LEPTIN, THE obese (ob) gene product, is a fat cell-derived

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1 X/97/$03.00/0 Vol. 82, No. 8 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1997 by The Endocrine Society Glucocorticoid Regulation of Leptin Synthesis and Secretion in Humans: Elevated Plasma Leptin Levels in Cushing s Syndrome* HIROAKI MASUZAKI, YOSHIHIRO OGAWA, KIMINORI HOSODA, TAKASHI MIYAWAKI, IKUKO HANAOKA, JUNKO HIRAOKA, AKIKO YASUNO, HARUO NISHIMURA, YASUNAO YOSHIMASA, SHIGEO NISHI, AND KAZUWA NAKAO Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606, Japan ABSTRACT Leptin, the obese (ob) gene product, is an adipocyte-derived satiety factor that is involved in the regulation of food ingestion and body weight. To investigate glucocorticoid regulation of leptin synthesis and secretion in humans, we measured plasma leptin levels in patients with Cushing s syndrome with adrenal or pituitary adenoma and in patients with iatrogenic Cushing s syndrome. Plasma leptin levels in patients with Cushing s syndrome were significantly elevated compared to those in nonobese healthy subjects and obese subjects without any metabolic or endocrine diseases at a given percentage of body fat by analysis of covariance. Received January 28, Revision received March 28, Accepted April 29, Address all correspondence and requests for reprints to: Yoshihiro Ogawa M.D., Ph.D., Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. ogawa@kuhp.kyoto-u.ac.jp. * This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture; the Japanese Ministry of Health and Welfare; the Yamanouchi Foundation for Research on Metabolic Disorders; the Uehara Memorial Foundation; the Japan Diabetes Foundation; and Otsuka Pharmaceutical Co. (Tokushima, Japan). In patients with adrenal or pituitary adenoma, after the tumor resection, plasma leptin levels were reduced, with a concurrent decrease in plasma cortisol levels. With no significant changes in body weight, plasma leptin levels were also elevated significantly in lean healthy volunteers 24 h after the admistration of 1 mg dexamethasone. Dexamethasone potently induced ob gene expression and leptin secretion in the organ culture of human adipose tissue. The data demonstrate that glucocorticoids act, at least in part, directly on the adipose tissue and increase leptin synthesis and secretion in humans. (J Clin Endocrinol Metab 82: , 1997) LEPTIN, THE obese (ob) gene product, is a fat cell-derived blood-borne satiety factor that is involved in the regulation of food intake and energy expenditure (1 3). The biological actions of leptin are mediated by interaction with its receptor in the hypothalamus (4). Adipose tissue expression of the ob gene and leptin secretion are markedly increased in several models of rodent obesity and human obesity (5 8). It is speculated that augmented production of leptin represents one of the deterrent mechanisms for the development of obesity. Plasma leptin levels have been shown to be correlated with the percentage of body fat in humans and total body lipid content in mice, suggesting that leptin serves as a good marker of adiposity (7, 9). Recent studies have demonstrated that leptin production is also regulated by a variety of hormones and chemicals both in vivo and in vitro; it is up-regulated by insulin, glucocorticoids, or neuropeptide Y (10 14) and down-regulated by camp or thiazolidinedione derivatives (13, 15, 16). There is also a gender difference in plasma leptin level in humans (17, 18). Glucocorticoids have been implicated in body weight regulation and the pathogenesis of obesity because of its orexigenic and adipogenic effects or its counterregulatory effects against insulin, such as gluconeogenesis and impaired glucose uptake (19 21). It was reported that glucocorticoids induce ob gene expression in rat adipose tissue both in vivo and in vitro (11, 12). De Vos et al. showed that sc injection of pharmacological doses of glucocorticoids for several days increases adipose tissue expression of the ob gene and postulated that leptin is involved in the concordant decrease in body weight and food ingestion (12). In the present study, to elucidate glucocorticoid regulation of leptin synthesis and secretion in humans, we measured plasma leptin levels in patients with Cushing s syndrome of various causes. We also examined the effects of glucocorticoids on leptin secretion in lean healthy subjects in vivo. To investigate whether glucocorticoids act directly on the human adipose tissue and regulate leptin production, we studied the effects of glucocorticoids on leptin synthesis and secretion in organ culture of human adipose tissue. Subjects Subjects and Methods We studied 17 patients with Cushing s syndrome of various causes (Table 1), 4 men (mean se, 36 5 yr; 3 with adrenal adenoma and 1 with pituitary adenoma) and 13 women [40 6 yr; 7 with adrenal adenoma, 3 with pituitary adenoma, and 5 who received oral prednisolone administration (20 60 mg/day) for at least 6 months and were diagnosed with iatrogenic Cushing s syndrome]. The diagnosis of adrenal and pituitary adenoma was confirmed by 2- and 8-mg dexamethasone suppression tests and computed tomographic analysis. Iatrogenic Cushing s syndrome was diagnosed by plasma cortisol levels and phys- 2542

