Huizenga, N.A.T.M.

A polymorphism in the glucocorticoid receptor gene, which decreases sensitivity to glucocorticoids in vivo, is associated with low insulin and cholesterol levels (Article)

2002-01-01T00:00:00Z

We investigated whether a polymorphism in codons 22 and 23 of the glucocorticoid (GC) receptor gene [GAGAGG(GluArg) 3GAAAAG(GluLys)] is associated with altered GC sensitivity, anthropometric parameters, cardiovascular risk factors, and sex steroid hormones. In a subgroup of 202 healthy elderly subjects of the Rotterdam Study, we identified 18 heterozygotes (8.9%) for the 22/23EK allele (ER22/23EK carriers). In the highest age group, the number of ER22/23EK carriers was higher (67-82 years, 12.9%) than in the youngest age group (53-67 years, 4.9%; P < 0.05). Two dexamethasone (DEX) suppression tests with 1 and 0.25 mg DEX were performed, and serum cortisol and insulin concentrations were compared between ER22/ 23EK carriers and noncarriers. After administration of 1 mg DEX, the ER22/23EK group had higher serum cortisol concentrations (54.8 ± 18.3 vs. 26.4 ± 1.4 nmol/l, P < 0.0001), as well as a smaller decrease in cortisol (467.0 ± 31.7 vs. 484.5 ± 10.3 nmol/l, P > 0.0001). ER22/23EK carriers had lower fasting insulin concentrations (P > 0.001), homeostasis model assess-ment?insulin resistance (IR) (index of IR, P > 0.05), and total (P > 0.02) and LDL cholesterol concentrations (P > 0.01). Our data suggest that carriers of the 22/ 23EK allele are relatively more resistant to the effects of GCs with respect to the sensitivity of the adrenal feedback mechanism than noncarriers, resulting in a better metabolic health profile. Diabetes 51:3128-3134,

A polymorphism in the glucocorticoid receptor gene, which decreases sensitivity to glucocorticoids in vivo, is associated with low insulin and cholesterol levels (Article)

2002-01-01T00:00:00Z

We investigated whether a polymorphism in codons 22 and 23 of the glucocorticoid (GC) receptor gene [GAGAGG(GluArg) --> GAAAAG(GluLys)] is associated with altered GC sensitivity, anthropometric parameters, cardiovascular risk factors, and sex steroid hormones. In a subgroup of 202 healthy elderly subjects of the Rotterdam Study, we identified 18 heterozygotes (8.9%) for the 22/23EK allele (ER22/23EK carriers). In the highest age group, the number of ER22/23EK carriers was higher (67-82 years, 12.9%) than in the youngest age group (53-67 years, 4.9%; P < 0.05). Two dexamethasone (DEX) suppression tests with 1 and 0.25 mg DEX were performed, and serum cortisol and insulin concentrations were compared between ER22/23EK carriers and noncarriers. After administration of 1 mg DEX, the ER22/23EK group had higher serum cortisol concentrations (54.8 +/- 18.3 vs. 26.4 +/- 1.4 nmol/l, P < 0.0001), as well as a smaller decrease in cortisol (467.0 +/- 31.7 vs. 484.5 +/- 10.3 nmol/l, P < 0.0001). ER22/23EK carriers had lower fasting insulin concentrations (P < 0.001), homeostasis model assessment- insulin resistance (IR) (index of IR, P < 0.05), and total (P < 0.02) and LDL cholesterol concentrations (P < 0.01). Our data suggest that carriers of the 22/23EK allele are relatively more resistant to the effects of GCs with respect to the sensitivity of the adrenal feedback mechanism than noncarriers, resulting in a better metabolic health profile.

