Effects of cortisol on lipid and lipoprotein synthesis in rat hepatocytes]

Under in vivo conditions cortisol induces moderate hyperlipidemia followed by an increase in the phospholipid and triglyceride concentrations in the blood and a decrease of cholesterol; similar changes were observed in the liver. At all time intervals studied cortisol inhibits the phospholipid and cholesterol syntheses and decreases the specific radioactivities of the lipids in the mitochondrial fraction. The hormone has an inhibiting effect on the fatty acid synthesis at early postinjection stages. The phospholipid synthesis is increased after adrenalectomy and is then inhibited after injection of the hormone. A single injection of ACTH or cortisol causes suppression of phospholipid and cholesterol syntheses and a decrease in their specific radioactivities in the mitochondria. A similar effect is observed under stress conditions. In addition, the hormone inhibits the synthesis of lipoprotein apoproteins of very low and high densities. After 5 hours following the hormone injection the lipoprotein apoprotein synthesis in the liver is activated; the activation of apoprotein synthesis is also observed after adrenalectomy. However, the injection of the hormone to adrenalectomized rats decreases the apoprotein synthesis. It was shown that in blood serum cortisol affects the conversions of very low density lipoproteins into low density lipoproteins, thus providing for hyperlipidemia.     

Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase.

Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.     

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.     

Cortisol, adrenocorticotropic hormone and apolipoprotein synthesis in rat hepatocytes]

The influence of cortisol (5 mg/kg body wt administered daily for 5 and 10 days) on biosynthesis of apoproteins of lipoproteins of very low density in the liver and on the synthesis of apolipoproteins of very low, low, and high density (VLDL, LDL, and HDL apoproteins, respectively) in the blood serum of adrenalectomized animals, and after replacement cortisol therapy was studied. Cortisol treatment during these periods resulted in the VLDL apoproteins biosynthesis inhibition in the rat liver. The synthesis of apolipoproteins was increased by adrenalectomy; this effect was eliminated after replacement cortisol treatment. The apoprotein synthesis was stimulated within 5 hours by single injection of cortisol or ACTH. Study of the blood serum apolipoproteins specific radioactivity indicated metabolic change of lipoproteins, such as disturbed conversion from VLDL to LDL. Single and prolonged cortisol administration led to the opposite results. The authors believe that the metabolic disturbances of lipoproteins in the blood play a more important role in the pathogenesis of cortisol-induced hyperlipidemia than lipoprotein syntesis stimulation in the liver.     

The effect of cortisol on the synthesis of rat plasma albumin, fibrinogen and transferrin

A decrease of absolute synthesis of albumin, no change in that of fibrinogen and an increased fractional synthesis of transferrin were observed 3h after intraperitoneal administration of a pharmacological dose of 5 mg of cortisol to 220g rats in the post-absorptive state and previously kept on a diet with 40% protein. The concentration in liver of total free amino acids was practically unchanged at this time. Intraperitoneal administration of a mixture of amino acids with the cortisol raised this concentration and was accompanied by an almost complete de-repression of the synthesis of albumin, with no real effect on that of fibrinogen. In considerable contrast, in rats studied at 24h after intraperitoneal administration of cortisol, and who had been fed once in the interim (but who had received no amino acids intraperitoneally), there was a marked increase in the absolute synthesis of albumin and fibrinogen, with an increase in fractional synthesis that was less proportionately but still very significant and which included transferrin. The amino acid concentrations had risen above the supplemented values at 3h but not as much proportionately as the fractional synthesis rates, and of course not as much as the absolute synthesis rates, of albumin and fibrinogen. These time-dependent effects of cortisol suggest to us that our studies resolve the apparently conflicting results of the effect of cortisol on the synthesis of albumin reported by others.     

