David D. Brandon

Enhanced cortisol production rates, free cortisol, and 11beta-HSD-1 expression correlate with visceral fat and insulin resistance in men: effect of weight loss.

Controversy exists as to whether endogenous cortisol production is associated with visceral obesity and insulin resistance in humans. We therefore quantified cortisol production and clearance rates, abdominal fat depots, insulin sensitivity, and adipocyte gene expression in a cohort of 24 men. To test whether the relationships found are a consequence rather than a cause of obesity, eight men from this larger group were studied before and after weight loss. Daily cortisol production rates (CPR), free cortisol levels (FC), and metabolic clearance rates (MCR) were measured by stable isotope methodology and 24-h sampling; intra-abdominal fat (IAF) and subcutaneous fat (SQF) by computed tomography; insulin sensitivity (S(I)) by frequently sampled intravenous glucose tolerance test; and adipocyte 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1) gene expression by quantitative RT-PCR from subcutaneous biopsies. Increased CPR and FC correlated with increased IAF, but not SQF, and with decreased S(I). Increased 11beta-HSD-1 gene expression correlated with both IAF and SQF and with decreased S(I). With weight loss, CPR, FC, and MCR did not change compared with baseline; however, with greater loss in body fat than lean mass during weight loss, both CPR and FC increased proportionally to final fat mass and IAF and 11beta-HSD-1 decreased compared with baseline. These data support a model in which increased hypothalamic-pituitary-adrenal activity in men promotes selective visceral fat accumulation and insulin resistance and may promote weight regain after diet-induced weight loss, whereas 11beta-HSD-1 gene expression in SQF is a consequence rather than cause of adiposity.

Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.

Enhanced cortisol production rates, free cortisol, and 11β-HSD-1 expression correlate with visceral fat and insulin resistance in men: effect of weight loss

Controversy exists as to whether endogenous cortisol production is associated with visceral obesity and insulin resistance in humans. We therefore quantified cortisol production and clearance rates, abdominal fat depots, insulin sensitivity, and adipocyte gene expression in a cohort of 24 men. To test whether the relationships found are a consequence rather than a cause of obesity, eight men from this larger group were studied before and after weight loss. Daily cortisol production rates (CPR), free cortisol levels (FC), and metabolic clearance rates (MCR) were measured by stable isotope methodology and 24-h sampling; intra-abdominal fat (IAF) and subcutaneous fat (SQF) by computed tomography; insulin sensitivity (S(I)) by frequently sampled intravenous glucose tolerance test; and adipocyte 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1) gene expression by quantitative RT-PCR from subcutaneous biopsies. Increased CPR and FC correlated with increased IAF, but not SQF, and with decreased S(I). Increased 11beta-HSD-1 gene expression correlated with both IAF and SQF and with decreased S(I). With weight loss, CPR, FC, and MCR did not change compared with baseline; however, with greater loss in body fat than lean mass during weight loss, both CPR and FC increased proportionally to final fat mass and IAF and 11beta-HSD-1 decreased compared with baseline. These data support a model in which increased hypothalamic-pituitary-adrenal activity in men promotes selective visceral fat accumulation and insulin resistance and may promote weight regain after diet-induced weight loss, whereas 11beta-HSD-1 gene expression in SQF is a consequence rather than cause of adiposity.

Inhibition of dexamethasone binding to human glucocorticoid receptor by New World primate cell extracts.

To determine if New World primates express an inhibitor that influences glucocorticoid receptor (GR) binding characteristics, we examined [3H]dexamethasone binding in cytosol prepared from B95-8 lymphoid cells, derived from the cotton top tamarin (Saguinus oedipus), in combination with cytosol prepared from human or rat tissues. B95-8 cytosol inhibited specific binding of [3H]dexamethasone (P < 0.01) when mixed with cytosol prepared from either a human lymphoid cell line (HL) or rat thymus. The inhibitory activity was heat labile and trypsin sensitive. Peak inhibitory activity was found in the 150-200 kd fractions after Sephacryl G-200 ultrafiltration. Scatchard analysis of [3H]dexamethasone binding using mixed cytosol showed a diminished GR apparent binding affinity when compared to HL cytosol. Kinetic studies using mixed cytosol indicated that B95-8 cytosol did not affect the apparent dissociation rate of [3H]dexamethasone. These data demonstrate that B95-8 cells contain a competitive inhibitor that prevents binding of dexamethasone to its cognate receptor.

Resistance to multiple steroids in two sisters

A 14-year-old Native American girl from the Iroquois Nation was referred as a potential patient with the syndrome of Apparent Mineralocorticoid Excess. Instead, her evaluation revealed resistance to glucocorticoids, mineralocorticoids, and androgens. She lacked Cushingoid features in spite of significantly high cortisol levels. Menstruation was regular and there was no clinical evidence of masculinization despite high serum androgen levels in the male range. The patients sister had similar clinical features. Partial resistance to exogenous glucocorticoid and mineralocorticoid administration was well demonstrated in both patients. It is proposed that these patients represent the first cases of partial resistance to multiple steroids, possibly owing to a coactivator defect.

