Introduction

Pigs are unusual mammals in a number of ways: they are the only ungulates that (1) build nests for birth (Figure 1A), and (2) have large litters, where (3) each young is behaviorally well developed but with a poor thermoregulatory ability [1,2]. It is also striking that piglets appear to lack brown adipose tissue (BAT) [3] and rely on shivering as the main mechanism for thermoregulation [4,5]. BAT is a specific type of fat that is widely expressed in neonatal animals as well as in hibernating rodents [6]. Its physiological role is to generate heat by uncoupling oxidative phosphorylation. Uncoupling protein 1 (UCP1) is exclusively expressed in BAT and plays a crucial role for thermogenesis. UCP1 is located in the inner membrane of the mitochondria, where it catalyzes a regulated proton leakage across the membrane. The established energy is then released as heat [6]. It has been proposed that the acquisition of BAT and UCP1 gave the early mammals an evolutionary advantage by allowing them to be active during periods of nocturnal or hibernal cold and to survive the cold stress at birth [6].

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Figure 1. Characterization of the UCP1 Locus in Pigs

(A) Wild boar sow with striped piglets in their nest.

(B) Dotpath alignments of genomic UCP1 sequences from pigs, humans, and cattle. The approximate positions of the six exons in human UCP1 are indicated. Gap1 and Gap2 indicate two gaps in the alignment between the pig and human sequences.

(C) Rate of nonsynonymous and synonymous substitutions in pairwise comparisons of UCP1 sequences from humans, mice, cattle, and pigs. The data are based on sequences from exon 1, 2, and 6. B, Bos taurus; H, Homo sapiens; M, Mus musculus; and S, Sus scrofa.

(Photo: Anneli Andersson, Linköping University, Sweden)

https://doi.org/10.1371/journal.pgen.0020129.g001

No conclusive evidence for the presence of BAT [7] or for the expression of UCP1 [3,8] has yet been demonstrated in pigs, despite considerable efforts to study this subject. It has therefore been questioned whether pigs express this tissue, and it has been suggested that UCP1 expression may have been down-regulated during domestication because of strong selection for an efficient energy metabolism. Here we provide an explanation for poor thermoregulation in pigs and the evolution of the unique features in maternal behavior and piglet development. We demonstrate that UCP1 became disrupted in the pig lineage about 20 million years ago.

Results

We took advantage of the recent release of a partial pig genome sequence [9] to investigate the porcine UCP1 locus. The human transcript was blasted against the Sus scrofa trace archive (http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?) by using discontiguous Mega BLAST (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/blast/tracemb.shtml). Two hits, pig trace sequences 812263277 and 782925243, corresponding to UCP1 exons 2 and 6, were obtained and used for primer design. The complete genome sequence of pig UCP1 was determined by long-range PCR and genome walking by using genomic DNA from a Large White domestic pig and the porcine bacterial artificial chromosome (BAC) clone PigE-117H8 [10] containing UCP1. An alignment with the corresponding human sequence revealed two gaps in the porcine sequence, reducing the total size from 10.1 kilobases in humans to 4.3 kilobases in the pig (Figure 1B). Alignments with cattle UCP1 showed that Gap1, located in intron 2, represents an insertion in the human lineage or a deletion that occurred before the split of the pig and cattle lineages. Gap2 is unique to the pig and eliminates exons 3 to 5, implying that UCP1 is disrupted. The presence of this deletion was confirmed by PCR amplification of the deletion breakpoint in pigs representing many different breeds, as well as in European wild boars, Bornean bearded pig (S. barbatus), wart hog (Phacochoerus africanus), and red river hog (Potamochoerus porcus).

Southern blot analysis using a porcine probe revealed a single restriction fragment consistent with the presence of a single UCP1 copy in the pig genome (Figure S1). The similarity between the end sequences of the porcine UCP1 BAC clone and sequences flanking the human UCP1 gene also supports the interpretation that we have analyzed the true UCP1 locus and not a defect duplicated version.

The UCP1 coding sequence in pigs contains three additional disrupting mutations: a two–base pair insertion in exon 1, a 16–base pair deletion in exon 2 (both causing frameshifts), and a nonsense mutation in exon 6 (Figure S2). In contrast, strong purifying selection is observed at the UCP1 locus in humans, cattle, and mice, as is evident from the 5- to 10-fold higher rate of synonymous (d_S) versus nonsynonymous (d_N) substitutions among these species (Figure 1C). As expected, d_S was nearly identical when comparing humans with pigs and humans with cattle. In contrast, d_N was markedly elevated in all pairwise comparisons involving the pig (Figure 1C). For example, human versus pig: d_N(H-S) = 13.9 ± 2.4%, human versus cattle: d_N(B-H) = 8.8 ± 1.9%, Z = 1.66, p < 0.05 in a one-sided test; Z=D/s(D) where D=d_N(H-S) - d_N(B-H), V(D)=V(d_N(H-S)) + V(d_N(B-H)), and s(D)=[V(D)]^1/2 [11]. This implies that porcine UCP1 was inactivated sometime subsequent to the divergence from the cattle lineage and that it has accumulated synonymous and nonsynonymous substitutions at the same rate since then. The inactivation of UCP1 was dated to have happened about 20 million years ago by using the estimated d_N rate for the human/cattle comparison and the estimated d_S rate for the pig/cattle comparison to explain the excess of nonsynonymous substitutions in the pig UCP1 sequence; the estimated time since the divergence of the human and pig/cattle lineages (94 million years) and the pig and cattle lineages (60 million years) were from Springer et al. [12]. The result is consistent with the observation that UCP1 is disrupted in several pig species.

Discussion

We have shown that UCP1 was inactivated in the pig lineage about 20 million years ago. UCP1 knockout mice show only a mild phenotype; they are cold sensitive but fully viable [13]. Furthermore, these UCP1-null mice develop BAT, which implies that the disruption of porcine UCP1 cannot by itself explain the absence of BAT. A possible evolutionary scenario is that a pig ancestor lost UCP1 function and the ability to use BAT for thermoregulation because of no or only weak selection for this mechanism in a warm climate; the wild boar is the only porcine species that has adapted to temperate climates, whereas all other Suidae live in tropical or subtropical environments. The wild boar has then evolved compensatory mechanisms to adapt to a cold environment. Newborn pigs use shivering for thermogenesis [4,5], and the wild boar is the only ungulate that builds a thermoprotective nest for giving birth (Figure 1A). A temperature around +20 °C has been estimated in winter farrowing nests of free-ranging domestic pigs despite outside temperatures down to −20 °C [14]. In modern pig production, heat-lamps are used to facilitate thermocontrol in piglets.

An interesting topic for future research will be to study UCP1 in more distantly related species to date more precisely the disruption of this gene in the pig lineage. It will be of considerable interest to investigate if the loss of gene expression closely correlates with changes in behavior and litter size among species. Our study does not resolve whether the disruption of UCP1 was the primary event leading to the loss of BAT or whether it was a secondary event. This question could possibly be resolved by a comparative study of different Suidae species, but it cannot be answered by analyzing sequence data from one species only, because of the poor precision in dating mutation events that happened 20 million years ago. Another important question is whether the loss of UCP1 and BAT is associated with other genetic changes during the evolution of the pig lineage. For instance, it is possible that the disruption of UCP1 has led to an upregulation of UCP2 expression, as observed in the UCP1 knockout mice [13].

Materials and Methods

Supporting Information

Acknowledgments