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* Department of Animal Sciences, University of Kentucky, Lexington 40546 and
and
Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport 71130
2 Correspondence:
808 W. P. Garrigus Bldg. (phone: 859-257-7513; fax: 859-257-3412; E-mail:
jmatthew{at}uky.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Amino Acids • Biochemical Transport • Glutamates • Growth • Sheep
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Given the importance of glutamate to ruminant energy and N metabolism, and given that plasma membrane transport capacity is thought to limit glutamate metabolism (Low et al., 1994; Nissim, 1999; Gegelashvili et al., 2001), knowledge of if and how glutamate transporter expression is altered in response to growth is necessary to design diets that appropriately influence glutamate transport capacity. Two high-affinity (µM) concentrative glutamate transporters (GLT-1 and EAAC1) are expressed by intestinal epithelial, liver, or kidney tissues of young sheep (Howell et al., 2001). Although poorly characterized, expression of GLT-1 and EAAC1 is likely sensitive to substrate, hormonal, and nutritional status stimuli (Nicholson and McGivan, 1996; Matthews et al., 1999; Munir et al., 2000). Accordingly, the three primary objectives of this study were to determine the influence of growth on the amount of GLT-1 and EAAC1 expressed by these tissues in growing vs. nongrowing wethers fed forage-based diets to identify potential metabolic indicators/affectors of transporter expression, and to relate glutamate transporter expression to plasma levels of the major interorgan N carriers, glutamine and alanine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Research protocols were approved by the University of Kentucky Institutional Animal Care and Use Committee. Eighteen crossbred wether lambs (mean BW = 30 ± 1.2 kg) were obtained from the University of Kentucky Agricultural Research Center Sheep Unit and randomly assigned to one of two dietary treatments. The lambs were housed and group-fed within dietary treatments in 2.4-m2 pens for at least 14 d and then placed in metabolism crates for at least 14 d in an environmentally controlled room (21°C) with 16-h light/8-h dark photo cycles. Dietary treatments consisted of forage-based diets (Table 1
) formulated to 1.2 (n = 9) or 2.0 (n = 9) times their NEm requirements (NRC, 1985). Diets were formulated so that all lambs would receive the same amount of metabolizable protein per day using calculations described by the NRC for beef cattle (NRC, 1996). Sheep were weighed twice a week and the amount of diet fed was adjusted accordingly. The ADG achieved was calculated over 10 of the 14 d that sheep were housed in metabolism crates. Feed and fecal samples were collected the final 5 d, composited within animal across sampling day, and stored at µ30°C. Blood samples were collected by jugular venipuncture 3 h after the morning feeding on the day prior to tissue collection, and plasma harvested after centrifugation (12,000 x g for 20 min) and stored at -30°C. At the conclusion of the feeding period, animals were killed by intrajugular administration of pentobarbital (80 mg/kg of BW), and the rumen, small intestine, liver, and kidney were removed. Samples of whole ruminal contents from the midventral region of the rumen were taken, strained through four layers of cheesecloth, and the pH of the liquor determined. Five milliliters of ruminal liquor was then acidified with 1 mL of 1.1 M metaphosphoric acid and stored at µ30°C. Representative samples (1 g) of liver (caudal lobe), kidney (cortex and medulla), and ileal epithelium were homogenized in 5% trichloroacetic acid (TCA) and stored at -20°C until analyzed for AA content.
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Analysis of Diet, Ruminal Fermentation, and Blood Metabolites
Diet samples were analyzed for DM, CP, NDF, ADF, ash, GE, and DE, whereas ruminal liquor samples were analyzed for VFA, and plasma samples for VFA and glucose, as described previously (Ratner, 1943). Glutamine, alanine, and L- and D-glutamate concentrations in plasma and tissue (TCA extracts) were determined by combined HPLC and enzymatic-fluorimetric analysis. Specifically, glutamine, alanine, and total glutamate (L- and D-isomers) were quantified by HPLC after precolumn derivatization with o-phthalaldehyde (Welbourne and Chevalier, 1997). L-glutamate was measured by fluorimetric-enzymatic assay, using L-glutamate dehydrogenase (Calbiochem, LaJolla, CA) to convert L-glutamate and NAD to 
-ketoglutarate, ammonium, and NADH + H+. The reduced form of NAD was measured using a digital filter fluorimeter (model 112, Sequoia-Turner, Dubuque, IA) with a primary excitation wavelength of 320 to 390 nm and a secondary maximal emission of 415 nm (Bernt, 1974). L-glutamate was assayed with a coefficient of variation of 4.1% and a recovery (2 nmol) of 96 ± 4%. D-glutamate concentrations were taken as the difference between the total glutamate determined by HPLC and the L-glutamate concentration determined by enzymatic assay (Schuldt et al., 1999).
