Estrogen sulfotransferase inhibits adipocyte differentiation

Abstract

The estrogen sulfotransferase (EST) is a phase II drug-metabolizing enzyme known to catalyze the sulfoconjugation of estrogens. EST is highly expressed in the white adipose tissue of male mice, but the role of EST in the development and function of adipocytes remains largely unknown. In this report, we showed that EST played an important role in adipocyte differentiation. EST was highly expressed in 3T3-L1 preadipocytes and primary mouse preadipocytes. The expression of EST was dramatically reduced in differentiated 3T3-L1 cells and mature primary adipocytes. Overexpression of EST in 3T3-L1 cells prevented adipocyte differentiation. In contrast, preadipocytes isolated from EST knockout (EST-/-) mice exhibited enhanced differentiation. The inhibitory effect of EST on adipogenesis likely resulted from the sustained activation of ERK1/2 MAPK and inhibition of insulin signaling, leading to a failure of switch from clonal expansion to differentiation. The enzymatic activity of EST was required for the inhibitory effect of EST on adipogenesis, because an enzyme-dead EST mutant failed to inhibit adipocyte differentiation. In vivo, overexpression of EST in the adipose tissue of female transgenic mice resulted in smaller adipocyte size. Taken together, our results suggest that EST functions as a negative regulator of adipogenesis.

Fig. 1.

The expression of EST decreased during adipogenesis. A, Tissues were harvested from 8-wk-old C57BL/6J mice (n = 3–4). Total RNA was isolated and the expression of EST mRNA was measured by real-time PCR. B, Epididymal adipose tissue was harvested from male C57BL/6J mice treated with chow or high-fat diet for 2–3 months (top panel), or 16-wk-old male ob/ob mice (bottom panel). Total RNA was isolated and the expression of EST mRNA was measured by real-time PCR (n = 4 for each group). C, 3T3-L1 cells were induced to differentiate with the standard protocol. The mRNA expression of EST and adipocyte-related genes PPARγ2 and aP2 was measured by real-time PCR. D, Epididymal fat was excised from 8-wk-old C57BL/6J mice (n = 3), and the tissue was fractionated into adipocytes and preadipocytes. Total RNA was isolated and the expression of EST, PPARγ2, and aP2 mRNA was measured by real-time PCR. The expression level of mRNA in preadipocytes was arbitrarily assigned a value of 1. *, P < 0.05; **, P < 0.01.

Fig. 2.

Overexpression of EST in 3T3-L1 cells inhibited adipogenesis, whereas ablation of EST accelerated the differentiation of primary preadipocytes. A, Stable 3T3-L1 cell lines expressing vector (Vector) or EST (clones EST1 and EST2) were generated by retroviral transduction. Total RNA was isolated, and the mRNA expression of EST was measured by real-time PCR. B, tk-ERE luciferase reporter gene and an ERα expression vector were transiently transfected into Vector and EST cells. Transfected cells were then treated with vehicle (EtOH) or E₂ (100 nm) for 20 h before luciferase assay. The transfection efficiency was normalized against the β-gal activity from the cotransfected CMX-β gal vector. Results shown are fold induction over vector control and represent the averages and sd from triplicate assays. C, Vector and EST cells were induced to differentiate using the standard differentiation medium. At d 6 after induction, the cells were fixed and stained with Oil-red O. D, Total RNA was extracted from cells before and after the induction of differentiation and analyzed for the expression of indicated genes by real-time PCR. E, Primary preadipocytes isolated from Wt and EST−/− mice and were treated with standard differentiation medium in the presence of 2 μm BRL49653. Oil-red O staining was performed, and the cells were microscopically examined on d 10 after induction. F, Total RNA was extracted from cells before and after induction of differentiation for 10 d and analyzed for gene expression by real-time-PCR analysis. *, P < 0.05; **, P < 0.01.

Fig. 3.