The acute phase response of Atlantic cod (Gadus morhua): humoral and cellular response

Intra-muscular injection of turpentine oil was used to induce acute phase response (APR) in Atlantic cod (Gadus morhua L.). The effects on the serum cortisol, total protein, IgM and pentraxin concentration were examined as well as the effects on natural antibody, anti-trypsin and leukocyte respiratory burst activity. The turpentine injection resulted in a 26 fold increase in the cortisol level after 72 h. Slightly reduced serum protein level in both groups was attributed to the restricted feeding during the experimental period. The IgM serum concentration was significantly reduced after 168 h in the turpentine treated fish while the natural antibody activity was not affected. The anti-trypsin activity was initially suppressed but recovered to normal levels at the end of the experiment. The turpentine injection had little effect on the serum level of the pentraxins, CRP-PI and CRP-PII. The respiratory burst activity was significantly suppressed after 72 h. It is concluded that 1) cod shows a relatively slow humoral and cellular response to APR induction, 2) the increase in serum cortisol level may be the key modulator of the mainly suppressive effects on the immune parameters and 3) pentraxins are not typical acute phase proteins in cod.     

The influence of hydrocortisone on the synthesis and turnover of microvillous membrane glycoproteins in suckling rat intestine.

Synthesis and degradation of intestinal mucosal and microvillous membrane glycoproteins were studied in control suckling rats, and suckling rats given cortisol acetate by intraperitoneal injection for 3 days. Cortisol acetate had no effect on total uptake of radioactive glucosamine by the protein free compartment of rat intestine. Early incorporation of [1(-14)C]glucosamine by intestinal glycoproteins was enhanced by cortisol, but stimulation was the same in membrane and homogenate fractions. Polyacrylamide gel electrophoresis of membrane proteins solubilized with 2% sodium dodecyl sulphate demonstrated a cortisol dependent change, characterized by loss of faster travelling glycoproteins, and a corresponding shift in maximum labelling at 3 h from these glycoproteins to more slowly migrating glycoproteins. Degradation was studied qualitatively with a double isotope technique. Glycoprotein degradative rates appeared to be stimulated by cortisol, but similarly in membrane and total homogenate fractions. On polyacrylamide gels, the areas occupied by glycoproteins with the highest apparent degradative rates, corresponded closely with the areas of most active labelling at 3 h. The rate of degradation in the most actively labelled zone appeared to be higher after cortisol than in the controls. The results indicate that cortisol does not alter membrane composition by inhibiting degradation of selected glycoproteins, and are consistent with a model in which cortisol stimulates the synthesis of specific membrane glycoproteins in suckling rats, while inhibiting synthesis of other glycoproteins.     

Regulation of Cerebroside and Sulfatide Metabolism in Glia Cells

Mouse oligodendroglioma cells, G-26 clone 20 and 24, contain galactosylceramide (cerebroside) and sulfogalactosylceramide (sulfatide) as determined by an HPLC technique. The synthesis of both these lipids was stimulated by 10(-6) M hydrocortisone (cortisol) and also by the removal of serum from the culture medium. Forty-eight hours after the addition of cortisol the incorporation of H235SO4 into sulfatide, the level of sulfatide and the specific activity of the enzyme 3'-phosphoadenosine 5'-phosphosulfate:galactosylceramide sulfotransferase in the cells increased three- to fourfold. The level of cerebroside and the specific activity of UDP-galactose:hydroxyacyl sphingosine galactosyltransferase also increased threefold in the cells on treatment with cortisol. The effect of the hormone on the synthesis of cerebroside preceded the increase in sulfatide synthesis. Experiments with cycloheximide and actinomycin D showed that the effect of the hormone on glycolipid synthesis in these cells were mediated through de novo messenger RNA and protein synthesis. Removal of serum from the culture medium resulted in an approximately twofold enhancement of H235SO4 incorporation into sulfatide within 24 h. The levels of sulfatide and cerebroside and the specific activity of the galactosyltransferase and sulfotransferase also increased significantly after serum removal. However, in contrast to the effect of the steroid, the sulfotransferase activity and the level of sulfatide increased prior to elevations in galactosyltransferase and cerebroside. The effect of serum removal was also found to be mediated by de novo RNA and protein synthesis. The effects of cortisol and serum removal on the synthesis of cerebroside and sulfatide were strictly additive.     

