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

ABSTRACT: To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress.Experimental study.University research center and private assisted reproductive technology centers.Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis.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.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.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.We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells.

SUBMITTER: Jesus TT

PROVIDER: S-EPMC4845725 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Jesus Tito T TT Oliveira Pedro F PF Silva Joaquina J Barros Alberto A Ferreira Rita R Sousa Mário M Cheng C Yan CY Silva Branca M BM Alves Marco G MG

Fertility and sterility 20151214 3

<h4>Objective</h4>To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress.<h4>Design</h4>Experimental study.<h4>Setting</h4>University research center and private assisted reproductive technology centers.<h4>Patient(s)</h4>Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis.<h4>Intervention(s)</h4>Testicular biopsies were used from patients under tre ...[more]

PMID: 26698679

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Project description:BackgroundThe AKT/mammalian target of rapamycin (mTOR) signaling pathway is regulated by 17α-estradiol (E2) signaling and mediates E2-induced proliferation and progesterone receptor (PgR) expression in breast cancer.Methods and resultsHere we use deep sequencing analysis of previously published data from The Cancer Genome Atlas to demonstrate that expression of a key component of mTOR signaling, rapamycin-insensitive companion of mTOR (Rictor), positively correlated with an estrogen receptor-α positive (ERα+) breast tumor signature. Through increased microRNA-155 (miR-155) expression in the ERα+ breast cancer cells we demonstrate repression of Rictor enhanced activation of mTOR complex 1 (mTORC1) signaling with both qPCR and western blot. miR-155-mediated mTOR signaling resulted in deregulated ERα signaling both in cultured cells in vitro and in xenografts in vivo in addition to repressed PgR expression and activity. Furthermore we observed that miR-155 enhanced mTORC1 signaling (observed through western blot for increased phosphorylation on mTOR S2448) and induced inhibition of mTORC2 signaling (evident through repressed Rictor and tuberous sclerosis 1 (TSC1) gene expression). mTORC1 induced deregulation of E2 signaling was confirmed using qPCR and the mTORC1-specific inhibitor RAD001. Co-treatment of MCF7 breast cancer cells stably overexpressing miR-155 with RAD001 and E2 restored E2-induced PgR gene expression. RAD001 treatment of SCID/CB17 mice inhibited E2-induced tumorigenesis of the MCF7 miR-155 overexpressing cell line. Finally we demonstrated a strong positive correlation between Rictor and PgR expression and a negative correlation with Raptor expression in Luminal B breast cancer samples, a breast cancer histological subtype known for having an altered ERα-signaling pathway.ConclusionsmiRNA mediated alterations in mTOR and ERα signaling establishes a new mechanism for altered estrogen responses independent of growth factor stimulation.

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

Publications

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

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