Mechanisms for increased myocardial fatty acid
utilization following short-term high-fat feeding
Jordan J. Wright1†, Jaetaek Kim1,2†, Jonathan Buchanan1, Sihem Boudina1, Sandra Sena1,
Kyriaki Bakirtzi3, Olesya Ilkun1, Heather A. Theobald1, Robert C. Cooksey1,
Kostantin V. Kandror3, and E. Dale Abel1*
1Division of Endocrinology, Metabolism and Diabetes and Program in Molecular Medicine, University of Utah School of
Medicine, 15 N 2030 East, Bldg 533, Rm 3110B, Salt Lake City, UT 84112, USA;2Division of Endocrinology and Metabolism,
Department of Internal Medicine, College of Medicine, Chung-Ang University, Seoul, Korea; and3Department of
Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
Received 29 July 2008; revised 4 January 2009; accepted 11 January 2009; online publish-ahead-of-print 15 January 2009
Time for primary review: 22 days
Aims Diet-induced obesity is associated with increased myocardial fatty acid (FA) utilization, insulin
resistance, and cardiac dysfunction. The study was designed to test the hypothesis that impaired
glucose utilization accounts for initial changes in FA metabolism.
Methods and results Ten-week-old C57BL6J mice were fed a high-fat diet (HFD, 45% calories from fat)
or normal chow (4% calories from fat). Cardiac function and substrate metabolism in isolated working
hearts, glucose uptake in isolated cardiomyocytes, mitochondrial function, insulin-stimulated protein
kinase B (Akt/PKB) and Akt substrate (AS-160) phosphorylation, glucose transporter 4 (GLUT4) translo-
cation, pyruvate dehydrogenase (PDH) activity, and mRNA levels for metabolic genes were determined
after 2 or 5 weeks of HFD. Two weeks of HFD reduced basal rates of glycolysis and glucose oxidation and
prevented insulin stimulation of glycolysis in hearts and reduced insulin-stimulated glucose uptake in
cardiomyocytes. Insulin-stimulated Akt/PKB and AS-160 phosphorylation were preserved, and PDH
activity was unchanged. GLUT4 content was reduced by 55% and GLUT4 translocation was significantly
attenuated. HFD increased FA oxidation rates and myocardial oxygen consumption (MVO2), which could
not be accounted for by mitochondrial uncoupling or by increased expression of peroxisome proliferator
activated receptor-a (PPAR-a) target genes, which increased only after 5 weeks of HFD.
Conclusion Rates of myocardial glucose utilization are altered early in the course of HFD because of
reduced GLUT4 content and GLUT4 translocation despite normal insulin signalling to Akt/PKB and AS-
160. The reciprocal increase in FA utilization is not due to PPAR-a-mediated signalling or mitochondrial
uncoupling. Thus, the initial increase in myocardial FA utilization in response to HFD likely results from
impaired glucose transport that precedes impaired insulin signalling.
Fatty acid metabolism;
Obesity and diabetes are associated with reduced myocar-
dial glucose utilization (transport, glycolysis, and oxi-
utilization and oxygen consumption (MVO2),1–3which are
believed to contribute to cardiac dysfunction.1,2,4,5In
ob/ob and db/db mouse models of severe obesity and
insulin resistance, abnormal cardiac metabolism precedes
the onset of hyperglycaemia and impaired cardiac function
in vivo.6–9Similarly, in Zucker fatty rat hearts, glucose util-
ization is depressed prior to evidence of cardiac dysfunc-
tion,10but at a time when there is left ventricular
expression of glucose transporters, myocardial insulin resist-
ance, and increased sarcolemmal localization of the FA
transporter CD36. Activation of peroxisome proliferator-
activated receptor-a (PPAR-a) may increase the expression
of target genes such as pyruvate dehydrogenase kinase
(PDK4), which will decrease flux through pyruvate dehydro-
genase (PDH), while increasing the expression of genes such
as acyl-CoA dehydrogenases, mitochondrial and cytosolic
thioesterases and uncoupling proteins that will promote FA
oxidation.1,2,4,12–15In addition, recent studies in ob/ob
and db/db mice have suggested that FA and ROS-mediated
mitochondrial uncoupling could contribute to the observed
increase in MVO2.2,16–18
The sequence by which these pathophysiological mechan-
isms develop and their inter-relationships early in the course
†These authors contributed equally.
