The present study aimed to determine whether porcine genotype and/or postnatal age influenced mRNA abundance or protein expression of uncoupling protein (UCP)2 or 3 in subcutaneous adipose tissue (AT) and skeletal muscle (SM) and the extent to which these differences are associated with breed-specific discordance in endocrine and metabolic profiles. Piglets from commercial and Meishan litters were ranked according to birth weight. Tissue samples were obtained from the three median piglets from each litter on either day 0, 4, 7, 14 or 21 of neonatal life. UCP2 protein abundance in AT was similar between genotypes on the first day of life, but it was elevated at all subsequent postnatal ages (P<0.05) in AT of Meishan piglets. In contrast, UCP2 mRNA abundance was lower in Meishans up to 14 days of age. UCP2 mRNA expression was not correlated with protein abundance in either breed at any age. UCP3 mRNA in AT was similar between breeds up to day 7; thereafter, expression was higher (general linear model, P<0.05) in Meishan piglets. Conversely, UCP3 mRNA expression in SM was higher in commercial piglets after day 7. Colonic temperature remained lower in Meishan than commercial piglets throughout the study; this was most obvious in the immediate post-partum period when Meishan piglets had lower (P<0.05) plasma triiodothyronine. In conclusion, we have demonstrated that porcine genotype influences the expression and abundance of UCP2 and 3, an influence which may, in part, be due to the distinctive endocrine profiles associated with each genotype.
Piglets, like most mammalian neonates, possess very little adipose tissue at birth (1% of body weight) (Mount 1968) and are therefore extremely sensitive to the drop in environmental temperature experienced (15–20 °C) during the transition from the intra- to extrauterine environment. This drop in ambient temperature results in a rapid fall in body temperature, from 39 °C to 37 °C (Herpin et al. 2002). Body temperature then gradually rises to 39 °C (the normal porcine temperature) within 48 h after birth, indicating that homeothermy is progressively achieved (Mount 1968). In order to adapt to this large change in environmental temperature, most mammalian species possess brown adipose tissue (Nedergaard & Cannon 1992), a tissue specialized for heat production via the mitochondrial uncoupling protein (UCP)-1, which is unique to brown adipose tissue. UCP1 is located in the inner mitochondrial membrane and rapidly generates heat by uncoupling the normal cellular production of ATP and dissipating the energy produced as heat (Lin & Klingenberg 1982). Pigs however, are one of the few species so far found to lack brown adipose tissue (Trayhurn et al. 1989). Consequently, piglets may utilize other strategies of heat production and conservation, including muscular shivering in order to maintain homeothermy.
UCP2 is a recently discovered member of the UCP family (Fleury et al. 1997) and has postulated roles in energy regulation (Walder et al. 1998, Boss et al. 2000), production of reactive oxygen species (ROS) (Negre-Salvayre et al. 1997, Kizaki et al. 2002) and apoptosis (Voehringer et al. 2000). However, research into the role of UCP2 has been hampered by a lack of specific antibodies (Pecqueur et al. 2001). The first evidence for the presence of UCP2 mRNA (and another member of the UCP family, UCP3) in porcine tissue was published by Damon et al.(2000), who found UCP2 mRNA to be expressed in both adipose tissue and skeletal muscle. Molecular regulation of UCP2 is highly complicated in that changes in mRNA expression do not always mirror changes in protein.
UCP3 is present in adipose tissue and skeletal muscle as well as cardiac muscle and is expressed in skeletal muscle of the neonatal pig (Damon et al. 2000). UCP3 has a number of proposed roles in the regulation of fatty acid metabolism (Brun et al. 1999, Himms-Hagen & Harper 2001, Schrauwen et al. 2001, Wang et al. 2003), protecting against obesity (Walder et al. 1998) and ROS accumulation (Vidal-Puig et al. 2000). These functions, however, have been disputed (Nedergaard & Cannon 2003), and to date their function has been described only in rodents.
The existence of UCP2 and 3 in adipose tissue and skeletal muscle of a number of species and their regulation by fatty acids (Ricquier 1999) and triiodothyronine (T3) (Lanni et al. 2003) has prompted the investigation of whether UCP2 or 3, like UCP1, have a role in heat production (Samec et al. 1998). All studies to date suggest that UCP2 and 3 do not have a major role in heat production (Nedergaard et al. 2001, Stuart et al. 2001), but this has not been investigated in a species that does not express UCP1, such as the pig.
