title: D-Aspartic acid stimulates steroidogenesis through the delay of LH receptor internalization in a mammalian Leydig cell line.
abstract: OBJECTIVE
  Recent experimental evidence on non-mammalian animal models showed that D-Aspartic acid (d-Asp) administration increases testosterone levels through upregulation of StAR in Leydig cells. In this study, we aimed to investigate in vitro the signaling pathway associated with d-Asp stimulation in MA-10 murine Leydig cells.
  
  
  METHODS
  MA-10 cells were stimulated with different concentrations of d-Asp, in presence or absence of hCG. Then total testosterone (T) levels in the culture medium were evaluated by electrochemiluminescence immunoassay, and StAR and LHR protein expressions were quantified by the means of Western blotting. LHR cellular localization after hormonal stimulation was assessed by immunofluorescence.
  
  
  RESULTS
  Stimulation with the sole d-Asp did not induce any relevant increase of T release from cultured cells. On the other hand, stimulation with hCG induced significant increase of T (P = 0.045). Concomitant stimulation with hCG and d-Asp, at the concentration of 0.1 and 1 nM, induced additional and significant increase of released T (P = 0.03 and P = 0.04, respectively). StAR protein levels increased after concomitant stimulation with hCG and d-Asp 0.1 nM, compared with stimulation with the sole hCG (P = 0.02), whereas no variation in LHR protein expression was observed. Finally, d-Asp attenuated displacement of LHR staining, from cell membrane to cytoplasm, subsequent to hCG stimulation.
  
  
  CONCLUSIONS
  In this study, we confirmed a steroidogenic role for d-Asp, in concert with hCG, on murine Leydig cells, which is mediated by an increase in StAR protein levels. In addition, we showed that the possible mechanism subtending the effect of d-Asp could rely on the modulation of LHR exposure on the cell membrane.
authors.affiliation: Department of Medicine, Section of Endocrinology and Centre for Human Reproduction Pathology, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Di Nisio
authors.affiliation: Department of Medicine, Section of Endocrinology and Centre for Human Reproduction Pathology, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy.
authors.forename: L
authors.initials: L
authors.lastname: De Toni
authors.affiliation: Department of Molecular Medicine, University of Padova, Padua, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Ferigo
authors.affiliation: Department of Medicine, Section of Endocrinology and Centre for Human Reproduction Pathology, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy.
authors.forename: M S
authors.initials: MS
authors.lastname: Rocca
authors.affiliation: Department of Medicine, Section of Endocrinology and Centre for Human Reproduction Pathology, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Speltra
authors.affiliation: Department of Medicine, Section of Endocrinology and Centre for Human Reproduction Pathology, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Ferlin
authors.affiliation: Department of Medicine, Section of Endocrinology and Centre for Human Reproduction Pathology, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy. carlo.foresta@unipd.it.
authors.forename: C
authors.initials: C
authors.lastname: Foresta
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000819:agonists* / Q000494:pharmacology; D002999:Clone Cells; D026603:D-Aspartic Acid / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D007985:Leydig Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D049990:Membrane Transport Modulators / Q000494:pharmacology*; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D010750:Phosphoproteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011974:Receptors, LH / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012102:Reproductive Control Agents / Q000819:agonists* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013739:Testosterone / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D006063:Chorionic Gonadotropin; D049990:Membrane Transport Modulators; D010750:Phosphoproteins; D011974:Receptors, LH; D012102:Reproductive Control Agents; D000098682:Steroidogenic Acute Regulatory Protein; D013739:Testosterone; D026603:D-Aspartic Acid
keywords: D-Aspartic acid; LH receptor internalization; Leydig cells; Steroidogenesis; Testosterone