HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jas.2007-0333v1 86/9/2321    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Regmi, P. R. Articles by Oba, M. Search for Related Content PubMed PubMed Citation Articles by Regmi, P. R. Articles by Oba, M.

Effects of ammonia load on glucose metabolism by isolated ovine duodenal mucosa^1,2

Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, Canada, T6G 2P5

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To determine the effects of ammonia load on glucose metabolism in ruminant small intestinal tissues, duodenal mucosal cells (DMC) were isolated from growing female sheep (n = 10; 46. 0 ± 0. 8 kg of BW) fed diets differing in CP content: high (19. 4%) vs. low (13. 1%). Ammonia concentration in the duodenal digesta fluid was greater for sheep fed a high CP diet compared with those fed a low CP diet (16. 4 ± 1. 0 vs. 9. 1 ± 1. 8 mM). The isolated primary mucosal cells were incubated for 90 min with [2-¹³C] glucose (3 mM) and ammonium chloride (0, 0. 1, 1, 5, 10, 20, or 50 mM) in Krebs-Ringer HEPES buffer. It was hypothesized that DMC would increase glucose carbon utilization for the synthesis of nonessential AA when the ammonia concentration in the incubation media increased. However, utilization of glucose carbon for alanine synthesis decreased linearly (P = 0. 03) as the ammonia concentration in the incubation media increased. Furthermore, glucose disappearance and utilization of glucose carbon for aspartate synthesis were not affected (P > 0. 47) by the ammonia concentration. Contrarily, in vitro glucose disappearance was greater (P = 0. 03) for DMC isolated from sheep fed a low CP diet vs. a high CP diet [14. 6 ± 1. 6 vs. 8. 6 ± 1. 3 nmol·(10⁶ cells)^–1·(90 min) ^–1], and hexokinase activity was greater (P = 0. 01) in the mucosa of sheep fed a low CP diet compared with a high CP diet (1. 22 ± 0. 05 vs. 1. 04 ± 0. 02 mUnit/mg of protein). These observations indicate that ammonia load does not affect the extent of glucose utilization by DMC, and that glucose carbon may not play a significant role for the synthesis of alanine, aspartate, or glutamate when DMC are exposed to increased concentrations of ammonia.

Key Words: ammonia • duodenal mucosal cell • glucose • ovine • nonessential amino acid

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Glucose is one of the limiting nutrients in high-producing ruminant animals (Fahey and Berger, 1988). Gluconeogenesis, the primary source of glucose in ruminants, may not be sufficient to meet the glucose demand of high-producing ruminant animals (Elliot, 1976; Hurtaud et al., 2000). Glucose absorbed in the intestine can contribute up to 30% of the total glucose supply for cattle fed high-concentrate diets (Huntington, 1997), and greater duodenal glucose supply increases milk and milk lactose yield in cows (Rigout et al., 2002). In addition, starch digestion in the small intestine is more energetically efficient compared with starch fermentation in the rumen (Huntington et al., 2006).

Ruminant duodenal mucosal cells (DMC) possess the metabolic capability to assimilate ammonia-N into nonessential AA (NEAA) such as alanine, glutamate, and aspartate (Oba et al., 2005). Alanine synthesis can be a metabolic pathway by which DMC can detoxify ammonia because of the net positive flux of alanine by the portal-drained viscera (El-Kadi et al., 2006). Depending on dietary CP content, duodenal flow of ammonia varies greatly. However, its effects on glucose metabolism in DMC have not been studied. In a previous study (Oba et al., 2005), ammonia-N assimilation into alanine by DMC increased in the presence of glucose, but it is not known whether glucose utilization for alanine synthesis increases when DMC are exposed to greater concentrations of ammonia. This information is important to develop feeding strategies to improve postruminal glucose and AA absorption and to reduce ammonia excretion in ruminant animals.

