Bilateral Adrenal Enucleation Research Articles

Adrenal Enucleation in MSG-Damaged Hyperleptinemic Male Rats Transiently Restores Adrenal Sensitivity to Leptin

It is known that the neonatal treatment of rats with monosodium L-glutamate (MSG) induces several metabolic abnormalities, resulting in enhanced adiposity and hyperleptinemia. Our study was designed to explore the consequences of MSG-induced chronic hyperleptinemia on adrenal sensitivity to the inhibitory effect of exogenous leptin. Neonatal male rats treated with MSG or vehicle (controls, CTR) were followed during 150 days in order to study changes observed over development in body weight, food consumption as well as in vivo hypothalamo-pituitary-adrenal (HPA) axis and adipocyte functions. During adulthood, adrenal response to adrenocorticotropin (ACTH) was evaluated both in vitro and in vivo in order to determine the adrenal sensitivity to the inhibitory effect of leptin. For this purpose, sham-operated as well as CTR and MSG rats with bilateral adrenal enucleation (AE) were used. Our results indicate that: (1) between 30 and 150 days of age, MSG animals developed hypophagia, accompanied by arrest in body weight gain, and concomitant enhanced basal levels of all HPA axis components and of leptin; (2) adrenals from of 150-day- old MSG rats displayed an in vitro adrenocortical hyperresponse to ACTH stimulation as well as an adrenal refractoriness to the physiological inhibitory effect of leptin on ACTH-stimulated glucocorticoid output, and (3) bilateral AE in adult MSG-treated rats transiently reversed the MSG-induced hyperleptinemia, restoring normal leptin levels as well as a normal adrenal sensitivity to the inhibitory effect of leptin. Our data indicate that adrenal exposure to the chronically high plasma leptin levels observed in MSG rats is involved in the loss of the inhibitory regulatory effect of leptin at the adrenal level, being therefore, at least in part, responsible for the increased total and free glucocorticoid production measured in MSG adult rats. Furthermore, this study strongly suggests that the adrenal overfunction, frequently associated with different phenotypes of obesity, could be due to an adrenal resistance to the leptin-negative regulation.

Bilateral adrenal enucleation-induced changes in adenohypophyseal pro-opiomelanocortin (POMC)-related peptides synthesis and secretion: A comparative study with adrenalectomized rats

The aim of the present study was to elucidate the modulatory effect of transient changes in endogenous glucocorticoids, occurring after bilateral adrenal enucleation (ENUC), on anterior pituitary (AP) proopiomelanocortin (POMC)-derived peptides synthesis and output in rats. For this purpose, adult female rats were either bilaterally ENUC, adrenalectomized (ADX), or sham-operated (SHAM) and killed by decapitation 2, 7, 14, and 21 days after surgery. Trunk blood was collected for measurements of ACTH, beta-endorphin (beta-END) and corticosterone (B) concentrations; APs were quickly dissected for the determination of ACTH, beta-endorphin (beta-END)-like (beta-END-LI) and gamma 3-MSH contents and adrenal glands were removed and submitted to histological study. The results indicate that ENUC and ADX increased AP POMC-related peptides synthesis and release in association with changes in the AP processing of peptides belonging to the N-terminal (gamma 3-MSH), mid (ACTH) and C-terminal (beta-LPH/ENDs) portions of POMC. While ADX abolished plasma B levels, ENUC induced a transient (day 2) decrease in plasma B concentrations which returned to SHAM levels at 7 days after surgery. These data tallied with the histological observations carried out, indicating a time-dependent regenerative process of the adrenal which was completed by three weeks after ENUC. There was a different pattern in plasma ACTH and beta-END levels between ENUC and ADX; maximal plasma peptide levels were found 7-14 days after ENUC, then falling down to SHAM values at 21 days post ENUC. Conversely, there was a constant increment in plasma peptide levels up to 21 days after ADX. At 2 days after both ENUC and ADX all peptides measured in the AP were lower than SHAM values, thus reflecting a rapid corticotrope secretion. Thereafter, 7 or more days after surgery, AP peptide content in ADX rats increased, in a time-related fashion, up to 21 days after surgery. Only beta-END-LI showed a similar AP content to that of the SHAM group, thereafter indicating a preferential cleavage of POMC to beta-END long after ADX (21 days). ENUC rats showed increased AP POMC peptides content throughout the whole time, and it was significantly different from SHAM and ADX values 14 days post-surgery. Interestingly, we found an increment in AP gamma 3-MSH, a peptide which is preferentially synthesized in the intermediate lobe of the rat pituitary, in both ENUC and ADX situations. Our results further indicate that: 1) glucocorticoids, from regenerating adrenal origin, induce a fast negative feedback mechanism on AP secretion, and 2) there might be a delayed inhibitory action of newly synthesized corticosteroids on higher levels of the central nervous system. The lack of glucocorticoids (ADX) clearly corroborates a persistent enhancement of AP POMC-related peptides synthesis and secretion. The differences in AP processing of POMC between ENUC and ADX might be due to qualitative/quantitative changes in hypothalamic ACTH secretagogues output.

