Glycosylphosphatidylinositol-anchored HDL-binding Protein Research Articles

Acquired Chylomicronemia Due to Autoantibodies Against GPIHBP1

Lead Author's Financial Disclosures Nothing to disclose. Study Funding None. Background/Synopsis We report a case of recurrent triglyceride-induced pancreatitis that was found to be due to autoantibodies to glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) in the setting of biopsy-proven membranous lupus nephritis. Objective/Purpose This case report highlights a very rare cause of acquired chylomicronemia and discusses the treatment of GPIHBP1 autoantibody-induced hypertriglyceridemia. Methods A 12-year-old female with no medical history was admitted to the hospital for triglyceride induced pancreatitis. Her initial lipids consisted of: triglycerides 4070, total cholesterol 290, HDLc 9, and non-HDL-c 281 mg/dL. After inpatient treatment, she was started on fenofibrate and referred to lipid clinic. Despite fenofibrate 160 mg daily, icosapent ethyl 2 g twice a day, and a strict low-fat diet, the patient continued to experience triglyceride-induced pancreatitis. Her triglycerides fluctuated between 1200 - 2300 mg/dL. Genotyping was negative for familial chylomicronemia (deficiency/mutation in LPL, APOC2, APOA5, LFM-1, and GPIHBP1). Therefore, the patient was presumed to have genotype-negative familial chylomicronemia syndrome. Over the next 2 years, the patient had 15 hospital admissions for triglyceride-induced pancreatitis. At age 14 she was started on alirocumab 75 mg injections every 14 days, which decreased, but did not eliminate, the frequency of pancreatitis episodes. Her triglyceride levels subsequently ranged 650 - 991 mg/dL. During a hospital admission at age 15, the patient was found to have nephrotic syndrome with an ANA titer >1:2560. Renal biopsy confirmed membranous lupus nephritis. She was treated with pulse-dose steroids, mycophenolate mofetil, and hydroxychloroquine. Her lupus diagnosis prompted testing for GPIHBP1 autoantibodies,which were strongly positive. Results Two months after our patient's diagnosis of GPIHBP1 autoantibody induced hypertriglyceridemia, she continues to receive treatment with prednisone 40 mg daily, mycophenolate mofetil 1000 mg twice a day, and hydroxychloroquine 200 mg daily and has received two doses of rituximab-pvvr 1000 mg. Her triglycerides remain elevated 1400 - 2200 mg/dL. Conclusions This case highlights a recently described entity in the literature, “GPIHBP1 autoantibody syndrome,” wherein patients with unexplained chylomicronemia are found to have autoantibodies to GPIHBP1, causing hypertriglyceridemia resistant to traditional medical therapy. Of the 33 known patients worldwide with GPIHBP1 autoantibody syndrome, immunosuppression with rituximab and mycophenolate are reported to be effective therapies.Our patient is currently undergoing immunosuppression, but triglyceride levels have yet to decrease. Additional time and/or rituximab doses may be needed in order to maximally suppress autoantibody production and, in turn, reduce triglyceride levels. Nothing to disclose.

On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity

Angiopoietin-like (ANGPTL) 8 is a secreted inhibitor of LPL, a key enzyme in plasma triglyceride metabolism. It was previously reported that ANGPTL8 requires another member of the ANGPTL family, ANGPTL3, to act on LPL. ANGPTL3, much like ANGPTL4, is a physiologically relevant regulator of LPL activity, which causes irreversible inactivation of the enzyme. Here, we show that ANGPTL8 can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states. In contrast to the augmented inhibitory effect of the ANGPTL3/ANGPTL8 complex on LPL activity, the ANGPTL4/ANGPTL8 complex is less active compared with ANGPTL4 alone. In our experiments, all three members of the ANGPTL family use the same mechanism to inactivate LPL, which involves dissociation of active dimeric LPL to monomers. This inactivation can be counteracted by the presence of glycosylphosphatidylinositol-anchored HDL binding protein 1, the endothelial LPL transport protein previously known to protect LPL from spontaneous and ANGPTL4-catalyzed inactivation. Our data demonstrate that ANGPTL8 may function as an important metabolic switch, by forming complexes with ANGPTL3, or with ANGPTL4, in order to direct the flow of energy from triglycerides in blood according to the needs of the body.

