GPIHBP1, an endothelial cell (EC) protein, captures lipoprotein lipase (LPL) within the interstitial spaces (where it is secreted by myocytes and adipocytes) and transports it across ECs to its site of action in the capillary lumen. GPIHBP1's 3-fingered LU domain is required for LPL binding, but the function of its acidic domain (AD) has remained unclear. We created mutant mice lacking the AD and found severe hypertriglyceridemia. As expected, the mutant GPIHBP1 retained the capacity to bind LPL. Unexpectedly, however, most of the GPIHBP1 and LPL in the mutant mice was located on the abluminal surface of ECs (explaining the hypertriglyceridemia). The GPIHBP1-bound LPL was trapped on the abluminal surface of ECs by electrostatic interactions between the large basic patch on the surface of LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the surface of ECs. GPIHBP1 trafficking across ECs in the mutant mice was normalized by disrupting LPL-HSPG electrostatic interactions with either heparin or an AD peptide. Thus, GPIHBP1's AD plays a crucial function in plasma triglyceride metabolism; it sheathes LPL's basic patch on the abluminal surface of ECs, thereby preventing LPL-HSPG interactions and freeing GPIHBP1-LPL complexes to move across ECs to the capillary lumen.
We recently showed that NOTUM, a liver-secreted Wnt inhibitor, can acutely promote browning of white adipose. We now report studies of chronic overexpression of NOTUM in liver indicating that it protects against diet-induced obesity and improves glucose homeostasis in mice. Adeno-associated virus (AAV) vectors were used to overexpress GFP or mouse Notum in the livers of male C57BL/6J mice and the mice were fed an obesifying diet. After 14 weeks of high fat, high sucrose diet feeding, the AAV-Notum mice exhibited decreased obesity and improved glucose tolerance compared to the AAV-GFP mice. Gene expression and immunoblotting analysis of the inguinal fat and brown fat revealed increased expression of beige/brown adipocyte markers in the AAV-Notum group, suggesting enhanced thermogenic capacity by NOTUM. A beta3 adrenergic receptor agonist-stimulated lipolysis test suggested increased lipolysis capacity by NOTUM. The levels of collagen and C-C motif chemokine ligand 2 (CCL2) in the epididymal white adipose tissue of the AAV-Notum mice were significantly reduced, suggesting decreased fibrosis and inflammation, respectively. RNA sequencing analysis of inguinal white adipose of 4-week chow diet-fed mice revealed a highly significant enrichment of extracellular matrix (ECM) functional cluster among the down-regulated genes in the AAV-Notum group, suggesting a potential mechanism contributing to improved glucose homeostasis. Our in vitro studies demonstrated that recombinant human NOTUM protein blocked the inhibitory effects of WNT3A on brown adipocyte differentiation. Furthermore, NOTUM attenuated WNT3A's effects on upregulation of TGF-beta signaling and its downstream targets. Overall, our data suggest that NOTUM modulates adipose tissue function by promoting thermogenic capacity and inhibiting fibrosis through inhibition of Wnt signaling.
PHARC (polyneuropathy, hearing loss, cerebellar ataxia, retinitis pigmentosa, and cataract) is a human neurological disorder caused by deleterious mutations in the ABHD12 gene, which encodes an integral membrane lyso-phosphatidylserine (lyso-PS) lipase. Pharmacological or genetic disruption of ABHD12 leads to higher levels of lyso-PS lipids in human cells and the central nervous system (CNS) of mice. ABHD12 loss also causes rapid rewiring of PS content, resulting in selective increases in the level of arachidonoyl (C20:4) PS and decreases in the levels of other PS species. The biochemical basis for ABHD12-dependent PS remodeling and its pathophysiological significance remain unknown. Here, we show that genetic deletion of the lysophospholipid acyltransferase LPCAT3 blocks accumulation of brain C20:4 PS in mice lacking ABHD12 and concurrently produces hyper-increases in the level of lyso-PS in these animals. These lipid changes correlate with exacerbated auditory dysfunction and brain microgliosis in mice lacking both ABHD12 and LPCAT3. Taken together, our findings reveal that ABHD12 and LPCAT3 coordinately regulate lyso-PS and C20:4 PS content in the CNS and point to lyso-PS lipids as the likely bioactive metabolites contributing to PHARC-related neuropathologies.
BACKGROUND: A protein that is expressed on capillary endothelial cells, called GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1), binds lipoprotein lipase and shuttles it to its site of action in the capillary lumen. A deficiency in GPIHBP1 prevents lipoprotein lipase from reaching the capillary lumen. Patients with GPIHBP1 deficiency have low plasma levels of lipoprotein lipase, impaired intravascular hydrolysis of triglycerides, and severe hypertriglyceridemia (chylomicronemia). During the characterization of a monoclonal antibody-based immunoassay for GPIHBP1, we encountered two plasma samples (both from patients with chylomicronemia) that contained an interfering substance that made it impossible to measure GPIHBP1. That finding raised the possibility that those samples might contain GPIHBP1 autoantibodies. METHODS: Using a combination of immunoassays, Western blot analyses, and immunocytochemical studies, we tested the two plasma samples (as well as samples from other patients with chylomicronemia) for the presence of GPIHBP1 autoantibodies. We also tested the ability of GPIHBP1 autoantibodies to block the binding of lipoprotein lipase to GPIHBP1. RESULTS: We identified GPIHBP1 autoantibodies in six patients with chylomicronemia and found that these autoantibodies blocked the binding of lipoprotein lipase to GPIHBP1. As in patients with GPIHBP1 deficiency, those with GPIHBP1 autoantibodies had low plasma levels of lipoprotein lipase. Three of the six patients had systemic lupus erythematosus. One of these patients who had GPIHBP1 autoantibodies delivered a baby with plasma containing maternal GPIHBP1 autoantibodies; the infant had severe but transient chylomicronemia. Two of the patients with chylomicronemia and GPIHBP1 autoantibodies had a response to treatment with immunosuppressive agents. CONCLUSIONS: In six patients with chylomicronemia, GPIHBP1 autoantibodies blocked the ability of GPIHBP1 to bind and transport lipoprotein lipase, thereby interfering with lipoprotein lipase-mediated processing of triglyceride-rich lipoproteins and causing severe hypertriglyceridemia. (Funded by the National Heart, Lung, and Blood Institute and the Leducq Foundation.).
