J. Anim Sci. 2006. 84:1761-1766. doi:10.2527/jas.2005-519
© 2006 American Society of Animal Science
The relationships among mitochondrial uncoupling protein 2 and 3 expression, mitochondrial deoxyribonucleic acid single nucleotide polymorphisms, and residual feed intake in Angus steers1
W. H. Kolath*,
M. S. Kerley*,2,
J. W. Golden*,
S. A. Shahid
and
G. S. Johnson
* Division of Animal Sciences and
and
Department of Veterinary Pathobiology, University of Missouri, Columbia 65211
 |
Abstract
|
|---|
The objective of this study was to determine the relationships of uncoupling protein 2 and 3 expression, SNP of mitochondrial DNA, and residual feed intake (RFI) in Angus steers selected to have high or low RFI. Individual feed intake was measured via the GrowSafe feed intake system over a 3-mo period and used to calculate RFI, a measure of efficiency. Based on these calculations, 6 low- (average RFI = –1.57 kg) and 6 high- (average RFI = 1.66 kg) RFI steers were selected for further study. Blood was collected via jugular venipuncture 1 wk before slaughter for the isolation of mitochondrial DNA. The steers were then killed to collect LM for the measurement of uncoupling protein 2 and 3 mRNA and protein expression. Protein and mRNA expression of uncoupling protein 2 and 3 were determined by Western blotting and quantitative PCR, respectively. To determine SNP of mitochondrial DNA, total DNA was isolated from blood via standard phenol/chloroform extraction; fragments were amplified with PCR and sequenced with an automated nucleotide sequencer. Average daily gain and carcass composition were not different (P > 0.13) between the high- and low-RFI steers; however, ADFI by the high-RFI animals was 3.77 kg greater (P < 0.001) than the low-RFI animals. No difference (P > 0.55) was observed between the high- and low-RFI animals in their expression of uncoupling protein 2 or 3 mRNA or protein. On average 9.8 and 8.9 polymorphisms were found per mitochondrial genome for the low- and high-RFI steers, respectively. None of these polymorphisms were related to RFI. It seems that the expression of uncoupling protein 2 and 3 and mitochondrial DNA sequence are not related to RFI status.
Key Words: feed efficiency • mitochondria • residual feed intake
|
INTRODUCTION
|
|---|
Mitochondria are the primary site of cellular energy production and produce the majority of ATP used to drive cellular processes. The electron transport chain is composed of 83 subunits, of which 70 and 13 are encoded by the nuclear and mitochondrial genome, respectively (Leonard and Schapira, 2000
). Mutations of mitochondrial DNA have been shown to alter mitochondrial energy production in humans, and a number of disease states are characterized by the presence of one or more mitochondrial DNA mutations (Wallace, 1999).
Many other nuclear-encoded proteins are involved in mitochondrial function including inner membrane transporters such as adenine nucleotide translocator and uncoupling protein 2 and 3, whose functions have yet to be elucidated. One of the hypothesized roles of uncoupling protein 2 and 3 is to "uncouple" oxidative phosphorylation from electron transport by transporting protons back into the mitochondrial matrix. Uncoupling protein 2 or 3 null mice have been shown to have reduced proton leak (Krauss et al., 2002), and therefore uncoupling proteins could modulate mitochondrial energy production.
Previous work in cattle (Kolath et al., 2005) and poultry (Bottje et al., 2002) has provided evidence of a link between mitochondrial respiration and feed efficiency. We hypothesized that 2 mechanisms could explain this observation. First, that increased expression of uncoupling protein 2, 3, or both in high-residual feed intake (RFI) steers would uncouple the proton gradient and thereby increase the energy requirements of the animal to produce the same quantity of ATP. Second, polymorphisms of mitochondrial DNA in high-RFI steers would reduce the function of the electron transport chain, altering the rate of mitochondrial respiration. Therefore, the objective of this study was to determine the relationships between uncoupling protein 2 and 3 expression and mitochondrial DNA sequence in Angus steers selected to have high or low RFI.
|
MATERIALS AND METHODS
|
|---|
Animal Management
The research protocols used in this study were approved by the University of Missouri Animal Care and Use Committee (No. 3278). Eighty Angus steers (average initial BW = 262.2 ± 21.75 kg) were used to select high- and low-RFI animals. Steers were obtained from a single herd enrolled in the MFA Health Track Beef Alliance, Columbia, MO, and had been previously vaccinated and preconditioned for 45 d before arrival at the University of Missouri Beef Research Farm.
Upon receiving the animals, electronic identification tags (Allflex USA Inc., Dallas-Ft. Worth Airport, TX) were attached to the exterior of the left ear for the measurement of individual feed intake with the GrowSafe feed intake system (GrowSafe Systems Ltd., Airdrie, AB, Canada). The GrowSafe system (model 4000E) consisted of a total of 16 nodes with 2 nodes per pen. Ten animals were housed in each of the 8 pens with 5 animals per node. Data were collected over the entire feeding period of approximately 160 d. Days in which there was a hardware malfunction or power failure or feed leaks exceeded 3% were removed from the analysis. Average daily leak throughout the experimental period, excluding days removed from the analysis, was 0.48 ± 0.66%.
Steers were placed on a receiving diet for 14 d to allow for acclimation to the feeding system. The composition of the experimental diet fed for the remainder of the experimental period is shown in Table 1. All steers had ad libitum access to both feed and water. Steers were weighed every 28 d, and RFI was calculated for the entire feeding period. Expected feed intake was calculated by regressing actual intake against ADG and metabolic midweight (Basarab et al., 2003). The RFI value for each animal was calculated as the difference between the actual and expected intakes.
Six high- and 6 low-RFI steers were selected based on their RFI values and were transported to the University of Missouri Abattoir where the animals were killed; tissue was obtained from the longissimus lumborum, frozen in liquid nitrogen, and stored at – 80° C until further study. Hot carcass weights were recorded for each animal, and the carcasses were chilled for a 24-h period at 5° C.
After the 24-h chill, a beef LM area dot grid was used to measure LM area of each carcass to the nearest 0.01 cm2. Fat thicknesses were determined using a USDA preliminary yield grade ruler (USDA, 1997) at an anatomical location perpendicular to the vertebral column and ³/3 of the distance caudal to the LM. To determine preliminary yield grades, the fat measurements were then adjusted, correcting for any atypical fat distribution. Marbling scores were identified by an experienced grader using the USDA marbling standards (USDA, 1997; Abundant, Moderately Abundant, Slightly Abundant, Moderate, Modest, Small, Slight, Traces, and Practically Devoid). Maturity scores were also assessed using the USDA standards (USDA, 1997) for animals older than A maturity.
RNA Isolation and Quantitative Real-Time PCR
Total RNA was isolated using the Trizol procedure (Invitrogen Life Technologies, Carlsbad, CA). After isolation, RNA was suspended in molecular biology-grade H2O. The RNA concentration of the samples was determined by measuring the absorbance at 260 nm. The purity of the isolated RNA was verified by measuring the ratio of absorbencies between 260 and 280 nm, and by separating 2.5 µg of RNA on a 0.8% agarose gel in 0.09 M Tris-borate, 0.002 M EDTA, with 0.5 µg of ethidium bromide/mL. Total RNA was then reverse-transcribed using the Superscript First Strand synthesis system for reverse transcription-PCR (Invitrogen Life Technologies).
Primers and TaqMan probes for uncoupling protein 2, uncoupling protein 3, and cyclophilin A (which was used as a housekeeping gene) were designed using the Primer Express software (Applied Biosystems, Foster City, CA; Table 2). Amplification was performed in triplicate using Taqman Universal PCR master mix (Applied Biosystems), and fluorescence was detected with the ABI Prism 7700 sequence detector (Applied Biosystems). The data were analyzed using the Sequence Detection Software (Applied Biosystems), and expression levels were calculated by subtracting the cycle threshold value for cyclophilin A from the gene of interest.
Western Blotting
Frozen tissue samples were homogenized in PBS (137 mM NaCl, 3 mM KCl, 6.5 mM Na2PO4, and 3.5 mM KH2PO4), centrifuged at 500 x g for 10 min, and the supernatant was aspirated. The protein concentration of the supernatant was determined with a BCA protein assay kit (Pierce Biotechnology Inc., Rockford, IL). Thirty micrograms of protein was fractionated on 10% SDS-Page gels and then blotted to polyvinylidene fluoride membranes overnight. An Enhanced NuGlo western blotting kit (Alpha Diagnostics Inc., San Antonio, TX) was utilized for blocking and development of the blots. Antibodies against uncoupling protein 2 and 3 were purchased from Alpha Diagnostics. The primary antibodies were diluted 1:750 and 1:1,000 for uncoupling protein 2 and 3, respectively. The blots were exposed to Hyperfilm ECL (GE Healthcare, Piscataway, NJ), and the density of the each band was determined using 1D Scan EX software (Scanalytics Inc., Fairfax, VA).
