The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer.
The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction.
In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. These are also available as one exposure on a standard 35mm slide or as a 17" x 23" black and white photographic print for an additional charge.
Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order.
University Microfilms International A Bell & Howell Information Company
300 North Ztjeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600
Order Number 1S36551
Effects of diet on amylase content and synthesis in cultured rat acinar cells
Justice, Jill Diane, M.S.
The University of Arizona, 1989
U M I 300 N. Zeeb Rd. Ann Arbor, MI 48106
EFFECTS OF DIET ON AMYLASE CONTENT AND SYNTHESIS IN CULTURED
RAT ACINAR CELLS
by
Jill Diane Justice
A Thesis Submitted to the Faculty of the
COMMITTEE ON NUTRITIONAL SCIENCES (Graduate)
In Partial Fulfillment of the Requirements For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
19 8 9
2
STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
This thesis has been approved on the date shown below:
SIGNED
APPROVAL BY THESIS DIRECTOR
/ P.M. Brannon Associate Professor of Nutrition
DEDICATION
This thesis is dedicated to my husband, Wade W.
Justice. His love, patience and continuous support have
made this thesis project possible.
4
ACKNOWLEDGEMENTS
I wish to thank Dr. Patsy Brannon for her inspiration
and careful guidance during this thesis project. I also
thank Drs. Edward Sheehan and Donald McNamara for their
participation in this project.
Finally, I wish to thank my laboratory and departmental
colleagues for their enthusiastic support.
5
TABLE OF CONTENTS
Page
LIST OF FIGURES 7
LIST OF TABLES 8
LIST OF ABBREVIATIONS 9
ABSTRACT 11
INTRODUCTION 12
LITERATURE REVIEW 14
The Endocrine Pancreas 14
The Exocrine Pancreas 16
Function and Morphology 16
Synthesis and Secretion of 18 Digestive Enzymes
Regulation of Secretion 21
Regulation of Digestive Enzyme 29 Synthesis
Dietary Adaptation of Digestive 34 Enzymes
Pancreatic Amylase 38
Characteristics 28
Enzymatic Mechanism 41
Amylase Purification 42
Regulation of Amylase... 44
Acinar Cell Culture 49
Pancreatic Acinar Cells 49
Pancreatic. Acini 52
6
TABLE OF CONTENTS—Continued
Page
MATERIALS AND METHODS 54
Materials 54
Methods . . 55
Experimental Obj ectives 55
Animals and Diets 56
Isolation and Culture of Acinar Cells 56
Analyses of Cellular Protein and 61 Amylase
Incorporation of [3H]-phenylalanine. 62 into Protein
Incorporation of [3H] -phenylalanine 63 into Amylase
Data Analyses 67
RESULTS 68
Characteristics of the a-GHI-Seph 68 Affinity Adsorbent
Incorporation of [3H]-phe into Total 71 and Amylase Protein by Cultured Cells
Effects of Diet on Amylase Activity and 71 Cellular Protein
Effect of Diet on Amylase Relative Synthesis 76
DISCUSSION 82
SUMMARY AND CONCLUSIONS 93
APPENDIX 95
REFERENCES 97
7
LIST OF FIGURES
Figure Page
1 Schematic of a Pancreatic Acinar Cell 17
2 Stimulus-Secretion Coupling in the 25 Pancreatic Acinar Cell
3 Isolation of Pancreatic Acinar Cells 60
4 SDS-PAGE Validation of Affinity 66 Adsorbent
5 SDS-PAGE Profile of a-GHI-affinity 70 Adsorbent Dissociated Acinar Protein
6 [3H]-phe Incorporation into Amylase 72 (A) and Total Protein (B) by Freshly-Isolated Acinar Cells
7 Effects of Diet on Amylase Relative 79 Synthesis in Cultured Acinar Cells
8 Regression Analysis of Amylase Activity 81 Versus Relative Synthesis in Cultured Acinar Cells
8
LIST OF TABLES
Table Page
1 Pancreatic Enzymes 30
2 Comparison of Pancreatic Amylase 40 in Five Species
3 Composition of Wayne Rodent Blox ..57
4 Composition of Purified Diets 58
5 Binding of Amylase to a-GHI-Sepharose 69 Affinity Adsorbent
6 [3H]-phe Incorporation into Amylase 73 Protein and Total Protein by Cultured Rat Acinar Cells
7 Effects of Antecedent Diet on Cellular 75 Amylase Activity and Total Cellular Protein in Cultured Acinar Cells
8 Effects of Diet on Relative Amylase 77 Synthesis, Cellular Protein and DNA in Cultured Acinar Cells
9 Comparative Effect of Diets High in 80 Fat and Carbohydrate on Amylase Activity and Relative Synthesis in Cultured Acinar Cells
SDS—PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
TCA trichloroacetic acid
TEMED N,N,N1,N•-tetramethyl-ethylenediamine
VIP vasoactive intestinal peptide
ABSTRACT
11
To study adaptation of pancreatic amylase to diet, an
affinity adsorbent, a-GHI-AH-Sepharose 4B, was used to
determine amylase synthesis in cultured pancreatic acinar
cells. This adsorbent exhibited a consistent binding
capacity and was specific for amylase. Acinar cells from
rats fed high fat (HF) or carbohydrate (HC) diets for 7 d
were cultured 1-48 h in serum-free medium. Amylase activity
remained significantly higher in HC cells than in HF cells
through 24 h in culture, despite its decrease with time in
culture. The relative synthesis of amylase (3H-phe
amylase/3H-phe total protein x 100) was significantly higher
in HC than in HF cells at isolation and remained higher
during culture. These results demonstrate that this
affinity adsorbent can be used to determine amylase
synthesis and suggest that the effect of diet on amylase
activity and relative synthesis persists in cultured
pancreatic acinar cells.
