Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT05569928 |
| Other study ID # |
0061/2022 |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
September 30, 2022 |
| Est. completion date |
January 30, 2025 |
Study information
| Verified date |
October 2022 |
| Source |
University of Roma La Sapienza |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
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.
Description:
Working hypothesis
The overall working hypothesis is that the normal physiological action of ANGPTL3 is to slow
lipolysis and inhibit remnant clearance by the liver. In this way, the protein causes an
increase in the flux of apoB particles transiting from the VLDL into LDL density range,
resulting in a net increase in LDL production. In previous studies we observed that the
extent of conversion of VLDL to IDL and then LDL varies from 25% to 75%.2 Where and how
ANGPTL3 might act remains to be discovered. A comparison of the rates of VLDL1 and VLDL2
production, rates of lipolysis of these particles, and the amount of apoB converted to IDL
and then LDL in control subjects and those heterozygous and homozygous for ANGPTL3
loss-of-function (LOF) variants will enable us to delineate where ANGPTL3 acts, and possibly
reveal a gene-dose related effect.
Alternative hypotheses will also be explored including the possibility that accelerated
chylomicron lipolysis in ANGPTL3 LOF carriers leads to a subnormal amount of dietary
triglyceride being delivered to the liver which as a result impairs VLDL assembly and
secretion. This would imply that the hypobetalipoproteinaemia phenotype is primarily due to
an abnormality in hepatic production. Or, as recently suggested from studies in familial
hypercholesterolemia, that there is a stimulation of LDL catabolism when ANGPTL3 activity is
reduced.
Primary Objectives
The primary objectives are to investigate in subjects heterozygous and homozygous for ANGPTL3
LOF variants compared to matched controls the:
- Kinetics of apoB48 in chylomicrons and their remnants in light of our earlier
investigations that revealed dramatic reductions in post-prandial chylomicronaemia in
homozygous LOF carriers.4
- Kinetics of apoB100 in VLDL1, VLDL2, IDL and LDL. This will reveal the basis for the
hypobetalipoproteinaemia seen in this condition 4-6 and clarify how partial or complete
loss of ANGPTL3 leads to lower levels of VLDL, remnants, and LDL.
- Kinetics of TG in VLDL1 and VLDL2. This will reveal how lipolysis is altered when
ANGPTL3 action is impaired or absent.
Secondary Objectives
Our secondary objectives are to investigate:
- The impact of ANGPTL3 on the kinetics of the key regulatory apolipoproteins apoC-III and
apoE, since apoC-III has been reported to be lower in concentration when ANGPTL3 is
reduced.
- The detailed composition of apoB-containing lipoprotein species. We will explore the
proteomic and lipidomic profiles of chylomicrons, VLDL1, VLDL2, IDL and LDL in this
unique cohort to understand further the compositional signatures in this specific 'low
ASCVD risk' condition.
Experimental Design and Approach Subjects recruitment: Subjects will be recruited on the
basis of their known status for the S17X LOF variant. The individuals are inhabitants of the
Campodimele village, near Rome, Italy. The investigators have over many years established a
close and mutually valued connection with the people of Camapodimele and have published key
characteristics of the lipid phenotype that is exhibited by those carrying the ANGPTL3 LOF,
which is a profound combined hypolipidemia associated with reduced plasma levels of total
cholesterol, TG, VLDL-TG and cholesterol, LDL cholesterol, apoB, and a blunted post-prandial
triglyceride response to a test fat meal. Our plan is to recruit the 4 available homozygotes
and identify 6-8 heterozygotes who exhibit low ANGPTL3 plasma levels (<150ng/ml) and low TG
(<120mg/dl). Age and sex matched healthy controls (n=6-8) with no known factors perturbing
lipid metabolism will also be recruited from the Campodimele population.
Metabolic protocol: The investigators have developed and validated novel methods allowing to
simultaneously follow the kinetics of TGs in large VLDL1 and smaller VLDL2, apoB48 in
chylomicrons, VLDL1 and VLDL2, apoB100 in VLDL1, VLDL2, IDL and LDL, and the production and
catabolic rates for apoC-III and apoE. 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 (68 g), 24.4 % carbohydrate (63 g) and 15.5 % protein (40 g) 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. The total blood volume to be withdrawn over the period of the kinetic
study (about 350 ml) has been reduced in recognition that some of the subjects (homozygotes
in particular) are elderly. (This volume of blood donation is considered acceptable and is
below what has been collected in earlier clinical studies in this population). The reduction
in blood drawn does not alter our ability to derive the full set of kinetic parameters - The
investigators will make best use of the highly sensitive analytical techniques. Dr Arca's
clinical team will perform the metabolic protocol on site at special clinical premises in
Campodimele. Samples will be taken to his laboratory in Rome for aliquoting and shipment.
Lipoprotein isolation: Isolation of lipoprotein fractions for kinetic analysis is time
consuming and requires specialised equipment (ultracentrifuges) and considerable expertise;
isolating TRL fractions from subjects with low plasma TG is even more challenging.
