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activator
1 Department of Medical Physiology, University of Tromsø, N-9037 Tromsø, Norway; 2 Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 4N1; and 3 Department of Cardiology and Medicine, University of Edinburgh, Edinburgh EH8 9XF, Scotland
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hearts from diabetic
db/db mice, a model of Type 2 diabetes, exhibit
left ventricular failure and altered metabolism of exogenous substrates. Peroxisome proliferator-activated receptor-
(PPAR-) ligands reduce plasma lipid and glucose concentrations and improve insulin sensitivity in db/db mice. Consequently,
the effect of 4- to 5-wk treatment of db/db mice
with a novel PPAR- ligand (BM 17.0744; 25-38
mg · kg
1 · day1),
commencing at 8 wk of age, on ex vivo cardiac function and metabolism
was determined. Elevated plasma concentrations of glucose, fatty acids,
and triacylglycerol (34.0 ± 3.6, 2.0 ± 0.4, and 0.9 ± 0.1 mM, respectively) were reduced to normal after treatment with BM
17.0744 (10.8 ± 0.6, 1.1 ± 0.1, and 0.6 ± 0.1 mM).
Plasma insulin was also reduced significantly in treated compared with untreated db/db mice. Chronic treatment of
db/db mice with the PPAR- agonist resulted in
a 50% reduction in rates of fatty acid oxidation, with a concomitant
increase in glycolysis (1.7-fold) and glucose oxidation (2.3- fold).
Correction of the diabetes-induced abnormalities in systemic and
cardiac metabolism after BM 17.0744 treatment did not, however, improve
left ventricular contractile function.
substrate oxidation; isolated working mouse hearts
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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INSULIN RESISTANCE describes an impaired response of glucose transport to insulin, which is an early characteristic in the development of type 2 (non-insulin-dependent) diabetes mellitus (NIDDM). Together with hyperinsulinemia, insulin resistance contributes to the development of risk factors for coronary heart disease, namely, obesity, dyslipidemia, hypertension, and atherosclerosis (15, 17, 27), commonly known as the "insulin resistance syndrome" or "syndrome X." Consequently, treatment of insulin resistance as a therapeutic goal for patients with NIDDM has led to the development of hypoglycemic drugs such as thiazolidinediones and metformin. Concern about the long-term effects of thiazolidinediones, however, has also stimulated the development of other insulin sensitizers.
Peroxisome proliferator-activated receptors (PPARs) belong to a
superfamily of nuclear, ligand-activated transcription factors. PPARs
play an important role in the transcriptional regulation of genes
coding for proteins involved in lipid utilization and storage,
lipoprotein metabolism, adipocyte differentiation, and insulin action
(34). At present, three major PPAR family members, with
distinct expression in various tissues, have been identified. PPAR-
is highly expressed in the liver and to a lesser extent in the heart,
skeletal muscle, and kidney. PPAR-
is predominately expressed in
adipose tissue, whereas PPAR-
is ubiquitously expressed. The
biological effect of PPARs can be studied by the use of selective ligands; PPAR- is the cellular target for the antidiabetic
thiazolidinediones, whereas PPAR- is the cellular target for
fibrates, Wy-14643, and the novel agent BM 17.0744 (22).
Ligands for PPARs also include naturally occurring fatty acids (FAs) as
well as arachidonic acid analogs. The role of PPAR- in the heart is
not clear, but it has been implicated as having a role in the metabolic
remodeling known to occur in several physiological (fasting and
postnatal development) and pathophysiological (diabetes, hypertrophy,
heart failure, and cardiomyopathy) conditions (3, 29, 36).
In vitro experiments with cultured neonatal cardiomyocytes have
demonstrated that activation of PPAR- increases transcription of
genes involved in FA transport and oxidation (8, 36, 37).
The effect on cardiac metabolism after in vivo treatment with PPAR-
ligands has, however, not been determined.
