Download Differential effects of saturated and unsaturated fatty - AJP

Am J Physiol Heart Circ Physiol 291: H38 –H44, 2006.
First published January 27, 2006; doi:10.1152/ajpheart.01295.2005.
TRANSLATIONAL PHYSIOLOGY
Differential effects of saturated and unsaturated fatty acid diets on
cardiomyocyte apoptosis, adipose distribution, and serum leptin
Isidore C. Okere,1 Margaret P. Chandler,1 Tracy A. McElfresh,1 Julie H. Rennison,1
Victor Sharov,4 Hani N. Sabbah,4 Kou-Yi Tserng,2 Brian D. Hoit,3
Paul Ernsberger,2 Martin E. Young,5 and William C. Stanley1,2
Submitted 8 December 2005; accepted in final form 24 January 2006
Okere, Isidore C., Margaret P. Chandler, Tracy A. McElfresh,
Julie H. Rennison, Victor Sharov, Hani N. Sabbah, Kou-Yi Tserng,
Brian D. Hoit, Paul Ernsberger, Martin E. Young, and William C.
Stanley. Differential effects of saturated and unsaturated fatty acid diets
on cardiomyocyte apoptosis, adipose distribution, and serum leptin. Am J
Physiol Heart Circ Physiol 291: H38 –H44, 2006. First published January
27, 2006; doi:10.1152/ajpheart.01295.2005.—Fatty acids are the primary
fuel for the heart and are ligands for peroxisome proliferator-activated
receptors (PPARs), which regulate the expression of genes encoding
proteins involved in fatty acid metabolism. Saturated fatty acids, particularly palmitate, can be converted to the proapoptotic lipid intermediate
ceramide. This study assessed cardiac function, expression of PPARregulated genes, and cardiomyocyte apoptosis in rats after
8 wk on either a low-fat diet [normal chow control (NC); 10%
fat calories] or high-fat diets composed mainly of either saturated (Sat)
or unsaturated fatty acids (Unsat) (60% fat calories) (n ⫽ 10/group). The
Sat group had lower plasma insulin and leptin concentrations compared
with the NC or Unsat groups. Cardiac function and mass and body
mass were not different. Cardiac triglyceride content was increased in the
Sat and Unsat groups compared with NC (P ⬍ 0.05); however, ceramide
content was higher in the Sat group compared with the Unsat group
(2.9 ⫾ 0.2 vs. 1.4 ⫾ 0.2 nmol/g; P ⬍ 0.05), whereas the NC group was
intermediate (2.3 ⫾ 0.3 nmol/g). The number of apoptotic myocytes,
assessed by terminal deoxynucleotide transferase-mediated dUTP nickend labeling staining, was higher in the Sat group compared with the
Unsat group (0.28 ⫾ 0.05 vs. 0.17 ⫾ 0.04 apoptotic cells/1,000 nuclei;
P ⬍ 0.04) and was positively correlated to ceramide content (P ⬍ 0.02).
Both high-fat diets increased the myocardial mRNA expression of the
PPAR-regulated genes encoding uncoupling protein-3 and pyruvate dehydrogenase kinase-4, but only the Sat diet upregulated medium-chain
acyl-CoA dehydrogenase. In conclusion, dietary fatty acid composition
affects cardiac ceramide accumulation, cardiomyocyte apoptosis, and
expression of PPAR-regulated genes independent of cardiac mass or
function.
ceramide; fatty acids; heart; lipotoxicity; mitochondria; obesity
RECENT STUDIES in transgenic mice (4, 5, 19, 42) and rat models
of genetic obesity and hyperlipidemia (43, 44) suggest that
cardiac lipotoxicity can occur because of the accumulation of
lipids in cardiomyocytes. Current dietary guidelines recom-
Address for reprint requests and other correspondence: W. C. Stanley, Dept.
of Physiology and Biophysics, School of Medicine, Case Western Reserve
Univ., 10900 Euclid Ave., Cleveland, OH 44106-4970 (e-mail:
[email protected]).
H38
mend a low saturated fat/high-carbohydrate diet for optimal
cardiac health (18). On the other hand, results of recent studies
suggest that consumption of a low-carbohydrate/high-fat diet
can be effective for weight loss (22, 30). At present, there is
little information regarding the effects of prolonged high-fat
diets on the heart.
Lipid accumulation in the heart has been observed under
conditions of elevated plasma free fatty acids, such as Type 2
diabetes mellitus and chronic high-fat feeding (10, 11, 16, 17,
32), and is linked with cardiomyocyte apoptosis, left ventricular (LV) hypertrophy, and contractile dysfunction (5). Excessive storage of metabolically active lipid products in the heart
can lead to the formation of ceramide, a proapoptotic lipid
intermediate formed primarily from palmitate (4, 31). Intracellular lipid accumulation and consequent apoptosis are dependent on the type of fatty acid in the milieu: studies in isolated
cells show that palmitate induces apoptosis but oleate does not
and that the addition of oleate to medium containing palmitate
protects against apoptosis (14, 21).
