Central and peripheral glucocorticoid receptor function in abdominal obesity.

Abdominal obesity seems to be associated with a moderately deranged feedback regulation of the hypothalamic-pituitary-adrenal (HPA) axis where central glucocorticoid receptors (GR) are involved. Therefore, functions of central and peripheral GR were compared in this study. Furthermore, since trinucleotide repeats in early exons of steroid hormone receptor genes influence transcription, and therefore may influence receptor density, this was also studied. Ten middle-aged men, 5 with abdominal obesity and 5 controls, were studied. The suppression of dexamethasone (dex) on serum cortisol was used in dose-response tests to assess the function of central GR. Abdominal adipose tissue biopsies were incubated and exposed to cortisol in different concentrations, and the function of the peripheral GR assayed as induction of lipoprotein lipase (LPL) activity. Aberrant expansion of exonic trinucleotide repeats in the first coding exon of the GR gene was studied by sequencing of genomic DNA. Results showed that men with abdominal obesity showed less inhibition of serum cortisol by dex, particularly at lower concentrations, while in the controls cortisol secretion was inhibited in an apparent dose-response manner. LPL activity in adipose tissue was lower in abdominal obese men than in controls. However, the sensitivity to cortisol was not different between the groups. There was no evidence for expansion of trinucleotide repeats. These results suggest that the central GR and the peripheral GR in adipose tissue exhibit functional differences in abdominal obesity.

The relationship of nasal polyps, infection, and inflammation.

The role of infection as cause or effect in nasal polyps is debated. In experimentally induced sinusitis in rabbits, polyps are frequent. The initial polyp formation sequence involves multiple epithelial disruptions with proliferating granulation tissue. Regenerating epithelial branches spread into the underlying connective tissue, where intraepithelial microcavities give rise to a polyp body from the adjacent mucosa. Clinical as well as experimental studies indicate that nasal polyp formation and growth are activated and perpetuated by an integrated process of mucosal epithelium, matrix, and inflammatory cells, which in turn may be initiated by both infectious and noninfectious inflammation. The complexity of the pathophysiologic events in nasal polyposis is reinforced by the finding that epithelial desquamation, combined with infection or inflammation, will initiate polyp formation. Systemic glucocorticosteroids inhibit polyp formation as well as growth of pathogenic bacteria in the sinuses of rabbits with experimental infection. Therapeutic use of corticosteroids in polyp disease, combined with antibiotics or surgery, should be modified in relation to long-term progression, intensity variations, and predisposing conditions.

In vivo modulation of glucocorticoid receptor mRNA by inhaled fluticasone propionate in bronchial mucosa and blood lymphocytes in subjects with mild asthma.

BACKGROUND: In vivo regulation of the glucocorticoid receptor (GR) by glucocorticoids provides a means of modulating sensitivity of targeted cells. OBJECTIVE: We sought to determine the in vivo modulation of GR mRNA expression by fluticasone propionate (FP) in subjects with mild asthma. METHODS: Ten atopic asthmatic subjects were treated with FP 250 microg twice daily for 4 weeks. Before and after treatment, the patients underwent fiberoptic bronchoscopy with endobronchial biopsy and sampling of venous blood for measurements of GR mRNA levels. A solution hybridization assay was used for quantitative analysis of GR mRNA. In addition, a 24-hour urinary cortisol excretion and an adrenocorticotropic hormone test before and after treatment with FP were performed. RESULTS: A high interindividual variation in GR mRNA expression was seen. However, we detected a significant reduction of the GR mRNA levels in the endobronchial biopsy specimens after FP treatment (36.6 +/- 23.1 and 25.0 +/- 10.9 amol GR mRNA/microg RNA, respectively; P <.01). In the peripheral blood lymphocytes an even more striking downregulation of the GR by its cognate ligand was documented (30.3 +/- 26.5 and 8.8 +/- 5 amol GR mRNA/microg RNA, respectively; P <.001), possibly reflecting differences in glucocorticoid sensitivity between tissues. A small but significant reduction of the 24-hour urinary cortisol excretion was observed (233 +/- 109 and 157 +/- 66 nmol/L, respectively; P <.01), whereas the feedback regulation of glucocorticoid synthesis by means of the hypothalamic-pituitary-adrenal axis as assessed by the adrenocorticotropic hormone test remained normal after treatment with FP. CONCLUSION: The results in this study confirm the potency of the inhaled corticosteroid FP and provide evidence for a considerable tissue-specific interindividual variation in the expression of the GR.