Effect of sympathetic and angiotensin-aldosterone systems on renal salt conservation in the rat.

During dietary salt deprivation, the sympathetic nervous system and the angiotensin-aldosterone system are stimulated. Both systems are thought to be essential for maximal salt conservation by the kidney. To study their relative contributions, we produced negative salt balance in rats by intraperitoneal injection of furosemide, followed by a low-salt diet (<0.008% NaCl). In a 1-wk metabolic study, the animals were unable to replace the drug-induced salt deficit. Six groups of rats were studied. A control group established baseline function, a second group of 6-hydroxydopamine (OHDA) rats were treated with OHDA to destroy sympathetic efferent nerve terminals, and a third group (losartan) were treated with the angiotensin-receptor antagonist losartan. The influence of catecholamines and aldosterone released from the adrenal gland was studied in a further three groups. Rats were sham-adrenalectomized (sham), subjected to bilateral adrenal enucleation (Enuc) to eliminate catecholamine secretion, or were bilaterally adrenalectomized (Adx), eliminating both catecholamine and corticosteroid release. Dexamethasone was used as glucocorticoid replacement in this group. Steady-state urinary salt excretion was not different between control and OHDA rats. The losartan group showed significantly increased sodium but not chloride excretion. Surprisingly, there were no differences in salt excretion among sham, Enuc, and Adx groups. We conclude that, during a state of chronic salt depletion, renal mechanism(s) independent of neuronally released or systemically circulating catecholamines or of adrenally released aldosterone can ensure maximal salt conservation by the kidney. Although our data show that losartan increased sodium excretion under these conditions, we suggest that the losartan effect can be explained by a reduction of bicarbonate reabsorption, obligating simultaneous excretion of the cation.

Biochemical evidence for peripheral neural regulation of adrenocortical regeneration in response to bilateral adrenal enucleation

Male sprague-Dawley rats (130–150 g) with bilateral adrenal enucleation were used in this study. Animals were sacrificed at 2, 7, and 11 days post enucleation and plasma corticosterone and adrenal gland acetylcholin-esterase (AChE), choline acetyltransferase (ChAT), corticosterone, epinephrine (EP) and norepinephrine (NE) were assayed. The results show a progressive and gradual increase in plasma and adrenal corticosterone levels, AChE, ChAT, EP, and NE levels in the regenerating adrenal cortex from day 2 to day 11 post bilateral adrenal enucleation. At day 11 post surgery, the activities of ChAT and AChE were 60% and 25%, respectively when compared to sham operated control. The EP and NE levels returned to normal levels after 11 days post surgery in the regenerating gland when compared to the sham control. The presence of both cholinergic enzymes and the availability of high levels of catecholamine strongly suggest a progressive development of sympathetic innervation in the regenerating adrenal cortex.

IGF1 and 2 in two models of adrenal growth

Insulin-like growth factors (IGFs) 1 and 2 were measured in the adrenal glands of rats undergoing either compensatory growth following left unilateral adrenalectomy or adrenal regeneration following bilateral adrenal enucleation. In normal rat adrenal gland, the tissue concentration of IGF2 (7.45 ± 0.99 pg/ μg protein) was higher than IGF1 (1.26 ± 0.23 pg/ μg protein), both peptides being more abundant in the inner zones of the adrenal gland compared to the capsule-glomerulosa. During compensatory growth of the right adrenal gland, IGF1 and 2 increased significantly compared with control right adrenal glands at 24 h following left unilateral adrenalectomy ( P < 0.001). At 68 h, the increase remained significant for IGF1 ( P = 0.012). The two peptides were measured in the regenerating adrenal gland at 7,14 and 21 days following bilateral enucleation. Whilst there was a trend towards an increase in the IGF1 and 2 content of regenerating adrenal glands, the increase was significant only for IGF2 in the left adrenal gland at 21 days following enucleation. Plasma IGF1 and 2 did not increase compared to controls during the experiments (110.97 ± 1.95 and 46.33 ng/ml, respectively), suggesting that the changes in tissue IGF reflect increased local production during rapid growth of the adrenal gland.

