Tight junctions regulate the transfer of ions

Tight junctions regulate the transfer of ions as well as small molecules across endothelial barriers (Li et al., 2015). We also investigated whether resistin affects tight junctions, which play an important role in the conformation of polarized endothelial barriers (Matsuzaki et al., 2010). A previous study indicated that resistin could induce the proliferation and migration of endothelial cells (Calabro et al., 2004). In our study, resistin had the same effect on EA.hy926 cells (Fig. 2A–E). This made us wonder whether resistin could also affect tight junctions because increasing numbers of cells would compress the spaces between the endothelial cells. Unexpectedly, the expression of the TJ-related proteins TJP1, OCLN and CLDN were unchanged by resistin treatment (Fig. 2F) and the paracellular pathway was also not affected (Fig. 2E). Our data show that resistin does not regulate tight junctions or the paracellular pathway, suggesting that resistin might mediate glucose transport in other ways. Next, the effect of resistin on transmembrane transport was analyzed. Our data show that resistin down-regulates expression of GLUT1 (Fig. 3A–B) and that overexpressing GLUT1 in EA.hy926 cells completely blocks the role of resistin (Fig. 3C–D). Based on these results, we hypothesized that resistin impairs glucose transport by suppressing the expression of GLUT1. A similar effect has been reported by Pifferi et al., who found that impaired glucose transport in the Ifosfamide of rats was due to decreased GLUT1 expression in the endothelial cells of the blood–brain barrier (Pifferi et al., 2007). Peroxisome proliferator-activated receptor gamma (PPARγ), encoded by the PPARG gene in humans, is a type II nuclear receptor that is primarily present in macrophages (Desvergne and Wahli, 1999), colon and adipocytes (Devine et al., 1999). A great deal of research has shown that PPARγ can regulate glucose metabolism (Mudaliar and Henry, 2001) and adipocyte differentiation (Rosen et al., 1999). It is also closely tied with cancer as well as arteriosclerosis (Marx et al., 2002). Rosiglitazone, widely used in the treatment of diabetes (Phielix et al., 2011), is a classical type of hypoglycemic drug which exerts its effect by activating PPARγ to lower plasma glucose (Hulstrom et al., 2008). It works by combining with the PPARs and activating the expression of related genes to promote glucose uptake in tissues, thus reducing plasma glucose. In our study, in silico analysis suggested that there were two putative PPARγ binding regions in GLUT1 gene (Fig. 4B). Since resistin is an adipocytokine, this prompted us to investigate whether resistin could regulate the expression of GLUT1 via transcriptional regulation. Our results suggest that resistin suppresses the expression of PPARγ (Fig. 4C). Both the dual luciferase assay and the ChIP assay confirmed that PPARγ can bind the two putative regions (Fig. 4D–F). Overexpressing PPARγ in EA.hy926 cells recovered the suppression effect of resistin on GLUT1 expression (Fig. 4G). This indicates that resistin down-regulates GLUT1 expression through the suppression of PPARγ. Rosiglitazone facilitates glucose uptake in adipose tissue, liver and muscle. Our research shows that rosiglitazone promotes the expression of GLUT1 (Fig. 4H). Given that PPARγ expresses in vascular endothelial cells, this suggests that rosiglitazone could also induce endothelial cells to transport plasma glucose and could thus constitute a novel anti-diabetes therapy. In conclusion, we show that resistin down-regulates the expression of GLUT1 by suppressing PPARγ, thus causing impaired glucose transportation in endothelial cell layers. Although our research was based on resistin, hyperglycemia induced by other factors might share a similar mechanism. This means that the disorder of endothelial cells could prevent glucose transferring across the blood–tissue barrier, and provides a novel explanation for hyperglycemia and a new therapeutic option for type 2 diabetes mellitus.