Defining the mechanisms of uncoupling protein 3-induced thermogenesis and metabolism in brown adipose tissue

Uncoupling 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.