Variations in glucocorticoid levels within the physiological range affect plasma leptin levels.

OBJECTIVE: Leptin, the obese gene product, is thought to regulate body fat through its action on hypothalamic receptors that influence satiety. The hormonal regulation of leptin is important, since it might affect adiposity. Leptin regulation in man is poorly understood. We studied the relation between endogenous cortisol and leptin levels as well as the acute and chronic effects of a low dose of dexamethasone (DEX) on plasma leptin levels in healthy male volunteers. SUBJECTS AND EXPERIMENTAL PROTOCOL: The correlation between basal plasma levels of leptin and cortisol and the chronic effect of DEX treatment were studied in 12 subjects. Plasma leptin and cortisol levels were determined every other hour for 24 h, before and after 2 weeks of oral administration of 0.1 mg DEX twice daily. The acute effect was studied in 20 subjects, who received 1 mg DEX at 2300 h. Fasting blood samples were taken at 0800 h on the same day (i.e. before DEX) and on the day after. RESULTS: Under basal conditions, we found a correlation between mean plasma levels of leptin and cortisol (r = 0.7, P<0.02). Mean plasma leptin levels had increased by 50% after 2 weeks of DEX treatment (P<0.05). The circadian rhythm of leptin was preserved, but the night peak occurred 2.5 h earlier (P<0.05). Fasting plasma leptin levels were 20% higher 9 h after 1 mg DEX orally than at the same time on the day before (P<0.002). CONCLUSION: Physiological variations in cortisol are involved in the regulation of leptin.

Evidence that the beta-isoform of the human glucocorticoid receptor does not act as a physiologically significant repressor.

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRalpha and hGRbeta, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRbeta message had a widespread tissue distribution. To demonstrate the presence of hGRbeta as protein we produced specific rabbit antisera to hGRbeta, as well as a hGRbeta-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRbeta is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRbeta form. We also showed that hGRbeta bound to hsp90 by immunoprecipitation of in vitro translated hGRbeta in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRbeta inhibited the effects of dexamethasone-activated hGRalpha on a glucocorticoid-responsive reporter gene. In conclusion, low hGRbeta expression levels and hGRbeta-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRalpha effects challenge the concept of the hGRbeta isoform as a proposed dominant negative inhibitor of hGRalpha activity.

Circadian cortisol rhythms in healthy boys and girls: relationship with age, growth, body composition, and pubertal development.

To provide basic information on the normal functioning of the hypothalamus-pituitary-adrenal axis in relation to pubertal development, growth (weight and height), body composition, and gender and to obtain reference data for serum cortisol concentrations in children, we investigated the basal circadian rhythm of serum cortisol in a group of 235 healthy children (162 boys and 73 girls). The age range was between 2.2-18.5 yr. Serum cortisol was analyzed from venous blood samples taken at 1400, 1800, 2200, 0200, 0400, 0600, and 1000 h. No evidence was found for differences in temporal placement or level of the circadian cortisol rhythm in relation to age, growth, or body composition. However, we found a broad range of cortisol levels in a healthy population, with individual mean diurnal levels ranging from 100-510 nmol/L. Regardless of high or low mean diurnal cortisol levels, repeated measurements within and between pubertal stages indicated that an individual remains in his or her cortisol range throughout pubertal development. In conclusion, the present study shows that 1) serum cortisol levels do not correlate with either age or gender; 2) there is a large and significant interindividual variability in endogenous mean diurnal cortisol levels; and 3) despite this variability between individuals, there is no correlation between cortisol levels and either body composition or growth rate. This suggests that the variability in cortisol levels is an expression of normal homeostasis rather than pathology.

