QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Swanson, K. C. Articles by Harmon, D. L. Search for Related Content PubMed PubMed Citation Articles by Swanson, K. C. Articles by Harmon, D. L.

---

Article

Dietary Carbohydrate Source and Energy Intake Influence the Expression of Pancreatic -Amylase in Lambs^{1 ,2 ,3}

Animal Sciences Department, University of Kentucky, Lexington, KY 40546-0215

⁵To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In ruminants, pancreatic -amylase is the primary enzyme responsible for the initial hydrolysis of -linked glucose in the small intestinal lumen. The objective of this experiment was to examine the effects of altered dietary starch and energy supply on the expression of pancreatic -amylase mRNA, protein and activity in lambs. Wether lambs (n = 24; 28 ± 0.5 kg body weight) were fed low or high starch diets at 1.2 or 1.8 x net energy of maintenance for at least 28 d before tissue collection. Lambs fed the high energy/high starch diet tended to have more pancreatic -amylase protein (54.5 kDa; P = 0.08) and had greater activity (P = 0.03), but -amylase mRNA (1.6 kb) tended to be lower (P = 0.17). Additionally, rumen fluid total short-chain fatty acid concentration was greater (P = 0.04) and plasma glucose concentration tended to be greater (P = 0.07) in lambs fed the high energy/high starch diet. However, pancreatic trypsinogen protein (25.5 kDa) and jejunal maltase activity were not influenced by dietary treatment, suggesting that different regulatory systems are involved in regulating the tissue protein or activity levels of these two enzymes compared with -amylase. These data suggest that dietary regulation of pancreatic -amylase expression in ruminants is complex and probably regulated by transcriptional and post-transcriptional events.

KEY WORDS: • sheep • gene expression • starch digestion • amylase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Feed grains, which containhigh levels of starch, are a major energy source for ruminants fed for meat and milk production. In diets that do not contain high starch, the majority of starch is digested by rumen microflora. In high concentrate diets, however, significant amounts (up to 400 g/kg) of dietary starch reach the small intestine (Orskov 1986 ). As the flow of starch increases to the small intestine, starch is digested with decreasing efficiency, resulting in a loss of potential energy available for growth or milk production. In the small intestine, luminal pancreatic -amylase and membrane-bound small intestinal disaccharidases are responsible for the breakdown of starch to glucose, which is absorbed by the mucosa primarily via the Na⁺/glucose cotransporter (SGLT1,⁶ Wright 1993 ). Increases in postruminal starch result in decreased pancreatic -amylase activity in pancreatic tissue and secretions (Chittenden et al. 1984 , Kreikemeier et al. 1990 , Swanson et al. 1998 , Walker and Harmon 1995 ), but have little influence on small intestinal disaccharidase and glucose transport activity (Bauer 1996 , Harmon 1992 ). Additionally, Kreikemeier et al. (1991) showed that increasing postruminal glucose infusion to steers resulted in a linear increase in net portal glucose absorption up to 60 g/h; however, increasing postruminal cornstarch caused a plateau in net portal glucose absorption at 20 g/h, suggesting that the breakdown of starch to glucose is limited. This is supported further by the fact that 15 times as much starch as glucose flows past the ileum when starch is infused postruminally at a rate of 60 g/h. This suggests that secretion of pancreatic -amylase activity limits small intestinal starch digestion. In contrast, dietary energy intake may be an important positive regulator of pancreatic -amylase activity (Kreikemeier et al. 1990 ). However, increases in energy intake were concomitant with increased dietary protein intake. This observation makes it difficult to discern whether the response is due to energy or protein because increases in small intestinal protein supply enhance secretion of pancreatic -amylase activity and small intestinal starch digestion (Richards et al. 1997 and 1998 , Wang and Taniguchi 1998 ).

Regulatory mechanisms involved in changes in -amylase production and secretion are largely unknown, especially in ruminants. In monogastrics, the regulation of pancreatic -amylase production with dietary adaptation occurs primarily at the transcriptional level (Brannon 1990 ). Increases in -amylase production in the developing preruminant are also primarily under transcriptional control (Le Huerou et al. 1990b ). However, very little is known concerning the influence of diet on the expression of -amylase in ruminants. Therefore, the objectives of this experiment were to determine the influence of dietary carbohydrate source and energy intake, with similar metabolizable protein intake, on the expression of steady-state levels of pancreatic -amylase mRNA, protein and activity, and then to collate these observations with total tract digestion, rumen fermentation and blood metabolites.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Dietary treatments and sample collection.

