J. Anim Sci. 2008. 86:2651-2656. doi:10.2527/jas.2007-0752
© 2008 American Society of Animal Science
Plasma amino acid profile and expression of the ubiquitin-mediated proteolytic pathway in lambs with induced metabolic acidosis1
S. L. Greenwood*,
N. E. Odongo*,2,
O. AlZahal*,
K. C. Swanson*,
A. K. Shoveller*,
J. C. Matthews
and
B. W. McBride*,3
* Department of Animal and Poultry Science, University of Guelph, Ontario, Canada N1G 2W1;and
Department of Animal and Food Sciences, University of Kentucky, Lexington, 40546
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Abstract
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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.
Key Words: metabolic acidosis • protein degradation • sheep • ubiquitin-mediated proteolytic pathway
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INTRODUCTION
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Feeding high starch diets to ruminants is common practice in production systems when there is a need to increase energy availability. However, increased fermentation rate in the rumen increases production of propionate and lactate, commonly resulting in ruminal acidosis (Nocek, 1997
). Consequently, increased absorption of lactate across the ruminal wall can lead to metabolic acidosis. Physiological compensation for decreased blood pH includes increased bicarbonate buffering, interorgan N transport, and ultimately H+ excretion via NH4Cl from the kidney. These actions are accomplished primarily through increased net glutamine output from the liver, increased N excretion from the kidney, and increased protein catabolism within muscle (Hollidge-Horvat et al., 1999; Garibotto et al., 2004; Taylor and Curthoys, 2004).
Metabolic acidosis has been widely studied in nonruminants because it is a sign of cachexia, renal failure, and trauma injuries (Caso et al., 2004; Lecker et al., 2006; Curthoys et al., 2007). Research using nonruminant animals has yielded strong evidence for the induction of the ubiquitin-mediated proteolytic pathway as the primary proteolytic mechanism stimulated under acidotic conditions to provide AA for N buffering (Du et al., 2005). The ATP-requiring ubiquitin pathway is unique for its usage of the 26S proteasome, which recognizes ubiquitin chains bound to target proteins by ubiquitin-conjugating enzymes (Hershko and Ciechanover, 1998; Ciechanover, 2006).
Despite detrimental effects of metabolic acidosis in livestock production systems, the effect of metabolic acidosis on intracellular protein degradation in ruminants has received little attention to date. Ruminants have unique compensatory mechanisms via N recycling in the rumen and shifts in nutrient absorption (Kingston-Smith and Theodorou, 2000), as well as repartitioning of nutrient utilization to restore acid-base balance (Lobley et al., 1995). Therefore, it is plausible that metabolic acidosis stimulates proteolysis differently in ruminants. In a previous companion study, our objective was to characterize blood acid-base variables in lambs as a result of nutritionally induced metabolic acidosis using an anionic supplement (Las et al., 2007). In continuance of our objective, the current study examines the effect of this nutritionally induced metabolic acidosis on plasma AA and tissue variables associated with protein catabolism to further characterize physiological response to decreased plasma pH and strong ion difference.
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MATERIALS AND METHODS
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Animals and Experimental Design
All animal procedures were approved by the University of Guelph Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care.
Ten fully fleeced yearling Rideau-Arcott wether lambs (54.3 ± 6.7 kg of BW) were allotted randomly to 2 treatments in a randomized complete block design as described previously by Las et al. (2007).
Experimental Treatments
As described previously by Las et al. (2007), lambs were offered 2 equal portions of dehydrated alfalfa pellets (1.2 kg/d total containing 90% DM, 22% CP as % of DM, 1.2 Mcal of NEg/kg of DM) at 1000 and 1300 h daily during the preexperimental period. During the experimental period, control lambs were supplemented with a canola meal supplement, whereas acidosis lambs were supplemented with an HCl-treated canola meal (NutriChlor, Nutritech Solutions, Abbotsford, Canada). Lambs were fed supplement from d 0 to d 10, receiving 0 g (d 0), 50 g (d 1), 100 g (d 2), 150 g (d 3), and 200 g/d (d 4 to 10) in 2 (0700 and 1100 h) equal portions mixed with 30 mL of molasses, whereas alfalfa pellets were offered at 1000 and 1500 h during the experimental period. Laboratory analyses of the diets were given by Las et al. (2007). Treatments were discontinued on d 11, and lambs were slaughtered by captive bolt stunning and exsanguination.
