Mechanism of reduced myocardial glucose utilization during acute hypertriglyceridemia in rats.

Abstract

The purpose of the research is to study the effect of acute inhibition of intravascular lipolysis on myocardial substrate selection during hypertriglyceridemia using in vivo radiotracer analysis and positron emission tomography.
We induced acute hypertriglyceridemia in vivo using an intravenous infusion of Intralipid 20% (IL) without and with acute inhibition of fatty acid delivery from circulating triglycerides with injection of Triton WR-1339 (TRI) during a euglycemic-hyperinsulinemic clamp in Wistar rats. We determined the effect of TRI on myocardial uptake of circulating triglycerides and free fatty acids using intravenous injection of [(3)H]-triolein and [(14)C]-bromopalmitate, respectively. Myocardial blood flow, oxidative metabolism, and metabolic rate of glucose (MMRG) were determined using micro-positron emission tomography (microPET) with [(13)N]-ammonia, [(11)C]-acetate, and 2-deoxy-2-[F-18]fluoro-D: -glucose (FDG).
TRI reduced myocardial incorporation of [(3)H]-triolein but not [(14)C]-bromopalmitate showing that it selectively reduces myocardial fatty acid delivery from circulating triglycerides but not from free fatty acids. IL reduced myocardial blood flow and MMRG by 37% and 56%, respectively, but did not affect myocardial oxidative metabolism. TRI did not abolish the effect of IL on myocardial blood flow and MMRG.
Hypertriglyceridemia acutely reduces myocardial blood flow and MMRG in rats, but this effect is not explained by increased myocardial fatty acid delivery through intravascular triglyceride lipolysis.

Full text

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B Academy of Molecular Imaging, 2008
Published Online: 4 September 2008DOI: 10.1007/s11307-008-0171-2
Mol Imaging Biol (2009) 11:6Y14
RESEARCH ARTICLE
Mechanism of Reduced Myocardial Glucose
Utilization During Acute Hypertriglyceridemia
in Rats
Sébastien L. Ménard,1Xiuli Ci,1Frédérique Frisch,1François Normand-Lauzière,2
Jules Cadorette,2René Ouellet,2Johannes E. Van Lier,2François Bénard,2
M’hamed Bentourkia,2Roger Lecomte,1André C. Carpentier1
1Department of Medicine, Division of Endocrinology, Centre de Recherche Clinique Etienne-Le Bel, Centre Hospitalier Universitaire de
Sherbrooke, Université de Sherbrooke, 3001, 12th Ave. North, Sherbrooke, QC, Canada J1H 5H4
2Sherbrooke Molecular Imaging Center, Centre de Recherche Clinique Etienne-Le Bel, Centre Hospitalier Universitaire de Sherbrooke,
Université de Sherbrooke, Sherbrooke, QC, Canada
Abstract
Purpose: The purpose of the research is to study the effect of acute inhibition of intravascular
lipolysis on myocardial substrate selection during hypertriglyceridemia using in vivo radiotracer
analysis and positron emission tomography.
Procedures: Weinducedacutehypertriglyceridemiain vivo usinganintravenousinfusionofIntralipid
20% (IL) without and with acute inhibition of fatty acid delivery from circulating triglycerides with
injection of Triton WR-1339 (TRI) during a euglycemic–hyperinsulinemic clamp in Wistar rats. We
determinedtheeffectofTRIonmyocardialuptakeofcirculatingtriglyceridesandfreefattyacidsusing
intravenous injection of [3H]-triolein and [14C]-bromopalmitate, respectively. Myocardial blood flow,
oxidative metabolism, and metabolic rate of glucose (MMRG) were determined using micro-positron
emission tomography (μPET) with [13N]-ammonia, [11C]-acetate, and 2-deoxy-2-[F-18]fluoro-D-
glucose (FDG).
Results: TRI reduced myocardial incorporation of [3H]-triolein but not [14C]-bromopalmitate
showing that it selectively reduces myocardial fatty acid delivery from circulating triglycerides but
not from free fatty acids. IL reduced myocardial blood flow and MMRG by 37% and 56%,
respectively, but did not affect myocardial oxidative metabolism. TRI did not abolish the effect of
IL on myocardial blood flow and MMRG.
Conclusions: Hypertriglyceridemia acutely reduces myocardial blood flow and MMRG in rats, but
this effect is not explained by increased myocardial fatty acid delivery through intravascular
triglyceride lipolysis.
