Regulation of cardiac and skeletal muscle malonyl-CoA decarboxylase by fatty acids

Martin E. Young, Gary W. Goodwin, Jun Ying, Patrick Guthrie, Christopher R. Wilson, Frank A. Laws, and Heinrich Taegtmeyer

Division of Cardiology, Department of Internal Medicine, University of Texas-Houston Medical School, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, an important modulator of fatty acid oxidation. We hypothesized that increased fatty acid availability would increase the expression and activity of heart and skeletal muscle MCD, thereby promoting fatty acid utilization. The results show that high-fat feeding, fasting, and streptozotocin-induced diabetes all significantly increased the plasma concentration of nonesterified fatty acids, with a concomitant increase in both rat heart and skeletal muscle MCD mRNA. Upon refeeding of fasted animals, MCD expression returned to basal levels. Fatty acids are known to activate peroxisome proliferator-activated receptor-alpha (PPARalpha ). Specific PPARalpha stimulation, through Wy-14643 treatment, significantly increased the expression of MCD in heart and skeletal muscle. Troglitazone, a specific PPARgamma agonist, decreased MCD expression. The sensitivity of MCD induction by fatty acids and Wy-14643 was soleus > extensor digitorum longus > heart. High plasma fatty acids consistently increased MCD activity only in solei, whereas MCD activity in the heart actually decreased with high-fat feeding. Pressure overload-induced cardiac hypertrophy, in which PPARalpha expression is decreased (and fatty acid oxidation is decreased), resulted in decreased MCD mRNA and activity, an effect that was dependent on fatty acids. The results suggest that fatty acids induce the expression of MCD in rat heart and skeletal muscle. Additional posttranscriptional mechanisms regulating MCD activity appear to exist.

heart; malonyl-coenzyme A decarboxylase; nonesterified fatty acids; peroxisome proliferator-activated receptor-alpha ; skeletal muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FATTY ACIDS have many essential functions in the cell. These include roles as fuels (30, 41), as mediators of signal transduction [e.g., activation of various protein kinase C (PKC) isoforms, initiation of apoptosis] (12, 38), as ligands for nuclear transcription factors [e.g., peroxisome proliferator-activated receptor-alpha (PPARalpha )] (7, 20), and as essential components of biological membranes (39). Because of these many functions, the levels of intracellular fatty acids and their derivatives are tightly regulated. Elevation of intracellular fatty acids and lipids (and abnormalities in lipid handling) has been associated with various pathologies, including insulin resistance (31, 32), pancreatic dysfunction (38, 42), and cardiotoxicity (46). One way in which mammalian organisms respond to elevations in fatty acid levels is by increasing the expression, in tissues such as cardiac and skeletal muscle of various proteins involved in fatty acid utilization (17, 23, 26). Fatty acid-induced genes known to be involved in fatty acid metabolism include the fatty acid transporter CD36, fatty acid binding proteins, muscle-specific carnitine palmitoyltransferase 1 (CPT-1), and various enzymes in beta -oxidation (e.g., the acyl-CoA dehydrogenase isoforms) (4, 17, 43).

The flux-generating step in long-chain fatty acid oxidation appears to be the transport of the activated, acyl-CoA derivative of fatty acids into the mitochondrial matrix (reviewed in Refs. 11 and 27). Three proteins are involved in this process, CPT-1, a translocase, and CPT-2. Of these, CPT-1 is rate limiting. Two isoforms of CPT-1 exist, the liver and muscle (m) isoforms. In fully differentiated cardiac and skeletal muscle, the mCPT-1 isoform predominates (5). mCPT-1 is highly sensitive to allosteric inhibition by malonyl-CoA, resulting in reduced fatty acid oxidation (27). Situations in which intracellular malonyl-CoA levels increase are associated with decreased fatty acid oxidation (14, 28, 34).