A number of clinical (13, 30, 35) and experimental studies
(4, 33) have documented altered cardiac metabolism as well
as impaired mechanical function of the heart in diabetes, a diabetic
cardiomyopathy that is not secondary to atherogenesis and
ischemic heart disease. Most experimental animal studies have, however, employed Type 1 insulin-deficient diabetic models; relatively few studies have been conducted with NIDDM animal models exhibiting insulin resistance (25). However, in a recent study by
Belke et al. (4), it was shown that isolated hearts from
the genetically diabetic C57BL/KsJ (db/db) mouse,
an accepted animal model of Type 2 diabetes mellitus, exhibited reduced
contractile function as well as decreased glucose utilization
(glycolysis and glucose oxidation) and increased FA oxidation. Although
PPAR ligands exhibit both hypoinsulinemic and hypoglycemic properties
(16, 39), little is known about their effect on myocardial
substrate metabolism. Therefore, the aim of this study was to examine
the effect of long-term treatment with the novel PPAR- agonist BM
17.0744 on cardiac metabolism and function in the genetically diabetic
C57BL/KsJ (db/db) mouse.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Female C57BL/KsJ diabetic (db/db) mice, as well as their nondiabetic littermates (db/+), were purchased from M&B A/S (Ry, Denmark) and used at the University of Tromsø for investigation of ventricular function. The same mouse strain from the Jackson Laboratory (Bar Harbor, ME) was used for measurements of myocardial metabolism, which were carried out at the University of Calgary. The animals arrived at an age of 6-7 wk and were housed at 23 ± 1°C on a 12:12-h light-dark cycle. They were given ad libitum access to food and water. The animals were allowed to adapt for at least 5 days after arrival. Animals (5 of 65) that did not show a positive weight gain during the 4- to 5-wk experimental period were excluded.Treatment
At an age of 8 wk, the animals were randomly divided in groups of four to five animals per cage; one group of diabetic mice received the PPAR- ligand BM 17.0744 (db/db + BM
group) (Roche Pharmaceuticals). This compound is an 
-substituted
alkyl carboxylic acid and is structurally unrelated to the
thiazolidinediones (23). Transactivation studies by Meyer
et al. (22) have shown that this compound activates
PPAR- with no effect on PPAR-; BM 17.0744 was a more potent
PPAR- ligand than Wy-14643 and 5,8,11,14-eicosatetraynoic acid. The experimental group was treated for a total period of 4-5 wk; the actual dose of the drug calculated from daily water intake ranged between 24.5 ± 1.35 and 37.9 ± 2.5 mg · kg1 · day1. Body
weight as well as food and water intake were measured twice a week
(food and water intake as average for the animals in each cage). The
experiments were approved by the Animal Welfare Committees of the
University of Tromsø and the University of Calgary and followed the
guidelines of the Norwegian and Canadian Councils on Animal Care.
Blood Samples and Analysis of Serum Metabolites
Blood glucose (after 4 h of fasting) was measured before and during treatment (weekly) using Precision Plus electrodes (Medisense and Abbott Laboratories; Bedford, MA). The blood samples were obtained from the leg of the animals. In addition, blood samples were taken on the day of euthanization from the vena cava before excision of the heart. These were collected immediately after the administration of heparin (~100 units iv), cooled on ice, and centrifuged (14,000 rpm for 2 min at 4°C) to avoid the rise in free FAs, due to the release of heparin-induced lipoprotein lipase. Serum glucose, nonesterified free FAs (FFA), and triacylglycerol (TG) levels were measured using kits from Boehringer-Mannheim (Cat. No. 1442449; Mannheim, Germany), Wako Chemicals (Cat. No. 994-75409; Neuss, Germany), and Roche Diagnostic Systems (Cat. No. 0736791; Basel, Germany), respectively. Insulin levels were determined using a radioimmunoassay kit from Linco Research (Cat. No. RI-13K) with rat insulin standards.Isolated Heart Perfusion
Solutions. The Krebs-Henseleit bicarbonate (KHB) buffer used for the initial Langendorff perfusion (pH 7.4) consisted of (in mM) 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 0.5 EDTA, and 11 glucose and was gassed with 95% O2-5% CO2. The buffer used for the working heart experiments was a modified KHB buffer supplemented with 0.4 mM palmitate bound to 3% BSA (fraction V, Cat. No. A-8022; Sigma) as a substrate in addition to 11 mM glucose (1). Measurement of the concentration of FFA in KHB buffer containing 3% BSA (but no added palmitate) revealed that 3% BSA contained 0.28 ± 0.02 mM FAs, so that the total FFA concentration in the buffer used during the working heart perfusions was 0.68 mM. In addition, measurement of the Ca2+ concentration in the buffer, using a blood gas analyzer (Rapidlab 800, Chiron Diagnostics; Halstead, UK) equipped with an ion-selective electrode, showed a value of 1.6 mM. The buffer was recirculated and filtered through an in-line filter. Large parts of the perfusion system were water jacketed and heated to maintain the heart temperature (monitored by a temperature probe in the pulmonary trunk) at 37°C.
Heart perfusion conditions. The animals were anesthetized with 10 mg pentobarbital sodium (intraperitoneal injection), and 100 units heparin was used as an anticoagulant. The hearts were quickly excised and cooled in ice-cold KHB buffer. Extraneous tissues were removed, the aorta was cannulated with an 18-gauge plastic cannula, and the heart thereafter underwent a Langendorff perfusion (60 mmHg of perfusion pressure) with KHB buffer for ~15 min to wash the blood out of the heart. During this time, the left atrium was cannulated with a 16-gauge steel cannula, which was connected to the preload reservoir (20).
