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-Amylase Expression in Calves1 ,2 ,3
Department of Animal Sciences, University of Kentucky, Lexington, KY 40546-0215
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-amylase mRNA, protein and activity in calves. Holstein calves [(n = 24) 88 ± 3 kg body weight (BW)], fitted with abomasal infusion cannulas, were randomly assigned within block (week of infusion) to one of four abomasal infusion treatments. Calves were fed an alfalfa-based diet, and SH [4 g/(kg BW · d)] and/or casein [0.6 g/(kg BW · d)] was infused abomasally for 10 d before tissue collection. There was a SH x casein interaction (P < 0.10) for pancreatic weight (g and g/kg BW) because casein increased pancreatic weight in the absence of SH but did not influence pancreatic weight in the presence of SH. Pancreatic -amylase mRNA tended to be lower (P = 0.06) and protein and activity (U/g pancreas and U/g protein) were lower (P = 0.02) in calves receiving abomasal SH. The concentration of pancreatic trypsin activity (U/g pancreas and U/g protein) was lower (P < 0.03) in calves receiving abomasal SH. There was a SH x casein interaction for total -amylase and trypsin activity [U/pancreas and U/(pancreas · kg BW)] because casein increased total activity in the absence of SH but not in the presence of SH. These data suggest that increases in small intestinal protein flow enhance pancreatic weight and thus total pancreatic -amylase and trypsin activity, yet small intestinal SH inhibits the increase in pancreatic weight resulting from increased small intestinal protein flow. Additionally, postruminal SH decreases -amylase expression largely by translational events.
KEY WORDS: • hydrolyzed starch • -amylase • trypsin • bovine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). As the flow of starch increases to the small intestine, starch is digested with decreasing efficiency (2
), 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 (3
). Increases in postruminal starch result in decreased pancreatic -amylase activity in pancreatic tissue and secretions (4
–7
), but have little influence on small intestinal disaccharidase and glucose transport activity (8
,9
). Pancreatic -amylase secretion is increased with increasing postruminal casein infusion in the presence of starch (10
,11
). However, information is lacking about how postruminal starch and protein interact to regulate pancreatic exocrine function in ruminants.
In monogastrics, the regulation of pancreatic -amylase production with dietary adaptation occurs primarily at the transcriptional level (12
). Increases in -amylase production in the developing preruminant are also primarily under transcriptional control (13
). However, in sheep, dietary regulation of pancreatic -amylase expression seems to be regulated by more complex mechanisms, possibly involving both transcriptional and post-transcriptional events (14
). Because the dynamic nature of ruminal fermentation results in variable types and amounts of nutrients flowing through the small intestine, it is difficult to examine precisely specific nutrient-gene interactions occurring within the gastrointestinal tract in feeding studies. Therefore, more precise information on the effect of postruminal nutrients on pancreatic enzyme expression and secretion is needed. Our objectives were to determine the interaction of small intestinal carbohydrate and protein supply on steady-state -amylase mRNA, protein and activity in the bovine pancreas.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Surgical procedures, postsurgical care and the experimental protocol were approved by the University of Kentucky Animal Care and Use Committee. Holstein steer calves [(n = 24) mean initial body weight (BW)6
= 88.0 ± 3 kg] were assigned randomly within block (week of infusion) to one of four abomasal infusion treatments. Calves were 
3 mo of age and had been adapted to a high forage diet from weaning (at 4 wk of age) until and throughout the experiment; thus, they were functioning ruminants. Abomasal catheters were made and placed in the calves under general anesthesia as described previously (15
). Calves were fed an alfalfa-based diet (Table 1
) at 25 g/kg BW. The diet was formulated to supply 1.2 x net energy of maintenance requirement and to exceed ruminally degradable intake protein and metabolizable protein requirements for a steer gaining 0.33 kg/d (16
). Daily feed allotments were divided into equal portions and fed every 2 h using automated feeders (SS100; Ankom, Fairport, NY). Calves were housed in a temperature- (23°C) and light- (16 h light: 8 h dark) controlled room with water available at all times. Calves were housed individually in 2.5 x 2.5 m pens and were tethered during abomasal infusion. Abomasal infusion treatments were water (control), 4 g/(kg BW · d) partially hydrolyzed starch (SH), 0.6 g/(kg BW · d) casein and SH + casein. Casein and SH were suspended in tap water and the infusion rate was 125 mL/h for all treatments. Partially hydrolyzed starch, raw cornstarch that had been partially hydrolyzed by a heat-stable -amylase (17
), was used because its digestion characteristics are similar to those of native starch passing through the small intestine. Homogeneity of the SH and casein solutions was maintained by rapid continuous mixing with a stir-bar and stir-plate. The SH and casein levels were chosen because similar starch flow would occur when moderate-to-high concentrate diets are fed and because negative and positive responses in pancreatic -amylase secretion had been observed when similar amounts of SH and casein, respectively, were infused abomasally (5
,6
,10
). Abomasal infusion periods were 10 d. The first 3 d were for adaptation in which 25, 50 and 75% of the final quantity was infused for d 1, 2 and 3, respectively. Body weights were obtained at the beginning and end of the 10-d period and average daily gain calculated.
