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Triglyceride-Rich Lipoprotein-Associated Apolipoprotein
C-III Production Is Stimulated by Plasma Free Fatty
Acids in Humans
Mirjana Pavlic, René Valéro, Hélène Duez, Changting Xiao, Linda Szeto,
Bruce W. Patterson, Gary F. Lewis
Downloaded from http://atvb.ahajournals.org/ by guest on May 12, 2017
Objective—Insulin resistant states are associated with increased fatty acid flux to liver and intestine, which stimulates the
production of triglyceride-rich lipoproteins (TRL). ApoC-III production and plasma and TRL concentrations are
increased in insulin resistance and may contribute to the hypertriglyceridemia of these conditions. The mechanism
underlying that increase is not known, but because apoC-III and VLDL production are closely linked we hypothesized
that FFAs may stimulate TRL apoC-III production.
Methods and Results—We used Intralipid/heparin (IH) to raise plasma FFA in 12 healthy men in the fed state, and stable
isotopes to examine apoC-III metabolism. TRL apoC-III concentration was significantly higher in the IH study, and this
increase was associated with higher production (PR) and fractional catabolic rate (FCR). The increase in production was
greater than in FCR (90% versus 30%, respectively), accounting for the elevated concentration. Glycerol infusion had
no effect on apoC-III concentration, PR, or FCR compared to saline, indicating that the effect was not attributable to
glycerol released from intralipid.
Conclusion—These findings confirm that TRL apoC-III production is stimulated by an acute elevation of plasma FFAs,
suggesting a novel regulatory pathway that may play a role in the overproduction of TRL apoC-III in insulin resistant
states. (Arterioscler Thromb Vasc Biol. 2008;28:1660-1665)
Key Words: apolipoprotein C3 䡲 free fatty acids 䡲 insulin resistance 䡲 lipoprotein
H
ypertriglyceridemia is a well described feature of insulin
resistance and type 2 diabetes and is commonly associated with other lipid abnormalities such as low plasma HDL
concentration and a shift toward smaller and denser LDL
particles, as well as an increased risk of atherosclerotic
cardiovascular disease.1 This hypertriglyceridemia is attributable predominantly to an increased rate of production of
triglyceride rich lipoproteins (TRL) by both the liver (reviewed in2) and intestine.3,4 Several factors contribute to the
overproduction of TRL particles in these conditions; including increased flux of fatty acids to liver and intestine,
increased de novo lipogenesis, increased stability of apoB,
and overexpression of microsomal transfer protein (MTP),
the latter contributing to the increased efficiency of lipoprotein assembly and secretion.5,6
Reduced clearance of the triglyceride moiety of TRL and
of the particles themselves also contributes to the hypertriglyceridemia (reviewed in2), particularly when the metabolic
control of type 2 diabetes is poor and in the postprandial state,
when there is a large influx of intestinally-derived TRL
particles. Functional deficiency of lipoprotein lipase (LPL) or
competition for a common, saturable, lipolytic pathway has
been shown to play an important contributing role in this
impaired TRL clearance.7 Additionally, elevated apoC-III in
insulin resistance and in type 2 diabetes8 –14 has also been
postulated to contribute to the impairment of TRL particle
clearance, possibly by inhibiting the activity of LPL15 or by
interfering with the interaction between TRL particles and
hepatic lipoprotein receptors.16,17 Major insight into the function of apoC-III has been obtained from genetically modified
mouse models, with overexpression of apoC-III resulting in
severe hypertriglyceridemia18 and apoC-III deficiency resulting in hypotriglyceridemia.19
ApoC-III is synthesized mainly by the liver and to a lesser
extent by the intestine (reviewed in20,21). Mature apoC-III is a
79 amino-acid glycoprotein present in VLDL, chylomicrons,
and HDL, and has the ability to exchange between TG-rich
lipoproteins and HDL.22 In normolipidemic subjects the
majority of plasma apoC-III is bound to HDL whereas in
hypertriglyceridemic subjects the majority is bound to TRL.21
Original received March 20, 2008; final version accepted June 5, 2008.
From the Departments of Medicine and Physiology, Division of Endocrinology and Metabolism (M.P., R.V., H.D., C.X., L.S., G.F.L.), University of
Toronto, Canada; and the Washington University School of Medicine (B.W.P.), St. Louis, Mo.
M.P. and R.V. contributed equally to this study.
