• abdominal obesity;
  • cortisol;
  • glucocorticoid receptors;
  • leptin;
  • metabolic syndrome


  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objective: Abdominal obesity has a key role in the pathogenesis of prevalent and serious diseases and has been shown to be associated with an altered hypothalamic-pituitary-adrenal (HPA) axis function, which is regulated by endocrine feedback mediated via hippocampal glucocorticoid receptors (GR).

Research Methods and Procedures: We examined the HPA axis function by repeated salivary samples for the assessment of cortisol, as well as other endocrine, anthropometric, metabolic, and circulatory variables in middle-aged Swedish men (n = 284). With the restriction enzyme BclI, variants of the GR gene (GRL) locus were identified and two alleles with fragment lengths of 4.5 and 2.3 kilobases (kb) were detected.

Results: The observed frequencies were 40.1% for the 2.3- and 2.3-kb, 46.2% for the 4.5- and 2.3-kb, and 13.7% for the 4.5- and 4.5-kb genotypes. The larger allele (4.5 and 4.5 kb) was associated with elevated body mass index (BMI; p < 0.001), waist-to-hip circumference ratio (p = 0.015), abdominal sagittal diameter (p = 0.002), leptin (p < 0.001), and systolic blood pressure (borderline, p = 0.058). The 4.5- and 4.5-kb allele was associated with leptin after adjustment for BMI. Moreover, salivary cortisol values, particularly after stimulation by a standardized lunch (p = 0.040 to 0.086), were elevated in the men with the larger allele.

Discussion: These results indicate that there is an association between a deficient GR function, defined as a poor feedback regulation of the HPA axis activity, and a polymorphic restriction site at the GR gene locus. An abnormal control of HPA axis function due to genetic alterations may contribute to the pathogenesis of abdominal obesity.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Human obesity is characterized by wide variation in the distribution of excess body fat, and fat distribution affects the risks associated with obesity. Over the last decades, research has revealed relationships between abdominal obesity and abnormalities in glucose, insulin, and lipid metabolism, including hypertension in men and women (1). Abdominally obese individuals have also been shown to display abnormalities in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, which seem to originate with a hypersensitivity developing into a poorly regulated activity (2, 3).

The pituitary stimulates adrenal cortex to produce and release cortisol, which in turn exerts a negative endocrine feedback regulation, mediated via centrally localized glucocorticoid receptors (GR). Once cortisol is bound to these receptors, the activity of the HPA axis is controlled (4). The response to exogenous glucocorticoid is usually required to detect abnormal feedback regulatory the GR function of cortisol. We have previously reported that this feedback regulation is perturbed in abdominal obesity (2, 5).

The regulation of the HPA axis is strongly genetically controlled. Monozygotic twins show almost identical secretory patterns of cortisol (6). There is also evidence of a genetic control of abdominal obesity as well as its associated metabolic disturbances (7). There is thus reason to believe that the susceptibility to perturbation of the HPA axis activity might be partially genetically determined.

Previous genetic studies in obese subjects have identified a GR gene (GRL) locus BclI restriction fragment length polymorphism localized at a known BclI site in intron 1 and a putative site in intron 2, which is discoverable as a 4.5-kilobase (kb) allele (8, 9). This 4.5-kb allele is known to be associated with abdominal obesity and insulin resistance (8, 9) but has not been examined in relation to direct functional measurements of the GR. Furthermore, its prevalence in the population is not known. Thus, the purpose of this study is to examine the role of this GR polymorphism on the HPA axis activity and to estimate the prevalence of the various genotypes in a sample of Swedish men.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References