副腎再生高血圧症

In young rats consuming 1% NaCl drinking solution, unilateral nephrectomy and bilateral adrenal enucleation caused a hypertension. Plasma corticosterone concentration in hypertensive rats was not significantly higher than that of normotensive control rats in early hypertensive or chronic hypertensive stage. At the end of experiment, each rat received an intravenous injection of 0.4 microCi/g of 3H-lysine and was sacrificed 2 hours after the injection. Incorporation of 3H-lysine into collagen or elastin of the mesenteric artery and heart in hypertensive rats was greater than that of normotensive rats. Administration of phenoxybenzamine hydrochloride lower the blood pressure of hypertensive rats and reduced the incorporation of 3H-lysine into collagen and elastin of the mesenteric artery and heart. From these findings, increased protein synthesis of collagen and elastin in hypertensive rats appears to play an important role for the maintenance of adrenal regeneration hypertension.

Adrenal regeneration in the rat is mediated by mitogenic N-terminal pro-opiomelanocortin peptides generated by changes in precursor processing in the anterior pituitary.

In order to investigate the role of N-terminal proopiomelanocortin (N-POMC) in adrenal regeneration after bilateral adrenal enucleation (hereafter referred to as enucleation) rats 13 days after enucleation were injected (200 microliters s.c. plus 200 microliters i.p.) at 08.00 and 20.00 h with normal rabbit serum (NRS), an ACTH antiserum or an N-POMC antiserum. On the next day the animals were injected with colchicine, killed and mitotic figures in adrenal histological sections counted. The same treatment was given to rats 20 days after enucleation. Only the N-POMC antiserum significantly diminished adrenal mitotic activity 14 and 21 days after enucleation (P less than 0.01 and 0.05 respectively) when compared with NRS-treated enucleated rats, whereas plasma corticosterone levels in rats 14 days after enucleation were significantly (P less than 0.005) decreased only by treatment with ACTH antiserum. To determine whether the mitogenic N-POMC peptides involved in adrenal regeneration originated from the pituitary intermediate lobe, 0.9% (w/v) NaCl or ergocryptine (10 mg/kg body weight) was administered s.c. twice daily to rats between 7 and 13 days after enucleation. On day 14, adrenal mitotic activity and plasma levels of ACTH and N-POMC were not significantly different between ergocryptine and saline-treated enucleated rats, whereas alpha-MSH levels in ergocryptine-treated enucleated rats were significantly (P less than 0.02) decreased. Increases in N-POMC content of the pituitary lobe accompanied those of ACTH in animals 7, 10, 14 and 21 days after enucleation (P less than 0.01 compared with sham-treatment). Anterior lobes from rats 10 days after enucleation or from adrenalectomized rats showed raised ACTH and N-POMC levels compared with sham-treated animals, whereas alpha-MSH content in the anterior lobe of enucleated rats was significantly (P less than 0.005) decreased. Adrenalectomized animals had raised (P less than 0.005) amounts of alpha-MSH compared with sham-treated animals. Plasma levels of ACTH and N-POMC were significantly (P less than 0.01) raised in rats 10 days after enucleation or in adrenalectomized rats compared with those in sham-treated animals, whereas alpha-MSH levels were raised (P less than 0.005) only in adrenalectomized rats. Sephadex G-75 chromatography of anterior lobe extracts obtained 10 days after surgery from sham-treated, enucleated and adrenalectomized rats was performed.(ABSTRACT TRUNCATED AT 400 WORDS)

Further evidence that N-terminal pro-opiomelanocortin peptides are involved in adrenal mitogenesis

In order to demonstrate the mitogenic effects of N-terminal pro-opiomelanocortin (N-POMC) peptides on the adrenal glands further, female rats with bilateral adrenal enucleation (hereafter referred to as enucleation) were hypophysectomized 11 days after enucleation and injected twice daily with 5 micrograms purified human N-POMC(1-28), ACTH(1-24) or 0.9% (w/v) NaCl. On day 14 after enucleation, rats were injected with colchicine and, after killing, their adrenal glands weighed, fixed and mitotic counts in histological sections assessed. Plasma corticosterone was measured fluorometrically. In other experiments, rats 7 days after enucleation were hypophysectomized and implanted with osmotic minipumps delivering 5 micrograms purified N-POMC(1-28) per day. On day 14 after enucleation, animals were treated as above. Collagenase-dispersed adrenal cells were incubated with purified or synthetic N-POMC(1-28) or synthetic N-POMC (1-36) (1-300 nmol/l) and [3H]thymidine incorporation into DNA was determined. Intact female rats were implanted with osmotic minipumps delivering 8 micrograms purified or synthetic N-POMC(1-28)/day, 8 micrograms synthetic N-POMC(1-36)/day or saline alone. Mitotic counts were performed on histological sections. Both s.c. injection or continuous delivery from minipumps of purified N-POMC(1-28) partially prevented the atrophy of regenerating adrenal glands after hypophysectomy (s.c. injection of N-POMC: 2.29 +/- 0.92 mitoses/section compared with 0.52 +/- 0.39 for controls; minipump delivery: 5.02 +/- 0.97 compared with 0.13 +/- 0.05; P less than 0.01 for both experiments). ACTH did not augment mitotic activity in enucleated-hypophysectomized rats but significantly increased plasma concentrations of corticosterone in s.c. injection experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced protein synthesis of heart in adrenal-regeneration hypertension and its reduction following antihypertensive treatment.

