Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.     

ANGPTL8 requires ANGPTL3 to inhibit lipoprotein lipase and plasma triglyceride clearance

Angiopoietin-like (ANGPTL)3 and ANGPTL8 are secreted proteins and inhibitors of LPL-mediated plasma triglyceride (TG) clearance. It is unclear how these two ANGPTL proteins interact to regulate LPL activity. ANGPTL3 inhibits LPL activity and increases serum TG independent of ANGPTL8. These effects are reversed with an ANGPTL3 blocking antibody. Here, we show that ANGPTL8, although it possesses a functional inhibitory motif, is inactive by itself and requires ANGPTL3 expression to inhibit LPL and increase plasma TG. Using a mutated form of ANGPTL3 that lacks LPL inhibitory activity, we demonstrate that ANGPTL3 activity is not required for its ability to activate ANGPTL8. Moreover, coexpression of ANGPTL3 and ANGPTL8 leads to a far more efficacious increase in TG in mice than ANGPTL3 alone, suggesting the major inhibitory activity of this complex derives from ANGPTL8. An antibody to the C terminus of ANGPTL8 reversed LPL inhibition by ANGPTL8 in the presence of ANGPTL3. The antibody did not disrupt the ANGPTL8:ANGPTL3 complex, but came in close proximity to the LPL inhibitory motif in the N terminus of ANGPTL8. Collectively, these data show that ANGPTL8 has a functional LPL inhibitory motif, but only inhibits LPL and increases plasma TG levels in mice in the presence of ANGPTL3.     

Angiopoietin-like 4 Modifies the Interactions between Lipoprotein Lipase and Its Endothelial Cell Transporter GPIHBP1

The release of fatty acids from plasma triglycerides for tissue uptake is critically dependent on the enzyme lipoprotein lipase (LPL). Hydrolysis of plasma triglycerides by LPL can be disrupted by the protein angiopoietin-like 4 (ANGPTL4), and ANGPTL4 has been shown to inactivate LPL in vitro. However, in vivo LPL is often complexed to glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) on the surface of capillary endothelial cells. GPIHBP1 is responsible for trafficking LPL across capillary endothelial cells and anchors LPL to the capillary wall during lipolysis. How ANGPTL4 interacts with LPL in this context is not known. In this study, we investigated the interactions of ANGPTL4 with LPL-GPIHBP1 complexes on the surface of endothelial cells. We show that ANGPTL4 was capable of binding and inactivating LPL complexed to GPIHBP1 on the surface of endothelial cells. Once inactivated, LPL dissociated from GPIHBP1. We also show that ANGPTL4-inactivated LPL was incapable of binding GPIHBP1. ANGPTL4 was capable of binding, but not inactivating, LPL at 4 °C, suggesting that binding alone was not sufficient for ANGPTL4''s inhibitory activity. We observed that although the N-terminal coiled-coil domain of ANGPTL4 by itself and full-length ANGPTL4 both bound with similar affinities to LPL, the N-terminal fragment was more potent in inactivating both free and GPIHBP1-bound LPL. These results led us to conclude that ANGPTL4 can both bind and inactivate LPL complexed to GPIHBP1 and that inactivation of LPL by ANGPTL4 greatly reduces the affinity of LPL for GPIHBP1.     

Genetic Variation in ANGPTL4 Provides Insights into Protein Processing and Function

Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that modulates the disposition of circulating triglycerides (TG) by inhibiting lipoprotein lipase (LPL). Here we examine the steps involved in the synthesis and post-translational processing of ANGPTL4, and the effects of a naturally occurring sequence variant (E40K) that is associated with lower plasma TG levels in humans. Expression of the wild-type and mutant proteins in HEK-293A cells indicated that ANGPTL4 formed dimers and tetramers in cells prior to secretion and cleavage of the protein. After cleavage at a canonical proprotein convertase cleavage site (¹⁶¹RRKR¹⁶⁴), the oligomeric structure of the N-terminal domain was retained whereas the C-terminal fibrinogen-like domain dissociated into monomers. Inhibition of cleavage did not interfere with oligomerization of ANGPTL4 or with its ability to inhibit LPL, whereas mutations that prevented oligomerization severely compromised the capacity of the protein to inhibit LPL. ANGPTL4 containing the E40K substitution was synthesized and processed normally, but no monomers or oligomers of the N-terminal fragments accumulated in the medium; medium from these cells failed to inhibit LPL activity. Parallel experiments performed in mice recapitulated these results. Our findings indicate that oligomerization, but not cleavage, of ANGPTL4 is required for LPL inhibition, and that the E40K substitution destabilizes the protein after secretion, preventing the extracellular accumulation of oligomers and abolishing the ability of the protein to inhibit LPL activity.Angiopoietin-like protein 4 (ANGPTL4)⁴ is a 50-kDa protein that is synthesized and secreted from several metabolically active tissues and has been implicated in the trafficking of circulating TG (1, 2). Triglycerides, either acquired from the diet or synthesized endogenously, circulate in blood as constituents of chylomicrons and very low density lipoproteins (VLDL). As these lipoproteins circulate in tissues they encounter lipoprotein lipase (LPL) at the vascular endothelial surfaces. LPL hydrolyzes the TG, producing free fatty acids that are taken up by the surrounding tissues. ANGPTL4 inhibits the activity of LPL, thereby limiting the uptake of TG-derived fatty acids by the underlying cells (3, 4). Overexpression of ANGPTL4 in mice causes severe hypertriglyceridemia, whereas mice lacking ANGPTL4 have increased LPL activity and low plasma levels of TG (5, 6). In mice, ANGPTL4 is predominantly expressed in adipose tissue and is strongly induced by fasting (2). Accordingly it has been proposed that ANGPTL4 inhibits LPL activity in adipose tissue to reroute fatty acids away from fat to muscle and other tissues when food intake is low (3, 4).ANGPTL4 belongs to a family of seven structurally similar secreted proteins (ANGPTL1-ANGPTL7) that contain a signal sequence followed by an α-helical region predicted to form a coiled-coil, and a globular fibrinogen-like domain at the C terminus (1). Gel filtration studies of recombinant ANGPTL4 indicate that the protein assembles into oligomers that are stabilized by disulfide bonds (7). Substitution of two highly conserved cysteine residues at positions 76 and 80 in the α-helical domain prevents oligomerization of ANGPTL4 and impairs the ability of the recombinant protein to increase plasma TG levels when overexpressed in the livers of rats (7).Upon secretion into the circulation, ANGPTL4 is cleaved into an N-terminal domain and a C-terminal fibrinogen-like domain (8). The N-terminal peptide circulates as an oligomer, and the fibrinogen-like domain circulates as a monomer (8). The N-terminal helical region of ANGPTL4 is necessary and sufficient for inhibition of LPL (9). A peptide corresponding to amino acids 1-187 of the protein binds LPL with high affinity and converts the enzyme from catalytically active dimers to inactive monomers, thereby inhibiting LPL activity (10). After disrupting the LPL dimer, ANGPTL4 is released. The LPL monomers remain folded and stable but fail to re-form active dimers. These data suggest that the N-terminal domain of ANGPTL4 interacts directly but transiently with LPL, triggering a stable conformational switch in LPL that irreversibly inactivates the enzyme.Recently, we used a population-based resequencing strategy to examine the metabolic role of ANGPTL4 in humans (11). Resequencing the coding region of ANGPTL4 in a large (n = 3,501), multiethnic sample revealed multiple rare sequence variations that alter an amino acid in the protein and are associated with low plasma TG levels. In addition, we identified a more common variant (E40K), that was present in ∼3% of European-Americans and was associated with significantly lower plasma levels of TG and low density lipoprotein-cholesterol (LDL-C), and higher levels of high density lipoprotein (HDL)-C in two large epidemiological studies (11). These association studies confirmed that ANGPTL4 is involved in TG metabolism in humans, and also revealed additional roles in humans in the metabolism of HDL and LDL, which were not apparent from studies in genetically modified mice.Here we examined the synthesis, secretion, and processing of ANGPTL4 and determine the mechanism by which substitution of a basic (lysine) for an acidic (glutamate) residue at residue 40 affects the function of the protein.     

