C. Richard Parker

Expression and Subcellular Distribution of the β-Isoform of the Human Glucocorticoid Receptor1

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGRα and hGRβ, that differ at their carboxy-termini. In contrast to the well characterized hGRα isoform, which modulates gene expression in a hormone-dependent fashion, the biological significance of hGRβ has only recently begun to emerge. We and others have shown that the hGRβ messenger RNA transcript is widely expressed in human tissues and that the hGRβ protein functions as a dominant negative inhibitor of hGRα in transfected cells. Unfortunately, these initial studies did not determine whether the hGRβ protein was made in vivo. Such analyses are hindered because available anti-hGR antibodies cannot discriminate between the similarly sized hGRα and hGRβ proteins. Therefore, to investigate the expression of the hGRβ protein, we have produced an antipeptide, hGRβ-specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the car...

Chronic hyperinsulinemia and the adrenal androgen response to acute corticotropin-(1–24) stimulation in hyperandrogenic women

OBJECTIVE Many women with androgen excess demonstrate elevated circulating insulin levels independent of obesity. In addition, in these women some investigators have demonstrated a negative correlation between the circulating levels of the adrenal androgens, dehydroepiandrosterone or dehydroepiandrosterone sulfate and insulin. The mechanism by which insulin decreases adrenal androgens is unclear. The objective of this study was to determine whether chronic hyperinsulinemia in hyperandrogenic women results in an alteration in the adrenocortical response to corticotropin, resulting in decreased androgen secretion. STUDY DESIGN We studied seven hyperandrogenic women with severe chronic hyperinsulinemia and eight hyperandrogenic normoinsulinemic patients. Nine healthy women served as controls for the basal hormonal levels and the response to a 3-hour, 100 gm oral glucose tolerance test. In all subjects insulin and glucose were measured hourly during the oral glucose tolerance test and the baseline sample was assayed for total testosterone, dehydroepiandrosterone sulfate, dehydroepiandrosterone, androstenedione, sex hormone-binding globulin, and free testosterone. In hyperandrogenic women cortisol, dehydroepiandrosterone, and androstenedione were also measured, before and 60 minutes, after acute intravenous administration of 0.25 mg corticotropin (1-24). RESULTS There was no difference in the response of cortisol, dehydroepiandrosterone, or androstenedione to corticotropin-(1-24) stimulation between normoinsulinemic and hyperinsulinemic hyperandrogenic patients. As defined, the hyperinsulinemic patients had higher basal and peak insulin levels and areas under the insulin response curve compared with the normoinsulinemic patients or controls. Total testosterone and dehydroepiandrosterone did not differ among study groups. As expected, hyperandrogenic patients demonstrated lower sex hormone-binding globulin activity and higher free testosterone, androstenedione, and dehydroepiandrosterone sulfate basal levels compared with controls. CONCLUSIONS The results of this study do not support the hypothesis that chronic hyperinsulinemia in hyperandrogenic patients significantly inhibits the andrenocortical secretion of dehydroepiandrosterone or androstenedione in response to corticotropin stimulation or the basal circulating adrenal androgen levels. Additional studies, including a greater number of patients, may be needed to fully establish these conclusions.

Regulation of Extraadrenal Steroid 21-Hydroxylase Activity: INCREASED CONVERSION OF PLASMA PROGESTERONE TO DEOXYCORTICOSTERONE DURING ESTROGEN TREATMENT OF WOMEN PREGNANT WITH A DEAD FETUS