A cohort of men (n = 1302) was recruited in 1992 from the National Population Registry in Göteborg, Sweden. These men were all born during the first 6 months of 1944 and were living in Göteborg. A questionnaire was sent to these men between January and June, and 1040 (80%) of them responded (10, 11). Based on the self-reported waist-to-hip circumference ratio (WHR), three subgroups were defined as follows: 150 men with the lowest (≤0.885), 150 with the highest (≥1.01), and 150 around the arithmetic mean (0.94 to 0.96) of WHR. These 450 men were invited to a health examination in 1995, and 284 (63.1%) volunteered to participate; 94 (62.7%) men with lowest, 94 (62.7%) with highest, and 96 (64.0%) around the mean value of WHR. None were excluded. The study was conducted according to the principles expressed in the Declaration of Helsinki, and all subjects participating provided written informed consent. Nine subjects refused to be involved in the genetic studies, and in 13 subjects the amount of blood samples was not sufficient to conduct genetic analysis. The study was approved by the ethics committee of the medical faculty of the University of Göteborg and by the Swedish Data Inspection Board.

Study Design

The same team of research nurses and technicians performed all the examinations in the morning after the subjects had been fasting overnight.


Body weight was measured to the nearest 0.1 kg, and height was measured to the nearest 0.01 m. The body mass index (BMI) was calculated as the weight in kilograms divided by the square of height in meters (12). The waist (W) circumference was measured half way between the lower rib and iliac crest, the hip (H) circumference was measured over the widest part in the gluteal region, and the WHR was calculated (13). The abdominal sagittal diameter (centimeters) was determined as the distance between the examination table and the highest point of the abdomen in a recumbent position (14).

Salivary Cortisol Measurements.

The assessment of cortisol was performed using a sampling device called Salivette (Sarstedt, Rommelsdorf, Germany), where salivary cortisol concentrations were determined. The Salivette consists of a small cotton swab inside a centrifugation tube (15). The participants were asked to chew on the cotton swab for 45 to 60 seconds and store the Salivettes at +8 °C until returning them to the laboratory when all samples had been obtained. On a random working day, the participants delivered a salivary cortisol sample in the morning before breakfast (8:00 to 9:00 am); at 11:45 am, at 30, 40, and 60 minutes after a standardized lunch at noon, at 5:00 pm; and finally just before bedtime. The lunch was provided by the laboratory and contained 266 to 277 calories (protein, 18.2 to 21.0 g; carbohydrate, 33.6 to 36.4 g; fat, 5.6 to 6.5 g). The cortisol peak after lunch was calculated as the arithmetic mean of the cortisol levels for the 30, 45, and 60 minutes values after the standardized lunch minus the cortisol level at 11:45 am. Careful oral and written instructions were provided to avoid misunderstanding, and the feasibility of the procedures was tested in about 40 men who were not included in the final analyses. Salivary cortisol was determined by radioimmunoassay (RIA; Orion Diagnostica, Turku, Finland).

The day after these measurements, a dexamethasone suppression test was performed. Preliminary examinations (16) showed that using a low dose (0.5 mg × 1) of dexamethasone reveals mild abnormalities in the ability of the central GRs to control the HPA axis by feedback suppression. These abnormalities are not uncovered with the conventionally used dose of 1 mg (17). Consequently, the participants were given one 0.5-mg tablet of dexamethasone (Decadron; MSD, Sollentuna, Sweden) and two Salivettes. A saliva sample was collected in the morning before breakfast (8:00 to 9:00 am). At 10:00 pm the dexamethasone tablet was taken, and the salivary sampling was repeated on the following morning. The decrease in salivary cortisol level after dexamethasone administration was calculated as the arithmetic mean of the noninhibited morning cortisol levels, including that of the day curve measurements, minus the cortisol level after dexamethasone intake.

Hormones, Leptin, Glucose, and Serum Lipids.

Venous blood was obtained in the morning after overnight fasting. Total serum testosterone was determined by RIA (Testosterone125I RIA; ICN Biomedicals, Costa Mesa, CA) using testosterone bound to bovine serum albumin at C-19 as the antigen (testosterone-19-carboxymethylether-bovine serum albumin). Insulin growth factor-I (IGF-I) was determined by an extraction RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA) that involves the separation of soluble IGF-I from binding proteins with hydrochloric acid-ethanol (18). Insulin was measured by RIA (Pharmacia Insulin RIA 100; Kabi Pharmacia Diagnostics, Uppsala, Sweden). Serum leptin concentrations were determined in duplicate using a human leptin RIA kit (Linco Research, St. Charles, MO). Glucose was determined using the automated glucose analyzer ESAT 6660 from Eppendorf (19), and serum lipids were measured with an enzymatic procedure in a Roche Molecular Biochemicals Cobas Fara II (20).