After unilateral nephrectomy and bilateral adrenal enucleation at 5 weeks of age, salt-loaded rats developed hypertension (ARH) at 6 weeks. Fibrous vascular protein and in vivo incorporation of 3H-lysine into this protein fraction were measured at 15 weeks of age in these animals. This study demonstrates: (1) that incorporation rates of 3H-lysine into cardiac collagen and elastin in ARH rats were greater than in control rats (p less than 0.001, respectively), and (2) that administration of phenoxy-benzamine hydrochloride decreased the incorporation of tritiated lysine into cardiac collagen in ARH rats, concomitant with a reduction in blood pressure. Based on these findings, increased synthesis of cardiac collagen and elastin appears to play an important role in the development of hypertension in ARH.

Return of pituitary-adrenal function after adrenal enucleation or transplantation: diurnal rhythms and responses to ether.

Adrenocortical diurnal rhythms and responses to ether vapor were studied in rats 1, 3, and 5 weeks after bilateral adrenal enucleation, autotransplantation, or sham transplantation in order to 1) determine whether diurnal rhythms in the plasma corticosterone concentration and adrenal responsiveness to ACTH are dependent on innervation of the adrenals, 2) compare regeneration of function of transplanted and enucleated adrenals, and 3) investigate adrenal mass-related nonsteroidal inhibition of ether-stimulated ACTH secretion. Rats in both enucleate and transplant groups exhibited significant morning-evening differences in adrenal and plasma corticosterone concentrations and significant adrenocortical responses to ether 3 and 5 weeks, but not 1 week, after surgery. The morning-evening differences in corticosterone concentration occurred in the absence of significant morning-evening variation in the plasma ACTH concentration, supporting our previous finding of a diurnal rhythm in adrenal responsiveness to ACTH. The responsiveness rhythm cannot be dependent on adrenal nerves unless transplanted adrenals receive functionally specific reinnervation within 3 weeks. The processes of regeneration of function after enucleation and after transplantation are similar; there were no differences in plasma or adrenal corticosterone values between rats in enucleate and transplant groups at any time or under any condition tested. As regeneration progressed, plasma ACTH responses to ether declined in both enucleate and transplant groups in the absence of changes in plasma corticosterone feedback. These results support our previous finding of adrenal mass-related nonsteroidal suppression of ACTH responses to ether.

Sodium balance after adrenal enucleation

To better define the relationship between adrenal enucleation (AE), sodium retention and adrenal regeneration hypertension (ARH), we measured daily sodium balance in rats after bilateral adrenal enucleation with (AEN) and without uninephrectomy (AE), and before and after development of ARH, along with the appropriate control groups. We found that both AE and AEN groups retained sodium during 7 days after surgery; AE 1.16 +/- 0.24 and AEN 1.32 +/- 0.33 meq/100 g body wt. Sham-operated, uninephrectomized rats and AE rats treated with spironolactone were in zero sodium balance. In the AEN rats after the development of ARH (BP greater than 150 Torr), zero sodium balance was also noted. Only AEN rats developed hypertension while sham-operated, uninephrectomized rats and AE rats with both kidneys remained normotensive for 28 wk. In summary, we have demonstrated that for 1 wk after AE, rats are in positive sodium balance and this is reversed by spironolactone but not by uninephrectomy.

Steroid secretion by the enucleated rat adrenal: measurements during salt retention and the development of hypertension.