Fatty acids are the primary metabolic fuel for the heart and
also act as endogenous ligands to the peroxisome proliferatoractivated receptors (PPARs) and induce genes that encode
proteins that regulate fatty acid metabolism (35). Studies (8) in
transgenic mice show that overexpression of PPAR␣ is associated with myocardial accumulation of triglyceride and ceramide, accelerated fatty acid oxidation, LV hypertrophy and
dilatation, and eventual systolic contractile dysfunction. A
similar effect is observed with cardiac-specific overexpression
of fatty acyl-CoA synthase (5), suggesting that accelerated
fatty acid uptake by the heart can result in “cardiac lipotoxicity” (4). On the other hand, our laboratory (26) recently
observed improved LV contractile function and attenuation of
LV hypertrophy and remodeling in hypertensive Dahl saltsensitive rats fed a high-fat diet compared with normal low-fat
chow despite similar levels of hypertension. Furthermore, cardiomyocyte-restricted PPAR␣ or PPAR␤/␦ deletion in mice
reduces the capacity for fatty acid oxidation and leads to
cardiomyocyte lipid accumulation, LV hypertrophy, and contractile dysfunction (3, 4, 31). These findings suggest that
exaggeration or diminution of fatty acid metabolism causes
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/06 $8.00 Copyright © 2006 the American Physiological Society
http://www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 14, 2017
Departments of 1Physiology and Biophysics, 2Nutrition, and 3Medicine, Case Western Reserve University,
Cleveland, Ohio; 4Department of Medicine, Division of Cardiovascular Medicine, Henry Ford Heart and Vascular
Institute, Detroit, Michigan; and 5Department of Pediatrics, Baylor College of Medicine, Houston, Texas
H39
CARDIAC EFFECTS OF HIGH-FAT DIETS
METHODS
Experimental design. All procedures for this study were conducted
according to Guide for the Care and Use of Laboratory Animals
published by the National Institutes of Health (NIH Publication No.
85-23, Revised 1996) and were approved by the Institutional Animal
Care and Use Committee of the Case Western Reserve University. All
measurements were performed with the investigator blinded to treatment. Twenty-eight male Wistar rats were obtained at 8 –9 wk of age
from Harlan (Indianapolis, IN) and maintained on a reverse 12:12-h
light-dark cycle. The high-fat diets used in the study were obtained
from Research Diets (New Brunswick, NJ).
Initial pretreatment measurements were made for systolic arterial
blood pressure, body weight, and LV function. Systolic pressure was
measured by using the tail-cuff method after habituation to the test
procedure. The rats were randomized into three groups: a normal
chow control group (NC; n ⫽ 9), a high long-chain saturated fatty acid
diet group (Sat; n ⫽ 10), and a high long-chain polyunsaturated fatty
acid diet group (Unsat; n ⫽ 9). The rats were maintained on the diets
for 8 wk and then euthanized. Echocardiography and tail-cuff blood
pressure measurements were repeated after 4 wk on the diets.
Diets. The control diet was a commercially available normal rodent
chow of which carbohydrate, protein, and fat provided 70, 20, and
10% of calories, respectively (Teklad). The carbohydrate was derived
from corn starch, maltodextrin, and sucrose, which made up 45, 5, and
50% of carbohydrate calories, respectively, and the fat was derived
from soybean oil and lard. The high long-chain saturated fatty acid
diet provided 60% of calories from cocoa butter, and the high
polyunsaturated diet was derived from safflower oil, which provided
60% of calories from fat. The fatty acid composition of the three
AJP-Heart Circ Physiol • VOL
Table 1. Fatty acid composition
Fatty Acid
NC
Sat
Unsat
16:0
18:0
18:1
18:2
4
3
1
3
17
39
3
1
12
14
10
24
Values are expressed as percentage of total calories in chow. NC, normal
chow control group; Sat, high long-chain saturated fatty acid diet group; Unsat,
high long-chain polyunsaturated fatty acid group.
chows was measured by gas chromatography-mass spectrometry as
previously described (1) and is reported in Table 1. The palmitate
concentration in the diets was 4, 17, and 12% of the total calories for
the NC, Sat, and Unsat diets, respectively.
Echocardiography. LV function was evaluated with a Sequoia
C256 system (Siemens Medica) with a 15-MHz linear array transducer as previously described (25). Briefly, rats were anesthetized
with 1.2–2.0% isoflurane by mask, the chest was shaved, the animal
was situated in the supine position on a warming pad, and ECG limb
electrodes were placed. Two-dimensional guided M-mode, two-dimensional, and Doppler echocardiographic studies of aortic and transmitral flows were performed from parasternal and foreshortened
apical windows (25). All data were analyzed offline with software
resident on the ultrasound system at the end of the study. LV
end-diastolic and end-systolic areas were planimetered from the
parasternal long-axis, and area of fractional shortening was calculated
as previously described by Morgan et al. (25) and Okere et al. (26).
Terminal hemodynamic measurements. All terminal studies were
performed between 3 and 6 h from the initiation of the dark phase of
the daily light-dark cycle. After 8 wk on the diets, fed animals were
weighed and anesthetized with 1.5–2% isoflurane, and measurements
of aortic blood and LV pressure were made as previously described
(25) with the use of a 3.5-Fr pressure transducer (Millar Mikrotip). At
the end of the measurements, 3 ml of blood were drawn from the
inferior vena cava for metabolic measurements, the LV was quickly
removed and weighed, and a portion of the LV was quickly embedded
in a histological matrix (OCT Tissue-Tek) and stored in dry ice while
the remainder was freeze clamped and stored at ⫺80°C. The liver and
kidneys were removed and weighed. Adipose tissue was isolated and
weighed from the intrathoracic and intra-abdominal cavities and the
epididymal fat pad.