Isolation of microvillus plasma membranes from suckling-rat intestine. The influence of premature induction of digestive enzymes by injection of cortisol acetate

1. Cortisone administration to suckling rats leads prematurely to induction of enzymes of the intestinal microvillus plasma membrane and lengthening of the intestinal microvilli. To investigate the membrane changes that might be involved, a method for the isolation of a fraction enriched with microvillus plasma membrane was developed in suckling rats. Plasma-membrane fractions were compared from 13-day-old control rats and from 13-day-old rats given cortisol acetate by subcutaneous injection for 3 days. 2. After cortisol injection, the activity of maltase, trehalase, sucrase and leucyl beta-naphthylamidase increased markedly, and to the same extent, in intestinal homogenates and plasma-membrane preparations. Purification, and recovery of five marker enzymes with respect to homogenate activity, and recovery of protein, were similar for both membrane preparations, particularly after correction for non-membrane activity, which was high in suckling rats and affected by cortisol. 3. In material released from the plasma membrane by digestion with papain, maltase protein was increased after cortisol injection at least as much as maltase activity. Sucrase activity increased at least 200-fold, and this increase was associated with the appearance of a new sucrase band on polyacrylamide-gel electrophoresis. 4. Sodium dodecyl sulphate electrophoresis of plasma-membrane proteins revealed at least four additional macromolecules after cortisol injection. Concurrently several proteins disappeared from the plasma membrane. The added proteins appeared in the main to be removed from the plasma membrane by papain, whereas the deleted proteins were in the papain-resistant fraction. 5. Enzymic stimulation induced by cortisol acetate in the suckling-rat plasma membrane therefore appears to involve the addition of new proteins, rather than activation of proteins in situ. Deletion of proteins from the membrane during induction of hydrolytic enzymes may reflect other phenomena such as protein reorganization associated with the change in microvillus shape.     

Corticotropin regulation of the synthesis of a specific rat adrenal cytosolic protein. Effects of hypophysectomy and actinomycin D

The mechanism of corticotropin stimulation of the synthesis of a specific rat adrenal cytosolic protein was investigated. This protein (protein E) has a mol.wt. of approx. 30000. It is detected by polyacrylamide-gel electrophoresis of cytosol prepared from adrenal slices from rats treated with corticotropin in vivo and control rats, the slices being incubated with [(3)H]- and [(14)C]-leucine respectively. In rats 1-15 days after hypophysectomy, corticotropin, like dibutyryl cyclic AMP, induces an increase in protein E similar to that induced in control rats, even though both compounds no longer stimulate total protein synthesis. Corticotropin stimulation of protein E synthesis is mediated by cyclic AMP but not by corticosterone, since aminoglutethimide, a steroidogenic inhibitor, does not affect corticotropin stimulation, and dexamethasone alone has no effect. Actinomycin D, when injected in vivo 1h before or after corticotropin injection, prevents the effect of corticotropin on protein E synthesis, which is interpreted as evidence that mRNA synthesis is necessary for the stimulation of protein E synthesis. When injected more than 2h after corticotropin, actinomycin D does not prevent corticotropin stimulation of protein E synthesis, but completely blocks corticotropin stimulation of total protein synthesis. This is interpreted as meaning that, after stimulation of mRNA coding for protein E, corticotropin has no effect on the synthesis of protein E. On the other hand, corticotropin stimulation of protein E synthesis persists after hypophysectomy even though it no longer stimulates total protein synthesis. These data suggest that the factor(s) involved in the synthesis of protein E are more stable than those involved in total protein synthesis.     

The effect of high dose of cortisol on glucose-6-phosphatase and fructose-1,6-bisphosphatase activity,and glucose and fructose-2,6-bisphosphate concentration in carp tissues (Cyprinus carpio L.)

