Influence of substrate and/or neurohormonal mimic on in vitro pancreatic
enzyme release from calves postruminally infused with
partially hydrolyzed starch and/or casein1
K. C. Swanson2, J. C. Matthews, C. A. Woods, and D. L. Harmon3
Department of Animal Sciences, University of Kentucky, Lexington 40546-0215
ABSTRACT: Our objectives were to determine the
effects of neuroendocrine challenge and substrates on
in vitro α-amylase and trypsin release in pancreatic
tissue collected from Holstein calves (n = 24; 88 ± 3 kg)
abomasally infused for 10 d with tap water (control),
partially hydrolyzed starch (SH; 4 g/[kg of BWⴢd]) and/
or casein (0.6 g/[kg of BWⴢd]). The caudal portion of the
pancreas was removed, rinsed with ice-cold saline, cut
into approximately 2 × 2-mm segments, and incubated
in oxygenated Krebs Ringer bicarbonate buffer containing no substrate (control), glucose, amino acids, or
VFA at 39°C. After 60 min of incubation, neurohormonal mimics (none; control), carbachol (acetylcholine analog; 10 ␮M final), or caerulein (cholecystokinin mimic;
100 nM final) were added to the flasks and tissue was
incubated for 60 min. Pancreatic tissue concentrations
and in vitro release of α-amylase and trypsin decreased
(P < 0.001) in calves abomasally infused with SH. Carbachol increased (P < 0.10) α-amylase and trypsin release in tissue collected from all calves. An effect of
caerulein to increase α-amylase release (P < 0.10) was
only observed with prior exposure to abomasal casein
infusion in vivo or with simultaneous incubation with
amino acids in vitro. Caerulein increased (P < 0.10)
trypsin release in tissue collected from all calves except
for those receiving SH + casein. Glucose decreased (P <
0.10) α-amylase release from pancreatic tissue collected
from calves receiving abomasal control and casein
treatments. Amino acids decreased (P < 0.10) α-amylase
and trypsin release from pancreatic tissue collected
from calves receiving the abomasal control treatment.
Glucose, amino acids, and VFA decreased (P < 0.10)
trypsin release from tissue collected from calves receiving abomasal SH. These data indicate that carbachol
can stimulate pancreatic enzyme release in vitro. Caerulein, however, is only effective in stimulating in vitro
pancreatic enzyme release in tissue from calves with
an increased postruminal protein supply or in tissue
incubated with amino acids. The results indicate that
postruminal and local nutrients might be important in
altering the responsiveness to a neuroendocrine challenge and could be an important regulatory event involved with dietary adaptation in ruminants.
Key Words: α-Amylase, Cattle, Hormones, In vitro, Pancreas, Trypsin
2003 American Society of Animal Science. All rights reserved.
Introduction
Ruminants differ from nonruminants in that ruminants have a near continuous flow of digesta entering
the duodenum (Merchen, 1988) and blood glucose primarily results from gluconeogenesis (Brockman and
Laarveld, 1986). Consistent with these differences in
digestive physiology is that there is little or no postpran-
1
Published as publication No. 02-07-28 of the Kentucky Agric.
Exp. Stn.
2
Present address: USDA, ARS, U.S. Meat Animal Research Center,
P.O. Box 166, Clay Center, NE 68933.
3
Correspondence: (phone: 859-257-7516; fax: 859-257-3412; Email: [email protected]).
Received February 27, 2002.
Accepted December 9, 2002.
J. Anim. Sci. 2003. 81:1323–1331
dial fluctuation in the flow of pancreatic juice and protein into the ruminant duodenum and in blood glucose
levels (Taylor, 1962; Walker and Harmon, 1995; Le
Drean et al., 1997). As a result, different regulatory
mechanisms are likely to be involved in dietary adaptation of pancreatic enzyme secretion in ruminants compared with monogastrics. Additionally, very little is
known about what mediates changes in pancreatic enzyme secretion.
