Research Article

Negative Effects of High Glucose Exposure in Human Gonadotropin-Releasing Hormone Neurons

Figure 2

High glucose effects in FNCB4 cells. (a–c) Quantitative RT-PCR analysis of mRNA expression for GnRH (a), KISS1R (b) and KISS1, (c) genes in FNC-B4 cells exposed to normal (NG, 5 mM), high (HG, 22 mM), and very high (VHG, 40 mM) glucose concentration or mannitol (M, 22 mM) for 24 hours. Results were calculated according to the comparative Ct method, using 18S rRNA subunit as the reference gene for normalization and were obtained from three separate experiments, each performed in triplicate . Data are reported as mean SEM and are expressed in percentage (%) of NG. * ; ** versus NG. (d–l) Immunofluorescent localization of GnRH (d, and e), KISS1R (g and h), and kisspeptin (j and k) proteins in FNC-B4 cells exposed to NG (d, g, and j) or VHG (e, h, and k). Dual labeling with the nuclear staining DAPI (blue color) and anti-KISS1R (green color; g and h) or anti-kisspeptin (green color; j and k) antibodies is also shown. Original magnification 20; scale bar = 50 μm. Computer-assisted image analysis for quantification of GnRH, KISS1R, and kisspeptin immunopositivity is shown in panels (f), (i), and (l), respectively. = number of analyzed cells.
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