The physiological role of mitochondrial thioesterase 1 (MTE1) is unknown. It was proposed that MTE1 promotes fatty acid (FA) oxidation (FAO) by acting in concert with uncoupling protein (UCP)3. We previously showed that ucp3 is a peroxisome proliferator-activated receptor-α (PPARα)-regulated gene, allowing induction when FA availability increases. On the assumption that UCP3 and MTE1 act in partnership to increase FAO, we hypothesized that mte1 is also a PPARα-regulated gene in cardiac and skeletal muscle. Using real-time RT-PCR, we characterized mte1 gene expression in rat heart and soleus muscles. Messenger RNA encoding for mte1 was 3.2-fold higher in heart than in soleus muscle. Cardiac mte1 mRNA exhibited modest diurnal variation, with 1.4-fold higher levels during dark phase. In contrast, skeletal muscle mte1 mRNA remained relatively constant over the course of the day. High-fat feeding, fasting, and streptozotocin-induced diabetes, interventions that increase FA availability, muscle PPARα activity, and muscle FAO rates, increased mte1 mRNA in heart and soleus muscle. Conversely, pressure overload and hypoxia, interventions that decrease cardiac PPARα activity and FAO rates, repressed cardiac mte1 expression. Specific activation of PPARα in vivo through WY-14643 administration rapidly induced mte1 mRNA in cardiac and skeletal muscle. WY-14643 also induced mte1 mRNA in isolated adult rat cardiomyocytes dose dependently. Expression of mte1 was markedly lower in hearts and soleus muscles isolated from PPARα-null mice. Alterations in cardiac and skeletal muscle ucp3 expression mirrored that of mte1 in all models investigated. In conclusion, mte1, like ucp3, is a PPARα-regulated gene in cardiac and skeletal muscle.
- nonesterified fatty acids
- uncoupling protein 3
- diurnal variations
acyl-coa thioesterases catalyze the hydrolysis of fatty acyl-CoA molecules into nonesterified fatty acid anions and free CoA. At least five thioesterases have been identified to date: a cytosolic isoform (CTE1), two peroxisomal isoforms (PTE1 and PTE2), and two mitochondrial isoforms (MTE1 and MT-ACT48) (16, 17, 27, 35, 42). Of these, MTE1 is abundantly expressed in tissues engaged in high rates of fatty acid oxidation, such as liver, brown adipose tissue (BAT), skeletal muscle, and heart (41). The tissue and subcellular distribution of MTE1, in addition to the observation that mte1 expression is increased in livers of fasted rats, led to early suggestions that this enzyme promotes β-oxidation of fatty acids (16). Consistent with this idea, the peroxisome proliferator-activated receptor-α (PPARα, a nuclear receptor known to induce key regulators of fatty acid oxidation in response to increased fatty acid availability) agonist clofibrate markedly induces mte1 expression in rat livers (16). Despite these observations, very little information is currently available with regard to MTE1 regulation in extrahepatic tissues, such as cardiac and skeletal muscle.
Only recently has a mechanism been proposed by which MTE1 may function to increase fatty acid oxidation. Uncoupling proteins (UCPs) are inner mitochondrial membrane-bound proteins classically known for uncoupling ATP synthesis from oxidative metabolism (34). UCPs, of which at least three closely related homologs have been cloned, appear to act as fatty acid anion transporters as opposed to transporters of protons (37). UCP1 (thermogenin) is highly expressed in BAT, where it is involved in nonshivering thermogenesis (36). In contrast, recent studies suggest that UCP2 (a ubiquitously expressed isoform) may act to minimize generation of mitochondrially derived reactive oxygen species (23, 31). UCP3, on the other hand, exhibits a similar tissue distribution pattern to that of MTE1, being highly expressed in BAT, heart, and skeletal muscle (6, 45). Furthermore, UCP3 expression increases during conditions associated with increased fatty acid oxidation, an effect mediated by PPARα (8, 50). These observations led to the suggestion that UCP3 promotes fatty acid oxidation (5). Himms-Hagen and Harper (15) recently published a hypothetical link between MTE1 and UCP3, suggesting that these two proteins function in concert to augment fatty acid oxidation. This hypothesis hinges on mitochondrial CoA. For the complete oxidation of fatty acids, mitochondrial CoA is required at two stages of β-oxidation (the formation of mitochondrial fatty acyl-CoA from fatty acyl-carnitine, and the thiolysis of the β-ketoacyl-CoA intermediate) and at one stage of the TCA cycle (the oxidative decarboxylation of α-ketoglutarate). If influx of fatty acyl groups into the mitochondrial matrix is dramatically increased, such that free CoA is exhausted by the formation of mitochondrial fatty acyl-CoAs, then the rate of β-oxidation will be limited. Hydrolysis of accumulating fatty acyl-CoAs by MTE1 will liberate free CoA required for continued β-oxidation. It is then hypothesized that the fatty acid anion generated is exported out of the mitochondrial matrix by UCP3, after which cytosolic fatty acyl-CoA can be regenerated. MTE1 and UCP3 may therefore act together to preserve high rates of fatty acid oxidation in the face of elevated fatty acid availability.