Novel GPIHBP1-independent pathway for clearance of plasma TGs in Angptl4−/−Gpihbp1−/− mice

Mice lacking glycosylphosphatidylinositol-anchored HDL-binding protein 1 (GPIHBP1) are unable to traffic LPL to the vascular lumen. Thus, triglyceride (TG) clearance is severely blunted, and mice are extremely hypertriglyceridemic. Paradoxically, mice lacking both GPIHBP1 and the LPL regulator, angiopoietin-like 4 (ANGPTL4), are far less hypertriglyceridemic. We sought to determine the mechanism by which Angptl4-/-Gpihbp1-/- double-knockout mice clear plasma TGs. We confirmed that, on a normal chow diet, plasma TG levels were lower in Angptl4-/-Gpihbp1-/- mice than in Gpihbp1-/- mice; however, the difference disappeared with administration of a high-fat diet. Although LPL remained mislocalized in double-knockout mice, plasma TG clearance in brown adipose tissue (BAT) increased compared with Gpihbp1-/- mice. Whole lipoprotein uptake was observed in the BAT of both Gpihbp1-/- and Angptl4-/-Gpihbp1-/- mice, but BAT lipase activity was significantly higher in the double-knockout mice. We conclude that Angptl4-/-Gpihbp1-/- mice clear plasma TGs primarily through a slow and noncanonical pathway that includes the uptake of whole lipoprotein particles.

The role of plasma lipoprotein lipase, hepatic lipase and GPIHBP1 in the metabolism of remnant lipoproteins and small dense LDL in patients with coronary artery disease

BackgroundThe relationship between plasma lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), glycosylphosphatidylinositol anchored HDL binding protein1 (GPIHBP1) concentration and the metabolism of remnant lipoproteins (RLP) and small dense LDL (sdLDL) in patients with coronary artery disease (CAD) is not fully elucidated. MethodsOne hundred patients who underwent coronary angiography were enrolled. The plasma LPL, HTGL and GPIHBP1 concentrations were determined by ELISA. The time dependent changes in those lipases, lipids and lipoproteins were studied at a time-point just before, and 15min, 4h and 24h after heparin administration. ResultsThe LPL concentration exhibited a significant positive correlation with HDL-C, and inversely correlated with TG and RLP-C. The HTGL concentration was positively correlated with RLP-C and sdLDL-C. The HTGL ratio of the pre-heparin/post-heparin plasma concentration and sdLDL-C/LDL-C ratio were significantly greater in CAD patients than in non-CAD patients. GPIHBP1 was positively correlated with LPL and inversely correlated with RLP-C and sdLDL-C. ConclusionThe HTGL concentration was positively correlated with RLP-C and sdLDL-C, while LPL and GPIHBP1 were inversely correlated with RLP-C and sdLDL-C. These results suggest that elevated HTGL is associated with increased CAD risk, while elevated LPL is associated with a reduction of CAD risk.

Apolipoprotein C-III inhibits triglyceride hydrolysis by GPIHBP1-bound LPL

apoC-III is often assumed to retard the intravascular processing of triglyceride-rich lipoproteins (TRLs) by inhibiting LPL, but that view is based largely on studies of free LPL. We now recognize that intravascular LPL is neither free nor loosely bound, but instead is tightly bound to glycosylphosphatidylinositol-anchored HDL-binding protein 1 (GPIHBP1) on endothelial cells. Here, we revisited the effects of apoC-III on LPL, focusing on apoC-III's capacity to affect the activity of GPIHBP1-bound LPL. We found that TRLs from APOC3 transgenic mice bound normally to GPIHBP1-bound LPL on cultured cells in vitro and to heart capillaries in vivo. However, the triglycerides in apoC-III-enriched TRLs were hydrolyzed more slowly by free LPL, and the inhibitory effect of apoC-III on triglyceride lipolysis was exaggerated when LPL was bound to GPIHBP1 on the surface of agarose beads. Also, recombinant apoC-III reduced triglyceride hydrolysis by free LPL only modestly, but the inhibitory effect was greater when the LPL was bound to GPIHBP1. A mutant apoC-III associated with low plasma triglyceride levels (p.A23T) displayed a reduced capacity to inhibit free and GPIHBP1-bound LPL. Our results show that apoC-III potently inhibits triglyceride hydrolysis when LPL is bound to GPIHBP1.

Mutating a conserved cysteine in GPIHBP1 reduces amounts of GPIHBP1 in capillaries and abolishes LPL binding

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease-chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.

Genetic Predisposition of Human Plasma Triglyceride Concentrations

The issue of plasma triglyceride levels relative to the risk of development of cardiovascular disease, as well as overall mortality, has been actively discussed for many years. Like other cardiovascular disease risk factors, final plasma TG values have environmental influences (primarily dietary habits, physical activity, and smoking), and a genetic predisposition. Rare mutations (mainly in the lipoprotein lipase and apolipoprotein C2) along with common polymorphisms (within apolipoprotein A5, glucokinase regulatory protein, apolipoprotein B, apolipo-protein E, cAMP responsive element binding protein 3-like 3, glycosylphosphatidylinositol-anchored HDL-binding protein 1) play an important role in determining plasma TG levels.

