Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 and the intravascular processing of triglyceride-rich lipoproteins


Correspondence: Anne P. Beigneux, Loren G. Fong, or Stephen G. Young, 650 Charles E. Young Dr. South, Los Angeles, CA 90095, USA.

(fax: (310) 206-0865; e-mails:,,


Lipoprotein lipase (LPL) is produced by parenchymal cells, mainly adipocytes and myocytes, but is involved in hydrolysing triglycerides in plasma lipoproteins at the capillary lumen. For decades, the mechanism by which LPL reaches its site of action in capillaries was unclear, but this mystery was recently solved. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, ‘picks up’ LPL from the interstitial spaces and shuttles it across endothelial cells to the capillary lumen. When GPIHBP1 is absent, LPL is mislocalized to the interstitial spaces, leading to severe hypertriglyceridaemia. Some cases of hypertriglyceridaemia in humans are caused by GPIHBP1 mutations that interfere with the ability of GPIHBP1 to bind to LPL, and some are caused by LPL mutations that impair the ability of LPL to bind to GPIHBP1. Here, we review recent progress in understanding the role of GPIHBP1 in health and disease and discuss some of the remaining unresolved issues regarding the processing of triglyceride-rich lipoproteins.


We have known for decades that the lipolytic processing of chylomicrons and very low-density lipoproteins (VLDL) by lipoprotein lipase (LPL) occurs at the capillary lumen, mainly in adipose tissue and striated muscle [1-4]. For a long time, it was assumed that LPL was bound to heparan sulfate proteoglycans (HSPGs) on the surface of capillary endothelial cells. This model (Fig. 1a) was based on the fact that LPL binds avidly to HSPGs in in vitro studies [5-7] and that LPL can be released into the plasma by heparin [8], a highly sulfated proteoglycan. However, some elements of the story were missing from this model. First, it was unclear why LPL, an enzyme that is secreted by myocytes and adipocytes, would bind preferentially to HSPGs at the capillary lumen rather than to HSPGs surrounding myocytes and adipocytes. Secondly, this model did not explain how LPL was transported across endothelial cells to the capillary lumen.

Figure 1.

Two models of the metabolism of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL). (a) Before the discovery of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1). It was known that LPL is synthesized and secreted by parenchymal cells (myocytes or adipocytes), and that LPL is active inside capillaries, but how LPL reaches the capillary lumen was unknown. Chylomicron particles and LPL were both assumed to bind to heparan-sulfate proteoglycans (HSPGs) inside capillaries. (b) An updated model of lipolysis. GPIHBP1, a cell-surface protein of capillary endothelial cells, ‘picks up’ LPL from the interstitial spaces, transports it across endothelial cells to the capillary lumen, and helps to keep LPL at the surface of endothelial cells. Modified, with permission, from Young et al. [66].

In the past few years, the accepted model of plasma triglyceride metabolism at the capillary lumen has changed significantly (Fig. 1b). It is now clear that glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a protein of capillary endothelial cells, is the principal binding site for LPL on endothelial cells and that it is responsible for transporting LPL to the capillary lumen [9]. It is not surprising that GPIHBP1 has proved to be important in human disease. GPIHBP1 mutations that interfere with LPL binding and transport cause severe hypertriglyceridaemia (chylomicronaemia) [10-15], while some LPL mutations cause hypertriglyceridaemia by interfering with the capacity of LPL to bind to GPIHBP1 [16]. In this review, we will discuss the role of GPIHBP1 in lipolysis and its involvement in hypertriglyceridaemia in humans. In addition to discussing recent progress in the understanding of lipolysis, we will highlight important topics for future research.

The discovery of GPIHBP1 and its role in lipolysis

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 was initially identified by Ioka et al., by expression cloning, as a molecule that conferred upon Chinese hamster ovary (CHO) cells the ability to bind high-density lipoproteins [17]. The authors noted that this protein has three important features [17]. First, GPIHBP1 has a very impressive acidic domain; 17 of 25 consecutive residues at the amino terminus of mouse GPIHBP1 are glutamate or aspartate. Secondly, GPIHBP1 has a cysteine-rich lymphocyte antigen 6 (Ly6) motif, similar to CD59 (which regulates complement activation) and urokinase-type plasminogen activator receptor. Thirdly, mature GPIHBP1 contains a glycosylphosphatidylinositol (GPI) anchor. GPIHBP1 can be released from the plasma membrane by cleaving the GPI anchor with a phosphatidylinositol-specific phospholipase C. In the initial report, the authors suggested that GPIHBP1 played a role in ‘the initial entry of HDL cholesterol into scavenger cells for further transportation of cholesterol' and added that understanding GPIHBP1 function would ‘require the development of knockout mice’ [17].