The steady-state concentration of any metabolite in the cell is regulated by its rate of synthesis as well as its rate of degradation. Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) and is degraded by malonyl-CoA decarboxylase (MCD) (1). ACCbeta , the isoform which predominates in cardiac and skeletal muscle, is regulated both allosterically and covalently (19, 32). Little is known about the regulation of MCD, although recent work suggests that MCD in the heart and skeletal muscle is regulated covalently (14, 36). Whether the expression of MCD is altered during various physiological or pathophysiological conditions in cardiac or skeletal muscle is not known, with the exception of insulin-dependent diabetes mellitus, in which cardiac MCD expression and activity have recently been shown to be increased (37).

The purpose of the present study was to test the hypothesis that situations associated with increased plasma fatty acid levels would increase the expression and activity of cardiac and skeletal muscle MCD. The latter would promote increased fatty acid oxidation. In all cases investigated, elevation of plasma fatty acid levels resulted in increased cardiac and skeletal [extensor digitorum longus (EDL) and soleus] muscle MCD expression. Changes in MCD mRNA were not always paralleled by MCD activity. Specific PPARalpha activation (by Wy-14643), but not PPARgamma activation (by troglitazone), increased MCD expression and activity. Cardiac hypertrophy, which is associated with both decreased PPARalpha expression (3) and decreased fatty acid oxidation (2, 21), resulted in decreased MCD expression and activity, an effect which was dependent on fatty acids. We provide evidence for the regulation of MCD both transcriptionally and posttranscriptionally.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Spraque-Dawley rats (200-225 g initial weight) were kept in the Animal Care Center of the University of Texas-Houston Medical School under controlled conditions (23 ± 1°C; 12:12-h light-dark cycle) and received standard laboratory chow and water ad libitum, unless otherwise stated.

Materials. Chemicals, enzymes, isotopes, primers, and probes were obtained from sources described previously (10, 16).

Dietary manipulation. Rats were fed either standard laboratory chow, a high-carbohydrate/low-fat (HC/LF) diet, or a low-carbohydrate/high-fat (LC/HF) diet (Purina Mills). The HC/LF and LC/HF diets were isocaloric and varied only in the proportion of energy obtained from carbohydrate and fat. The contribution of carbohydrate, fat, and protein to total energy available was 71, 6, and 23% for the HC/LF diet and 24, 53, and 23% for the LC/HF diet, respectively. The source of carbohydrate was a combination of sucrose and dextrin, and the source of fat was a combination of corn oil and lard. Nonnutritive fiber was also increased in the LC/HF diet. The length of time in which the rats were fed the special diets is specified for individual experiments.

The effects of high-fat feeding on cardiac and skeletal muscle MCD expression were investigated. Rats were fed the LC/HF diet for either 1, 4, or 8 days. Rats were then anesthetized with chloral hydrate (300 mg/kg), and the heart, EDL, and soleus muscles were isolated. The isolated muscles were immediately freeze-clamped in liquid N2.

The effects of fasting and refeeding were also investigated. Rats were fasted for either 1 or 2 days, after which the heart and EDL and soleus muscles were isolated as above. A subset of fasted rats was refed with the HC/LF diet for an additional 4 days.

Induction of diabetes. Diabetes was induced through a single injection of streptozotocin (STZ; 55 mg/kg iv). Control animals received buffer (Hanks' buffer; GIBCO BRL Life Sciences) only. Two weeks after the initial injection, the animals were anesthetized, and hearts and skeletal muscles were removed, freeze-clamped, and stored at -80°C. Animals were considered diabetic if their blood glucose level was >300 mg/dl (16.7 mM).

Specific PPARalpha and PPARgamma activation. To test the effects of PPARalpha activation, Wy-14643 was added to standard powdered Purina rodent chow at a concentration of 0.01% (wt/wt). Rats were fed the Wy-14643-containing diet for 4 days. To test the effects of PPARgamma activation, troglitazone was added to standard powdered Purina rodent chow at a concentration of 0.1% (wt/wt). Rats were fed the troglitazone-containing diet for 4 days. In both sets of experiments, control animals received powdered rodent chow only.