Blood Pressure.

The blood pressure was measured twice on the right arm with the participants sitting, using a random-zero mercury sphygmomanometer (21), with a 5-minute rest for the subject before and between readings, and with the auscultation site at heart level, a peak inflation level of 30 mm Hg above radial pulse disappearance, a cuff-deflation rate of 2 to 3 mm Hg/second, and pressure value recorded to the nearest even digit. The heart rate was recorded simultaneously, and the individual mean systolic and diastolic blood pressures were calculated as the mean of the two measurements.

Determination of the Genotypes.

DNA was extracted using Qiagen kits (Qiagen Inc., GmbH, Germany). One μg of DNA from each subject was digested with 20 units of BclI restriction enzyme (New England Biolabs) at 50 °C for 16 to 20 hours in 20 μL of total volume. The digested DNA was separated in a 1.2% agarose gel for 16 to 20 hours at 50 mA and transferred to a nylon membrane by alkaline blotting (22) for hybridization. Prehybridization was carried out in 3 mL of a solution of 0.25 M Na2HPO4 (pH 7.4), 1 mM EDTA, 7% sodium dodecyl sulfate, and 100 μg/mL salmon sperm (SSPE) DNA (Stratagene, La Jolla, CA) for 4 to 18 hours at 65 °C in a hybridization incubator (Robins Scientific Corp., Sunnyvale, CA). The membranes were then hybridized with a 4.3-kb human GRL cDNA probe (23), which was labeled with [α-32P]dCTP by random priming (24) using a T7 DNA polymerase kit (T7 Quick Prime; Pharmacia Biotech Inc., Baie dÚrfe, QC) to ≥1 × 109 cpm/μg specific activity. After hybridization at 65 °C for 18 to 20 hours, nonspecific binding was washed off two to four times in a solution of 0.1 M SSPE (20 mM NaCl, 1 mM Na2HPO4, 0.1 mM EDTA) plus 0.1% sodium dodecyl sulfate. Occasionally, the membranes were washed in a more stringent solution containing 10 times less salt. Subsequently, the membranes were exposed to Kodak X-Omat AR film (New Haven, CT) for 70 to 120 hours at −70 °C.

Statistical Analysis

Data comparisons were carried out with one-way ANOVA. Spjotvoll–Stoline post hoc tests (25) were performed to test for differences between each genotype. All metabolic variables, including leptin, were adjusted for BMI and WHR by ANCOVA. The results are presented as mean ± SD. For the relationship between pairs of variables, Spearman rank-correlation coefficients were calculated (Hommel post hoc test (26)).

p values are two-sided throughout, and a p < 0.05 was considered to be significant. The statistical analyses were performed with SPSS for Windows, release 7.5 (SPSS, Chicago, IL)


  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Table 1 shows the results of measurements of the GRL polymorphism in the men by genotype. The 4.5- and 4.5-kb homozygotes showed significantly higher BMI, WHR, abdominal sagittal diameter, leptin, and systolic blood pressure (borderline) than the 2.3- and 2.3-kb homozygotes. In heterozygotes (4.5 and 2.3 kb), testosterone was borderline significantly higher than in the 2.3- and 2.3-kb homozygotes. In comparison with heterozygotes, the men with the 4.5- and 4.5-kb genotype had higher BMI, WHR, abdominal sagittal diameter, and leptin levels. When adjusted for BMI and WHR, the results of leptin, insulin, and metabolic variables remained unchanged.