Metabolic measurements. Plasma free fatty acid and triglycerides
concentrations were measured by using enzymatic spectrophotometric
assays (Wako and Sigma, respectively). Blood glucose concentration
was also measured by enzymatic spectrophotometric assays from
perchloric acid deproteinized whole blood samples (Stanbio). Serum
levels of leptin and adiponectin and plasma concentration of insulin
were measured by ELISA (ALPCO Diagnostics, Salem, NH). Myocardial activities of medium-chain acyl-CoA dehydrogenase (MCAD)
and citrate synthase were measured spectrophotometrically as previously described (27). Tissue triglyceride content was measured from
homogenate extracts using enzymatic spectrophotometric assay of
triglycerides (2). Palmitoyl ceramide (C16 ceramide) content was
measured by gas chromatography with flame ionization detector using
C17 ceramide as an internal standard, as previously described (36).
All tissue measurements are expressed per gram wet mass.
Histological assessments of apoptosis. The presence of nuclear
DNA fragmentation, a marker of apoptosis, was assessed in frozen LV
sections with the use of ApopTag in situ fluorescein apoptosis detection kit (Oncor). The DeadEnd fluorometric terminal deoxynucleotide
transferase-mediated dUTP nick-end labeling system measures the
fragmented DNA of apoptotic cells by incorporating fluorescein-12dUTP at 3⬘-OH DNA ends using the terminal deoxynucleotidyl
transferase. Sections were also stained with ventricular anti-myosin
291 • JULY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 14, 2017
myocardial hypertrophy and dysfunction. On the other hand,
our laboratory (24) recently found that feeding a high-fat diet
or administration of a PPAR␣ agonist does not affect LV
function or remodeling in rats with infarct-induced heart failure. This neutral impact occurred despite upregulation of the
fatty acid metabolic pathway and accumulation of myocardial
triglyceride.
Little is known about the effects of high-fat diets on cardiac
function, apoptosis, and the expression of PPAR-regulated
genes in normal animals. In addition, the effects of potential
differences in long-chain saturated fatty acids and unsaturated
fatty acids have not been distinguished. In the present study,
we assessed the effects of feeding rats either standard low-fat
chow (10% of calories from fat as 3% unsaturated and 7%
saturated fatty acids) or high-fat diets (60% of calories from
fat) composed primarily of either saturated or unsaturated fatty
acids. We hypothesized that a diet high in saturated fatty acids
would increase myocardial ceramide content, trigger apoptosis,
and result in cardiac dysfunction, whereas a high-fat diet
composed of unsaturated fatty acids would lack this effect. We
further hypothesized that the expression of PPAR-regulated
genes would be increased by a saturated or unsaturated high-fat
diet. Studies were performed in normal Wistar rats subjected to
8 wk of dietary treatment. Rats were fed ad libitum; however,
there were no differences among groups in body weight, and
thus the confounding effects of obesity observed in previous
feeding studies were avoided (23, 39, 40). LV function was
assessed by echocardiography and direct measurements of LV
pressure. Because insulin and the adipokine hormones leptin
and adiponectin can affect cardiac metabolism and arterial
blood pressure and potentially trigger cardiac hypertrophy (7,
33, 41), we also assessed the effects of the high-fat diets on
regional fat deposits and circulating hormone levels.
H40
CARDIAC EFFECTS OF HIGH-FAT DIETS
Table 3. Plasma lipid concentrations
Plasma free fatty acid, mM
Plasma triglyceride, mg/ml
NC
Sat
Unsat
0.42⫾0.04
1.52⫾0.16
0.52⫾0.04
1.31⫾0.12
0.61⫾0.05*
0.57⫾0.06†
Values are means ⫾ SE. *P ⬍ 0.05 vs. NC; †P ⬍ 0.05 vs. NC and Sat.
0.9 ␮g/ml for NC, Sat, and Unsat groups, respectively), nor
were blood glucose levels different (3.1 ⫾ 0.1, 3.2 ⫾ 0.01, and
3.1 ⫾ 0.1 mM, respectively).
The Sat group had significantly higher cardiac triglyceride
content when compared with the NC group (Fig. 3). Palmitoyl
ceramide (C-16 ceramide) content was not different between
the NC and Sat treatment groups but was significantly higher in
the Sat group compared with the Unsat group (Fig. 3).
Cardiac mRNA expression of atrial natriuretic factor, a
marker of cardiac hypertrophy and heart failure, was similar
among treatment groups (Fig. 4). Cardiac expression of the
RESULTS
Body and visceral adipose tissue mass. There were no
differences in body mass before or after treatment, nor was
there any effect of diet on weight gain (Table 2). Food
consumption was not different among groups, with the average
daily consumption of 184 ⫾ 15, 170 ⫾ 5, and 187 ⫾ 22 kcal
for NC, Sat, and Unsat groups, respectively. The fat mass in the
intrathoracic space was greater in the Sat group than the NC or
Unsat group (Table 2). On the other hand, the mass of intraabdominal and epididymal fat was lower in the Sat group than
the Unsat group.