Measurements of Ventricular Function
Two needle electrodes were attached to the right atrium for electrical pacing of the heart using a stimulator (model 9D, Grass Instruments; Quincy, MA) delivering 2- to 4-V pulses of 4-ms duration. A steel cannula (23 gauge) was inserted into the left ventricle through the apex of the heart to measure intraventricular pressure (20). This cannula was connected to a 2.5-Fr miniature pressure transducer (Millar Micro-Tip, Millar Instruments; Houston, TX) as well as to a conventional Statham pressure transducer via a fluid-filled two-way adapter (Transpac IV, Abbott Ireland; Sligo, Ireland). After instrumentation, the heart was switched from Langendorff to working mode, the left atrium was filled at a preload pressure of 12.5 mmHg (height of preload column above the heart), and the output from the left ventricle was ejected into an afterload column with a height corresponding to a pressure of 50 mmHg. While in the working mode, the hearts were paced at 6 Hz. Pressure signals were recorded on-line (10-s duration of each recording, sample frequency of 500 Hz) and analyzed using LabView-based software designed by Knut Steinnes (EDB consultant, University of Tromsø). Heart rate (HR) and left ventricular systolic (LVSP) and end-diastolic pressure (LVEDP) as well as the first derivative of the LV pressure (±dP/dt) were derived from the pressure traces. LV developed pressure (LVDP) was calculated as the difference between LVSP and LVEDP, whereas coronary flow (CF) and aortic flow (AF) were measured by timed collections of the effluent dropping from the heart and flow in the afterload line, respectively, using graduated cylinders (5). Cardiac output (CO) was calculated as the sum of AF and CF. Cardiac power was calculated as the product of LVDP and CO and converted to milliwatts per gram dry weight (32). Values derived from the pressure traces were averaged over no less than 30 beats.Measurements of Cardiac Metabolism
In these experiments, the working heart apparatus was made air tight by sealing the heart within the apparatus (to allow for collection of 14CO2 as a result of glucose oxidation) according to the methods outlined in Belke et al. (5). The total volume of perfusate buffer was 40 ml. Hearts were perfused in working mode for 60 min as described above except that hearts were not paced and pressure changes and peak systolic pressures (PSP) were recorded in the aortic (afterload) line using the 2.5-Fr miniature pressure transducer. AF and CF were also measured as previously described (5). Throughout the 60-min perfusion for the metabolic protocol, pressure and flow measurements were obtained every 10 min. At 20-min intervals, a 2.5-ml sample of buffer was withdrawn from the buffer reservoir through an injection port.The metabolism of glucose and palmitate was measured according to the methods outlined in detail by Belke et al. (5). Glycolysis and glucose oxidation was measured simultaneously in one set of hearts; glycolytic flux was determined by measuring the amount of 3H2O released from the metabolism of [5-3H]glucose, and glucose oxidation was determined by trapping and measuring 14CO2 released by the metabolism of [U-14C]glucose. Palmitate oxidation was determined in separate perfused hearts by measuring the amount of 3H2O released from [9,10-3H]palmitate. At the end of perfusion, hearts were quickly frozen between precooled metal clamps, weighed, and stored in liquid nitrogen for later analysis. A small sample of the heart (10-20 mg) was used to determine the wet-to-dry weight ratio of the hearts, which was used to calculate the total dry mass of the heart. Metabolic rates were calculated based on the 3H2O/14CO2 production and the specific activities of the tracers in the perfusate, using the total dry mass of the heart to correct for slight variations in heart size.
The yield of ATP that could be expected from glucose and palmitate metabolism was calculated using a stoichiometric ratio of 2 mol ATP per 1 mol glucose passing through glycolysis, 30 mol ATP per 1 mol glucose being oxidized, and 105 mol ATP per 1 mol palmitate being oxidized (24).
RNA Preparation and Analysis
Frozen cardiac tissue was homogenized in TRIzol reagent, and total RNA was prepared as follows: extraction with chloroform, precipitation with isopropanol, and washing once with 70% ethanol. Extracted RNA was then dissolved in RNAase-free water. The quantity of total RNA was determined by the absorption ratio at 260 to 280 nm. RT-PCR was performed to generate sscDNA templates from extracted RNA. Briefly, RNA (1 µg) was incubated with bulk first strand reaction mix (Amersham Biosciences) containing the primer dp(N)6 (0.2 µg) with the addition of NotI-d(T)18 (0.2 µg) and dithiothreitol. Samples were incubated at 37°C for 60 min. From the RT-PCR incubation, 2 µl sscDNA template was then used for subsequent PCR amplification with sequence specific primers. Each 50 µl PCR reaction contained 2 µl sscDNA template, 1 µl deoxynucleotide mix (2.5 mM dNTPs), DNA polymerase (2 IU DyNAse), and 1 µl each of upper and lower primers (50 µM). The sequences of upper and lower primer pairs were selected using gene sequence information from the NCBI nucleotide sequence database. Primer design was conducted using Primer Select software. The primer sequences were as follows: acyl CoA oxidase (ACO), upper 5'-CTGTGGCCTGGCTGACGTTATTTG-3' and lower 5'-ATGCCATTGCCAGGAAGACCAGAG-3'; muscle type carnitine palmitoyl transferase (mCPT-1), upper 5'-ATTGGGCACCTCTGGGAGTTTGTC-3' and lower 5'-CGGTACATGTTTTGGTGCTTTTCGG-3'; and
-actin, upper 5'-CACCACAGCTGAGAGGGAAATCG-3' and lower
5'-GAAAGGGTGTAAAACGCAGCTCAGTAAC-3'. Amplification was conducted in a
GeneAmp cycler (PCR system 9700). After an initial denaturation step of
94°C for 1,500 s, 25 cycles of 95°C for 45 s, 68°C for
15 s, and 72°C for 20 s were used, followed by a final step
at 72°C for 1 min. PCR products were cooled to 4°C and thereafter
kept on ice. PCR products (10 µl) were loaded onto a 1.5% horizontal
agarose gel and electrophoresed at 100 V for 105 min. Gels were stained
with ethidium bromide (0.5 µg/ml) and quantified using fluorescence
image analysis (Scion image analysis software). Amplification of each
template yielded a single band of the expected size. The results for
expression of specific mRNAs are always presented relative to the
expression of the control gene (-actin).
