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In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens - as it does in mammals - despite an apparent absence of GPIHBP1.

In mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), the LPL secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why isn't the freshly secreted LPL in wild-type mice captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries? We hypothesized that LPL-HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: 1) soluble GPIHBP1 in the cell culture medium; 2) GPIHBP1-coated agarose beads; and 3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained a Ly6 domain missense mutation (W109S), but was almost normal when GPIHBP1's acidic domain was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells.

LPL contains two principal domains: an amino-terminal catalytic domain (residues 1-297) and a carboxyl-terminal domain (residues 298-448) that is important for binding lipids and binding glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL's carboxyl terminus (residues ∼403-438) were crucial. Here, we tested the ability of LPL-specific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL's carboxyl terminus (residues ∼403-438) were not sufficient for 88B8 binding; upstream sequences (residues 298-400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPIHBP1. In conclusion, we identified an LPL mAb that binds to LPL's GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL's carboxyl-terminal domain.

LPL hydrolyzes triglycerides in triglyceride-rich lipoproteins along the capillaries of heart, skeletal muscle, and adipose tissue. The activity of LPL is repressed by angiopoietin-like 4 (ANGPTL4) but the underlying mechanisms have not been fully elucidated. Our objective was to study the cellular location and mechanism for LPL inhibition by ANGPTL4. We performed studies in transfected cells, ex vivo studies, and in vivo studies with Angptl4(-/-) mice. Cotransfection of CHO pgsA-745 cells with ANGPTL4 and LPL reduced intracellular LPL protein levels, suggesting that ANGPTL4 promotes LPL degradation. This conclusion was supported by studies of primary adipocytes and adipose tissue explants from wild-type and Angptl4(-/-) mice. Absence of ANGPTL4 resulted in accumulation of the mature-glycosylated form of LPL and increased secretion of LPL. Blocking endoplasmic reticulum (ER)-Golgi transport abolished differences in LPL abundance between wild-type and Angptl4(-/-) adipocytes, suggesting that ANGPTL4 acts upon LPL after LPL processing in the ER. Finally, physiological changes in adipose tissue ANGPTL4 expression during fasting and cold resulted in inverse changes in the amount of mature-glycosylated LPL in wild-type mice, but not Angptl4(-/-) mice. We conclude that ANGPTL4 promotes loss of intracellular LPL by stimulating LPL degradation after LPL processing in the ER.

GPIHBP1, a GPI-anchored protein in capillary endothelial cells, is crucial for the lipolytic processing of triglyceride-rich lipoproteins (TRLs). GPIHBP1 shuttles lipoprotein lipase (LPL) to its site of action in the capillary lumen and is essential for the margination of TRLs along capillaries - such that lipolytic processing can proceed. GPIHBP1 also reduces the unfolding of the LPL catalytic domain, thereby stabilizing LPL catalytic activity. Many different GPIHBP1 mutations have been identified in patients with severe hypertriglyceridemia (chylomicronemia), the majority of which interfere with folding of the protein and abolish its capacity to bind and transport LPL. The discovery of GPIHBP1 has substantially revised our understanding of intravascular triglyceride metabolism but has also raised many new questions for future research.

RationaleGPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1 missense mutations that interfere with LPL binding cause familial chylomicronemia.ObjectiveWe sought to understand mechanisms by which GPIHBP1 mutations prevent LPL binding and lead to chylomicronemia.Methods and resultsWe expressed mutant forms of GPIHBP1 in Chinese hamster ovary cells, rat and human endothelial cells, and Drosophila S2 cells. In each expression system, mutation of cysteines in GPIHBP1's Ly6 domain (including mutants identified in patients with chylomicronemia) led to the formation of disulfide-linked dimers and multimers. GPIHBP1 dimerization/multimerization was not unique to cysteine mutations; mutations in other amino acid residues, including several associated with chylomicronemia, also led to protein dimerization/multimerization. The loss of GPIHBP1 monomers is relevant to the pathogenesis of chylomicronemia because only GPIHBP1 monomers-and not dimers or multimers-are capable of binding LPL. One GPIHBP1 mutant, GPIHBP1-W109S, had distinctive properties. GPIHBP1-W109S lacked the ability to bind LPL but had a reduced propensity for forming dimers or multimers, suggesting that W109 might play a more direct role in binding LPL. In support of that idea, replacing W109 with any of 8 other amino acids abolished LPL binding-and often did so without promoting the formation of dimers and multimers.ConclusionsMany amino acid substitutions in GPIHBP1's Ly6 domain that abolish LPL binding lead to protein dimerization/multimerization. Dimerization/multimerization is relevant to disease pathogenesis, given that only GPIHBP1 monomers are capable of binding LPL.