Metabolic parameters. Plasma free fatty acid concentration
was significantly higher in the Unsat group when compared
with the NC group, and plasma triglyceride level was significantly lower in the Unsat group compared with both of the
other groups (Table 3). Serum insulin levels were significantly
lower in the Sat group compared with the NC group, and there
was no significant difference between the Sat and Unsat or the
NC and Unsat groups (Fig. 1). Serum leptin was significantly
lower in the Sat diet group when compared with both the NC
and Unsat groups (Fig. 1). Serum leptin concentration was
positively correlated to the mass of the epididymal and intraabdominal adipose and was inversely related to the mass of
intrathoracic adipose (Fig. 2). Serum adiponectin levels were
not different among groups (5.7 ⫾ 1.0, 4.7 ⫾ 0.8, and 6.1 ⫾
Table 2. Body and left ventricle mass measurements
Pretreatment body mass, g
Terminal body mass, g
Abdominal fat/body mass, g/kg
Epididymal fat/body mass, g/kg
Intrathoracic fat/body mass, g/kg
LV mass, g
RV mass, g
LV mass/body mass, g/kg
Biventricular mass index, g/kg
NC
Sat
Unsat
335⫾13
560⫾13
42.5⫾4.7
25.9⫾2.3
0.78⫾0.11
1.01⫾0.03
0.28⫾0.02
1.80⫾0.05
2.29⫾0.07
327⫾3
531⫾7
33.5⫾1.5
20.2⫾1.5*
1.31⫾0.09†
0.96⫾0.02
0.27⫾0.01
1.80⫾0.03
2.31⫾0.03
326⫾13
535⫾23
49.8⫾3.2‡
25.2⫾1.4
0.83⫾0.12
0.99⫾0.04
0.28⫾0.03
1.80⫾0.04
2.29⫾0.07
Values are means ⫾ SE. LV, left ventricular; RV, right ventricular. *P ⬍
0.05 Sat vs. Unsat; †P ⬍ 0.05 Sat vs. NC and Unsat; ‡P ⬍ 0.05 Unsat vs. Sat.
AJP-Heart Circ Physiol • VOL
Fig. 1. Serum insulin and leptin concentrations under unfasted conditions after
8 wk of dietary treatment. Unsat, unsaturated fatty acid. *P ⬍ 0.05 compared
with normal chow control (NC) group; #P ⬍ 0.05 compared with NC and
saturated fatty acid (Sat) groups.
291 • JULY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 14, 2017
antibody to identify cells of cardiomyocyte origin as previously
described (12, 29).
mRNA measurements. RNA extraction and quantitative RT-PCR
were performed on frozen, powdered LV tissue using previously
described methods (6, 9, 13). Specific quantitative assays were designed from rat sequences available in GenBank for expression of
atrial natriuretic factor and genes that are known to be regulated by
PPAR␣: MCAD, pyruvate dehydrogenase kinase-4 (PDK-4), and
uncoupling protein-3 (UCP-3). Standard RNA was made for all assays
by the T7 polymerase method (Ambion), using total RNA isolated
from rat hearts. The correlation between the number of PCR cycles
required for the fluorescent signal to reach a detection threshold and
the amount of standard was linear over at least a 5-log range of RNA
for all assays. mRNA concentration was normalized to cyclophilin,
which was quantitatively measured in each sample and was not
different among the experimental groups (data not shown).
Statistical analysis. A one-way ANOVA was used to compare mass
measurements, hemodynamic function, plasma free fatty acids and
triglyceride, and tissue contents of triglyceride among diet groups. A
two-way ANOVA was used for the comparison of echocardiographic
data among groups. All values are recorded as means ⫾ SE, and a
0.05 level of significance was used.
CARDIAC EFFECTS OF HIGH-FAT DIETS
H41
NC, Sat, and Unsat groups, respectively), suggesting that the
upregulation of MCAD mRNA was not translated into a
greater amount of active protein. The activity of citrate synthase was similar among groups (164 ⫾ 7, 181 ⫾ 7, and 164 ⫾
1 ␮mol䡠min⫺1 䡠g⫺1 in the NC, Sat, and Unsat groups, respectively), suggesting that there was no difference in mitochondrial content among groups.
Cardiac function and blood pressure. There were no significant differences among groups in the prediet echocardiograms
or tail-cuff blood pressures (data not shown). Eight weeks of
dietary treatment did not change arterial systolic blood pressure, heart rate, or LV pressure (Table 4). Echocardiography
measurements showed similar values for all treatment groups
(Table 5).
Apoptosis. The number of cardiomyocyte apoptotic events
was higher in the Sat group compared with the Unsat group
(0.28 ⫾ 0.05 vs. 0.17 ⫾ 0.04 apoptotic cells/1,000 nuclei; P ⬍
Fig. 2. Serum leptin concentration plotted as function of mass of epididymal,
intra-abdominal, and intrathoracic fat.