The physiological function of GPIHBP1 became clearer with the characterization of Gpihbp1 knockout mice (Gpihbp1−/−) [18]. When fed a low-fat chow diet, Gpihbp1−/− mice have plasma triglyceride levels of approximately 2000–5000 mg/dL and plasma cholesterol levels of 300–900 mg dL−1 [18]; most of this lipid is found in the VLDL/chylomicron fraction. As shown by electron microscopy, the lipoprotein particles in Gpihbp1−/− mouse plasma are far larger than those of wild-type mice [18]. Furthermore, the clearance of retinyl palmitate from Gpihbp1−/− mice was markedly delayed [18], and the plasma levels of apolipoprotein (apo)-B48 were elevated. These findings suggest that the lipolytic processing of triglyceride-rich lipoproteins (TRLs) in Gpihbp1−/− mice is defective.

Three additional observations lent support to the notion that GPIHBP1 is important for lipolysis. The first was that GPIHBP1 is highly expressed in heart and adipose tissue, the same tissues that express high levels of LPL. The second observation was that GPIHBP1, when expressed in cultured cells, binds LPL avidly. And third, GPIHBP1 is found exclusively in capillary endothelial cells, where lipolysis occurs. These findings led us to speculate that GPIHBP1 was a binding site for LPL within capillaries [18].

Beigneux et al. [18] had initially reported that GPIHBP1 binds apo-AV and TRL particles. Later, Gin et al. [19] investigated these findings in more detail. The interaction between GPIHBP1 and apo-AV depends on the heparin-binding domain of apo-AV and on GPIHBP1's acidic domain [20] but not on its Ly6 domain [19]. These authors also showed that TRL binding to GPIHBP1-expressing CHO cells is mediated by hamster LPL secreted by CHO cells [19]. Hamster LPL binds to GPIHBP1, and the LPL promotes binding of TRLs.

Because the plasma of Gpihbp1−/− mice contains elevated levels of apo-B48, we suggested that GPIHBP1 might play an important role in processing apo-B48–containing lipoproteins [18]. However, subsequent studies showed that this is not the case; plasma triglyceride levels in Gpihbp1−/− mice that produce only apo-B48 (Gpihbp1−/−Apob48/48) and Gpihbp1−/− mice that produce only apo-B100 (Gpihbp1−/−Apob100/100) are elevated to the same degree [21].

Metabolic consequences of impaired lipolysis in Gpihbp1−/− mice

Recently, Weinstein et al. [22] demonstrated that the ratio of 18 : 2 and 18 : 3 fatty acids to 16 : 1 fatty acids in adipose tissue was lower in Gpihbp1−/− mice than in wild-type mice, consistent with increased de novo lipogenesis. The opposite was observed in the liver: the 18 : 2 and 18 : 3 to 16 : 1 fatty acid ratio of liver triglycerides was higher in Gpihbp1−/− than in wild-type mice [22]. The expression of lipid biosynthetic genes was significantly higher in adipose tissue of Gpihbp1−/− than wild-type mice, both in animals fed chow and high-fat diets [22]. In the liver, the expression of lipid biosynthetic genes was lower in Gpihbp1−/− than wild-type mice. These findings suggest that impaired delivery of lipids to peripheral tissues of Gpihbp1−/− mice is associated with increased uptake of lipids by the liver [22].

GPIHBP1 transports LPL to the capillary lumen

Based on two experimental findings, we considered the possibility that GPIHBP1 is involved in LPL transport. First, tissue stores of LPL are essentially identical in wild-type and Gpihbp1−/− mice [18, 23] and, secondly, Gpihbp1−/− mice had high levels of catalytically active LPL in the plasma after intraperitoneal injection of heparin [18]. These observations raised an obvious question: why do Gpihbp1−/− mice have chylomicronaemia, given that they have large amounts of catalytically active LPL? Weinstein et al. [23] proposed that the LPL in Gpihbp1−/− mice was probably mislocalized away from the intravascular compartment. In support of this idea, the entry of LPL into the plasma compartment after an injection of heparin was delayed in Gpihbp1−/− mice [23]. Furthermore, an injection of triglyceride emulsion particles failed to release LPL into the plasma of Gpihbp1−/− mice.

Davies et al. [9] used microscopy to investigate the possibility that LPL was mislocalized in Gpihbp1−/− mice. They found that the LPL in wild-type mice is largely associated with capillaries, whereas the LPL in Gpihbp1−/− mice was mislocalized to the interstitial spaces surrounding parenchymal cells (Fig. 2) [9]. This was the case in many tissues, including heart, skeletal muscle, mammary gland, and brown adipose tissue. Imaging cross-sections of capillaries containing endothelial cell nuclei was also informative. In wild-type mice, LPL was present, along with GPIHBP1, in the capillary lumen; in Gpihbp1−/− mice, LPL was absent from the capillary lumen (Fig. 3) [9]. These findings support the idea that GPIHBP1 functions as an LPL transporter.

