The role of catecholamines and glucagon on serum and liver metallothionein response to restraint stress

The role of glucagon and catecholamines on serum and liver metallothionein (MT) concentrations in basal and stress conditions has been studied. Glucagon showed no effect on serum MT either in basal (unstressed) or stress (20 hours of restraint) conditions. In contrast, glucagon administration increased both unstressed and stressed levels of liver MT. No effect of the alpha + beta-catecholamine blocker labetalol on serum MT levels was observed in unstressed rats. However, the administration of labetalol abolished the increase in serum MT levels caused by stress. These data suggest that catecholamines might be involved in serum MT regulation during stress, while they might not be important in the maintenance of basal serum MT levels. Finally, no significant effect of adrenergic blockade was found on basal and stress levels MT, in agreement with previous data from this laboratory.     

Differential effect of adrenalectomy on rat liver metallothionein mRNA levels in basal and stress conditions.

Liver metallothionein (MT) mRNA and serum MT levels of adrenalectomized (ADX) and sham-ADX rats in basal and stress (1, 3 or 6 h of restraint) conditions have been measured. Serum MT levels were overall lower in ADX than in sham-ADX rats. Basal liver MT mRNA levels were increased in ADX rats, suggesting that glucocorticoids have an inhibitory role on the regulation of liver MT synthesis. In contrast, liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats, suggesting a stimulatory role for glucocorticoids. These results suggest that glucocorticoids have a different role in liver MT regulation depending on the physiological situation.     

Effect of stress,adrenalectomy and changes in glutathione metabolism on rat kidney metallothionein content: comparison with liver metallothionein

Eighteen hours of immobilization stress, accompanied by food and water deprivation, increased liver metallothionein (MT) but decreased kidney MT levels. Food and water deprivation alone had a significant effect only on liver MT levels. In contrast, stress and food and water deprivation increased both liver and kidney lipid peroxidation levels, indicating that the relationship between MT and lipid peroxidation levels (an index of free radical production) is unclear. Adrenalectomy increased both liver and kidney MT levels in basal conditions, whereas the administration of corticosterone in the drinking water completely reversed the effect of adrenalectomy, indicating an inhibitory role of glucocorticoids on MT regulation in both tissues. Changes in glutathione (GSH) metabolism produced significant effects on kidney MT levels. Thus, the administration of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased kidney GSH and increased kidney MT content, suggesting that increased cysteine pools because of decreased GSH synthesis might increase kidney MT levels through an undetermined mechanism as it appears to be the case in the liver. However, attempts to increase kidney MT levels by the administration of cysteine or GSH were unsuccesful, in contrast to what is known for the liver. The present results suggest that there are similarities but also substantial differences between liver and kidney MT regulation in these experimental conditions.     

Glucocorticoid independent mediation of interleukin-1 induced changes in serum zinc and liver metallothionein levels

Interleukin-1 (IL-1) causes changes in zinc metabolism which have been attributed to mediation, at least in part, by glucocorticoids. However, IL-1 was found to actually lower serum corticosterone levels in rats. In addition, adrenalectomy only partially inhibited the ability of IL-1 to depress serum zinc levels and increase the amount of zinc associated with hepatic metallothionein. Furthermore, IL-1 increased total liver metallothionein protein to similar levels in both adrenalectomized and normal rats. Administering the synthetic glucocorticoid dexamethasone with IL-1 to adrenalectomized rats produced additive, but not synergistic effects on serum zinc and metallothionein concentrations. Studies with actinomycin D suggested that IL-1 induction of metallothionein might involve glucagon.     

Sex and restraint stress differences in rat metallothionein and Zn levels

Sex differences in serum and liver metallothionein (MT) levels were studied in adult male and female rats. Whereas it was found that female rats had higher hepatic MT levels than male rats in basal, unstressed conditions, no significant differences were found in serum MT levels. Restraint stress increased both serum and liver MT in both sexes. The increase in serum MT was greater in male than in female rats, whereas no significant differences between sexes were found in liver MT content after restraint stress. It is suggested that MT regulation might be sex-dependent and that MT might play some extrahepatic function during stress.     

Effect of gamma irradiation on liver metallothionein synthesis and lipid peroxidation in rats.