Tissue TG Content
Tissue lipids were extracted from ~30 mg (wet wt) heart tissue by the method of Folch and dried under N2 atmosphere. The dried lipids were mixed with 300 µl tert-butyl alcohol and 150 µl Triton X-100-methyl alcohol mixture (1:1, vol/vol), and the TG content was measured with a Triglyceride 25 kit from ABX Diagnostics (Montpellier, France). The same method was used to measure TG content in liver biopsies taken from the mice at the day of euthanization.Statistical Analysis
Data are expressed as means ± SE. Differences in cardiac function and substrate metabolism were determined by ANOVA, followed by an unpaired Student's t-test or a Mann-Whitney rank sum test. Differences between means were regarded statistically significant when the P values were <0.05.| |
RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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General Features of Experimental Animals
Changes in body weights of the various experimental groups during the experimental period are given in Fig. 1. Nondiabetic (db/+) mice showed a 20 ± 2% increase in body weight over the experimental period (8 to 12-13 wk of age), although their body weight (22.0 ± 0.5 g) at euthanization was only one-half of that measured for diabetic (db/db) mice. The latter group increased their body weight by 25 ± 3% (from 34.4 ± 0.6 to 42.9 ± 0.8 g), whereas diabetic mice treated with BM 17.0744 (db/db +BM) increased their body weight by only 15 ± 2% (from 35.7 ± 0.7 to 40.8 ± 1.0 g), despite a marked enlargement of the liver (5.4 ± 0.2 vs. 1.8 ± 0.1 g in untreated mice). The effect of BM 17.0744 on liver weight has previously been shown to be species dependent, occurring in rats and mice but not in so-called low responders to peroxisome proliferators (22). Furthermore, BM 17.0744 treatment did not result in any changes in heart weight or the heart-to-body weight ratio (Table 1).
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Increasing hyperglycemia was observed in untreated diabetic mice
(db/db) from 8 to 12 wk of age, with blood
glucose levels three- to fourfold higher than those in nondiabetic
controls (Fig. 2A). This graph
also shows that BM 17.0744 effectively reduced blood glucose in
diabetic mice, so that glucose concentrations after 1 wk of treatment
were comparable to that of nondiabetics. Changes in water intake over
the experimental period are also shown in Fig. 2. Water intake was
considerably higher in untreated diabetic mice
(db/db) compared with nondiabetic
(db/+) littermates. In BM 17.0744-treated diabetic mice
(db/db + BM), there was a rapid decline in
water intake during the first week parallel to the decline in blood
plasma glucose. The average water intake for the last 2 wk of the
experiment was 17.6 ± 0.7, 4.5 ± 0.3, and 3.9 ± 0.2 ml · animal1 · day1 for
untreated diabetics (db/db), diabetic mice
treated with BM 17.0744 (db/db + BM), and
nondiabetic controls (db/+), respectively. The corresponding
food intake values were 7.4 ± 0.3, 5.7 ± 0.3, and 3.6 ± 0.1 g · animal1 · day1.
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Table 2 shows plasma concentrations of
glucose, nonesterified FAs, TG, and insulin at the day of
euthanization. Untreated diabetic (db/db) mice
demonstrated markedly elevated values for all these parameters compared
with nondiabetic (db/+) animals, consistent with previous
results (4). Treatment of diabetic animals with BM 17.0744 normalized plasma glucose, FAs, and TG concentrations and significantly
reduced the plasma insulin concentration.
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Tissue analysis of TG content revealed that there was a marked accumulation of TG in hearts from db/db compared with db/+ mice. Furthermore, treatment of diabetic animals with BM 17.0744 reduced tissue TG by 46% (Table 1).
Cardiac Function
Cardiac function of the isolated working hearts was measured during 60 min of working heart perfusion. Figure 3 shows the perfusion time course for CO and cardiac power values, the latter calculated as LVDP times CO. LV function improved during the first 10-20 min of the perfusion period and was relatively stable thereafter. Table 3 lists the average values of several functional parameters for the 10- to 60-min perfusion period. Cardiac power was markedly reduced (43%) in diabetic (db/db) hearts compared with nondiabetic (db/+) controls. The greatest contribution to this difference occurred in overall CO, which was 7.3 ml/min in nondiabetic control hearts and 5.3 ml/min in diabetic hearts. The diabetic hearts also expressed significantly higher LVEDP as well as lower LVDP. Overall, the present study confirmed our previous observations (4) that cardiac mechanical performance was significantly reduced in perfused diabetic (db/db) hearts compared with hearts from lean nondiabetic controls (db/+). Under the current perfusion conditions, the parameters of cardiac function obtained in hearts from diabetic mice treated with BM 17.0744 (db/db + BM) were not statistically different from those obtained from untreated diabetics (Fig. 3 and Table 3). Table 4 shows several parameters of cardiac function (HR, CO, PSP, and PSP × CO) in the unpaced hearts used for the metabolic measurements. On the basis of these measurements, BM 17.0744 treatment for 4 wk did not change contractile function in diabetic (db/db) mice.
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Cardiac Metabolism
Average rates of glycolysis, glucose oxidation, and palmitate oxidation for untreated and BM 17.0744-treated diabetic hearts are shown in Fig. 4. The rates of glycolysis and palmitate oxidation were calculated over the 0- to 60-min range, whereas the rate of glucose oxidation was calculated over the 20- to 60-min range (to achieve steady state). The rate of glycolysis in hearts from diabetic mice treated with BM 17.0744 (db/db + BM) was 1.7-fold higher than the rate observed in untreated diabetic (db/db) hearts (6,329 ± 466 vs. 3,839 ± 374 nmol · min1 · g dry wt1,
P < 0.05). Likewise, myocardial glucose oxidation was
increased by 2.3-fold compared with the rate of untreated diabetics
(1,946 ± 176 vs. 847 ± 184 nmol · min1 · g dry wt1,
P < 0.05). On the other hand, the rate of palmitate
oxidation in BM 17.0744-treated diabetic hearts was reduced by 50%
compared with untreated diabetic hearts (405 ± 76 vs. 796 ± 139 nmol · min1 · g dry
wt1, P < 0.05).
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When rates of substrate metabolism were converted to calculated ATP
production, we found that myocardial FA oxidation accounted for 72% of
the total ATP production from exogenous substrates in untreated
diabetic (db/db) mice (Fig.
5). Glucose oxidation and glycolysis
accounted for 22% and 7% of total ATP production, respectively. In
hearts from BM 17.0744-treated mice, however, the amount of ATP derived
from glucose oxidation and glycolysis was significantly higher,
accounting for 51% and 11% of the total ATP production, respectively,
whereas the ATP production from FA oxidation was reduced to only 37%.
Thus our results demonstrate that treatment of diabetic mice with BM
17.0744 alters cardiac metabolism from a predominant reliance on FA
oxidation toward an increased reliance on glucose utilization for
energy production.
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Although ACO and mCPT-1 have been identified as direct PPAR- targets
in experiments with isolated neonatal cardiomyocytes (2,
8), treatment of db/db mice with BM
17.0744 did not alter the cardiac expression of either ACO or mCPT-1
(Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The role of PPAR- in the heart is far from clear. Recent
studies with cultured neonatal cardiomyocytes have demonstrated that
activation of PPAR- increased transcription of genes coding for
proteins involved in FA transport and oxidation (8, 36, 37). In contrast, the present study for the first time reports that long-term in vivo treatment with a novel PPAR- agonist results in decreased myocardial FA oxidation with a concomitant increase in
glucose utilization in hearts from Type 2 diabetic
(db/db) mice.
In accordance with previous results (4, 9, 38), the
present study shows that the genetically diabetic C57BL/KsJ
(db/db) mouse exhibits the classical
characteristics of the metabolic syndrome: hyperinsulinemia,
hyperglycemia, hyperlipidemia, and obesity. Treatment of these mice for
4-5 wk with BM 17.0744, starting at an age of 8 wk, resulted in
marked changes in systemic metabolism. Administration of BM 17.0744 resulted in reduced body weight gain over the experimental period,
confirming previous reports on PPAR- agonist treatment in both
normal (26) and obese (16) experimental models.
PPAR- is highly expressed in the liver, regulating the expression of
genes involved in hepatic lipid and lipoprotein metabolism (12). The hypolipidemic effect of PPAR- treatment has
been associated with increased hepatic -oxidation (22)
and increased very low-density lipoprotein (VLDL) degradation
(18) as well as reduced hepatic VLDL-TG secretion
(12). Finally, increased insulin sensitivity of adipose
tissue (26) should also inhibit lipolytic activity via
insulin-mediated inhibition of the hormone-sensitive lipase
(15). Treatment with BM 17.0744 also normalized blood glucose concentrations and, consequently, water intake. Moreover, plasma analysis at the end of the treatment period confirmed that not
only plasma glucose but also insulin levels were reduced after BM
17.0744 treatment. Most likely, PPAR- ligands reduce plasma glucose
via increased sensitivity to insulin (16, 26, 39). Circulating FAs are reported to contribute to insulin resistance in
Type 2 diabetes (7), and, therefore, the observed
lipid-lowering effect of BM 17.0744 may also contribute to increased
glucose uptake in peripheral tissues. Pill and Kuhnle (26)
previously reported a glucose-lowering effect of the compound in
db/db mice but saw no changes in plasma insulin
after 5 days of treatment. Furthermore, 12 days of treatment of
db/db mice with a different PPAR- ligand,
Wy-14643 (10 mg · kg1 · day1), produced
minimal effects on glucose levels (6). These findings demonstrate the importance of performing long-term studies to reveal
the potency of antidiabetic compounds.