Pressure overload-induced hypertrophy. Cardiac pressure overload was induced by banding the ascending aorta as described previously (24). In control animals, sham operations were performed without banding of the aorta. Nine days after aortic constriction, the animals were anesthetized, and hearts were removed, freeze-clamped, and stored at -80°C. Seven days before surgery, the animals had received either standard laboratory chow (control diet), the HC/LF diet, or the LC/HF diet. Specific feeding was maintained until the completion of the experiment (i.e., isolation of hearts).

Plasma glucose and nonesterified fatty acid level determination. Immediately before muscle isolation from the rats, 1 ml of blood was withdrawn. The sample was placed on ice before centrifugation for 10 min at full speed with a desktop microfuge. The supernatant was retained and stored at -80°C until glucose and nonesterified fatty acid (NEFA) levels were determined. Plasma glucose levels were measured for control and diabetic rats by means of a glucose/L-lactate analyzer (2300 STAT, Yellow Springs Instruments). Plasma NEFA levels were measured spectrophotometrically with a commercially available kit (Wako Chemicals, Richmond, VA). Specimen blanks were prepared for all samples to allow for possible hemolysis.

Measurement of MCD activity. MCD activity in homogenates of pulverized heart and skeletal muscle was measured by following the rate of 14CO2 generation from [3-14C]malonyl-CoA, as described previously (14). The concentration of malonyl-CoA in the assay was 200 µM; at this concentration of malonyl-CoA, the enzymatic rate is near maximal (14). All activity values are expressed as nanomoles of malonyl-CoA decarboxylated per minute per milligram of protein. Homogenate protein concentrations were determined using the Bradford assay.

RNA extraction and quantitative RT-PCR. RNA extraction and quantitative RT-PCR of samples were performed using previously described methods (6, 10, 13). Specific quantitative assays for 36B4, cyclophilin, and MCD were designed from the rat sequences available in GenBank. Primers and probes (shown in Table 1) were designed around specific splice junctions, preventing the recognition of any contaminating genomic DNA. 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 a 5-log range of RNA for all assays (data not shown). The level of transcripts for the constitutive housekeeping gene products 36B4 and cyclophilin were quantitatively measured in each sample to control for sample-to-sample differences in RNA concentration. Expression is reported as either the number of MCD transcripts per number of 36B4 molecules or the number of MCD transcripts per number of cyclophilin molecules.

                              
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Table 1.   Primer and probe sequences used for cyclophilin and MCD quantitative PCR

Statistical analysis. Data are presented as means ± SE. Statistically significant differences between groups were calculated by the Student's t-test. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Higher MCD expression and activity in heart vs. skeletal muscle. Figure 1 shows the differential expression and activity of MCD among the heart, EDL, and soleus muscles. When normalized to the housekeeping gene encoding for ribosomal 36B4 (whose level of expression, when normalized to total RNA content, is the same among the heart, EDL, and soleus muscles; data not shown), the level of MCD expression is 2.1- and 2.7-fold greater in the heart compared with EDL and soleus muscles, respectively (Fig. 1A). When normalized to the housekeeping gene encoding for cyclophilin, the level of MCD expression is similar among the three muscles (Fig. 1B), due to differential expression of cyclophilin among the heart, EDL, and soleus (data not shown). However, unlike 36B4, the expression of cyclophilin does not change upon any of the interventions within this study (data not shown). Thus the remaining MCD expression data are normalized to cyclophilin. The maximal activity of MCD measured in the heart was 9.0- and 4.6-fold greater compared with the activity in EDL and soleus muscles, respectively (Fig. 1C).


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Fig. 1.   Comparison of malonyl-CoA decarboxylase (MCD) expression (A and B) and activity (C) between heart, extensor digitorum longus (EDL), and soleus muscle. Values shown are means ± SE for 6 separate observations. MCD expression values are normalized either against 36B4 (A) or cyclophilin (B). Units for enzyme activity are nmol · min-1 · mg protein-1.

High-fat feeding increases plasma NEFA levels and alters cardiac and skeletal muscle MCD expression and activity. The time-dependent effects of increased dietary fatty acids on cardiac and skeletal (EDL and soleus) muscle MCD mRNA levels, MCD activity, and plasma NEFA levels were investigated. Feeding on the LC/HF diet caused a rapid elevation in plasma NEFA levels (within 1 day), an effect that was sustained for the duration of the experiment (Fig. 2A). LC/HF feeding significantly increased MCD mRNA in heart, EDL, and soleus muscle within 1 day (Fig. 2B). The sensitivity of MCD mRNA induction was soleus > EDL > heart. EDL and soleus muscle MCD mRNA remained significantly elevated throughout the time course of the experiment. Although heart MCD mRNA was elevated 4 and 8 days after the initiation of high-fat feeding, this increase was not statistically significant at these time points. The effects of high-fat feeding on MCD activity are shown in Table 2. In contrast, feeding a LC/HF diet decreased MCD activity in the heart by 17%, increased the activity in the soleus muscle by 30%, and had no effect on the EDL muscle.


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Fig. 2.   Time course for altered plasma nonesterified fatty acid (NEFA) levels (A), as well as for heart, EDL, and soleus muscle MCD expression (B), during feeding on the low-carbohydrate/high-fat (LC/HF) diet. Values shown are means ± SE for 5 separate observations. All MCD expression values are normalized against the housekeeping gene cyclophilin. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control (day 0).


                              
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Table 2.   Effects of high-fat feeding on heart, EDL, and soleus muscle MCD activity

Fasting and refeeding alter plasma NEFA levels and cardiac and skeletal muscle MCD expression and activity. Fasting significantly increased plasma NEFA levels within 24 h (Fig. 3A). Although plasma NEFA levels were still significantly elevated after 2 days of fasting (compared with basal levels), they were markedly reduced compared with the 24-h time point. Refeeding on the HC/LF diet lowered plasma NEFA levels to basal values. Fasting also significantly increased cardiac and skeletal muscle MCD mRNA at both time points investigated (Fig. 3B). The sensitivity of MCD mRNA induction was soleus = EDL > heart. Refeeding returned MCD mRNA to near-basal levels in all three muscles. In contrast, fasting had no significant effects on MCD activity in any of the muscles investigated (Table 3). Refeeding on the HC/LF diet significantly decreased cardiac, but not EDL or soleus, muscle MCD activity (Table 3).


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Fig. 3.   Effects of fasting and refeeding on plasma NEFA levels (A), as well as on heart, EDL, and soleus muscle MCD expression (B). Animals were refed the high-carbohydrate/low-fat (HC/LF) diet. Values shown are means ± SE for 4 or 5 separate observations. All MCD expression values are normalized against the housekeeping gene cyclophilin. **P < 0.01 and ***P < 0.001 vs. control (day 0).


                              
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Table 3.   Effects of fasting and refeeding on heart, EDL, and soleus muscle MCD activity

STZ-induced diabetes alters plasma NEFA levels and cardiac and skeletal muscle MCD expression and activity. STZ induction of diabetes significantly increased plasma glucose (3.18-fold; P < 0.001) and NEFA (3.7-fold; Fig. 4A) levels. Induction of diabetes increased MCD mRNA levels in both cardiac and skeletal muscle, particularly in the soleus (Fig. 4B). Diabetes was also associated with increased MCD activity in both EDL and soleus muscles (Table 4). No change in cardiac MCD activity was observed 2 wk after diabetes induction (Table 4).


