© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Decreased Production Rates of VLDL Triglycerides and ApoB-100 in Subjects Heterozygous for Familial Hypobetalipoproteinemia
From the Division of Atherosclerosis, Lipid Research, and Nutrition and the Division of Gastroenterology (B.W.P.), Department of Internal Medicine, Washington University School of Medicine, St. Louis, Mo.
Correspondence to Gustav Schonfeld, MD, Department of Internal Medicine, Washington University School of Medicine, 660 S Euclid, Box 80466, St. Louis, MO 63110. E-mail gschonfe{at}imgate.wustl.edu
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Abstract |
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Abstract—Familial hypobetalipoproteinemia (FHBL) is an autosomal codominant disorder characterized by low levels of apolipoprotein (apo) B and low-density lipoprotein (LDL) cholesterol. Decreased production rates of apoB have been demonstrated in vivo in FHBL heterozygotes. In the present study, we wished to investigate whether the transport of triglycerides was similarly affected in these subjects. Therefore, we studied the in vivo kinetics of very-low-density lipoprotein (VLDL) triglycerides and VLDL apoB-100 simultaneously in 7 FHBL heterozygotes from 2 well-characterized kindreds and 7 healthy normolipidemic subjects. In both kindreds, hypobetalipoproteinemia is caused by mutations in the 5' portion of the apoB gene specifying short truncations of apoB undetectable in plasma. A bolus injection of deuterated palmitate and a primed constant infusion of deuterated leucine were given simultaneously, and their incorporation into VLDL triglycerides and VLDL apoB, respectively, were determined by gas chromatography–mass spectrometry. Kinetic parameters were calculated by using compartmental modeling. VLDL apoB fractional catabolic rates (FCRs) in FHBL heterozygotes and controls were similar (11.6±3.9 and 10.9±2.4 pools per day, respectively, P=0.72). On the other hand, FHBL heterozygotes had a 75% decrease in VLDL apoB production rates compared with normal subjects (5.8±1.8 versus 23.4±7.1 mg/kg per day, P<0.001). The decreased production rates of VLDL apoB accounts for the very low concentrations of plasma apoB found in heterozygotes from these kindreds (24% of normal). Mean VLDL triglyceride FCRs in FHBL subjects and controls were not significantly different (1.06±0.74 versus 0.89±0.50 pools per hour, respectively, P=0.61). There was a good correlation between VLDL apoB FCR and VLDL triglyceride FCR in the 2 groups (r=0.84, P<0.001). VLDL triglyceride production rates were decreased by 60% in FHBL heterozygotes compared with controls (9.3±6.0 versus 23.0±9.6 µmol/kg per hour, P=0.008). Thus, the hepatic secretion of VLDL triglycerides is reduced in FHBL heterozygotes but to a lesser extent than the decrease in apoB-100 secretion. This is probably achieved by the secretion of VLDL particles enriched with triglycerides.
Key Words: familial hypobetalipoproteinemia • apolipoprotein B • triglycerides • stable isotopes
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Introduction |
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Familial hypobetalipoproteinemia (FHBL) is an autosomal codominant disorder characterized by abnormally low plasma levels of apoB and LDL cholesterol. Heterozygotes are usually asymptomatic, whereas homozygotes may manifest signs of fat malabsorption and complications related to deficiency of fat-soluble vitamins.1 Some forms of FHBL are caused by mutations in the apoB gene on chromosome 2, which interfere with the translation of the full-length protein and produce truncated forms of apoB detectable in plasma.1 2 The truncations are designated according to a centile nomenclature using the full-length apoB, consisting of 4536 amino acids as apoB-100. In FHBL heterozygotes with mutations in the 5' region of the apoB gene predicted to produce truncations shorter than apoB-27, no truncated apoB forms are detectable in plasma. It is assumed that this is due to failure of these short truncations in the assembly and secretion of triglyceride-rich lipoproteins. Four such mutations have been identified to date.3 4 5
To define the mechanisms responsible for the decreased plasma apoB levels in FHBL heterozygotes, our group and others used stable isotope tracer methodology to study the in vivo kinetics of apoB. These studies have demonstrated that the truncated apoB forms were produced at lower rates than were the normal apoB-100 forms.6 7 The clearance of some truncations from plasma, especially those containing the LDL receptor–binding domain (ie, apoB-75 and apoB-89), was faster than apoB-100 clearance, as reflected by higher fractional catabolic rates (FCRs).8 9 FHBL heterozygotes also produce apoB-100 at a production rate that is less than the 50% of normal expected from the presence of one normal allele,10 11 further contributing to the low plasma apoB levels.
