Massive weight loss decreases corticosteroid-binding globulin levels and increases free cortisol in healthy obese patients: an adaptive phenomenon?

OBJECTIVE—Obesity, insulin resistance, and weight loss have been associated with changes in hypothalamic-pituitary-adrenal (HPA) axis. So far, no conclusive data relating to this association are available. In this study, we aim to investigate the effects of massive weight loss on cortisol suppressibility, cortisol-binding globulin (CBG), and free cortisol index (FCI) in formerly obese women. RESEARCH DESIGN AND METHODS—Ten glucose-normotolerant, fertile, obese women (BMI >40 kg/m2, aged 38.66 ± 13.35 years) were studied before and 2 years after biliopancreatic diversion (BPD) when stable weight was achieved and were compared with age-matched healthy volunteers. Cortisol suppression was evaluated by a 4-mg intravenous dexamethasone suppression test (DEX-ST). FCI was calculated as the cortisol-to-CBG ratio. Insulin sensitivity was measured by an euglycemic-hyperinsulinemic clamp, and insulin secretion was measured by a C-peptide deconvolution method. RESULTS—No difference was found in cortisol suppression after DEX-ST before or after weight loss. A decrease in ACTH was significantly greater in control subjects than in obese (P = 0.05) and postobese women (P ≤ 0.01) as was the decrease in dehydroepiandrosterone (P ≤ 0.05 and P ≤ 0.01, respectively). CBG decreased from 51.50 ± 12.76 to 34.33 ± 7.24 mg/l (P ≤ 0.01) following BPD. FCI increased from 11.15 ± 2.85 to 18.16 ± 6.82 (P ≤ 0.05). Insulin secretion decreased (52.04 ± 16.71 vs. 30.62 ± 16.32 nmol/m−2; P ≤ 0.05), and insulin sensitivity increased by 163% (P ≤ 0.0001). Serum CBG was related to BMI (r0 = 0.708; P = 0.0001), body weight (r0 = 0.643; P = 0.0001), body fat percent (r0 = 0.462; P = 0.001), C-reactive protein (r0 = 0.619; P = 0.004), and leptin (r0 = 0.579; P = 0.007) and negatively to M value (r0 = −0.603; P = 0.005). CONCLUSIONS—After massive weight loss in morbidly obese subjects, an increase of free cortisol was associated with a simultaneous decrease in CBG levels, which might be an adaptive phenomenon relating to environmental changes. This topic, not addressed before, adds new insight into the complex mechanisms linking HPA activity to obesity.

N utritional status and activity of the hypothalamic-pituitary-adrenal (HPA) axis are strictly associated (1,2). However, little is known about the effect of starvation and intentional calorie deprivation on the HPA axis (3,4). The HPA axis controls the secretion of cortisol with excessive secretion being inhibited by negative feedback. Several studies suggest that abnormalities in cortisol action and HPA axis control may be a factor that links disturbances of carbohydrate metabolism (which characterize insulin resistance) and obesity (5)(6). In obesity, estimation of plasma cortisol does not reflect the function of the HPA axis (7), and levels of cortisol in obese patients have been reported to be normal (8), low (9), or increased (10). On the contrary, response to different stimuli (high secretion of cortisol after laboratory stress tests or after different exogenous neuropeptides) has been found to be altered (11). Compared with these tests, the dexamethasone suppression test (DEX-ST), mainly used in the diagnosis of Cushing's disease, appears to be inadequate. Nevertheless, it has been widely used to evaluate the HPA axis in obesity, and the outcomes are contradictory (12)(13)(14). The free cortisol index (FCI)-the total cortisol-to-cortisolbinding globulin ratio (CBG) -has also been proposed to be able to evaluate HPA axis activity (7,15) since it reflects the biologically active fraction of total cortisol (7). Moreover, FCI is significantly associated with several features of insulin resistance syndrome (15). CBG levels are generally decreased in obese and diabetic subjects, although genetic factors are also known to play a role in the interindividual variation in CBG levels (16,17). Reduced levels of CBG favor locally increased cortisol disposal with no change in circulating cortisol; thus, fat accumulatates. Therefore, in obesity, tissues may be exposed to a relative excess of cortisol without any increase in circulating cortisol.
Biliopancreatic diversion (BPD), a malabsorptive bariatric surgical technique, causes massive weight loss (18,19), restoration of insulin sensitivity, and amelioration of insulin secretion (18). It restores the regular diurnal rhythm and pulsatility of insulin, leptin, and adiponectin but does not influence those of cortisol (19).