Dental maturity in children of short stature, with or without growth hormone deficiency.

The aim of this investigation was to study dental maturity in healthy prepubertal children of short stature (height<-2 SD), with or without growth hormone (GH) deficiency, compared to healthy controls. The GH-deficient group (GH level<10.0 microg/l) included 29 children (11 female, 18 male) with a mean age of 10.2+/-2.2 years. The GH non-deficient group consisted of 17 children (5 female, 12 male) with a mean age of 8.5+/-2.1 years. All the children were evaluated for serum concentrations of IGF-1, alkaline phosphatase (ALP), triiodthyronin (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), and fasting plasma insulin; height and bone age were also recorded. Dental maturity was determined from panoramic radiographs. The mean difference between the dental and chronological ages was -0.67+/-0.89 years in the GH-deficient group compared to 0.23+/-1.07 years in their controls: in the GH non-deficient group the difference was -0.95+/-0.82 years compared to controls 0.16+/-1.06 years in their controls. Compared to chronological age, both bone and dental age were lower in the GH-deficient and GH non-deficient groups. It is concluded that children of short stature, both GH-deficient and GH non-deficient. exhibit a delayed dental age compared to their chronological age- and sex-matched controls. A multiple stepwise regression analysis showed that the sitting height and GH level were the only significant factors associated with dental maturity.

Glucocorticoid resistant syndromes--molecular basis and clinical presentations.

The mechanisms of action of glucocorticoid hormones are mediated via specific intracellular receptor proteins. The glucocorticoid receptor (GR) regulates expression of specific target genes or gene networks by ligand-dependent transcriptional activation, i.e. ligand-dependent activation of the receptor with subsequent dimer formation and DNA binding. There are a number of factors, such as the receptor concentration, receptor associated proteins, receptor alterations and the effects on the gene network including hormonal regulation of transcription, mRNA splicing and translation, that might influence glucocorticoid responsiveness in a normal and healthy population as well as in different diseases. Several categories of glucocorticoid resistance have been described including inherited GR resistance which has been explained in terms of specific mutations and offers an important model for genetic and clinical studies of steroid sensitivity, and relative glucocorticoid resistance, which occurs naturally in the course of cellular differentiation, cell to cell or tissue to tissue, since all cells possess receptors for glucocorticoids but do not show the same response to them. From a clinical point of view, it is also interesting to consider preexisting genetic susceptibility to glucocorticoids, acquired changes in the GR gene structure and organization, including alterations of noncoding sequences, and the importance of mutations, deletions and other changes in the GR gene affecting receptor function. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance, both generalized inherited glucocorticoid resistance (GIGR) and directed mutagenesis, has identified two regions of clustered mutations in the proximity of previously identified affinity labeled residues directly affecting the steroid binding function. Finally, studies of New Words primates and cell lines derived from hematologic malignancies constitute animal and human models for the molecular basis of glucocorticoid resistance where a number of inherited and acquired mutations in the GR gene have been demonstrated.

Molecular basis of glucocorticoid-resistant syndromes.

Generalized inherited glucocorticoid resistance (GIGR) is a rare syndrome characterized by elevated levels of plasma cortisol but lacking the symptoms of Cushing's syndrome. Biochemically, the condition is characterized by a relative resistance to glucocorticoids that can be compensated for by the elevated levels of cortisol. The inheritance pattern of GIGR is incompletely understood, and one of the central questions is whether there is a correlation between genotype and phenotype. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance has identified two regions of clustered mutations in the proximity of previously identified affinity-labeled residues, the putative steroid-binding site. In the majority of cases, the mutation affects steroid binding and transactivation to the same degree, with the exceptions suggesting an explanation for the variability of the clinical manifestations. From a clinical point of view, in addition to preexisting genetic resistance to glucocorticoids, it is important to consider acquired changes in glucocorticoid receptor (GR) gene structure and organization, including alterations of noncoding sequences, and the importance of the resultant mutations, deletions, and other changes affecting receptor function. Finally, studies of New World primates and cell lines derived from hematologic malignancies constitute animal and human models for the molecular basis of glucocorticoid resistance where a number of inherited and acquired mutations in the GR gene have been demonstrated.