Determination of cortisol production rates with contemporary liquid chromatography―mass spectrometry to measure cortisol-d3 dilution after infusion of deuterated tracer

OBJECTIVES Measurement of 24-h cortisol production rate (CPR) using steady-state infusion of deuterated cortisol and analysis of stable-isotope dilution by MS is a valuable tool to examine hypothalamic-pituitary-adrenal axis activity in humans. We have developed and validated an improved method for measuring cortisol dilution with contemporary LC-MS technology. DESIGN AND METHODS Plasma samples and calibrators were extracted with ethylacetate. LC-MS was performed with a Surveyor HPLC and TSQ Quantum triple-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source. RESULTS Selectivity was improved over previous methods via elimination of an interferent identified as 20β-dihydrocortisol. The LLOQ for cortisol-d(3) was 2.73nmol/L and LOD 1.37nmol/L. Plasma calibrators were linear over the concentration range 1.5-10% cortisol-d(3), with correlation coefficients >0.995. CONCLUSIONS This APCI LC-MS method offers simplified sample work-up and analysis and enables selective detection of the low concentration of cortisol-d(3) infused for determination of 24-h CPR.

A Subnormal Peak Cortisol Response to Stimulation Testing Does Not Predict a Subnormal Cortisol Production Rate

INTRODUCTION The decision to commence lifelong glucocorticoid replacement therapy is often based on a cortisol stimulation test. We investigated the relationship between the peak cortisol response to insulin-induced hypoglycemia and daily cortisol production rate (CPR) to ascertain whether provocative tests are accurate in indicating the need to initiate lifelong glucocorticoid replacement. PATIENTS AND METHODS Ten patients (five male; mean age, 44 +/- 13 yr) with pituitary disease and with demonstrably suboptimal peak cortisol response (350-500 nmol/liter) to insulin-induced hypoglycemia, underwent CPR measurement by isotope dilution using gas chromatography-mass spectrometry and 24-h urinary free cortisol (UFC). RESULTS The median baseline and peak cortisol attained with hypoglycemia were 284 (164-323) and 473.5 (366-494) nmol/liter, respectively. A strong positive correlation was seen between peak stimulated cortisol and CPR (adjusted for body surface area) (r = 0.75; P = 0.02), and in all patients CPR [4.6 (2.9-15.1) mg/d x m(2)] was within the reference range (2.1-12 mg/d x m(2)) or elevated (one patient). A wide range was found for 24-h UFC [116.5 (20.5-265.9) nmol/liter] in this group of patients, and this parameter lacked significant correlation with either serum cortisol concentration or CPR. CONCLUSION This is the first study to demonstrate a significant correlation between CPR and peak cortisol values during hypoglycemic challenge. An inadequate cortisol response to hypoglycemia suggests the need for glucocorticoid cover at times of stress, but these data indicate that a suboptimal peak cortisol does not equate to a low CPR and should not be an automatic indication for lifelong glucocorticoid replacement therapy. UFC bears no relation to serum cortisol or CPR and is therefore unhelpful in assessment of such patients.

Enhanced cortisol production rates, free cortisol, and 11β-HSD-1 expression correlate with visceral fat and insulin resistance in men

Controversy exists as to whether endogenous cortisol production is associated with visceral obesity and insulin resistance in humans. We therefore quantified cortisol production and clearance rates, abdominal fat depots, insulin sensitivity, and adipocyte gene expression in a cohort of 24 men. To test whether the relationships found are a consequence rather than a cause of obesity, eight men from this larger group were studied before and after weight loss. Daily cortisol production rates (CPR), free cortisol levels (FC), and metabolic clearance rates (MCR) were measured by stable isotope methodology and 24-h sampling; intra-abdominal fat (IAF) and subcutaneous fat (SQF) by computed tomography; insulin sensitivity (S(I)) by frequently sampled intravenous glucose tolerance test; and adipocyte 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1) gene expression by quantitative RT-PCR from subcutaneous biopsies. Increased CPR and FC correlated with increased IAF, but not SQF, and with decreased S(I). Increased 11beta-HSD-1 gene expression correlated with both IAF and SQF and with decreased S(I). With weight loss, CPR, FC, and MCR did not change compared with baseline; however, with greater loss in body fat than lean mass during weight loss, both CPR and FC increased proportionally to final fat mass and IAF and 11beta-HSD-1 decreased compared with baseline. These data support a model in which increased hypothalamic-pituitary-adrenal activity in men promotes selective visceral fat accumulation and insulin resistance and may promote weight regain after diet-induced weight loss, whereas 11beta-HSD-1 gene expression in SQF is a consequence rather than cause of adiposity.