Mammalian target of rapamycin controls glucose consumption and redox balance in human Sertoli cells

Abstract

Objective: To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress.

Design: Experimental study.

Setting: University research center and private assisted reproductive technology centers.

Patient(s): Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis.

Intervention(s): Testicular biopsies were used from patients under treatment for recovery of male gametes. Primary hSCs cultures were established from each biopsy and divided into a control group and one treated with rapamycin, the inhibitor of mTOR, for 24 hours.

Main outcome measure(s): Cytotoxicity of hSCs to rapamycin was evaluated by sulforhodamine B assay. The glycolytic profile of hSCs was assessed by proton nuclear magnetic resonance and by studying protein expression of key glycolysis-related transporters and enzymes. Expression of mitochondrial complexes and citrate synthase activity were determined. Protein carbonylation, nitration, lipid peroxidation, and sulfhydryl protein group contents were quantified. The mTOR signaling pathway was studied.

Result(s): Rapamycin increased glucose consumption by hSCs, maintaining lactate production. Alanine production by rapamycin-exposed hSCs was affected, resulting in an unbalanced intracellular redox state. Rapamycin-exposed hSCs had decreased expression of mitochondrial complex III and increased lipid peroxidation, whereas other oxidative stress markers were unaltered. Treatment of hSCs with rapamycin down-regulated phospho-mTOR (Ser-2448) levels, illustrating an effective partial inhibition of mTORC1. Protein levels of downstream signaling molecule p-4E-BP1 were not altered, suggesting that during treatment it became rephosphorylated.

Conclusion(s): We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells.

Keywords: Sertoli cells; mTOR; rapamycin; spermatogenesis; testis.

FIGURE 1

Effect of rapamycin in glucose uptake and consumption by human Sertoli cells. The consumption of glucose by human Sertoli cells was determined (A) and is represented in nanomoles per microgram of protein. The expression of glucose transporters (GLUT) GLUT1 (B), GLUT2 (C), GLUT3 (D) are also represented, as well as illustrative Western blot bands. The bands were divided by the respective tubulin and the results are represented as fold variation to control. Results are expressed as mean + SEM (n = 6 for each condition). *Significantly different results relative to control (P<.05).

FIGURE 2

Effect of rapamycin in intracellular glucose metabolism and in oxidative profile of human Sertoli cells. The production of lactate (A) and alanine (B) by human Sertoli cells was determined. The results are expressed as nanomoles per microgram of protein. The lactate-to-alanine ratio (C) is represented as an indicator of intracellular redox state. Oxidative damages were measured in proteins carbonylation (D) and nitration (E) and by sulfhydryl (SH) groups content (F). Results are expressed as mean ± SEM (n = 6 for each condition). *Significantly different results relative to control (P<.05).

FIGURE 3

Effect of rapamycin in the oxidative profile, mitochondrial complexes proteins levels and in mitochondrial integrity of human Sertoli cells. Oxidative damages were measured in lipid peroxidation (A). The protein levels of mitochondrial complex II (B), complex III (C), complex IV (D), and complex V (E) are represented, as well as illustrative Western blot bands. The bands were divided by the respective actin and the results are represented in fold variation to control. The presence of/or intact mitochondria was measured by citrate synthase activity (F). The results are expressed as nanomoles of coenzyme A per minute per milligram of protein. Results are expressed as mean ± SEM (n = 6 for each condition). *Significantly different results relative to control (P<.05).

FIGURE 4

Effect of rapamycin in the protein expression levels of target of rapamycin (TOR) signaling pathway. The protein expression levels of phosphatidylinositol 3-kinase (PI3K) (A); glycogen synthase kinase 3 (GSK3) (B); phosphorylated mammalian TOR (P-mTOR) (C), and phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (P-4E-BP1) (D) in human Sertoli cells were determined. The bands were divided by the respective tubulin and the results are represented in fold variation to control. Results are expressed as mean ± SEM (n = 6 for each condition). *Significantly different results relative to control (P<.05).