Investigation of the physiological and biochemical function of mitochondrial uncoupling protein 3

Uncoupling proteins (UCPs) are highly conserved inner mitochondrial membrane proteins that have been found in plants, nematodes, flies, and vertebrates. UCPs dissipate the proton gradient formed by the electron transport chain in an energy-expending process that generates heat. In mammals, the brown fat-specific UCP1 is thought to be the dominant, if not the only significant mediator of thermogenic responses. However, adult humans express only negligible amounts of brown fat and UCP1, yet still show significant non-shivering thermogenic responses (e.g. amphetamine-induced hyperthermia, diet induced thermogenesis, fever). Thus, the fact that human thermogenic mechanisms haven't been identified is a huge gap in our understanding of human thermoregulation. UCP3 is primarily expressed in skeletal muscle, an established thermogenic organ which is a major target of amphetamine-induced pathology. UCP3 knockout mice have a near complete loss (~80%) of amphetamine-induced thermogenesis and are completely protected from amphetamine-induced death over a range of lethal doses. With regard to mechanisms of UCP3 activation, we observed that norepinephrine and free fatty acids are elevated in the bloodstream prior to peak amphetamine-induced hyperthermia. However, little is known about the anatomic location of UCP3-dependent thermogenesis or the mechanisms by which fatty acids regulate UCP function. Thus, we sought to investigate the physiology and biochemical activation of UCP3 to establish the thermogenic potential of skeletal muscle uncoupling and elucidate the mechanisms of UCP3 function. The overall goal of this research was to identify the tissue target(s) and mechanisms involved in amphetamine-induced UCP3-dependent thermogenesis. Herein, we show that in addition to a deficit in induced thermogenesis, UCP3-null mice also lack responses to other physiologically-relevant stimuli (i.e. catecholamines and bacterial pathogens). Conversely, UCP3 knockout mice, engineered to express UCP3 only in skeletal muscle have an augmented thermogenic response to amphetamines. In order to explore UCP3's mechanism of activation, we performed a modified yeast two-hybrid analysis and identified [Delta][superscript 3,5][Delta][superscript 2,4]dienoyl-CoA isomerase (DCI) as a UCP3 binding partner. DCI, an auxiliary fatty acid oxidation enzyme, protects cells from the accumulation of toxic lipid metabolites. Using immunoprecipitation and fatty acid oxidation (FAO) assays, we determined that UCP3 and DCI directly bind in the mitochondrial matrix in order to augment lipid metabolism. These findings support a novel model in which skeletal muscle UCP3 is responsible for inducible thermogenesis through cooperation with binding partners such as DCI which enhance oxidation of fatty acids. Together, these studies shed light on thermogenic pathways in rodents that are likely to be relevant to humans.