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Dietary Macronutrient Content Alters Cortisol Metabolism Independently of Body Weight Changes in Obese Men

Endocrinology Unit (R.H.S., D.J.W., N.M.M., R.A., B.R.W.), Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom; Division of Obesity and Metabolic Health (A.M.J., G.E.L.), Rowett Research Institute, Aberdeen AB21 9SB, United Kingdom; and Mass Spectrometry Core Laboratory (N.Z.M.H., R.A., B.R.W.), Wellcome Trust Clinical Research Facility, Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Roland H. Stimson, University of Edinburgh, Endocrinology Unit, Centre for Cardiovascular Science, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: roland.stimson{at}ed.ac.uk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Dietary macronutrient composition influences cardiometabolic health independently of obesity. Both dietary fat and insulin alter glucocorticoid metabolism in rodents and, acutely, in humans. However, whether longer-term differences in dietary macronutrients affect cortisol metabolism in humans and contribute to the tissue-specific dysregulation of cortisol metabolism in obesity is unknown.

Objective: The objective of the study was to test the effects of dietary macronutrients on cortisol metabolism in obese men.

Design: The study consisted of two randomized, crossover studies.

Setting: The study was conducted at a human nutrition unit.

Participants: Participants included healthy obese men.

Interventions, Outcome Measures, and Results: Seventeen obese men received 4 wk ad libitum high fat-low carbohydrate (HF-LC) (66% fat, 4% carbohydrate) vs. moderate fat-moderate carbohydrate (MF-MC) diets (35% fat, 35% carbohydrate). Six obese men participated in a similar study with isocaloric feeding. Both HF-LC and MF-MC diets induced weight loss. During 9,11,12,12-[²H]₄-cortisol infusion, HF-LC but not MF-MC increased 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity (rates of appearance of cortisol and 9,12,12-[²H]₃-cortisol) and reduced urinary excretion of 5- and 5ß-reduced [²H]₄-cortisol metabolites and [²H]₄-cortisol clearance. HF-LC also reduced 24-h urinary 5- and 5ß-reduced endogenous cortisol metabolites but did not alter plasma cortisol or diurnal salivary cortisol rhythm. In sc abdominal adipose tissue, 11ß-HSD1 mRNA and activity were unaffected by diet.

Conclusions: A low-carbohydrate diet alters cortisol metabolism independently of weight loss. In obese men, this enhances cortisol regeneration by 11ß-HSD1 and reduces cortisol inactivation by A-ring reductases in liver without affecting sc adipose 11ß-HSD1. Alterations in cortisol metabolism may be a consequence of macronutrient dietary content and may mediate effects of diet on metabolic health.

	Introduction

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THE OBESITY PANDEMIC heralds dramatic increases in type 2 diabetes and, potentially, in cardiovascular disease, but not everyone with obesity will develop these complications. Factors that predict adverse outcomes, over and above the effect of obesity, include dietary fat and carbohydrate content (1, 2). We hypothesized that the effects of macronutrient content are mediated by alterations in cortisol action.

In obesity, the metabolic clearance rate of cortisol is increased in the liver (3), secondary to enhanced inactivation by 5- and 5ß-reductases (4) and impaired regeneration of cortisol from cortisone by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) (5, 6). Enhanced cortisol clearance putatively leads to compensatory activation of the hypothalamic-pituitary-adrenal axis (7) to maintain normal plasma cortisol concentrations. In addition, intra-adipose cortisol generation by 11ß-HSD1 is increased in obesity (6, 8, 9). However, the basis for dysregulation of cortisol metabolism in obesity is unknown.

Both the hypothalamic-pituitary-adrenal axis (10) and metabolism of cortisol in extraadrenal tissues may be influenced by dietary macronutrients. In rats, high-fat overfeeding recapitulates the combination of decreased 11ß-HSD1 and increased 5ß-reductase in the liver observed in human obesity (11). High-fat overfeeding in mice (12) and rats (11) decreases 11ß-HSD1 mRNA and activity in adipose tissue. In humans, either a mixed meal (13) or infusions with insulin or lipid (14) increase 11ß-HSD1 activity acutely.

