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Regulated Expression of Genes/Proteins Critical to Anionic Amino Acid N Metabolism by Developing and Aging Beef Cattle
J.C. Matthews, J.A. Boling
Department of Animal and Food Sciences
The anionic amino acids aspartate and glutamate are critical for, and extensively metabolized to support, whole-animal energy and N metabolism in ruminants. The overall hypothesis being tested in this project is that expression of membrane transporters and metabolizing enzymes of glutamate and aspartate is sensitive to stage of life cycle and subtherapeutic levels of chlortetracycline.
2009 Project Description
The overall goal of this project is to optimize the dietary protein load to absorption and metabolic capacities of gastrointestinal and peripheral tissues throughout the life cycle of beef cattle, by developing the means to manipulate amino acid N metabolic capacity. Significant progress has been made this year towards meeting research goals of Objective 1.
Experiment 1: In many cool-season forage systems, microbial-derived amino acids typically supply adequate or surplus sources of N whereas energy supply can limit growth. The uptake and metabolism of glutamate and glutamine is critical for epithelial metabolism, as their common carbon skeleton (alpha-ketoglutarate) is the primary metabolic fuel of the intestine. Previously we reported (see the 2008 report below) the effect of increased intestinal supply of rumen-derived microbes (hence, transporter substrates), energy, or both, in growing Angus steers on glutamate transporter gene expression (mRNA) by duodenal (D), jejunal (J), and ileal (I) epithelia. To complement these findings, the effect of these treatments in the same tissues on expression of mRNA for 7 glutamine transporters (ATA1, ATA2, ATA3, SN1, SN2, LAT1, LAT2) was evaluated by real-time RT-PCR. Basal expression of all mRNA but ATA3 differed (P≤0.06) among D, J, and I: ATA1, D=J>I; ATA2, D=J, D=I, J>I; SN1, D>J=I; SN2, J>D=I; LAT1, D=J>I; LAT2, D=J, J=I, D0.15) mRNA content except that expression of SN1 and 2 mRNA by I was increased (P≤0.08) 57 and 51%, respectively, with increased energy supply. This latter finding suggests that luminal energy supply may limit I expression of SN1 and SN2 mRNA.
Experiment 2: To complete our in vivo study of whether basal expression of 20 amino acid (AA) transporter genes by D, J, and I epithelia is affected by the increased luminal supply of rumen-derived microbes (hence, AA substrates), starch-derived energy, or both, the effect of substrate and energy supply on expression of mRNA encoding AA transport systems x-c (xCT), T (TAT1), H+-dependent AA (PAT1), and H+-dependent peptide (PepT1) was conducted to quantify the relative expression of AA transporter mRNA:18S rRNA. Basal expression of mRNA differed (0.001 ≤ P ≤ 0.09) among the 3 small intestinal epithelia. For xCT, TAT1, and PepT1, the expression pattern was D = J > I. For PAT1, however, the pattern was J > D = I. Increased luminal AA supply did not affect transporter mRNA content. Although D and J expression were unaffected by increased luminal energy supply, I expression of xCT mRNA was increased by 41% (P = 0.04), PepT1 by 162% (P = 0.03), and TAT1 by 56% (P = 0.08). The findings that PAT1, TAT1, and xCT mRNA are expressed by cattle small intestinal epithelia are novel. Given the known localization and function of the proteins encoded by the evaluated mRNA, this study suggests that the potential for I apical uptake of luminal peptide-bound AA by PepT1, basolateral transfer of aromatic AA into blood by TAT1, and cystine uptake from blood by xCT, is enhanced by the increased luminal starch-derived energy supply to forage-fed growing cattle.
The findings of this research will give insight into when and how amino acid metabolism is altered to support growth and fattening of steers and milk production capacity of mature and aging beef cows. Specifically, these studies will provide data to determine differences in gene expression that account for different levels of metabolic capacity in the life-cycle of cattle. The extent to which protein and amino acid-N is utilized and retained in the tissues contributes to the efficiency of growth, maintenance, and production of cattle.
The application of this research is to optimize the dietary protein load to absorption and metabolic capacities of gastrointestinal and peripheral tissues as calves develop and cows age so that management tools and agents to control/achieve optimal functional capacity of these proteins can be developed. Having the ability to manipulate metabolic capacity will enable feeding strategies and systems to be developed that improve the efficiency of N utilization for maintenance and production, while minimizing excretion of metabolic N where differences are determined to exist in the young versus old mature beef cow.