Figure 2.

Confocal immunofluorescence microscopy images showing that lipoprotein lipase (LPL) is tightly bound within the interstitial spaces in skeletal muscle of Gpihbp1−/− mice. Images show β-dystroglycan (a marker of skeletal myocytes; green), CD31 (a marker of endothelial cells; purple), and LPL (red) in muscle from a wild-type (Gpihbp1+/+) mouse and a Gpihbp1−/− mouse. LPL is largely bound to capillaries in the Gpihbp1+/+ mouse but is mislocalized to the interstitial spaces surrounding myocytes in the Gpihbp1−/− mouse. Reproduced, with permission, from Davies et al. [9].

Figure 3.

Confocal micrographs demonstrating that lipoprotein lipase (LPL) is absent from the capillary lumen in Gpihbp1−/− mice. Micrographs show glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) (purple), LPL (red), and CD31 (green) in brown adipose tissue. Nuclei were stained with DAPI (blue). The luminal (arrowhead) and basolateral (arrow) surfaces of capillaries can be distinguished in cross-sections of capillaries containing an endothelial cell nucleus. GPIHBP1, LPL, and CD31 are found at both the luminal and basolateral surfaces of capillaries of wild-type (Gpihbp1+/+) mice; however, LPL was not found along the luminal surface of capillaries of Gpihbp1−/− animals. Note the absence of LPL, but not CD31, in the lumen of the Gpihbp1−/− capillary. Reproduced, with permission, from Davies et al. [9].

Direct support for the role of GPIHBP1 as a transporter was obtained with transport assays across confluent monolayers of endothelial cells. GPIHBP1-expressing endothelial cells transported a GPIHBP1-specific monoclonal antibody from the basolateral to the apical face of cultured endothelial cells [9]. Antibody transport could be abolished when GPIHBP1 at the basolateral face of cells was released by cleaving the glycosylphosphatidylinositol anchor of GPIHBP1 with a phosphatidylinositol-specific phospholipase C. GPIHBP1-expressing endothelial cells also transported LPL from the basolateral to the apical face of cells [9].

Phenotypic differences in GPIHBP1-deficient and LPL-deficient mice

Because LPL cannot reach the capillary lumen in the absence of GPIHBP1, one might have predicted that Gpihbp1−/− mice would closely resemble Lpl knockout mice (Lpl−/−). However, this is not the case. Newborn Lpl−/− mice have markedly elevated plasma triglyceride levels (~20 000 mg dL−1) and die within 24 h [24]. By contrast, suckling Gpihbp1−/− mice are healthy with plasma triglyceride levels of ~120 mg dL−1 [18].

Transient expression of LPL (via an injection of LPL adenovirus) rescues LPL-deficient mice from perinatal lethality, allowing them to survive into adulthood [25, 26]. Adult Lpl−/− mice have markedly elevated triglyceride levels, very much like adult Gpihbp1−/− mice.

Why are the phenotypes of suckling Lpl−/− and Gpihbp1−/− pups so different, whereas the phenotypes of adult Lpl−/− and Gpihbp1−/− mice are so similar? The answer likely relates to the fact that suckling mice, but not adult mice, produce large amounts of LPL in the liver [18]. The fenestrated capillaries of the liver would allow unrestricted access of LPL to TRLs in the plasma, even when GPIHBP1 is absent.

Atherosclerosis in Gpihbp1−/− mice

Chylomicrons are often considered to be non-atherogenic, in part because of early observations indicating minimal atherogenicity of very large lipoproteins in rabbits and also because of a limited ability of very large lipoproteins to enter the arterial wall [27]. Surprisingly, however, chow-fed Gpihbp1−/− mice developed lipid- and macrophage-rich atherosclerotic lesions in the aortic root and coronary arteries [21]. These lesions, which were relatively small at 11–12 months but larger by 16–22 months of age, were present in both male and female mice. These lesions were far smaller than those in Apoe−/− mice [28-31], but their existence demonstrated that large TRLs can be atherogenic. These findings were consistent with the presence of aortic lesions in Lpl-deficient mice [26] and reports of atherosclerotic disease in LPL-deficient humans [32].

Regulation of GPIHBP1 expression

Fasting leads to increased levels of Lpl expression in heart, but reduced expression in white adipose tissue [33]. This physiological response would serve to promote triglyceride delivery to the heart and away from adipose tissue during fasting [34-36]. By contrast, fasting leads to increased Gpihbp1 expression in both heart and adipose tissue [33]. Why Gpihbp1 expression increases in white adipose tissue during fasting is unclear, but one possibility is that increased expression of GPIHBP1 somehow facilitates LPL regulation by angiopoietin-related protein (ANGPTL)4.