Correspondence to Dr Gary F. Lewis, Toronto General Hospital, 200 Elizabeth Street, EN12-218, Toronto, Ontario, M5G 2C4. E-mail
[email protected]
© 2008 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1660
DOI: 10.1161/ATVBAHA.108.169383
Pavlic et al
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Although apoC-III is a relatively minor component of HDL
protein, approximately 50% of total plasma apoC-III may be
in the HDL fraction.21 In humans, the APOA1/C3/A4/A5
gene cluster is located on chromosome 11q23.23 ApoC-III
gene expression is normally downregulated by insulin24 via
Foxo1.25 A variant promoter that is defective in its response
to insulin results in overexpression of the apoC-III gene,
contributing to the development of hypertriglyceridemia.26
These observations have led some to hypothesize that overexpression of apoC-III in insulin resistant states leads to an
elevation of TRL apoC-III concentration and that this phenomenon is an important contributor to the highly prevalent
hypertriglyceridemia that occurs in insulin resistance.26 In
support of this notion, insulin sensitizing therapy with the
PPAR␥ agonist pioglitazone in patients with type 2 diabetes
reduced apoC-III production, plasma apoC-III concentration,
and lowered plasma triglycerides,27 although it should be
noted that pioglitazone also has some PPAR␣ agonist activity
and the drug may have mediated its effect on apoC-III
production and triglyceride metabolism by a mechanism that
is not dependent on insulin sensitization. Insulin resistance
and apoC-III production are correlated in humans,11 and
increased VLDL apoC-III concentration resulting from its
overproduction is strongly associated with the delayed catabolism of triglycerides and apoB in VLDL.28
Fatty acid flux from adipose tissue is increased in insulin
resistant states and plays an important role in stimulating the
production of TRL particles by the liver.6,29 More recently we
have made the observation that acute elevation of plasma
FFAs by Intralipid/heparin increased intestinal lipoprotein
production in the Syrian Golden hamster30 and in humans.31
Because increased apoC-III production is closely linked with
VLDL-triglyceride production,32 we hypothesized that TRL
apoC-III production may also be stimulated by elevated
plasma FFAs, providing an alternative mechanism explaining
the increased rate of production of TRL apoC-III in insulin
resistance. In the present study we examined the effect of an
acute elevation of plasma FFAs on TRL apoC-III production
in healthy men and found that TRL apoC-III production rate
is indeed increased by an acute elevation of plasma FFAs.
Materials and Methods
Subjects
The subjects participating in the present study and the study protocol
have been reported previously.31 The previous report of these
subjects focused exclusively on the kinetics of TRL-apoB100 and
TRL-apoB48 in response to an acute elevation of plasma FFA.31 The
demographic characteristics and fasting biochemical profiles of the
12 study participants were described in detail in that publication,31
and only mean values are presented in Table 1 of the present article.
All were healthy male subjects with no previous history of cardiovascular, gastrointestinal, or systemic illness, or surgical intervention
within 6 months before the studies. No subject was taking medications, and they were all normoglycemic. To exclude those with
gross dyslipidemia from the study, individuals whose total cholesterol was ⬎5.3 mmol/L, TG ⬎3 mmol/L, HDL-c ⬍0.9 mmol/L,
LDL-c ⬎3.2 mmol/L were excluded.
The Research Ethics Board of the University Health Network,
University of Toronto, approved the study and all subjects gave
written informed consent prior to their participation.
Fatty Acids Stimulate TRL–ApoC-III Production
1661
Table 1. Demographic Characteristics and Mean Fasting
Biochemical Profiles of Study Participants
Mean⫾SEM
Range
Age, y
43.1⫾4.9
30–57
Weight, kg
76.4⫾8.4
66.8–101
Waist, cm
98.0⫾2.8
87–112
BMI, kg/m2
24.5⫾2.7
22.6–31.7
Glucose, mmol/l
4.5⫾0.48
Insulin, pmol/l
52.1⫾7.4
FFA, mmol/L
4.3–5.5
27.2–89.5
0.5⫾0.01
0.2–1.0
Plasma TC, mmol/L
4.08⫾0.45
3.40–5.25
Plasma TG, mmol/L
1.24⫾0.21
0.72–2.87
LDL-c, mmol/L
2.59⫾0.16
1.6–3.17
TRL apoC-III, mg/dl
4.72⫾0.84
1.57–14.7
Experimental Protocol for Lipoprotein
Kinetic Studies
The experimental protocol has previously been described in detail.31
Briefly, 12 subjects underwent 2 separate 12-hour lipoprotein turnover studies, both conducted during a constant fed state, in random
order, 4 to 6 weeks apart. One study was performed during
intravenous infusion of Intralipid (a synthetic triglyceride emulsion,
suitable for intravenous infusion, that provides a source of mainly
polyunsaturated FFAs) and heparin (to activate lipoprotein lipase,
which stimulates intravascular lipolysis of the intralipid triglycerides), and the other during intravenous saline infusion as a control
study. Because the infusion of Intralipid raises both FFAs and
glycerol, a subset of 5 subjects also underwent a third kinetics study
during i.v. infusion of glycerol as an additional control study to
differentiate the effects of FFA from potential effects of glycerol
released by intravascular lipolysis of Intralipid. In this case, all 3
studies were performed in random order, 4 to 6 weeks apart.
