A radioimmunoassay for human plasma corticosterone.

A radioimmunoassay for human plasma corticosterone has been developed. Antiserum against corticosterone was produced in rabbits immunized with corticosterone-21-hemisuccinate conjugated to bovine serum albumin. The antiserum cross-reacted with progesterone, DOC and dehydrocorticosterone more than 20%. After the extraction with ether, and the separation by Sephadex LH-20 microcolumn chromatography, recovery was 51.2 +/- 12.1% in 50 assays. The mean coefficient of variation between assays was 7.7% and within assays was 8.6%. Human plasma corticosterone is measured readily by assaying aliquots of an ether extract of 0.05 to 0.1 ml of plasma after microcolumn chromatography. The mean plasma corticosterone concentration at 9 a.m. was 7.1 +/- 3.2 ng/ml in 45 normal subjects. Plasma corticosterone increased 5.2 times as much as basal values after ACTH injection, whereas radioimmunoassayed cortisol increased 2.4 times. On the other hand, plasma corticosterone decreased to 22.6% of basal values at four hours after 1 mg dexamethasone, whereas radioimmunoassayed cortisol decreased to 12.3% of basal values.     

Interleukin 1: a regulatory role in glucocorticoid-regulated hepatic metabolism

The purpose of this study was to investigate the effect of interleukin 1 (IL 1) on glucocorticoid-regulated hepatic metabolism. Steroid binding in liver cytosol, plasma glucose, plasma corticosterone, and phosphoenolpyruvate carboxykinase (PEPCK) activity were assayed in C3H/HeJ mice after IL 1 administration. Mice received 5 pyrogenic U (PU) of rabbit IL 1 i.p. and were sacrificed 4 hr later. In adrenal-intact mice, steroid binding and plasma glucose were significantly decreased (63 and 64% of control) and plasma corticosterone was significantly elevated threefold. In adrenalectomized mice, IL 1 (5 PU) treatment produced similar results in steroid binding (66% of control) and plasma glucose (71% of control). PEPCK was measured in intact mice fasted overnight and treated with 5 PU of IL 1. PEPCK was induced in fasted control animals (23.1 +/- 1.4 U/mg) vs fed control animals (15.9 +/- 0.7 U/mg). IL 1 treatment inhibited the induction of PEPCK in fasted animals (13.4 +/- 2.0 U/mg) and caused a significant decrease in steroid binding (78% of fasted control) and plasma glucose (82% of fasted control). No difference in plasma corticosterone was seen in IL 1-treated mice and fasted control mice. These data indicate that IL 1 decreases intracellular steroid receptors, resulting in decreased induction of PEPCK and subsequent reduced gluconeogenesis and plasma glucose. We propose that IL 1 plays a regulatory role in glucocorticoid-regulated hepatic metabolism.     

Radioimmunoassay of human plasma corticosterone: method, measurement of episodic secretion and adrenal suppression and stimulation

A radioimmunoassay for human plasma corticosterone has been developed. Antisera were obtained by immunizing rabbits with corticosterone-21-hemisuccinate-BSA. An antiserum titer of 1:4000 was used for standard curves ranging from 0–1000 pg. Interfering steroids were removed from plasma extracts by paper chromatography. Plasma blanks obtained from adrenalectomized or Addisonian patients ranged from 29 to 42 ng/dl. Recovery of radioactive corticosterone through the entire method was 67.6 ± 5.2%. The coefficient of variation within assays was 19% and between assays 13%. The average 8 a.m. value in males was 396 ± 228 ng/dl and in females it was 655 ± 271 ng/dl. Corticosterone was found to be secreted episodically, in parallel with cortisol. Secretion of this steroid was suppressed by dexamethasone and stimulated by ACTH infusion.     

Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

Adrenal function and the effect of a high-fat diet on C57BL/6J and C57BL/6J-ob/ob mice.

In C57BL/6J mice and the ob/+ and ob/ob mutants total plasma corticosterone levels were found to be statistically different. In C57BL/6J mice the level was 1.9 +/- 0.2 mug/100 ml plasma, in ob/+ mice 8.6 +/- 1.6 mug/100 ml and in ob/ob mice 13.7 +/- 1.5 mug/100 ml. The percentage of protein-bound corticosterone as well as the free endogenous corticosterone levels were also different. Feeding a high-fat diet to young C57BL/6J and C57BL/6J-ob/ob mice for a period of 4 weeks had no effect upon blood glucose, plasma insulin and plasma corticosterone levels. The significantly higher increase in body weight of the high-fat diet groups of both lines of mice was mainly due to fat cell hypertrophy.     