The experimental protocol complied with the published guidelines (Federation of Animal Science Society 1999 ). Crossbred wether lambs [n = 24; mean body weight (BW) ± SEM, 28 ± 0.5 kg] were assigned randomly to one of four dietary treatments. Lambs were fed diets (Table 1 ) containing low or high starch concentrations (50 and 400 g/kg, respectively) at 1.2 or 1.8 x net energy of maintenance (NE_m) requirements (NRC 1985 ) for at least 28 d (average 36 d). The low energy intake (1.2 x NE_m) was chosen to ensure that lambs would maintain BW. The high energy intake (1.8 x NE_m) was chosen so that lambs would be gaining weight and consume all of the feed offered. Lambs were fed diets twice daily (0700 and 1700 h) in two equal portions. Dietary energy intake was adjusted according to BW every 14 d. In the low starch diets, the majority of the carbohydrate was supplied by the fiber portion of the diet. Diets were formulated so that all lambs would receive the same amount of metabolizable protein per day⁷ using the calculations described by the NRC (1996) for beef because changes in small intestinal protein supply alter pancreatic -amylase secretion (Richards et al. 1998 , Wang and Taniguchi 1998 ). Diets also were formulated to meet or exceed the daily requirements for Ca, P (the Ca:P ratio was 2:1) and vitamins A, D and E for a lamb with gain equivalent equal to 1.8 x NE_m. Lambs were housed in a temperature- (23°C) and light- (16 h light:8 h dark) controlled room with water available at all times. Lambs were group-fed within treatment until 2 wk before tissue collection; they were then placed in individual metabolism crates for the remainder of the experiment. Feed and fecal samples were collected on the final 5 d, composited within animal across sampling day and stored at -30°C until further processing. Blood samples were collected via jugular venipuncture 3 h after the morning feeding on the day before tissue collection, and plasma was harvested by centrifugation (12,000 x g for 20 min) and stored at -30°C until analysis.

Diet digestibility, rumen fermentation and blood metabolites.

Diet and fecal samples were dried in a 55°C oven, ground to pass through a 2-mm screen and analyzed for dry matter (DM) and organic matter (OM) by standard procedures (AOAC 1990 ). Diet and fecal nitrogen concentrations were determined using the Leco FP-2000 (Leco Corporation, St. Joseph, MI); the percentage of crude protein (CP) was calculated by multiplying the percentage of nitrogen x 6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) concentrations were determined by the method of Robertson and Van Soest (1981) . Feed and fecal starch were analyzed using the method of Herrera-Saldana and Huber (1989) . Gross energy was determined by bomb calorimetry (Model 1261, Parr Instrument, Moline, IL).

Ruminal fluid samples were prepared for short-chain fatty acid (SCFA) analysis as described previously (Harmon et al. 1985 ). Samples were analyzed using gas chromatography on a HP 5890 Series II (Hewlett Packard, Palo Alto, CA) and a column packed with 10% SP1200, 1% H₃PO₄ on 80/100 W AW Chromsorb (Supelco, Bellefonte, PA) maintained at 125°C. Inlet and detector were maintained at 200 and 250°C, respectively, with carrier gas (N₂) at 20 mL/min. Plasma was analyzed for SCFA concentrations by ion exchange clean-up and gas chromatography (Reynolds et al. 1986 ) on a HP 6890 series (Hewlett Packard) and a 2 m x 2 mm glass column packed with 80/120 Carbopak B-DA 4% Carbowax, 20 M (Supelco) maintained at 175°C. Inlet and detector were maintained at 200 and 225°C, respectively, with carrier gas (N₂) at 24 mL/min. Plasma was analyzed for glucose concentration using the hexokinase method (Slein 1963 ; Sigma Chemical, St. Louis, MO).

RNA isolation and Northern blot.

Total RNA was isolated by the guanidium thiocyanate/acid/phenol extraction procedure as described previously (Matthews et al. 1996 ) with modifications. Because of the high levels of RNAse in sheep pancreas (Stevens and Hume 1995 ), pancreatic tissue was homogenized at 1 g/30 mL extraction buffer instead of 1 g/10 mL, and the RNA pellet was resuspended and stored (-80°C) in formamide.

Total RNA (10 µg/lane) was separated by electrophoresis through a 10 g/L agarose, 20 mmol/L formaldehyde gel. The RNA was transferred to a 0.45-µm nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) by downward capillary action (Zhou et al. 1994 ), covalently cross-linked by UV light, and hybridized with ³²P-labeled bovine -amylase cDNA in 10 g/L bovine serum albumin (BSA), 243 mmol/L SDS, 500 mmol/L Na₂HPO₄ (pH 7.2) and 1 mmol/L EDTA for 15 h at 65°C (Matthews et al. 1998 ). The radiolabeled cDNA probe was prepared from a 0.89-kb segment of bovine pancreatic -amylase (Le Huerou et al. 1990b ) by random priming extension (Gibco BRL, Grand Island, NY) using [³²P]dCTP (NEN Life Science Products, Boston, MA). After hybridization, the blots were washed 4 x 15 min at 65°C in 40 mmol/L Na₂PO₄ (pH 7.2), 3.5 mmol/L SDS, and 1 mmol/L EDTA. The hybridization bands were visualized by autoradiography. Blots were stripped, later probed for 18S rRNA using a mouse 18S cDNA probe (Ambion, Austin, TX), and hybridization bands were visualized by autoradiography using procedures similar to those described above. Densitometric data for -amylase were corrected for unequal loading by expressing data relative to 18S rRNA expression. The 18S rRNA has been found to be a superior internal standard compared with other housekeeping genes commonly used in the pancreas (Yamada et al. 1997 ). Similar results were obtained when densitometric analysis of the entire lane from the photographic negative of the ethidium bromide–stained gel was used to correct for unequal loading.

Immunoblot.