INTRODUCTION
12
The pancreatic acinar cell, the main exocrine component
of the pancreas, has the ability to alter the content of
specific digestive enzymes in response to dietary
composition. For example, a diet high in carbohydrate
results in a secretion rich in pancreatic amylase; whereas a
diet high in protein leads to pancreatic secretion enriched
in proteases. This adaptation of pancreatic enzymes to diet
appears to be the result of changes in enzyme synthesis and
levels of mRNA coding for the enzymes. While the
characteristics of these adaptations have been studied
extensively, the mechanisms remain unknown.
Pancreatic amylase is one of the major digestive
enzymes of the exocrine pancreas. It hydrolyzes the ct-1,4-
glucose linkages of dietary starch in the duodenum to form
branched dextrins. There are several effectors implicated
in the dietary adaptation of amylase; of these, glucose and
insulin are the most likely. The effects of glucose and
insulin on the regulation of pancreatic amylase are
noteworthy when considering that alterations in glucose,
insulin and amylase are all present in the diabetic disease
state. In diabetes, a relative insulin insufficiency exists
and this leads to hyperglycemia and decreased tissue glucose
utilization. Pancreatic amylase is also decreased in
diabetes. Understanding the possible interaction of glucose
13
and insulin in the regulation of amylase would not only
elucidate pancreatic exocrine adaptation, but could possibly
advance our knowledge of diseases such as diabetes.
In the past, studies examining the adaptation of
amylase to diet and its mechanism(s) have been done in whole
animals. Thus, it has been difficult to interpret the
direct effects of various diets or hormones on the acinar
cells because of the homeostatic interactions in the intact
organism. In a primary culture of rat acinar cells, various
nutrients, hormones or combinations thereof can be
manipulated? and their direct effects on the acinar cells
can be determined without the confounding variables of an in
vivo study.
These studies herein examined the effects of antecedent
diet on amylase activity and relative synthesis in cultured
rat pancreatic acinar cells in order to understand better
the direct effects of dietary adaptation of amylase. First,
a method for the determination of amylase synthesis was
developed and validated. Amylase synthesis was determined
by the incorporation of 3H-phenylalanine into amylase
protein that was measured by an affinity adsorbent. Next,
pancreatic acinar cells were isolated and cultured from rats
fed various diets, and the effects of these antecedent diets
on relative amylase synthesis and activity were determined.
LITERATURE REVIEW
14
The pancreas consists of two components: the endocrine
pancreas, which secretes hormones, and the exocrine pancreas
which secretes digestive enzymes, fluid and electrolytes.
Both of these components are influenced by neural, hormonal
and dietary factors. Although the pancreas is usually
considered two separate organ systems, one endocrine and one
exocrine in nature; it is actually an integrated organ that
coordinates the digestion and utilization of food.
THE ENDOCRINE PANCREAS
The endocrine component of the pancreas, the islets of
Langerhans, is essential in the regulation of the exocrine
pancreas, the acini and ducts. There are approximately 1 x
106 islets distributed throughout the human pancreas and
about 5,000 in the rat pancreas (1). Regardless of the
absolute number of islets, they comprise about 1-2% of the
pancreatic volume in all mammalian species and are typically
composed of 5,000 endocrine cells each (1). There are four
major types of endocrine cells, the most well-defined being
the B cells which secrete insulin. In addition, the A cells
produce glucagon, the D cells produce somatostatin, and the
PP cells produce pancreatic polypeptide. All of these
hormones are synthesized in the rough endoplasmic reticulum
(RER) of the endocrine cell and are stored in cytoplasmic
15
granules until they are released in response to stimuli (2).
In addition to their other metabolic effects, these
endocrine secretions are functionally related to one another
with respect of their effects on the exocrine pancreas.
Insulin appears to have long-term effects on regulation of
the biosynthesis of pancreatic exocrine digestive enzymes
(3-5) and short-term effects on the potentiation of exocrine
secretory response to gastrointestinal hormones and
neurotransmitters (6-9) . Glucagon appears to have an
inhibitory effect on exocrine secretion (10-12), although
whether this is a direct or indirect effect is unknown.
Pancreatic polypeptide is also presumed to inhibit exocrine
secretion (13). Somatostatin has indirect effects on the
exocrine pancreas as it has effects on regulators of
exocrine function. It has been shown to inhibit the release
of glucagon (14), insulin (15) and pancreatic polypeptide
(ie).
Strong evidence for the integration of endocrine and
exocrine functions lies in the structural relationship
between the islets and the exocrine (acinar) cells. Because
there is no substantial capsule or basement membrane
surrounding the islets (1), pancreatic acinar cells lie in
close contact with the islets. This relationship results in
two types of acinar cells: the periinsular acini or
"halos," those cells in proximity to the islets, and the
teliinsular acini, those cells removed from the islets. The
16
periinsular acini contain larger nuclei and more zymogen
granules than the teliinsular acini (17) and this appears to
be related to locally high insulin levels. When compared to
teliinsular acini, the periinsular acini also contain higher
concentrations of digestive enzymes (18). The halo
phenomenon may be the result of the distinct pattern of
blood-flow from the islets to the exocrine cells. Almost
all efferent blood flow goes into acinar capillaries before
leaving the pancreas, which creates a gradient of islet
hormones that is greatest in the periinsular acini and
decreases as the distance from the islets increases (13).
This structural relationship leads to a locally-mediated
regulation of exocrine function by islet hormones.