Attenuated corticosterone response to chronic ACTH stimulation in hepatic lipase-deficient mice: evidence for a role for hepatic lipase in adrenal physiology

Hepatic lipase (HL), a liver-expressed lipolytic enzyme, hydrolyzes triglycerides and phospholipids in lipoproteins and promotes cholesterol delivery through receptor-mediated whole particle and selective cholesterol uptake. HL activity also occurs in the adrenal glands, which utilize lipoprotein cholesterol to synthesize glucocorticoids in response to pituitary ACTH. It is likely that the role of adrenal HL is to facilitate delivery of exogenous cholesterol for glucocorticoid synthesis. On this basis, we hypothesized that HL deficiency would blunt the glucocorticoid response to ACTH. Furthermore, because exogenous cholesterol also is derived from the LDL receptor (LDLR) pathway, we hypothesized that LDLR deficiency would blunt the response to ACTH. To test these hypotheses, we compared the corticosterone response to eight daily ACTH injections in HL-deficient (hl-/-), LDLR-deficient (Ldlr-/-), and HL- and LDLR-doubly deficient (Ldlr-/- hl-/-) mice with that in wild-type (WT) mice. Plasma corticosterone levels were measured on days 2, 5, and 8. Differences in plasma corticosterone levels between genotypes were analyzed by Kruskal-Wallis one-way ANOVA on ranks and pairwise multiple comparisons by Dunn's test. Our results demonstrate a trend toward reductions in plasma corticosterone levels on day 2 and significant reductions on day 5 and day 8 in the knockout models. Thus, on day 5, plasma corticosterone levels were reduced by 57, 70, and 73% (all P < 0.05) and on day 8 by 76, 59, and 63% (all P < 0.05) in hl-/-, Ldlr-/-, and Ldlr-/- hl-/- mice, respectively. These results demonstrate that HL deficiency, like LDLR deficiency, blunts the adrenal response to chronic ACTH stimulation and suggest a novel role for HL in adrenal physiology.     

The effect of teratogens on maternal corticosterone levels and cleft incidence in A/J mice.

It is unknown whether orofacial clefting, one consequence of teratogenic exposure, results from a direct interaction between the teratogen and the embryonic palate, or indirectly from maternal alterations caused by the teratogen. In the current study pregnant A/J mice were exposed to one of three cleft-inducing agents in order to examine the relationship between drug-induced clefting and the response of maternal plasma corticosterone to drug administration. The agents used, haloperidol (HAL), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or phenytoin (PHT), were administered in teratogenic doses between 0800 and 0930 on gestational day 10 (GD 10). For corticosterone determinations, mice were dosed on GD 10, and blood was collected at 1, 4, 24, or 48 hr after dosing. For fetal evaluation of cleft lip and/or cleft palate, mice were dosed on GD 10 and killed on GD 18. Phenytoin was the most potent inducer of cleft lip and palate and induced a sustained elevation of plasma corticosterone in maternal animals. The other treatments, in order of decreasing potency to induce clefting and/or cause an elevation of corticosterone in plasma were 2,4,5-T > HAL > controls. Correlations between maternal corticosterone levels and clefting incidence were very high at all time points examined; total exposure (area under the curve) was also highly correlated. A linear relationship between drug-induced increases in maternal corticosterone levels and the incidence of clefting in A/J mice was evident. Based on these findings, we believe that increased maternal corticosterone levels may play a role in orofacial clefting in A/J mice.     

Increased plasma corticosterone levels in bovine growth hormone (bGH) transgenic mice: Effects of ACTH,GH and IGF-I onin vitro adrenal corticosterone production

Previous work from our laboratory provided evidence for increased plasma corticosterone levels in mice transgenic for human and bovine growth hormone (GH). Corticosterone was elevated in both sexes, under both basal and ether-induced stress conditions. The objectives of the present study were to investigate thein vitro adrenal sensitivity to ACTH, GH and/or IGF-I in normal and bGH transgenic mice, to examine plasma corticosterone levels at different times of the day, and to determine plasma levels of ACTH in these animals. For the measurement of plasma corticosterone and ACTH levels, transgenic and normal siblings were housed 2 per cage and decapitated simultaneously within 20 seconds of the first disturbance of the cage. The corticosterone production byin vitro adrenal incubations did not differ between adrenals from normal and transgenic mice at the basal level or in the presence of different doses of ACTH. Growth hormone or IGF-I did not have any effect on corticosterone productionin vitro when given alone, and did not modify the effects of ACTH on the accumulation of corticosterone in the media. Plasma corticosterone concentrations were higher in transgenic than in normal animals in both morning and evening. Plasma concentrations of ACTH in animals killed in the morning were sharply increased in transgenic males as compared with their normal siblings. The results indicate that increased circulating levels of corticosterone in transgenic mice are not due to a potentiation of ACTH actions by GH or IGF-I, but rather to a chronic increase in plasma ACTH levels. The increase in ACTH is presumably a reflection of GH actions in the hypothalamic-pituitary system.     