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At the conclusion of the infusion period, calves were weighed and anesthetized by intravenous administration of pentobarbital sodium (170 mg/kg BW; Sigma Chemical, St. Louis, MO). The caudal portion of the pancreas was removed, weighed and subsampled for Northern, immunoblot and enzyme activity analyses. The remainder of the pancreas was removed and total pancreatic weight determined. Additionally, the liver was removed and weighed and the small intestine was removed and trimmed from the mesentery. For rapid determination of small intestinal length, the small intestine was laid out on a board and looped around pegs that were securely anchored to each end of the board.
RNA isolation and Northern blot.
Total RNA was isolated by the guanidium thiocyanate/acid/phenol extraction procedure as previously described (14
). Briefly, because of the high levels of RNAse in cattle pancreas (18
), pancreatic tissue was homogenized at 1 g/30 mL of extraction buffer instead of 1 g/10 mL immediately upon collection, 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 (19
), covalently cross-linked by UV light, and hybridized with 32P-labeled bovine -amylase or 18S cDNA in a solution containing 10 g/L bovine serum albumin (BSA), 243 mmol/L SDS, 500 mmol/L Na2HPO4 (pH 7.2), and 1 mmol/L EDTA (20
) for 15 h at 65°C (14
). The radiolabeled cDNA probe was prepared from a 0.89-kb segment of bovine pancreatic -amylase (13
) and from mouse 18S cDNA (Ambion, Austin, TX) by random priming extension (Gibco BRL, Grand Island, NY) using [32P]dCTP. 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. Densitometric data were corrected for unequal loading by expressing data relative to 18S rRNA expression. The 18S rRNA has been found to be a superior constitutively expressed standard compared with other genes expressed by the pancreas (21
). Northern blots were run after each experimental period (block) so that six blots were run with each treatment represented once per blot. This approach was taken because bovine pancreatic RNA stability can be difficult to maintain over time, and to allow for analysis of RNA across treatments after approximately the same period of storage.
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 5 µL/mL of a protease inhibitor cocktail for mammalian cell and tissue extracts (Sigma Chemical) to prevent proteolysis (22
). Homogenates were stored at -30°C until further analysis. Protein concentration of the homogenate was determined using the Lowry method (23
) with BSA as the standard. Proteins (10 µg/lane) were separated by 12% SDS PAGE (24
), and electrotransferred to a 0.45-µm nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was then hybridized with an anti--amylase polyclonal antibody (rabbit anti-human pancreatic -amylase; 1:500 dilution; Biodesign International, Kennebuk, ME) in blocking solution (20 g/L nonfat dry milk in 10 mmol/L Tris-Cl, pH 7.5, 300 mmol/L NaCl). Immunoreactive products were visualized by chemiluminescence (Pierce, Rockford, IL) with a horseradish peroxidase-conjugated secondary antibody (donkey anti-rabbit immunoglobulin; 1:5000 dilution; Amersham Pharmacia Biotech). Competitive inhibition experiments with human salivary -amylase (Sigma Chemical) demonstrated the specificity of the -amylase immune reaction (data not shown). The immunoblots were run at the end of the experiment on two gels (3 blocks/gel).
Densitometry and molecular size determination.
Digital images of the autoradiographic film from immunoblot and Northern analyses were scanned (HP DeskScan II; Hewlett-Packard, Palo Alto, CA) and the intensities of the bands determined using the UN-SCANIT software program (Silk Scientific, Orem, UT). Data are reported as arbitrary units. Apparent migration weights (Mr) of -amylase protein and mRNA were calculated by regression of the distance migrated against the Mr of molecular weight (or size) markers. The molecular weight (or size) markers used for Northern and immunoblot analyses were purchased from Sigma Chemical (0.2–10 kb RNA Ladder) and Gibco BRL (10-kDa protein ladder; 10–200 kDa), respectively.
Pancreatic -amylase and trypsin activity.
Pancreatic tissue (1 g) was homogenized in 154 mmol/L NaCl (10 mL) using a polytron and stored at -30°C until analysis. Protein concentrations were determined using the method of Lowry (23
). Activity of -amylase was determined using the procedure of Rauscher et al. (25
) with the aid of a commercial kit (Sigma Chemical). To assay for trypsin activity, the method described by Geiger and Fritz (26
) was used after activation with 100 U/L enterokinase (Sigma Chemical) (27
). One unit (U) of enzyme activity equals 1 µmol product produced per minute.