We hypothesized that longer-term variations in dietary macronutrient composition influence cortisol metabolism in humans, and that abnormal liver and/or adipose cortisol metabolism in obesity occurs in response to dietary constituents. We compared effects of diets with contrasting macronutrient content on glucocorticoid metabolism in obese men undergoing weight loss.

	Subjects and Methods

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Participants and protocols

Healthy men aged 20–65 yr with body mass index (BMI) 30–40 kg/m² were recruited to a residential study. Exclusion criteria included chronic illness, alcohol intake greater than 27 U/wk, regular medication or special diet, and abnormality on physical examination or laboratory blood tests. Written informed consent and local ethical committee approval were obtained.

Ad libitum dietary manipulation

As detailed elsewhere (15), 17 men (BMI 35.1 ± 0.9 kg/m², aged 38 ± 10 yr) completed both phases of a balanced randomized crossover study. Each phase comprised a 3-d weight maintenance diet followed by 4 wk of either an high fat-low carbohydrate (HF-LC) or moderate fat-moderate carbohydrate diet (MF-MC) diet. The proportions of energy from each macronutrient group in each meal were as follows: HF-LC, protein 30%, fat 66%, carbohydrate 4%; and MF-MC, protein 30%, fat 35%, carbohydrate 35%. HF-LC and MF-MC meals were energy isodense (1315 kcal/kg) with similar proportions of saturated fats, and food was available ad libitum. Food offered and food left uneaten was weighed to generate intake data in Table 1.

A separate group of six men (BMI 38.0 ± 1.3 kg/m², aged 41 ± 5 yr) completed a protocol identical to the ad libitum study, except: 1) participants were offered a fixed energy intake of 2000 kcal/d; 2) fewer subjects were included, following power calculations using data from the ad libitum study; 3) a longer weight maintenance period of 7 d was adopted at the start to obtain detailed cortisol measurements at baseline; and 4) no weight maintenance period was used between the two diet phases.

Clinical measurements

Food intake and body weight were monitored daily. At the beginning and end of each diet phase, blood was obtained after an overnight fast and body composition was measured by a four-compartment model (15).

In the ad libitum study, glucocorticoid metabolism was assessed at the end of each 4-wk dietary phase. Subjects collected urine for 24 h and then fasted overnight. At 0700 h, approximately 500 mg sc periumbilical adipose tissue was obtained under local anesthetic by needle aspiration and immediately frozen at –80 C. Intravenous cannulae were sited in antecubital veins for infusion and sampling. After 3.5 mg priming, 9,11,12,12-[²H]₄-cortisol (d4-cortisol; Cambridge Isotopes, Andover, MA) was infused at 20% molar percent excess in hydrocortisone-21-succinate at 1.74 mg/h for 4 h (16). Plasma and urine were collected at intervals as described (16).

In the isocaloric study, d4-cortisol infusions were performed at the end of the 7-d weight maintenance diet (baseline) and after 1 and 4 wk of each diet. Adipose biopsies were obtained at baseline and after 4 wk of each diet. In addition, salivary samples were collected into Salivette tubes (Starstedt, Nümbrecht, Germany) at 0800, 1200, 1800, and 2200 h each week for measurement of cortisol.

Laboratory analyses

Endogenous and deuterated cortisol and cortisol metabolites in plasma and urine were analyzed by electron impact gas chromatography/ mass spectrometry (16). Cortisol kinetics was calculated using the mean of five measurements in steady state (16). Urinary indices of 11ß-HSD1, 11ß-HSD2, and 5- and 5ß-reductase activities were calculated as described (4).

Adipose tissue was analyzed for 11ß-HSD1 activity and mRNA using the ABI PRISM 7700 sequence detection system with commercial primers and probes (PE Applied Biosystems, Cheshire, UK) as described (6, 8). Results are expressed as a ratio relative to the sum of cyclophyllin A and 18S transcript levels as internal control.