Decreased ligand affinity rather than glucocorticoid receptor down-regulation in patients with endogenous Cushing's syndrome (Article)

2000-01-01T00:00:00Z

OBJECTIVE: Glucocorticoids (GCs) serve a variety of important functions throughout the body. The synthesis and secretion of GCs are under the strict influence of the hypothalamo-pituitary-adrenal axis. The mechanisms of action of GCs are mediated by the intracellular glucocorticoid receptor (GR). Over the years, many studies have been performed concerning the regulation of GR expression by GC concentrations. METHODS: In the present study, we determined the characteristics of the GR in peripheral mononuclear blood leukocytes (PBML) from thirteen patients with endogenous Cushing's syndrome and fifteen control subjects, using a whole cell dexamethasone binding assay. Furthermore, cortisol concentrations were determined in order to investigate a possible relationship between serum cortisol levels and receptor characteristics. RESULTS: There were no differences in mean receptor number between patients and controls. On the other hand, a significantly lower ligand affinity was identified in cells from patients with Cushing's syndrome compared with controls. A complete normalisation of the ligand affinity was observed after treatment in the only patient tested in this respect, whereas the receptor number was not affected. In patients, there was a statistically significant negative correlation between cortisol concentrations and ligand affinity, which was not found in controls. CONCLUSION: Receptor down-regulation does not occur in PBML from patients with endogenous Cushing's syndrome. On the other hand, there seems to be a diminished ligand affinity which possibly reflects receptor modification in response to exposure to the continuously high cortisol levels in patients with Cushing's syndrome. This assumption is substantiated by the fact that in one patient a normalisation of the ligand affinity after complete remission of the disease was seen.

Five patients with biochemical and/or clinical generalized glucocorticoid resistance without alterations in the glucocorticoid receptor gene (Article)

2000-01-01T00:00:00Z

Cortisol resistance (CR) is a rare disease characterized by a generalized reduced sensitivity of end-organs to the actions of glucocorticoids (GCs). GC effects are mediated by the GC receptor (GR). The molecular alterations in CR described thus far were located in the hormone-binding domain of the GR gene. Recent reports of a considerable prevalence of abnormalities in the GR in patients attending the endocrine clinic prompted us to carry out further investigations with respect to GR protein and GR gene in patients attending the endocrine clinic for a broad spectrum of complaints and biochemical evidence suggesting a CR. In the present study, we describe five patients with biochemical and clinical CR. All patients showed a diurnal rhythm of serum cortisol concentrations (albeit at a high level), an insufficient suppression of serum cortisol concentration in reaction to 1 mg dexamethasone (DEX), and variable degrees of androgen overproduction, in the absence of clinical signs and symptoms of Cushing's syndrome. Three of the four female patients presented with complaints of androgen overproduction, two of them in combination with fatigue. The other female patient had severe steroid-resistant asthma. The only male patient and his son were asymptomatic. In four patients, we investigated receptor protein characteristics on mononuclear leukocytes in a whole cell DEX binding assay and studied the ability of DEX to inhibit mitogen-induced cell proliferation in mononuclear leukocytes in vitro. In all patients investigated, we found alterations in receptor number or ligand affinity and/or the ability of DEX to inhibit mitogen-induced cell proliferation. To investigate the molecular defects leading to the clinical and biochemical pictures in these patients, we screened the GR gene using PCR/single-strand conformational polymorphism/sequence analysis. No GR gene alterations were found in these patients. In conclusion, the five patients described had clinical and biochemical evidence of CR, but no abnormalities were demonstrated in the GR gene. Probably, as yet undefined alterations somewhere in the cascade of events starting with ligand binding to the GR protein, and finally resulting in the regulation of the expression of GC responsive genes, or postreceptor defects or interactions with other nuclear factors form the pathophysiologic basis of CR in these patients.