After an overnight fast, an intravenous catheter was inserted into
a superficial vein in each forearm, one for infusion and one for blood
sampling. At 4 AM the subject was instructed to start ingesting the
first aliquot of a liquid food supplement, hourly for the first 3 hours
(Hormel Great Shake Plus, Hormel Health Labs, 49% calories from
fat, 38% from carbohydrates, 13% from proteins), each hourly
aliquot equivalent to 1/17th of their total daily caloric needs. After
the first 3 hours (after 7 AM), the subjects ingested the same formula
every half hour for the remainder of the study, ie, 1/34th of their
daily caloric intake every half hour. Total energy requirements to
achieve a constant fed state were determined using the Harris
Benedict Equation. At 4 AM, at the same time as the start of the
hourly formula ingestion, an i.v. infusion with either Intralipid/
heparin (IH; Baxter; Intralipid 20% at 20 mL/h plus heparin 250U/h)
or saline (65 mL/h) or glycerol (2.25 g/h) was started and was
constantly infused for the full 17-hour duration of the study. Five
hours after starting the liquid formula ingestion (at 9 AM), a primed
constant infusion of deuterium-labeled leucine33 ([D3]L-leucine
98%, Cambridge Isotope Laboratories) was administered
(10 ␮mol/kg bolus followed by 10 ␮mol/kg.hr for 12 hours) to
enrich apoC-III to calculate production and clearance rates, as
previously described.34 Blood samples were collected at regular
intervals as previously described.31 The total blood volume drawn
during the entire study was ⬍350 mL. The hematocrit does not
change appreciably after removal of this amount of blood.
For details on laboratory methods, calculation of lipoprotein
clearance and production rates by multicompartmental modeling,
and statistical analysis, please see the data supplement (available
online at http://atvb.ahajournals.org).
Results
Glycerol Control Study
We have recently demonstrated that glycerol infusion has no
effect on plasma FFA, TG, TRL-apoB100, or TRL apoB48
Arterioscler Thromb Vasc Biol
September 2008
Table 2. Individual Subject’s Mean Triglyceride-Rich
Lipoprotein (TRL) ApoC-III Concentration, Production Rate,
and FCR During the Intralipid/Heparin and the Saline
Infusion Studies
TRL ApoC-III-(fed)
mg/dl
TRL ApoC-III FCR
pools/d
TRL ApoC-III PR
mg/kg 䡠 d
1
5.0
1.256
2.800
2
7.2
1.329
4.315
3
3.7
1.649
2.747
4
6.3
1.227
3.472
Subject
Intralip/Heparin
5
7.3
0.887
2.932
6
12.3
1.994
11.055
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7
7.7
1.071
3.716
8
14.7
0.911
6.019
9
13.5
0.748
4.559
10
5.7
0.654
1.657
11
5.2
0.650
1.521
12
11.7
1.318
6.961
1.14⫾0.12
4.31⫾1.25
Mean⫾SEM
8.37⫾2.41
Control
1
2.9
0.985
1.287
2
4.8
0.841
1.800
3
3.2
1.196
1.744
4
4.8
0.838
1.827
5
7.5
0.753
2.986
6
7.6
1.399
4.800
7
7.5
0.890
3.003
8
11.5
0.723
3.734
9
7.0
0.851
2.684
10
2.9
0.484
0.634
11
3.5
0.441
0.703.
12
4.5
0.951
1.931
5.65⫾0.8
0.86⫾0.08
2.26⫾0.36
0.002
0.0001
0.003
Mean⫾SEM
P value
TRL-apoCIII mean values for the period 5 hrs after the start of IH infusion,
ie for the duration of the subsequent 12 hr turnover study.
