title: 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.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  University research center and private assisted reproductive technology centers.
  
  
  METHODS
  Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis.
  
  
  METHODS
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells.
authors.affiliation: Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã.
authors.forename: Tito T
authors.initials: TT
authors.lastname: Jesus
authors.affiliation: Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; Institute of Health Research an Innovation, Portugal.
authors.forename: Pedro F
authors.initials: PF
authors.lastname: Oliveira
authors.affiliation: Centre for Reproductive Genetics Prof. Alberto Barros, Porto, Portugal.
authors.forename: Joaquina
authors.initials: J
authors.lastname: Silva
authors.affiliation: Institute of Health Research an Innovation, Portugal; Centre for Reproductive Genetics Prof. Alberto Barros, Porto, Portugal; Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: Alberto
authors.initials: A
authors.lastname: Barros
authors.affiliation: Organic Chemistry, Natural and Agrofood Products Centre, Department of Chemistry, University of Aveiro, Aveiro, Portugal.
authors.forename: Rita
authors.initials: R
authors.lastname: Ferreira
authors.affiliation: Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; Centre for Reproductive Genetics Prof. Alberto Barros, Porto, Portugal.
authors.forename: Mário
authors.initials: M
authors.lastname: Sousa
authors.affiliation: Center for Biomedical Research, Population Council, New York, New York.
authors.forename: C Yan
authors.initials: CY
authors.lastname: Cheng
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã.
authors.forename: Branca M
authors.initials: BM
authors.lastname: Silva
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã. Electronic address: alvesmarc@gmail.com.
authors.forename: Marco G
authors.initials: MG
authors.lastname: Alves
mesh_terms: D002478:Cells, Cultured; D004734:Energy Metabolism / Q000187:drug effects; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis; D006801:Humans; D015227:Lipid Peroxidation; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010766:Phosphorylation; D061251:Primary Cell Culture; D050050:Protein Carbonylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D066244:Proton Magnetic Resonance Spectroscopy; D012708:Sertoli Cells / Q000187:drug effects* / Q000201:enzymology*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D013091:Spermatogenesis; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D005947:Glucose; D020123:Sirolimus
keywords: Sertoli cells; mTOR; rapamycin; spermatogenesis; testis