Key words: Intravascular lipolysis, Triglyceride-rich lipoproteins, Fatty acid metabolism,
Myocardial metabolism, Lipoprotein lipase, Triton WR-1339, FDG
Introduction
A
oxidation of fatty acids in the adult heart, while the
pproximately 60% to 70% of energy is derived from
rest is being provided mostly by glucose and lactate. Insulin
resistant states and type 2 diabetes are associated with
increased circulatory free fatty acid (FFA) and triglyceride
fluxes [1] and with a very important increase in myocardial
fatty acid utilization at the expense of reduced glucose
utilization [2]. More reliance on fatty acid oxidation with
reduced oxidation of glucose is associated with reduced
1
2
Correspondence to: André C. Carpentier; e-mail: Andre.Carpentier@
USherbrooke.ca

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energetic efficiency, more oxygen being required from the
same cardiac workload [3]. This may pose a problem in
critical situations such as during ischemia.
Increased FFA delivery to lean tissues, including the heart,
reduces glucose utilization [4] (see [1] for review). Another
potentially very important source of fatty acids to the heart is
circulating triglyceride-rich lipoproteins. Because triglycer-
ides circulate in the millimolar range and contain three fatty
acids per molecule and because the heart highly expresses
lipoprotein lipase (LpL) [5], this has been proposed as the
major source of fatty acids to the heart [6]. Experimental
evidence in perfused heart models [7, 8] and from in vivo
studies in rodents [9, 10] have suggested that the heart is a
major user of fatty acids derived from circulating triglycer-
ides. Several genetically modified mouse models with either
reduced [11–13] or increased [14, 15] myocardial LpL
activityhavedemonstratedthe potential ofchronic modulation
of myocardial fatty acid delivery from circulating triglycerides
to change substrate selection and induce cardiomyopathy.
However, changes in circulating triglyceride levels occur
acutely after dietary fat intake. The impact of very acute
changes in fatty acid delivery from circulating triglycerides on
myocardial energy substrate selection is currently unknown.
The aim of the present study was to study the impact of
myocardial fatty acid delivery from circulating triglycerides
on myocardial glucose utilization in the presence or absence
of intravenous infusion of Intralipid, a triglyceride emulsion,
to simulate a hypertriglyceridemic state. Our hypothesis was
that inhibition of myocardial fatty acid delivery from circulat-
ing triglycerides would lead to reversal of Intralipid-induced
reduction of myocardial glucose utilization if this is a major
source of fatty acid delivery to the heart in vivo (Fig. 1).
Materials and Methods
Animals
Male Wistar rats (Charles River, Quebec, Canada) weighting 350–
400 g were acclimatized to 12 h/day light cycle at constant temperature
(22°C) for 7 days. A total of 63 rats were used for the experiments
describedherein.Aftera12-hfastingperiod,animalswereanesthetized
with Isoflurane (Abbott laboratories, Montreal, Canada) delivered
through a nose cone at a concentration of 2.0% (volume:volume).
Anesthesia was maintained for the duration of the in vivo experiments.
Catheterswereplacedintoacarotidarteryforbloodsamplingandintoa
jugular and two tail veins for intravenous infusions. The catheters were
kept open with infusion of 0.9% saline. All animal protocols were
approved by the Animal Ethics Committee of the Faculty of Medicine
of the Université de Sherbrooke in accordance with the guidelines
of the Canadian Council on Animal Care.
Experimental Protocols
All in vivo experiments described herein were initiated 60-min
after insertion of catheters and performed under euglycemic–
hyperinsulinemic conditions, as previously described [4]. At time
0 of the experiment, a primed (180 mU/kg) constant insulin infusion
[12 mU/kg/min in 0.1% bovine serum albumin (BSA) in normal
saline; Novolin®ge, Toronto, Canada] was started and continued for
2 h to standardize the metabolic condition in all in vivo protocols.