Statistics are by paired t test for comparison of Intralipid/heparin and saline
infusion studies.
levels, production and clearance rates compared to saline.31
Here we found no difference between glycerol and saline
infusion on TRL apoC-III concentration (saline 5.52⫾0.9
versus glycerol 6.0⫾0.46 mg/dL, P⫽0.464), production (saline 2.54⫾0.63 versus glycerol 2.14⫾0.20 mg/kg 䡠 d,
P⫽0.617), and clearance (saline 0.99⫾0.11 versus glycerol
0.80⫾0.06 pool/d, P⫽0.300). For clarity, and because power
calculation estimated n⫽64 subjects would be needed to
detect a significant difference between the kinetics parameters for TRL apoC-III in the saline and glycerol studies at
P⫽0.05 with 80% power, the following results and illustration will therefore compare only the Intralipid/heparin and
saline studies.
TRL and HDL apoC-III conc (mg/dl)
1662
14
IH HDL
Saline HDL
IH TRL
Saline TRL
12
10
8
*
6
4
*
2
0
0
5
10
15
20
Time (hr)
Figure 1. TRL and HDL apoC-III concentrations over the time
course of the kinetics study. ApoC-III concentrations were measured after an overnight fast, (time 0 hour), and throughout the
12-hour lipoprotein turnover study (5 to 17 hours) in subjects
receiving either Intralipid/heparin or saline (n⫽12). Values are
mean⫾SEM for each group. The overall significance over the
time course of the kinetics study for difference in TRL apoC-III
concentration between the 2 studies was *P⬍0.0046. HDL
apoC-III concentration decreased in the IH study, and the overall significant difference over time course of the kinetics study
was *P⬍0.05.
Effect of Acute Elevation of FFAs on Plasma and
TRL Lipid and TRL and HDL Apolipoprotein
Concentrations in the Constant Fed State
We have previously shown that plasma FFA, plasma TG
levels, and TRL-TG increased during feeding, to a greater
extent with Intralipid/heparin infusion compared to saline and
plasma concentrations, remained stable in both the saline and
IH studies for the duration of the 12-hour kinetics study
(illustrated in31). Similary, TRL apoC-III concentrations increased with feeding, remained stable during the 12-hour
kinetics study, and were significantly higher in the IH study
compared to saline (IH 8.37⫾2.41 mg/dL versus Saline
5.65⫾0.75 mg/dL, P⫽0.002) (Table 2 and Figure 1). HDL
apoC-III concentration decreased with feeding, remained
stable during the kinetics study, and was significantly lower
in IH compared to saline control (IH 2.50⫾0.48 mg/dL
versus Saline 3.17⫾0.48, P⫽0.012; Figure 1).
Effects of Acute Elevation of FFAs on TRL
ApoC-III Lipoprotein Production and
Clearance Rates
Individual subject’s TRL apoC-III pool sizes, production
rates, and FCRs for the IH and the saline infusion studies are
provided in Table 2, and mean production rate and FCR
results are depicted graphically in Figure 2. The mean TRL
apoC-III fractional catabolic rate was 30% higher in the IH
compared to the saline control study (IH 1.14⫾0.12 versus
saline 0.86⫾0.08 pool/d, P⫽0.001; Figure 2A). The production rate of TRL apoC-III increased by 90% in subjects
infused with IH compared to saline (IH 4.31⫾0.77 versus
saline 2.26⫾0.36 mg/kg 䡠 d, P⫽0.003; Figure 2B).
Discussion
There is a positive association between TRL apoC-III and
TRL triglyceride concentrations and production rates2,11 and
also TRL apo-B concentrations and production rates,28,32,35
B
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TRL-apo-CIII Production Rate (mg/kg.day)
A
TRL-apo-CIII Fractional Catabolic Rate
(pools/day)
Pavlic et al
2
Saline Control
* p=0.001
1.6
IH
1.2
0.8
0.4
0
6
5
Saline Control
* p=0.003
IH
4
3
2
1
0
Figure 2. Effect of Intralipid/heparin infusion on mean TRL
apoC-III fractional catabolic (FCR) and production rates (PR).
