Plasma ACTH in Rats Following Medical Adrenalectom Robert B. Mims, MD and Wilma Beard, BS Los Angeles, California
We have previously reported that surgically adrenalectomized nonstressed rats showed delayed secretion of the expected increase in plasma ACTH concentrations following adrenalectomy.1 However, increased plasma ACTH concentrations were always detected when these animals were subjected to severe stress, and their responsiveness increased again seven days or more following surgical adrenalectomy. We are now reporting similar results which were obtained following prolonged systemic glucocorticoid administration (medical adrenalectomy) in similar groups of rats. Materials and Methods Several groups of Holtzman albino female rats weighing 200 to 250 gm began as experimental subjects at the same time for this study. The animals were housed three per cage with constant light and room temperature and were fed rat chow (Purina) and water ad libitum. All animals received tap water prior to adrenalectomy, but 0.9 percent saline was used afterwards, which helped maintain a high survival rate in each group. All rats were weighed daily. Other details concerning adrenalectomy, anesthesia, stressed Supported by USPH Training Grant AM-05176. Requests for reprints should be addressed to Dr. Robert B. Mims, Director, Home Health Services, USC School of Medicine and the LAC/USC Medical Center, Los Angeles, Calif 90033.
and non-stressed blood collection, animal sacrifice, and the biological ACTH assay have previously been described. 2 In a preliminary experiment, equivalent results were obtained by peritoneal, intramuscular, and subcutaneous administration of the same type and dose of glucocorticoids. Subsequently, all animals received glucocorticoids by the subcutaneous route, and injections were given at 1600 daily for 14 days. Glucocorticoid preparations were mixed with normal saline so that each rat received a 10 mg dose in a 0.5 ml volume. Control rats received only 0.5 ml of saline. This protocol had previously caused severe adrenal atrophy in similar groups of rats, but smaller doses proved unsatisfactory. The adrenal glands were re-
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, VOL. 69, NO. 3, 1977
moved, rapidly trimmed, and weighed wet on a torsion balance after collecting blood in each group of rats.
Results The Effects of Various Glucocorticoid Preparations Table 1 shows the effects of various glucocorticoid preparations given 10 mg/day subcutaneously for 14 days, on adrenal and body weight, plasma corticosterone, and plasma ACTH. Cortisone acetate was the most effective, and hydrocortisone succinate or phosphate the least effective in causing adrenal atrophy and adrenal pituitary suppression. Cortisone and hydrocortisone acetate deposits were obvious at the injection sites up to two to three weeks following the injections. These deposits were removed in sacrificed animals and confirmed as cortisone or hydrocortisone by chemical analysis. Succinate and phosphate glucocorticoid injections did not cause visible deposits as described above for acetate preparations. Adrenal weights decreased 48 percent and 68 percent following hydrocortisone and cortisone acetate injections, but only 23 percent for hydrocortisone succinate and phosphate preparations. Body 145
weight increased 12 percent in control rats and 4 percent in succinatephosphate injected animals during the two injection intervals, but decreased 20 percent and 35 percent in hydrocortisone and cortisone acetate injected animals. Adrenal mass and body weights did not return to basal values or those of the control animals until four to six weeks after discontinuing the glucocorticoid acetate injections.
Plasma ACTH Following Surgical Adrenalectomy in Rats Figure 1 shows plasma ACTH results in a typical group of stressed and non-stressed rats following surgical adrenalectomy. Plasma ACTH was unexpectedly low initially in the nonstressed surgical adrenalectomized groups, but it increased gradually and stabilized during the next four to six weeks. Plasma ACTH was always elevated in the groups which were subjected to severe stress. The severely stressed group of rats also appeared to have two major secretory phases, ie, an immediate and a more exaggerated delayed phase.
Plasma ACTH Following Medical
Adrenalectomy in Rats Figure 2 shows plasma ACTH results in groups of stressed and nonstressed rats following two weeks of subcutaneous cortisone acetate injections (medical adrenalectomy). The 146
stressed group of medically adrenalectomized rats showed a sluggish plasma ACTH response during the first two weeks after discontinuing the injections, but they were able to respond adequately or excessively to stress thereafter. In contrast, the groups of non-stressed medically adrenalectomized rats had no detectable plasma ACTH up to six weeks after discontinuing cortisone acetate injections.