Proprotein convertases [corrected] are responsible for proteolysis and inactivation of endothelial lipase

Plasma lipoprotein metabolism is tightly regulated by several members of the triglyceride lipase family, including endothelial lipase (EL) and lipoprotein lipase (LPL). Our previous work suggested that EL is proteolytically processed. In this report, we have used a combination of epitope tagging, mutagenesis, and N-terminal sequencing to determine the precise location of the cleavage site within EL. The cleavage occurs immediately after the sequence RNKR, a known recognition sequence for the proprotein convertase (PC) family. We demonstrate that some PCs, but not all, can proteolytically cleave EL at this site and thereby directly regulate EL enzymatic activity through modulating EL cleavage. Furthermore, specific knockdown of individual PCs proves that PCs are the proteases that cleave EL in human endothelial cells. Interestingly, a homologous site in LPL is also cleaved by PCs. This action is unusual for PCs, which are traditionally known as activators of pro-proteins, and highlights a potential role of PCs in lipid metabolism through their proteolytic processing of lipases.     

Angiopoietin-like Protein 4 Inhibition of Lipoprotein Lipase: EVIDENCE FOR REVERSIBLE COMPLEX FORMATION*

Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator.     

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.     

Hepatic proprotein convertases modulate HDL metabolism

The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.     

The acute phase response stimulates the expression of angiopoietin like protein 4

The acute phase response is characterized by elevations in serum triglyceride levels due to both an increase in hepatic VLDL production and a delay in the clearance of triglyceride rich lipoproteins secondary to a decrease in lipoprotein lipase (LPL) activity. Recently there has been a marked increase in our understanding of factors that regulate LPL activity. GPIHBP1 facilitates the interaction of LPL and lipoproteins thereby allowing lipolysis to occur. Angiopoietin like proteins (ANGPTL) 3 and 4 inhibit LPL activity. In the present study, treatment of mice with LPS, an activator of TLR4 and a model of Gram-negative infections, did not alter the expression of GPIHBP1 in heart or adipose tissue. However, LPS decreased the expression of ANGPTL3 in liver and increased the expression of ANGPTL4 in heart, muscle, and adipose tissue. Serum ANGPTL4 protein levels were markedly increased at 8 and 16 h following LPS treatment. Administration of zymosan, an activator of TLR2 and a model of fungal infections, also increased serum ANGPTL4 protein and mRNA levels in liver, heart, muscle, and adipose tissue. Finally, treatment of 3T3-L1 adipocytes with LPS or cytokines (TNF alpha, IL-1 beta, and interferon gamma) stimulated ANGPTL4 expression. These studies demonstrate that ANGPTL4 is a positive acute phase protein and the increase in ANGPTL4 could contribute to the hypertriglyceridemia that characteristically occurs during the acute phase response by inhibiting LPL activity.     

Regulation of triglyceride metabolism by Angiopoietin-like proteins

Plasma triglyceride concentrations are determined by the balance between production of the triglyceride-rich lipoproteins VLDL and chylomicrons in liver and intestine, and their lipoprotein lipase-mediated clearance in peripheral tissues. In the last decade, the group of Angiopoietin-like proteins has emerged as important regulators of circulating triglyceride (TG) levels. Specifically, ANGPTL3 and ANGPTL4 impair TG clearance by inhibiting lipoprotein lipase (LPL). Whereas ANGPTL4 irreversibly inactivates LPL by promoting conversion of active LPL dimers into inactive monomers, ANGPTL3 reversibly inhibits LPL activity. Studies using transgenic or knockout mice have clearly demonstrated the stimulatory effect of Angptl3 and Angptl4 on plasma TG, which is further supported by human genetic data including genome wide association studies. Whereas ANGPTL3 is mainly active in the fed state, ANGPTL4 is elevated by fasting and mediates fasting-induced changes in plasma TG and free fatty acid metabolism. Both proteins undergo oligomerization and are subject to proteolytic cleavage to generate N- and C-terminal fragments with highly divergent biological activities. Expression of ANGPTL3 is exclusive to liver and governed by the liver X receptor (LXR). In contrast, ANGPTL4 is expressed ubiquitously and under sensitive control of the Peroxisome proliferator-activated receptor (PPAR) family and fatty acids. Induction of ANGPTL4 gene expression by fatty acids and via PPARs is part of a feedback mechanism aimed at protecting cells against lipotoxicity. So far there is very little evidence that other ANGPTLs directly impact plasma lipoprotein metabolism. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Calorimetric approach for comparison of Angiopoietin-like protein 4 with other pancreatic lipase inhibitors

Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP – ε-polylysine (EPL), (−)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system.     