Statistical analysis.
Data were analyzed as a randomized complete block design with a 2 x 2 factorial arrangement of treatments using the General Linear Models procedure of SAS (28
). The model included block (group of 4 steers) and the effects of starch hydrolysate, casein and their interaction. Differences were considered significantly different when P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). There was a starch hydrolysate x casein interaction (P = 0.006) for pancreatic weight (g and g/kg BW), reflecting an increase in pancreatic weight with casein infusion in the absence of SH but not in the presence of SH. Liver weight and small intestinal length, however, were not influenced by abomasal infusion treatment.
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. However, there was a SH x casein interaction (P = 0.001) for total pancreatic protein and total pancreatic protein per kg BW, reflecting an increase in pancreatic protein with casein infusion in the absence of SH but not in the presence of SH.
Pancreatic -amylase mRNA and protein were expressed by all calves (Figs. 1
, 2). The size of the -amylase mRNA and protein was calculated to be 1.6 kb and 55.6 kDa, respectively. Pancreatic -amylase mRNA tended (P = 0.06) to be lower in calves receiving abomasal SH but was not influenced by abomasal casein (Table 3
). Also, there was a tendency (P = 0.09) for a SH x casein interaction because casein increased -amylase mRNA in the absence of SH but not in the presence of SH. Pancreatic -amylase protein was lower (P = 0.02) in calves receiving abomasal SH but was not influenced by abomasal casein. Similarly, the concentration of pancreatic -amylase activity (U/g pancreas and U/g protein) was lower (P = 0.001) in calves receiving abomasal SH but was not influenced by abomasal casein. There was a SH x casein interaction (P 0.05) for total -amylase activity (U/pancreas and U/kg BW) because casein increased total activity to a much greater extent in the absence of SH than in the presence of SH.
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-amylase/trypsin activity was lower (P = 0.01) with abomasal SH infusion. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,29
) and that this limitation could be the result of inadequate secretion of -amylase into the small intestine (9
,30
). Therefore, knowledge concerning the regulatory mechanisms involved with -amylase expression and secretion is critical so that diets can be manipulated to enhance -amylase secretion, small intestinal starch digestion and whole-body energetic efficiency. Increasing small intestinal protein flow appears to enhance pancreatic -amylase secretion and small intestinal starch digestion (31
). Therefore, this experiment was designed to specifically examine the interaction of postruminal starch and protein on pancreatic -amylase expression. This was accomplished by infusing SH and/or casein into the abomasum and measuring steady-state -amylase mRNA, protein and activity. This experimental model allows for precise information about nutrient-gene interaction because the intestinal flow of nutrients can be closely controlled.
A major goal of our research group has been to improve starch digestion in growing and finishing calves. The calves used in the present experiment were functional ruminants and it is assumed that regulatory systems coincide with those of larger growing calves once the transition from preruminant to ruminant is complete. Activity of pancreatic -amylase decreased in this experiment, as has been observed previously with larger calves (4
–7
). However, the reader should be cautioned that age and weight may influence the response in -amylase expression to changes in small intestinal nutrient flow.
The regulation of pancreatic enzyme expression can be very complex. Possible mechanisms include changes in tissue weight, changes in tissue mRNA concentration of enzyme, changes in tissue protein concentration of enzyme, post-translational modification of enzyme, and changes in the rate of synthesis and/or secretion of enzyme. The results of this experiment begin to identify mechanisms that influence adaptation of pancreatic -amylase expression in response to nutrient load challenges in the small intestine.
In this experiment, calves receiving abomasal casein alone had much greater pancreatic weights than calves receiving abomasal water (control), SH, or SH + casein. Interestingly, liver weight and small intestinal length were not influenced by abomasal infusion treatment, suggesting that pancreatic weight may be more responsive to changes in nutrient supply, particularly protein. Dietary protein is thought to be the major dietary intestinal stimulant for cholecystokinin release in monogastrics (32
). Cholecystokinin, when administered exogenously for various periods of time, appears to be the most powerful stimulator of pancreatic growth (33
). Therefore, it is possible that the increased pancreatic weight observed in calves receiving the abomasal casein treatment was mediated through changes in cholecystokinin release. Interestingly, however, SH inhibited the increase in pancreatic weight with abomasal casein. The mechanisms involved in this inhibition are unknown. More research regarding the influence of small intestinal starch and protein on pancreatic growth seems warranted.