Preparation of lipoprotein fractions will therefore be undertaken in the laboratory of Dr
Taskinen in Helsinki (after shipment on ice from Rome). Chylomicrons, VLDL1, VLDL2, IDL and
LDL will be isolated in a well-established stepwise centrifugal procedure. First, total TRL
will be concentrated by ultracentrifugation (d <1.006 g/ml for 18 hrs) and then this 'top'
fraction will be harvested and used to isolate chylomicrons (Sf >400), VLDL1 (Sf 60-400), and
VLDL2 (Sf 20-60) by density gradient ultracentrifugation. The d>1.006 g/ml ('bottom')
fraction will be used to separate IDL and LDL in further sequential centrifugation steps.
These methods are employed routinely in the Helsinki lab. The isolated lipoprotein fractions
are then prepared for transfer to Gothenburg for enrichment analyses. This preparation
involves measurements of TG, cholesterol, phospholipids and protein concentration, and
delipidation, precipitation and measurement of apoB. Analyses of enrichments of isotopes in
apoproteins and lipids, and mathematical modelling: The next step in the kinetic
investigation is the analysis of the enrichment of stable isotopes using highly sensitive
mass spectrometry methods. The concentrations of lipids and apolipoproteins (apoB100, apoB48,
apoC-III and apoE) will be determined using immunoassay and mass spectrometry techniques 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.
All of these methods, including sample transfers, are robust and well established in the
Helsinki and Gothenburg labs, and have been the subject of a series of publications. The
isotope enrichment results and TG/apoprotein concentrations in all the isolated fractions
form the data set on which the multicompartmental modelling is conducted. The investigators
have successfully developed and applied our models to data derived from subjects with a
variety of conditions (hypertriglyceridemia, hypotriglyceridaemia, diabetes) as well as on
and off lipid-lowering treatment. The models are applicable to all settings encountered and
have generated novel insights into lipoprotein metabolism.12x-14 Kinetic parameters derived
from multicompartmental models are: Production rates for apoB48 particles in the chylomicron,
VLDL1 and VLDL2 fractions. Production rates for apoB100 in VLDL1, VLDL2, IDL and LDL.
Lipolysis rates for TG in VLDL1 and VLDL2 Fractional clearance rates for apoB48-containing
particles in chylomicrons, VLDL1, VLDL2, and for apoB100-containing particles in VLDL1,
VLDL2, IDL and LDL. Rates of conversion of chylomicrons to VLDL1 and VLDL2, rates of
conversion of VLDL1 to VLDL2 to IDL and to LDL. Production and clearance rates of apoC-III
and apoE.
Main Outcome Measures and Study Deliverables. How ANGPTL3 regulates lipoprotein metabolism in
humans is unknown and, therefore, it is important to keep an open mind as to its precise mode
of action on apoB and TG kinetics as explored in this study. Our working hypothesis based on
observations to date in LOF carriers is that it has an impact on all apoB-containing
lipoproteins that is distinct from other regulatory factors such as those that alter
lipoprotein production - microsomal triglyceride transfer (MTP) protein activity or mutations
that inhibit VLDL1 assembly (TM6SF2), or those that alter lipoprotein receptor-mediated
clearance such as PCSK9.
The investigators have recently published research of factors that alter apoB and TG
production such as TM6SF2, PNPLA3 and the role of PCSK9 in controlling the catabolism of
apoB-containing lipoproteins. Further, and importantly, the investigators are just completing
a kinetic study in a group of apoCIII LOF carriers who exhibit low TG levels and other
phenotypic traits comparable to those seen in ANGPTL3 LOF (as yet unpublished). The
availability of these comparator studies provides a metabolic background against which we can
evaluate the specific actions of ANGPTL3.
The primary evaluation will focus on the differences in VLDL1, VLDL2, IDL and LDL clearance
rates, the rates of TG lipolysis in VLDL1 and VLDL2, and the rates of conversion of VLDL1/
VLDL2 to IDL and LDL in the homozygous S17X carriers compared to heterozygotes and controls.
The key research question is "does ANGPTL3 deficiency or absence result in increased
lipolysis and increased fractional clearance rates for VLDL2 and IDL (the precursors of LDL)
which then leads to reduced intravascular generation of LDL particles, and possibly reduction
in the circulating level of remnant lipoproteins". the investigator will also explore a
potential gene-dose effect by including heterozygotes in the statistical analysis. The key
kinetic parameters will be tested in ANOVA models, with inclusion of potential confounding
factors such as sex and BMI.
the secondary hypothesis that ANGPTL3 acts to reduce overall lipoprotein assembly and
secretion in the liver will be tested directly by comparing the production rates of VLDL1,
VLDL2, IDL and LDL apoB100, and the secretion rate of VLDL1-TG and VLDL2-TG in the three
subject groups.
Further outcomes will be to align the proteomic and lipidomic compositional data with
variation in kinetic parameters. If it is the case that ANGPTL3 LOF carriers have very low
levels of remnant particles (and other atherogenic lipoproteins) then the composition of VLDL
fractions, IDL and LDL may reveal a 'low risk signature' that is helpful when evaluating the
efficacy of agents designed to lower or inhibit ANGPTL3 action.