Peroxisome proliferator-activated receptor (PPAR)γ agonists increase Gpihbp1 expression in adipose tissue, heart and skeletal muscle, whereas PPARα and PPARδ agonists have little or no effect. Sequences upstream of exon 1 in Gpihbp1 contain a PPAR-binding site that exhibits activity in a luciferase reporter assay [33]. In addition, a knockout of PPARγ in endothelial cells lowers Gpihbp1 transcript levels in brown and white adipose tissue, suggesting that PPARγ regulates Gpihbp1 expression levels in vivo [33]. However, whether GPIHBP1 expression in humans is altered by PPARγ agonists is not known.

In mice, the expression of LPL in the liver can be induced by dietary cholesterol [37]. Consistent with this observation, plasma triglyceride levels were lower in Gpihbp1−/− mice fed a high-cholesterol diet compared with those fed a low-cholesterol diet [38]. Conversely, plasma triglyceride levels in Gpihbp1−/− mice were significantly increased following treatment with ezetimibe, which lowers cholesterol absorption and leads to lower levels of Lpl expression in the liver. Again, the responsiveness of plasma triglyceride levels to changes in LPL expression in the liver is probably explained by fenestrated capillaries in the liver, which would give LPL ready access to TRLs in the bloodstream.

GPIHBP1 expression in different tissues

In general, the tissue pattern of GPIHBP1 expression is similar to that of LPL. For example, Gpihbp1 and Lpl transcripts are both found at high levels in heart and brown adipose tissue and at lower levels in skeletal muscle [18]. There are, however, exceptions to this rule. First, GPIHBP1 is absent from the capillaries of the brain, even though certain regions, for example the hippocampus, express large amounts of LPL [39-43]. The reason for this discrepancy is not clear, but one possibility is that LPL actually has an extravascular function in the brain.

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 and LPL expression levels are also different in the lung [44]. Lpl transcript levels are negligible whereas Gpihbp1 transcript levels are very high (in the same range as brown adipose tissue and heart). Consistent with high Gpihbp1 transcript levels, GPIHBP1 protein levels are high in lung capillaries. Indeed, when GPIHBP1-specific monoclonal antibodies are injected into mice, antibody binding to lung capillaries is very high, similar to levels of antibody binding in heart and brown adipose tissue [44]. Interestingly, while Lpl transcript levels in the lung are extremely low, LPL protein can be detected in the lung. We suspect that GPIHBP1 in lung capillaries scavenges LPL produced by other tissues. While most of the LPL secreted by myocytes and adipocytes is bound by GPIHBP1 or HSPGs, we suspect that some LPL escapes and finds its way into the lymph and eventually into the venous circulation. That LPL, we believe, is likely to be captured by GPIHBP1 in lung capillaries. Consistent with this idea, we found that the amount of LPL protein in the lung was lower in Gpihbp1−/− mice than in wild-type mice [44]. In addition, there are relatively high levels of human LPL in the lungs of transgenic mice that express human LPL exclusively in skeletal muscle [44].

LPL–GPIHBP1 interactions

It has long been recognized that LPL has positively charged domains that bind to heparin [45-48], and we hypothesized that those same domains would interact with the amino-terminal acidic domain of GPIHBP1. In support of this, wild-type GPIHBP1 avidly binds LPL, but an acidic domain GPIHBP1 mutant (in which the aspartates and glutamates in the second half of the acidic domain were replaced by alanines) was unable to bind LPL. Also, a rabbit antiserum against the GPIHBP1 acidic domain markedly reduced LPL binding to cells expressing wild-type GPIHBP1 [20]. These findings suggest that electrostatic interactions play a role in LPL–GPIHBP1 interactions. Consistent with this, the binding of LPL to GPIHBP1 can be blocked by polyaspartate, polyglutamate, and heparin [20]. Furthermore, binding of LPL to GPIHBP1 was reduced when positively charged residues in the carboxyl-terminal heparin-binding domain of LPL (K403, R405, K407, K413, and K414) were replaced by alanine [20].

In addition to the acidic domain, the Ly6 domain of GPIHBP1 is crucial for LPL–GPIHBP1 interactions. The Ly6 domain is an ~80–amino acid motif containing 10 cysteines arranged in a characteristic spacing pattern. Each of the 10 cysteines is disulfide-bonded, resulting in five disulfide bonds that create a three-finger structural motif [49]. The involvement of the Ly6 domain in LPL binding is supported by several observations. First, a chimeric protein containing the acidic domain of GPIHBP1 and the Ly6 domain of another Ly6 family member (CD59) lacks the ability to bind LPL [20]. Secondly, replacing any of the 10 cysteines in the Ly6 domain of GPIHBP1 by alanine reduces the ability of GPIHBP1 to bind LPL [50]. Thus, intact disulfide bonds and maintenance of the three-finger structure of GPIHBP1 are important for LPL binding. After mutating each amino acid of the Ly6 domain (Cys-65 to Cys-136), we tested the ability of each mutant GPIHBP1 protein to bind LPL. In addition to the conserved cysteines, 12 residues were found to be essential for LPL binding, nine of which were clustered in finger 2 of the three-finger Ly6 domain [51]. The mutant GPIHBP1 proteins that could not bind LPL in cell-based binding assays also lacked the ability to transport LPL across cultured endothelial cells [51].