Pancreatic tissue (1 g) was homogenized using a polytron (Brinkman, Westbury, NY) in 9 mL of 250 µmol/L sucrose, 10 mmol/L Hepes-KOH (pH 7.5), 1 mmol/L EGTA, and 2 mg/L each of N-tosyl-L-phenylalanine chloromethyl-ketone, N--p-tosyl-L lysine ketone, leupetinin hemisulfate, aproptinin and pepstatin A to prevent proteolysis (Matthews et al. 1998 ). Homogenates were stored at -30°C until further analysis. Protein concentration of the homogenate was determined according to the method of Lowry et al. (1951) with BSA as the standard. Proteins (30 µg/lane) were separated by 7.5% SDS PAGE (Laemmli 1970 ) and electrotransferred to a 0.45-m nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane then was hybridized with an anti--amylase polyclonal antibody (rabbit anti-human pancreatic -amylase; 1:500 dilution; Biodesign International, Kennebunk, ME) in blocking solution (20 g/L nonfat dry milk in 10 mmol/L Tris-Cl, pH 7.5, 300 mmol/L NaCl) for 1 h at room temperature with agitation. Immunoreactive products were visualized by chemiluminescence (Pierce, Rockford, IL) with a horseradish peroxidase–conjugated secondary antibody (donkey anti-rabbit immunoglobulin; 1:5000 dilution; Amersham). Competitive inhibition experiments with human salivary -amylase (Sigma Chemical) demonstrated the specificity of the immune reaction (data not shown).

For the detection of trypsinogen, similar procedures were used except proteins (10 µg) were separated by 12% SDS PAGE. The antibody used was an anti-trypsinogen polyclonal antibody (rabbit anti-bovine pancreatic trypsinogen; 1:5000 dilution; Biodesign International) and the blocking buffer consisted of 20 g/L nonfat dry milk in 10 mmol/L Tris-Cl, pH 7.5, and 200 mmol/L NaCl. Competitive inhibition experiments with bovine pancreatic trypsinogen (Sigma Chemical) demonstrated the specificity of the immune reaction (data not shown).

Densitometry and molecular size determination.

Digital images of the autoradiographic film from Northern and immunoblot analyses were scanned (HP DeskScan II, Hewlett Packard) and the intensities of the bands determined using the UN-SCANIT software program (Silk Scientific, Orem, UT). Northern and immunoblot analyses were repeated once and densitometric results from individual lambs were averaged across blots. Northern analysis data from the first four lambs (one from each treatment) were not included because substantial RNA degradation occurred. Data are reported as arbitrary units. Apparent migration weights (M_r) of -amylase protein and mRNA were calculated by regression of the distance migrated against the M_r of molecular weight (or size) markers. The molecular weight (or size) markers used for -amylase Northern and immunoblot analyses were purchased from Gibco (0.24–9.5 kb RNA Ladder) and Bio-Rad (Broad Range; 7–203 kDa), respectively. The molecular weight marker used for trypsinogen immunoblot analysis was purchased from Gibco (10 kDa protein ladder; 10–200 kDa).

Pancreatic -amylase and jejunal maltase activity.

Pancreatic tissue (1 g) was homogenized in 154 mmol/L NaCl (9 mL) using a polytron and stored at -30°C until analysis. Protein concentrations were determined using the method of Lowry et al. (1951) . Activity of -amylase was measured using potato amylopectin as the substrate and measuring the amount of reducing sugars liberated with maltose as the standard (Walker and Harmon 1996 ). Samples were analyzed weekly so that loss of enzyme activity over time was kept to a minimum. One unit (U) of -amylase activity equals 1 µmol reducing sugar produced per minute. Similar results were obtained when samples were stored as a 1 g/10 mL homogenate at -30°C for up to 30 d.

The jejunal samples were thawed and the mucosa collected by scraping with a glass slide. The mucosa was homogenized in cold 154 mmol/L NaCl; maltase activity was measured using maltose as the substrate (Dahlqvist 1964 , Kreikemeier et al. 1990 ) and measuring the amount of glucose liberated using the hexokinase method (Slein 1963 ). One unit of maltase activity equals 1 µmol of maltose hydrolyzed per minute.

Statistical analysis.

Data were analyzed as a completely randomized design with a 2 x 2 factorial arrangement of treatments using the General Linear Models procedures of SAS (SAS 1988 ). Because the high energy/high starch treatment may have resulted in the only sheep with substantial starch flow to the small intestine, comparisons were made between the high energy/high starch treatment and the mean of the other treatments using a contrast statement. Differences were considered significant when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Final BW was greater (P = 0.02) in lambs fed 1.8 x NE_m but was not influenced by dietary carbohydrate source (Table 2 ). Because fecal starch concentrations were < 1 g/100 g DM for the high energy/high starch treatment (data not shown), we assumed that total tract starch digestion was nearly complete for all treatments. Digestibility of DM and OM was greater (P < 0.04) in lambs fed 1.8 x NE_m and in lambs fed high starch diets. Digestibility of CP was greater for lambs fed high starch diets, although the lowest values were always obtained for the high intake of low starch (energy x starch, P = 0.01). Digestibility of energy was lower (P = 0.01) and NDF tended to be lower (P = 0.07) in lambs fed 1.8 x NE_m. Digestibility of energy and NDF also were greater (P < 0.02) in lambs fed high starch diets. Digestibility of ADF was greater (P = 0.01) in lambs fed high starch diets.

View this table: [in this window] [in a new window]   Table 5. Pancreatic weight, steady-state levels of pancreatic -amylase mRNA, protein and activity, and jejunal maltase activity in lambs fed diets that were 1.2 and 1.8 ;\x> NEm and differing in starch concentration12

View larger version (53K): [in this window] [in a new window]   Figure 1. Northern blot analysis of pancreatic -amylase mRNA and 18S rRNA expression in lambs fed diets at 1.2 and 1.8 x net energy of maintenance (NEm) and differing in starch concentration. Data represent a typical blot using 16 representative animals (4/dietary treatment; L, low; H, high; E, energy; S, starch). Lambs were fed treatment diets for at least 28 d. Pancreatic tissue was collected and total RNA isolated and analyzed for -amylase mRNA and 18 rRNA expression using Northern analysis.

