THERE IS a strong association between nutritional status

Transcription

1 X/98/$03.00/0 Vol. 83, No. 6 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1998 by The Endocrine Society Relationship between Plasma Adrenocorticotropin, Hypothalamic Opioid Tone, and Plasma Leptin* GARY S. WAND AND HENRY SCHUMANN Departments of Medicine and Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland ABSTRACT The purpose of the present study was to further the understanding of the relationship between plasma leptin concentrations, hypothalamic opioid tone, and plasma ACTH secretory dynamics. ACTH(1 24) challenges (250 g) produced the expected increase in plasma cortisol levels but did not alter plasma leptin levels. Activation of the entire hypothalamic-pituitary-adrenal (HPA) axis was induced by employing the opioid receptor antagonist, naloxone. By blocking opioidergic inhibitory input to hypothalamic CRH neurons, naloxone induced the expected increase in plasma ACTH and cortisol. Plasma ACTH levels peaked 30 min after naloxone administration, whereas plasma cortisol levels peaked 60 min after opioid receptor blockade. Once again, plasma leptin concentrations were not altered by this THERE IS a strong association between nutritional status and the activity of the hypothalamic-pituitary-adrenal (HPA) axis (1, 2). Obesity (3 5), as well as calorie deprivation (6), modify the functioning of the HPA axis. Leptin, a newly discovered adipostatic hormone (7), inhibits appetite and also has an inhibitory effect on the HPA axis (8 10). For example, administration of leptin to fasting rodents (9) or to rodents stressed by immobilization (10) inhibits plasma ACTH and corticosterone levels. Administration of leptin reduces corticosterone levels in the genetically obese, ob/ob mice (11). Leptin also inhibits hypoglycemia-induced CRH secretion from isolated, perifused rat hypothalami (10). More evidence supporting inhibitory actions of leptin on the HPA axis includes studies showing a reciprocal relationship between plasma leptin and plasma corticosterone concentrations (9). Leptin may modulate the diurnal rhythm of corticosterone secretion. The mechanism(s) through which leptin inhibits HPA axis activity remains unknown. A clue to this mechanism may be the interaction of leptin and opioidergic (POMC) neurons of the arcuate nucleus. Recent studies have shown that leptin s action is, in part, mediated through release of melanocortin from arcuate nucleus POMC neurons (12). These studies indicate that leptin-induced melanocortin release reduces food intake via signaling through the melanocortin-4 (MC4) Received February 17, Revision received March 12, Accepted March 17, Address all correspondence and requests for reprints to: Gary S. Wand, M.D., The Johns Hopkins University School of Medicine, Ross Research Building, Room 850, 720 Rutland Avenue, Baltimore, Maryland gwand@welchlink.welch.jhu.edu. * This work was supported by NIH Grant RO1-AA (to G.S.W.), The Alcohol Medical Research Foundation (to G.S.W.), and a generous gift from The Kenneth Lattman Foundation (to G.S.W.). manipulation. However, there was a positive correlation between fasting, integrated plasma leptin concentrations, and plasma ACTH responses to naloxone (peak r 0.822, P ; and area under curve r 0.832, P ). The correlation was stronger when leptin was normalized to body mass index and expressed as the leptin/body mass index ratio (peak r 0.878, P ; and area under curve r 0.882, P ). In summary, these findings indicate that activation of the HPA axis does not acutely alter plasma leptin concentrations. However, plasma leptin levels may influence hypothalamic opioidergic tone and thus modulate the magnitude of CRH release. The acute interaction of the HPA axis and leptin is unidirectional. (J Clin Endocrinol Metab 83: , 1998) receptor (12). The arcuate nucleus POMC neurons also have another function. This opioidergic system inhibits CRH neuron activity through -endorphin release (13 17). Because plasma leptin levels stimulate arcuate nucleus POMC activity and, in turn, POMC neurons (through -endorphin) inhibit CRH release, it is plausible that ambient plasma leptin concentrations may influence HPA axis secretory dynamics through acute and chronic effects on this opioidergic pathway. Although there is mounting evidence that leptin has an inhibitory effect on the HPA axis in rodents and humans, the nature of the reciprocal relationship is uncertain. A recent study showed no change in plasma leptin levels after the administration of CRH (18). However, it remains unclear whether activation of the HPA axis by other means will