PPAR␣-regulated fatty acid oxidation enzymes, PDK-4, and
UCP-3 were elevated to similar extents (2.5-fold increases) by
Sat and Unsat feeding compared with the NC-fed group (Fig.
4). Sat feeding led to significant elevation in MCAD gene
expression; however, the Unsat group was not different from
the NC group. The activity of MCAD was not different among
groups (13 ⫾ 1 , 12 ⫾ 1, and 11 ⫾ 1 ␮mol䡠min⫺1 䡠 g⫺1 in the
AJP-Heart Circ Physiol • VOL
Fig. 4. mRNA expression in left ventricular tissue for atrial natriuretic factor
(ANF) and peroxisome proliferator-activated receptor-␣ (PPAR␣)-regulated
genes after 8 wk of dietary treatment. Values are normalized to cyclophillin
mRNA and expressed as percentage of mean of NC group. PDK-4, pyruvate
dehydrogenase kinase-4; UCP-3, uncoupling protein-3; MCAD, medium-chain
acyl-CoA dehydrogenase. *P ⬍ 0.05 compared with NC group.
291 • JULY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 14, 2017
Fig. 3. Left ventricular tissue triglyceride and C-16 ceramide content after 8
wk of dietary treatment. *P ⬍ 0.05 compared with NC group; **P ⬍ 0.05
compared with NC and Sat groups.
H42
CARDIAC EFFECTS OF HIGH-FAT DIETS
Table 4. Tail-cuff systolic arterial blood pressure and
terminal LV pressure measurements
Systolic blood pressure, mmHg
Heart rate, beats/min
LV systolic pressure, mmHg
LVEDP, mmHg
Duration of contraction, ms
Duration of relaxation, ms
Peak LV ⫹dP/dt, mmHg/s
Peak LV ⫺dP/dt, mmHg/s
␶
NC
Sat
Unsat
128⫾1
449⫾7
117⫾5
7⫾0
49⫾2
70⫾3
7,328⫾470
6,414⫾438
12⫾0
127⫾1
466⫾9
128⫾4
9⫾1
62⫾2*
64⫾3
7,711⫾245
7,249⫾277
12⫾1
126⫾1
431⫾12
115⫾10
7⫾1
54⫾6
73⫾7
6,637⫾634
5,956⫾608
12⫾1
0.04), whereas the NC group was not different from either of
the high-fat diet groups (0.20 ⫾ 0.03 apoptotic cells/1,000
nuclei). There was a significant positive correlation between
the number of apoptotic events and the LV ceramide content
(P ⬍ 0.02) (Fig. 5).
Fig. 5. Number of cardiomyocyte apoptotic events plotted as function of C-16
ceramide content after 8 wk of dietary treatment.
DISCUSSION
The results of this study showed that the fatty acid composition of high-fat diets differentially affected PPAR-regulated
gene expressions, fat distribution, and cardiomyocyte apoptosis. In addition, serum leptin and insulin levels were reduced
only in the long-chain saturated fat-fed rats. On the other hand,
neither high-fat diet altered cardiac mass or adversely affected
cardiac function; thus 2 mo on a high-fat diet does not cause
any obvious toxicity in the heart. Taken together, these findings
suggest divergent effects of high-fat diets composed of saturated and unsaturated fatty acids on cardiac metabolic phenotype and apoptosis.
A commonly proposed mechanism for lipid-induced cardiomyocyte death is via the activation of caspases by ceramide,
a potent proapoptotic by-product of lipid metabolism (15, 37,
38). Palmitate is the primary fatty acid moiety in cardiac
ceramide (34), and thus one would expect to induce a higher
rate of cardiac cell death in a diet high in palmitate compared
with a diet low in palmitate but high in unsaturated fatty acid,
as observed in the present investigation (Fig. 1). Ceramide is
either produced via de novo synthesis from palmitate or by the
hydrolysis of membrane sphingomyelin by the action of sphingomyelinase. Our results suggest that feeding a diet high in
palmitate increases myocardial palmitoyl-ceramide content,
whereas a high unsaturated fatty acid diet results in significantly reduced ceramide content. Moreover, the number of
Table 5. Echocardiography results after 8 wk on diet
LV end-diastolic diameter, cm
LV end-systolic diameter, cm
Area of fractional shortening, %
Velocity of circumferential
shortening, s⫺1
Myocardial performance index
Cardiac index, ml䡠min⫺1䡠kg⫺1
Relative wall thickness
NC
Sat
Unsat
0.82⫾0.02
0.42⫾0.02
65⫾2
7.0⫾0.3
0.85⫾0.02
0.47⫾0.02
62⫾3
6.9⫾0.4
0.78⫾0.02
0.36⫾0.03
67⫾2
8.5⫾0.6
0.40⫾0.02
91⫾5
0.45⫾0.01
0.43⫾0.02
110⫾6
0.40⫾0.03
0.43⫾0.03
89⫾5
0.46⫾0.02
Values are means ⫾ SE. * P ⬍ 0.05 Sat vs. NC and Unsat.
