Insights into the Evolution of Non‐shivering Thermogenesis from UCP1 of the Naked Mole Rat (Heterocephalus glaber)

Abstract

Recognized as the primary location of non‐shivering thermogenesis (NST) in eutherian mammals, brown adipose tissue (BAT) functions to accelerate rewarming in hibernators and defend body temperature among newborns and small‐bodied species enduring cold stress. Uncoupling protein 1 (UCP1) is highly expressed in BAT and underlies the molecular mechanism of NST. UCP1 resides in the inner mitochondrial membrane where, upon functional induction by free fatty acids, it catalyzes mitochondrial proton leak, collapsing the proton gradient that stores oxidation energy. The ensuing futile cycling of protons increases substrate oxidation and thus heat production. Central questions remain regarding the exact mechanism by which UCP1 facilitates proton leak, stemming from the lack of a crystal structure of the protein. It is also unknown whether UCP1 function has been shaped by evolution to meet differing thermoregulatory demands of various lineages. We adopt a comparative approach, targeting species with extreme ecophysiological characteristics expected to culminate in drastic differences in thermoregulatory demands and thus UCP1 function, to provide insights into protein structure‐function relationships. For instance, the naked mole rat (NMR; Heterocephalus glaber) is well‐suited to survive in hypoxic burrows that exhibit high year‐round temperatures (30‐34°C). Hinting at a reduced NST capacity, NMRs also display poikilothermy, failing to maintain constant body temperatures when artificially cold challenged. Moreover, NMR UCP1 displays several unique amino acid substitutions, one of which, H145Q (histidine to glutamine), occurs at the well‐known “histidine pair motif”. This motif, typically comprised of histidines at positions 145 and 147, has been hypothesized to be a critical final proton acceptor/donor site shuttling protons into the mitochondrial matrix. The exact mutation that occurs in the NMR, H145Q, was previously shown to diminish hamster UCP1‐mediated proton conductance in proteoliposomes by 90%, calling the functional performance of NMR UCP1 into question. We functionally tested wildtype NMR UCP1 as well as histidine pair mutant combinations at positions 145 and 147, not in the highly artificial system of proteoliposomes, which may promote artefactual protein behaviour, but instead in the more native mammalian environment of HEK293 cells. Using plate‐based respirometry techniques, we demonstrate that wildtype NMR UCP1 uncouples, thus NMRs retain this mechanism of NST. While we hypothesized that reversion of the H145Q mutation would increase UCP1‐mediated proton leak, surprisingly both variants function similarly when activated with free fatty acids and inhibited with guanosine diphosphate (GDP). Likewise, mutation of the histidine at position 147, the other half of the “histidine pair motif”, does not drastically alter UCP1 function. Overall, these data suggest that the “histidine pair motif” may not be crucial for protein function, contrary to previous claims. Comparative functional assessments of UCP1 variants provide a deeper understanding of structure‐function relationships of this enigmatic protein and the evolution of mammalian NST in response to ecophysiological constraints.