👤 G Olivecrona

Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid (FA) uptake from triglyceride (TG)-rich lipoproteins such as very low density lipoproteins (VLDL). While pharmacological inhibition of ANGPTL3 is being evaluated as a lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aims to compare the therapeutic potential of liver-specific Angptl3 and Angptl4 silencing to attenuate hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established humanized model for lipoprotein metabolism. Mice were subcutaneously injected twice per week with saline or liver-targeted antisense oligonucleotides against Angptl3, Angptl4, both, or a scrambled oligonucleotide. Plasma lipid levels, VLDL clearance, and hepatic VLDL production were determined, and atherosclerosis development was assessed. For toxicological evaluation, cynomolgus monkeys were treated with three dosages of liver-targeted ANGPTL4-silencing oligonucleotides. Liver-targeted Angptl4 silencing reduced plasma TGs (-48%) and total cholesterol (-56%), explained by higher VLDL-derived FA uptake by brown adipose tissue and lower VLDL production by the liver. Accordingly, Angptl4 silencing reduced atherosclerotic lesion size (-86%) and improved lesion stability. Hepatic Angptl3 silencing similarly attenuated hyperlipidemia and atherosclerosis development. While Angptl3 and Angptl4 silencing lowered plasma TGs in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma TGs independent of the nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects. Liver-targeted Angptl4 silencing potently attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, and liver-targeted ANGPTL4 silencing is well tolerated in non-human primates. These data warrant further clinical development of liver-targeted ANGPTL4 silencing. Show less

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns. Show less

Studies in mice have shown that the decrease in lipoprotein lipase (LPL) activity in adipose tissue upon fasting is mediated by induction of the inhibitor ANGPTL4. Here, we aimed to validate this concept in humans by determining the effect of a prolonged fast on ANGPTL4 and LPL gene and protein expression in human subcutaneous adipose tissue. Twenty-three volunteers ate a standardized meal at 18.00 h and fasted until 20.00 h the next day. Blood was drawn and periumbilical adipose tissue biopsies were collected 2 h and 26 h after the meal. Consistent with previous mouse data, LPL activity in human adipose tissue was significantly decreased by fasting (-60%), concurrent with increased ANGPTL4 mRNA (+90%) and decreased ANGPTL8 mRNA (-94%). ANGPTL4 protein levels in adipose tissue were also significantly increased by fasting (+46%), whereas LPL mRNA and protein levels remained unchanged. In agreement with the adipose tissue data, plasma ANGPTL4 levels increased upon fasting (+100%), whereas plasma ANGPTL8 decreased (-79%). Insulin, levels of which significantly decreased upon fasting, downregulated ANGPTL4 mRNA and protein in primary human adipocytes. By contrast, cortisol, levels of which significantly increased upon fasting, upregulated ANGPTL4 mRNA and protein in primary human adipocytes as did fatty acids. ANGPTL4 levels in human adipose tissue are increased by fasting, likely via increased plasma cortisol and free fatty acids and decreased plasma insulin, resulting in decreased LPL activity. This clinical trial was registered with identifier NCT03757767. Show less

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex. Show less

Angiopoietin-like protein (ANGPTL)4 regulates plasma lipids, making it an attractive target for correcting dyslipidemia. However, ANGPTL4 inactivation in mice fed a high fat diet causes chylous ascites, an acute-phase response, and mesenteric lymphadenopathy. Here, we studied the role of ANGPTL4 in lipid uptake in macrophages and in the above-mentioned pathologies using Show less

Activity of lipoprotein lipase (LPL) is high in mouse kidney, but the reason is poorly understood. The aim was to characterize localization, regulation, and function of LPL in kidney of C57BL/6J mice. We found LPL mainly in proximal tubules, localized inside the tubular epithelial cells, under all conditions studied. In fed mice, some LPL colocalized with the endothelial markers CD31 and GPIHBP1 and could be removed by perfusion with heparin, indicating a vascular location. The role of angiopoietin-like protein 4 (ANGPTL4) for nutritional modulation of LPL activity was studied in wild-type and Angptl4 Show less