View larger version (60K): [in this window] [in a new window]   Figure 2. Immunoblot analysis of pancreatic -amylase (A) and trypsinogen (B) protein expression in lambs fed diets at 1.2 and 1.8 x net energy of maintenance (NEm) and differing in starch concentration. Data represent a typical set of four animals (1/dietary treatment; L, low; H, high; E, energy; S, starch). Lambs were fed treatment diets for at least 28 d. Pancreatic tissue was collected and analyzed for -amylase and trypsinogen protein expression using immunoblot analysis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Relationships between pancreatic -amylase production and diet digestibility, rumen fermentation and blood metabolites have received little attention. In agreement with our digestibility results (Table 2) , others have reported improvements in digestion when increasing the concentrate in the diet and when restricting dietary intake in lambs (Murphy et al. 1994 ). Although total tract digestibility of starch was nearly complete for all treatments, significant starch flow to the small intestine in the high energy/high starch diet was likely (Orskov 1986 ).

Greater rumen total SCFA concentrations and molar proportions of butyrate and propionate in lambs fed the high energy/high starch diet (Table 3) also may affect pancreatic -amylase protein and activity. Increased ruminal SCFA concentrations result in decreased ruminal pH. Decreased intestinal digesta pH has been correlated with increased pancreatic secretion in sheep (Krzeminski et al. 1990 ). Although the exact mechanisms are not known, it is possible that gastrointestinal hormones are being secreted in response to decreases in digesta pH. Another possibility could be a direct effect of SCFA on the pancreas after absorption and transfer through the blood. Intravenous infusion or in vitro incubation with SCFA has been shown to increase -amylase secretion in sheep (Harada and Kato 1983 , Katoh and Tsuda 1984 ). The secretory response was greater with intravenous infusion or in vitro incubation with butyrate and isovalerate (a mixture of 2- and 3-methylbutyrate), respectively, than with other SCFA. Although plasma total SCFA concentration was lower in lambs fed high starch diets (Table 4) , the concentration of 2-methylbutyrate was greater. Also, 2-methylbutyrate was greater when comparing the high energy/high starch treatment with other treatments. It remains to be determined whether 2-methylbutyrate, a ruminal fermentation product of amino acids, is a major regulator of pancreatic -amylase expression.

Changes in blood metabolites also result in changes in hormone release throughout the body. For example, in ruminants, increases in blood glucose or certain SCFA increase insulin release from the pancreas (Brockman and Laarveld 1986 ). Although the role of insulin on pancreatic -amylase production and secretion is poorly defined in ruminants, insulin has been implicated as an important component of pancreatic -amylase regulation in monogastrics (Brannon 1990 ).

Pancreatic weight could be an important regulator of -amylase production and secretion (Wang et al. 1998 ). Changes in pancreatic weight could result in differences in total -amylase content without changes in -amylase concentration (Table 5) . Pancreatic weight tended to be greater in lambs fed 1.8 x NE_m. However, no differences were observed when expressed as g/kg BW. This suggests that pancreatic weight was a function of BW differences.

The sizes of the -amylase mRNA and protein were calculated to be 1.6 kb and 54.5 kDa, respectively. These values are similar to those observed in cattle (Le Huerou et al. 1990b ), chickens (Benkel et al. 1997 ), mice (Hagenbuchle et al. 1980 ), pigs (Lhoste et al. 1993 ), rats (MacDonald et al. 1980 ) and humans (Nakamura et al. 1984 ). The similarity of molecular size among species and the specificity of the primary antibody for -amylase protein (determined through competitive inhibition experiments) suggest that the mRNA and protein detected in Northern and immunoblot analyses was indeed -amylase. The size of the sheep pancreatic trypsinogen (25.5 kDa) also is similar to trypsinogens from cattle (Le Huerou et al. 1990a ) and monogastrics (Lowe 1994 ).

Previous research has shown that high dietary starch (or postruminal infusions of starch) results in the depression of -amylase activity in the pancreas or pancreatic secretions of ruminants (Chittenden et al. 1984 , Kreikemeier et al. 1990 , Swanson et al. 1998 , Walker and Harmon 1995 ). Similarly, intravenous infusion of glucose inhibits -amylase secretion in sheep (Call et al. 1975 ). In contrast, lambs fed the high energy/high starch diet had a greater concentration of pancreatic -amylase activity and tended to have greater -amylase protein in this experiment. It is possible that, in earlier experiments, metabolizable protein was inadequate to maintain and(or) stimulate pancreatic enzyme synthesis or secretion (Richards et al. 1998 , Wang and Taniguchi 1998 ). The benefits of ruminal fermentation are that as ruminal fermentable organic matter increases (e.g., increased starch intake), the synthesis and intestinal flow of microbial protein increases. This can lead to an inherent confounding of studies such as these. In addition, in earlier experiments, protein intake differed between dietary treatments (Kreikemeier et al. 1990 ) or protein intake was not increased to meet the dietary protein requirement when partially hydrolyzed starch was infused postruminally (Swanson et al. 1998 , Walker and Harmon 1995 ). The data from this experiment indicate that increasing starch at high energy intake, when intestinal flow of protein is not changed, increases plasma glucose and pancreatic -amylase protein and activity. It should be pointed out, however, that although the experimental diets were formulated so that equal quantities of protein were flowing through the small intestine, differences in small intestinal protein flow are possible. Therefore, because increases in postruminal protein are associated with increasing pancreatic -amylase secretion in ruminants (Richards et al. 1998 , Wang and Taniguchi 1998 ), it cannot be ruled out that the responses observed resulted because of differences in metabolizable protein flow to the small intestine. A more precise experimental model in which small intestinal flow of nutrients could be monitored and controlled more closely would allow for more precise information about nutrient gene interaction. However, differences in metabolizable protein flow among dietary treatments likely were small and probably did not greatly influence pancreatic -amylase production or secretion.