ApoB-100 is secreted from the liver in VLDL particles associated with
lipids, mainly triglycerides, which serve as an important
source of energy delivery to peripheral tissues in the form
of fatty acids.12 It is unknown to what extent the hepatic
secretion of triglycerides is affected by the defect in
apoB secretion observed in FHBL heterozygotes. The secretion of apoB
and triglycerides is associated in some
hypertriglyceridemic states.13
However, dissociation of VLDL apoB and triglyceride
secretion rates has been reported in other
conditions,14 15 resulting in VLDL particles with
variable triglyceride to apoB ratios. A marked
reduction in VLDL triglyceride export would limit the
delivery of fatty acids to peripheral tissues, which would
result in accumulation of triglycerides in
hepatocytes and could eventually lead to the development of
fatty liver. Because FHBL heterozygotes are usually
asymptomatic and in a good nutritional state, we
hypothesized that the production of VLDL
triglycerides may not be affected to the same extent as
apoB production in these subjects. To test this hypothesis, we
evaluated the in vivo metabolism of VLDL
triglyceride and VLDL apoB simultaneously in
FHBL heterozygotes from 2 well-characterized FHBL kindreds. We have
previously described the genetic defects responsible for
hypobetalipoproteinemia in both
kindreds.16 17 The affected members of the C kindred have
a non-sense mutation (Arg412
stop) in exon 10 of the apoB
gene, which is predicted to produce apoB-9. In the D kindred, a
mutation in the splice donor site in intron 5 of the apoB gene expected
to produce apoB-2 was found in affected subjects. We could not find the
truncated protein in plasma in heterozygotes from these 2 kindreds, and
their VLDL particles contain only apoB-100 produced by the normal
allele.
In the present study, we used a novel approach for studying the in vivo kinetics of VLDL triglycerides and apoB simultaneously: endogenous labeling with stable isotopically labeled tracers. Deuterated palmitate and deuterated leucine were infused, and their incorporation into VLDL triglycerides and VLDL apoB, respectively, were determined. The data were analyzed by compartmental modeling to determine the kinetic parameters.
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Subjects
Three heterozygotes from the C kindred, 4 heterozygotes from the D kindred, and 7 normal healthy controls participated in the present study. The clinical characteristics of the study subjects are shown in Table 1
. All FHBL
heterozygotes were asymptomatic and taking no medications.
The subjects in the control group were all normolipidemic, had no
evidence of hepatic, renal, or endocrine disorders, and were not taking
any medications. Detailed physical examinations, blood counts, liver
function tests, and lipid profiles were performed on enrollment. All
participants gave written informed consent.
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Study Protocol
The study protocol was approved by the Human Studies Committee
of Washington University School of Medicine. The subjects were
instructed not to change their diet 10 days before the study. For the
kinetic study, subjects were admitted to the General Clinical Research
Center after a 10-hour fast. An intravenous catheter was
inserted into the antecubital vein of one arm for the infusion of
tracers, and a second catheter was inserted into a vein in the other
arm for blood sampling. A bolus of
[2H2]palmitate
(potassium salt, 98% isotopic purity, Isotec) complexed to 5% human
albumin at a dose of 6 µmol/kg was injected over 1
minute, immediately followed by a primed constant infusion of
deuterated leucine.
[2H3]Leucine (98%
isotopic purity, Cambridge Isotopes) was administered as a priming dose
of 6.3 µmol/kg, followed by 6.3 µmol/kg per hour for 8
hours through a calibrated syringe pump (Harvard
Apparatus). The subjects remained fasting during the
infusion period and for another 8 hours after the termination of the
infusion. Noncaloric drinks were permitted throughout the study. A
total of 21 blood samples for leucine and palmitate enrichment
measurements were drawn during the study, starting at time 0 and
continuing up to 96 hours. Blood was collected into EDTA-containing
tubes, and plasma was separated immediately by low-speed
centrifugation.
Analytical Methods
Measurements of Plasma Lipids and Apolipoproteins
By use of commercially available enzymatic kits (Technicon
Instruments), total cholesterol, triglycerides,
LDL cholesterol, and HDL cholesterol were
determined on baseline plasma samples by the Lipid Research Center Core
Laboratory at Washington University School of Medicine. Plasma apoB and
apoA-I were determined by immunonephelometry (Behring).
Isolation of VLDL
VLDL (d<1.006 g/mL) was isolated from 5 mL of fresh plasma by
ultracentrifugation at 40 000 rpm for 18 hours and
stored at –70°C until further analysis.
Triglyceride and cholesterol concentrations in
VLDL fractions were determined by enzymatic assays. VLDL apoB
concentrations were determined by enzyme-linked immunosorbent
assay7 10 using the monoclonal antibody C1.4.