We measured deoxycorticosterone (DOC) and progesterone (P) in plasma of 47 women pregnant with a dead fetus and sequentially throughout gestation in 35 women pregnant with a live fetus. When P levels in plasma were low, the plasma levels of DOC in women pregnant with a dead fetus varied but usually were similar to those in women pregnant with a live fetus. However, when P levels were high, the levels of DOC in some women pregnant with a dead fetus were considerably lower than those in women pregnant with a live fetus. To test whether this finding was due to loss of transfer of DOC from fetus to mother or else loss of extraadrenal steroid 21-hydroxylase activity in the mother after death of the fetus, we conducted several studies. The levels of P and DOC in plasma of one woman remained constant from 30 min after fetal death until delivery occurred 13 h later. Estrogen treatment of four women pregnant with a dead fetus brought about an increase in plasma levels of DOC in three of the women. In one woman the ratio of plasma DOC to P was 0.015, a value similar to that found before fetal death, but was 0.003 after fetal death but before estrogen treatment. In two women pregnant with a dead fetus the transfer constants of conversion of plasma P to DOC were 0.011 and 0.005 before, and 0.024 and 0.013, respectively, during estrogen treatment. In one woman pregnant with a deformed fetus with adrenal agenesis, the metabolic clearance rates of DOC before and during estrogen treatment were similar, whereas the plasma production rates of DOC were 2.75 before and 4.31 mg/24 h during estrogen treatment. We suggest that (a) the DOC in plasma of near-term pregnant women arises in part by extraadrenal 21-hydroxylation of plasma P and (b) estrogen stimulates steroid 21-hydroxylase activity in extraadrenal tissues.

Biopsychological Markers of Distress in Informal Caregivers

Background. Thirty caregiving wives participated in a study of caregiving distress and negative mood (depressive symptoms) by making diary entries on stressful caregiving situations and collecting saliva samples 4 times a day. At the end of the 7-day study period, caregivers’ salivary cortisol production was compared with their diary entries and correlated with pencil and paper self-report scores of caregiver distress and depressive symptoms. Findings. Despite the inability to control a number of factors thought to confound cortisol production (exercise, smoking, alcohol ingestion, and prescription medications), there was a statistically significant difference between No Caregiving and Caregiving cortisol, F(1,739) = 7.67, P = 0.006, with cortisol production higher when caregiving events occurred. However, efforts to code specific types of caregiving situations (e.g., 1 = indirect care; 4 = AD problem behavior care) did not further differentiate cortisol production. Although caregivers’ self-reports for the same 7-day period indicated they were depressed, pencil-and-paper measures of distress and negative affect were not significantly correlated with cortisol production. Conclusions and Recommendations. The finding that this caregiving group was significantly stressed by caregiving, as evidenced by increased cortisol production during caregiving episodes, verifies the importance of further exploration of specific caregiving situations as contributory factors in caregiver health and well-being. In that saliva is a relatively economical and comparatively noninvasive biological data source for community-based stress studies, methodological limitations of the study are identified and 5 recommendations are made for future biological marker studies of caregiver distress in community-based settings.

Adrenocortical secretion of dehydroepiandrosterone in healthy women: highly variable response to adrenocorticotropin.

Excess adrenal androgen (AA) levels are observed in 25--50% of women with the polycystic ovary syndrome (PCOS), and AA excess in PCOS may represent selection bias. Thus, it is possible that AA secretion among the general population is highly variable, and that those women who are predisposed to secreting greater amounts of AA have a greater probability of having PCOS. We now hypothesize that the levels of AAs are highly variable among normal nonhyperandrogenic women, and that this heterogeneity is the result of a variable response of AAs to ACTH stimulation. To test this hypothesis we prospectively studied the response of dehydroepiandrosterone (DHA) and cortisol (F) to a 60-min acute stimulation with ACTH-(1--24) in 56 healthy eumenorrheic nonhirsute healthy women with a mean age of 28.9 yr (range, 20--37 yr.) and a mean body mass index (BMI) of 29.2 kg/m(2) (18.2--46.2 kg/m(2)). Baseline samples and poststimulation samples were assayed for DHA and F. The basal and ACTH-stimulated levels of DHA, but not those of F, were negatively correlated with age, although neither the basal nor ACTH-stimulated responses of DHA and F varied with BMI. After controlling for age, the basal F level was negatively correlated to its net increment (i.e. Delta F; r = -0.54; P < 0.001), whereas there was no significant relationship between basal DHA and Delta DHA. We also compared the intersubject variability (coefficient of variation) for basal and stimulated levels of DHA and F. For basal (DHA(0)), 60 min (DHA(60)), and net increment in (Delta DHA) DHA levels, the coefficients of variation were 67.9%, 61.4%, and 76.0%, respectively; for F(0), F(60), and Delta F, they were 40.4%, 16.9%, and 31.3%, respectively. The variance in Delta DHA was significantly higher, and the variance in F(60) was significantly lower than that in all other variables; DHA(0), DHA(60), F(0), and Delta F had similar variances. In conclusion, in our population of healthy reproductive-aged women we observed that both basal and ACTH-stimulated levels of DHA after ACTH-(1--24) stimulation had significantly greater intersubject variance (approximately 60--70%) compared with the basal and poststimulation levels of F (approximately 15--40%). These data support the hypothesis that among normal women, AA (i.e. DHA) levels are highly variable compared to those of F. In addition, the intersubject variability in DHA levels is at least in part due to a variable response of AAs to ACTH stimulation. Whether the AA excess frequently observed in PCOS is due to the greater risk of those women with higher AA levels, basally and after ACTH stimulation, remains to be confirmed.

