Sex- and Tissue-Specific Role of Estrogen Sulfotransferase in Energy Homeostasis and Insulin Sensitivity

Abstract

Estrogen sulfotransferase catalyzes the sulfoconjugation and deactivation of estrogens. Previously, we showed that loss of Est in male ob/ob mice, but not in female ob/ob mice, exacerbated the diabetic phenotype, but the underlying mechanism was unclear. In this study, we show that transgenic reconstitution of Est in the adipose tissue, but not in the liver, attenuated diabetic phenotype in Est-deficient ob/ob mice (obe mice). Mechanistically, adipose reconstitution of Est in obe mice (oae mice) resulted in reduced local and systemic inflammation, improved insulin sensitivity, and increased energy expenditure. At the molecular level, adipose induction of lipocalin-2 (Lcn2) in oae males may have contributed to the inhibition of inflammation because the level of Lcn2 was negatively associated with tumor necrosis factor (Tnf) α expression, and treatment of differentiated adipocytes with Lcn2 antagonized Tnfα-responsive inhibition of insulin signaling. The metabolic benefit of adipose reconstitution of Est was sex specific, because adipose reconstitution of Est in obe females had little effect. Interestingly, despite their improved metabolic functions, obe male mice with reconstituted Est in their adipose tissue failed to ameliorate the impairment of the structure and function of the pancreatic islets. In summary, our study uncovers a crucial adipose- and male-specific role of Est in maintaining the whole-body energy homeostasis.

Figure 1.

Transgenic reconstitution of Est in the adipose tissue and liver of the obe mice. (A) The oae mice that bear the expression of Est in the adipose tissue in the background of obe were created by crossing the obe mice with the adipose-specific aP2-Est transgenic mice. The ole mice that bear the expression of Est in the liver in the background of obe were created by crossing the obe mice with the liver-specific Lap-Est transgenic mice. (B and C) The endogenous and transgenic Est protein expression in the WAT, liver, and skeletal muscle of (B) ob/ob, obe, and oae female and male mice and (C) ob/ob and ole male mice was measured by Western blot analysis. n.s., nonspecific bands.

Figure 2.

Adipose reconstitution of Est improves the metabolic function of obe mice in a male-specific manner. (A) Body weight and body composition analysis by magnetic resonance imaging in male mice. n = 6 to 8. (B) Rectal temperature (tem) in adult obe and oae male mice. (C) Oil Red O staining of liver sections from 17-week-old male obe and oae mice. (D) Hepatic triglyceride (TG) levels in male obe and oae mice. (E) Liver to body ratio in male obe and oae mice. n = 5 to 8. (F) The expression of genes involved in lipogenesis, fatty acid oxidation, and triglyceride export in the liver was measured by real-time PCR. n = 6. (G) Oxygen consumption was measured by metabolic cages. n = 4. (H) H&E staining of brown adipose tissue of male obe and oae mice. (I) Expression of transcription factors involved in the browning of brown adipose tissue, as measured by real-time PCR. n = 6. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Adipose reconstitution of Est improves the glucose tolerance and insulin sensitivity of obe mice in a male-specific manner. (A) GTT and (B) ITT in male obe and oae mice. n = 4 to 6. (C) Insulin resistance calculated and expressed as HOMA-IR score in male obe and oae mice. (D) Western blotting measurement of Akt phosphorylation in the WAT, liver, and skeletal muscle of male mice that were treated with phosphate-buffered saline or insulin. Shown on the right are densitometric quantifications of the blots. (E) The expression of Glut4 in the skeletal muscle of male obe and oae mice was measured by real-time PCR. n = 6. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4.

Adipose reconstitution of Est in obe mice attenuates adipose and systemic inflammation in a male-specific manner. (A) The weight of visceral fat shown as a percentage of body mass in male obe and oae mice. n = 4 to 6. (B) H&E staining of sections of visceral fat in male mice. Bar = 5 μm. Shown on the right are Image J quantifications of the crownlike structures. (C) The quantifications of adipocyte sizes are shown as relative sizes. (D) Immunohistochemical staining of Cd68 in visceral fat sections of male mice. Bar = 100 μm. (E) The serum levels of Il-6 and Tnfα in male obe and oae mice were measured by ELISA. (F and G) The expression of (F) inflammatory marker genes and (G) estrogen-responsive genes in the visceral fat of male mice was measured by real-time PCR. n = 7. *P < 0.05; **P < 0.01.

Figure 5.

The reduced WAT local and systemic inflammation in oae males may have been accounted for by the upregulation of Lcn2. (A) The expression of Lcn2 in the visceral WAT of obe and oae male mice was measured by real-time PCR. n = 7. (B) Immunostaining of Lcn2 in visceral fat of male obe and oae mice. Arrows indicate the positive staining. (C) The serum level of Lcn2 protein in male obe and oae mice was measured by Western blotting. Shown at the bottom is the densitometric quantification of the blots. (D) The hepatic mRNA expression of Lcn2 was measured by real-time PCR. n = 4 to 7. (E) Association between the mRNA expression of Lcn2 and Tnfα in the visceral fat of male oae mice. (F) The mRNA expression of insulin-responsive genes in differentiated 3T3-L1 cells was measured by real-time PCR. (G) Insulin-induced Akt phosphorylation in differentiated 3T3-L1 cells was measured by Western blotting. Shown are the representative blot and densitometric quantification of the results from three independent experiments. *P < 0.05; **P < 0.01.

Figure 6.

Adipose reconstitution of Est fails to rescue the pancreatic β cell damage in obe males. (A) GSIS in male obe and oae mice. n = 7 to 8. (B) Homeostatic model assessment (HOMA) of β cell function in male mice. (C) H&E staining (upper two panels) and immunostaining of insulin (lower two panels) of pancreatic sections from male obe and oae mice. Shown on the right are Image J quantifications of pancreatic islets and insulin-positive areas. Bar = 100 μm. (D) Immunostaining of Cd68 in pancreatic sections of male obe and oae mice. Dashed line borders pancreatic islet area from the acini area. Shown at the bottom are Image J quantifications of the Cd68 staining. Bar = 2 μm. (E and F) The mRNA and protein expression of mouse Est in isolated pancreatic islets was measured by (E) Northern blot analysis and (F) Western blot analysis, respectively. *P < 0.05. n.s., statistically not significant; WT, wild-type.

Figure 7.

Liver reconstitution of Est fails to ameliorate inflammation and diabetic phenotype in obe male mice. (A) GTT and (B) GSIS in male obe and ole mice. n = 6 to 7. (C and D) H&E staining of (C) pancreatic and (D) visceral fat sections in male obe and ole mice. Bar = 100 μm. (E) The serum levels of Tnfα and Il-6 in male obe and ole mice were measured by enzyme-linked immunosorbent assay. n.s., not significant.