Objective:

Our goal was to assess the awakening cortisol response (ACR) in obese and reduced obese men and women.

Research Methods and Procedures:

Fifty-one men (16 lean, 19 abdominally obese, and 16 reduced obese) and 31 women (12 lean, 10 subcutaneously obese, and 9 reduced obese) were selected to participate to this study. Strict ranges of BMI and waist circumference were used to select the participants. Medical examination, psychological assessment, anthropometric measurements, and blood sampling were undergone at the laboratory. Cortisol response to awakening was determined with saliva cortisol sampling being taken immediately at the time of awakening and 30 minutes thereafter over 3 days within a period of 2 months.

Results:

Men with visceral obesity exhibited an enhanced ACR, whereas this response tends to return to normal in a reduced obese state. In women, peripheral fat accumulation does not modify ACR, but weight loss increased the response.

Discussion:

These results highlight gender effects on ACR of obese and reduced obese subjects, which could be accounted for by the different fat distribution profiles that characterize men and women. They also provide further support for the usefulness of ACR in assessing the hypothalamic-pituitary-adrenal axis activity status.

Introduction

There is a positive association between stress and obesity. In fact, obesity has been linked to disturbances of the hypothalamic-pituitary-adrenal (HPA)¹ axis and to an elevated cortisol output (1). This association seems to be more pronounced and prevalent in individuals, both men and women, with abdominal obesity (2). These individuals, in contrast to those with peripheral subcutaneous obesity, exhibit an inadequate cortisol suppression after a dexamethasone test, an exaggerated urinary cortisol output, an excessive cortisol secretion after psychological stress tests, and a hyperreactivity to the administration of cortisol secretagogues such as corticotropin-releasing factor (CRF), adrenonocorticotropic hormone, or CRF combined with arginine vasopressin (3, 4, 5, 6, 7, 8, 9, 10). In addition, prospective studies have demonstrated that chronic stress and cortisol reactivity lead to preferential deposition of abdominal fat (11). Although the brain mechanisms involved in the alteration of the HPA axis in obesity remain unknown, there is nonetheless evidence showing that cortisol might be involved in the stimulation of appetite (12, 13).

Studies on stress and obesity are confronted with the difficulty inherent to the valid assessment of the HPA axis activity. One potentially valuable way to assess the functionality of the HPA axis is by measuring the awakening cortisol response (ACR). ACR may be influenced by numerous factors including gender and the use of oral contraceptives but apparently not by age, time of awakening (as long as individuals wake up in the morning), sleep duration and quality, physical activity, smoking habits, and morning routines (14, 15). Moreover, this indicator of the HPA axis responsiveness is partially determined by genetic factors (16) and appears to be lowered by chronic fatigue (17) and amplified by depressive symptoms (18) and bipolar illness (19). ACR appears to be elevated in men with abdominal obesity (20, 21), but little is known about ACR in obese women, and the impact of weight reduction on the HPA axis activity has yet to be unequivocally described.

The present study was designed to investigate ACR in obese and reduced obese men and women. Men and women show different patterns of fat deposition, with men being characterized by visceral adiposity, whereas women generally distribute fat toward peripheral subcutaneous depots. Weight loss is a condition liable of generating relevant information about the relationship between obesity and the HPA axis activity status. In obese Zucker rats, Timofeeva et al. (22) observed an important activation of the HPA axis after energy restriction. However, only a few studies have examined the effects of weight loss on the HPA axis activation in humans. Weight reduction by starvation elevates plasma cortisol levels (23), whereas studies assessing the effect of weight loss by very low caloric diet have led to conflicting results regarding morning plasma cortisol levels and the stimulated cortisol response to CRF (24, 25).

Research Methods and Procedures

Subjects

A total of 87 healthy men (n = 54) and women (n = 33), 23 to 51 years old, were recruited through newspapers and radio advertisements and through advertising posters put on billboards at Laval University. Women who were selected were premenopausal with a normal menstrual cycle and were not using oral contraceptives. Exclusion criteria were any history of depression or psychiatric disorders, cardiovascular problems, smoking, and regular alcohol consumption. Subjects had to be totally medication free from the beginning until the end of the study and were required to abstain from alcohol consumption and physical activity 24 hours before sampling days and from caffeine during each day of the study.

