The FASEB Journal • Hypothesis
Insulin resistance improves metabolic and contractile
efficiency in stressed rat heart
Romain Harmancey, Truong N. Lam, Genna M. Lubrano, Patrick H. Guthrie,
Deborah Vela, and Heinrich Taegtmeyer1
Department of Internal Medicine, Division of Cardiology, University of Texas Medical School at
Houston, Houston, Texas, USA
ABSTRACT Insulin resistance is a prominent feature
in heart failure, while hyperglycemia impairs cardiac
contraction. We propose that decreased insulin-medi-
ated glucose uptake by the heart preserves cardiac
function in response to metabolic and hemodynamic
stress. To test this hypothesis, we fed rats a high-
sucrose diet (HSD). Energy substrate metabolism and
cardiac work were determined ex vivo in a sequential
protocol simulating metabolic and hemodynamic
stress. Compared to chow-fed, control rats, HSD im-
paired myocardial insulin responsiveness and induced
profound metabolic changes in the heart, characterized
by reduced rates of glucose uptake (7.91ⴞ0.30 vs.
10.73ⴞ0.67 ␮mol/min/g dry weight; P<0.001) but
increased rates of glucose oxidation (2.38ⴞ0.17 vs.
1.50ⴞ0.15 ␮mol/min/g dry weight; P<0.001) and
oleate oxidation (2.29ⴞ0.11 vs. 1.96ⴞ0.12 ␮mol/
min/g dry weight; P<0.05). Tight coupling of glucose
uptake and oxidation and improved cardiac efficiency
were associated with a reduction in glucose 6-phosphate
and oleoyl-CoA levels, as well as a reduction in the
content of uncoupling protein 3. Our results suggest
that insulin resistance lessens fuel toxicity in the
stressed heart. This calls for a new exploration of the
mechanisms regulating substrate uptake and oxidation
in the insulin-resistant heart.—Harmancey, R., Lam,
T. N., Lubrano, G. M., Guthrie, P. H., Vela, D.,
Taegtmeyer, H. Insulin resistance improves metabolic
and contractile efficiency in stressed rat heart. FASEB J.
26, 3118 –3126 (2012). www.fasebj.org
Key Words: high-sucrose diet 䡠 metabolism 䡠 hyperglycemia 䡠
glucotoxicity
Insulin resistance is an independent predictor of
heart failure (1). Impaired insulin stimulation of glu-
cose transport is a defining feature of type 2 diabetes
and is thought to be the cause of contractile dysfunc-
tion in the heart, especially when the heart is stressed
(2). In fact, the incidence of heart failure is 5-fold
Abbreviations: H&E, hematoxylin and eosin; HSD, high-
sucrose diet; MVo2, myocardial oxygen consumption; PAS,
periodic acid-Schiff; PDH, pyruvate dehydrogenase; PDK,
pyruvate dehydrogenase kinase; UCP, uncoupling protein
3118
higher in patients with diabetes than in patients with-
out diabetes (3).
While hyperglycemia may compensate for impaired
insulin-stimulated glucose transport in muscle, there is
mounting evidence that supraphysiologic concentra-
tions of glucose are deleterious to cardiac function (4).
Akin to the molecular derangements induced by excess
glucose entry into the ␤-cells of the pancreas and
skeletal myotubes, cardiac glucotoxicity may also de-
velop in the heart by rerouting of hexose 6-phosphates
into the hexosamine biosynthetic and pentose phos-
phate pathways (5). In support of this hypothesis,
extensive experimental work performed on animal
models suggests that hyperglycemia per se is associated
with a modification of contractile proteins and altered
calcium homeostasis in cardiac myocytes, and impaired
diastolic and systolic function (6 – 8).
In light of these observations, there is still uncertainty
about whether reduced glucose uptake has deleterious
or protective effects for the heart subjected to hyper-
glycemia. We propose that myocardial insulin resis-
tance may preserve the heart’s function when glucose
supply is abnormally elevated. To address this question,
we fed rats a high-sucrose diet (HSD), a dietary manip-
ulation known to rapidly impair systemic and myocar-
dial insulin sensitivity (9). We then tested our hypoth-
esis in working hearts perfused ex vivo using a
sequential protocol simulating metabolic and hemody-
namic stress.
MATERIALS AND METHODS
Animals and diets
Male Sprague Dawley rats (200 –224 g) were obtained from
Harlan Laboratories (Indianapolis, IN, USA) and housed as
described previously (10). Rats were fed an HSD (sucrose
67% of total kilocalories; diet D11725; Research Diets, Inc.,
1
Correspondence: University of Texas Medical School at
Houston, 6431 Fannin, MSB 1.246, Houston, TX 77030, USA.
E-mail: heinrich.taegtmeyer@uth.tmc.edu
doi: 10.1096/fj.12-208991
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.
0892-6638/12/0026-3118 © FASEB
New Brunswick, NJ, USA) or maintained on standard labora-
tory chow (LabDiet Laboratory Rodent Diet 5001; PMI Nu-
trition International, St. Louis, MO, USA) for up to 8 wk.
Heparinized plasma samples were obtained from the tail vein
of conscious rats maintained in the fed state or following 18
h of food withdrawal. The protocol was approved by the
Animal Welfare Committee of the University of Texas Health
Science Center at Houston.
Histology
Hearts were rinsed with saline and sectioned into 2-mm-thick
slices from apex to base. Two equatorial slices were taken
from each heart. One slice was fresh-frozen, embedded in
optimal cutting temperature compound, and stored at
⫺20°C. The other slice was fixed in 10% neutral buffered
formalin. Frozen tissue was sliced into 5-␮m-thick sections
and stained with oil red O for triglyceride detection. Forma-
lin-fixed tissue was embedded in paraffin and serially cut into
5-␮m-thick sections. Sections were stained with hematoxylin
and eosin (H&E), Masson’s trichrome, and periodic acid-
Schiff (PAS) for morphometric analysis and for detection of
fibrosis and glycogen, respectively.
Working heart perfusions
Hearts were perfused as working heart ex vivo (11). In brief,
