Basal and glucocorticoid induced changes of hepatic glucocorticoid receptor during aging: relation to activities of tyrosine aminotransferase and tryptophan oxygenase

The characteristics of glucocorticoid receptors, their sensitivity to glucocorticoid as well as the basal and glucocorticoid induced thyrosine aminotranferase (TAT) and tryptophan oxygenase (TO) activities were studied in rat liver during aging. The concentration (N) and dissociation constant (K_d) of glucocorticoid receptor (GR) significantly change during the aging both in untreated and dexamethasone treated animals. The level of receptors was lower in dexamethasone treated rats of all analyzed aged groups compared to untreated animals. In comparison to untreated groups, there was no correlation between the changes of N and K_d during the lifespan. According to immunochemical analysis, the decline of receptor protein content occurs during lifespan. Dexamethasone treatment reduced the level of receptor protein compare to respective age group of untreated rats. The glucocorticoid-receptor (G-R) complexes from both untreated and treated animals underwent thermal activation, although the extent of activation was more pronounced in the case of untreated groups compared to treated animals. The magnitude of heat activation of receptor complexes was more pronounced in the liver of the youngest untreated rats compared to elderly ones, while the receptor activation between treated groups of studied ages has shown less significant differences. Besides, basal as well as induced TAT and TO activities after dexamethasone injection also showed age-related alterations. The observed alterations in GR might play a role in the changes of the cell responses to glucocorticoid during the age. This presumption is supported by detected changes in basal and dexamethasone induced TAT and TO activities during aging.     

米非司酮与孕激素受体及糖皮质激素受体结合力的研究

目的探讨米非司酮的抗孕激素及抗糖皮质激素作用。方法用放射配体结合实验测定米非司酮与猕猴子宫胞浆孕激素受体及大鼠肝胞浆糖皮质激素受体的结合力(BA)。结果米非司酮和孕酮与孕激素受体的BA分别为175.58%±19.20%、100%,50%抑制浓度(IC50)分别为(17.20±1.20)nm o l/L、(30.20±2.31)nm o l/L,两者相比,P均<0.01;米非司酮和地塞米松与糖皮质激素受体的BA分别为344.41%±57.41%、100%,IC50分别为(4.21±1.02)nm o l/L、(14.50±1.89)nm o l/L,两者相比,P均<0.01。结论米非司酮具有强的抗孕激素作用和一定的抗糖皮质激素作用。     

Determination of glucocorticoid receptors in leukemic myeloblasts by isoelectric focusing in polyacrylamide gel

We investigated the optimal conditions for measuring glucocorticoid receptor in blast cells from patients with acute nonlymphocytic leukemia. Cytosol receptor measured with isoelectric focusing was saturated after 60 min of incubation at 0 degrees C with 100 nM of either dexamethasone or triamcinolone. Saturation was achieved when cytosol from at least 7 X 10(6) cells was used for incubation. Trypsin treatment of the cytosol resulted in a sharpened peak of receptor focusing at pH 5.6 with no loss of receptor-bound radioactivity. The two physical forms of glucocorticoid receptor were isolated with DEAE cellulose chromatography. They were both found to focus at pH 5.6 during isoelectric focusing.     

Modulation of glucocorticoid action and the treatment of type-2 diabetes

The global epidemic of obesity and type-2 diabetes has heightened the need to understand the mechanisms that contribute to its pathogenesis and also to design and trial novel treatments. Patients with glucocorticoid (GC) excess--'Cushing's syndrome'--are phenotypically similar to patients with simple obesity. As such, much research has focused on the manipulation of local GC action as a therapeutic strategy. The majority of the classical actions of GCs are mediated via activation of the glucocorticoid receptor (GR). 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inactive cortisone to cortisol and therefore amplifies local GC action. There is now a wealth of data from rodent and clinical studies implicating this conversion in the pathogenesis of obesity, type-2 diabetes, and the metabolic syndrome. Selective 11beta-HSD1 inhibitors (selective in that they block the activity of 11beta-HSD1 and not 11beta-HSD2 which inactivates cortisone to cortisol in mineralocorticoid target tissues) are currently in development although not yet available for use in clinical studies. Rodent studies utilizing these compounds have shown dramatic improvements in insulin sensitivity as well as improvements in lipid profiles and atherogenesis. A further experimental approach has been to design drugs that antagonize GR activation, and again these compounds appear to improve insulin sensitivity and lower glucose production rates. The key test for both of these research strategies is whether they will translate into clinical studies, and results from these trials are now eagerly awaited.     

