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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 9 3125-3128
Copyright © 1999 by The Endocrine Society

Original Studies

Levels of Leptin during Hydrocortisone Infusions that Mimic Normal and Reversed Diurnal Cortisol Levels in Subjects with Adrenal Insufficiency1

Jonathan Q. Purnell and Mary H. Samuels

Division of Metabolism, Endocrinology, and Nutrition (J.Q.P.), Department of Medicine, University of Washington, Seattle, Washington 98195; Division of Endocrinology, Diabetes, and Clinical Nutrition (M.H.S.), Oregon Health Sciences University, Portland, Oregon 97201

Address all correspondence to: Jonathan Q. Purnell, M.D., University of Washington, Division of Metabolism, Endocrinology, and Nutrition, Box 356426, Seattle, Washington 98195. E-mail: purnell{at}u.washington.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Levels of leptin throughout the day follow a circadian pattern, with a trough in the late morning/early afternoon and a peak at midnight. This pattern of appearance of leptin correlates inversely with the circadian appearance of cortisol. Pharmacological doses of cortisol increase leptin messenger RNA expression in vitro and raise plasma leptin levels in animals and humans. To determine whether the circadian appearance of leptin might be accounted for by delayed effects from physiological cortisol secretion on fat cells, seven subjects with confirmed adrenal failure were admitted to the Clinical Research Center, on three separate dates, to receive 48-h infusions of: continuous normal saline (NS), a normal daily amount and diurnal pattern of cortisol (ND), and a normal daily amount but reversed diurnal pattern of cortisol. Blood samples were taken every 15 min during the second 24 h of infusion and pooled for hourly measurements of leptin. The circadian pattern of leptin appearance was unchanged during all of the infusion protocols. Area-under-the-curve analysis showed no differences in the total amount of leptin during the NS and ND protocols (20,565 ng/mL·24 h vs. 20,637 ng/mL·24 h during NS and ND protocols, respectively; P = 0.94). Acute changes in physiological levels of cortisol do not affect the circadian appearance of leptin in subjects with adrenal failure, nor is cortisol required to maintain normal leptin levels for up to 72 h. The circadian variation of leptin levels cannot be accounted for by normal activity of the hypothalamic-pituitary-adrenal axis.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
LEPTIN IS A hormone secreted by adipose tissue that circulates in the blood in proportion to the amount of body fat (1). Leptin binds to its receptor in hypothalamic weight regulatory centers, and peripheral or central administration of leptin have been demonstrated to affect appetite, energy expenditure, and hypothalamic-pituitary hormone production (2). Daily blood levels of leptin have been demonstrated to follow a circadian pattern in healthy lean and obese subjects and in subjects with type 2 diabetes (3, 4, 5). This circadian pattern of leptin levels, with a trough in the morning and peak at midnight, is similar to levels of TSH (6) but inverse to levels of ACTH and cortisol (4). To date, possible factors that may control the circadian pattern of leptin levels have not been elucidated.

It is possible that appearance of leptin affects the secretion of cortisol through feedback signaling to its receptors in the hypothalamus. Evidence for this mechanism comes from studies demonstrating that administration of leptin to obese animals lowers ACTH and corticosterone levels (7) and blunts the rise in stress-induced corticosterone levels in normal rodents (8). Therefore, circadian feedback of leptin to the central nervous system may influence hourly cortisol appearance, with a delay resulting from transport of leptin through the CSF and to hypothalamic centers.

On the other hand, administration of glucocorticoids to in vitro cell cultures stimulates leptin messenger RNA expression (within hours) and leptin secretion from cells (12–24 h later) (9, 10, 11). Similarly, glucocorticoid administration to humans results in increased leptin levels as early as 12–24 h afterward (10). It is possible, therefore, that circadian changes in cortisol may account, in part, for the circadian appearance of leptin and that the 12-h time lag in appearance of the two hormones is the result of delayed cellular signaling by cortisol on the fat cell, through gene transcription. However, previous studies of leptin levels in humans have used pharmacological doses of glucocorticoids. The relevance of these pharmacological doses to normal fat cell physiology and whether physiological levels of cortisol affect leptin levels are unknown.

