Effect of hydrocortisone and dexamethasone on xylose uptake by isolated rat soleus muscle

To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10⁻⁴ M hydrocortusine, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10⁻² M, insulin-stimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inihibit basal or insulin-stimulated sugar uptake.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.     

Effect of protein synthesis inhibitors on xylose uptake and 125I-insulin binding by rat soleus muscle

Prolonged exposure (90–180 min) to cycloheximide (0.2 mg/ml), puromycin (0.2 mg/ml) or chloramphenicol (0.1 mg/ml) did not affect ¹²⁵I-insulin binding by rat soleus muscle. Chloramphenicol (2 mg/ml) depressed insulin binding and insulin-stimulated xylose uptake; these effects were attributed to the “toxic” effect of chloramphenicol on muscle ATP levels. Cycloheximide and puromycin inhibited insulin-stimulated xylose uptake without affecting ATP. Puromycin and chloramphenicol, but not cycloheximide, also inhibited basal sugar transport. This difference, and the rapid onset of all these inhibitory effects, suggest that they are not due to the inhibition of protein synthesis, but rather to some more direct effect on sugar transport itself.     

Direct interaction of phenylarsine oxide with hexose transporters in isolated rat adipocytes

It has previously been shown that phenylarsine oxide (PhAsO), an inhibitor of protein internalization, also inhibits stereospecific uptake of D-glucose and 2-deoxyglucose in both basal and insulin-stimulated rat adipocytes. This inhibition of hexose uptake was found to be dose-dependent. PhAsO rapidly inhibited sugar transport into insulin-stimulated adipocytes, but at low concentrations inhibition was transient. Low doses of PhAsO (1 microM) transiently inhibit stereospecific hexose uptake and near total (approx. 90%) recovery of transport activity occurs within 20 min. Interestingly, once recovered, the adipocytes can again undergo rapid inhibition and recovery of transport activity upon further treatment with PhAsO (1 microM). In addition, PhAsO is shown to inhibit cytochalasin B binding to plasma membranes from insulin-stimulated adipocytes in a concentration-dependent manner which parallels the dose-response inhibition of hexose transport by PhAsO. The data presented suggest a direct interaction between the D-glucose transporter and PhAsO, resulting in inhibition of transport. The results are consistent with the current recruitment hypothesis of insulin activation of sugar transport and indicate that a considerable reserve of intracellular glucose carriers exists within fat cells.     

Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum

Clostridium acetobutylicum exhibited diauxie growth in the presence of mixtures of glucose and xylose. Both glucose- and xylose-grown cells had a glucose uptake activity. On the other hand, growth on xylose was associated with the induction of a xylose permease activity, which was repressed by glucose in xylose-induced cells. The rate of sugar uptake with increasing sugar concentrations showed saturation kinetics with an apparent Km of 1.25 X 10(-5) M for glucose and 5 X 10(-3) M for xylose. Concomitant with the production of solvents, the activities of the glucose and xylose transport systems decreased. Among the main products of fermentation, butanol was shown to be a potent inhibitor of the growth of the organism and of the rate of sugar uptake as well as of sugar incorporation into cell materials. These inhibitory effects of butanol were more pronounced in xylose-grown cells than in glucose-grown cells. Butanol completely inhibited growth at a concentration of 14 g/liter for cultures growing on glucose and 8 g/liter for cultures growing on xylose. Concentrations of 7 and 10.5 g/liter of butanol caused a 50% inhibition of the xylose and glucose incorporations into cell materials. These inhibitory levels of butanol were found in typical glucose or xylose fermentation.     

Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum.