Table 1.  Results of measurements in relation to the genotypes (2.3 and 2.3, 4.5 and 2.3, and 4.5 and 4.5 kb) of the GRL gene identified by BclI
 Genotypes in kb  
Variables2.3 and 2.3 (N = 105)4.5 and 2.3 (N = 121)4.5 and 4.5 (N = 36)p valuesAdjusted p values*
  • Values (means ± SD) with different letters are significant at the 0.05 level.

  • *

    Adjusted for BMI and WHR.

BMI (kg/m2)26.5 ± 3.9a25.4 ± 3.5b28.3 ± 4.5c<0.001 
WHR0.94 ± 0.07a0.93 ± 0.06a0.97 ± 0.08b0.015 
Abdominal sagittal diameter (cm)22.7 ± 3.6a22.1 ± 3.3a24.6 ± 4.2b0.002 
Diurnal cortisol secretion (nM)6.9 ± 1.97.8 ± 5.07.8 ± 2.7>0.20 
Dexamethasone suppression test11.9 ± 5.512.0 ± 5.412.5 ± 5.1>0.20 
Testosterone (nM)18.8 ± 5.4a20.6 ± 5.3b19.4 ± 6.30.055 
IGF-I (μg/liter)206.3 ± 68.6209.1 ± 55.8197.6 ± 82.8>0.20 
Leptin (μg/liter)6.1 ± 3.8a5.5 ± 3.9a9.0 ± 5.8b<0.0010.039
Insulin (mU/liter)12.8 ± 9.312.1 ± 12.814.9 ± 9.1>0.20>0.20
Glucose (mM)4.6 ± 1.14.5 ± 0.74.8 ± 1.4>0.20>0.20
Triglycerides (mM)1.9 ± 1.11.8 ± 1.11.9 ± 0.8>0.20>0.20
Cholesterol (mM)6.2 ± 1.26.1 ± 1.06.3 ± 0.8>0.20>0.20
High-density lipoprotein cholesterol (mM)1.2 ± 0.41.2 ± 0.31.3 ± 0.3>0.20>0.20
Low-density lipoprotein cholesterol (mM)4.1 ± 1.14.0 ± 1.04.2 ± 0.9>0.20>0.20
Systolic blood pressure (mm Hg)127.4 ± 18.0a129.7 ± 17.1135.5 ± 17.2b0.058 
Diastolic blood pressure (mm Hg)82.2 ± 10.684.3 ± 10.485.4 ± 10.80.194 
Heart rate (beats/minute)67.8 ± 10.069.5 ± 11.270.8 ± 9.4>0.20 

Correlations between the variables among 2.3- and 2.3-kb homozygote men are shown in Table 2. The BMI, WHR, and abdominal sagittal diameter correlated strongly with each other and with leptin, insulin, glucose, triglycerides, high-density lipoprotein cholesterol (negative), blood pressures, and heart rate. Testosterone was negatively related, whereas IGF-I showed no relationship to the anthropometric measurements. The dexamethasone suppression test result correlated positively with diurnal cortisol secretion and negatively with variability of diurnal cortisol secretion, WHR, glucose, and systolic blood pressure. There were other interrelationships between circulatory, metabolic, and endocrine measurements as seen in Table 2. When a similar correlation matrix was analyzed for the men who were homozygotes for the 4.5-kb allele or heterozygotes (4.5- and 2.3-kb allele) similar results were essentially obtained (not shown).

Table 2.  Correlations between measured variables in the 2.3- and 2.3-kb homozygotes (N = 105)
  • *

    Correlation coefficients significant at the 0.05 level.

  • Abbreviations: D. abdominal sagittal diameter; DCS. diurnal cortisol secretion; T. testosterone; TG. triglycerides; CH. cholesterol; HDL. high-density lippoprotein cholesterol; LDL. low-density lipoprotein cholesterol; SBP. systolic blood pressure; DBP. diastolic blood pressure; HR. heart rate.


