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Journal of Lipid Research, Vol. 48, 1511-1517, July 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology
agonist increases mitochondrial thioesterase I activity in heart
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* Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030
Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, OH 44106
** Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital at Huddinge, Stockholm, Sweden
Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106
Department of Medicine, Division of Cardiology, University of Maryland School of Medicine, Baltimore, MD 21201
Published, JLR Papers in Press, April 16, 2007.
1 To whom correspondence should be addressed. e-mail: wstanley{at}medicine.umaryland.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PPAR) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of protein expression of MTE-I and UCP3 or about MTE-I activity; thus, we investigated the effects of diabetes and treatment with a PPAR agonist on these parameters. Rats were either made diabetic with streptozotocin (55 mg/kg ip) and maintained for 10–14 days or treated with the PPAR agonist fenofibrate (300 mg/kg/day) for 4 weeks. MTE-I and UCP3 protein expression, MTE-1 activity, palmitate export, and oxidative phosphorylation were measured in isolated cardiac mitochondria. Diabetes and fenofibrate increased cardiac MTE-I mRNA, protein, and activity (
4-fold compared with controls). This increase in activity was matched by a 6-fold increase in palmitate export in fenofibrate-treated animals, despite there being no effect in either group on UCP3 protein expression. Both diabetes and fenofibrate caused significant decreases in state III respiration of isolated mitochondria with pyruvate + malate as the substrate, but only diabetes reduced state III rates with palmitoylcarnitine. Both diabetes and specific PPAR activation increased MTE-I protein, activity, and palmitate export in the heart, with little effect on UCP3 protein expression.
Supplementary key words cardiac • fatty acids • lipotoxicity
Abbreviations: IFM, interfibrillar mitochondria; MTE-I, mitochondrial thioesterase I; P/O, ADP-to-oxygen ratio; PPAR, peroxisome proliferator-activated receptor ; RCR, respiratory control ratio; SSM, subsarcolemmal mitochondria; UCP3, uncoupling protein 3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PPAR), a ligand-activated nuclear receptor that increases the expression of key proteins that regulate cellular fatty acid uptake and oxidation (2). It was recently demonstrated that the mRNA expression for two mitochondrial proteins involved in fatty acid metabolism, mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), is increased in the heart by either diabetes or activation of PPAR (3–5). UCP3 resides on the inner mitochondrial membrane and can export fatty acid anions across the inner mitochondrial membrane (6–8). MTE-I mRNA and protein are most strongly expressed in tissues with high rates of fatty acid oxidation, such as brown adipose tissue, liver, kidney, skeletal muscle, and heart (9, 10). MTE-I catalyzes the formation of fatty acid anions from long-chain acyl-CoA in the mitochondrial matrix, which are then exported to the cytosol, suggesting that UCP3 and MTE-I act together to help regulate the long-chain fatty acyl-CoA concentration in the mitochondrial matrix (3, 7, 11). Little is known about the effects of diabetes and PPAR activation on UCP3 and MTE-I expression or on mitochondria respiration and fatty acid export from the mitochondria.
Recent studies suggest that diabetes and the subsequent increase of plasma fatty acids can cause excessive accumulation of lipids in the heart, resulting in cardiac dysfunction and cardiomyopathy (12, 13), and that this condition can be mimicked by cardiac-specific overexpression of PPAR (14). Gerber, Aronow, and Matlib (3) recently observed that diabetes upregulates MTE-I protein expression and activity in the heart; however, it is not known whether selective activation of PPAR mimics this effect. In addition, the consequences of alterations in MTE-I protein expression on mitochondrial function are not well understood (11, 15). Altered mitochondrial respiration in liver, skeletal muscle, and heart has been observed in response to perturbations that alter UCP3 and/or MTE-I expression (e.g., treatment with fenofibrate or thyroid hormone, caloric restriction, fasting, or diabetes) (3, 16–22). Induction of MTE-I should facilitate the export of fatty acid anions and reduce the accumulation of toxic long-chain acyl-CoAs (3, 7, 23–25) and thus might help prevent the lipotoxic cardiomyopathy that can develop in diabetes (12, 13), with accelerated lipid uptake or impaired fatty acid metabolism (26–29).