TRL apoC-III FCR (A) and PR (B) were determined at steady
state in subjects receiving either Intralipid/heparin (black bars) or
saline infusion (white bars). Both TRL apoC-III FCR and PR were
significantly increased by the Intralipid/heparin infusion compared to saline (*P⫽0.001 and *P⫽0.003, respectively).
which are increased in insulin resistant states. Elevated FFA
flux from extrahepatic tissues is one important cause of the
overproduction of VLDL in insulin resistance,6,29,31,36,37 and
we have recently shown in the Syrian Golden hamster30 and
in humans31 that intestinal lipoprotein production is also
stimulated by an acute elevation of plasma FFAs. Here, we
examined whether an acute elevation of FFAs also stimulates
TRL apoC-III production in healthy humans. Our findings
confirm that TRL-associated apoC-III production is stimulated by an acute elevation of plasma FFAs, suggesting a
novel regulatory pathway that may play a role in the overproduction of TRL apoC-III in insulin resistant states.
Kinetics studies in human subjects have shown that increased production rather than decreased clearance of apoCIII is the most important determinant of the elevated plasma
apoC-III levels that characterize patients with hypertriglyceridemia and features of insulin resistance.11,32 We used Intralipid/heparin infusion to raise plasma FFA concentration in
the fed state and well established stable isotope enrichment
techniques to examine TRL apoC-III metabolism. The TRL
apoC-III concentration was significantly higher in the IH
infusion study compared with saline control study, and this
increase was associated with both higher production rates of
TRL apoC-III as well as higher FCR, with the increase in
production rate being of greater relative magnitude (90%
higher production rate in IH versus saline) than the increase
in FCR (30% higher FCR in IH versus saline), providing an
explanation for the increase in concentrations. Glycerol had
no effect on apoC-III concentration, production or clearance
rates when compared to saline infusion, indicating that the
effect of IH was attributable to the FFAs released from the
Fatty Acids Stimulate TRL–ApoC-III Production
1663
synthetic TG emulsion by the action of heparin-stimulated
LPL, and not attributable to the release of glycerol.
Unlike apoB, apoC-III and other apolipoproteins are able
to exchange actively between lipid surfaces, and apoC-III
redistribution to intralipid particles must be considered in the
interpretation of our findings. Larger intralipid particles
would be expected to contain apoC-III and other exchangeable apolipoproteins, as has been demonstrated by others in
the past.38,39 Whereas newly secreted apoC-III is very likely
to be rapidly transferred to intralipid particles, there is no
known mechanism for apolipoprotein feeding back on its own
gene transcription, and therefore by far the most likely culprit
responsible for enhancing apoC-III production by 90% in our
studies is still the FFA released by intravascular lipolysis of
intralipid. As shown in Figure 1, there was a slight decline in
HDL apoC-III during the Intralipid/heparin infusion experiment. This decline likely reflects the transfer of HDL apoCIII to the increased pool of TRL and intralipid particles.
Because the residence time of HDL apoC-III has been shown
to be 3 times longer than VLDL apoC-III,34 and if intralipid
particle-associated apoC-III is also cleared faster than HDL
apoC-III because of enhanced lipolysis of intralipid by
heparin-stimulated LPL, this transfer of apoC-III from HDL
to TRL could account for the 30% higher FCR that we
documented during Intralipid/heparin versus saline infusion.