This study examines the possibility that alterations in the physiological levels and infusion pattern of cortisol affect leptin levels in subjects with complete adrenal failure (Addison’s disease). Because these subjects lack endogenous cortisol secretion, varying both the amount and pattern of cortisol within the physiological range, through variable hydrocortisone (HC) infusions, and measuring resulting leptin levels, are possible for study purposes.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Experimental subjects

Seven healthy subjects (three women, four men) with the diagnosis of primary adrenal failure were recruited for this study. Complete adrenal failure was defined as spontaneous serum cortisol of levels less than 5 µg/dL, after 12 h without glucocorticoid replacement, and peak serum cortisol of less than 5 µg/dL, 60 min after a 250-µg ACTH stimulation test. Each subject signed a consent form approved by the Oregon Health Sciences University institutional review board before starting the study. All subjects had Addison’s disease for at least 1 yr before study, and all were receiving stable replacement doses of glucocorticoids and fludrocortisone. Two subjects also had primary hypothyroidism, on stable doses of T4 replacement, with normal TSH levels. Two subjects had premature ovarian failure on stable oral contraceptive doses.

Studies

Each subject underwent three clinical studies during admissions, separated by at least one month, at the Oregon Health Sciences University Clinical Research Center (CRC). For each study, subjects discontinued oral glucocorticoid medications 24 h before admission. Other medications were given as usual for the patient. Each of the three studies lasted 2 days. Starting at 0800 h on the day of admission, the subject received one of the three infusions given for an initial 24 h, to wash out effects of their oral glucocorticoid medications. The infusion was then continued for a second 24-h period, during which time blood samples were withdrawn from a separate iv every 15 min. Infusions consisted of either: 1) a baseline study, during which they received iv normal saline (NS) but no glucocorticoids; 2) a so-called normal physiological study, during which they received iv HC designed to normalize 24-h serum cortisol levels, pulses, and diurnal variation (ND) [this infusion protocol was kindly supplied by investigators at the NIH, based on initial studies they performed to optimized serum cortisol levels in subjects with adrenal insufficiency (12); the total amount of cortisol infused over 24 h was 19 mg. Resulting serum cortisol levels from this study were visually compared with 24-h serum cortisol levels obtained in healthy subjects receiving saline infusions. In five of the subjects, the serum cortisol levels were indistinguishable from normal levels. In two subjects, resulting serum cortisol levels were low. These subjects underwent repeat infusion with higher doses of HC (38 mg/24 h), which resulted in serum cortisol levels in the normal range]; or 3) a reverse study, in which they received iv HC in the same dose and number of pulses as in the physiological study but with the diurnal rhythm reversed from the physiological study (RD).

Cortisol

Cortisol was measured in duplicate, in each sample, by two-site chemiluminescent assay (Nichols Institute Diagnostics, San Juan Capistrano). Assay sensitivity was 0.8 µg/dL and intra- and interassay variation were less than 8% at cortisol levels measured in the study.

Leptin

Leptin was measured using a commercial kit (Linco Research, Inc., St. Charles, MO) using the double antibody/PEG technique. The sensitivity for this assay is 0.5 ng/mL and has 100% specificity for human leptin, with less than 0.2% specificity for rat or mouse leptin. The within-assay coefficient of variation is 4.98%. The between-assay coefficient of variation is 5.5%. All samples from all of the infusion protocols from a single individual were run in duplicate in same assay.

Data analysis

Because of the large range of individual leptin levels, 24-h profiles for leptin are represented as the mean ± SEM percent change from the 0800-h value. Differences among hourly leptin values on different infusion protocols were tested using t testing. To compare total daily leptin and cortisol levels, area-under-the-curve (AUC) amounts over 24 h were calculated in six subjects who had 20 or more, out of 24, samples available for measurement; and differences were tested using paired t testing.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The mean for age and body mass index (BMI) of the subjects was 42 (range, 29–67) yr old and 29 (range, 22–43) kg/m2, respectively. The mean fasting 0800-h leptin, while receiving the ND infusion protocol, was 13 (range, 2.1–23) ng/mL. When compared with a group of healthy controls taking part in an unrelated study matched for range of age and BMI, leptin levels in the subjects with adrenal failure are appropriate for their weights (Fig. 1Go).