Isolation of VLDL ApoB and Plasma Amino Acids
ApoB was isolated from VLDL fractions by precipitation with
butanol isopropyl ether18 and hydrolyzed in 6N HCl for 24
hours at 110°C. Amino acids were isolated from 0.25 mL of plasma by
cation exchange chromatography.19 The
amino acids obtained from apoB hydrolysates and plasma amino acids were
derivatized to n-heptafluorobutyryl–n-propyl
esters and dissolved in ethyl acetate for enrichment
measurements.20 Leucine enrichment was determined by
negative chemical ionization gas chromatography–mass
spectrometry on a Hewlett-Packard model 5988 equipped with a DB-17
capillary column. Molecular ions at mass-to-charge ratio (m/z) 349
(m+0) and 352 (m+3) were selectively monitored. The enrichment data
were converted to tracer/tracee ratio (TTR) by analysis of
isotopic enrichment standards.
Isolation of VLDL Triglycerides and Plasma Free
Fatty Acids
Lipids were extracted from VLDL fractions by chloroform-methanol
(1:2 [vol/vol]) and separated by thin-layer
chromatographic plates (Whatman Inc).
Triglyceride bands were scraped from the plates,
derivatized with acetyl chloride–methanol (1:20 [vol/vol]) at 70°C
for 30 minutes, and the resulting methyl esters were extracted with
heptane.
Lipids were extracted from 0.25 mL plasma by hexane, reacted with iodomethane (dissolved in dichloromethane 1:10 [vol/vol]) to convert free fatty acids (FFAs) to their methyl esters, and then passed onto solid-phase extraction cartridges (Lc-Si SPE Tubes, Supelco). Methyl esters were eluted from the cartridges with 2% ethyl acetate in hexane.21 The samples were dried under N2 and dissolved in heptane for enrichment measurements.
Methyl palmitate enrichment in VLDL triglycerides and plasma was determined by electron impact ionization gas chromatography–mass spectrometry on a Hewlett-Packard model 5971 MSD equipped with an Omegawax 250 capillary column. Molecular ions at m/z 270 (m+0) and 272 (m+2) were selectively monitored. Isotopic enrichment standards were used as previously described,21 and the data were expressed as TTR.
For determination of plasma FFA concentrations, heptadecanoic acid was added as an internal standard to 4 plasma samples (time points 0, 4, 8, and 16 hours) from each study. Plasma FFAs were derivatized and extracted as described above and analyzed by gas chromatography. The mean of the 4 values was used to describe plasma FFA concentrations.
Kinetic Analysis
VLDL apoB and VLDL triglyceride kinetics were
analyzed by compartmental modeling using the SAAM II program
(SAAM Institute, University of Washington, Seattle). Several
assumptions were made that are common to all linear, time-invariant
compartmental models as used in the present study. It is assumed
that transfer of material between compartments follows first-order
kinetics with respect to tracer content, which is always a valid
assumption when the tracer does not perturb the pool
size,22 as for apoB and triglycerides. It is
also assumed that the rate constants do not vary with time, such that
the system is at metabolic steady state. The model
structure represents the minimal complexity that is necessary
and sufficient to describe the kinetic data.
The model used for apoB kinetics (Figure 1) has been used previously to describe
VLDL apoB kinetics for apoB-100 and various truncated forms of
apoB.6 7 8 9 10 23 The model consists of (1) a forcing function
to describe plasma leucine enrichment during and after the termination
of the 8-hour primed continuous tracer infusion period, (2) a delay for
hepatic VLDL apoB synthesis, assembly, and secretion, and (3) a
homogeneous VLDL apoB compartment. It was not necessary to
postulate the presence of "fast" and "slow" VLDL compartments
for any subject in the present study.
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The model used for VLDL triglyceride kinetics is
structurally similar to that used for apoB (Figure 1
). In
preliminary analyses, it was determined that a sum of 3
exponential terms was necessary and sufficient to describe the bolus
plasma [2H2]palmitate
kinetics. Therefore, a 3-compartment catenary model structure was
arbitrarily chosen for this purpose; a 3-compartment mammillary
model or a "forcing function" defined as a sum of 3
exponentials would have provided identical descriptions of plasma
palmitate kinetics. Since the focus of the present study involves
the turnover of VLDL triglycerides, the model structure
used to define plasma palmitate kinetics is irrelevant. After a delay
for VLDL synthesis and secretion, the
[2H2] palmitate appeared
in VLDL triglycerides. As for apoB, it was not necessary to
postulate the existence of heterogeneous "fast" and
"slow" VLDL compartments to account for the VLDL
triglyceride palmitate kinetics, nor was it necessary to
postulate the existence of a second "slow" biosynthetic pathway to
provide a source of intrahepatically recycled
tracer.24
VLDL apoB and VLDL triglyceride production rates were calculated as the product of their respective FCRs and pool sizes, assuming a plasma volume 0.045 L/kg.
