Identification of two forms of glutamine synthetase in barley (Hordeum vulgare).

Hepatocytes of 14-day-old rats have no detectable glucokinase activity $\text{in}$$\text{vivo}$, but it was induced by insulin (10^?8M) in primary cultures of these hepatocytes. The glucokinase induced by insulin was separated by electrophoresis on a cellulose acetate membrane and identified by its low affinity for glucose. This precocious induction of glucokinase was completely prevented by the presence of either actinomycin D or cycloheximide. Glucagon also inhibited its induction by insulin. Dexamethasone and testosterone, which alone had no inductive effect, strongly enhanced the induction by insulin. When hepatocytes of 14-day-old rats were cultured with 10^?7M insulin, 10^?6M dexamethasone and 10^?7M testosterone for 48 hr, their glucokinase activity increased to the non-induced level in hepatocytes of adult rats. Estrogen, thyroxine or growth hormone did not induce glucokinase precociously. Testosterone did not enhance induction of glucokinase by insulin in cultured hepatocytes of adult rats.     

Factors that prevent the premature appearance of glucokinase in neonatal rat liver.

1. The physiological factors that prevent the precocious appearance of glucokinase activity in the 13-day-old rat that can be induced by oral glucose administration were explored. 2. Evidence is presented that the galactose component of milk sugar is inhibitory. In the absence of this inhibitory galactose, the amount of glucose necessary to effect appreciable induction is greater than that present in milk. 3. The induction is prevented both by administration of mannoheptulose, which inhibits insulin release, and by excess insulin; the amount of insulin available therefore seems to be critical. 4. The inhibition of induction by galactose does not appear to be via competition with glucose but by enhancing insulin release and thereby making this excessive. The relative amounts of glucose and insulin appear to be important in regulating glucokinase induction. 5. The precocious induction of glucokinase by glucose is inhibited by simultaneous treatment with approriate amounts of adrenaline, glucagon, dibutyryl cyclic AMP or isoprenaline but not by vasopressin or angiotensin II. 6. No single cause of glucokinase induction in neonatal rat liver can be recognized. The process is subject to regulation by many factors at a time subsequent to when competence to synthesize the enzyme has been established.     

Induction of glucokinase by insulin under the permissive action of dexamethasone in primary rat hepatocyte cultures.

Glucokinase, the organ specific key enzyme of glucose metabolism in liver, was studied in primary cultures of adult rat hepatocytes during the first two days after cell preparation. In the presence of dexamethasone low concentrations of insulin (10^?9 mol/l) prevented the observed time dependent decrease of glucokinase activity while higher insulin concentrations (10^?8 and 10^?7 mol/l) led to a twofold increase of enzyme activity. The enhancement of glucokinase activity was completely blocked by either actinomycin D or cycloheximide. The degree of this insulin dependent induction was correlated with the concentration of added dexamethasone, which seemed to perform a permissive function. The induction of glucokinase activity could be prevented by addition of glucagon (2 × 10^?7 mol/l).     

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.     

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.     

Control of phenylalanine hydroxylase synthesis in tissue culture by serum and insulin.

Stationary-phase, minimal deviation hepatoma H4-II-E-C3 cell cultures that are serum-deprived respond with a biphasic time course of phenylalanine hydroxylase induction when dialyzed fetal calf serum or insulin is added. These two agents induce phenylalanine hydroxylase additively, during both the initial 3-hour and the delayed 24-hour phases. The initial phase of induction by insulin is inhibited by cycloheximide but not by actinomycin D. The delayed induction by both dialyzed fetal calf serum and insulin is inhibited by 10(-6) M cycloheximide and 0.20 mug/ml actinomycin D. H4-II-E-C3 cells in culture do not synthesize the factor(s) in serum that induce phenylalanine hydroxylase.     

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.     

Regulation of the gene expression of glucokinase and L-type pyruvate kinase in primary cultures of rat hepatocytes by hormones and carbohydrates

The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3 h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic regulation of steroidogenesis in isolated adrenal cells of rat. Relationship of adrenocorticotropin-, adenosine 3':5'-monophosphate-and guanosine 3':5'-monophosphate-stimulated steroidogenesis with the activation of protein kinase.