An example of a western blot assay to determine the binding of LPL to mutant forms of GPIHBP1 is shown in Fig. 4. In this experiment, CHO cells were transfected with a wild-type or a mutant human GPIHBP1 expression vector; 24 h later, the cells were incubated with LPL for 2 h at 4 °C in the presence or absence of heparin. After washing the cells, the amount of LPL bound to cells was assessed by western blotting. Wild-type GPIHBP1 avidly bound LPL, but virtually no LPL bound to cells expressing GPIHBP1-C110A. Other GPIHBP1 mutants, for example GPIHBP1-D112A and GPIHBP1-W109S, did not affect LPL binding.

Figure 4.

A western blot assay of lipoprotein lipase (LPL) binding to wild-type glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) and mutant GPIHBP1 proteins harbouring specific missense mutations (D112A, W109S, and C110A). CHO cells were electroporated with expression vectors encoding S-protein–tagged versions of wild-type GPIHBP1 or the GPIHBP1 mutants. After 24 h, the cells were incubated for 2 h with V5-tagged human LPL ± heparin (500 U mL−1). After washing the cells, the level of GPIHBP1 expression in cells and the amount of bound LPL were assessed by western blotting. Actin was used as a loading control. LPL bound avidly to cells expressing wild-type GPIHBP1, GPIHBP1-D112A, and GPIHBP1-W109S; LPL did not bind to cells expressing GPIHBP1-C110A.

Mouse GPIHBP1 contains a single N-linked glycosylation site, which is important for the transport of mature, GPI-anchored GPIHBP1 to the cell surface [52]. However, an absence of N-linked glycosylation has little or no effect on the secretion of a soluble version of GPIHBP1 (i.e. a version lacking the GPI anchor) from cells [52]. The absence of the N-linked glycan does not prevent GPIHBP1 from binding LPL [52].

A study in 2011 had suggested the possibility LPL binding to GPIHBP1 might require intact LPL homodimers [19], but subsequent studies showed that this was not the case [53]. The carboxyl-terminal of LPL (residues 298–448), acting as a monomer and free of the amino-terminal catalytic domain of LPL, binds avidly to GPIHBP1 [53].

GPIHBP1 mutations and chylomicronaemia in humans

The finding of chylomicronaemia in Gpihbp1−/− mice [18] raised the possibility that human GPIHBP1 mutations might have a role in the pathogenesis of hypertriglyceridaemia. In recent years, a number of clinically significant GPIHBP1 mutations have been revealed, as discussed below. However, these mutations are not a common cause of hypertriglyceridaemia; even in highly selected chylomicronemia patients where mutations in LPL, APOC2, and APOA5 have been excluded, GPIHBP1 mutations are quite uncommon [11, 12, 15, 54].

The first GPIHBP1 mutation, a Q115P missense mutation, was identified by screening 60 patients with severe hypertriglyceridaemia who lacked mutations in LPL, APOC2, and APOA5 [12]. The affected patient had lifelong chylomicronaemia and was homozygous for the mutation. When GPIHBP1-Q115P was expressed in CHO cells, it reached the cell surface normally but had a markedly reduced capacity to bind LPL [12]. When the same mutation was introduced into mouse GPIHBP1, LPL binding was also markedly reduced [12]. Replacing Q115 in human GPIHBP1 by a lysine (the residue found in canine GPIHBP1) or a glutamate (found in platypus GPIHBP1) did not affect LPL binding [51].

A C65Y mutation in GPIHBP1 was identified in a 3-year-old boy with chylomicronaemia [11]. The child had plasma triglyceride levels of >1500 mg dL−1 and a history of pancreatitis. When GPIHBP1-C65Y was expressed in cultured cells, it reached the cell surface but could not bind LPL. Later, Coca-Prieto et al. [13] analysed five patients with childhood-onset chylomicronaemia and found that one had a C68Y mutation in GPIHBP1. Two more GPIHBP1 cysteine mutations were identified in Sweden. Three members of a single family had chylomicronaemia, and all three were compound heterozygotes for C65S and C68G alleles [10]. Studies in transfected cells revealed that GPIHBP1-C65S and GPIHBP1-C68G reached the cell surface but were unable to bind LPL. LPL mass and activity levels were normal in the adipose tissue of affected subjects, consistent with normal LPL stores in tissues of Gpihbp1−/− mice [23]. Interestingly, breast milk from one of the C65S/C68G compound heterozygotes contained normal amounts of LPL [10].