*Corresponding author. Tel: þ1 801 585 0727; fax: þ1 801 585 0701.
E-mail address: email@example.com
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009.
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Cardiovascular Research (2009) 82, 351–360
of obesity-related cardiac dysfunction are not well under-
stood, because most studies have been performed after
obesity is established or after prolonged periods of
high-fat feeding. Few studies have attempted to elucidate
early events. At 4 weeks of age, leptin-deficient or -resistant
models such as ob/ob and db/db mice exhibit decreased
rates of glucose utilization, increased rates of myocardial
FA utilization and increased MVO2 in the absence of
increased expression of PPAR-a target genes.6Eight weeks
of high-fat feeding in rats precipitated cardiac dysfunction,
which was associated with impaired insulin signal transduc-
tion and increased myocardial FA uptake and triglyceride
(TG) esterification, which was attributed to increased sarco-
lemmal translocation of CD36.15,19Although these animals
were not obese, they manifested hepatic steatosis, skeletal
muscle TG accumulation, and impaired glucose tolerance.
Ten days of high-fat feeding in C57BL6 mice reduced insulin-
mediated glucose uptake in proportion to reduced glucose
transporter 4 (GLUT4), and reduced protein kinase B
(Akt/PKB) phosphorylation.20In this study, the metabolic
fate of FA and glucose in the heart and expression of
PPAR-a targets were not determined. Thus it is not known
if short-term high-fat feeding alters myocardial FA metab-
olism or MVO2and if these changes can be attributed to
the activation of PPAR-a target genes.
The goal of our study was to determine myocardial sub-
strate metabolism after short-term high-fat feeding in the
mouse and the molecular mechanisms responsible for
observed changes. Two weeks of high-fat feeding increased
myocardial FA utilization and MVO2 and decreased basal
and insulin-stimulated rates of glycolysis and glucose oxi-
dation. These metabolic changes were associated with
impaired GLUT4 translocation but normal PDH activity.
Importantly, there was no increase in PPAR-a target gene
expression, no reduction in malonyl CoA concentrations,
and no evidence of mitochondrial uncoupling. Thus,
impaired glucose utilization might be the initial defect
that precipitates altered myocardial substrate utilization
following short-term high-fat feeding.
2.1. Animals and diets
The investigation conforms to the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of
Health (NIH Publication No. 85–23, revised 1996) and was approved
by the Institutional Animal Care and Use Committee of the Univer-
sity of Utah. Male C57BL/6J mice were fed ad libitum either a stan-
dard chow diet which provided 24.5% calories from protein, 4.4%
calories from fat, and 54.5% calories from carbohydrate (8656,
Harlan Teklad, Madison, WI, USA) or a high-fat, high-sucrose diet
(D12451, Research Diets, New Brunswick, NJ, USA), which provided
20% calories from protein (200 gm% casein), 45% calories from fat
(178 gm% lard, 25 gm% soybean oil), and 35% of calories from carbo-
hydrate (50% sucrose) (73 gm% corn starch, 100 gm% maltodextrin,
and 173 gm% sucrose) for 2 or 5 weeks, starting at ten weeks of
age. Serum levels of glucose, insulin, TGs, and free fatty acids
(FFA), glucose tolerance tests, and tissue TG levels were measured
after a 6 h fast as previously described.7,21The area under the curve
is calculated by deriving the sums of the area of a trapezoid for each
adjacent time point and glucose measurement, using Microsoft
excel. Hearts were excised following anaesthesia with chloral
hydrate. For insulin-stimulated signal transduction, mice were
deprived of food for 6 h, anaesthetized with intraperitoneal
chloral hydrate, and then injected with either saline or 3 U of
human regular insulin (Novolin R; Novo Nordisk, Bagsvaerd,
Denmark) via the inferior vena cava. Hearts were harvested 5 min
after saline or insulin injection and immediately frozen in liquid
2.2. Substrate metabolism, MVO2, and cardiac
function in isolated working hearts
Rates of glucose oxidation, glycolysis, palmitate oxidation, and
myocardial oxygen consumption (MVO2) were measured in isolated
working hearts obtained from random-fed high-fat fed (HFD) and
normal chow (NC) mice, perfused with Krebs Henseleit buffer sup-
plemented with 5 mM glucose and 0.4 mM palmitate bound to 3%
BSA as previously described.6,7
2.3 Measurement of malonyl CoA concentrations
and pyruvate dehydrogenase activity
Malonyl CoA concentrations were measured by HPLC22,23and PDH
activity determined using a radioactive enzymatic method17in
hearts that were isolated after a 6 h fast.