Clostridium acetobutylicum exhibited diauxie growth in the presence of mixtures of glucose and xylose. Both glucose- and xylose-grown cells had a glucose uptake activity. On the other hand, growth on xylose was associated with the induction of a xylose permease activity, which was repressed by glucose in xylose-induced cells. The rate of sugar uptake with increasing sugar concentrations showed saturation kinetics with an apparent Km of 1.25 X 10(-5) M for glucose and 5 X 10(-3) M for xylose. Concomitant with the production of solvents, the activities of the glucose and xylose transport systems decreased. Among the main products of fermentation, butanol was shown to be a potent inhibitor of the growth of the organism and of the rate of sugar uptake as well as of sugar incorporation into cell materials. These inhibitory effects of butanol were more pronounced in xylose-grown cells than in glucose-grown cells. Butanol completely inhibited growth at a concentration of 14 g/liter for cultures growing on glucose and 8 g/liter for cultures growing on xylose. Concentrations of 7 and 10.5 g/liter of butanol caused a 50% inhibition of the xylose and glucose incorporations into cell materials. These inhibitory levels of butanol were found in typical glucose or xylose fermentation.     

Glucocorticoid-induced heat resistance in mammalian cells

Chinese hamster ovary cells were incubated for 24 h in a variety of steroid hormones (testosterone, progesterone, hydrocortisone, dexamethasone, and ecdysterone) to test their effect on the subsequent heat resistance of the cells. Only the glucocorticoids, hydrocortisone and dexamethasone, consistently induced heat resistance. Heat resistance induced by hydrocortisone at 10(-6)M developed after a lag of 2-3 h and was maximal by 20 h. Resistance was expressed in both asynchronous and plateau phase cells and was maintained for several days in medium without added hormone. Incubation of cells with hydrocortisone and a 100-fold excess of progesterone (a glucocorticoid antagonist) partially inhibited the development of resistance. Prior exposure to hydrocortisone did not inhibit the subsequent development of heat induced thermotolerance. However, cells made thermotolerant by prior heat shock did not display further heat resistance with hydrocortisone treatment. There was no evidence for the induction of heat shock proteins (HSP) by these steroid hormones although the 28 kDHSP was further enhanced by combined heat and hydrocortisone. Our results indicate that heat resistance in mammalian cells may be induced by physiological concentrations of glucocorticoids and that the characteristics of this resistance are consistent with a receptor mediated event.     

Inhibitory effect of adrenaline and hydrocortisone on the growth of Allium cepa roots

The roots of Allium cepa were allowed to grow in distilled water containing 10(-4) M adrenaline hydrochloride or 2 X 10(-3) M hydrocortisone sodium succinate. Adrenaline inhibited the growth of roots; they decreased in length, number and total dry weight. The total amount of DNA in the roots was reduced much less than that of extracellular root components after adrenaline. Also hydrocortisone treatment resulted in a considerable decrease of the length and dry weight of Allium cepa roots. Both DNA and extracellular root components were influenced.     

Glucocorticoids inhibit eosinophil responses to granulocyte-macrophage colony-stimulating factor

The role of the eosinophil as an active proinflammatory cell in asthma and other allergic disorders has been well established. Glucocorticosteroids have long been used therapeutically as antiinflammatory agents in a variety of disease states where eosinophilia is a prominent feature. Although glucocorticoids are known to reduce tissue and circulating eosinophil numbers, the mechanisms by which they do so have not been clearly elucidated. Culture of eosinophils in vascular endothelial cell supernatants (VEC SUP) induces phenotypic and functional changes and prolongs the survival of the eosinophils. The survival-promoting activity in VEC SUP was shown to be granulocyte-macrophage CSF (GM-CSF) by neutralization with specific antibody. The potent glucocorticosteroid, dexamethasone (DEX), inhibited the prolongation of eosinophil survival caused by culture in either VEC SUP or human rGM-CSF. DEX (10(-6) M) exerted a direct survival-inhibitory effect on the eosinophil by the 4th day in culture in VEC SUP. This survival-inhibitory effect was dependent on the concentration of DEX (10(-10)-10(-6) M). Other glucocorticoids, including prednisolone (10(-7), 10(-6) M) and hydrocortisone (10(-7), 10(-6) M) also inhibited survival. The rank order of potency of the steroids indicates that this effect is mediated by a glucocorticoid receptor. This conclusion is supported by the failure of the sex steroids testosterone (10(-8)-10(-6) M) or beta-estradiol (10(-6) M) to inhibit eosinophil survival in the presence of VEC SUP. The effect of glucocorticoids on eosinophils is not a simple direct toxic effect because it was reversed by higher concentrations of GM-CSF. DEX shifted the GM-CSF dose-response curve for survival approximately fivefold to the right. GM-CSF induced a shift in eosinophil buoyant density which was partially blocked by DEX. These results suggest that glucocorticoids may inhibit elements of cytokine "priming" of eosinophils and that the eosinophilopenic effects of glucocorticoids may result in part from a direct effect on the eosinophil within a regulatory system involving cytokines.     

