Plasma Leptin Levels Do not Change in Patients with Cushing's ...

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ABSTRACT. In the present study, we characterized the changes in plasma leptin levels in patients with pituitary Cushing's disease and in age- and sex-matched ...

0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 8 Printed in U.S.A.

COMMENTS Plasma Leptin Levels Do not Change in Patients with Cushing’s Disease Shortly after Correction of Hypercortisolism GIOVANNI CIZZA, ANGELA J. LOTSIKAS, JULIO LICINIO, PHILIP W. GOLD, GEORGE P. CHROUSOS

AND

Developmental Endocrinology Branch, National Institute of Child Health and Human Development (G.C., A.J.L., G.P.C.), and Clinical Neuroendocrinology Branch (G.C., J.L., P.W.G.), National Institute of Mental Health, Bethesda, Maryland 20892 ABSTRACT In the present study, we characterized the changes in plasma leptin levels in patients with pituitary Cushing’s disease and in age- and sex-matched controls. Plasma levels of ACTH, cortisol, and leptin were measured before and after iv administration of ovine CRH in controls once and in patients twice (while they had active hypercortisolism and 10 days after successful surgery). Cushing’s patients had elevated body mass indexes (34 6 1.9 vs. 22.9 6 0.8) and plasma leptin levels (35.6 6 3.4 vs. 9.2 6 1.9 ng/mL) compared to controls, which

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NE OF THE hallmarks of Cushing’s syndrome is severe centripetal obesity associated with visceral adiposity (1). Recently, leptin, a hormone that plays a pivotal role in the regulation of body weight, was isolated from adipose tissue (2). CRH regulates body weight and adiposity by causing suppression of appetite and food intake and by stimulating sympathetic system activity (3). Central administration of leptin increases messenger ribonucleic acid levels of CRH in the paraventricular nucleus of the hypothalamus and decreases messenger ribonucleic acid levels of neuropeptide Y (NPY) in the arcuate nucleus (4). The latter is the most potent appetite stimulant known; however, it also stimulates CRH production, probably as a counterregulatory signal (5). The circadian secretion of leptin, with a peak around 2000 h, is the inverse of those of ACTH and cortisol (6). Glucocorticoids stimulate in vitro expression and secretion of leptin (7) as well as hypothalamic NPY secretion (8, 9). Therefore, several potential interrelations between leptin and the hypothalamicpituitary-adrenal axis exist at different levels. In the present study, we examined the effects of acute and chronic changes in hypothalamic-pituitary-adrenal axis activity on leptin levels in patients with Cushing’s disease before and after correction of hypercortisolism by transsphenoidal adenomectomy. In parallel, we studied age- and gender-matched lean controls.

Received January 6, 1997. Revision received April 1, 1997. Rerevision received May 1, 1997. Accepted May 5, 1997. Address all correspondence and requests for reprints to: Giovanni Cizza, M.D., Ph.D., National Institutes of Health, Building 10, Room 10N-262, 10 Center Drive, Bethesda, Maryland 20892-1862.

remained unchanged 10 days after successful transsphenoidal surgery and directly proportional to the body mass index. Plasma leptin levels were not affected by CRH infusion in either the controls or the patients despite clear-cut elevations in plasma ACTH and cortisol. These findings suggest that although acute changes in plasma cortisol do not affect plasma leptin, chronic hypercortisolism results in elevated leptin levels, probably by causing visceral obesity. (J Clin Endocrinol Metab 82: 2747–2750, 1997)

Subjects and Methods Study subjects The charts of patients referred within a 3-yr period with a diagnosis of ACTH-dependent Cushing’s syndrome to the NIH Clinical Center were reviewed. These patients had history and physical examination compatible with Cushing’s syndrome, elevated 24-h urinary free cortisol and/or 17-hydroxysteroid excretion, abnormal diurnal rhythm of ACTH and cortisol, ovine CRH (oCRH) test compatible with Cushing’s disease, and pituitary microadenoma identified by magnetic resonance imaging scan and/or by inferior petrosal sinus sampling (10). This diagnosis was confirmed in 17 patients, who subsequently underwent transsphenoidal resection of their pituitary adenoma. In 13 patients, surgery was successful, as confirmed by baseline morning cortisol levels of 2.0 mg/dL or less and a suppressed oCRH in a test performed 10 days after surgery (11). In 4 patients, surgery was unsuccessful. In addition, 3 normal men and 6 normal women were selected from a pool of subjects recruited through the normal volunteer office of the NIH Clinical Center for oCRH infusion studies.