High-resolution imaging of dietary lipids in cells and tissues by NanoSIMS analysis

Nanoscale secondary ion MS (NanoSIMS) imaging makes it possible to visualize stable isotope-labeled lipids in cells and tissues at 50 nm lateral resolution. Here we report the use of NanoSIMS imaging to visualize lipids in mouse cells and tissues. After administering stable isotope-labeled fatty acids to mice by gavage, NanoSIMS imaging allowed us to visualize neutral lipids in cytosolic lipid droplets in intestinal enterocytes, chylomicrons at the basolateral surface of enterocytes, and lipid droplets in cardiomyocytes and adipocytes. After an injection of stable isotope-enriched triglyceride-rich lipoproteins (TRLs), NanoSIMS imaging documented delivery of lipids to cytosolic lipid droplets in parenchymal cells. Using a combination of backscattered electron (BSE) and NanoSIMS imaging, it was possible to correlate the chemical data provided by NanoSIMS with high-resolution BSE images of cell morphology. This combined imaging approach allowed us to visualize stable isotope-enriched TRLs along the luminal face of heart capillaries and the lipids within heart capillary endothelial cells. We also observed examples of TRLs within the subendothelial spaces of heart capillaries. NanoSIMS imaging provided evidence of defective transport of lipids from the plasma LPs to adipocytes and cardiomyocytes in mice deficient in glycosylphosphatidylinositol-anchored HDL binding protein 1.

Postprandial inhibition of gastric ghrelin secretion by long-chain fatty acid through GPR120 in isolated gastric ghrelin cells and mice

Ghrelin is a gastric peptide hormone that controls appetite and energy homeostasis. Plasma ghrelin levels rise before a meal and fall quickly thereafter. Elucidation of the regulation of ghrelin secretion has been hampered by the difficulty of directly interrogating ghrelin cells diffusely scattered within the complex gastric mucosa. Therefore, we generated transgenic mice with ghrelin cell expression of green fluorescent protein (GFP) to enable characterization of ghrelin secretion in a pure population of isolated gastric ghrelin-expressing GFP (Ghr-GFP) cells. Using quantitative RT-PCR and immunofluorescence staining, we detected a high level of expression of the long-chain fatty acid (LCFA) receptor GPR120, while the other LCFA receptor, GPR40, was undetectable. In short-term-cultured pure Ghr-GFP cells, the LCFAs docosadienoic acid, linolenic acid, and palmitoleic acid significantly suppressed ghrelin secretion. The physiological mechanism of LCFA inhibition on ghrelin secretion was studied in mice. Serum ghrelin levels were transiently suppressed after gastric gavage of LCFA-rich lipid in mice with pylorus ligation, indicating that the ghrelin cell may directly sense increased gastric LCFA derived from ingested intraluminal lipids. Meal-induced increase in gastric mucosal LCFA was assessed by measuring the transcripts of markers for tissue uptake of LCFA, lipoprotein lipase (LPL), fatty acid translocase (CD36), glycosylphosphatidylinositol-anchored HDL-binding protein 1, and nuclear fatty acid receptor peroxisome proliferator-activated receptor-γ. Quantitative RT-PCR studies indicate significantly increased mRNA levels of lipoprotein lipase, glycosylphosphatidylinositol-anchored HDL-binding protein 1, and peroxisome proliferator-activated receptor-γ in postprandial gastric mucosa. These results suggest that meal-related increases in gastric mucosal LCFA interact with GPR120 on ghrelin cells to inhibit ghrelin secretion.

Triglycerides and Cardiovascular Disease

A long-standing association exists between elevated triglyceride levels and cardiovascular disease* (CVD).1,2 However, the extent to which triglycerides directly promote CVD or represent a biomarker of risk has been debated for 3 decades.3 To this end, 2 National Institutes of Health consensus conferences evaluated the evidentiary role of triglycerides in cardiovascular risk assessment and provided therapeutic recommendations for hypertriglyceridemic states.4,5 Since 1993, additional insights have been made vis-a-vis the atherogenicity of triglyceride-rich lipoproteins (TRLs; ie, chylomicrons and very low-density lipoproteins), genetic and metabolic regulators of triglyceride metabolism, and classification and treatment of hypertriglyceridemia. It is especially disconcerting that in the United States, mean triglyceride levels have risen since 1976, in concert with the growing epidemic of obesity, insulin resistance (IR), and type 2 diabetes mellitus (T2DM).6,7 In contrast, mean low-density lipoprotein cholesterol (LDL-C) levels have receded.7 Therefore, the purpose of this scientific statement is to update clinicians on the increasingly crucial role of triglycerides in the evaluation and management of CVD risk and highlight approaches aimed at minimizing the adverse public health–related consequences associated with hypertriglyceridemic states. This statement will complement recent American Heart Association scientific statements on childhood and adolescent obesity8 and dietary sugar intake9 by emphasizing effective lifestyle strategies designed to lower triglyceride levels and improve overall cardiometabolic health. It is not intended to serve as a specific guideline but will be of value to the Adult Treatment Panel IV (ATP IV) of the National Cholesterol Education Program, from which evidence-based guidelines will ensue. Topics to be addressed include epidemiology and CVD risk, ethnic and racial differences, metabolic determinants, genetic and family determinants, risk factor correlates, and effects related to nutrition, physical activity, and lipid medications. In the United States, the National Health and …