Steroid secretion by ACTH-stimulated human fetal adrenal tissue during the first week in organ culture

Fetal adrenal tissue has been reported to lose its in vivo secretory pattern by virtue of a loss of fetal zone cells after the first week in culture. Consequently, we studied the steroidogenic capacity and the responsiveness to ACTH of human fetal adrenal tissue during the first week in organ culture. The culture medium was removed daily and assayed for cortisol and dehydroisoandrosterone sulfate (DS). First, as the concentration of ACTH in the medium was increased from 0 to 1 micrograms/ml steroid secretion increased. When tissue fragments were maintained in the absence of ACTH for 3 to 4 days, there was a striking increase in steroid secretion upon addition of ACTH to the medium, with larger rates of secretion of cortisol than DS being observed. Second, the steroidogenic capacity of the separate zones of the fetal adrenal gland was assessed. Tissue from the fetal zone secreted large amounts of DS and small amounts of cortisol, whereas neocortex tissue secreted similar quantities of DS and cortisol. Third, fetal zone tissue was maintained the absence of ACTH for 4 days and thereafter ACTH was added to the media for an additional 6 days. In this experiment, there was a marked increase in DS secretion rate after the addition of ACTH and a smaller increase in cortisol secretion.

Release of immunoreactive α-MSH by synaptosome-enriched fractions of homogenates of hypothalami

Immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be concentrated in a synaptosome-enriched fraction prepared by differential centrifugation of rat hypothalamic homogenates. The release of the hormone from this preparation was investigated. After incubation, the synaptosomes were isolated by ultrafiltration and alpha-MSH in the ultrafiltrate was determined by radioimmunoassay. Particle-bound alpha-MSH, recovered by extraction with acid ethanol, and alpha-MSH released from the synaptosome preparation, were immunologically similar to synthetic alpha-MSH and had an accompanying melanotropic activity. Less than 10% of the particle-bound alpha-MSH was released during incubation in 0.32 M sucrose. However, in the presence of 2 mM Ca2+, alpha-MSH release increased with increasing concentrations (30-150 mM) of K+. The stimulatory effect of 60 mM K+ was complete within 2 min and was potentiated by increasing Ca2+ concentrations over the range of 0 to 2 mM. K+-induced release of alpha-MSH was independent of temperature from 1 to 30 degrees C, and neither glucose (10 mM) nor dopamine (10(-10)-10(-2) M) had any effect on the release of the peptide. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains a releasable pool of immunoreactive alpha-MSH that is mobilized by depolarizing concentrations of K+ in a Ca2+-dependent manner.

