Hormonal control of ornithine decarboxylase in isolated liver cells and the effect of ethanol oxidation

The regulation of ornithine decarboxylase activity was studied in freshly isolated rat hepatocytes incubated in a chemically defined medium for 5 h. Glucagon, dibutyryl cyclic AMP, insulin and dexamethasone produced dramatic increases in ornithine decarboxylase activity, 6–100-times the basal activity. Actinomycin D inhibited completely the stimulatory action of these substances. With glucagon, dibutyryl cyclic AMP and insulin, the rise in ornithine decarboxylase activity was rapid but transient, peaking at 200 min and then declining rapidly. By contrast, the response to dexamethasone was gradual and sustained in the 5 h incubation. The transient nature of the response to glucagon was unaltered by repeated additions of optimally effective doses of glucagon suggesting the development of ‘refractoriness’ to the actions of this hormone. Ethanol oxidation inhibited by 50% the stimulation of ornithine decarboxylase by glucagon and dexamethasone and this effect was blocked by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Acetate (2.5–20 mM), the metabolic product of hepatic ethanol oxidation, was also effective. The data indicate that glucagon, insulin and glucocorticoids are all effective in stimulating the activity of ornithine decarboxylase in isolated hepatocytes but they differ in their duration and time of peak of action. Additionally, the inhibitory effect of ethanol on the hormonal stimulation of ornithine decarboxylase is dependent on its oxidation and may be mediated by acetate.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Insulin and glucagon regulate the activation of two distinct membrane-bound cyclic AMP phosphodiesterases in hepatocytes.

Glucagon (10 nM) caused a transient elevation of intracellular cyclic AMP concentrations, which reached a peak in around 5 min, and slowly returned to basal values in around 30 min. When 1 mM-3-isobutyl-1-methylxanthine (IBMX) was present, this process yielded a Ka of 1 nM for glucagon. The addition of insulin (10 nM) after 5 min exposure to glucagon (10 nM) caused intracellular cyclic AMP concentrations to fall dramatically, attaining basal values within 10 min. The regulation of this process was dose-dependent, exhibiting a Ka of 0.4 nM for insulin. If insulin and glucagon were added together to hepatocytes, then insulin decreased the magnitude of the cyclic AMP response to glucagon. IBMX (1 mM) prevented insulin antagonizing the action of glucagon in both of these instances. A gentle homogenization procedure followed by a rapid subcellular fractionation of hepatocytes on a Percoll gradient was developed. This was used to resolve subcellular membrane fractions and to identify cyclic AMP phosphodiesterase activity in both membrane and cytosol fractions. Glucagon and insulin only affected the activity of two distinct membrane-bound species, a plasma-membrane enzyme and a 'dense vesicle' enzyme. Glucagon (10 nM), insulin (10 nM), IBMX (1 mM), dibutyryl cyclic AMP (10 microM) and cholera toxin (1 microgram/ml) all elicited the activation of the 'dense vesicle' enzyme. The plasma-membrane enzyme was not activated by glucagon, IBMX or dibutyryl cyclic AMP, although insulin and cholera toxin both led to its activation. The degree of activation of the plasma-membrane enzyme produced by insulin was increased in the presence of IBMX or dibutyryl cyclic AMP. Glucagon pretreatment (5 min) of hepatocytes blocked the ability of insulin to activate the plasma-membrane enzyme. The activity state of these phosphodiesterases is discussed in relation to the observed changes in intracellular cyclic AMP concentrations. It is suggested that insulin exerts its action on the plasma-membrane phosphodiesterase through a mechanism involving a guanine nucleotide-regulatory protein.     

Dexamethasone restores hormonal inducibility of ornithine decarboxylase in primary cultures of rat hepatocytes

Induction of ornithine decarboxylase by various hormones was studied in quiescent primary cultures of adult rat hepatocytes maintained in a chemically defined medium. The following results were obtained: Enzyme activity rose transiently during the first day of cultivation in hormone-untreated cells. During this phase, insulin increased ornithine decarboxylase activity. Inducibility by insulin was maintained for more than 40 h only after pretreatment with 0.1 microM dexamethasone. Enzyme activity could be induced by 1 nM insulin and peaked after 7 h. Inducibility by glucagon and growth hormone required pretreatment with the glucocorticoid hormone. Ornithine decarboxylase activity was maximal 5 h after glucagon addition. Concentrations down to 0.1 nM were effective. Pretreatment with dexamethasone was most effective, when the hormone was present during the first 20 h of cultivation. The effect of the glucocorticoid during the pretreatment phase was diminished by colchicine and to a lesser extent by cytochalasine B. We suggest that part of the permissive effect of dexamethasone could be mediated by changes in the cytoskeleton and the function of hormone receptors. The fact that induction of ornithine decarboxylase was exerted by several hormones despite the absence of cell proliferation and DNA synthesis may indicate that polyamine biosynthesis has an important role in the quiescent hepatocyte.     