PPAR- is believed to regulate metabolic remodelling in the heart in
both physiological and pathophysiological conditions (3, 29,
36). Changes in the metabolic profile of the myocardium after BM
17.0744 treatment presumably reflect changes in the expression or
catalytic activity of the enzymes involved in FA (and carbohydrate) metabolism, because metabolic measurements were performed under conditions with a fixed substrate supply. Because experiments with
cultured neonatal cardiomyocytes have shown that activation of PPAR-
increases the transcription of genes coding for proteins involved in FA
transport and oxidation (8, 36, 37), it might be
anticipated that in vivo treatment with PPAR- ligands also should
result in increased myocardial FA oxidation because of enhanced
expression of PPAR- target proteins. The present study, however,
clearly shows that BM 17.0744 treatment of db/db mice caused a 50% reduction in myocardial FA oxidation with a concomitant increase in glucose utilization (1.7-fold increase in
glycolysis and 2.3-fold increase in glucose oxidation). Furthermore, the expression of two PPAR- target genes in the heart, mCPT-1 and
ACO, was not changed after BM 17.0744 treatment of
db/db mice. These findings are in accordance with
previous studies showing that treatment of mice with PPAR- ligands
(Wy-14643 or ciprofibrate) did not change cardiac mCPT-1
(11) and ACO (10, 11) expression. Several
factors can explain the differences between the present results and
those obtained with cultured myocytes. First, studies on cultured
myocytes permit only direct effects of PPAR- activation to be
observed. In addition, FA oxidation is very low in neonatal cardiomyocytes (21), which might imply that the potential
for PPAR- activation is much higher in these preparations compared with intact hearts. This is illustrated by the fact that uncoupling protein-2, one of the PPAR--regulated cardiac genes, was found to be
upregulated by Wy-14643 in neonatal cultured myocytes (36) but not in hearts from adult rats (40). It is
therefore reasonable to suggest that the reduction in myocardial FA
oxidation in response to BM 17.0744 treatment is mediated via indirect
mechanisms, perhaps related to the lipid-lowering effect of the drug
that regulate activity of key proteins rather than their expression.
The BM 17.0744-induced increase in glucose oxidation could, for
example, result in elevated tissue levels of malonyl CoA that could
inhibit mCPT-1 activity and thus reduce FA oxidation. It should be
noted that BM 17.0744 treatment resulted in a 20% decline in food
intake; the extent to which this change in food intake contributed to alternations in cardiac metabolism will need to be evaluated in future experiments.
Zhou et al. (41) recently reported elevated myocardial TG values in Zucker diabetic fatty (ZDF) rats and concluded that this finding was coupled to underexpression of enzymes of FA oxidation. The present study also demonstrated elevated myocardial TG accumulation in hearts from db/db mice, but clearly this finding cannot be explained in terms of low FA oxidation rates; a high capacity for FA oxidation is a hallmark of the diabetic myocardium (4, 28, 31). Instead, our results indicate that the circulating lipid burden (elevated plasma FFAs and TG) that prevails in the diabetic state cannot be adequately handled by the myocardium, so that some of the FAs taken up by the db/db heart are converted to TG. It should also be noted that the reduction of plasma lipids after treatment with BM 17.0744 partly restored the normal, steady-state level of myocardial TG, again underlining the importance of the supply of FAs in determining net changes in the myocardial TG content.
The present study demonstrates ventricular dysfunction in heart from
diabetic db/db mice, confirming previous results
by Belke et al. (4). Echocardiographic measurements in the
diabetic ZDF rat by Zhou et al. (41) have also revealed
ventricular dysfunction, which was associated with elevation in
myocardial TG and ceramide. Treatment of the ZDF rats with
troglitazone, a PPAR- activator, reduced TG and ceramide content and
improved contractile function (41). In contrast to these
results, the BM 17.0744-induced reduction in myocardial TG levels was
not associated with any improvement of contractile function. One reason
why the present intervention (BM 17.0744 treatment) failed to improve
cardiac function could be that irreversible structural changes
(14, 19) had occurred before the treatment was initiated.
Zhou et al. (41) began trogitazone treatment at 6 or 7 wk
of age, when the rats were still normal. Moreover, Belke et al.
(4) reported that both contractile function and metabolism
were normalized in hearts from db/db mice
overexpressing hGLUT4 glucose transporters, but of course the
"intervention" for a transgenic mouse is life long.
Additional studies are required to determine the first appearance of
cardiac dysfunction in db/db mice, so that the
question of whether a more prolonged treatment with BM 17.0744 could
have prevented mechanical dysfunction in db/db
hearts can be investigated. Furthermore, given the metabolic effect of
PPAR- treatment, it will also be of great interest to examine how
these hearts perform during physiological stressful conditions, such as
ischemia-reperfusion or elevated work loads. Finally, the
mechanism explaining the decrease in myocardial FA oxidation after in
vivo treatment with a PPAR- agonist should be investigated.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Pill (Roche, Germany) for providing BM 17.0744. The technical assistance from the bioengineers Thale Henden and Elisabeth Boerde is greatly appreciated.
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FOOTNOTES |
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This study was supported by Norwegian Research Council for Science and the Humanities Grant 129769/310 and by the Canadian Diabetes Association.
Address for reprint requests and other correspondence: E. Aasum, Dept. of Medical Physiology, Institute of Medical Biology, Faculty of Medicine, Univ. of Tromsø, N-9037 Tromsø, Norway (E-mail: ellenaa{at}fagmed.uit.no).