Statistical Analysis
Data are presented as mean±SD. Unpaired t
tests were used to compare results obtained in control subjects and
FHBL heterozygotes. The Pearson correlation coefficient was used to
analyze the correlation between apoB and
triglyceride parameters. A value of
P<0.05 was considered to be significant.
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Results |
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Lipid and Apo Levels
The subjects in the control group and the FHBL subjects were well matched for sex, age, and body mass index (Table 1
). By
definition, plasma levels of total cholesterol, LDL
cholesterol, triglycerides, and apoB were
significantly lower in FHBL heterozygotes than in controls
(P<0.001 for all parameters). There were no
differences in mean plasma HDL cholesterol and apoA1 levels
between the 2 groups (Table 1). Plasma total FFA concentrations
were lower in FHBL heterozygotes than in controls (0.55±0.14 versus
0.72±0.15 mmol/L, P=0.04).
Levels of VLDL apoB and VLDL triglycerides were determined
at 4 different time points during each study. The coefficients of
variation for both apoB and triglycerides were <10%
between different samples, indicating that each subject remained in a
steady state throughout the kinetic study. The concentrations of VLDL
triglycerides, VLDL cholesterol, and VLDL apoB
were significantly lower in FHBL heterozygotes than in control subjects
(P
0.01 for all variables). The mean ratio of
triglyceride to apoB concentrations in VLDL fractions was
higher in FHBL subjects than in normal controls, although the
difference was not statistically significant (16.9±4.9 versus
12.1±4.8, P=0.09) (Table 2).
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Kinetic Parameters
Plasma and VLDL apoB leucine TTR curves from 2
representative studies are shown in Figure 2. As the figure shows, there was a good
agreement between the observed tracer data and the model-derived fits
for VLDL apoB. Mean VLDL apoB FCRs were not significantly different
between the FHBL and control subjects (11.6±3.9 versus 10.9±2.4 pools
per day, P=0.72), whereas the mean production rate
of VLDL apoB in FHBL heterozygotes was 24.8% of that in control
subjects (5.8±1.8 versus 23.4±7.1 mg/kg per day, P<0.001)
(Table 3). The decreased
production rates account for the 75% reduction in VLDL apoB
pool size observed in FHBL subjects.
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) and VLDL apoB
(•) in subject 5 of the control group (A) and subject 6 of FHBL
heterozygotes (B) (see Table 1
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Figure 3 shows plasma and VLDL
triglyceride palmitate TTR curves from 2
representative studies. In all subjects, the enrichment
of palmitate in VLDL trigycerides reached a peak at 60 to 90 minutes
after the bolus injection was given. As was observed for apoB, there
was a good agreement between the observed tracer data and the
model-derived fits. No significant difference in VLDL
triglyceride FCRs was observed between FHBL heterozygotes
and normal subjects (1.06±0.74 versus 0.89±0.50 pools per hour,
P=0.61). On the other hand, the production rates of
VLDL triglycerides were significantly lower in the FHBL
heterozygotes than in the normal controls (9.3±6.0 versus
23.0±9.6 µmol/kg per hour, P=0.008) (Table 4). Most of the reduction in VLDL
triglyceride pool size could be attributed to the decrease
in production rates. However, the decrease in VLDL
triglyceride production rates in FHBL subjects
(59.6%) was less pronounced than the decrease in VLDL apoB
production rates (75.2%).
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There was a good correlation between the FCRs of VLDL apoB and VLDL
triglycerides in the study subjects, as shown in Figure 4 (r=0.84,
P<0.001). The positive significant correlation persisted
when the FCRs of FHBL heterozygotes and controls were analyzed
separately. The correlation between VLDL apoB and VLDL
triglyceride production rates was relatively weaker
(r=0.58, P=0.03) (Figure 5). No significant correlation was
observed when the production rates of each study group were
analyzed separately (r=0.42, P=NS for
FHBL subjects; r=0.11, P=NS for controls).