We hypothesized that glucose carbon utilized for NEAA synthesis in DMC would increase when the ruminant duodenal mucosa are exposed to a greater ammonia load. Thus, the objective of the study was to evaluate the effects of dietary CP content and ammonia load in the incubation media on glucose metabolism in DMC isolated from growing sheep.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and Diets

The experimental protocol was approved by Faculty Animal Policy and Welfare Committee at the University of Alberta.

Sheep were used as a ruminant model. Ten Suffolk crossbred growing female sheep (36. 9 ± 1. 5 kg of BW, 4 mo old) were fed a high CP (19. 4%) or a low CP (13. 1%) diet (Table 1) during the experiment. High and low CP diets were fed primarily to achieve greater and lesser ammonia loads in the duodenum, respectively. Total DM offered daily was 3. 5% of the BW, and the amount was adjusted every week according to the change in BW. Animals were fed twice daily at 0800 and 1800 h. The diets contained 3. 32 and 3. 39 Mcal of ME/kg of DM, respectively, for high and low CP diets, both of which met the energy requirements of growing sheep at ADG of 400 g/d (NRC, 1985). The dietary concentration of CP in high and low CP diets was determined to provide approximately 15% above and below the CP requirement for growing sheep, respectively (NRC, 1985).

Ammonia Measurement

Duodenal digesta were collected from the proximal 50 cm of the duodenum immediately after slaughter, and stored at –20°C until analysis. Samples were centrifuged for 10 min at 1,000 x g, and the supernatant was analyzed for ammonia concentration spectrophotometrically (Hewlett Packard 8452A Diode Array, American Laboratory Trading, Groton, CT) as described by Fawcett and Scott (1960). The samples were collected once at slaughter, which may not account for diurnal variation, but they were expected to sufficiently characterize the treatment effects due to the large difference between treatments in dietary CP content (13. 1 vs. 19. 4%).

Cell Isolation

The DMC were isolated within 30 min after slaughter according to a method described by Okine et al. (1995) with some modifications. Briefly, 1-m-long duodenal segments from 50 cm distal to the pylorus were collected immediately after slaughter. The excised duodenal tissue was flushed with Krebs-Ringer HEPES buffer (120 mM NaCl, 4. 7 mM KCl, 1. 2 mM MgSO₄, 1. 2 mM KH₂PO₄, 25 mM NaHCO₃, and 25 mM HEPES; oxygenated with O₂:CO₂ [95:5], pH 7. 4; solution A) to remove digesta and mucus. The flushed tissue was filled with solution A containing 2. 5 mg/mL BSA (Sigma Chemical Co., St. Louis, MO; solution B), and both ends of the duodenal segment were clamped. The duodenal tissue filled with solution B was incubated in a container that contained solution A for 15 min in a shaking water bath (Julabo SW 22, Labortechniek, Seelbach, Germany) at 37°C. The duodenal contents were discarded and the segment was again filled with the solution B containing 1. 5 mg/mL hyaluronidase (Worthington Biochemical Corporation, Lakewood, NJ). The refilled segments containing the digestion solution were kept on crushed ice in a container, and gently patted with the fingertips for 2 min to release the DMC into the lumen. The resulting DMC suspension was washed 3 times with solution A followed by centrifugation (1,000 x g for 3 min each time). Yield of isolated cells was 98 ± 0. 9 x 10⁷ per isolation, and their viability as determined by trypan blue dye exclusion was 77. 6 ± 3. 2% (n = 10). The loss of viability after a 90-min incubation at 37°C was <5%.

Incubation and Metabolite Analysis

Immediately after cell yield and viability were determined, the isolated primary cells were incubated for 90 min with glucose (3 mM) and ammonium chloride (0, 0. 1, 1, 5, 10, 20, or 50 mM) in solution A in a heated (37°C) shaking water bath. The incubation time of 90 min was chosen according to Oba et al. (2004), and metabolic conversion of glucose to other metabolites was expected to be linear during the 90-min incubation. Ammonia concentrations of the incubation media were selected to reflect the wide range of ammonia concentrations predicted to exist in the duodenum (Winnicka et al., 1992; Parker et al., 1995). The glucose concentration in the incubation media represents blood glucose concentration observed in ruminants (Fernandez et al., 2001). The study was conducted by using parallel incubations; the first set of flasks contained [2-¹³C] glucose (Cambridge Isotope Laboratories Inc., Andover, MA) in the media to determine isotopic enrichment of NEAA and lactate, whereas the second set of flasks contained [U-¹⁴C] glucose (Sigma; 0. 78 µCi/flask; 13. 3 µCi/mmol of glucose) in the media to determine ¹⁴CO₂ production from glucose. Incubation was initiated by adding 0. 5 mL of the cell suspension (1 x 10⁷ viable cells) to the media in 25-mL Erlenmeyer flasks. The flasks, containing 3 mL of total incubation volume, were oxygenated and capped immediately. The flasks in the first set were capped with glass caps, whereas the flasks in the second set were sealed with a rubber serum cap fitted with a suspended center well (Kontes, Vineland, NJ) containing a folded piece of filter paper. Incubation was terminated in both sets of flasks after 90 min by adding 0. 2 mL of concentrated HClO₄ to the media. Each treatment was carried out in triplicate, and all flasks were incubated together. Two additional sets of flasks in triplicate were also prepared for the assessment of endogenous metabolite concentrations and as a 0-min control; the media were acidified immediately after the addition of the cell suspension (endogenous metabolites) or following the addition of 3 mM glucose and cell suspension (0-min control).

After termination of incubation, the incubation media in the first set of flasks were neutralized with 0. 275 mL of 5. 8 M K₂CO₃, and clarified supernatant was obtained by centrifugation (2,300 x g for 7 min). Glucose concentration of the supernatant was determined using glucose oxidase/peroxidase enzyme and dianisidine dihydrochloride (Sigma) as described previously (Raabo and Terkildsen, 1960), and the absorbance was determined with a microplate spectrophotometer (Spectra Max 190, Sunnyvale, CA). Net glucose disappearance was calculated by subtracting the amount of glucose in incubation media of treatment flasks from that of 0-min control.

A known weight (0. 5 g) of the internal standard mixture containing tracers ([U-¹³C] labeled alanine, aspartate, glutamate, and lactate; 250 nmol/g each) was added to a known weight (2 g) of clarified medium for determination of metabolite concentrations by the isotope dilution technique. To enhance gas chromatography-mass spectrometry (GC-MS) measurements at low substrate concentrations, the internal standard mixture also contained known amounts of unlabeled alanine, aspartate, glutamate and lactate to raise unlabelled substrate concentrations to within the standard curve range. Samples were applied to 1. 0 g of cation-exchange resin (AG-50W- x 8, 100–200 mesh, H⁺-form; Bio-Rad Laboratories, Hercules, CA) to separate AA and lactic acid. Standard curves were generated by adding different concentrations of unlabelled substrates to the internal standard. Alanine, aspartate, glutamate, and lactate were converted to tertiary butyldimethylsilyl derivatives (Calder and Smith, 1988). Concentration of metabolites and their isotopic enrichment with ¹³C were determined using GC-MS under electron impact mode (Agilent 6890 coupled to an Agilent 5973 Mass Selective Detector, Agilent, Palo Alto, CA). The following masses were monitored by GC-MS: alanine 260, 261, 263; lactate 261, 262, 264; aspartate 418, 419, 422; and glutamate 433, 434, 437. To make corrections for spill-over of each ion, ratios of following masses were individually determined for [U-¹³C] labeled and unlabeled alanine, lactate, aspartate, and glutamate (from the same lots that were used to prepare internal standard): alanine 260:261, 260:262, 262:263, 260:263; lactate 261:262, 261:264; aspartate 418:419, 418:422; and glutamate 433:434, 433:437.

For the second set of flasks, after termination of incubation, 0. 3 mL of benzethonium hydroxide was injected into the center wells using a syringe and left for 1 h at room temperature to capture CO₂. Center wells were transferred to scintillation vials and filled with 4 mL of scintillant (Ecolite, Research Product Division, Irvine, CA). Radioactivity was determined by liquid scintillation spectroscopy (Beckman LS Beta Counter 5801, Minneapolis, MN), and the amount of glucose oxidized to CO₂ was calculated after subtracting the background nonspecific activity (0-min control).

Enzyme Assay

Additional duodenal tissue (approximately 10 cm in length) was collected from the duodenum, proximal to the previously collected segment, immediately after slaughter. The tissue was rinsed with solution A before freezing at –80°C. Duodenal mucosal cells were scraped from the frozen duodenal tissue using a glass slide. The scraped mucosal cells were homogenized in a buffer containing 0. 15 M KCl, 5 mM MgSO₄, and 5 mM EDTA. Cell disintegration was carried out using a sonicator (Sonic 300 Dismembrator, Manassas, VA) for 3 min. The homogenate was centrifuged at 21,000 x g at 0°C for 1 h, and the supernatant was used for the measurement of hexokinase (EC 2. 7. 1. 1) and pyruvate kinase (EC 2. 7. 1. 40) activity as described by Darrow and Colowick (1962) and Cardenas and Dyson (1973), respectively. Briefly, for the glucokinase assay, 2. 5 mL of a reaction cocktail (pH 8. 5; prepared by mixing 5. 0 mL of 100 mM glycylglycine buffer, 5. 0 mL of 200 mM ATP, 6. 0 mL of 0. 01% cresol red, and 33. 4 mL of deionized water) was added with 0. 4 mL of 200 mM glucose and 0. 1 mL of the supernatant in a cuvette, and the absorbance of the solution was measured at 560 nm at 25°C for 5 min using a spectrophotometer (Beckman DU50, Beckman Analytical, Palo Alto, CA). For the pyruvate kinase assay, a reaction cocktail composed of 1. 3 mL of deionized water, 0. 8 mL of 100 mM potassium phosphate buffer (pH 7. 6 at 37°C), 0. 16 mL of 8 mM phosphoenol pyruvate, 0. 2 mL of 3 mM β-NADH, 0. 2 mL of 100 mM MgSO₄, 0. 1 mL of 40 mM ADP, 0. 04 mL of L-lactic dehydrogenase, and 0. 1 mL of 30 mM fructose diphosphate was prepared. The cocktail was mixed with 0. 1 mL of the supernatant in a cuvette, and the absorbance of the solution was determined at 340 nm at 37°C for 5 min using the same spectrophotometer.

Statistical Analysis

The effect of dietary CP was determined using the MIXED procedure (SAS Institute Inc., Cary, NC) using the following model: Y_ijk = µ + P_i + T_j + P_i x T_j + _ijk, where Y_ijk was the observed value, µ was the overall mean, P_i was the effect of dietary CP concentration, and T_j was the effect of slaughter time. The linear and quadratic effects of ammonia concentration in the incubation media was determined using the following model: Y_ijkl = µ + P_i + T_j + P_i x T_j + S_k(P_i x T_j) + A_l + T_j x A_l + P_i x T_j x A_l + A_l x A_l + _ijkl,, where Y_ijkl was the observed value, µ was the overall mean, P_i was the fixed effect of dietary CP concentration, T_j was the random effect of slaughter time, S_k was the random effect of sheep, and A_l was the fixed effect of ammonia concentration in the incubation media. The second statistical model had originally included interactions between the effects of ammonia and dietary CP concentration, but they were removed as they were not significant (P > 0. 27) for any response variable. Linear and quadratic contrasts based on unequally spaced treatments were used to evaluate effects of ammonia concentration. Treatment effects were considered significant at P < 0. 05.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Dietary treatment did not affect (P 0. 17) DMI, BW gain, and ADG at slaughter (Table 2). The CP intake (P < 0. 001) and duodenal ammonia concentration (P = 0. 005) were greater for the high CP treatment compared with the low CP treatment.

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

It was observed previously that ammonia-N assimilation into NEAA by DMC increased in the presence of glucose (Oba et al., 2005). However, the previous study did not examine how the extent of exposure of DMC to ammonia affects glucose carbon utilization for NEAA synthesis by DMC. This information is important because glucose is one of the limiting nutrients for high-producing ruminant animals (Fahey and Berger, 1988). A significant amount of dietary starch reaches the duodenum when ruminant animals are fed a high-grain diet, and some starch is absorbed as glucose in the small intestine (Owens et al., 1986; Huntington, 1997). In addition, dietary CP content affects ammonia concentration in the ruminant duodenum as shown in this study. Using ovine DMC as a model, the current study was conducted to determine if ammonia load affects glucose metabolism by DMC.

Results of this study indicate that ammonia concentration in the incubation media did not affect glucose disappearance or glucose carbon utilization for the production of CO₂ and aspartate by DMC. As the ammonia concentration in the incubation media increased, glucose carbon utilization for alanine production by DMC decreased. This did not support our hypothesis that glucose carbon utilized to synthesize NEAA in DMC would increase when the duodenal mucosa are exposed to greater ammonia concentration. The present results indicated that glucose can provide carbon for NEAA synthesis in DMC, but glucose carbon utilization for alanine and aspartate synthesis does not change as ammonia concentration in the incubation media increases.

In agreement with the present results, Topping and Visek (1977) indicated that ammonia had no effect on glucose oxidation in rat enterocytes, whereas Prior et al. (1974) observed that ammonia increased glucose oxidation in chick enterocytes. In contrast to the present results that lactate production from glucose increased up to 0. 1 mM and decreased to 50 mM ammonia concentration during a 90-min incubation, Prior et al. (1974) indicated that 10 mM ammonia increased lactate synthesis from glucose in chick enterocytes during a 60-min incubation. A previous study on glucose metabolism in isolated chick enterocytes (Porteous, 1980) indicated that AA such as glutamate, aspartate, and glutamine, which can produce ammonia in the incubation media, had no effect on glucose oxidation and use of glucose carbon for the synthesis of lactate, aspartate, glutamate, and glutamine. However, AA increased the use of glucose carbon for alanine synthesis. Kight and Fleming (1995) reported that glutamine had a suppressive effect on glucose metabolism by altering the amount of pyruvate carbon entering the TCA cycle in rat enterocytes. The reasons for the discrepancies between the current study and previous studies are not clearly understood, but might be attributed to the differences in incubation conditions and animal species.

The present study showed that the DMC isolated from sheep fed a low CP diet utilized more glucose compared with those from sheep fed a high CP diet. This is attributed to greater hexokinase activity observed in the duodenal mucosa from sheep fed low dietary CP. The greater hexokinase activity indicated that sheep fed a low CP diet might have relied on glucose as a metabolic fuel to a greater extent compared with those fed a high CP diet. Although we used urea to increase dietary CP concentration, we expected that AA absorption at the duodenum was greater for sheep fed a high CP diet because of the amount of microbial protein. According to the dairy NRC (2001) model, microbial protein produced in sheep fed the high CP diet was greater by 57 g/d than those fed the low CP diet. Thus, it is speculated that the greater amount of AA absorbed spared glucose use in the DMC of sheep fed a high CP diet compared with those of sheep fed a low CP diet. Furthermore, the interaction between the effects of in vitro ammonia concentrations and dietary CP contents was not significant for any of the response variables observed in the present study, indicating that the effects of dietary treatment may not be attributed to the difference in the ammonia load between the diets. Previous studies showed that glucose metabolism in DMC can be increased when AA such as glutamate, glutamine (Oba et al., 2004), or leucine (Reiser et al., 1975) are limiting. Further, Taniguchi et al. (1995) showed that the net absorption of glucose across the portal-drained viscera was reduced with decreased protein supply, indicating that low protein supply can enhance glucose demand by the intestinal tissues. However, in contrast to our finding that hexokinase activity is decreased with high dietary CP, hexokinase activity in rat intestinal mucosa was not affected by high dietary casein intake (Shakespeare et al., 1969).