Although -amylase mRNA levels typically increase in response to dietary starch in monogastric species (Brannon 1990 ), a tendency for a reduction in steady-state levels of mRNA was observed in lambs fed the high energy/high starch diet. This decrease in -amylase mRNA suggests that dietary regulation of pancreatic -amylase expression in ruminants is complex and likely regulated by both transcriptional and post-transcriptional events. Increases in steady-state levels of pancreatic -amylase protein and activity in the presence of decreased mRNA could result from a decreased rate of transcription (or decreased mRNA stability) with increased efficiency of translation. Alternatively, transcription could be inhibited (or mRNA stability reduced) with a parallel inhibition on secretory processes, resulting in the accumulation of enzyme in the tissue.

The regulation of the rate of transcription and stability of -amylase mRNA have not been studied extensively. Increases in -amylase mRNA transcription that fully account for the increase in -amylase mRNA have been reported for immortalized rat acinar cells (AR42J) when treated with glucocorticoids (Logsdon et al. 1987 ). This observation suggests that the rate of transcription and mRNA levels are related directly. Also, hybrid constructs in transgenic mice have been performed to define a DNA sequence in the 5'-flanking region of the -amylase gene, which was taken to be a functional dietary response unit (Schmid and Meisler 1992 ). However, Carreira et al. (1997) reported decreases in pancreatic -amylase mRNA stability in rats fed a protein-free diet compared with those fed a control or high protein diet. This result suggests that -amylase mRNA stability can be altered and that dietary protein is critical in maintaining -amylase mRNA stability. Although dietary crude protein and metabolizable protein intakes were formulated to be similar across treatments in this experiment, changes in mRNA stability cannot be ruled out.

Little is known about the regulation of -amylase protein translation. In monogastric species, differences in -amylase translation are thought to be related directly to mRNA levels (Logsdon et al. 1987 ). In contrast, the results from this experiment suggest that protein translation may be one of the major regulatory steps in the control of -amylase gene expression in sheep. Interestingly, with regard to the relationship between substrate availability and transport capacity, the expression of intestinal SGLT1 in sheep appears to be regulated primarily by translational events. Specifically, the expression of SGLT1 protein and activity are increased to a much greater extent than mRNA in response to development, weaning or postruminal glucose infusion (Dyer et al. 1997 , Lescale-Matys et al. 1993 ).

Traditionally, it has been thought that pancreatic enzyme synthesis, tissue content and secretion are related directly. Although we did not measure secretion in this experiment, the possibility of both transcriptional and secretory regulation occurring seems feasible. As knowledge regarding cellular regulation of pancreatic enzyme secretion accumulates, it seems likely that nonparallel secretion of digestive enzymes occurs and that there are different pools of vesicles that contain different proportions of enzymes (Case 1998 ). It also is likely that there are alternative routes of secretion that do not involve exocytosis of typical zymogen granules (Rothman et al. 1999 ). Because of the complexity of the secretory regulation and the differences in the digestive characteristic between monogastric and ruminant species, it seems reasonable to suggest that regulation could be occurring at both the transcriptional and secretory levels.

The -amylase protein response observed in this experiment seems to be specific for -amylase because trypsinogen protein was not influenced by dietary treatment. This suggests that different regulatory systems are involved in regulating the tissue protein levels of these two enzymes. Whether the specific regulation is at the transcriptional, translational or secretory level remains unclear.

Small intestinal disaccharidases are responsible for the final hydrolysis of starch to glucose in the small intestine. Maltase attacks the -1,4 linkage of maltose (Goda and Koldovsky 1988 ). The effect of energy source and level on the expression of jejunal disaccharidase mRNA and protein was not evaluated in this experiment. However, jejunal maltase activity was not influenced by dietary treatment, in agreement with previous research, suggesting that small intestinal disaccharidase activity is unaffected by dietary treatment in ruminants (Harmon 1992 ). Therefore, small intestinal maltase likely is not involved in dietary adaptation to small intestinal starch digestion.

To our knowledge, this experiment is the first set of data that describes dietary effects on pancreatic -amylase expression in ruminants. Dietary adaptation of pancreatic -amylase expression in ruminants seems complex, possibly involving both transcriptional and post-transcriptional regulation. Possible regulators could include plasma glucose, SCFA, gastrointestinal hormones released in response to changes in digesta content or pH, or hormones released in response to changes in blood metabolite concentrations. A better understanding of the mechanisms regulating digestive enzyme expression and secretion could lead to the development of feeding strategies that would enhance enzyme production and secretion, small intestinal digestibility and overall efficiency of animal production.