Several studies have recently shown that metallothionein (MT), a protein characterized by a high thiol content and that binds Zn2+ and Cu+, might be involved in the protection against oxidative stress and can act as a free radical scavenger. Oxidative stresses, such as irradiation, increase lipid peroxidation (LP) and subsequent tissue damage through free radical production. The induction of hepatic MT synthesis by gamma-irradiation (20 Gy) at 8, 24, 30 and 48 hrs. post-irradiation in two different age groups of Sprague-Dawley rats (39-40 and 48-49 days old) was studied. LP measured by the thiobarbituric acid reactive substances (TBARS) assay and Cu and Zn levels in liver have also been determined. In the younger group, the gamma-irradiation induced hepatic MT synthesis and increased LP that peaked 24 hrs. after irradiation. During the first 30 hrs. post-irradiation, a positive and statistically significant correlation between hepatic MT content and LP level in liver was found. In the older group, liver MT synthesis was only increased 1.7-fold and LP levels were not altered at 24 hrs. post-irradiation compared with sham-irradiated rats.Therefore it appears that LP is not necessary for induction of MT synthesis by gamma-irradiation.     

Liver, Brain, and Heart Metallothionein Induction by Stress

To date, stress has been reported to induce metallothionein (MT) synthesis in the liver only. In the present experiment, the effects of food and water deprivation alone or of immobilization stress plus food and water deprivation on liver, brain, and heart MT have been studied in adult male rats. Liver and brain MT levels were increased by immobilization stress as soon as 6 h after the onset of stress. Eighteen hours of immobilization, which is accompanied by food and water deprivation, further increased liver and brain MT levels and significantly increased heart MT content. A specific effect of immobilization was evident in all three tissues, because the effect of food and water deprivation alone was significantly lower than that of immobilization plus starvation. Changes in MT apparently were not related to changes in cytosolic Zn.     

Metallothionein-I induction by stress in specific brain areas

The distribution of metallothionein-I (MT) in several areas of the brain and its induction by immobilization stress has been studied in the rat. MT content was highest in hippocampus and midbrain and lowest in frontal cortex and pons plus medulla oblongata. Immobilization stress for 18 hours (which was accompanied by food and water deprivation) significantly increased MT levels in the frontal cortex, pons plus medulla oblongata and hypothalamus, but not in midbrain and hippocampus. The effect of stress on MT levels was specific as food and water deprivation along had no significant effect on MT levels in any of the brain areas studied. The effect of stress on MT levels was independent of changes in cytosolic Zn content; this was generally unaffected by stress or food and water deprivation but decreased in pons plus medulla oblongata from stressed rats. The results suggest that MT is induced more significantly in the brain areas that are usually involved in the response of animals to stress.     

Dissociation between adrenocorticotropin and corticosterone responses to restraint after previous chronic exposure to stress

The effect on emotional reactivity produced by a model for chronic stress in which different types of acute stresses were randomly combined for 29 days was studied in adult male rats. Chronically stressed rats showed a slight decrease in body weight gain and an increase in relative adrenal weight. Chronic stress did not modify defecation rate but reduced exploratory activity in the holeboard. Neither basal nor acute-stress induced levels of adrenocorticotropin (ACTH) were modified by previous chronic stress. Likewise, basal corticosterone levels were similar in both groups. However, corticosterone response to acute restraint stress was higher in chronically stressed than in control rats. The mechanisms underlying the dissociation between ACTH and corticosterone as well as its possible implications are discussed.     

The effect of chronic food and water restriction on open-field behaviour and serum corticosterone levels in rats

In operant conditioning experiments, two methods are commonly used to motivate laboratory rats to perform designated tasks. The first is restricting food so that rats are forced to lose 20% of body weight within one week, followed by maintenance at 80% of the baseline weight for the remainder of the experiment. The second is restricting access to water to 15 min in each 24 h period. These methods are effective in motivating the animals. There is, however, little information available on the effects on performance in tests of behaviour that are not related to operant conditioning. In addition, it is not clear if these commonly used methods of food and water restriction will lead to physiological stress as indicated by an elevation of serum corticosterone. Male rats were either food-restricted to reduce and maintain their weight at 80% of baseline weight, or were restricted to 15 min access to water every 24 h. Activity in the open field was significantly greater in food-restricted rats than in water-restricted or control rats, but freezing behaviour was similar in all experimental groups. Food-restricted rats had a higher mean serum corticosterone level than water-restricted and control rats 37 days after the start of the experimental period. These data suggested that chronically restricting food and maintenance of body weight at 80% of baseline body weight led to significant behavioural changes and physiological stress. In contrast, water restriction did not lead to changes in behaviour or corticosterone levels. A second experiment was conducted to compare the effects of food restriction to 80% of baseline body weight, as described above, with a less stringent protocol in which test rats were initially reduced to 80% of baseline weight, but were then maintained at 80% of an ad libitum fed control rat's weight. Serum corticosterone levels and adrenal gland weights were measured after the initial week of forced weight loss and after maintenance for 21 days. Forced loss of 20% of body weight in the first week led to significantly increased serum corticosterone levels and adrenal gland weights compared to ad libitum fed controls. Serum corticosterone levels and adrenal gland weights in rats maintained at 80% of their initial body weight for 21 days remained higher than ad libitum fed control rats. However, rats maintained at 80% of an ad libitum fed control rat's weight did not differ from control rats in serum corticosterone levels or adrenal gland weights at the end of the 21-day study period. Adjustment of the feeding regimen in this manner eliminated physiological evidence of chronic stress.     

