Uncoupling protein 3 as a mitochondrial fatty acid anion exporter

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Abstract
SPECIFIC AIMS There is increasing evidence that uncoupling protein 3, in contrast to brown adipose tissue-specific UCP1, is not playing a major role in energy dissipation but rather is involved in fatty acid metabolism. We and others have recently postulated the hypothesis that UCP3 is involved in the export of nonesterified fatty acid anions, which cannot be oxidized, from the mitochondrial matrix to prevent accumulation of these fatty acid anions inside the matrix. Here, we test the feasibility of this hypothesis by systematically interfering at distinct steps in the pathway of fatty acid metabolism, thereby creating conditions in which the entry of (nonesterified) fatty acids into the mitochondrial matrix is enhanced and UCP3 is anticipated to be induced. PRINCIPAL FINDINGS 1. H-FABP null mice are characterized by increased UCP3 protein content UCP3 protein levels in skeletal gastrocnemius muscle of H-FABP null mice were increased 5.3-fold compared with their wild-type littermates (529±99 vs. 100±38 arbitrary units in H-FABP null (n=5) and wild-type littermates (n=7) respectively, mean ± se, P < 0.05, Fig. 1 ⤻ A). Figure 1. The effect of A) absence of H-FABP, B) administration of etomoxir, C) feeding of a high-fat diet rich in long-chain fatty acids, and D) feeding of a high-fat diet rich in medium-chain fatty acids on UCP3 protein content. Control values are normalized to 100 arbitrary units. UCP3 is systematically up-regulated when the entry of long-chain fatty acids into the mitochondrial matrix is enhanced. *P < 0.05 compared with control. Download figure Download PowerPoint 2. Etomoxir reduces skeletal muscle CPT1 activity and increases UCP3 protein content Etomoxir significantly decreased CPT1 activity by 58% in the etomoxir group, indicating effective blockade of mitochondrial uptake of long-chain fatty acyl-CoA esters (8.8±2.1 vs. 3.3±2.2 mU/g wet mass in control and etomoxir, respectively, n=6, Pn=6, PB⤻ ). 3. High-fat feeding induces skeletal muscle UCP3 protein content We fed Male Wistar rats either a control, low-fat diet (7 en% as fat) or a high-fat diet entirely composed of long-chain triacylglycerol (46 en% as fat of which 79% C16:0 and 20% C18:0) for 2 wk. The high-fat diet resulted in an up-regulation of UCP3 protein content by twofold (200±35 vs. 100±21 arbitrary units in high-fat LCT vs. low-fat, respectively, n=10, PC⤻ ). 4. High-fat diets consisting of medium-chain fatty acids do not induce UCP3 protein content A unique property of medium-chain fatty acids is that they do not need to be esterified in the cytoplasm for subsequent oxidation. We fed male Wistar rats a high-fat diet entirely composed of medium-chain triacylglycerol (46 en% as fat of which 60% C8 and 40% C10) for 2 wk. The up-regulation of UCP3 as observed on a high-fat long-chain fatty acid diet, was completely absent when the high-fat diet was provided in the form of medium-chain fatty acids (85±17 vs. 100±21 arbitrary units in high-fat MCT vs. low-fat, respectively, n=10, P>0.05, Fig. 1D⤻ ). The lack of up-regulation of UCP3 in the medium-chain group vs. the long-chain group could not be explained by differences in plasma free fatty acid concentrations (200±19, 249±34, and 273±26 μmol/L in low-fat, high-fat MCT and high-fat LCT, respectively, n=10, P>0.05). CONCLUSIONS AND SIGNIFICANCE Although the physiological function of UCP3 is still under debate, its function is more closely related to fatty acid metabolism compared with energy dissipation. Based on the available literature on UCP3 regulation, we have postulated the hypothesis that UCP3 acts as an outward transporter of long-chain fatty acid anions from the mitochondrial matrix into the soluble cytoplasm (Fig. 2 ⤻ ). Thus, in situations where the fatty acid delivery to mitochondria exceeds the oxidative capacity, long-chain fatty acids will accumulate inside the myocyte. These long-chain fatty acids are able to enter the mitochondrial matrix by a so-called flip-flop mechanism, and due to the higher pH inside the mitochondrial matrix, part of these long-chain fatty acids will be deprotonated at the matrix side. In this way, fatty acid anions can reach the mitochondrial matrix. It is important to note that, once inside the matrix, these long-chain fatty acid anions can neither be diverted to β-oxidation (due to lack of ACS inside the matrix) nor cross the inner mitochondrial membrane, and are thus trapped inside the mitochondria (Fig. 2)⤻ . Here, they can have deleterious effects on mitochondrial function, for example due to their amphiphilic nature and proneness to peroxidation. Here, UCP3 could become involved in facilitating outward transport of these fatty acid anions, since it has been shown that UCP3 is able to transport fatty acid anions. We tested this hypothesis by creating situations in which the entry of specifically nonesterified long-chain fatty acids is most likely enhanced and examined the effect on UCP3 induction. Figure 2. Schematic model of the putative function of UCP3. UCP3 protein content is increased in conditions in which the entry of long-chain fatty acids into the mitochondrial matrix is enhanced: 1) in H-FABP null mice, which are characterized by enhanced unbound cytoplasmic fatty acid concentrations and decreased capacity to activate fatty acids via ACS, 2) after inhibition of the carnitine shuttle system using etomoxir, and 3) on a high-fat diet, associated with increased supply of fatty acids to the mitochondria. Note that a high-fat diet rich in medium-chain fatty acids does not increase UCP3 protein content. Medium-chain fatty acids can enter the mitochondrial matrix in their nonactivated form, but can still be oxidized due to the presence of a medium-chain acyl-CoA synthetase inside the matrix. In the latter situation, UCP3 would not be needed for export of fatty acids. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; FA, fatty acid; TCA, tricarboxylic acid cycle. Download figure Download PowerPoint After being taken up into the muscle cell, the majority of fatty acids inside the soluble cytoplasm are bound to heart-type fatty-acid binding protein (H-FABP or FABPc). The water solubility of fatty acids (4–6 μM) exceeds the affinity of H-FABP for fatty acids (4–14 nM) by far, indicating an important role for H-FABP in controlling the intracellular unbound fatty acid concentration, in order to maintain a fatty acid gradient between plasma/interstitium and cytoplasm to facilitate fatty acid uptake. Lack of H-FABP would thus lead to an intracellular increase of unbound fatty acids, particular because plasma fatty acid levels are elevated in these mice. In addition, H-FABP null mice have impaired mitochondrial oxidation of fatty acids, possibly because H-FABP is essential for activation of fatty acids by interacting with long-chain acyl-CoA synthethase (ACS). Unbound fatty acids rapidly incorporate into (mitochondrial) membranes and can thus enter the mitochondrial matrix in their nonesterified form, where they will be deprotonated and cannot be oxidized. The pronounced up-regulation of UCP3 in H-FABP null mice is consistent with our hypothesis and could well serve to protect mitochondria against accumulation of nonesterified fatty acid anions inside the matrix. Another way to increase the entry of (nonesterified) fatty acids into the mitochondrial matrix is by blocking the main route for mitochondrial transport of long-chain fatty acyl-CoA esters. It is anticipated that by doing so nonesterified fatty acids will accumulate in the cytoplasm, which could then enter the mitochondrial matrix. The observed up-regulation of UCP3 upon inhibition of CPT1 activity, again is in accordance with a role of UCP3 in fatty acid anion export. Furthermore, it further dissociates the physiological role of UCP3 from energy dissipation and/or stimulation of fatty acid oxidation, since the latter two are decreased upon blocking the mitochondrial entry of long-chain fatty acids. With high-fat feeding, the continuous delivery of fatty acids exceeds the oxidative capacity and not all fatty acids can be oxidized, resulting in a transient accumulation of fatty acids inside the muscle cell. Under such conditions, the entry of nonesterified fatty acids into the mitochondrial matrix will increase and the observed high-fat induced up-regulation of UCP3 may serve to facilitate outward transport of these fatty acids from the matrix. In contrast to long-chain fatty acids, medium-chain fatty acids do not need to be esterified to CoA esters in the cytoplasm. Medium-chain fatty acids bypass the control of mitochondrial transport by CPT1 and, in contrast to long-chain fatty acids, medium-chain fatty acids can be esterified to their respective CoA esters inside the mitochondrial matrix and can therefore still be diverted to β-oxidation. On a high-fat diet consisting of medium-chain fatty acids, there would be no need to up-regulate UCP3, as the majority of fatty acids that enter the matrix will be of medium-chain length and thus can still be diverted to β-oxidation once inside the matrix. The lack of up-regulation of UCP3 upon the medium-chain high-fat diet vs. the long-chain high-fat diet, despite similar plasma nonesterified fatty acid levels, therefore provide evidence for the hypothesis that UCP3 is specifically involved in the export of nonesterified long-chain fatty acid anions from the mitochondrial matrix into soluble cytoplasm. Moreover, upon entering the β-oxidation, the metabolism of fatty acids is independent of chain length, suggesting that the physiological function of UCP3 is not related to fatty acid metabolism distal from β-oxidation. Export of nonesterified fatty acids from the mitochondrial matrix might be of physiological importance to prevent mitochondrial damage, as nonesterified fatty acids are prone to lipid peroxidation. In this context, it is important to note that it was recently found that mice lacking UCP3 indeed have increased lipid peroxidation. Furthermore, UCP3 protein content is decreased in type 2 diabetic subjects, who were found to be more susceptible to mitochondrial DNA damage in skeletal muscle. Moreover, the decreased UCP3 protein content in diabetic patients together with the generally observed accumulation of lipids inside the myocytes of type 2 diabetics, might be an explanation for the impaired mitochondrial functioning in type 2 diabetes, that could lead to the development of skeletal muscle insulin resistance, although further studies are needed to test this concept. In addition, accumulation of fatty acids inside the mitochondrial matrix might lead to disturbances in fatty acid oxidation, explaining the reported aberrations in fat oxidation in humans with the exon 6 splice donor mutation and in UCP3 knockout mice. In summary, we have provided evidence that the physiological function of UCP3 is the outward transport of fatty acid anions. Interference in successive steps in fatty acid handling, transport and oxidation revealed that UCP3 is not directly involved in fatty acid oxidation. Rather, we showed that UCP3 protein increases if the supply of fatty acids to the mitochondria exceeds fat oxidative capacity, making it feasible that the physiological function of UCP3 indeed is in fatty acid anion export. Although we realize that none of the experiments presented provide direct and definitive proof for our hypothesis, we interpret these data as compelling circumstantial evidence for our hypothesis on the function of UCP3. Definitive proof for the hypothesis awaits the development of methodology to measure mitochondrial fatty acid anion accumulation and/or transport. 1To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0515fje; 3Present address: Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, Raymond Stotzer Pkwy, College Station TX, 77843-4467, USA.

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