THE EXOCRINE PANCREAS
Function and Morphology. The exocrine pancreas is one
of the body's major digestive organs. It synthesizes more
protein per gram of tissue than any other organ, and
secretes between 6 and 20 grams of digestive enzymes and
zymogens (digestive pro-enzymes) in approximately 2.5 liters
of fluid daily (1). The major structural components
responsible for the exocrine function of the pancreas are
the acini, which secrete digestive enzymes, and the ductal
cells, which secrete fluid and electrolytes.
Acini are the enzyme-synthesizing and secreting units
of the pancreas and are comprised of clusters of acinar
FIGURE 1: SCHEMATIC OF A PANCREATIC ACINAR CELL
A typical acinar cell is shown with the nucleus (N) and rough endoplasmic reticulum (RER) localized in the basal portion of the cell and the Golgi complex (GC), condensing vacuoles (CV) and zymogen granules (ZG) located in the apical pole of the cell.
18
cells surrounding a central duct. A typical acinar cell
(represented in Figure 1) is polarized. The basal portion
of the cell consists of the nucleus surrounded by the RER,
while the Golgi complex (GC) and zymogen granules (ZG) are
found predominantly in the apical pole of the cell.
Synthesis and Secretion of Digestive Enzvmes. The
secretory cycle in the exocrine pancreas consists of six
streptomycin, and 5 Mg/ml Fungizone), and resuspended in 3
60
Aseptic removal of pancreas
Y Chelation of divalent cations
Y Digestion with collagenase-hyaluronidase
Repeat chelation and digestion
Y Dispersal of cells
Separation of exocrine acinar cells in ficoll
Y Culture 1 x 10-6 cells in 2 ml of serum-free medium
FIGURE 3 i ISOLATION OF PANCREATIC ACINAR CELLS
61
ml of Waymouth's medium. Cell number and viability were
determined by trypan blue dye exclusion using a cell
suspension containing 0.08% trypan blue dye. Cells (1 x
106/well) were plated in 24-we11 clusters in 2.0 ml of
Waymouth's serum-free medium and incubated at 37°C with 5%
humidified C02 for up to 48 h. Serum-free medium (181,193)
was composed of Waymouth's medium with 10 mg/ml bovine serum
albumin, 1 x 10*"8 M dexamethasone and 42 pM epidermal growth
factor (EGF). Cells were harvested and centrifuged at 500xg
at 4°C for 2 min; the supernatant was discarded, and cell
pellets were washed twice with ice-cold phosphate-buffered
saline (PBS) and frozen for subsequent analysis.
Analyses of Cellular Protein and Amylase. Frozen cell
pellets were homogenized in 0.50 ml PBS by sonication on ice
for 15 s. Aliquots of homogenates were assayed for cellular
protein by the method of Lowry (194) with bovine serum
albumin as the standard. This method requires complexing
the protein with copper under alkaline conditions. This
complex is then reduced by the addition of the Folin-
Ciocalteu (phosphomolybdic-phosphotungstic) reagent, and the
resulting blue product is measured spectrophotometrically at
660 nanometers (nm). Homogenates were also assayed for
cellular amylase activity by the Phadebas blue starch method
(195,196) using Sigma Enzyme 2E standard. In this assay,
the amylase substrate is a cross-linked starch polymer to
62
which a blue dye is covalently coupled. When a-amylase
hydrolyzes the (l-4)-glycan bonds of this water-insoluble
susbstrate, soluble starch-dye moieties are formed that are
measured spectrophotometrically at 620 nm. The amount of
blue dye in solution is proportional to the a-amylase
activity in the sample. Amylase activity was expressed as
units (U, /timoles maltose released per minute).
Incorporation of r^-HI-phenylalanine into Protein. To
measure the incorporation of [3H]-phenylalanine ([3H]-phe)
into TCA-precipitable protein, 10.0 nCi of [3H]-phe was
added to each well in 50 nl aliquots of Waymouth's medium.
(Final concentration 5 /iCi/ml medium.) Cells were incubated
and harvested in duplicate at the times indicated after the
addition of 0.4 ml of ice cold 100 mM phenylalanine. The
cells were centrifuged at 500xg at 4°C for 2 min, rinsed
twice, then homogenized in 0.6 ml ice-cold PBS. Aliquots
(30 fil each) were taken for protein determination. An
aliquot was removed for determination of [3H]-phe
incorporation into amylase protein as described in the
following section. A final aliquot (100 fil) of the
homogenate was incubated 20 min of ice in 10% TCA with 20 /xg
bovine serum albumin, centrifuged at 4°C for 4 min at
2000xg. Pellets were washed twice with 10% TCA, dissolved
in 0.1 N NaOH and counted in ACS liquid scintillation
cocktail. Counts incorporated into cellular protein were
63
expressed as net [3H]-phe incorporation (dpm)/mg cellular
protein.
Incorporation of r3-H1-Phenylalanine into Amylase.
Incorporation of t3H]-phe into amylase protein was
determined in cell homogenates using an affinity adsorbent
for amylase (156). This affinity adsorbent, a-GHI-Sepharose
4B (a-GHI-Seph) was prepared as previously described (158).
Briefly, w-aminohexyl-Sepharose (4 g) was swelled in 0.2 M
phosphate buffer (pH 7.0) for 24 h at 4°C. After filter
washing with an excess of phosphate buffer, the Sepharose
was combined with a-GHI (Bay g5421, 200 mg) and phosphate
buffer and shaken at 4°C. After 30 minutes, sodium
borohydride (100 mg) was added and the mixture was shaken.
After 6 h, additional sodium borohydride (100 mg) was added
and the mixture was shaken for 22 h. Finally, the mixture
was washed with deionized water and brought to volume (50%
a-GHI-Seph/50% phosphate buffer); sodium azide (0.02%) was
added, and the affinity adsorbent was stored at 4°C.