hearts were perfused at 37°C with nonrecirculating Krebs-
Henseleit buffer equilibrated with 95% O2-5% CO2 and
supplemented with glucose and sodium oleate bound to 1%
BSA (Fraction V, fatty acid free; Millipore, Billerica, MA,
USA). The filling pressure was 15 cmH2O, with an initial
afterload pressure of 100 cmH2O. Cardiac power (watts) was
calculated as the product of cardiac output (coronary plus
aortic flow, m3/s) times the afterload (pascals). Myocardial
oxygen consumption (MVo2; ␮mol/min) was measured with
a YSI 5300A biological oxygen monitor (YSI, Yellow Springs,
OH, USA), using 1.06 mM for the concentration of dissolved
O2 at 100% saturation (12). Cardiac efficiency was expressed
as the ratio of cardiac power to MVo2. Glucose transport and
phosphorylation were determined by [3H]2O production
from [2-3H]glucose (0.05 ␮Ci/ml) (13). In parallel experi-
ments, glucose oxidation and oleate oxidation were deter-
mined by quantitative collection of [14C]O2 and [3H]2O
released in the coronary effluent using [U-14C]glucose (0.08
␮Ci/ml) and [9,10-3H]oleate (0.1 ␮Ci/ml), respectively (14).
Samples of the coronary effluent were assayed for lactate
levels using a YSI 2300 STAT Plus glucose and lactate ana-
lyzer. At the end of perfusion, the hearts were freeze-clamped
on their cannula with aluminum tongs cooled in liquid N2
and then stored at ⫺80°C.
Tissue metabolite analyses
The extraction of glycogen and total lipids was performed as
reported previously (10). Lipids were emulsified by sonica-
tion for 30 s in buffer containing 28.75 mM 1,4-piperazinedi-
ethanesulfonic acid, 57.76 mM MgCl2·6H2O, 8.76 ␮M BSA
(fatty acid free), and 0.1% SDS (15), and triacylglycerol
content was quantified using the L-Type TG H assay (Wako
Chemicals, Richmond, VA, USA). Oleyl-CoA levels were mea-
sured by HPLC (10). Glucose 6-phosphate was measured
according to Lang and Michal (16).
Immunoblot analyses
Tissue was homogenized in the presence of a cocktail of
phosphatase and protease inhibitors. Levels of total and/or
INSULIN RESISTANCE PROTECTS THE STRESSED HEART
phosphorylated proteins were detected by immunoblotting
using horseradish peroxidase-conjugated secondary antibod-
ies and chemiluminescence (Santa Cruz Biotechnology,
Santa Cruz, CA, USA). A list of the primary antibodies used is
given in Supplemental Table S1.
Quantitative real-time PCR
Total RNA was extracted from heart muscle with an RNeasy
fibrous tissue kit (Qiagen, Valencia, CA, USA) and treated
with DNA-free (Life Technologies, Grand Island, NY, USA).
Quantification of transcripts was based on known amounts of
synthetic DNA standard using TaqMan probes (Sigma-Al-
drich, St. Louis, MO, USA). All mRNA expression levels were
normalized to cyclophilin A and expressed in fold change vs.
chow-fed animals. Primer and probe sequences are given in
Supplemental Table S2.
Statistical analysis
The statistical significance between means was assessed by
unpaired Student’s t test or by ANOVA followed by a New-
man-Keuls or Bonferroni test.
RESULTS
HSD induces hyperlipidemia and systemic insulin
resistance
On the first day of HSD feeding, rats ate significantly
more than the control group (Fig. 1A). However, the
HSD-fed rats subsequently reduced their food intake to
levels lower than the chow-fed animals, resulting in a
normalization of calorie intake (Fig. 1B). Weight gain
was similar between chow- and HSD-fed rats (Fig. 1C).
After 3 wk of HSD consumption, plasma total choles-
terol, triglyceride, and free fatty acid levels rose by 77,
178, and 36%, respectively (Table 1). Fasting insulin
levels were also higher, although euglycemia was main-
tained (Table 1). At the time of sacrifice for the heart
perfusion, systemic insulin sensitivity was significantly
impaired with HSD (Fig. 1D), while glucose tolerance
was not altered (Fig. 1E). Fed plasma insulin levels and
fed blood glucose levels remained similar to controls at
the time of sacrifice (Fig. 1F, G).
HSD reduces cardiac glycogen stores but has no
effect on cardiac structure
Heart weights did not differ (Fig. 2A, B), and histolog-
ical analyses did not reveal any difference in cardiac cell
size or fibrosis (Fig. 2C). Glycogen staining patterns
were similar for the hearts of chow- and HSD-fed
animals (Fig. 2D), although subsequent enzymatic
quantification revealed a reduction in tissue glycogen
content with sucrose feeding (Fig. 2E). Myocardial
triglyceride levels were the same in both groups (Fig.
2D, F).
3119
Figure 1. The HSD-fed rat as a nonobese rodent model of insulin resistance. A) Food intake of chow-fed (solid squares with solid
trace) and HSD-fed (open circles with dotted trace) rats was recorded over the first 24 h of feeding, and then weekly until the
end of the protocol. *P ⬍ 0.05 and †††P ⬍ 0.001 vs. chow from 1 wk until 8 wk. B) Calorie intake was determined according to
the energy content of the standard rodent chow (3.04 kcal/g) and of the HSD (3.90 kcal/g). ***P ⬍ 0.001 vs. chow at d 1. C)
Body weight was measured for the same time points. Data are means ⫾ se from 12 animals/group. D) An insulin tolerance test
was performed starting 5 wk after the beginning of the feeding protocol by performing a subscapular subcutaneous injection
of 0.5 U insulin/kg body weight in rats that were unfed for 18 h. Data are means ⫾ se from 11 chow- and 9 HSD-fed rats.
**P ⬍ 0.01 vs. chow. E) A glucose tolerance test was performed on rats that were unfed for 18 h after 5 wk on diet by
administering 1g glucose/kg body weight by oral gavage of a 50% (w/v) glucose solution. Data are means ⫾ se from 4 chow-fed
and 5 HSD-fed rats. F, G) Fed plasma insulin levels (F) and fed blood glucose levels (G) were measured at time of sacrifice prior
to heart perfusion. Data represent means ⫾ se from 6 animals/group.

