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Nutrient-Gene Expression

Poly(A)⁺ RNA Encoding Proteins Capable of Transporting L-Methionine and/or DL-2-Hydroxy-4-(Methylthio) Butanoic Acid Are Present in the Intestinal Mucosa of Broilers¹

Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 and ^* Novus International, Incorporated, St. Charles, MO 63304

²To whom correspondence should be addressed. E-mail: webbk{at}vt.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
To investigate the presence of poly(A)⁺ RNA that encode proteins capable of transporting L-methionine (L-Met) and/or DL-2-hydroxy-4-(methylthio) butanoic acid (HMB), Xenopus oocytes were injected with poly(A)⁺ RNA isolated from broiler intestinal mucosa. Healthy oocytes at stage V or VI were collected from Xenopus laevis and microinjected with water, poly(A)⁺ RNA or size-fractioned poly(A)⁺ RNA. The ability of the injected oocytes to take up either L-Met or HMB was examined by incubating oocytes with [methyl-³H]-L-Met or [5-¹⁴C]-HMB. A greater uptake of L-Met (P < 0.01) and HMB (P < 0.05) by oocytes injected with poly(A)⁺ RNA from the duodenum, jejunum and ileum of the small intestine was observed compared with water-injected oocytes. The greatest (P < 0.05) uptake occurred when poly(A)⁺ RNA from the jejunum or ileum was injected. Injections from four different pools of sucrose gradient–fractionated poly(A)⁺ RNA from all three intestinal segments induced (P < 0.01) L-Met uptake. There were three to four different pools of sucrose gradient–fractionated poly(A)⁺ RNA from the duodenum, jejunum and ileum that induced (P < 0.05) HMB uptake. Uptake of HMB was greater at pH 5.5 than at pH 7.5 and was independent of Na⁺. Uptake of L-Met induced by all four poly(A)⁺ RNA pools decreased dramatically when Na⁺ was removed from the uptake buffer, which indicated that the majority of L-Met uptake was Na⁺-dependent. These results indicate that there are multiple sized poly(A)⁺ RNA that encode proteins capable of mediated transport of L-Met and/or HMB present in broiler intestinal mucosa.

KEY WORDS: • Xenopus • chickens • absorption • DL-2-hydroxy-4-(methylthio) butanoic acid • methionine

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Methionine and DL-2-hydroxy-4-(methylthio) butanoic acid (HMB) are commonly used as supplements in diet formulation in poultry feeding because of insufficient quantities of Met in commercial feedstuffs. For years, several models have been employed in attempts to reveal the absorption mechanisms for Met and/or HMB in chickens (1 –7 ). These studies suggested that mechanisms of methionine absorption included Na⁺-dependent transport, Na⁺-independent transport and/or diffusion. Mechanisms suggested for HMB absorption included Na⁺-independent but H⁺-dependent transport, and/or diffusion. However, because of the inherent characteristics of the models used in the studies, the actual presence of mRNA that encode proteins capable of Met or HMB uptake or the presence of specific transport proteins for these substrates in chicken intestine has not been described.

The application of molecular biotechnology to nutrition research has provided tools for researchers attempting to resolve the nature of potential absorption mechanisms. One of the unique techniques that has resolved many questions related to transport proteins is expression cloning using Xenopus laevis oocytes. Xenopus oocytes are a robust system for the expression of many different proteins of animal or plant origin (8 ). The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis. Xenopus oocytes at stage V have the complete machinery to translate exogenous RNA faithfully. The most important aspect is that the oocyte expression system can make post-translational modifications, which preserves the native biological activities of the expressed proteins. The oocyte microinjected with poly(A)⁺ RNA isolated from exogenous tissue sources will faithfully synthesize and assemble a variety of functional proteins. Utilizing this approach, we have been able to characterize the molecular and electrophysiologic properties of a cloned ovine peptide transporter (9 ).