	ACKNOWLEDGMENTS

 
We thank Isabelle Le Huerou-Luron and Paul Guilloteau (Laboratoire Du Jeune Ruminant, I.N.R.A., Rennes, France) for the -amylase cDNA probe and helpful comments on the manuscript. We thank Alma True for plasma SCFA analysis.

	FOOTNOTES

 
¹ Published as publication no. 00–07-40 of the Kentucky Agricultural Experiment Station.

² Presented in part at Experimental Biology 99, April 1999, Washington, DC[Swanson, K. C., Matthews, J. C., Wilson, A. D., Howell, J. A., Richards, C. J. & Harmon, D. L. (1999) Influence of dietary carbohydrate source and energy intake on pancreatic -amylase expression in lambs. FASEB J. 13: A257 (abs.)].

³ Supported by state and federal funds appropriated to the Kentucky Agricultural Experiment Station, University of Kentucky.

⁴ Current address: University of Tennessee, P.O. Box 1071, Knoxville, TN 37901-1071.

⁶ Abbreviations used: ADF, acid detergent fiber; BW, body weight; CP, crude protein; DM, dry matter; M_r, apparent migration weight; NDF, neutral detergent fiber; OM, organic matter; NE_m, net energy of maintenance; SCFA, short-chain fatty acids; SGLT1, Na⁺/glucose cotransporter.

⁷ Soypass and blood meal were used as sources of ruminally undegraded intake protein to balance for metabolizable protein intake

Manuscript received March 1, 2000. Initial review completed March 17, 2000. Revision accepted May 8, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Association of Official Analytical Chemists Official Methods of Analysis 15th ed. 1990 AOAS Arlington, VA.

2. Bauer M. L. Nutritional Regulation of Small Intestinal Glucose Absorption in Ruminants 1996 University of Kentucky Lexington, KY. Doctoral thesis

3. Benkel B. F., Nguyen T., Ahluwalia N., Benkel K. I., Hickey D. A. Cloning and expression of a chicken -amylase gene. Gene 1997;192:261-270[Medline]

4. Brannon P. M. Adaptation of the exocrine pancreas to diet. Annu. Rev. Nutr. 1990;10:85-105[Medline]

5. Brockman R. P., Laarveld B. Hormonal regulation of metabolism in ruminants; a review. Livest. Prod. Sci. 1986;14:313-334

6. Call J. L., Mitchell G. E., Jr, Little C. O. Response of ovine pancreatic amylase to elevated blood glucose. J. Anim. Sci. 1975;41:1717-1721

7. Carreira S., Fueri C., Chaix J.-C., Puigserver A. Stability of the mRNA encoding some pancreatic hydrolases is modulated by dietary protein intake in the rat. Br. J. Nutr. 1997;78:833-843[Medline]

8. Case R. M. Pancreatic exocrine secretion: mechanisms and control. Beger H. G. Warshaw A. L. Buchler M. W. Carr-Locke D. L. Neoptolemos J. P. Russell C. Sarr M. G. eds. The Pancreas 1998:63-100 Blackwell Science Malden, MA.

9. Chittenden L. W., Johnson D. D., Mitchell G. E., Jr, Tucker R. E. Ovine pancreatic amylase response to form of carbohydrate. Nutr. Rep. Int. 1984;29:1051-1060

10. Dahlqvist A. Method for assay of intestinal disaccharidases. Anal. Biochem. 1964;7:18-25[Medline]

11. Dyer J., Barker P. J., Shirazi-Beechey S. P. Nutrient regulation of the intestinal Na⁺/glucose co-transporter (SGLT1) gene expression. Biochem. Biophys. Res. Commun. 1997;230:624-629[Medline]

12. Federation of Animal Science Society Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching 1st rev. ed. 1999 Federation of Animal Science Societies Savoy, IL.

13. Goda T., Koldovsky O. Dietary regulation of small intestinal disaccharidases. World Rev. Nutr. Diet. 1988;57:275-329[Medline]

14. Hagenbuchle O., Bovey R., Young R. A. Tissue-specific expression of mouse -amylase genes: nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland. Cell 1980;21:179-187[Medline]

15. Harada E., Kato S. Effect of short-chain fatty acids on the secretory response of the ovine exocrine pancreas. Am. J. Physiol. 1983;244:G284-G290[Abstract/Free Full Text]

16. Harmon D. L. Dietary influences on carbohydrases and small intestinal starch hydrolysis capacity in ruminants. J. Nutr. 1992;122:203-210

17. Harmon D. L., Britton R. A., Prior R. L., Stock R. A. Net portal absorption of lactate and volatile fatty acids in steers experiencing glucose-induced acidosis or fed a 70% concentrate diet ad libitum. J. Anim Sci. 1985;60:560-569

18. Herrera-Saldana R., Huber J. T. Influence of varying protein and starch degradabilities on performance of lactating cows. J. Dairy Sci. 1989;72:1477-1483

19. Katoh K., Tsuda T. Effects of acetylcholine and short-chain fatty acids on acinar cells of the exocrine pancreas in sheep. J. Physiol. 1984;356:479-489[Abstract/Free Full Text]

20. Kreikemeier K. K., Harmon D. L., Brandt R. T., Jr, Avery T. B., Johnson D. E. Small intestinal starch digestion in steers: effect of various levels of abomasal glucose, corn starch and corn dextrin infusion on small intestinal disappearance and net glucose absorption. J. Anim. Sci. 1991;69:328-338[Abstract]