Insulin signaling through Akt is blunted in the hearts
of HSD-fed rats
The insulin responsiveness of the isolated working
hearts was assessed by adding escalating doses of insulin
to the perfusion buffer (Fig. 3A). In the hearts of
chow-fed rats, rates of glucose uptake gradually in-
creased with subphysiologic and physiological concen-
trations of the hormone, and then leveled out at 1 nM
insulin (Fig. 3B). In addition, the increase in insulin
gradually stimulated glucose oxidation (Fig. 3C). This
response to insulin was blunted in the hearts of HSD
rats. During the perfusion protocol, cardiac power was
steady and identical in both groups (data not shown).
We next investigated the effect of HSD on cardiac
insulin signaling at the level of Akt. As expected, Akt
phosphorylation at Thr308 and Ser473 was increased in
the hearts of chow-fed rats with insulin in the perfusion
medium (Fig. 3D). Basal Akt phosphorylation at both
Thr308 and Ser473 was higher in the nonperfused
hearts of HSD-fed rats. Following insulin stimulation ex
vivo, the phosphorylation of Akt at Thr308 increased to
a level similar to controls, but the amplitude of the
response was consequently reduced. Because Akt phos-
phorylation at Ser473 was maximal in nonperfused
hearts, the stimulatory effect of insulin on that phos-
phorylation site was completely lost. There was no
change in total Akt levels in the hearts of HSD-fed rats.
Reduced glucose uptake is associated with improved
contractile function in HSD-fed rat hearts subjected
to metabolic and hemodynamic stress

