Mol Metab 6:1137-1149 (2017) 

ANGPTL8 promotes the ability of ANGPTL3 to bind and inhibit lipoprotein lipase

Brandon S.J. Davies


摘要:

FLAG-tagged human LPL was concentrated from the medium of a Chinese hamster ovary cell line (CHO-K1) stably expressing FLAG-tagged human LPL as previously described [26], [27]. LargeBiT-human LPL (pEB12) was generated by cloning the linker and largeBiT sequence from pBiT1.1 (Promega) to the C-terminus of our FLAG-tagged LPL (pAH1) [27] using InFusion cloning (Clontech, catalog #638909). Lentiviruses containing this construct were produced by transfecting 293T cells with pEB12 and the lentiviral packaging vectors pMD2.G (Addgene #12259), pRSV-Rev (Addgene #12253), and pMDLg/pRRE (Addgene #12251). Conditioned media containing largeBiT-LPL expressing lentiviruses were collected and concentrated using Lenti-X Concentrator (Clontech, catalog #631231). 293T cells were then transduced with these lentiviruses and subjected to selection with puromycin (3 μg/ml) for 5 days. Conditioned media containing largeBiT-LPL was produced by culturing selected cells in serum-free DMEM for 72 h and then collecting media. LargeBiT-LPL conditioned media was concentrated before use with Amicon Ultra-15 Centrifugal Filter Units (EMD Millipore, catalog #UFC901024). The presence of LPL in the conditioned media was assessed by western blotting using a mouse antibody against the FLAG-tag (1:5000 Sigma–Aldrich; catalog #F1804). LPL activity was assessed by a lipase activity assay (see below).Strep-tagged mouse ANGPTL3 conditioned media was generated from 293T cells as we have described previously for ANGPTL4 [27]. As expected, when incubated with LPL at 37 °C, ANGPTL3 inhibited LPL in a time- and dose-dependent manner (Figure 1A). While high concentrations of ANGPTL3 abolished LPL activity (Figure 1A and Figure S1A), LPL protein levels were left unchanged (Figure S1B). We next tested the ability of ANGPTL3-inactivated LPL to bind the endothelial cell LPL receptor/transporter GPIHBP1. LPL inactivated by heat or high concentrations of ANGPTL3 was not able to bind GPIHBP1 on endothelial cells (Figure S1C), whereas control treated LPL or LPL treated with a concentration of ANGPTL3 insufficient to robustly inhibit lipase activity (Figure S1A) bound GPIHBP1 avidly (Figure S1C). Thus, just as we have shown previously for ANGPTL4-inactivated LPL [27], ANGPTL3-inactivated LPL had little capacity to bind GPIHBP1 on the surface of endothelial cells.ANGPTL3 is expressed only in the liver and, thus, could only act on peripheral tissues in an endocrine manner [14], [17]. In this context, ANGPTL3 would primarily encounter LPL bound to GPIHBP1 on the luminal wall of capillaries [8], [9]. A previous study, which used soluble GPIHBP1 and a cell-free system, found that GPIHBP1-bound LPL was protected from ANGPTL3 inhibition [33]. We asked if this protection was still observed when using full-length GPIHBP1 anchored to live endothelial cells. LPL-GPIHBP1 complexes were formed on the surface of endothelial cells and then treated with 0–750 nM (0–40 μg/ml) ANGPTL3 conditioned media. After incubation, LPL was released from the endothelial cells surface by heparin (100 U/ml) and assayed immediately for lipase activity. ANGPTL3 inhibited GPIHBP1-bound LPL in a dose-dependent manner; however, the ANGPTL3 concentration required for inhibition was substantially higher for GPIHBP1-bound LPL than for unbound LPL (Figure 1B; compare to Figure 1A).Because the concentrations of ANGPTL3 required to inhibit LPL were high, especially when LPL was bound to GPIHBP1, we assessed the physiological concentrations of ANGPTL3 in the plasma of fed and fasted mice. Employing the same ELISA used to determine the concentration of ANGPTL3 in conditioned media, we found that plasma ANGPTL3 concentrations in fasted male C57BL/6 mice ranged from 2.11 to 3.28 nM (mean 2.66 nM). Concentrations in fed mice were slightly higher (3.14–3.75 nM; mean 3.48 nM) (Figure 1C). Based on our inhibition data, these concentrations of ANGPTL3 would have minimal impact on LPL activity, yet ANGPTL3-deficient mice clearly have reduced plasma triglyceride levels and increased LPL activity [10], [11], [14]. Thus, we sought to determine what modifying factors might allow ANGPTL3 to inhibit LPL at physiological concentrations.We thank Catherine Musselman for expert technical assistance. This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (R01HL130146 to BSJD, R01HL134787 to RZ) and a Carver Medical Research Initiative grant from the Carver Trust (to BSJD).Appendix ASupplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molmet.2017.06.014.
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