On the basis of the assumption that MTE1 and UCP3 function in partnership, we hypothesize that these nuclear-encoded mitochondrial proteins are influenced by common transcriptional regulatory mechanisms. We (50) and others (8) have shown that ucp3 is a PPARα-regulated gene in cardiac and skeletal muscle. The purpose of the present study was therefore to test the hypothesis that mte1, like ucp3, is a PPARα-regulated gene in cardiac and skeletal muscle. Here we report cardiac and skeletal muscle mte1 and ucp3 expression in multiple models known to modulate PPARα activity, as well as fatty acid oxidation (FAO). The data support the hypothesis that mte1 is a PPARα-regulated gene in cardiac and skeletal muscle and are consistent with a role for this enzyme in the promotion of FAO.
MATERIALS AND METHODS
All animal research was carried out in accordance with the American Physiological Society’s Guiding Principles in the Care and Use of Animals, following institutional protocol review and approval. A total of 207 male Wistar rats (Charles River, Wilmington, MA; 200 g initial weight) were housed either at the Animal Care Center of the University of Texas Health Science Center at Houston (diabetes, PPARα agonist, pressure overload, and cardiomyocyte studies), at the Animal Care Center of the Children's Nutrition Research Center at Baylor (high-fat feeding and fasting studies), or at the Animal Care Center of the Hatter Institute for Cardiology Research (hypoxia studies). Eighteen wild-type (Svlmj) and 18 PPARα-null mice (The Jackson Laboratory, Bar Harbor, ME; 20 g initial weight) were housed at the Animal Care Center of the University of Texas Health Science Center at Houston. All animals were housed under controlled conditions (23 ± 1°C; 12:12-h light-dark cycle) and received standard laboratory chow and water ad libitum unless otherwise stated. Ten days before they were killed, animals were housed in a separate environment-controlled room, within which a strict 12:12-h light-dark cycle regime was enforced [lights on at 7:00 AM; zeitgeber time (ZT) 0]. On the day of the experiment, hearts and soleus muscles were isolated from anesthetized rodents (pentobarbital sodium; 100 mg/kg ip), freeze clamped in liquid nitrogen, and stored at −80°C before RNA extraction. We have previously shown that the responsiveness of the PPARα system within rat heart and soleus muscle is greatest during the middle of the dark phase, when the rodent is most active (40). Therefore, all animals in the present study were killed between 9:00 PM (ZT15) and 3:00 AM (ZT21) to optimize validity of the data obtained.
Rats were fed either a standard rodent chow, a low-fat diet (LFD), or a high-fat diet (HFD), as described previously (40).
High-fat feeding, fasting, induction of diabetes, and specific PPARα activation.
Experiments were performed in which rats were subjected to high-fat feeding (HFD; duration of 4 wk), fasting (duration of 18 h), or streptozotocin (STZ)-induced diabetes (65 mg/kg STZ iv; killed after 4 wk) or administered with a specific PPARα agonist (50 mg/kg WY-14643 ip; killed after 4 h), as described previously (40).
Pressure overload-induced hypertrophy.
Cardiac hypertrophy was induced by constriction of the ascending aorta, as described previously (21). The diameter of the lumen of the aorta after constriction was equivalent to that of an 18-gauge needle. In control animals, sham operations were performed without constriction of the aorta. Because gene expression can be affected by multiple factors, including physical activity, hormone levels, and food intake, animals were allowed to recover from surgery for 7 wk. On the day of the experiment, control and hypertrophied hearts were isolated, weighed, rapidly frozen in liquid nitrogen, and stored at −80°C before RNA isolation.