Hearts were first perfused at a normal workload for 20

TABLE 1. Fasting plasma parameters after 3 wk of HSD
min with near-physiological concentrations of glucose,
Parameter Chow HSD P
oleate, and insulin (Fig. 4A). Rates of glucose uptake were
lower, but not statistically significant, in hearts from
Cholesterol (mg/dl) 66.1 ⫾ 1.9 116.9 ⫾ 7.9 ⬍0.001 HSD-fed rats. This small decrease in glucose uptake may
Triacylglycerol (mg/dl) 38.1 ⫾ 10.1 105.8 ⫾ 10.6 ⬍0.001 partly account for the decrease in glycolytic flux (de-
Free fatty acids (mM) 0.14 ⫾ 0.01 0.19 ⫾ 0.02 N.S. creased lactate release). However, rates of glucose oxida-
Glucose (mg/dl) 99.8 ⫾ 3.5 107.2 ⫾ 7.5 N.S. tion and oleate oxidation, as well as cardiac performance,
Insulin (ng/ml) 0.40 ⫾ 0.04 1.05 ⫾ 0.19 ⬍0.01
were similar in both groups (Table 2). For the next 20
Data represent means ⫾ se of 6 animals/group. N.S., not min, hearts were subjected to a metabolic stress simulat-
significant. ing hyperglycemic, hyperlipidemic, and hyperinsulinemic

3120 Vol. 26 August 2012 The FASEB Journal 䡠 www.fasebj.org HARMANCEY ET AL.
Figure 2. Effects of HSD on tissue structure and substrate storage in the heart. A, B) Cardiac size of chow-fed (solid bars) and
HSD-fed (open bars) rats was assessed by normalizing heart weight to body weight (A) or heart weight to tibial length (B). C)
Cardiac cell size and extracellular fibrosis were analyzed on H&E- and Masson’s trichrome (MT)-stained cardiac tissue paraffin
sections, respectively. D) Intracardiac glycogen and triglyceride levels were compared by PAS and oil red O (ORO) staining,
respectively. E, F) Intracardiac glycogen (E) and triglycerides (F) were further quantified by enzymatic methods. Data represent
means ⫾ se from 6 animals/group. **P ⬍ 0.01 vs. chow.

conditions (Fig. 4A). This intervention resulted in rela-
tively higher rates of myocardial glucose uptake in both
groups, although the increase was significantly lower for
the insulin-resistant hearts (⫹65% with HSD vs. ⫹81%
with chow). Rates of glucose and fatty acid oxidation, as
well as lactate release, all increased similarly in both
groups. Overall, the amount of glucose going through the
glycolytic pathway was identical in both groups, and
cardiac power and efficiency remained steady under
metabolic stress. Last, we added a hemodynamic stress to
metabolic stress for the last 20 min of perfusion (Fig. 4A).
The new stress condition led to an additional increase in
the rates of glucose uptake by the hearts of chow-fed
animals (Table 2). Conversely, rates of glucose uptake did
not increase further for the hearts of HSD-fed rats.
However, rates of glucose and fatty acid oxidation in-
creased in both groups. Unexpectedly, the increase was
greater in the hearts of HSD-fed rats, for rates of oleate
oxidation (⫹45% with HSD vs. ⫹28% with chow), but
especially for rates of glucose oxidation (⫹164% with
HSD vs. ⫹43% with chow). The amount of glucose going
through the glycolytic pathway was still identical in both
groups, suggesting an increased flux of pyruvate into
mitochondria in the hearts of HSD-fed rats. Interestingly,
cardiac contractile performance of control animals failed
to increase in response to the acute increase in workload.
However, cardiac power increased by 8%, and cardiac
efficiency was better preserved, for the hearts of HSD-fed
rats (Table 2).
The stressed hearts of HSD-fed rats display increased
flux of glucose through the pyruvate dehydrogenase
complex and reduced accumulation of intermediary
metabolites
We next investigated the cause and consequence of this
increased coupling between glucose uptake and oxida-
tion at the molecular level. We observed that HSD was
associated with the reduced inhibitory phosphorylation
of the pyruvate dehydrogenase (PDH) E1␣ subunit on
Ser300 (Fig. 4B), thereby explaining the higher capac-
ity of the insulin-resistant heart to oxidize glucose.
Intracellular levels of glucose 6-phosphate were signif-
icantly lower (Fig. 4C). However, glycogen content did