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Fig. 4.   Effects of streptozotocin (STZ)-induced diabetes on plasma NEFA levels (A), as well as on heart, EDL, and soleus muscle MCD expression (B). Values shown are means ± SE for 6 separate observations. All MCD expression values are normalized against the housekeeping gene, cyclophilin. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.


                              
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Table 4.   Effects of STZ-induced diabetes on heart, EDL, and soleus muscle MCD activity

PPARalpha , but not PPARgamma , activation increases the mRNA and enzymatic activity of MCD in cardiac and skeletal muscle. To investigate further the mechanism by which the expression and activity of cardiac and skeletal muscle MCD are regulated, rats were treated either with the specific PPARalpha agonist Wy-14643 or the specific PPARgamma agonist troglitazone. Wy-14643 increased the expression of MCD in both cardiac and skeletal muscle (Fig. 5). The sensitivity of MCD mRNA induction was the same as that seen when plasma fatty acid levels were increased (soleus > EDL > heart; Fig. 5). In addition, Wy-14643 increased the activity of MCD in both the heart and soleus but not in the EDL muscle (Table 5). In contrast, troglitazone decreased MCD mRNA in both cardiac and skeletal muscles (Fig. 6). Troglitazone had no effect on MCD activity in any of the muscles investigated (Table 6).


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Fig. 5.   Effects of specific peroxisome proliferator-activated receptor-alpha (PPARalpha ) activation on heart, EDL, and soleus muscle MCD expression. Animals were fed either control food or powdered food containing Wy-14643 (0.01% wt/wt). Values shown are means ± SE for 6 separate observations. All MCD expression values are normalized against the housekeeping gene, cyclophilin. ***P < 0.001 vs. control.


                              
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Table 5.   Effects of specific PPARalpha activation on heart, EDL, and soleus muscle MCD activity



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Fig. 6.   Effects of specific PPARgamma activation on heart, EDL, and soleus muscle MCD expression. Animals were fed either control food or powdered food containing troglitazone (0.1% wt/wt). Values shown are means ± SE for 5 separate observations. All MCD expression values are normalized against the housekeeping gene, cyclophilin. *P < 0.05 and **P < 0.01 vs. control.


                              
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Table 6.   Effects of specific PPARgamma activation on heart, EDL, and soleus muscle MCD activity

Pressure overload-induced alterations in cardiac MCD expression and activity: dependence on fatty acids. We investigated whether pressure overload-induced cardiac hypertrophy affected the expression of MCD. Aortic constriction resulted in cardiac hypertrophy, as indicated by an increase in the heart weight-to-body weight ratio (4.02 ± 0.09 vs. 3.13 ± 0.06 g/kg for experimental and control groups; P < 0.001). Cardiac hypertrophy caused a significant decrease in the levels of both MCD mRNA and activity (Fig. 7A and Table 7, respectively).


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Fig. 7.   Effects of pressure overload-induced hypertrophy on cardiac MCD expression. Rats were fed 1 of 3 diets: standard laboratory chow (normal; A), HC/LF (B), or LC/HF (C) diets. Duration of diet feeding was a total of 16 days (7 days before and 9 days after surgery). Values shown are means ± SE for between 5 and 13 separate observations. All MCD expression values are normalized against the housekeeping gene, cyclophilin. *P < 0.05 and **P < 0.01 vs. HC/LF control. $$P < 0.01 vs. LC/HF control.


                              
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Table 7.   Effects of pressure overload on heart MCD activity