Hypobaric chamber-induced hypoxia.
Rats were placed in a hypobaric chamber, where they were exposed to 11% O2 (45 kPa) for 7 days. Age-matched animals exposed to normoxic conditions were used as controls. On the day of the experiment, left ventricles were dissected from control and hypoxic hearts, weighed, rapidly frozen in liquid nitrogen, and stored at −80°C before RNA extraction.
The potential role of PPARα in regulating cardiac and skeletal muscle mte1 expression was investigated using the previously established PPARα-null mouse (25). On the day of the experiment, wild-type and PPARα-null hearts were isolated, rapidly frozen in liquid nitrogen, and stored at −80°C before RNA isolation.
Isolated adult rat cardiomyocytes.
Isolated adult rat cardiomyocytes were prepared using protocols as described previously for mouse cardiomyocytes (4). Freshly isolated cardiomyocytes were cultured overnight in DMEM-containing laminin-ciated plates. The cells were then challenged with various concentrations of the PPARα agonist WY-14643 (0, 1, 10, or 100 μM). WY-14643 was prepared in DMSO (with a final DMSO concentration in the culture medium of 0.1%). After 12 h, cardiomyocytes were harvested in Tri Reagent and stored at −80°C before RNA isolation.
RNA extraction and quantitative RT-PCR.
RNA extraction and quantitative RT-PCR of samples were performed using previously described methods (10, 12, 14). Specific quantitative assays were designed from rat and mouse sequences available in GenBank. Taqman assays for rat and mouse mte1 are presented in Table 1, and assays for pparα, ucp3, β-actin, and cyclophilin have been reported previously (40, 50). Standard RNA was made for all assays by the T7 polymerase method (Ambion, Austin, TX) with the use of total RNA isolated from either rat or mouse hearts. The correlation between the number of PCR cycles required for the fluorescent signal to reach a detection threshold (CT) and the amount of standard was linear over at least a five-log range of RNA for all assays (data not shown). The level of transcripts for the constitutive housekeeping gene products β-actin and cyclophilin was quantitatively measured in each sample to control for sample-to-sample differences in RNA concentration. PCR data are reported as the number of transcripts per number of β-actin molecules, with the exception of the fasting and adult rat cardiomyocyte studies. Here, data are reported as the number of transcripts per number of cyclophilin molecules (as β-actin expression significantly decreased by 33% in hearts and soleus muscles isolated from fasted vs. fed rats, and β-actin expression changed on treatment of cardiomyocytes with WY-14643; data not shown). Expression of β-actin did not change with the other experimental interventions in rodent hearts and soleus muscles (data not shown). In Fig. 1, data are normalized to nanograms of total RNA, allowing a direct comparison of the level of mte1 and ucp3 expression between heart and soleus muscle that is unbiased by tissue-specific differences in housekeeping gene expression (β-actin expression is 2.4-fold higher in heart vs. soleus muscle, and cyclophilin expression is 1.7-fold higher in heart than in soleus muscle; data not shown).
Plasma nonesterified fatty acid level determination.
Immediately before muscle isolation, 1 ml of blood was withdrawn from all rats. The sample was placed on ice before centrifugation for 10 min at full speed using a desk-top microfuge. The supernatant was retained and stored at −80°C until nonesterified fatty acid (NEFA) levels were measured spectrophotometrically with a commercially available kit (Wako Chemicals, Dalton, GA). Specimen blanks were prepared for all samples to allow for possible hemolysis.
Two-way analysis of variance (ANOVA) was conducted to investigate the main effects of group (tissue or experimental condition) and time. The general linear model procedure in SAS software, version 8.2, was used for this analysis (SAS Institute, Cary, NC). A full model including second-order interactions was conducted for each experiment. Significant differences were determined using Type III sums of squares. Tukey's post hoc test was conducted for all significant two-way ANOVAs. The null hypothesis of no model effects was rejected at P < 0.05. Repeated-measures analysis was not utilized because samples in each group were from different animals.