Age-Dependent Dietary Regulation of Glucocorticoid Receptors in the Liver of Mice

Dietary restriction (DR) increases the resistance to different stresses, retards various age-related diseases and extends life span in a variety of animals. Here we have investigated the effect of DR (alternate days of feeding for 3 months) on glucocorticoid receptors (GRs) in the liver of adult (5 months) and old (20 months) male mice. A significant decrease was observed in the level of receptors in old mice (25%) as compared to the adult ones. DR subjected mice of both age groups showed a marked increase in the GR concentration (37% in adult and 31% in old mice) as compared to the ad libitum (AL) fed mice, whereas the affinity remained the same in both groups of animals at both ages. Scatchard analyses and the protein slot blot experiment confirmed the increase in the receptor level in AL and DR fed animals for both age groups. The magnitude of heat and salt activation of GR was higher in the adult mice as compared to the old mice who were fed AL. DR, however, significantly increased (40%) the magnitude of activation of GR in the older mice as compared to the AL fed animals, whereas no such change was observed in the adult animals. Further, DNase I digestion and extraction of nuclear bound GR-complexes showed a higher degree of extraction in adult animals (57%-59%) as compared to the old (31%-33%) animals. Mice subjected to DR revealed no significant change at either age. These findings indicate that DR regulates GR in an age-dependent manner and that it may allow animals to better adapt to metabolic regulation in older ages.     

强啡肽对实验性神经细胞老化过程中M胆碱能受体位点数影响的研究

目的　探讨强啡肽 (DYN)对实验性神经细胞 M受体位点数的影响。方法　采用小鼠神经母细胞瘤细胞 (NBA2 )无血清培养建立神经细胞老化实验研究模型。以 M受体放射性配基分析术研究 DYN对实验性神经细胞的 M受体位点数的影响。结果　两种不同浓度的 DYN皆可使M受体位点数下降 (P<0 .0 1 ) ,以高浓度组更显著。结论　 DYN使 M受体位点数下降的作用可能是延缓实验性神经细胞老化的机制之一。(最终浓度为 0 .1nmol/L) ,以测定非特异性结合。各管加入2 0 0 μl膜蛋白液 ,37℃振荡温育 4 0 min。冰冷缓冲液终止反应。6 9型纤维滤膜三层抽滤并淋洗 ,滤膜烘干 ,投入 5 ml闪烁液(含 0 .4 % PPO的三甲苯 )中 ,以 YSL- 76型液闪计数器做固相测定。1.8　 数据处理　实验得到特异性结合的技术率 ,被测定效率除 ,等于特异性结合衰变率 ,再除以放射性配基活度 ,即等于特异性结合位点数〔7〕。采用 POMS方差分析 ,经 IBM微机处理数据。2　结果与讨论DYN对 NBA2 细胞 M胆碱能受体位点数的影响 ,见表 1。表 1　 DYN对 M受体位点数的影响 (nmol/10 6 细胞 )组别 n M受体位点数 (x± s)BSD组 5 449.796± 356.730AID组 5 486.941± 2 4 7.1 65DYN- 7组 5 1 4 5.71 4± 77.82 61 )DYN- 8组 5 1 51 .873± 95.80 61 )　　注 :DY     

Interaction of glucocorticoid receptors from lymphoid cell lines with their nuclear acceptor sites.