The subjects were selected to fit into three groups: lean, BMI <27 kg/m², waist circumference (WC) <100 cm for men and <90 cm for women; obese, BMI 30 to 35 kg/m², WC >100 cm for men and <100 cm for women; and reduced obese, BMI >30 kg/m², WC >100 cm for men and <100 cm for women before weight loss, minimal weight loss of 5 kg, still losing weight or just stabilized. Weight loss resulted from changes in eating and physical activity habits. We made sure that our recruited physically active subjects did not have more than three 30-minute periods of mild- to moderate-intensity exercise per week. Subjects on medication to loose weight and on drastic diets were excluded.

All of the subjects were asked to come to the laboratory twice. During the first visit, subjects were submitted to a medical examination, blood sampling, psychological assessment, and anthropometric measurements. The participants were then instructed on the procedure for morning cortisol self-sampling at home and for bringing saliva samples to the laboratory on the second visit. This study protocol was approved by the medical ethic committee of Laval University (Project 152-99).

Medical Examination and Psychological Assessment

During the first visit to the laboratory, subjects underwent a medical examination to ascertain their health status. Blood samples were collected to determine serum lipids (total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, and triglycerides), fasting levels of glucose, and leptin for each participant. All subjects were asked to answer questionnaires about eating disorders [Eating Disorders Inventory (26), Binge Scale (27), Three-Factor Eating Questionnaire (28) ], body esteem [Body Esteem Scale (29) ], and depression symptoms [Beck Depression Inventory (30) ]. The participants were also screened for the presence of psychiatric and/or personality disorders by an interview with a trained research assistant [Structured Clinical Interview for DSM-III-R version Non-patient (31) ]. The scores for all these questionnaires were assessed and served as excluding factors for some of the subjects with eating behaviors or psychiatric disorders that could affect the HPA axis activity. On each sampling day, subjects were asked to answer questionnaires about depression [Beck Depression Inventory (30) ] and anxiety [State-Trait Anxiety Inventory (32) ]. In addition, participants were asked to provide specific information about the quality and the quantity of their sleep the night before the ACR test, the time of awakening, and the self-report of health and stress status. When the state of a participant was perturbed by one of these factors, the sampling day was postponed.

Anthropometric Measurements

Body weight was assessed on a balance beam scale to the nearest 0.1 kg with participants in underwear, and height was measured to the nearest centimeter. The BMI was calculated as weight (kilograms) divided by height (meters squared). WC was measured twice at the midpoint between the lowest rib and iliac crest, and the hip circumference was measured twice at the maximum width of the buttocks (33). The waist-to-hip ratio was calculated as the ratio between the waist and the hip circumferences. The abdominal sagittal diameter was determined at the nearest 0.1 cm as the distance between abdomen and back at the level of the midpoint between the lowest rib and iliac crest. Body density was determined by hydrodensitometry (34), and the Siri formula was used to estimate the percentage body fat from body density (35). The closed-circuit helium dilution method was used to assess residual lung volume (36).

ACR

Cortisol is secreted according to a circadian rhythm, and its level increases rapidly in the 30 minutes after awakening (14, 37). Salivary cortisol contains the free active fraction of cortisol and correlated tightly with cortisol in circulation (plasma cortisol) (38, 39). Measuring salivary cortisol allows for easy sampling of the hormone in everyday conditions and is much less invasive than measuring blood cortisol (40). ACR has been reported to show good intra-individual stability over time, at least over short periods of time (14, 15), and has been demonstrated to be a marker of HPA axis status (41). When measured repeatedly with strict reference to the time of awakening, ACR gives an insight of the effects of chronic stress (16, 42, 43, 44, 45). Throughout the 2 months of the study, patients had to sample their saliva on three different occasions. Subjects were instructed to start sampling immediately when awakening and 30 minutes thereafter. They were further informed to complete sampling before breakfast to avoid the effect of food intake on cortisol levels and contamination of saliva with food or drinks. Subjects were allowed to drink water between the two samples but not during the 5 minutes before sampling. Subjects were invited to store their saliva samples at 4 °C and to bring them to the laboratory before 11 AM on the same day. Participants were notified about the necessity of strictly following the time schedule to obtain valuable data. The subjects were contacted the day after the sampling to verify whether the sampling protocol was done according to our specifications (46). The Salivette sampling device (Sarstedt, Rommelsdorf, Germany) was used to collect saliva. The Salivette consists of a small cotton swab inside a standard centrifugation tube. ACR was determined by calculating the percentage of increase in cortisol levels between time of awakening (Time 0) and 30 minutes thereafter (Time 30). For each subject, the mean of the 3 days of sampling was assessed.

