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Nutrient-Gene Interactions
Research Communication

Chronic Metabolic Acidosis Increases mRNA Levels for Components of the Ubiquitin-Mediated Proteolytic Pathway in Skeletal Muscle of Dairy Cows¹

Department of Animal and Poultry Science and ^* Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1

³To whom correspondence should be addressed. E-mail: bmcbride{at}uoguelph.ca.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Ruminants fed high-grain diets often are subjected to ruminal acidosis, which can lead to excessive absorption of lactate into the blood stream, thereby causing metabolic acidosis. Metabolic acidosis leads to body protein loss, mainly due to increased skeletal muscle degradation. Our objective was to determine the effects of metabolic acidosis on the messenger RNA (mRNA) abundance of genes encoding components of the ubiquitin-mediated proteolytic pathway in the skeletal muscle of lactating Holstein cows. Cows (n = 20) were assigned to one of two treatments: 1) control; or 2) NutriChlor 18–8, an HCl-treated supplement, which was fed to induce chronic metabolic acidosis. The longissimus muscle was biopsied before and after 10 d of treatments. Total RNA isolated from muscle tissue was hybridized with ³²P-labeled cDNA probes encoding for 14-kDa ubiquitin carrier protein E2 (14-kDa E2), ubiquitin, and C8 and C9 subunits of the 20S proteasome. Induction of metabolic acidosis increased (P < 0.05) skeletal muscle mRNA levels for ubiquitin (25%), 14-kDa E2 (34%), and the C8 subunit (20%); however, mRNA abundance for the C9 subunit was unaffected (P > 0.05). These results suggest that up-regulation of the ubiquitin-proteasome pathway is the mechanism by which metabolic acidosis stimulates muscle wasting in ruminants.

KEY WORDS: • dairy cow • metabolic acidosis • gene expression • ubiquitin-mediated proteolytic pathway • skeletal muscle proteolysis

In early lactation, high-yielding dairy cows are often fed diets that are high in readily fermentable carbohydrates so as to maximize energy intake. This can cause accumulation of volatile fatty acids (VFAs)⁴ and lactate in the rumen, thereby lowering ruminal pH, a digestive disorder commonly referred to as ruminal acidosis (1). Depending on a variety of interactive factors, such as the rates of VFA and lactate production and absorption, excessive levels of lactate and VFAs can be absorbed into the bloodstream, resulting in metabolic acidosis (1). Chronic metabolic acidosis is characterized by decreased plasma bicarbonate (HCO₃^–), which is accompanied by a decrease in blood pH.

The regulation of extracellular pH during chronic metabolic acidosis requires coordinated changes in amino acid metabolism within muscle, liver, and kidney tissue (2). Liver ureagenesis is decreased, whereas glutamine synthesis from NH₄⁺ in perivenous hepatocytes is increased (2), such that the liver becomes a net exporter of glutamine. Because ureagenesis consumes HCO₃^–, the shift in liver nitrogen metabolism during chronic metabolic acidosis causes the retention of HCO₃^– in the body, which helps to buffer excesses of H⁺ (3). In synchrony with changes in liver N metabolism, kidney consumption of glutamine increases in sheep (4), likely as a mechanism to maintain acid-base homeostasis via kidney ammoniagenesis, which leads to urinary excretion of excess H⁺ as NH₄⁺. In addition, chronic metabolic acidosis is linked to reduced N balance and increased urinary excretion of N and 3-methylhistidine in rats (5,6) and humans (7), indicating increased skeletal muscle degradation. Williams et al. (8) reported that NH₄Cl-induced chronic metabolic acidosis in rats stimulated muscle protein degradation by activating the ubiquitin-mediated proteolytic pathway, which is the predominant biological pathway for the catabolism of myofibrillar proteins in skeletal muscle (9). In ruminants, however, the underlying mechanisms responsible for decreased N balance have not been delineated. Lobley et al. (10) did not report any changes in protein synthesis or degradation during NH₄Cl-induced chronic metabolic acidosis in sheep, although whole-body leucine oxidation was increased.