Variability in Glucocorticoid Sensitivity: The role of the glucocorticoid receptor (Doctoral Thesis)

1998-10-07T00:00:00Z

The adrenal glands are paired organs localized superomedially to the kidneys. Each gland is composed of a cortex and a medulla which are embryologically and functionally distinct. The cells in the medulla are the principal site of adrenaline production in the body. In the adrenal cortex, the adrenal steroids are synthesized. The cortex is composed of three histologically different zones, the zona glomemlosa, which is the outermost portion, the zona fasciculata and the zona reticularis, which is the innermost part. The adrenal cortex produces aldosterone, which is the principal mineralocorticoid, the glucocorticoid cortisol and the adrenal androgens. All steroid honnones produced by the adrenal cortex, are derived from cholesterol. About 80% of the cholesterol used for steroid synthesis is provided by circulating plasma lipoproteins (1- 3). The cells of steroidogenic tissues can also synthesize cholesterol de novo from acetate or mobilize intracellular cholesteryl ester pools (1-3). A series of enzymatic steps convert cholesterol into steroids with glucocorticoid, mineralocorticoid or androgen activity.

Interperson variability but intraperson stability of baseline plasma cortisol concentrations, and its relation to feedback sensitivity of the hypothalamo-pituitary-adrenal axis to a low dose of dexamethasone in elderly individuals (Article)

1998-01-01T00:00:00Z

In the present study, we investigated whether the negative feedback action of glucocorticoids (GCs) on the hypothalamo-pituitary-adrenal (HPA) axis changes with age. We performed a 1-mg dexamethasone (DEX) suppression test in 216 healthy elderly individuals. To investigate individual variability of feedback sensitivity in more detail, 2.5 yr later a 0.25-mg DEX suppression test was carried out in 164 of the same individuals. We investigated whether there was an effect of age or gender on both basal and post-DEX cortisol levels, as well as on the concentration of DEX. Furthermore, we examined whether the reactions to the two doses of DEX differed, and whether indications for an intraperson stability of baseline cortisol levels could be found. Neither the pre- nor the post-1-mg DEX plasma cortisol concentrations showed a correlation with age, and there were no differences between men and women. The same was true for the pre- and post-0.25-mg DEX cortisol concentrations. In reaction to 1 mg DEX, over 90% of the subjects investigated showed a cortisol suppression to levels below 50 nmol/L. After the administration of 0.25 mg DEX, there was a much wider range in post-DEX cortisol concentrations. After the administration of 1 mg DEX, there was a significant correlation between liver function parameters and plasma DEX concentrations in males, and there was a correlation between body mass index and plasma DEX concentration in females. Plasma DEX concentrations after the administration of 1 mg and 0.25 mg DEX were closely correlated within subjects (P < 0.001). There was an intraindividual stability of serum cortisol levels determined at an interval of 2.5 yr. Furthermore, the individuals with the highest baseline cortisol concentrations also had the highest post-0.25-mg DEX cortisol concentrations, indicating a close relationship between basal cortisol levels and the feedback sensitivity of the HPA axis to a low dose of DEX. These observations suggest a genetic influence on the set point of the HPA axis. Aging does not seem to lead to a change in HPA activity as measured by early morning total cortisol levels. Also, no changes in the sensitivity of the feedback system to DEX were observed with age. DEX metabolism is influenced by liver function (in males) and by body mass index (in females).

A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo (Article)

