Plasma Free Fatty Acids and Peroxisome Proliferator–Activated Receptor α in the Control of Myocardial Uncoupling Protein Levels

This investigation conformed with U.K. Home Office and National Institutes of Health guidelines on the care and use of laboratory animals. All mice were studied at 6–7 months of age.

The PPARα^−/− mouse was generated a little over 10 years ago by disruption of the ligand-binding domain of PPARα using homologous recombination (19) and has since become a much-used and invaluable tool for studying the role of the receptor in fatty acid metabolism (5,20,21). Mice homozygous for the mutation do not express PPARα protein and fail to display the peroxisome proliferator pleiotropic response when challenged with WY-14,643 (19). Here we used male wild-type SVEV129 and PPARα^−/− mice, which were a gift from Frank Gonzalez (National Cancer Institute, Bethesda, MD).

Wild-type and PPARα^−/− mice were treated with the PPARα-specific agonist, WY-14,643, and/or etomoxir, an inhibitor of β-oxidation that raises intracellular levels of intermediary fatty acid metabolites (22). Direct activation of PPARα with WY-14,643, or indirect stimulation by high intracellular fatty acid concentrations resulting from etomoxir treatment, were expected to increase UCP levels in wild-type, but not in PPARα^−/−, mouse hearts. Hence, PPARα^−/− mice and SVEV129 wild-type mice were fed for 7 days on one of four diets: 1) standard chow diet alone, 2) standard chow diet supplemented with WY-14,643 (0.1%), 3) etomoxir (0.02%), or 4) both WY-14,643 (0.1%) and etomoxir (0.02%).

When injected into rodents, STZ destroys pancreatic β-cells leading to a hypoinsulinemic state and high levels of circulating FFAs (23). Male wild-type SVEV129 and PPARα^−/− mice were given 210 mg/kg STZ (Sigma, St. Louis, MO) dissolved in 50 mmol/l citrate, pH 4.5. The STZ was administered in three daily intraperitoneal injections of 85 mg/kg, 70 mg/kg, and 55 mg/kg (24). Wild-type animals were injected with vehicle. All STZ-injected animals were assessed for the development of diabetes as described previously (25) and were killed 6 weeks after the initial injection.

Although diabetes in humans rarely occurs as a result of a defect in the leptin receptor, the db/db mouse, which has a single leptin receptor gene mutation, is a well-characterized model of diabetic cardiomyopathy, with metabolic changes similar to those in human type 2 diabetes (26). The db/db mouse has peripheral insulin resistance and increased pancreatic β-cell insulin secretion. Hyperglycemia eventually develops when enhanced insulin secretion can no longer compensate for peripheral and hepatic insulin resistance. Body weights of db/db mice increase progressively and plateau at 40–50 g at about 2 months of age, almost double the weight of wild-type mice (26), with greater lipolysis of endogenous adipose stores leading to increased levels of circulating FFAs (27). As diabetic patients have abnormally high levels of plasma FFAs (1,28), we measured plasma metabolites and cardiac UCP2 and UCP3 levels in the db/db mouse. Male C57BL/KsJ-lepr^db db/db mice and their nondiabetic (db/+) controls were obtained from Harlan U.K. (Bicester, U.K.).

For each experiment, whole mouse hearts (∼100 mg) were isolated, minced, washed, and suspended in 5 ml of isolation medium (0.3 mol/l sucrose, 10 mmol/l Na-HEPES, and 0.2 mmol/l EDTA, pH 7.2). The tissue was subjected to mild trypsin digestion (0.7 mg trypsin, 15 min, 4°C) and diluted with 5 ml of isolation medium containing 1 mg/ml BSA and 3.25 mg trypsin inhibitor. The suspension was stirred and the supernatant discarded. The partially digested tissue was resuspended in 10 ml isolation medium/BSA and homogenized with a handheld Teflon-glass homogenizer. The homogenate was centrifuged (10 min, 600g, 4°C). The supernatant solution was decanted and centrifuged (15 min, 8,000g, 4°C). The supernatant solution was discarded, and the pellet was twice resuspended in 10 ml isolation medium/BSA and centrifuged (15 min, 8,000g, 4°C), each time discarding the supernatant solution. The final, washed mitochondrial pellet was suspended in 1 ml of isolation medium/BSA. The mitochondrial solution was stored at −80°C for the preparation of protein lysates for UCP2 and UCP3 immunoblotting (29).