Isolation of Membrane Protein and Immunoblot Analysis
Tissue homogenates were centrifuged for 2 min at 400 x g to remove cellular debris. A plasma membrane-enriched fraction of cellular proteins was generated by centrifugation of the resulting supernatant at 100,000 x g for 30 min (Matthews et al., 1997). The membrane pellet was resuspended in SEB and proteins were separated by 7.5% SDS-PAGE, followed by electrotransfer to a 0.45--m nitrocellulose membrane (Bio-Rad, Hercules, CA). The GLT-1 and EAAC1 protein expression was evaluated by immunoblot analyses as previously described (Matthews et al., 1998; Howell et al., 2001). Briefly, EAAC1 was detected by hybridizing blots with 43 ng of IgG/mL of EAAC1 polyclonal antibody (Matthews et al., 1998) in blocking solution (1% nonfat dry milk and 2% casein hydrolysate in 10 mM Tris-Cl, pH 7.5, 300 mM NaCl) for 2 h at room temperature with agitation. Detection of GLT-1 was achieved by hybridization with 68 ng of IgG/mL of anti-GLT-1 polyclonal antibody (Jefferey D. Rothstein, John Hopkins University, Baltimore, MD) in blocking solution (1% nonfat dry milk in 10 mM Tris-Cl, pH 7.5, 200 mM NaCl) for 1.5 h at room temperature with agitation. Horseradish peroxidase-conjugated donkey anti-rabbit Ig (1:5000; Amersham, Arlington Heights, IL) was used to detect immunoreactive bands of both EAAC1 and GLT-1 antibodies by visualization with a chemiluminescence kit (Pierce, Rockford, IL). After exposure to autoradiographic film (Amersham), a digital image of the radiographic bands was recorded and quantified as described elsewhere (Swanson et al., 2000). Briefly, autoradiographic film was scanned (HP DeskScan II, Hewlett Packard, Mississauga, Ontario) into Adobe Photoshop (Adobe Systems Inc., San Jose, CA) and band intensities were determined using the UN-SCANIT software program (Silk Scientific, Orem, UT) and reported as arbitrary units. All observed immunoreactive species (one to three for GLT-1, two for EAAC1) within a sample were quantified. Densitometric data then were corrected for unequal (less than 20%) loading and/or transfer of GLT-1 and EAAC1 proteins by normalization to relative amounts of Fast-Green-stained (Fisher Scientific, Pittsburgh, PA) proteins common to all immunoblot lanes/samples. The relative amount of stained protein per lane for each sample of the blots was determined by densitometric analyses (as just described). Digital images were prepared with PowerPoint (Microsoft, Bellvue, WA). Apparent migration weights (Mr) were calculated by regression of the distance migrated against the Mr of a 10- to 200-kDa standard (Gibco BRL, Grand Island, NY).
Extraction of Total RNA, Isolation of Messenger RNA, and Northern Analysis
Total RNA was obtained by an acidic phenol-chloroform extraction and 15 µg of total RNA was size-separated in a 1% agarose gel containing 0.02 M formaldehyde, transferred to a nylon membrane, and crosslinked by UV light (Matthews et al., 1998). Expression of GLT-1 and EAAC1 messenger RNA (mRNA) were evaluated using [32P]-labeled rat complementary DNA (cDNA) probes specific for GLT-1 and EAAC1 as described (Matthews et al., 1998; Howell et al., 2001), except that hybridization and wash temperatures were 60 and 62°C and 55 and 65°C, respectively. After exposure to autoradiographic film, capture and reproduction of hybridization band images were as described above for immunoblots. Blots were stripped and probed (hybridization/wash, 60/64°C) for 18S ribosomal RNA (rRNA) using a [32P]-labeled mouse 18S cDNA probe (Ambion, Austin, TX). Hybridization bands were visualized and quantified by autoradiography as described above. Densitometric data for GLT-1 and EAAC1 mRNA were then corrected for unequal loading (less than 15%) by normalization to relative 18S rRNA mRNA expression.