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Discussion |
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In the present study, we wished to evaluate whether the secretion rates of VLDL apoB and VLDL triglycerides were associated in FHBL heterozygotes and to what extent triglyceride transport was affected in these subjects. Therefore, we investigated simultaneously the in vivo metabolism of the triglyceride and apoB moieties of VLDL in FHBL heterozygotes. The studies were performed in affected members of 2 families with well-characterized mutations in the apoB gene on chromosome 2. Both mutations give rise to truncated apoB forms that are not detectable in plasma because of their location in the 5' region of the apoB gene. Hence, affected members have only one functioning allele and only apoB-100–containing VLDL particles. Our results show that in these FHBL heterozygotes the production rates of VLDL apoB-100 are
25% of normal,
whereas the production rates of triglycerides are
40% of normal. On the other hand, the catabolism of these 2 VLDL
constituents is not altered in FHBL heterozygotes. We have used a novel approach of simultaneous endogenous labeling of VLDL apoB and triglycerides with stable isotope tracers. Stable isotopically labeled leucine has been widely used in the last decade by us6 7 8 9 10 23 and others11 25 26 to study the in vivo metabolism of apoB-100 and various truncated forms of apoB, and compartmental models have been developed to calculate the kinetic parameters of the different apoB-containing lipoproteins. On the other hand, the use of stable isotopically labeled precursors of triglycerides is a relatively new tool in kinetic research. Traditionally, studies of in vivo triglyceride metabolism involved the infusion of radiolabeled precursors such as [3H]glycerol, [14C]glycerol, and [14C]palmitate,13 27 28 29 which have the clear disadvantage of exposing healthy human subjects to radioactive compounds. Recently, Aarsland et al30 and Schwarz et al31 have used mass isotopomer distribution analysis for the measurement of total VLDL triglyceride production rates. They measured the incorporation of [13C]acetate into newly synthesized VLDL fatty acids during a 10-hour infusion of the former. This approach obviates the need for measurement of the precursor pool enrichment. However, it should be borne in mind that this method generates data that depend on de novo synthesis of VLDL triglyceride fatty acids and that in conditions with minimal de novo lipogenesis (ie, fasting state) the measured enrichments can be too low to allow a reliable analysis. The use of stable isotopically labeled precursors that incorporate directly into VLDL triglycerides such as palmitate and glycerol may be advantageous in these settings. We have recently conducted a study comparing the use of deuterated palmitate and deuterated glycerol for the study of VLDL triglyceride kinetics in normal volunteers and found that the kinetic parameters obtained with the 2 tracers were not significantly different (B.W. Patterson, N. Elias, S. Klein, unpublished data, 1999).
One interesting finding in the present study was that VLDL apoB-100
production rates in FHBL heterozygotes were only 25% of
normal, lower than would be expected from the presence of one
functioning allele. This is in accordance with previous studies
performed both under fasting conditions and in the fed state in FHBL
subjects heterozygous for other apoB truncations. Aguilar-Salinas et
al10 have studied the kinetics of apoB-100 in FHBL
heterozygotes with 5 different truncations ranging from apoB-31 to
apoB-89 and found that VLDL apoB-100 production rates were
35% of those observed in normal controls closely matched for age,
sex, and weight. Welty et al11 have found that VLDL
apoB-100 production rates, determined with subjects in the fed
state, were 26% of normal in apoB-67/apoB-100 heterozygotes. The
cellular mechanisms underlying the decreased VLDL apoB-100
production rates in FHBL heterozygotes are not fully
understood. ApoB-100 secretion studied in vitro in hepatoma cell lines
and rat hepatocytes appears to be under posttranscrptional
regulation.32 33 The amount secreted is determined by the
partitioning of newly synthesized apoB-100 between degradation in the
endoplasmic reticulum and progression to secretory pathways.
Theoretically, the presence of abnormal truncated apoB forms within the
hepatocyte may interfere with these processes, leading to
increased intracellular degradation and decreased secretion of
apoB-100. This is supported by the findings of a recent study in HepG2
cells, which were genetically modified to express apoB-82 in addition
to the naturally occurring apoB-100. A greater extent of presecretory
degradation of newly synthesized apoB-100 was demonstrated in these
cells compared with normal "wild-type" cells, which led to lower
than expected secretion of apoB-100.34 Although there is
still no evidence that intracellular degradation plays a major role in
the regulation of apoB secretion in humans, these in vitro observations
are in accordance with our in vivo findings. The decreased
production rates explain the very low plasma apoB
concentrations in affected members of these 2 families, which were 24%
of those found in normal controls. The clearance of the
apoB-100–containing VLDL particles by the lipolytic pathway or
receptor-dependent uptake seems to be unaffected, as reflected by
normal VLDL apoB FCRs.
