Prolactin Receptor Signaling Is Essential for Perinatal Brown Adipocyte Function: A Role for Insulin-like Growth Factor-2
Figure 3
PRLR is crucial for brown adipocyte differentiation.
A) Schematic representation of the null mutation of the PRLR gene by homologous recombination and gene targeting techniques. Black boxes represent exon 4, 5 and 6. NEO: neomycin gene ; arrows indicate the position of primers used for PCR. B) PCR genotyping of immortalized brown adipocyte cell lines. C) Morphologic features of wild-type (WT), PRLR KO (KO) and rescued-PRLR (KO+PRLR) brown adipocytes on day 0 and day 6 of the differentiation program. All cell lines presented fibroblast-like morphology (day 0). Inset: immunodetection of the SV40 large T antigen in the nucleus of WT and PRLR KO brown adipocytes. WT, KO and KO+PRLR cell lines were cultured in the presence of 20 nM insulin and 2 nM T3 for 6 days. Light microscopic examination revealed the presence of numerous intracytoplasmic lipid droplets in the WT brown adipocyte cell line whereas PRLR KO brown adipocytes were unable to accumulate lipid droplets in their cytoplasm. Overexpression of PRLR in PRLR KO brown adipocytes (KO+PRLR) by stable transfection of a murine PRLR expression vector restores their ability to differentiate into mature brown adipocytes. D) RT-PCR analyses of PRLR, PPARγ2, UCP1 mRNA expression and 18S RNA used as internal control in WT, KO and rescued-PRLR (KO+PRLR) brown adipocytes. Note the reappearance of a strong PPARγ2 expression in rescued-PRLR differentiated brown adipocytes.