The goal of this investigation was to determine whether the initiation of diabetes and selective PPAR activation increases the protein expression of MTE-I and/or UCP3, the mitochondrial activity of MTE-I, or the function of isolated mitochondria from the rat heart. We hypothesized that PPAR stimulation with diabetes, and pharmacologically with fenofibrate, would increase the protein expression and activity of MTE-I and the export of fatty acid anions from isolated mitochondria. Because diabetes decreases cardiac mechanical efficiency by increasing myocardial oxygen consumption (30), we also investigated whether diabetes or treatment with fenofibrate would decrease the ADP-to-oxygen ratio (P/O) or increase state IV respiration in the mitochondria (31, 32). Experiments were performed in established rat models of fenofibrate treatment and streptozotocin-induced diabetes that have previously shown upregulation of the mRNA of MTE-I and UCP3. It is well documented that cardiac mitochondria exist in two distinct populations, with the interfibrillar mitochondria (IFM) subpopulation having a higher respiratory capacity per milligram of mitochondrial protein than the subsarcolemmal mitochondria (SSM) subpopulation (33); thus, both populations were isolated and studied.
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MATERIALS AND METHODS |
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Study design
Three series of studies were performed. In series I, diabetes was induced with streptozotocin (55 mg/kg ip) (n = 9) and maintained for 10–14 days; these animals were then compared with a parallel untreated control group of rats matched for age (n = 8). In the diabetic rats, plasma glucose was monitored to ensure that it was >250 mg/dl. In series II, rats were fed a standard rat chow or treated with the PPAR agonist fenofibrate [300 mg/kg/day in the chow (34)] (n = 12/group). The cardiac mitochondria from series I and II were used to measure respiration, the expression of UCP3 and MTE-I mRNA and protein, and the activity of MTE-I. In series III, rats were either untreated or treated with fenofibrate as in series II (n = 9/group), and the rate of fatty acid export and MTE-1 activity were measured in SSM. All groups were in a reverse light/dark cycle for at least 1 week before euthanasia (35) and were euthanized in the fed state 2 h after initiation of the dark phase to ensure peak changes in MTE-I and UCP3 mRNA (4). Rats were anesthetized with isoflurane (1.5–2.0%), the chest was open by sternotomy, and the left ventricle was removed. Cardiac mitochondria were isolated into SSM and IFM fractions as described below, and aliquots were frozen for Western blot and enzyme activities. A separate tissue sample for mRNA analysis was immediately frozen in liquid nitrogen.
Isolation of mitochondria from cardiac tissue
Tissue was placed in buffer A [100 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM MgSO4(7H2O), and 1 mM ATP, pH 7.4] at 4°C. Cardiac mitochondria were then isolated according to Hoppel and colleagues (33, 36) with minor modifications. Tissue was finely minced and placed in buffer B (buffer A with 0.2% BSA), homogenized with a Polytron tissue processor (Brinkman Instruments, Westbury, NY) for 2.5 s at a rheostat setting of 6.5, and further homogenized with a Potter-Elvejhem homogenizer at a setting of 3. The homogenate was centrifuged at 600 g, with the supernatant being saved for SSM isolation and the pellet being saved for IFM isolation. For the IFM, the pellet underwent Polytron treatment and digestion with trypsin (5 mg/g wet weight) for 10 min at 4°C, and the mitochondria were collected as described above. Both fractions were centrifuged at 3,100 g and resuspended in 100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA. Protein was determined using the method of Lowry et al. (37) with BSA as a standard.
Measurement of mitochondrial oxygen consumption
Mitochondrial oxygen consumption was measured using a Clark-type oxygen electrode at 30°C in 80 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM KH2PO4, and 1 mg/ml BSA. The substrates pyruvate + malate, palmitoylcarnitine + malate, succinate, durohydroquinone, and N,N,N',N'-tetramethyl-p-phenylenediamine ascorbate were used to measure specific sites in the electron transport chain, state III (ADP-stimulated) respiration, state IV (ADP-limited) respiration, maximal oxidative phosphorylation with high ADP (2 mM), and uncoupled respiration with dinitrophenol. The respiratory control ratio (RCR) (state III/state IV) and P/O (ratio of oxygen consumed after the addition of a known amount of ADP) were also determined.
Measurement of mitochondrial palmitate export
Palmitate export was measured in isolated SSM from fenofibrate–treated rats according to Gerber, Aronow, and Matlib (3) The export of [1-14C]palmitic acid was measured after incubating isolated mitochondria for 6 min with 20 µM [1-14C]palmitoylcarnitine (specific activity, 10,000 dpm/nmol) at 37°C. The reaction was terminated as described previously (3), and the extramitochondrial fluid was separated by centrifugation and filtration (0.45 µm). Lipids were extracted and fatty acid moieties were separated by thin-layer chromatography, and the palmitic acid spot was visualized and counted (3).