Other measurements of the HPA axis function besides dexamethasone suppression and total cortisol secretion (Table 1) were also subjected to comparisons among the three GRL genotypes. These measurements included the absolute values of salivary cortisol after dexamethasone suppression, the variability of diurnal cortisol secretion, estimated as described in Rosmond et al. (2), and the separate values of cortisol 30, 45, and 60 minutes after lunch, as well as the lunch peak (arithmetic mean of the sum of 30-, 45-, and 60-minute values). The significant results are presented in Table 3.

Table 3.  Comparisons among the three GRL genotypes in selected measurements of salivary cortisol
 Genotypes in kb  
Variables2.3 and 2.3 (N = 105)4.5 and 2.3 (N = 121)4.5 and 4.5 (N = 36)p valuesAdjusted p values*
  • Values (means ± SD) with different letters are significant at the 0.05 level.

  • *

    Adjusted for BMI and WHR.

Cortisol level (nM) at 30 minutes after lunch7.1 ± 2.7a8.6 ± 7.910.8 ± 15.4b0.0860.063
Cortisol level (nM) at 45 minutes after lunch6.4 ± 2.2a8.0 ± 6.7b8.8 ± 7.0b0.0400.033
Cortisol level (nM) at 60 minutes after lunch5.8 ± 2.0a7.4 ± 6.18.3 ± 8.3b0.0480.037
Average cortisol (nM) of 30, 45, and 60 minutes6.4 ± 2.1a8.0 ± 6.89.3 ± 10.0b0.0590.044

The homozygotes for the 4.5-kb allele showed generally significantly higher values at 30, 45, and 60 minutes after lunch and a higher total lunch peak of cortisol than the other genotypes. These results remained after adjustments for BMI and WHR. The salivary cortisol concentrations at 5:00 pm and at bedtime were not significantly different among the genotypes.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The subjects examined were selected from a larger population of men born in 1944 (10, 11). These men responded to a questionnaire and performed self-measurements of body weight, height, and circumferences (10, 11). The subsample subjected to more detailed measurements was stratified by WHR measurement classes. The nonrespondents in this second phase of the examination did not differ from the respondents, and the rate of nonrespondents was similar across the three WHR subgroups (2). Therefore, the men of the present report can be considered as representative of the population of middle-aged men from the city of Göteborg, Sweden.

The assessment of cortisol in saliva provides several advantages over blood cortisol measurements, becausethe collection procedure is noninvasive and stress-free (15, 27, 28). Because salivary cortisol sampling is laboratory-independent, it can be applied under a variety of field settings. The concentration of cortisol in saliva is independent of the saliva flow and represents the unbound (“free”) hormone fraction, which reflects accurately the free fraction of cortisol in plasma, despite the conversion of cortisol to cortisone in saliva by 11β-hydroxysteroid dehydrogenase (28). There are strong correlations with serum-free cortisol (15), which has also been confirmed in our laboratory (r > 0.90, unpublished). Moreover, because leptin, insulin, and metabolic variables are highly dependent on BMI and WHR (1, 2, 3), these measurements were adjusted for the influence of the anthropometric factors.

The previously described polymorphism of the GRL, localized on chromosome 5, and its association with abdominal obesity were confirmed (9). The observed frequencies were 40.1% for the 2.3- and 2.3-kb, 46.2% for the 4.5- and 2.3-kb, and 13.7% for the 4.5- and 4.5-kb genotypes. These frequencies are different from those reported previously (8, 9), probably due to differences in ethnicity as well as sampling selection.

Correlation matrices showed the expected, previously described associations (1) between BMI, WHR, and abdominal sagittal diameter, on the one hand, and endocrine, metabolic, and circulatory variables, on the other, among men with any of the three GRL genotypes. The associations of diurnal cortisol secretion and dexamethasone inhibition with the other variables have been described previously (2, 3) in the total material and are thus apparently also present for the various GRL genotypes. The lack of correlations between BMI, WHR, and abdominal sagittal diameter and diurnal cortisol secretion is in agreement with previous reports (2, 3) and is probably due to the circumstance that both elevated and low cortisol secretions seem to show such associations (2).