alter plasma leptin levels. Therefore, the first aim of the study was to determine whether activation of the HPA axis would acutely alter plasma leptin levels. To this end, plasma leptin levels were monitored at baseline and for 120 min after activation of the HPA axis after administration of ACTH(1 24) or naloxone. The second aim of the study was to determine whether there was an association between plasma leptin levels and hypothalamic opioid activity. Our hypothesis was that plasma leptin concentrations would correlate with peak plasma ACTH levels induced by opioid receptor blockade. The hypothesis was tested by comparing the magnitudes of plasma ACTH release, in response to opioid receptor blockade, as a function of an individual s integrated plasma leptin level. Subjects Subjects and Methods Sixteen healthy, nonobese volunteers (9 women and 7 men), yr old, were recruited by newspaper advertisement. All subjects gave informed consent. General health status was assessed by medical history, 2138

2 LEPTIN AND THE HPA AXIS 2139 TABLE 1. Mean fasting plasma leptin concentrations Time 0 Placebo ACTH(1-24) Naloxone Leptin (ng/ml) Leptin (ng/ml)/bmi (kg/m 2 ) Fasting plasma leptin concentrations were calculated by 1) taking the mean basal (time 0) leptin level derived from each session (placebo, naloxone, and ACTH) or 2) taking the mean integrated leptin level derived from the placebo or ACTH or naloxone session. In subsequent experiments, leptin levels reflect the integrated value derived from the placebo session. physical examination, and standard laboratory tests (complete blood cell counts, electrolytes, liver and renal function tests, and glucose). To control for hormonal fluctuations, female subjects were studied only during the follicular phase of the menstrual cycle. Exclusion criteria were: 1) meeting Diagnostic and Statistical Manual-IV criteria for any psychoactive substance use disorder, including nicotine dependence; 2) meeting Diagnostic and Statistical Manual-IV criteria for a major axis I disorder and being in need of, or currently undergoing, pharmacotherapy; 3) being pregnant; 4) experiencing a serious medical condition; 5) having abnormal liver functions; 6) having central nervous system or endocrine disorders; and 7) being treated within the last 10 yr with:antidepressants, neuroleptics, mood stabilizers, sedative hypnotic medications, isoniazid, glucocorticoids, or psychostimulant appetite suppressants. Neuroendocrine protocol Subjects reported for sessions at 1230 h, having fasted since 0900 h breakfast. At each session, an iv catheter was inserted into a forearm vein at 1300 h. One hour after IV line placement, naloxone (125 g/kg), ACTH(1 24) (250 g) dissolved in 0.9% saline or Placebo (0.9% saline), was administered over 1 min as a bolus dose. Baseline blood samples were obtained 15 min before, and immediately before drug administration. After naloxone, ACTH(1 24), or Placebo administration, blood samples were drawn at 15, 30, 45, 60, 90, and 120 min. Sessions were administered (double-blind) in randomized order. Study days were separated by at least 48 h. Hormone assays Plasma concentrations of cortisol were measured by RIA (Diagnostic Products Corporation, Inc.; Los Angeles, CA). Intraassay and interassay coefficients of variation were 5.2% and 8.0%, respectively. Plasma concentrations of ACTH were measured by 2-site immunoradiometric assay (Nichols, San Capistrano, CA); intraassay and interassay coefficients of variance were 9% and 10%, respectively. Plasma concentrations of Leptin were measured by RIA (Linco Research, St. Charles, MO); intraassay and interassay coefficients of variance were 5% and 7%, respectively. Statistical analyses Hormone response was measured by two indicators: 1) Peak response was defined as the highest value after stimulation; and 2) Area under the cortisol-time curve was calculated over the 2-h time interval using the trapezoidal rule. Means plus and minus sem are reported. The effect of naloxone, cortrosyn, and placebo were analyzed by repeated-measures ANOVA with time of sampling as a within-subject factor. Correlation analysis between leptin and ACTH was performed by linear regression. Significance was accepted at P Each subject was assigned a plasma leptin concentration. Leptin concentrations were determined by two methods: 1) Leptin levels were expressed as the mean time 0 value (before injection), determined by averaging time 0 leptin concentrations recorded for each session (mean of time 0 placebo, naloxone, and cortrosyn); 2) Leptin levels were also expressed as 2-h integrated values, determined by taking the average