Levels of cardiac UCP2 and UCP3 were measured in mitochondrial preparations by immunoblotting, as described previously (29), using polyclonal rabbit anti-UCP2 and anti-UCP3 antibodies (Alpha Diagnostic, San Antonio, TX) and an anti-rabbit IgG peroxidase conjugate polyclonal antibody (Autogen Bioclear, Wiltshire, U.K.). Membranes were covered in enhanced chemiluminescence detection solution (Amersham, U.K.) and exposed to X-ray film for visualization of protein bands. Developed films were digitized using a scanner and quantified using Quantiscan 32 (Biosoft, U.K.).

Blood samples, taken from mice immediately after killing, were centrifuged and the supernatant frozen at −80°C for determination of glucose, cholesterol, triacylglycerol, β-hydroxybutyrate, and insulin levels or frozen with a final concentration of 30 μg/ml lipoprotein lipase inhibitor (tetrahydrolipstatin Xenical; Roche) for determination of FFA concentrations. Plasma FFAs were measured using the spectrophotometric NEFA C kit (Wako Chemicals, Neuss, Germany), and insulin levels were determined using an enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden). All other plasma metabolites were quantified using a spectrophotometric plasma analysis system (Monarch, Maumee, OH).

Data are presented as means ± SE. Statistical significance was assessed using two-way ANOVA followed by a modified t test (Instat v 3.05; GraphPad Software, San Diego, CA). Differences were considered significant at P < 0.05.

There were no significant differences in the fasting plasma lipid or insulin concentrations between chow-fed wild-type and PPARα^−/− mice (Table 1). Levels of myocardial UCP2 and UCP3 were 44 and 33% lower, respectively, in PPARα^−/− than in wild-type mice (P < 0.05) (Fig. 1).

In wild-type mice, feeding with the PPARα activator, WY-14,643, decreased plasma triacylglycerol levels by 32% (P < 0.05) and plasma insulin levels by 66% (P < 0.05) with no changes in plasma FFA levels (Table 1). Feeding the β-oxidation inhibitor etomoxir increased plasma FFA levels by 34% (P < 0.05) and triacylglycerol levels by 26% (P < 0.05). Treatment with both WY-14,643 and etomoxir decreased triacylglycerol levels by 42% (P < 0.01) and plasma insulin levels by 66% (P < 0.05). Levels of cholesterol were the same for all wild-type mice (Table 1). In the PPARα^−/− mice, treatment with WY-14,643, etomoxir, or WY-14,643 plus etomoxir resulted in no significant changes in levels of plasma FFAs, triacylglycerol, cholesterol, or insulin (Table 1).

Levels of myocardial UCP2 in wild-type mice increased by 42% with WY-14,643 treatment (P < 0.01), by 29% with etomoxir treatment (P < 0.05), and by 44% with both WY-14,643 and etomoxir (P < 0.001) (Fig. 1). Feeding PPARα^−/− mice with WY-14,643 did not alter cardiac UCP2 levels, although etomoxir increased UCP2 levels by 46% (P < 0.01), and the combination of WY-14,643 plus etomoxir increased UCP2 levels by 58% (P < 0.001). UCP2 levels in PPARα^−/− mouse hearts always remained lower than in untreated wild-type mice (Fig. 1).

