Analysis of ANGPTL8 promoter activity and screening of related transcription factors in bovine.

Analysing the molecular regulation mechanism of fat deposition in yellow cattle can provide a theoretical basis for the breeding of excellent beef cattle. ANGPTL8 (angiopoietin-like protein 8) promotes the formation of lipid droplets during adipocyte differentiation. To explore the promoter active region of ANGPTL8 and predict potential transcription factors, we further provide a theoretical basis for the functional analysis and regulatory mechanism of ANGPTL8 in adipogenesis. The promoter region of bovine ANGPTL8 was cloned by overlap extension PCR. Online software was used to predict potential transcription factor binding sites, and it identified PPARγ, SREBP1, C/EBPα, and Znf423 transcription factor binding sites in ANGPTL8 promoter region. A luciferase reporter gene vector which contained different deletion fragments of the ANGPTL8 promoter was constructed. Then, the vectors were cotransfected into 293 T cells with the internal control plasmid pRL-TK by cationic liposomes, and the relative fluorescence intensity was detected by a microplate reader. The results of the luciferase activity analysis showed that the core promoter area of ANGPTL8 was in the -885/-227 bp region of the 5' flanking sequence, while just two SREBP1 binding sites occurred in this area. When SREBP1 was knocked down by siRNA, the expression level of ANGPTL8 was reduced, and we speculated that SREBP1 may be an important transcription factor regulating ANGPTL8 transcription.

The Angiopoietin-Like Protein 3 and 8 Complex Interacts with Lipoprotein Lipase and Induces LPL Cleavage.

Lipoprotein lipase (LPL) is the key enzyme that hydrolyzes triglycerides from triglyceride-rich lipoproteins. Angiopoietin-like proteins (ANGPTL) 3, 4, and 8 are well-characterized protein inhibitors of LPL. ANGPTL8 forms a complex with ANGPTL3, and the complex is a potent endogenous inhibitor of LPL. However, the nature of the structural interaction between ANGPTL3/8 and LPL is unknown. To probe the conformational changes in LPL induced by ANGPTL3/8, we found that HDX-MS detected significantly altered deuteration in the lid region, ApoC2 binding site, and furin cleavage region of LPL in the presence of ANGPTL3/8. Supporting this HDX structural evidence, we found that ANGPTL3/8 inhibits LPL enzymatic activities and increases LPL cleavage. ANGPTL3/8-induced effects on LPL activity and LPL cleavage are much stronger than those of ANGPTL3 or ANGPTL8 alone. ANGPTL3/8-mediated LPL cleavage is blocked by both an ANGPTL3 antibody and a furin inhibitor. Knock-down of furin expression by siRNA significantly reduced ANGPT3/8-induced cleavage of LPL. Our data suggest ANGPTL3/8 promotes furin-mediated LPL cleavage.

ANGPLT3 in cardio-metabolic disorders.

Dyslipidemia is associated with numerous health problems that include the combination of insulin resistance, hypertension and obesity, which is always grouped together asmetabolic syndrome. Given that metabolic syndrome leads to a high mortality and poses serious risks to human health worldwide, it is vital to explore the mechanisms whereby dyslipidemia modulates the risk and the severity of cardio-metabolic disorders. Recently, a specific secretory protein family, named angiopoietin-like protein (ANGPTL), is considered as one of the significant biomarkers which facilitate the development of angiogenesis. Among the eight proteins of ANGPTL family, ANGPTL3 has been demonstrated as an essential modulator of lipid catabolism within circulation by inhibiting the activity of lipoprotein lipase (LPL) and endothelial lipase (EL). Consistent with these notions, mice with ANGPTL3 gene-deficiency presented reduced circulating levels of low density lipoprotein cholesterol (LDL-C) and lower risk of atherosclerosis. On the other hand, participants carrying homozygous loss-of function (LOF) mutation in ANGPTL3 gene also displayed lower circulating LDL-C levels and atherosclerotic risk. In the current review, we summarized the recent understanding of ANGPTL3 in controlling the risk and the development of dyslipidemia and its related cardio-metabolic disorders. Moreover, we also provided the perspectives which potentially suggested that ANGPTL3 could be considered as a promising target in treating metabolic syndrome.

Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities.

Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4). However, the mechanism by which ANGPTL3 inhibits LPL is unclear, in part due to challenges in obtaining pure protein for study. We used a new purification protocol for the N-terminal domain of ANGPTL3, removing a DNA contaminant, and found DNA-free ANGPTL3 showed enhanced inhibition of LPL. Structural analysis showed that ANGPTL3 formed elongated, flexible trimers and hexamers that did not interconvert. ANGPTL4 formed only elongated flexible trimers. We compared the inhibition of ANGPTL3 and ANGPTL4 using human very-low-density lipoproteins as a substrate and found both were noncompetitive inhibitors. The inhibition constants for the trimeric ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were only 2-fold different. Heparin has previously been reported to interfere with ANGPTL3 binding to LPL, so we questioned if the negatively charged heparin was acting in a similar fashion to the DNA contaminant. We found that ANGPTL3 inhibition is abolished by binding to low-molecular-weight heparin, whereas ANGPTL4 inhibition is not. Our data show new similarities and differences in how ANGPTL3 and ANGPTL4 regulate LPL and opens new avenues of investigating the effect of heparin on LPL inhibition by ANGPTL3.

A lipoprotein lipase-GPI-anchored high-density lipoprotein-binding protein 1 fusion lowers triglycerides in mice: Implications for managing familial chylomicronemia syndrome.

Lipoprotein lipase (LPL) is central to triglyceride metabolism. Severely compromised LPL activity causes familial chylomicronemia syndrome (FCS), which is associated with very high plasma triglyceride levels and increased risk of life-threatening pancreatitis. Currently, no approved pharmacological intervention can acutely lower plasma triglycerides in FCS. Low yield, high aggregation, and poor stability of recombinant LPL have thus far prevented development of enzyme replacement therapy. Recently, we showed that LPL monomers form 1:1 complexes with the LPL transporter glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) and solved the structure of the complex. In the present work, we further characterized the monomeric LPL/GPIHBP1 complex and its derivative, the LPL-GPIHBP1 fusion protein, with the goal of contributing to the development of an LPL enzyme replacement therapy. Fusion of LPL to GPIHBP1 increased yields of recombinant LPL, prevented LPL aggregation, stabilized LPL against spontaneous inactivation, and made it resistant to inactivation by the LPL antagonists angiopoietin-like protein 3 (ANGPTL3) or ANGPTL4. The high stability of the fusion protein enabled us to identify LPL amino acids that interact with ANGPTL4. Additionally, the LPL-GPIHBP1 fusion protein exhibited high enzyme activity in in vitro assays. Importantly, both intravenous and subcutaneous administrations of the fusion protein lowered triglycerides in several mouse strains without causing adverse effects. These results indicate that the LPL-GPIHBP1 fusion protein has potential for use as a therapeutic for managing FCS.

Structures of Angptl3 and Angptl4, modulators of triglyceride levels and coronary artery disease.

Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease.

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.