OBJECTIVE: Women with visceral obesity may have hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Since glucocorticoids are involved in the ...
International Journal of Obesity (1997) 21, 708±711 ß 1996 Stockton Press All rights reserved 0307±0565/96 $12.00
Short Report
Leptin and the hypothalamic-pituitary-adrenal axis activity in women with different obesity phenotypes R Vettor1, V Vicennati2, A Gambineri2, C Pagano1, F Calzoni2 and R Pasquali2 1
Endocrine-Metabolic Laboratory, Department of Internal Medicine, University of Padua; and 2Endocrine Unit, Department of Internal Medicine and Gastroenterology, University of Bologna, Italy
OBJECTIVE: Women with visceral obesity may have hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Since glucocorticoids are involved in the expression of the ob gene, this study was carried out to investigate the relationship between serum leptin and the activity of the HPA axis in women with different obesity phenotypes. DESIGN: Cross sectional clinical study. SUBJECTS: Fifteen obese (Body Mass Index BMI28 kg=m2) women and ten normal weight control women (BMI26 kg=m2) were included in the study. MEASUREMENTS: Body fat distribution was de®ned by CT scan at the L4±L5 level. Baseline blood samples were obtained for hormone concentrations. The activity of the HPA axis was evaluated by measuring ACTH and cortisol blood levels after combined iv administration of corticotropin releasing factor (100 mg) arginine-vasopressin (0.3 IU). RESULTS: Baseline cortisol, ACTH, and androgen levels were similar in all groups, whereas leptin levels were signi®cantly higher in the obese groups than in normal weight controls, without any signi®cant difference between women with different obesity phenotypes. Incremental areas of ACTH and cortisol were signi®cantly higher in women with visceral obesity than in those with subcutaneous obesity and controls. No signi®cant correlation was found between the activity of the HPA axis and leptin concentrations. Leptin showed a highly signi®cant correlation with BMI and subcutaneous fat and a weak but signi®cant correlation with visceral fat and the visceral-to-subcutaneous fat ratio. CONCLUSION: Women with different obesity phenotypes had similar serum leptin concentrations but different HPA axis activity, and there was no correlation between them. Keywords: obesity, HPA axis, leptin.
Introduction The ob gene is expressed in the adipose tissue and encodes for a protein, leptin, that is thought to be the putative afferent satiety signal from the adipose mass to the central nervous system (CNS).1,2 In humans, leptin concentration is closely related to body weight and total fat mass.3 Leptin expression and production by adipose tissue is regulated by several factors in¯uencing glucose metabolism such as insulin and glucocorticoids.2 In particular glucocorticoids are suggested as being involved in the development of central body fat distribution and related hormonal and metabolic abnormalities.4 The authors5,6 and others7 have demonstrated that subjects with visceral obesity may have hyperactivity of the HPA axis which could re¯ect a primary neuroendocrine abnormality. InterCorrespondence: Prof R Pasquali, UnitaÁ di Endocrinologia, Dipartimento di Medicina Interna e Gastroenterologia, Policlinico S. Orsola-Malpighi, Via Massarenti 9, 40138 Bologna, Italy. Received 13 January 1997; revised 18 March 1997; accepted 4 April 1997
estingly in physiological conditions the HPA axis response to neuroendocrine stimuli appears to be under close genetic control.8 Moreover, since a disregulation of the glucocorticoid pathway at the receptor level has been associated with predisposition to obesity, at least in animal models, the glucocorticoid receptors have been evaluated among many gene products likely to play a role in obesity.9 However preliminary data obtained in human studies have yielded contradictory results.10,11 This study was therefore carried out to investigate the relationship between the activity of the HPA axis and serum leptin concentrations in a group of women with different obesity phenotypes.