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  		Figure 1. Plot of fasting 0800-h leptin levels, as a function of BMI, in seven subjects with adrenal failure ({square}) and a group of healthy normal subjects (men and women, n = 37) matched for a similar range in age and BMI (•).

 
Serum cortisol levels, measured every hour during the three studies, are shown in Fig. 2Go. Cortisol levels obtained during the ND infusion protocol were indistinguishable from those of healthy, nonstressed volunteers (data not shown).



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  		Figure 2. Mean hourly cortisol levels in subjects with adrenal failure (n = 7) during the second day of a 48-h infusion of normal saline (•), HC infused to mimic normal diurnal variation of cortisol ({square}), or HC infused to mimic RD ({blacktriangleup}).

 
Hourly leptin levels demonstrated a diurnal pattern, with a trough in the early-to-late morning and a peak just after midnight, and did not differ between the three infusion protocols (Fig. 3Go). To determine whether the absence of HC affects total daily leptin levels, rather than pattern of leptin appearance, 24-h integrated leptin levels were determined during both the NS and ND protocols. No difference in mean 24-h leptin AUC levels was found, in the absence or presence of normal HC replacement (mean ± SEM 24-h AUC leptin: 20,565 ± 6573 ng/mL·24 h vs. 20,637 ± 7034 ng/mL·24 h, during NS and ND protocols, respectively; P = 0.94), despite a large difference in the total amounts of cortisol infused on these days (mean ± SEM 24-h AUC cortisol: 133 ± 13 µg/dL·24 h vs. 9892 ± 1525 µg/dL·24 h, during NS and ND protocols, respectively; P < 0.001) (Fig. 4Go).



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  		Figure 3. Mean ± SEM hourly leptin levels, as percent change from the 0800-h value, in subjects with adrenal failure (n = 7) during the second day of a 48-h infusion of either normal saline (•), HC infused to mimic normal diurnal variation of cortisol ({square}), or HC infused to mimic RD ({blacktriangleup}).

 


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  		Figure 4. Individual 24-h AUC levels for cortisol (µg/dL·24 h) (graph A) and leptin (ng/mL·24 h) (graph B) in subjects with adrenal failure (n = 6), while receiving a 48-h infusion of NS and while receiving a physiologic replacement infusion of HC designed to reproduce ND.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Little is known about the regulatory system responsible for the circadian variation of leptin levels in humans. Leptin levels in these subjects with Addison’s disease show a diurnal pattern of appearance similar to that demonstrated previously in healthy subjects, with the lowest levels during the early-to-late morning hours and highest around midnight (3, 5). Such a pattern is similar to the appearance of TSH (6) and is inverse to the appearance of ACTH and cortisol (4).

In the present study, not only did altering the pattern of HC infusion fail to affect the circadian appearance of leptin, but acute withdrawal of all glucocorticoid replacement for up to 72 h also failed to affect either the circadian pattern of leptin appearance or the total amount of measured plasma leptin over 24 h. These data strongly argue that acute changes in cortisol levels that are within the normal physiological range do not affect the pattern of fat cell secretion of leptin. Nor does the presence of cortisol seem to be a requirement for the secretion of a normal daily amount of leptin to occur. Support for these conclusions comes from a study by Schoeller et al. (13), who showed that rapid changes in the diurnal levels of leptin occurred even before changes were seen in the diurnal levels of cortisol, when subjects experienced a reversal of their day/night cycle.

This study was not designed to test whether leptin may modulate cortisol levels through feedback inhibition of the hypothalamic-pituitary-adrenal axis (14) or directly through inhibition of cortisol secretion by the adrenal gland (15); such effects remain possible and await further studies. Finally, although short-term changes in cortisol levels within the normal range do not affect leptin levels, it is still possible that chronic increases in cortisol levels, whether within what is considered the physiological range or when given in supraphysiological doses, do increase leptin levels as a result of trophic effects on the fat cell.