Immediately prior to use, 0.5 ml of the adsorbent was
transferred to a microfuge tube and centrifuged at 15,600xg
for 8 s in an Eppendorf microfuge. The supernatant was
discarded, and the adsorbent was rinsed with PBS and
resuspended in 0.5 ml PBS. All subsequent steps were
performed at 4°C unless otherwise indicated. The aliquot of
the cellular homogenate for amylase was centrifuged at
100,00xg for 60 min. An aliquot of the supernatant was
64
applied to the affinity adsorbent, and this mixture was
shaken for 30 min. This sample was centrifuged at 15,600xg
for 8 s and the post-incubation supernatant was removed. The
adsorbent was rinsed once with 1.0 ml of 0.15 M NaCl buffer
followed by 1.0 ml of 0.5 M NaCl buffer (156). Amylase
protein was dissociated from the adsorbent with 0.75 ml of
0.1% glycogen in 0.15 M NaCl buffer, pH 7.4. An aliquot
(0.7 ml) of the eluted protein was counted in ACS liquid
scintillation cocktail in a Packard scintillation counter
with a wide 3H-channel. Counts incorporated into amylase
protein were expressed as net [3H]-phe incorporation
(dpm)/mg cellular protein. Relative amylase synthesis was
expressed as the ratio of net [3H]-phe incorporation into
amylase protein to net [3H]-phe incorporation into cellular
protein x 100.
Additional studies were done to determine the binding
capacity and the specificity of this affinity adsorbent.
To determine binding capacity, various known amounts of
amylase activity were applied to the adsorbent. Nonbound
amylase activity was measured in the post-incubation
supernatant and in the subsequent washes. Bound amylase
activity was the difference between the total activity
applied to the adsorbent and the nonbound activity recovered
in the in the post-incubation supernatant and washes. The
bound activity was expressed as a percentage of total
amylase activity applied to the adsorbent.
65
Specificity of this affinity adsorbent was examined
using SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
This validation experiment is summarized in Figure 4.
Pancreatic acinar cells were isolated as described above.
The cells were then incubated with [3H]-phe (5 /aCi/ml) for
24 h, harvested and pooled (six wells per sample), as
previously described. The resulting pellet was homogenized
in 0.40 ml PBS and centrifuged at 100,000xg at 4°C for 60
min. An aliquot of the cellular homogenate was incubated
with the affinity adsorbent as described. A 100 n1 aliquot
of the glycogen-dissociated material was combined with
sample buffer (187.5 mM tris, 6% SDS, 30% glycerol and
0.003% bromophenol blue) and mercaptoethanol (1.3%). A 75
Hi aliquot of this sample preparation, a purified amylase
sample and low molecular weight standards were
electrophoresed on a 7.5% SDS polyacrylamide-gel (197). The
dissociated sample lanes were cut into 0.5 cm pieces,
digested in 30% hydrogen peroxide at 60°C overnight, and
counted in ACS liquid scintillation cocktail. The amylase
and standards were stained in 0.25% Coomassie Blue. The Rf
(the ratio of the distance from origin to the protein to the
distance from origin to dye front) of the amylase and
dissociated materials were determined.
66
Incubate acinar cells with 3H-phe 24 h
T Harvest acinar cells y
Homogenize in PBS, pH 7.4 (4*C)
Y Centrifuge for 60 min at 100,000xg at 4*C (discard pellet)
Y Incubate supernatant with 50% suspension of a-GHI-Seph
in PBS for 30 min at 4*C
Y Centrifuge at 15,600xg 8s; discard supernatant
Y Wash adsorbent with PBS then 0.5 M NaCl-5 mM P04, pH 7.4
Y Elute with 0.1% glycogen-PBS for 15 min at 4*C
Y Centrifuge at 15,600xg for 8 s
Y
Remove supernatant
Y Electrophorese on SDS-PAGE
Y Cut Gel into 0.5 cm pieces
Y Dissociate with H2O2 at 60*C overnight
Y Count 3H-protein
FIGURE 4; SDS-PAGE VALIDATION OF AFFINITY ADSORBENT
67
Data Analyses. Data were analyzed by one- or two-way
analysis of variance (ANOVA) and least significant
differences (LSD, 198). In studies of the rates of
incorporation, data were analyzed by least-squares linear
regression (198). Finally, the relationship between
cellular amylase content and relative amylase synthesis was
analyzed by curve fitting analysis (198).
RESULTS
68
Characteristics of the a-GHI-Seph Affinity Adsorbent.
The binding capacity and the specificity of the a-GHI-Seph
affinity adsorbent for amylase were examined. To ascertain
the adsorbent's binding capacity, the binding of amylase to
the adsorbent was determined over a range of amylase
activity (Table 5). Specifically, the percent of total
amylase activity applied to the adsorbent that was actually
bound was determined. The binding at the lowest level of
applied amylase activity (5.8 U) was significantly higher
(82%) than the binding at higher levels of applied activity
(11.4 to 46.2 U) at76%; however, the overall difference in
binding among the various applied activities was small (less
than 10%).
To determine the specificity of the adsorbent for
amylase, dissociated 3H-protein from the adsorbent was
subjected to SDS-polyacrylamide gel electrophoresis. As
illustrated in Figure 5, the 3H-protein comigrated (Rf=0.30)
with purified amylase (Rf=0.31). One hundred percent of the
radioactivity applied to the gel was recovered, and the
major peak contained 90% of the radioactivity. A minor peak
containing 9% of the applied radioactivity was seen at a
lower molecular weight than the major amylase peak.
69
TABLE 5: BINDING OF AMYLASE TO a-GHI-SEPHAROSE AFFINITY ADSORBENT-
Amylase Activity Applied (U)
5.8 ± 0.3
11.4 ± 0.6
23.1 ± 1.7
34.6 ± 2.5
46.2 ± 3.5
% Bound^.
82 ± 3a
73
A3 CM +1
76 ± 3b
77 ± 2ab
76 ± 3b
•'•Freshly isolated pancreatic acinar cells from rats fed CU diet were homogenized in PBS and these samples were centrifuged at 100,000xg for 60 minutes. The resulting supernatant was assayed for amylase activity, and various amounts of this homogenate were applied to the a-GHI-Seph affinity adsorbent. Amylase protein was purified as described earlier.
2Percent bound was calculated as total amylase activity applied - unbound amylase activity * total amylase activity applied x 100. Results are expressed as the mean ± SEM for duplicate samples from two experiments.
aWalues not sharing a superscript differed significantly (p<0.05) by ANOVA and LSD (198).
70
Amylase
FIGURE 5: SDS-PAGE PROFILE OF a-GHI-AFFINITY ADSORBENT DISSOCIATED ACINAR PROTEIN.
Dissociated 3H-acinar protein from the a-GHI-affinity adsorbent was electrophoresed on 7.5% polyacrylamide gel. The gel was cut into 0.5 cm pieces, digested in 30% H202 at 60*C overnight and counted. 100% of the applied radioactivity was contained in the major peak (Rf=0.30) which comigrated with purified amylase (Rf=0.31).
o 71
Incorporation of rlHI-phe Into Total and Amylase
Protein bv Cultured Cells. A time-course study of the
incorporation of [3H]-phe into amylase protein and TCA-
precipitable (total) protein was performed in order to
determine a period of linear phenylalanine incorporation
into protein. Incorporation of labelled amino acid into
amylase protein (Figure 6A) and total protein (Figure 6B)
was linear from 0 to 240 min (r=0.97 for both amylase and
total protein) in freshly isolated acinar cells from rats
fed a CU diet. Incorporation of label into amylase and
total protein (r=0.91 and r=0.98, respectively) was also
linear from 0 to 240 min in acinar cells cultured 24 h (data
not shown). From these data, a 180 min incubation with
[3H]-phe was selected to determine phenylalanine
incorporation in both freshly isolated and cultured cells
from rats fed CU, HF and HC diets in subsequent experiments.
Relative rates of amylase synthesis in cells from CU-
fed animals decreased through 48 h in culture (Table 6).
Absolute [3H]-phe incorporation into amylase protein
decreased with time in culture (p<0.05). Absolute [3H]-phe
incorporation into TCA-precipitable protein also
significantly decreased with time in culture, so the
relative rates of amylase synthesis were not altered in
cells cultured 24 h, and decreased in cells cultured 48 h.
Effects of Diet on Amylase Activity and Cellular
Protein. To determine the effect of diet on cellular
72
A. Amylase
y =-0.2 + 0.028 x, r = 0.97
CL
O)
CO
Q. •o B. Total Protein
40
y =-2.3 + 0.19 x, r = 0.97 q. 30
20
to
60 120 240
Time (min)
FIGURE 6: [3H]-PHE INCORPORATION INTO AMYLASE (A) AND TOTAL PROTEIN (B) BY FRESHLY ISOLATED ACINAR CELLS.
[3H]-phenylalanine incorporation was determined at the indicated intervals in freshly isolated acinar cells from rats fed a CU diet. Data were analyzed by least squares linear regression (198).
73
TABLE 6: [3H]-PHE INCORPORATION INTO AMYLASE PROTEIN AND TOTAL PROTEIN BY CULTURED RAT ACINAR CELLS.1
[3H]-phe incorporation2 (dpm)/mg protein
Amylase Relative Time (d) Amylase Total Protein Synthesis^. (%)
0 13909±2812a 87085±9336a 15.2a
24 7764±1196b 46323±5357b 17.7a
48 1131±407c 7872±1816c 8.3b
•'•Cultured pancreatic acinar cells were isolated from rats fed a CU diet and cultured 48 h in serum-free medium. Cells were then incubated 180 minutes with 5 fiCi/ml [3H]-phe.
2Values represent the mean ± SEM of triplicate samples from 3 experiments.
3Amylase relative synthesis is the ratio of [3H]-phe incorporation into amylase protein to [3H]-phe incorporation into total protein x 100.
abcValues not sharing a superscript for each parameter differed significantly (p<0.05) by ANOVA and LSD (198).
74
amylase activity and cellular protein, acinar cells were
isolated from rats fed HF or HC diets and cultured.
Antecedent diet had a dramatic effect on amylase activity
(Table 7). Total amylase activity (U/well) in cells
isolated from HC-fed rats was significantly (p<0.05) higher
(7-fold) than amylase activity in cells from HF-fed rats at
isolation and remained significantly higher (6-fold) through
24 h in culture. Amylase activity in the HC cells
significantly decreased (80%) with time in culture, so that
the difference between HC and HF cells was not significant
by 48 h in culture. Amylase activity in HC cells still
tended to be higher (24-fold) than in HF cells at 48 h.
There was no significant effect of time on amylase activity
in cells from HF-fed rats. There was, however, a
significant interactive effect of time and diet on amylase
activity in these cells. However, amylase activity in HF
cells tended to decrease with time in culture from 21 U/well
to 1.4 U/well. There was a significant (p<0.00001)
independent effect of time on total amylase activity.
Activity was greatest in freshly isolated cells and
decreased with time in culture. There was also a
significant (p<0.00001) independent effect of diet on total
amylase activity, which was higher in cells isolated from
HC-fed rats when compared to cells isolated from HF-fed
rats. Finally, there was an interactive effect of time and
diet on total amylase activity.