Materials and methods Fifteen obese women and ten normal weight control women were included in the study. Their characteristics are reported in Table 1. The obese women had been referred to the Endocrine Unit of the Department
Leptin and the hypothalamic-pituitary-adrenal axis in obesity R Vettor et al Table 1 Characteristics (mean s.d.) of obese women with visceral (V-BFD) and subcutaneous (S-BFD) body fat distribution and of normal weight control women Characteristics
Number Age (y) Body weight (kg) Body Mass Index (Kg=m2) Waist-to-hip ratio VAT (cm2) SAT (cm2) VAT=SAT ratio
Obese women V-BFD
S-BFD
Control women
7 33.8 7.4 104.2 9.9b 40.8 4.7b 0.88 0.11b 186.5 69.9a,c 593.7 86.9a 0.31 0.11d
8 28.3 7.1 100.1 24.8b 37.6 9.1b 0.80 0.09b 72.5 31.3c 554.8 144.5c 0.12 0.03
10 26.8 5.8 54.0 5.5 21.2 1.0 0.72 0.04 20.9 8.0 120.5 77.6 0.20 0.08
ANOVAa P values NS < 0.001 < 0.001 < 0.002 < 0.001 < 0.001 < 0.001
VAT: visceral adipose tissue, SAT: subcutaneous adipose tissue. a Refers to the statistical differences between the three groups (ANOVA). Student's t-test: a < 0.05 V-BFD and SBFD vs controls; b < 0.01 V-BFD and S-BFD vs controls; c < 0.05 V-BFD vs S-BFD.
of Internal Medicine and Gastroenterology, University of Bologna, as outpatients, for evaluation and treatment of their obesity, whereas controls were selected from doctors and nurses of the staff. All obese women had normal menses and ten of them had previously been pregnant. Other endocrine and metabolic diseases were excluded on the basis of physical examination and adequate laboratory tests. Eight women had mild hypertension but they had never been treated for this. None took drugs for at least one month before the study, nor were any dieting. Normal weight women were regularly menstruating and none of them had a history of overweight or obesity. All obese women and ®ve controls had participated in previous studies on the activity of the HPA axis in obesity.6 Written and informed consent was obtained for all women included in the study. Body height was measured without shoes to the nearest 0.5 cm, and body weight without clothes. All obese women had a body mass index (BMI) (weight, kg, divided by height, m2) value greater than 28. The waist (W) and hip (H) circumferences (WHR) were also measured as previously described5 and their ratio was calculated. Body fat distribution was de®ned by computerized axial tomography (at the L4±L5 level), and total visceral adipose tissue area (VAT), subcutaneous adipose tissue areas (SAT), and their ratios (V=S) were calculated. Women were examined during day 5±10 of the follicular phase of the menstrual cycle while following their usual diet, providing at least 250±300 g of carbohydrates were ingested. Blood tests were performed in the morning (8.00±9.00), after overnight fasting, while subjects had been quietly lying down for at least 15±20 min after an indwelling cannula had been placed in an antecubital vein of an arm kept patent with slowly-infused normal saline. A single blood sample was obtained for baseline hormone concentrations in all subjects. The activity of the HPA axis was examined by administering a combined stimulus of corticotropin releasing hormone (CRF) and arginine-vasopressin (AVP) as previously described.6 Only ®ve control women were submitted to this test. Human CRF (hCRF, Novabiochem, Lau-
fel®ngen, Switzerland) and synthetic 8-arginine-vasopressin (AVP) (Pitressin, Parke Davis Co, Germany) were injected as iv bolus, at doses of 100 mg and 0.3 IE, respectively. Blood samples for ACTH and cortisol determination were drawn in basal conditions (15 and 0 min) and after 15, 30, 60, 90, 120 min thereafter. Immediately after taking the blood samples, they were placed in different tubes containing EDTA without or with aprotinin (500 U=mL), for hormone determinations, and maintained on ice until being stored at 780 C until assayed. All assays of each women were performed in duplicate. Cortisol, ACTH and androgen levels were assayed as previously described.5 Plasma leptin was measured by radioimmunoassay (Linco Research Inc., St. Charles, MO, USA). In our laboratory, sensitivity is 0.5 ng=ml and the intra and interassay coef®cients of variations are 3% and 10%, respectively. Results are expressed as mean s.d. Incremental areas under the curves (AUC-l) of ACTH and cortisol were calculated by the trapezoidal method, after subtracting baseline values. All comparisons were performed by means of ANOVA and the Student's t-test, when appropriate. Simple and multiple regression analysis were also used to evaluate the correlations between the variables. The value of P < 0.05 was used to de®ne statistical signi®cance.
