Insulin and dexamethasone dynamically regulate adipocyte 11beta-hydroxysteroid dehydrogenase type 1

Abstract

The adipocyte enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) amplifies local glucocorticoid action by generating active glucocorticoids from inactive metabolites and has emerged as a key player in the pathogenesis of central obesity and metabolic syndrome. However, the regulation of adipocyte 11beta-HSD1 is incompletely understood. Therefore, the present study was designed to investigate the effects of insulin and glucocorticoid as well as their underlying molecular mechanisms on 11beta-HSD1 activity and expression in 3T3-L1 adipocytes and determine whether the in vitro findings could be confirmed in vivo. Our main in vitro findings are 1) insulin stimulated whereas dexamethasone inhibited 11beta-HSD1 activity and expression in a time- and concentration-dependent manner; 2) the effect of dexamethasone was mimicked by both cortisol and corticosterone but blocked by the glucocorticoid receptor antagonist RU486; 3) the p38 MAPK inhibitor SB220025, but not the ERK inhibitor U0126 or the phosphatidylinositol 3-kinase inhibitor LY294002, prevented insulin stimulation of 11beta-HSD1 activity; and 4) although dexamethasone did not alter the half-life of 11beta-HSD1 mRNA, insulin doubled it. Taken together, these in vitro results demonstrate that insulin stimulates adipocyte 11beta-HSD1 through a posttranscriptional mechanism that involves activation of the p38 MAPK signaling pathway, whereas dexamethasone exerts an opposite effect by a glucocorticoid receptor-mediated transcriptional mechanism. In contrast, both insulin and dexamethasone augmented 11beta-HSD1 activity and expression in rat white adipose tissue in vivo, thus confirming the role of insulin but revealing a fundamental difference regarding the role of dexamethasone in regulating adipocyte 11beta-HSD1 between the two model systems.

Figure 1

Time-dependent effects of insulin and dexamethasone on 11β-HSD1 activity and 11β-HSD1 mRNA. Differentiated 3T3-L1 adipocytes were treated with insulin (100 nm) or dexamethasone (Dex; 100 nm) for various times. For each time point, controls were included and treated with an appropriate volume of vehicle. Levels of 11β-HSD1 activity in intact cells (A) were determined by a standard radiometric conversion assay. Total cellular RNA was extracted, and the steady-state level of 11β-HSD1 mRNA (B) was measured by qRT-PCR. Data are expressed as a percentage of control and presented as mean ± sem of four independent experiments, each performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control.

Figure 2

Concentration-dependent effects of insulin and dexamethasone on 11β-HSD1 activity and 11β-HSD1 mRNA. Differentiated 3T3-L1 adipocytes were treated with various concentrations of insulin or dexamethasone (Dex) for 48 h. Controls were treated at the same time with an appropriate volume of vehicle. Levels of 11β-HSD1 activity in intact cells (A) were determined by a standard radiometric conversion assay. Total cellular RNA was extracted, and the steady-state level of 11β-HSD1 mRNA (B) was measured by qRT-PCR. Data are expressed as a percentage of control and presented as mean ± sem of four independent experiments, each performed in triplicate. **, P < 0.01; ***, P < 0.001 vs. control.

Figure 3

Combined effects of insulin and dexamethasone on 11β-HSD1 activity and 11β-HSD1 mRNA. Differentiated 3T3-L1 adipocytes were treated with insulin (10 nm), dexamethasone (Dex; 10 nm), or in combination (10 nm each) for 48 h. Controls were treated at the same time with an appropriate volume of vehicle. Levels of 11β-HSD1 activity in intact cells (A) were determined by a standard radiometric conversion assay. Total cellular RNA was extracted, and the steady-state level of 11β-HSD1 mRNA (B) was measured by qRT-PCR. Data are expressed as a percentage of control and presented as mean ± sem of four independent experiments, each performed in triplicate. **, P < 0.01; ***, P < 0.001 vs. control.

Figure 4

Effects of insulin and dexamethasone on 11β-HSD1 mRNA degradation. Differentiated 3T3-L1 adipocytes were treated with vehicle (controls), insulin (100 nm), or dexamethasone (Dex; 100 nm) for 24 h and then with DRB (100 μm) for various times. At the indicated time points, total cellular RNA was extracted and the steady-state level of 11β-HSD1 mRNA was measured by qRT-PCR. Data are presented as mean ± sem of four independent experiments, each performed in triplicate.

Figure 5

Effects of various kinase inhibitors on 11β-HSD1 activity. Differentiated 3T3-L1 adipocytes were preincubated for 1 h with U0126 (ERK 1/2 inhibitor; 10 μm), LY294002 (PI3K inhibitor; 30 μm), or SB220025 (p38 inhibitor; 10 μm), followed by the addition of insulin (100 nm) for 48 h. Controls were treated at the same time with an appropriate volume of vehicle. Levels of 11β-HSD1 activity in intact cells were determined by a standard radiometric conversion assay. Data are expressed as a percentage of control and presented as mean ± sem of four independent experiments, each performed in triplicate. **, P < 0.01; ***, P < 0.001 vs. control.

Figure 6

Effects of RU-486 on dexamethasone-induced decreases in 11β-HSD1 activity. Differentiated 3T3-L1 adipocytes were pretreated for 1 h with the GR antagonist RU-486 (1 μm), followed by the addition of dexamethasone (Dex; 100 nm) for 48 h. Controls were treated at the same time with an appropriate volume of vehicle. Levels of 11β-HSD1 activity in intact cells were determined by a standard radiometric conversion assay. Data are expressed as a percentage of control and presented as mean ± sem of four independent experiments, each performed in triplicate. ***, P < 0.001 vs. control.

Figure 7

Effects of insulin and dexamethasone on H6PD mRNA. Differentiated 3T3-L1 adipocytes were treated for 48 h with various concentrations of insulin or dexamethasone (A) or for 48 h in combination with each at 10 nm concentration (B). Controls were treated at the same time with an appropriate volume of vehicle. Total cellular RNA was extracted, and the steady-state level of H6PD mRNA was measured by qRT-PCR. Data are expressed as a percentage of control and presented as mean ± sem of four independent experiments, each performed in triplicate. *, P < 0.05; ***, P < 0.001 vs. control.

Figure 8

Effects of insulin and dexamethasone in vivo. Male Wistar rats at 7 wk of age were injected sc with the long-acting insulin analog glargine (0.5 IU/kg·d) or the synthetic glucocorticoid dexamethasone (120 μg/kg·d) for 7 d. At the end of treatment, epididymal adipose tissues were collected. Levels of 11β-HSD1 activity (A) and mRNA (B) as well as H6PD mRNA (C) in adipose tissues were determined by a standard radiometric conversion assay and qRT-PCR, respectively. Data are expressed as a percentage of control and presented as mean ± sem (n = 4 rats). *, P < 0.05; ***, P < 0.001 vs. control; a vs. b, P < 0.05; a vs. c, P < 0.001.