It is well documented that Meishan piglets, an ancient oriental breed, have a significantly lower neonatal mortality rate than commercial breeds (Le Dividich et al. 1991), despite being smaller at birth and having larger litters (Le Dividich et al. 1991, Herpin et al. 1993). Meishan sows are obese and produce milk with a higher fat content than commercial sows (Alston-Mills et al. 2000), and this may be one factor responsible for the apparent resistance to hypoglycaemia and hypothermia observed in their piglets. These factors have prompted pig producers to introduce the Meishan genome into commercial breeds of pig, such as the Large White. It is not known, however, whether there are breed-related differences in UCP2 or 3 expression and abundance in adipose tissue or skeletal muscle of these pigs. UCP1 and, more recently, UCP2 and 3 have been shown to be regulated by free fatty acids (Brun et al. 1999, Samec et al. 1999). With the observed differences in milk composition, it is possible that fatty acids may play a role in the regulation of uncoupling activity in pigs. The aim of this study, therefore, was to investigate the differences in UCP2 and 3 in adipose tissue and skeletal muscle between two breeds of pig, and whether either is involved in heat production. We hypothesize that UCP expression will be related to genotype, temperature and endocrine profile in the newborn pig.
Materials and Methods
Thirteen Meishan sows and 17 commercial sows of known mating date and parity were used in the study. At around 4 h after birth, all piglets in the litter were weighed, and the three closest to the median weight were randomly assigned to be tissue sampled on either day 0 (Meishan n=7, commercial n=5), 4 (Meishan n=7, commercial n=6), 7 (Meishan n=6, commercial n=5), 14 (Meishan n=9, commercial n=6) or 21 (Meishan n=7, commercial n=6) of postnatal age. At the same time of each day, colonic temperature was measured, piglets were weighed and a venous blood sample taken. On the assigned sampling day, piglets were humanely killed with an overdose of barbiturate anaesthetic (200 mg/kg pentobarbital sodium: Euthatal: RMB Animal Health, Stoke, Staffs, UK). The tissues were rapidly dissected, weighed, placed in liquid nitrogen and stored at −80 °C for subsequent analyses. All operative procedures and experimental protocols had the required Home Office approval as designated by the Animals (Scientific Procedures) Act (1986).
Plasma concentrations of glucose (CV=5.3%), triacylglycerol (CV=6.9%) (Sigma Chemical, St Louis, MO, USA) and non-esterified fatty acids (NEFA) (CV=2.8%) (Wako NEFA-C, Alpha Labs, Eastleigh, Hants, UK) in plasma were determined enzymatically (Schermer et al. 1996). Total plasma triiodothyronine (T3) (CV=4.9) and thyroxine (T4) (CV=8.4) concentrations were assessed by radio-immunoassay (ICN Pharmaceuticals, Basingstoke, UK).
Mitochondrial fractions were prepared from subcutaneous adipose tissue as described by Schermer et al.(1996). Protein content of each preparation was determined by the Lowry assay (Lowry et al. 1951) and abundance of UCP2 was determined in 10 μg of mitochondria by the same antibody as described by Pecqueur et al.(2001) at a dilution of 1 in 10 000. This antibody has recently been validated for use in mitochondria from another large mammal, the sheep (Mostyn et al. 2003). Specificity of detection was confirmed with non-immune rabbit serum and tissue from a UCP2 knockout mouse model. Attempts were made to obtain a porcine-specific UCP3 antibody, but all antibodies tested (anti-human and anti-rat) produced a number of non-specific bands. Therefore, no UCP3 abundance measurements are available. A range of molecular weight markers were included on all gels. Densitometric analysis was performed on each membrane following image detection with a Fujifilm LAS-1000 cooled CCD camera (Fuji Photo Film Co., Tokyo, Japan), and results were expressed in densitometric units. All gels were run in duplicate and a reference sample (a 4-day-old adipose tissue mitochondrial sample) was included on each to allow comparison between gels.
Total RNA was isolated from adipose and muscle tissue with Tri-Reagent (Sigma, Poole, UK), as described by Mostyn et al.(2002). To maximize sensitivity, a two-tube approach to reverse transcription (RT) was adopted, and the conditions used to generate first-strand cDNA were 70 °C (5 min), 4 °C (5 min), 25 °C (5 min), 25 °C (10 min), 42 °C (1 h), 72 °C (10 min) and 4 °C (5 min). The RT reaction (final volume, 20 μl) contained 1 μg total RNA, 5×cDNA (first-strand) buffer (250 mM Tris–HCl, 40 mM MgCl2, 150 mM KCl, 5 mM dithioerythritol, pH 8.5), 2 mM dNTPs, 1 × hexanucleotide mix, 10 units RNase inhibitor and 10 units M-MLV reverse transcriptase. All of these commercially available products were purchased from Roche Diagnostics Ltd (Lewes, UK).
The expression of mRNA for UCP2 was determined by utilizing the following set of cDNA primers to the porcine UCP2 and UCP3 genes: UCP2 forward: 5′-cttctgcggttcctctgtgt-3′ and reverse: 5′-cataggtcaccagctcagca-3′ (Genbank AF036757); UCP3 forward: 5′-gacgtggtgaaggttcgatt-3′ and reverse: 5′-cgagttcatgtaccgggtct-3′ (Genbank AF128837). Intron-spanning products of 641 and 330 base pairs respectively were generated to exclude amplification of genomic DNA. QuantumRNA alternate 18S internal standards (Ambion, Abingdon, UK) were also used to check for equal loading. Briefly, the incubation conditions were 94 °C (2 min) 1 cycle; 94 °C (30 s), 60.3 °C (30 s), 72 °C (1 min) 30 cycles, and 72 °C (7 min) 1 cycle. The PCR reaction (final volume, 20 μl) contained 10×PCR buffer (100 mM Tris–HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3), 500 μM dNTPs, 1 mM of each UCP2 primer, 3.75 U Taq polymerase. Agarose gel electrophoresis (2.0%) and ethidium bromide staining confirmed the presence of both UCP2/3 and 18S products of the expected sizes. All gels were run in duplicate with a standard run on all gels. Densitometric analyses were carried out as Western blotting, and the results, in arbitrary units, were expressed as the ratio of an 18S rRNA internal control and internal standard.
All statistical evaluations were performed by using SPSS 10.0 for Windows using the General Linear Model procedure with correction for repeated measures. Differences between postnatal age and genotype were investigated. All values presented are means with their standard errors.
Temperature regulation, piglet growth, UCP mRNA and protein abundance
Colonic temperature was higher (P<0.05) in the commercial piglets throughout the study (Fig. 1A). This was most pronounced on the first day of life when Meishan piglets had a colonic temperature almost 1.5 °C lower than commercial piglets. Although Meishan piglets exhibited an initially lower body temperature, all piglets attained a normal body temperature of approximately 39 °C by day 4 of postnatal age. Commercial piglets were heavier (P<0.05) throughout the study (Fig. 1B) and had an approximate growth rate of 0.34 kg/day compared with 0.13 kg/day (P<0.01) in Meishan piglets.
UCP2 mRNA and protein in adipose tissue were similar between breeds on day 0, with mRNA rising in the commercial group, but not Meishan group, to be significantly different (P<0.05) on days 4, 7 and 14 (Fig. 2A). In contrast, the Meishan group exhibited a pronounced (P<0.05) upregulation of UCP2 protein not observed in the commercial group, whose abundance remained low (Fig. 2B). UCP3 mRNA expression in adipose tissue was similar between breeds up to day 7; thereafter, expression was greater (P<0.05) in Meishan piglets (Fig. 3). Conversely, in skeletal muscle, UCP3 mRNA expression was similar up to day 7 when it was lower (P<0.0.5) in the Meishan group (Fig. 4).
Plasma hormones and metabolites
Plasma glucose concentrations were similar during the immediate postnatal period (days 0 and 4) between breeds, after which commercial piglets exhibited elevated (P<0.05) plasma glucose (Fig. 5A). Circulating NEFA concentrations were similar between breeds during the first week of life (Fig. 5B), but thereafter were upregulated (P<0.05) in the Meishan group. Plasma triacylglycerol was also regulated differentially between the piglet breeds; concentrations were higher in the Meishan group on day 0 and then lower throughout the study, although this reached statistical significance only on days 0, 4 and 14 (Fig. 5C).
Commercial piglets exhibited higher plasma triiodothyronine (T3) on day 0 than Meishan piglets, resulting in a threefold difference in plasma T3 (Fig. 6A), but these differences were not maintained due to a dramatic decline (P<0.01) in T3 in the commercial group. Plasma thyroxine (T4) values, however, were similar between breeds throughout the study period (Fig. 6B).
A number of significant age-dependent relationships were observed between metabolic, molecular and hormonal parameters throughout the study, as outlined in Table 1. The strongest relationships were observed on day 0 in the Meishan group. Adipose tissue UCP3 mRNA expression was found to be positively correlated to triacylglycerols and NEFA but negatively correlated to colonic temperature. A strong relationship was also present between body weight and colonic temperature in the commercial piglets on day 0.
The major findings of the present study were the differential influences of porcine breed and postnatal age on UCP2 and 3 mRNA expression in adipose tissue and skeletal muscle. A novel and significant finding was the marked discrepancy between protein abundance and mRNA expression of UCP2 in porcine adipose tissue.
Discrepancy between UCP2 mRNA and protein
This is the first study to demonstrate the presence of UCP2 protein in the pig, although previous authors have reported the presence of UCP2 mRNA in adipose tissue and skeletal muscle of pigs (Damon et al. 2000). As previously described in the mouse, we have shown a clear discrepancy between UCP2 mRNA expression and protein abundance (Pecqueur et al. 2001), confirming the complex regulation of UCP2 in a large mammal, the pig. A short open reading frame (ORF) in exon 2 of the human and mouse UCP2 gene has been described (Pecqueur et al. 1999) that substantially increases in UCP2 protein production in vitro (Pecqueur et al. 2001) in the absence of any changes in UCP2 mRNA. ATG sequences in the ORF strongly inhibit translation of UCP2 mRNA (Pecqueur et al. 2001). The homology (88%) between porcine and human UCP2 is high; therefore, it is predicted that a similar ORF will be present in the porcine gene, although this has yet to be confirmed. The mechanism by which UCP2 protein is upregulated with no change in mRNA remains to be established but appears to be strongly influenced by genotype.
Influence of breed on UCP2 and 3
We have shown differential regulation for both UCP2 and UCP3 by genotype and tissue type. UCP2 abundance was higher in Meishan. Raised protein, but not mRNA, in Meishan piglets could have a number of implications with respect to the physiological regulation of lipids, fatty acid transport out of the mitochondria and protection against ROS buildup. Both UCP2 and 3 are postulated to allow extrusion of protonated fatty acids out of the mitochondrial matrix (Schrauwen et al. 2001). Reduced UCP2 protein in commercial piglets may suggest a greater risk of peroxidative damage due to fatty acid build up in the mitochondria. However, as discussed below, this may be offset by a reduced lipid intake by the commercial piglets.
UCP2 and 3 have been proposed to have a role in thermoregulation, although this is disputed (Nedergaard & Cannon 2003), particularly in species that express UCP1, whose role in heat production is undisputed. As pigs do not express UCP1, it was postulated that UCP2 or 3 may have this role. However, we found no relationship between UCP2/3 abundance and temperature, because, despite an almost twofold increase in UCP2 protein abundance in fat, Meishan piglets remained colder throughout the study. It seems likely that the increased UCP2 and 3 abundance and mRNA expression respectively in fat may promote lipid catabolism as suggested by previous work mimicking conditions of high plasma NEFA, as during fasting or consumption of a high-fat diet (Samec et al. 1998). This would be predicted to protect against fatty acid buildup within the mitochondria, which can be peroxidative. Enhanced UCP2 protein abundance in Meishan piglets could, therefore, be a response to the greater intake of fat through maternal milk. UCP2 and UCP3 abundance were negatively correlated to colonic temperature on days 0 and 21, further disproving the hypothesis that UCP2/3 are involved in temperature regulation in the pig.
Influence of breed on temperature regulation and endocrine profile
There was a divergence in colonic temperature between breeds throughout the study. Immediately after birth, commercial piglets exhibited a higher colonic temperature and achieved a normorthermic body temperature (39 °C) by 4 days of age, a response which was delayed in the Meishan piglets. This result is not unexpected given that Meishan piglets are known to be resistant to hypothermia (Le Dividich et al. 1991). Thus, the lower body temperature observed in Meishan piglets may be of benefit in reducing mobilization of limited body fat reserves. A lower metabolic rate would reduce the amount of fat required for oxidation to produce heat for the newborn piglet, and thus improve their survival chances.
