2B, RBP4 treatment markedly decreased cytosolic SREBP-1 but elevated nuclear SREBP-1 levels. We further determined the messenger RNA (mRNA) amounts of SREBP-1a, SREBP-1c, and SREBP-2 by real-time polymerase chain reaction (PCR). Moreover, the mRNA levels of SREBP-1c but not SREBP-1a were significantly increased in a dose-dependent manner in response to RBP4 treatment (Fig. 2C), despite the fact that the SREBP-1c to SREBP-1a ratio (1:2) of human HepG2 cells was much less than that of mouse hepatocytes (9:1).[27] However, RBP4 did not significantly

affect the degree of SREBP-2 nuclear form (Fig. S3A) and its mRNA expression (Fig. S3B). To determine the functional effects of increased nuclear SREBP-1 translocation by RBP4, the gene expression of key target enzymes of SREBP-1 in the HepG2 cells was evaluated by quantitative reverse-transcription (RT)-PCR. As expected in Erlotinib molecular weight the case of dynamically altered nuclear SREBP-1c, RBP4 dose-dependently increased the expression of endogenous lipogenic genes, including FAS, ACC1, and DGAT2, involved in fatty acid and TAG synthesis in HepG2 cells. Similar to the lack of an effect on nuclear SREBP-2, the expression of mRNAs encoding two key enzymes of cholesterol biosynthesis, 3′-hydroxylmethyl glutaryl coenzyme A reductase (HMGCR) selleck screening library and low-density lipoprotein

receptor (LDLR), was not altered in RBP4-treated HepG2 cells (Fig. 3A). Buspirone HCl In contrast, RBP4 exerted less effect on the nuclear SREBP-2-mediated transcriptional activation of SRE-containing

target genes, including the 4×SRE-Lucand LDLR-Luc reporter genes (Fig. 3B). We next mapped the human SREBP-1c promoter and identified the element responsible for RBP4 action. Transcriptional activation of the wildtype SREBP-1c promoter was markedly induced by RBP4 in HepG2 cells. Disruption of the LXRE and SRE motif in the same promoter diminished the level of basal transcription and prevented the further induction caused by RBP4 (Fig. 3C). These data show that the LXRE and SRE motifs are necessary for the RBP4-dependent induction of SREBP-1c transcription. PGC-1β is a recently identified transcriptional coactivator closely related to lipid metabolism.[28] To evaluate the effects of RBP4 on PGC-1β expression, we exposed HepG2 cells to recombinant RBP4 for different time periods and examined the effects on PGC-1β expression. RBP4 treatment was found to cause a time-dependent increase in the expression of Ppargc1b mRNA as determined by northern blot (Fig. 4A) and quantitative RT-PCR (Fig. 4B). The levels of Ppargc1b mRNA, over the control at 8 hours, increased as early as 2 hours after the addition of RBP4 to the cells, increased as early as 2 hours after RBP4 treatment. Moreover, the PGC-1β transcript levels remained high throughout the 24-hour treatment period. The effects of RBP4 were further found to be dose-dependent (Fig. 4C).