Procedures have been developed which provide simple means of determining binding constants of steroid receptors for glucocorticoids in mouse lymphoid cell lines and of characterizing the interaction of the steroid--receptor complex with the nucleus. An average of 70% of the steroid--receptor complexes is found associated with the nuclear fraction in three investigated cell lines, whereas 30% of the steroid--receptor complexes is found in the cytosol fraction. This distribution of the steroid-receptor complex within the cell is independent of whether steroid uptake of the cells is performed at low or at high steroid concentration. Part of the binding of the steroid receptor to the nuclear fraction is sensitive to high ionic strength and to high pH. A larger fraction of the steroid--receptor complex binding to the nuclear fraction is insensitive to high ionic strength and pH when the steroid uptake is performed at low steroid concentrations than when performed at high steroid concentrations. Steroid--receptor complex is released from the nuclear fraction by DNAase treatment but not by RNAase treatment. The possible correlation between the sensitivity to ionic strength and pH and the specificity of the binding is discussed.     

糖皮质激素作用机制的研究进展

糖皮质激素广泛应用于肾脏疾病的治疗,既往几十年中,糖皮质激素更多为被经验性应用于临床治疗,其作用机制尚不明确.以往认为糖皮质激素是通过免疫抑制和抗炎来发挥作用,近年,随着对肾小球足细胞的认识进一步加深,发现糖皮质激素还具有直接保护足细胞的作用.本文就糖皮质激素受体信号通路、免疫抑制机制和对足细胞保护机制作一综述.     

Low-dose dexamethasone alleviates lipopolysaccharide-induced acute lung injury in rats and upregulates pulmonary glucocorticoid receptors

Background and objective: The major causes of mortality among patients who survive acute lung injury/ARDS (ALI/ARDS) are due to the extensive tissue remodelling and fibrosis. Use of high‐dose glucocorticoids to reduce these inflammatory and fibroproliferative responses has been shown to do more harm than good. Recently, Meduri et al. found that the early use of low‐dose prolonged methylprednisolone in patients with severe ALI/ARDS significantly relieved the systemic inflammatory response and improved pulmonary and extrapulmonary organ function. This study investigated the therapeutic effect of low‐dose dexamethasone (Dex) on inflammation and fibrosis in LPS‐induced ALI in rats and its influence on the expression of the pulmonary glucocorticoid receptor (GR). Methods: Eighty Wistar rats were randomly divided into four groups: a control group (intraperitoneal normal saline injection (5 mL/kg) throughout experiment, n = 24); the LPS model group (LPS injection (5 mg/kg) for 3 days and normal saline thereafter, n = 24); the LPS + Dex group (LPS injection for 3 days and Dex solution (5 mg/kg) thereafter, n = 16); and the Dex group (normal saline injection (5 mL/kg) for 3 days and Dex solution (5 mg/kg) thereafter, n = 16). Levels of tumor necrosis factor‐α, matrix metallopeptidase‐9 and procollagen N‐terminal propeptide type I in BAL fluid were examined by ELISA on the third, seventh and fourteenth days after injection. Pulmonary hydroxyproline content was measured and histological examination was performed with haematoxylin–eosin and Victoria blue‐ponceau. Pulmonary distribution of GR‐positive cells was examined immunohistochemically, and expression of GR mRNA and protein was determined by RT‐PCR and western blot analysis. Results: Histological assessments showed that pulmonary fibrosis occurred in parallel with inflammation in the rat ALI model. Compared with the LPS group, the inflammation and fibrosis parameters were significantly improved in the LPS + Dex group at different periods after injection (P < 0.05 or P < 0.01), although parameters in the LPS + Dex group were not as good as those of the control group. GR mRNA and protein expression in the LPS + Dex group were markedly higher than that of the LPS group on the seventh and the fourteenth days (both P < 0.01). Western blotting showed that Dex also promoted the nuclear translocation of GR protein. Conclusion: Low‐dose Dex can reduce pulmonary inflammation and fibrosis after LPS‐induced ALI in rats and can elevate GR expression in the lung, probably through upregulating GR levels and promoting the nuclear translocation of GR protein.     

Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor

Crystallography and computer modeling have been used to exploit a previously unexplored channel in the glucocorticoid receptor (GR). Highly potent, nonsteroidal indazole amides showing excellent complementarity to the channel were designed with the assistance of the computational technique AlleGrow. The accuracy of the design process was demonstrated through crystallographic structural determination of the GR ligand-binding domain–agonist complex of the D-prolinamide derivative 11. The utility of the channel was further exemplified through the design of a potent phenylindazole in which structural motifs, seen to interact with the traditional GR ligand pocket, were abandoned and replaced by interactions within the new channel. Occupation of the channel was confirmed with a second GR crystal structure of this truncated D-alaninamide derivative 13. Compound 11 displays properties compatible with development as an intranasal solution formulation, whereas oral bioavailability has been demonstrated with a related truncated exemplar 14. Data with the pyrrolidinone amide 12 demonstrate the potential for further elaboration within the “meta” channel to deliver compounds with selectivity for the desired transrepressive activity of glucocorticoids. The discovery of these interactions with this important receptor offers significant opportunities for the design of novel GR modulators.     

Raised urinary glucocorticoid and adrenal androgen precursors in the urine of young hypertensive patients: possible evidence for partial glucocorticoid resistance

OBJECTIVE—To evaluate urinary glucocorticoid excretion profiles in a cohort of recently diagnosed young hypertensive patients.
METHODS—After excluding patients with secondary causes, 60 individuals with premature hypertension were recruited (diagnosed by ambulatory blood pressure monitoring before the age of 36 years). In addition, 30 older hypertensive controls (age of onset > 36 years, "middle aged hypertensive controls"), and 30 normal controls (age matched to the young hypertensive group) were studied. All provided 24 hour urine collections for mass spectrometry for total cortisol metabolites and total androgen metabolites by gas chromatography.
RESULTS—Among male patients, those with premature hypertension had higher total urinary excretion of cortisol metabolites (mean (SD), 13 332 (6472) µg/day) than age matched normal controls (7270 (1788) µg/day; p = 0.00001) or middle aged hypertensive controls (8315 (3565) µg/day; p = 0.002). A similar increase was seen among the female patients, although the absolute concentrations were lower. There was no significant difference between middle aged hypertensive patients and normal controls. Urinary total androgen excretion profiles in female patients also showed an unusual increase in the premature hypertension group (2958 (1672) µg/day) compared with the other groups (middle aged hypertensive controls, 1373 (748) µg/day, p = 0.0003; normal controls, 1687 (636) µg/day, p = 0.002). In all subjects, serum sodium and creatinine concentrations were within the normal range; serum potassium concentrations were found to be low before the start of treatment.
CONCLUSIONS—Individuals presenting with premature hypertension have an abnormally high excretion of glucocorticoid metabolites in the urine. While the mechanism remains uncertain, these findings are compatible with partial resistance of the glucocorticoid receptors, with a compensatory increase in cortisol and androgen metabolites. The mineralocorticoid effects of the latter (sodium and water retention) may contribute to an abnormally high blood pressure and may have implications for targeted selection of first line treatment in young hypertensive patients.


Keywords: premature hypertension; glucocorticoid resistance; cortisol metabolites; glucocorticoid receptor resistance     

大剂量糖皮质激素下调血管内皮细胞GR表达的意义

目的观察LPS和Dex对人脐静脉内皮细胞糖皮质激素受体(GR)的表达情况。方法利用内毒素脂多糖LPS"致伤"血管内皮细胞,用地塞米松进行"治疗",然后通过RT-PCR和免疫组化的方法检测血管内皮细胞糖皮质激素受体(GR)的表达情况。结果内皮细胞本身能明显表达GRmRNA,运用不同浓度Dex和100 ng/ml LPS刺激后,可使人脐静脉内皮细胞内GRmRNA表达明显下降,应用大剂量(10-6mol/L)的Dex刺激细胞3h,GRmRNA的表达无明显变化,6 h开始明显下降,12 h达高峰,24 h回升近刺激前水平。结论大剂量的Dex(10-6mol/L)可下调GR的表达,表明严重创伤时血管内皮细胞中糖皮质激素作用存在其他的作用机制。     

Longevity and aging of mice from linesdivergently selected for body weightfor over 90 generations

We have analysed the differences between longevity and aging process in two lines of mice selected divergently for body weight. As a result of long-term selection,the mice from two lines varied significantly in bodyweight during their life span. The differencesregarded maturation rate, the length of the life time,reproduction performance, and fertility. Females fromthe light line (L) were fertile for a longer period oftime than those from the heavy line (C), while theprocess of spermatogenesis disappearance startedearlier in the light males than in the heavy ones. Therewere also differences in the relative weight ofadrenal glands and histopathological changes connectedwith the hyperplasia of their spindle cells. Theadrenal glands in females from both lines were heavierthan in males. A significantly higher percentage of thecortex cell hyperplasia was observed in adrenals ofmice from the C line than the L line. Thedifferences were also scored between mice from theselection lines in relation to the length of theirlife. On average, the L mice lived 30 days longerthan the heavy ones. Body weight loss was observedearlier in the old heavy males than the L ones andthis was correlated with the earlier mortality in thisgroup of animals.     

