Comparative analysis of the thermogenic protein UCP1 across the mammalian phylogeny

Sammanfattning: Uncoupling protein 1 (UCP1) drives mitochondrial inefficiency to produce heat in mammalian brown adipose tissue (BAT). Many mammalian species rely on this form of adaptive non-shivering thermogenesis (NST) to defend high body temperatures in the cold. Little is known regarding how evolution may have shaped UCP1 function to reflect distinct thermoregulatory requirements of various lineages. This thesis merges genetic and functional data while using a comparative approach to gain insights into the evolutionary rise of thermogenic UCP1, as well as its repeated loss of function among several eutherian lineages. UCP1 structure-function relationships and mechanistic insights are gained by examining natural mutations among the orthologues of different species.  In Paper I, we reveal that, like eutherian mammals, marsupial UCP1 is expressed in adipose tissue of developing young gray short-tailed opossums (Monodelphis domestica) and coincides with the onset of thermoregulatory competence. Transcriptomic analyses reveal partial browning signatures in adipose tissue of young opossums, resembling eutherian beige adipose tissue. Overexpression of marsupial UCP1 in a mammalian test system (HEK293 cells), however, reveals its lack of thermogenic functionality. I then performed ancestral reconstruction of UCP1 and demonstrate that the thermogenic function arose in the stem eutherian ancestor. In Paper II, I uncover that UCP1 not only became pseudogenized in pigs (e.g. Sus spp.), but in a common ancestor of both pigs and peccaries (e.g. Catagonus wagneri) as indicated from a shared inactivating mutation, re-calibrating the timeline of this inactivation and our understanding of how it may limit the geographic distribution of modern peccaries. In Paper III, I uncover a novel UCP1 pseudogene unique to the largest seals, elephant seals (Mirounga spp.), showing that UCP1 is retained within most members of the seal lineage for neonatal defense of body temperatures, but its loss coincides with the extreme body sizes attained by elephant seals. In Paper IV, we functionally verify that N-terminal truncation or frameshift mutation repair cannot rescue the thermogenic function of elephant seal UCP1. By contrast, we verify the thermogenic capacity of UCP1 of the small-bodied harbor seal (Phoca vitulina), matching that of terrestrial eutherians.In Paper V, we examine UCP1 in the naked mole-rat (Heterocephalus glaber), a species that displays a natural mutation to the histidine pair motif that has been previously deemed crucial for UCP1 function. We hypothesized that this may underlie the poor thermoregulatory abilities of the species. Our assessment of UCP1 mutants, however, reveal that the naked mole-rat retains UCP1 function and that the histidine pair motif is unnecessary for the GDP-sensitive thermogenic function of the protein, providing important structure-function information of UCP1 and questioning a proposed mechanistic model. In summary, this thesis utilizes UCP1 as a biomarker to trace the evolution of mammalian NST and thermoregulation. Insights gained provide clues to the various factors influencing mammalian endothermy and hints of structure-function relationships in this thermogenic protein.