Discussion The first part of this study demonstrated that subcutaneously administered cortisone acetate was the most effective in causing severe adrenal atrophy, adrenal failure, pituitary suppression, and general body catabolism. These effects appeared to be more related to the acetate radical which apparently caused delayed absorption at the injection sites for a few weeks. In contrast, succinate and phosphate preparations caused less catabolic and suppressive effects, left no visible deposits at the injection sites, and were probably easier to absorb and/or metabolize. The reversal of these effects was more rapid with succinate and phosphate than with acetate preparations. Although these results were obtained in the rat, they may be applicable to human subjects in clinical situations. Plasma ACTH concentrations were initially undetectable or very low in surgically and medically adrenalectomized groups of rats not subjected to stress. Plasma ACTH gradually increased within days in non-stressed surgically adrenalectomized rats, but was undetectable up to six weeks in non-stressed medically adrenalectomized rats. The medically adrenalectomized rats appeared to have prolonged suppression of their hypothalamic pituitary adrenal axis, which
undoubtedly was due to axis suppression by systemic glucocorticoids,3'4 and, in part, caused by the active cortisone deposits which remained at the injection sites. Although the surgically and medically adrenalectomized groups of animals could respond to severe stress within hours or days post-adrenalectomy, it required a few weeks to elevate their basal ACTH concentrations under non-stressful conditions despite absent or very low plasma corticosterone concentrations. Similar results have been observed in medically5 and surgically adrenalectomized humans (unpublished observa-
tions). Although surgically adrenalectomized rats subjected to severe stress showed rapid ACTH responsiveness within hours after surgery, medically adrenalectomized rats showed suppression and delayed secretion of plasma ACTH for up to two weeks following medical adrenalectomy. Rats exhibit three phases of ACTH secretion following adrenalectomy, ie, the acute stress response, a decline during the next 24 to 36 hours (if not severely stressed), and a subsequent increase in basal ACTH concentration associated with chronic adrenal insufficiency. In this study, stressed adrenalectomized rats appeared to have an additional plasma ACTH secretory phase within two weeks after the third phase of ACTH secretion had begun. Dallman, et al,6 did in vitro studies of rat pituitaries during the first 72 hours post-adrenalectomy and showed decreased responsiveness to median eminence extracts, presumably due to an altered pituitary capacity to secrete ACTH during sustained stimulation by CRF. However, weeks after adrenalectomy, other investigators4 have shown increased median eminence activity in stressed adrenalectomized rats which is consistent with the delayed exaggerated response (a fourth phase) shown in our study. In conclusion, these results suggest the existence of a different hypothalamic pituitary ACTH control mechanism which is gradually activated in response to chronic glucocorticoid insufficiency under nonstressful conditions. This mechanism may be present in addition to the well-known ACTH mechanism for responding rapidly to acute glucocorticoid insufficiency and acute, or severe, stress.
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, VOL. 69, NO.
3, 1977
Table 1. The Effects of 10 mg/day of Subcutaneous Glucocorticoid Preparations for 14 Days in Rats
50,
SO°
40 PLASMA ACTH
Stressed Nonssressed
mUO/di
Procedures
Body weight Adrenal weight/ Number (gm) 100gm of Rats Before After body weight
20,
Comp B ,g/1O min
ACTH mU/dl 7 14 28 3 21 DAYS AFTER SURGICAL ADRENALECTOMY
Normal saline
(controls)
10
211
240
31 mg
17.2
9.6
Hydrocorticone succinate & P04
20
248
257
24 mg
16.2
3.4
Hydrocortisone acetate
10
246
196
16 mg
0.3
0.7
Cortisone acetate
10
238
155
10 mg
0.2
0.0
Summary Cortisone acetate was more effective than succinate or phosphate preparations in producing adrenal atrophy and failure, pituitary suppression, and general body catabolism following subcutaneous administration in rats. Nonstressed groups of adrenalectomized rats initially had very low or undetectable plasma ACTH concentrations, whether adrenalectomized by medical or surgical means. Basal plasma ACTH concentrations increased gradually during the next four to six weeks in the surgical, but not in the medical groups, despite presumably low plasma corticosterone. Surgically adrenalectomized rats always showed immediate responsiveness following severe stress, but such a response was delayed in the medically adrenalectomized rats for up to two weeks after cortisone injections. The delayed ACTH responsiveness was probably caused by prolonged pituitary hypothalamic suppression by systemic cortisol which also remained for two to three weeks at the injection sites. Another peak of plasma ACTH was observed about two weeks after the expected post-adrenalectomy elevation, particularly in the stressed, surgically adrenalectomized rats. These results suggest that a dif-
ferent hypothalamic pituitary mechanism may exist for control of ACTH secretion during chronic glucocorticoid insufficiency and non-stressful conditions, than that used during glucocorticoid insufficiency and acute stress, at least in the adrenalectomized rat.
