Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
Bench to Bedside
Poster Board SAT-154
Objective:To examine whether GLUT1 overexpression modulates gonadotrope function.
Methods: LßT2 gonadotrope cells were stably transfected with a CMV vector carrying the mouse Glut1 gene or CMV vector alone. Stable clones of LßT2-CMV and LßT2-GLUT1 cells were selected using antibiotic resistance. RNA was isolated from stable cells (basal condition without GnRH stimulation) and used for real-time PCR analysis to examine changes in gene expression (GLUT1, luteinizing hormone (LHß), follicle stimulating hormone (FSHß), GnRH receptor (GnRHr), and early growth response gene 1 (Egr-1). Whole cell extracts were isolated from stable cells (basal condition without GnRH stimulation) and used in Western blot analysis with GLUT1 antibody. Immunofluorescence analyses were performed to detect subcellular localization of GLUT1 and LH. Glucose-uptake assays were also performed.
Results: Real-time PCR analysis of LßT2-GLUT1 cells showed a 7.6-fold increase in GLUT1 gene expression compared to LßT2-CMV cells (P< 0.05). LßT2-GLUT1 cells also showed a 4.6-fold increase in expression of LHß mRNA, and a 22.1-fold increase in FSHß mRNA compared to LßT2-CMV cells (P < 0.05 ). There were no changes in expression of GnRHr or Egr-1 genes in LßT2-Glut1 compared to LßT2-CMV. Western blot analysis showed increased levels of GLUT1 protein in LßT2-GLUT1 compared to LßT2-CMV cells. Immunofluorescence analysis showed that GLUT1 overexpression was associated with increased levels of LHß protein in LßT2-Glut1 cells compared to LßT2-CMV cells. LßT2-GLUT1 cells showed increased glucose uptake compared to LßT2-CMV cells.
Conclusions: Glut1 overexpression in LßT2 cells increases gonadotropin gene expression and protein synthesis in the absence of GnRH. Our data suggest that GLUT1 may play a novel role in GnRH signaling which can modulate gonadotrope function and may provide new insights into the regulation of the H-P-G axis and puberty.
Nothing to Disclose: CB, OB, AEYE, FG
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