75
TABLE 7: EFFECTS OF ANTECEDENT DIET ON CELLULAR AMYLASE ACTIVITY AND TOTAL CELLULAR PROTEIN IN CULTURED ACINAR CELLS.1
Time in Culture (h) Antecedent Diet
EE HC
Total Cellular Amylase Activity (U/well)2'3
0 21.1 ± 10.0C (3) 148.1 ± 71.2a (4)
24 13.8 ± 5.2C (3) 79.7 ± 24.7b (3)
48 1.4 ± 0.3C (2) 33.9 ± 6.4C (3)
Total Cellular Protein (mg/well)2'4
0 0.23±0.06ab (3) 0.28±0.07a (4)
24 0.17±0.10bc (3) 0.22±0.04ab (3)
48 0.24±0.01a (2) 0.15±0.06c (3)
^•Acinar cells were isolated from rats fed HC (high carbohydrate) or HF (high fat) diets for 7 d and cultured in serum-free medium for 48 h. Values represent the mean ± SEM for at least triplicate samples from the number of experiments indicated in parentheses.
2Each sample well contained approximately 1 x 106 cells.
3There was a significant (p<0.00001) independent effect of time on total amylase activity: 0 h > 24 h > 48 h. There was also a significant (p<0.00001) independent effect of diet on total amylase activity: HC > HF.
4There was no effect of diet on total cellular protein. There was a significant (p<0.001) independent effect of time on total cellular protein: 0 h > 24 h = 48 h.
abcValues not sharing superscripts differed significantly (p<0.05) by ANOVA and LSD (198).
76
Antecedent diet had no effect on total cellular protein
(Table 7); while time in culture had a significant
independent effect resulting in an overall decrease in
cellular protein (p<0.001). There was also an interaction
between time and diet on cellular protein. Cells from HF-
fed rats demonstrated no significant change in protein from
0 to 48 h while cells from HC-fed rats had a 50% decrease in
protein content during 48 h in culture.
Effect of Diet on Amylase Relative Synthesis. To
determine the effect of diet on relative amylase synthesis,
acinar cells were isolated from rats fed HC or HF diets for
7 d; and de novo amylase synthesis was measured by the
incorporation of [3H]-phe into amylase and total protein.
Amylase relative synthesis was 192% higher (p<0.05) in
freshly isolated cells from rats fed HC diet, and remained
significantly higher (162%) through 48 h in culture, than HF
cells (Table 8). Although amylase relative synthesis
significantly decreased during culture in both HF and HC
cells (39% and 49%, respectively), the decrease was similar
in both groups such that the initial difference in relative
amylase synthesis was maintained throughout the culture
period (Figure 7). There was no independent effect of diet
on cellular protein (Table 8); however, there was a
significant (p<0.001) overall decrease in cellular protein
with time in culture. Cellular DNA was not affected by
77
TABLE 8: EFFECTS OF DIET ON RELATIVE AMYLASE SYNTHESIS, CELLULAR PROTEIN AND DNA IN CULTURED ACINAR CELLS.1
Time in Culture (ch Antecedent Diet HE HC
Amylase Relative Synthesis (%)2
0 (3) 11.7±0.6a (4)
24 6.0±1.0b (3) 9.8±0.9a (3)
48 3.7±1.2C (3) 6.0±0.8b (3)
Cellular Protein (mg/well)3 0 0.47±0.07 (3) 0.53±0.18 (4)
24 0.45±0.39 (3) 0.40±0.10 (3)
48 0.19±0.03 (2) 0.33±0.09 (3)
Cellular DNA (fig/well)4 0 7.4±1.5 (3) 7.1±0.6 (2)
24 6.811.3 (3) 7.4±1.1 (2)
48 6.3±1.5 (2) 6.0±0.7 (2)
•'•Acinar cells were isolated from rats fed HC (high carbohydrate) or HF (high fat) diets for 7 d and cultured (lxlO6 cells) in serum-free medium 48 h. Values represent the mean ± SEM for at least triplicate samples from the number of experiments indicated in parentheses.
2There was a significant (p<0.00001) independent effect of time on amylase synthesis: 0 h = 24 h > 48 h. There was also a significant (p<0.00001) independent effect of diet on amylase synthesis: HC > HF. There was no interactive effect of time and diet on amylase synthesis.
3There was no effect of diet on cellular protein. There was a significant (p<0.001) independent effect of time on cellular protein: D0=D1 > D2. There was no interactive effect of time and diet on cellular protein.
4 There were no significant effects of diet or time.
abcvaiues not sharing a superscript differed significantly (p<0.05) by ANOVA and LSD (198).
78
either time in culture or diet (Table 8).
The comparative effect of the HF and HC diets on
amylase activity (U/mg protein) and amylase relative
synthesis is presented in Table 9. With respect to relative
amylase synthesis, the ratio of HC/HF values remained
constant throughout 48 h in culture, while the ratio HC/HF
with respect to amylase activity increased with time in
culture.
The relationship between amylase relative synthesis and
cellular amylase activity was not linear (Figure 8). By
curve-fitting regression analysis, this relationship was
best described by an exponential function, y=0.88x2*2. The
correlation of this best fit exponential was strong
(r=0.86).
79
Time in Culture (day)
FIGURE 7: EFFECTS OF DIET ON AMYLASE RELATIVE SYNTHESIS IN CULTURED ACINAR CELLS.
Values represent the mean ± SEM for triplicate samples from at least 2 experiments. Values not sharing a superscript differed significantly (p<0.05) by ANOVA and LSD (198).
