The Uncoupling Protein 1 Gene (UCP1) Is Disrupted in the Pig Lineage: A Genetic Explanation for Poor Thermoregulation in Piglets

Andersson L (2006) The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: A genetic explanation for poor thermoregulation in piglets. PLoS Genet 2(8): e129. DOI: 10.1371/journal.pgen.0020129 The Uncoupling Protein 1 Gene (UCP1) Is Disrupted in the Pig Lineage: A Genetic Explanation for Poor Thermoregulation in Piglets Frida Berg 0 1 Ulla Gustafson 0 1 Leif Andersson Leif.Andersson@imbim 0 1 0 Editor: Gregory S. Barsh, Stanford University School of Medicine , United States of America 1 1 Department of Medical Biochemistry and Microbiology, Uppsala University , Uppsala , Sweden , 2 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences , Uppsala , Sweden Piglets appear to lack brown adipose tissue, a specific type of fat that is essential for nonshivering thermogenesis in mammals, and they rely on shivering as the main mechanism for thermoregulation. Here we provide a genetic explanation for the poor thermoregulation in pigs as we demonstrate that the gene for uncoupling protein 1 (UCP1) was disrupted in the pig lineage. UCP1 is exclusively expressed in brown adipose tissue and plays a crucial role for thermogenesis by uncoupling oxidative phosphorylation. We used long-range PCR and genome walking to determine the complete genome sequence of pig UCP1. An alignment with human UCP1 revealed that exons 3 to 5 were eliminated by a deletion in the pig sequence. The presence of this deletion was confirmed in all tested domestic pigs, as well as in European wild boars, Bornean bearded pigs, wart hogs, and red river hogs. Three additional disrupting mutations were detected in the remaining exons. Furthermore, the rate of nonsynonymous substitutions was clearly elevated in the pig sequence compared with the corresponding sequences in humans, cattle, and mice, and we used this increased rate to estimate that UCP1 was disrupted about 20 million years ago. - 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]. 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. 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 Brown adipose tissue (BAT) is unique to mammals. It is rich in mitochondria and generates heat to maintain body temperature during cold stress, referred to as nonshivering thermogenesis. BAT is found in abundance in rodents as well as in newborns of larger mammals, including humans. Uncoupling protein 1 (UCP1) is exclusively expressed in BAT and is localized to the inner membrane of the mitochondria. Its physiological role is to uncouple oxidative phosphorylation so that most of the energy in fat stores is dissipated as heat rather than being converted to ATP. Piglets are sensitive to cold stress and rely on shivering as the main mechanism for thermoregulation. Furthermore, pigs are the only hoofed mammals that build nests for birth; in modern pig production, heat-producing lamps are used to keep the piglets warm. It is also striking that pigs appear to lack BAT. Here the authors show that the UCP1 gene is disrupted in domestic pigs and their wild ancestors. The inactivation of UCP1 was estimated to have happened about 20 million years ago. The finding provides an explanation for the poor thermoregulation in piglets that may have led to the evolution of the unique maternal behavior in this species. 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 twobase pair insertion in exon 1, a 16base pair deletion in exon 2 (both causing frameshifts), and a nonsense mutation in exon 6 (Figure (...truncated)