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To determine the effect of metabolic acidosis on expression of L-Gln, L-Glu, and L-Asp metabolizing enzymes and transporters, the relative content of mRNA, protein, or mRNA and protein, of 6 enzymes and 5 transporters was determined by real-time reverse transcription-PCR and immunoblot analyses in homogenates of kidney, skeletal muscle, and liver of growing lambs fed a common diet supplemented with canola meal (control; n = 5) or HCl-treated canola meal (acidosis; n = 5). Acidotic sheep had a 790% greater (P = 0.050) expression of renal Na(+)-coupled neutral AA transporter 3 mRNA and a decreased expression of renal glutamine synthetase mRNA (47% reduction, P = 0.037) and protein (57% reduction, P = 0.015) than control sheep. No change in renal cytosolic phosphoenolpyruvate carboxykinase (protein and mRNA), glutaminase (mRNA), or L-Glu dehydrogenase (protein) was found. In skeletal muscle, acidotic sheep had 101% more (P = 0.026) aspartate transaminase protein than did control sheep, whereas no change in the content of 3 Na(+)-coupled neutral AA transporters (mRNA) or 2 high-affinity L-Glu transporter proteins was found. In liver, no change in the content of any assessed enzyme or transporter was found. Collectively, these findings suggest that tissue-level responses of sheep to metabolic acidosis are different than for nonruminants. More specifically, these results indicate the potential capacity for metabolism of L-Asp and L-Glu by skeletal muscle, and L-Gln absorption by kidneys, but no change in hepatic expression of L-Gln metabolism, elaborates previous metabolic studies by revealing molecular-level responses to metabolic acidosis in sheep. The reader is cautioned that the metabolic acidosis model employed in this study differs from the increased plasma lactate-induced metabolic acidosis commonly observed in ruminants fed a highly fermentable grain diet.

Provision of AA has shown success in attenuating proteolytic activity in monogastrics suffering from metabolic acidosis. However, it is unknown whether AA supplementation can provide any beneficial effects to ruminants with nutritionally induced metabolic acidosis. The objective of the current study was to examine the effects of glutamine infusion on various protein degradation components across several tissues in sheep with induced metabolic acidosis. Sheep were assigned to a randomized complete block design with 2 x 2 factorial arrangement of treatments (n = 6 sheep/treatment) consisting of a control or acidosis diet, and receiving a saline or L-glutamine infusion. Sheep were fed diets for 10 d and slaughtered on d 11. Liver, kidney, and muscle samples were collected at slaughter and examined for relative messenger RNA (mRNA) expression of ubiquitin, C8, E2, cathepsin L, cathepsin B, caspase-3, and m-calpain, as well as protein expression of ubiquitin. Relative mRNA expression of C8 (P = 0.02), E2 (P = 0.06), and ubiquitin (P = 0.07) was less in kidney in acidotic vs. control sheep. Additionally, mRNA expression of m-calpain in kidney was greater (P = 0.01) as a result of glutamine infusion. There were no significant alterations (P > 0.10) in mRNA of any component as a result of acidosis in the liver or muscle. This study demonstrates the inability of metabolic acidosis to increase expression of the ubiquitin-mediated proteolytic pathway in skeletal muscle; however, downregulation of renal mRNA expression of these components is apparent during the induction of metabolic acidosis.

This study characterized the effects of nutritionally induced metabolic acidosis with or without Gln infusion on acid-base balance, plasma AA, and plasma NEFA in sheep. In a randomized complete block design with a 2 x 2 factorial arrangement of treatments, 24 fully fleeced sheep (Rideau-Arcott, 63.6 +/- 5.9 kg of BW) were fed a control supplement (CS; 300 g/d of canola meal) or an acidosis supplement (AS; 300 g/d of NutriChlor; HCl-treated canola meal), offered twice daily at 0700 and 1100 h. Sheep were infused at 1400 h daily with 0.3 g of L-glutamine per kg of BW or saline via jugular vein catheters for 7 d. The sheep were individually housed and limit-fed a basal diet of dehydrated alfalfa pellets (1.75 kg/d; 90% DM, 22% CP, and 1.2 Mcal of NE(g)/kg on a DM basis) offered twice daily at 1000 and 1300 h. Blood and urine was sampled daily between 1100 and 1130 h, and blood samples were analyzed for hematocrit, plasma pH, gases, strong ions, AA, and NEFA, whereas urine was analyzed for pH. The AS reduced (P < 0.01) DMI, urine and plasma pH, blood urea, partial pressure of CO(2), strong ion difference, and plasma HCO(3)(-), and increased (P < 0.01) plasma K(+), Ca(2+), and Cl(-). The AS with saline infusion increased (P

Metabolic acidosis is a condition often induced by ruminal acidosis. Identification of the specific proteolytic pathways affected by metabolic acidosis and characterization of AA concentration changes induced by metabolic acidosis in ruminants has yet to be confirmed. The objective of this study was to examine the effect of nutritionally induced metabolic acidosis on lamb plasma AA and tissue variables, including mRNA and protein expression of components of the ubiquitin-mediated proteolytic pathway. Lambs (n = 10) were divided evenly into treatment groups receiving alfalfa pellets supplemented with 1) a control canola meal supplement, or 2) HCl-treated canola meal supplement for a 10-d treatment period. On d 11, lambs were slaughtered and liver, muscle, and kidney samples were collected to determine mRNA expression of components of the ubiquitin-mediated proteolytic pathway and ubiquitin protein expression. Plasma concentrations of serine (P = 0.06), glycine (P = 0.002), and glutamine (P = 0.04) were greater in acidotic lambs compared with control animals, indicating that protein catabolism may be occurring. However, no alteration (P > 0.1) in messenger RNA expression of the proteasome subunit C8, ubiquitin-conjugating enzyme E2, or ubiquitin or in ubiquitin protein expression were observed. These results suggest that ubiquitin-mediated proteolysis is not the primary pathway of protein degradation in lambs afflicted with metabolic acidosis.