The hepatic secretion of VLDL triglycerides was decreased in our FHBL heterozygotes but to a lesser extent than VLDL apoB secretion, the latter being the factor determining the number of VLDL particles secreted by the liver. Thus, the particles secreted in FHBL heterozygotes are relatively enriched with triglycerides. The ability of the liver to secrete VLDL particles with different triglyceride content under different conditions has been established in previous studies.35 In vitro observations in rat hepatocyte cell lines led to the hypothesis that the assembly of VLDL proceeds in 2 steps, the first of which involves the synthesis of a lipid-poor apoB–containing particle. In the second step, there is a fusion of this particle with a preformed apoB-free lipid core (synthesized in the smooth endoplasmic reticulum) to become a mature VLDL particle.36 37 38 Recently, Hamilton et al,39 studying genetically modified mice lacking apoB in intestinal cells, have shown by electron microscopy that these lipid cores can grow larger in the absence of apoB. They hypothesized that the larger size of these particles is due to a retarded rate of transport through the endoplasmic reticular compartment as a consequence of apoB deficiency, which allows them to accumulate more lipids. It is possible that in FHBL heterozygotes with sparse amounts of apoB in hepatocytes, the amount of lipid added to the core particles is higher than usual. Some of these particles would then fuse with lipid-poor apoB particles to produce triglyceride-enriched VLDL. However, any supporting evidence that these processes play a role in VLDL assembly in vivo is still lacking.
Although there appears to be some compensation in triglyceride secretion by the liver, the rates are still reduced by 60% in FHBL heterozygotes compared with normal subjects. This defect in triglyceride export may lead to accumulation of triglycerides in hepatocytes and raises a question about the occurrence of fatty liver in these subjects. There is little information concerning the prevalence of fatty liver in FHBL heterozygotes. In a search of the literature, we could find only one case report of fatty liver in an FHBL heterozygote with well-documented apoB truncation (apoB-38.9).40 In 2 other reported cases of fatty liver associated with hypocholesterolemia, the diagnosis of FHBL was suspected, but no apoB truncations were found.41 42 The occurrence of fatty liver in FHBL depends on the balance between hepatic triglyceride synthetic rates and VLDL triglyceride secretion rates; in other words, it depends on what proportion of triglyceride synthesized can be exported from the liver in VLDL particles. Hepatic triglyceride synthesis is controlled by nutritional and hormonal factors and depends largely on the supply of fatty acids to the liver. These originate from 3 main sources: plasma FFA, hydrolysis of lipoproteins, and de novo lipogenesis.12 Although we found a modest decrease in plasma FFA concentrations in our FHBL heterozygotes compared with normal controls, the levels were still within normal limits. It will not be feasible to assess hepatic triglyceride synthetic rates in human subjects by in vivo metabolic studies. Animal models of FHBL and in vitro studies in genetically modified cultured hepatocytes may prove to be useful in testing this point.
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Acknowledgments |
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This study was supported by National Institutes of Health grant R01 HL-42460, General Clinical Research Center grant USPHS MO1 RROOO36, and Biomedical Mass Spectrometry Resource grant RR 00954. The authors thank the personnel of the Core Laboratory at Washington University School of Medicine for their expert technical assistance and the staff of the General Clinical Research Center for help in performing the metabolic studies. We express our gratitude to Sherry Banez-Muth for recruiting the study subjects and Tom Kitchens for his expert technical help.
Received February 11, 1999; accepted March 24, 1999.
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References |
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- Schonfeld G. The hypobetalipoproteinemias. Annu Rev Nutr.. 1995;15:23–34.[Medline] [Order article via Infotrieve]
- Linton MF, Farese RV, Young SG. Familial hypobetalipoproteinemia. J Lipid Res.. 1993;34:521–541.[Medline] [Order article via Infotrieve]
-
Collins DR, Knott TG, Pease RJ, Powell LM, Wallis
SC, Robertson S, Pullinger CR, Milne RM, Marcel YL, Humphries SE,
Talmud PJ, Lloyd JK, Miller NE, Muller D, Scott J. Truncated variants
of apolipoprotein B cause
hypobetalipoproteinemia. Nucleic
Acids Res. 1988;16:8361–8375.
[Abstract/Free Full Text] -
Huang LS, Ripps ME, Korman SH, Deckelbaum RJ,
Breslow JL. Hypobetalipoproteinemia due
to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu
recombination. J Biol Chem.. 1989;264:11394–11400.
[Abstract/Free Full Text] - Huang LS, Kayden H, Sokol RJ, Breslow JL. ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia. J Lipid Res.. 1991;32:1341–1348.[Abstract]
- Parhofer KG, Barrett PHR, Bier DM, Schonfeld G. Positive linear correlation between the length of truncated apolipoprotein B and its secretion rate (in vivo studies in apoB-89, apoB-75, apoB-54.8, and apoB-31 heterozygotes). J Lipid Res.. 1996;37:844–852.[Abstract]
- Srivastava N, Noto D, Averna M, Pulai J, Srivastava RAK, Cole TG, Latour MA, Patterson BW, Schonfeld G. A new apolipoprotein B truncation (apoB 43.7) in familial hypobetalipoproteinemia: genetic and metabolic studies. Metabolism.. 1996;45:1296–1304.[Medline] [Order article via Infotrieve]
- Parhofer KG, Barrett PHR, Bier DM, Schonfeld G. Lipoproteins containing the truncated apolipoprotein, apoB-89, are cleared from human plasma more rapidly than apoB-100 containing lipoproteins in vivo. J Clin Invest.. 1992;89:1931–1937.