Whole-blood glucose level was maintained using a variable 25%
dextrose intravenous infusion according to the glucose level,
determined every 10 min using a blood glucose monitor (Accusoft
Advantage™, Roche, USA) [16]. Also at time 0, Triton WR-1339
(Tylaxapol,SigmaChemicals,400mg/kg;referredtoastheTRIgroup)
vs. normal saline (referred to as the SAL group) was injected
intravenously to inhibit intravascular triglyceride lipolysis during the
experiment [17]. Our first set of in vivo experiments (Fig. 2a) was
performed to validate the use of Triton WR-1339 to reduce fatty acid
availability to tissues specifically derived from circulating triglycer-
ides and not from plasma FFA. [1-14C]-2-bromopalmitate (a FFA
tracer—injected in n=18 animals) in 5% BSA (Moravek Biochem-
icals, California), 5 μCi, and 60 μCi of [1–3H]-triolein (a triglyceride
tracer—injected in n=15 animals) sonicated in 0.5 ml of Intralipid
20% were given intravenously at time 120 and 125 min, respectively,
during the euglycemic–hyperinsulinemic clamp, as previously de-
scribed [16], with and without intravenous administration of Triton
WR-1339 as described above. Arterial blood samples were taken at
time −0 and 0 min and at times 90, 100, 110, and 120 min to
determine blood glucose and plasma insulin, FFA, and triglyceride
levels. Blood samples were also taken at times 121, 123, and 125 to
determine plasma [1-14C]-2-bromopalmitate and at times 126, 128,
and 130 to determine plasma [1-3H]-triolein radioactivity, and organs
were removed and processed to determine tissue tracer uptake as
described below.
Fig. 1. Myocardial fatty acid uptake from circulating trigly-
cerides (TG) vs. circulating free fatty acids (FFA). TG contained
in lipoproteins are hydrolyzed by lipoprotein lipase (LpL), a
process that may contribute to myocardial fatty acid delivery
with consequent reduction in myocardial glucose metabolism
during acute hypertriglyceridemia. Triton WR-1339 is a deter-
gent that binds circulating TG and prevents LpL-mediated
triglyceride hydrolysis (intravascular lipolysis). The physiolog-
ical processes that are described by the multi-compartmental
analysis of fluoro-deoxyglucose (FDG) (i.e., K1, k2, k3, and
myocardial metabolic rate of glucose—MMRG) are also
depicted.
S.L. Ménard et al.: Circulating Triglycerides and Myocardial Glucose Utilization
7

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Imaging Protocols
Figure 2b depicts the imaging protocol used in our second set of
in vivo experiments. To determine whether our in vivo imaging
methods could detect change in myocardial metabolism with
change in circulating lipids, we performed experiments using
intravenous infusion of Intralipid 20% (20 μl/min) (IL) vs. saline
(SAL) from time 0 and continued for the 2 h of the experiment.
Intralipid is a chylomicron-like emulsion that may be employed to
increase plasma triglyceride levels in vivo [18] and has been shown
by others to be metabolized similarly as endogenous lipoproteins
by the heart in rodents [6, 15]. In a third group of rats, we
administered intravenous injection of Triton WR-1339 together
with IL infusion (IL + TRI group) to determine the role of
intravascular triglyceride lipolysis on any effect of IL on myocar-
dial metabolism. Imaging experiments were performed with the
avalanche photodiode-based small animal PET scanner (μPET) of
the Sherbrooke Molecular Imaging Centre [19]. Beforeimaging,the
heart position was localized with a Doppler probe (0.64 cm [1/4 in.],
9 MHz; Parks Medical Electronics). During imaging, the animals
rested supine on the scanner bed and were kept warm with a
heating pad. At time 50-min, approximately 5 mCi [13N]-ammonia
in 0.5 ml of saline was administered intravenously over a period of
30 s using an automated injection system to determine myocardial
blood flow [20]. This method had been shown to give similar
estimates of myocardial blood flow when compared to the [15O]-
water technique [21]. Image acquisition was performed using a
10-min list-mode dynamic acquisition. At time 70 min, an
intravenous injection of approximately 3 mCi [11C]-acetate in
0.5 ml of saline was administered over 30 s to determine myocardial
oxidative metabolic rate [22]. Image acquisition was also
performed using a 10-min list-mode dynamic acquisition. At
time 90-min, an intravenous injection of approximately 1 mCi
2-deoxy-2-[F-18]fluoro-D-glucose (FDG) in 0.5 ml of saline was
given over a period of 30 s to determine myocardial glucose
utilization. Image acquisition was performed using a 30-min list-
mode dynamic acquisition. The PET imaging protocols were
previously validated in other studies [23, 24]. Blood samples were
taken at time 90, 100, 110, and 120-min to determine blood
glucose and plasma FFA and TG.