Corticosterone does not change open elevated plus maze-induced antinociception in mice

It has been demonstrated that the exposure of rodents to the standard elevated plus-maze (sEPM: 2 open and 2 enclosed arms) elicits defensive behavioral reactions and antinociception and also activates the hypothalamo-pituitary-adrenal (HPA) axis. We have recently reported that EPM-induced antinociception is particularly observed when rats and mice are exposed to a totally open EPM (oEPM: 4 open arms). Given that the oEPM seems to be a more aversive situation than the sEPM, we hypothesized that oEPM exposure would induce higher plasma levels of corticosterone than sEPM exposure in mice. In this study, we investigated the influence of exposure to eEPM (enclosed EPM: 4 enclosed arms), sEPM or oEPM on plasma corticosterone levels in mice, with or without prior nociceptive stimulation (2.5% formalin injection into the right hind paw). We also tested whether the nociceptive response in the formalin test and oEPM-induced antinociception are altered by adrenalectomy. Results showed that oEPM-exposed mice spent less time licking the injected paw than sEPM- and eEPM-exposed animals. All three types of EPM exposure increased plasma corticosterone when compared to the basal group, but sEPM- and oEPM-exposed mice showed higher corticosterone levels than eEPM-exposed mice. Prior nociceptive stimulation (formalin injection) did not enhance the plasma corticosterone response induced by the three types of EPM exposure. Indeed, formalin injection appeared to provoke a ceiling effect on plasma corticosterone concentration. Furthermore, neither the nociceptive response in the formalin test nor oEPM-induced antinociception was changed by adrenalectomy. Present results suggest that oEPM antinociception does not depend on corticosterone release in mice.     

Role of maternal plasma corticosterone elevation in the teratogenicity of secalonic acid D in mice

Secalonic acid D (SAD) is a teratogenic mycotoxin that causes cleft palate in the offspring of treated pregnant mice. To investigate the role of maternal corticosterone in the teratogenicity of SAD, pregnant CD-1 mice were treated with 30 mg/kg of SAD i.p. on day 11 of pregnancy in either 5% (w/v) NaHCO3 or 20% (v/v) dimethyl sulfoxide (DMSO) in NaHCO3. Radioimmunoassay (RIA) was performed to determine plasma corticosterone at 24, 48, 72, and 96 hr after dosing. No interference by EDTA, SAD, DMSO, or pentobarbital was noticed on the RIA. Significant (P less than .01) elevations in plasma corticosterone concentrations were seen 24 and 48 hr following dosing of SAD in NaHCO3 with concentrations reaching a peak just prior to the onset of shelf elevation and fusion. Simultaneous treatment with DMSO, an agent known to antagonize the teratogenic effect of SAD, completely abolished the SAD-induced corticosterone elevation at the 24 hr time point and significantly (P less than .01) reduced it at the 48 hr time point. To evaluate the specificity of the role of corticosterone in the teratogenicity of SAD, plasma samples from mature males similarly treated with either single or multiple doses of SAD ranging from 15 to 45 mg/kg were assayed for corticosterone. A dose of SAD comparable to that used in the pregnant females failed to significantly change plasma corticosterone concentrations in the males. An elevation corresponding only to 75% of that in the females was seen in males receiving multiple doses of SAD totaling three times the dose used in the females. As with females, DMSO completely abolished plasma corticosterone elevation by SAD in the males. These results demonstrate, for the first time, the effect of SAD on a mammalian endocrine system and provide evidence for a specific involvement of elevated maternal plasma corticosterone concentrations in SAD teratogenicity.     

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A₂ (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A₂ (sPLA₂), but not a catalytically inactive mutant form of GX sPLA₂, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA₂ inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA₂-overexpressing Y1 cells, ruling out a role for this sPLA₂ receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA₂. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA₂ antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA₂ is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.     