Processing and sorting of the prohormone convertase 2 propeptide

The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion.     

Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln²⁹–His⁵³, which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu³²–His⁵⁵. We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3^-/- mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.Lipoprotein lipase (LPL)⁵ plays a pivotal role in lipid metabolism by catalyzing the hydrolysis of plasma triglycerides (TGs). LPL is likely to be regulated by mechanisms that depend on nutritional status and on the tissue in which it is expressed (1–3). Two secreted proteins, angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), play important roles in the regulation of LPL activity (4, 5). ANGPTL3 and ANGPTL4 consist of a signal peptide, an N-terminal segment containing coiled-coil domains, and a C-terminal fibrinogen-like domain. The N-terminal segment as well as full-length ANGPTL3 and ANGPTL4 have been shown to inhibit LPL activity, and deletion of the N-terminal segment of ANGPTL3 and ANGPTL4 resulted in total loss of LPL-inhibiting activity (6, 7). These observations clearly indicate that the N-terminal region of ANGPTL4 contains the functional domain that inhibits LPL and affects plasma lipid levels. The coiled-coil domains have been proposed to be responsible for oligomerization (8); however, it is not known whether the coiled-coil domains directly mediate the inhibition of LPL activity.To define the physiological role of ANGPTL4 more clearly, we characterized the pharmacological consequences of ANGPTL4 inhibition in mice treated with the ANGPTL4-neutralizing monoclonal antibody (mAb) 14D12 (9). Injection of mAb 14D12 significantly lowered fasting TG levels in C57BL/6J mice relative to levels in C57BL/6J mice treated with an isotype-matched anti-KLH control (KLH) mAb (9). These reduced TG values were similar to decreases in fasting plasma TG levels measured in Angptl4 knock-out (-/-) mice. This study demonstrated that mAb 14D12 is a potent ANGPTL4-neutralizing antibody that is able to inhibit systemic ANGPTL4 activity and thereby recapitulate the reduced lipid phenotype found in Angptl4^-/- mice. The readily apparent pharmacological effect of mAb 14D12 prompted new questions about the epitope recognized by mAb 14D12 and how this antibody-antigen binding event affected ANGPTL4 function as an LPL inhibitor.Although ANGPTL4 is able to interact directly with LPL (10), it is not clear which amino acids within ANGPTL4 mediate this interaction. Here we show that amino acids Gln²⁹–His⁵³ of mANGPTL4 contain the epitope for mAb 14D12. This region, hereby designated specific epitope 1 (SE1), also defines a domain that mediates the interaction between ANGPTL4 and LPL and the subsequent inactivation of LPL. With this information we present evidence that ANGPTL3 also contains an SE1 region, and with antibodies specifically reactive with ANGPTL3 SE1 we examine whether the ANGPTL3 SE1 region is involved in LPL binding and inhibition. We also determined whether treatment of C57BL/6 mice with an anti-ANGPTL3 SE1 mAb can recapitulate the phenotype of lower serum TG and cholesterol levels found in Angptl3^-/- mice. Finally we tested the therapeutic potential of an anti-ANGPTL3 SE1 mAb for treatment of hyperlipidemia in apolipoprotein E^-/- (ApoE^-/-) or low density lipoprotein receptor^-/- (LDLr^-/-) mice.     