Thus far, no data are reported in literature concerning the effects of surgically induced massive and stable weight and fat mass loss on FCI and CBG concentrations and response to DEX-ST. The present study aimed to investigate 10 glucose-normotolerant morbidly obese (BMI Ͼ40 kg/m 2 ) women before and 2 years after BPD for 1) changes of FCI and CBG levels and 2) suppression of morning cortisol, ACTH, and DHEA levels after intravenous dexamethasone (DEX).

RESEARCH DESIGN AND
METHODS -The study group included 10 normotensive obese women (BMI Ͼ40 kg/m 2 ) evaluated before and 2 years after BPD. Postobese subjects were admitted monthly to our unit in the first 3 months after surgery and were controlled every 3 months thereafter. During the second year, they were admitted every 6 months. Patients were required to record their weight monthly. A stable weight (fluctuation Ͻ5%) is generally achieved by 12-15 months. Ten normal weight (BMI Յ24 kg/m 2 ) volunteers recruited among nurses and students in the hospital staff served as healthy control subjects to evaluate suppressibility of the HPA axis after DEX-ST. Subjects were nonsmokers and were 20 -35 years of age. Glucose tolerance was evaluated by a 75-g oral glucose tolerance test (OGTT) (20). They were evaluated in the early follicular phase by undergoing the OGTT and the euglycemic-hyperinsulinemic clamp study before the DEX-ST. None of the study participants had endocrine or nonendocrine diseases. They were not taking any medications except subjects after BPD, who were prescribed oral supplementation of sulfate iron (525 mg daily) calcium carbonate (1 g daily), multivitamins (Supradyn Roche, Milan, Italy) (1 tablet a day), and ergo-calciferol (400,000 UI intramuscular) (Ostelin fl, Teofarma, Italy) every 2 weeks. Medical histories, physical examinations, electrocardiogram, and blood screening showed that patients were in good health. None were taking anticonvulsant medications or corticosteroids. Depression was excluded by the Italian version of the Epidemiological Studies Depression Rating Scale (21).
The ethical committee of Catholic University approved the study, and subjects signed an informed consent document before participation. Body composition was estimated by the isotopic dilution method (22). Fat-free mass (FFM) was measured in kilograms and obtained by dividing total body water by 0.73 (22). Subjects underwent BPD, which is a malabsorptive surgical procedure (23).
Insulin sensitivity was estimated by a euglycemic-hyperinsulinemic clamp as previously described (24). Whole-body glucose uptake, normalized by FFM (M value in mmol ⅐ kg FFM -1 ⅐ min Ϫ1 ), was determined during a primed constant infusion of insulin (at the rate of 6 pmol ⅐ min Ϫ1 ⅐ kg Ϫ1 ).
␤-Cell function was estimated on 2-h OGTT values of glucose, insulin, and Cpeptide. Samples were withdrawn every 30 min. ␤-Cell function was assessed using a model describing the relationship between insulin secretion and glucose concentration, which has been previously described in detail (18). The model expresses insulin secretion as a sum of two components: ␤-cell glucose sensitivity and the rate sensitivity. Basal and total insulin secretions during the OGTT were calculated from the estimated model parameters. Total insulin secretion was calculated as the integral over the first 2 h of the OGTT.

Statistical methods
Data are presented as means Ϯ SD unless otherwise stated. Before statistical analysis, normal distribution and homogeneity of the variances were tested. All parameters fulfilled these conditions. Comparisons between groups were performed using paired t test while comparisons among groups were performed using oneway ANOVA followed by Bonferroni's post-hoc test for multiple comparisons whenever appropriate. Relationships between variables were sought by linear correlation analysis (Spearman's r 0 ). Levels of statistical significance were set at P Ͻ 0.05. Data analyses were performed with SPSS statistical software (SPSS V12.0; SPSS, Chicago, IL).
CONCLUSIONS -In the present study, we observed a significant decrease in circulating CBG levels and a concomitant increase of the metabolically active free cortisol fraction in obese women after surgically induced weight loss (BPD). Interestingly, despite the complete suppression of cortisol secretion by DEX infusion in all subjects independent of their body weight, the midnight fall in ACTH levels was significantly greater and the suppression of DHEA more enhanced in normal weight subjects compared with obese and post-BPD women (Fig. 1). A subtle primary dysfunction of the HPA axis might be envisaged in morbid obesity and after BPD. In post-BPD women, the HPA axis may adapt to a changing environment leading to decreased CBG levels and an overall increase in FCI. Several hypotheses can be formulated to explain the decrease of CBG levels and the different response to the DEX-ST.