Cortisol and Leptin Assays

Saliva samples were centrifuged at 6000 rpm for 30 minutes and then stored at -27 °C until they were assayed. Salivary cortisol levels were determined by radioimmunoassay (Medicorp Inc., Montreal, Canada). Immediately after sampling, blood was kept at 4 °C for 1 hour, after which time it was centrifuged at 3000 rpm for 10 minutes at 4 °C. Serum was obtained from these samples and immediately stored at -75 °C until assayed. Leptin concentrations were assayed with a commercial radioimmunoassay kit.

Statistical Analysis

Data presented in text, Table 1 , and figures are means standard error. The ANOVA followed by post hoc paired Student's t tests was used to reveal differences between groups. The morning cortisol slopes were analyzed by multivariate analysis of variance. Levels of cortisol-binding globulin are likely to be influenced by sex hormone levels in women. For that reason, cortisol values were adjusted for estradiol levels by using estimated values from the regression model between estradiol levels and cortisol levels at awakening and 30 minutes thereafter. All statistical analyses were performed with JMP software (version 3.2.2; SAS Institute, Inc., Cary, NC). Tukey-Kramer post hoc tests were used to reveal differences between groups.

Table 1. - General characteristics of subjects.

Full table

Results

Descriptive characteristics of the participants in the three groups of subjects are presented in Table 1. Among the 88 participants recruited for this study, four women (two obese and two reduced obese) were excluded because their WC exceeded 90 cm. In addition, two men (one lean and one obese) were excluded from the analyses because they were not able to provide saliva samples, or they did not follow the saliva sampling protocol. In 21 subjects, ACR was assessed from two sampling sessions instead of three because one of the sessions did not provide enough samples. Our analyses revealed intra-individual variability (r = 0.09 for absolute and r = 0.14 for relative increases in cortisol levels in response to awakening), most likely because our measurements elapsed over a long period of time. Age was not different between groups and genders. In the reduced obese group, mean body weight loss was 10.8 1.0 kg (men, 12.0 1.4 kg; women, 8.8 0.7 kg), which corresponded to a decrease of 10.8 0.9% of initial weight (11.6 1.3% for men; 9.6 0.8% for women).

Figure 1 illustrates salivary cortisol levels of the three groups at the time of awakening and 30 minutes thereafter. Salivary cortisol concentrations rose in 85% of the subjects over the first 30 minutes after awakening. In men, ANOVA revealed a significant group-by-time interaction [F(2,48) = 4.31; p = 0.019]. Immediately after awakening, cortisol levels in reduced obese men were greater than those of obese men [F(2,48) = 5.22; p = 0.009], whereas there was no difference among the three groups 30 minutes thereafter [F(2,48) = 1.48; p = 0.237]. In women, ANOVA also revealed a significant group-by-time interaction effect [F(_2,24) = 8.67; p = 0.001]. When analyzed separately, cortisol levels were significantly different among the three groups only 30 minutes after awakening [F(2,24) = 3.82; p = 0.037]; reduced obese women values were greater than those of the two other groups. Overall, there was no sex effect in the ACR [F(1,76) = 0.01; p = 0.939]. However, when analyses were made for each weight status group, we found no sex effect in lean [F(1,25) = 0.09; p = 0.768], obese [F(1,26) = 1.45; p = 0.239], or reduced obese [F(1,21) = 2.51; p = 0.128] but a sex-by-time interaction in reduced obese [F(1,21) = 27.89; p < 0.001]. Compared with reduced obese men, cortisol levels in reduced obese women were decreased and increased immediately after awakening (t = 2.71; p = 0.0130) and 30 minutes after awakening (t = 3.72; p = 0.001), respectively.