In a separate experiment, we investigated whether fatty acids play a role in hypertrophy-associated changes in MCD expression and activity by placing rats on diets with differing fatty acid contents. After 7 days on the special diets (either HC/LF or LC/HF), one-half of the rats underwent aortic banding, whereas the other one-half were sham operated. Rats were maintained on their special diets for 9 days after surgery, at which time the hearts were isolated. Aortic constriction caused similar increases in the heart weight-to-body weight ratio whether rats were fed the HC/LF diet (3.91 ± 0.24 vs. 3.16 ± 0.08 g/kg for experimental and control groups; P < 0.05) or the LC/HF diet (3.90 ± 0.13 vs. 3.17 ± 0.07 g/kg for experimental and control groups; P < 0.05). Hearts isolated from sham-operated rats fed the LC/HF diet compared with sham-operated rats fed the HC/LF diet possessed significantly higher levels of MCD expression (Fig. 7B). As observed during standard laboratory chow feeding, banding significantly reduced the MCD mRNA when rats were fed the LC/HF diet. In contrast, pressure overload did not decrease cardiac MCD expression for rats fed the HC/LF diet (Fig. 7B). Similar results were observed for MCD activity, in which aortic banding significantly decreased heart MCD activity only for rats fed the LC/HF diet (Table 7).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that fatty acids regulate the expression and activity of cardiac and skeletal muscle MCD. High-fat feeding, fasting, and STZ-induced diabetes all increased MCD mRNA in cardiac and skeletal muscle, with concomitant elevations in plasma fatty acid levels. However, changes in MCD mRNA were not always paralleled by MCD activity. The specific PPARalpha agonist Wy-14643 increased cardiac and skeletal muscle MCD mRNA as well as activity. PPARgamma activation did not induce MCD. The sensitivity of MCD mRNA induction by fatty acids (and Wy-14643) was soleus > EDL > heart. In addition, cardiac hypertrophy, which is associated with decreased PPARalpha expression (3) and decreased fatty acid oxidation (2, 21), results in decreased MCD expression and activity, an effect which is dependent on fatty acids. These results suggest that MCD is regulated both transcriptionally and posttranscriptionally. In the former case, this study suggests that fatty acids induce MCD gene expression. Whether this is due to PPARalpha activation requires further investigation.

Malonyl-CoA is a key regulator of fatty acid oxidation in both the heart and skeletal muscle by inhibiting CPT-1 and the entry of long-chain fatty acyl-CoAs (LCFAs) into the mitochondrial matrix (27). Because malonyl-CoA is synthesized by ACC, much work has focused on the mechanisms of regulation of ACC in both cardiac and skeletal muscle. ACCbeta , the major isoform in muscle, is regulated both allosterically (by citrate and LCFAs) and through covalent modification (reversible phosphorylation by AMP-activated protein kinase) (19, 32). MCD, the enzyme which degrades malonyl-CoA, has only recently been investigated. Initial studies suggest that MCD is regulated at the levels of both expression and covalent modification (14, 37), although the exact nature of these regulatory mechanisms is unknown.

An inverse correlation often, but not always, exists between intracellular malonyl-CoA levels and rates of fatty acid oxidation in both heart and skeletal muscle (8, 14, 15, 28, 29, 34). For example, in skeletal muscle, the levels of malonyl-CoA decrease during fasting, whereas the rate of fatty acid oxidation increases (44). This decreased malonyl-CoA level cannot be accounted for by assayable changes in ACC activity (44). This observation could be explained through alterations in the intracellular concentrations of the substrate and/or allosteric effectors for ACC (32) and/or alterations in the activity of MCD. We report here that fasting increases the expression of skeletal muscle MCD more than fourfold (Fig. 3B). Although assayable MCD activity did not significantly change, there was a trend for increased activity in both EDL and soleus muscles (Table 3). In addition to fasting, diabetes and high-fat feeding also increase plasma fatty acid levels (Figs. 2A, 3A, and 4A). All three conditions are associated with increased muscle MCD mRNA, suggesting that fatty acids may be involved in the regulation of MCD gene expression. It could be hypothesized that the mechanism by which fatty acids potentially induce MCD is mediated by PPARalpha , because specific PPARalpha activation led to increased skeletal muscle (and heart) MCD expression (as well as activity; Fig. 5 and Table 5). In contrast, specific PPARgamma activation decreased muscle MCD expression. This might be due to the lipid-lowering properties of troglitazone, thus lowering the stimulus for MCD expression.