Thyroid hormones play an important role in the initiation of independent temperature regulation at birth in a number of species such as the sheep and human (Gunn & Gluckman 1995, Schermer et al. 1996). This is due to the positive thermogenic influence of T3 on UCP1 (Rabelo et al. 1995). We found that genotype had a marked effect on postnatal plasma hormones and metabolites. T3 was particularly sensitive to genotype on day 0, with Meishan piglets possessing 30% less circulating T3 than commercial piglets, despite similar T4 concentrations. As demonstrated previously in Polish Landrace piglets (Slebodzinski et al. 1981), T4 concentrations were highest during the first 24 h of life in both commercial and Meishan piglets. The commercial piglets followed the pattern of T4 previously demonstrated (Slebodzinski et al. 1981); however, Meishan piglets did not increase plasma T4 between days 7 and 14. It is possible that the lower plasma concentrations of adrenaline previously described in Meishan piglets (Le Dividich et al. 1991) may be partly responsible for the lower T3, as catecholamines have a stimulatory effect on 5′deiodinase, which catalyses the conversion of T4 to T3.
Piglet breed also appeared to influence the regulation of plasma triacylglycerol and NEFA. On the first day of postnatal age, plasma NEFA were very low and similar between breeds, as expected since piglets are born with little fat (Mount 1968). However, plasma triacylglycerol was significantly elevated in the Meishan piglets, a finding which may be partly explained by the provision of higher milk fat content from Meishan sows (Alston-Mills et al. 2000). By day 21, plasma NEFA were higher in Meishan piglets and are likely to reflect the greater rate of fat deposition in Meishans (Kouba et al. 1999).
As described above, UCP3 may be involved in the regulation of fatty acids. Regression analysis demonstrated a number of relationships between UCP3 and factors known to influence metabolism such as T3, NEFA and triacylglycerols (Jezek et al. 1994, Winkler & Klingenberg 1994, Branco et al. 1999, Jekabsons et al. 1999). On day 0, UCP3 mRNA expression in adipose tissue was positively correlated to plasma triacylglycerol and NEFA in Meishan piglets, but negatively related to temperature; on day 4, plasma NEFA was positively correlated with skeletal muscle UCP3 mRNA expression. This gives further evidence for the role of UCP3 in regulating lipid metabolism. Excess NEFA has been shown to increase UCP3 mRNA expression (Jezek 1999). The regulation of UCP3 by NEFA may be more important in Meishan piglets, who acquire more fat through maternal milk.
Interestingly, a negative relationship was observed between plasma T3 and skeletal muscle UCP3 mRNA expression on days 0 and 21 in the commercial piglets. Previous studies have demonstrated a stimulatory effect of T3 on UCP3 expression (Gong 1997, Lanni et al. 1999); however, it is difficult to make direct comparisons as these experiments were carried out on adult rodents.
In conclusion, we have demonstrated for the first time the existence of UCP2 protein in porcine adipose tissue and its differential regulation by breed and postnatal age. We have further shown that genotype and postnatal age have a significant influence on the expression and abundance of UCP2 and 3 via the distinctive endocrine profiles associated with each genotype, and have further confirmed the complex regulation of UCP2. The influence of genotype is most prevalent after 1 week of age when homoeo-thermy is established in the pig. Finally, we have further demonstrated that UCP2 and 3 appear not to be involved in heat production in the neonatal pig.
Significant associations between endocrine, physiological and metbolic parameters measured during the study
|Breed||X axis||Y axis||R2||n||Pvalue||Curve|
|NEFA, non-esterified fatty acids; T3, tri-iodothyronine; aUCP3 mRNA, adipose tissue UCP3 messenger RNA; mUCP3 mRNA, skeletal muscle UCP3 messenger RNA; C, commercial; M, Meishan.|
This work was supported by Biotechnology and Biological Sciences Research Council Grant S15331. J.C. Litten was supported by a Wye College studentship, and B. Miroux’s contribution by CNRS Centre de la Recherche Scientifique.
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