Myocardial levels of UCP3 in wild-type mice increased by 54% with WY-14,643 (P < 0.05), by 126% with etomoxir (P < 0.001), and by 164% with the combined WY-14,643 plus etomoxir treatment (P < 0.01) (Fig. 1). UCP3 levels were not altered in PPARα^−/− mouse hearts by any treatment (Fig. 1).

Six weeks after STZ injections, plasma FFAs were increased by 91% in wild-type mice (P < 0.01) and by 196% in PPARα^−/− mice (P < 0.001), and plasma triacylglycerol levels were increased by 61% in the wild-type mice (P < 0.05) and by 71% in the PPARα^−/− mice (P < 0.05). Cholesterol levels were increased by 27% in both wild-type mice (P < 0.001) and the PPARα^−/− mice (P < 0.01). Levels of plasma glucose were increased by 99% in the wild-type mice (P < 0.01) and by 143% in the PPARα^−/− mice (P < 0.01). Insulin levels were 50% lower in the wild-type mice (P < 0.01) and 45% lower in the PPARα^−/− mice (P < 0.05) (Table 2). Cardiac UCP2 levels were not altered by STZ treatment in wild-type or in PPARα^−/− mice, but UCP3 levels increased by 50% in wild-type mouse hearts with no increase in UCP3 levels in the PPARα^−/− mice (Fig. 2). Cardiac levels of UCP3 correlated positively with plasma FFA levels in control and STZ-treated mice (r = 0.84, P < 0.01) (Fig. 4).

The db/db mice were 90% heavier than db/+ mice (P < 0.01), with no significant increase in heart weight (Table 3). The db/db mice had 58% higher plasma FFA, 59% higher triacylglycerol, 31% higher cholesterol, and 248% higher glucose concentrations than the db/+ mice. Plasma insulin levels were 310% higher in the db/db mice (P < 0.05) (Table 3). Cardiac levels of UCP2 and UCP3 were, respectively, 37 (P < 0.01) and 38% (P < 0.01) higher in db/db mice than in db/+ mice (Fig. 3). Cardiac UCP3 levels correlated positively with plasma FFA levels in db/db and db/+ mice (r = 0.66; P < 0.05) (Fig. 4).

This study represents a comprehensive analysis of changes in cardiac UCP protein levels occurring with abnormal metabolic conditions, specifically those associated with high plasma FFA concentrations. A number of studies have measured changes in UCP mRNA expression in the heart (5,13,14) and other tissues (2,3,30,31) in response to circulating metabolite changes. Often, however, changes in UCP mRNA are not accompanied by similar changes at the protein level (15–17). The aim of the current study was to demonstrate how cardiac UCPs are regulated at the protein level by changes in FFA concentrations through PPARα activation. This is an extensive investigation into control of cardiac UCP protein levels and its importance to our understanding of the physiological role of these intriguing proteins.

In human heart, levels of cardiac UCP2 and UCP3 protein correlated positively with levels of plasma FFAs (6), the natural ligands of PPARα. A link between FFAs, PPARα activation, and UCP mRNA levels has been suggested in mouse heart. Reduced dietary fatty acid availability decreased cardiac expression of both UCP2 and UCP3 (5), and cardiac overexpression of PPARα increased UCP2 and UCP3 mRNA levels twofold and fivefold, respectively (32).

In the PPARα^−/− mouse, hepatic fatty acid oxidation is inhibited due to decreased β-oxidation enzyme activity (21). Cardiac expression of β-oxidation enzymes, including medium-chain acyl-CoA dehydrogenase and carnitine palmitoyltransferase-2, are lower in PPARα^−/− mice (33). Levels of UCP3 mRNA were lower in the PPARα^−/− mouse heart, whereas UCP2 mRNA levels were unchanged (5). In contrast, we found that both UCP2 and UCP3 protein concentrations were lower, by 44 and 33%, respectively, in the PPARα^−/− mouse heart compared with the wild-type mouse. Thus, changes in mRNA and protein levels may be discordant, although PPARα exercises some control over protein levels of both UCPs in heart.