Statistical Analysis
Data were analyzed as a completely randomized design using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC). Analysis of variance for measured variables was performed using intake in the model. Intake effects were considered significant at the = 0.05 level, unless otherwise indicated. Within intake treatments, differences between D- and L-glutamate tissue concentrations were evaluated using isomer in the model. Unequal experimental observations resulted from loss of sample integrity during collection and/or storage.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Initial BW of sheep did not differ (P < 0.540, SEM = 1.2) between dietary treatment groups. Final BW of the sheep fed the 2.0 x NEm diet was 4 ± 1.4 kg greater (P < 0.001, SEM = 1.4) than lambs fed the 1.2 x NEm diet. Final BW of lambs fed the 1.2 x NEm diet did not differ (P < 0.139) from initial BW, whereas lambs fed the 2.0 x NEm diet gained (P < 0.001) 0.26 ± 0.05 kg/d.
Tissue Content of High-Affinity Glutamate Transporters
Immunoblot analysis was performed on membrane proteins isolated from ileal epithelium, liver, and kidney tissues to compare relative levels of GLT-1 and EAAC1 expression by growing vs. nongrowing sheep (Figure 1). Two predominant EAAC1 species (93 and 67 kDa) were detected in the ileum, liver, and kidney tissues of sheep of both treatment groups. For GLT-1, ileum epithelial and kidney tissues expressed predominant species of about 188 and 142 kDa. In addition, a third immunoreactive species of >203 kDa was expressed by liver tissue. No treatment effect in the size of immunoreactive species was observed. As qualitatively observed by autoradiography (Figure 1) and quantitatively determined by densitometric analyses (Table 2), the amount of EAAC1 protein expressed by liver and kidney tissues between growing or nongrowing sheep did not differ. In contrast, the ileal epithelium of growing sheep expressed 313% more (P < 0.04) EAAC1 protein than did ileal tissue isolated from the maintained sheep. For GLT-1, the relative amount of protein expressed by ileum epithelia or kidney collected from growing and non-growing sheep did not differ. However, the liver of growing sheep expressed 240% more (P < 0.001) GLT-1 protein than did sheep that maintained their weight over the experimental period.
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To determine whether the differential expression of EAAC1 and GLT-1 protein was correlated with steady-state mRNA levels, Northern blot analysis was performed on RNA isolated from ileal and liver tissues (Figure 2). A single EAAC1 transcript of 2.8 kb was detected in the ileum of sheep from both dietary treatments, whereas a single GLT-1 transcript of about 12 kb was detected in liver tissue isolated from both groups of sheep. Equal loading and transfer of dietary treatment RNA to the blots was demonstrated by the detection of similar amounts of [32P]-labeled 18S rRNA cDNA hybridization to 18S rRNA between treatment groups (Figure 2). In contrast to the greater expression of ileal epithelium EAAC1 and liver GLT-1 protein of growing animals, densitometric analyses demonstrated that the amount of EAAC1 (P < 0.79) or GLT-1 (P < 0.45) mRNA did not differ between dietary treatment groups (Table 3).
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Concentrations of L-glutamate and D-glutamate within ileal epithelium, liver, or kidney tissues were compared within nongrowing and growing animals to establish the relationship between glutamate isoform concentrations and system X-AG glutamate transporter content (Table 4). For nongrowing animals, the ileal epithelium contained about 47% more (P < 0.001) D-glutamate than L-glutamate (43 vs. 29 nmol/mg protein), whereas liver D-glutamate concentrations were only 16% that of L-glutamate (2.7 vs. 17.4 nmol/mg). Within growing animals, ileal epithelium concentrations of L- and D-glutamate (43 vs. 36 nmol/g) did not differ (P < 0.161), whereas liver D-glutamate concentrations were 36% that of L-glutamate (P < 0.001; 5.9 vs. 16.4 nmol/mg protein). In contrast to the intestine and liver, D-glutamate was not detected in the kidneys.