There are 1.12 million cows grazing forage lands in Kentucky, and 32.8 million throughout the United States (National Agricultural Statistical Service Report, USDA, January 1, 2004). By identifying proteins that are altered in commercially-relevant production stages throughout the life cycle of cattle, this research fills a critical void in our current growth prediction models used by the livestock industry. For Kentucky alone, if only a 0.9 kg gain/calf (0.4% of body weight) is realized from this research, then an increase in direct farm receipts of $1.2 million dollars from the sale of weanling calves and a total economic gain of $6 million annually will be realized. Analogously, if the productive life of aged cows (about 200,000 cows) can be increased by only one calf, then a net savings of $4,000,000 ($20/cow) per year in heifer replacement costs will be realized.
Another significant problem of many of Kentucky's 30,000 cow/calf producers that our research addresses is the lack of a rational basis for culling an old cow from the herd, other than whether she breeds back. However, very little data exists regarding the effects of age on the metabolic capacity of the beef cow. Therefore, the clinical blood profiles of old vs young cows being generated by our correlating clinical blood profiles to hepatic and skeletal muscle protein profiles of old vs young beef cows will provide unique and extremely pertinent data. This data will be useful for producers to make management decisions (when to cull) and to veterinarians as a diagnostic tool.
In addition, this data describing the metabolic decline of beef cows will be highly relevant to the effect of senescence in other animal species (including humans).
Greenwood, S. L., O. AlZahal, K. C. Swanson, J. C. Matthews, and B. W. McBride. 2009. Influence of Glutamine Infusion on Ubiquitin, Caspase-3, Cathepsins L and B, and m-Calpain Expression in Sheep with Nutritionally Induced Metabolic Acidosis. Journal of Animal Science 87:2073-2079, doi:10.2527/jas.2008-1748.
Liao, S. F., D. L. Harmon, E. S. Vanzant, K. R. McLeod, J. A. Boling, and J. C. Matthews. 2010. The Small Intestinal Epithelia of Beef Steers Differentially Express Sugar Transporter mRNA in Response to Abomasal vs Ruminal Infusion of Starch Hydrolysate. Journal of Animal Science 88:306-314, doi.10.2527jas.2009-1992.
Xue, Y., S. F. Liao, K. Son, S. L. Greenwood, B. W. McBride, J. A. Boling, and J. C. Matthews. 2010. Metabolic Acidosis in Sheep Alters Expression of Renal and Skeletal Muscle Amino Acid Enzymes and Transporters. In press; published online by the Journal of Animal Science on October 9, 2009 as doi.10.2527jas.2009-2101.
Koontz, A. F., S. W. El-Kadi, D. L. Harmon, E. S. Vanzant, J. C. Matthews, J. A. Boling, and K. R. McLeod. 2010. Effect of Ractopamine on Whole Body and Splanchnic Energy Metabolism in Holstein Steers. Canadian Journal of Animal Science (accepted, December, 2009).
Liao, S. F., Y. Xue, J. A. Boling, and J. C. Matthews. 2009. Expression of System N Protein mRNA, but not System A or L, Is Upregulated by Ileal Epithelia of Growing Beef Cattle in Response to Increased Luminal Supply of Energy but not Amino Acids. FASEB J. 23:LB416.
Matthews, J. C., S. F. Liao, and J. A. Boling. 2009. Basal Expression of 27 Nucleoside and Amino Acid Transporter mRNA by Small Intestinal Epithelia of Forage-fed Growing Beef Steers Is Differentially Affected by Increased Luminal Substrate or Energy Supply. J. Anim. Sci. Vol. 87, E-Suppl. 2/J. Dairy Sci. Vol. 92, E-Suppl. 1, pgs. 378-379.
Liao, S. F., J. A. Boling, and J. C. Matthews. 2009. Luminal Energy Supply (but not Substrate) Affects Expression of mRNA for Three Proteins Capable of Amino Acid Transport by Ileal Epithelium (but not Duodenal or Jejunal) of Forage-fed Growing Beef Cattle. J. Anim. Sci. Vol. 87, E-Suppl. 2/J. Dairy Sci. Vol. 92, E-Suppl. 1, pg 238.