2.4. Respiration and ATP production in
Mitochondrial respiratory parameters were studied in saponin-
permeabilized fibres17,18using four independent substrates (in
mM): (i) glutamate 5 and malate 2, (ii) pyruvate 10 and malate 5,
(iii) palmitoyl-carnitine 0.02 and malate 2, or (iv) succinate 5 and
rotenone 10 mM. The measured mitochondrial respiratory par-
ameters were: basal respiration before the addition of ADP (State
2 or V0), maximally ADP-stimulated (1 mM) respiration (State 3 or
VADP), and respiration in the presence of oligomycin (1 mg/mL),
which inhibits ATP synthase activity (State 4 or VOligo). ATP concen-
tration was determined by a bioluminescence assay using the
Enliten Luciferase/Luciferin Reagent (Promega, Madison, WI, USA).
2.5. Isolation of cardiac myocytes and
determination of glucose uptake
Insulin-stimulated 2-deoxyglucose uptake was measured in collagen-
ase dissociated mouse cardiomyocytes as previously described.7
2.6. Western blot analysis
Total protein lysates were extracted from frozen hearts for immuno-
blot analysis as described;24see Supplementary material online for
2.7. Analysis of glucose transporter 4 translocation
by sucrose gradient centrifugation
GLUT4 translocation was determined by examining insulin-mediated
redistribution of GLUT4 vesicles from intracellular compartments to
the plasma membrane using sucrose gradients;25see Supplementary
material online for details.
2.8. Glucose transporter 4 immunofluorescence
Hearts were rapidly removed and fixed in 4% paraformaldehyde,
protected in a sequential series of 10, 20, and 30% sucrose/PBS sol-
utions, oriented in OCT-filled molds (Tissue-Tek, Hatfield, PA, USA),
rapidly frozen, and then sectioned at 6 mm on a Cryostat microtome
(Leica Instruments, Bannockburn, IL, USA) at 2208C.26Sections
were washed with PBS, blocked in 1% bovine serum albumin
without permeabilization, to analyze the fluorescence associated
with the cell surface, and then incubated with a polyclonal rabbit
GLUT4 antibody (Millipore) at 1:400 dilution for 2 h at room temp-
erature.27,28Slides were washed with PBS and then incubated
J.J. Wright et al.
with Alexa Fluor 488-labelled goat anti-rabbit IgG (Invitrogen) at
1:500 dilution in PBS with 0.1% bovine serum albumin for 1 h and
were examined using an Olympus IX71 inverted microscope
(Olympus, Center Valley, PA, USA) equipped with a fluorescence
filter at 40? magnification.
2.9. Quantitative RT–PCR
Total RNA was extracted from hearts and reverse transcribed
cDNA was analyzed by quantitative real-time PCR as previously
described.18,24Samples from six hearts per condition were analyzed
in triplicate. Data were normalized by expressing them relative to
b-actin expression and are reported as fold change relative to age-
matched NC-fed mice.
2.10. Statistical analysis
Data expressed as mean+SEM, were analyzed by ANOVA, and sig-
nificant differences assessed by Fisher’s protected least significant
difference or the unpaired t-test, with P , 0.05 accepted as signifi-
cant (Statview 5.0.1 software package, SAS Institute Inc., Cary, NC,
3.1. Metabolic characterization and cardiac
Body weights were increased by 8.0 and 9.4%, respectively,
in HFD animals relative to NC mice at 2 and 5 weeks,
respectively, and cardiac hypertrophy did not develop in
HFD mice (see Supplementary material online, Table S2).
After 2 weeks of HFD, fasting serum glucose, TG and FFA
concentrations were not different in NC and HFD mice, but
insulin concentrations were modestly increased (see Sup-
plementary material online, Table S2). Intraperitoneal
glucose tolerance was mildly impaired, as evidenced by a
25% increase in the area under the GTT curve in HFD
animals (P , 0.03); however, there was no statistical differ-
ence in glucose concentrations at the end of the GTT,
although values trended higher in HFD mice (179+24 vs.