The objectives of this study, therefore, were to determine the effects of chronic metabolic acidosis on messenger RNA (mRNA) abundance for genes that encode components [i.e., ubiquitin, 14-kDa ubiquitin carrier protein E2 (14-kDa E2), and C8 and C9 subunits] of the ubiquitin-mediated proteolytic pathway in the skeletal muscle of lactating Holstein dairy cows. In this experiment, we used an HCl-treated commercial dietary supplement, rather than NH₄Cl, as an experimental model for induction of chronic metabolic acidosis. This approach avoided the additional NH₃ load from NH₄Cl intake, which, in turn, would alter rates of liver ureagenesis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Animals and experimental design.

Multiparous Holstein dairy cows (n = 20; 684.5 ± 88.5 kg body wt; 117.2 ± 51.0 d in milk; 34.0 ± 6.1 kg milk/d) were used in this study. Cows were housed individually in tie stalls. Cows were paired based on milk yield and days in milk and randomly assigned to one of two treatments in a randomized complete-block design. For ease of management, the initiation of cows to treatment was staggered such that a maximum of 4 cows (i.e., 2 pairs) were being treated at any one time. The experiment was conducted at the Ponsonby Dairy Research Center (University of Guelph, Guelph, ON) from November 2002 through February 2003. Animals were cared for and handled in accordance with the Canadian Council on Animal Care regulations, and the University of Guelph Animal Care Committee approved their use in this experiment.

Experimental treatments, induction of metabolic acidosis and sample collection.

Cows were fed a total mixed ration (TMR) formulated to meet their nutrient requirements (11). The TMR contained 499 g dry matter/kg, 179 g crude protein/kg dry matter, and 6.6 MJ NE_L (net energy for lactation)/kg dry matter, and it consisted of (g/kg, as-fed basis): corn silage, 517; high-moisture corn, 149; mixed haylage, 131; mixed hay, 44; and a protein supplement for lactating cows, 159. Cows were given unrestricted access to feed twice daily at 0900 and 1500 h. The feed intake of the individual cows was recorded daily. The amounts of TMR fed were adjusted daily such that there were <5% orts.

On the first day of the experiment (d 0), between 0900 and 1000 h, muscle tissue was obtained by needle biopsy from the longissimus muscle as described by Bergström et al. (12). Briefly, a 10- x 10-cm skin area 10 cm behind the last rib and 8 cm from the backbone was shaved and then scrubbed with iodine. A local anesthetic (20 g/L of lidocaine without epinephrine) was injected subcutaneously without penetrating the muscle tissue. A 3-cm scalpel incision was made through the skin, a sterile biopsy needle was inserted into the muscle to 5 cm, and muscle tissue samples (100 to 200 mg wet wt) were removed. Muscle tissue samples were immediately (<1 min after sampling) frozen in liquid N₂ and stored at -70°C pending RNA analysis. After sampling, the incision was stapled. These muscle tissue samples provided background mRNA abundance levels before the induction of metabolic acidosis.

A temporary vinyl catheter (0.86 mm i.d., 1.32 mm o.d.; Scientific Commodities) was then inserted into the left jugular vein to facilitate blood sampling. A blood sample was then withdrawn anaerobically, using a sterile 3-mL nonventing blood gas syringe with lithium heparin as an anticoagulant (Marquest Medical Products). Blood was analyzed for plasma pH and pCO₂ using a Nova StatProfile 5 blood gas analyzer (Nova Biomedical), which automatically calculated plasma bicarbonate (HCO₃^–) and base excess (BE) levels. A urine sample was obtained by vulval stimulation, and urine pH was measured using an Accumet pH meter (Fisher Scientific). These d 0 blood and urine samples were used for baseline measurements of the acid-base status.