1998-01-01T00:00:00Z

We investigated whether a polymorphism at nucleotide position 1220, resulting in an asparagine-to-serine change at codon 363 in the glucocorticoid receptor (GR) gene is associated with an altered sensitivity to glucocorticoids. In a group of 216 elderly persons, 13 heterozygotes for the N363S polymorphism were identified by PCR/single strand conformation polymorphism analysis. In 2 dexamethasone (DEX) suppression tests (DSTs), using 1 and 0.25 mg DEX, the circulating cortisol and insulin concentrations were compared between N363S carriers and controls. In the 1-mg DST, there were no differences between N363S carriers and controls, with respect to adrenal suppression, but there was a significantly higher (P < 0.05) insulin response in N363S carriers. In the 0.25-mg DST, a significantly larger (P < 0.05) cortisol suppression and higher (P < 0.05) insulin response were seen in N363S carriers. Comparison of blood pressure, body mass index (BMI), and bone mineral density (BMD) between the N363S carriers and controls showed that N363S carriers had a higher (P < 0.05) BMI but normal blood pressure. There was an obvious trend towards lower age-, BMI-, and sex-adjusted BMD in the lumbar spine in N363S carriers. GR characteristics measured in 41 controls and 9 N363S carriers in peripheral mononuclear leucocytes showed no differences between N363S carriers and controls, with respect to GR number and ligand binding affinity. However, there was a trend towards greater sensitivity to DEX in the carriers' lymphocytes, in a mitogen-induced cell proliferation assay. In transfection assays, the capacity of the codon 363 variant to activate mouse mammary tumor virus promotor-mediated transcription in COS-1 cells was unaltered, when compared with the wild-type GR. We conclude that in 6.0% of our study population, a polymorphism in codon 363 of the GR gene was found. Individuals carrying this polymorphism seemed healthy at clinical examination but had a higher sensitivity to exogenously administered glucocorticoids, with respect to both cortisol suppression and insulin response. Life-long exposure to the mutated allele may be accompanied by an increased BMI and a lowered BMD in the lumbar spine but does not affect blood pressure.

Human adrenocorticotropin-secreting pituitary adenomas show frequent loss of heterozygosity at the glucocorticoid receptor gene locus (Article)

1998-01-01T00:00:00Z

Corticotropinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance. As cortisol resistance can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotropinomas to cortisol is also caused by de novo mutations in the GR gene. We screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing's disease for mutations using PCR/single strand conformation polymorphism analysis. In a previous study, we identified 5 polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus. Except for 1 silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotropinomas from these patients. Of the 22 patients, 18 were heterozygous for at least 1 of the polymorphisms. In 6 of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only 1, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically nonfunctioning, 8 prolactinomas, and 9 GH-producing adenomas) and found evidence for LOH in only 1 prolactinoma. In six patients LOH was found at the GR gene locus (chromosome 5) in DNA derived from adenoma cells. Our observations indicate for the first time that LOH at the GR gene locus is a relatively frequent phenomenon in pituitary adenomas of patients with Cushing's disease. This might explain the relative resistance of the adenoma cells to the inhibitory feedback action of cortisol on ACTH secretion. The specificity of the GR LOH to corticotropinomas supports this concept. Somatic mutations of the GR are not a frequent cause of relative cortisol resistance in these cells.

Differential hormone-dependent transcriptional activation and -repression by naturally occurring human glucocorticoid receptor variants (Article)

1997-01-01T00:00:00Z

The molecular mechanisms underlying primary glucocorticoid resistance or hypersensitivity are not well understood. Using transfected COS-1 cells as a model system, we studied gene regulation by naturally occurring mutants of the glucocorticoid receptor (GR) with single-point mutations in the regions encoding the ligand-binding domain or the N-terminal domain reflecting different phenotypic expression. We analyzed the capacity of these GR variants to regulate transcription from different promoters, either by binding directly to positive or negative glucocorticoid-response elements on the DNA or by interfering with protein-protein interactions. Decreased dexamethasone (DEX) binding to GR variants carrying mutations in the ligand-binding domain correlated well with decreased capacity to activate transcription from the mouse mammary tumor virus (MMTV) promoter. One variant, D641V, which suboptimally activated MMTV promoter-mediated transcription, repressed a PRL promoter element containing a negative glucocorticoid-response element with wild type activity. DEX-induced repression of transcription from elements of the intercellular adhesion molecule-1 promoter via nuclear factor-kappaB by the D641V variant was even more efficient compared with the wild type GR. We observed a general DEX-responsive AP-1-mediated transcriptional repression of the collagenase-1 promoter, even when receptor variants did not activate transcription from the MMTV promoter. Our findings indicate that different point mutations in the GR can affect separate pathways of gene regulation in a differential fashion, which can explain the various phenotypes observed.