Sensitivity to corticosterone-induced cleft palate is not associated with H-2

The frequency of cleft palate (CP) and corticosterone levels in maternal plasma and amniotic fluid were determined in pregnant C57BL/10 (H-2b) and congenic B10.A (H-2a) mice after the ip injection of repository ACTH, corticosterone acetate, or desoxycorticosterone acetate (DOCA) on the 11th through 14th day of gestation. It was found that ACTH (a) induces CP in B10.A but not C57BL/10 mice, (b) induces CP in B10.A mice at about the same frequency noted when diluent alone is injected, and (c) produces an elevation in maternal plasma corticosterone levels 1.5- to 2.0-fold lower and amniotic fluid levels 3- to 5-fold lower than those found after the injection of 5 mg corticosterone acetate, a dose which induces a comparable frequency of CP in B10.A mice. The injection of 5 mg corticosterone acetate produced CP frequencies in C57BL/10 and B10.A mice of 4.9 and 3.3%, respectively, and increasing the dose to 9.2 mg resulted in significant increases in CP to 23.8 and 24.7%, respectively. DOCA at two dose levels induced CP in B10.A fetuses at about the frequency noted when diluent alone has been given. These findings show that susceptibility to corticosterone induced CP is not associated with the major histocompatibility complex of the mouse, H-2, as is the case with glucocorticoids (e.g., cortisone, dexamethasone), and they raise the possibility that factors other than or in addition to corticosterone may be involved in spontaneous or ACTH- or stress-induced CP.     

High-performance liquid chromatographic separation of corticosteroids in plasma of rats. Its application to the determination of the circadian rhythm of 18-hydroxycorticosterone related to aldosterone and corticosterone

An efficient separation of corticosteroids in plasma of rats was obtained by reversed-phase high-performance liquid chromatography (HPLC). Plasma corticosteroid assays with HPLC separation were used to determine the circadian rhythm of 18-hydroxycorticosterone (18-OHB) and its possible relationship to aldosterone or corticosterone in conscious rats under standard conditions (regular diet; 12-hour light and 12-hour dark cycle). Significant circadian rhythms of plasma corticosterone, 18-OHB and aldosterone were observed with peak values at 20.00 h and nadir values at 08.00 h. The mean ratio of plasma 18-OHB to aldosterone during 24 h was 2.4. The circadian rhythm of 18-OHB was also correlated with that of plasma aldosterone or corticosterone.     

Plasma and adrenal corticosterone concentrations and liver glycogen content in premature mice at birth and during neonatal development

The concentrations of corticosterone in the plasma and adrenal glands and the content of glycogen in the liver were estimated from birth to day 6 after birth in surviving premature mice removed by Caesarean section on day 19 of pregnancy and submitted to reanimation during 30 min; the neonates were nourished by nursing mothers from 30 min after birth. A group of full-term newborns was removed by Caesarean section on day 20 of pregnancy and killed 30 min after reanimation. Premature mice were characterized by neonatal changes of three parameters used. The plasma corticosterone level reached a peak in the first 6 h after birth, then decreased until day 6. The adrenal corticosterone level did not vary significantly 30 min after birth, then decreased progressively until day 2. The liver glycogen content, very high on day 19 of pregnancy, increased 30 min after birth, then fell sharply until day 2. In full-term newborns removed by Caesarean section and killed 30 min after reanimation the plasma corticosterone level increased, whereas the adrenal corticosterone level and the liver glycogen content did not decrease. The adrenal gland of surviving premature mice was able to respond to the stress induced by the reanimation; the stimulation of glucocorticoid function was similar in both neonates.     

Absence of HDL cholesteryl ester uptake in mice via SR-BI impairs an adequate adrenal glucocorticoid-mediated stress response to fasting

Receptor-mediated cholesterol uptake has been suggested to play a role in maintaining the adrenal intracellular free cholesterol pool and the ability to produce hormones. Therefore, in the current study, we evaluated the importance of scavenger receptor class B type I (SR-BI)-mediated cholesteryl ester uptake from HDL for adrenal glucocorticoid hormone synthesis in vivo. No difference was observed in the plasma level of corticosterone between SR-BI-deficient and wild-type mice under ad libitum feeding conditions. Overnight fasting ( approximately 16 h) stimulated the plasma level of corticosterone by 2-fold in wild-type mice. In contrast, no effect of fasting on plasma corticosterone levels was observed in SR-BI-deficient mice, leading to a 44% lower plasma corticosterone level compared with their wild-type littermate controls. In parallel, an almost complete depletion of lipid stores in the adrenal cortex of fasted SR-BI-deficient mice was observed. Plasma adrenocorticotropic hormone levels were increased by 5-fold in fasted SR-BI-deficient mice. SR-BI deficiency induced marked changes in the hepatic expression of the glucocorticoid-responsive genes cholesterol 7alpha-hydroxylase, HMG-CoA synthase, apolipoprotein A-IV, corticosteroid binding globulin, interleukin-6, and tumor necrosis factor-alpha, which coincided with a 42% decreased plasma glucose level under fasting conditions. In conclusion, we show that the absence of adrenal HDL cholesteryl ester uptake in SR-BI-deficient mice impairs the adrenal glucocorticoid-mediated stress response to fasting as a result of adrenal glucocorticoid insufficiency and attenuated liver glucocorticoid receptor signaling, leading to hypoglycemia under fasting conditions.     