leptin concentration measured during the 120-min placebo session or naloxone session or cortrosyn session. Tables 1 and 2 indicate that for each subject, plasma leptin concentrations were extremely stable within and between sessions. Integrated placebo leptin concentrations were used for linear regression studies. TABLE 2. Stability of plasma leptin concentrations Between Session (% CV) With-in Session (% CV) Placebo Naloxone Cortrosyn Coefficient of variation (CV) was calculated for between and within-session plasma leptin concentrations for each subject. Results To determine whether direct adrenal activation of cortisol secretion is sufficient to acutely alter plasma leptin levels, ACTH(1 24) challenges were performed. Figure 1 shows that ACTH(1 24) produced the expected increase in plasma cortisol levels but did not alter the plasma leptin concentration. Plasma leptin levels were identical throughout placebo and ACTH(1 24) sessions. Activation of the entire HPA axis was induced by employing the opioid receptor antagonist, naloxone. By blocking hypothalamic opioidergic inhibitory input to CRH-secreting neurons, naloxone induced the expected increase in plasma ACTH and cortisol (Fig. 2A). Plasma ACTH levels peaked 30 min after naloxone administration. Plasma cortisol levels peaked 60 min after the naloxone challenge. Once again, plasma leptin concentrations were not altered by this manipulation (Fig. 2B). As indicated in Tables 1 and 2, subjects had no significant within-session or between-session changes in plasma leptin levels. Linear regression was performed to test whether there is an association between hypothalamic opioid activity and integrated plasma leptin levels. There was a positive correlation between the 2-h integrated plasma leptin concentrations and plasma ACTH response to naloxone (peak r 0.822, P ; and area under curve r 0.832, P ) (Fig. 3, A and C). The correlation was stronger when leptin was normalized to body mass index (BMI) and expressed as leptin/bmi ratio (Fig. 3, B and D) (peak r 0.878, P ; and area under curve r 0.882, P ). The mean BMI was 25 1 kg/m 2 (men, ; women, ). The correlations held true when women and men were analyzed separately. There was no correlation between baseline ACTH levels and leptin, nor was there a correlation between peak ACTH levels and BMI. Discussion Leptin decreases food intake and increases metabolism (19 21). This signaling peptide is synthesized and secreted by adipose cells with circulating levels reflecting total body white adipose tissue (20). Several leptin receptor splice variants have been identified in hypothalamus and in other tissues (22 26). The principal action of leptin is to reduce appetite through an action on the hypothalamus (27). Leptin s

3 2140 WAND ET AL. JCE&M 1998 Vol 83 No 6 FIG. 1. Mean SE plasma cortisol (A) and leptin (B) responses to ACTH(1 24) or to placebo in seven normal men and nine normal women. FIG. 2. Mean SE plasma ACTH (A), cortisol (A), and leptin (B) responses to naloxone (125 g/kg) or to placebo in seven normal men and nine normal women. hypothalamic actions involve signaling through opioidergic, CRH, and neuropeptide Y pathways (11, 28). In addition to modulating food intake and energy expenditure, leptin interacts with a number of endocrine systems (29 34), including the HPA axis (9, 35). Most studies indicate that leptin restrains and inhibits HPA axis activity. The current study was performed to determine whether activation of the HPA axis would acutely alter plasma leptin levels and to determine whether there was an association between hypothalamic opioid tone and integrated plasma leptin levels. Our results firmly demonstrate that activation of the HPA axis does not acutely effect plasma leptin concentrations. Thus, the acute interaction of the HPA axis and leptin is unidirectional. Because we did not measure plasma leptin levels beyond 2 h after naloxone or ACTH(1 24) administration, it is possible that plasma leptin levels increased or decreased at later time points. In fact, a recent study showed that plasma leptin levels were greater 24 h after receiving 1 mg dexamethasone than before taking the corticosteroid (36). Additionally, our small sample size increases the possibility for type II error and the unequivocal acceptance of the null hypothesis. However, if the HPA axis does alter plasma leptin levels over this 2-h interval, it is a very minor effect and probably would have no physiological relevance. Interestingly, there was a strong positive correlation between integrated fasting plasma leptin levels and the magnitude of plasma ACTH release after opioid