Aim: Uncoupling protein 1 (UCP1) is the crucial protein for non-shivering thermogenesis in placental mammals, but the molecular mechanism of thermogenic proton transport is still unknown. Its histidine pair motif (H145 and H147) has been claimed as a critical element for proton translocation, leading to the paradigmatic “cofactor model” of the UCP1 thermogenic mechanism. The histidine pair motif is mutated (H145Q) in the naked mole-rat (NMR, Heterocephalus glaber) UCP1, suggesting disrupted thermogenic function in line with NMR's poor thermoregulatory abilities. Here, we investigated the functionality NMR versus mouse UCP1 to scrutinized the importance of the histidine pair motif. Methods: Respiratory analyses for UCP1 function were performed in isolated brown adipose tissue mitochondria from NMR and mouse. The histidine pair motif of NMR UCP1 was manipulated through mutations, ectopically overexpressed in HEK293 cells and subjected to plate-based respirometry for functional comparison. Results: Isolated BAT mitochondria of NMRs display guanosine diphosphate-sensitive respiration, indicative of thermogenically competent UCP1. Overexpressed wildtype NMR UCP1 demonstrates proton leak activity comparable to mouse UCP1. Neither restoration of the histidine pair motif nor full ablation of the motif through a double mutation affects UCP1-dependent respiration. Conclusions: The UCP1 variant of the NMR, a warm-adapted fossorial species, excludes the histidine pair motif as crucial for UCP1 thermogenic function. Collectively, we show that functional investigation into natural sequence variation of UCP1 not only casts new light on the thermophysiology of NMRs but also represents a powerful tool to delineate structure-function relationships underlying the enigmatic thermogenic proton transport of UCP1.

Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5′ truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation. 

The molecular evolution of the mammalian heater protein UCP1 is a powerful biomarker to understand thermoregulatory strategies during species radiation into extreme climates, such as aquatic life with high thermal conductivity. While fully aquatic mammals lost UCP1, most semiaquatic seals display intact UCP1 genes, apart from large elephant seals. Here, we show that UCP1 thermogenic activity of the small-bodied harbor seal is equally potent compared to terrestrial orthologs, emphasizing its importance for neonatal survival on land. In contrast, elephant seal UCP1 does not display thermogenic activity, not even when translating a repaired or a recently highlighted truncated version. Thus, the thermogenic benefits for neonatal survival during terrestrial birth in semiaquatic pinnipeds maintained evolutionary selection pressure on UCP1 function and were only outweighed by extreme body sizes among elephant seals, fully eliminating UCP1-dependent thermogenesis.

Brown adipose tissue (BAT) is a heater organ that expresses thermogenic uncoupling protein 1 (UCP1) to maintain high body temperatures during cold stress. BAT thermogenesis is considered an overarching mammalian trait, but its evolutionary origin is unknown. We show that adipose tissue of marsupials, which diverged from eutherian mammals ~150 million years ago, expresses a nonthermogenic UCP1 variant governed by a partial transcriptomic BAT signature similar to that found in eutherian beige adipose tissue. We found that the reconstructed UCP1 sequence of the common eutherian ancestor displayed typical thermogenic activity, whereas therian ancestor UCP1 is nonthermogenic. Thus, mammalian adipose tissue thermogenesis may have evolved in two distinct stages, with a prethermogenic stage in the common therian ancestor linking UCP1 expression to adipose tissue and thermal stress. We propose that in a second stage, UCP1 acquired its thermogenic function specifically in eutherians, such that the onset of mammalian BAT thermogenesis occurred only after the divergence from marsupials. 

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.  