The effect of a high dose of cortisol (200 mg kg(-1) body mass) on juvenile carp was investigated. The activity of glucose-6-phosphatase in liver and of fructose-1,6-bisphosphatase in liver, kidney and muscle, the serum glucose and fructose-2,6-bisphosphate concentration as well as the serum concentration of the injected hormone were measured after 24, 72 and 216 h after intraperitoneal cortisol injection. The activities of fructose-1,6-bisphosphatase in liver and kidney and glucose-6-phosphatase in liver were elevated in comparison with the control, while the fructose-1,6-bisphosphatase activity in the muscle tissue was unchanged. After cortisol injection, the serum glucose level was nearly two times higher after 24 and 72 h and was still 50% higher after 216 h compared with controls. In contrast, the liver fructose-2,6-bisphosphate concentration was unchanged after 24 h. More than two times higher fructose-2,6-bisphosphate concentration was observed in liver after 72 h and it was still elevated after 216 h after the cortisol injection.     

Cortisol 21-mesylate exerts glucocorticoid action on the induction of milk protein synthesis in cultured mammary gland

Cortisol 21-mesylate, an alkylating derivatives of cortisol, was previously shown to exert an anti-glucocorticoid action in rat hepatoma cell culture (Simons, Thompson and Johnson 1980). In this study the effect of cortisol 21-mesylate on milk protein synthesis induced in cultured mouse mammary gland by glucocorticoid, insulin, and prolactin was investigated. Addition of cortisol 21-mesylate at concentrations ranging from 10(-8) M to 10(-6) M produced no inhibition of casein synthesis that was induced by glucocorticoid, insulin and prolactin in mammary explants from midpregnant mice. On the other hand, cortisol 21-mesylate in combination with insulin and prolactin stimulated casein synthesis in cultured tissue. The potency of cortisol mesylate was about 1/10 to 1/30th of that of cortisol. Cortisol 21-mesylate, like cortisol, also augmented the accumulation of alpha-lactalbumin in midpregnant rat mammary tissue cultured in the presence of insulin and prolactin. A cell-free competition study of glucocorticoid receptors using cytoplasmic extracts from mouse mammary tissue showed that cortisol 21-mesylate competitively inhibited the binding of dexamethasone on glucocorticoid receptors. The apparent affinity of cortisol 21-mesylate for glucocorticoid receptors is about 1/10th of that of cortisol. These results indicate that cortisol 21-mesylate acts as a glucocorticoid but not as an antiglucocorticoid in the mammary gland.     

The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase.

The microsomal activities of the hepatic enzymes hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase exhibit a diurnal rhythm with maximum activities observed during the dark period and minimum activities around noon (12:00h). This diurnal rhythm was maintained for both enzymes after adrenalectomy, but the amplitude of variation for the activity of both enzymes was greatly decreased. A single injection of cortisol administered to adrenalectomized rats 3h before the expected maximum in enzyme activity resulted in a twofold increase in the activity of both enzymes 3h later, at values similar to those observed for control rats killed at the same time. This response appeared to require protein synthesis, since it was blocked by actinomycin D. However, the administration of cortisol to adrenalectomized rats 3 h before the expected minimum did not result in significant change in the activity of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase 3 h later. Kinetic studies of cholic acid metabolism in vivo demonstrated that adrenalectomy results in a significant decrease in the rate of synthesis of cholic acid and a considerable decrease in the pool size of cholic acid and its metabolic products. Treatment of adrenalectomized rats with cortisol increased the rate oonsistent with the effects of adrenalectomy and cortisol treatment on the activity of cholesterol 7alpha-hydroxylase.     