In nonruminants, and likely in ruminants, the autonomic system of the pancreas is thought to interact
with hormonal controls, and possibly with substrate
controls, to provide well-coordinated responses to stimuli associated with eating and digestion (Bockman,
1998). However, research describing effects of humoral
mediators has been minimal. It is unclear how changes
in pancreatic enzymes incurred through dietary manip-
1323
1324
Swanson et al.
Table 1. Composition of experimental diet fed to calves
infused abomasally with water (control),
starch hydrolysate, and/or caseina
Ingredient
DM, %
Alfalfa cubes
Fine-ground corn
Molasses
Corn oil
Sodium phosphate
Trace mineral premixb
Vitamin A, D, and E premixc
90.00
7.96
0.50
0.50
0.50
0.50
0.04
a
Diet contained 7.9% ash, 16.1% CP, 44.6% NDF, and 33.2% ADF
(DM basis).
b
12% Ca, 6.2% P, 17% NaCl, 3% Mg, 1% S, 0.8% K, 2,300 ppm of
Zn, 175 ppm of Fe, 2,200 ppm of Mn, 1,070 ppm of Cu, 55 ppm of I,
11 ppm of Co, 29 ppm Se, 13.6 IU/g of vitamin A, and 0.06 IU/g of
vitamin E.
c
8,800 IU/g of vitamin A, 1,760 IU/g of vitamin D, and 1.1 IU/g of
vitamin E.
ulation interact with humoral mediators. Our hypothesis was that changes in postruminal and local nutrients
influence the ability of the pancreas to respond to a
neuroendocrine challenge in vitro. Therefore, the objectives of this experiment were to determine the effects
of in vitro neuroendocrine challenge and substrates on
α-amylase and trypsin release from pancreata collected
from calves postruminally infused with altered small
intestinal carbohydrate and protein supply.
Materials and Methods
at 2.5% of BW. Daily feed allotments were divided into
equal portions and fed every 2 h using automated feeders (SS100; Ankom Co., 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 × 2.5m pens and were tethered during abomasal infusion.
Abomasal infusion treatments were tap water (control),
4 g/(kg of BWⴢd) of partially hydrolyzed starch (SH),
0.6 g/(kg BWⴢd) of casein, or SH plus casein. Casein
and SH were suspended in tap water and the infusion
rate was 125 mL/h for all treatments. Partially hydrolyzed starch is raw cornstarch that has been partially hydrolyzed by a heat-stable α-amylase (Bauer et
al., 1995; Walker and Harmon, 1995) and was used
because its digestion characteristics are similar to native starch passing through the small intestine. The
SH and casein levels were chosen because similar
starch flow would occur when moderate to high concentrate diets were fed and because negative and positive
responses in pancreatic α-amylase secretion had been
observed previously using a similar experimental regimen (Walker and Harmon, 1995; Richards et al., 1998;
Swanson et al., 2002b). Abomasal infusion periods were
10 d. The first 3 d were used for adaptation with 25,
50, and 75% of the final quantity infused. At the conclusion of the infusion period, calves were weighed and
anesthetized by intravenous administration of pentobarbital sodium (Sigma Chemical, St. Louis, MO; 170
mg/kg of BW). The caudal portion of the pancreas was
removed and processed as described below.
Animals and Abomasal Infusion Treatments
In vitro Enzyme Release
The University of Kentucky Institutional Animal
Care and Use Committee approved the experimental
protocol. A full description of experimental procedures,
excluding in vitro enzyme release methodology, has
been presented previously (Swanson et al., 2002a).