AJP-Heart Circ Physiol • VOL
apoptotic events was significantly lower in the Unsat-fed rats
than the Sat-fed animals. This is consistent with findings in
cultured neonatal cardiomyocytes, which showed that addition
of oleate to the medium prevented palmitate-induced apoptosis
(14, 34). Taken together, these findings suggest that consuming
a diet rich in unsaturated fatty acids may reduce loss of
cardiomyocytes due to apoptosis. The present investigation did
not address the lifelong effects of consuming a diet rich in
unsaturated fatty acids; however, one might speculate that such
a diet might prevent cardiac dysfunction and/or LV remodeling
during senescence due to cumulative apoptosis. Additional
studies are required to examine the more long-term effects of
dietary lipid on cardiac apoptosis and function.
In the present study, we observed a similar increase in the
cardiac expression of the PPAR genes PDK-4 and carnitine
palmitoyltransferase 1 in both high-fat-fed groups; however,
the expression of MCAD mRNA was only significantly elevated by saturated fat feeding. In addition, the activity of
MCAD was not affected by high-fat feeding, nor was the
activity of citrate synthase, an index of mitochondrial content.
Thus the increase in MCAD mRNA did not translate into
greater enzyme activity. As we previously observed with
metabolic genes and proteins in dogs and rats with heart failure
(20, 24), alterations in gene expression frequently do not
translate into changes in protein expression or activity. The
mechanism for the differential regulation of MCAD mRNA by
the two diet remains to be elucidated.
The results of this study showed that the fatty acid composition of the high-fat diet differentially affected fat distribution
and plasma leptin levels but had no effect on serum adiponectin
concentration. Sat feeding induced greater intrathoracic adiposity and reduced epididymal and abdominal fat compared
with the Unsat group, suggesting that long-chain saturated fat
is not as readily stored as triglyceride in adipocytes in these
regions. The reduction in serum leptin level in the Sat group
and the significant positive correlation with epididymal and
abdominal fat stores suggest that long-chain saturated fatty
acids do not induce leptin secretion from adipose and thus
291 • JULY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 14, 2017
Values are means ⫾ SE. LVEDP, LV end-diastolic pressure; ⫹dP/dt and
⫺dP/dt, maximal positive and negative first derivative of pressure, respectively. *P ⬍ 0.05 Sat vs. NC.
CARDIAC EFFECTS OF HIGH-FAT DIETS
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
ACKNOWLEDGMENTS
We thank Emily Griffith, Janean Johnson, and Theodore Kung for assistance with the animal studies, Hazel Huang and Dr. Monika Duda for
assistance with the data analysis, and Daniel Brunengraber for the GC-MS
analysis of fatty acids in the chows.
19.
GRANTS
This research was supported by National Heart, Lung, and Blood Institute
Grants HL-074237 (to W. Stanley and H. Sabbah) and HL-074259 (to M.
Young).
20.
REFERENCES
1. Brunengraber DZ, McCabe BJ, Kasumov T, Alexander JC, Chandramouli V, and Previs SF. Influence of diet on the modeling of adipose
tissue triglycerides during growth. Am J Physiol Endocrinol Metab 285:
E917–E925, 2003.
2. Chandler MP, Huang H, McElfresh TA, and Stanley WC. Increased
nonoxidative glycolysis despite continued fatty acid uptake during demand-induced myocardial ischemia. Am J Physiol Heart Circ Physiol 282:
H1871–H1878, 2002.
3. Cheng L, Ding G, Qin Q, Huang Y, Lewis W, He N, Evans RM,
Schneider MD, Brako FA, Xiao Y, Chen YE, and Yang Q. Cardiomyocyte-restricted peroxisome proliferator-activated receptor-delta deletion
perturbs myocardial fatty acid oxidation and leads to cardiomyopathy. Nat
Med 10: 1245–1250, 2004.
4. Chiu HC, Kovacs A, Blanton RM, Han X, Courtois M, Weinheimer
CJ, Yamada KA, Brunet S, Xu H, Nerbonne JM, Welch MJ, Fettig
NM, Sharp TL, Sambandam N, Olson KM, Ory DS, and Schaffer JE.
Transgenic expression of fatty acid transport protein 1 in the heart causes
lipotoxic cardiomyopathy. Circ Res 96: 225–233, 2005.
5. Chiu HC, Kovacs A, Ford DA, Hsu FF, Garcia R, Herrero P, Saffitz
JE, and Schaffer JE. A novel mouse model of lipotoxic cardiomyopathy.
J Clin Invest 107: 813– 822, 2001.
6. Chomczynski P and Sacchi N. Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156 –159, 1987.
7. Correia ML, Morgan DA, Sivitz WI, Mark AL, and Haynes WG.
Leptin acts in the central nervous system to produce dose-dependent
changes in arterial pressure. Hypertension 37: 936 –942, 2001.
8. Finck BN, Han X, Courtois M, Aimond F, Nerbonne JM, Kovacs A,
Gross RW, and Kelly DP. A critical role for PPAR␣-mediated lipotoxAJP-Heart Circ Physiol • VOL
21.
22.
23.
24.
25.
26.
27.
icity in the pathogenesis of diabetic cardiomyopathy: modulation by
dietary fat content. Proc Natl Acad Sci USA 100: 1226 –1231, 2003.
Gibson UE, Heid CA, and Williams PM. A novel method for real time
quantitative RT-PCR. Genome Res 6: 995–1001, 1996.