Unexpected Expression Pattern for Glycosylphosphatidylinositol-anchored HDL-binding Protein 1 (GPIHBP1) in Mouse Tissues Revealed by Positron Emission Tomography Scanning

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), a GPI-anchored endothelial cell protein, binds lipoprotein lipase (LPL) and transports it into the lumen of capillaries where it hydrolyzes triglycerides in lipoproteins. GPIHBP1 is assumed to be expressed mainly within the heart, skeletal muscle, and adipose tissue, the sites where most lipolysis occurs, but the tissue pattern of GPIHBP1 expression has never been evaluated systematically. Because GPIHBP1 is found on the luminal face of capillaries, we predicted that it would be possible to define GPIHBP1 expression patterns with radiolabeled GPIHBP1-specific antibodies and positron emission tomography (PET) scanning. In Gpihbp1(-/-) mice, GPIHBP1-specific antibodies were cleared slowly from the blood, and PET imaging showed retention of the antibodies in the blood pools (heart and great vessels). In Gpihbp1(+/+) mice, the antibodies were cleared extremely rapidly from the blood and, to our surprise, were taken up mainly by lung and liver. Immunofluorescence microscopy confirmed the presence of GPIHBP1 in the capillary endothelium of both lung and liver. In most tissues with high levels of Gpihbp1 expression, Lpl expression was also high, but the lung was an exception (very high Gpihbp1 expression and extremely low Lpl expression). Despite low Lpl transcript levels, however, LPL protein was readily detectable in the lung, suggesting that some of that LPL originates elsewhere and then is captured by GPIHBP1 in the lung. In support of this concept, lung LPL levels were significantly lower in Gpihbp1(-/-) mice than in Gpihbp1(+/+) mice. In addition, Lpl(-/-) mice expressing human LPL exclusively in muscle contained high levels of human LPL in the lung.

Triglyceride lipases and atherosclerosis

There are strong epidemiologic connections between plasma triglycerides and atherosclerosis. We will consider to what extent this goes back to derangements of the lipoprotein lipase (LPL) system. The roles of hepatic lipase and endothelial lipase will also be touched upon. Understanding of LPL action has taken major steps with the discovery of lipase maturation factor 1 as a specific endoplasmic reticulum chaperon needed for proper folding of the lipases, glycosylphosphatidylinositol-anchored HDL-binding protein 1 as an endothelial cell protein needed for transport and binding of LPL and some angiopoietin-like proteins that can modulate LPL activity. Studies of genetic variants continue to support the important roles of the lipases in lipoprotein metabolism and in atherosclerosis. There are several ways by which derangement of the lipases may contribute to atherogenesis. Lipase actions are major determinants of plasma lipoprotein patterns. LPL activity must be modulated in relation to the physiological situation (feeding, fasting, exercise, etc.). Fatty acids and monoglycerides generated must be efficiently removed so that they do not endanger the integrity of the endothelium, cause lipotoxic reactions or both. In addition, the lipases may cause binding and endocytosis of lipoprotein particles in the artery wall.

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

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.

Plasma apolipoprotein AV levels in mice are positively associated with plasma triglyceride levels

Apolipoprotein AV (apoAV) overexpression causes a decrease in plasma triglyceride (TG) levels, while deficiency of apoAV causes hypertriglyceridemia in both men and mice. However, contrary to what would be expected, plasma apoAV and TG levels in humans are positively correlated. To address this apparent paradox, we determined plasma apoAV levels in various mouse models with median TG levels ranging from 30 mg/dl in wild-type mice to 2089 mg/dl in glycosylphosphatidylinositol-anchored HDL binding protein 1-deficient mice. The data show that apoAV and TG levels are positively correlated in mice (r = +0.798, P < 0.001). In addition, we show that LPL gene transfer caused a simultaneous decrease in TG and apoAV in LPL-deficient mice. The combined data suggest that apoAV levels follow TG levels due to an intimate link between the apoAV molecule and TG-rich lipoproteins, comprising both secretion and removal of these lipoproteins. Taken together, the data suggest that higher plasma apoAV levels reflect an increased demand for plasma TG hydrolysis under normal physiological conditions.