Tests for differences in cortisol levels between the GRL genotypes revealed that there were higher cortisol levels after stimulation by a standardized lunch in men with the 4.5- and 4.5-kb genotype than in those with the 2.3- and 2.3-kb genotype. Such differences were observed for both the individual time point measurements as well as the average cortisol level after lunch. These results indicate that there is an association between the BclI GRL polymorphism and the regulation of the HPA axis activity.

The HPA axis is regulated by central inputs via hypothalamic centers, which are balanced by an inhibitory feedback function exerted by GRs localized mainly in the hippocampal region of the brain (4). The high cortisol levels observed in the 4.5- and 4.5-kb individuals indicate that there is an elevated input of hypothalamic signals, which are not sufficiently controlled by the feedback regulatory system.

Previous studies of the men examined in this research have shown that cortisol assessments after stimulation of the HPA axis either by perceived stress or by physiological stress (lunch) have revealed central and peripheral consequences of elevated cortisol secretion that are more extreme than the diurnal curve in the steady state or in the dexamethasone suppression test (2, 3). Such consequences include abdominal obesity, decreased secretions of testosterone and growth hormone, and perturbed metabolic and circulatory variables (2, 3). The assessment of cortisol after stimulation of the HPA axis may therefore be considered to be of particular importance for various indicators of endocrine, metabolic, and circulatory health. These measurements are probably more precise and sensitive than the dexamethasone test, which is characterized by a relatively larger error due to the pronounced variation of the noninhibited morning cortisol levels. The lack of associations between the dexamethasone test and the GRL polymorphism, while more sensitive measurements show relationships, is presumably a consequence of the higher discriminative power of the other tests used herein.

The fact that stimulated cortisol secretion after lunch was related to the GRL polymorphism, while other measurements of cortisol were not, is compatible with previous findings showing that the GR controls stimulated cortisol secretion, while the mineralcorticoid receptor determines basal activity of cortisol secretion (4).

In other groups of men with similar background, the GR function was examined with a dose-response curve of a dexamethasone test (16). Perturbations of GR function were found to suggest a diminished sensitivity. The present results may therefore suggest a malfunction of the GR as far as sensitivity is concerned. This might be in part accounted for by the GRL polymorphism or another mutation in linkage disequilibrium with the BclI polymorphism.

Leptin levels were higher in the homozygotes for the 4.5-kb allele than in the men with the 2.3- and 2.3-kb genotype. Leptin levels were correlated with BMI (r = 0.68, p < 0.001, in the total group of men) as previously described (29). This finding suggests a form of leptin resistance in men with a combination of elevated leptin and BMI. Such a leptin resistance was more pronounced in the men with the larger GR allele who had elevated values of both BMI and leptin and remained after adjustments for BMI and WHR. A previous report indicated that there was a direct association between HPA axis perturbations and the leptin resistance in the men examined here (30). Evidence has recently been presented to the effect that glucocorticoids induce overeating and obesity, despite elevated leptin levels (31), suggesting that an elevation of the HPA axis activity could in fact desensitize the energy intake control system of leptin and thus induce obesity. Consequently, an association between perturbed functions of the GR and leptin resistance could be present, thus explaining the strong relationships between obesity, the GR function, and the gene polymorphism as evidenced herein.

The GRL polymorphism presented in this report is localized in the BclI site in intron 1 or possibly in intron 2 near the upstream part of the coding exons of the GRL (9). Because this polymorphism is not located in a coding, regulatory, or splicing region of the GRL, the functional role, if any, is uncertain. However, it may indicate functionally important polymorphisms in the vicinity. Exon 2 does not seem to be abnormal in men with abdominal obesity (unpublished observations), but other neighboring regions of the gene, including the promotor region, may harbor polymorphisms in linkage disequilibrium with the restriction site of the present study. This is currently under investigation.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by grants from the Swedish Medical Research Council (K97-19X-00251-35A) and the Medical Research Council of Canada (MT-13960).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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