INSULIN RESISTANCE PROTECTS THE STRESSED HEART 3121

Figure 3. Impaired insulin responsiveness in the working heart of HSD-fed rats. A) Isolated hearts were perfused in the working mode
for 48 min in Krebs-Henseleit buffer supplemented with 5 mM glucose and 0.8 mM oleate. After allowing for stabilization of baseline
parameters, the hearts were perfused in presence of 10⫺11 M insulin. The insulin concentration was then increased 10-fold every 8
min and up to 10⫺7 M. Cardiac function and myocardial glucose utilization rates were monitored 5 min after each change in the
insulin concentration (arrows). B, C) Rates of glucose uptake (B) and glucose oxidation (C) for the perfused hearts of chow-fed (solid
squares with solid trace; n⫽5) or HSD-fed (open circles with dotted trace; n⫽7) rats were measured for each concentration of insulin
tested. D) Immunoblot analysis of phospho-Akt in hearts from chow-fed (C; solid bars) and sucrose-fed (S; open bars) rats. Akt
phosphorylation at Thr308 and Ser473 was assessed at baseline in nonperfused hearts and in hearts that were perfused ex vivo with
insulin with the protocol described in A. Data are shown as means ⫾ se. *P ⬍ 0.05 vs. chow.

not differ between groups at the end of the perfusion
protocol (Fig. 4D), suggesting that the increase of
pyruvate oxidation, rather than an increase in glucose
storage as glycogen, causes the reduction in intracellu-
lar glucose 6-phosphate levels. The intracellular levels
of the fatty acid intermediate oleoyl-CoA also decreased
(Fig. 4E), and this decrease was independent of intra-
cellular triglyceride content (Fig. 4F).
of the other fatty acid oxidation-related enzymes were not
altered, with the notable exception of UCP3, the expres-
sion of which was markedly increased (Fig. 5A). Surpris-
ingly, UCP3 protein levels were decreased (Fig. 5B),
suggesting the activation of specific post-transcriptional
regulatory mechanisms associated with HSD.

Uncoupling protein 3 (UCP3) is post-transcriptionally DISCUSSION

down-regulated in the hearts of HSD-fed rats
To gain insight into the molecular basis of enhanced
efficiency of the stressed hearts of HSD-fed rats, we
investigated potential changes in the expression levels of
enzymes involved in substrate partitioning. The mRNA
and protein levels of GLUT1 and GLUT4 were similar in
both groups (Fig. 5A, B). However, the transcript levels of
the fatty acid transporter CD36, the pyruvate dehydroge-
nase kinase (PDK) 2, and of the muscle type carnitine
palmitoyl transferase-1 were decreased. Transcript levels
We have shown that reduced glucose uptake and in-
creased rates of glucose oxidation are associated with
improved contractile function of the heart subjected to
metabolic and hemodynamic stress. The improvement
of contractile efficiency for the insulin-resistant heart of
HSD-fed rats can be explained as follows. First, glucose
uptake is reduced in response to excess fuel supply,
which may limit the rerouting of glycolytic intermedi-
ates into nonoxidative pathways. Second, the coupling
between glycolysis and pyruvate oxidation is improved.

