In this study, we show that adult Gpihbp1−/− mice have severe hypertriglyceridemia, associated with a marked delay in the clearance of retinyl palmitate from the plasma. Gpihbp1 is expressed at high levels in heart, adipose tissue, and skeletal muscle—the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. Together, these findings strongly suggest that GPIHBP1 is a key platform for the LpL-mediated processing of chylomicrons in capillaries.
We found a large difference in GPIHBP1 expression in different capillary beds. While GPIHBP1 was expressed highly in the capillaries of lipolytic tissues such as adipose tissue, heart, and skeletal muscle, it was virtually absent in the brain. These findings raise the possibility that differences in endothelial cell gene expression could play an active role in regulating the delivery of lipid nutrients to different tissues.
The ability of GPIHBP1 to bind both to LpL and chylomicrons favors the concept that GPIHBP1 serves as an important “platform” for lipolysis by drawing chylomicrons and LpL into close proximity. However, it is unlikely that Gpihbp1
deficiency completely eliminates LpL-mediated processing of lipoproteins. Lpl-
deficient mice die shortly after birth with plasma triglyceride levels as high as 20,000 mg/dl (Weinstock et al., 1995
), whereas Gpihbp1
−/− mice survive and have far lower triglyceride levels. These differences between Gpihbp1
- and Lpl
-deficient mice strongly suggest that some LpL-mediated lipolysis occurs in the absence of GPIHBP1. Without question, LpL is present in Gpihbp1
−/− mice; we clearly identified mouse LpL—with normal enzymatic activity—in the plasma of Gpihbp1
−/− mice after an injection of heparin.
The fact that LpL was identified in the post-heparin plasma of Gpihbp1
−/− mice indicates that, in vivo
, there are additional binding sites for LpL—aside from GPIHBP1. Cell culture experiments have shown, quite convincingly, that LpL binds to HSPGs on the surface of endothelial cells (Cheng et al., 1981
). However, at this point, we do not know with certainty whether the heparin-releasable LpL in Gpihbp1
−/− mice is attached to HSPGs, and indeed whether the LpL in Gpihbp1
−/− mice that is mobilized by heparin is actually located within the lumen of capillaries. At this time, is not known with certainty how much of the LpL found into the bloodstream after an injection of heparin is actually released from the luminal surface of capillaries versus
other sites (e.g
., parenchymal cells, basement membranes, and subendothelial spaces). Further, the percentage of post-heparin LpL that arises from endothelial cells versus
those other sites could differ in Gpihbp1
−/− and Gpihbp1
+/+ mice. In any case, since the post-heparin plasma LpL levels are higher in wild-type mice than in Gpihbp1
−/− mice, it seems possible that a significant fraction of the heparin-releasable LpL in wild-type mice is bound by GPIHBP1. In future studies, it will be of interest to determine the fraction of the total LpL pool on endothelial cells that is bound to GPIHBP1 and HSPGs, and whether LpL bound to GPIHBP1 is catalytically more efficient than LpL bound to HSPGs.
The degree of hypertriglyceridemia in Gpihbp1
−/− pups before weaning was milder than that in 4—6 week-old Gpihbp1
−/− mice, which in turn was milder than that in 7–16-week-old Gpihbp1
−/− mice. The explanation for this finding is not known with certainty, but we suspect that it relates to the fact that Lpl
mRNA levels are much higher in the liver during the suckling phase (Langner et al., 1989
). We have confirmed higher Lpl
expression levels by quantitative RT-PCR: suckling C57BL/6 mice have hepatic Lpl
mRNA levels that are 50-times greater than those of 10-week-old mice, and hepatic Lpl
mRNA levels in 4-week-old mice are approximately double those in 10-week-old mice (Supplementary Online Figure 7
). Although we have not yet proven that the differences in hepatic expression of Lpl
are responsible for the lower triglyceride levels in the younger mice, the time course of Lpl
expression would be consistent with this possibility. In any case, we emphasize that the defect in LpL activity in adult Gpihbp1
−/− mice is severe. When newborn Lpl
knockout mice were injected with an LpL adenovirus, some survived the suckling period, owing to the adenoviral-mediated LpL expression in the liver (Strauss et al., 2001
). However, this adenoviral-mediated LpL expression disappeared by 30 days of age. After the extinction of LpL expression, the plasma triglyceride levels in the rescued mice were 2,000–5,000 mg/dl—very similar to those in chow-fed Gpihbp1
−/− mice in this study.
The plasma levels of apo-B48 were markedly elevated in Gpihbp1
−/− mice. In the mouse, apo-B48 is synthesized by both the liver and the intestine (Hirano et al., 1996
). Given the markedly delayed clearance of the retinyl palmitate in Gpihbp1
−/− mice, we suspect that much of the apo-B48 accumulation is due to delayed clearance of intestinal lipoproteins. However, the lipolytic processing of chylomicrons and hepatic VLDL are similar, and there is no reason to believe that the accumulation of apo-B48 is exclusively due to an accumulation of intestinal lipoproteins.
The molecular basis for chylomicron binding to GPIHBP1 requires further study. Chylomicrons contain several apolipoproteins (e.g
., apo-B48, apo-E, apo-AV) that have positively charged domains that bind to heparin sulfate and/or HSPGs (Cardin et al., 1984
; Cardin et al., 1986
; Lookene et al., 2005
), so it is easy to imagine that one or more of these apolipoproteins could mediate the binding of chylomicrons to the strongly negatively charged domain of GPIHBP1. In particular, apo-AV would make a good candidate as a ligand for GPIHBP1, given that Apoav
deficiency causes hypertriglyceridemia associated with decreased LpL-mediated lipolysis (Calandra et al., 2006
; Grosskopf et al., 2005
; Pennacchio et al., 2001
). In support of this idea, our studies showed that apo-AV-phospholipid disks bind avidly to Gpihbp1
-transfected proteoglycan-deficient cells (Supplementary Online Figure 6
). Another interesting hypothesis would be that GPIHBP1 could act as a receptor for apo-B48, a key structural protein of large, triglyceride-rich lipoproteins. It would make sense that a receptor for apo-B48 would be located in the capillary endothelium of muscle and fat, where lipolysis occurs. Also, this hypothesis is in line with evolutionary considerations. Both apo-B48 and chylomicrons (defined as intestinal lipoproteins that are secreted into the lymph) are unique to mammals (Teng and Davidson, 1992
). Similarly, GPIHBP1—defined as a GPI-anchored Ly-6-motif protein with a very strong acidic domain—is present in all mammals but is absent in fish, amphibians, and birds.
Transfection of a Gpihbp1
cDNA into cultured cells promotes the binding of both LpL and chylomicrons, but it is not yet clear whether a single GPIHBP1 molecule is capable of binding chylomicrons and LpL simultaneously. If a single GPIHBP1 molecule binds only a single ligand, chylomicrons and LpL could still be drawn together if the GPIHBP1 molecules were clustered together on the cell surface. This possibility seems plausible given that other GPI-anchored proteins are concentrated within lipid rafts on the plasma membrane (Varma and Mayor, 1998
). Also, GPIHBP1 could exist as a homodimer on the cell surface and therefore bind to more than one ligand. Of note, urokinase-type plasminogen activator receptor, a related GPI-anchored Ly-6-motif protein, exists as a dimer on the cell surface (Cunningham et al., 2003
In summary, GPIHBP1 is crucial for the lipolytic processing of triglyceride-rich lipoproteins. It is located on the luminal surface of the capillary endothelium and binds both LpL and chylomicrons. It likely forms a platform for lipolysis and plays an important role in the delivery of lipid nutrients to cells.