To determine the presence of membrane proteins capable of transporting L-Met and/or HMB in intestinal mucosa of broilers, functional expression of chicken poly(A)⁺ RNA fractions in Xenopus laevis oocytes was employed as the model system in the present study. Surveying for the presence of mRNA that encode proteins capable of Met or HMB transport by functional expression of size-fractioned mRNA provides a valuable first step in the eventual identification and characterization of specific transport proteins. Our results indicate that there are multiple poly(A)⁺ RNA fractions that encode proteins capable of mediated transport of L-Met and/or HMB present in broiler intestinal mucosa.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Unless noted otherwise, all chemicals, substrates and reagents were of either molecular biology– or cell culture–tested chemical grades. For RNA extraction and processing, all water, solutions and equipment were appropriately treated to control nuclease activity (10 ). Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). All buffers used for oocyte culture were sterilized by filtration (0.2-µm tissue culture filter unit from Nalgene, Rochester, NY). [5-¹⁴C] DL-HMB was provided by Novus International (St. Charles, MO) and [methyl-³H] L-Met was purchased from ICN Biomedicals, (Irvine CA).

Birds.

Male broilers [42 d old; body weight, 2.03 kg] were killed by electrocution. The gastrointestinal tract was removed and cut open to clean residual digesta by rinsing in cold 9 g/L saline. The mucosa was scraped using a glass slide and all of the collected scrapings were quickly frozen in liquid nitrogen and later transferred to -80°C for storage. All animal procedures were approved by Virginia Tech’s Animal Care Committee.

Extraction of total RNA and isolation of size-fractionated poly(A)⁺ RNA.

Total RNA was extracted from homogenized mucosa using an acidic phenol/chloroform solution (Tri Reagent, Molecular Research Center, Cincinnati, OH). Aqueous supernatants (containing the total RNA) were pooled and stored at -80°C as a sodium acetate (0.3 mol/L, pH 5.2) and ethanol (2.5 volumes) precipitate. Poly(A)⁺ RNA was isolated from total RNA by chromatography on oligo(dT) cellulose (Type 7, Pharmacia Biotech, Piscataway, NJ) as described by Sambrook et al. (11 ). The poly(A)⁺ RNA isolation was performed at least once on an oligo(dT) cellulose column and then the purity of poly(A)⁺ RNA was examined on a 10 g/L agarose gel containing 2.2 mol/L formaldehyde. If both 18s and 28s rRNA were still visible, a second enrichment step was performed on a separate column of oligo(dT) cellulose. Appropriate eluates were collected and the poly(A)⁺ RNA precipitated with isopropanol. The poly(A)⁺ RNA precipitate was recovered after centrifugation for 30 min at 48,000 x g at 4°C.

When appropriate, poly(A)⁺ RNA were size-fractionated (11 ) by centrifugation through a 13-mL, 80–200 g/L linear sucrose gradient for 15.5 h at 80,000 x g at 4°C. Typically, 48 fractions (0.27 mL each) were collected. An equal volume of 5.0 mmol/L ß-mercaptoethanol was added to each fraction to reduce the methyl mercuric hydroxide (Johnson Matthey, Ward Hill, MA) in the gradient buffer, and the size-fractionated poly(A)⁺RNA were stored as an ethanol precipitate at -20°C.

Preparation of oocytes and microinjection of poly(A)⁺ RNA.

Typically, several ovarian lobes were removed (12 ) from anesthetized female Xenopus laevis frogs and placed in a Ca²⁺-free medium (96 mmol/L NaCl, 2 mmol/L KCl, 1 mmol/L MgCl₂, 5 mmol/L HEPES, pH 7.5; 13 ). Oocytes were rinsed in Ca²⁺-free medium, and Stage V and VI oocytes (14 ) were manually defolliculated after being treated with Collagenase A (Boehringer Mannheim, Indianapolis, IN). The defolliculated oocytes were rinsed and allowed to heal at 18°C for 18–24 h in Ca²⁺-containing medium (96 mmol/L NaCl, 2.5 mmol/L sodium pyruvate, 2 mmol/L KCl, 1.8 mmol/L CaCl₂, 1 mmol/L MgCl₂, 1.0 x 10⁵ U/L of penicillin-G, 1.67 x 10⁵ U/L of streptomycin and 5 mmol/L HEPES, pH 7.5).