Oleoyl-estrone increases adrenal corticosteroid synthesis gene expression in overweight male rats

Oleoyl-estrone (OE) induces a marked loss of body fat in rats by maintaining energy expenditure, body protein and blood glucose despite decreasing food intake. OE increases glucocorticoids, but they arrest OE lipid-mobilization. We studied here whether OE induces a direct effect on adrenal glands function as part of this feedback regulation. Dietary overweight male rats were given oral 10 nmol/g OE gavages for ten days. A group (PF) of pair-fed to OE rats, and controls received vehicle-only gavages. OE rats lost slightly more body than PF, but had larger adrenal glands. Tissue corticosterone levels, and gene expressions for glucocorticoid-synthesizing enzymes were increased in OE versus controls and PF; thus, we assumed that adrenal growth affected essentially its cortex since OE also lowered the expression of the medullar catecholamine synthesis enzyme genes. Serum corticosterone was higher in PF than in OE and controls, but liver expression of corticosteroid-disposing steroid 5α-reductase was 3× larger in OE than PF and controls. Circulating glucocorticoids changed little under OE, in spite of higher adrenal gland and liver content, hinting at modulation of glucocorticoid turnover as instrumental in their purported increased activity. In conclusion, we have observed that OE considerable enhanced the expression of the genes controlling the synthesis of glucocorticoids from cholesterol in the rat and increasing the adrenal glands’ corticosterone, size and cellularity, but also the liver disposal of corticosteroids, suggesting that OE increases corticosterone synthesis and degradation (i.e. serum turnover), a process not driven by limited energy availability but directly related to the administration of OE.     

Capsaicin effects on stress pathology and gastric acid secretion in rats

The effects of capsaicin on stress ulcer formation, plasma corticosterone levels and gastric acid secretion were examined in rats. Capsaicin desensitization did not affect restraint stress ulcer formation but was associated with markedly elevated corticosterone levels, even in non-stressed rats. Acute, orally administered capsaicin augmented ulcer formation only if its administration was followed immediately by restraint stress. Delays of 1, 2, or 3 h between drug administration and stress produced ulceration comparable to control values. Capsaicin did not affect basal (non-stimulated) gastric acid secretion but substantially decreased pentagastrin-stimulated acid output.     

Glucocorticoid regulation of metallothionein during murine development

During the second half of gestation in the mouse there is a rise in both fetal (4-fold) and maternal (10-fold) metallothionein-I (MT-I) mRNA in the liver (but not in the kidney). There is a large increase in plasma corticosterone (the predominant murine glucocorticoid hormone), as well as an increase in hepatic zinc, which is coincident with the induction of MT-I mRNA. Considering that both of these compounds are known to be effective inducers of MT-I mRNA, we set out to determine whether either one or both were involved in the developmental regulation of MT-I genes. Several lines of evidence suggest that corticosterone is the principal inducer of fetal MT-I mRNA: The induction of MT-I mRNA in the liver, but not in the kidney, mimics glucocorticoid regulation but not metal regulation. Reduction of maternal corticosterone levels by treating mice with metyrapone lowered MT-I mRNA levels but had no effect on zinc levels. A line of transgenic mice carrying a metallothionein-growth hormone fusion gene that is responsive to metals but unresponsive to glucocorticoids was not developmentally regulated. Based on these observations, we propose that corticosterone is responsible for the induction of MT-I mRNA and that the resulting MT sequesters zinc and copper which may be used later in development.     

Illuminating the interrelated immune and endocrine adaptations after multiple exposures to short immobilization stress by in vivo blocking of IL-6

Intermittent psychological stress was induced in adult rats by 2 h/day of immobilization stress for 4 days, with or without blocking the function of IL-6 by using an anti-IL-6 antibody. Basal concentrations of serum corticosterone, IL-1beta, IL-6, and TNF-alpha were assessed 24 h after the last intervention, as were levels of glucocorticoid receptors (GR) and activities of glucocorticoid-inducible enzymes (tyrosine aminotransferase and glutamine synthetase) in muscle and liver. Whole blood cultures were used to assess both spontaneous and LPS-induced reactivity of peripheral blood mononuclear cells. Stress increased corticosterone concentration in a manner partially modulated by IL-6. Serum IL-1beta concentration was downregulated during stress when IL-6 was blocked (P < 0.01). LPS-induced IL-6 secretion by peripheral blood mononuclear cells in vitro correlated positively with serum IL-1beta concentration in antibody-treated groups, independently of stress (R = 0.70 in nonstressed and R = 0.78 in stressed rats; both P < 0.05), whereas serum corticosterone concentration correlated positively with LPS-induced secretion of IL-6 only in control rats (R = 0.66; P < 0.05). Reductions in liver GR levels indicated independent effects of stress (34.5%) and anti-IL-6 antibody (16.7%) and additive effects for both (62.5%). Similar results are reported for vastus muscle. Conversely, stress increased tyrosine aminotransferase and glutamine synthetase activities in muscle and liver with a significant (P < 0.05) effect of anti-IL-6 antibody only seen in stressed livers. In conclusion, IL-6 plays a role in maintaining circulating IL-1beta concentration after multiple exposures to stress, thus promoting a continued elevation of corticosterone release; in peripheral tissues, IL-6 antagonizes the effects of glucocorticoids, especially at the level of GR concentration.     

Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats.

We have examined the role of glucocorticoids in the stress-induced inhibition of testicular steroidogenesis. Immobilization (3 hr) reduced plasma testosterone (T) levels to 24% of control values but did not affect plasma LH levels. This reduction was partially reversed by in vivo injections of the antiglucocorticoid, RU486, prior to the stress session at a dose of 10 mg/kg BW, but not at 1.0 or 50 mg/kg BW. Stressed rats that were treated with 10 mg/kg BW RU486 had twofold higher plasma T levels than vehicle-treated stressed animals. Injections of RU486 did not affect plasma LH levels in control or stressed rats and did not affect T levels of unstressed rats. Stressed rats had eightfold higher plasma corticosterone levels than controls, and RU486 had no effect on control or stress levels of corticosterone. The possible role of glucocorticoids in mediating the effect of stress on testicular T production was investigated also in vitro by incubating testicular interstitial cells from unstressed rats for 3 hr with corticosterone (0, 0.01, 0.1, or 1.0 microM) or dexamethasone (0, 0.001, 0.01, or 0.1 microM), followed by an additional 2 hr with hCG (0, 25, 50, or 100 microIU). Both corticosterone and dexamethasone inhibited hCG-stimulated T production in a dose-dependent manner. Cells incubated with the highest concentration of either of the glucocorticoids showed significantly reduced responses to hCG stimulation. In the absence of hCG, in vitro T production was not affected by dexamethasone or 0.01 and 0.1 microM corticosterone. However, the highest dose of corticosterone (1.0 microM) produced a 63% elevation in basal T production. Coincubation of testicular interstitial cells with corticosterone (1.0 microM) or dexamethasone (0.1 microM) and RU486 (0.01, 0.1, and 1.0 microM) reversed the glucocorticoid-induced suppressions of T production in a dose-dependent manner. Our results suggest that during stress increases in plasma levels of glucocorticoids in male rats act via glucocorticoid receptors on testicular interstitial cells to suppress the testicular response to gonadotropins, and that the decline of testosterone production during immobilization stress is in part mediated by a direct action of glucocorticoids on the testis.     