Sample Collection
Lamb BW were recorded 2 d before commencement of the experiment. As described in Las et al. (2007), vinyl catheters were fitted into the left jugular vein of each lamb for repeated blood sampling during the experimental period. Baseline blood measurements were determined on d 0, followed by daily jugular blood sample collections (between 1100 and 1130 h) from d 1 to 10. Sterile 3-mL nonventing tubes containing lithium-heparin (Gaslyte, Vital Signs Inc., Englewood, CO) were used for blood collection. Within 3 min of blood collection on each day, blood acid-base variables were measured and have been reported in Las et al. (2007), along with plasma variables measured after blood separation by centrifugation.
Approximately 3 g each of liver (center of right lobe), kidney (cross section including cortex and medulla), and muscle (sections of sternomandibularis) samples were collected within 10 min of slaughter and snap frozen in liquid N2 and stored at –70°C until analysis.
Plasma AA Concentration
Reverse-phase HPLC was performed to determine AA concentrations in plasma samples from each lamb. Samples were first pooled within animal from d 4 to 10 of the experimental period, and subsequently prepared using the method of Bidlingmeyer et al. (1984) with modifications for biological samples as described by House et al. (1994).
Messenger RNA (mRNA) Expression of C8, E2, and Ubiquitin in Liver, Kidney, and Muscle Tissue
Relative real-time PCR was performed to examine fluctuations in mRNA expression of components of the ubiquitin-mediated proteolytic pathway. Total RNA was isolated from the kidney, liver, and muscle tissue using the TRIzol method (Invitrogen, Burlington, Canada). Total RNA was resuspended in diethylpyrocarbonate-treated water before quantification using absorbance readings at 260 nm/280 nm. Samples were treated for removal of genomic contamination (DNase, Invitrogen), and analyzed for RNA integrity (2100 Bioanalyzer, Agilent Technologies, Brockville, Canada). All intact RNA was reverse-transcribed (5 µg of RNA per sample), and complementary DNA was diluted to 1:50 for gene amplification by TaqMan real-time PCR (Prism 7000, Applied Biosystems, Foster City, CA). Exon-spanning primers and FAM-labeled probes (Table 1) were custom designed (Applied Biosystems) for the proteasome subunit C8, the ubiquitin-conjugating enzyme E2, ubiquitin, and β-Actin (housekeeping gene) genes using bovine sequences listed on GenBank (National Center Biotechnology Information, Bethesda, MD) that were observed to have no similarities to other gene sequences through BLAST (National Center Biotechnology Information). Standard dilutions were amplified on each plate (1:10, 1:40, 1:160, 1:640, 1:2,560), and all samples were tested in triplicate. Standard dilutions were used to generate plate efficiency values (E = 10–1/slope) and incorporated into determination of mRNA (Pfaffl, 2001). Control animals were pooled to generate calibrator values for all genes within each tissue type.
Ubiquitin Protein Expression
Expression of the ubiquitin protein was analyzed in liver, kidney, and muscle samples collected from each animal. One gram of wet tissue was combined with 250 mM sucrose, 10 mM HEPES-KOH, 1 mM EGTA, and protease inhibitor cocktail (Sigma-Aldrich, Oakville, Canada), and homogenized before being stored at –70°C until analysis. Protein concentration was determined (Bradford, 1976) using BSA as the standard. Equal parts Laemmli buffer (Sigma-Aldrich) and sample, containing 15 µg of protein, were combined and heated before being loaded into wells of premade 18% resolving Tris-HCl gels (BioRad, Mississauga, Canada) for protein separation by SDS-PAGE. Proteins were transferred from gels to polyvinylidene fluoride membranes (Millipore, Billerica, MA) before blocking for 1.5 h with blocking reagent (2% instant milk powder dissolved in 200 mM NaCl low stringency Tris buffer saline and Tween 20 solution). Membranes were then incubated with blocking reagent containing 1:1,000 of bovine ubiquitin monoclonal antibody of mouse origin (Fitzgerald Ind. Intl., Inc., Concord, MA) for 1.5 h at room temperature, washed, and subsequently incubated in 1:5,000 horseradish peroxidase linked secondary whole antibody of sheep origin (Amersham Biosciences, Piscataway, NJ) and blocking reagent at 4°C overnight. Antibodies have been validated for use on bovine samples and considered appropriate for use with ovine samples due to high sequence conservation between bovine and ovine species. Membranes were washed before chemiluminescence determination using an Enhanced Chemiluminescence detection reagent kit (Amersham Biosciences). Membranes were stained with fast-green, and an internal control was loaded on each gel. Density of bands and fast-green stained membranes of each lane were determined (Northern Eclipse, Empix Imaging, Mississauga, Canada). The band value was then normalized to the density of the entire fast-green stained lane as described by Howell et al. (2003).