- Krul ES, Parhofer KG, Barrett PHR, Wagner RD, Schonfeld G. ApoB-75, a truncation of apolipoprotein B associated with familial hypobetalipoproteinemia: genetic and kinetic studies. J Lipid Res.. 1992;33:1037–1050.[Abstract]
-
Aguilar-Salinas CA, Barrett PHR, Parhofer KG, Young SG,
Tessareau D, Bateman J, Quinn C, Schonfeld G. Apolipoprotein B-100
production is decreased in subjects heterozygous for
truncations of apoprotein B. Arterioscler Thromb Vasc Biol.. 1995;15:71–80.
[Abstract/Free Full Text] -
Welty FK, Lichtenstein AH, Barrett PHR, Dolnikowski GG,
Ordovas JM, Schaefer EJ. Decreased production and increased
catabolism of apolipoprotein B-100 in apolipoprotein B-67/B-100
heterozygotes. Arterioscler Thromb Vasc Biol.. 1997;17:881–888.
[Abstract/Free Full Text] - Lewis GF. Fatty acid regulation of very low density lipoprotein production. Curr Opin Lipidol.. 1997;8:146–153.[Medline] [Order article via Infotrieve]
- Kissebah AH, Alfarsi S, Adams PW. Integrated regulation of very low density lipoprotein triglyceride and apolipoprotein-B kinetics in man: normolipidemic subjects, familial hypertriglyceridemia and familial combined hyperlipidemia. Metabolism.. 1981;30:856–868.[Medline] [Order article via Infotrieve]
-
Melish G, Le NA, Ginsberg H, Steinberg D, Brown WV.
Dissociation of apoprotein B and triglyceride
production in very low density lipoproteins. Am J
Physiol.. 1980;239:E354–E362.
[Abstract/Free Full Text] - Steiner G, Reardon MF. A new model of human VLDL metabolism based on simultaneous studies of its apoB and triglyceride. Metabolism.. 1983;32:342–347.[Medline] [Order article via Infotrieve]
-
Wu J, Kim J, Li Q, Kwok PY, Cole TG, Cefalu B, Averna
M, Schonfeld G. Known mutations of apoB account for only a small
minority of hypobetalipoproteinemia.
J Lipid Res.. 1999;40:955–959.
[Abstract/Free Full Text] - Pulai JI, Hamideh Z, Kwok PY, Kim JH, Wu J, Schonfeld G. Donor splice mutation (665+1 G-T) in familial hypobetalipoproteinemia with no detectable apoB truncation. Am J Med Genet.. 1998;80:218–220.[Medline] [Order article via Infotrieve]
- Klein RL, Zilversmit DB. Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether. J Lipid Res.. 1984;25:1380–1386.[Abstract]
- Adams RF. Determination of amino acid profiles in biological samples by gas chromatography. J Chromatogr.. 1974;95:189–212.[Medline] [Order article via Infotrieve]
- Patterson BW, Hachey DL, Cook GL, Amann JM, Klein PD. Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins. J Lipid Res.. 1991;32:1063–1072.[Abstract]
- Patterson BW, Zhao G, Klein S. Improved accuracy and precision of gas chromatography/mass spectrometry measurements for metabolic tracers. Metabolism.. 1998;47:706–712.[Medline] [Order article via Infotrieve]
- Carson ER, Cobelli C, Finkelstein L. The Mathematical Modeling of Metabolic and Endocrine Systems. New York, NY: John Wiley & Sons Inc; 1983.
- Parhofer KG, Barrett PHR, Bier DM, Schonfeld G. Determination of kinetic parameters of apolipoprotein B metabolism using amino acids labeled with stable isotopes. J Lipid Res.. 1991;32:1311–1323.[Abstract]
- Zech LA, Grundy SM, Steinberg D, Berman M. Kinetic model for production and metabolism of very low density lipoprotein triglycerides: evidence for a slow production pathway and results for normolipidemic subjects. J Clin Invest.. 1979;63:1262–1273.
- Cohn JS, Wagner DA, Cohn SD, Millar JS, Schaefer EJ. Measurement of very low density and low density lipoprotein apolipoprotein (apo) B-100 and high density lipoprotein apo A-I production in human subjects using deuterated leucine: effect of fasting and feeding. J Clin Invest.. 1990;85:804–811.