From d 1 to 10, cows were subjected to one of two experimental dietary treatments: 1) canola meal (control); or 2) NutriChlor 18–8 (Nutritech Biochemicals), which was fed to induce chronic metabolic acidosis. According to the manufacturer, NutriChlor 18–8 consists of HCl-treated canola meal. For this reason, canola meal was used as the control treatment. The daily amounts of canola meal or NutriChlor 18–8 fed were 0.3 kg (d 1), 0.6 kg (d 2), 0.9 kg (d 3), 1.2 kg (d 4) and 1.5 kg/d (d 5 to 9). The daily amounts of canola meal and NutriChlor 18–8 were divided into two equal portions, which were mixed thoroughly by hand with the morning and afternoon TMR allocations. The dietary cation-anion balance [calculated as (Na⁺ + K⁺) - (Cl^– + S^2–) meq] of the TMR and NutriChlor 18–8 was 213.5 and -2102 meq/kg, respectively. After feeding at 0900 and 1500 h daily from d 1 to 9, jugular venous blood samples were obtained from each cow, and blood indicators of acid-base status (i.e., pH, HCO₃^–, and BE) were determined as already described. Urine samples were also obtained, by vulval stimulation, and urine pH was measured as already described. On d 10, experimental treatments were discontinued, and at 0900 h muscle tissue samples were obtained from the longissimus muscle (within 3 cm of the previous biopsy site) using the procedures already described. During development of the experimental model for the induction of metabolic acidosis, it was calculated that cows fed NutriChlor 18–8 at the stated levels would reach the desired endpoint (i.e., plasma pH of 7.38 to 7.39) by d 10.

Muscle RNA isolation and Northern hybridizations.

Total RNA was extracted from muscle tissue using TRI reagent (Sigma Chemical Co.) according to the manufacturer’s instructions. A total of 15 µg of RNA was separated by electrophoresis on a 1% agarose-formaldehyde gel, followed by capillary transfer to positively charged nylon membranes (Roche Diagnostics) and cross-linking to membranes using a Stratalinker (Stratagene). ³²P-Labeled cDNA probes were made by random priming, using Redivue ³²P dCTP (specific activity 3000 µCi/mmol, Amersham Biosciences) with the Rediprime II DNA labeling kit (Amersham Biosciences) according to the manufacturer’s instructions, with 25 ng of cDNA. Rat cDNA probes that encoded for a 896-bp fragment of 14-kDa E2 (GenBank accession #M62388; 13), a 562-bp fragment of the C8 proteasome subunit (GenBank accession #D90258; 14,15), and a 606-bp fragment of the C9 proteasome subunit (GenBank accession #NM 017281; 14,15) were used because the equivalent bovine genes have not been identified. A rat cDNA probe for ubiquitin (GenBank accession #Z18245; 16) that shares 93% homology with bovine ubiquitin was used. Membranes were prehybridized with Rapid Hyb Buffer (Amersham Biosciences) for 30 min at 65°C, then hybridized for 2 h at 65°C with the ³²P-labeled cDNA probes.

After hybridization, membranes were washed first at low stringency for 20 min in 2x SSC and 0.1% SDS at room temperature, then twice at high stringency for 15 min each, in solutions containing 0.1% SDS, with the stringency of these washes ranging from 1X to 0.1X SSC at 65°C depending on the probe. Blots were then visualized by autoradiography by exposing membranes at -70°C for 24 to 48 h on X-OMAT-AR film (Kodak), using intensifying cassettes. Blots were scanned for densitometry analysis using Gene Snap software (Version 6.00.20; Synaptics). All membranes were then stripped and reprobed with ³²P-labeled cDNA probe for bovine ß-actin, to normalize for RNA loading. The 374-bp ß-actin probe was obtained by RT-PCR of bovine liver RNA, using the ß-actin primers, 5'-CGTGACATTAAGGAGAAGCTGTGC-3' and 5'-CTCAGGAGGAGCAATGATCTTGAT-3' (GenBank accession #BC008633). The fragment obtained from RT-PCR was sent for sequencing at the Guelph Molecular Supercenter (University of Guelph, Guelph, ON); it showed homology to bovine ß-actin.