Limitations of the present study must be addressed. First,
for practical reasons the study was designed to examine the
effect of a short-term elevation of plasma FFAs on lipoprotein metabolism. It is not feasible nor is it technically
possible, considering issues of stable isotope recycling that
complicate interpretation of data with prolonged infusion, to
examine longer term elevations of plasma FFAs on
lipoprotein-associated apolipoprotein kinetics. Extrapolation
of our findings to clinical conditions that are associated with
chronic elevations of plasma FFAs, such as insulin resistance
and type 2 diabetes, should therefore be made with appropriate caution. The levels of FFA achieved in the present study
were well within the physiological range that occurs in those
with insulin resistance or type 2 diabetes. Second, our study
was conducted in the fed state to facilitate the assessment of
TRL apoB48 kinetics (reported in31). This was necessary
because of the technical challenge of measuring very low
concentrations of apoB-48-containing particles. Our finding
that TRL apoC-III production rates were increased in the IH
study and stimulated by acute elevation of plasma FFA were
made in the setting of high-fat feeding and ongoing fat
absorption. We cannot say with certainty that the same would
have been observed in the fasted state. This would have to be
specifically examined in future studies. Third, because apoCIII is produced in both liver and intestine, we were unable to
determine whether the increase in production occurred primarily in the liver, primarily in the intestine, or in both organs
to a relatively similar extent. Fourth, an in vivo human study
of this nature cannot examine the molecular mechanisms of
the increased apoC-III production and cannot determine
whether the increase occurred as a result of upregulation of
apoC-III gene transcription or whether the effect occurred at
the posttranslational level. In addition we cannot determine
whether the increase in production of TRL apoC-III occurred
1664
Arterioscler Thromb Vasc Biol
September 2008
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as a direct result of fatty acids stimulating the hepatocyte or
intestinal enterocyte, was mediated through one or a number
of intermediary regulators of apoC-III secretion, or whether
the effect occurred secondary to the well described insulin
resistance that occurs with elevation of plasma FFAs.40 Given
our findings of a simultaneous upregulation of TRL apoB48,
apoB100,31 and apoC-III production in response to the
elevation of plasma FFAs, and because the regulation of
apoB48 and apoB100 by fatty acids has been shown to occur
primarily at the posttranslational level by inhibition of apoB
degradation and enhancement of lipoprotein assembly,41 it is
quite possible that the increase in apoC-III production seen in
the present study also occurred posttranslationally. This
interesting issue remains to be determined in future in vitro or
ex vivo mechanistic studies.
As can be seen in Figure 1, TRL apoC-III concentration
(pool size, on which our kinetic modeling was based) was not
in perfect steady state during the 12-hour kinetics study (5 to
17 hours). We turned to model simulations to assess the
potential impact of a nonsteady state on the analysis of
apoC-III turnover kinetics, as we have described and illustrated in the online Data Supplement. Based on these model
simulations and for the reasons described in the online Data
Supplement, we are confident that our calculated production
rates are valid and are not affected by the small perturbations
of steady state that occurred in this study.
In conclusion, the present report provides evidence that
TRL apo-CIII production is stimulated by a short-term
elevation of plasma FFAs in healthy men. It is reasonable to
assume that chronic elevation of plasma FFAs seen in type 2
diabetes and insulin resistance could play an important role in
TRL apoC-III overproduction, which in turn could aggravate
the hypertriglyceridemia of insulin resistant states and type 2
diabetes. Given the complexity of the mechanisms that have
been shown to contribute to the overproduction of intestinal
and hepatic lipoproteins in these conditions, it is unlikely that
TRL apoC-III overproduction is the sole or even the dominant mechanism accounting for the hypertriglyceridemia, but
it certainly could contribute and should be considered a
potential therapeutic target for ameliorating this atherogenic
condition.
Acknowledgments
We are indebted to Patricia Harley, RN for her assistance with
subject recruitment and conducting the clinical protocol.
Sources of Funding
This work was supported by funding from the Canadian Institutes for
Health Research (MOP-43839). G.F.L. holds a Canada Research
Chair in Diabetes and is a Career Investigator of the Heart and Stroke
Foundation of Canada. H.D. is the recipient of a Postdoctoral
Fellowship Award from the Heart and Stroke Foundation of Canada.
B.W.P. was supported by NIH P30 DK56341 (Clinical Nutrition
Research Unit). C.X. is supported by a Postdoctoral Fellowship
Award from Canadian Diabetes Association.
Disclosures
None.
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Triglyceride-Rich Lipoprotein-Associated Apolipoprotein C-III Production Is Stimulated
by Plasma Free Fatty Acids in Humans
Mirjana Pavlic, René Valéro, Hélène Duez, Changting Xiao, Linda Szeto, Bruce W. Patterson
and Gary F. Lewis
Arterioscler Thromb Vasc Biol. 2008;28:1660-1665; originally published online June 12, 2008;
doi: 10.1161/ATVBAHA.108.169383
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Online Data Supplement
Laboratory methods
Plasma was separated from blood within 2 hours in a refrigerated centrifuge at
2000 rpm for 15 min at 4°C. TRL were isolated at each time point at d=1.006 for
16 hrs, 39,000 rpm at 12°C. Plasma amino acids were recovered from 0.25ml
plasma after precipitation of plasma proteins with 1.8ml acetone and extraction of
the aqueous phase with hexane1. The aqueous phase was dried under vacuum
and amino acids derivatised and enrichments determined as described below.