136+8, P ¼ 0.1). In perfused hearts, heart rate, developed
pressure, and cardiac output in HFD hearts were similar to
controls even after 5 weeks, and the inotropic effect of
insulin was preserved in HFD hearts after 2 or 5 weeks
3.2. Glucose utilization in isolated working hearts
Cardiac substrate metabolism was determined in the pre-
sence and absence of 1 nM insulin. After 2 weeks of HFD,
basal rates of glycolysis and glucose oxidation were
reduced by 25–30% and declined further (by 30–40%,
respectively, vs. normal diets) after 5 weeks of HFD
(Figure 1). In control hearts, insulin increased glycolysis by
46%, but this response was completely abolished after 2
and 5 weeks of HFD. In control hearts, insulin increased
glucose oxidation by 43%. Although basal rates of glucose
oxidation were reduced at 2 weeks, the ability of insulin
to increase glucose oxidation was still preserved. However,
by 5 weeks, the ability of insulin to stimulate glucose
oxidation was also abolished.
Basal and insulin-stimulated cardiac performance in isolated working hearts after 2 and 5 weeks of high-fat diet
Basal NC, n ¼ 10
Basal HF, n ¼ 8
Ins NC, n ¼ 11
Ins HF, n ¼ 7
Basal NC, n ¼ 8
Basal HF, n ¼ 8
Ins NC, n ¼ 10
Ins HF, n ¼ 10
Heart rate (b.p.m.)
Coronary flow (mL/min)
Aortic flow (mL/min)
Cardiac output (mL/min)
Cardiac power (mW/g)
Ins, 1 nM insulin stimulation. NC, normal chow; HF, high fat; SP, systolic pressure; DP, diastolic pressure; DevP, developed pressure (SP minus DP). Cardiac output ¼ coronary flow þ aortic flow; cardiac power is derived
from cardiac output and DevP, normalized to heart weight.
*P , 0.05 and **P , 0.01 vs. normal chow under similar heart perfusion conditions.†P , 0.05 and‡P , 0.01 vs. no insulin, under similar dietary conditions.#P ¼ 0.07 vs. no insulin under similar dietary conditions.
High-fat feeding and cardiac metabolism353
3.3. Insulin-stimulated glucose uptake in isolated
We hypothesized that impaired cellular uptake of glucose
accounted for the absence of insulin-stimulated glycolysis.
Indeed, insulin-stimulated 2-deoxyglucose uptake in isolated
cardiomyocytes was significantly reduced after 2 and 5 weeks
of HFD, respectively (Figure 2). We then determined if this
could be accounted for by impaired insulin signalling to
Akt/PKB. Following in vivo administration of insulin, we
stream target AS-160 after 2 or 5 weeks of HFD (Figure 3).
3.4. Glucose transporter 4 translocation
Impaired insulin-mediated glucose uptake despite normal
signalling to AS-160 led us to hypothesize that GLUT4
content or translocation was impaired following HFD. Total
GLUT4 content was reduced by 55% following 2 weeks of
HFD (Figure 4A). Using sucrose gradient centrifugation we
observed that insulin reduced the content of intracellular
GLUT4 vesicles in NC-fed, but not in HFD mice (Figure 4B).
In NC-fed animals, there was a 20–30% increase in plasma
membrane GLUT4 in insulin-treated mice, which was
absent in the HFD animals (Figure 4C). Using GLUT4
(NC) hearts after 2 and 5 weeks, respectively. *P , 0.05 and **P , 0.01 vs. normal chow, at the same insulin concentration.†P , 0.05 vs. no insulin under similar
Insulin-stimulated glucose uptake in isolated cardiomyocytes. Data (mean+SEM) were obtained from five high-fat (HF) hearts and five normal chow
SEM) were obtained from perfusion of four high-fat (HF) hearts and five to six normal chow (NC) hearts after 2 weeks and three to five high-fat hearts and four to
five normal chow hearts after 5 weeks. **P , 0.01 vs. normal chow perfused under the same conditions.†P , 0.05 vs. basal under similar dietary conditions.
