The exact mechanism by which ANGPTL3 deficiency lowers LDL and VLDL-TG is not completely understood. To get more insight into these mechanisms, we planned to measure in vivo turnover of TG- and apoB-containing lipoproteins (VLDL and LDL) in subjects with complete and partial ANGPTL3 deficiency (caused by homozygous or heterozygous LoF mutations in ANGPTL3 gene) in comparison with healthy controls. To this aim, 30 subjects will receive infusion of d3-leucine to assess the kinetics of apoB-containing lipoproteins and a bolus injection of d5-glycerol to assess the kinetics of triglycerides associated with different lipoprotein fractions.
As the deficiency of ANGPTL3 is a rare condition, we have planned to include in this study 10 individuals already identified by Dr. Stitziel at Washington University at St. Louis (USA). We estimated that this sample size will give an 80% power to detect a significant difference in lipoprotein turnover.
As we have recently observed an association between ANGPTL3 deficiency and lower plasma levels of PCSK9 (a major player in the regulation of LDLR activity), we will also investigate in two hepatoma cell lines, Huh7 and HepG2, the effect of ANGPTL3 silencing on expression and activity of PCSK9 and LDLR.
Our overall hypothesis is that 1) ANGPTL3-deficient subjects have a decreased production rate of VLDL and LDL associated with a decreased TG synthesis and increased catabolism of LDL than control and 2) ANGPTL3-deficient cells show a decreased transport and secretion of PCSK9 and this is associated with increased expression of LDLR at the cell surface.
If these hypotheses will be confirmed, our results, not only, will further support the importance to develop new anti-ANGPTL3 therapeutic strategies but also will provide potential indications for these compounds into the different hyperlipemic phenotypes.
A recent study discovered that nonsense (null) mutations in the gene ANGPTL3 (encoding the angiopoietin-like 3 protein) cause familial combined hypolipidemia, characterized by very low levels of all three major lipoprotein fraction. In an analysis of all reported FHBL2 families, we found that individuals with complete ANGPTL3 deficiency had mean low-density lipoprotein cholesterol (LDL-C) of 41 mg/dL, mean triglycerides (TG) of 35 mg/dL, and mean high-density lipoprotein cholesterol (HDL-C) of 23 mg/dL. In addition, population-based sequencing of ANGPTL3 has revealed that missense mutations in ANGPTL3 may also associate with lower plasma glucose. Thus, targeting ANGPTL3 has emerged as a new therapeutic opportunity to lower two causal risk factors (LDL-C and TG) for coronary heart disease (CHD) with potentially favorable metabolic effects.
The processing of TG-rich lipoproteins by the enzyme LPL is a key event in plasma lipid metabolism. LPL-mediated lipolysis results in the clearance of TG-rich particles from plasma, production of LDL, and provides lipids for the biosynthesis and maturation of HDL. Rare LoF mutations of LPL cause chylomicronemia, severe hypertriglyceridemia, very low HDL-C, and premature CHD; conversely, a gain-of-function variant in the LPL gene is associated with lower TG, higher HDL-C, and reduced CHD risk. LPL-mediated TG lipolysis occurs on the luminal surface of capillary endothelial cells, where LPL activity is extensively regulated by cell-surface receptors, angiopoietin-like molecules, apolipoproteins, and other cofactors.
As summarized above, the available human genetic data suggest that therapeutic manipulation of LPL activity should affect CHD risk in a manner by which increasing LPL activity should reduce risk of CHD. Developing agents that directly induce LPL activity may prove to be difficult; an obvious alternative would be to target natural inhibitors of LPL (such as ANGPTL3) with the notion that removing the natural inhibition will result in increased LPL activity.
In addition, ANGPTL3 is emerging as an attractive therapeutic target in the LPL pathway due to its association with both LDL and triglycerides (as opposed to APOC3 and ANGPTL4 which are not associated with alterations in LDL). However, to fully appreciate the therapeutic potential of ANGPTL3 inhibition, additonal questions must be clarified. In particular, it must be determined by which mechanisms ANGPTL3 (or its absence ) may influence the production and the catabolic rated of VLDL and LDL and if there is a relationship between this protein and the synthesis an function of PCSK9 and LDLR proteins, which have been demonstrated to be pivotal role in the regulation of plasma levels of LDL-C. A better understanding of these aspects will share more lights on the master regulatory activity of ANGTL3 on lipid and lipoprotein metabolism. This will, not only, further support the importance to develop new anti-ANGPTL3 medications but also will provide potential indications for the use of these compounds in different hyperlipemic phenotypes.
Beyond large-scale randomized controlled drug trials, naturally occurring human genetic variation offers an opportunity to understand the effects of loss of gene function in humans. Humans with homozygous (or compound heterozygous) or heterozygous ANGPTL3 mutations have lifelong complete or partial deficiency of ANGPTL3, respectively, and currently offer a unique insight into the long-term dose dependent effects of inhibiting this protein.