The effect of oral contraceptive treatment on the serum concentration of dehydroisoandrosterone sulfate

Dehydroisoandrosterone sulfate (DS), the major C19-steroid in the human circulation, was measured in serum obtained from blood samples collected daily (8 to 10 A.M.) throughout the menstrual cycles of eight normal, presumably ovulatory women and daily throughout the treatment cycles in four women taking an oral contraceptive (norethindrone, 1 mg., plus mestranol, 80 mcg.). The serum concentrations of DS in the ovulatory women ranged from 1,025 to 4,200 ng. per milliliter; mean, 2,062 +/- 137 ng. per milliliter (mean and standard error; n = 213). Serum DS concentrations during the follicular and luteal phases of the menstrual cycles of these women were similar. In women taking the oral contraceptive, the plasma DS concentrations ranged from 475 to 1,400 ng. per milliliter (mean, 895 +/- 83; n = 119). The 24 hour secretory pattern of DS was evaluated in one subject during a nontreatment cycle and again after 20 days of oral contraceptive treatment. In this subject, the mean serum DS level was 34 per cent lower during oral contraceptive treatment than the level before treatment. The decrease in the serum concentration of DS during oral contraceptive treatment likely results from a reduction in adrenal DS secretion since DS secretion by the normal human ovary is negligible and ovarian dehydroisoandrosterone secretion is small. Therefore, it is likely that the reduced serum DS levels in women taking oral contraceptives are the consequence of reduced adrenal secretion of DS resulting from reduced release of adrenocorticotropic hormone.

Effects of intrapartum stress on fetal adrenal function

OBJECTIVE Our purpose was to characterize the fetal adrenal response to acute intrapartum stress in otherwise uncomplicated pregnancies. STUDY DESIGN Term infants (n = 61) diagnosed as having fetal distress during labor, as indicated by heart rate abnormalities, and delivered of women having normal pregnancies were pair-matched with 61 infants showing no signs of fetal distress or acidemia (controls) on the basis of gestational age and delivery method. Umbilical cord serum dehydroepiandrosterone sulfate and cortisol were measured, and the data were analyzed by two-tailed t test, Fishers exact test, analysis of variance, and Tukeys multiple range test. RESULTS Distressed infants had lower serum levels of dehydroepiandrosterone sulfate (3992 +/- 246 nmol/L, mean +/- SE) than control infants (4853 +/- 283 nmol/L); distressed infants also had higher levels of cortisol (412 +/- 17 nmol/L) than did control infants (299 +/- 16 nmol/L). The dehydroepiandrosterone sulfate/cortisol ratios in control infants (17.7 +/- 1.2) were almost twice those of distressed infants (10.8 +/- 0.9). These same relationships also were noted when the infant pairs were segregated according to whether delivered vaginally (21 pairs) or by cesarean section (40 pairs). The abnormalities in steroid levels in the distressed infants were independent of the presence or absence of acidemia. CONCLUSION Intrapartum stress acutely alters fetal adrenal steroidogenesis, leading to increased production of cortisol and decreased secretion of dehydroepiandrosterone sulfate.

The adrenal genetic puzzle: how do the fetal and adult pieces differ?

The basis for the steroidogenic differences between the human fetal adrenal (HFA) and adult adrenal is not well defined. However, gene expression clearly plays a critical role in defining their distinct steroidogenic and structural phenotypes. We used DNA microarrays to compare expression levels of several thousand transcripts between the HFA and adult adrenal gland. Gene profiling was done using seven independent microarrays that contained between 7075 and 9182 cDNA elements. Twenty-five transcripts were found to have a greater than 5-fold difference in expression between HFA and adult adrenals. The largest differences were observed for transcripts that encode insulin-like growth factor-II (IGF-II) (25-fold higher in HFA) and 3β hydroxysteroid dehydrogenase (3βHSD) (21-fold higher in adult). The vast majority of the 25 transcripts have not been studied with regard to adrenal function. We also determined the transcripts that had the highest signal intensities, which is an approximate measure of expression level, for both the fetal and adult adrenal RNA samples. The enzyme 24-dehydrocholesterol reductase, which is involved in cholesterol biosynthesis, exhibited the highest signal intensity for fetal adrenal RNA. For adult adrenal mRNA, the expression of 11β-hydroxylase transcripts was found to have the highest signal intensity ranking. Overall, 10 of the top 20 highest signal intensities were similar for adult and fetal adrenal transcripts. The gene profile data for fetal vs. adult adrenal glands should provide valuable information that could help define mechanisms involved in adrenal growth and development.