2 LEPTIN AND GLUCOCORTICOIDS 2543 TABLE 1. Patient data Patients Age (yr) ical findings. All patients were characterized by centripetal fat accretion with abdominal striae, facial plethora, and skin acnes. Patients with adrenal or pituitary adenomas showed considerable elevations in plasma cortisol levels and urinary 17-hydroxycorticosteroid (17-OHCS) excretion. On the other hand, in patients with iatrogenic Cushing s syndrome, plasma cortisol levels and urinary 17-OHCS excretion were suppressed. No diurnal rhythm in plasma cortisol levels was found in these patients (data not shown). As control subjects, we also studied nonobese healthy subjects, 48 men [aged yr; mean se,31 7 yr; 16.0 kg/m 2 body mass index (BMI) 23.0 kg/m 2 ] and 34 premenopausal women (aged yr; 33 6 yr; 16.0 kg/m 2 BMI 23.0 kg/m 2 ), and obese subjects without any metabolic and endocrine diseases, 28 men (aged yr; 33 6 yr; 27.3 kg/m 2 BMI 50.0 kg/m 2 ) and 20 premenopausal women (aged yr; 29 5 yr; 27.8 kg/m 2 BMI 56.0 kg/m 2 ). Among those studied, the percentages of body fat in 13 women with Cushing s syndrome, 57 men (aged yr; mean 32 yr), and 49 premenopausal women (aged yr; mean 32 yr) were determined by dual energy x-ray absorptiometry method (22) using Hologic QDR-2000 (Hologic, Waltham, MA) on the day of plasma samplings. The present study was conducted with informed consent and was approved by the ethical committee on human research of Kyoto University Graduate School of Medicine. Plasma samplings Causes In all subjects studied, blood was withdrawn at 0600 h from the antecubital vein in a recumbent position after an overnight fast, immediately transferred to chilled siliconized glass tubes containing ethylenediamine tetraacetate (1 mg/ml), and centrifuged at 4 C. Plasma samples were immediately frozen and stored at 20 C until the assays for leptin and cortisol levels. Dexamethasone administration in healthy volunteers Ten lean healthy individuals (eight men and two women; 27 1 yr; BMI, kg/m 2 ; percentage of body fat, %) volunteered for the following study. One milligram of dexamethasone was orally administered at 0800 h after an overnight fast. Blood was withdrawn at the time of administration and 4 (before lunch), 8, 12 (before dinner), and 24 (next morning, 0800 h) h later. Food was delivered as breakfast ( h), lunch ( h), and dinner ( h). Plasma leptin levels without dexamethasone was also determined. No individuals took any medications or consumed an unusual diet. Organ culture of human adipose tissue Plasma cortisol ( g/dl) Human sc abdominal fat pads were obtained from three nonobese male subjects without endocrine or metabolic diseases at the time of Urinary 17-OHCS excretion (mg/day) surgery for hepatic cancer. Adipose tissue samples were rinsed and incubated with phosphate-buffered saline at 37 C for 30 min. Five hundred milligrams of well minced adipose tissue fragments were placed in 100-mm dish each containing 10 ml DMEM (Flow Laboratories, Costa Mesa, CA) supplemented with 0.5% FBS and 5 mg/ml BSA as described previously (8). After a 24-h preconditioning, cultured media were changed, and tissue fragments were subsequently incubated with or without 10 7 mol/l dexamethasone (Sigma Chemical Co., St. Louis, MO) for 3 days. Triplicate experiments were performed in each case. One milliliter of conditioned medium was obtained for the RIA for leptin every 24 h, and media were added to correct for the final concentration of dexamethasone. Total ribonucleic acid (RNA) was extracted 12 h after the dexamethasone treatment. Genomic DNA was quantitated to monitor the amount of adipose tissue fragments in each dish. Hormone assays The leptin levels in human plasma and culture media from the human adipose tissue were determined by use of the RIA for human leptin as described previously (8). Measurement of plasma cortisol levels and urinary 17-OHCS excretion levels were carried out as described previously (23). Northern blot analysis Northern blot analysis was performed using the 32 P-labeled fulllength human ob complementary DNA fragment as a probe (24). Transferred membranes were rehybridized with a human -actin genomic probe (Wako Pure Chemical Industries, Osaka, Japan) to confirm the integrity of RNA in each sample. Autoradiography was performed for 3hat 70 C with intensifying screens and quantitated by densitometric scanning. Statistical analysis BMI (kg/m 2 ) % of body