Glucose utilization in isolated working hearts. Glucose metabolism in isolated working hearts after 2 and 5 weeks of high-fat feeding. Data (mean+
J.J. Wright et al.
immunofluorescence, we observed that insulin led to a
clearly visible increase in cell surface GLUT4 staining in
NC-fed animals (Figure 4D). In contrast, in HFD mice,
there was no consistent change in membrane fluorescence
above background fluorescence in insulin-treated heart
samples isolated from HFD mice. Thus, three independent
approaches support the existence of an HFD-induced
defect in GLUT4 translocation.
3.5. Pyruvate dehydrogenase activity
Total PDH activity measured after 3 weeks of NC and HFD
were 3.65+0.45 and 3.75+0.27 nmol/min/mg, respect-
ively (P ¼ 0.85), and percentage in the active form was
39.6+5.0 and 35.8+4.1%, respectively (P ¼ 0.58).
3.6. Fatty acid metabolism in isolated working
Two weeks of HFD increased basal rates of palmitate oxi-
dation by 40% and MVO2 by 20%. After 5 weeks, MVO2
increased further and was 40% higher in hearts from HFD
mice relative to NC animals and palmitate oxidation rates
remained elevated. In contrast to glucose utilization, the
ability of insulin to suppress FA oxidation was not altered
by HFD, with insulin reducing palmitate oxidation rates
and MVO2in control and HFD hearts by 15 and 10%, respect-
ively (Figure 5). After 5 weeks, tissue TG content in hearts
was similar in HFD and NC-fed mice (7.7+0.3 vs.
7.9 + 0.4nmol/mg wet heart weight, P . 0.2, respect-
ively), and there were no differences in malonyl CoA
content (0.702+0.032—HFD vs. 0.767+0.064 pmol/mg/
wet weight—NC, P . 0.3).
3.6. Mitochondrial function in
To determine if mitochondrial uncoupling contributed to the
increase in MVO2following HFD, we examined mitochondrial
function in saponin-permeabilized cardiac muscle fibres.
After 2 and 5 weeks of HFD, no evidence of mitochondrial
dysfunction (normal state 3 respirations and ATP production
rates) or increased mitochondrial uncoupling (normal state 4
respirations and ATP/O ratios) was observed (Figure 6).
Similar results were obtained in permeabilized fibres
obtained after perfusing hearts with 1 mM palmitate,
which we previously reported unmasked mitochondrial
uncoupling in ob/ob and db/db mouse hearts17,18(data not
3.7. Transcriptional adaptations to high-fat feeding
After 2 weeks of HFD, there were no changes in expression
levels of the 10 PPAR-a targets examined. There was also
no change in mRNA levels of PGC-1a or b, PPAR-a, GLUT1,
or GLUT4. After 5 weeks, expression of MCD, MTE1, PDK4,
after 2 and 5 weeks of high-fat feeding. Graphs represent densitometry obtained from five mice per group. (B) Representative immunoblots for total and
phospho-AS-160 in the same animals as panel A.
Akt/PKB and AS-160 phosphorylation. (A) Representative immunoblots for total and phospho-Akt/PKB following intravenous saline (2) or insulin (þ),
High-fat feeding and cardiac metabolism355
suggesting that transcriptional activation of PPAR-a targets
occurred after changes in FA metabolism had developed.
This study demonstrates that hearts rapidly adapt to caloric
excess, developing patterns of substrate utilization that
mimic changes observed in longstanding obesity or diabetes
(type 1 or type 2).1,2,4–7They occur in the absence of signifi-
cant obesity or hyperlipidaemia, but are associated with
mildly abnormal glucose tolerance and a two-fold increase
in fasting insulin concentrations. As early as 2 weeks of
HFD, MVO2and FA oxidation are increased, whereas rates
of glucose oxidation and glycolysis are reduced. PDH activity
is not altered, but GLUT4 protein content is reduced via
post-transcriptional mechanisms. GLUT4 translocation is
also impaired independently of changes in insulin-mediated
activation of the upstream regulators of glucose transport
Akt/PKB and AS-160. Increased FA utilization occurs prior
to the activation of PPAR-a signalling, without any changes
in malonyl CoA content and in the absence of mitochondrial
uncoupling. Taken together, these data suggest that the
initial molecular defect that alters myocardial substrate
metabolism early in the course of high-fat feeding is
impaired GLUT4-mediated myocardial glucose utilization.