- Lichtenstein AH, Hachey DL, Millar JS, Jenner JL, Booth L, Ordovas J, Schaefer EJ. Measurement of human apolipoprotein B-48 and B-100 kinetics in triglyceride-rich lipoproteins using [5,5,5-2H3] leucine. J Lipid Res.. 1992;33:907–914.[Abstract]
- Quarfordt SH, Frank A, Shames DM, Berman M, Steinberg D. Very low density lipoprotein triglyceride transport in type IV hyperlipoproteinemia and the effects of carbohydrate-rich diets. J Clin Invest.. 1970;49:2281–2297.
- Grundy SM, Mok HYI, Zech L, Steinberg D, Berman M. Transport of very low density lipoprotein triglycerides in varying degrees of obesity and hypertriglyceridemia. J Clin Invest.. 1979;63:1274–1283.
- Shumak SL, Zinman B, Zuniga-Guarjardo S, Poapst M, Steiner G. Triglyceride-rich lipoprotein metabolism during acute hyperinsulinemia in hypertriglyceridemic humans. Metabolism.. 1988;37:461–466.[Medline] [Order article via Infotrieve]
- Aarsland A, Chinkes D, Wolfe RR. Contributions of de novo synthesis of fatty acids to total VLDL-triglyceride secretion during prolonged hyperglycemia/hyperinsulinemia in normal man. J Clin Invest.. 1996;98:2008–2017.[Medline] [Order article via Infotrieve]
- Schwarz JM, Neese RA, Turner S, Dare D, Hellerstein MK. Short-term alterations in carbohydrate energy intake in humans. J Clin Invest. 1995;96:2735–2743.
- Gibbons GF. Assembly and secretion of hepatic very low density lipoprotein. Biochem J.. 1990;268:1–13.[Medline] [Order article via Infotrieve]
- Ginsberg HN. Synthesis and secretion of apolipoprotein B from cultured liver cells. Curr Opin Lipidol.. 1995;6:275–280.[Medline] [Order article via Infotrieve]
-
Srivastava RAK, Srivastava N, Averna M, Cefalu AB,
Schonfeld G. Molecular bases of low production rates of
apolipoprotein B-100 and truncated apoB-82 in a mutant HepG2 cell line
generated by targeted modification of the apolipoprotein B gene.
J Lipid Res.. 1999;40:901–912.
[Abstract/Free Full Text] -
Sniderman AD, Cianflone K. Substrate delivery as a
determinant of hepatic apoB secretion. Arterioscler Thromb.. 1993;13:629–636.
[Abstract/Free Full Text] -
Boren J, Rustaeus S, Olofsson SO. Studies on the
assembly of apolipoprotein B100 and apoB48-containing very low density
lipoprotein in McARH7777 cells. J Biol Chem.. 1994;269:25879–25888.
[Abstract/Free Full Text] -
Gordon DA, Jamil H, Gregg RE, Olofsson S-O, Boren
J. Inhibition of the microsomal triglyceride transfer
protein blocks the first step of apolipoprotein B lipoprotein assembly
but not the addition of bulk core lipids in the second step.
J Biol Chem.. 1996;271:33047–33053.
[Abstract/Free Full Text] - Hamilton RL, Erickson SK, Havel RJ. Nascent VLDL assembly occurs in two steps in the endoplasmic reticulum (ER) of hepatocytes. In: Woodford XFP, Davignon J, Sniderman A, eds. Atherosclerosis. Amsterdam, Netherlands. Elsevier Science; 1995:414–418.
-
Hamilton RL, Wong JS, Cham CM, Nielsen LB, Young SG.
Chylomicron-sized lipid particles are formed in the setting of
apolipoprotein B deficiency. J Lipid Res.. 1998;39:1543–1557.
[Abstract/Free Full Text] - Tarugi P, Lonardo A, Ballarini G, Grisendi A, Pulvirenti M, Bagni A, Calandra S. Fatty liver in heterozygous hypobetalipoproteinemia caused by a novel truncated form of apolipoprotein B. Gastroenterology.. 1996;111:1125–1133.[Medline] [Order article via Infotrieve]
- Hagve TA, Myrseth LE, Schrumpf E, Blomhoff JP, Christophersen B, Elgjo K, Gjone E, Prydz H. Liver steatosis in hypobetalipoproteinemia. J Hepatol.. 1991;13:104–111.[Medline] [Order article via Infotrieve]
- Wishingrad M, Paaso B, Garcia G. Fatty liver due to heterozygous hypobetalipoproteinemia. Am J Gastroenterol.. 1994;89:1106–1107.[Medline] [Order article via Infotrieve]
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