Regulation of ornithine decarboxylase activity by amino acids, cyclic AMP and luteinizing hormone in cultured mammalian cells

Any one of five amino acis (alanine, asparagine, glutamine, glycine, and serine) is an essential requirement for the induction of ornithine decarboxylase (EC 4.1.1.17) in cultured chinese hamster ovary (CHO) cells maintained with a salts/glucose, medium. Each of these amino acids induced a striking activation of ornithine decarboxylase in the presence of dibutyryl cyclic AMP and luteinizing hormone. The effect of the other amino acids was considerably less or negligible. The active amino acids at optimal concentrations (10 mM) induced only a 10-20 fold enhancement of enzyme activity alone, while in the presence of dibutyryl cyclic AMP, ornithine decarboxylase activity was increased 40-50 fold within 7-8 h. Of the hormones and drugs tested, luteinizing hormone resulted in the highest (300-500 fold) induction of ornithine decarboxylase with optimal concentrations of dibutyryl cyclic AMP and asparagnine. Omission of dibutyryl cyclic AMP reduced this maximal activation to one half while optimal levels of luteinizing hormone alone caused no enhancement of ornithine decarboxylase activity. The induction of ornithine decarboxylase elicited by dibutyryl cyclic AMP, amino acid and luteinizing hormone was diminished about 50% with inhibitors of RNA and protein synthesis. The specific amino acid requirements for ornithine decarboxylase induction in chinese hamster ovary cells was similar to the requirements for induction in two other transformed cell lines. Understanding the mechanism of enzyme induction requires an identification of the essential components of the regulatory system. The essential requirement for enzyme induction is one of five amino acids. The induction of ornithine decarboxylase by dibutyryl cyclic AMP and luteinizing hormone was additive in the presence of an active amino acid.     

Stimulation by glucocorticoids of protein degradation in hepatocyte monolayers.

1. Protein degradation in rat hepatocytes in stationary monolayer culture was measured as release of radioactive trichloroacetic acid-soluble material from intracellular proteins labelled with [3H]leucine. 2. Glucocorticoids, but not other steroids, stimulated protein breakdown in the hepatocyte monolayers. The effects observed were greater when the cells were preincubated with the hormones, indicating that the stimulation was not immediate. In addition, the stimulation by glucocorticoids persisted for up to 4 h after hormone removal. 3. Cycloheximide and the lysosomotropic agents leupeptin and ammonia effectively blocked glucocorticoid stimulation of protein degradation. 4. Insulin blocked dexamethasone stimulation when added at the same time as the steroid, but not when added 3 h later. 5. Stimulation of protein breakdown by dexamethasone was additive with that by glucagon or dibutyryl cyclic AMP, suggesting that its mechanism of action is different from that of the latter two agents. 6. Total activities of several lysosomal enzymes were unaffected under conditions where protein breakdown was stimulated by either glucagon or dexamethasone. 7. It is suggested that, whereas glucagon, dibutyryl cyclic AMP and insulin modulate protein breakdown in these cells via changes in autophagocytosis, the stimulation by glucocorticoids is exerted independently, perhaps by stimulating the synthesis of membrane proteins essential to the autophagic process.     

Age-related decrease in sensitivity to glucagon and dibutyryl cyclic AMP inhibition of fatty acid synthesis in hepatocytes isolated from obese female Zucker rats

Hepatocytes were isolated from 3 and 5 month old female genetically obese Zucker rats and their lean littermate controls. An age-dependent loss in sensitivity of fatty acid synthesis to inhibition by both glucagon and dibutyryl cyclic AMP was observed with hepatocytes from the obese rats. Hepatocytes from lean animals were much more sensitive to these agents, regardless of age. Low concentrations of glucagon and dibutyryl cyclic AMP actually produced some stimulation of fatty acid synthesis with hepatocytes prepared from the older obese rats. 5-Tetradecyloxy-2-furoic acid, a compound which inhibits fatty acid synthesis, was a very effective inhibitor of fatty acid synthesis by hepatocytes isolated from all rats used in the study. An inhibition of lactate plus pyruvate accumulation and a strong stimulation of glycogenolysis occurred in response to both glucagon and dibutyryl cyclic AMP with hepatocytes from both age groups of lean and obese rats. The results suggest that with aging of the obese female Zucker rat some step of hepatic fatty acid synthesis becomes progressively less sensitive to inhibition by glucagon and dibutyryl cyclic AMP. This may play an important role in maintenance of obesity in these animals.     