21. Kreikemeier K. K., Harmon D. L., Peters J. P., Gross K. L., Armendariz C. K., Krehbiel C. R. Influence of dietary forage and feed intake on carbohydrase activities and small intestinal morphology of calves. J. Anim. Sci. 1990;68:2916-2929[Abstract]

22. Krzeminski R., Mikolajczyk M., Kulasek G. The effect of intraduodenal infusion of 0.1 N HCl on the volume and composition of pancreatic juice and bile in wethers. J. Anim. Physiol. Anim. Nutr. 1990;64:139-142

23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 1970;227:680-685[Medline]

24. Le Drean G., Le Huerou-Luron I., Chayvialle J. A., Philouze-Rome V., Gestin M., Bernard C., Toullec R., Guilloteau P. Kinetics of pancreatic exocrine secretion and plasma gut regulatory peptide release in response to feeding in preruminant and ruminant calves. Comp. Biochem. Physiol. 1997;117A:245-255

25. Le Huerou I., Wicker C., Guilloteau P., Toullec R., Puigserver A. Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family. Eur. J. Biochem. 1990a;193:767-773[Medline]

26. Le Huerou I., Wicker C., Guilloteau P., Toullec R., Puigserver A. Specific regulation of the gene expression of some pancreatic enzymes during postnatal development and weaning in the calf. Biochim. Biophys. Acta 1990b;1048:257-264[Medline]

27. Lescale-Matys L., Dyer J., Scott D., Freeman T. C., Wright E. M., Shirazi-Beechey S. P. Regulation of the ovine intestinal Na⁺/glucose co-transporter (SGLT1) is dissociated from mRNA abundance. Biochem. J. 1993;291:435-440

28. Lhoste E. F., Fiszlewicz M., Gueugneau A.-M., Wicker-Planquart C., Puigserver A., Corring T. Effects of dietary proteins on some pancreatic mRNAs encoding digestive enzymes in the pig. J. Nutr. Biochem. 1993;4:143-152

29. Logsdon C. D., Perot K. J., McDonald A. R. Mechanism of glucocorticoid-induced increase in pancreatic amylase gene transcription. J. Biol. Chem. 1987;262:15765-15769[Abstract/Free Full Text]

30. Lowe M. E. The structure and function of pancreatic enzymes. Johnson L. R. eds. Physiology of the Gastrointestinal Tract 3rd ed. 1994:1531-1542 Raven Press New York, NY.

31. Lowry O. H., Rosenbrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265-275[Free Full Text]

32. MacDonald R. J., Crerar M. M., Swain W. F., Pictet R. L., Thomas G., Rutter W. J. Structure of a family of rat amylase genes. Nature (Lond.) 1980;287:117-122[Medline]

33. Matthews J. C., Beveridge M. J., Malandro M. S., Rothstein J. D., Campbell-Thompson M., Verlander J. W., Kilberg M. S., Novak D. A. Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta. Am. J. Physiol. 1998;274:C603-C614[Abstract/Free Full Text]

34. Matthews J. C., Wong E. A., Bender P. K., Bloomquist J. R., Webb K. E., Jr Demonstration and characterization of dipeptide transport system activity in sheep omasal epithelium by expression of mRNA in Xenopus laevis oocytes. J. Anim. Sci. 1996;74:1720-1727[Abstract]

35. Merchen N. R. Digestion, absorption and excretion in ruminants. Church D. C. eds. The Ruminant Animal—Digestive Physiology and Nutrition 1988:172 Prentice-Hall Englewood Cliffs, NJ.

36. Murphy T. A., Loerch S. C., Smith F. E. Effects of feeding high-concentrate diets at restricted intakes on digestibility and nitrogen metabolism in growing lambs. J. Anim. Sci. 1994;72:1583-1590[Abstract]

37. Nakamura Y., Ogawa M., Nishide T., Emi M., Kosaki G., Himeno S., Matsubara K. Sequences of cDNAs for human salivary and pancreatic -amylases. Gene 1984;28:263-270[Medline]

38. National Research Council Nutrient Requirements of Sheep 6th ed. 1985 National Academy Press Washington, DC.

39. National Research Council Nutrient Requirements of Beef Cattle 7th ed 1996 National Academy Press Washington, DC.

40. Orskov E. R. Starch digestion and utilization in ruminants. J. Anim. Sci. 1986;63:1624-1633

41. Reynolds P. J., Huntington G. B., Reynolds C. K. Determination of volatile fatty acids, lactate and ß-hydroxybutyrate in blood by ion exchange cleanup and gas chromatography. J. Anim. Sci. 1986;63(suppl. 1):424(abs.)

42. Richards C. J., Bohnert D. W., Harmon D. L., Huntington G. B., Larson B. T., McLeod K. R. Intestinal starch disappearance in steers abomasally infused with varying levels of protein. J. Anim. Sci. 1997;75(suppl. 1):97(abs.)

43. Richards C. J., Swanson K. C., Bohnert D. W., Lewis S. J., Harmon D. L., Huntington G. B. Effect of post-ruminal protein infusion on pancreatic exocrine secretion in beef steers. J. Anim. Sci. 1998;76(suppl. 1):312(abs.)

44. Robertson J. B., Van Soest P. J. The detergent system of analysis and its application to human foods. James W.P.T. Theander O. eds. The Analysis of Dietary Fiber 1981:123-158 Marcel Dekker New York, NY.