oCRH test Two peripheral oCRH tests (1 mg/kg BW) were performed in patients, one before surgery (pre-op CRH) and another 10 days after surgery (post-op CRH), as previously described (11). Starting on the day of surgery, eight doses of dexamethasone (0.5 mg every 6 h) were administered iv, and then dexamethasone was withheld for 2–3 days. At that time, successful surgery was defined as morning plasma cortisol levels equal to or lower than 2 mg/dL; unsuccessful surgery was defined as morning plasma cortisol levels greater than 2 mg/dL (1). Patients in whom surgery was successful were placed on oral glucocorticoid replacement with hydrocortisone hemisuccinate (12–15 mg/m2zday) (1). On day 10, hydrocortisone was withheld in the morning, and an oCRH test was performed as previously reported (1). One peripheral oCRH (1 mg/kg BW) test was performed in normal subjects.

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COMMENTS

TABLE 1. Clinical characteristics of 2 men and 11 women with Cushing’s disease who underwent successful transsphenoidal resection of their pituitary adenomas Patient no.

Age (yr)

Sex

Ht (cm)

Wt pre-op (kg)

Wt post-op (kg)

BMI pre-op (kg/m2)

BMI post-op (kg/m2)

1 2 3 4 5 6 7 8 9 10 11 12 13

26 34 34 36 36 32 43 33 36 42 36 39 27

M M F F F F F F F F F F F

169 179 160 160 166 163 172 155 160 160 164 161 170

73.2 96 137 68.1 81.5 84.1 102.5 89.6 95.9 76.4 88.2 97.4 94.2

70.7 100.4 133 69.2 80 81.7 102.4 86.6 98.5 73.5 89 98.2 104.6

25.5 29.9 53.5 26.6 29.7 31.7 34.8 37.3 37.5 29.9 32.8 33.7 32.5

24.6 31.4 51.9 27 29.1 30.8 34.8 36.1 38.5 28.7 33.1 33.9 36.1

35 61.4

2/11

165 6 1.8

91 6 4.8

91 6 4.9

33 6 1.9

34 61.9

Mean 6

SEM

TABLE 2. Clinical characteristics of three normal men and six normal women Subject no.

Age (yr)

Sex

Ht (cm)

Wt (kg)

Body mass index (kg/m2)

1 2 3 4 5 6 7 8 9

30 35 47 33 39 41 44 26 29

M M M F F F F F F

175 180 180 172 159 162 160 159 166

75 76.6 75 55 66.5 59 60.8 62 53

24.5 23.6 23.1 18.6 26.3 22.5 23.8 24.5 19.2

3662

3/6

16863.0

6563

22.960.8

Mean 6

SEM

Assays ACTH and cortisol were measured in serum samples obtained during the CRH test by specific RIAs (10). Leptin was measured at the same times by a commercially available RIA (Linco Research, St. Charles, MO). Controls were used in the low and high sections of the standard curve. Samples were run in duplicate, and standards were run in triplicate. The intra- and interassay coefficients of variation were both below 5%.

Statistical analyses All data are expressed as the mean 6 se. The effect of CRH injection on hormonal responses was analyzed by repeated measures ANOVA, with time of sampling as a within-subject factor. The total area under the curve (AUC) for all hormones was then calculated (12). Comparisons were made using one-way ANOVA, followed by Scheffe’s test. Correlation analysis between plasma leptin and body mass index (BMI) was performed by linear regression. Significance was accepted at P # 0.05.

Results

Patients and controls had similar age and height, but significantly different body weight (P # 0.0004) and BMI (P # 0.0003; Tables 1 and 2). The body weight and BMI of patients with successful surgery did not change 10 days after surgery. Basal ACTH and cortisol plasma levels were about 9- and 5-fold lower (P # 0.0001), respectively, in controls than in Cushing’s patients before surgery and were significantly (P # 0.0001) stimulated by oCRH injection (Fig. 1, A and B). Baseline plasma ACTH and cortisol were dramatically (P # 0.0001) decreased in patients with Cushing’s disease 10 days after curative surgery. oCRH injection stimulated ACTH and cortisol secretion over baseline before, but not after, surgery (Fig. 2, A–D). Thus, the post-op ACTH AUC (7926 6 1451