糖皮质激素受体阻断后对大鼠阿霉素肾病的影响

用微量热法研究了Ｄ－氨基葡萄糖西佛碱ＳＧ及其配合物对金黄色葡萄球菌的作用。发现不同化合物对金黄色葡萄球菌的作用效果不同，其效果为：Ｚｎ（Ⅱ）－ＳＧ＞Ｃｕ（Ⅱ）－ＳＧ＞Ｃ（Ⅲ）－ＳＧ＞ＳＧ（配体），半抑制浓度Ｃ１／２依次增大，不同化合物作用下其代谢热谱有所不同，说明与细菌作用的方式也不同。     

Effect of aging on cold tolerance and thyroid activity in CBA/Ca inbred mice

Relationships between cold tolerance, serum levels of thyroxine (T₄), thyronine (T₃), and thyrotropine (TSH), and thyroid morphometry have been investigated in male CBA/Ca inbred mice at various ages through their life span. From the data obtained it appeared that there was an age-related decrease in cold tolerance up to 18 months of age which was followed by an increase, the age effect being most apparent in relation to cold resistance and cold tolerance during the recovery period following cold exposure. The age-related changes in cold tolerance appeared to be associated with changes in the serum concentrations of T₃, T₄, and TSH. In contrast to the T₃ serum levels which showed a decrease at 36 months, the thyroxine contents showed a perceptible decrease from the age of 12–18 months onwards. A similar pattern was observed for the TSH levels, with a peak at 21 months, followed by a decline at 30 months. A relationship with age between serum T₄ level and thyroid weight was indicated together with structural changes in the thyroid gland, particularly during senescence, for example the size and number of thyroid epithelial cells had become enlarged by 30 months of age.     

Physicochemical analysis of glucocorticoid receptor in the immature pituitary gland: Evidence for lability and aggregation of the heat-activated form

The present study was aimed at characterizing the deficiency of the glucocorticoid-receptor system in the immature pituitary gland of neonatal rats. Under conditions of both constant protein and DNA concentrations, [³H]dexamethasone binding to nuclei was significantly lower in the immature, compared with the mature, glands, despite the comparable density of receptor sites. Chromatography on DEAE-Sephacel columns revealed the failure of the neonatal receptor to form stable activated complexes, although the dissociation of tracer from binding sites, as well as the magnitude of receptor activation, remained closely similar to those found for the adult binder. Also, a striking observation was that the immature receptor exhibited the property of forming more than twice as much inactive aggregate as the mature component, when heat-activated in presence of KCl. It thus seems that development of full expression of glucocorticoid-binding activity in the pituitary involves qualitative changes of both the receptor molecules and the cytoplasmic environment, that tend to enhance stability of the activated form of the binder.     

Multipotent stem cell (CFU-S) numbers and circadian variations in aging mice

The multipotent stem cell (CFU-S) numbers were studied in aging female C3H mice (16, 21 and 26 months old, respectively) versus young controls (3 months old). Using the spleen colony technique, the d-8 CFU-S numbers were measured every 3 h during the 24-h period at three different times of the year. Prominent circadian variations were found in young mice. The peak and trough values were significantly different also in aging mice, although the peak-trough differences were declining. When comparing young and old mice at different times of the 24-h period, the CFU-S numbers were sometimes significantly different, but often not. The 24-h mean values were consistently declining during aging. Young mice had different circadian variation patterns and 24-h mean values when examined at different times of the year. It is concluded that the d-8 CFU-S numbers decline in aging mice. Conflicting reports may partly be due to neglect of physiological variations.