Depot-specific regulation of the conversion of cortisone to cortisol in human adipose tissue

Abstract

Objective: Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue.

Methods and procedures: Om and Abd sc adipose tissue were obtained at surgery from subjects with a wide range of BMI. Hydroxysteroid dehydrogenase (HSD) activity ((3)H-cortisone and (3)H-cortisol interconversion) and expression were measured before and after organ culture with insulin and/or dexamethasone.

Results: Type 1 HSD (HSD1) mRNA and reductase activity were mainly expressed within adipocytes and tightly correlated with adipocyte size within both depots. There was no depot difference in HSD1 expression or reductase activity, while cortisol inactivation and HSD2 mRNA expression (expressed in stromal cells) were higher in Om suggesting higher cortisol turnover in this depot. Culture with insulin decreased HSD reductase activity in both depots. Culture with dexamethasone plus insulin compared to insulin alone increased HSD reductase activity only in the Om depot. This depot-specific increase in reductase activity could not be explained by an alteration in HSD1 mRNA or protein, which was paradoxically decreased. However, in Om only, hexose-6-phosphate dehydrogenase (H6PDH) mRNA levels were increased by culture with dexamethasone plus insulin compared to insulin alone, suggesting that higher nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) production within the endoplasmic reticulum (ER) contributed to the higher HSD reductase activity.

Discussion: We conclude that in the presence of insulin, glucocorticoids cause a depot-specific increase in the activation of cortisone within Om adipose tissue, and that this mechanism may contribute to adipocyte hypertrophy and visceral obesity.

Figure 1

Comparison of HSD1 and 2 mRNA levels and interconversion of ³H-cortisol and ³H-cortisone in omental (Om, open bars) and abdominal subcutaneous (sc, black bars) adipose tissues. (a) Paired Om and sc human adipose tissue samples from 26 subjects (7 men and 19 women, BMI: 39.8 ± 2.6 (23–64). Age: 45.6 ± 2.5 (19–63)) were obtained and relative expression levels of HSD1 and 2 quantified relative to cyclophilin A with quantitative real-time PCR. Data were analyzed by two-way ANOVA followed by paired t-test. *P < 0.05, depot effect. (b and c) HSD1 reductase activity was assessed in intact fragments of paired Om and sc adipose tissues by measuring the conversion of ³H-cortisone to cortisol during a 90-min incubation in a subset of subjects (6 men, 15 women, BMI: 42.4 ± 3.1 (23–64), age: 43.9 ± 3.1 (19–63)). Data were expressed per (b) 50 mg adipose tissue or (c) 10⁵ cells and analyzed by two-way ANOVA followed by paired t-test. **P < 0.01 (depot effect). (d) Comparison of the correlation between HSD1 mRNA levels and ³H-cortisone to ³H-cortisol conversion in Om and sc human adipose tissue (age: 45.4 ± 3.6 (34–63), BMI: 39.8 ± 3.7 (23–64), 4 men and 11 women). The slopes of the relationship were significantly greater in Om than sc (Om; 4.55 ± 1.67 (mean ± s.d.) vs. sc; 1.26 ± 0.38, P < 0.05, n = 15). HSD1, type 1 hydroxysteroid dehydrogenase; PPIA, cyclophilin A.

Figure 2

Comparison of (a) HSD1 and (b) HSD 2 mRNA levels in adipose tissue, adipocytes (fat cells (FCs)) or stromal vascular cells (SVCs). Adipocytes and SVCs were prepared by collagenase digestion of human omental and subcutaneous adipose tissue (1 man and 6 women, BMI = 35.3 ± 2.4 (25–41), age = 47.7 ± 6.3 (26–63)). Relative expression levels of (a) HSD1 and (b) HSD2 were quantified relative to the control cyclophilin A by quantitative real-time PCR. (c) HSD reductase activity in tissue fragments, adipocyte or SVCs. Reductase activity of HSD1 was assessed by measuring the conversion of ³H-cortisone to ³H-cortisol in tissue fragments and cell fractions (n = 2). Data for tissue and cell fractions were normalized to tissue or adipocyte lipid. HSD1, type 1 hydroxysteroid dehydrogenase.