Regulation of glucocorticoid receptor mRNA in nasal mucosa by local administration of fluticasone and budesonide.

The glucocorticoid receptor (GR) is downregulated by glucocorticoids (autoregulation). In contrast, the metallothionein gene (MTIIa) is positively regulated by glucocorticoids, which requires a functional receptor protein. We have investigated the expression of GR and MTIIa mRNA in nasal mucosal biopsy specimens, nasal brush-lavage samples, and peripheral blood lymphocytes from 14 healthy volunteers after local treatment with one of two different glucocorticoids: fluticasone propionate or budesonide. In nasal mucosal biopsy specimens, a significant decrease in GR mRNA occurred with increasing doses of both steroids, whereas a significant and parallel increase in MTIIa mRNA was observed. We found nasal brush-lavage less suitable for studies of GR mRNA and MTIIa mRNA regulation by locally administered glucocorticoids. In mucosal biopsy specimens, but not in peripheral blood lymphocytes, we found a correlation between basal GR mRNA and MTIIa mRNA levels, where low GR mRNA levels were associated with low MTIIa mRNA levels, and vice versa. In conclusion, this study shows that locally administered glucocorticoids significantly affect the expression of specific genes and that there is an interindividual and tissue-specific variation in GR mRNA and MTIIa mRNA expression, which may be used in studies of variations in clinical responses to nasal glucocorticoids.

Cardiac nuclear hormone receptor mRNA in heart failure in man.

Eight donor hearts and six explanted hearts due to dilated cardiomyopathy, normal skeletal muscle and liver were analysed. Glucocorticoid receptor (GR) and thyroid hormone receptor (T3R) isoforms beta 1, beta 2, alpha 1 and alpha 2 mRNA abundance were determined by solution hybridization. Both GR and T3R receptor mRNA isoforms were lower in the myocardium as compared to skeletal muscle and in particular to liver. GR mRNA abundance was higher than that of any T3R isoform while the sum of ligand-binding isoforms (beta 1, beta 2 and alpha 1) were similar in the myocardium and in skeletal muscle as opposed to the liver where GR mRNA was higher. GR mRNA abundance was similar in right and left ventricles from donor hearts and in cardiomyopathy. T3R beta 1 showed higher levels in the right ventricle with higher levels in cardiomyopathy as compared to donor heart. T3R isoform alpha 1 and especially the alpha 1/alpha 2 ratio were lower in left ventricle in cardiomyopathy compared to donor hearts. In conclusion, GR and T3R isoforms mRNA abundance are low in the human myocardium. In the failing myocardium GR and T3R beta 2 and alpha 2 show no signs of reactivity while T3R beta 1 and alpha 1 mRNA adapt.

Effect of glucocorticosteroid treatment on glucocorticoid receptor expression in human adipocytes.

The influence of glucocorticoid excess on expression of the glucocorticoid receptor (GR) and beta-adrenoceptor subtype was studied in isolated adipocytes obtained by sc fat biopsies from 17 healthy individuals. The biopsies were taken before and after 7 days of treatment with 25 mg prednisolone, given orally. GR and beta 1- and beta 2-adrenoceptor messenger ribonucleic acid (mRNA) levels were measured with a solution hybridization assay, and the number of beta 1- and beta 2-adrenoceptor binding sites was determined in radioligand binding experiments. GR protein levels were determined by Western blot analysis using an anti-GR antibody. Both GR protein and GR mRNA levels decreased significantly (P < 0.05) by about 50% after treatment, whereas no significant changes were demonstrated in either beta 1- or beta 2-adrenoceptor mRNA levels. The number of beta 2-adrenoceptor-binding sites, however, increased by 70% after treatment (P < 0.05), whereas the number of beta 1-adrenoceptor binding sites was not affected. The affinity of each receptor subtype was not significantly altered by steroid treatment. In conclusion, an in vivo glucocorticoid excess decreases GR mRNA as well as GR protein levels and selectively increases beta 2-adrenoceptor density in sc fat cells of healthy individuals. This indicates that glucocorticoids modulate human adipose metabolism by altering the expression of regulatory proteins at various mRNA and post-mRNA levels.