Goodpaster BH and Kelley DE. Skeletal muscle triglyceride: marker or
mediator of obesity-induced insulin resistance in Type 2 diabetes mellitus?
Curr Diab Rep 2: 216 –222, 2002.
Goodpaster BH, Stenger VA, Boada F, McKolanis T, Davis D, Ross R,
and Kelley DE. Skeletal muscle lipid concentration quantified by magnetic resonance imaging. Am J Clin Nutr 79: 748 –754, 2004.
Goussev A, Sharov VG, Shimoyama H, Tanimura M, Lesch M,
Goldstein S, and Sabbah HN. Effects of ACE inhibition on cardiomyocyte apoptosis in dogs with heart failure. Am J Physiol Heart Circ
Physiol 275: H626 –H631, 1998.
Heid CA, Stevens J, Livak KJ, and Williams PM. Real time quantitative
PCR. Genome Res 6: 986 –994, 1996.
Hickson-Bick DL, Buja ML, and McMillin JB. Palmitate-mediated
alterations in the fatty acid metabolism of rat neonatal cardiac myocytes.
J Mol Cell Cardiol 32: 511–519, 2000.
Hickson-Bick DL, Sparagna GC, Buja LM, and McMillin JB. Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the
generation of ROS. Am J Physiol Heart Circ Physiol 282: H656 –H664,
2002.
Kelley DE. Skeletal muscle triglycerides: an aspect of regional adiposity
and insulin resistance. Ann NY Acad Sci 967: 135–145, 2002.
Kelley DE, McKolanis TM, Hegazi RA, Kuller LH, and Kalhan SC.
Fatty liver in Type 2 diabetes mellitus: relation to regional adiposity, fatty
acids, and insulin resistance. Am J Physiol Endocrinol Metab 285: E906 –
E916, 2003.
Krauss RM, Eckel RH, Howard B, Appel LJ, Daniels SR, Deckelbaum
RJ, Erdman JW Jr, Kris-Etherton P, Goldberg IJ, Kotchen TA,
Lichtenstein AH, Mitch WE, Mullis R, Robinson K, Wylie-Rosett J, St
Jeor S, Suttie J, Tribble DL and Bazzarre TL. AHA Dietary Guidelines: revision 2000: a statement for healthcare professionals from the
Nutrition Committee of the American Heart Association. Circulation 102:
2284 –2299, 2000.
Lee Y, Naseem RH, Duplomb L, Park BH, Garry DJ, Richardson JA,
Schaffer JE, and Unger RH. Hyperleptinemia prevents lipotoxic cardiomyopathy in acyl CoA synthase transgenic mice. Proc Natl Acad Sci USA
101: 13624 –13629, 2004.
Lei B, Lionetti V, Young ME, Chandler MP, D’Agostino C, Kang E,
Altarejos M, Matsuo K, Hintze TH, Stanley WC, and Recchia FA.
Paradoxical downregulation of the glucose oxidation pathway despite
enhanced flux in severe heart failure. J Mol Cell Cardiol 36: 567–576,
2004.
Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr, Ory DS
and Schaffer JE. Triglyceride accumulation protects against fatty acidinduced lipotoxicity. Proc Natl Acad Sci USA 100: 3077–3082, 2003.
McAuley KA, Hopkins CM, Smith KJ, McLay RT, Williams SM,
Taylor RW, and Mann JI. Comparison of high-fat and high-protein diets
with a high-carbohydrate diet in insulin-resistant obese women. Diabetologia 48: 8 –16, 2005.
Mickelsen O, Takahashi S, and Craig C. Experimental obesity. I.
Production of obesity in rats by feeding high-fat diets. J Nutr 57: 541–554,
1955.
Morgan EE, Rennison JH, Young ME, McElfresh TA, Kung TA,
Tserng K-Y, Hoit BD, Stanley WC, and Chandler MP. Effects of
chronic activation of peroxisome proliferator-activated receptor-␣ or highfat feeding in a rat infarct model of heart failure. Am J Physiol Heart Circ
Physiol 290: H1899 –H1904, 2006.
Morgan EE, Faulx MD, McElfresh TA, Kung TA, Zawaneh MS,
Stanley WC, Chandler MP, and Hoit BD. Validation of echocardiographic methods for assessing left ventricular dysfunction in rats with
myocardial infarction. Am J Physiol Heart Circ Physiol 287: H2049 –
H2053, 2004.
Okere IC, Chess DJ, McElfresh TA, Johnson J, Rennison J, Ernsberger P, Hoit BD, Chandler MP, and Stanley WC. High-fat diet
prevents cardiac hypertrophy and improves contractile function in the
hypertensive Dahl salt-sensitive rat. Clin Exp Pharmacol Physiol 32:
825– 831, 2005.
Panchal AR, Stanley WC, Kerner J, and Sabbah HN. Beta-receptor
blockade decreases carnitine palmitoyl transferase I activity in dogs with
heart failure. J Card Fail 4: 121–126, 1998.
291 • JULY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 14, 2017
reduce circulating leptin concentration. These changes in leptin
were observed in the absence of any changes in body weight.
