Obesity is currently a worldwide pandemic. Leptin resistance is a main mechanism of obese human and rodents. The downregulation of the long form of the leptin receptor (Lrb) was involved in leptin resistance in diet-induced obese rats. In the studies, we investigated whether arcuate nucleus (ARC) silencing of Lrb would promote diet-induced obesity in rats. Lentiviral vectors expressing Lrb-shRNA were administered to 5-week-old male rats by ARC injection. Following viral delivery, the rats were provided with a high-fat diet (HFD) or a chow diet (CD). After 8 weeks of the diet, serum leptin, and insulin concentrations were measured by RIA, gene expression of Lrb in the ARC was detected by a real-time RT-PCR, and leptin signaling was examined by western blot. The Lrb-shRNA knocked down the expression of Lrb mRNA in infected regions by 54% for the HFD rats and 47% for the CD rats respectively. The Lrb knockdown reduced Stats3 activation and increased expression of Npy mRNA. The rats with reduced Lrb in the ARC showed a significant increase in energy intake and body weight (BW) again when fed with a HFD. By contrast, there were no effects of Lrb reduction on energy intake or BW when rats maintained on a low-fat chow. Our results provide evidence that Lrb knockdown selectively in the ARC promotes diet-induced obesity and associated metabolic complications in rats.
J Bian, X M Bai, Y L Zhao, L Zhang and Z J Liu
T Zhao, H Zhang, C Jin, F Qiu, Y Wu and L Shi
Melatonin, synthesized primarily by the pineal gland, is a neuroendocrine hormone with high membrane permeability. The vascular effects of melatonin, including vasoconstriction and vasodilation, have been demonstrated in numerous studies. However, the mechanisms underlying these effects are not fully understood. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed broadly on smooth muscle cells and play an important role in vascular tone regulation. This study explored the mechanisms of myocyte BKCa channels and endothelial factors underlying the action of melatonin on the mesenteric arteries (MAs). Vascular contractility and patch-clamp studies were performed on myocytes of MAs from Wistar rats. Melatonin induced significant vasodilation on MAs. In the presence of Nω-nitro-l-arginine methyl ester (l-NAME), a potent endothelial oxide synthase (eNOS) inhibitor, melatonin elicited concentration-dependent relaxation, with lowered pIC50. The effect of melatonin was significantly attenuated in the presence of BKCa channel blocker iberiotoxin or MT1/MT2 receptor antagonist luzindole in both (+) l-NAME and (−) l-NAME groups. In the (+) l-NAME group, iberiotoxin caused a parallel rightward shift of the melatonin concentration–relaxation curve, with pIC50 lower than that of luzindole. Both inside-out and cell-attached patch-clamp recordings showed that melatonin significantly increased the open probability, mean open time and voltage sensitivity of BKCa channels. In a cell-attached patch-clamp configuration, the melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole. These findings indicate that in addition to the activation of eNOS, melatonin-induced vasorelaxation of MAs is partially attributable to its direct (passing through the cell membrane) and indirect (via MT1/MT2 receptors) activation of the BKCa channels on mesenteric arterial myocytes.
Y-L Zhao, W-D Han, Q Li, Y-M Mu, X-C Lu, L Yu, H-J Song, X Li, J-M Lu and C-Y Pan
LRP16 gene expression is induced by 17-βestradiol (E2) via estrogen receptor alpha (ERα) in MCF-7 human breast cancer cells. A previous study also demonstrated that ectopic expression of LRP16 gene promoted MCF-7 cell proliferation. To explore the mechanism of hormone-induced LRP16 gene expression, the LRP16 gene promoter region (−2600 to −24 bp upstream of the LRP16 gene translation starting site) was analyzed in the present study by using different 5′-truncated constructs, and a luciferase reporter. The 5′-flanking sequence of −676 to −24 bp (pGL3-S5) was found to be E2-responsive. After exchange of the fragment from −213 to −24 bp with the TK gene proximal promoter region in pGL3-S5, E2 still induced reporter gene activity in MCF-7 and HeLa cells. Sequence analysis showed that the pGL3-S6 (−676 to −214) sequence contains two motifs that may contribute to E2-induced transactivation; namely, an estrogen-responsive element (ERE) half-site/Sp1 at −246 to −227 bp and an E-box site at −225 to −219 bp. Further deletion and mutation analysis of these two motifs indicated that both the 1/2 ERE and Sp1 binding sites were required for E2 action, while E-box deletion did not affect the luciferase activity in MCF-7 and HeLa cells. The results of gel mobility shift and chromatin immunoprecipitation assays confirmed that both ERαand Sp1 were required for hormone-induced transactivation, which involved both ERαand Sp1 directly binding to DNA. Taken together, these findings suggest that ERαand Sp1 play a role in activation of the human LRP16 gene promoter.