Only healthy oocytes with a resting membrane potential more negative than -30 mV 1 d after removal of follicular tissue were used for injection. Oocytes were placed in a petri dish containing injection buffer (96 mmol/L NaCl, 2 mmol/L KCl, 1.8 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5 mmol/L HEPES/NaOH, pH 7.5) and injected into their vegetal pole near the polar interface. Individual fractions of the stored, sucrose gradient–fractionated poly(A)⁺ RNA were recovered as described above, suspended in RNase-free water, quantified by measuring absorbance at 260 nm, and either injected immediately or stored at -20°C for no >1 wk before injection. After receiving their injection treatments, oocytes were cultured for an appropriate time (usually 4–7 d) at 18°C in Ca²⁺-containing medium. Oocytes injected with water served as controls. Oocytes of the same injection treatment were cultured in a common 20-mL borosilicate vial. The medium was changed daily and damaged oocytes were discarded.

L-Met and DL-HMB uptake experiments.

As the initial step to identify chicken intestinal mucosa tissue mRNA encoding proteins capable of transport of HMB and Met, oocytes were injected with 50 nL of polyA⁺ RNA or water and incubated for a varying number of days. A time-course trial was also conducted to determine the optimal length of uptake time to measure the transport of HMB or methionine. Then, individual fractions of sucrose-gradient fractionated poly(A)⁺ RNA were examined for their ability to induce HMB and L-Met Na⁺-dependent, Na⁺-independent and H⁺-dependent uptake. Only those fractions that were able to induce uptake were used for more detailed evaluations of the transport process.

Only those oocytes that were healthy and of the same size and stage of maturation were selected for uptake experiments on the day of assay. Before assay, oocytes were washed four times in the appropriate uptake buffer to remove any residual buffer salts and antibiotics. Uptake experiments were initiated by placing all oocytes of a given injection treatment into 7-mL glass vials that contained 0.5 mL of experimental uptake buffer. The uptake buffer consisted of appropriate uptake buffer containing 2.9 mCi/L of [¹⁴C] HMB (38 mCi/mmol) or 3 mCi/L [³H] L-Met (60 mCi/mmol).

Uptake was terminated by the addition of 4 mL of 4°C stop solution (uptake buffer plus 5 mmol/L HMB or L-Met). Treatment groups of oocytes were serially washed six times in 1.5 mL of the appropriate 4°C stop solution in a 24-well tissue culture plate. Aliquots (30 µL) of the last wash of each treatment group were collected for determination of background radioactivity. Oocytes were individually transferred in 30 µL of stop solution into 7-mL plastic scintillation vials containing 0.25 mL of 10% SDS and allowed to digest at room temperature (21°C). Then, 4 mL of scintillation fluid (Scintiverse BD; Fisher Scientific, Fairlawn, NJ) was added to each vial and these were allowed to equilibrate before the [¹⁴C] or [³H] content was quantified by liquid scintillation counting (LS 5000TA Scintillation Counter; Beckman Instruments, Fullerton, CA).

Uptake experiments were conducted to determine whether there was expression of Na⁺-dependent and/or Na⁺-independent transport of HMB and L-Met by the oocytes. Total uptake of substrates was determined using an uptake buffer containing 96 mmol/L NaCl, 2 mmol/L KCl, 1.8 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5 mmol/L HEPES/KOH, pH 5.5 and appropriate concentrations of HMB or L-Met. The Na⁺-independent transport of HMB and L-Met was determined using a buffer containing 96 mmol/L choline chloride, 2 mmol/L KCl, 1.8 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5 mmol/L HEPES/KOH, pH 5.5, and appropriate concentrations of HMB or L-Met. The difference between total uptake and Na⁺-independent uptake was an estimate of Na⁺-dependent uptake. Similarly, uptake experiments were conducted to evaluate whether there was expression of H⁺-dependent transport of HMB and L-Met. Uptake of substrates was determined using a buffer containing 96 mmol/L NaCl, 2 mmol/L KCl, 1.8 mmol/L CaCl₂, 1 mmol/L MgCl₂, 3 mmol/L 2-(N-morpholino)ethanesulfonic acid (MES), 3 mmol/L HEPES, pH 5.5 or 7.5, and appropriate concentrations of HMB or L-Met.

Statistical analysis.