pg/mLzmin) and cortisol AUC (1372 6 251 mg/dLzmin) were dramatically lower than the pre-op ACTH AUC (9528 6 72 pg/mLzmin; P # 0.0001) and cortisol AUC (132 6 13 mg/ dLzmin; P # 0.0001), respectively. Patients in whom surgery was not curative exhibited similar basal and oCRH-stimulated plasma levels of ACTH and cortisol before and after surgery (data not shown). At time zero, leptin levels were 4-fold (P # 0.0001) higher in patients than controls. After oCRH administration, leptin levels did not change in controls (Fig. 1C) or in patients before (Fig. 2E) or after correction of hypercortisolism (Fig. 2F). Consequently, leptin AUC was unchanged in patients after surgery (pre-op, 2340 6 214 ng/mLzmin; post-op, 2038 6 185 ng/mLzmin). In patients in whom surgery was unsuccessful, leptin levels did not change (data not shown). There was an expected positive correlation between BMI and plasma leptin concentrations in controls and patients with active Cushing’s syndrome (r 5 0.611; P # 0.0001). A similar correlation was observed when women were considered as a subgroup (r 5 0.791; P # 0.0003). The correlation between BMI and leptin was stronger in Cushing’s patients (r 5 0.571; P # 0.06) before surgery than in controls (r 5 0.376; P # 0.31). After surgery, there were no changes in the BMI/ leptin correlation (r 5 0.63; P # 0.07). Discussion

In the current study, we found that acute changes in plasma ACTH and cortisol did not affect circulating leptin levels in either lean healthy volunteers or obese Cushing’s

COMMENTS

FIG. 1. Mean 6 SE plasma ACTH (A), cortisol (B), and leptin (C) responses to oCRH (1 mg/kg) in three normal men and six normal women.

FIG. 2. Mean 6 SE plasma ACTH, cortisol, and leptin responses to oCRH in 2 men and 11 women with Cushing’s disease before (left panels. solid circles) and after (right panels. open circles) curative surgery.

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patients. Thus, neither the rapid increases in plasma ACTH and cortisol induced by oCRH stimulation in normal subjects and Cushing’s patients before surgery nor the acute decreases in cortisol observed in Cushing’s patients after curative surgery were associated with changes in plasma leptin. Leptin represents a signal of a negative feedback loop from the adipocyte to the satiety- and energy-regulating centers in the brain; however, the factors that regulate leptin production in vivo are largely unknown. Glucocorticoids were considered potential regulators of leptin production, as they stimulate appetite and increase body weight. Acute increases in cortisol levels did not affect leptin production in normal subjects, in agreement with a study reporting no effects of short term administration of a pharmacological dose of methylprednisolone (30-min iv infusion of 125 mg) on leptin levels in normal men, but in disagreement with a study showing a 2-fold increase in fasting leptin levels after short term treatment with a high dose of dexamethasone (3.0 mg, twice daily, for 48 h) in healthy women (13, 14). Plasma leptin concentrations were 4-fold higher in Cushing’s patients than in lean healthy volunteers, in agreement with a recent study reporting 2-fold increases in serum leptin in patients with Cushing’s syndrome (15). These patients had a markedly elevated BMI, and their high leptin levels were consistent with the idea that leptin levels are proportional to the amount of body fat. There was a significant positive correlation between BMI and plasma leptin when the volunteer and patient groups were combined, but this correlation lost its significance when the two groups were analyzed separately. To reconcile the fact that we observed no short term effect

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of hypercortisolism on leptin secretion with the stimulatory effects of glucocorticoids on leptin reported in vitro, we hypothesize that the possible effects of glucocorticoids are counterbalanced by other humoral and/or neural factors that are as yet unclear. The sympathetic system represents a potential candidate for such counterregulation. The adipocyte is richly innervated by catecholaminergic terminals, and cold exposure rapidly induces catecholamine-mediated inhibition in the expression of the ob gene (16). Catecholamine levels are decreased in Cushing’s patients, and glucocorticoids are known to inhibit the systemic sympathetic and sympatho-adrenal system at different levels (17, 18). However, the sensitivity of peripheral tissues to cathecholamines is quite increased in Cushing’s patients, and this is a major mechanism by which glucocorticoids cause hypertension (19, 20). Therefore, even though leptin levels may receive positive glucocorticoid input in patients with Cushing’s disease, they may also receive increased inhibitory sympathetic input, with the two inputs canceling each other. Increases in cortisol levels during the CRH test did not affect leptin levels in Cushing’s patients before surgery. Therefore, in the context of chronically elevated basal cortisol levels, further acute elevations of cortisol do not influence leptin levels. We also observed no changes in the basal leptin levels of these patients 10 days after curative surgery. Because no changes in body weight were observed in this period, we conclude that changes in cortisol levels per se do not directly affect leptin levels, but, rather, exert their influence indirectly by promoting adiposity over the long term. References 1. Cizza G, Chrousos GP. 1997 ACTH-dependent Cushing syndrome. In: Endocrine neoplasms. Boston: Kluwer; 25– 40. 2. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JF. 1994 Positional cloning of the mouse obese gene and its human homologue. Nature. 372:425– 432. 3. Chrousos GP, Gold PW. 1992 The concept of stress and stress system disorders. JAMA. 267:1244 –1252. 4. Schwartz MW, Seeley RJ, Campfield LA, Burn PA, Baskin DG. 1996 Iden-

