Uncoupling protein 3 and the protection of skeletal muscle mitochondrial...


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Title Uncoupling protein 3 and the protection of skeletal muscle mitochondrial function; a transgenic approach
Period 05 / 2006 - 04 / 2011
Status Completed
Dissertation Yes
Research number OND1318185
Data Supplier ZonMw


The rapidly growing prevalence of type 2 diabetes mellitus in our western society will progressively affect public health. Type 2 diabetes is a major risk factor for cardiovascular diseases and contributes to increased mortality. One of the earliest hallmarks of type 2 diabetes is resistance of the skeletal muscle to the hormone insulin, resulting in impaired glucose uptake and hyperglycemia. It is nowadays recognized that accumulation of fat inside the muscle (both in the form of intramuscular triglycerides and as free fatty acids) is causally related to the development of diabetes. In muscle cells, fatty acids are prone to lipid peroxidation, which can damage mitochondria. Interestingly, skeletal muscle of type 2 diabetic patients is indeed characterized by increased mitochondrial damage and an impaired mitochondrial function, which in turn will lead to a further accumulation of fatty acids, resulting in a vicious circle in which mitochondrial function rapidly deteriorates. Under normal physiological conditions, mitochondria are protected against this lipid-induced mitochondrial damage, and based on the results of substantial (mainly human) physiological studies we hypothesize that the skeletal muscle specific mitochondrial uncoupling protein-3 (UCP3), for which the exact physiological function is still under debate, fulfills this role by exporting non-metabolizable fatty acids from the mitochondria. Importantly, in the (pre-)diabetic state, the protein content of UCP3 is reduced by 40-50%, and is restored under conditions that improve insulin sensitivity. Therefore, we propose that UCP3 plays a central role in protecting mitochondria against lipid-induced damage and that this defense mechanism is disturbed in pre-diabetic subjects, thereby contributing to the development of skeletal muscle insulin resistance. As the putative causality of this relation cannot be unraveled using human studies, we here propose to use in vitro and in vivo animal models to test the novel hypothesis that UCP3 is essential in the protection against lipid-induced mitochondrial damage and to test whether a lack of UCP3 accelerates the development of muscular insulin resistance under diabetogenic conditions. To this end, we will perform detailed experiments, using genetic-manipulated mice over- and underexpressing UCP3, to investigate if a) UCP3 can indeed export non-metabolizable fatty acids from the mitochondria, b) a lack of UCP3 will lead to lipid-induced mitochondrial damage and c) a reduced content of UCP3 accelerates the development of muscular insulin resistance. In the present project, we will establish a unique collaboration between well-recognized experts in the field of mitochondrial uncoupling and the field of cellular fatty acid transporters. We feel that such approach will warrant a major progress towards the understanding of the function of UCP3. If our hypothesis turns out to be true, UCP3 would be an important novel target for (pharmacological and/or nutritional) up-regulation in order to counteract lipid-induced mitochondrial dysfunction as an effective therapy for type 2 diabetes mellitus. In 1997, the discovery of a human homologue of the mitochondrial uncoupling protein-1 (UCP1), named UCP3 was received as a major breakthrough in the battle against the worldwide obesity epidemic. Like UCP1, UCP3 was assumed to dissipate energy as heat and play a major role in body weight regulation in humans. In a NWO-postdoctoral fellowship we however showed that UCP3 has no major function in the regulation of energy metabolism in humans (reviewed in: (1)). For example, we showed that a 60% up-regulation of UCP3 did not affect in vivo mitochondrial uncoupling in humans (2). In search for the real physiological function of UCP3, we and others observed that UCP3 is up-regulated under conditions of an abundant fatty acid supply to the mitochondria and is down-regulated when fatty acid oxidation is increased or plasma fatty acid levels are lowered. When fatty acid delivery mismatches oxidative capacity, those fatty acids that cannot be oxidized will accumulate in the muscle cell. Subsequently, the increased load of fatty acids on the mitochondrial membranes will lead to the entrance of neutral fatty acids into the mitochondrial matrix (3). However, in the matrix neutral fatty acids cannot be oxidized (only fatty acids in the acyl-CoA form can undergo beta-oxidation) but will be deprotonated (due to the proton gradient across the inner mitochondrial membrane). Since mitochondrial membranes are impermeable to fatty acid anions (4), these