Altered Entrainment to the Day/Night Cycle Attenuates the Daily Rise in Circulating Corticosterone in the Mouse

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT_1B receptors, decreasing responsiveness to light. Consequently, 5-HT_1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light∶dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT_1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.     

Reproductive phase dependent circadian variations of plasma melatonin, testosterone, thyroxine and corticosterone in Indian palm squirrel, Funambulus pennanti

To explain the complex mechanism of environmental influence along with internal hormonal (factors) milieu on daily variations in the circulating levels of melatonin, testosterone, thyroxine and corticosterone were analyzed with the help of inferential statistics (Cosinor rhythmometry) in a seasonally breeding tropical rodent, F. pennanti during the reproductively active (RAP) and inactive phases (RIP). Plasma melatonin, thyroxine and corticosterone levels exhibited a significant circadian oscillation during both the active and inactive phases of the annual reproductive cycle. Melatonin showed higher amplitude during RIP in the circulating plasma. Testosterone presented a peak level during evening hours (16:00 - 18:00 h) during RAP only. The phase of thyroxine was noted ∼09:76 h and ∼10.35 h during active and inactive phases, respectively. Corticosterone showed a peak level at ∼12.00 h during both phases of the reproductive cycle. Further, in this tropical rodent, the minimum difference in photoperiod (∼3 - 4 hours) and maximum variation in temperature (max. 18°C - min. 10°C during RIP and max. 45°C - min. 32°C during RAP) and humidity (85% during RIP and 35% during RAP) regulated the diurnal rhythm of circulating melatonin circadian rhythm by ∼1 hour phase advance during RIP. In conclusion, the studied hormonal rhythms may be part of an integrative system to coordinate reproduction and physiological processes successfully with environmental factors.     

Adrenalectomy, Corticosteroid replacement and their importance for drug-induced memory-enhancement in mice

Adrenalectomy blocks the memory-improving effect of piracetam-like compounds in mice. If this blockade is due to the removal of endogenous corticosteroids, replacement therapy with exogenous corticosteroids should reinstate the effects on memory. The present experiments were designed to determine the appropriate replacement dose (concentration in the drinking fluid) for corticosterone and aldosterone, the main corticosteroids in mice. Based on the effects of corticosterone on thymus weight, replacement with 3 μg/ml corticosterone given in the drinking fluid (0.9% NaCl) for one week was found to be appropriate. The appropriate replacement dose for aldosterone was found by giving aldosterone to adrenalectomized (ADX) mice in the drinking fluid in combination with 3 μg/ml corticosterone. The combination of 3 μg/ml corticosterone+30 ng/ml aldosterone resulted in a plasma ratio of corticosterone/aldosterone which most closely approximated the ratio seen in sham-ADX control animals. The physiologic adequacy of the corticosteroid replacement doses resulting from this study were clearly demonstrated in subsequent behavioral experiments where blockade of the memory-enhancing effects of piracetam by adrenalectomy were overcome by replacement with either 3 μg/ml corticosterone or 30 ng/ml aldosterone given in the drinking fluid.     

MRNA and enzyme activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 are elevated in C57BL/KsJ-db/db mice.

To evaluate the importance of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in insulin resistant diabetic C57BL/KsJ-db/db mice, we measured the activity and mRNA level of 11beta-HSD1 in the liver of db/db mice and their heterozygote litter mates, db/+m mice. The blood glucose, plasma insulin, and corticosterone levels of db/db mice were significantly higher than those of db/+m mice. Despite hyperinsulinemia, the activity level of this enzyme was significantly higher in db/db mice, and the mRNA level of hepatic 11beta-HSD1 was also significantly higher in db/db mice. Since hepatic 11beta-HSD1 in vivo mainly functions as 11-keto-reductase and does not work as 11beta-oxidase, these results suggest that the rate of hepatic conversion of 11-dehydrocorticosterone to corticosterone is increased in db/db mice, resulting in higher glucocorticoid activity in the liver. The increased hepatic corticosterone concentration due to the elevation of 11beta-HSD1 and high plasma corticosterone concentration may antagonize the action of insulin and cause insulin resistance. These findings have a potentially important implication for relationships between increased hepatic 11beta-HSD1 and insulin resistance in db/db mice. The present paper is the first to demonstrate the increased activities and mRNA level of hepatic 11beta-HSD1 in db/db mice.