Statistical analysis.

Data were analyzed using the SAS General Linear Models ANOVA procedure (17), with the following general model: Y_ijk = µ + _i + ß_j + _ijk, where Y_ijk = dependent variable, µ = overall mean, _i = effect of block (i = 1... 10), ß_j = effect of treatment (j = 1, 2), and _ijk = random residual error. Initial analysis showed that blocking was not significant, so it was removed from the model and block df was pooled with error df. Values of P < 0.05 were considered significant, unless otherwise indicated.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Induction of chronic metabolic acidosis.

As expected, baseline (pretreatment) measurements of acid-base status (i.e., plasma pH, plasma HCO₃^– and BE concentrations, and urine pH) did not differ (P > 0.05) between treatment groups before treatments commenced (d 0; Table 1). By d 10 of the experiment, plasma pH, plasma HCO₃^– and BE concentrations, and urine pH were lower (P < 0.05) in cows treated with NutriChlor than in control cows (Table 1), indicating that dietary treatment with NutriChlor induced chronic metabolic acidosis. These observations are consistent with results from previous studies with dairy cows in which the dietary cation-anion balance was altered by including HCl in the diet (18).

The objective of this study was to determine whether chronic metabolic acidosis in lactating dairy cows increases the abundance of mRNAs that encodes for 14-kDa E2, ubiquitin, and C8 and C9 proteasome subunits, which are components of the ubiquitin-mediated proteolytic pathway in skeletal muscle. We measured the abundance of mRNAs that encode for 14-kDa E2 and ubiquitin, which are involved in ubiquitin conjugation of proteins that are targeted for proteolysis (13), and C8 and C9 subunits of the 20S proteasome, which is the proteolytic core of the 26S proteasome (9). In rat muscle, increased mRNA abundance for these components of the ubiquitin-mediated pathway is associated with increased muscle protein degradation in vitro (19,20), so mRNA abundance appears to be a valid indirect indicator of muscle proteolysis. As expected, mRNA levels did not differ between control and acidotic cows before treatments commenced (P > 0.05; Figs. 1and 2).

View larger version (50K): [in this window] [in a new window]   FIGURE 1 Effects of chronic metabolic acidosis on relative mRNA levels of the 14-kDa E2 and ubiquitin components of the ubiquitin-mediated proteolytic pathway in skeletal muscle of lactating dairy cows. Values are means ± SEM, n = 10. Bars without a common letter differ, P < 0.05. Representative northern blots are shown below the corresponding histograms. Bar labels: control-B, control cows before commencement of treatment; NutriChlor-B, acidotic cows before commencement of treatment; Control-A = control cows 10 d after commencement of treatment; and NutriChlor-A, acidotic cows 10 d after commencement of treatment.

View larger version (50K): [in this window] [in a new window]   FIGURE 2 Effects of chronic metabolic acidosis on mRNA levels of the C8 and C9 subunits of the 20S proteasome components of the ubiquitin-mediated proteolytic pathway in skeletal muscle of lactating dairy cows. Values are means ± SEM, n = 10. Bars without a common letter differ, P < 0.05. Representative northern blots are shown below the corresponding histograms. Bar labels: control-B, control cows before commencement of treatment; NutriChlor-B, acidotic cows before commencement of treatment; Control-A, control cows 10 d after commencement of treatment; and NutriChlor-A, acidotic cows 10 d after commencement of treatment.