Influence of thyroparathyroidectomy and thyroxine replacement on CU and ZN cellular distribution and on the metallothionein level and induction in rats

Thyroid hormones are involved in copper and zinc distribution in rat tissues. We examined the influence of thyroparathyroidectomy (TPTY) and of a replacement therapy by T4 on Cu and Zn organ distribution. MT levels were also measured both in basal conditions and after induction by cadmium. The results confirm that a lack of T4 modified Cu and Zn in serum and tissues. In serum, TPTY increased Cu (+15%) and ceruloplasmin (+18%), and decreased Zn (−18%). In tissues, Cu was altered in liver (+13%), kidney (−24%), heart (−16%) duodenum (−18%), and Zn in liver (+25%) and kidney (−10%). The soluble fractions (100,000 g supernatant) were mainly affected in liver and kidney, and the subcellular fractions in heart and duodenum. MT levels were modified in basal conditions only in liver (+57%) and kidney (−36%). T4 administration partially prevented the effect of TPTY on both elements and MT concentrations. Therefore, no evidence is provided for a direct role of T4 in the metabolism of MT in a way comparable to the effects of glucocorticoids. However, MT could mediate the consequences of TPTY on metal distribution in certain organs, such as liver and kidney.     

Hepatic cyclic AMP generation and ornithine decarboxylase induction by glucagon and beta adrenergic agonists

The relationship of hepatic ornithine decarboxylase (ODC) activity to cyclic AMP levels and nutritional status was studied in the pre-weanling rat. Previous studies demonstrated that 2 hr without food causes a loss of hepatic ODC induction after glucagon or catecholamine injection. Isoproterenol or glucagon administration produced increased hepatic cyclic AMP and tyrosine aminotransferase activity which were not prevented by nutritional deprivation. Blockade of hepatic beta 2 receptors by the selective antagonist ICI 118,551 prevented increased cAMP levels and ODC activity after isoproterenol administration. Blockade of beta 1 receptors by atenolol did not prevent increased cAMP levels or ODC induction by isoproterenol although it did block activation of cardiac ODC. The phosphodiesterase inhibitor RO20-1724 increased hepatic cAMP levels as well as ODC and TAT activities, although the increase in ODC activity was attenuated by nutritional deprivation. RO20-1724 also potentiated the induction of hepatic ODC after glucagon or isoproterenol administration. Administration of 8-bromo cAMP elevated hepatic ODC activity regardless of nutritional status but also elevated serum levels of growth hormone and corticosterone. Hepatic ODC induction by glucagon or beta 2 agonists can be dissociated from changes in cAMP levels during nutritional deprivation.     

Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats

Previous studies have indicated that neonatal handling influences development of hypothalamic-pituitary-adrenal (HPA) control of corticosterone. In addition, corticosterone influences memory consolidation processes in contextual fear conditioning. Therefore, neonatal handling may affect hippocampal-dependent memory processes present in contextual fear conditioning by influencing the development of HPA control of corticosterone. To investigate the effects of neonatal handling on early learning, rat pups were either handled (15-min removal from home cage) on the first 15 days after birth or left undisturbed in their home cage. Handled rats and nonhandled rats were fear conditioned at 18, 21, or 30 days of age and then tested at two time points--24 h following conditioning and at postnatal day 45. Subsequently, at approximately postnatal day 60, rats were exposed to restraint stress and corticosterone levels were assessed during restraint and recovery. Handled and nonhandled rats did not differ significantly in their freezing response immediately following footshock on the conditioning day. However, when tested for contextual fear conditioning at 24 h following conditioning and at postnatal day 45, handled rats showed more freezing behavior than nonhandled rats. When exposed to restraint stress, handled rats had a more rapid return of corticosterone to basal levels than nonhandled rats. These results indicate that neonatal handling enhances developmentally early memory processes involved in contextual fear conditioning and confirms previously reported effects of neonatal handling on HPA control of corticosterone.     

Role of glucocorticoids and catecholamines on hepatic thiobarbituric acid reactants in basal and stress conditions in the rat.

Thiobarbituric acid-reactants (TBARs) are considered to be an index of lipid peroxidation. In the present experiments, the effect of stress and hormones on hepatic TBARs levels was studied in Sprague-Dawley rats. In unstressed conditions adrenalectomized rats showed higher TBARs levels than sham-adrenalectomized rats. The effect of adrenalectomy was reverted by the administration of corticosterone but not by that of aldosterone, indicating that glucocorticoids exert a negative role on the regulation of liver TBARs. The effect of these hormones appears to be a permissive one, since the administration of a long lasting ACTH preparation did not reduce liver TBARs. In contrast to that observed in unstressed rats, glucocorticoids appeared to increase liver TBARs in stressed rats. Nevertheless, other alternative explanations are possible. Finally, no evidence for a role of catecholamines in the regulation of hepatic TBARs was found.