Contraction-mediated GLUT4 translocation in beating
hearts might be the major mediator of basal myocardial
glucose utilization. Support for this comes from studies in
mice with cardiomyocyte deletion of GLUT4 (G4H2/2).29
These animals have increased expression levels of GLUT1,
yet after an overnight fast, basal rates of glucose uptake
in perfused hearts were negligible.30Moreover, in mice
with cardiomyocyte-restricted KO of insulin receptors
(CIRKO), basal rates of glycolysis in perfused hearts were sig-
nificantly increased, despite .50% reduction in levels of
GLUT1 protein, but a two-fold increase in the GLUT4
protein.21In contrast, in isolated cardiomyocytes, GLUT1
is the major contributor to basal glucose uptake. Thus in
CIRKO mice, GLUT1 protein content and basal glucose
uptake in cardiomyocytes were proportionately reduced,21
whereas in GLUT4-deficient cardiomyocytes, basal rates of
glucose uptake were unchanged.31Thus, the normal rate
of basal glucose uptake in isolated cardiomyocytes in the
present study was not unexpected given that expression
levels of GLUT1 were unchanged.
PDH flux is an important regulator of glucose oxidation in
the heart.32Prior studies in HFD rats demonstrated reduced
pared after 2 weeks of high-fat diet (HFD) or normal chow (NC). Lower panel is densitometric analysis of the immunoblot shown. (B) Representative western blot
of glucose transporter 4 in sucrose gradients prepared from hearts of normal chow or high-fat mice. Hearts were obtained from animals that received 3 U of
insulin, which was injected via the inferior vena cava (þinsulin, right panels) or an equivalent volume of normal saline (left panel). Blots are representative
of three independent sucrose gradients per condition. (C) Representative immunoblots of plasma membrane GLUT4 or caveolin content in hearts from NC or
HF mice that received saline or insulin (þI). Densitometry of the GLUT4 band was normalized to caveolin content and the relative ratio of GLUT4 to caveolin
is shown beneath each panel. The GLUT4/caveolin ratio from each saline-treated animal is normalized to 1, and the þinsulin value is the fold change relative
to saline. Blots are representative of results obtained from three hearts per condition. (D) Representative immunohistochemistry of GLUT4 in left ventricle sec-
tions isolated from saline or insulin-injected mice on normal chow or high-fat diets. Data are representative of three independent experiments.
Myocardial glucose transporter 4 content and glucose transporter 4 translocation. (A) Glucose transporter 4 content in whole heart homogenates pre-
J.J. Wright et al.
activity of the active fraction of PDH (PDHa) after 28 days
but not after 10 days of high-fat feeding. Moreover, PDH
kinase activities were increased at 28 days, but not at 10
days.33,34Other studies in skeletal muscle of humans and
rodents have also suggested that the decline in PDH activity
with high-fat feeding parallels an increase in PDH kinase
activity.32,35,36In our study, we observed no change in the
expression levels of pyruvate dehydrogenase kinase (PDK4)
high-fat feeding. Data (mean+SEM) were obtained from perfusion of three to four high-fat hearts and five normal chow hearts after 2 weeks and five
high-fat hearts and four to five normal chow hearts after 5 weeks. *P , 0.05 and **P , 0.01 vs. normal chow perfused under the same conditions.†P , 0.05
vs. basal under similar dietary conditions.
Fatty acid utilization and MVO2. Palmitate oxidation and myocardial oxygen consumption (MVO2) in isolated working hearts after 2 and 5 weeks of
panels) in saponin-permeabilized mitochondrial fibres isolated from hearts of high-fat and normal chow mice after 2 and 5 weeks. Mitochondria were incubated
with palmitoyl-carnitine (PC), pyruvate (Pyr), Glutamate (Glut), or succinate in the presence of rotenone (SR), as described in Methods. Data are mean+SEM
and were obtained from six hearts per condition.