Proteins in the TRL fraction were delipidated using methanol/diethyl ether. ApoCIII was isolated from TRL by preparative isoelectric focusing (IEF) on
polyacrylamide-urea gels (pH gradient 4-7)2, stained with Coomasie blue stain
and destained with 10% acetic acid. The band corresponding to the major
isoform of apoC-III, apoC-III1, was excised and added to a borosilicate sample
vial for further processing (representative gels from the IH and saline studies with
clear separation of the apoC-III bands are shown in Fig S1B.)
Gels were hydrolysed with 6N HCl for 24h at 110°C, cooled to -20°C for 20 min
and centrifuged at 3500rpm for 10 min. Samples were dried under vacuum and
then derivatized by adding 100µl 1:1 acetonitrile (ACN):MTBSTFA (N-tertbutyldimethyl-N-methyltrifluoracetamide; Sigma-Aldrich) mixture and incubating
30 min at 70°C3 tBDMS derivatives were determined by electron impact
ionization GC/MS (Agilent 5975/6890N GC/MS, Agilent Technologies Canada
INC, Mississauga, Ontario, Canada). Chromatographic separations were
preformed on a 30mx0.25mm Agilent DB-17MS column with the helium carrier
1
gas flow 1ml/min, and the temperature programmed, following an initial hold at
80°C for 1 min to raise from 80°C to 150°C at 70°C/min then to 250°C at
20°C/min and then ramped to 300°C at 70°C/min. Selective ion monitoring at m/z
= 200 and 203 (ionic species corresponding to derivatized non deuterium-labeled
and deuterium-labeled leucine respectively) was performed, and the enrichment
data were converted to tracer-to-tracee ratios by measurement of appropriate
isotopic enrichment standards. To minimize artifacts, calibration with standards of
known isotopic enrichements was performed with each set of samples and a
blank gel was run to ensure leucine peak is always <5% (blank TTR mean ±SEM
2.32±0.07%).
Insulin, glucose and lipid concentrations in plasma, TRL and HDL were
determined as we have previously described4. Total and TRL-apoC-III
concentrations were determined by an automated (MODULAR P automatonRoche-France) immunoturbidimetric assay5. Antihuman apoC-III polyclonal
antibodies
from
goat
were
used
according
to
the
manufacturer's
recommendations (K-Assay, Kamiya biomedical company, USA).
Calculation
of
lipoprotein
clearance
and
production
rates
by
multicompartmental modeling
Stable isotope enrichment time course curves for apoC-III were fitted to a three
compartment model using SAAM II computer software (version 1.2; University of
Washington, Seattle, WA)6. Compartment 1 represents the plasma amino acid
precursor pool. Compartment 2 is an intracellular delay compartment, which
accounts
for
apoC-III
synthesis
lipoprotein
assembly
and
secretion.
2
Compartment 3 represents circulating plasma lipoproteins (Figure S1 A). We
assumed constant enrichment of the precursor pool and also that TRL apoC-III
attained similar enrichment plateau as TRL apoB48, which we previously
estimated to be approximately 60% of the plasma amino acid enrichment
4,6
.
Therefore, the peak enrichment attainable by TRL apoC-III in this study was set
to 60% of the plasma amino acid enrichment. This approach is not likely to affect
the overall conclusions of the study regarding the effect of plasma FFA on apoCIII PR. Each subject was in steady state with respect to TRL apoC-III
concentrations during the 12-hr kinetic study, so fractional catabolic rate (FCR)
was equivalent to fractional synthetic rate. Kinetics parameters were derived from
individual enrichment time course curves (Figure S1 C). Production rates were
calculated using the FCR of TRL apoC-III multiplied by pool size measured over
the 12 hours of the study where pool size = average plasma concentration
(mg/dl) between t5hr and t17h of the experimental protocol x plasma volume/kg
body weight (plasma volume is estimated as 0.045 liter/kg).