fat Plasma leptin (ng/ml) Females 1 41 Adrenal adenoma Adrenal adenoma Adrenal adenoma Adrenal adenoma Adrenal adenoma Adrenal adenoma Pituitary adenoma Pituitary adenoma SLE (30 mg) Behc et s disease (30 mg) SLE (30 mg) SLE (60 mg) SLE (20 mg) Males Adrenal adenoma ND Adrenal adenoma ND Adrenal adenoma ND Pituitary adenoma ND OHCS, 17-Hydroxycorticosteroid; BMI, body mass index; SLE, systemic lupus erythematosus; ND, not determined. All values were expressed as the mean se. Relations between plasma leptin levels and percentage of body fat were evaluated by Pearson s correlation test. To assess differences in plasma leptin levels for the same percentage of body fat according to hypercorticoidism, the slopes and elevations of the linear regression lines between two groups were statistically compared by analysis of covariance (18, 25). Other statistical analysis was performed with ANOVA and t test, where applicable.

3 2544 MASUZAKI ET AL. JCE&M 1997 Vol 82 No 8 Results Plasma leptin levels in Cushing s syndrome Because of the sexual dimorphism in plasma leptin levels (17, 18), we studied male and female patients with Cushing s syndrome separately. In the present study, plasma leptin levels in 34 nonobese women ranged from ng/ml, with a mean se of ng/ml. In 20 obese women without endocrine or metabolic disorders, plasma leptin levels ranged from ng/ml, with a mean se of ng/ml; these values were significantly higher (P 0.001) than those in nonobese women (Fig. 1A). Plasma leptin levels in 13 women with Cushing s syndrome (16.6 kg/m 2 BMI 30.3 kg/m 2 ) ranged from ng/ml, with a mean se of ng/ml (Table 1). Plasma leptin levels in patients with Cushing s syndrome were approximately 5-fold higher than those in nonobese subjects (P 0.001), which were comparable to those in obese subjects (Fig. 1A). There were positive correlations between plasma leptin levels and percentage of body fat in patients with Cushing s syndrome and control subjects (Fig. 1B). When the regression lines of the two groups were compared by analysis of covariance, plasma leptin levels in patients with Cushing s syndrome were elevated significantly relative to those in control subjects (both nonobese and obese subjects) at a given percentage of body fat (P 0.05). No significant correlations were observed between plasma leptin levels and cortisol levels in patients with Cushing s syndrome (Table 1). We also measured plasma leptin levels in 4 men with Cushing s syndrome (19.8 kg/m 2 BMI 29.6 kg/m 2 ; Table 1). Plasma leptin levels in men with Cushing s syndrome were also elevated significantly compared with those in control men (P 0.001). Plasma leptin levels in men with Cushing s syndrome ranged from ng/ml, with a mean se of ng/ml; those in 48 nonobese healthy men ranged from ng/ml, with a mean se of ng/ml; and those in 28 obese men without endocrine or metabolic disorders ranged from ng/ml, with a mean se of ng/ml. Plasma leptin levels before and after adenoma resection In three patients with adrenal adenoma (patients 1, 2, and 3 in Table 1) and one patient with pituitary adenoma (patient 8 in Table 1), we also examined plasma leptin levels before and after adenoma resection (Table 2). After tumor resection, plasma leptin levels in these patients were decreased, with a concurrent decrease in plasma cortisol levels. There were no obvious changes in food intake during the observation period. Effects of glucocorticoids on leptin secretion in humans To assess the effects of glucocorticoids on leptin secretion in humans, 1 mg dexamethasone was orally administered to 10 lean healthy volunteers. Figure 2 illustrates the changes in plasma leptin levels 24 h after a single administration of dexamethasone. The plasma leptin level in each case was expressed as a percentage of the initial value. A significant FIG. 1. A, Plasma leptin levels in 13 women with Cushing s syndrome (closed column) and 49 premenopausal control women, nonobese healthy subjects, and obese subjects with no endocrine or metabolic diseases (open columns). *, P vs. the value in nonobese healthy subjects. B, Correlation between plasma leptin levels and percentage of body fat in 13 women with Cushing s syndrome (closed circles) and 49 premenopausal control women (open circles). Plasma leptin levels were correlated with the percentage of body fat in patients with Cushing s syndrome (r 0.66; P 0.001) and in premenopausal controls (r 0.75; P 0.001). A solid line indicates the linear regression line for patients with Cushing s syndrome: y 2.478x A broken line indicates the linear regression line for control subjects: y 1.633x Comparing the slopes of the linear regression lines between the two groups, plasma leptin levels in patients with Cushing s syndrome were elevated significantly (P 0.05) compared to those in control subjects at a given percentage of body fat.