Rios et al. [14] identified two family members with chylomicronaemia and a homozygous deletion of the entire GPIHBP1 gene. The proband was an Asian Indian boy who had severe chylomicronaemia at 2 months of age. Array-based copy-number analysis of the genomic DNA uncovered a 17.5-kb deletion that included GPIHBP1. A 44-year-old aunt with a history of hypertriglyceridaemia and pancreatitis was homozygous for the same deletion [14].

Charrière et al. [15] identified two patients with GPIHBP1 mutations among 376 patients with chylomicronaemia. A young child with markedly elevated plasma triglyceride levels and a history of pancreatitis had a C89F mutation in one GPIHBP1 allele; the other allele had a deletion of the entire GPIHBP1 gene [15]. The second patient, a 35-year-old man with a plasma triglyceride level of >2200 mg dL−1, was homozygous for a G175R mutation. The G175R mutation was located in the carboxyl-terminal domain and probably interfered with the addition of the GPI anchor. In cell transfection experiments, it was found that the G175R mutation reduced the amount of GPIHBP1 at the cell surface [15].

Recently, Surendran et al. [54] found a T108R mutation in GPIHBP1 in a 1-year-old child with hypertriglyceridaemia and a history of pancreatitis. Given the clinical presentation, it seems likely that this mutation abolished the ability of GPIHBP1 to bind LPL binding, but this was not tested. In an earlier study, we found that a T108A mutation had no effect on LPL binding [51].

The levels of LPL in pre-heparin plasma are extremely low in humans with GPIHBP1 defects [10]. Similarly, plasma LPL levels after a bolus injection of heparin are low. In the Q115P homozygote, postheparin LPL levels were ~10% of those in controls [18]. The postheparin LPL levels in the three C65S/C68G compound heterozygotes were only ~5% of those in control subjects [10]. Postheparin LPL activity levels were also extremely low or undetectable in the G175R homozygote, the patient with the C89F mutation, the C65Y homozygote and the C68Y homozygote [11, 13, 15]. In all kindreds examined to date, GPIHBP1 deficiency is a recessive syndrome; heterozygotes have normal plasma lipid levels [11, 14, 15]. The same is the case in the knockout mice, where one can show that a single GPIHBP1 knockout allele lowers tissue levels of GPIHBP1 expression by 50%. It appears that, half-normal levels of GPIHBP1 are sufficient to transport LPL across endothelial cells, both in humans and in mice.

LPL mutations that interfere with LPL binding to GPIHBP1

The discovery of GPIHBP1 point mutations that inhibit the ability of GPIHBP1 to bind to LPL suggested that there might be ‘mirror image’ LPL mutations that inhibit the ability of LPL to bind to GPIHBP1. To investigate this possibility, Voss et al. [16] re-examined previously described LPL mutations associated with chylomicronaemia. Two mutations, C418Y and E421K [55, 56], were of particular interest because they were distant from the catalytic domain of LPL and had been reported to have little effect on catalytic activity. The C418Y mutation was discovered in a 30-year-old man with severe hypertriglyceridaemia and a history of pancreatitis [56]; the E421K mutation was found in a 24-year-old woman who died of pancreatitis during pregnancy [55].

Voss et al. [16] hypothesized that the C418Y and E421K mutations might cause hypertriglyceridaemia by interfering with the ability of LPL to bind to GPIHBP1. They showed that the enzymatic specific activities of the mutant LPLs were normal and that the mutations did not affect the binding of LPL to heparin. However, both mutations markedly reduced the capacity of LPL to bind to wild-type GPIHBP1 [16]. When these mutations were introduced into mouse or chicken LPL (cLPL), they also reduced binding to GPIHBP1.

The C418Y mutation was interesting because Cys-418 has been reported to form a disulfide bond with Cys-438 [57]. The inability of LPL-C418Y to bind to GPIHBP1 raised the possibility that the disulfide bond is essential for GPIHBP1 binding. However this is apparently not the case; LPL-C438A and LPL-C438Y are able to bind to GPIHBP1 (although they appear to bind less avidly than wild-type LPL).

The reduced ability of C418Y-LPL and E421K-LPL to bind to GPIHBP1 suggested that the carboxyl-terminal region of LPL plays an important role in GPIHBP1 binding. In support of that idea, a monoclonal antibody against chicken LPL (cLPL) with an epitope between residues 416 and 435 blocked binding of cLPL to GPIHBP1 while having little effect on cLPL binding to heparin. Mutagenesis studies showed that changing cLPL residues 421–425, 426–430, or 431–435 to alanine reduced cLPL binding to GPIHBP1 and did so without affecting cLPL binding to heparin [16].