Characterization of cardiac and skeletal muscle mte1 and ucp3 gene expression.
Diurnal variations in the expression of mte1 and ucp3 were investigated in both rat heart and soleus muscle (Fig. 1). A modest rhythm was observed in cardiac mte1 expression, with a 1.4-fold change from peak (ZT21) to trough (ZT9; Fig. 1A). In contrast, soleus muscle mte1 expression did not fluctuate over the course of the day (Fig. 1A). The data also show that mte1 expression is on average 3.2-fold higher in the heart compared with soleus muscle (Fig. 1A). Messenger RNA encoding for ucp3 exhibited striking diurnal variations in both muscle types; highest levels of expression were observed during the dark (4.2-fold higher) and light (5.4-fold higher) phases for the heart and soleus muscle, respectively (Fig. 1B). In contrast to mte1 expression, ucp3 expression is on average 1.5-fold higher in soleus muscle compared with the heart over the course of the day, with greatest differences in expression observed during the light phase (4.3-fold difference at ZT9; Fig. 1B). However, heart ucp3 expression was significantly higher at ZT15 compared with that of soleus muscles isolated at the same time point (Fig. 1B). It is also noteworthy that mte1 expression is markedly higher than ucp3 expression over the course of the day in both cardiac and skeletal muscle (35.7- and 7.4-fold, respectively; Fig. 1).
Effects of elevated fatty acid availability on cardiac and skeletal muscle mte1 expression.
We investigated whether conditions known to increase circulating fatty acid levels, and to increase PPARα activity and rates of FAO in cardiac and skeletal muscle, affected mte1 expression in these muscle types. High-fat feeding, fasting, and STZ-induced diabetes were utilized to increase circulating NEFA levels (Fig. 2).
Feeding rats the HFD for 4 wk resulted in increased mte1 expression in both cardiac and skeletal muscle (Fig. 3A).Expression of ucp3 was also increased in hearts and soleus muscles isolated from high fat-fed rats (Fig. 3B).
Fasting for 18 h significantly induced mte1 expression in both heart and soleus muscle (Fig. 4A).These changes in mte1 expression were mirrored by increased expression of ucp3 in muscles isolated from fasted rats (Fig. 4B).
Induction of diabetes in rats through STZ administration significantly increased cardiac and soleus muscle mte1 expression (Fig. 5A).This was again associated with ucp3 induction in both muscle types investigated (Fig. 5B).
Effects of pressure overload and hypoxia on cardiac mte1 and ucp3 expression.
Both pressure overload-induced hypertrophy and hypobaric chamber-induced hypoxia have been shown to decrease PPARα activity and rates of FAO in the heart (1, 2, 18, 33, 43). We therefore tested the hypothesis that mte1 expression is decreased in both the hypertrophied and hypoxic heart. For the hypoxia studies, left ventricles were utilized, as opposed to whole hearts, because of the hypertrophic effects of sustained hypoxia on the right ventricle (33).
Seven weeks of aortic constriction significantly increased the heart weight-to-body weight ratio (2.84 × 10−3 ± 0.02 × 10−3 vs. 3.69 × 10−3 ± 0.05 × 10−3 for control vs. experimental groups, respectively; P < 0.001). Consistent with results of previously published reports, cardiac pparα expression was decreased in the hypertrophied heart (Table 2) (2, 50). Expression of mte1 was also significantly lower in hypertrophied hearts compared with sham-operated controls (Table 2). A similar pattern was observed for ucp3, wherein expression was significantly decreased after pressure overload (Table 2).
One week of hypoxia did not affect the left ventricular weight-to-body weight ratio (2.7 × 10−3 ± 0.2 × 10−3 vs. 2.9 × 10−3 ± 0.6 × 10−3 for control vs. experimental groups, respectively). Hypoxia significantly decreased left ventricular expression of pparα, mte1, and ucp3 (Table 2).
Induction of mte1 after PPARα activation in vivo or in vitro.
We next initiated studies to test the hypothesis that mte1 is a PPARα-regulated gene. Administration of the PPARα agonist WY-14643 to rats rapidly (within 4 h) induced mte1 in both hearts and soleus muscles (Fig. 6A).Induction of ucp3 was also observed in these muscle types after WY-14643 administration (Fig. 6B). Similarly, treatment of isolated adult rat cardiomyocytes with WY-14643 resulted in a dose-dependent induction of both mte1 and ucp3 (Fig. 7).