4 LEPTIN AND GLUCOCORTICOIDS 2545 TABLE 2. Data for patients 1 4 before and after surgery Patient no. Age (yr) Causes Plasma cortisol ( g/dl) Urinary 17-OHCS excretion (mg/day) % of body fat Plasma leptin (ng/ml) 1 41 Adrenal adenoma months after surgery Adrenal adenoma months after surgery months after surgery Adrenal adenoma months after surgery Pituitary adenoma months after surgery FIG. 2. Changes in plasma leptin levels in 10 lean healthy volunteers after administration of 1 mg dexamethasone (right) or without dexamethasone (left). *, P 0.01 vs. the initial values; **, P vs. the values without dexamethasone. DEX, Dexamethasone; N.S., not significant. rise ( 170%; P 0.01) in plasma leptin levels occurred 24 h after dexamethasone administration. Twenty-four hours after dexamethasone administration, plasma leptin levels were significantly higher than those without dexamethasone (P 0.005). Without dexamethasone, plasma leptin levels were highest at midnight during the time course studied (data not shown), which is consistent with a previous report (26, 27). Effects of glucocorticoids on leptin synthesis and secretion in cultured human adipose tissue To elucidate whether glucocorticoids act directly on the human adipose tissue, we examined the effects of glucocorticoids on ob gene expression and leptin secretion in organ culture of human adipose tissue. In the absence of dexamethasone, leptin secretion was increased time dependently (Fig. 3A). On the other hand, leptin secretion was markedly FIG. 3. A, Time course of leptin levels in culture medium of the human adipose tissue with or without 10 7 mol/l dexamethasone. Subcutaneous abdominal fat pads were obtained at the time of surgery in three cases. The results were combined into one figure. Closed circles indicate those with dexamethasone, and open circles represent those without dexamethasone. Leptin levels in medium with dexamethasone were elevated significantly (P 0.01 on day 1; P on days 2 and 3) compared to those in medium without dexamethasone. Each point represents the mean SE (n 9). DEX, Dexamethasone. B, Northern blot analysis of ob mrna in cultured human adipose tissue in the same three cases as in A. Seven micrograms of total RNA were fractionated on 1% agarose gel and analyzed. elevated in adipose tissue treated with 10 7 mol/l dexamethasone compared to that in tissue without dexamethasone. Seventy-two hours after the incubation, leptin levels in medium from adipose tissue treated with dexamethasone were approximately 3 times higher than those in tissue with-

5 2546 MASUZAKI ET AL. JCE&M 1997 Vol 82 No 8 out dexamethasone. The secretory rate of leptin in the presence of dexamethasone was ng/g tissue day (mean se), which was significantly higher than that in the absence of dexamethasone ( ng/g tissue day) h after the beginning of culture (P 0.001). Northern blot analysis revealed that ob gene expression is markedly augmented in adipose tissue treated with 10 7 mol/l dexamethasone 12 h after the beginning of culture (Fig. 3B). Fold increases in ob messenger RNA (mrna) levels were 4.2, 5.2, and 8.9 in patients 1, 2, and 3, respectively. Discussion The present study demonstrates that plasma leptin levels are significantly elevated in patients with Cushing s syndrome of various causes. The plasma levels were comparable to those in obese subjects. These observations are consistent with recent reports (28). Cushing s syndrome is an in vivo state of glucocorticoid excess that is often associated with increased body fat mass (23, 29). This might explain the increase in leptin secretion in these patients. In the present study, however, plasma leptin levels in patients with Cushing s syndrome were elevated significantly compared to those in control subjects at a given percentage of body fat. Furthermore, in patients with Cushing s syndrome with adrenal or pituitary adenoma, tumor resection caused a marked reduction in plasma leptin levels with a concomitant decrease in plasma cortisol levels and with only a slight reduction in body weight. These