Mitochondrial respiration and ATP production rates. State 3 and state 4 respirations (upper panels) and ATP production rates and ATP/O ratios (lower
High-fat feeding and cardiac metabolism 357
after 2 weeks of high-fat feeding, and consistent with this,
we observed no differences in the total or active fraction
of PDH, measured at this early time point. We did observe
a significant increase in PDK4 activity after 5 weeks of
high-fat feeding and would expect that PDHa(if measured)
would be reduced after 5 weeks of high-fat feeding. Taken
together, these findings are consistent with the conclusion
that reduced GLUT4-mediated glucose uptake may rep-
resent the critical mechanism for reduced basal rates of gly-
colysis and glucose oxidation early in the course of high-fat
feeding (2 weeks), but as the duration of high-fat feeding
becomes more prolonged, reduced PDH flux will likely
contribute to the impairment in glucose oxidation.
High-fat feeding attenuated insulin-mediated glucose
uptake in isolated cardiomyocytes and prevented insulin-
mediated increases in glycolysis and glucose oxidation in
isolated working hearts despite normal insulin signalling to
AS-160. We believe this reflects a distal defect in GLUT4
translocation. Evidence for this was obtained by analyzing
to the sarcolemma and qualitatively by GLUT4 immuno-
histochemistry. The dissociation of insulin-mediated GLUT4
translocation from Akt/PKB and AS-160 signalling suggests
that high-fat feeding initially impairs key steps in the move-
ment of GLUT4 vesicles from their intracellular compartment
tothe sarcolemma.Studies inpalmitate-exposed L6myotubes
and skeletal muscles of HFD mice illustrated that impaired
insulin-mediated glucose uptake can occur in the absence
of defects in insulin-stimulated phosphorylation of Akt/PKB
or AS-160.37Moreover, increased sarcolemmal cholesterol
(phosphatidylinositol-3,4-bisphosphate) content or disrup-
tion of cortical F-actin can impair insulin-mediated GLUT4
translocation in skeletal muscle without reducing insulin-
mediated phosphorylation of Akt/PKB.38–40The molecular
mechanisms that are responsible for the transit of GLUT4
from the intracellular compartment to the sarcolemma are
complex and incompletely understood, but involve vesicle
budding, actin polymerization, and movement of GLUT4
vesicles on microtubules by myosin motors.41Moreover,
there are regulated steps involved in GLUT4 vesicle docking
and fusion, which could be perturbed by high-fat feeding in
the heart.41For example, increased expression of the
SNARE protein Munc-18, a negative regulator of GLUT4
vesicle docking, was described in skeletal muscle with
of lipoprotein lipase.42Thus, additional studies will be
required to elucidate the mechanisms by which short-term
high-fat feeding impairs GLUT4 trafficking in the heart.
An intriguing aspect of this study is the difference in
tempo of impaired insulin-stimulated glycolysis relative to
the ability of insulin to stimulate glucose oxidation. Insulin-
stimulated glycolysis was completely absent after 2 weeks of
HFD at a time when the ability of insulin to simulate glucose
oxidation was relatively preserved, whereas the ability
of insulin to stimulate glucose oxidation was abrogated
after 5 weeks of HFD. Increased glycolysis following insulin
stimulation is due in part to increased GLUT4 translocation,
which was clearly impaired as early as 2 weeks of HFD. The
oxidative metabolism of glucose while partially dependent
on glycolytic flux is also regulated by flux through PDH,
which is regulated by PDH phosphatases and kinases whose
activities are modulated by allosteric interactions with
nucleotides, acetyl CoA, NAD(H), and intracellular [Mg2þ]
and [Ca2þ].43Thus, the regulatory mechanisms for glycolysis
might exhibit differential insulin sensitivity relative to
mechanisms that regulate glucose oxidation.
