The first objective in this study was to learn whether the reduction in GH proteins (29
) or mRNA (30
) reported by other workers in rats and humans was due to losses in GH antigen-bearing or GH mRNA-bearing cells. Our studies agree with those of previous workers who showed that pituitary GH protein levels decline to about 50% in middle-aged female rats (29
). This decline in pituitary GH is associated with a decline in serum GH levels (29
), which, in turn, affects GH-dependent gene expression in other organs in the aging rat (48
) and eventually results in a decline in GH cell number by 20 months of age (32
). The new findings in our study include the fact that this decline in GH cell number can be seen as early as 12–14 months of age. We also report a 50% drop in the expression of GHRH R mRNA and a 60–70% drop in target cells for GHRH R. Middle age in female rats corresponds to the perimenopausal age group in women, when earlier reports had shown a maximal loss of somatotropes in human pituitaries (27
After confirmation that reduced GH functions reported by others were caused in part by fewer GH antigen- or mRNA-bearing cells, the second objective in our study was to learn whether DHEA could reverse these aging effects by direct actions on pituitary cells. DHEA treatment in vitro significantly increased the percentages of immunolabeled soma-totropes only in aging animals and restored percentages of GH-bearing cells to levels seen in young animals.
Once the in vitro
data showed restorative effects of DHEA on aging GH cells, the studies sought in vivo
evidence that this adrenal hormone had similar effects. The objective was to learn whether a brief exposure period to DHEA would stimulate the aging GH cell population so that they would be better able to respond to a bolus of GHRH in vivo
. The GHRH bolus was given because of the pulsatile nature of GHRH secretion and the recognition that endogenous pulses of GHRH could mask the results in either DHEA- or vehicle-treated animals. This study reports that DHEA administration for 2.5 d significantly enhanced serum GH (1 h after GHRH exposure), and this correlated well with the significantly increased expression of GH proteins and GHRH receptivity (detected by binding to biotinylated GHRH). Our in vivo
studies agree with a recent report by Suarez et al.
), who showed that DHEA potentiated GHRH-induced GH release (not basal GH release) by modulating cAMP production in cultured young rat AP cells.
DHEA did not enhance GH mRNA levels in vivo
. This may reflect in vivo
influences by other factors that affect GH cells, including GHRH, somatostatin, ghrelin, IGF-I, and sex steroids (including metabolites of DHEA) (36
). The endogenous levels of the some of these factors, the expression of their specific receptors (51
), and the sensitivity of the pituitary to them may also change during aging (52
). The interpretation of an in vivo
study must recognize that DHEA treatment may also affect each of these above-mentioned factors either directly or indirectly.
The tests of DHEA actions on GHRH R expression were limited to the in vivo study. The reason for this was because of the rapid loss in transcripts with time in culture in these defined medium. In this context, it should be noted that tests of stability of the GH transcript in culture were made in parallel to those in which GHRH R mRNA was tested. There was an approximately 15% loss in GH transcripts detected during the first 3 h of culture, with no significant losses during the subsequent 24-h culture period.
Thus, the first sets of studies agreed that DHEA restored GH cell numbers in vivo
and in vitro
, especially if they were detected by immunolabeling. Under the experimental conditions of our study, DHEA did not affect the percentages of somatotropes from young diestrous animals in the wide dose range tested, which agrees with one in vivo
). Halmy et al.
) reported that a 30-d implantation of 80 mg DHEA in mature female rats produced no change or a decrease in GH content.
However, two studies have reported enhancing effects of DHEA on the young pituitary population (43
). Suarez et al.
) showed that either DHEA or estrogen given to young female rats in vivo
caused hyperplasia in the pituitary. Simard et al.
) also reported DHEA enhancement of GH protein expression. This could be explained by different times of treatment and also the fact that the diestrous rats in our study were already expressing the highest levels of GH normally seen during the cycle (24
) and might be considered to be in an estrogen-rich environment. Simard et al.
) used a group of mixed cycling female rats, which might include metestrous rats, which express about 50–70% of the GH proteins and mRNA seen in the other stages (24
). Collectively, these studies suggest that DHEA may have a more potent enhancing effect on GH in rats when administered in a low estrogen state. The reason our study compared aging animals with diestrous animals was because diestrus is a stage when ER expression is maximal (56
). This hypothesis is supported by recent reports in humans that showed that responses to DHEA varied with the estrogenic environment (58
). In this study DHEA enhanced GH secretion only in patients who were not receiving estrogen replacement therapy. Thus, it is possible that the effects of DHEA would be seen more often in estrogen-deprived conditions.