As can be seen in figure 1 of the manuscript, TRL apoC-III concentration
(pool size, upon which our kinetic modeling was based) was not in perfect steady
state during the 12hr kinetics study (5 to 17hrs). We turned to model simulations
to assess the potential impact of a non-steady state on the analysis of apoC-III
turnover kinetics. The model assumed a homogeneous single pool for apoC-III,
and production from a source of labeled leucine that was held to a constant
TTR=2%. In the first solution we simulated a metabolic steady state, with
constant absolute production and clearance rates and a fractional clearance rate
3
of 0.5 h-1. In the second solution, the absolute clearance rate was kept constant
and the absolute production rate increased at a linear rate over the time course,
resulting in a final pool size of apoC-III after 10 hours that was triple the starting
value. In the third solution, the absolute production rate was kept constant and
the absolute clearance rate decreased in a linear rate over the time course, such
that the final pool size again was 3 times the initial pool size (note that the
fractional clearance rates changed non-linearly in the latter two solutions). Both
non-steady state solutions show a rise toward the same TTR plateau (see data
supplement for figure S2). The time course for change in pool size in the two
non-steady state solutions was identical; thus, it would not be possible to
distinguish between these non-steady state solutions even if time-dependent
values for apoC-III concentration were included in the model. Note that if perfect,
noiseless TTR values could be obtained, it is theoretically possible to distinguish
between these 3 solutions (theoretical identifiability). Given the noise that is
inherent in measuring apoC-III TTR, however, it would not be possible to
distinguish
between
these
solutions
(they
lack
practical
identifiability).
Considering that the change in pool size simulated in these solutions (3-fold) was
far greater than we observed for our actual apoC-III study (far less than 3-fold),
we therefore feel justified in using a steady-state model for apoC-III kinetics. The
apoC-III system may not actually be at a true steady state, but due to the lack of
practical identifiability it would not be possible to resolve a more complicated
non-steady state model. In summary, we are confident that our calculated
4
production rates are valid and are not affected by the small perturbations of
steady state that occurred in this study.
Statistics
Results are presented as mean ± SEM. Paired t-test was used to compare
results from the Intralipid/heparin infusion and the saline control studies. ANOVA
with post-hoc Tukey’s test was used for the n=5 participants that underwent all
three studies (saline, Intralipid plus heparin and glycerol infusion studies). Since
no differences for any of the parameters of interest were found between the
saline and glycerol infusion studies, all further statistical analyses focused
exclusively on the Intralipid/heparin vs saline infusion studies. Two-way ANOVA
for repeated measures was used to analyze the kinetic experiments and was
performed using SAS software (Version 8.0, Cary, NC, USA). All other analyses
were performed with the SPSS version14. Sample size calculation was using the
gpower3 software 7.
5
References for online data supplement.
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enrichment. J Lipid Res 1999;40:2118-2124.
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1977;18:613-622.
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4. Duez H, Lamarche B, Valero R, Pavlic M, Proctor S, Szeto L, Lewis GF.
Intestinal as well as hepatic lipoprotein production is stimulated by an acute
elevation of plasma free fatty acids in humans. Circulation (in press) 2007.
5. Nicolay A, Lombard E, Arlotto E, Saunier V, Lorec-Penet AM, Lairon D,
Portugal H. Evaluation of new apolipoprotein C-II and apolipoprotein C-III
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6. Batal R, Tremblay M, Barrett PH, Jacques H, Fredenrich A, Mamer O,
Davignon J, Cohn JS. Plasma kinetics of apoC-III and apoE in normolipidemic
and hypertriglyceridemic subjects. J Lipid Res 2000;41:706-718.
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Behav Res Methods 2007;39:175-191.
6
Figure S1
Legend to Figure S1. (A) Multicompartmental model describing TRL apoCIII1 tracer kinetics. Compartment 1 represents the plasma amino acid precursor
pool, compartment 2 is an intracellular delay compartment, which accounted for
the synthesis, assembly and secretion of apoC-III, and compartment 3 is the
plasma lipoproteins. Subjects were in steady-state during the kinetics study so
FCR was equivalent to fractional synthetic rate, (B) Representative IEF gels
showing clear separation of apoC-III1, (C) Isotopic enrichment of TRL apoC-III
with deuterium-labeled leucine. Representative enrichment curves are
expressed as tracer to tracee ratio during the 12hr of d3-leucine infusion in one
individual during constant enteral feeding.
7
Figure S2
TTR in product during non-steady state that results in tripling
of pool size
0.025
0.020
0.015
TTR
Starting soln
Faster production
0.010
Slow er clearance
0.005
0.000
0
2
4
6
8
10
12
Hour s
Legend to Figure S2.
The figure illustrates the rise-to-plateau TTR in the apoC-III:
The starting steady-state solution is shown as a solid line, while the two nonsteady state solutions are shown at discrete time points. Both non-steady state
solutions show a rise toward the same TTR plateau. The time course for change
in pool size in the two non-steady state solutions was identical (not shown); thus,
it would not be possible to distinguish between these non-steady state solutions
even if time-dependent values for apoC-III concentration were included in the
model.
8