Testosterone insulin-like effects: an in vitro study on the short-term metabolic effects of testosterone in human skeletal muscle cells

Abstract

Purpose: Testosterone by promoting different metabolic pathways contributes to short-term homeostasis of skeletal muscle, the largest insulin-sensitive tissue and the primary site for insulin-stimulated glucose utilization. Despite evidences indicate a close relationship between testosterone and glucose metabolism, the molecular mechanisms responsible for a possible testosterone-mediated insulin-like effects on skeletal muscle are still unknown.

Methods: Here we used undifferentiated proliferating or differentiated human fetal skeletal muscle cells (Hfsmc) to investigate the short-term effects of testosterone on the insulin-mediated biomolecular metabolic machinery. GLUT4 cell expression, localization and the phosphorylation/activation of AKT, ERK, mTOR and GSK3β insulin-related pathways at different time points after treatment with testosterone were analyzed.

Results: Independently from cells differentiation status, testosterone, with an insulin-like effect, induced Glut4-mRNA expression, GLUT4 protein translocation to the cytoplasmic membrane, while no effect was observed on GLUT4 protein expression levels. Furthermore, testosterone treatment modulated the insulin-dependent signal transduction pathways inducing a rapid and persistent activation of AKT, ERK and mTOR, and a transient inhibition of GSK3β. T-related effects were shown to be androgen receptor dependent.

Conclusion: All together our data indicate that testosterone through the activation of non-genomic pathways, participates in skeletal muscle glucose metabolism by inducing insulin-related effects.

Keywords: Human skeletal muscle cells; Insulin; Metabolism; Testosterone.

Fig. 1

Effect of testosterone on Glut4 mRNA expression. Glut1, Glut3 and Glut4 mRNA expression was evaluated in undifferentiated or differentiated Hfsmc treated for 24 h with T (100 nM) or I (100 nM) for comparison. I or T treatment significantly increased Glut4 mRNA vs. control (c) (*P < 0.05; **P < 0.01); no difference was observed in Glut subtype 1 and 3 mRNA expression. Results (mean ± SE) are derived from three experiments with different cell preparations and expressed as fold increase vs. ctr, taken as 1

Fig. 2

Effect of testosterone on GLUT4 translocation in Hfsmc, before and after differentiation. Immunofluorescence analysis (a) revealed no signal for GLUT4 membrane expression in control (ctr) untreated undifferentiated or differentiated Hfsmc; positive staining for GLUT4 was observed after 30-min incubation with I (100 nM) or T (100 nM) both in undifferentiated or differentiated conditions (upper panels). Cells were incubated with antibody probes specific for GLUT4, followed by incubations with fluorescent secondary antibody. Middle panels represent DAPI blue staining of nuclei; lower panels depict GLUT4/DAPI staining merge. Pictures are representative. Results are derived from four separate experiments, using distinct cell preparations. Percentage of GLUT4 positive undifferentiated or differentiated cells (**P < 0.01 vs. ctr) are represented in panels of figure b, respectively. Data are expressed as mean ± SE

Fig. 3

Effect of testosterone on I-related metabolic pathways. The treatment of Hfsmc for 15 min with T (100 nM) did not activate any of the tested intracellular paths, at variance with I (100 nM), used as positive control, (a–d); however, T after 30 min significantly induced activation of phospho (p)-AKT, ERK1/2, mTOR and GSK3β (*P < 0.05 or **P < 0.01 vs. T0), as depicted by the densitometric analysis, reported below each representative blot. Specific total proteins were used as loading controls. Results of densitometric analysis (mean ± SE) are expressed as ratio p-/total-protein, fold increase vs. T0, taken as 1; data derived from three/four experiments using different cell preparations. Protein analysis revealed no time-dependent changes of GLUT4 expression (e)

Fig. 4

Effect of bicalutamide pre-treatment on testosterone-induced GLUT4 translocation in Hfsmc. Immunofluorescence analysis (a) revealed no signal for GLUT4 membrane expression in control (ctr) untreated Hfsmc; positive staining for GLUT4 was observed after 30-min incubation with I (100 nM) or T (100 nM) (upper panels). 1-h pre-treatment with Bic 100 nM completely counteracted T-induced GLUT4 translocation. Cells were incubated with antibody probes specific for GLUT4, followed by incubations with fluorescent secondary antibody. Middle panels represent DAPI blue staining of nuclei; lower panels depict GLUT4/DAPI staining merge. Pictures are representative. Results are derived from four separate experiments, using distinct cell preparations. Percentage of GLUT4 positive cells (*P < 0.05, ***P < 0.001 vs. ctr and ^$$$ P < 0.001 vs. T) are represented in b. Data are expressed as mean ± SE

Fig. 5

Effect of bicalutamide on Testosterone-induced I-related metabolic pathways. The pre-treatment with bicalutamide (100 nM, Bic) prevented T-induced phosphorylation (p) of AKT, ERK1/2, mTOR and GSK3β (*P < 0.05, **P < 0.01 or ***P < 0.001 vs. T0), as depicted by the densitometric analysis, reported below each representative blot. Specific total proteins were used as loading controls. Results of densitometric analysis (mean ± SE) are expressed as ratio p-/total-protein, fold increase vs. T0, taken as 1; data derived from three/four experiments using different cell preparations