At this point, it would appear that there are two LPL domains that are important for GPIHBP1 binding. The first is the main heparin-binding domain (K403, R405, K407, K413, and K414) and the second is an independent downstream domain encompassing residues ~416–435. It seems likely that the positively charged heparin-binding domain of LPL interacts with the negatively charged acidic domain of GPIHBP1. The LPL residues that interact with the Ly6 domain of GPIHBP1 have not been definitively identified, but it is tempting to speculate that this domain interacts with LPL residues ~416–435.

Unresolved issues

The identification of GPIHBP1 solved the long-standing question of how LPL reaches the capillary lumen. However, many other aspects of the physiology of lipolysis remain unclear. With regard to GPIHBP1, there are eight issues that we believe need further investigation.

Aside from the rare mutations that disrupt LPL binding to GPIHBP1, are LPL–GPIHBP1 interactions clinically relevant?

Most patients with hypertriglyceridaemia do not have mutations in GPIHBP1, even when other potential culprit genes have been excluded (e.g. LPL, GPIHBP1, APOC2, APOA5) [11, 12, 15, 54, 58]. However, this may not mean that GPIHBP1-mediated LPL transport is irrelevant to the pathogenesis of common forms of hypertriglyceridaemia. It is possible, for example, that some cases of hypertriglyceridaemia are caused by defects in the cellular machinery for moving GPIHBP1 and LPL across endothelial cells. Also, some cases of hypertriglyceridaemia might be caused by inaccessibility of LPL–GPIHBP1 complexes on the surface of endothelial cells. For example, if GPIHBP1–LPL complexes were somehow buried within the glycocalyx along the capillary lumen, the clinical consequences might be similar to GPIHBP1 deficiency, where LPL is mislocalized to the subendothelial spaces.

Understanding LPL–GPIHBP1 interactions on both sides of endothelial cells

How GPIHBP1 manages to ‘pick up’ LPL from the subendothelial spaces is still unclear. LPL binds avidly to HSPGs [59, 60]. Even when GPIHBP1 is absent, LPL is tightly bound within the interstitial spaces, presumably attached to HSPGs (the LPL within the subendothelial spaces of Gpihbp1−/− mice is readily released by heparin). It is important to understand how GPIHBP1 recruits LPL from HSPG binding sites within the interstitial spaces. It seems possible that the acidic domain of GPIHBP1 acts as a ‘lasso’, wresting LPL away from HSPG binding sites (Fig. 5a,b). Once bound to the acidic domain, it seems possible that interactions with the Ly6 domain of GPIHBP1 strengthen binding, effectively driving LPL to the surface of endothelial cells (Fig. 5c). Testing this concept will require comparing the affinity of LPL for HSPGs and GPIHBP1.

Figure 5.

Lipoprotein lipase– Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (LPL–GPIHBP1) interactions at the basolateral face of endothelial cells. Even in the absence of GPIHBP1, LPL is immobilized in the subendothelial spaces, probably bound to heparan sulfate proteoglycans (HSPGs) (a). We hypothesize that the acidic domain of GPIHBP1 (orange-coloured domain; b) is responsible for competing with HSPGs and wresting LPL away from HSPGs. Once the LPL is detached from HSPGs, LPL can interact fully with both binding domains of GPIHBP1 (the acidic and the Ly6 domains), resulting in high-affinity binding (c).

The situation at the luminal face of endothelial cells also remains unclear. It is known that the carboxyl-terminal domain of LPL is important for its ability to bind to triglyceride emulsion particles [23, 61]. It is also clear that an infusion of triglyceride emulsion particles releases LPL into the plasma. It would be interesting to determine whether triglyceride-rich lipoproteins, by binding to LPL's lipid-binding sequences, are capable of detaching some LPL from GPIHBP1 (Fig. 6a,b), or whether the LPL–GPIHBP1 interaction is simply disrupted by the fatty acid products of triglyceride hydrolysis (Fig. 6c). In addition, it will be important to determine whether protein regulators of lipolysis influence LPL–GPIHBP1 interactions. ANGPTL4 inactivates LPL by converting LPL homodimers to monomers [36], but it is not known whether ANGPTL4 plays a role in detaching LPL from GPIHBP1.

Figure 6.

Lipoprotein lipase– Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (LPL–GPIHBP1) interactions at the capillary lumen. (a) LPL is normally bound to GPIHBP1 on the cell surface, but LPL is released into the circulation following injection of a triglyceride emulsion (intralipid) [23, 61]. (b) Because LPL binds to triglyceride-rich particles [5], it is possible that triglyceride-rich lipoproteins may cause some LPL molecules to become detached from GPIHBP1. (C) It is also possible that the fatty acid products of lipolysis weaken LPL–GPIHBP1 interactions and cause LPL to become detached from GPIHBP1.