Briefly, 24 Holstein steer calves (88.0 ± 3.41 kg) were
randomly assigned within block (week of infusion; four
calves per week) to one of four abomasal infusion treatments. Calves were fed an alfalfa-based diet (Table 1)
The techniques described are based on similar procedures used for other species or purposes (Williams and
Lee, 1974; Williams, 1975; Williams and Chandler,
1975; Carson et al., 1981; Katoh and Tsuda, 1984; Katoh and Yajima, 1989). Briefly, approximately 10 g of
pancreas was rinsed with and transported to the lab in
ice-cold saline (0.9% NaCl). Preliminary experiments
indicated that rinsing and transfer with ice-cold saline
gave greater viability than saline at room temperature,
Table 2. In vitro pancreatic α-amylase and trypsin tissue concentration and release in calves
infused abomasally with water (control), starch hydrolysate, and/or caseina,b
Treatment
Item
Control
Starch
hydrolysate
Tissue α-amylase, U/gb
Tissue trypsin, U/gb
α-Amylase release, U/g tissue
Trypsin release, U/g tissue
α-Amylase release/trypsin release
185.4
7.60
67.0
1.81
38.4
80.8
5.38
22.3
1.34
18.7
P-Value
Casein
Starch
hydrolysate
+ casein
195.4
8.77
70.3
2.60
28.0
70.7
4.67
26.7
1.68
18.9
SEM
Starch
hydrolysate
Casein
Starch
hydrolysate
× casein
24.9
0.87
13.3
0.43
5.29
<0.001
0.003
<0.001
0.03
<0.001
0.99
0.79
0.69
0.08
0.23
0.69
0.30
0.96
0.47
0.22
a
Values are means and pooled SEM for starch hydrolysate × casein interaction, n = 6. Calves were abomasally infused with treatments for
10 d. Pancreatic tissue was collected and processed for in vitro enzyme release analysis.
b
Initial tissue concentrations before in vitro incubation with substrates or neurohormonal mimics.
1325
In vitro bovine pancreatic enzyme release
14
160
12
10
8
6
y = 31.176x + 0.0396
r2 = 0.962
4
2
0
0
0.1
0.2
0.3
0.4
0.5
120
Treatm ent
addition
100
80
60
C ontrol
40
A cetylcholine
C arbachol
0
120
Amylase release, U/g tissue
140
20
Tissue weight, g
B.
Amylase release, U/g tissue
Amylase release, U/flask
A.
0
20
40
60
80
100
120
140
Incubation tim e, m in
100
80
Figure 2. Influence of acetylcholine (5.5 ␮M) or carbachol (10 ␮M) on α-amylase release from bovine pancreas
(n = 3).
60
40
20
0
0
60
120
180
240
Incubation time, min
Figure 1. Influence of tissue weight (a) and incubation
time (b) on α-amylase release from bovine pancreas (n
= 3).
or 39°C, or Krebs Ringer bicarbonate buffer (KRB; Umbreit et al., 1964) with 25 mM HEPES either ice-cold,
room temperature, or 39°C (data not shown). The piece
was transferred to ice-cold KRB and cut into small segments (approximately 2 × 2 mm) with a pair of fine
scissors. Tissue segments (approximately 100 mg) were
blotted dry on a paper towel, weighed using a glass
weigh funnel, and transferred by rinsing the tissue with
1 mL of KRB into 25-mL flasks containing 2 mL of KRB
and various substrates (total volume of KRB = 3 mL).
The flasks were gassed with 95% O2/5% CO2, capped,
and placed in a 39°C shaking water bath at 90 oscillations/min for one of two incubation times (60 or 120
min). For time 0 controls, tissue was added to flasks on
ice. Preliminary experiments indicated that α-amylase
release was linear with tissue amount from 20 to 400
mg and time from 60 to 180 min of incubation after an
initial lag period (Figure 1). After incubation, the flasks
were placed on ice and the buffer was removed and
stored at −30°C until analysis for α-amylase and trypsin
activity. A separate aliquot of tissue was homogenized
in saline and stored at −30°C until analysis of α-amylase and trypsin activity. Activity of α-amylase in buffer
and homogenate was determined using the procedure
of Rauscher et al. (1986), with the aid of a commercial
kit (Sigma Chemical). Trypsin activity was determined
using the procedure of Geiger and Fritz (1986) after
activation with enterokinase (200 U/L; Glazer and
Steer, 1977; Sigma Chemical).