The heart, like skeletal muscle, is able to adapt to alterations in its environment. For example, with pressure overload, the heart undergoes coordinated morphological and genomic changes, including reexpression of the fetal gene program and the development of myocyte hypertrophy (10, 35, 45). In metabolic terms, the hypertrophied heart decreases fatty acid utilization and increases its reliance on glucose as a fuel (2, 21). The mechanisms involved in this substrate switching are complex. As already mentioned, one important transcriptional regulator of this phenomenon appears to be PPARalpha . PPARalpha expression decreases with hypertrophy, as do several PPARalpha -regulated genes that are known to be involved in fatty acid metabolism (e.g., mCPT-1 and medium- and long-chain acyl-CoA dehydrogenase) (3, 10, 33). The present study has found that pressure overload-induced hypertrophy results in decreased MCD expression and activity (Fig. 7A and Table 7). Furthermore, when animals are fed a low-fat (HC/LF) diet, during which time the availability of the ligand for PPARalpha (namely fatty acids) will be reduced, the decrease in MCD expression with hypertrophy is prevented (Fig. 7B). Therefore, hypertrophy-induced changes in MCD expression are dependent on fatty acids. This is further evidence in support of the hypothesis that PPARalpha regulates the expression of MCD. From these results, we speculate that decreased MCD expression with pressure overload may play a role in decreased fatty acid oxidation by the hypertrophied heart (through increased inhibition of mCPT-1 by malonyl-CoA).

As mentioned above, both the heart and skeletal muscle are able to adapt to changes in its environment. The heart adapts to changes in both workload and substrate availability, resulting in switching of substrate preference, enabling constant ATP generation and therefore maintenance of contractile performance. For example, during insulin-dependent diabetes, the heart adapts to increased fatty acid utilization in the presence of both increased fatty acid availability and decreased glucose utilization (40). In contrast, not only does skeletal muscle require adaptation for the maintenance of ATP generation in different activity states, but, because of its sheer mass, skeletal muscle is important in whole body fuel homeostasis. It is essential for skeletal muscle to respond to alterations in plasma fuel levels, either directly (e.g., fatty acids through PPARalpha ) or indirectly (e.g., glucose through insulin), thereby preventing detrimental accumulation of intermediates, as observed during various pathologies, especially insulin resistance (9). These differences in the roles of substrate sensing by heart and skeletal muscle are exemplified by the present study. The sensitivity of MCD induction by either fatty acids or specific PPARalpha activation (by Wy-14643) was soleus > EDL > heart. The skeletal muscle therefore responded to increased fatty acid availability much more dramatically than did the heart, presumably to prevent the detrimental accumulation of fatty acids. Furthermore, the highly insulin-sensitive, oxidative soleus muscle was the most sensitive to MCD induction at the level of both transcription and activity. Because fatty acids are aerobically metabolized through beta -oxidation in the mitochondrial matrix, it is not surprising that this muscle induced MCD to the greatest extent.

Accumulation of intracellular fatty acid (and lipid) levels is associated with various pathologies, including insulin resistance (31, 32), beta -cell dysfunction (38, 42), and cardiotoxicity (46). Although the precise mechanisms by which fatty acid accumulation is detrimental are not known, several possibilities exist, including initiation of apoptosis (38) and chronic stimulation of PKC (25). A link has been proposed between intracellular malonyl-CoA levels and insulin resistance, as recently reviewed by Ruderman et al. (32). Increased malonyl-CoA levels, through either increased synthesis or decreased degradation, would inhibit fatty acid oxidation, resulting in increased intracellular LCFA levels and insulin resistance. One possibility is that impaired induction of MCD in response to fatty acids would result in intracellular LCFA accumulation. Whether impaired MCD induction occurs in insulin-resistant skeletal muscle requires elucidation. In support of this hypothesis is the finding that cardiac muscle isolated from the insulin-resistant obese Zucker rat possesses decreased PPARalpha expression (46).