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.
OBJECTIVE: Gpihbp1-deficient (Gpihbp1-/-) mice lack the ability to transport lipoprotein lipase to the capillary lumen, resulting in mislocalization of lipoprotein lipase within tissues, defective lipolysis of triglyceride-rich lipoproteins, and chylomicronemia. We asked whether GPIHBP1 deficiency and mislocalization of catalytically active lipoprotein lipase would alter the composition of triglycerides in adipose tissue or perturb the expression of lipid biosynthetic genes. We also asked whether perturbations in adipose tissue composition and gene expression, if they occur, would be accompanied by reciprocal metabolic changes in the liver. METHODS AND RESULTS: The chylomicronemia in Gpihbp1-/- mice was associated with reduced levels of essential fatty acids in adipose tissue triglycerides and increased expression of lipid biosynthetic genes. The liver exhibited the opposite changes: increased levels of essential fatty acids in triglycerides and reduced expression of lipid biosynthetic genes. CONCLUSIONS: Defective lipolysis in Gpihbp1-/- mice causes reciprocal metabolic perturbations in adipose tissue and liver. In adipose tissue, the essential fatty acid content of triglycerides is reduced and lipid biosynthetic gene expression is increased, whereas the opposite changes occur in the liver.
Interest in lipolysis and the metabolism of triglyceride-rich lipoproteins was recently reignited by the discovery of severe hypertriglyceridemia (chylomicronemia) in glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1)-deficient mice. GPIHBP1 is expressed exclusively in capillary endothelial cells and binds lipoprotein lipase (LPL) avidly. These findings prompted speculation that GPIHBP1 serves as a binding site for LPL in the capillary lumen, creating "a platform for lipolysis." More recent studies have identified a second and more intriguing role for GPIHBP1-picking up LPL in the subendothelial spaces and transporting it across endothelial cells to the capillary lumen. Here, we review the studies that revealed that GPIHBP1 is the LPL transporter and discuss which amino acid sequences are required for GPIHBP1-LPL interactions. We also discuss the human genetics of LPL transport, focusing on cases of chylomicronemia caused by GPIHBP1 mutations that abolish GPIHBP1's ability to bind LPL, and LPL mutations that prevent LPL binding to GPIHBP1.
OBJECTIVE: To determine whether plasma triglyceride levels in adult Glycosylphosphatidylinositol HDL-binding protein 1 (GPIHBP1)-deficient (Gpihbp1(-/-)) mice would be sensitive to cholesterol intake. METHODS AND RESULTS: Gpihbp1(-/-) mice were fed a Western diet containing 0.15% cholesterol. After 4 to 8 weeks, their plasma triglyceride levels were 113 to 135 mmol/L. When 0.005% ezetimibe was added to the diet to block cholesterol absorption, Lpl expression in the liver was reduced significantly, and the plasma triglyceride levels were significantly higher (>170 mmol/L). We also assessed plasma triglyceride levels in Gpihbp1(-/-) mice fed Western diets containing either high (1.3%) or low (0.05%) amounts of cholesterol. The high-cholesterol diet significantly increased Lpl expression in the liver and lowered plasma triglyceride levels. CONCLUSIONS: Treatment of Gpihbp1(-/-) mice with ezetimibe lowers Lpl expression in the liver and increases plasma triglyceride levels. A high-cholesterol diet had the opposite effects. Thus, cholesterol intake modulates plasma triglyceride levels in Gpihbp1(-/-) mice.
Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a protein in the lymphocyte antigen 6 (Ly-6) family, plays a key role in the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 binds lipoprotein lipase and chylomicrons and is expressed along the luminal surface of microvascular endothelial cells. Lipolysis is known to be regulated by metabolic factors and is controlled at multiple levels, including the number of LPL binding sites on capillaries. Here, we tested the possibility that GPIHBP1 expression could be regulated by dietary perturbations and by peroxisome proliferator-activated receptors (PPARs). Gpihbp1 transcript levels in the heart and in brown and white adipose tissue increased with fasting and returned toward baseline after refeeding. A PPARgamma agonist increased Gpihbp1 expression in adipose tissue, heart, and skeletal muscle, whereas PPARalpha and PPARdelta agonists had no effect. Gpihbp1 was expressed in endothelial cells of embryoid bodies generated from mouse embryonic stem cells, and Gpihbp1 expression in embryoid bodies was up-regulated by a PPARgamma agonist. Sequences upstream from exon 1 of Gpihbp1 contain a strong PPAR binding site, and that site exhibited activity in a luciferase reporter assay. Gpihbp1 transcript levels in brown and white adipose tissue were lower in endothelial cell PPARgamma knockout mice than in littermate control mice, suggesting that PPARgamma regulates Gpihbp1 expression in vivo. We conclude that GPIHBP1 is regulated by dietary factors and by PPARgamma.