Statistical Analysis
All results were analyzed using PROC MIXED (SAS Inst. Inc., Cary, NC) using the following model: Yij = µ + 
i + βj + 
ij, where Yi was the dependent variable, µ was the overall mean, i was the effect of dietary treatment, βj was the effect of block (blocked by weight), and ij was the random residual error. Block was insignificant (P > 0.05) for all measured variables and thus was removed from the model. Results were declared significant at P < 0.05 unless otherwise stated.
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RESULTS AND DISCUSSION
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As described by Las et al. (2007), blood and urinary pH were decreased, whereas plasma bicarbonate and calcium concentrations were increased in acidotic lambs vs. control lambs. Though plasma evidence of metabolic acidosis was observed in acid-base variables (Las et al., 2007), only greater plasma concentrations of serine (P = 0.06), glycine (P = 0.002), and glutamine (P = 0.04) were observed in acidotic lambs (Table 2). Previous research has demonstrated that renal serine synthesis can occur via 2 different pathways, either via glycine catabolism or gluconeogenic precursors, and that glycine could be a major contributor to renal ammonia production (Lowry et al., 1987). In agreement with these observations, arterial concentrations of both glycine and serine were increased with the induction of metabolic acidosis in nonruminants (Lowry et al., 1987; Garibotto et al., 2004), which was consistent with our current findings in lambs.
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Table 2. Plasma AA concentrations in lambs offered control canola supplement (control) or HCl-treated canola supplement (acidotic)
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No change (P > 0.1) in mRNA expression of C8, E2, or ubiquitin was observed in kidney (Figure 1), liver (Figure 2), or muscle (Figure 3) tissue collected from acidotic lambs compared with control lambs using real-time PCR in agreement with previous Northern hybridizations using these same tissue samples (Greenwood et al., 2007). Also, no fluctuations in ubiquitin protein expression were observed in the kidney (P = 0.34; Figure 4), liver (P = 0.56; Figure 5), or muscle (P = 0.26; Figure 6) in acidotic lambs compared with control lambs.
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Figure 1. Relative mRNA expression of C8, E2, and ubiquitin in kidney tissue collected from control lambs (control, n = 5) and acidotic lambs (acidotic, n = 5). All genes were calibrated against β-actin and are relative to the pooled control group values. Depicted as least squares means ± SEM.
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Figure 2. Relative mRNA expression of C8, E2, and ubiquitin in liver tissue collected from control lambs (control, n = 5) and acidotic lambs (acidotic, n = 5). All genes were calibrated against β-actin and are relative to the pooled control group values. Depicted as least squares means ± SEM.
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Figure 3. Relative mRNA expression of C8, E2 and ubiquitin in muscle tissue collected from control lambs (control, n = 5) and acidotic lambs (acidotic, n = 5). All genes are calibrated against β-actin and are relative to the pooled control group values. Depicted as least squares means ± SEM.
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Figure 4. Immunoblot (A) and densitometric comparison (B) of ubiquitin protein expression in kidney tissue collected from control lambs (control, CS; n = 5) and acidotic lambs (acidotic, AS; n = 5). Densitometric levels were corrected by creating a ratio between ubiquitin and fast-green stained lane for each sample. Depicted as least squares means ± SEM.
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Figure 5. Immunoblot (A) and densitometric comparison (B) of ubiquitin protein expression in liver tissue collected from control lambs (control, CS; n = 5) and acidotic lambs (acidotic, AS; n = 5). Densitometric levels were corrected by creating a ratio between ubiquitin and fast-green stained lane for each sample. Depicted as least squares means ± SEM.