receptor blockade with naloxone. The higher the leptin concentration, the greater the ACTH response to naloxone stimulation. This finding cannot be attributed to differences in BMI, for two reasons. First, there was no correlation between BMI and the ACTH response; second, the association between plasma leptin and stimulated ACTH release was even stronger when leptin levels were normalized to each subject s BMI. This observation suggests that ambient leptin concentrations are associated with, and perhaps influence, the magnitude of ACTH release, at least when ACTH secretion is provoked by modulating the opioid system. How might this come about? The hypothalamic POMC system in the arcuate nucleus is implicated in energy homeostasis. Recent studies have shown that leptin-induced reductions in food intake are mediated through the opioidergic pathway (37). For example, agonists of the MC4 receptor reduce food intake (37), and targeted mutation of the MC4 receptor causes obesity (38). There are high levels of leptin receptor expression on POMC neurons in the arcuate nucleus (39). In addition to being involved in appetite control, arcuate nucleus opioidergic neurons impose inhibitory constraint on CRH neurons of the paraventricular nucleus (14). The greater the opioid tone, the greater the inhibition of the CRH neuron. The observation that the hypothalamic POMC system is one mediator of leptin s action is interesting because recent findings show

4 LEPTIN AND THE HPA AXIS 2141 FIG. 3. Correlation of plasma leptin and naloxone-stimulated plasma ACTH levels. A, Peak ACTH vs. leptin (peak r 0.822, P ); B, peak ACTH vs. leptin/bmi (peak r 0.878, P ); C, AUC ACTH vs. leptin (AUC r 0.832, P ); D, AUC ACTH vs. leptin/bmi (AUC r 0.882, P ). that human obesity and hyperleptinemia are linked to a segment of chromosome 2 near the POMC gene locus (40). It is plausible that plasma leptin concentrations alter opioidergic activity in the arcuate nucleus, which then, in turn, influences CRH-ACTH interactions. We speculate that individuals with higher plasma leptin concentrations have higher hypothalamic opioid tone. The effect of plasma leptin concentrations on opioidergic tone is unmasked during opioid receptor blockade (Fig. 4). In summary, these findings indicate that activation of the HPA axis does not acutely alter plasma leptin concentrations. However, plasma leptin levels may influence central nervous system opioidergic tone and thus modulate the magnitude of CRH release. FIG. 4. Proposed model for how plasma leptin concentration may influence the secretory dynamics of the HPA axis. Plasma leptin modulates arcuate nucleus opioidergic activity by signaling through leptin receptors on POMC neurons. Acute action: We speculate that an acute rise in plasma leptin induces melanocortin release, which (in conjunction with neuropeptide Y) inhibits appetite. Simultaneously, leptin induces -endorphin release, which inhibits CRH secretion. Chronic action: Whereas acute leptin exposure increases melanocortin and -endorphin release, we speculate that chronic leptin exposure modulates POMC gene expression and/or biosynthesis and thereby modulates opioid tone. The greater an individual s plasma leptin concentration, the greater the hypothalamic opioid activity. The greater the opioid activity, the greater the tonic restraint on the CRH neuron. Therefore, the magnitude of plasma ACTH response to opioid receptor blockade with naloxone positively correlates with the amount of opioid activity. Acknowledgments We thank Dr. Daniel Berkowitz for thoughtful discussions on this topic, and June Dameron for preparation of the manuscript. References 1. Bjorntorp P Endocrine abnormalities of obesity. Metabolism. 44: Akana SF, Strack AM, Hanson ES, Dallman MF Regulation of activity in the hypothalamo-pituitary-adrenal axis is integral to a larger hypothalamic system that determines caloric flow. Endocrinology. 135: Cunningham JJ, Calles-Escandon J, Garrido F, Carr D.B, Bode HH Hypercorticosteronuria and diminished pituitary responsiveness to CRF in obese Zucker rats. Endocrinology. 118: Tokuyama K, Himms-Hagen J Increased sensitivity of the genetically obese mouse to corticosterone. Am J Physiol. 252:E202 E Kopelman PG Obesity in Europe. In: Bjorntorp P, Rossner S, eds. London: Libey; 88: Chowers I, Einat R, Feldman S Effects of starvation on levels of corti-

5 2142 WAND ET AL. JCE&M 1998 Vol 83 No 6 cotrophin releasing factor, corticotrophin and plasma corticosterone in rats. Acta Endocrinol (Copenh). 61: Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM Positional cloning of the mouse obese gene and its human homologue. Nature. 372: Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG Identification of targets of leptin action in rat hypothalamus. J Clin Invest. 98: Ahima RS, Prabakaran D, Mantzoros C, et al Role of leptin in the neuroendocrine response to fasting. Nature. 382: Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW, Flier JS Leptin inhibition of the HPA axis in response to stress. Endocrinology. 138: Stephens TW, Basinski M, Bristow PK, et al The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 377: Seeley RJ, Yagaloff KA, Thiele TE, Van Dijk G, Baskin DG, Schwartz MW Melanocortin receptors in leptin effects. Nature. 390: Del Campo MAF, Dowson JH, Herbert J, Paykel ES Effects of naloxone on diurnal rhythms in mood and endocrine function: a dose-response study in man. Psychopharmacology. 144: Delitala G, Trainer PJ, Oliva O, Fanciulli G, Grossman AB Opioid peptide and -adrenoceptor pathways in the regulation of the pituitary-adrenal axis in man. J Endocrinol. 141: Torpy DJ, Grice JE, Hockings GI, Walters MM, Crosbie GV, Jackson RV Alprazolam blocks the naloxone-stimulated hypothalamo-pituitary-adrenal axis in man. J Clin Endocrinol Metab. 76: Inder WJ, Hellemans J, Ellis MJ, Evans MJ, Livesey JH, Donald RA Elevated basal adrenocorticotropin and evidence for increased central opioid tone in highly trained male athletes. J Clin Endocrinol Metab. 80: Facchinetti F, Fioroni L, Martignoni E, Sances G, Costa A, Genazzani AR Changes of opioid modulation of the hypothalamo-pituitary-adrenal axis in patients with severe premenstrual syndrome. Psychosom Med. 56: Cizza G, Lotsikas AJ, Licinio J, Gold PW, Chrousos GP Plasma leptin levels do not change in patients with Cushing s disease shortly after correction of hypercortisolism. J Clin Endocrinol Metab. 82: Romsos DR, Swick AG, Chrunyk BA, Cunningham D, Mistry AM Intracerebroventricular recombinant leptin decreases food-intake and increases metabolic rate in ob/ob mice. FASEB J. 10: Halaas JL, Gajiwala KS, Maffei M, et al Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 269: Pelleymounter MA, Cullen MJ, Baker MB, et al Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 269: Emilsson V, Liu Y-L, Cawthorne MA, Morton NM, Davenport M Expression of the functional leptin receptor mrna in pancreatic islet and direct inhibitory action of leptin on insulin secretion. Diabetes. 46: Cioffi JA, Shafer AW, Zupancic TJ Novel B219/OB receptor isoformspossible role of leptin in hematopoiesis and reproduction. Nature Med. 2: Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P Localization of leptin receptor messenger-rna and the long form splice variant (OB-Rb) in mouse hypothalamus and adjacent brain-regions by in-situ hybridization. FEBS Lett. 387: Tartaglia LA, Dembski M, Weng X, et al Identification and expression cloning of a leptin receptor, OB-R. Cell. 83: Lee GH, Proenca R, Montez JM, et al Abnormal splicing of the leptin receptor in diabetic mice. Nature. 379: Barinaga M Obese protein slims mice. Science. 269: White BD, Dean RG, Edwards GL, Martin RJ Type II corticosteroids receptor stimulation increases NPY gene expression in basomedial hypothalamus of rats. Am J Physiol. 266:R1523 R Considine RV, Considine EL, Williams CJ, et al Evidence against either a premature stop codon or the absence of obese gene mrna in human obesity. J Clin Invest. 95: De Vos P, Saladin R, Auwerx J, Staels B Induction of ob gene expression by corticosteroids is accompanied by body weight loss and reduced food intake. J Biol Chem. 270: Chehab FE, Lim ME, Lu RH Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet. 12: Deleted in proof. 33. Barash IA, Cheung CC, Weigle DS, et al Leptin is a metabolic signal to the reproductive-system. Endocrinology. 137: Collins S, Kuhn CM, Petro AE, Swick AG, Chrunyk BA, Surwit RS Role of leptin in fat regulation. Nature. 380: Sleicker LJ, Sloop KW, Surface PL, et al Regulation of expression of ob messenger-rna and protein by glucocorticoids and camp. J Biol Chem. 271: Masuzaki H, Ogawa Y, Hosoda K, et al Glucocorticoid regulation of leptin synthesis and secretion in humans: elevated plasma leptin levels in Cushing s syndrome. J Clin Endocrinol Metab. 82: Fan W, Boston BA, Keterson RA, Hruby VJ, Cone R Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature. 385: Huszar D, Lynch CA, Fairchild-Huntress V Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell. 88: Cheung CC, Clifton DK, Steiner RA Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology. 138: Comuzzie AG, Hixson JE, Almasy L, et al A major QTL determining serum leptin levels and fat mass is located on human chromosome 2. Nat Genet. 15: