Pig has no uncoupling protein 1

doi:10.1016/j.bbrc.2017.04.118

Biochemical and Biophysical Research Communications

Volume 487, Issue 4, 10 June 2017, Pages 795-800

https://doi.org/10.1016/j.bbrc.2017.04.118 Get rights and content

Highlights

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Unlike most mammalian UCP1 genes, which have 6 exons, pig UCP1 gene has only 3 exons.
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Only exons 1 and 2 of the UCP1 gene are transcribed in pig adipose tissues.
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Truncated pig UCP1 mRNA is not translated in pig adipose tissues.
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Uncoupled respiration was not detected in pig adipocytes. Therefore, pig has no BAT.

Abstract

Brown adipose tissue (BAT) is critical for mammal's survival in the cold environment. Uncoupling protein 1 (UCP1) is responsible for the non-shivering thermogenesis in the BAT. Pig is important economically as a meat-producing livestock. However, whether BAT or more precisely UCP1 protein exists in pig remains a controversy. The objective of this study was to ascertain whether pig has UCP1 protein. In this study, we used rapid amplification of cDNA ends (RACE) technique to obtain the UCP1 mRNA 3′ end sequence, confirmed only exons 1 and 2 of the UCP1 gene are transcribed in the pig. Then we cloned the pig UCP1 gene exons 1 and 2, and expressed the UCP1 protein from the truncated pig gene using E. coli BL21. We used the expressed pig UCP1 protein as antigen for antibody production in a rabbit. We could not detect any UCP1 protein expression in different pig adipose tissues by the specific pig UCP1 antibody, while our antibody can detect the cloned pig UCP1 as well as the mice adipose UCP1 protein. This result shows although exons 1 and 2 of the pig UCP1 gene were transcribed but not translated in the pig adipose tissue. Furthermore, we detected no uncoupled respiration in the isolated pig adipocytes. Thus, these results unequivocally demonstrate that pig has no UCP1 protein. Our results have resolved the controversy of whether pigs have the brown adipose tissue.

Introduction

Mammals contain mainly two types of adipose tissues, white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores excess energy in the form of triglycerides when an animal's energy intake exceeds its energy expenditure. During starvation, WAT lipolysis provides energy to animals [1]. Brown adipose tissue contains multilocular fat droplets and many mitochondria, and is distinguished by its ability to dissipate energy in the form of heat [2]. BAT plays a significant role in newborns; they use this tissue for non-shivering thermogenesis to defend themselves against the cold environment [3].

BAT-dependent non-shivering thermogenesis is attributed to the uncoupling protein 1 (UCP1) [4], [5], [6]. UCP1 is a member of the of mitochondrial anion carrier proteins family and is a transmembrane protein with the size of approximately 35 KD. UCP1 disengages ATP synthesis from oxidative phosphorylation and dissipates energy as heat [7].

Although BAT has been found in many mammals such as mice, human, goat, and dog [8], [9], [10], [11]. However, whether brown adipose tissue exists in the pig is still a controversy. Brown adipocytes were found in different pig adipose tissues by microscopic and electron microscopic methods [12]. But this result was not supported by the immunoblotting study in another laboratory using the rabbit anti-rat UCP1 antibody [13]. The authors concluded that pigs have no brown adipose tissue, and all adipose tissues in the pig appear to be exclusively “white” [13]. More recently, Berg et al. (2006) determined the complete genome sequence of pig UCP1 gene by long-range PCR and genome walking. In this study, they found that exons 3 to 5 of the pig UCP1 gene were lost 20 million years ago; more than twenty pigs from different breeds of domestic pigs and wild boar were included in their study [14]. Thus, pig UCP1 gene contains only three exons: exons 1, 2 and 3, which are corresponding to the exons 1, 2 and 6 of the mouse UCP1 gene, respectively. By using a goat UCP1 antibody in their immunoblotting assay Mostyn et al. (2014) showed that UCP1 protein is present in pig adipose tissue and is responsive to postnatal leptin treatment [15]. However, Jastroch & Anderson (2015) believe the increased UCP1 protein in Mostyn's study (2014) may be explained by non-specific hybridization with other mitochondrial anion carrier proteins, such as UCP2 or UCP3 by the goat UCP1 antibody [16]. Berg et al. (2006) only examined the UCP1 gene at genome level; they did not measure the UCP1 protein. By contrast, Mostyn and his colleagues measured only UCP1 protein by a rabbit anti-goat UCP1 antibody; they did not study the UCP1 gene structure or its transcript. Therefore, in this study, we examined the UCP1 gene structure and determined whether pig UCP1 gene is being transcribed, translated and functional.

Section snippets

Materials and methods

All animal use protocols were approved by the College of Animal Science, South China Agricultural University. All experiments were performed in accordance with relevant guidelines and regulations of ‘the instructive notions with respect to caring for laboratory animals’ issued by the Ministry of Science and Technology of the People's Republic of China.

Pig UCP1 gene contains only three exons

Since whether UCP1 exists in pig has never been conclusively established, we first examined UCP1 gene structure and its transcript in the pig. We designed four pairs of primers based on pig UCP1 pseudogene sequence obtained from Berg’ research to determine the UCP1 gene structure and its transcript in pigs, and the primer locations on the UCP1 gene are shown in Fig. 1A. The four primer pairs produced four corresponding PCR products using genome DNA as template (Fig. 1B). We sequenced the PCR

Discussion

Increasing energy expenditure represents an efficient means for animal resistance to cold environment and combating obesity [17], [18]. Increasing non-shivering thermogenic capacity through activating BAT or obtaining BAT-like characteristics in WAT might be an excellent strategy for piglets defending against the cold environment or treating obesity in human [19], [20]. However, little is known about the mechanism of piglet's poor thermogenic ability. Our results clearly indicate pig has no

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National High Technology Research and Development Program 863 (#2013AA102502); the National Natural Science Foundation of China (#31372283); The Team Project of Guangdong Agricultural Bureau (#2016LM2148) and the Natural Fund Key Projects of Guangdong Province (#2015A030311006). C.H. is supported by the USDA National Institute of Food and Agriculture, Hatch project HAW-H2037, managed by the College of Tropical Agriculture and Human Resources, University of Hawaii

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Pig has no uncoupling protein 1

Highlights

Abstract

Introduction

Section snippets

Materials and methods

Pig UCP1 gene contains only three exons

Discussion

Conflict of interest

Acknowledgements

Cell

Cell Metab.

Cell Metab.

Cell

Res. Vet. Sci.

Res. Vet. Sci.

Mol. Metab.

Biochim. Biophys. Acta

J. Biol. Chem.

The different shades of fat

Nature

Influencing factors of thermogenic adipose tissue activity

Front. Physiol.

Adaptive thermogenesis in adipocytes: is beige the new brown?

Genes & Dev.