Angiopoietin like protein 4 expression is decreased in activated macrophages

Angiopoietin like protein 4 (ANGPTL4) inhibits lipoprotein lipase (LPL) activity. Previous studies have shown that Toll-like Receptor (TLR) activation increases serum levels of ANGPTL4 and expression of ANGPTL4 in liver, heart, muscle, and adipose tissue in mice. ANGPTL4 is expressed in macrophages and is induced by inflammatory saturated fatty acids. The absence of ANGPTL4 leads to the increased uptake of pro-inflammatory saturated fatty acids by macrophages in the mesentery lymph nodes due to the failure of ANGPTL4 to inhibit LPL activity, resulting in peritonitis, intestinal fibrosis, weight loss, and death. Here we determined the effect of TLR activation on the expression of macrophage ANGPTL4. LPS treatment resulted in a 70% decrease in ANGPTL4 expression in mouse spleen, a tissue enriched in macrophages. In mouse peritoneal macrophages, LPS treatment also markedly decreased ANGPTL4 expression. In RAW cells, a macrophage cell line, LPS, zymosan, poly I:C, and imiquimod all inhibited ANGPTL4 expression. In contrast, neither TNF, IL-1, nor IL-6 altered ANGPTL4 expression. Finally, in cholesterol loaded macrophages, LPS treatment still decreased ANGPTL4 expression. Thus, while in most tissues ANGPTL4 expression is stimulated by inflammatory stimuli, in macrophages TLR activators inhibit ANGPTL4 expression, which could lead to a variety of down-stream effects important in host defense and wound repair.     

Modulation of plasma TG lipolysis by Angiopoietin-like proteins and GPIHBP1

There is evidence that elevated plasma triglycerides (TG) serve as an independent risk factor for coronary heart disease. Plasma TG levels are determined by the balance between the rate of production of chylomicrons and VLDL in intestine and liver, respectively, and their rate of clearance in peripheral tissues. Lipolytic processing of TG-rich lipoproteins is mediated by the enzyme lipoprotein lipase (LPL), which is tethered to the capillary endothelium via heparin sulphate proteoglycans. In recent years the Angiopoietin-like proteins ANGPTL3 and ANGPTL4 have emerged as novel modulators of LPL activity. Studies in transgenic animals supported by in vitro experiments have demonstrated that ANGPTL3 and ANGPTL4 impair plasma TG clearance by inhibiting LPL activity. In humans, genetic variation within the ANGPTL3 and ANGPTL4 genes contributes to variation in plasma TG and HDL levels, thereby validating the importance of ANGPTLs in the regulation of lipoprotein metabolism in humans. Combined with the discovery of GPIHBP1 as a likely LPL anchor, these findings have led to a readjustment of the mechanism of LPL function. This review provides an overview of our current understanding of the role and regulation of ANGPTL3, ANGPTL4 and GPIHBP1, and places the newly acquired knowledge in the context of the established function and mechanism of LPL-mediated lipolysis.     

Genetic Mimicry Analysis Reveals the Specific Lipases Targeted by the ANGPTL3-ANGPTL8 Complex and ANGPTL4

Angiopoietin-like proteins, ANGPTL3, ANGPTL4, and ANGPTL8, are involved in regulating plasma lipids. In vitro and animal-based studies point to LPL and endothelial lipase (EL, LIPG) as key targets of ANGPTLs. To examine the ANGPTL mechanisms for plasma lipid modulation in humans, we pursued a genetic mimicry analysis of enhancing or suppressing variants in the LPL, LIPG, lipase C hepatic type (LIPC), ANGPTL3, ANGPTL4, and ANGPTL8 genes using data on 248 metabolic parameters derived from over 110,000 nonfasted individuals in the UK Biobank and validated in over 13,000 overnight fasted individuals from 11 other European populations. ANGPTL4 suppression was highly concordant with LPL enhancement but not HL or EL, suggesting ANGPTL4 impacts plasma metabolic parameters exclusively via LPL. The LPL-independent effects of ANGPTL3 suppression on plasma metabolic parameters showed a striking inverse resemblance with EL suppression, suggesting ANGPTL3 not only targets LPL but also targets EL. Investigation of the impact of the ANGPTL3-ANGPTL8 complex on plasma metabolite traits via the ANGPTL8 R59W substitution as an instrumental variable showed a much higher concordance between R59W and EL activity than between R59W and LPL activity, suggesting the R59W substitution more strongly affects EL inhibition than LPL inhibition. Meanwhile, when using a rare and deleterious protein-truncating ANGPTL8 variant as an instrumental variable, the ANGPTL3-ANGPTL8 complex was very LPL specific. In conclusion, our analysis provides strong human genetic evidence that the ANGPTL3-ANGPTL8 complex regulates plasma metabolic parameters, which is achieved by impacting LPL and EL. By contrast, ANGPTL4 influences plasma metabolic parameters exclusively via LPL.     

Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg₄₁↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg₄₁XXXXArg₄₆↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg₄₆↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.