The data presented with the isolated adrenal cells, in the present study, show that adrenocorticotropin in the physiological concentration range stimulates the synthesis of guanosine 3':5'-monophosphate(cyclic GMP), protein kinase activity, and steroidogenesis in a concentration-dependent manner without detectable rise in the levels of adenosine 3':5'-monophosphate (cyclic AMP). Millimolar concentrations of cyclic AMP and cyclic GMP, which stimulate corticosterone synthesis, also activate kinase activity and steroidogenesis in a sigmoid concentration-response manner. The process of phosphorylation activated by corticotropin, cyclic AMP and cyclic GMP is not inhibited by cycloheximide or actinomyin D. It is therefore proposed that the hormonal responses mediated by cyclic GMP and cyclic AMP are via the protein kinase enzymatic steps, and the inhibitory effect of cycloheximide and actinomycin D in corticotropin-stimulated steroidogenesis follows this step. In conjuction with our previous observations that the biosynthetic steps from (20S)-20-hydroxycholesterol to corticosterone are neither inhibited by cycloheximide nor affected by cyclic GMP, it is inferred that the rate-limiting step of adrenal steroidogenesis is the transformation of cholesterol to (20S)-20hydroxycholesterol and this very step is regulated by cyclic GMP and cyclic AMP. Of further significance are the findings that micromolar cincentrations of cyclic AMP and cyclic GMP, which do not stimulate steroidogenesis, effectively stimulate protein kinase activity in a concentration-dependent manner. It is therefore concluded that all cyclic-nucleotide-dependent protein kinase activities of the cell are not necessarily related to steroidogenesis.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Insulin and tri-iodothyronine induce glucokinase mRNA in primary cultures of neonatal rat hepatocytes.

Glucokinase (EC 2.7.1.2) first appears in the liver of the rat 2 weeks after birth and increases after weaning on to a high-carbohydrate diet. We investigated the hormonal regulation of glucokinase (GK) mRNA in primary cultures of hepatocytes from 10-12-day-old suckling rats. GK mRNA was undetectable in such cells after 48 h of culture in serum-free medium devoid of hormones. Addition of insulin or tri-iodothyronine (T3) to the medium resulted in induction of GK mRNA. The effects of insulin and T3 were dose-dependent and additive. Dexamethasone alone did not induce GK mRNA, but enhanced the response to insulin and decreased the response to T3. Induction of GK mRNA by insulin was not affected when the medium glucose concentration was varied between 5 and 15 mM, nor when culture was conducted in glucose-free medium supplemented with lactate and pyruvate or galactose. The time course of initial accumulation of GK mRNA in response to insulin was characterized by a lag of 12 h and an induction plateau reached after 36 h. If hepatocytes were then withdrawn from insulin for 24 h and subsequently subjected to a secondary stimulation by insulin, GK mRNA re-accumulated with much faster kinetics and reached the fully induced level within 8 h. Both primary and secondary responses to insulin were abolished by actinomycin D. These results provide insight into the role of hormonal stimuli in the ontogenic development of hepatic glucokinase.     

Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver.

1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably less than the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.     

Epidermal growth factor mimics insulin effects in rat hepatocytes.

Epidermal growth factor (EGF) mimicked the effect of insulin to activate glycogen synthase and stimulate glycogen synthesis in isolated rat hepatocytes. Both agents required glucose (greater than 5 mM) and had similar time courses of action. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were 9 nM and 4 nM respectively. Combinations of the two agents produced additive responses. EGF also resembled insulin in its ability to inhibit the effects of 0.1-1.0 nM-glucagon on cyclic AMP and glycogen phosphorylase in hepatocytes. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were approx. 5 nM and 0.5 nM respectively. EGF and insulin inhibited phosphorylase activation by exogenous cyclic AMP, and inhibited cyclic AMP accumulation induced by forskolin. They also inhibited phosphorylase activation provoked by phenylephrine, but not by vasopressin. EGF added alone rapidly activated phosphorylase and increased cytosolic [Ca2+], but the effects were no longer apparent at 5 min and were smaller than those of vasopressin. Insulin did not induce these changes. In hepatocytes previously incubated with myo-[3H]inositol, EGF did not significantly increase myo-inositol 1,4,5-trisphosphate. However, its ability to increase cytosolic [Ca2+] was blocked by neomycin, an inhibitor of phosphatidylinositol bisphosphate hydrolysis. It is concluded that some, but not all, of the effects of EGF in liver are strikingly similar to those exerted by insulin, suggesting that these agents may have some similar mechanisms of action in this tissue.     

Insulin regulates the expression of the enhancer of split- and hairy-related protein-2 gene via different pathways in 3T3-L1 adipocytes and L6 myotubes.

We investigated the effect of insulin on the expression of the enhancer of split- and hairy-related protein-2 gene in 3T3-L1 adipocytes and L6 myotubes. The level of enhancer of split- and hairy-related protein-2 mRNA was increased by insulin in both cells. While both wortmannin and LY294002 blocked the increase in 3T3-L1 adipocytes, and only PD98059 was effective in L6 myotubes. Although the increase by insulin in these cells was inhibited by treatment with actinomycin D, this was enhanced by treatment with cycloheximide. Furthermore, cyclic AMP increased the level of enhancer of split- and hairy-related protein-2 mRNA in both cells in an additive manner. Thus, we conclude that insulin and cyclic AMP induce the expression of the enhancer of split- and hairy-related protein-2 gene in both 3T3-L1 adipocytes and L6 myotubes, and that the gene expression enhanced by insulin is regulated by the cell type-specific pathway. The former requires a phosphoinositide 3-kinase pathway and the latter a mitogen-activated protein kinase pathway.