The treatment structure for in vitro release experiments was a 4 × 3 factorial with four substrates and
three neurohormonal mimics as the factors. Each in
vitro treatment was done in triplicate flasks with tissue
from each individual calf. The four substrate treatments were 1) control (no additions), 2) 3.3 mM glucose
(Huntington and Reynolds, 1986), 3) 0.25 × the recommended concentration of minimum essential media
amino acids solution (Gibco BRL, Grand Island, NY,
product No. 11130-051) for in vitro cell or tissue incubation and 0.26 mM glutamine (Eisemann and Huntington, 1994), or 4) VFA (2 mM acetate, 0.5 mM propionate,
0.2 mM isobutyrate, 0.4 mM butyrate, 0.05 mM 2methyl butyrate, 0.10 mM 3-methyl butyrate, and 0.15
mM valerate). Substrate levels were chosen because
they are similar to physiological blood levels in cattle
(Bergen, 1979; Eisemann and Huntington, 1994). The
pH of all buffers was adjusted to 7.4 so that pH would
not influence enzyme release. Substrates were present
the entire incubation period.
After 60 min of incubation, 100 ␮L of neurohormonal
mimic solution was added. The three neurohormonal
mimic treatments were 1) control (saline), 2) 10 ␮M
carbachol (final; Sigma Chemical), or 3) 100 nM caerulein (final; Sigma Chemical). The concentrations of carbachol and caerulein were chosen because dose response experiments indicated these concentrations elicited maximal secretory responses (Swanson et al.,
2000a). Carbachol is an acetylcholine analog and caerulein is a cholecystokinin (CCK) mimic with a similar
structure and action as CCK (Williams, 1975; Beretta
et al., 1981). After adding stimulant, pancreatic tissue
was incubated for 60 min and the buffer frozen and
later analyzed for α-amylase and trypsin activity as
described above. The times for neurohormonal mimic
addition and buffer collection were chosen because enzyme release was linear from 60 to 120 min and changes
in enzyme release could be measured over a 60-min
period (Figure 2).
1326
Swanson et al.
Statistical Analysis
90
b
Results
Pancreatic tissue concentrations of α-amylase decreased (P < 0.001) by 60% and trypsin decreased by
39% (P < 0.001) in calves abomasally infused with SH,
but were not influenced by abomasal casein (Table 2).
Similarly, in vitro pancreatic α-amylase release decreased (P < 0.001) by 64% and trypsin release decreased (P = 0.03) by 32% in calves abomasally infused
with SH. Release of α-amylase was not influenced by
abomasal casein infusion, but in vitro trypsin release
increased (P = 0.08) by 36% in calves abomasally infused
with casein. The ratio of α-amylase:trypsin secreted
was decreased (P < 0.001) by 43% in calves abomasally
infused with SH reflecting a greater depression of αamylase than trypsin release.
Because there were interactions (P < 0.10) between
in vivo (SH and casein) and in vitro (substrate and
neurohormonal mimic) factors, the effects of hormone
and substrate were compared within each individual
in vivo treatment (i.e., control, SH, casein, and SH +
casein) for all of the variables measured. Carbachol
increased (P < 0.10) α-amylase release by 7, 25, 19, and
27% compared with the control in tissue collected from
calves from the control, SH, casein, and SH + casein
treatments, respectively (Figure 3). In contrast, caerulein increased (P < 0.10) α-amylase release by 13%, compared with the control, only in tissue collected from
calves abomasally infused with casein. Carbachol increased (P < 0.10) trypsin release by 27, 39, 40, and
18% compared with the control in tissue collected from
calves from the control, SH, casein, and SH + casein
treatments, respectively (Figure 4). Caerulein increased (P < 0.10) trypsin release by 18, 17, and 28%,
compared with the control (no neuroendocrine challenge), in tissue collected from all calves from the control (water infusion), SH, and casein treatments. The
ratio of α-amylase:trypsin secreted decreased (P < 0.10)
with carbachol addition by 18 and 14% or with caerulein
addition by 16 and 10% in tissue from calves abomasally
infused with the control or casein treatments, respec-
c
a
b
a
Amylase release, U/g tissue
70
a
c o n tro l
c a rb a c h o l
c a e r u le in
60
50
40
b
b
30
a
a
a
ab
20
10
0
C o n tr o l
S ta r c h H y d r o ly s a te
C a s e in
S ta r c h
H y d r o ly s a te + C a s e in
In v iv o tr e a tm e n t
Figure 3. Neurohormonal mimic effects on in vitro αamylase release from bovine pancreas. Data are the means
± SEM for neurohormonal mimic × starch hydrolysate ×
casein interaction (n = 24). Starch hydrolysate (P < 0.001);
hormone (P < 0.001); casein × hormone (P = 0.02); starch
hydrolysate × casein × hormone (P = 0.07). Bars with
different letters differ within abomasal infusion treatment
(P < 0.10).
tively (Figure 5). However, no differences were detected
in the ratio of α-amylase:trypsin release between neurohormonal treatments in tissue from calves abomasally
infused with SH or SH + casein.
Incubation with glucose decreased (P < 0.10) α-amylase release by 7 and 18% from tissue collected from
calves receiving the abomasal control and casein treat-
3 .5
b
3
Trypsin release, U/g tissue
Secretion from 60 to 120 min was calculated for triplicate flasks, averaged, and analyzed as a randomized
complete block split-plot design using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). The whole
plot consisted of block (week of infusion), SH, casein,
and the SH × casein interaction. The subplot consisted
of substrate, neurohormonal mimic, and their interaction. All interactions between whole plot (abomasal infusion) and subplot factors (substrate and neurohormonal mimic) also were tested. The effects of whole plot
variables were tested with period × SH × casein as the
error term. The effects of subplot variables were tested
using the residual error. Means were compared using
the protected LSD method. Differences were considered
statistically significant when P < 0.10.
80
c
c o n tro l
c a rb a c h o l
c a e ru le in
2 .5
a
b
b
b
2
a
b
a
a
c
1 .5
a
1
0 .5
0
C o n tro l
S ta rc h H yd ro lys a te
C a s e in
S ta rc h
H yd ro lys a te + C a s e in
In v iv o tre a tm e n t
Figure 4. Neurohormonal mimic effects on in vitro trypsin release from bovine pancreas. Data are the means ±
SEM for neurohormonal mimic × starch hydrolysate ×
casein interaction (n = 24). Starch hydrolysate (P = 0.03);
casein (P = 0.08); hormone (P < 0.001); starch hydrolysate
× hormone (P = 0.005); starch hydrolysate × casein × hormone (P = 0.01). Bars with different letters differ within
abomasal infusion treatment (P < 0.10).
In vitro bovine pancreatic enzyme release
50
a
control
carbachol
caerulein
45
Amylase/Trypsin
40
b
b
35
a
b
30
25
NS
b
NS
20
15
10
5
0
Control
Starch Hydrolysate
Casein
Starch
Hydrolysate+Casein
In vivo treatment
Figure 5. Neurohormonal mimic effects on in vitro αamylase/trypsin release from bovine pancreas. Data are
the means ± SEM for neurohormonal mimic × starch hydrolysate × casein interaction (n = 24). Starch hydrolysate
(P = 0.003); hormone (P < 0.001); starch hydrolysate ×
hormone (P = 0.02). Bars with different letters differ
within abomasal infusion treatment (P < 0.10). NS indicates P > 0.10 for neurohormonal mimic effect within
abomasal infusion treatment.
ments, respectively (Figure 6). Amino acids decreased
(P < 0.10) α-amylase release by 7% from tissue collected
1327
from calves abomasally infused with water, whereas no
differences in α-amylase release were observed between
substrate treatments in calves receiving abomasal SH
or SH + casein. Amino acids decreased (P < 0.10) trypsin
release by 14% from tissue collected from calves receiving the abomasal control treatment (Figure 7). Glucose,
amino acids, and VFA decreased (P < 0.10) trypsin release by 18, 18, and 14%, respectively, from tissue collected from calves receiving abomasal SH. No differences in trypsin release were observed between substrate treatments in calves receiving abomasal casein
or SH + casein. Amino acids increased (P < 0.10) the
ratio of α-amylase:trypsin secreted by 16%, compared
with the control, in calves receiving the abomasal control treatment (data not shown). There were no differences in α-amylase:trypsin release between substrate
treatments in calves receiving abomasal SH, casein, or
SH + casein.