Results Baseline cortisol, ACTH and androgen concentrations were similar in all groups. On the contrary, compared to normal weight controls, leptin levels were signi®cantly (P0.01) higher in obese groups than in controls, without any further difference between visceral and sub-cutaneous fat distribution groups (Table 2). The main results of the test CRF=AVP have been previously reported.6 Brie¯y, there were no signi®cant differences in baseline hormone levels between the groups. The absolute increases of ACTH and cortisol after stimulation (at times 15, 30 and 60 min) were
709
Leptin and the hypothalamic-pituitary-adrenal axis in obesity R Vettor et al
710
Table 2 Hormonal parameters (mean s.d.) in obese women with visceral (V-BFD) and subcutaneous (S-BFD) body fat distribution and of normal weight control women Parameters
Obese women
ANOVAa P value
V-BFD
S-BFD
Control women
Basal values Cortisol (ng=mL) ACTH (pg=mL) Testosterone (ng=mL) Androstenedione (ng=dL) DHEAS (mg=mL) Leptin (ng=mL)
108.1 29.4 19.1 6.2 0.41 0.17 148.2 80.9 1.43 0.25 30.5 14.6a
103.5 39.2 13.1 5.9 0.45 0.20 147.4 65.1 2.66 1.36 35.2 11.9a
154.6 51.8 18.3 5.9 0.45 0.17 151.0 28.2 2.79 1.32 4.5 1.8
ns ns ns ns ns < 0.001
CRF=AVP test CortisolAUC-l(ng=L.min) ACTHAUC-l (pg=L.min)
11994 4799b 6623 3490b
8016 5206 3680 2161
6511 5626 3418 1632
0.05 < 0.1 < 0.05
Refers to the statistical differences between the three groups (ANOVA). Student's t-test: a P < 0.01 V-BFD and S-BFD vs controls; b < 0.05 V-BFD vs S-BFD and controls.
signi®cantly higher in visceral than in subcutaneous fat distribution or control women. Moreover, CortisolAUC-l and ACTHAUC-l were signi®cantly (P < 0.05) higher in visceral than in sub-cutaneous fat distribution or control women (see Table 2). Leptin concentrations were signi®cantly correlated with body weight (r2 0.648; P < 0.001), BMI (r2 0.582; P < 0.001), and visceral (r2 0.213; P < 0.04) and subcutaneous (r2 0.670; P < 0.001) fat depots. After adjusting for BMI, no correlation was found between leptin levels and WHR, whereas a negative weak but signi®cant correlation with the V=S ratio (t 2.327; P < 0.05) was found. There were no signi®cant correlations between leptin and hormone values, including basal and stimulated cortisol and ACTH.
leptin on HPA axis in human obesity, but our present ®ndings do not support the existence of a clear regulatory interplay. As previously described by others4 we found that leptin concentrations were highly signi®cantly correlated with BMI and total and subcutaneous fat and signi®cantly less with visceral fat mass. The differences in the correlation coef®cients between leptin and subcutaneous and visceral fat are intriguing but may be in accordance with previous studies performed in animal models reporting that the ob mRNA levels in the subcutaneous adipose tissue are higher than those observed in the omental retroperitoneal and mesenteric depots.17 This may be consistent with the hypothesis that total body fat, rather than the amount of regional fat, is the major determinant of circulating leptin in humans.
Discussion
References
These data fail to provide evidence for a relationship between the activity of HPA axis and serum leptin concentrations in women, although they do not exclude it. On the other hand, data coming from in vitro and in vivo studies in both animals and humans reported a clear in¯uence of glucocorticoids on adipose tissue ob gene expression and leptin production.12,13 The interaction between glucocorticoids and leptin may operate at the neuroendocrine levels and in peripheral tissues, such as the adipose tissue. In the human brain glucocorticoid receptors are widely expressed, particularly in the limbic-hypothalamic system, and are regulated by many factors, including CRF and other neuropeptides, such as NPY, and catecholamines.14 NPY is a potent regulatory factor of ACTH secretion, acting on the CRF containing neurons in the paraventricular nucleus15 and stimulates leptin secretion, which in turn act at the central level by inhibiting NPY gene expression.16 This system appears to be unbalanced in human obesity. At present little is known about a possible effect of
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