RNA extraction and quantitative RT-PCR
RNA extraction and real-time quantitative RT-PCR were performed, as described previously, on frozen powdered left ventricle tissue (38–40). Previously published rat sequences for the expression of MTE-I and UCP3 were found using GenBank, with specific quantitative assays designed for each (4, 5). The primer sequences were as follows: 5'-GTGACCTATGACATCATCAAGGA-3' (forward), 5'-GCTCCAAAGGCAGAGACAAAG-3' (reverse), and 5'-FAM-CTGGACTCTCACCTGTTCACTGACAACTTCC-TAMRA-3' (probe) for UCP3; and 5'-TGTTGGGAACACCGTATGCT-3' (forward), 5'-CCACGACATCCAAGAGACCA-3' (reverse), and 5'-FAM-TTTGGTCATTTTGACTTTGTCTCTCAGAAGGG-TAMRA-3' (probe) for MTE-1. The T7 polymerase method (Ambion) was used to make standard RNA using total RNA isolated from rat hearts. The correlation between the amount of standard and the number of PCR cycles required for the fluorescent signal to reach a detection threshold was linear over at least a 5 log range of RNA for all assays. The mRNA level for cyclophilin was quantitatively measured in each sample to control for sample-to-sample differences in RNA concentration. Because the expression of cyclophilin was not different among the groups, the PCR data are reported as the number of transcripts per nanogram of total RNA.
Western blot analysis
Western blot analysis for MTE-I and UCP3 was performed on frozen SSM and IFM aliquots in duplicate at 75 and 150 µg of total protein, respectively. The protein was separated by electrophoresis on 4–12% Bis-Tris SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with TBS-Tween containing 5% milk and then incubated with primary antibodies to MTE-I (1:2,000) and UCP3 (1:1,000; Alpha Diagnostic). A polyclonal antibody for MTE-I was raised in rabbits immunized with purified rat MTE-I as described previously (9). After treatment with the secondary antibody, the membranes were developed in an enhanced chemiluminescence substrate solution (Amersham) and bands were quantified using NIH software. Variability among gels was corrected for by loading a standard sample of isolated skeletal muscle mitochondria (100 µg of protein) in duplicate on each gel. Results are expressed as a fraction of the mean of the control SSM group. To verify that the blot at 30 kDa was UCP3, isolated cardiac mitochondria from UCP3 knockout and wild-type mice (a gift from Dr. E. Dale Abel, University of Utah) were run alongside samples from all treatment groups, and the expected band at 30 kDa for UCP3 was completely absent for the UCP3 knockout mouse (data not shown).
Measurement of MTE-I and citrate synthase activities
Citrate synthase activity was assessed on frozen SSM and IFM as described previously (41). MTE-I activity was measured spectrophotometrically on SSM and IFM with palmitoyl-CoA as the substrate by following the increase in absorbance at 412 nm in the presence of 5,5'-dithiobis 2-nitrobenzoic acid (22, 42).
Statistical analysis
Effects of treatment and differences between SSM and IFM were determined using a two-way ANOVA with the Bonferroni post hoc test. Differences in mRNA and plasma measurements were determined with a two-tailed t-test. Data are presented as means ± SEM, and p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA and protein expression
Diabetes or activation of PPAR with fenofibrate resulted in significant increases in MTE-1 and UCP3 mRNA compared with control animals (Fig. 1
). MTE-I protein content in SSM and IFM increased by >5-fold with diabetes or fenofibrate treatment compared with controls; however, there were no significant changes in UCP3 protein expression (Fig. 2
). There were no differences between SSM and IFM in MTE-I or UCP3 protein content. Similar results for MTE-I and UCP3 protein expression were found when data were normalized to mitochondrial citrate synthase activity (data not shown).
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with fenofibrate significantly increased palmitate export from cardiac mitochondria compared with control animals (Fig. 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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increases the protein expression and activity of MTE-I and the rate of fatty acid export from cardiac mitochondria. Neither diabetes nor specific activation of PPAR significantly affected the protein expression of UCP3 despite a >4-fold increase in mRNA for UCP3. These findings add support to the concept that MTE-I functions to regulate the concentration of long-chain fatty acyl-CoAs in the mitochondrial matrix through the generation and export of free fatty acid anions. Therefore, MTE-I appears to play a key role in the adaptation of cardiac mitochondria to the increase in long-chain fatty acids that occurs in diabetes.