Substrate uptake by oocytes injected with fractionated poly(A)⁺ RNA was compared with substrate uptake by water-injected oocytes (15 ). The experiments were carried out with five or six replicates, and results are presented as the mean ± SEM. Data were evaluated using one-way ANOVA, and the Least Significant Difference test ( < 0.05) was used for post-hoc comparisons with control only (15 ). When unbalanced data sets were produced, uptake was evaluated using PROC MIX procedures (16 ).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
The expression time course of poly(A)⁺ RNA encoding for proteins transporting Met and HMB was examined by uptake assays using [5-¹⁴C] HMB or [Methyl-³H] L-Met. The ability of jejunal poly(A)⁺ RNA–injected oocytes to transport HMB and Met was compared with that of water-injected oocytes on d 1, 2, 3, 4 and 5 after injection in uptake buffer at pH 5.5 (Fig. 1 ). Uptake of both HMB and Met induced in poly(A)⁺ RNA-injected oocytes increased over time. On the contrary, neither HMB nor Met uptake in water-injected oocytes increased over 5 d. Greater (P < 0.01) uptake of Met by oocytes injected with poly(A)⁺ RNA was observed on d 3, 4 and 5 compared with water-injected oocytes. Greater (P < 0.01) uptake of HMB by oocytes injected with poly(A)⁺ RNA was also observed on d 4 and 5 compared with water-injected oocytes. A period of 4–5 d after poly(A)⁺ RNA injection was necessary for induction of Met or HMB absorption.

View larger version (13K): [in this window] [in a new window]   Figure 1. Time-dependent expression of uptake activity by Xenopus oocytes injected with broiler jejunal poly(A)+ RNA or water (control). The ability of RNA-injected oocytes to transport DL-2-hydroxy-4-(methylthio) butanoic acid (HMB) and methionine was compared with that of water-injected oocytes on days 1, 2, 3, 4 and 5 after injection. Values are means ± SEM, n = 6; *different from control, P < 0.05.

View larger version (12K): [in this window] [in a new window]   Figure 2. Time course for uptake activity by Xenopus oocytes injected with poly(A)+ RNA or water (control). The absorption of both DL-2-hydroxy-4-(methylthio) butanoic acid (HMB) and methionine from pH 5.5 buffer by oocytes injected with polyA+ RNA or water was measured over the course of 75 min. Values are means ± SEM, n = 6; *different from control, P < 0.05.

View larger version (28K): [in this window] [in a new window]   Figure 3. Uptake by Xenopus oocytes injected with poly(A)+ RNA from different intestinal segments (Duo, duodenum; Jej, jejunum; Ile, ileum) of broilers or water (control). The uptake of DL-2-hydroxy-4-(methylthio) butanoic acid (HMB) and methionine was evaluated in oocytes injected with poly(A)+ RNA from different intestine segments or water 4 d after injection. Values are means ± SEM, n = 6; *different from control, P < 0.05.

View larger version (18K): [in this window] [in a new window]   Figure 4. Uptake of [methyl-3H]-Met by Xenopus oocytes injected with size-fractionated poly(A)+ RNA from different intestinal segments (Duo, duodenum; Jej, jejunum; Ile, ileum) of broilers or water (control, H2O). Fractions 19–36 were injected into oocytes to measure methionine uptake. Values are means ± SEM, n = 6; *different from control, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Figure 5. Uptake of [5-14C]-DL-2-hydroxy-4-(methylthio) butanoic acid (HMB) by Xenopus oocytes injected with size-fractionated poly(A)+ RNA from different intestinal segments (Duo, duodenum; Jej, jejunum; Ile, ileum) of broilers or water (control, H2O). Fractions 19 to 36 were injected into oocytes to measure HMB uptake. Values are means ± SEM, n = 6; *different from control, P < 0.05.

View larger version (13K): [in this window] [in a new window]   Figure 6. Na+-dependent uptake of [methyl-3H]-Met by Xenopus oocytes injected with broiler jejunal poly(A)+ RNA of broilers or water (control, H2O). Fractions 19 to 36 from the jejunal segment were injected into oocytes and uptake was measured in Na+-buffer or Na+-free buffer. Values are means ± SEM, n = 6; *different from control, P < 0.05.

View larger version (38K): [in this window] [in a new window]   Figure 7. H+-dependent uptake of [5-14C]- DL-2-hydroxy-4-(methylthio) butanoic acid (HMB) by Xenopus oocytes injected with broiler jejunal poly(A)+ RNA of broilers or water (control, H2O). Fractions 21 to 24, 29 to 31, and 33 to 35 from jejunal segment were pooled for injection into oocytes for all treatments. Values are means ± SEM, n = 6; *different from control, P < 0.05.

In summary, mRNA encoding proteins capable of transporting both Met and HMB are present in intestinal mucosa of chickens. Multiple transport systems appear to be involved in transporting both Met and HMB. Both Na⁺-dependent and Na⁺-independent transport systems for Met are present in intestinal mucosa of chickens, whereas the transport systems for HMB appear to be primarily H⁺ dependent and Na⁺ independent. HMB does not seem to be transported as readily as Met. Differences in the amount of L-Met and DL-HMB transported might be related to the differences in the mechanism by which HMB and L-Met are transported. In vivo chicken experiments have shown that the small intestine had similar capacities to absorb DL-HMB and L-Met (4 ,18 ,19 ). However, when the mechanisms of transport were examined, L-Met transport appeared to be Na⁺- and energy-dependent carrier mediated and HMB transport appeared to be comprised of Na⁺-independent and proton-dependent carrier mediated and diffusion (3 ,4 ,20 ). The discrepancy between the results from the in vivo chicken studies and the present study might be due to the presence of other transport proteins involved in HMB absorption, but more likely, the diffusion component of HMB absorption was not examined in the present study.

Our present study provides the first evidence of the presence of poly(A)⁺ RNA that encode proteins capable of mediated transport for Met and HMB in the intestine of chickens. To further identify mRNA that encode proteins responsible for transporting Met or HMB, cDNA libraries can then be constructed using those fractions that induced uptake activity in oocytes. The specific cDNA encoding proteins responsible for Met or HMB uptake could then be identified by screening these libraries using functional assays. Understanding the mechanisms involved in the absorption of Met and HMB will help us use them appropriately as valuable feed supplements.

	FOOTNOTES

 
¹ This material is based upon work supported in part by the Virginia Agricultural Experiment Station under project no. 6129990. Support was also provided by Novus International, Inc.

Manuscript received 12 September 2001. Initial review completed 10 October 2001. Revision accepted 22 December 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 

1. Brachet, P. & Puigserver, A. (1989) Na+-independent and nonstereospecific transport of 2-hydroxy 4-methylthiobutanoic acid by brush border membrane vesicles from chick small intestine. Comp. Biochem. Physiol. B 94:157-163.[Medline]

2. Dibner, J. J., Atwell, C. A. & Ivey, F. J. (1992) Effect of heat stress on 2-hydroxy-4-(methylthio)butanoic acid and DL-methionine absorption measured in vitro. Poult. Sci. 71:1900-1910.[Medline]

3. Dibner, J. J., Knight, C. D., Swick, R. A. & Ivey, F. J. (1988) Absorption of ¹⁴C-2-hydroxy-4-(methylthio) butanoic acid (Alimet) from the hindgut of the broiler chick. Poult. Sci. 67:1314-1321.[Medline]

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5. Knight, C. D., Wuelling, C. W., Atwell, C. A. & Dibner, J. J. (1994) Effect of intermittent periods of high environmental temperature on broiler performance responses to sources of methionine activity. Poult. Sci. 73:627-639.[Medline]

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7. Soriano-Garcia, J. F., Torras-Llort, M., Ferrer, R. & Moreto, M. (1998) Multiple pathways for L-methionine transport in brush-border membrane vesicles from chicken jejunum. J. Physiol. 509:527-539.[Abstract/Free Full Text]

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9. Pan, Y-X., Wong, E. A., Bloomquist, J. R. & Webb, K. E., Jr (2001) Expression of a cloned ovine gastrointestinal peptide transporter (oPepT1) in Xenopus oocytes induces uptake of oligopeptides in vitro. J. Nutr. 131:1264-1270.[Abstract/Free Full Text]

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14. Dumont, J. N. (1972) Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136:153-179.[Medline]

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17. Soriano-Garcia, J. F., Torras-Llort, M., Moreto, M. & Ferrer, R. (1999) Regulation of L-methionine and L-lysine uptake in chicken jejunal brush-border membrane by dietary methionine. Am. J. Physiol. 277:R1654-R1661.[Abstract/Free Full Text]

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20. Brachet, P. & Puigserver, A. (1987) Transport of methionine hydroxy analog across the brush border membrane of rat jejunum. J. Nutr. 117:1241-1246.

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