Incorporation of Tracers into Plasma,
Intracellular Lipids, and Mitochondria
Determination of uptake of plasma [1-3H]-triolein and [1-14C]-2-
bromopalmitate into tissue lipids was performed as described
previously [16]. Briefly, the heart was removed immediately and
snap-frozen after thorough washing with 0.9% NaCl and blotting to
remove excess water. Samples of heart (100 mg) were homoge-
nized in a 0.9% NaCl solution. Plasma and tissue lipids were
extracted according to the method described by Folch et al. [25]
and were applied onto thin-layer chromatography plates (Silica Gel
60, F-254, Selecto Scientific, Suwanee, GA, USA) with standards
for free fatty acids (FFA), triglycerides (TG), diglycerides (DG),
monoglycerides (MG), cholesterol esters, and phospholipids. The
plates were eluted in a hexane/diethyl ether/acetic acid (80:20:2)
solution and stained with dichlorofluorescein (1 mg/mL ethanol).
Relative uptake (%) of [14C] and [3H] in different tissue lipids was
calculated by dividing the activity in each fraction by the total
recovered in all fractions multiplied by 100.
Mitochondria from the heart were extracted by using the method
previously described in [16, 25]. Briefly, 500 mg of the heart were
homogenized on ice (PowerGen 125, Fisher Scientific International
Inc., USA) in an ice-cold homogenization solution (0.075 M
sucrose, 0.225 M sorbitol, 1 mM EGTA, 0.1% fatty-acid-free BSA,
and 10 mM Tris–HCl, pH 7.4). The homogenates were centrifuged
at 1,000×g for 10 min at 4°C, and the supernatants were
centrifuged again at 12,000×g for 10 min at 4°C. The supernatants
were then removed, and 1,000 μL of an incubation solution
(10 mM Tris–HCl, 25 mM sucrose, 75 mM sorbitol, 100 mM KCl,
10 mM K2HPO4, 0.05 mM EDTA,5 mM MgCl2, and 1 mg/mL FFA-
free BSA) was added to the pellet containing mitochondria. Fifty
microliters of the mitochondrial samples was kept frozen at −80°C
for the measurement of glutamate dehydrogenase (GDH) activity to
correct for the fractional recovery of mitochondria extracts from
tissues [25]. The GDH activity was measured according to the
Sigma quality control test procedure (EC 1.4.1.3, Enzymatic Assay
of L-GDH) modified as previously described [16]. The fractional
recovery of mitochondria was then calculated by dividing mitochon-
drial GDH activity (units per gram of tissue) by whole-tissue GDH
activity (units per gram of tissue). Mitochondrial [14C] and [3H]
activity were corrected by dividing by the fractional recovery to
determine mitochondrial uptake of [1-14C]-2-bromopalmitate and
[1-3H]-oleate from labeled triolein, respectively.
Fig. 2. a Experimental protocol for the in vivo validation
of Triton WR-1339 method. b Experimental protocol of the
in vivo experiments using micro-positron emission tomogra-
phy (μPET).
8
S.L. Ménard et al.: Circulating Triglycerides and Myocardial Glucose Utilization

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Imaging Data Analysis
For [13N]-ammonia and [11C]-acetate, dynamic series of 23 frames
each were sorted out from the list mode data, using the following
sequence: 1×30 s, 12×5 s, 8×30 s, 2×150 s. For FDG, a dynam-
ic sequence series of 29 frames was sorted out using the following
sequence: 1×30 s, 12×5 s, 8×30 s, 6×150 s, 2×300 s. Image
planes were reconstructed on a 128×128 matrix with a 0.475×
0.475 mm pixel size using the maximum likelihood expectation
maximization algorithm with 15 iterations [26, 27]. Regions of
interest (ROI) were drawn manually over the myocardium in the
left ventricle and on the left ventricular cavity from the best frame
image acquired with [13N]-ammonia acquisition and then used on
all frames of the three tracers. Tissue time–activity curves were
generated from the ROI on the myocardium, and the arterial tracer
activity curve was obtained from the ROI drawn over the left
ventricular cavity. The PET kinetic parameter values were assessed
with the input function derived from the images. We used a three-
compartment kinetic model for [13N]-ammonia that provides an
estimate of blood flow from k1value [27]. For [11C]-acetate, we
used a three-compartment kinetic model that estimates the
generation of CO2from the citric cycle in the myocardium using
k2value [26]. For the modeling of myocardial uptake of FDG, we
used the classical three-compartment model of Sokoloff [28].