These results showed a dramatic upregulation of MTE-1 mRNA, protein expression, and activity by diabetes, which was largely mimicked by pharmacological stimulation of PPAR, suggesting that increased plasma fatty acids in diabetes upregulate MTE-I via ligand activation of PPAR. Upregulation of MTE-I activity in cardiac mitochondria may help prevent the lipotoxic cardiomyopathy that can develop in diabetes (12, 13), as it would reduce the accumulation of toxic long-chain acyl-CoA in the mitochondrial matrix and facilitate the export of fatty acid anions (7, 11, 23–25). Upregulation of MTE-1 in diabetes should also prevent depletion of the free CoA pool and thus help maintain the oxidation of pyruvate and -ketoglutarate. UCP3 is known to transport fatty acid anions across the inner mitochondrial membrane (43); however, the results of this study demonstrate that although MTE-I protein and activity, and palmitate export, are clearly upregulated with diabetes or PPAR activation, UCP3 protein is not (Fig. 2). Perhaps there is either a sufficient amount of UCP3 in the membrane to support the increased rate of fatty acid export observed after chronic treatment with fenofibrate or other protein(s) are involved in this process. Recent studies show that the plasma membrane fatty acid transport protein CD36 also resides in skeletal muscle mitochondria, suggesting the possibility that it could participate in this process in cardiac mitochondria (44–46). Additional work is needed to determine the specific protein responsible for the export of the fatty acid anion generated by MTE-I in the matrix.
Although several previous studies have established that there is upregulation of the mRNA for UCP3 with fasting, diabetes, or stimulation of PPAR (5), less is known about the regulation of protein expression. The increase in UCP3 mRNA with severe diabetes or treatment with a PPAR agonist is >3-fold (4, 5); however, Murray et al. (15) recently observed a more modest increase of 50% in UCP3 protein levels in cardiac mitochondria isolated from streptozotocin-induced and db/db diabetic mice and in mice treated with the PPAR agonist WY-14,643. Gerber, Aronow, and Matlib (3) observed a similar increase in UCP3 protein in streptozotocin-induced diabetic rats. In this study, we did not observe a significant increase in protein expression in either the diabetic or fenofibrate group; however, there was a strong trend for an increase in UCP3 in the SSM fraction of the diabetic animals (54% increase compared with the control group) (Fig. 2), and when only the SSM fraction was compared using a t-test, the difference was significant (p < 0.03). There was no significant increase in UCP3 protein expression in the IFM fraction of either group. In the studies by Murray et al. (15) and Gerber, Aronow, and Matlib (3), the procedure for the isolation of mitochondria would yield mainly SSM, suggesting that any increase in UCP3 is primarily in this fraction. Together, it appears that the increase in mRNA for UCP3 is not proportional to the increase in UCP3 protein expression and that the increase in MTE-I protein expression far exceeds the increase in UCP3.
The effects of diabetes on the expression of MTE-I were emulated by pharmacological activation of PPAR; however, mitochondrial respiration was impaired to a greater extent in diabetes, with a reduction in state III rates with both lipid and nonlipid substrates, whereas PPAR activation only affected nonlipid substrates (Tables 1, 2). Previous studies demonstrated a similar reduction in state III respiration with either glutamate and succinate (31, 32) or pyruvate (30) with diabetes. The results of this study extended these finding by demonstrating reduced respiratory function of electron transport chain complexes II–IV (Table 3). We hypothesized that state IV respiration would be increased, as demonstrated recently by Lashin and Romani (31, 32) in ketotic diabetic rats; however, we observed a significant decrease with pyruvate + malate but not with palmitoylcarnitine (Table 1), consistent with the well-documented decreased activity of pyruvate dehydrogenase in the diabetic heart (47). How et al. (30) did not find any difference in state IV respiration between normal and diabetic mice with either pyruvate or palmitoylcarnitine as substrate, yet they observed decreased cardiac mechanical efficiency (external power generation/myocardial energy expenditure) in working hearts at both low and high fatty acid concentrations. The mechanism for this effect remains unclear.
In conclusion, treatment with a PPAR agonist increases MTE-1 mRNA, protein expression, and activity and the rate of palmitate export from isolated cardiac mitochondria, suggesting that MTE-I functions to regulate the concentration of long-chain fatty acyl-CoA in the mitochondrial matrix through the generation and export of free fatty acid anions. Thus, upregulation of MTE-I activity may prevent the accumulation of long-chain fatty acyl-CoA in the mitochondrial matrix when the heart is exposed to high levels of fatty acids.
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ACKNOWLEDGMENTS |
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Manuscript received August 16, 2006 and in revised form December 15, 2006 and in re-revised form March 26, 2007.
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REFERENCES |
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