Serum insulin, glucose, and homeostasis model of assessment of insulin resistance index were measured as described (15). Salivary cortisol was analyzed by ELISA (Salimetrics LLC, State College, PA).

Statistical analysis

All parameters were normally distributed by Kolmogorov-Smirnov testing and are presented as mean ± SEM. Comparisons between the two diets are by paired t tests. Comparisons with baseline are by repeated-measures ANOVA with post hoc Fisher’s least significant differences test. P < 0.05 was considered significant.

	Results

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Energy intake, body composition, and biochemistry

In the ad libitum study, energy intakes were 154 kcal/d lower on the HF-LC than the MF-MC diet (P < 0.05) (Table 1), and weight loss was correspondingly greater (6.3 ± 2.2 vs. 4.4 ± 2.6 kg in 4 wk, P < 0.01). In the isocaloric study, despite being served food of the same energy content, intake was slightly lower (66 kcal/d) and weight loss greater (7.2 ± 2.3 vs. 4.7 ± 1.0 kg in 4 wk, P < 0.05), on the HF-LC diet after correction for unconsumed food. However, loss of fat mass was similar on the HF-LC and MF-MC diets in both studies. Fasting plasma glucose and insulin concentrations were reduced more markedly by the HF-LC than the MF-MC diet.

Cortisol metabolism

In both the ad libitum and isocaloric studies, rates of appearance (Ra) of endogenous cortisol and d3-cortisol during steady state of the deuterated cortisol infusion were higher on the HF-LC than the MF-MC diet (Fig. 1). The isocaloric study showed that these differences were explained by increases from baseline during the HF-LC, which were significant within 1 wk for Ra cortisol and by 4 wk for Ra d3-cortisol. In the ad libitum study, plasma cortisol at steady state was higher on the HF-LC diet (266 ± 15 vs. 224 ± 12 nmol/liter, P < 0.001), consistent with slower d4-cortisol clearance rate (0.37 ± 0.03 vs. 0.39 ± 0.03 liters/min, P = 0.05) and reduced urinary excretion of both 5- and 5ß-reduced deuterated cortisol metabolites (Table 2). In addition, 24-h urinary excretion of endogenous 5- and 5ß-reduced cortisol metabolites decreased on each diet from baseline (Table 2) and were 24% lower on the HF-LC diet than the MF-MC diet.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Whole-body 11ß-HSD1 activity in ad libitum and isocaloric diet studies. Data are mean ± SEM for n = 17 (ad libitum study) and n = 6 (isocaloric study) during HF-LC diet (filled columns) and MF-MC diet (open columns). Comparisons by paired t tests (ad libitum study) or repeated-measures ANOVA followed by post hoc Fisher’s least significant differences tests (isocaloric study): *, P < 0.05, **, P < 0.01, compared with MF-MC diet; #, P < 0.05, ##, P < 0.01, compared with baseline. A, Ra endogenous cortisol. Ra cortisol was higher on the HF-LC diet in both studies and increased from baseline at both 1 and 4 wk only on the HF-LC diet. B, Ra of d3-cortisol. Ra d3-cortisol was higher in both studies after 4 wk.

In the isocaloric study, there were no effects of diet on salivary cortisol or its diurnal variation (data not shown).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
These data show for the first time that chronic manipulation of dietary macronutrient composition influences glucocorticoid metabolism in humans. In obese men, an HF-LC diet increased whole-body regeneration of cortisol by 11ß-HSD1 and reduced the rate of inactivation of cortisol by 5- and 5ß-reductases. As in previous studies, discrepancy between cortisol regeneration measured during dynamic testing and the more conventional index of 24-h urinary endogenous cortisol/cortisone metabolite ratios (Table 2) reflects the confounding effects of 5- and 5ß-reductase activities on ratios of steroids excreted in urine. The increased 11ß-HSD1 activity on the HF-LC vs. MF-MC diet was independent of differences in energy consumption and weight loss: the same effect was observed under fixed feeding (approximately isocaloric) conditions; and the MF-MC diet induced substantial weight loss without altering 11ß-HSD1 activity. Moreover, the effect of HF-LC was already apparent after 1 wk of the diet when weight loss was minimal.