Reduced myocardial glucose utilization and increased
myocardial FA utilization after 2 weeks of HFD, although
reminiscent of changes described in severe diabetes and
obesity occurred in the absence of major changes in the
serum concentrations of glucose, FFA, or TGs. It is widely
believed that an important mediator of altered myocardial
substrate metabolism in obesity and diabetes is activation
of PPAR-a signalling via increased delivery of FA ligands to
the heart.44The present study suggests that the activation
of PPAR-a signalling does not occur early in the course of
high-fat feeding at a time when myocardial FA utilization
is increased. We propose that the initial increase in FA util-
ization likely results from reduced basal rates of myocardial
glucose utilization, which is secondary to reduced GLUT4
content and translocation, which according to Randle’s
hypothesis would be predicted to increase FA oxidation.45
CD36 translocation to the sarcolemma has been described
respectively, was quantified by real-time PCR and normalized to b-actin. Values represent fold change in mRNA relative to normal chow, which was assigned as
1. Data are mean+SEM. *P , 0.05 and **P , 0.01 vs. normal chow of similar age. Gene names are: PGC1-a or b-PPAR gamma coactivator 1-alpha or beta;
PPAR-a, peroxisome proliferator activated receptor-alpha; HADHa or b-hydroxy acyl CoA dehydrogenase alpha or beta; MTE1, mitochondrial thioesterase-1;
CPT1b, carnitine palmitoyl transferase-1 beta; CPT2, carnitine palmitoyl transferase-2; PDK4, pyruvate dehydrogenase kinase-4; UCP2 or 3, uncoupling
protein 2 or 3; MCAD/LCAD, medium or long chain acyl CoA dehydrogenase; ACCb, acetyl CoA carboxylase-beta; MCD, malonyl CoA decarboxylase; GLUT1/4,
glucose transporter isoform 1 or 4.
Expression levels of genes that regulate fatty acid and glucose metabolism. Total RNA from six high-fat and six normal chow hearts at 2 and 5 weeks,
J.J. Wright et al.
in the hearts of rats after 8 weeks of high-fat feeding.15We
did not determine sarcolemmal CD36 content in the present
study, thus this mechanism cannot be ruled out. Perfusion of
hearts with FA alone could also clarify the mechanism. If
impaired glucose uptake was the sole basis for initial meta-
bolic defects within 2 weeks of high-fat feeding, then FA
utilization rates in the absence of glucose in the perfusate
would not be expected to be changed.
Convincing evidence for the increased activation of
PPAR-a signalling pathways was evident only after 5 weeks
of HFD. Thus it is likely that the activation of the PPAR-a
pathway may sustain the increase in myocardial FA utiliz-
ation only when increased dietary lipid intake persists.
These results are similar to those of Buchanan et al., who
noted increased myocardial FA utilization and decreased
glucose utilization in hearts from obese 4-week-old ob/ob
and db/db mice prior to the onset of diabetes, which was
not associated with the activation of PPAR-a signalling in
young mice. However, PPAR-a signalling increased as
animals aged and after hyperglycaemia developed.6
HFD caused an early increase in MVO2, which commonly
accompanies increased FA metabolism. Previous studies
from our laboratory suggested that mitochondrial uncoupling
may contribute to increased MVO2 in severe obesity.17,18
However, the present study demonstrated that following
short-term high-fat feeding, changes in FA oxidation and
oxygen consumption occurred in the absence of mitochon-
drial uncoupling. Thus, increased MVO2is likely a conse-
quence of altered substrate metabolism. FA is a less
efficient substrate than glucose, producing less ATP per
oxygen consumed. Thus an increase in FA utilization in HFD
sumption.16,46It is likely that as caloric excess becomes more
prolonged, mitochondrial uncoupling could occur as could be
sustained by the increased expression of uncoupling proteins
(UCP2 and UCP3), which was evident after 5 weeks of HFD. In
addition, increased expression of mitochondrial thioes-
efficiency by promoting futile ATP-wasting FA cycling
between the mitochondria and cytosol.47,48
In conclusion, high-fat feeding causes an early reduction in
glucose utilization on the basis of reduced GLUT4 content
and GLUT4 translocation, which is independent of coordinate
oxidation (Randle effect) is initially independent of PPAR-a
activation, and the increase in MVO2is not attributable to
mitochondrial uncoupling. Thus, cardiac metabolism rapidly
adapts to high-fat feeding. These changes precede the devel-
opment of obesity or diabetes, but recapitulate changes that
have classically been associated with longstanding obesity
Supplementary material is available at Cardiovascular
We thank James Cox in the Metabolomics Facility of the University
of Utah for Malonyl CoA measurements.
Conflict of interest: none declared.
This work was supported by the National Institutes of Health
(RO1HL73167, UO1HL70525, and UO1087947) to E.D.A. who
is an Established Investigator of the American Heart
Association. J.J.W. was supported by undergraduate student
awards from the American Heart Association (Western Affili-
ates), the Endocrine Society, and the University of Utah
Undergraduate Research Opportunity Program (UROP) and
S.B. by postdoctoral fellowships from the Juvenile Diabetes
Research Foundation and the American Heart Association.
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