The decrease in CBG levels following BPD may alter the sensitivity of the pituitary gland to cortisol. The recently reported (26) CBG-deficient mouse model is in agreement with the crucial role of the binding globulin in the ACTH feedback regulation of glucocorticoids. Additionally, a decrease in CBG levels would act to amplify the availability of metabolically free cortisol at peripheral target organs. Increased FCI might be a counteracting mechanism to the risk of hypoglycemia (stressor). In fact, in healthy subjects, hypoglycemia is reversed by a small permissive amount of glucocorticoids (27). Thus, postobese women may be able to maintain an elevated level of free cortisol available to all tissues without increasing total cortisol as demonstrated in freeliving animals (28). Increased free cortisol appears to promote food-seeking behavior in animals (28).
CBG may also act as a proper hormone (28,29), and its levels (4) or its binding capacity (28,30) decrease in condition of restrain, stress, and reduced food intake. Nevertheless, in anorectic women, we have previously found normal levels of CBG but rapid escape to the effect of DEX-ST (31). However, little is known about the transcriptional regulation of the CBG gene in response to FFA or insulin sensitivity, and this may represent a simplistic mechanism by which CBG levels are decreased. A reduced CBG liver synthesis might also occur after bariatric surgery, but in our series, liver enzymes and protein levels were unchanged after massive weight reduction. Neverthe-less, changes in hepatic CBG expression at the gene level might take place after surgery. Changes in CBG and FCI levels might also be related to the improvement of the low-grade inflammatory status (28) occurring in post BPD-patients (32), as suggested by the decrease of CRP. The observed dissociation between cortisol and DHEA response to the DEX-ST in post-BPD women also supports this hypothesis, with higher concentrations of DHEA (the biologically active form of the hormone) in postobese women compared with obese and control subjects.
Finally, other pleiotropic effects of cortisol cannot be excluded. In fact, enterocytes experience an adaptational hyperplasia after jejunoileal bypass (33), and glucocorticoids represent an important factor in the modulation of intestinal physiological functions, contributing to a more functional cell morphology and production of a potent regulator of crypt cell proliferation (34). The increase in FCI might contribute to this long termadaptational hyperplasia. In contrast with previous reports (17), we did not find any relation between fasting CBG and insulin secretion, likely because of the small sample size of patients or as a distinctive effect of the surgery.
Concerning the effect of the DEX-ST, the dissociation between cortisol (which was fully suppressed in all subjects) and DHEA in obese and postobese women compared with healthy control subjects, as well as differences in ACTH levels, suggest a different adrenal sensitivity and/or pituitary response as seen in the presence of a stress situation among the three groups. Previous studies, which have used an oral DEX-ST to indirectly test activity of the HPA axis in obesity, found no impairment of cortisol suppression (12)(13)(14). We used intravenous DEX infusion (25) to minimize the possible alteration in oral DEX absorption and/or liver metabolism since postobese women had severe lipid malabsorption. Any attempt to explain these results should take into account that in our series, data on cortisol clearance and/or distribution volume, which can affect results, are lacking.
Thus, we combined the DEX-ST with a single measurement of CBG and calculation of FCI. DEX-ST reliably explores the negative feedback regulation of cortisol on ACTH and relies on multiple time points. FCI is strongly associated with CBG-binding activity and with adrenal cortisol production, but one morning point alone for CBG and cortisol might not be representative of the whole-day FCI rhythm. Moreover, estimation of free cortisol by equilibrium dialysis would be more accurate than a derivative method to assess the amount of free hormone. Additionally, we used an intravenous DEX-ST (25) to minimize the possible variations in DEX absorption in postobese women who had severe lipid malabsorption.
The wide variability in the HPA axis balance in weight loss subjects may be related to the stress of starvation and/or to specific metabolic alterations rather than to the weight loss per se. For instance, in starving subjects, Yanovski et al. (14) reported that a relatively high dietary carbohydrate intake prevents the disruption of the HPA axis by reducing the reliance of ketones (4,14).
We are aware that the present findings cannot be applied to all obese subjects as our patients were a subset of "relatively healthy" obese women who were minimally affected by their obesity. They were not affected by hypertension, diabetes, or severe dyslipidemia. Thus, they may be the ones who have the fewest abnormalities of their HPA axis. However, future studies addressing this issue will be important to our understanding of dysregulation of the HPA axis.
In conclusion, we observed that after weight loss in morbidly obese subjects, free cortisol increased with an associated decrease in CBG levels. From the DEX infusion data, it is suggested that pituitary response and/or adrenal sensitivity differ between massively obese post-BPD patients and normal weight subjects. The long-lasting CBG decreases after drastic weight loss might be an adaptive phenomenon relating to environmental changes. This topic, not addressed before, adds new insight to the complexity of the intricate mechanisms linking HPA activity to obesity. The challenge remains to understand why CBG decreases and which consequences it exerts on the bioactivity of cortisol.