Pathway-specific response to cortisol in the metabolism of catfish

Cortisol produced biochemical pathway-specific effects on metabolic enzymes and other macromolecules in the freshwater catfish, Clarias batrachus. Injection of cortisol increased 1.6-fold activity of citrate synthase (CS) in brain, liver and skeletal muscle of the fish over vehicle-injected control, while administration of metyrapone (a cortisol synthesis inhibitor) reduced CS activity by 52%. Cortisol treatment of metyrapone-treated fish induced CS activity by approximately 2.5-fold, which was blocked after administration of actinomycin D or cycloheximide. This shows de novo synthesis of CS to enhance aerobic capacity of fish. In contrast the activities of glucose-6-phosphate dehydrogenase (G6-PDH) and lactate dehydrogenase (LDH) increased in response to metyrapone and decreased after administration of cortisol in all the three tissues. The cortisol-mediated decrease in G6-PDH and LDH activities reflects reduction in biosynthetic and anaerobic capacity of fish. Administration of metyrapone significantly increased RNA/DNA ratio and protein but cortisol decreased these macromolecular contents in brain, liver and skeletal muscle. It shows cortisol-induced decrease in protein synthesis capacity of fish. The present study suggests that cortisol-induces catabolic and aerobic but inhibits anabolic and anaerobic processes in freshwater catfish. The cortisol-dependent metabolic responses may also be associated with the permissive effect of cortisol on other hormone(s) in fish.     

Cloning of the glucocorticoid receptor (GR) in gilthead seabream (Sparus aurata). Differential expression of GR and immune genes in gilthead seabream after an immune challenge

In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.     

Dynamics of cortisol and the development of the glucocorticoid function in the early ontogenesis of Atlantic salmon Salmo salar

The dynamics of cortisol were studied during the embryonic and early postembryonic development of Atlantic salmon Salmo salar (in brood obtained from the same pair of spawners, but with differing hatching dates). The initial level of maternal cortisol in the eggs after fertilization comprised of 25.6 ± 2.2 ng/g, and decreased continuously in the development process to 1.1 ± 0.2 ng/g by the 95th day after fertilization (t1) or the 18th day after hatching (t2). The first increase in the concentration of endogenous cortisol to 6.1 ± 1.2 ng/g was recorded at t2 = 24 days. Then the level of cortisol increased constantly and reached 32.8 ng/g by t1 = 149 (t2 = 72). A comparison of the hormone dynamics in two offspring groups from the same pare of spawners, differing in the longevity of embryogenesis, demonstrated that the level of cortisol increased after hatching more intensely in fishes with shorter embryogenesis. An injection of DL-aminoglutethimide, a compound preventing the synthesis of cortisol, significantly decreased the concentration of cortisol for the first time at the day of t1 = 101 (t2 = 24), i.e. at the first moment of the increase of endogenous cortisol. The effect from the adrenaline injection as a stress hormone was observed only at the day of t1 = 149 (t2 = 72), i.e., during the transition to the fry stages of development. Problems concerning when steroidogenesis begins, of the formation of the hypothalamic-hypophyseal-interrenal axis as a single hormone-competent ensemble, and of the development the stressed response in salmon juveniles are discussed.     

Increased ACTH and cortisol secretion after interleukin-alpha injection in the common marmoset (Callithrix jacchus jacchus)

We have studied the effect of intravenous injection of interleukin-1 (dose range: from 0.25 to 4.5 microg/kg of body weight) on plasma ACTH and cortisol levels in the marmoset, a primate paradygm of peripheral glucocorticoid resistance. Blood sampling were collected and body temperature recorded 0, 15, 30, 60, 120, 180, 240 and 300 min after injection. Interleukin-1 stimulated secretion of ACTH in a dose-dependent fashion. Maximal secretion occurred 120 min after injection, and lasted up to 240 min. Plasma ACTH levels returned to baseline 300 min after interleukin-1 injection. Plasma cortisol levels were related to ACTH levels. Body temperature elevation, which occurred 10-15 min after injection was dose-dependent, and lasted 3 h. Results suggest that the pyrogenic effect of interleukin is associated, in the marmoset, with integrated activation of the hypothalamic-pituitary-adrenal axis. In light of the proneness of marmosets to hyperimmune disorders, our data are consistent with the hypothesized central biological role of IL-1, as well as the pathophysiological relevance of the neuro-endocrine-immune cross-talk during the acute phase response.     