Previous in vivo studies (Castlebury and Preston, 1993; Taniguchi et al., 1995) showed that abomasal or duodenal protein supply increased intestinal starch disappearance or glucose absorption into the portal vein. In a steer infused with 800 g/d corn starch into the abomasum, abomasal supply of 800 g/d sodium caseinate increased the net glucose absorption across portal-drained viscera by 2-fold compared with the ruminal supply of the same quantity of sodium caseinate (Taniguchi et al., 1995). Similarly, in a study with sheep, small intestinal starch disappearance and glucose absorption across the portal-drained viscera were increased with greater casein infusion into the abomasum (Castlebury and Preston, 1993). The reasons for the increased glucose absorption with greater CP supply in these studies could be that the presence of additional AA reduced glucose metabolism in the intestinal tissues (Oba et al., 2004; Reiser et al., 1975). Another study showed that abomasal infusion of casein increased Na⁺ glucose co-transporter 1 activity in the small intestinal mucosa of sheep (Mabjeesh et al., 2003), indicating potentially enhanced glucose absorption from the intestinal lumen.

In contrast to the results of the present study, some studies (Guerino et al., 1991; El-Kadi et al., 2006) have reported that increased CP supply had either no or negative effects on net glucose absorption across portal-drained viscera. Increased casein infusion into the duodenum (up to 105 g/d) had no effect on arterial glucose utilized by mesenteric-drained viscera in sheep fed a low starch (3. 9%) diet (El-Kadi et al., 2006). In the El-Kadi et al. (2006) study, glucose absorption in the duodenum was expected to be negligible because of the extremely low dietary starch intake, which might be the reason for the lack of protein effects on glucose metabolism. Intestinal protein supply increased the amount of starch that disappeared in the intestine (Richards et al., 2002) and increased glucose absorption across the portal-drained viscera (Taniguchi et al., 1995) when a large quantity of starch was available in the duodenum. However, in a study with steers fed a high concentrate (78% corn) diet, abomasal infusion of casein (150 and 300 g/d) decreased the amount of glucose absorbed across the portal-drained viscera (Guerino et al., 1991). The exact mechanism responsible for the discrepancies among the literature warrants further investigation.

In the present study, dietary CP intake had no effect on amount of glucose oxidized and glucose carbon utilized for lactate, alanine, aspartate, and glutamate production in isolated DMC. Thus, the metabolic pathways that account for greater in vitro glucose disappearance by DMC for the low CP treatment were not identified in the current study. However, the greater glucose disappearance for the DMC obtained from the sheep fed a low CP diet might be attributed to its utilization for the synthesis of intermediates of the pentose phosphate pathway, one of the major metabolic pathways in enterocytes (Wu, 1996).

In conclusion, under the incubation conditions employed in this study, a greater ammonia load in the incubation media did not change glucose utilization by DMC, and the interaction between the effects of in vitro ammonia concentrations and dietary CP contents was not significant for any of the response variables observed in the present study. These results indicate that glucose carbon may not play a significant role for the synthesis of alanine, aspartate, or glutamate when the DMC are exposed to greater ammonia load. However, high dietary CP intake reduced glucose metabolism in DMC probably because of decreased hexokinase activity. Further investigation is warranted to understand the physiological basis that regulates glucose utilization in the DMC.

	Footnotes

 
¹ This work was supported by Natural Sciences and Engineering Research Council of Canada.

² The assistance provided by B. J. Bequette of the University of Maryland for GC-MS analysis is gratefully acknowledged.

³ Corresponding author: masahito.oba{at}ualberta.ca

Received for publication June 7, 2007. Accepted for publication May 3, 2008.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


Baldwin, R. L. VI, and K. R. McLeod. 2000. Effects of diet forage:concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro. J. Anim. Sci. 78:771–783.[Abstract/Free Full Text]

Calder, A. G., and A. Smith. 1988. Stable isotope ratio analysis of leucine and ketoisocaproic acid in blood plasma by gas chromatography/mass spectrometry: Use of tertiary butyldimethylsilyl derivatives. Rapid Commun. Mass Spectrom. 2:4–16.[Medline]

Cardenas, J. M., and R. D. Dyson. 1973. Bovine pyruvate kinase. II. Purification of the liver isozyme and its hybridization with skeletal muscle pyruvate kinase. J. Biol. Chem. 248:6938–6944.[Abstract/Free Full Text]

Castlebury, R. E., and R. L. Preston. 1993. Effect of dietary protein level on nutrient digestion in lambs duodenally infused with cornstarch. J. Anim. Sci. 71(Suppl. 1):264. (Abstr. )

Darrow, R. A., and S. P. Colowick. 1962. Hexokinase from baker’s yeast. Pages 226–227 in Methods in Enzymology No. 5. S. P. Colowick and N. O. Kaplan, ed. Academic Press Inc., New York, NY.

El-Kadi, S. W., R. L. Baldwin VI, N. E. Sunny, S. L. Owens, and B. J. Bequette. 2006. Intestinal protein supply alters amino acid, but not glucose, metabolism by the sheep gastrointestinal tract. J. Nutr. 136:1261–1269.[Abstract/Free Full Text]

Elliot, J. M. 1976. The glucose economy of the lactating dairy cow. Pages 59–66 in Proc. Cornell Nutr. Conf. Feed Manuf., Ithaca, NY.

Fahey, G. C., Jr., and L. L. Berger. 1988. Carbohydrate nutrition of ruminants. Page 269 in The Ruminant Animal: Digestive Physiology and Nutrition. D. C. Church, ed. Prentice Hall, Englewood Cliffs, NJ.

Fawcett, J. K., and J. E. Scott. 1960. A rapid and precise method for the determination of urea. J. Clin. Pathol. 13:156–160.[Abstract/Free Full Text]

Fernandez, J. M., T. Sahlu, S. P. Hart, M. J. Potchoiba, H. M. E. Shaer, N. Jacquemet, and H. Carneiro. 2001. Experimentally induced subclinical hyperammonemia in dairy goats. Small Rumin. Res. 42:5–20.[CrossRef]

Guerino, F., G. B. Huntington, and R. A. Erdman. 1991. The net portal and hepatic flux of metabolites and oxygen consumption in growing beef steers given postruminal casein. J. Anim. Sci. 69:387–395.[Abstract]

Huntington, G. B. 1997. Starch utilization by ruminants: From basics to the bunk. J. Anim. Sci. 75:852–867.[Abstract/Free Full Text]

Huntington, G. B., D. L. Harmon, and C. J. Richards. 2006. Sites, rates, and limits of starch digestion and glucose metabolism in growing cattle. J. Anim. Sci. 84(E Suppl. ):E14–E24.[Abstract/Free Full Text]

Hurtaud, C., S. Lemosquet, and H. Rulquin. 2000. Effect of graded duodenal infusions of glucose on yield and composition of milk from dairy cows. 2. Diets based on grass silage. J. Dairy Sci. 83:2952–2962.[Abstract]

Kight, C. E., and S. E. Fleming. 1995. Oxidation of glucose carbon entering the TCA cycle is reduced by glutamine in small intestine epithelial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 268:G879–G888.[Abstract/Free Full Text]

Mabjeesh, S. J., D. Guy, and D. Sklan. 2003. Na+/glucose co-transporter abundance and activity in the small intestine of lambs: Enhancement by abomasal infusion of casein. Br. J. Nutr. 89:570–580.

NRC. 1985. Nutrient Requirements of Sheep. 6th rev. ed. Natl. Acad. Press, Washington, DC.

NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Press, Washington, DC.

Oba, M., R. L. Baldwin VI, and B. J. Bequette. 2004. Oxidation of glucose, glutamate, and glutamine by isolated ovine enterocytes in vitro is decreased by the presence of other metabolic fuels. J. Anim. Sci. 82:479–486.[Abstract/Free Full Text]

Oba, M., R. L. Baldwin VI, S. L. Owens, and B. J. Bequette. 2005. Metabolic fates of ammonia in ruminal epithelial and duodenal mucosal cells isolated from growing sheep. J. Dairy Sci. 88:3963–3970.[Abstract/Free Full Text]

Okine, E. K., D. R. Glimm, J. R. Thompson, and J. J. Kennelly. 1995. Influence of stage of lactation on glucose and glutamine metabolism in isolated enterocytes from dairy cattle. Metabolism 44:325–331.[CrossRef][Medline]

Owens, F. N., R. A. Zinn, and Y. K. Kim. 1986. Limits to starch digestion in the ruminant small intestine. J. Anim. Sci. 63:1634–1648.[Abstract/Free Full Text]

Parker, D. S., M. A. Lomax, C. J. Seal, and J. C. Wilton. 1995. Metabolic implications of ammonia production in the ruminants. Proc. Nutr. Soc. 54:549–563.[CrossRef][Medline]

Porteous, J. W. 1980. Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush border cells. Biochem. J. 188:619–632.[Medline]

Prior, R. L., D. C. Topping, and W. J. Visek. 1974. Metabolism of isolated chick small intestinal cells: Effects of ammonia and various salts. Biochemistry 13:178–183.[CrossRef][Medline]

Raabo, E., and T. C. Terkildsen. 1960. On the enzymatic determination of blood glucose. Scand. J. Clin. Lab. Invest. 12:402–407.[Medline]

Reiser, S., O. E. Michaelis 4th, and J. Hallfrisch. 1975. Effects of sugars on leucine and lysine uptake by intestinal cells from rats fed sucrose and stock diets. Proc. Soc. Exp. Biol. Med. 150:110–114.[CrossRef][Medline]

Richards, C. J., A. F. Branco, D. W. Bohnert, G. B. Huntington, M. Macari, and D. L. Harmon. 2002. Intestinal starch disappearance increased in steers abomasally infused with starch and protein. J. Anim. Sci. 80:3361–3368.[Abstract/Free Full Text]

Rigout, S., S. Lemosquet, J. E. Van Eys, J. W. Blum, and H. Rulquin. 2002. Duodenal glucose increases glucose fluxes and lactose synthesis in grass silage-fed dairy cows. J. Dairy Sci. 85:595–606.[Abstract]

Shakespeare, P., L. M. Srivastava, and G. Hubscher. 1969. Glucose metabolism in the mucosa of the small intestine: The effect of glucose on hexokinase activity. Biochem. J. 111:63–67.[Medline]

Taniguchi, K., G. B. Huntington, and B. P. Glenn. 1995. Net nutrient flux by visceral tissues of beef steers given abomasal and ruminal infusions of casein and starch. J. Anim. Sci. 73:236–249.[Abstract]

Topping, D. C., and W. J. Visek. 1977. Synthesis of macromolecules by intestinal cells incubated with ammonia. Am. J. Physiol. 233:E341–E347.[Medline]

Winnicka, A. W., K. P. Ostaszewski, E. Miernik-Degnrska, and A. Degnrski. 1992. Effect of subclinical hyperammonaemia on phagocytic activity of polymorphonuclear leukocytes isolated from peripheral blood of sheep. Small Rumin. Res. 7:253–264.[CrossRef]

Wu, G. 1996. An important role of pentose cycle in the synthesis of citrulline and proline from glutamine in porcine enterocytes. Arch. Biochem. Biophys. 336:224–230.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: jas.2007-0333v1 86/9/2321    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Regmi, P. R. Articles by Oba, M. Search for Related Content PubMed PubMed Citation Articles by Regmi, P. R. Articles by Oba, M.