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Immune system stimulation by repeated lipopolysaccharide injections alters longissimus dorsi sarcoplasmic protein profile in pigs
178
Abstracts
Undergraduate Research Competition
Undergraduate Research Competition
162 Immune system stimulation by repeated lipopolysaccharide injections alters longissimus dorsi sarcoplasmic protein profile in pigs A. Outhousea,⁎, J. Grubbsa, C. Tugglea, N. Gablera, A. Rakhshandehb, S. Lonergana, aDepartment of Animal Science, Iowa State University, Ames, b Texas Tech University, Lubbock, United States
163 Small heat shock protein 27 may be related to toughness in loins of callipyge lambs T. Cramera,⁎, Y.H.B. Kima, M. Penicka, J. Waddellb, C. Bidwella, aAnimal Sciences, Purdue University, West Lafayette, bU, Stephenville, United States
Objectives: The objective of this study was to investigate the effects of immune system stimulation (ISS) in pigs divergently selected for residual feed intake (RFI) on the longissimus dorsi (LD) sarcoplasmic protein profile of pigs. Defining these changes will provide insight into how lean tissue growth is impacted by feed efficiency and ISS. Materials and Methods: Twelve pigs (6 per line, BW 63 ± 4 kg) from the Iowa State RFI project were used. Low RFI pigs are more efficient than their high RFI counterparts, providing a tool to study the underlying biology of feed efficiency in pigs. Three gilts from each line were injected intramuscularly every 48 h, for 7 d, with increasing amounts of E.coli lipopolysaccharide (ISS; initial dose: 30 μg/kg BW). The remaining three pigs from each line were injected with saline. Pigs were euthanized (day 7); LD tissue was collected and frozen. The sarcoplasmic protein fraction was then isolated and two-dimensional difference in gel electrophoresis (2D-DIGE) determined changes in the protein profile between ISS and Control and line (high verses low RFI). Protein identities were determined by mass spectrometry and were confirmed through 2D Western blots. Results: A total of 354 protein spots were resolved using 2D-DIGE. Fifty-five, and 29, protein spots were different in response to ISS, and selection for RFI, respectively (P b 0.05). Fourteen spots were selected for identification. Aldose reductase decreased in response to ISS by 32% (P = 0.002). Aldose reductase catalyzes the conversion of glucose to sorbitol. Fructose produced from sorbitol can be used as an energy source for glycolysis and glyconeogenesis. A decrease in abundance of aldose reductase may be due to a shift in energy utilization during ISS. Tripartite motif-containing protein 72 (TRIM-72) and alpha-enolase were both found to be changed in abundance due to RFI selection. Both TRIM-72 and alpha-enolase were increased in low RFI pigs by 88% (P = 0.03) and 45% (P = 0.005), respectively. A central role of TRIM-72 is cellular membrane repair. The observed increase in TRIM-72 may indicate low RFI pigs are better equipped to handle membrane damage due to cellular stress. Additionally, relative to the high RFI line, expression of the enzyme alpha-enolase, was higher in low RFI pigs indicating an increase in the metabolic activity of low RFI pigs. Bisphosphoglycerate mutase (BPGM), was affected by both ISS and genetic line (P = 0.0074). BPGM decreased 21% in response to ISS (P = 0.0036) and was increased 11% in low RFI pigs (P = 0.063). BPGM is responsible for the catalytic synthesis of 2,3-bisphosphoglycerate (2,3-BPG). When 2,3-BPG binds with to hemoglobin oxygen is released. An increase in BPGM shows a potential change in oxygen release in the muscle between the RFI lines and ISS. Conclusion: Based on protein abundance, ISS alters the capacity for energy metabolism in the muscle. Moreover, selection for reduced RFI alters the abundance of membrane repair proteins and enzymes involved in energy metabolism. Selection for RFI did not alter the response of and enzymes involved in energy metabolism. Selection for RFI did not alter the response of porcine muscle to ISS. This work was supported by the Agriculture and Food Research Initiative Competitive Grant no. 2011-68004-30336 from the USDA National Institute of Food and Agriculture.