fatty acid anions would be trapped inside the mitochondrial matrix. As uncoupling proteins, after reconstitution in model membranes, were shown to be able to transport fatty acid anions over mitochondrial membranes (5), we postulated that UCP3 is an exporter of fatty acid anions to prevent their accumulation inside the matrix (6) (fig 1). Subsequently, we tested this putative function of UCP3 by interfering at several steps in fatty acid metabolism, thereby influencing the balance between fatty acid supply and fat oxidative capacity. All gathered data indeed fitted our hypothesis, as we showed among others that a) acute lowering of fat oxidative capacity strongly up-regulates UCP3 (7), b) UCP3 is inversely related to fat oxidative capacity of muscle (8) and c) medium-chain fatty acids, which do not accumulate in the matrix, do not need UCP3 (8) (for more detail see: strategy).
An important, unanswered question remains what the physiological relevance of UCP3 as a fatty acid anion exporter could be. The consequence of insufficient amounts of UCP3 is the accumulation of fatty acid anions inside the mitochondrial matrix. These fatty acid anions are vulnerable to oxidative damage because the matrix is also the major site of production of reactive oxygen species (ROS). Fatty acids that have become oxidized by ROS can lead to damage to mitochondrial DNA, RNA and proteins in the mitochondrial matrix (9), ultimately leading to mitochondrial damage and dysfunction. Very interestingly, it has recently been shown that UCP3 can be activated by 4-hydroxy-2-nonenal (10), a by-product of lipid peroxidation. This finding indicates a positive feedback between lipid peroxidation and UCP3, which fits with our idea that UCP3 protects mitochondria against mitochondrial damage induced by fatty acids or lipid peroxides.
Based on (our) previous findings that UCP3 is: The prevalence of type 2 diabetes mellitus is rapidly increasing worldwide, with an estimated number of 171 million people suffering from diabetes in 2000, and the number of people being in the pre-diabetic glucose intolerant state is even more worrisome (estimated at 317 million people in 2003). It is well recognized that high-fat diets in combination with a reduced physical activity, leading to obesity, play a major role in this rapid increase in diabetes prevalence (19). One of the earliest hallmarks of type 2 diabetes is skeletal muscle insulin resistance (19). Since skeletal muscle is responsible for a major part of postprandial glucose uptake, resistance of this tissue to the hormone insulin will lead to a reduced glucose disposal and ultimately hyperglycemia. So far, there is ample evidence that high levels of plasma fatty acids (as is observed in diabetic patients) leads to accumulation of fat in non-adipose tissues, such as liver, heart and skeletal muscle and this contributes to the development of insulin resistance (20). With respect to skeletal muscle, the debate on the mechanism through which intramuscular fat accumulation leads to insulin resistance is still ongoing, but in recent years, mitochondrial dysfunction has been identified as a key player in the development of skeletal muscle insulin resistance (14-16). Mitochondrial dysfunction coincides with fat accumulation in the muscle cell and it has been suggested that these lipids can cause damage to mitochondria (14, 16). In that context, type 2 diabetic patients have indeed been characterized by increased mitochondrial damage (14) while endurance training, which is recognized for its capability to stimulate mitochondrial biogenesis, is (one of) the best strategies to prevent type 2 diabetes (21). Given the essential role of mitochondria for cellular life, it is of utmost importance (not only with regard to diabetes) to further understand how mitochondrial dysfunction is caused, and more important, can be prevented. General experimental approach: In the present project, we aim to perform detailed and fundamental experiments that can provide the necessary evidence for our hypothesis that UCP3 protects mitochondria against lipid-induced damage (18). First, we will perform experiments aimed at demonstrating that UCP3 can function as a fatty acid anion exporter. For this, we will collaborate with experts from the field of the fatty acid transporters and apply the techniques that have been used to prove fatty acid transport capacity for the fatty acid transporters. We feel that this approach, which is unique for the mitochondrial uncoupling protein field, will provide important information with respect to the question whether UCP3 can act as a fatty acid anion exporter.

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Supervisor Prof.dr. J.F.C. Glatz
Supervisor Prof.dr. P.A.J. Schrauwen
Co-supervisor Dr. J. Hoeks
Doctoral/PhD student Dr. M.W. Nabben


D21700 Physiology
D23220 Internal medicine

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