Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, gene). 17β-estradiol (E) modulates /GLUT4 expression, but the involved mechanisms are unclear. Although E exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) /GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased /GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, /GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.

Background: Estrogens are involved in glycemic regulation, playing an important role in the development and/or progression of insulin resistance. For that, estrogens regulate the expression of the glucose transporter protein GLUT4 (codified by the solute carrier family 2 member 4 gene, Slc2a4), thus modulating adipose and muscle glucose disposal. This regulation is a balance between ESR1-mediated enhancer effect and ESR2-mediated repressor effect on Slc2a4 gene. However, molecular mechanisms involved in these effects are poorly understood. Since the specificity protein 1 (SP1) participates in several ESR-mediated genomic regulations, the aim of the present study is to investigate the participation of SP1 in the ESR1/2-mediated regulation of Slc2a4 gene.Methods: Differentiated 3T3-L1 adipocytes were 24-hour challenged with 10 nM estradiol (E2) and 10 nM ESR1 agonist (PPT) or 100 nM ESR2 agonist (DPN), added or not with E2. Slc2a4 and Sp1 mRNAs (RT-qPCR), total GLUT4 and nuclear ESR1, ESR2 and SP1 proteins (Western blotting), SP1 binding activity into Slc2a4 promoter (EMSA), and nuclear complexation of SP1/ESR1 (immunoprecipitation) were analyzed.Results: E2 and PPT increased (25-50%) whereas DPN reduced (20-45%) Slc2a4 and GLUT4 expression. Nuclear content of ESR1 and ESR2 remained unchanged. Nuclear content of SP1 increased (50 to 90%) by PPT and DPN added or not with E2; the highest effect observed with PPT alone. PPT also increased the nuclear content of SP1/ESR1 complex and the SP1 binding into the Slc2a4 promoter.Conclusions: ESR1 activation in adipocytes increased the nuclear content of SP1 protein, the SP1/ESR1 interaction and SP1 binding into the Slc2a4 gene promoter, culminating with increased Slc2a4/GLUT4 expression. No involvement of SP1 seems to occur in ESR2-mediated repressor effect on Slc2a4. We expect that this ESR1/SP1 cooperative effect can contribute to the development of new approaches for prevention or treatment of insulin resistance and diabetes mellitus.

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