The present results not only provide evidence for transcriptional control of MCD but also suggest that posttranscriptional mechanisms operate to regulate MCD activity. For example, high-fat feeding resulted in increased MCD activity only in the soleus muscle, despite the observation that MCD mRNA increased in all muscles investigated. High-fat feeding actually decreased cardiac MCD activity. Furthermore, fasting dramatically increased MCD mRNA levels in all muscles but had only modest effects on skeletal muscle MCD activity. These results clearly suggest that posttranscriptional mechanisms also operate to regulate MCD activity. From the present results, it could be hypothesized that fatty acids, in addition to MCD induction, cause inhibition of MCD activity. Possible mechanisms of posttranscriptional regulation might include alternative splicing, translational control, covalent modification, or the presence of an unidentified MCD-inhibitory protein. In support of the hypothesis that an MCD binding protein exists, a 40-KDa protein has been found that co-purifies with dog heart MCD through seven separate purification steps (Goodwin GW, unpublished observations). Recent studies in both the heart and skeletal muscle have shown that increased contractility is associated with increased MCD activity through covalent modification (14, 36). This alteration in MCD activity appears to be due to phosphorylation (36).

The present study has investigated whether alterations in substrate availability or cardiac workload are associated with changes in heart and skeletal muscle MCD expression and activity in vivo. When plasma fatty acid levels were elevated through high-fat feeding, fasting, and diabetes, MCD mRNA levels increased in both cardiac and skeletal muscle. However, these situations are associated with changes not only in plasma fatty acid levels, but also in levels of other fuels (such as glucose) and hormones, which may potentially play a role in the induction of MCD. Furthermore, the present results can only speculate as to the involvement of PPARalpha in the regulation of MCD expression. Further experiments will be required to investigate in more depth the role of this nuclear receptor in MCD induction. Although the present study has investigated both the expression and the activity of MCD in several in vivo models, no measurements of ACC (or AMP kinase) expression or activity have been made, nor have intracellular levels of malonyl-CoA or fatty acid oxidation rates been determined. Therefore, it can only be speculated whether alterations in MCD activity, as measured in the present study, result in expected changes in malonyl-CoA levels and fatty acid oxidation in these same samples. However, several previously published studies have focused on malonyl-CoA levels and/or fatty acid oxidation rates during similar conditions (e.g., fasting, diabetes, hypertrophy) (22, 37, 44). The present results complement these previously published studies. Furthermore, interpretation of whole cell malonyl-CoA level measurements can be clouded by the likely compartmentation of malonyl-CoA within the cell (18).

In conclusion, elevation of plasma fatty acids was associated with increased expression of MCD in both heart and skeletal muscle. We provide evidence in support of the hypothesis that fatty acid-induced MCD expression is mediated through PPARalpha , because specific PPARalpha activation stimulates MCD expression in a manner similar to that seen by fatty acids. Thus MCD might be part of the feed forward mechanism, in which fatty acids stimulate their utilization by muscle. Whether this feed forward mechanism is impaired in insulin-resistant muscle is presently unknown. In addition to altered substrate availability, pressure overload-induced cardiac hypertrophy (which is associated with decreased PPARalpha expression and decreased fatty acid utilization) results in decreased MCD expression and activity.


    ACKNOWLEDGEMENTS

The troglitazone was a kind gift from Parke, Davis, Morris Plains, NJ.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants HL-43133 and HL-61483 and by grants from the American Heart Association, National Center and Texas Affiliate.

Address for reprint requests and other correspondence: H. Taegtmeyer, Dept. of Internal Medicine, Div. of Cardiology, Univ. of Texas-Houston Medical School, 6431 Fannin, MSB 1.246, Houston, TX 77030 (Email Heinrich.Taegtmeyer{at}uth.tmc.edu).

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.

Received 11 May 2000; accepted in final form 8 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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