The data indicate that metabolic acidosis-induced protein degradation in ruminants is likely the result of upregulation of gene expression for components of the ubiquitin-proteasome proteolytic pathway. Other intracellular proteolytic systems, namely the lysosomal and Ca²⁺-dependent pathways, are also present in skeletal muscle, although we did not measure the mRNA levels for these systems; however, the ubiquitin-proteasome system is the predominant proteolytic pathway for skeletal muscle protein breakdown (9). Although metabolic acidosis increased the mRNA levels for the ubiquitin, 14-kDa E2, and C8 subunit components, there was no effect on the mRNA levels for the C9 subunit. All components of the ubiquitin-proteasome system are involved in various steps that regulate this pathway, but their relative importance in these regulatory steps has not been elucidated (9). Therefore, the physiological importance of the lack of response in mRNA abundance for the C9 subunit in parallel with the changes in mRNA abundance for the other components of the system is unknown. However, Adegoke et al. (21) also reported changes in mRNA abundance for ubiquitin, 14-kDa E2, and the C9 subunit, but not the C8 subunit, in pig intestinal mucosa. There are no published data describing gene expression for the various protein degradation systems in the skeletal muscle of acidotic dairy cows; however, the acidosis-induced upregulation of mRNA levels for major components of the ubiquitin-mediated pathway is consistent with results reported for rats (8). In sheep, NH₄Cl-induced metabolic acidosis stimulates whole-body BCAA oxidation (10), and the likely source of these BCAAs is increased skeletal muscle protein degradation.

The present study has practical implications for early-lactating dairy cows. Because feed intake is decreased during early lactation, the shortfall in nutrient supply necessitates the mobilization of body reserves (fat and protein), causing negative energy and nitrogen balance (22). A common feeding management strategy to increase energy intake is to increase the dietary content of readily fermentable nonfiber carbohydrates; however, this may cause ruminal acidosis. If ruminal acidosis is prolonged, excessive absorption of lactate into the bloodstream can induce metabolic acidosis (1), which, according to the present results, can upregulate gene expression of the major proteolytic pathway in skeletal muscle, leading to undesirable muscle protein wasting. Muscle protein mass decreases in early-lactating dairy cows (22), likely mediated via increased skeletal muscle protein degradation. In addition, cows are commonly fed anionic salts [e.g., (NH₄)₂SO₄] during the dry period to stimulate bone resorption and reduce the incidence of milk fever during early lactation (18). In this case, a urine pH of <6.5 is recommended to minimize the risk of milk fever, lower than the pH of 7.55 attained in the present study. A more severe chronic metabolic acidosis would likely result in a greater induction of protein degradation systems in skeletal muscle, further exacerbating muscle protein wasting.

In summary, our results demonstrated that the induction of chronic metabolic acidosis in lactating dairy cows upregulated skeletal muscle mRNA levels for ubiquitin, 14-kDa E2 and the C8 subunit, which are components of the ubiquitin-proteasome proteolytic pathway. This likely is the mechanism by which metabolic acidosis stimulates muscle wasting in ruminants, as has been reported for other species.

	ACKNOWLEDGMENTS

 
The authors thank the staff of the Ponsonby Dairy Research Centre (University of Guelph) for their technical assistance. The authors are grateful to Keiji Tanaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) for the gift of the plasmids encoding the rat C8 and C9 proteasome subunits, to Simon Wing (McGill University, Montreal, Canada) for the gift of the plasmid encoding the rat 14-kDa E2, and to Susan Samuels (Food, Nutrition and Health, University of British Columbia, Vancouver, Canada) for the gift of the plasmid encoding ubiquitin.

	FOOTNOTES

 
¹ Supported by grants from the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) and the Natural Sciences and Engineering Research Council of Canada (NSERC).

² Present address: Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8.

⁴ Abbreviations used: BE, base excess; 14-kDa E2, 14-kDa ubiquitin carrier protein E2; mRNA, messenger RNA; TMR, total mixed ration; VFA, volatile fatty acid.

Manuscript received 27 October 2003. Initial review completed 21 November 2003. Revision accepted 3 December 2003.

	LITERATURE CITED

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