Inhibition of the glycogenolytic effects of alpha-adrenergic stimulation and glucagon by cobalt ions in perfused rat liver

Cobalt ions (2 mM) inhibited the glycogenolysis induced by phenylephrine and glucagon in perfused rat liver. Cobalt ions also inhibited 45Ca++ efflux from prelabelled livers induced by phenylephrine and glucagon. In addition, they inhibited the rise in tissue levels of cyclic AMP caused by glucagon, but did not inhibit the stimulation of 45Ca++ efflux or glycogenolysis by cyclic AMP or dibutyryl cyclic AMP. The specific binding of glucagon and alpha-agonist to hepatocytes was not inhibited by cobalt ions. These data suggest that cobalt ions, presumably through their high affinity for calcium binding sites on membranes inhibit the stimulation of glycogenolysis by phenylephrine and glucagon in distinct ways; one by inhibiting calcium mobilization and the other by inhibiting cyclic AMP production. Therefore, it is conceivable that membrane-bound calcium plays an important role in stimulating Ca++ mobilization by phenylephrine, and cyclic AMP production by glucagon.     

Hormonal control of liver regeneration

Two peaks in cyclic AMP production in rat livers 4 and 12h after partial hepatectomy (MacManus et al., 1972) were confirmed and a third peak established at 22h, which is the peak of DNA synthesis. The increases in cyclic AMP were prevented by beta-adrenergic blocking agents, propranolol and pindolol, without affecting ornithine decarboxylase induction or DNA synthesis. The alpha-blocking agents, phenoxybenzamine and phentolamine, given at the time of partial hepatectomy, delayed the rise in ornithine decarboxylase normally found 4h after operation, but did not affect DNA synthesis. If the alpha-blocking agents were given at 9-12h or 18h, the onset of DNA synthesis was delayed. Phenoxybenzamine did not affect the induction of ornithine decarboxylase in intact rat livers by glucagon or growth hormone, but did inhibit induction by dexamethasone. The induction of ornithine decarboxylase produced by dexamethasone was inhibited by 17alpha-hydroxy-progesterone; this compound also blocked the induction of ornithine decarboxylase in livers of partially hepatectomized rats.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

Cell-type differences in the serum and cyclic AMP-dependent regulation of ornithine decarboxylase activity in two cultured fibroblast types.

Serum re-feeding stimulated ornithine decarboxylase (ODC) activity 8 to 10-fold in FS fibroblasts and 5 to 8-fold in 3T3 fibroblasts. Addition of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine at the time of serum re-feeding further stimulated ODC activity in 3T3 fibroblasts but inhibited the serum stimulation of ODC activity in FS fibroblasts. It is suggested that serum and cyclic AMP independently regulate ODC activity in cultured fibroblasts.     

Regulation of coenzyme A biosynthesis by glucagon and glucocorticoid in adult rat liver parenchymal cells

We studied the effects of glucagon, dibutyryl cyclic AMP and dexamethasone on the rate of [(14)C]pantothenate conversion to CoA in adult rat liver parenchymal cells in primary culture. The presence of 30nm-glucagon increased the rate by about 1.5-fold relative to control cultures (range 1.4-2.3) and 2.4-fold relative to cultures containing 1-3m-i.u. of insulin/ml. The half-maximal effect was obtained at 3nm-glucagon. Dibutyryl cyclic AMP plus theophylline also enhanced the rate by about 1.5-fold. Dexamethasone acted synergistically with glucagon; glucagon at 0.3nm had no effect when added alone, but resulted in a 1.7-fold enhancement when added in the presence of dexamethasone (maximum effect at 50nm). The 1.4-fold enhancement caused by the addition of saturating glucagon concentrations was increased to a 3-fold overall enhancement by the addition of dexamethasone. However, dexamethasone added alone over the range 5nm to 5mum had no effect on the rate of [(14)C]pantothenate conversion to CoA. The stimulatory effect of dibutyryl cyclic AMP plus theophylline was also enhanced by the addition of dexamethasone. Changes in intracellular pantothenate concentration or radioactivity could not account for the stimulatory effects of glucagon, dibutyryl cyclic AMP or dexamethasone. Addition of 18mum-cycloheximide, an inhibitor of protein synthesis, decreased the rate of incorporation of [(14)C]pantothenate into CoA and the enhancement of this rate by glucagon and dibutyryl cyclic AMP plus theophylline in a reversible manner. These results demonstrate an influence of glucagon, dibutyryl cyclic AMP and glucocorticoids on the intracellular mechanism regulating total CoA concentrations in the liver.     

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.     

Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.     

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats: in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP) in the liver of adult rats increased 4-5 times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, in controlled by both glucagon and glucocorticoid.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP⁺) in the liver of adult rats increased 4–5-times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached the adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, is controlled by both glucagon and glucocorticoid.