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12. 13. 14. 15. 16. 17.

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tification of targets of leptin action in rat hypothalamus J Clin Invest. 5:1101–1106. Liu JP, Clarke IJ, Funder JW, Engler D. 1994 Studies on the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophyseal portal circulation of the conscious sheep. II. The central noradrenergic Y pathways cause immediate and prolonged hypothalamic-pituitary-adrenal activation: potential involvment in the pseudoCushing syndrome of endogenous depression and anorexia nervosa. J Clin Invest. 93:1439 –1450. Licinio J, Montzoros C, Negrao AB, et al. 1997 Periodicity and pulsatility of circulating leptin levels in men. Nat Med. 3:575–579. Slieker LJ, Sloop KW, Surface PG, et al. 1996 Regulation of expression of ob mRNA and protein by glucocorticoids and cAMP. J Biol Chem. 271:5301–5304. White BD, Dean RG, Edwards GL, Martin RJ. 1994 Type II corticosteroids receptor stimulation increases NPY gene expression in basomedial hypothalamus of rats. Am J Physiol 266:R1523–R1529. Stephens, TW, Basinski, M, Bristow, PK, et al. 1995 The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 377:530 –532. Cizza G, Nieman L, Doppman J, et al. 1996 Factitious Cushing syndrome. J Clin Endocrinol Metab. 81:3573–3577. Avgerinos PC, Chrousos GP, Nieman LK, Oldfield EH, Loriaux LD, Cutler GB. 1987 The corticotropin-releasing hormone test in the postoperative evaluation of patients with Cushing’s syndrome. J Clin Endocrinol Metab. 65:906 –913. Cizza G, Pacak K, Kvetnansky R, et al. 1995 Decreased stress responsivity of central and peripheral catecholaminergic systems in aged 344/N Fischer rats. J Clin Invest. 95:1217–1224. Tataranni PA, Pratley R, Maffei M, Ravussin E. Acute and prolonged administration of glucocorticoids (methylprednisolone) does not affect plasma leptin concentrations in humans. Int J Obesity. In press. Larsson H, Ahren B. 1996 Short-term dexamethasone treatment increases plasma leptin independently of changes in insulin sensitivity in healthy women. J Clin Endocrinol Metab. 81:4428 – 4432. Leal-Cerro A, Considine RV, Peino R, et al. 1996 Serum immunoreactive leptin levels are increased in patients with Cushing’s syndrome Horm Metab Res. 12:711–713. Trayhurn P, Duncan JS, Rayner DV. 1995 Acute cold-induced suppression of ob gene expression in white adipose tissue of mice: mediation by the sympathetic system. Biochem J. 311:729 –733. Pacak K, Papanicolaou DA, Tsigos C, Goldstein DS, Chrousos GP. Plasma elevation of catecholamines during acute glucocorticoid deficiency after surgical treatment for Cushing syndrome [Abstract]. Proc of the 10th Int Congr of Endocrinol. 1996. Papanicolaou DA, Petrides JS, Tsigos C, et al. 1996 Exercise stimulates interleukin-6 secretion: inhibition by glucocorticoids and correlation with catecholamines. Am J Physiol 271:E601–E605. Saruta T, Suzuki H, Handa M, Igarashi Y, Kondo K, Senba S. 1986 Multiple factors contribute to the pathogenesis of hypertension in Cushing’s syndrome. J Clin Endocrinol Metab. 62:275–279. Lenders JW, Golczynska A, Goldstein DS. 1995 Glucocorticoids, sympathetic activity, aand presynaptic a2-adrenoceptor function in humans. J Clin Endocrinol Metab. 80:1804 –1808.

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