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). Ileal epithelium concentrations of D-glutamate, glutamine, and alanine did not differ. In contrast, the concentration of L-glutamate in ileal epithelium, which had increased EAAC1 content (Table 2), was 49% greater (P < 0.015) in growing vs. nongrowing animals. In liver, which had increased GLT-1 content (Table 2), the concentration of D-glutamate was 119% greater (P < 0.042) in growing animals, whereas L-glutamate concentrations did not differ. In kidney tissue, no differences in AA concentrations were observed between treatment animals. Plasma Concentrations of Amino Acids Differed Between Treatments
To establish the relationships between tissue and plasma concentrations of AA, plasma concentrations of glutamine, alanine, and glutamate were determined (Table 5). Plasma glutamine concentration of growing animals was 28% greater (P < 0.009) than for nongrowing animal, whereas alanine concentrations tended (P < 0.072) to be elevated by 22%. In contrast, plasma concentrations of L-glutamate or D-glutamate did not differ between growing and nongrowing animals. Within animal treatments, D-glutamate accounted for 12.4% of the total plasma glutamate concentration in nongrowing and 7.7% in growing sheep.
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To document that differences in tissue D-glutamate levels likely resulted from an increased bacterial protein load, and as an indicator of whole-animal energy status, the VFA profiles of ruminal digesta and plasma were evaluated as a relative index of microbial fermentation (Table 6). Although ruminal liquor pH did not differ (P < 0.663) between the two dietary treatments, the total VFA concentration was 50% greater (P < 0.005) in sheep fed at 2.0 x NEm. Contributing to this increase, molar proportion of acetate was increased (P < 0.002) by 5.6% and that of propionate by 7.9% (P < 0.013) in sheep fed at 2.0 x NEm, although the ratio of acetate to propionate production did not differ (3.52 vs. 3.55; P < 0.920). In contrast to the elevated levels of acetate and propionate in ruminal liquor of growing vs. maintained sheep, the molar proportion of butyrate was 11% lower (P < 0.047). Similarly, sheep fed the 2.0 x NEm diet possessed (numerically) lower concentrations of isobutyrate, isovalerate, and valerate.
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). In contrast, butyrate and isobutyrate concentrations did not differ, 2-methylbutyrate and 3-methylbutyrate concentrations were below detectable limits (0.001 mM), and glucose concentrations did not differ. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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In terms of glutamate metabolism by ruminant tissues, glutamate oxidation by sheep small intestinal mucosa is extensive since only about 4% of gut-infused [14C]glutamate appears in the portal blood as [14C]-labeled glutamate and/or glutamine (Tagari and Bergman, 1978), and about 65 to 75% of enterally-derived glutamate is oxidized to CO2 in a single pass. In contrast, gluconeogenesis by the liver accounts for about 25% of total glutamate turnover in sheep, constituting a metabolically significant, if relatively small (4 to 6.2%), contribution to total plasma glucose (Heitmann et al., 1973; Heitmann and Bergman, 1981). In terms of whole-animal reserve gluconeogenic capacity, glutamate-derived gluconeogenesis by the kidney is important, accounting for 33 to 50% of glutamate-derived glucose in fasted and starved sheep, respectively (Heitmann and Bergman, 1981).
Despite differences in the metabolic fate of glutamate in these tissues, all of these epithelial tissues need the ability to concentrate glutamate since extracellular glutamate concentration modulates glutaminase-, glutamine synthetase-, and alanine aminotransferase-dependent metabolic pathways (Welbourne and Nissim, 2001). In addition, cellular proliferation and apoptosis events are influenced by extracellular glutamate. Given the importance of glutamate transport capacity, we tested the hypothesis that the relative content of EAAC1 and GLT-1 by these tissues would be greater in lambs that were growing (2.0 x NEm treatment) vs. maintaining (1.2 x NEm treatment) BW. These forage-based diets were formulated to yield quantities of protein (whether of dietary or microbial origin) equal to the small intestine and were similar to those used previously by this group to establish differences in growth of sheep (Swanson et al., 2000). The use of these diets to establish growing vs. nongrowing animal models was successful. Compared to nongrowing animals, lambs fed enough diet (2.0 x NEm) achieved a moderate rate of growth, increased rumen and plasma concentrations of acetate and propionate, and increased glutamine and alanine plasma concentrations. Thus, the growing sheep possessed elevated levels of nutrient metabolites, which is consistent with ruminants in a higher nutritional status. In addition, the numerically higher concentrations of ruminal liquor isoacids measured from nongrowing sheep are indicative of lower fermentable carbohydrate supply. Overall, plasma L-glutamate concentrations reported in this study are comparable to what others have observed in the plasma of fed sheep. Although the plasma values do not take into account the glutamate contained in the red blood cells, the exchange of glutamate between plasma and blood cells is reported to be minimal (Heitmann and Bergman, 1981).