WY-14,643, a highly specific agonist for PPARα, promotes binding of PPARα and 9-cis-retinoic acid receptor heterodimers to PPAR response elements on DNA, upstream of target genes (8). Incubation of rat cardiomyocytes with fatty acids or WY-14,643 increased only UCP2 mRNA levels (9); however, feeding WY-14,643 increased only UCP3 mRNA levels in rat heart (5) and WY-14,643 incubation increased UCP3 mRNA levels in rat preadipocytes, with no change in UCP2 mRNA levels (30). Here, WY-14,643 feeding, alone or with etomoxir, increased both cardiac UCP2 and UCP3 protein levels in wild-type but not PPARα^−/− mice.

Etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase-1 originally developed for treating type 2 diabetes (34), has no acute cardiovascular effects in rats, as shown by unaltered heart rate and blood pressure (35). Etomoxir feeding increases cardiac lipid droplet number (36) and, due to systemic inhibition of β-oxidation, increases plasma FFA levels (22,37). Etomoxir increased UCP3 protein levels in mouse and human skeletal muscle (4,38), and was found to increase UCP3 mRNA levels in rat preadipocytes, with no change in UCP2 mRNA (39). We found that etomoxir feeding, with or without WY-14,643, increased protein levels of UCP2 and UCP3 in mouse heart, suggesting that increased circulating FFA concentrations can increase protein levels of both UCPs in wild-type mouse heart.

Furthermore, it would appear from this finding that β-oxidation is not required for UCP levels to increase. Should the UCPs uncouple electron transport from ATP synthesis in cardiac mitochondria, then the increase in UCPs with etomoxir treatment suggests that partial inhibition of fatty acid oxidation, using such inhibitors as etomoxir, trimetazidine, perhexiline, and ranolazine, may have negative effects if used to treat heart failure (40,41). Activation of PPARα, due to the build up of fatty acid intermediary metabolites, may increase cardiac UCP levels, potentially increasing the energy deficit associated with higher mortality in heart failure patients (42).

In PPARα^−/− mouse heart, neither WY-14,643 nor etomoxir feeding increased UCP3 levels, suggesting that fatty acid control of UCP3 occurred solely via PPARα. WY-14,643 did not alter, but etomoxir increased, cardiac UCP2 levels, which suggests a PPARα-independent mechanism, possibly triggered by high intracellular fatty acid levels. Increased hepatic UCP2 mRNA levels have been reported in PPARα^−/− mice treated with the modified fatty acid tetradecylthioacetic acid, further supporting the case for a fatty acid–dependent, PPARα-independent regulation of UCP2 (43). As PPARβ/δ may partially compensate for a lack of PPARα (20), and as PPARβ/δ is present in the heart (44), it is possible that cardiac UCP2 protein can be increased in PPARα^−/− mice via fatty acid activation of PPARβ/δ.

STZ-induced diabetes has been associated with increased UCP2 and UCP3 mRNA levels in rat skeletal muscle (3) and increased UCP3 mRNA in the rat heart (5) (13). Consequently, we measured UCP2 and UCP3 in the hearts of STZ-treated wild-type and PPARα^−/− mice to establish whether such changes occur at the protein level and whether they depend on PPARα.

Whereas cardiac UCP2 protein levels did not change after STZ treatment in wild-type or PPARα^−/− mice, cardiac UCP3 levels increased by 50% in wild-type but not in the PPARα^−/− mice, again suggesting that UCP3 is under the control of PPARα. These findings, though consistent with previous studies, are hard to reconcile with our hypothesis that high plasma FFAs can increase myocardial UCP2 levels, especially given the clear increase in UCP3 levels in the same mice. The explanation for a lack of an increase in UCP2 in hearts of STZ-treated mice is unknown. In contrast, STZ-induced diabetes was associated with increased mRNA levels of both UCP2 and UCP3 in rat skeletal muscle (3).