Influence of the adrenal glucocorticoids on the stimulation of synthesis of hepatic ribonucleic acid and plasma acute-phase globulins by leucocytic endogenous mediator.

An injection of unpurified leucocytice endogenous mediator into rats results in an increased incorporation of [6(-14)C]orotate into hepatic RNA, an increase in the concentration of RNA associated with the bound ribosomal fraction of liver, and increases in the concentrations in serum of acute-phase proteins such as alpha2-macrofoetoprotein and haptoglobin. If given 3 days after adrenalectomy or 7 days after hypophysectomy,, leucocyte factor did not induce the increase in RNA synthesis or alpha2-macrofoetoprotein concentrations but did stimulate an increase in serum haptoglobin. When hypophysectomized or adrenalectomized rats received daily subcutaneous injections of 0.5mg of cortisol, leucocyte factor again induced a significant increase in the synthesis of hepatic RNA and an increase in the concentration of serum alpha2-macrofoetoprotein. These observations suggest that leucocyte factor can regulate acute-phase-protein synthesis at several different sites, one or more of which requires permissive action of the glucocorticoid hormones. Futher, leucocyte factor will stimulate an increase rate of incorporation of orotate into hepatic ribosomes when added in vitro in the presence of cortisol to a liver-perfusion system. Thus the stimulatory effect of leucocyte factor may be directy on liver but may require the presence of other hormones to stimulate the incorporation of orotate into RNA.     

Glucocorticoid action on rat thymic lymphocytes. Experiments utilizing adenosine to support cellular metabolism lead to a reassessment of catabolic hormone actions.

Inhibition of glucose uptake has been proposed as a primary cause of many of the subsequent inhibitory effects of glucocorticoids. This hypothesis has been tested in experiments where adenosine is substituted for glucose. Like glucose, adenosine maximally supports glycolytic and oxidative ATP generation, and by its use the hormonal inhibition of glucose uptake is circumvented. With adenosine, inhibition by cortisol is seen at at least one other metabolic site, respiratory ATP synthesis. This action can be observed by hormone-induced increases in levels of lactate, pyruvate, and AMP that accompany a lowering of ATP. Evidence for this metabolic action is also seen when cells are provided with a limiting amount of glucose; despite inhibition of glucose uptake, a cortisol-induced increase in lactate accompanies the reduction in levels of ATP. Decreased respiratory ATP synthesis is also suggested by a hormonal reduction in the metabolism of labeled exogenous pyruvate to 14CO2. Several experimental approaches suggest that inhibition of oxidative ATP production, rather than of glucose uptake, is the event most responsible for glucocorticoid-induced changes in the balance of adenine nucleotides, which in turn contribute to effects on protein synthesis and uridine uptake. First, the characteristic inhibitory cortisol effects on adenine nucleotides and protein synthesis are undiminished when adenosine is substituted for glucose. Second, in adenosine-supported cells the onset of the hormone-induced increase in levels of lactate corresponds closely to the appearance of measurable reductions in ATP. In contrast, when cells are supported by glucose, the hormonal inhibition of glucose uptake is maximal by 30 to 35 min, nearly an hour before effects on levels of ATP are detectable. Third, when cells are made strongly dependent upon glucose for ATP production by deprivation of exogenous substrate and cortisol is added at 90 min, a characteristic inhibition of the uptake of glucose added 40 min later is seen; nevertheless, this is insufficient to prevent added glucose from immediately and fully restoring ATP, rates of protein synthesis, and uridine uptake. Inhibitory effects on ATP, protein synthesis, and uridine do appear after an additional hour or so, a time commensurate with the development of an inhibition of oxidative metabolism. Fourth, limiting added glucose can reduce uptake more than cortisol, without reducing levels of ATP.