Current treatments for obesity fail to significantly impact body weight and protect against its devastating health consequences. A promising approach to induce weight loss is to increase energy expenditure and nutrient disposal. However, the incomplete understanding of the mitochondrial mechanisms and physiological factors that regulate energy expenditure and fuel disposal has hindered progress. Uncoupling protein-3 (UCP3) is a skeletal muscle-enriched member of the widely conserved class of mitochondrial anion / solute carrier superfamily of proteins linked in a variety of clinical genetic studies to obesity in prone human populations. UCP3 activation increases insulin sensitivity, fatty acid oxidation, and thermogenesis, and loss of UCP3 promotes obesity under high caloric load. Targeting UCP3 (and UCP1) for increasing energy expenditure, while highly promising, has been confounded by (A) the lack of understanding of how it regulates fat oxidation, (B) how it is regulated at a molecular level, and (C) what it actually transports. Work in this application addresses each of these gaps in uncoupling protein biology and importantly, significantly delves into how UCP1 functions as well. We discovered that UCP3 regulates mitochondrial C4 substrate (malate, aspartate) transport directly (when reconstituted into liposomes and in yeast) and in muscle mitochondria from wild type but not UCP3 knockout mice. Further we have identified two UCP3 transport-defective mutants that will be critical for work in this application. We also found that UCP3 forms a complex with mitochondrial malate dehydrogenase (MDH2), which converts malate to oxaloacetate, the mitochondrial metabolite necessary for complete fatty acid oxidation. Finally, we show that skeletal muscle-specific UCP3 expression rescues drug- induced thermogenesis (a response that requires fatty acids) in global UCP3 knockout mice, showing that muscle may be a novel site of UCP3 thermogenesis. The overall working hypothesis of this proposal is that UCP3 coordinates the maximal capacity for skeletal muscle thermogenesis and fat oxidation through the control of mitochondrial malate and potentially other C4 metabolite mitochondrial import (anaplerotic flux of malate and likely aspartate, among others). We will test this in the following Aims: (1) Define the role of UCP3 in C4 metabolite mitochondrial transport and the molecular mechanisms involved. (2) Examine the mechanisms and physiological relevancy of UCP3-dependent metabolite transport for fat oxidation and UCP3-mediated metabolite transport in muscle in vivo using state of the art metabolic tracer experiments. Significance summary: This work will provide fundamental and novel insights into the molecular mechanisms regulating UCP3 (and likely UCP1), and will identify novel UCP-modulating ?druggable? targets and mechanisms.
Work in this proposal aims to determine the molecular mechanisms by which mitochondrial uncoupling protein 3 (UCP3) increases fat oxidation and thermogenesis, especially in skeletal muscle based on our team's recent discoveries that UCP3 has a novel anaplerotic function to import malate into mitochondria and that is controls not only drug-induced but also physiologically mediated thermogenesis. Experiments are designed to identify the repertoire of solutes that UCP3 transports, and they will also characterize a novel interaction between UCP3 and mitochondrial malate dehydrogenase 2 (mMDH2) and its functional importance for UCP3 function, malate transport, and thermogenesis. A greater mechanistic understanding of the regulation and functions of UCP3 as well as other thermogenic uncoupling proteins is greatly needed if we are to exploit these promising energy-expending proteins therapeutically, therefore this work will lay a foundation for the development of anti-obesity drugs that target UCP3-induced energy wasting and fat metabolism in muscle.
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