45. Rothman S., Liebow C., Grendell J. Nonparallel transport and mechanisms of secretion. Biochim. Biophys. Acta 1999;1071:159-173

46. SAS Institute, Inc. SAS Language Guide for Personal Computers (release 6.03 edition) 1988 SAS Institute Cary, NC.

47. Schmid R. M., Meisler M. H. Dietary regulation of pancreatic amylase in transgenic mice mediated by a 126-base pair DNA fragment. Am. J. Physiol. 1992;262:G971-G976[Abstract/Free Full Text]

48. Slein M. W. D-Glucose determinations with hexokinase and glucose-6 phosphate dehydrogenase. Bergmeyer H. U. eds. Methods of Enzymatic Analysis 1963:117 Academic Press New York, NY.

49. Stevens C. E., Hume I. D. Comparative Physiology of the Vertebrate Digestive System 2nd ed. 1995:176 Cambridge University Press New York, NY.

50. Swanson K. C., Richards C. J., Harmon D. L. Influence of abomasal infusion of glucose or starch hydrolysate on pancreatic exocrine secretion in beef steers. J.Anim.Sci. 1998;76(suppl. 1):313(abs.)

51. Taylor R. B. Pancreatic secretion in the sheep. Res. Vet. Sci. 1962;3:63-77

52. Walker J. A., Harmon D. L. Influence of ruminal or abomasal starch hydrolysate infusion on pancreatic exocrine secretion and blood glucose and insulin concentrations in steers. J. Anim. Sci. 1995;73:3766-3774[Abstract]

53. Walker J. A., Harmon D. L. Technical note: a simple, rapid assay for -amylase in bovine pancreatic juice. J. Anim. Sci. 1996;74:658-662[Abstract]

54. Wang X. B., Ogawa T., Suda S., Taniguchi K., Uike H., Kumagai H., Mitani K. Effects of nutritional level on digestive enzyme activities in the pancreas and small intestine of calves slaughtered at same body weight. Asian-Australas. J. Anim. Sci. 1998;11:375-380

55. Wang X. B., Taniguchi K. Activity of pancreatic digestive enzymes in sheep given abomasal infusion of starch and casein. Anim. Sci. Technol. (Japan) 1998;69:870-874

56. Wright E. M. The intestinal Na⁺/glucose cotransporter. Annu. Rev. Physiol. 1993;55:575-589[Medline]

57. Yamada H., Chen D., Monstein H. J., Hakanson R. Effects of fasting on the expression of gastrin, cholecystokinin, and somatostatin genes and of various housekeeping genes in the pancreas and upper digestive tract of rats. Biochem. Biophys. Res. Commun. 1997;231:835-838[Medline]

58. Zhou M. Y., Xue D., Gomez-Sanchez P., Gomez-Sanchez C. E. Improved downward capillary transfer for blotting of DNA and RNA. BioTechniques 1994;16:58-59[Medline]

This article has been cited by other articles:

				K. C. Swanson, N. Kelly, H. Salim, Y. J. Wang, S. Holligan, M. Z. Fan, and B. W. McBride Pancreatic mass, cellularity, and {alpha}-amylase and trypsin activity in feedlot steers fed diets differing in crude protein concentration J Anim Sci, April 1, 2008; 86(4): 909 - 915. [Abstract] [Full Text] [PDF]

				S. M. Rodriguez, K. C. Guimaraes, J. C. Matthews, K. R. McLeod, R. L. Baldwin VI, and D. L. Harmon Influence of abomasal carbohydrates on small intestinal sodium-dependent glucose cotransporter activity and abundance in steers J Anim Sci, October 1, 2004; 82(10): 3015 - 3023. [Abstract] [Full Text] [PDF]

				M. Z. Fan, J. C. Matthews, N. M. P. Etienne, B. Stoll, D. Lackeyram, and D. G. Burrin Expression of apical membrane L-glutamate transporters in neonatal porcine epithelial cells along the small intestinal crypt-villus axis Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G385 - G398. [Abstract] [Full Text] [PDF]

				K. C. Swanson, J. C. Matthews, C. A. Woods, and D. L. Harmon Influence of substrate and/or neurohormonal mimic on in vitro pancreatic enzyme release from calves postruminally infused with partially hydrolyzed starch and/or casein J Anim Sci, May 1, 2003; 81(5): 1323 - 1331. [Abstract] [Full Text] [PDF]

				J. A. Howell, A. D. Matthews, T. C. Welbourne, and J. C. Matthews Content of ileal EAAC1 and hepatic GLT-1 high-affinity glutamate transporters is increased in growing vs. nongrowing lambs, paralleling increased tissue D- and L-glutamate, plasma glutamine, and alanine concentrations J Anim Sci, April 1, 2003; 81(4): 1030 - 1039. [Abstract] [Full Text] [PDF]

				C. J. Richards, K. C. Swanson, S. J. Paton, D. L. Harmon, and G. B. Huntington Pancreatic exocrine secretion in steers infused postruminally with casein and cornstarch J Anim Sci, April 1, 2003; 81(4): 1051 - 1056. [Abstract] [Full Text] [PDF]

				K. C. Swanson, J. C. Matthews, C. A. Woods, and D. L. Harmon Postruminal Administration of Partially Hydrolyzed Starch and Casein Influences Pancreatic {alpha}-Amylase Expression in Calves J. Nutr., March 1, 2002; 132(3): 376 - 381. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Swanson, K. C. Articles by Harmon, D. L. Search for Related Content PubMed PubMed Citation Articles by Swanson, K. C. Articles by Harmon, D. L.