Figure 1.

Mean salivary cortisol levels (means standard error) at awakening time and 30 minutes later in () lean, () obese, and () reduced obese subjects. * Indicates a different level for reduced obese men vs. lean and obese men (p = 0.0089). ** Indicates a different level for reduced obese women vs. lean and obese women (p = 0.0370).

Full figure and legend (57K)

ACRs are presented in Figure 2 in terms of absolute (micrograms per deciliter) and relative (percentage) increase. Obese men secreted significantly more cortisol in response to awakening than lean and reduced obese men [F(2,48) = 4.71, p = 0.014 for absolute increase and F(2,48) = 5.70, p = 0.006 for relative increase]. In women, we observed a significant difference between groups [F(2,24) = 6.90, p = 0.004 for absolute increase and F(2,24) = 4.86, p = 0.017 for relative increase] because reduced obese women secreted significantly more cortisol in response to awakening compared with lean and obese women. No difference between men and women was observed (t = 1.19, p = 0.238 for absolute increase and t = 0.57, p = 0.570 for relative increase). When the analyses were performed by group, there was no difference between lean men and lean women (t = 0.36, p = 0.725 for absolute increase and t = 0.01, p = 0.994 for relative increase). A difference appeared between obese men and obese women, with obese men having a larger increase of cortisol than obese women but only when the response was expressed in terms of absolute increase in cortisol levels (t = 2.17, p = 0.039 for absolute increase and t = 1.74, p = 0.094 for relative increase). An important gender difference was seen in the reduced obese groups, in which women had a higher cortisol response than men (t = 5.21, p < 0.001 for absolute increase and t = 3.98, p = 0.001 for relative increase).

Figure 2.

Cortisol secretion (means standard error) in response to awakening in () lean, () obese, and () reduced obese men and women, expressed in g/dl of increase (A, B) and in percentage of increase (C, D). * p < 0.05; ** p < 0.01.

Full figure and legend (157K)

As shown in Figure 3 , leptin values in women were greater than those of men (t = 7.40; p < 0.001). In men, mean leptin levels of lean participants were smaller than those of obese and reduced obese groups [F(2,46) = 9.25; p < 0.001]. In women, leptin levels of obese women were significantly higher than those of the other two groups [F(2,23) = 7.73; p = 0.003].

Figure 3.

Leptin levels (means standard error) in men and women, () lean, () obese, and () reduced obese. Asterisks represent significant differences: ** p < 0.01; *** p < 0.001.

Full figure and legend (157K)

Discussion

The present study was aimed at assessing the ACR in obese and reduced obese men and women. Men who are characterized with visceral obesity exhibited an enhanced ACR, whereas this response tended to be comparable with lean subjects in reduced obese individuals. Reduced obese women exhibited a significantly elevated ACR compared with lean subjects, but there was no difference in this variable between lean and obese subjects. No sex difference was observed in ACR between lean men and lean women, in agreement with previous reports with subjects similar to ours (47, 48, 49). From this observation, one can argue that a great part of the gender difference between the obese and reduced obese groups came from a different pattern of body fat distribution. However, we cannot exclude the possibility that gender-specific differences in sex hormones may have contributed to the results. Although the exact physiological significance of ACR has yet to be delineated (49), the high ACRs seen in men with abdominal obesity could reflect a dysregulation in the HPA axis activity in those subjects.