Cardiac and skeletal muscle mte1 expression in PPARα-null mice.
We investigated further the hypothesis that mte1 is a PPARα-regulated gene by measuring the expression of mte1 in hearts and soleus muscles isolated from PPARα-null mice. Messenger RNA encoding for mte1 was markedly lower in hearts (68% lower) and soleus muscles (44% lower) isolated from PPARα-null mice compared with the same muscle types isolated from age-matched wild-type mice (Fig. 8A).Basal expression of cardiac ucp3 was similarly diminished in hearts isolated from PPARα-null mice (Fig. 8B). In contrast, ucp3 expression was not significantly different between soleus muscles isolated from PPARα-null and wild-type mice (Fig. 8B).
The purpose of the present study was to test the hypothesis that mte1, like ucp3, is a PPARα-regulated gene in cardiac and skeletal muscle. We therefore measured mte1 expression in hearts and soleus muscles isolated from rodent models known to either increase (high-fat feeding, fasting, STZ-induced diabetes, WY-14643 administration) or decrease (pressure overload-induced hypertrophy, hypobaric chamber-induced hypoxia, PPARα-null mice) PPARα activity. Activation of PPARα using these models was associated with induction of cardiac and skeletal muscle mte1 expression. Induction of mte1 through specific activation of PPARα was also observed in isolated adult rat cardiomyocytes. Conversely, downregulation of PPARα activity decreased cardiac and skeletal muscle mte1 expression. These changes in mte1 expression were mirrored by similar alterations in ucp3 expression. We therefore conclude that mte1, like ucp3, is a PPARα-regulated gene in cardiac and skeletal muscle. The induction of both MTE1 and UCP3 during periods of increased fatty acid availability is consistent with a role for these proteins in the promotion of FAO.
PPARα regulates mte1 gene expression.
The nuclear receptor PPARα plays a central role in metabolic adaptation of tissues such as cardiac and skeletal muscle (3, 49). On activation by its ligand, PPARα, in concert with its heterodimerization partner retinoid X receptor, binds to the fatty acid response element within the promoter of various target genes (22). The latter include those genes encoding for enzymes promoting FAO (e.g., malonyl-CoA decarboxylase, muscle-specific carnitine palmitoyltransferase, medium- and long-chain acyl-CoA dehydrogenases, and UCP3), as well as those encoding enzymes that repress carbohydrate metabolism (e.g., pyruvate dehydrogenase kinase 4) (7, 9, 13, 44, 46, 47, 50). The ligand for PPARα appears to be either fatty acids themselves or a fatty acid derivative (11). PPARα is therefore part of a feed-forward mechanism in which fatty acids are able to promote their own utilization. Disruption of this mechanism leads to accumulation of detrimental fatty acid derivatives, which have been associated with various pathologies, including insulin resistance, β-cell dysfunction, and cardiomyopathy (28, 38, 51).
Given the assumption that MTE1 acts in concert with UCP3 to increase FAO, we hypothesized that the mte1 gene is regulated by PPARα in cardiac and skeletal muscle, tissues known to engage in high rates of FAO. We tested this hypothesis in a number of ways. Expression of mte1 was investigated in cardiac and skeletal muscle during situations known to increase fatty acid availability, muscle PPARα activity, and rates of FAO. These included high-fat feeding, fasting, and uncontrolled type 1 diabetes mellitus (19, 20, 26, 32, 39, 48, 49). Consistent with our hypothesis, all three interventions resulted in induction of mte1 expression in rat heart and soleus muscle (Figs. 3–5). Similarly, administration of rats with the specific PPARα agonist WY-14643 rapidly induced mte1 expression in both muscle types (Fig. 6). The same agonist induced mte1 expression in isolated adult rat cardiomyocytes in a dose-dependent manner (Fig. 7). Expression of mte1 was also investigated during conditions associated with decreased cardiac PPARα activity and FAO rates. These included pressure overload-induced hypertrophy and hypobaric chamber-induced hypoxia (1, 2, 18, 33, 43). Both interventions significantly decreased cardiac mte1 expression (Table 2). To investigate directly the contribution of PPARα toward cardiac and skeletal muscle mte1 expression in vivo, PPARα-null mice were employed. Expression of mte1 was markedly decreased in both muscle types isolated from mice devoid of functional PPARα (Fig. 6). Taken together (Table 3), these observations lead to the conclusion that mte1 is a PPARα-regulated gene in cardiac and skeletal muscle.