There was an interaction (P < 0.10) between substrate
and neurohormonal mimic for α-amylase release. Carbachol increased α-amylase release by 18, 26, and 17%,
respectively, compared with the control, in the abomasal control, glucose, and amino acid treatments (data
not shown). However, caerulein only increased (P <
0.10) α-amylase release by 10%, compared with the
control, in the amino acid treatment. There were no
differences in α-amylase release between neurohormonal mimic treatments in the presence of VFA.
Figure 6. Substrate effects on in vitro α-amylase release from bovine pancreas. Data are the means ± SEM for
substrate × starch hydrolysate × casein interaction (n = 18). Starch hydrolysate (P < 0.001); substrate (P = 0.008); starch
hydrolysate × substrate (P = 0.02). Bars with different letters differ within abomasal infusion treatment (P < 0.10). NS
indicates P > 0.10 for substrate effect within abomasal infusion treatment.
1328
Swanson et al.
Figure 7. Substrate effects on in vitro trypsin release from bovine pancreas. Data are the means ± SEM for substrate
× starch hydrolysate × casein interaction (n = 18). Starch hydrolysate (P = 0.03); casein (P = 0.08); substrate (P = 0.06);
casein × substrate (P = 0.01). Bars with different letters differ within abomasal infusion treatment (P < 0.10). NS
indicates P > 0.10 for substrate effect within abomasal infusion treatment.
Discussion
A major problem facing beef and dairy producers is
how to most efficiently utilize feedstuffs to optimize
beef or milk production and minimize the excretion of
nutrients. These challenges are exacerbated given the
typically high microbial fermentation of most dietary
nutrients. Increasing starch flow through the small intestine decreases α-amylase production and secretion,
which subsequently results in inefficiencies of small
intestinal starch digestion. To overcome these inefficiencies of small intestinal starch digestion, we must
identify whether small intestinal nutrients (products
of forestomach microbial fermentation and abomasal
hydrolytic digestion) influence pancreatic enzyme production and secretion. The interactions occurring in
vivo complicate the interpretation of experimental data;
thus, understanding of direct interactions and molecular mechanisms of regulation of the exocrine pancreas
can be aided by the use of in vitro systems (Logsdon,
1989).
Decreased in vitro enzyme release from tissue collected from calves abomasally infused with SH largely
was the result of differences in tissue concentrations of
the enzyme. This may indicate that concentration of
enzyme in the tissue and the total mass of the tissue
are very important determinants of enzyme release.
Although trypsin concentration in tissue was not influ-
enced by abomasal casein, in vitro trypsin release increased with abomasal casein infusion. This may indicate that tissue from calves receiving abomasal casein
had a greater capacity to secrete trypsin and may indicate that pancreatic release has multiple avenues of
regulation. A further discussion of the in vivo effects
on pancreatic weight and α-amylase expression have
been reported previously (Swanson et al., 2002a).
Irrespective of abomasal infusion treatment, carbachol always stimulated in vitro α-amylase and trypsin
release as compared to control. Carbachol, an acetylcholine analog, has also been shown to stimulate in vitro
α-amylase release from sheep pancreatic tissue using
a similar model (Harada and Kato, 1983). These data
together indicate that the ruminant pancreas responds
to neural stimulation. Our data also suggest that increasing postruminal supply of starch or protein does
not influence neural control of the pancreas.
Caerulein, a CCK mimic, differentially influenced enzyme release of tissue collected from calves on different
abomasal infusion treatments. Caerulein stimulated αamylase and trypsin release to a greater extent in tissue
from calves abomasally infused with casein. These observations indicate that pancreatic tissue from calves
with increased small intestinal protein flow is more
responsive to CCK stimulation. However, caerulein did
not improve enzyme release in tissue collected from
calves receiving abomasal casein infusion in the pres-
In vitro bovine pancreatic enzyme release
ence of SH, suggesting that SH obviates the ability of
the pancreas to respond to a neuroendocrine challenge.
Changes in pancreatic responsiveness to hormones may
be involved in dietary adaptation of α-amylase secretion
in vivo. The mechanisms behind these differences in
responsiveness to caerulein are unknown, but increases
in pancreatic CCK receptors or affinity could be involved. Differential expression of A- and B-subtypes of
CCK/gastrin receptors occurs in the developing calf (Le
Meuth et al., 1993) and both subtypes are involved
in exogenous CCK stimulation of exocrine secretion in
calves (Le Drean et al., 1999). However, the importance
of these CCK receptors relative to dietary or small intestinal adaptation to nutrients is unknown. Data quantifying CCK receptor number and affinity relative to dietary treatment is needed to further our understanding
about tissue responsiveness to stimuli. Additionally,
better information about how diet and postruminal nutrients influence CCK secretion is needed. In nonruminants, CCK is considered to be one of the major gastrointestinal hormones released in response to a meal (Solomon, 1994). However, in ruminants, the importance
of CCK as an important regulator of pancreatic enzyme
secretion is uncertain because of the near continuous
flow of digesta entering the duodenum (Merchen, 1988).
Additionally, the role of feedback regulation by CCKreleasing peptides released by the small intestine or
pancreas (Miyasaka and Funakoshi, 1998) in ruminants is unknown.
It is interesting that carbachol (neural mimic) consistently elicited greater release of enzymes (Figures 3
and 4) than caerulein (hormonal mimic), particularly
since CCK and acetylcholine receptors are thought to
activate the same inositol triphosphate/diacylglycerol
second messenger cascade (Yule and Williams, 1994).
Preliminary experiments (Swanson et al., 2000a) with
tissue from calves of similar age and diet indicated that
the concentrations of carbachol and caerulein used in
this experiment elicited maximal secretory responses.
It cannot be ruled out that abomasal infusion of nutrients could influence the concentration at which the
maximal release is observed. Differences in the magnitude of the response to carbachol and caerulein could,
therefore, be related to differences in the concentration
of carbachol or caerulein necessary to cause the maximal secretory response. However, others have speculated that neural mechanisms may be more important
than hormonal mechanisms in the regulation of pancreatic enzyme secretion in ruminants (Croom et al., 1992)
because of the continuous nature of digesta flow from
the rumen.
Pancreatic α-amylase and trypsin release from tissue
from calves abomasally infused with SH generally was
less responsive to neurohormonal stimulation as compared to those not receiving abomasal SH. Additionally,
the ratio of α-amylase:trypsin secreted was not influenced by neurohormonal mimic in calves abomasally
infused with SH, but carbachol and caerulein decreased
secreted α-amylase:trypsin compared to the control in
1329
the abomasal control and casein treatments. Differences in the ratio of α-amylase:trypsin secreted in the
abomasal control and casein treatments also indicates
that α-amylase and trypsin release are differentially
regulated by neurohormonal mimic. This would suggest
that nonparallel release of pancreatic enzymes is possible. Others have suggested that nonparallel secretion
of digestive enzymes occurs and that there are different
pools of vesicles that contain different proportions of
enzymes (Case, 1998).
Substrate treatments generally had less of an effect
than did neurohormonal mimic on in vitro pancreatic
enzyme release. This is not surprising because luminal
substrate effects could be mediated via peptide hormones or neural stimulation. However, glucose decreased in vitro pancreatic α-amylase release from
calves abomasally infused with the control or casein
treatment. Kreikemeier et al. (1991) reported that abomasal infusion of raw cornstarch linearly increased arterial glucose concentrations in growing beef steers.
Also, Walker and Harmon (1995) reported increases in
portal glucose concentrations in response to abomasal
SH infusion. In wether lambs, Call et al. (1975) reported
that increasing the blood glucose by infusion of a 16%
glucose solution intravenously for two 3-h periods/d for
7 d sharply reduced secretion of α-amylase activity.
