2008

2008. whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by Darusentan caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent. (Invitrogen #CAV1RSS352112). Transfected cells were grown on transwell filters for 48 h, and LPL transport was quantified as described earlier. To measure LPL mass in pre- and postheparin plasma, blood samples from wild-type and = 0.0003; duplicate samples for each dose) and genistein (= 0.0009; two sets of experiments each done in duplicate) significantly reduced transport as judged by one-way ANOVA. To further investigate the possibility of vesicular trafficking, we examined GPIHBP1 localization by immunogold EM. We cultured endothelial cells on transwell filters until they formed tight monolayers, and gold-conjugated antibody 11A12 was added to the apical face of cells. After 2 h at 37C, the cells were fixed and processed for EM. We found gold beads clustered in 60C100 nm invaginations at both the apical and basolateral faces of cells (Fig. 3A): 64.1% of gold particles were within invaginations or internalized vesicles; 22.4% were at the necks of invaginations; and 13.5% were on the plasma membrane but not associated with invaginations. No gold-conjugated antibody 11A12 was found on endothelial cells that expressed CD59 rather than GPIHBP1 (supplementary Fig. III). Open in a separate window Fig. 3. Electron micrographs showing GPIHBP1 and LPL on plasmalemmal invaginations. (A, B) Localization of gold-labeled antibodies against GPIHBP1 (A) or V5-LPL (B) after their addition to the apical face of GPIHBP1-expressing endothelial cells grown on filters. Gold particles are indicated by white arrows. (C, D) Localization of gold-conjugated antibody 11A12 in endothelial cells 2 h after injection of the antibody Darusentan into the quadriceps of a wild-type mouse Darusentan (C) or a siRNA and those transfected with a control siRNA (Fig. 5A). However, because we were able to achieve only a 50% knockdown of caveolin-1 expression, as judged by Western blotting (Fig. 5B), we next attempted to quantify LPL transport with Darusentan endothelial cells isolated from the lungs of wild-type and siRNA. Bar graphs are composites of two independent experiments done in triplicate and show the fold change (mean SEM) in LPL transport compared with cells transfected only with control siRNA. (B) Caveolin-1 protein levels (mean SEM) in control and siRNA-transfected cells as judged by quantitative Western blotting (control set to 100%). (C) LPL internalization by GPIHBP1-expressing mouse lung endothelial cells derived from wild-type or = 0.6759). (D) Localization of gold-labeled antibodies against V5-LPL after their addition to the apical face of GPIHBP1-expressing = 3/genotype). (B) Immunohistochemical detection of caveolin-1 (green) in BAT of deficiency has been reported to reduce the number of invaginations and vesicles in endothelial cells (11, 25), a finding that we have verified. However, we did not detect a defect in GPIHBP1 or LPL movement across The Metabolic and Molecular Bases of Inherited Disease. C. R. Scriver, A. L. Beaudet, W. S. Sly et al., editors. McGraw-Hill, New York. 2705C2716. 3. Olivecrona T., Olivecrona G.2009. The ins and outs of adipose tissue. Cellular Lipid Metabolism. C. Ehnholm, editor. Springer Berlin Heidelberg. 315C369. Darusentan 4. Wang H., Eckel R. H. 2009. Lipoprotein lipase: from gene to obesity. Am. J. Physiol. Endocrinol. Metab. 297: E271CE288 [PubMed] [Google Scholar] 5. Beigneux A. P., Davies B. S., Gin P., Weinstein M. M., Farber E., Qiao X., Peale F., Bunting S., Walzem R. L., Wong J. S., et al. 2007. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons. Cell Metab. 5: 279C291 [PMC free article] [PubMed] [Google Scholar] Muc1 6. Davies B. S., Beigneux A. P., Barnes R. H.,.