Overexpression of EST resulted in a sustained activation of ERK1/2 (p42/p44) MAPK. A, Vector and EST overexpressing 3T3-L1 cells were grown to confluence. The cells were then treated with the standard differentiation medium. Cell numbers were determined by MTT assay. Each time point represents a triplicate determination. B, Cell lysates were prepared before and after the induction of differentiation and subjected to Western blot analysis to detect phospho-ERK1/2 and total ERK1/2. C, Cells were treated with standard differentiation medium in the absence or presence of PD98059 at the indicated concentrations. On d 5, cells were fixed and stained with Oil-red O. D, Lysates from cells as described in panel C were subjected to Western blot analysis to detect the expression of P-ERK1/2 and total ERK1/2. E, Total RNA from cells as described in panel C was subjected to real-time PCR analysis to detect the expression PPARγ2 and aP2. F, The mRNA expression of IGF-I before and after induction of differentiation was measured by real-time PCR analysis. *, P < 0.05; **, P < 0.01.

Fig. 4.

Overexpression of EST attenuated the insulin signaling pathway. A, Cell lysates prepared from Vector and EST cells before and after induction of differentiation were subjected to Western blot analysis to detect the levels of P-Akt and total Akt. B, Total RNA from cells described in panel A was subjected to real-time PCR analysis to detect the expression of insulin receptor (IR) and IRS-1. C and D, Vector and EST overexpression 3T3-L1 preadipocytes (C), or primary preadipocytes derived from Wt and EST−/− mice (D) were preincubated in Krebs Ringer-HEPES buffer containing 5 mm glucose and 2% BSA for 2 h before being treated with 100 nm insulin for 10 min at 37 C. Cell lysates were prepared and subjected to immunoprecipitation by an anti-IRS-1 antibody and Western blot analysis to detect the expression of P-IRS-1 and total IRS-1. The protein expression of P-Akt and total Akt was detected from cell lysate by Western blot analysis without immunoprecipitation. E, The expression of PTP1B in Vector and EST overexpression 3T3-L1 preadipocytes (left panel), or primary preadipocytes derived from Wt and EST−/− mice (right panel) was measured by real-time PCR analysis. F, The expression of STAMP2 in undifferentiated Vector and EST cells (left panel), and chow-fed or high-fat diet (HFD)-fed Wt and EST−/− mice was measured by real-time PCR analysis. *, P < 0.05; **, P < 0.01.

Fig. 5.

The enzymatic activity of EST was required for the inhibitory effect of EST on adipocyte differentiation. A, The expression of EST AAK mutant, as compared with Vector and EST clones, was measured by real-time PCR analysis. B, tk-ERE luciferase reporter gene and an ERα expression vector were transiently transfected into Vector, EST Wt, and EST AAK cells. Transfected cells were then treated with vehicle (EtOH) or E₂ (10 of 100 nm) for 20 h before luciferase assay. The transfection efficiency was normalized against the β-gal activity from the cotransfected CMX-β-gal vector. Results shown are fold induction over vector control and represent the averages and sd from triplicate assay. C, Cells were treated with the standard differentiation medium. At d 6 after induction, cells were fixed and stained with Oil-red O. D, Total RNA was extracted at the indicated time points, and the gene expression was measured by real-time PCR analysis. The expression level of mRNA in control preadipocytes was arbitrarily assigned a value of 1.

Fig. 6.

Overexpression of EST in the adipose tissue of Tg mice resulted in smaller adipocyte size and compromised insulin responses. A, Schematic representation of the Tet-off aP2-tTA/TetRE-EST transgenic system. P_CMV, minimal CMV promoter. B, Female mice (12 wk of age) maintained on chow diet were subjected to the measurement of the expression of the EST transgene in WAT by Western blot analysis using an anti-HA antibody. When applicable, Tg mice were treated with DOX for 2 wk. Lanes represent individual mice. C, Hematoxylin and eosin staining on paraffin sections of abdominal fat of 12-wk-old female mice fed with chow diet. D, Mice received insulin treatment (0.5 U/kg of body weight ip injection) for 10 min before Western blot analysis to detect the levels of P-Akt and total Akt in the abdomen fat of 12-wk-old female mice fed with chow diet. E, The expression of ATGL and HSL in the abdominal fat of female mice was measured by real-time PCR analysis.