Inhibition of pancreatic enzyme secretion with intravenous glucose infusion also has been reported in dogs
(Nakajima and Magee, 1970). These data suggest that
glucose alone may be partly responsible for down-regulation of α-amylase secretion when increased starch
flows through the small intestine. However, in calves
receiving abomasal starch, no differences with substrate addition were observed. This could indicate that
the pancreas can adapt to postruminal starch in that
glucose does not influence α-amylase secretion after the
down-regulation of α-amylase occurs. Amino acids also
decreased in vitro pancreatic α-amylase release from
calves on the abomasal control treatment and decreased
in vitro pancreatic trypsin release from calves on the
abomasal control and SH treatments. Although it has
typically been thought that blood amino acids have little
influence on pancreatic enzyme secretion (Lavau et al.,
1974), amino acids can have an inhibitory influence
on secretagogue-induced exocrine secretion from the
isolated perfused rat pancreas (Harada et al., 1984).
Glucose and VFA also had negative effects on in vitro
pancreatic trypsin release in calves abomasally infused
with SH. VFA have generally been thought to be a
stimulator of α-amylase release in ruminants (Harada
and Kato, 1983; Katoh and Tsuda, 1984; Katoh and
Yajima, 1989). It is unclear why VFA did not influence
α-amylase release and decreased trypsin release in
calves abomasally infused with SH. The importance of
VFA as a regulator of pancreatic enzyme release is
questionable, however, because the peripheral concentrations of VFA are very low because of extensive metabolism in the portal-drained viscera and liver (Bergman and Wolff, 1971). Others reporting positive results
1330
Swanson et al.
with VFA addition have often used supraphysiological
concentrations (Katoh and Yajima, 1989). The concentrations of VFA used in this experiment were near physiological concentrations, at least for portal blood, and
the general inability of VFA treatment to influence secretion of α-amylase and trypsin supports the theory
that VFA are not major physiological regulators of pancreatic enzyme release.
The increased responsiveness of pancreatic tissue to
caerulein stimulation in calves abomasally infused with
casein discussed above also occurred in vitro. Caerulein
stimulated in vitro α-amylase release in tissue incubated with amino acids but not with control, glucose,
or VFA. This suggests that amino acids may be required
locally for CCK to positively influence α-amylase release. Neurohormonal mimics did not influence α-amylase release when incubated with VFA suggesting that
VFA reduce the responsiveness of the tissue to neurohormonal stimulation. Others have suggested that, in
sheep, the cellular secretory process evoked by acetylcholine is qualitatively similar to that of VFA, and that
Ca2+ ions might be an important mediator for these
secretagogues in the acinar cells of the pancreas of
sheep (Katoh and Tsuda, 1984). Therefore, it is possible
that VFA interact with the receptors involved with neurohormonal stimulation of pancreatic enzyme release
in cattle so that binding between the neurohormone
and receptor is altered.
In summary, these data indicate that carbachol (acetylcholine analog) can stimulate pancreatic enzyme release in vitro. Caerulein (CCK mimic) also can stimulate pancreatic trypsin release. However, caerulein is
only effective in stimulating in vitro pancreatic enzyme
release in tissue from calves with increased postruminal protein flow or in tissue incubated with amino acids,
suggesting that postruminal and local nutrients can
alter the responsiveness of the pancreas to caerulein.
However, caerulein did not improve enzyme release in
tissue collected from calves receiving abomasal casein
infusion in the presence of SH, suggesting that SH obviates the positive effect elicited by casein. Postruminal
and local nutrients may be important in altering the
responsiveness of the pancreas to respond to stimuli
and could be an important regulatory event involved
with dietary adaptation in ruminants.
Implications
Our results show that bovine pancreatic tissue is responsive to neural and hormonal mediators and that
hormonal regulation is influenced by postruminal and
local nutrients. Changes in pancreatic responsiveness
to hormones may be involved in dietary adaptation of
α-amylase secretion. 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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