In monogastrics, pancreatic enzyme mRNA expression generally is related to the amount of the specific substrate passing through the small intestine (12
). However, in this experiment, steady-state -amylase mRNA tended to decrease in calves abomasally infused with SH. This suggests that an inverse relationship exists between starch flow and -amylase mRNA expression. In agreement with the mRNA data, -amylase protein and activity (U/g) were lower with abomasal SH infusion. However, the magnitude of the response to SH was much greater for -amylase protein and activity than for -amylase mRNA, suggesting that regulation occurs at least in part by post-transcriptional events. Similarly, in lambs, dietary adaptation of -amylase mRNA expression appears to be regulated in part by post-transcriptional events (14
). In contrast, in monogastrics, changes in mRNA levels generally mediate the observed alterations in protein synthesis of pancreatic -amylase in response to changes in small intestinal carbohydrate levels (34
). Accordingly, regulatory mechanisms of -amylase expression appear to differ between ruminants and nonruminants.
-Amylase protein and activity responded similarly to abomasal infusion treatment in this experiment. -Amylase protein and activity also responded similarly to alterations in dietary energy and starch levels in lambs (14
). Together, these two studies indicate that regulation by post-translational modification of -amylase is not responsible for dietary or small intestinal adaptation of -amylase expression in ruminants. Together with the observation that -amylase mRNA and protein do not respond to diet or postruminal nutrients in a directly proportional manner, this suggests that dietary/small intestinal adaptation of -amylase expression is regulated primarily by translational events.
The concentration of trypsin activity also was lower in calves abomasally infused with SH. However, the ratio of the concentration of pancreatic -amylase/trypsin activity was lower with abomasal SH infusion, suggesting that small intestinal SH had a greater negative effect on -amylase expression than trypsin expression. These differences between treatments in the ratio of pancreatic -amylase/trypsin activity also suggest that different regulatory mechanisms could be involved in the production and expression of these two enzymes.
Interestingly, when enzyme activity is expressed on a per pancreas basis, results differed from those expressed on a concentration basis. The total content of enzyme activity within the pancreas should be positively correlated with secretion of enzyme activity into the small intestine if synthesis and secretion rates are similar. Calves receiving the abomasal casein treatment had much greater amounts of total pancreatic -amylase and trypsin activity than calves from other treatments. Increases in total enzyme activity were largely the result of differences in pancreatic tissue weight because calves abomasally infused with casein had heavier pancreata. Others have also shown that pancreatic size can be a major regulator of enzyme production. Wang et al. (35
) found no difference in enzyme concentration but observed differences in total activity when calves were fed to achieve high daily gain compared with those fed to achieve low daily gain. Again, as with pancreatic weights, SH inhibited the increase in total -amylase and trypsin observed with abomasal casein infusion.
Richards et al. (10
) reported increases in pancreatic -amylase secretion but no change in trypsin secretion from calves abomasally infused with raw cornstarch + casein vs. those infused with only raw cornstarch. The reasons why increases were not observed in tissue content of -amylase in calves abomasally infused with SH + casein compared with SH in the present study is unclear. There could be differences in the ability of raw cornstarch and SH that block the ability of casein to stimulate -amylase production or tissue content and enzyme secretion may not be directly related. Similarly designed experiments examining pancreatic enzyme expression and secretion have to be conducted to determine whether postruminal nutrients influence pancreatic enzyme content and secretion differently.
To our knowledge, this experiment provides the first set of data to describe the effects of postruminal nutrients on the expression of pancreatic -amylase mRNA, protein and activity in cattle. These data suggest that increases in small intestinal protein flow enhance pancreatic weight and thus total pancreatic -amylase and trypsin activity, whereas small intestinal starch inhibits this protein-dependent increase in pancreatic weight. Additionally, postruminal starch decreases -amylase expression largely by translational events. Because postruminal protein infusion did not increase -amylase expression in the presence of SH, feeding greater amounts of escape protein may not be a viable method to increase pancreatic -amylase secretion and small intestinal starch digestion in young growing calves. A better understanding of the mechanisms regulating digestive enzyme production 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.
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ACKNOWLEDGMENTS |
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-amylase cDNA probe. |
FOOTNOTES |
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-amylase expression in calves. J. Anim. Sci. (suppl. 1) 79: 80 (abs.)]. 
2 Published as publication no. 01–07-101 of the Kentucky Agricultural Experiment Station.
3 Supported by state and federal funds appropriated to the Kentucky Agricultural Experiment Station and award no. 00–35206-9380 National Research Institute Competitive Grants Program/United States Department of Agriculture.
4 Present address: USDA, ARS, U.S. Meat Animal Research Center, P. O. Box 166, Clay Center, NE 68933.
5 To whom correspondence should be addressed. E-mail: dharmon{at}ca.uky.edu
6 Abbreviations used: BSA, bovine serum albumin; BW, body weight; SH, starch hydrolysate.
Manuscript received 4 September 2001. Initial review completed 29 October 2001. Revision accepted 18 December 2001.
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