Cellular mechanisms of LPL transport across endothelial cells

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 is essential for shuttling LPL across endothelial cells, but the cellular mechanisms of this transport process are unclear. Do GPIHBP1–LPL complexes cluster in caveolar invaginations and move across cells in vesicles? Is caveolin-1 important for LPL transport? Is the transport process unidirectional (i.e. towards the capillary lumen), or do GPIHBP1 and LPL move across endothelial cells in both directions? If LPL and GPIHBP1 also move towards the basolateral face of cells, do they move in the same vesicles that carry the fatty acid products of lipolysis? Finally, it is unclear whether the transport process is regulated.

Mechanisms for the margination of TRLs along capillaries

The field needs a better understanding of why TRLs stop at the luminal face of capillaries (so that lipolysis can occur) rather than simply ‘flowing by’ in the bloodstream. Many studies have investigated leukocyte margination in blood vessels, but few have examined the margination of TRL particles. One possibility, proposed in several reviews [62, 63], is that interactions between apolipoproteins and endothelial cell HSPGs are crucial for the margination of TRLs. However, it is also possible that the LPL–GPIHBP1 complex is an important factor for TRL margination in capillaries, particularly because LPL binds avidly to TRLs. It will be important to define which molecular interactions cause TRLs to stop along the capillary wall—so that lipolysis can proceed.

Interactions between GPIHBP1 and LPL and apo-AV

Lipoprotein lipase binds to GPIHBP1 with great specificity, and some of the amino acids required for this interaction have been revealed, both for LPL and GPIHBP1 [16, 50, 51]. However, precisely how the two molecules interact remains unclear. Does the acidic domain of GPIHBP1 interact solely with the carboxyl-terminal heparin-binding domain of LPL? Does the Ly6 domain of GPIHBP1 interact directly with the carboxyl-terminal ~30 amino acids of LPL? In order to provide definitive answers to these questions, the structures of GPIHBP1, the carboxyl terminal region of LPL, and ultimately the LPL–GPIHBP1 complex will need to be determined.

The stoichiometry of LPL–GPIHBP1 interactions also needs to be resolved. LPL is secreted as a homodimer, and is thought to contain two binding sites for GPIHBP1. However, it is unclear whether LPL is bound and escorted across endothelial cells by two GPIHBP1 molecules, or only one. The binding of LPL by two GPIHBP1 molecules would presumably contribute to high-affinity interactions, but whether this occurs is unknown. It is conceivable that the binding of a single GPIHBP1 to one LPL monomer would change the conformation of the partner LPL monomer in such a way that it would lose its ability to bind a second GPIHBP1 molecule.

Does GPIHBP1 affect the efficiency of triglyceride hydrolysis?

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 serves as a binding site for LPL at the capillary lumen, but does it have additional functions? For example, could GPIHBP1 assist in lipolysis? Is GPIHBP1-bound LPL more efficient than unbound LPL in hydrolysing triglycerides? Does GPIHBP1 participate in the regulation of LPL activity? GPIHBP1 has been reported to protect LPL from inactivation by ANGPTL4 [64], but another group found that ANGPTL3 inactivates LPL in the presence of soluble GPIHBP1 [65]. These issues need to be explored in greater detail.

Which factors control GPIHBP1 expression in capillary endothelial cells?

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 is located exclusively in capillary endothelial cells and is not found in larger blood vessels. Indeed, when examining GPIHBP1 expression in heart or brown adipose tissue by immunofluorescence microscopy, it is clear that GPIHBP1 expression is silenced as soon as the capillary expands into the very smallest venule. Is the restricted expression of GPIHBP1 in capillaries governed by cis-acting regulatory elements within the GPIHBP1 gene? If so, these elements need to be defined. Also, it is unclear whether GPIHBP1 expression is regulated by metabolic cues from surrounding tissues (i.e. paracrine factors secreted by parenchymal cells).

Is LPL transported to the capillary lumen in all vertebrates?

Lipoprotein lipase is present in all vertebrate species, including fish, amphibians, birds, and mammals. However, to date, GPIHBP1 has only been identified in mammals. Why is GPIHBP1 uniquely important for mammals? Might this relate to the fact that mammals nurse their young? Perhaps GPIHBP1-mediated transport of LPL to the capillary lumen is essential for the production of lipid-rich maternal milk by the mammary gland. The nature of lipolysis in fish, amphibians and birds, which lack GPIHBP1, is also intriguing. Do the endothelial cells of these species have a different LPL transporter? Or is it possible that LPL in these other vertebrates simply stays in the interstitial spaces surrounding parenchymal cells? Rather than transporting LPL to the capillary lumen, perhaps these vertebrates transport lipoproteins across capillaries to the interstitial spaces where LPL resides. All these possibilities need to be investigated.

Conflict of interest statement

No conflicts of interest to declare.


This work was supported by National Institutes of Health grants R01 HL094732 (to APB), P01 HL090553 (to SGY), and R01 HL087228 (to SGY), and a postdoctoral fellowship award from the American Heart Association, Western States Affiliate (CNG).