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Research has demonstrated the importance of the ubiquitin-mediated proteolytic pathway under conditions of severe metabolic acidosis in nonruminants (Mitch et al., 1994; Hollidge-Horvat et al., 1999; Garibotto et al., 2004); however, the current study now provides evidence that the same proteolytic pathway is not the primary response pathway in mildly acidotic lambs. Exploration of the possibility that the ubiquitin-mediated proteolytic pathway is not the predominant pathway in metabolically acidotic lambs will lead to examination of other proteases, such as cathepsins and calpains. The Ca2+-dependent calpains have been proposed to be precursors binding to the Z-disc, and subsequently releasing actin and myosin fragments (Bartoli and Richard, 2005). Calcium concentrations were increased in lambs offered the acidosis diet (Las et al., 2007), potentially providing the Ca2+ binding required for calpain activation. In addition, caspase-3 has also been described as a precursor for preliminary breakdown of muscle before proteasomes can successfully degrade the smaller filaments (Du et al., 2005). Furthermore, cathepsin L has been identified as a lysosomal protease that is upregulated in muscle as a result of fasting, tumor bearing, uremia, and diabetes mellitus (Lecker et al., 2004). Cathepsins B and D have also been observed to be upregulated during muscle wasting, which is symptomatic of several ailments including diabetes, trauma, fasting, cancer, and sepsis (Bechet et al., 2005). However, these cathepsin isoforms have also been observed to be downregulated in the kidney due to NH4Cl loading (Fawcett et al., 2000), indicating potential differential regulation depending on tissue.
A second possibility is that ruminants can spare protein from degradation using other biological mechanisms to cope with increased N requirements without sacrificing their own tissue protein. It has been previously observed that, during metabolic acidosis, ruminant metabolism has the unique capacity to favor increasing perivenous hepatocyte glutamine synthesis, in lieu of sacrificing HCO3– for carbamoyl phosphate synthesis for increased ureagenesis in periportal hepatocytes (Lobley et al., 1995; Milano et al., 2000). Renal glutamine deamination is also increased to dispose of excess ammonia as ammonium ions in urine (Heitmann and Bergman, 1980). As a result, these unique mechanisms could prevent significant protein degradation as a result of metabolic acidosis.
Model variables must also be considered because the current study utilized an anionic supplement to induce metabolic acidosis instead of inducing increased lactate and VFA absorption through offering a highly fermentable diet. However, the primary objective of the current research was to examine the blood variables associated with use of a dietary cation-anion difference. Despite differences in how blood pH depression is achieved, the level of anionic supplement used in the current study elicits a drop in blood pH observed for other acidotic animal models (Lobley et al., 1995; Mutsvangwa et al., 2004; Moret et al., 2007). Also, the length of treatment was typical of experiments previously cited (Heitmann and Bergman, 1980; Lobley et al., 1995).
In conclusion, the results herein indicate that ubiquitin-mediated proteolysis was not the primary pathway of tissue protein degradation in lambs with induced metabolic acidosis. Further investigations will focus on different proteolytic pathways, such as the cathepsins and calpains, to determine if protein degradation is increased via these proteases under conditions of metabolic acidosis in ruminants.
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Figure 6. Immunoblot (A) and densitometric comparison (B) of ubiquitin protein expression in muscle tissue collected from control lambs (control, CS; n = 5) and acidotic lambs (acidotic, AS; n = 5). Densitometric levels were corrected by creating a ratio between ubiquitin and fast-green stained lane for each sample. Depicted as least squares means ± SEM.
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Footnotes
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1 The authors thank the staff of the Ponsonby Research Station, University of Guelph for technical assistance provided during the experimental period. We would also like to thank J. E. Las for assistance throughout the experimental trial. Financial support provided by the Ontario Ministry of Agriculture, Food and Rural Affairs and the Natural Sciences and Engineering Research Council of Canada (B.W. McBride) is also gratefully acknowledged. 
2 Present address: Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada T1J 4B1.
3 Corresponding author: bmcbride{at}uoguelph.ca
Received for publication November 23, 2007.
Accepted for publication May 27, 2008.
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Abstract
INTRODUCTION