Effects of three proposed inhibitors of adipocyte glucose transport on the reconstituted transporter

Three compounds which inhibit glucose transport in rat adipocytes have been proposed to act directly on the glucose transporter protein. We tested these proposals by examining the effects of the compounds on the stereospecific glucose uptake catalyzed by adipocyte membrane proteins after reconstitution into liposomes. Effects on the transport activity reconstituted from human erythrocyte membranes were also examined. Glucose 6-phosphate, which was suggested to inhibit the transporter noncompetitively (Foley, J.E. and Huecksteadt, T.P. (1984) Biochim. Biophys. Acta 805, 313-316), had no effect on either type of reconstituted transporter, even when present at 5 mM on both sides of the liposomal membranes. Thus, it is unlikely to act directly on the transporter. The metalloendoproteinase substrate dipeptide Cbz-Gly-Phe-NH2, which inhibited insulin-stimulated but not basal glucose uptake in adipocytes (Aiello, L.P., Wessling-Resnick, M. and Pilch, P.F. (1986) Biochemistry 25, 3944-3950), inhibited the reconstituted erythrocyte transporter noncompetitively with a Ki of 1.5-2 mM. The inhibition of the erythrocyte transporter was identical in liposomes of soybean and egg lipid. Transport reconstituted using adipocyte membrane fractions was also inhibited by the dipeptide, with the activity from basal microsomes more sensitive than that from insulin-stimulated plasma membranes. These results indicate that the dipeptide interacts directly with the transporter, and may be a potentially useful probe for changes in transporter structure accompanying insulin action. Phenylarsine oxide, which was suggested to act directly on the adipocyte transporter (Douen, A.G., and Jones, M.N. (1988) Biochim. Biophys. Acta 968, 109-118), produced only slight (about 10%) inhibition of the reconstituted adipocyte and erythrocyte transporters, even when present at 100-200 microM and after 30 min of pretreatment. These results suggest that the major actions of phenylarsine oxide observed in adipocytes are not direct effects on the transporter, but rather effects on the pathways by which insulin regulates glucose transport activity (Frost, S.C. and Lane, M.D. (1985) J. Biol. Chem. 260, 2646-2652).     

Lipid Peroxidation-Induced Inhibition of γ-Aminobutyric Acid Uptake in Rat Brain Synaptosomes: Protection by Glucocorticoids

Incubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) resulted in an inhibition of gamma-aminobutyric acid (GABA) uptake. The inhibitory effects of X/XO were temperature- and time-dependent, and were characterized by an increased Km for GABA and a decreased Vmax. Inhibition of GABA uptake by X/XO was associated with both the formation of malonyldialdehyde (MDA) and conjugated dienes, indicating that lipid peroxidation was involved. Studies with catalase, superoxide dismutase (SOD), mannitol, and chelated iron suggested that hydroxyl radical (OH X) was probably responsible for the initiation of lipid peroxidation. Both the peroxidation of synaptosomal membranes and the inhibition of GABA uptake by X/XO were enhanced by the addition of ADP and FeCl2. The X/XO-induced inhibition of GABA uptake by synaptosomes could be prevented by preincubation of synaptosomes with certain glucocorticoids prior to X/XO exposure. Methylprednisolone sodium succinate (MPSS), dexamethasone sodium phosphate (DMSP), and prednisolone sodium succinate (PSS) all prevented the inhibition of GABA uptake by X/XO. MPSS was most effective at concentrations around 100 microM, DMSP was slightly more potent, and PSS was optimal at around 300 microM. On the other hand, hydrocortisone sodium succinate (HCSS) was ineffective at preventing X/XO-induced inhibition of GABA uptake at concentrations up to 3 mM. The steroids are presumed to work through a mechanism that blocked the formation of lipid peroxides, as MPSS inhibited the formation of conjugated dienes in synaptosomes exposed to X/XO at a concentration that also protected GABA uptake.     