According to this model, K1 is an index of membrane glucose
transport, k2is an index of FDG escape, k3is an index of glucose
phosphorylation. The myocardial metabolic rate of glucose (MMRG)
can be derived from these constants, the arterial blood glucose
concentration, and the lumped constant (LC):
?
MMRG ¼
glycemiammol=l
LC
ðÞ
?
? Km
ðEq:1Þ
Km¼
K1? k3
k2þ k3
ð
ð
Þ
Þ
ðEq:2Þ
where LC is a term made up of six constants that relate the
transport and phosphorylation kinetics of FDG to that for
glucose [29].
Statistical Analysis
All data are reported as mean ± SE. Plasma glucose, insulin, FFA,
and TG levels at steady state (baseline and between 90 to 120 min
during the euglycemic clamp) were averaged for statistical analysis.
Unpaired Student t test or one-way ANOVA with Tukey’s post hoc
test were performed using GraphPad Prism version 5.00 for
Windows (San Diego California USA). A P value of less than
0.05 was considered significant.
Results
Triton WR-1339 Selectively Reduces Myocardial
Uptake of Circulating Triglycerides
but not Circulating FFA
Bloodglucoseandplasmainsulin,FFA,andtriglyceridelevels
at baseline and during euglycemic–hyperinsulinemic clamp in
rats injected with SAL vs. TRI are shown in Table 1. There
was no significant difference in these parameters at baseline
between the two groups. During the clamp, blood glucose and
plasma FFA levels were similar between the two groups. As
expected, plasma triglyceride levels were significantly in-
creased approximately fourfold in TRI (PG0.001). However,
plasma insulin levels were lower in TRI vs. SAL (P=0.04).
This effect was explained by an in vitro interference of Triton
WR1339 with the insulin immunoassay. This was further
suggested by glucose infusion rates (GINF) that were
virtually identical with and without TRI injection (Table 1).
Table 2 depictsthe effect of injectionof TritonWR-1339on
area under curve (AUC) of plasma3H-triolein activity and on
myocardial uptake and incorporation of3H-triolein-derived3H
in cellular lipids and in mitochondria. TRI was associated with
an ~168% increase of plasma3H-triolein activity AUC (P=
0.02) and with ~48% (P = NS) and ~44% (P=0.03) reductions
in myocardial3H-triolein-derived label uptake in cellular lipids
and in mitochondria, respectively. However, TRI had no effect
on relative uptake of3H-triolein in the various cellular lipid
fractions. Thus, Triton WR-1339 reduced myocardial fatty
acid uptake from circulating triglycerides without change in
partitioning into the different intracellular lipids.
In contrast, TRI had no significant effect on plasma
[1-14C]-2-bromopalmitate AUC activity and on myocardial
uptake of this tracer in cellular lipids and in mitochondria
(Table 3). Furthermore, TRI did not affect relative uptake of
[1-14C]-2-bromopalmitate into the various cellular lipid
factions.
μPET Studies
Weight of the animals was similar in SAL, IL, and IL + TRI
experiments (387±12, 387±11, and 418±14 g, respectively,
P=0.14). After surgery, heart rate and body temperature were
also similar in all three groups (heart rate: 354±14, 350±21,
and 303±14 beats/min, respectively, P=0.09; body tem-
perature: 33.9±0.7, 34.5±0.4, and 35.1±0.4°C, respectively,
P=0.30). Baseline blood glucose and plasma FFA levels
Table 1. Plasma glucose, insulin, FFA and TG levels at baseline and
during the normoglycemic–hyperinsulinemic clamp
ParameterSaline (SAL)
(n=11)
Triton WR-1339
(TRI) (n=9)
Pa
Baseline
Glucose (mmol/l)
Insulin (pmol/l)
FFA (mmol/l)
TG (mmol/l)
Normoglycemic–hyperinsulinemic clamp
Glucose (mmol/l)
Insulin (pmol/l)
FFA (mmol/l)
TG (mmol/l)
GINF (μmol/min)
7.4±0.3
329±76
1.23±0.13
0.45±0.06
7.2±0.6
373±144
1.23±0.14
0.34±0.08
0.77
0.77
0.97
0.30
4.1±0.5
4,140±616
0.67±0.06
0.26±0.07
42.3±3.4
4.7±0.4
1,941±713
0.79±0.24
1.40±0.13
40.4±1.9
0.44
0.04
0.56
G0.001
0.68
aThese P values are from unpaired Student t tests; a P value of less than
0.05 is considered significant. Data are mean ± SE.
