GPIHBP1 stabilizes lipoprotein lipase and prevents its inhibition by angiopoietin-like 3 and angiopoietin-like 4

Abstract

Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4(-/-)/Gpihbp1(-/-) mice were lower than those of Gpihbp1(-/-) mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3(-/-)/Gpihbp1(-/-) mice were only slightly lower than those of Gpihbp1(-/-) mice. Treating Gpihbp1(-/-) mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability.

Fig. 1.

Soluble GPIHBP1 binds with LPL. Bovine LPL was adsorbed to wells of a mouse anti-LPL monoclonal IgG-coated 96-well plate and then incubated with varying concentrations of soluble mouse GPIHBP1 at concentrations ranging between 0 and 40 nM. The amount of soluble mouse GPIHBP1 bound to LPL was then quantitated by incubating with rabbit anti-GPIHBP1 primary antibodies, then HRP-conjugated goat anti-rabbit secondary antibodies, and finally, TMB substrate (see Experimental Procedures). The amount of soluble mouse GPIHBP1 bound to LPL is expressed as absorbance at 450 nm. Values are the mean ± SEM of triplicate determinations.

Fig. 2.

Soluble GPIHBP1 stabilizes LPL. Recombinant human LPL (approximately 10 nM) was preincubated at room temperature in LPL assay buffer for up to 124 min with either no supplement (♦), 0.1 U/ml heparin added at time = 0 min (□), 0.1 U/ml heparin added at time = 40 min (□), 100 nM soluble mouse GPIHPB1 added at time = 0 min (•), or 100 nM soluble mouse GPIHBP1 added at time = 40 min (○). The preincubated samples were then assayed for LPL activity at room temperature with fluorogenic substrate DGGR. LPL activity is expressed as relative fluorescence units (RFUs) per minute. Values are the mean ± SEM of triplicate determinations.

Fig. 3.

Concentration dependence of heparin or soluble GPIHBP1 on LPL stability. Recombinant human LPL (approximately 10 nM) was preincubated with (A) heparin ranging between 0 and 0.1 U/ml or with (B) soluble mouse GPIHBP1 ranging between 0 and 400 nM in LPL assay buffer at room temperature for 60 min. LPL activity was then assayed at room temperature with DGGR substrate. LPL activity is expressed as RFUs per minute. Values are the mean ± SEM of triplicate determinations.

Fig. 4.

Soluble GPIHBP1 but not heparin prevents inactivation of LPL by ANGPTL4. Recombinant human LPL (approximately 10 nM) with (A) no supplement, (B) 0.1 U/ml heparin, or (C) 100 nM soluble mouse GPIHBP1 was preincubated at room temperature in LPL assay buffer containing human ANGPTL4 at 0 nM (♦), 2.5 nM (○), 5 nM (□), 10 nM (□), or 20 nM (•). LPL activity was then assayed at room temperature with DGGR substrate. LPL activity is expressed as RFUs per minute. Values are the mean ± SEM of triplicate determinations.

Fig. 5.

Soluble GPIHBP1 and heparin prevents inhibition of LPL by ANGPTL3. Recombinant human LPL (approximately 10 nM) with (A) no supplement, (B) 0.1 U/ml heparin, or (C) 100 nM soluble mouse GPIHBP1 was preincubated at room temperature in LPL assay buffer containing human ANGPTL3 at 0 nM (♦), 62.5 nM (○), 125 nM (□), 250 nM (□), or 500 nM (•). LPL activity was then assayed at room temperature with DGGR substrate. LPL activity is expressed as RFUs per minute. Values are the mean ± SEM of triplicate determinations.

Fig. 6.

Concentration dependence of inhibition of LPL by ANGPTL3 or ANGPTL4 in the presence of stabilizing concentrations of heparin or soluble GPIHBP1. Recombinant human LPL with no supplement (♦), 0.1 U/ml heparin (□), 100 nM soluble mouse GPIHBP1 (•), or 400 nM soluble mouse GPIHBP1 (○) was preincubated at room temperature in LPL assay buffer containing either (A) human ANGPTL4 for 20 min or (B) human ANGPTL3 for 60 min at concentrations ranging between 0 and 2000 nM. LPL activity was then assayed at room temperature with DGGR substrate. LPL activity is expressed as RFUs per minute. Values are the mean ± SEM of triplicate determinations.

Fig. 7.

Disrupting Angptl4 gene expression normalizes TG levels in hyperlipidemic Gpihbp1^−/− mice. A: Fasting serum TG levels were determined in 8-week-old male mice lacking zero, one, or two copies of Angptl4 in Gpihbp1^+/+ or Gpihbp1^−/− backgrounds. TG levels of Angptl4^−/−/Gpihbp1^−/− mice were 93% lower than those of Angptl4^+/+/Gpihbp1^−/− mice. B: Fed serum TG levels were determined in 18 to 20-week-old male mice lacking zero, one, or two copies of Angptl3 in the Gpihbp1^+/+ or Gpihbp1^−/− background. TG levels of Angptl3^−/−/Gpihbp1^−/− mice were 26% lower than those of Angptl3^+/+/Gpihbp1^−/− mice. The bars represent the mean total serum TG level, which is also given below the genotype labels for each group. The number of mice in each group (n) is also shown.

Fig. 8.

Neutralizing ANGPTL4 with mAb 14D12 reduces serum TGs in hyperlipidemic Gpihbp1^−/− mice. A: Serum TG levels were determined in Gpihbp1^−/− mice treated with either ANGPTL4-neutralizing mAb 14D12 or control mAb KLH. Mice were injected intraperitoneally with 30 mg/kg mAb 14D12 (n = 8) or mAb KLH (n = 10) on day 0. Blood was drawn from fasted mice for serum TG measurements on day 4. B: Serum TG levels were determined in Gpihbp1^−/− mice treated with either ANGPTL3-neutralizing mAb 5.50.3 or control mAb KLH. Mice were injected intraperitoneally with 30 mg/kg mAb 5.50.3 (n = 7) or control mAb KLH (n = 7) on day 0 and day 4. Blood was drawn from fed mice for serum TG measurements on day 8. The bars represent the mean total serum TG level for each group. The percent reduction in TG levels is also shown.