3122 Vol. 26 August 2012 The FASEB Journal 䡠 www.fasebj.org HARMANCEY ET AL.
Figure 4. Increased flux through PDH and reduced levels of intermediary metabolites in the stressed hearts of HSD-fed rats.
A) Isolated hearts from chow-fed (C; solid bars; n⫽11) or sucrose-fed (S; open bars; n⫽9) rats were perfused successively in 3
different conditions for a total of 60 min. The hearts were perfused with 5 mM glucose, 0.4 mM oleate, and 0.5 ng/ml insulin
(near-physiological milieu) during the first 20 min, before being perfused with a buffer containing 25 mM glucose, 0.8 mM
oleate, and 5 ng/ml insulin (metabolic stress) for the next 20 min. A hemodynamic stress (afterload raised from 100 to 140
cmH2O; 1 ␮M epinephrine) was superimposed to the metabolic stress for the last 20 min of perfusion. Cardiac function and
myocardial rates of glucose and fatty acid oxidation were monitored every 5 min and averaged for each perfusion condition.
Data are reported in Table 2. B) At the end of the perfusion protocol, the levels of inhibitory phosphorylation of PDH on serine
residues 232, 293, and 300 were determined by immunoblot. C–F) Intracardiac levels of the glycolytic intermediate glucose
6-phosphate (C), glycogen (D), the fatty acid intermediate oleoyl-CoA (E), and triglycerides (F) were quantified from freeze-clamped,
perfused heart tissue. All data are presented as means ⫾ se. *P ⬍ 0.05, **P ⬍ 0.01 vs. chow.

Third, cardiac efficiency is improved because there is
increased utilization of glucose for oxidative phosphor-
ylation in a state of increased energy demand.
A key element of the present study was to utilize an
animal model where myocardial glucose uptake would
be impaired in response to insulin. The HSD rapidly
induces systemic insulin resistance in rats. The impair-
ment of insulin action in skeletal muscle and liver
following short-term high-sucrose feeding has already
been studied (17). However, the effects on the heart
are less documented and sometimes conflicting (9). In
our study, we observed a mild reduction in cardiac
glycogen levels, which may reflect an increased inhibi-
tion of glycogen synthase due to reduced myocardial
insulin sensitivity. Interestingly, basal Akt activity was
increased and corresponded to a blunted response to
insulin stimulation in the isolated working hearts. This
observation is in accordance with previous findings
made by others in the liver of rats fed with the same
HSD for 3 wk, where increased basal activity of the
PI3K/Akt axis was proposed to contribute to the main-
tenance of normal rates of basal glucose production
(18). The alteration of Akt activity had a modest
inhibitory effect on myocardial glucose uptake when
the hearts were perfused with normal glucose levels
(Fig. 3B). This observation (made ex vivo) and the
maintenance of normoglycemia in vivo suggest that the
animals were in a phase of compensated insulin resis-
tance at the time the experiments were performed.
The reduction in myocardial glucose uptake became
significant when simulating a phase of decompensated
insulin resistance by raising insulin and glucose to
supraphysiologic concentrations ex vivo. The role of
impaired insulin signaling through Akt in this decrease
is supported by the fact that GLUT1 and GLUT4
transporter levels were not altered by the HSD. Inter-

INSULIN RESISTANCE PROTECTS THE STRESSED HEART 3123

TABLE 2. Improved metabolic and contractile efficiency for working hearts of HSD-fed rats subjected to metabolic and hemodynamic stress

Near-physiological milieu: 5 Metabolic stress: 25 mM Metabolic and hemodynamic

mM glucose, 0.4 mM oleate, glucose, 0.8 mM oleate, stress: 40% rise in afterload ⫹ 1
0.5 ng/ml insulin 5 ng/ml insulin ␮M epinephrine