In summary, alteration in physiological levels of cortisol does not affect the total amount or pattern of appearance of leptin levels in humans. The hypothalamic-pituitary-adrenal axis, therefore, is not responsible for the circadian variation in leptin levels. Chronic changes in cortisol levels could, however, still affect leptin levels as a consequence of cortisol’s ability to induce changes in fat cell size or number.


   Footnotes
 
1 This work was supported by NIH Grant R29-DK-48366 (M.H.S.), the Oregon Health Sciences University CRC (NIH GCRC Grant M01-RR-00334), and through the CRC Facility at the University of Washington and supported by the NIH Grant M01-RR-00037 (J.Q.P.). Back

Received April 1, 1999.

Accepted June 9, 1999.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Considine RV, Madhur KS, Heiman ML, et al. 1996 Serum immunoreactive-leptin concentrations in normal-weight, and obese humans. N Engl J Med. 334:292–295.[Abstract/Free Full Text]
  2. Matson CA, Wiater MF, Weigle DS. 1996 Leptin and the regulation of body adiposity. Diabetes Rev. 4:488–508.
  3. Sinha MK, Ohannesian JP, Heiman ML, et al. 1996 Nocturnal rise of leptin in lean, obese, and non-insulin-dependent diabetes mellitus subjects. J Clin Invest. 97:1344–1347.[Medline]
  4. Licinio J, Mantzoros C, Negrao AB, et al. 1997 Human leptin levels are pulsatile, and inversely related to pituitary-adrenal function. Nat Med. 3:575–579.[CrossRef][Medline]
  5. Saad MF, Riad-Gabrial MG, Khan A, et al. 1998 Diurnal, and ultradian rhythmicity of plasma leptin: effects of gender and adiposity. J Clin Endocrinol Metab. 83:453–459.[Abstract/Free Full Text]
  6. van Coevorden A, Laurent E, Decoster C, et al. 1989 Decreased basal, and stimulated thyrotropin secretion in healthy elderly men. J Clin Endocrinol Metab. 69:177–185.[Abstract]
  7. 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.[CrossRef][Medline]
  8. Ahima RS, Prabakaran D, Mantzoros C, et al. 1996 Role of leptin in the neuroendocrine response to fasting. Nature. 382:250–252.[CrossRef][Medline]
  9. Murakami T, Iida M, Shima K. 1995 Dexamethasone regulates obese expression in isolated rat adipocytes. Biochem Biophys Res Commun. 214:1260–1267.[CrossRef][Medline]
  10. Kolaczynski JW, Goldstein BJ, Considine RV. 1997 Dexamethasone, OB gene, and leptin in humans; effect of exogenous hyperinsulinemia. J Clin Endocrinol Metab. 82:3895–3897.[Abstract/Free Full Text]
  11. Halleux C, Servais I, Reul BA, Detry R, Brichard SM. 1998 Multihormonal control of ob gene expression and leptin secretion from cultured human visceral adipose tissue: increased responsiveness to glucocorticoids in obesity. J Clin Endocrinol Metab. 83:902–910.[Abstract/Free Full Text]
  12. Esteban NV, Loughlin T, Yergey AL, et al. 1991 Daily cortisol production rate in man determined by stable isotope dilution/mass spectrometry. J Clin Endocrinol Metab. 71:39–45.
  13. Schoeller DA, Cella LK, Sinha MK, Caro JF. 1997 Entrainment of the diurnal rhythm of plasma leptin to meal timing. J Clin Invest. 100:1882–1887.[Medline]
  14. Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW, Flier JS. 1997 Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress. Endocrinology. 138:3859–3863.[Abstract/Free Full Text]
  15. Bornstein SR, Uhlmann K, Haidan A, Ehrhart-Bornstein M, Scherbaum WA. 1997 Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly. Diabetes. 46:1235–1238.[Abstract]



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