Brown adipose tissue (BAT), present in many placental mammals, provides adaptive nonshivering thermogenesis (NST) for body temperature regulation and has facilitated survival in diverse thermal niches on our planet. Intriguingly, several key details on the molecular mechanisms of NST and their potential ecophysiological adaptations are still unknown. Comparative studies at the whole animal level are unpragmatic, due to the diversity and complexity of thermoregulation among different species. We propose that the molecular evolution of mitochondrial uncoupling protein 1 (UCP1), a central component for BAT thermogenesis, represents a powerful opportunity to unravel key questions of mammalian thermoregulation. Comparative analysis of UCP1 may elucidate how its thermogenic function arose, how environmental selection has shaped protein function to support ecophysiological requirements, and how the enigmatic molecular mechanism of proton leak is governed. Several approaches for the assessment of UCP1 function in vitro have been introduced over the years. For comparative characterization of UCP1, we put forward the overexpression of UCP1 orthologues and mutated variants in a mammalian cell system as a primary strategy and discuss advantageous aspects in contrast to other experimental systems. In turn, we suggest how remaining experimental caveats can be solved by complimentary test systems before physiological consolidation in the animal model. Furthermore, we highlight the appropriate bioenergetic techniques to perform the functional analyses on UCP1. The comparative characterizations of diverse UCP1 variants may enable key insights into open questions surrounding the molecular basis of NST.

The thermogenic mechanisms supporting endothermy are still not fully understood in all major mammalian subgroups. In placental mammals, brown adipose tissue currently represents the most accepted source of adaptive non-shivering thermogenesis. Its mitochondrial protein UCP1 (uncoupling protein 1) catalyzes heat production, but the conservation of this mechanism is unclear in non-placental mammals and lost in some placentals. Here, we review the evidence for and against adaptive non-shivering thermogenesis in marsupials, which diverged from placentals about 120-160 million years ago. We critically discuss potential mechanisms that may be involved in the heat-generating process among marsupials.

Uncoupling protein 1 (UCP1) governs non-shivering thermogenesis in brown adipose tissue. It has been estimated that pigs lost UCP1 similar to 20 million years ago (MYA), dictating cold intolerance among piglets. Our current understanding of the root causes of UCP1 loss are, however, incomplete. Thus, examination of additional species can shed light on these fundamental evolutionary questions. Here, we investigated UCP1 in the Chacoan peccary (Catagonus wagneri), a member of the Tayassuid lineage that diverged from pigs during the late Eocene-mid Oligocene. Exons 1 and 2 have been deleted in peccary UCP1 and the remaining exons display additional inactivating mutations. A common nonsense mutation in exon 6 revealed that UCP1 was pseudogenized in a shared ancestor of pigs and peccaries. Our selection pressure analyses indicate that the inactivation occurred 36.2-44.3 MYA during the mid-late Eocene, which is much earlier than previously thought. Importantly, pseudogenized UCP1 provides the molecular rationale for cold sensitivity and current tropical biogeography of extant peccaries.

In eutherian mammals, brown adipose tissue (BAT) permits non-shivering thermogenesis (NST) through high metabolic rates catalyzed by the unique mitochondrial uncoupling protein 1 (UCP1). The tissue has recently gained remarkable attention due to its discovery in adult humans. Approaching BAT and UCP1 as therapeutic targets to combust surplus energy bares high potential to combat the epidemic of the metabolic syndrome that has precipitated in our society as a result of our modern lifestyles. Our understanding of the physiological and molecular control of BAT may benefit tremendously from consideration of its evolution that basically outlines the blueprint of how to construct a fat burning tissue. Here, we discuss the evolutionary history of UCP1 and BAT, from its origins and emergence to its downfall in several mammalian lineages. Additionally, we delineate the annotation of UCPs in vertebrates by analyzing genomic organization and summarize the phylogeny of UCP1 within the closest relatives of humans, the great apes. Outlining whether the molecular networks controlling thermogenesis in adipose tissue (commonly known as the browning potential) pre-dated the classical thermogenic function of BAT and UCP1, and whether the evolutionary inactivation of UCP1 enhanced compensatory thermogenic mechanisms, should be of major interest to those who aim to access adipose tissue thermogenesis in a biomedical context.