findings suggest that glucocorticoids themselves are involved in the regulation of leptin secretion in humans. We previously showed that ob mrna levels differ according to the anatomical locations of fat pads in rats and humans (5, 24). Since Cushing s syndrome is characterized by the relocation of body fat or centripetal fat accretion (23, 29), it is interesting to know the leptin production in different fat pads from patients with Cushing s syndrome. The pathophysiological significance of leptin overproduced in Cushing s syndrome is unclear at present. It has been shown that plasma ACTH and corticosterone levels are elevated in two rodent models of leptin deficiency (fasted mice and ob/ob mice) (3, 30), which are corrected by exogenous administration of leptin (30). Furthermore, leptin has been shown to inhibit the hypothalamic CRH release from isolated perfused hypothalamic tissues (31). It is, therefore, tempting to speculate that leptin, which is induced by cortisol excess, acts as a negative regulator of adrenal cortisol production in Cushing s syndrome by its inhibitory effects on the CRH and ACTH production. Leptin also decreases mrna for neuropeptide Y, a potent stimulator of food intake, in the hypothalamic arcuate nucleus (32, 33), which might contribute to the antiobesity action of leptin (32 34). Patients with Cushing s syndrome might exhibit increased appetite because of the increased neuropeptide Y production by cortisol excess (35). In this regard, leptin may also act to antagonize cortisol by its inhibitory effects on the neuropeptide Y production in Cushing s syndrome. The present study also demonstrates that plasma leptin levels are elevated 24 h after a single administration of dexamethasone in vivo. The results are consistent with recent reports published during the preparation of the current study (36, 37). It has been demonstrated that sc injection of pharmacological doses of glucocorticoids (1 100 g hydrocortisone/g BW) potently induces ob gene expression in rats (12). In the present study, the dose of dexamethasone used (1 mg) was within the physiological range ( g/kg BW) (23). These observations strongly suggest that glucocorticoids are involved physiologically in the regulation of leptin secretion in vivo. The present study demonstrates that glucocorticoids increase leptin synthesis and secretion in cultured human adipose tissue in vitro, indicating that glucocorticoids act directly on the adipose tissue and regulate leptin production. The dose of dexamethasone used in the culture (10 7 mol/l) was equivalent to 13 g/dl cortisol in potency, which is also within the physiological range. These results are consistent with in vitro experiments using primary cultured rat and human adipocytes (11, 38, 39). It has been demonstrated that a half-site of the glucocorticoid response element is present in the 5 -flanking region of the human ob gene (40). It is likely that glucocorticoids activate the ob gene transcription through interaction with the glucocorticoid response element. In summary, the present study demonstrates that glucocorticoids increase leptin synthesis and secretion in human adipose tissue both in vivo and in vitro; this provides a better understanding of glucocorticoid regulation of leptin synthesis and secretion in humans. Acknowledgments We thank Drs. T. Nomura and Y. Fujisawa (Molecular Pharmacology Laboratory, Takeda Chemical Industries, Osaka, Japan) for helpful discussions, and Dr. S. Arii (Department of Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan); Drs. M. Murata, A. Sugawara, and T. Matsuo (Department of Internal Medicine, Osaka Saiseikai Nakatsu Hospital, Osaka, Japan); and Dr. S. Nakaishi (Department of Internal Medicine, Osaka Red Cross Hospital, Osaka, Japan) for sampling adipose tissue and plasma. We also acknowledge K. Hiramatsu and C. Kawahara for their excellent secretarial assistance. References 1. Caro JF, Shinha MK, Kolaczynski JW, et al Leptin: the tale of an obesity gene. 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