Figure 3

HSD1 expression and reductase activity are positively associated with adipocyte size and obesity (BMI). (a) Correlation between adipocytes size (FCS, expressed as average adipocyte weight (μg lipid/adipocyte)) and HSD1 mRNA levels in omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. (b) Correlation between fat cell size (FCS) and % conversion of ³H-cortisone to ³H-cortisol expressed per 10⁵ adipocytes in Om and Abd sc. (c) Correlation of adipocyte size and % conversion of ³H-cortisone to ³H-cortisol per 50 mg of tissue in Om and Abd sc. (d) Correlation between BMI (kg/m²) and ³H-cortisone to ³H-cortisol per 10⁵ adipocytes in Om and Abd sc. Data are from 22 subjects (6 men and 16 women), BMI: 40.6 ± 2.8 (23–64), age: 46 ± 2.8 (19–63). Individual numbers (n) are given on each figure, because not all parameters were measured in all subjects. HSD1, type 1 hydroxysteroid dehydrogenase.

Figure 4

Depot difference in the effect of culture with insulin and/or dexamethasone on type 1 hydroxysteroid dehydrogenase reductase activity in omental and subcutaneous human adipose tissue. Adipose tissue from total 20 subjects (7 men, 13 women, BMI: 45.7 ± 3.4 (25–64), age: 43.5 ± 3.4 (19–63)) was placed in organ culture in the conditions of basal (B), without any hormones), 7 nmol/l insulin (I), 25 nmol/l dexamethasone (D), or the combination of insulin plus dexamethasone (ID) for 6–7 days. % conversion of ³H-cortisone to ³H-cortisol or ³H-cortisol to ³H-cortisone was determined before (fresh; 0′) and after culture during a 90-min incubation (in the absence of I and D). Data were analyzed by two-way ANOVA followed by paired t-test. Bars with different letters are significantly different from each other (P < 0.05). ^#P < 0.05 basal vs. insulin alone by paired t-test. After culture, the depot differences in activity within hormonal condition were all significant by paired t-tests, P < 0.01, except for the insulin alone condition.

Figure 5

Effect of culture with insulin and dexamethasone on type 1 hydroxysteroid dehydrogenase (HSD1) and 2 mRNA levels in omental and abdominal subcutaneous adipose tissue. Adipose tissue from a subset of subjects described in Figure 4 (total 12 subjects (6 men, 6 women, BMI averaged 42.9 ± 3.6; range 25.2–58, age averaged 46.7 ± 4.3, range 19–63)) was used to assess relative expression levels of HSD1 and 2 before (0′) and after culture with quantitative real-time PCR. Data were analyzed by two-way ANOVA followed by paired t-test. Bars with different letter are significantly different from each other (P < 0.05). B, basal; D, dexamethasone; I, insulin; ID, insulin plus dexamethasone.

Figure 6

Effect of culture with insulin and dexamethasone on type 1 hydroxysteroid dehydrogenase (HSD1) protein expression in omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. Adipose tissue homogenates were prepared from Om and Abd sc adipose tissue after culture with insulin and/or dexamethasone (as described in the legend to Figure 4) and subjected to immunoblot analysis using rabbit anti-human 11β-HSD1 antibody and mouse anti-human α-tubulin antibody. Data are mean ± s.e.m. of five paired (Om and Abd sc) samples from severely obese subjects (a subset of subject described in Figure 4, 1 man, 4 women). ANOVA revealed significant main effects of dexamethasone (P < 0.005) and depot (P < 0.005), and a trend toward an insulin effect, with no interactions. *Dex effect (B vs. D or I vs. ID) by post-hoc t-test, P ≤ 0.05; ^$insulin effect B vs. I, P < 0.1; ^†P < 0.05, insulin effect—D vs. ID). Depot differences were significant by post-hoc tests for B (P < 0.05), I (P < 0.02), and ID (P < 0.001) conditions. B, basal; D, dexamethasone; I, insulin; ID, insulin plus dexamethasone.

Figure 7

Depot difference in the regulation of hexose-6-phosphate dehydrogenase (H6PDH) mRNA levels in omental and abdominal subcutaneous human adipose tissue. Relative expression levels of H6PDH mRNA levels were determined with quantitative real-time PCR after organ culture (basal (B), without any hormones), 7 nmol/l insulin (I), 25 nmol/l dexamethasone (D), or the combinations of insulin plus dexamethasone (ID)) in a subset of subjects described in Figure 4 (5 men, 3 women, BMI = 49 ± 3.7; 28–58, age = 40 ± 4.7; 19–59). *P < 0.05, paired t-test on log-transformed values, insulin alone vs. insulin plus dex.