42
Figure 1. Plasma ACTH in surgically adrenalectomized rats. The open and closed circles represent mean plasma ACTH in stressed and non-stressed rats after surgical adrenalectomy. Each point represents separate groups of six to eight rats, and the brackets are SEM.
S~~~~~ Stressed "- Nonstressed
o-o
20 PLASMA
ACTH mU/dI
2 WEEKS
3 4 OFF CORTISONE
5
6
Figure 2. Plasma ACTH in medically adrenalectomized rats. The open and closed circles represent mean plasma ACTH in stressed and non-stressed rats after medical adrenalectomy. Each point represents separate groups of 10 to 12 rats, and the brackets are SEM.
Acknowledgements We are deeply indebted to Ms. Susan Messner, Ms. Ursula A. Finck, and Mr. Stephen Haims for help in preparing this manuscript.
Literature Cited 1. Mims RB: Plasma ACTH in the adrenalectomized rat. Horm Metab Res 5:368-371, 1973 2. Cooper CE, Nelson DH: ACTH levels in plasma in pre-operative and surgically stressed patients. J Clin Invest 41:15991605, 1967 3. Mims RB, Nelson DH: Suppression of ACTH release following discontinuance of corticosteroid therapy in rats. Clin Res 13:246-246, 1965
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, VOL. 69, NO. 3, 1977
4. Vern ikos-DanellIis J: Effects of stress, adrenalectomy, hypophysectomy and hydrocortisone on the corticotroph in releasing activity of rat median eminence. Endocrinology 76:122-126, 1965 5. Graber AL, Ney RL, Nicholson WE, et al: Natural history of pituitary-adrenal recovery following long-term suppression with corticosteroids. J Clin Endocrinol Metab 25:1 1-16, 1965 6. Dallman MF, DeManincor D, Shinsako J: Dimibiishing corticotropin capacity to release ACTH during sustained stimulation: The 24 hours after bilateral adrenalectomy in the rat. Endocrinology 95:65-73, 1974
147
We have previously reported that surgically adrenalectomized nonstressed rats showed delayed secretion of the expected increase in plasma ACTH concentrations following adrenalectomy.1 However, increased plasma ACTH concentrations were always detected when these animals were subjected to severe stress, and their responsiveness increased again seven days or more following surgical adrenalectomy. We are now reporting similar results which were obtained following prolonged systemic glucocorticoid administration (medical adrenalectomy) in similar groups of rats. Materials and Methods Several groups of Holtzman albino female rats weighing 200 to 250 gm began as experimental subjects at the same time for this study. The animals were housed three per cage with constant light and room temperature and were fed rat chow (Purina) and water ad libitum. All animals received tap water prior to adrenalectomy, but 0.9 percent saline was used afterwards, which helped maintain a high survival rate in each group. All rats were weighed daily. Other details concerning adrenalectomy, anesthesia, stressed Supported by USPH Training Grant AM-05176. Requests for reprints should be addressed to Dr. Robert B. Mims, Director, Home Health Services, USC School of Medicine and the LAC/USC Medical Center, Los Angeles, Calif 90033.
and non-stressed blood collection, animal sacrifice, and the biological ACTH assay have previously been described. 2 In a preliminary experiment, equivalent results were obtained by peritoneal, intramuscular, and subcutaneous administration of the same type and dose of glucocorticoids. Subsequently, all animals received glucocorticoids by the subcutaneous route, and injections were given at 1600 daily for 14 days. Glucocorticoid preparations were mixed with normal saline so that each rat received a 10 mg dose in a 0.5 ml volume. Control rats received only 0.5 ml of saline. This protocol had previously caused severe adrenal atrophy in similar groups of rats, but smaller doses proved unsatisfactory. The adrenal glands were re-
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, VOL. 69, NO. 3, 1977
moved, rapidly trimmed, and weighed wet on a torsion balance after collecting blood in each group of rats.