Beta 1 and beta 2 adrenoceptor ligand and mRNA expression in dilated cardiomyopathy.

beta 1 and beta 2 adrenoceptor ligand activity has been shown to be down-regulated in failing myocardium. It is the aim of this study to test the hypothesis that also mRNA levels are down-regulated in dilated cardiomyopathy. beta 1 and beta 2 adrenoceptor ligand activities and mRNA expressions were analyzed in left ventricular biopsies from six organ donor hearts, in papillary muscles from seven patients operated on for mitral regurgitation, and in six explanted hearts as the result of dilated cardiomyophathy. mRNA levels were determined by solution hybridization. beta 1 ligand activity was decreased in the cases of mitral regurgitation (p < 0.01) and dilated cardiomyopathy (p < 0.001). beta 2 ligand activity did not differ between the three groups. mRNA expression was depressed in mitral regurgitation regarding both beta 1 (p < 0.001) and beta 2 (p < 0.01), while no differences were observed in dilated cardiomyopathy as compared to the donor hearts. The regulation of beta 1 and beta 2 adrenoceptor ligand activity and mRNA expression appears to follow a specific pattern in dilated cardiomyopathy. The specific down-regulation of beta 1 ligand activity seems to occur at a posttranslational level.

Growth hormone (GH) treatment up-regulates GH receptor mRNA levels in adipocytes from patients with GH deficiency and Prader-Willi syndrome.

We have investigated the effect of growth hormone (GH) treatment on GH receptor mRNA expression in five prepubertal children with Prader-Willi syndrome and in eight patients with GH deficiency. An adipose tissue needle biopsy was taken before and after 2-4 mo of GH treatment, and RNA was isolated from adipose tissue and from adipocytes. GH receptor mRNA levels were determined by an RNase protection/solution hybridization assay. To further assess the specificity of the assay for GH receptor mRNAs, RNA extracted from human adipose tissue was subjected to Northern blot analysis. GH treatment significantly increased GH receptor mRNA levels in adipose tissue and isolated adipocytes. Our results indicate that GH may have an important role in regulating the GH receptor in humans.

The genetic basis of glucocorticoid resistance.

Familial glucocorticoid resistance is a rare syndrome characterized by elevated levels of plasma cortisol but lacking the symptoms of Cushing's syndrome. Biochemically, the condition is characterized by a relative resistance to glucocorticoids that can be compensated for by the elevated levels of cortisol. Analysis of mutations within the receptor resulting in relative glucocorticoid resistance, both familial glucocorticoid resistance and directed mutagenesis, has identified two regions of clustered mutations in the proximity of previously identified affinity-labeled residues. In the majority of cases, the mutation affects steroid binding and transactivation to the same degree, but this is not always the case.

Na,K-ATPase receptor subunits alpha 1, alpha 2 and alpha 3 mRNA in dilated cardiomyopathy.

Na,K-ATPase receptor density has been shown to be down-regulated with decreasing ejection fraction in patients with chronic heart failure. It was the aim of the present study to determine whether down-regulation is detected also at the mRNA level. Six donor hearts and six explanted hearts due to dilated cardiomyopathy (ejection fraction 23 +/- 5%) were analyzed. RNA was extracted. Quantitative Na,K-ATPase receptor catalytic subunit alpha 1, alpha 2 and alpha 3 mRNA expression was determined by solution hybridization. No cross-reactivity occurred between the three probes. alpha 1 mRNA was expressed at about 5 and 10 times higher (p < 0.001) concentrations than alpha 2 and alpha 3 mRNA, respectively, and alpha 2 mRNA higher (p < 0.001) than alpha 3. There were no differences between right and left ventricles and between donor hearts and patients with dilated cardiomyopathy. In conclusion, Na,K-ATPase alpha 1 mRNA is the predominant subunit expressed in human myocardium. Depressed ejection fraction in dilated cardiomyopathy is not associated with changed mRNA subunit expression. Documented downregulation of Na,K-ATPase activity, therefore, may be associated with the structural and membrane-related beta subunit or posttranscriptional modification of the catalytic subunits.