Effect of insulin and adrenergic agonists on glucose transport of porcine adipocytes

1. Insulin increased basal 2-deoxyglucose uptake in isolated swine adipocytes by 75%. In the absence of insulin, isoproterenol did not inhibit basal 2-deoxyglucose transport. 2. Adenosine deaminase plus isoproterenol or theophylline alone reduced insulin effect by 10 and 40%, respectively. Isoproterenol alone or with 2-chloroadenosine did not inhibit insulin effect on glucose transport activity. 3. Insulin effect was inhibited by isoproterenol in the presence of theophylline but not in the presence of adenosine deaminase. 4. These results suggest that catecholamines do not counter-regulate basal and insulin-stimulated glucose transport in swine adipocytes.     

ACTH1-24 stimulates muscle cell glucose uptake

3H-2-deoxyglucose (2-DG) uptake was measured in L6A-1 rat skeletal muscle cells (a rapidly fusing subclone of L6), following addition of several concentrations (10(-16) to 10(-9)M) of the N-terminal fragment of ACTH1-24 to cells deprived of serum and insulin for 21 hours, but maintained in the presence of (5 micrograms/ml) insulin (stimulated state). There was a marked dose-dependent increase of 2-DG uptake at the various ACTH1-24 (P less than 0.001). There was no correlation between the time of exposure of the cells to serum-free conditions and the rate of uptake of 2-DG at the various ACTH1-24 concentrations both in the basal and insulin-stimulated states. Addition of catochalasin B (50 microM) to the cells, which inhibited both basal and insulin-stimulated uptake of 2-DG (by 70% and 91%, respectively) completely eliminated the enhancement of both of these uptake rates to 10(-12)M ACTH1-24. The results suggest that: 1) ACTH1-24 stimulates carrier-mediated uptake of glucose in skeletal muscle cells. 2) The site of action of ACTH1-24 is on the non-insulin mediated glucose uptake (NIMGU) system. 3) ACTH1-24 may be a useful probe to delineate some of the events associated with the NIMGU pathway.     

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and on 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661-668). Brief exposure (1 min) to N-ethylmaleimide (0.3-10 mM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin action was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockade on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20-30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effect of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 mM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.     

Early postnatal effects of prenatal exposure to glucocorticoids on testosterone metabolism and biogenic monoamines in discrete neuroendocrine regions of the rat brain

We investigated the effects of hydrocortisone acetate and dexamethasone administered to pregnant rats during the last gestational week on sexual differentiation of testosterone metabolism and biogenic monoamine contents and turnover in the discrete brain regions in 10-day-old offspring. In the preoptic area, sex-dependent differences in aromatase activity were attenuated by prenatal glucocorticoids. Prenatal dexamethasone but not hydrocortisone acetate caused the inversion of sexual dimorphism of 5alpha-reductase activity in the preoptic area. In the brain preoptic area of the male pups prenatally exposed to hydrocortisone acetate, a decrease in noradrenaline turnover was found. Dopamine turnover in the preoptic area and 5-hydroxytryptamine metabolism in the preoptic area and medial basal hypothalamus increased in females as a result of hydrocortisone acetate treatment. Our results indicate that excess glucocorticoids in prenatal life modifies the basic neurochemical and neurophysiological mechanisms of sexual brain differentiation and might contribute to behavioral and reproductive disorders in adulthood.     