Parameter Chow HSD Chow HSD Chow HSD

Glucose uptake (␮mol/min/gdw) 5.24 ⫾ 0.14 4.81 ⫾ 0.24 9.51 ⫾ 0.36 7.96 ⫾ 0.39** 10.73 ⫾ 0.67 7.91 ⫾ 0.30***
Glucose oxidation (␮mol/min/gdw) 0.40 ⫾ 0.04 0.43 ⫾ 0.03 1.05 ⫾ 0.12 0.90 ⫾ 0.10 1.50 ⫾ 0.15 2.38 ⫾ 0.17***
Lactate release (␮mol/min/gdw) 5.20 ⫾ 0.43 3.52 ⫾ 0.21** 9.68 ⫾ 0.41 8.81 ⫾ 0.51 13.11 ⫾ 0.53 12.40 ⫾ 0.39
Glucose utilized (␮mol/min/gdw) 5.60 ⫾ 0.46 3.94 ⫾ 0.22** 10.73 ⫾ 0.44 9.71 ⫾ 0.56 14.61 ⫾ 0.57 14.79 ⫾ 0.48
Oleate oxidation (␮mol/min/gdw) 1.13 ⫾ 0.06 1.13 ⫾ 0.06 1.52 ⫾ 0.07 1.58 ⫾ 0.07 1.96 ⫾ 0.12 2.29 ⫾ 0.11*
Cardiac power (mW) 8.0 ⫾ 0.4 8.8 ⫾ 0.4 8.2 ⫾ 0.4 9.1 ⫾ 0.3 8.3 ⫾ 0.5 9.8 ⫾ 0.4*
Cardiac efficiency 0.83 ⫾ 0.03 0.83 ⫾ 0.04 0.82 ⫾ 0.03 0.85 ⫾ 0.04 0.54 ⫾ 0.02 0.61 ⫾ 0.03*

Isolated hearts were perfused as described in Fig. 4A. Lactate release is given in micromole glucose equivalents. Glucose utilized represents
the sum of glucose released as lactate and being oxidized. Data are presented as means ⫾ se from 5 chow-fed and 6 HSD-fed animals/group
for rate of glucose uptake; 11 chow-fed and 9 HSD-fed animals/group for all other parameters. gdw, grams dry weight. *P ⬍ 0.05, **P ⬍ 0.01,
***P ⬍ 0.001 vs. chow.

estingly, the quantities of glucose oxidized or released
as lactate remained the same, which demonstrates that,
in presence of hyperglycemia, the flux of glucose
through the glycolytic and oxidation pathways can be
maintained despite a decrease in glucose transport.
Even more surprising is the fact that substrate oxidation
was higher in the hearts of HSD-fed rats when a
hemodynamic stress was superimposed to the meta-
bolic stress. Increased oxidation of glucose by the
insulin-resistant, stressed hearts was an unpredicted
finding since rats fed the HSD rapidly developed hy-
perlipidemia, a condition that usually inhibits myocar-
dial glucose utilization due to increased lipid delivery to
the heart (Randle cycle; ref. 19). It is of note that we
observed a small, but significant, reduction in the
intracellular levels of the fatty acid intermediate oleoyl-
CoA. Moreover, the expression of the fatty acid trans-
porter CD36 was also decreased at the mRNA level.
Insulin has been shown to stimulate fatty acid uptake in
cardiac myocytes (20), and although we did not mea-
sure the rates of myocardial fatty acid uptake, the data
suggest that these rates may also be reduced in the
hearts of HSD-fed rats. Therefore, glucose oxidation
may be stimulated by a reduction of fatty acid-mediated
inhibition of glucose oxidation, and fatty acid oxidation
may have been, in turn, stimulated by a lesser accumu-
lation of fatty acid intermediates. The reduced inhibi-
tory phosphorylation of PDH at high workload is con-
sistent with a switch toward preponderant glucose
utilization, increased cardiac efficiency, and improved
contraction of the stressed heart. This rapid increase in
pyruvate oxidation was associated with a decrease in
glucose 6-phosphate levels. Conversely to the hearts of
HSD-fed rats, heart work failed to increase for control
rats in response to the increase in workload, and this
despite higher rates of glucose uptake. Reduced cou-
pling of glucose metabolism is linked to poor mechan-
ical function of the heart in diabetes, most likely
because the spillover of excess glucose in nonoxidative
pathways reduces the activity of the sarco(endo)plasmic

Figure 5. Post-transcriptional down-regulation of UCP3 in the heart of HSD-fed rats. A) Total RNA was prepared from the hearts
of chow-fed (solid bars) or HSD-fed (open bars) rats and analyzed by real-time PCR to determine the abundance of transcripts
encoding metabolic enzymes. The proteins analyzed included the glucose transporters GLUT1 and GLUT4; the PDK isoforms
1, 2, and 4; the fatty acid transporters CD36 and fatty acid transporter 1 (FATP1); the uncoupling protein UCP3; peroxisome
proliferator-activated receptor ␣ (PPAR␣); malonyl-CoA decarboxylase (MCD); muscle carnitine palmitoyl transferase-1
(mCPT1); and the medium-chain and long-chain acyl-CoA dehydrogenases (MCAD and LCAD, respectively). B) Protein levels
of GLUT1, GLUT4, and UCP3 were measured by immunoblot and normalized to ␤-tubulin levels. Data represent means ⫾ se
from 6 animals/group. *P ⬍ 0.05 vs. chow.

