In Vivo Metabolism of apoB-containing Lipoproteins in ANGPTL3 Deficient Subjects

Rationale. ANGPTL3 has been identified as important regulator of lipolysis as well as a determinant of plasma levels of apolipoprotein B-containing lipoproteins. However, the precise mechanisms by which ANGPTL3 influences the flux of apoB particles transiting from the VLDL into LDL density range or affect LDL synthesis by modulating chylomicron remnants removal are unclear. It has been reported that the genetically determined ANGPTL3 absence or deficit is linked to lower plasma levels of apoB-containing lipoproteins. Therefore, a way to address the action of ANGPTL3 is to evaluate the in vivo lipoprotein metabolism in subjects carrying genetic mutations lowering ANGPTL3 as compared to normal controls.

Overall goal. The aim of this proposal is to uncover the role of ANGPTL3 in chylomicron, VLDL and LDL metabolism in humans (with a particular focus on its impact on the VLDL to LDL conversion pathway) using well-established in vivo lipoprotein kinetic methodologies.

Target population. For the present study, we will recruit subjects carrying the loss-of function mutation S17X in ANGPTL3 gene. Of them, 4 will be homozygotes, showing undetectable plasma levels of ANGPTL3 and hypobetalipoproteinemia and 8 will be heterozygotes with low ANGPTL3 plasma levels (<150 ng/ml), low TG (<120mg/dl) and LDL-C <160 mg/dl. Gender, age and BMI matched controls (n= 8) with plasma TG<180 mg/dl and LDL-C <160 mg/dl, and no known factors perturbing lipid metabolism will also be recruited. Cases and controls will be recruited from the Campodimele population.

Methods. Subjects will receive a 500 mg injection of deuterated glycerol to assess triglyceride kinetics, and an injection of deuterated leucine (7 mg/kg body weight) to assess the kinetics of apoB100, apoB48, apoC-III, and apoE. To evaluate chylomicron metabolism, subjects will be served a standard fat rich meal that contain 927 kcal (59.5 % fat, 24.4 % carbohydrate and 15.5 % protein) 2 hrs after injection of isotopes. Blood samples will be taken at frequent time points for 8 hrs followed by further blood samples collected next morning (24 hours) and on days 2, 3 and 4 after tracer administration. Chylomicrons, VLDL1, VLDL2, IDL and LDL will be isolated in a well-established stepwise centrifugal procedure. The concentrations of lipids and apolipoproteins will be determined using immunoassay and mass spectrometry in whole plasma and in lipoprotein fractions at each time point in the metabolic protocol. Isotope enrichment in apoproteins and lipids (triglycerides) will be performed using GC/MS.

Assessment. The main kinetic parameters derived from multicompartmental models will be:

1. Production and fractional clearance rates for apoB48-containing particles in chylomicrons, VLDL1, VLDL2, and for apoB100-containing particles in VLDL1, VLDL2, IDL and LDL.
2. Lipolysis rates for TG in VLDL1 and VLDL2.
3. Rates of conversion of chylomicrons to VLDL1 and VLDL2, rates of conversion of VLDL1 to VLDL2 to IDL and to LDL.