Results The Effects of Various Glucocorticoid Preparations Table 1 shows the effects of various glucocorticoid preparations given 10 mg/day subcutaneously for 14 days, on adrenal and body weight, plasma corticosterone, and plasma ACTH. Cortisone acetate was the most effective, and hydrocortisone succinate or phosphate the least effective in causing adrenal atrophy and adrenal pituitary suppression. Cortisone and hydrocortisone acetate deposits were obvious at the injection sites up to two to three weeks following the injections. These deposits were removed in sacrificed animals and confirmed as cortisone or hydrocortisone by chemical analysis. Succinate and phosphate glucocorticoid injections did not cause visible deposits as described above for acetate preparations. Adrenal weights decreased 48 percent and 68 percent following hydrocortisone and cortisone acetate injections, but only 23 percent for hydrocortisone succinate and phosphate preparations. Body 145
weight increased 12 percent in control rats and 4 percent in succinatephosphate injected animals during the two injection intervals, but decreased 20 percent and 35 percent in hydrocortisone and cortisone acetate injected animals. Adrenal mass and body weights did not return to basal values or those of the control animals until four to six weeks after discontinuing the glucocorticoid acetate injections.
Plasma ACTH Following Surgical Adrenalectomy in Rats Figure 1 shows plasma ACTH results in a typical group of stressed and non-stressed rats following surgical adrenalectomy. Plasma ACTH was unexpectedly low initially in the nonstressed surgical adrenalectomized groups, but it increased gradually and stabilized during the next four to six weeks. Plasma ACTH was always elevated in the groups which were subjected to severe stress. The severely stressed group of rats also appeared to have two major secretory phases, ie, an immediate and a more exaggerated delayed phase.
Plasma ACTH Following Medical
Adrenalectomy in Rats Figure 2 shows plasma ACTH results in groups of stressed and nonstressed rats following two weeks of subcutaneous cortisone acetate injections (medical adrenalectomy). The 146
stressed group of medically adrenalectomized rats showed a sluggish plasma ACTH response during the first two weeks after discontinuing the injections, but they were able to respond adequately or excessively to stress thereafter. In contrast, the groups of non-stressed medically adrenalectomized rats had no detectable plasma ACTH up to six weeks after discontinuing cortisone acetate injections.
Discussion The first part of this study demonstrated that subcutaneously administered cortisone acetate was the most effective in causing severe adrenal atrophy, adrenal failure, pituitary suppression, and general body catabolism. These effects appeared to be more related to the acetate radical which apparently caused delayed absorption at the injection sites for a few weeks. In contrast, succinate and phosphate preparations caused less catabolic and suppressive effects, left no visible deposits at the injection sites, and were probably easier to absorb and/or metabolize. The reversal of these effects was more rapid with succinate and phosphate than with acetate preparations. Although these results were obtained in the rat, they may be applicable to human subjects in clinical situations. Plasma ACTH concentrations were initially undetectable or very low in surgically and medically adrenalectomized groups of rats not subjected to stress. Plasma ACTH gradually increased within days in non-stressed surgically adrenalectomized rats, but was undetectable up to six weeks in non-stressed medically adrenalectomized rats. The medically adrenalectomized rats appeared to have prolonged suppression of their hypothalamic pituitary adrenal axis, which
undoubtedly was due to axis suppression by systemic glucocorticoids,3'4 and, in part, caused by the active cortisone deposits which remained at the injection sites. Although the surgically and medically adrenalectomized groups of animals could respond to severe stress within hours or days post-adrenalectomy, it required a few weeks to elevate their basal ACTH concentrations under non-stressful conditions despite absent or very low plasma corticosterone concentrations. Similar results have been observed in medically5 and surgically adrenalectomized humans (unpublished observa-
tions). Although surgically adrenalectomized rats subjected to severe stress showed rapid ACTH responsiveness within hours after surgery, medically adrenalectomized rats showed suppression and delayed secretion of plasma ACTH for up to two weeks following medical adrenalectomy. Rats exhibit three phases of ACTH secretion following adrenalectomy, ie, the acute stress response, a decline during the next 24 to 36 hours (if not severely stressed), and a subsequent increase in basal ACTH concentration associated with chronic adrenal insufficiency. In this study, stressed adrenalectomized rats appeared to have an additional plasma ACTH secretory phase within two weeks after the third phase of ACTH secretion had begun. Dallman, et al,6 did in vitro studies of rat pituitaries during the first 72 hours post-adrenalectomy and showed decreased responsiveness to median eminence extracts, presumably due to an altered pituitary capacity to secrete ACTH during sustained stimulation by CRF. However, weeks after adrenalectomy, other investigators4 have shown increased median eminence activity in stressed adrenalectomized rats which is consistent with the delayed exaggerated response (a fourth phase) shown in our study. In conclusion, these results suggest the existence of a different hypothalamic pituitary ACTH control mechanism which is gradually activated in response to chronic glucocorticoid insufficiency under nonstressful conditions. This mechanism may be present in addition to the well-known ACTH mechanism for responding rapidly to acute glucocorticoid insufficiency and acute, or severe, stress.