Our data suggest that fat mass may not be the only determinant
of circulating leptin. Studies in isolated cardiomyocytes demonstrate that elevated leptin levels stimulate hypertrophy (41)
and chronic leptin administration in vivo increases arterial
blood pressure (7, 28, 33), suggesting that a dietary-induced
reduction in leptin may be beneficial to the heart. Clearly,
additional work is needed to elucidate the mechanism for the
reduced leptin levels with the Sat diet and the effects of chronic
alterations in leptin on the heart.
In summary, feeding a high-fat diet for 2 mo did not affect
body mass, cardiac function, or LV mass. On the other hand,
feeding a high saturated fat diet resulted in lower insulin and
leptin concentrations in the plasma compared with either a
low-fat diet or a high unsaturated fat diet. The high-fat diet
differentially upregulated the expression of PPAR-regulated
genes, but neither diet affected the activity of the mitochondrial
enzymes citrate synthase or MCAD. The high unsaturated fat
diet reduced cardiac ceramide content compared with the high
saturated fat diet, which corresponded with a reduced frequency of cardiomyocyte apoptosis. Thus these results suggest
that over a 2-mo period, high-fat diets do not adversely affect
cardiac function in the absence of effects on weight gain.
H43
H44
CARDIAC EFFECTS OF HIGH-FAT DIETS
AJP-Heart Circ Physiol • VOL
37. Tserng KY and Griffin RL. Ceramide metabolite, not intact ceramide
molecule, may be responsible for cellular toxicity. Biochem J 380: 715–
722, 2004.
38. Unger RH and Orci L. Lipoapoptosis: its mechanism and its diseases.
Biochim Biophys Acta 1585: 202–212, 2002.
39. West DB, Boozer CN, Moody DL, and Atkinson RL. Dietary obesity in
nine inbred mouse strains. Am J Physiol Regul Integr Comp Physiol 262:
R1025–R1032, 1992.
40. Woods SC, Seeley RJ, Rushing PA, D’Alessio D, and Tso P. A
controlled high-fat diet induces an obese syndrome in rats. J Nutr 133:
1081–1087, 2003.
41. Xu FP, Chen MS, Wang YZ, Yi Q, Lin SB, Chen AF, and Luo JD.
Leptin induces hypertrophy via endothelin-1-reactive oxygen species
pathway in cultured neonatal rat cardiomyocytes. Circulation 110: 1269 –
1275, 2004.
42. Yagyu H, Chen G, Yokoyama M, Hirata K, Augustus A, Kako Y, Seo
T, Hu Y, Lutz EP, Merkel M, Bensadoun A, Homma S, and Goldberg
IJ. Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases
lipid uptake and produces a cardiomyopathy. J Clin Invest 111: 419 – 426,
2003.
43. Young ME, Guthrie PH, Razeghi P, Leighton B, Abbasi S, Patil S,
Youker KA, and Taegtmeyer H. Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart. Diabetes 51:
2587–2595, 2002.
44. Zhou YT, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens
D, Orci L, and Unger RH. Lipotoxic heart disease in obese rats:
implications for human obesity. Proc Natl Acad Sci USA 97: 1784 –1789,
2000.
291 • JULY 2006 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 14, 2017
28. Rahmouni K, Correia ML, Haynes WG, and Mark AL. Obesityassociated hypertension: new insights into mechanisms. Hypertension 45:
9 –14, 2005.
29. Sabbah HN, Sharov VG, Gupta RC, Todor A, Singh V, and Goldstein
S. Chronic therapy with metoprolol attenuates cardiomyocyte apoptosis in
dogs with heart failure. J Am Coll Cardiol 36: 1698 –1705, 2000.
30. Samaha FF, Iqbal N, Seshadri P, Chicano KL, Daily DA, McGrory J,
Williams T, Williams M, Gracely EJ, and Stern L. A low-carbohydrate
as compared with a low-fat diet in severe obesity. N Engl J Med 348:
2074 –2081, 2003.
31. Schaffer JE. Lipotoxicity: when tissues overeat. Curr Opin Lipidol 14:
281–287, 2003.
32. Sharma S, Adrogue JV, Golfman L, Uray I, Lemm J, Youker K, Noon
GP, Frazier OH, and Taegtmeyer H. Intramyocardial lipid accumulation
in the failing human heart resembles the lipotoxic rat heart. FASEB J 18:
1692–1700, 2004.
33. Singhal A, Farooqi IS, Cole TJ, O’Rahilly S, Fewtrell M, Kattenhorn
M, Lucas A, and Deanfield J. Influence of leptin on arterial distensibility:
a novel link between obesity and cardiovascular disease? Circulation 106:
1919 –1924, 2002.
34. Sparagna GC, Hickson-Bick DL, Buja LM, and McMillin JB. Fatty
acid-induced apoptosis in neonatal cardiomyocytes: redox signaling. Antioxid Redox Signal 3: 71–79, 2001.
35. Stanley WC, Recchia FA, and Lopaschuk GD. Myocardial substrate
metabolism in the normal and failing heart. Physiol Rev 85: 1093–1129,
2005.
36. Tserng KY and Griffin R. Quantitation and molecular species determination of diacylglycerols, phosphatidylcholines, ceramides, and sphingomyelins with gas chromatography. Anal Biochem 323: 84 –93, 2003.