Information regarding substrate regulation of system X-AG transporters is limited, especially with regard to whole-animal studies. A pertinent observation from this study was the increased expression of EAAC1 and GLT-1 by ileal epithelium and liver tissue membranes, respectively, of growing sheep. Coincident with increased glutamate transporter content was an increased concentration of L-glutamate in ileal, and D-glutamate in hepatic, cellular membranes. That EAAC1 content was elevated in ileal epithelia that contained elevated L-glutamate, but GLT-1 was not, suggests that in vivo EAAC1 expression may have been stimulated by the presence of L-glutamate, whereas GLT-1 was not. Alternatively, the elevated glutamate levels could have resulted from elevated EAAC1 function, which in turn was increased in response to some other cellular signal.
With regard to intracellular signals, the influence of intracellular L-glutamate on EAAC1 and GLT-1 contents has been investigated using primary and immortalized cell cultures. In contrast to elevated EAAC1 amounts that were coincident with increased L-glutamate concentrations (29.3 vs. 43.7 nmol/mg) in ileal epithelium of growing sheep, expression of EAAC1 protein by immortalized bovine renal tubule epithelial cells was increased when intracellular L-glutamate was decreased from approximately 30 to 15 nmol/mg of protein (Nicholson and McGivan, 1996). In another apparent difference between whole-animal and in vitro cell culture models, expression of EAAC1 by primary "astrocyte-poor" neuronal cultures was decreased in the presence of L-glutamate (Munir et al., 2000). However, further research led these investigators to conclude that intracellular glutamate level is not a sufficient stimulus for alteration of plasma membrane expression of EAAC1. Instead, only when the protein kinase C pathway was activated, would increased intracellular L-glutamate result in decreased EAAC1 expression (Robinson, 2002).
An obvious candidate for this stimuli and difference between experimental models is the influence of hormones. However, little is known regarding hormonal control of system X-AG transporter expression. Specifically, culture of C6 glioma cells with platelet-derived growth factor (PDGF) induces increased system X-AG activity and EAAC1 content in plasma membranes, which is consistent with glutamate supporting PDGF-stimulated proliferation responses (Sims et al., 2000). Research with transgenic mice (Matthews et al., 1999) indicates that placental expression of GLT-1 is stimulated by atypically high growth hormone levels and that IGF-II is required for GLT-1 and EAAC1 expression. Preliminary data from our laboratory (J. C. Matthews, unpublished data) indicate that expression of system X-AG activity by primary cultures of steer hepatocytes is stimulated by insulin. Common to both PDGF- and insulin-induced secondary messenger pathways is the dependence on tyrosine kinase and phosphatidylinositol-3 kinase activation. Therefore, given the elevated nutritional status of the growing vs. nongrowing lambs of the present study, it is likely that both insulin/PDGF and L-glutamate levels were elevated in the ileal epithelium in growing lambs and that a combination of these regulatory proteins factors resulted in the increased expression of EAAC1.
An important question of gene expression is on what level the regulation occurs. In this study, post-transcriptional regulation likely occurred because increased expression of EAAC1 protein in ileal epithelium and GLT-1 protein (Table 2) in the liver of growing animals was not paralleled by increased mRNA levels (Table 3). Several other studies also indicate that steady-state levels of glutamate transporters do not correlate with mRNA levels. For example, increased amounts of EAAC1 protein expression by AA-deprived NBL-1 cells is accompanied by a decrease in EAAC1 mRNA levels (Plakidou-Dymock and McGivan, 1993). Similarly, the expression of EAAT4 mRNA in placental tissue is not coincident with protein expression (Matthews et al., 1998; 1999). In contrast to these findings, expression of GLT-1 mRNA by primary astrocytes was paralleled by increased GLT-1 protein content when the medium was supplemented with dibutyryl-cAMP (dbcAMP) (Schlag et al., 1998). Along with the recent identification of membrane-binding proteins that separately regulate EAAC1 (Lin et al., 2001) and EAAT4 (Jackson et al., 2001) activity, these data indicate that regulation of system X-AG proteins is complex and likely involves transcriptional, post-transcriptional, and post-translational regulation, depending on the particular affects.