It has been shown that rats have an inability to excrete a sodium load following bilateral adrenal enucleation. Rat adrenal vein plasma was analyzed for 18-hydroxy-ll-deoxycorticosterone (18-0H-D0C),1 corticosterone, deoxycorticosterone (DOC), and aldosterone following bilateral adrenal enucleation. All steroids analyzed were secreted in low amounts during the two weeks following enucleation. It appears unlikely that any of these steroids play an important role in the inability to excrete a sodium load which occurs immediately after enucleation. Rat adrenal vein plasma was also analyzed during various stages of adrenal regeneration hypertension. Secretion of all steroids was markedly decreased within 24 hr of enucleation. DOC secretion started to rise at one week and returned to near control levels at three weeks. 18-0H-D0C secretion was markedly diminished during the first three weeks, but greater than control six weeks after enucleation. Corticosterone secretion returned to normal at six weeks, and aldo...

A Sodium-Retaining Influence of Enucleate Rat Adrenal Glands1

The response of rats to a light fluid and sodium load (5 ml/100 g of 0.2 or 0.9% NaCl) was determined after bilateral adrenal enucleation. For the first week, and particularly the first 72 hr, after operation these animals had a marked inability, relative to either shamoperated or adrenalectomized controls, to excrete sodium. The effect was not mimicked by adrenal trauma not involving enucleation but was blocked by ligation of the adrenal pedicle prior to enucleation, by the presence of one intact adrenal, by corticosterone or spironolactone. Although small doses of aldosterone in adrenalectomized animals essentially duplicated the effects of enucleation, indirect evidence, including failure to demonstrate its presence in adequate amounts, does not implicate aldosterone as the probable and as yet unidentified Naretaining agent involved. Whatever its cause, this effect of adrenal enucleation could possibly be involved in initiating adrenal regeneration hypertension. (Endocrinology81: 1331, 1967)

Complementary role of adrenal cortex and salt in adrenal-regeneration hypertension.

The relationship between salt ingestion and regenerating adrenal cortical tissue in the genesis of adrenal-regeneration hypertension has been studied by varying the content of sodium chloride in the diet from 2.5 to 10%. Results indicate that adrenal-regeneration hypertension is the product of an interplay between the hypertensive potentials of sodium chloride and regenerating adrenal cortical tissue. In young, female rats, enucleation and subsequent regeneration of the adrenal cortex has a hypertensive potential of itself which may be inhibited by low salt ingestion, overshadowed by excessively high salt ingestion and optimally demonstrated by salt consumption approximating 5% of the daily food intake. On the other hand, sodium chloride has an independent hypertensive potential, which is consistently augmented by the presence of a regenerating adrenal gland. Whatever adrenal cortical factors are involved, they are not altered by bilateral adrenal enucleation nor are they mimicked by unilateral adrenalectomy or sham adrenal enucleation.

STUDIES ON ADRENAL ENUCLEATION

Bilateral adrenal enucleation (AE) was performed on rats, and at 24 hours, 3 days, 1, 2, 3, 4, 6, 8, 10, 15 and 20 weeks after the operation, changes in the anterior pituitary were histologically observed. Also the changes at 10 weeks after right adrenalectomy plus left AE, and the changes at 3 and 7 days after adrenalectomy were respectively compared with shamoperated controls.After AE, enlargement and increase in number of β-cells in the wide sense were observed in the anterior pituitary. This change was most remarkable at 14 postoperative weeks, and at this period, nuclei in enlarged spherical β-cells were generally shrunken, conspicuously showing signet rings. Further, characteristic cells which showed relatively large colloidal substance and vacuolization appeared. In this case, some changes were observed also in α-and γ-cells, but these were generally slight. These changes returned nearly to the normal after 10 weeks. Findings at 10 weeks after right adrenalectomy plus left AE were approximately equal to those of AE at 10 weeks.At 3 days after adrenalectomy, slight enlargement of β-cells was observed, whereas after 7 days, atrophy and degeneration of α-and β-cells were remarkable. This was considered to be the results of diminished reaction due to weakening of the body.From these results, probability was estimated that ACTH would be produced by β-cells in the wide sense in the anterior pituitary.

Gastric secretion in the adrenalectomized rat.

TUERKISCHER and Wertheimer (1945) using a modification of the technique for the study of gastric secretion first reported by Roe and Dyer (1939) found a significant decrease in gastric secretion in adrenal-ectomized rats. They particularly emphasized the point that this decrease in gastric secretion could not be accounted for by the surgical trauma imposed upon the animal. A normal volume of secretion was obtained after bilateral adrenal-enucleation. The decrease in gastric secretion following adrenalectomy could be corrected with adrenal cortical extract but not with desoxycorticosterone acetate or normal saline. It seemed important to attempt to confirm these findings. The results herein reported extend and confirm those published elsewhere (Madden and Ramsburg, 1951).