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.
May 9, 2002;10.1152/ajpheart.00226.2001
Received 22 March 2001; accepted in final form 3 May 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Aasum, E,
and
Larsen TS.
Pyruvate reverses fatty-acid-induced dpression of ventricular function and calcium overload after hypothermia in guinea pig hearts.
Cardiovasc Res
33:
370-377,
1997
2.
Barger, PM,
Brandt JM,
Leone TC,
Weinheimer CJ,
and
Kelly DP.
Deactivation of peroxisome proliferator-activated receptor-alpha during cardiac hypertrophic growth.
J Clin Invest
105:
1723-1730,
2000[ISI][Medline].
3.
Barger, PM,
and
Kelly DP.
PPAR signaling in the control of cardiac energy metabolism.
Trends Cardiovasc Med
10:
238-245,
2000[ISI][Medline].
4.
Belke, DD,
Larsen TS,
Gibbs EM,
and
Severson DL.
Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice.
Am J Physiol Endocrinol Metab
279:
E1104-E1113,
2000
5.
Belke, DD,
Larsen TS,
Lopaschuk GD,
and
Severson DL.
Glucose and fatty acid metabolism in the isolated working mouse heart.
Am J Physiol Regul Integr Comp Physiol
277:
R1210-R1217,
1999
6.
Berger, J,
Leibowitz MD,
Doebber TW,
Elbrecht A,
Zhang B,
Zhou GC,
Biswas C,
Cullinan CA,
Hayes NS,
Li Y,
Tanen M,
Ventre J,
Wu MS,
Berger GD,
Mosley R,
Marquis R,
Santini C,
Sahoo SP,
Tolman RL,
Smith RG,
and
Moller DE.
Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPAR delta ligands produce distinct biological effects.
J Biol Chem
274:
6718-6725,
1999
7.
Bergman, RN,
and
Ader M.
Free fatty acids and pathogenesis of type 2 diabetes mellitus.
Trends Endocrinol Metab
11:
351-356,
2000[ISI][Medline].
8.
Brandt, JM,
Djouadi F,
and
Kelly DP.
Fatty acids activate transcription of the muscle carnitine palmitoyltransferase I gene in cardiac myocytes via the peroxisome proliferator-activated receptor alpha.
J Biol Chem
273:
23786-23792,
1998
9.
Coleman, DL,
and
Hummel KP.
The effects of hypothalamics lesions in genetically diabetic mice.
Diabetologia
6:
263-267,
1970[ISI][Medline].
10.
Cook, WS,
Yeldandi AV,
Rao MS,
Hashimoto T,
and
Reddy JK.
Less extrahepatic induction of fatty acid beta-oxidation enzymes by PPAR alpha.
Biochem Biophys Res Commun
278:
250-257,
2000[ISI][Medline].
11.
Finck, BN,
Lehman JJ,
Leone TC,
Welch MJ,
Bennett MJ,
Kovacs A,
Han X,
Gross RW,
Kozak R,
Lopaschuk GD,
and
Kelly DP.
The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus.
J Clin Invest
109:
121-130,
2002[ISI][Medline].
12.
Fruchart, JC,
Duriez P,
and
Staels B.
Peroxisome proliferator-activated receptor-alpha activators regulate genes governing lipoprotein metabolism, vascular inflammation and atherosclerosis.
Curr Opin Lipidol
10:
245-257,
1999[ISI][Medline].
13.
Galderisi, M,
Anderson KM,
Wilson PW,
and
Levy D.
Echocardiographic evidence for the existence of a distinct diabetic cardiomyopathy (the Framingham Heart Study).
Am J Cardiol
68:
85-89,
1991[ISI][Medline].
14.
Giacomelli, F,
and
Wiener J.
Primary myocardial disease in the diabetic mouse. An ultrastructural study.
Lab Invest
40:
460-473,
1979[ISI][Medline].
15.
Ginsberg, HN.
Insulin resistance and cardiovascular disease.
J Clin Invest
106:
453-458,
2000[ISI][Medline].
16.
Guerre-Millo, M,
Gervois P,
Raspe E,
Madsen L,
Poulain P,
Derudas B,
Herbert JM,
Winegar DA,
Willson TM,
Fruchart JC,
Berge RK,
and
Staels B.
Peroxisome proliferator-activated receptor alpha activators improve insulin sensitivity and reduce adiposity.
J Biol Chem
275:
16638-16642,
2000
17.
Hayden, JM,
and
Reaven PD.
Cardiovascular disease in diabetes mellitus type 2: a potential role for novel cardiovascular risk factors.
Curr Opin Lipidol
11:
519-528,
2000[ISI][Medline].
18.
Kersten, S,
Desvergne B,
and
Wahli W.
Roles of PPARs in health and disease.
Nature
405:
421-424,
2000[Medline].
19.
Kuo, TH,
Giacomelli F,
Wiener J,
and
Lapanowski Netzel K.
Pyruvate dehydrogenase activity in cardiac mitochondria from genetically diabetic mice.
Diabetes
34:
1075-1081,
1985[Abstract].