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. Show less

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant ( Show less

Apolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients. Show less

Patients at increased cardiovascular risk commonly display high levels of plasma triglycerides (TGs), elevated LDL cholesterol, small dense LDL particles and low levels of HDL-cholesterol. Many remain at high risk even after successful statin therapy, presumably because TG levels remain high. Lipoprotein lipase (LPL) maintains TG homeostasis in blood by hydrolysis of TG-rich lipoproteins. Efficient clearance of TGs is accompanied by increased levels of HDL-cholesterol and decreased levels of small dense LDL. Given the central role of LPL in lipid metabolism we sought to find small molecules that could increase LPL activity and serve as starting points for drug development efforts against cardiovascular disease. Using a small molecule screening approach we have identified small molecules that can protect LPL from inactivation by the controller protein angiopoietin-like protein 4 during incubations in vitro. One of the selected compounds, 50F10, was directly shown to preserve the active homodimer structure of LPL, as demonstrated by heparin-Sepharose chromatography. On injection to hypertriglyceridemic apolipoprotein A-V deficient mice the compound ameliorated the postprandial response after an olive oil gavage. This is a potential lead compound for the development of drugs that could reduce the residual risk associated with elevated plasma TGs in dyslipidemia. Show less

Since its discovery, apolipoprotein A-V has been considered to be a potent factor affecting plasma triglycerides (TG) in humans and mice. Several single nucleotide polymorphisms in the APOA5 gene are associated with increased TG levels in humans, and some nonsense mutations affecting protein structure predispose for familial hypertriglyceridemia and late onset chylomicronemia. It is not clear, how apoA-V decreases plasma TG. There are three major hypotheses: apolipoprotein A-V could work through (1) an intracellular mechanism affecting VLDL production in the liver, (2) stimulation of proteoglycan-bound lipoprotein lipase at the endothelium of capillaries in peripheral organs, or (3) enhancing the clearance of TG-rich lipoproteins via lipoprotein receptors in the liver. There is good evidence for a role of apoA-V in extracellular TG metabolism and increasing support for an additional function of ApoA-V as a receptor ligand. The intracellular role of apoA-V for lipoprotein assembly and secretion is still speculative. This review discusses these possible mechanisms. Show less

Severe hypertriglyceridaemia due to chylomicronemia may trigger an acute pancreatitis. However, the basic underlying mechanism is usually not well understood. We decided to analyze some proteins involved in the catabolism of triglyceride-rich lipoproteins in patients with severe hypertriglyceridaemia. Twenty-four survivors of acute hypertriglyceridaemic pancreatitis (cases) and 31 patients with severe hypertriglyceridaemia (controls) were included. Clinical and anthropometrical data, chylomicronaemia, lipoprotein profile, postheparin lipoprotein lipase mass and activity, hepatic lipase activity, apolipoprotein C II and CIII mass, apo E and A5 polymorphisms were assessed. Only five cases were found to have LPL mass and activity deficiency, all of them thin and having the first episode in childhood. No cases had apolipoprotein CII deficiency. No significant differences were found between the non-deficient LPL cases and the controls in terms of obesity, diabetes, alcohol consumption, drug therapy, gender distribution, evidence of fasting chylomicronaemia, lipid levels, LPL activity and mass, hepatic lipase activity, CII and CIII mass or apo E polymorphisms. However, the SNP S19W of apo A5 tended to be more prevalent in cases than controls (40% vs. 23%, NS). Primary defects in LPL and C-II are rare in survivors of acute hypertriglyceridaemic pancreatitis; lipase activity measurements should be restricted to those having their first episode during childhood. Show less

The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression. Show less

Apolipoprotein A-V (apoA-V) is present in low amounts in plasma and has been found to modulate triacylglycerol levels in humans and in animal models. ApoA-V displays affinity for members of the low density lipoprotein receptor (LDL-R) gene family, known as the classical lipoprotein receptors, including LRP1 and SorLA/LR11. In addition to LDL-A binding repeats, the mosaic receptor SorLA/LR11 also possesses a Vps10p domain. Here we show that apoA-V also binds to sortilin, a receptor from the Vsp10p domain gene family that lacks LDL-A repeats. Binding of apoA-V to sortilin was competed by neurotensin, a ligand that binds specifically to the Vps10p domain. To investigate the biological fate of receptor-bound apoA-V, binding experiments were conducted with cultured human embryonic kidney cells transfected with either SorLA/LR11 or sortilin. Compared with nontransfected cells, apoA-V binding to SorLA/LR11- and sortilin-expressing cells was markedly enhanced. Internalization experiments, live imaging studies, and fluorescence resonance energy transfer analyses demonstrated that labeled apoA-V was rapidly internalized, co-localized with receptors in early endosomes, and followed the receptors through endosomes to the trans-Golgi network. The observed decrease of fluorescence signal intensity as a function of time during live imaging experiments suggested ligand uncoupling in endosomes with subsequent delivery to lysosomes for degradation. This interpretation was supported by experiments with (125)I-labeled apoA-V, demonstrating clear differences in degradation between transfected and nontransfected cells. We conclude that apoA-V binds to receptors possessing LDL-A repeats and Vsp10p domains and that apoA-V is internalized into cells via these receptors. This could be a mechanism by which apoA-V modulates lipoprotein metabolism in vivo. Show less

Apolipoprotein A-V is a potent modulator of plasma triacylglycerol levels. To investigate the molecular basis for this phenomenon we explored the ability of apolipoprotein A-V, in most experiments complexed to disks of dimyristoylphosphatidylcholine, to interact with two members of the low density lipoprotein receptor family, the low density lipoprotein receptor-related protein and the mosaic type-1 receptor, SorLA. Experiments using surface plasmon resonance showed specific binding of both free and lipid-bound apolipoprotein A-V to both receptors. The binding was calcium dependent and was inhibited by the receptor associated protein, a known ligand for members of the low density lipoprotein receptor family. Preincubation with heparin decreased the receptor binding of apolipoprotein A-V, indicating that overlap exists between the recognition sites for these receptors and for heparin. A double mutant, apolipoprotein A-V (Arg210Glu/Lys211Gln), showed decreased binding to heparin and decreased ability to bind the low density lipoprotein receptor-related protein. Association of apolipoprotein A-V with the low density lipoprotein receptor-related protein or SorLA resulted in enhanced binding of human chylomicrons to receptor-covered sensor chips. Our results indicate that apolipoprotein A-V may influence plasma lipid homeostasis by enhancing receptor-mediated endocytosis of triacylglycerol-rich lipoproteins. Show less

Transgenic and gene disruption experiments in mice have revealed that apolipoprotein (apo) A-V is a potent regulator of plasma triglyceride (TG) levels. To investigate the molecular basis of apoA-V function, the ability of isolated recombinant apoA-V to modulate lipoprotein lipase (LPL) activity was examined in vitro. With three distinct lipid substrate particles, including very low-density lipoprotein (VLDL), a TG/phospholipid emulsion, or dimyristoylphosphatidylcholine liposomes, apoA-V had little effect on LPL activity. In the absence or presence apolipoprotein C-II, apoA-V marginally inhibited LPL activity. On the other hand, apoA-V-dimyristoylphosphatidylcholine disc particles bound to heparin-Sepharose and were specifically eluted upon application of a linear gradient of NaCl. The interaction of apoA-V with sulfated glycosaminoglycans was further studied by surface plasmon resonance spectroscopy. ApoA-V showed strong binding to heparin-coated chips, and binding was competed by free heparin. ApoA-V enrichment enhanced binding of apoC-II-deficient chylomicrons and VLDL to heparin-coated chips. When LPL was first bound to the heparin-coated chip, apoA-V-enriched chylomicrons showed binding. Finally, human pre- and post-heparin plasma samples were subjected to immunoblot analysis with anti-apoA-V IgG. No differences in the amount of apoA-V present were detected. Taken together, the results show that apoA-V lipid complexes bind heparin and, when present on TG-rich lipoprotein particles, may promote their association with cell surface heparan sulfate proteoglycans. Through such interactions, apoA-V may indirectly affect LPL activity, possibly explaining its inverse correlation with plasma TG levels. Show less