Browsing by Subject "Amphetamine"
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Item Investigation of the physiological and biochemical function of mitochondrial uncoupling protein 3(2010-12) Kenaston, Monte Alexander; Mills, Edward M.; Bratton, Shawn B.; Gore, Andrea C.; Hursting, Stephen D.; Sprague, Jon E.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.Item Regulation of FOSB MRNA Isoforms by Drugs of Abuse(2006-05-15) Alibhai, Imran Nizamudin; Nestler, Ericdelta FosB, a truncated splice isoform of FosB, is a transcription factor that accumulates within a subset of neurons after chronic administration of drugs of abuse or other chronic stimuli. Due likely to its structure and post-translational modifications, delta FosB protein is uniquely stable relative to the transiently expressed full-length FosB and all other Fos family proteins. The goal of this study was to determine if the relative expression of the two fosB isoforms is regulated at the mRNA level, thereby further contributing to the accumulation of delta FosB. First, unlike the protein, the half-life of delta fosB mRNA is only slightly longer than that of full-length fosB mRNA both in cultured cells in vitro and in the brain in vivo. Additionally, similar to c-fos, both fosB isoforms are induced abundantly in striatum after acute administration of amphetamine and partially desensitize after chronic dosing. Surprisingly, the relative ratio of the fosB to delta fosB mRNA (normally 16:1 in saline controls)decreases significantly only after acute doses or at doses that elicit the greatest induction of both transcripts. When acute amphetamine doses are incrementally increased, fosB levels are induced to roughly equivalent levels regardless of dose; however, delta fosB levels increase as the drug dose increases. A similar pattern of fosB and delta fosB mRNA induction was seen in cell culture. These findings suggest that the splicing of fosB RNA may be regulated by the quantity of unspliced transcript available to the splicing machinery. That is, above a certain threshold of full-length fosB, the remaining primary transcript is alternatively spliced into delta fosB. This splicing phenomenon is likely regulated by the Polypyrimidine Tract Binding (PTB1) protein. PTB1 protein has been shown to inhibit the U2AF splicing complex and thus prevent alternative splicing of regions in close proximity to where it is bound. It has previously been demonstrated that PTB1 protein binds the fosB transcript in vitro. Here, it is shown that overexpression of PTB1 in PC12 cells alters the ratios of the fosB isoforms by increasing the amount of fosB transcript relative to delta fosB transcript. Therefore, this study concludes that under basal conditions PTB1 protein binds the majority of the fosB premRNA, thereby inhibiting the generation of the delta fosB transcript. Only when PTB1 protein is saturated with transcript does the ratio of fosB to delta fosB decrease significantly, because the unbound pre-mRNA is spliced into delta fosB. These data provide fundamental information concerning the generation of delta fosB mRNA and indicate the selective accumulation of delta FosB protein with chronic drug exposure does not involve its preferential generation by splicing.