Abnormal Patterns of Lipoprotein Lipase Release into the Plasma in GPIHBP1-deficient Mice

Michael M Weinstein,Liya Yin,Anne P Beigneux,Brandon S.J Davies,Peter Gin,Kristine Estrada,Kristan Melford,Joseph R Bishop,Jeffrey D Esko,Geesje M Dallinga-Thie,Loren G Fong,André Bensadoun,Stephen G Young

doi:10.1074/jbc.m806067200

Abstract

GPIHBP1-deficient mice (Gpihbp1(-/-)) exhibit severe chylomicronemia. GPIHBP1 is located within capillaries of muscle and adipose tissue, and expression of GPIHBP1 in Chinese hamster ovary cells confers upon those cells the ability to bind lipoprotein lipase (LPL). However, there has been absolutely no evidence that GPIHBP1 actually interacts with LPL in vivo. Heparin is known to release LPL from its in vivo binding sites, allowing it to enter the plasma. After an injection of heparin, we reasoned that LPL bound to GPIHBP1 in capillaries would be released very quickly, and we hypothesized that the kinetics of LPL entry into the plasma would differ in Gpihbp1(-/-) and control mice. Indeed, plasma LPL levels peaked very rapidly (within 1 min) after heparin in control mice. In contrast, plasma LPL levels in Gpihbp1(-/-) mice were much lower 1 min after heparin and increased slowly over 15 min. In keeping with that result, plasma triglycerides fell sharply within 10 min after heparin in wild-type mice, but were negligibly altered in the first 15 min after heparin in Gpihbp1(-/-) mice. Also, an injection of Intralipid released LPL into the plasma of wild-type mice but was ineffective in releasing LPL in Gpihbp1(-/-) mice. The observed differences in LPL release cannot be ascribed to different tissue stores of LPL, as LPL mass levels in tissues were similar in Gpihbp1(-/-) and control mice. The differences in LPL release after intravenous heparin and Intralipid strongly suggest that GPIHBP1 represents an important binding site for LPL in vivo.

Highlights

Illaries, mainly in heart, skeletal muscle, and adipose tissue [1, 2]
Delayed Appearance of lipoprotein lipase (LPL) into the Plasma after an Intravenous Injection of Heparin—We hypothesized that the GPIHBP1 in capillaries could play a physiologic role in binding LPL in vivo, and further hypothesized that the kinetics of LPL appearance in the plasma after an injection of heparin might differ in Gpihbp1Ϫ/Ϫ and control mice
Plasma LPL levels in Gpihbp1Ϫ/Ϫ mice were significantly lower than those in Gpihbp1ϩ/Ϫ and Gpihbp1ϩ/ϩ mice at baseline (p ϭ 0.015) and 1 and 3 min after heparin (p ϭ 0.000001 and 0.00003, respectively) (Fig. 1A), the plasma LPL levels were similar at the 15-min time point

Summary

EXPERIMENTAL PROCEDURES

Modified Mice—Gpihbp1Ϫ/Ϫ mice (Ͼ90% C57BL/6, Ͻ10% 129/Sv) have been described previously [8]. The samples were incubated overnight at 4 °C and the microtiter wells were washed with an automated plate washer six times with phosphate-buffered saline containing 0.05% Tween 20 This was followed by an incubation with biotinylated goat anti-mouse LPL immunoglobulins overnight at 4 °C. Subcutaneous heparin (100 units) was given at baseline and 8 and 16 h later, and plasma triglyceride levels were measured at multiple time points. Assessing LPL Specific Activity—To determine the specific activity of LPL, 200 ␮l of plasma was collected 1 or 15 min after an injection of heparin (50 units), diluted with an equal volume of buffer A (0.25 M NaCl, 10 mM sodium phosphate, pH 6.5) containing 30% glycerol and 1% bovine serum albumin, and applied to a 1-ml heparin-Sepharose column (GE Healthcare).

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS