The role of ANGPTL8 in metabolism and cardiovascular diseases: Consensus and controversy.

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.

Introduction and Objective: Angiopoietin-like protein 3 (ANGPTL3) is a hepatocyte-secreted protein that plays a crucial role in lipid metabolism by inhibiting lipoprotein lipase (LPL). Loss-of-function variants of ANGPTL3 are associated with lower triglyceride and cholesterol levels, as well as reduced risks of obesity, diabetes, and cardiovascular diseases, making it an attractive therapeutic target. Despite its potential, the intracellular biogenesis of ANGPTL3, particularly within the endoplasmic reticulum (ER), remains poorly understood. The ER-associated degradation (ERAD) pathway is responsible for clearing misfolded ER proteins. However, the role of ERAD in lipid metabolism remains largely unexplored. In this study, we aim to: (1) investigate the physiological role of hepatic SEL1L-HRD1 ERAD in lipid metabolism, and (2) uncover how this pathway regulates the biogenesis of ANGPTL3. Methods: A hepatocyte-specific Sel1L knockout mouse model is used to examine the impact of ERAD dysfunction on ANGPTL3 biogenesis. Plasma lipid levels and lipoprotein lipase activity were measured. Biochemical analyses, including Western blotting and immunofluorescence, were performed to assess ANGPTL3 levels in the ER and its secretion. Mechanistic studies evaluated the folding state and stability of ANGPTL3, and interaction with ERAD components. Results: Our results indicate that ANGPTL3 is a substrate of the SEL1L-HRD1 ERAD pathway. In hepatocyte-specific Sel1L-deficient mice, we observed lower plasma triglyceride levels and increased LPL activity, correlating with impaired ANGPTL3 secretion. Further analysis revealed that in the absence of SEL1L-HRD1 ERAD, nascent ANGPTL3 accumulated in the ER due to improper folding and retention, disrupting lipid metabolism. Conclusion: This study not only clarifies the role and molecular mechanism of ERAD in regulating lipid metabolism but also provides new insights into ANGPTL3 biogenesis, with potential clinical implications for developing novel treatments for hyperlipidemia and cardiovascular diseases. Disclosure L.E. Zhou: None. S. Sun: None. L. Qi: None. Funding American Heart Association (AHA) Predoctoral Fellowship (25PRE1375196)

BackgroundIt is unclear why primary nephrotic syndrome (PNS) patients often have dyslipidemia. Recent studies have shown that angiopoietin-like protein 3 (ANGPTL3) is an important regulator of lipid metabolism. In this study, we explored how ANGPTL3 impacts dyslipidemia during PNS development.MethodsWe measured the serum levels of ANGPTL3 in PNS patients (n=196). Furthermore, the degree of proteinuria and lipid metabolism were examined in angptl3-overexpressing transgenic (angptl3-tg) mice at different ages. Moreover, in this study, we used the clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system to create angptl3-knockout (angptl3-/-) mice to investigate lipopolysaccharide (LPS)-induced nephrosis.ResultsCompared with that in the healthy group, the serum level of ANGPTL3 in the PNS group was significantly increased (32 (26.35-39.66) ng/ml vs. 70.44 (63.95-76.51) ng/ml, Z =-4.81, P < 0.001). There were significant correlations between the serum level of ANGPTL3 and the levels of cholesterol (r=0.34, P < 0.001), triglycerides (r= 0.25, P = 0.001) and low-density lipoprotein (r= 0.50, P < 0.001) in PNS patients. With increasing age, angptl3-tg mice exhibited increasingly severe hypertriglyceridemia and proteinuria. The pathological features of angptl3-tg mice included rich lipid droplet deposition in hepatocytes and diffuse podocyte effacement. Compared to wild-type mice, angptl3-/- mice showed significantly lower degrees of lipid dysfunction and proteinuria after stimulation with LPS. The effects of ANGPTL3 on nephrotic dyslipidemia were confirmed in cultured hepatocytes subjected to angptl3 knockdown or overexpression. Finally, significant alterations in lipoprotein lipase (LPL) levels were observed in liver tissues from Angptl3-/- and wild-type mice stimulated with LPS.ConclusionsANGPTL3 could be involved in the development of dyslipidemia, as well as proteinuria, during PNS pathogenesis. Inhibition of LPL expression may the mechanism by which ANGPTL3 induces hyperlipidemia in PNS.

Cardiovascular disease has been the leading cause of death throughout the world for nearly 2 decades. Hypertriglyceridemia affects more than one-third of the population in the United States and is an independent risk factor for cardiovascular disease. Despite the frequency of hypertriglyceridemia, treatment options are primarily limited to diet and exercise. Lipoprotein lipase (LPL) is an enzyme responsible for clearing triglycerides from circulation, and its activity alone can directly control plasma triglyceride concentrations. Therefore, LPL is a good target for triglyceride-lowering therapeutics. One approach for treating hypertriglyceridemia may be to increase the amount of enzymatically active LPL by preventing its inhibition by angiopoietin-like protein 4 (ANGPTL4). However, little is known about how these two proteins interact. Therefore, we used hydrogen-deuterium exchange MS to identify potential binding sites between LPL and ANGPTL4. We validated sites predicted to be located at the protein-protein interface by using chimeric variants of LPL and an LPL peptide mimetic. We found that ANGPTL4 binds LPL near the active site at the lid domain and a nearby α-helix. Lipase lid domains cover the active site to control both enzyme activation and substrate specificity. Our findings suggest that ANGPTL4 specifically inhibits LPL by binding the lid domain, which could prevent substrate catalysis at the active site. The structural details of the LPL-ANGPTL4 interaction uncovered here may inform the development of therapeutics targeted to disrupt this interaction for the management of hypertriglyceridemia.

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.

Background/Introduction Dyslipidemia is a significant risk factor for cardiovascular disease (CVD), and a residual risk of CVD remains despite the current standard of care. While combination therapies are often employed, some patients experience safety concerns and adverse side effects. Angiopoietin-like protein 3 (ANGPTL3) has emerged as a promising therapeutic target for the treatment of dyslipidemia. Genome-wide association studies have identified ANGPTL3 as a critical regulator of blood lipid levels in humans. It modulates lipid and lipoprotein metabolism by inhibiting lipoprotein lipase (LPL) and endothelial lipase (EL). BW-00112 is a fully synthetic, chemically optimized, double-stranded ANGPTL3 siRNA conjugated with N-acetylgalactosamine (GalNAc) for targeted delivery. This innovative approach shows great potential in addressing unmet needs in the management of dyslipidemia. Purpose The primary objective of this study was to assess the safety and tolerability of a single dose of BW-00112 in Chinese healthy subjects with 100 mg/dL ≤ LDL-C &amp;lt; 190 mg/dL and 100 mg/dL≤ TG &amp;lt;500 mg/dL up to 12 weeks. The secondary objectives of this study were to characterize pharmacokinetics (PK) profile and evaluate the pharmacodynamics (PD) effect (ANGPTL3, TG, LDL-C, non-HDL-C) of a single dose of BW-00112 up to 12 weeks. Methods This was a phase 1, randomized, double-blind, placebo-controlled, single dose study to evaluate the safety, tolerability, PK, and PD of subcutaneous doses of BW-00112 in Chinese healthy subjects with elevated LDL-C who were not receiving lipid-lowering therapy. Results BW-00112 was well tolerated with subcutaneous administration as a single dose ranging from 150 mg to 600 mg in Chinese healthy subjects. There were no serious adverse events, and no death or study discontinuations due to treatment-emergent adverse events (TEAEs). Most of the TEAEs were mild in severity. Adverse events of special interest, including injection site reactions and abnormalities in liver function, did not raise safety concerns. Exposure (AUC0-last and Cmax) increased in a dose-dependent manner within the linear range of 150 mg to 600 mg. However, the increase was slightly more than dose proportional. BW-00112 at doses of 150 mg to 600 mg resulted in substantial reductions in ANGPTL3 levels (mean -76% to -89%) 12 weeks after dosing. BW-00112 at doses of 150 mg to 600 mg also led to reductions in triglyceride (mean -62% to -76%), LDL-C (mean -31% to -34%), ApoB (mean -26% to -31%), Non-HDL-C (mean -32% to -36%), and remnant-C (mean -38% to -41%). Conclusion BW-00112 was generally well tolerated when administered subcutaneously as a single dose ranging from 150 mg to 600 mg. Substantial PD effects were observed with single subcutaneous doses of BW-00112 ranging from 150 mg to 600 mg, including reductions in ANGPTL3, triglycerides, LDL-C, ApoB, non-HDL-C, and remnant-C. No big difference was observed on safety, PK, and PD in Australian and Chinese healthy subjects.Reductions in serum ANGPTL3 and lipids

BackgroundModulators of triglyceride metabolism include lipoprotein lipase (LPL), angiopoietin-like protein 4 (ANGPTL4), and apolipoprotein C-3 (ApoC3). There is evidence on the influence of this triangle of molecules on an increased risk of atherosclerotic cardiovascular disease (CV) in the general population. Patients with rheumatoid arthritis (RA) present changes in lipid profiles and accelerated CV disease. In the present study, we set out to study whether the ANGPTL4, ApoC3, and LPL axis differs in subjects with RA compared to controls. In a further step, we investigated the relationship of this axis with subclinical atherosclerosis in patients with RA.MethodsCross-sectional study that included 569 individuals, 323 patients with RA and 246 age-matched controls. ANGPTL4, ApoC3 and LPL, and standard lipid profiles were analyzed in patients and controls. Carotid intima-media thickness (cIMT) and carotid plaques were assessed in RA patients. A multivariable analysis was performed to assess whether the ANGPTL4, ApoC3, and LPL axis was altered in RA and to study its relationship with RA dyslipidemia and subclinical carotid atherosclerosis.ResultsMost lipid profile molecules did not differ between patients and controls. Despite this, and after fully multivariable analysis including CV risk factors, use of statins, and changes in the lipid profile caused by the disease itself, patients with RA showed higher serum levels of ANGPTL4 (beta coef. 295 [95% CI 213–376] ng/ml, p<0.001) and ApoC3 (beta coef. 2.9 [95% CI 1.7–4.0] mg/dl, p<0.001), but lower circulating LPL (beta coef. −174 [95% CI −213 to −135] ng/ml, p<0.001). ANGPTL4 serum levels were positively and independently associated with a higher cIMT in patients with RA after fully multivariable adjustment.ConclusionThe axis consisting in ANGPTL4, ApoC3, and LPL is disrupted in patients with RA. ANGPTL4 serum levels are positively and independently associated with a higher cIMT in RA patients.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/β-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

Pathological cardiac hypertrophy is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the role of angiopoietin-like protein 8 (ANGPTL8) in pathological cardiac hypertrophy. We found that serum ANGPTL8 levels were significantly increased in hypertensive patients with cardiac hypertrophy and in mice with cardiac hypertrophy induced by Ang II or TAC. Furthermore, the secretion of ANGPTL8 from the liver was increased during hypertrophic processes, which were triggered by Ang II. In the Ang II- and transverse aortic constriction (TAC)-induced mouse cardiac hypertrophy model, ANGPTL8 deficiency remarkably accelerated cardiac hypertrophy and fibrosis with deteriorating cardiac dysfunction. Accordingly, both recombinant human full-length ANGPTL8 (rANGPTL8) protein and ANGPTL8 overexpression significantly mitigated Ang II-induced cell enlargement in primary neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. Mechanistically, the antihypertrophic effects of ANGPTL8 depended on inhibiting Akt and GSK-3β activation, and the Akt activator SC-79 abolished the antihypertrophic effects of rANGPTL8 in vitro. Moreover, we demonstrated that ANGPTL8 directly bound to the paired Ig-like receptor PIRB (LILRB3) by RNA-seq and immunoprecipitation-mass screening. Remarkably, the antihypertrophic effects of ANGPTL8 were largely blocked by anti-LILRB3 and siRNA-LILRB3. Our study indicated that ANGPTL8 served as a novel negative regulator of pathological cardiac hypertrophy by binding to LILRB3 (PIRB) and inhibiting Akt/GSK3β activation, suggesting that ANGPTL8 may provide synergistic effects in combination with AT1 blockers and become a therapeutic target for cardiac hypertrophy and heart failure.

BackgroundTocilizumab is a humanized immunoglobulin G antibody to the interleukin 6 (IL-6) receptor that is used in the treatment of patients with rheumatoid arthritis (RA). Patients receiving TCZ are more...

Angiopoietin like protein 4 expression is decreased in activated macrophages

Effect of ANGPTL3 Inhibition With Solbinsiran in Preclinical and EarlyHumanStudies.

BackgroundElevated triglycerides or triglyceride-rich lipoproteins are an additional cause of cardiovascular (CV) disease. Given that patients with systemic lupus erythematosus (SLE) have a high prevalence of premature CV disease and show an altered lipid profile, our objective was to study whether three molecules that play a central role in the triglyceride metabolism: apolipoprotein C-III (ApoC3), angiopoietin-like protein 4 (ANGPLT4), and lipoprotein lipase (LPL) differ between SLE patients and controls, and how they are related to disease characteristics, including disease damage.MethodsCross-sectional study that included 347 women, 185 of them diagnosed with SLE and 162 age-matched controls. ANGPTL4, ApoC3 and LPL, and standard lipid profiles were analyzed in SLE patients and controls. A multivariable analysis was performed to assess whether ANGPTL4, ApoC3 and LPL molecules differ between patients and controls and to study their relationship with SLE disease damage.ResultsAfter fully multivariable analysis that included classic CV risk factors, and the modifications that the disease itself produces over the lipid profile, it was found that ApoC3 was significantly lower (beta coef. -1.2 [95%CI -1.6- -0.8) mg/dl, <0.001), and ANGPTL4 (beta coef. 63 [95%CI 35-90] ng/ml, <0.001) and LPL (beta coef. 79 [95%CI 30-128] ng/ml, p=0.002) significantly higher in patients with SLE compared to controls. Disease damage score was significantly and independently associated with higher serum levels of LPL (beta coef. 23 [95%CI 10-35] ng/ml, p=0.001). Mediation analysis suggested that the relationship between disease damage and LPL was direct and not mediated by ApoC3 or ANGPLT4.ConclusionThe ApoC3, ANGPLT4 and LPL axis is disrupted in patients with SLE. Disease damage explains this disturbance.

Long-term stress exposure can lead to disturbed homeostasis and cause many life-style diseases. Phloridzin possesses various bioactivities, but the understanding of the effects of phloridzin on stress-related lipid metabolism disorder is limited. Our results demonstrate that phloridzin improved plasma lipoprotein lipase (LPL) activity and triglyceride metabolism in restrained mice. A decrease of angiopoietin-like protein 4 (ANGPTL4) mRNA expression and an increase of AMP-activated protein kinase (AMPK) phosphorylation were observed after phloridzin treatment. After inhibiting AMPK phosphorylation, the effects of phloridzin on the amelioration of plasma LPL activity and suppression of ANGPTL4 expression were blocked. In addition, cardiac AMPK phosphorylation, plasma LPL activity and ANGPTL4 expression were also affected by phloridzin, even if the glucocorticoid receptor was blocked. Taken together, the down-regulation of ANGPTL4 expression by phloridzin was probably via a direct activation of AMPK pathway. This discovery can provide a biochemical and nutritional basis for the use of phloridzin-containing food and beverage in daily life.

Angiopoietin-like protein 4 (ANGPTL4) is a circulating protein predominantly expressed in adipose tissue and liver. Several recent studies demonstrated that ANGPTL4 is the target gene of peroxisome proliferation activators, the agonists of which are widely used as the antidiabetic and lipid-lowering drugs. Here we provide evidence that ANGPTL4 is a blood-borne hormone directly involved in regulating glucose homeostasis, lipid metabolism, and insulin sensitivity. Adenovirus-mediated expression of ANGPTL4 potently decreased blood glucose and improved glucose tolerance, whereas it induced hyperlipidemia, fatty liver, and hepatomegaly in C57 mice. In db/db diabetic mice, ANGPTL4 treatment reduced hyperglycemia to a normal level, and markedly alleviated glucose intolerance and hyperinsulinemia. Ex vivo studies on primary rat hepatocytes revealed that ANGPTL4 significantly decreased hepatic glucose production and enhanced insulin-mediated inhibition of gluconeogenesis. Serum levels of ANGPTL4 in human subjects inversely correlated with plasma glucose concentrations and HOMA IR, the homeostasis model assessment of insulin resistance. In patients with type 2 diabetes, serum levels of ANGPTL4 were significantly lower than those in healthy subjects, suggesting that the decreased ANGPTL4 could be a causative factor of this disease. These results collectively indicate that ANGPTL4 exerts distinct effects on glucose and lipid metabolism, and that its beneficial effect on glucose homeostasis might be useful for the treatment of diabetes.