min. The transit of newly synthesized DPP IV to the cell surface displayed identical or very similar kinetics in both cell types with the major portion of DPP IV appearing at the cell surface after 60 min. DPP IV molecules were very slowly degraded in hepatocytes as well as in hepatoma cells with half-lives of approximately 45 h. Inhibition of oligosaccharide processing with 1-deoxymannojirimycin led to the formation of DPP IV molecules containing N-glycans of the oligomannosidic type. This glycosylation variant was degraded with the same half-life as complex-type glycosylated DPP IV. By contrast, inhibition of N-glycosylation with tunicamycin resulted into rapid degradation of non-N-glycosylated DPP IV molecules in both cell types. Non-N-glycosylated DPP IV could not be detected at the cell surface indicating an intracellular proteolytic process soon after biosynthesis.     

Glucagon-stimulated but not isoproterenol-stimulated glucose formation inhibition by interleukin-6 in primary cultured rat hepatocytes.

During prolonged sepsis, impairment of glucose supply by the liver leads to hypoglycemia. Our aim was to investigate whether proinflammatory cytokine interleukin-6, a major mediator of the hepatic acute phase reaction, could contribute to this impairment by inhibiting hepatic glucose production stimulated by glucagon or isoproterenol in rat hepatocytes. Interleukin-6 inhibited the stimulation of glucose formation from glycogen by glucagon but not by isoproterenol in cultured rat hepatocytes. This was confirmed in the perfused rat liver. In cultured hepatocytes, the increase in cyclic adenosine-3',5'-monophosphate formation by glucagon was inhibited by interleukin-6, which was probably due to attenuation of glucagon binding to the glucagon receptor. The increase in cyclic adenosine-3',5'-monophosphate stimulated by isoproterenol was not affected by interleukin-6. However, the cytokine inhibited both expression of the key gluconeogenic control enzyme, phosphoenolpyruvate carboxykinase, stimulated by glucagon and isoproterenol. Thus, while increased glucose demand during the acute-phase reaction might initially be accomplished by catecholamine-mediated stimulation of glucose formation from glycogen, inhibition of gluconeogenesis by interleukin-6 may contribute to the impairment of glucose homeostasis during the prolonged acute phase reaction.     

Excretion of glutathione conjugates by primary cultured rat hepatocytes

Conjugation of xenobiotics with glutathione occurs commonly within the liver, and these glutathione conjugates are then preferentially excreted into bile. We have characterized this excretory process using primary cultured hepatocytes (24 h). 1-Chloro-2,4-dinitrobenzene rapidly entered the cells and formed a glutathione conjugate, S-(dinitrophenyl)glutathione, irrespective of the temperature of incubation. In contrast, the efflux of the glutathione conjugate was essentially absent in the cold but recovered rapidly upon rewarming of the cells. Therefore, initial rates of efflux of the conjugate at 37 degrees C were measured from cells preloaded biosynthetically at 10 degrees C. Efflux was a saturable process with respect to intracellular S-(dinitrophenyl)glutathione with an apparent Km of 0.58 +/- 0.12 mM and Vmax of 0.15 +/- 0.05 nmol/min/mg of protein. The excretion of S-(dinitrophenyl)glutathione had an energy of activation of 15.3 kcal/mol. The glutathione conjugate of p-nitrobenzylchloride when formed within the hepatocytes acted as a competitive inhibitor of S-(dinitrophenyl)glutathione efflux. Cultured hepatocytes, therefore, appeared to have a specific transport process for the excretion of glutathione conjugates. The addition of S-(dinitrophenyl)glutathione, but not GSH, GSSG, or methionine, to the medium caused a decrease in the rate of efflux of radiolabeled S-(dinitrophenyl)glutathione. The hepatocytes were able, however, to excrete the glutathione conjugate against an excess of extracellular S-(dinitrophenyl)glutathione. This observation suggested that extracellular S-(dinitrophenyl)glutathione, although capable of binding to the carrier, entered the hepatocytes quite slowly relative to rates of efflux. This carrier may function in a manner that would minimize the reuptake by hepatocytes of conjugates that have been excreted into the bile.     

Dietary fat type alters glucose metabolism in isolated rat hepatocytes

Dietary fat type can influence the regulation of carbohydrate metabolism in multiple tissue types. The influence of feeding high-fat (40% of kilocalories) diets containing either menhaden oil (MO) or coconut oil (CO) on hepatic glycogenolytic and gluconeogenic capacities was studied in isolated rat hepatocytes. Estimates of both glycogenolytic and gluconeogenic capacities were performed on hepatocytes isolated from fed and fasted animals, respectively. In MO-fed animals, both basal and hormone-stimulated rates of glucose production were significantly greater than those in CO-fed animals. However, both groups displayed a similar maximal increase in glucose production above basal for glucagon and epinephrine (2.3- and 1.9-fold, respectively). Basal rates of adenosine 3′,5′-cyclic phosphate (cAMP) production were not different between groups whereas glucagon-stimulated cAMP production was increased twofold in the MO-fed group. In both MO and CO groups, the addition of 10 nM insulin reduced glucose production in fed animals to similar absolute rates. In animals fasted for 24 hours, gluconeogenic capacity was estimated using 10 mM pyruvate, lactate, or glycerol. Glucose production from all substrates was significantly greater in CO-fed animals. In addition to increased gluconeogenic rates, maximal phosphoenolpyruvate carboxykinase (PEPCK) activity was increased in the CO-fed group. Insulin reduced glucose production in both dietary groups, but the absolute rate of glucose production was 28% greater in the CO-fed group relative to the MO-fed group. In summary, dietary fat type can markedly influence the regulation of hepatic glucose metabolism in multiple metabolic pathways. MO feeding promoted glycogenolysis and sensitivity to insulin whereas CO feeding favored gluconeogenesis and reduced insulin sensitivity.     

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'.