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, VOL. 69, NO.
3, 1977
Table 1. The Effects of 10 mg/day of Subcutaneous Glucocorticoid Preparations for 14 Days in Rats
50,
SO°
40 PLASMA ACTH
Stressed Nonssressed
mUO/di
Procedures
Body weight Adrenal weight/ Number (gm) 100gm of Rats Before After body weight
20,
Comp B ,g/1O min
ACTH mU/dl 7 14 28 3 21 DAYS AFTER SURGICAL ADRENALECTOMY
Normal saline
(controls)
10
211
240
31 mg
17.2
9.6
Hydrocorticone succinate & P04
20
248
257
24 mg
16.2
3.4
Hydrocortisone acetate
10
246
196
16 mg
0.3
0.7
Cortisone acetate
10
238
155
10 mg
0.2
0.0
Summary Cortisone acetate was more effective than succinate or phosphate preparations in producing adrenal atrophy and failure, pituitary suppression, and general body catabolism following subcutaneous administration in rats. Nonstressed groups of adrenalectomized rats initially had very low or undetectable plasma ACTH concentrations, whether adrenalectomized by medical or surgical means. Basal plasma ACTH concentrations increased gradually during the next four to six weeks in the surgical, but not in the medical groups, despite presumably low plasma corticosterone. Surgically adrenalectomized rats always showed immediate responsiveness following severe stress, but such a response was delayed in the medically adrenalectomized rats for up to two weeks after cortisone injections. The delayed ACTH responsiveness was probably caused by prolonged pituitary hypothalamic suppression by systemic cortisol which also remained for two to three weeks at the injection sites. Another peak of plasma ACTH was observed about two weeks after the expected post-adrenalectomy elevation, particularly in the stressed, surgically adrenalectomized rats. These results suggest that a dif-
ferent hypothalamic pituitary mechanism may exist for control of ACTH secretion during chronic glucocorticoid insufficiency and non-stressful conditions, than that used during glucocorticoid insufficiency and acute stress, at least in the adrenalectomized rat.
42
Figure 1. Plasma ACTH in surgically adrenalectomized rats. The open and closed circles represent mean plasma ACTH in stressed and non-stressed rats after surgical adrenalectomy. Each point represents separate groups of six to eight rats, and the brackets are SEM.
S~~~~~ Stressed "- Nonstressed
o-o
20 PLASMA
ACTH mU/dI
2 WEEKS
3 4 OFF CORTISONE
5
6
Figure 2. Plasma ACTH in medically adrenalectomized rats. The open and closed circles represent mean plasma ACTH in stressed and non-stressed rats after medical adrenalectomy. Each point represents separate groups of 10 to 12 rats, and the brackets are SEM.
Acknowledgements We are deeply indebted to Ms. Susan Messner, Ms. Ursula A. Finck, and Mr. Stephen Haims for help in preparing this manuscript.
Literature Cited 1. Mims RB: Plasma ACTH in the adrenalectomized rat. Horm Metab Res 5:368-371, 1973 2. Cooper CE, Nelson DH: ACTH levels in plasma in pre-operative and surgically stressed patients. J Clin Invest 41:15991605, 1967 3. Mims RB, Nelson DH: Suppression of ACTH release following discontinuance of corticosteroid therapy in rats. Clin Res 13:246-246, 1965
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, VOL. 69, NO. 3, 1977
4. Vern ikos-DanellIis J: Effects of stress, adrenalectomy, hypophysectomy and hydrocortisone on the corticotroph in releasing activity of rat median eminence. Endocrinology 76:122-126, 1965 5. Graber AL, Ney RL, Nicholson WE, et al: Natural history of pituitary-adrenal recovery following long-term suppression with corticosteroids. J Clin Endocrinol Metab 25:1 1-16, 1965 6. Dallman MF, DeManincor D, Shinsako J: Dimibiishing corticotropin capacity to release ACTH during sustained stimulation: The 24 hours after bilateral adrenalectomy in the rat. Endocrinology 95:65-73, 1974
147
