Browsing by Subject "Thermogenesis"
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Item Defining the mechanisms of uncoupling protein 3-induced thermogenesis and metabolism in brown adipose tissue(2014-12) Veron, Sonya Maria; Mills, Edward MichaelUncoupling proteins (UCPs) constitute a highly conserved subset of mitochondrial solute carriers. Discovered in small rodents in the early 1970’s, UCPs and their homologs have since been found in nematodes, plants, birds, and, most recently, in significant depots within humans (Krauss et al. 2005, Van marken Lichtenbelt 2009). Following activation by long chain fatty acids (LCFA, e.g. oleic acid) and reactive oxygen species (ROS, e.g. 4-hydroxynonenal (4HNE)), UCPs form a proton channel within the inner mitochondrial membrane and permit the influx of hydrogen ions from the inter membrane space into the mitochondrial matrix. UCPs effectively uncouple oxidative phosphorylation (OX-PHOS) from ATP generation, resulting in increasing oxygen consumption and dissipating the chemical energy in the form of heat. Found primarily in brown adipose tissue (BAT) of small hibernating mammals, the canonical role of uncoupling protein 1 (UCP1) in mammalian adaptive thermogenesis has been thoroughly studied. However, UCP1 is not the only member of the uncoupling family found within BAT. Also playing a key role in this tissue is uncoupling protein 3 (UCP3), which is a close homolog to UCP1. However, in spite of the fact that UCP3 shares more than 50% amino acid homology and tissue localization with UCP1, the true function UCP3 is very poorly elucidated. Part of the difficulty in determining this function lies in the expression levels of the UCP3 protein, which are hundreds of folds less than UCP1 in this tissue. In addition, their homologous structure makes teasing apart UCP3-specific phenomena from UCP1-mediated mechanisms very difficult using conventional techniques in cell and molecular biology. While UCP1 is almost exclusively found in BAT, UCP3 is expressed primarily in skeletal muscle (SKM), which lacks UCP1 completely (Krauss et al. 2005). Because UCP3 is so enriched in SKM, many studies have focused on its role in that tissue and have then tried to transpose these functions into BAT. As a result, UCP3 has been implicated in facilitating numerous biological processes, including non-adaptive facultative thermogenesis, affecting SKM oxidative capacity by modulating LCFA export, and ameliorating elevated levels of ROS-mediated stress within the tissue via glutathionine (GSH) interacting moieties. Ultimately, however, little consensus exists on the function of UCP3 within SKM, and subsequently, even less is known about its purpose in BAT. Previous data has shown that murine UCP1 has the capacity to bind to itself and form homo-tetramers when expressed in vitro in recombinant E. coli (Hoang T. et al. 2013). Here we show that UCP1 interacts with UCP3 in BAT in vivo, supporting Hoang’s research above by showing that UCP1 has the capacity to not only homodimerize but potentially oligomerize with other UCP homologs. While many groups using UCP3-null mice have reported no gross changes in physiologic responses, data previously published in the lab showed that mice lacking UCP3 were protected from potentially fatal hyperthermic effects when administered sympathomimetic agents such as 3,4-Methylenedioxymethamphetamine (MDMA), methamphetamine (METH), lipopolysaccharide (LPS), or norepinephrine (NE) (Mills et al. 2003, Kenaston et al. 2010). This implies that UCP3 plays an intimate role in sympathetic nervous system (SNS) mediated thermogenesis. Based upon the foregoing, the primary goal of the research discussed in this thesis was to elucidate the functions of UCP3 within BAT. In this study, we recapitulated results seen by other students in this lab: that global UCP3-null mice do indeed exhibit a blunted thermogenic response when treated with sympathomimetic agonists. In addition, despite the near-ubiquitous expression of UCP2 throughout the mammalian organism, this UCP is not involved in SNS-mediated thermogenesis (Arsenijevic et al. 2000). Our data shows that UCP3 is vital to the catecholamine-mediated thermogenic responses following sympathomimetic drug administration. When challenged by METH, UCP3-null mice were able to respond, albeit with a blunted increase in body temperature. Furthermore, when challenged by NE, a key neurotransmitter involved in mediating the responses initiated by the SNS following METH exposure, UCP3-null mice were able to mount half the hyperthermic response seen in WT littermates. However, UCP1/UCP3 double-null animals exhibited an almost four-fold hypothermic effect compared to WT littermates when challenged with NE. In addition, UCP1/UCP3 double-null mice were unable to restore body temperatures back to baseline values, an effect seen in all the other genotypes. This implies that UCP3 plays an important role in restoring body temperatures to physiological norms. Therefore, while the mechanism underlying the decreased responsiveness to NE remains unclear, it is clear that whether localized to SKM or BAT, UCP3 is a major player in the mammalian response to SNS-mediated thermogenesis and global thermoregulation.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 The underlying mechanisms of UCP3-dependent thermogenesis in skeletal muscle(2015-12) Dao, Christine Ky Linh; Mills, Edward Michael; Wright, Casey; Bratton, Shawn; Mukhopadhyay, Somshuvra; Ivy, JohnMitochondrial uncoupling proteins (UCPs) are anion / solute transporters that dissipate the proton gradient used to drive ATP generation. By allowing protons to flow down their electrochemical gradient, UCP activation releases the energy generated from mitochondrial substrate oxidation as heat. This thermogenic process is important in normal thermoregulation (i.e. non-shivering thermogenesis), and also serves as an attractive target in the treatment of obesity by lowering metabolic efficiency. The skeletal muscle (SKM) enriched UCP homologue, UCP3, is associated with increased energy expenditure, fatty acid metabolism, and insulin sensitivity. Unlike the cold-induced prototypical pathway of UCP1-mediated non-shivering thermogenesis in brown adipose tissue (BAT), the mechanisms underlying the thermogenic actions of UCP3 in SKM are not well characterized. Although global UCP3 knockout mice exhibit normal thermoregulatory responses to cold under fed conditions, they exhibit an attenuated hyperthermic response when administered amphetamine-type drugs. In our initial investigation, we show that selective overexpression of UCP3 in SKM by the human α-skeletal actin promoter restored methamphetamine (Meth)-induced hyperthermia in the UCP3⁻/⁻ background (TgSKM UCP3⁻/⁻), but not in the UCP1/UCP3 double knockout background (TgSKM UCP1⁻/⁻+UCP3⁻/⁻). Taken together, these findings further bolster the role of UCP3 as a thermogenic mediator in SKM, and suggest a novel mechanism of crosstalk between BAT UCP1 and SKM UCP3 in Meth-induced hyperthermia. In the second aspect of my project, we characterized the underlying mechanisms of UCP3-dependent thermogenesis within SKM by utilizing an immunoprecipitation- based mass spectrometry approach to identify interacting partners of UCP3. These analyses corroborated previous work performed by our lab, and demonstrated that UCP3 interacts with a subset of fatty acid metabolizing enzymes. Interestingly, one such enzyme, enoyl-CoA hydratase-1 (ECH1), is involved in the metabolism of oleic acid, a known ligand activator of UCP3. This work reveals that ECH1:UCP3 complex formation enhances uncoupled-respiration and fatty acid metabolism, and that genetic mouse models in vivo show that UCP3 and ECH1 participate in a common pathway of thermogenesis. These findings support a new model by which UCP3-dependent thermogenesis in SKM is mediated in part through its cooperation with ECH1, and suggest new approaches for treatment of obesity and related metabolic diseases.