GINF Glucose infusion rate during the clamp, FFA free fatty acids, TG
triglycerides
S.L. Ménard et al.: Circulating Triglycerides and Myocardial Glucose Utilization
9

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were not significantly different between SAL, IL, and IL +
TRI (blood glucose: 6.5±0.7, 6.4±0.7, and 5.9±0.4 mmol/l,
respectively, P=0.81; baseline plasma FFA: 0.68±0.12,
0.78±0.06, and 0.72±0.10 mmol/l, respectively, P=0.81).
Baseline plasma triglyceride levels were also not significantly
different between the three groups (0.21±0.02, 0.37±0.09,
and 0.25±0.04 mmol/l, respectively, P=0.17).
Table 4 shows the metabolic parameters of interest during
the euglycemic–hyperinsulinemic clamp and μPET study. By
design, plasma glucose levels were similar between the three
experimental groups. Plasma FFA levels were significantly
higher in IL and IL + TRI vs. SAL. Plasma triglyceride levels
in IL and IL + TRI experiments were also significantly higher
than SAL (PG0.001). FDG K1and k2were similar in all
three groups, whereas k3was significantly reduced in IL and
IL + TRI compared with SAL (P=0.02). MMRG (Fig. 3a)
was significantly lower in IL and IL + TRI vs. SAL (PG0.05).
IL also significantly reduced myocardial blood flow ([13N]-
ammonia k1—Fig. 3b) vs. SAL (PG0.05), whereas TRI did
not significantly improve this IL-mediated reduction in
blood flow. Total myocardial oxidative rate (11C-acetate
k2—Fig. 3c) was not significantly different between the three
groups (P=0.06), although it tended to be lower in the IL +
TRI group vs. the two others. Figure 4 shows representative
μPET scans after FDG injection in the three groups of rats.
Discussion
In the present study, IL resulted in increased plasma
triglyceride and FFA levels together with significant reduc-
tion in MMRG and reduction in myocardial blood flow but
no change in myocardial oxidative capacity, heart rate, or
body temperature. In contrast to our initial hypothesis, we
found no significant reversal of IL-induced reduction in
Table 2. Effect of Triton WR-1339 on plasma AUC of3H-triolein activity and on myocardial uptake of3H-triolein
ParameterSaline (SAL) (n=4) Triton WR-1339 (TRI) (n=3)Pa
AUCplasma(dpm/ml×5 min)
Cellular lipid uptake (dpm/g×5 min)
Mitochondrial uptake (dpm/g×
5 min)
RUPPL heart(% total lipids)
RUMG heart(%total lipids)
RUDG heart(%total lipids)
RUTG heart(%total lipids)
RUFFA heart(%total lipids)
RUCHOL heart(%total lipids)
3,898±1,078
31,838±14,913
3,502±259b
10,433±1,859
16,689±10,342
1,953±540c
0.02
0.44
0.03
25.3±11.6
19.3±6.0
2.8±0.3
28.5±7.7
24.0±4.9
0.3±0.3
25.8±9.2
19.5±5.1
2.5±0.3
29.3±6.6
22.3±4.8
0.3±0.3
0.97
0.98
0.54
0.94
0.81
1.0
Data are mean ± SE. Plasma and tissue lipid 3H-triolein activity was determined only in a subset of the animals because of error in the dual label counting
procedure in n=8 rats at the time of these experiments.
AUC Area under curve, CHOL cholesterol esters, DG diglycerides, MG monoglycerides, FFA free fatty acids, PPL phospholipids, RU relative uptake, TG
triglycerides.
aP values are from unpaired Student t tests.
bn=7
cn=8.