Once the in vivo
and in vitro
studies showed DHEA enhancement, we began tests designed to learn whether DHEA worked directly through its own receptor or after metabolism to estrogens. In the first of these studies, we tested estrogen directly on the expression of GH proteins and mRNA by aging pituitary cells. Our studies showed a potent restorative effect of estrogens on aging GH cells. This agrees with a previous study (55
) in young female rats that showed that an antiestrogen, LY156758, prevented DHEA enhancement of GH protein expression in vitro
after 72 h. Comparing DHEA and estrogen showed that the latter steroid was effective at lower doses. However, DHEA was effective at concentrations comparable to physiological circulating levels of DHEA in the rat (~1 nM) (59
). The series of inhibitors tested supported the hypothesis that DHEA did not work directly on its receptor (44
) and that it must be metabolized to estrogens before it restores the expression of GH cell proteins. The enzymes necessary for DHEA conversion to downstream metabolites [3β-HSD (60
) and 17β-HSD (61
)] have been previously reported to be expressed in the pituitaries of young female rats. Furthermore, aromatase enzyme, which mediates the final conversion of any of the DHEA metabolites to estrogens, is expressed in both pituitary cells and endothelial cells of the pituitary of young (62
) and aged rats (63
The results of these studies of estrogenic effects correlate well with those of other studies that show estrogen involvement in GH gene expression. Aromatase knockout mice have decreased expression of GH, GHRH R, and Pit-1 mRNA, and estrogen replacement enhanced the expression of all of these genes (64
). There is a coincident surge in GH along with the LH surge at ovulation in sheep, indicating that rising estrogen levels enhance GH secretion (65
). Studies from our laboratory in young cycling female rats across the estrous cycle showed an increase in GH mRNA during diestrus and proestrus, when serum estrogen is rising (24
Thus, collectively, our data suggest that DHEA metabolites are aromatized and act on ERs. However, there are reports suggesting that DHEA could act on ERs without conversion to estrogens. Nephew et al.
) have shown that DHEA can directly bind to ERs in vivo
(even inhibiting estrogen binding to its own receptor) and cause dimerization of ERs, which are functionally active in yeast. However, these actions required approximately 1000 times higher concentrations of DHEA than E2
. Another study has shown direct binding by DHEA to ERβ in transfected human embryonic kidney 293 cells, which do not have the required converting enzymes for its bioconversion to estrogen (67
). Other studies show direct binding of DHEA to ERα (68
) and DHEA mediating ER-estrogen response element-dependent transcriptional activity, independent of its conversion to estradiol (69
). Estrogen has been shown to be stimulatory on the 5′-promoter activity of GH gene using the MtT/S rat pure somatotrope cell line (70
). Somatotropes are known to express both ERα and ERβ (57
). Because ICI 182,780 blocks both ERα and ERβ, it cannot be inferred from this study whether one or both of these ER subtypes are involved in DHEA actions. Because our studies used mixed cultures of pituitary cells, the possibility of DHEA acting through ERs expressed in gonadotropes, lactotropes, or other cell types to mediate these changes in a paracrine fashion must also be considered.
Finally, these data do not rule out conversion to androgens, because DHEA-mediated restoration of somatotropes could occur with minimal conversion of DHEA to estrogen, and estrogen mediates the same effect at doses that are 1/10th or 1/100th the concentration of DHEA. Although androgen receptors have been shown to be expressed in rat AP (71
), the role of androgens in GH expression in females has not been studied extensively. A 72-h incubation with dihydrotestosterone (0.01–50 nM) and testosterone (0.01–1000 nM) did not seem to affect spontaneous GH release in pituitary cultures from pituitaries of cycling female rats taken from random stages of the cycle (55
). Some of the metabolites of DHEA, such as androstenediol (byproduct of DHEA by 17β-HSD), 3α-androstenediol, and 3β-androstenediol (derivatives from dihydrotestosterone), have weak estrogenic activity (72
). Thus, although we cannot rule out actions mediated by androgen receptors at this point, the total blocking effects of the aromatase inhibitor in our study strongly supports the hypothesis that the enhancing actions of DHEA on aging somatotropes are dependent on its aromatization to estrogen metabolites.
Circulating levels of DHEA are approximately 1 nM in rats (59
). DHEA may be synthesized de novo
in various regions of the brain as a neurosteroid. DHEA levels are three times higher in the hypothalamus than in the cortical regions of the brain (74
), which might provide a route for DHEA-mediated regulation of the pituitary. It is interesting to note that the precursor for DHEA, pregnenolone sulfate, declines in the aging rodent brain (75
), and it is not known whether this decline plays a role in the decline of GH gene expression in aging.
In summary, this is the first study in female rats to demonstrate a potential pathway within the aging pituitary by which DHEA or its estrogen metabolite may restore some of the expression of GH in aging somatotropes to levels seen during peak periods of the cycle in young animals. The results indicate that both estrogen and its precursor, DHEA, positively influence GH gene expression in aging. Collectively, this study shows a therapeutic potential for DHEA in the restoration of GH cell functions that might benefit frail elderly with low GH levels.