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5-25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL-GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.

GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia.

The S447X polymorphism in lipoprotein lipase (LPL), which shortens LPL by two amino acids, is associated with low plasma triglyceride levels and reduced risk for coronary heart disease. S447X carriers have higher LPL levels in the pre- and post-heparin plasma, raising the possibility that the S447X polymorphism leads to higher LPL levels within capillaries. One potential explanation for increased amounts of LPL in capillaries would be more avid binding of S447X-LPL to GPIHBP1 (the protein that binds LPL dimers and shuttles them to the capillary lumen). This explanation seems plausible because sequences within the carboxyl terminus of LPL are known to mediate LPL binding to GPIHBP1. To assess the impact of the S447X polymorphism on LPL binding to GPIHBP1, we compared the ability of internally tagged versions of wild-type LPL (WT-LPL) and S447X-LPL to bind to GPIHBP1 in both cell-based and cell-free binding assays. In the cell-based assay, we compared the binding of WT-LPL and S447X-LPL to GPIHBP1 on the surface of cultured cells. This assay revealed no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1. In the cell-free assay, we compared the binding of internally tagged WT-LPL and S447X-LPL to soluble GPIHBP1 immobilized on agarose beads. Again, no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1 were observed. We conclude that increased binding of S447X-LPL to GPIHBP1 is unlikely to be the explanation for more efficient lipolysis and lower plasma triglyceride levels in S447X carriers.

Triglyceride-rich lipoproteins (TRLs) undergo lipolysis by lipoprotein lipase (LPL), an enzyme that is transported to the capillary lumen by an endothelial cell protein, GPIHBP1. For LPL-mediated lipolysis to occur, TRLs must bind to the lumen of capillaries. This process is often assumed to involve heparan sulfate proteoglycans (HSPGs), but we suspected that TRL margination might instead require GPIHBP1. Indeed, TRLs marginate along the heart capillaries of wild-type but not Gpihbp1⁻/⁻ mice, as judged by fluorescence microscopy, quantitative assays with infrared-dye-labeled lipoproteins, and EM tomography. Both cell-culture and in vivo studies showed that TRL margination depends on LPL bound to GPIHBP1. Notably, the expression of LPL by endothelial cells in Gpihbp1⁻/⁻ mice did not restore defective TRL margination, implying that the binding of LPL to HSPGs is ineffective in promoting TRL margination. Our studies show that GPIHBP1-bound LPL is the main determinant of TRL margination.

Abstract Background Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins and enables uptake of lipolysis products for energy production or storage in tissues. Our aim was to study the localization of LPL and its endothelial anchoring protein glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) in mouse pancreas, and effects of diet and leptin deficiency on their expression patterns. For this, immunofluorescence microscopy was used on pancreatic tissue from C57BL/6 mouse embryos (E18), adult mice on normal or high-fat diet, and adult ob/ob-mice treated or not with leptin. The distribution of LPL and GPIHBP1 was compared to insulin, glucagon and CD31. Heparin injections were used to discriminate between intracellular and extracellular LPL. Results In the exocrine pancreas LPL was found in capillaries, and was mostly co-localized with GPIHBP1. LPL was releasable by heparin, indicating localization on cell surfaces. Within the islets, most of the LPL was associated with beta cells and could not be released by heparin, indicating that the enzyme remained mostly within cells. Staining for LPL was found also in the glucagon-producing alpha cells, both in embryos (E18) and in adult mice. Only small amounts of LPL were found together with GPIHBP1 within the capillaries of islets. Neither a high fat diet nor fasting/re-feeding markedly altered the distribution pattern of LPL or GPIHBP1 in mouse pancreas. Islets from ob/ob mice appeared completely deficient of LPL in the beta cells, while LPL-staining was normal in alpha cells and in the exocrine pancreas. Leptin treatment of ob/ob mice for 12 days reversed this pattern, so that most of the islets expressed LPL in beta cells. Conclusions We conclude that both LPL and GPIHBP1 are present in mouse pancreas, and that LPL expression in beta cells is dependent on leptin.