Our results suggest that UCP3 is more sensitive than UCP2 to PPARα control in heart (5). In contrast to the STZ-treated animals, however, diabetic db/db mice, which had higher plasma levels of FFAs, triacylglycerol, glucose, cholesterol, and insulin, had higher levels of both cardiac UCP2 and UCP3 levels than the db/+ mice. Against the background of a completely altered metabolic state, it is impossible to attribute the UCP changes to a single factor; however, other work suggests that increased levels of circulating FFAs increase UCP mRNA (14) and protein (6) levels in the heart.

In summary, our results are remarkably consistent in that high plasma FFA concentrations increased UCP3 levels through PPARα activation. This appears to be a very sensitive control mechanism, as we found significant positive correlations between cardiac UCP3 levels and plasma FFA concentrations, in both types of diabetic animals and their controls (Fig. 4). The mechanism for control of cardiac UCP2 levels appears to be far less straightforward with an alternative, PPARα-independent mechanism for upregulating cardiac UCP2 levels, and the link between plasma FFA levels and UCP2 expression being less sensitive than for UCP3, as exemplified by the STZ-treated mouse heart.

The precise role of the UCPs in the heart remains to be resolved, and the effect of the changes that we report here is, as yet, unknown. There is emerging evidence that UCP3 may function as a mitochondrial fatty acid anion transporter because, when upregulated, it is associated with a greater capacity for lipid utilization (31). There are conflicting reports on whether high UCP levels impair the mitochondrial proton electrochemical potential, with UCP2 (43) but perhaps not UCP3 (2,30,45) eliciting such an effect. Although an effect of increased UCP levels on cardiac energetics remains to be demonstrated, high plasma FFA concentrations are associated with low cardiac PCr-to-ATP ratios in diabetic patients (1), a decreased myocardial PCr-to-ATP ratio being a strong predictor of mortality in patients with dilated cardiomyopathy (42). The hyperthyroid rat heart, which has a decreased PCr-to-ATP ratio (46), has increased myocardial UCP2 and UCP3 levels, increased mitochondrial uncoupling, and reduced cardiac efficiency (29). Furthermore, PPARγ agonist treatment, which lowers plasma FFA levels, has a beneficial effect on cardiac energy metabolism in diabetic rodents (47,48).

It remains to be established whether changes in cardiac levels of UCP2 and/or UCP3, such as those reported here, would lead to altered mitochondrial respiration, or indeed to changes in cardiac function. Further studies correlating changes in UCP levels with functional effects will define the role of mitochondrial uncoupling in the heart. Similarly, whereas a strength of this study over previous investigations is the measurement of cardiac UCP protein levels, the study is somewhat limited by the lack of data on UCP mRNA. Such data may have permitted clarification as to whether the discrepancies between changes in UCP mRNA in other studies and protein in our own are due to posttranscriptional control mechanisms. The small amount of ventricular tissue available from a single mouse was sufficient only for preparation of mitochondrial protein for the Western blots. A larger animal model, in which UCP mRNA and protein levels could both be measured, alongside mitochondrial respiration, may allow links between UCP gene expression and cardiac energy metabolism to be determined.

Our results show that UCP2 and UCP3 protein levels are regulated in heart by different mechanisms. Levels of both proteins are sensitive to plasma fatty acid concentrations and appear to be controlled by PPARα. UCP2, however, may also be controlled by another, PPARα-independent mechanism, possibly via PPARβ/δ. We suggest that there are discrete roles for UCP2 and UCP3 in the heart, with more work required to fully define their physiological importance.

This study was supported by grants from the British Heart Foundation and the Medical Research Council.

We thank Frank Gonzalez (National Institutes of Health) for the gift of PPARα^−/− mice and Gillian Watson for plasma insulin and metabolite assays. A.J.M. thanks the British Heart Foundation, and M.P. thanks the Rhodes Trust for their DPhil studentships.