Original Article Plasma Free Fatty Acids and Peroxisome Proliferator–Activated Receptor  in the Control of Myocardial Uncoupling Protein Levels Andrew J. Murray, 1 Marcello Panagia, 1 David Hauton, 2 Geoffrey F. Gibbons, 2 and Kieran Clarke 1 Diabetic patients have abnormal cardiac energy metabo- lism associated with high plasma free fatty acid (FFA) concentrations. We investigated whether high plasma FFAs increase mitochondrial uncoupling protein (UCP) levels in the mouse heart by activating the nuclear transcription factor peroxisome proliferator–activated receptor (PPAR). We used Western blotting to measure UCP protein levels in isolated cardiac mitochondria from PPAR / and diabetic mice. Cardiac UCP2 and UCP3 were significantly lower in the PPAR / mouse than in the wild type. Treatment with the PPAR-specific agonist, WY-14,643, increased cardiac UCP2 and UCP3 levels in wild-type mice but did not alter UCP levels in PPAR / mice. Inhibition of -oxidation with etomoxir increased cardiac UCP2 and UCP3 levels in wild-type mice and UCP2 levels in PPAR / mice but did not alter UCP3 levels in PPAR / mice. Streptozotocin treatment, which increased circulating FFAs by 91%, did not alter cardiac UCP2 levels in wild-type or PPAR / mice but increased UCP3 levels in wild-type, and not in PPAR / , mice. The diabetic db/db mouse had 50% higher plasma FFA concentrations and elevated cardiac UCP2 and UCP3 protein levels. We conclude that high plasma FFAs activated PPAR to increase cardiac UCP3 levels, but cardiac UCP2 levels changed via PPAR-dependent and -independent mechanisms. Diabetes 54:3496 –3502, 2005 T he phosphocreatine (PCr)-to-ATP ratio, an index of myocardial energetic status, has been shown to correlate negatively with plasma free fatty acid (FFA) concentrations in patients with type 2 diabetes (1), but the cellular link between energy metab- olism and circulating FFA concentrations has yet to be defined. Conditions that increase plasma FFA levels, such as high-fat feeding, fasting, and streptozotocin (STZ)- induced diabetes, increase cardiac and skeletal muscle mitochondrial uncoupling protein (UCP)3 levels in the rat (2–5). A positive correlation between circulating FFA concentrations and both UCP2 and UCP3 protein levels occurs in human heart (6), suggesting that plasma FFAs may control UCP levels in the heart. The UCPs are believed to dissipate the proton electrochemical gradient by allowing protons to reenter the mitochondrial matrix without the concomitant synthesis of ATP (7). Long-chain FFAs are natural ligands for the peroxisome proliferator–activated receptors (PPARs) (8 –10), and the UCP genes have PPAR response elements in their pro- moter regions (11,12). Thus, plasma FFAs control cardiac UCP levels via PPAR activation. The PPAR link has been shown by decreased UCP3 mRNA levels in the PPAR -/- mouse heart and increased UCP3 mRNA levels in rat heart after treatment with the specific PPAR agonist, WY- 14,643 (5). Most studies of changes in cardiac UCPs, associated with alterations in circulating metabolite con- centrations, have reported UCP mRNA levels (5,10,13,14), yet UCP mRNA and protein do not necessarily change in parallel (15,16). Studies in brown adipose tissue (15,16) and gastrocne- mius muscle (16) have demonstrated that dramatic changes in levels of UCP2 or UCP3 transcripts were translated to much smaller changes, or no significant changes, at the protein level. Dietary conjugated linoleic acid was found to increase skeletal muscle UCP3 protein in mice, despite unchanged UCP3 mRNA levels (17). These findings indicate complex posttranslational control of UCP levels and expose a limitation in the conclusions drawn from studies that have demonstrated changes only in UCP mRNA. Furthermore, changes in mRNA are not necessar- ily concordant between studies; for example, treatment with WY-14,643 increased UCP2 mRNA in rat cardiomyo- cytes (9) but not in rat heart (5). To draw conclusions about UCP function, it is important to use protein level measurements. In this study, we have investigated the links between plasma FFA concentrations, PPAR, and UCPs by measur- ing cardiac UCP2 and UCP3 protein levels in mutant PPAR -/- mice and two mouse models of diabetes known to have high plasma FFA levels, the STZ-treated mouse and the diabetic (db/db) mouse. Thus, we have determined how plasma FFAs, which have been associated with changes in UCP mRNA levels, affect cardiac UCP2 and UCP3 protein levels in vivo, thereby highlighting post- translational control of UCP levels. Part of this work has been published in abstract form (18). From the 1 University Laboratory of Physiology, University of Oxford, Oxford, U.K.; and the 2 Metabolic Research Laboratory, Oxford Centre for Diabetes, Endocrinology and Metabolism, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, U.K. Address correspondence and reprint requests to Professor Kieran Clarke, University Laboratory of Physiology, University of Oxford, Parks Road, Oxford, OX1 3PT, U.K. E-mail: kieran.clarke@physiol.ox.ac.uk. Received for publication 25 April 2005 and accepted in revised form 9 September 2005. FFA, free fatty acid; PCr, phosphocreatine; PPAR, peroxisome proliferator– activated receptor; STZ, streptozotocin; UCP, uncoupling protein. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3496 DIABETES, VOL. 54, DECEMBER 2005