3124 Vol. 26 August 2012 The FASEB Journal 䡠 www.fasebj.org HARMANCEY ET AL.

reticulum Ca2⫹-ATPase and impairs calcium homeosta-
sis (7, 8). Therefore, the increase in contractile perfor-
mance in control hearts may have been hampered by
glucotoxic effects.
We acknowledge that further evidence is needed to
demonstrate that reduced glucose uptake is directly
linked to the improvement of efficiency of the stressed
heart, as the decreased expression of UCP3 was also
likely to increase cardiac efficiency in our model. The
striking discordance between UCP3 mRNA and protein
levels has been reported in the gastrocnemius muscle
of rodents (21, 22). While the UCP3 protein turns over
rapidly, the factors regulating its turnover are unknown
(23). However, both plasma insulin and glucose levels
are negatively correlated to UCP3 protein level in
human skeletal muscle (24). Therefore, it is not impos-
sible that both synthesis and degradation of UCP3 in
the heart is under the control of insulin signaling.
Additional models of myocardial insulin resistance will
have to be studied in order to confirm our hypothesis,
but interestingly, impaired myocardial glucose uptake
induced by high-fat feeding is associated with preserved
contractile function in rats undergoing coronary artery
ligation (25).
In summary, the results suggest an adaptive role for
impaired myocardial insulin sensitivity in a state of “fuel
overload. ” The novelty of the findings is 2-fold. First,
we demonstrate that in hyperglycemic, hyperlipidemic,
and hyperinsulinemic conditions, a moderate impair-
ment in glucose uptake does not reduce the glycolytic
flux or the rates of glucose oxidation. Second, a normal
INSULIN RESISTANCE PROTECTS THE STRESSED HEART
Figure 6. Proposed mechanisms leading to in-
creased contractile performance for the insulin-
resistant, stressed hearts of HSD-fed rats. In pres-
ence of hyperglycemia and hyperinsulinemia,
impaired insulin-stimulated phosphorylation of Akt
limits glucose transport into cardiac myocytes. Be-
cause insulin is known to stimulate the transloca-
tion of the fatty acid transporter CD36 at the
sarcolemma ( 20), insulin resistance is also likely to
limit fatty acid uptake in the heart of HSD-fed rats.
Both rates of glucose and fatty acid oxidation are
increased in response to an acute increase in work-
load. However, glucose oxidation increases more
than fatty acid oxidation, and combines to an
improved mitochondrial energy coupling (reduced
UCP3 levels) to increase contractile efficiency. The
energetic response of the stressed heart may have
been preserved by the impairment of insulin-stim-
ulated fatty acid uptake, and the reduction of fatty
acid-mediated inhibition of glucose oxidation. The
increased coupling between glucose uptake and
glucose oxidation limits the accumulation of glyco-
lytic intermediates and their rerouting into the
pentose phosphate pathway (PPP) and the hexo-
samine biosynthetic pathway (HBP). This could
increase the mechanical function of the heart by
preserving the activity of contractile proteins and
calcium homeostasis in cardiac myocytes. Dashed
lines represent pathways with a reduced activity.
Dotted lines represent possible consequences of
insulin resistance that have not been tested directly
in the present model.
rat heart fails to respond to an acute increase in
workload when subjected to this metabolic stress,
whereas cardiac performance increases for the hearts of
HSD-fed rats. The preserved contractile response of the
insulin-resistant heart is likely the result of reduced
accumulation of intracellular metabolites and of in-
creased contractile efficiency (Fig. 6). Taken together,
our data call for a new exploration of the mechanisms
regulating substrate uptake and oxidation in the insu-
lin-resistant heart.
The authors thank the Mouse Metabolism Core (Baylor
College of Medicine, Houston, TX, USA) for the plasma
analyses and Roxy Tate for editorial assistance. Figure 6 was
produced using Servier Medical Art (http://www.servier.
com/servier-medical-art). This work was supported by grants
from the U.S. National Heart, Lung, and Blood Institute and
the American Heart Association.
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Received for publication March 30, 2012.
Accepted for publication May 1, 2012.
HARMANCEY ET AL.