Table 3. Effect of Triton WR-1339 on plasma AUC of [1-14C]-2-bromopalmitate activity and on myocardial uptake of [1-14C]-2-bromopalmitate
Parameter Saline (SAL) (n=9) Triton WR-1339 (TRI) (n=9) Pa
AUCplasma(dpm/ml×10 min)
Cellular lipid uptake (dpm/g×5 min)
Mitochondrial uptake (dpm/g×
5 min)
RUPPL heart(% total lipids)
RUMG heart(%total lipids)
RUDG heart(%total lipids)
RUTG heart(%total lipids)
RUFFA heart(%total lipids)
RUCHOL heart(%total lipids)
6,453±915
5,887±1,109
5,811±1,976b
4,649±819b
3,935±723
3,450±987c
0.81
0.17
0.36
40.0±6.1
4.8±1.6
10.6±1.6
15.3±4.9
28.9±2.4
0.8±0.1
44.0±7.0
4.3±1.7
5.8±2.3
14.1±3.0
30.0±2.8
1.6±0.6
0.67
0.85
0.11
0.83
0.77
0.24
Data are mean ± SE.
AUC Area under curve, CHOL cholesterol esters, DG diglycerides, MG monoglycerides, FFA free fatty acids, PPL phospholipids, RU relative uptake, TG
triglycerides
aP values are from unpaired Student t tests.
bn=8
cn=6
10
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MMRG with Triton WR-1339. Triton WR-1339 also did not
reverse IL-induced reduction in myocardial blood flow. We
demonstrated that Triton WR-1339 reduced clearance of
circulating triglycerides and reduced myocardial uptake of
triglycerides without significantly changing plasma FFA
clearance and myocardial uptake. Thus, our results suggest
that IL-induced reduction in blood flow and MMRG is not
primarily caused by increased fatty acid delivery through in-
creased intravascular triglyceride lipolysis during euglycemic–
hyperinsulinemia in rats. Triton WR-1339 was not associated
withchangeininsulinsensitivity,asassessedbylackofchange
in glucose infusion rates during euglycemic–hyperinsulinemic
clamps. Plasma insulin levels were lower after injection of
Triton WR-1339, but this effect was caused by interference of
Triton WR-1339 with the insulin immunoassay used in the
present study.
Table 4. Metabolic parameters during the μPET imaging protocol with euglycemic–hyperinsulinemic clamp
ParameterSaline (SAL) (n=12)Intralipid (IL) (n=11) Intralipid + Triton (IL + TRI) (n=14)Pa
Glucose (mmol/l)
Insulin (pmol/l)
FFA (mmol/l)
TG (mmol/l)
Heart rate (beats/min)
Body temperature (°C)
FDG K1(ml/g/min)
FDG k2(min−1)
FDG k3(min−1)
KmFDG (ml/g/min)
MMRG (μmol/g/min)
[13N]–NH3k1(ml/g/min)
[11C]-Acetate k2(min−1)
6.3±0.1
4,407±558d
1.41±0.49c,d
1.57±0.69c,d
339±4d
33.6±0.2d
0.073±0.014
1.143±0.291
0.042±0.007c,d
0.0033±0.0008c,d
0.017±0.003c,d
6.16±0.52c,d
2.09±0.29
6.4±0.2
3,277±546
3.10±0.20b
5.09±0.32b,d
327±5
34.2±0.2
0.060±0.011
1.011±0.291
0.020±0.003b
0.0012±0.0002b
0.008±0.002b
3.90±0.38b
1.99±0.20
6.3±0.1
1,962±161b
3.91±0.41b
10.11±0.67c,d
313±6b
34.8±0.2b
0.082±0.021
1.153±0.183
0.017±0.006b
0.0012±0.0003b
0.006±0.001b
4.28±0.47b
1.42±0.13
0.69
G0.001
G0.001
G0.001
0.005
G0.001
0.67
0.89
0.02
0.009
0.002
0.005
0.06
Data are mean ± SE
MMRG Myocardial metabolic rate of glucose, FFA free fatty acids, TG triglycerides
aThese P values are from one factor ANOVAs with Tukey’s post hoc tests.
bPG0.05 vs. Salin by Tukey’s post hoc test.
cPG0.05 vs. Intralipid by Tukey’s post hoc test
dPG0.05 vs. Intralipid + Triton by Tukey’s post hoc test
Fig. 3. a Myocardial metabolic rate of glucose (MMRG), b myocardial blood flow, and c total myocardial oxidative rate during
euglycemic clamp in rats treated with saline (SAL, open bars), Intralipid 20% (IL, closed bars), and Intralipid 20% + Triton WR-
1339 (IL + TRI, grey bars). a PG0.05 vs. SAL group by ANOVA with Tukey’s post hoc test. Data are mean ± SE.