80
TABLE 9: COMPARATIVE EFFECT OF DIETS HIGH IN FAT AND CARBOHYDRATE ON AMYLASE ACTIVITY AND RELATIVE SYNTHESIS IN CULTURED ACINAR CELLS.1
Ratio HC/HF
Time in Culture (h) Activity Relative Synthesis
0 7.0 1.9
24 5.7 1.6
48 22.3 1.6
•'•Acinar cells were isolated from rats fed HC (high carbohydrate) or HF (high fat) diets for 7 d and cultured in serum-free medium 48 h as described in Tables 7 and 8.
81
500
>» C-400 »
> G> +* o S Q. 300
2 .2 y=0.88 X r=0.86
)D D)
« 5 200 «
c < p 1 0 0
Amylase Relative Synthesis (°/
FIGURE 8: REGRESSION ANALYSIS OF AMYLASE ACTIVITY VERSUS RELATIVE SYNTHESIS IN CULTURED ACINAR CELLS.
Cellular amylase activity (U/mg protein) was analyzed as a function of amylase relative synthesis (data from Table 6) by curve-fitting regression analysis (198).
DISCUSSION
82
These results confirm the adaptation of pancreatic
amylase to dietary carbohydrate. Feeding a HC diet
increased cellular amylase activity in freshly-isolated
pancreatic acinar cells 7-fold over feeding a HF diet.
Although this type of study has not previously been done in
isolated acinar cells, these results parallel the results of
other investigators (123,124,169) who observe increases from
3-5 fold in tissue amylase activity after feeding rats HC
diets. Antecedent diet also affected intracellular amylase
activity in cultured acinar cells. The difference in
amylase activity between cells from rats fed HC and HF diets
was maintained through 24 h in culture. After 48 h in
culture, cells from HC-fed rats tended to have a higher
cellular amylase activity than cells from HF-fed rats, but
this difference was not statistically significant. These
results demonstrate that antecedent diet affects cellular
amylase activity in freshly isolated rat acinar cells and
that these differences are maintained in vitro.
Despite differences in cellular amylase activity in the
acinar cells isolated from rats fed different antecedent
diets, amylase activity decreased significantly with time in
culture in both diet groups. In cells from HF-fed animals,
amylase activity decreased by 35% within 24 h and by 93%
within 48 h of culture. Similarly, amylase activity in
83
cells from HC-fed rats decreased by 46% within 24 h and by
77% within 48 h. This decrease in amylase content has been
a consistent observation in this particular acinar cell
culture. One interpretation of this large decrease in
amylase activity observed in cultured acinar cells is that
it results from de-differentiation of the acinar cell in
culture with a subsequent loss of exocrine function. An
alternative interpretation of the decrease in amylase
content in cultured cells, however, is that the turnover of
this enzyme changes in culture because of changes in the
rates of synthesis, degradation, secretion or all of these
that result from other factors.
A variety of evidence contradicts the interpretation
that these acinar cells do not remain differentiated in
culture and are not functional as pancreatic exocrine cells.
Brannon and coworkers (ISA) demonstrate that these cells
maintain the ultrastructural integrity of acinar cells
through 72 h in culture, as evidenced by the presence of ZG
and copious amounts of RER. In addition, these cells
respond to a pancreatic secretagogue, carbamyl choline, with
an increase in secretion of amylase by both freshly isolated
and cultured acinar cells. Finally, the results of the
present studies demonstrated that cultured acinar cells
synthesized de novo amylase protein through 48 h in culture.
Stimulated secretion and amylase synthesis are both
characteristics of the terminally differentiated pancreatic
84
acinar cell. Thus, considerable evidence argues against the
interpretation that the decrease in amylase during culture
results from de-differentiation of the cell.
The alternative interpretation suggests that culture
conditions result in alterations of the rate of amylase
synthesis, secretion or degradation compared to those rates
in vivo. The relative synthesis of amylase did decrease
with time in culture in the present studies; such a decrease
would contribute to lower cellular content of amylase.
Although stimulated secretion is maintained in cultured
cells, no data are available comparing the basal or non-
stimulated secretion rate of acinar cells in vivo and in
culture. It is possible that the basal secretion rate of
cultured acinar cells is increased because of a disruption
of gap junctions between these cells. Recently, Meda and
colleagues (199) and Bruzzone and coworkers (200)
demonstrate that, when gap junctions between pancreatic
acini are destroyed or decreased, the resulting blockage of
cell-to-cell communication leads to an increased basal
release of amylase. Both studies report that uncoupling
acinar gap junctions with an alkanol, heptanol, results in a
corresponding increase (2-3 fold) in the unstimulated
release of amylase, but has no effect on carbamyl choline-
stimulated amylase release. It is likely that isolation of
dispersed acinar cells as primarily single cells destroys
existing gap junctions, thus disrupting cell-to-cell
85
regulation. This disruption may result in increased rates
of basal enzyme secretion. An increased rate of basal
secretion coupled with the observed stable or decreased
rates of amylase synthesis would result in a rapid decrease
of amylase content, such as that observed. Amylase is
secreted continuously by cultured acinar cells as evidenced
by the increasing accumulation in the media (181). Further,
this secretion is basal, because the serum-free medium
contains no secretagogues. However, additional studies
addressing the effects of the acinar cell isolation and
culture on gap junctions and their functions are necessary
to evaluate the role of gap junctions and basal secretion
rates in the regulation of cellular amylase content.
Finally, no data are available on the rate of degradation of
amylase either in vivo or in cultured cells, so a role for
degradation in the regulation of amylase content in cultured
cells can not be proposed. Generally, degradation of
exocrine ZG proteins is not believed to occur; however,
amylase is the most slowly transported exocrine protein
(201) and may therefore be exposed to reticular or Golgi
proteases for longer than other secreted proteins. Future
studies should determine whether amylase is degraded in
cultured cells.