Modulation of glucose uptake in adipose tissue by nitric oxide-generating compounds

There is increasing evidence that endogenous nitric oxide (NO) influences adipogenesis, lipolysis and insulin-stimulated glucose uptake. We investigated the effect of NO released from S-nitrosoglutathione (GSNO) and S-nitroso N-acetylpenicillamine (SNAP) on basal and insulin-stimulated glucose uptake in adipocytes of normoglycaemic and streptozotocin (STZ)-induced diabetic rats. GSNO and SNAP at 0.2, 0.5, and 1 mM brought about a concentration-dependent increase in basal and insulin-stimulated 2-deoxyglucose uptake in adipocytes of normoglycaemic and STZ-induced diabetic rats. SNAP at 1.0 mM significantly elevated basal 2-deoxyglucose uptake (115.8 ± 10.4%) compared with GSNO at the same concentration (116.1 ± 9.4%;P 0.05) in STZ-induced diabetic rats. Conversely, SNAP at concentrations of 10 mM and 20 mM significantly decreased basal 2-deoxyglucose uptake by 50.0 ± 4.5% and 61.5 ± 7.2% respectively in adipocytes of STZ-induced diabetic rats (P 0.05). GSNO at concentrations of 10 mM and 20 mM also significantly decreased basal 2-deoxyglucose uptake by 50.8 ± 6.4% and 55.2 ± 7.8% respectively in adipocytes of STZ-induced diabetic rats (P 0.05). These observations indicate that NO released from GSNO and SNAP at 1 mM or less stimulates basal and insulin-stimulated glucose uptake, and at concentrations of 10 mM and 20 mM inhibits basal glucose uptake. The additive effect of GSNO or SNAP, and insulin observed in this study could be due to different mechanisms and warrants further investigation.     

The absence of any effect of alpha-human atrial natriuretic peptide on sodium transport and osmotic water flow in the toad bladder

Synthesized alpha-human atrial natriuretic peptide (alpha-hANP), at 10(-6) M, failed to inhibit short-circuit current and basal and 10 mU/ml vasopressin-stimulated osmotic water flow in the bladder either pretreated with cyclooxygenase inhibitor, or preincubated with arachidonic acid, a precursor of PGE2. These results indicate alpha-hANP to have no direct effect on sodium transport and water permeability in the bladder, and no evidence was obtained indicating that alpha-hANP suppresses vasopressin-stimulated water flow by increasing PGE2 production.     

A comparison of hydrocortisone and triamcinolone inhibition of scale and feather development

Hydrocortisone (30-40 micrograms on day 10) and triamcinolone (10-20 ng on day 7-8) both inhibit or alter morphogenesis of scales and feathers. However, there are marked temporal and region-specific differences in the effects induced by these two glucocorticoids. Triamcinolone (TAC) is most teratogenic on day 7 or 8, inhibiting formation of spurs and feathers and inducing club feather formation. Hydrocortisone is most teratogenic later in development, on day 10. Unique hydrocortisone-induced responses are complete inhibition of scutellate scale formation, bent feathers, and apteria around the external auditory meatus. Altered synthesis of keratin polypeptides follows inhibition of scale morphogenesis by hydrocortisone and TAC. These in vivo data suggest that heterogeneity of glucocorticoid binding occurs in embryonic chick metatarsal skin. Survival data indicate that TAC is 2,000 times more embryotoxic than hydrocortisone.     

The inhibitory effect of hydrocortisone on interferon production by rat spleen cells

The effect of hydrocortisone on interferon r(IFN-r) production by rat spleen cells and its mechanism were studied. The results showed that hydrocortisone inhibited IFN-r production at concentrations as low as 5.52 x 10(-10) M, with complete suppression at 5.52 x 10(-8) M, and the total number and survival rate of the cultured spleen cells were not apparently affected by 5.52 x 10(-8) M hydrocortisone. The inhibitory effect was dose-dependent when the concentration was from 5.52 x 10(-10) M to 5.52 x 10(-8) M and could be blocked by RU38486, a competitive antagonist of glucocorticoid. Our results suggested that glucocorticoid may inhibit IFN-r production through a receptor-mediated mechanism.