S.L. Ménard et al.: Circulating Triglycerides and Myocardial Glucose Utilization
11

Page 7

Our results are consistent with those of other investigators
showing reduced myocardial glucose utilization with intra-
venous infusion of Intralipid in vivo [4, 30]. Intravenous
infusion of lipids has also been previously shown to alter
myocardial blood flow in other animal models [31, 32]. This
lipid-induced reduction in myocardial blood flow could also
be mediated by increased blood viscosity [33]. Interestingly,
human LpL overexpression in aorta vascular smooth
muscles in mice alters vascular reactivity [34], suggesting
that lipolysis of triglycerides leads to endothelial dysfunction
in vivo. However, injection of Triton WR-1339 did not blunt
IL-induced reduction in myocardial blood flow. The lack of
Intralipid-induced change in total myocardial oxidative
metabolism that we observed in the present study is also
consistent with results from other investigators [35].
One possible explanation for this finding may be compen-
satory increase in myocardial plasma FFA utilization in the
presence of inhibition of myocardial utilization of fatty acids
derived from circulating triglycerides. Indeed, this phenome-
nonhasbeenshowntooccurinmiceusingLangendorffex vivo
myocardial perfusion protocols in some [6, 15] but not all
studies [7]. We found that blocking myocardial availability of
fatty acids from circulating triglycerides did not affect
myocardial
uptake. Thus, elevation of FFA levels seen with IL infusion
may have been the primary cause of IL-mediated reduction in
myocardial blood flow and MMRG. It is also possible that the
hyperinsulinemic condition in our experiments reduced
myocardial LpL activity [36], thus, minimizing the relative
contribution of fatty acid uptake from circulating triglycerides.
Finally, it is also possible that fatty acids delivered from
circulating triglycerides do not acutely mix into the same
intracellular fatty acid pool than that from plasma FFA [10],
with different acute metabolic consequences. Intriguingly,
total myocardial oxidative rate tended to be lower in the IL +
TRI vs. IL group, an effect that may have been expected on
the basis of reduced myocardial fatty acid delivery from
circulating triglycerides in the face of no significant change in
myocardial FFA uptake and similar MMRG.
Cardiac-specific LpL knockout and cardiac-specific LpL
overexpression mouse models have illustrated the potential
importance of prolonged lack or excess of fatty acid delivery
from circulating triglycerides for the development of
cardiomyopathy associated with change in myocardial
glucose utilization [11–15]. However, the relative contribu-
tion of fatty acids derived from circulating triglycerides vs.
plasma FFA pool as a substrate for the heart is controversial
in physiological conditions. Results from some investigators
have suggested that utilization rate of circulating triglycer-
ide-derived fatty acids in the mouse heart is quantitatively
lower [37], similar [7], or greater [6] to plasma FFA. These
disparities are likely explained by different experimental and
physiological conditions since myocardial fatty acid delivery
from circulating triglycerides may depend on its lipoprotein
source, and cardiac LpL function is modified in various
conditions [38]. Interestingly, a recent human study has
shown that plasma FFA contribute to more than 80% of fatty
acids utilized by the heart in vivo in humans with coronary
artery disease during the fasting state, whereas circulating
triglycerides contribute to less than 20% [39].
14C-bromopalmitate cellular and mitochondrial
Conclusion
In conclusion, the results from the present study show that
intravenous infusion of fat emulsion acutely reduces myo-
cardial glucose utilization and blood flow in vivo during
euglycemic–hyperinsulinemia in rats. However, these effects
do not result from increased delivery of fatty acids from
intravascular triglyceride lipolysis. Future studies will be
needed to determine whether myocardial fatty acid delivery
from circulating triglycerides may play a role in reduced
Fig. 4. Representative myocardial transaxial μPET scan
images after injection of [18F]FDG in the saline (SAL - upper
panel), Intralipid 20% (IL - middle panel), and Intralipid 20% +
Triton WR-1339 (IL + TRI - lower panel) groups.
12
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Page 8

myocardial glucose utilization in pathophysiological states
such as type 2 diabetes.
Acknowledgements. A.C.C. was supported by a Junior 2 Scholarship from
the Fonds de la recherche en santé du Québec (FRSQ). The Centre de
recherche clinique Etienne-Le Bel is a FRSQ-funded research centre. This
work was supported by grants from Association Diabète Québec, from the
Heart and Stroke Foundation of Canada (the Jonathan-Ballon Award), and
from the Canadian Institutes of Health Research (MOP 53094 and MOP
15348).
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