Most whole-body cortisol regeneration by 11ß-HSD1 occurs in the splanchnic circulation (17), and the liver is the major source of 5- and 5ß-reduced cortisol metabolites. Consistent with actions predominantly in the liver, we did not find effects of diet on 11ß-HSD1 or 5-reductase type 1 in sc adipose tissue. Previous studies showed no change in hepatic or sc adipose 11ß-HSD1 after weight loss in obese humans (9, 18), although one group found increased 11ß-HSD1 mRNA in isolated gluteal adipocytes (18), but these studies were not controlled for dietary macronutrient composition.

Low-carbohydrate intake appears to be the key factor responsible for alterations in glucocorticoid metabolism. Protein intake was similar between diets. Compared with baseline, fat intake was only marginally higher (160 kcal/d) on the HF-LC diet, whereas carbohydrate intake was substantially lower (1400 kcal/d). However, carbohydrate intake was also lower than baseline on the MF-MC diet (800 kcal/d), suggesting a threshold of reduced carbohydrate intake to mediate the effect. This is supported by fasting insulin concentrations (Table 1), which were decreased by the HF-LC but not the MF-MC diet in the ad libitum study and might directly alter hepatic 11ß-HSD1 (19) and 5- and 5ß-reductase activities (20). However, other unmeasured effects of the diets may be important, and additional studies are required to dissect the pathways responsible for altering cortisol metabolism.

The lack of effect of dietary manipulation on 11ß-HSD1 in adipose tissue might seem at odds with evidence in mice that overfeeding with a high saturated fat diet down-regulates 11ß-HSD1 in adipose tissue but not liver (12). However, in the current studies, the total fat intake was not substantially increased between the baseline and HF-LC diets, the fat content was a mixture of saturated, mono- and polyunsaturated forms, and subjects were losing rather than gaining weight, so that the paradigms are not comparable.

In conclusion, extraadrenal regeneration of cortisol is responsive to the macronutrient content of the diet. In these obese men, a low-carbohydrate diet reversed the increase in metabolic clearance of cortisol (3), increase in 5- and 5ß-reductase (4), and decrease in hepatic 11ß-HSD1 (5, 6) previously described in obesity. Thus, chronic changes in dietary macronutrients may be a primary driver for altered hepatic, but not adipose, cortisol metabolism in obesity. The increase in 11ß-HSD1 activity, and hence intrahepatic cortisol concentrations, caused by a ketogenic low carbohydrate diet has implications for the efficacy of different dietary strategies in reversing the metabolic consequences of obesity.

	Acknowledgments

 
We thank David Bremner, Scott Denham, Alessandra Gambineri, Jill Harrison, Dawn Livingstone, Rachel McDonnell, and Alison McNeilly and the Mass Spectrometry Core Laboratory of the Wellcome Trust Clinical Research Facility for their assistance.

	Footnotes

 
This work was supported by the British Heart Foundation and Scottish Executive Environment and Rural Affairs Department.

Disclosure Summary: R.H.S., A.M.J., N.Z.M.H., D.J.W., N.M.M., R.A., and G.E.L. have nothing to declare. B.R.W. has consulted recently for Astra-Zeneca, Incyte, Johnson & Johnson, Merck, Roche, Syrrx, and Vitae and has received lecture fees from Abbott and Bristol Myers Squibb. B.R.W. is an inventor on relevant patents held by the University of Edinburgh.

First Published Online September 4, 2007

Abbreviations: BMI, Body mass index; d4-cortisol, 9,11,12,12-[²H]₄-cortisol; HF-LC, high fat-low carbohydrate; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; MF-MC, moderate fat-moderate carbohydrate; Ra, rate of appearance.

Received March 27, 2007.

Accepted August 23, 2007.

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