Support for an MTE1-UCP3 interaction.
Evidence is accumulating in support of the hypothesis that MTE1 and UCP3 act in concert to promote FAO. Manipulation of ucp3 expression results in parallel changes in mte1 expression. For example, transgenic mice overexpressing ucp3 exhibit increased mte1 expression (29). Pharmacological induction of UCP3 in the liver through treatment of rats with fenofibrate resulted in a concomitant increase in mte1 expression and rates of hepatic FAO (24). More recently, triiodothyronine administration has been shown to induce both ucp3 and mte1 expression in rat gastrocnemius muscle (30). Consistent with coordinated regulation of these two genes, the present study has shown that mte1, like ucp3, is a PPARα-regulated gene in cardiac and skeletal muscle. For the animal models investigated presently, expression of ucp3 and mte1 always changed in parallel with one another (although fluctuations in ucp3 expression are generally greater; Table 3). We therefore propose that mte1 be added to the list of PPARα-regulated genes responsible for promoting FAO in cardiac and skeletal muscle during periods of increased fatty acid availability.
In addition to exposing regulation of the mte1 gene by PPARα, the present study reports a number of additional observations worthy of further discussion. Using real-time quantitative RT-PCR in a large sample set, we have very accurately characterized circadian- and tissue-dependent expression of both mte1 and ucp3 (Fig. 1). The data show that mte1 expression is on average 3.2-fold higher in heart vs. skeletal muscle over the course of the day (Fig. 1A). In addition, mte1 expression is on average 35.7- and 7.4-fold greater than ucp3 expression over the course of the day in heart and soleus muscle, respectively (Fig. 1). Assuming that the level of expression of these genes correlates with protein and activity levels, one can hypothesize that UCP3, as opposed to MTE1, is more likely to be a limiting factor. This may explain why ucp3 is more highly induced during periods of increased fatty acid availability, as mte1 levels are relatively high at baseline. It is also apparent that diurnal variations in mte1 expression are markedly less dramatic compared with those observed for ucp3 expression (Fig. 1). In addition, basal levels of expression of skeletal muscle ucp3 appear less dependent on PPARα compared with mte1 (Fig. 8). Such observations suggest that although both mte1 and ucp3 are PPARα-regulated genes, additional transcription factors differentially affect the expression of these two genes in cardiac and skeletal muscle.
The present study has shown that mte1 is a PPARα-regulated gene in cardiac and skeletal muscle. However, we have not determined whether the changes in mte1 gene expression observed result in comparable changes in MTE1 protein or activity. This is due in large part to the focus of the present study, that being investigation into the mechanism(s) of transcriptional regulation of the mte1 gene. Furthermore, given the large number of samples utilized for these gene expression measurements (233 whole heart, 233 soleus muscles, and 20 cardiomyocyte samples), the lack of a commercially available MTE1 antibody, and the difficulties associated with the specific measurement of MTE1 activity (as opposed to the other thioesterases found within the cell, including MT-ACT48, a second mitochondrial isoform), assessment of MTE1 protein and activity levels in the same sample set would be a major undertaking that is beyond the scope of the present study.
In conclusion, the gene encoding for mte1 is regulated by PPARα in cardiac and skeletal muscle. Conditions known to be associated with increased fatty acid availability, and increased FAO, caused induction of mte1 mRNA in cardiac and skeletal muscle. Conversely, conditions associated with decreased FAO caused repression of mte1 mRNA in both muscle types. These observations support the hypothesis that MTE1 promotes FAO, perhaps in concert with UCP3.
This work was supported by the American Heart Association Texas Affiliate Beginning Grant-In-Aid Award 0365028Y and National Heart, Lung, and Blood Institute Grant HL-074259-01.
We thank Drs. William C. Stanley, Jason R. Dyck, and Mary-Ellen Harper for constructive comments before submission.
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.
- Copyright © 2004 by American Physiological Society