Downregulation of thyroid hormone receptor subtype mRNA levels by amiodarone during catecholamine stress in vitro.

Amiodarone, a powerful antiarrhythmic drug, likely exerts its major effect by antagonism of thyroid hormone (T3), probably at the receptor level. T3 is known to regulate beta-adrenergic receptor density in the heart but the effects of sympathomimetic drugs on thyroid hormone receptors (T3R) is not known. The aim of this study was to investigate how amiodarone and isoproterenol affect T3R-mRNA in cultured cardiomyocytes. Confluent, isoproterenol pretreated, AT-1 cardiomyocytes were treated with isoproterenol free medium, amiodarone, T3 and amiodarone together with T3 for 48 hours. Solution hybridization for the determination of mRNA for T3R alpha 1, alpha 2, beta 1 and beta 2 were performed. In itself isoproterenol upregulated T3R alpha 1, T3R beta 1, T3R beta 2 (p < 0.05), but did not affect the levels of T3R alpha 2. Amiodarone and T3, respectively, downregulated T3R alpha 1, T3R beta 1, T3R beta 2 (p < 0.05), but did not affect the levels of T3R alpha 2. Amiodarone and T3, added together, upregulated T3R alpha 2 and T3R beta 1 (p < 0.05) as compared to amiodarone or T3 alone. There was an antagonistic effect between amiodarone and T3 for the regulation T3R beta 1. This is the first evidence showing that amiodarone regulates T3R-mRNA concentrations during cathecholamine stress. Isoproterenol regulation of T3R-mRNA levels provides further evidence for the close interaction between the thyroid hormone and the beta-adrenergic systems.

Evidence for the presence of functional thyrotropin receptor in cardiac muscle.

There is increasing evidence that the membrane-bound thyrotropin receptor (TSHR) may be mediating clinically important direct effects of thyrotropin (TSH) and of TSHR antibodies (TSHRab) in extra-thyroidal tissues. TSHR mRNA has formerly been detected in thyroid, retroorbital muscle and fibroblasts, peripheral lymphocytes and rodent fat. It is well known that thyroid disease may aggravate or induce heart disease, but the pathophysiological role of TSH and TSHRab is not clear. The aim of this study was to investigate if TSHR is present in cardiac muscle. Reverse transcriptase polymerase chain reactions revealed TSHR in human heart and Northern blot on extracted RNA showed a RNA species of 4.4 kb. TSH stimulation of cultured mouse AT-1 cardiomyocytes elevated the levels of intracellular second messenger 3',5'-cyclic AMP. This effect of TSH could be inhibited by TSHR antibodies. In solution hybridization levels of TSHR mRNA in AT-1 cells were 50% of mRNA in crude mouse heart. In conclusion functional TSHR is present in cardiomyocytes.

Effects of stimulatory and inhibitory thyrotropin receptor antibodies on lipolysis in infant adipocytes.

TSH is a potent lipolytic hormone for isolated human adipocytes from neonatal subjects. Crude immunoglobulin fractions from sera of patients with Graves' disease, containing stimulatory TSH receptor (TSHR) autoantibodies, significantly increased lipolysis in fat cells from infants, whereas immunoglobulin fraction from a patient with inhibitory TSHR autoantibodies (TBab) blocked TSH-induced lipolysis in a dose-dependent manner. Although TBab totally blocked the maximum lipolysis induced by TSH (10(5) mU/L), no effect was seen on isoprenaline-induced lipolysis. The maximum lipolytic response to TSH was similar to that seen with the beta-adrenoceptor agonist isoprenaline, and there was a similar cAMP increase in response to both stimulators. From these results, it is concluded that the TSHR in infant adipocytes is likely to be coupled to the adenylate cyclase system, and the lipolytic effect of TSH can be simulated by stimulatory TSHR autoantibodies or inhibited by TBab.