20.
Larsen, TS,
Dani R,
Giles RW,
Severson DL,
Lopaschuk GD,
Belke DD,
and
Tyberg JV.
The isolated working mouse heart: methodological considerations.
Pflügers Arch
437:
979-985,
1999[ISI][Medline].
21.
Lopaschuk, GD,
Collins Nakai RL,
and
Itoi T.
Developmental changes in energy substrate use by the heart.
Cardiovasc Res
26:
1172-1180,
1992
22.
Meyer, K,
Volkl A,
Endele R,
Kuhnle HF,
and
Pill J.
Species differences in induction of hepatic enzymes by BM 17.0744, an activator of peroxisome proliferator-activated receptor alpha (PPAR alpha).
Arch Toxicol
73:
440-450,
1999[ISI][Medline].
23.
Meyer, K,
Voss E,
Neidlein R,
Kuhnle HF,
and
Pill J.
omega-Substituted alkyl carboxylic acids as antidiabetic and lipid-lowering agents.
Eur J Med Chem
33:
775-787,
1998.
24.
Opie, LH.
The Heart: Physiology, from Cell to Circulation. New York: Lippincott-Raven, 1998, p. 295-342.
25.
Pierce, GN,
Maddaford TG,
and
Russell JC.
Cardiovascular dysfunction in insulin-dependent and non-insulin-dependent animal models of diabetes mellitus.
Can J Physiol Pharmacol
75:
343-350,
1997[ISI][Medline].
26.
Pill, J,
and
Kuhnle HF.
BM 17.0744: a structurally new antidiabetic compound with insulin-sensitizing and lipid-lowering activity.
Metabolism
48:
34-40,
1999[ISI][Medline].
27.
Reaven, GM.
Pathophysiology of insulin resistance in human disease.
Physiol Rev
75:
473-486,
1995
28.
Rodrigues, B,
Cam MC,
and
McNeill JH.
Metabolic disturbances in diabetic cardiomyopathy.
Mol Cell Biochem
180:
53-57,
1998[ISI][Medline].
29.
Sack, MN,
and
Kelly DP.
The energy substrate switch during development of heart failure: gene regulatory mechanisms.
Int J Mol Med
1:
17-24,
1998[ISI][Medline].
30.
Shehadhe, A,
and
Regan TJ.
Cardiac consequences of diabetes mellitus.
Clin Cardiol
18:
301-305,
1995[ISI][Medline].
31.
Stanley, WC,
Lopaschuk GD,
and
McCormack JG.
Regulation of energy substrate metabolism in the diabetic heart.
Cardiovasc Res
34:
25-33,
1997
32.
Suga, H.
Ventricular energetics.
Physiol Rev
70:
247-277,
1990
33.
Tomlinson, KC,
Gardiner SM,
Hebden RA,
and
Bennett T.
Functional consequences of streptozotocin-induced diabetes mellitus, with particular reference to the cardiovascular system.
Pharmacol Rev
44:
103-150,
1992[ISI][Medline].
34.
Torra, IP,
Chinetti G,
Duval C,
Fruchart JC,
and
Staels B.
Peroxisome proliferator-activated receptors: from transcriptional control to clinical practice.
Curr Opin Lipidol
12:
245-254,
2001[ISI][Medline].
35.
Uusitupa, M,
Mustonen J,
Laakso M,
Vainio P,
Lansimies E,
Talwar S,
and
Pyorala K.
Impairment of diastolic function in middle-aged type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetic patients free of cardiovascular disease.
Diabetologia
31:
783-791,
1988[ISI][Medline].
36.
Van der Lee, KA,
Vork MM,
De Vries JE,
Willemsen PH,
Glatz JF,
Reneman RS,
Van der Vusse GJ,
and
Van Bilsen M.
Long-chain fatty acid-induced changes in gene expression in neonatal cardiac myocytes.
J Lipid Res
41:
41-47,
2000
37.
Van der Lee, KA,
Willemsen PH,
Van der Vusse GJ,
and
Van Bilsen M.
Effects of fatty acids on uncoupling protein-2 expression in the rat heart.
FASEB J
14:
495-502,
2000
38.
Wyse, BM,
and
Dulin WE.
The influence of age and dietary conditions on diabetes in the db mouse.
Diabetologia
6:
268-273,
1970[ISI][Medline].
39.
Ye, JM,
Doyle PJ,
Iglesias MA,
Watson DG,
Cooney GJ,
and
Kraegen EW.
Peroxisome proliferator-activated receptor (PPAR)-alpha activation lowers muscle lipids and improves insulin sensitivity in high fat-fed rats: comparison with PPAR-gamma activation.
Diabetes
50:
411-417,
2001
40.
Young, ME,
Patil S,
Ying J,
Depre C,
Ahuja HS,
Shipley GL,
Stepkowski SM,
Davies PJA,
and
Taegtmeyer H.
Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor alpha in the adult rodent heart.
FASEB J
15:
833-845,
2001
41.
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
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), untreated
diabetic (db/db, n = 23;
), and BM 17.0744-treated diabetic
(db/db + BM, n = 27;
) mice. Combined data from Tromsø and Calgary are
shown. Values are means ± SE.