In terms of between-tissue comparisons of D-glutamate concentrations, a salient observation from the current study is that the liver of growing sheep contained about twice as much D-glutamate as that of nongrowing animals (Table 4). Although the concentration of D-glutamate in digesta was not determined, the greater amount of microbial fermentation byproducts found in rumen fluid (Table 6) indicates that a greater amount of microbial protein, and hence D-glutamate, was presented to the small intestine for digestion and absorption. Whether the increased hepatic D-glutamate reflects a relative saturation of hepatic oxidative capacity in animals with enough energy to grow or an enhanced expression of oxidizing capacity by animals of a nutritional status that did not support growth remains to be determined. Also unknown is the physiological significance of parallel increases in GLT-1 and D-glutamate content in liver tissue of growing animals. However, that hepatic glutamine synthetase exhibits similar affinities for the L- and D-isomer of glutamate (Curthoys and Watford, 1995) indicates that the interorgan flux of glutamine may be significantly influenced by hepatic D-glutamate concentrations.
In contrast to differences in hepatic D-glutamate concentrations, D-glutamate concentrations of ileum epithelial homogenates did not differ between homogenates of growing and non-growing sheep. However, for both growing and non-growing sheep, ileal concentrations were about ten times higher than that measured in the liver. The relatively lower liver D-glutamate level is consistent with high hepatic activities of the D-glutamate-utilizing enzymes: glutamine synthetase, D-AA oxidases, and D-glutamate cylotransferase. Comparatively, across all tissues, this relative ileal epithelium > liver > kidney D-glutamate concentration profile is consistent with D-glutamate concentrations measured in the tissues of nonruminants and is consistent with the concept that D-glutamate concentrations are inversely proportional to D-aspartate oxidase activities (D’Aniello et al., 1993; Kera et al., 1995). D-aspartate oxidase is expressed by hepatic and renal tissue of cattle (Zaar, 1996). Furthermore, although neither D-aspartate oxidase activity nor content was measured in the present study, it is of interest to determine if intestinal epithelium of young sheep expresses a low capacity to metabolize D-glutamate as is the case in young nonruminants.
With regard to interspecies comparison of D-glutamate metabolism, nonruminant hepatic and renal enzymes metabolize D-glutamate, whereas intestinal epithelia are not considered to have a great (if any) capability to metabolize D-isomers (D’Aniello et al., 1993). That D-glutamate concentration in sheep ileal epithelium was highest compared to liver and kidney suggests that the intestinal epithelia of ruminants, like nonruminants, possess a relatively low D-glutamate metabolic capacity. D-glutamate concentrations of 36.9 and 138 nmol/g of tissue have been measured in adult male rat liver and kidney, respectively (Kera et al., 1995). Because these data were reported as nmol/g of tissue, values from rat and sheep cannot be directly compared. However, if it is assumed that 1 g of tissue contains 10% protein, then D-glutamate concentration in sheep and rat liver can be compared by using a coefficient factor of 100: nmol/mg of protein x 1,000 mg of protein/g of protein * 0.1 g of protein/g of tissue = 100 nmol/g of tissue.
Accordingly, sheep liver contained 270 to 590 nmol of D-glutamate/g, which is two to four times the concentration of D-glutamate in rat liver. That kidney levels of D-glutamate (138 nmol/g) in rat exceeded that of rat liver (Kera et al., 1995), whereas D-glutamate was not detected in sheep kidney, also indicates that fundamentally different levels of D-glutamate supply and capacity for metabolism may exist between ruminant and nonruminant species. That is, animals exposed to large amounts of D-glutamate in the digesta (e.g., ruminants) may have evolved to possess increased capacities to metabolize D-glutamate after absorption.
In summary, the expression of EAAC1 and GLT-1, two system X-AG glutamate transporters, was evaluated in membranes isolated from young lambs that gained or maintained BW on forage-fed diets. No difference in transporter isoform expression was observed in the kidney between treatment groups, coincident with no difference in tissue AA concentrations. In contrast, both EAAC1 and L-glutamate content were increased in ileal epithelium of growing vs. nongrowing wethers, whereas in liver, GLT-1 and D-glutamate content were increased. In plasma, glutamine and alanine concentrations also were greater in growing animals, as were ruminal and plasma VFA concentrations. These results indicate that sheep growing at a modest rate of gain up-regulate the expression of EAAC1 and GLT-1 high-affinity glutamate transporters in ileum epithelia and liver to support growth by increasing the capacity for glutamate substrate to facilitate the unique metabolic functions of these tissues.
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Footnotes |
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Received for publication August 5, 2002. Accepted for publication November 14, 2002.
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-Glutamic acid to pyrrolidone carboxylic acid by the rat. J. Biol. Chem. 152:559–564.
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