In obese male subjects, ACR was twice that of lean subjects. These results are consistent with previous studies reporting a dysregulation of the HPA axis in men with abdominal obesity (8, 9). Women with visceral obesity also exhibit HPA axis dysregulation (3, 4, 5, 6, 7, 10). Furthermore, two recent studies found a positive association between ACR and central adiposity (20, 21). It is noteworthy that low morning cortisol levels (5, 9, 50) have also been reported in men with abdominal obesity in studies in which cortisol levels were measured at a fixed time without any reference to the awakening time. The reason that cortisol levels can be reduced in the presence of high ACRs remains to be determined. This phenomenon could be accounted for by differences in the metabolic clearance rates of cortisol. A reduced cortisol clearance rate in obese men (51) could explain the reduced basal rate while having a minor impact on the ACR.

The mechanism leading to an increased ACR in obese subjects has yet to be determined. An increase in ACR has been suggested to reflect an increased response of the HPA axis aimed at preparing the body for the day (14). Some studies have reported patterns of enhanced or blunted ACR under chronic stress (16, 42, 45, 52), burnout (45), and posttraumatic stress disorder (53). Depression is certainly one of the conditions that most enhances ACR (18, 54). It is noteworthy that the relationship between central obesity and the enhanced HPA axis activity in men is, however, independent of depression scores (55). Our observation that visceral fat loss normalizes ACR tends to support the view that visceral fat stimulates the HPA axis (56). Visceral fat has been reported to secrete cytokines such as interleukin-6, which is a recognized stimulator of the HPA axis (57). Deposition of visceral fat seems to be not only a consequence of high levels of cortisol but also a factor that causally dysregulates the HPA axis activity. It has been known for years that chronic activation of the HPA axis provokes an excessive cortisol secretion that, when combined with the decrease of growth hormone and sex steroids levels, leads to the development of a hormonal milieu very propitious to intra-abdominal fat deposition.

In women, ACR was influenced by body weight loss, which contrasts with what was observed in men. Obesity, however, did not enhance ACR. In fact, obese women had ACRs similar to those of lean subjects, whereas reduced obese women exhibited enhanced ACR compared with lean women. The observation that obese women did not exhibit an enhanced ACR further suggests the importance of visceral fat in influencing this variable. This study was designed to recruit a cohort of subjects that was representative of the general population. We, therefore, recruited men with abdominal obesity and women with peripheral obesity. Nevertheless, it is worth mentioning that we tested four women with abdominal fat, two in the obese group and two in the reduced obese group. Similar to abdominally obese men, obese women with abdominal fat exhibited enhanced ACR (mean absolute increase, 0.50 g/dl; mean relative increase, 106.4%), whereas the reduced obese women demonstrated a lowered ACR (mean absolute increase, 0.11 g/dl; mean relative increase, 45.2%). The data from those women, who did not fulfill our anthropometric criteria, were excluded from the analyses.

The mechanism where weight loss may enhance ACR in women is not known. The loss of weight in reduced obese women is seemingly attributable to a reduction of peripheral fat. Although it is as yet premature to conclusively suggest that peripheral fat can influence ACR, it is nonetheless tempting to underline a potential link between leptin, which is preferentially secreted by subcutaneous adipose tissue, and ACR. Leptin was significantly reduced in obese women and associated with an increase in ACR. Leptin is an adipocyte-derived cytokine, which, in addition to inhibiting appetite and stimulating thermogenesis (58), is capable of reducing the HPA axis (59) activity by inhibiting CRF synthesis (60) and secretion (61, 62). There is evidence that leptin is predominantly secreted by subcutaneous adipocytes (63, 64) and the possibility that leptin can prevent an enhanced ACR in obese women cannot be disregarded.

In conclusion, the present results highlight gender effects in the ACR of obese and reduced obese subjects, which could be accounted for by the different fat distribution profiles that characterize men and women. They also provide further support for the usefulness of ACR in assessing the HPA axis activity status.

Notes

¹ Nonstandard abbreviations: HPA, hypothalamic-pituitary-adrenal; CRF, corticotropin-releasing factor; ACR, awakening cortisol response; WC, waist circumference.

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Acknowledgments

We thank the nurse team and Jean Doré, who performed medical examinations of subjects. This study was supported by grants from the Research Center on Energy Metabolism (Centre de Recherche sur le Métabolisme Énergétique), the Laval Hospital Research Center, and the Canadian Institutes for Health Research.