Objectives: Callipyge lambs have a genetic mutation that produces tough meat. The responsible gene causes upregulated expression of calpastatin, which blocks the proteolytic activity of μcalpain during aging. Callipyge lambs have the genotype CN, with a paternal callipyge allele and a maternal allele, and produce tougher meat than the normal genotypes CC, NC, and NN. Small heat shock proteins (sHSP) are chaperone proteins that have a protective role in cell death. This process could be reversely related to the postmortem meat tenderization by hindering proteolytic enzymes from breaking down structural myofibrillar proteins. While several proteomic studies found that higher sHSP expression were observed in tougher meat compared to tender counterparts, the role of sHSP in toughness of CN lambs has never been investigated. Therefore, the objective of this study was to determine an involvement of sHSP in tenderness development of loins from callipyge lambs during postmortem aging. Materials and Methods: A total of 16 lambs, four from each genotype, was slaughtered. Muscle samples (M. longissmus dorsi) were collected at 15 min, 3, 6, and 9 days postmortem for protein extraction and tenderness measurement. Western blots were performed to determine the extent of degradation of the myofibrillar proteins desmin and troponin T over aging, as well as μ-calpain autolysis, calpastatin, HSP27 and aB-crystallin. Warner- Bratzler shear force (WBSF) measurements of loin chops across these aging times were conducted. The experimental design was a randomized complete block design, and data were analyzed by using mixed procedure of SAS to compare the traits across different genotypes and aging periods. Means were separated (F-test, P b 0.05) by least significant differences. Results: A significantly higher shear force value was observed in loins from CN compared to other genotypes regardless of aging time (P b0.05), as expected. Western blots for desmin and troponin T showed increased degradation products of those myofibrillar proteins with an increase in aging time, with less degradation products in the CN genotype than in the others (P b0.05). The extent of μ-calpain autolysis was also affected by genotype, where loins from CN exhibited more intact μ-calpain than loins from other genotypes throughout each aging time (P b0.05). Higher calpastatin expression in CN compared other genotypes was also found. A significant decrease in the extent of intact HSP27 with an increase in aging time was observed, while higher levels of intact HSP27 in CN compared to others was found in loins from CN regardless of aging time (P = 0.09). Further, the lower extent of degraded HSP27 in CN compared other genotype was also found over time (P b 0.001). No significant effects of genotypes and aging were found for aB-crystallin. Conclusion: A higher level of HSP27 was found in the CN genotype, which coincided with greater intact desmin and troponin T, less μ-calpain autolysis, and eventually higher shears force values as compared to other genotypes. This observation leads us to postulate that the up-regulation of HSP27 could be related to meat toughness in callipyge lamb meat by delaying muscle protein degradation during aging through its anti-apoptotic function. Further studies for the elucidation of the specific mechanisms and possible differences in sHSP behaviors across different muscle fiber types would be highly warranted.
Keywords: 2D-DIGE, immune system stimulation, protein profile, residual feed intake
Keywords: Callipyge, Small Heat Shock Proteins, Tenderness
doi:10.1016/j.meatsci.2015.08.173
doi:10.1016/j.meatsci.2015.08.174
Abstracts
Undergraduate Research Competition
Undergraduate Research Competition
162 Immune system stimulation by repeated lipopolysaccharide injections alters longissimus dorsi sarcoplasmic protein profile in pigs A. Outhousea,⁎, J. Grubbsa, C. Tugglea, N. Gablera, A. Rakhshandehb, S. Lonergana, aDepartment of Animal Science, Iowa State University, Ames, b Texas Tech University, Lubbock, United States
163 Small heat shock protein 27 may be related to toughness in loins of callipyge lambs T. Cramera,⁎, Y.H.B. Kima, M. Penicka, J. Waddellb, C. Bidwella, aAnimal Sciences, Purdue University, West Lafayette, bU, Stephenville, United States
Objectives: The objective of this study was to investigate the effects of immune system stimulation (ISS) in pigs divergently selected for residual feed intake (RFI) on the longissimus dorsi (LD) sarcoplasmic protein profile of pigs. Defining these changes will provide insight into how lean tissue growth is impacted by feed efficiency and ISS. Materials and Methods: Twelve pigs (6 per line, BW 63 ± 4 kg) from the Iowa State RFI project were used. Low RFI pigs are more efficient than their high RFI counterparts, providing a tool to study the underlying biology of feed efficiency in pigs. Three gilts from each line were injected intramuscularly every 48 h, for 7 d, with increasing amounts of E.coli lipopolysaccharide (ISS; initial dose: 30 μg/kg BW). The remaining three pigs from each line were injected with saline. Pigs were euthanized (day 7); LD tissue was collected and frozen. The sarcoplasmic protein fraction was then isolated and two-dimensional difference in gel electrophoresis (2D-DIGE) determined changes in the protein profile between ISS and Control and line (high verses low RFI). Protein identities were determined by mass spectrometry and were confirmed through 2D Western blots. Results: A total of 354 protein spots were resolved using 2D-DIGE. Fifty-five, and 29, protein spots were different in response to ISS, and selection for RFI, respectively (P b 0.05). Fourteen spots were selected for identification. Aldose reductase decreased in response to ISS by 32% (P = 0.002). Aldose reductase catalyzes the conversion of glucose to sorbitol. Fructose produced from sorbitol can be used as an energy source for glycolysis and glyconeogenesis. A decrease in abundance of aldose reductase may be due to a shift in energy utilization during ISS. Tripartite motif-containing protein 72 (TRIM-72) and alpha-enolase were both found to be changed in abundance due to RFI selection. Both TRIM-72 and alpha-enolase were increased in low RFI pigs by 88% (P = 0.03) and 45% (P = 0.005), respectively. A central role of TRIM-72 is cellular membrane repair. The observed increase in TRIM-72 may indicate low RFI pigs are better equipped to handle membrane damage due to cellular stress. Additionally, relative to the high RFI line, expression of the enzyme alpha-enolase, was higher in low RFI pigs indicating an increase in the metabolic activity of low RFI pigs. Bisphosphoglycerate mutase (BPGM), was affected by both ISS and genetic line (P = 0.0074). BPGM decreased 21% in response to ISS (P = 0.0036) and was increased 11% in low RFI pigs (P = 0.063). BPGM is responsible for the catalytic synthesis of 2,3-bisphosphoglycerate (2,3-BPG). When 2,3-BPG binds with to hemoglobin oxygen is released. An increase in BPGM shows a potential change in oxygen release in the muscle between the RFI lines and ISS. Conclusion: Based on protein abundance, ISS alters the capacity for energy metabolism in the muscle. Moreover, selection for reduced RFI alters the abundance of membrane repair proteins and enzymes involved in energy metabolism. Selection for RFI did not alter the response of and enzymes involved in energy metabolism. Selection for RFI did not alter the response of porcine muscle to ISS. This work was supported by the Agriculture and Food Research Initiative Competitive Grant no. 2011-68004-30336 from the USDA National Institute of Food and Agriculture.
Objectives: Callipyge lambs have a genetic mutation that produces tough meat. The responsible gene causes upregulated expression of calpastatin, which blocks the proteolytic activity of μcalpain during aging. Callipyge lambs have the genotype CN, with a paternal callipyge allele and a maternal allele, and produce tougher meat than the normal genotypes CC, NC, and NN. Small heat shock proteins (sHSP) are chaperone proteins that have a protective role in cell death. This process could be reversely related to the postmortem meat tenderization by hindering proteolytic enzymes from breaking down structural myofibrillar proteins. While several proteomic studies found that higher sHSP expression were observed in tougher meat compared to tender counterparts, the role of sHSP in toughness of CN lambs has never been investigated. Therefore, the objective of this study was to determine an involvement of sHSP in tenderness development of loins from callipyge lambs during postmortem aging. Materials and Methods: A total of 16 lambs, four from each genotype, was slaughtered. Muscle samples (M. longissmus dorsi) were collected at 15 min, 3, 6, and 9 days postmortem for protein extraction and tenderness measurement. Western blots were performed to determine the extent of degradation of the myofibrillar proteins desmin and troponin T over aging, as well as μ-calpain autolysis, calpastatin, HSP27 and aB-crystallin. Warner- Bratzler shear force (WBSF) measurements of loin chops across these aging times were conducted. The experimental design was a randomized complete block design, and data were analyzed by using mixed procedure of SAS to compare the traits across different genotypes and aging periods. Means were separated (F-test, P b 0.05) by least significant differences. Results: A significantly higher shear force value was observed in loins from CN compared to other genotypes regardless of aging time (P b0.05), as expected. Western blots for desmin and troponin T showed increased degradation products of those myofibrillar proteins with an increase in aging time, with less degradation products in the CN genotype than in the others (P b0.05). The extent of μ-calpain autolysis was also affected by genotype, where loins from CN exhibited more intact μ-calpain than loins from other genotypes throughout each aging time (P b0.05). Higher calpastatin expression in CN compared other genotypes was also found. A significant decrease in the extent of intact HSP27 with an increase in aging time was observed, while higher levels of intact HSP27 in CN compared to others was found in loins from CN regardless of aging time (P = 0.09). Further, the lower extent of degraded HSP27 in CN compared other genotype was also found over time (P b 0.001). No significant effects of genotypes and aging were found for aB-crystallin. Conclusion: A higher level of HSP27 was found in the CN genotype, which coincided with greater intact desmin and troponin T, less μ-calpain autolysis, and eventually higher shears force values as compared to other genotypes. This observation leads us to postulate that the up-regulation of HSP27 could be related to meat toughness in callipyge lamb meat by delaying muscle protein degradation during aging through its anti-apoptotic function. Further studies for the elucidation of the specific mechanisms and possible differences in sHSP behaviors across different muscle fiber types would be highly warranted.
Keywords: 2D-DIGE, immune system stimulation, protein profile, residual feed intake
Keywords: Callipyge, Small Heat Shock Proteins, Tenderness
doi:10.1016/j.meatsci.2015.08.173
doi:10.1016/j.meatsci.2015.08.174