Mitochondrial DNA Sequencing
Blood samples were collected via jugular venipuncture from the 12 selected animals, into Vacutainers (Becton, Dickinson and Company, Franklin Lakes, NJ) containing EDTA as an anticoagulant, 1 wk before the transport of the steers to the University of Missouri Abattoir for tissue collection. Standard phenol/chloroform extraction was used to extract DNA from the blood samples. The primers used for PCR amplification were based on the GenBank Bos taurus mitochondrial genome (GenBank Accession No. NC_001567) and were designed with Primer Premier 5 software (Premier Bio-soft International, Palo Alto, CA). Twenty-one primer pairs were used to amplify fragments that overlapped adjacent fragments by approximately 100 bp. Eleven additional single primers were used to resolve regions that the original 21 primer pairs could not.
Amplified PCR products were verified by agarose gel electrophoresis. Most amplicons were purified using QI-Aquick PCR purification columns (Qiagen Inc., Valencia, CA). In some cases in which byproducts were detected in the agarose gel, the fragment of interest was purified by preparative polyacrylamide gel electrophoresis (Shibuya et al., 1993). A 377A automatic nucleotide sequencer (Applied Biosystems) and the BigDye kit (Applied Biosystems) were used to sequence all purified PCR products. The resulting sequences were edited and assembled using GeneTool 2.0 (BioTools Inc., Edmonton, AB, Canada) to produce a single contiguous ~16,400-bp sequence for each steer. The assembled mitochondrial DNA sequences were compared with one another and to the original sequence in GenBank to discover polymorphic sites with GeneTool 2.0’s multialign feature.
Statistical Analysis
The data were analyzed using the GLM Procedure (SAS Inst. Inc., Cary, NC) as a completely randomized design, with animal as the experimental unit and RFI group as a fixed effect. An alpha level of 0.05 was used for the determination of statistical significance.
|
RESULTS AND DISCUSSION
|
|---|
The performance of the high- and low-RFI steers is shown in Table 3. There were no differences (P > 0.13) in initial or final BW or ADG between the 2 groups. However, G:F was increased (P < 0.001) for the low-RFI steers, and ADFI was greater (P < 0.001) for the high-RFI steers, which consumed 3.77 kg/d more feed than the low-RFI steers. Basarab et al. (2003) and Kolath et al. (2005) have reported similar data in which feed intake was greater (P < 0.001) for the high-RFI animals and G:F was increased (P < 0.001) in low-RFI steers, but ADG and BW of high- and low-RFI steers were not different (P > 0.80). Carcass composition as assessed by LM area, fat thickness over the 12th rib, HCW, USDA yield grade, and marbling score were not different (P > 0.45) between the high- and low-RFI groups. These data agree with previous reports from our laboratory (Kolath et al., 2005) in which carcass composition was not altered by RFI status. Other authors (Richardson et al., 2001; Basarab et al., 2003) have reported increased fat deposition in steers selected to have high-RFI.
The locations of SNP in the mitochondrial DNA sequence of high- and low-RFI steers are shown in Tables 4 to 8. On average, 9.8 and 8.9 mutations in the mitochondrial DNA sequence of high- and low-RFI steers, respectively, were found compared with the Genbank Bos taurus mitochondrial complete genome (GenBank Accession: NC_001567). At 3 locations (587, 9,682, and 13,310 bp) all 12 steers differed from the published sequence, indicating a possible error in the Genbank sequence. Multiple polymorphisms were found in the D-loop region in both the high- and low-RFI animals with the majority of the polymorphisms being found in at least one steer of both the high- and low-RFI groups. Only 1 steer in the low-RFI group contained a given polymorphism found in the genes of cytochrome c oxidase subunits I and III, NADH dehydrogenase subunit 4L, cytochrome B, and the serine transfer RNA. None of the animals in the high-RFI group contained a mutation in these genes. One steer in the high-RFI group contained a polymorphism in the leucine transfer RNA. At least 1 steer in both the high- and low-RFI groups contained a mutation in the following genes: NADH dehyrdrogenase subunits 1, 2, 4, 5, and 6, cytochrome c oxidase subunit 2, ATP synthase F0 subunit 6, and both ribosomal RNA. Only 2 of the 13 protein genes, NADH dehydrogenase subunit 3 and ATP synthase subunit 8 were not found to contain any polymorphisms. Nineteen of the transfer RNA genes also did not contain any polymorphisms. The lack of mutations across animals in either group indicated that polymorphisms of mitochondrial DNA are not related to the RFI status in a contemporary group of Angus steers.
View this table:
[in this window]
[in a new window]
|
Table 5. Locations of SNP in the mitochondrial DNA sequence of high or low residual feed intake (RFI) steers (continued from Table 4 )
|
|
View this table:
[in this window]
[in a new window]
|
Table 6. Locations of SNP in the mitochondrial DNA sequence of high or low residual feed intake (RFI) steers (continued from Table 5)
|
|
View this table:
[in this window]
[in a new window]
|
Table 7. Locations of SNP in the mitochondrial DNA sequence of high or low residual feed intake (RFI) steers (continued from Table 6)
|
|
View this table:
[in this window]
[in a new window]
|
Table 8. Locations of SNP in the mitochondrial DNA sequence of high or low residual feed intake (RFI) steers (continued from Table 7)
|
|
No difference (P > 0.55) was observed in the expression of uncoupling protein 2 and 3 mRNA or protein between the high- or low-RFI groups (Table 9). This result would indicate no difference in the amount of uncoupling of oxidative phosphorylation and electron transport by uncoupling protein 2 and 3. Recent evidence (Krauss et al., 2005) indicated that these proteins have roles in modulating reactive oxygen species production rather than an uncoupling role. Echtay et al. (2002) have shown that uncoupling protein 2 and 3 increase proton leak when superoxide is present thereby protecting the cell from rampant superoxide production. Also, the 100-fold lower expression of uncoupling protein 2 and 3 compared with uncoupling protein 1 would point to a limited role in altering energy expenditure (Pecquer et al., 2001). This evidence along with the lack of differences in superoxide production (Kolath et al., 2005) between high- and low-RFI animals would indicate that uncoupling protein 2 and 3 do not play a role in altering RFI status.
View this table:
[in this window]
[in a new window]
|
Table 9. Expression of uncoupling protein 2 and 3 mRNA and protein of high or low residual feed intake (RFI) steers1
|
|
|
Footnotes
|
|---|
1 Acknowledgements: This research was supported in part by a USDA special programs grant (No. 2003-34450-14578). 
2 Corresponding author: kerleym{at}missouri.edu
Received for publication September 14, 2005.
Accepted for publication February 15, 2006.
|
LITERATURE CITED
|
|---|
Basarab, J. A., M. A. Price, J. L. Aalhus, E. K. Okine, W. M. Snelling, and K. L. Lyle. 2003. Residual feed intake and body composition in young growing steers. Can. J. Anim. Sci. 83:189–204.
Bottje, W., Z. X. Tang, M. Iqbal, D. Cawthon, R. Okimoto, T. Wang, and M. Cooper. 2002. Association of mitochondrial function with feed efficiency within a single genetic line of male broilers. Poult. Sci. 81:546–555.[Abstract/Free Full Text]
Echtay, K. S., D. Roussel, J. St-Pierre, M. B. Jekabsons, S. Cadenas, J. A. Stuart, J. A. Harper, S. J. Roebuck, A. Morrison, S. Pickering, J. C. Clapham, and M. D. Brand. 2002. Superoxide activates mitochondrial uncoupling proteins. Nature 415:96–99.[CrossRef][Medline]
Kolath, W. H., M. S. Kerley, J. W. Golden, and D. H. Keisler. 2006. The relationship between mitochondrial function and residual feed intake in Angus steers. J. Anim. Sci. 84:861–865.[Abstract/Free Full Text]
Krauss, S., C. Zhang, and B. B. Lowell. 2002. A significant portion of mitochondrial proton leak in intact thymocytes depends on expression of UCP2. Proc. Natl. Acad. Sci. USA 99:118–122.[Abstract/Free Full Text]
Krauss, S., C. Zhang, and B. B. Lowell. 2005. The mitochondrial uncoupling-protein homologues. Nature Reviews 6:248–261.[Medline]
Leonard, J. V., and A. H. V. Schapira. 2000. Mitochondrial respiratory chain disorders I: Mitochondrial DNA defects. Lancet 355:299–304.[CrossRef][Medline]
NRC. 1996. Nutrient Requirements of Beef Cattle. 7th ed. Natl. Acad. Press, Washington, DC.
Pecquer, C., M. Alves-Guerra, C. Gelly, C. Levi-Meyrueis, E. Couplan, S. Collins, D. Ricquier, F. Bouillaud, and B. Miroux. 2001. Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation. J. Biol. Chem. 276:8705–8712.[Abstract/Free Full Text]
Richardson, E. C., R. M. Herd, V. H. Oddy, J. M. Thompson, J. A. Archer, and P. F. Arthur. 2001. Body composition and implications for heat production of Angus steer progeny of parents selected for or against residual feed intake. Aust. J. Exp. Agric. 41:1065–1072.[CrossRef]
Shibuya, H., D. J. Nonneman, T. H. M. Huang, V. K. Ganjam, F. A. Mann, and G. S. Johnson. 1993. Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene. Anim. Genet. 24:345–348.[Medline]
USDA. 1997. USDA Official United States standards for grading of carcass beef. Agric. Marketing Serv. USDA, Washington, DC.
Wallace, D. C. 1999. Mitochondrial diseases in man and mouse. Science 283:1482–1488.[Abstract/Free Full Text]
Top
Abstract
INTRODUCTION