There was no effect of diet on total cellular protein,
even though cellular protein decreased with time in culture.
The effect of time on cellular protein in this acinar cell
86
culture is also documented by Brannon and coworkers (181).
While the decrease in cellular protein in this study was
statistically significant, the difference between freshly
isolated cells and cells cultured 48 h was not large (30%).
As discussed above, a change in the rate of basal secretion
even with the observed stable rates of total protein
synthesis (193) would lead to a lower content of protein in
the cultured cells. There was also an interactive effect of
time and diet on cellular protein, such that cellular
protein in cells cultured 48 h from rats fed HF diets was
greater than that in cells cultured 24 h from HF-fed rats
and 48 h from rats fed HC diets. This interactive effect of
diet on cellular protein can not be readily explained.
However, Brannon and coworkers (193) report greater total
protein synthesis in cells from rats fed HF diets when
compared to that in cells from rats fed HC diets. An
increased rate of protein synthesis in HF cells could
explain the observed interactive effect of diet and time on
cellular protein content if secretion rates were comparable.
In these studies, the effects of antecedent diet on
relative rates of amylase synthesis were also examined in
freshly isolated and cultured rat acinar cells, using an
affinity adsorbent procedure. The characteristics of the
affinity adsorbent for amylase demonstrated that it was
useful for purifying pancreatic amylase from isolated and
cultured cells because of its binding capacity and
87
specificity. The binding capacity of this affinity
adsorbent was examined over the range of amylase activity
applied in subsequent experiments. There was a
significantly higher percentage of amylase bound at the
lowest level of amylase activity applied. However, this
difference was small (6 U) and could have been the result of
the error that was inherent in measuring such low levels of
amylase activity. In addition, these experiments required
measuring amylase activity in the presence of 0.1% glycogen
in the elution buffer. Glycogen (at 0,1, 1.0 and 5.0%)
interfered with the amylase assay by competing with the
blue-starch substrate in the assay (data not shown). Thus,
the presence of 0.1% glycogen may have introduced another
source of error that was magnified at the lower
concentrations of amylase activity, even though this
glycogen concentration was controlled for by adding glycogen
at similar concentrations to the amylase assay standard
curve. This affinity adsorbent was also specific for
amylase as validated by SDS-PAGE. The major peak of
radioactive bound and dissociated protein comigrated with a
purified sample of amylase. There was, however, a small
peak of radioactivity (Rf=0.54) that migrated at a faster
rate than amylase. This component was of a smaller apparent
molecular weight than amylase. It was possible that this
was either a degraded product of amylase or a small amount
of contaminating protein that could not be completely
88
removed from the purified amylase sample. Whatever the
nature of this species, it comprised a small proportion
(<10%) of the total radioactivity recovered. Based on its
reproducible binding capacity and consistent specificity,
this affinity adsorbent was appropriate for purifying
amylase and was used in subsequent experiments examining the
effects of antecedent diet on cultured acinar cells.
A time-course study of the incorporation of [3H]-phe
into amylase protein and TCA-precipitable (total) protein
was done, in order to establish a period of linear
incorporation into protein. When synthetic rates of a
protein are measured by pulse-labelling as they were in
these studies, the results can be confounded by simultaneous
degradation of the protein. Under these conditions it would
be difficult to measure the incorporation rate of
radioactive label into de novo protein because degradation
may substantially affect the observed radioactivity in the
protein. In these experiments it was important to establish
a labelling time that was short relative to the half-life of
the protein. In the case of acinar cell amylase synthesis,
it was also important to have a labelling period that was
short enough to avoid the effects of secretion on cellular
amylase activity. Thus, a pulse-label period of linear
incorporation is a time period in which amylase and total
protein incorporation are increasing at linear rates,
implying that the effects of protein degradation and
89
secretion are small relative to the appearance of newly
labelled protein (202). In these experiments incorporation
of [3H]-phe into amylase and total protein was linear for
240 min. From these results, a 180 min incorporation period
was then used to examine amylase relative synthesis in
subsequent experiments.
The absolute incorporation of [3H]-phe into amylase and
total protein in cells from CU-fed rats decreased with time
in culture, while the relative rate of amylase synthesis
did not change through 24 h in culture. This observed
decrease in absolute [3H]-phe incorporation may have been
the result of a decrease in the specific activity of the
total intracellular phe pool, rather than the result of a
decreased protein synthetic rate. Brannon and coworkers
(193) report a 70% decrease in the specific activity of
intracellular phe in cells cultured 48 h. Brannon and Scott
(203) also report a decrease in intracellular phe specific
activity - a 50% decrease in cells cultured 24 h. In both
of these reports and in the current study cells were
isolated from rats fed CU diets. However, it is difficult
to compare the changes in specific activity (SA) in the two
studies by Brannon because the cells were cultured for
different lengths of time (48 h vs. 24 h) and from different
sub-strains of male Sprague-Dawley rats (one from University
of Arizona Department of Animal Resources and the other from
Harlan, Indianapolis, IN). Despite differences in the
90
magnitude of change in the SA of phe in these two studies,
both report decreases in the SA of phe with time in culture.
Specific activity of total intracellular phe was not
determined in the present studies; however, the cell culture
system used in the present studies is identical to that used
by Brannon and coworkers (193,203). Further, the sub
strain of rats used in the present studies is the same as
that used in the study by Brannon and Scott (203).
Therefore, the observed decrease of the absolute
incorporation of [3H]-phe into amylase and total protein in
the present study was similar to those previously reported
and most likely resulted from a decrease in the SA of
intracellular phe during culture in acinar cells.
Why the SA of intracellular phe changes in cultured
cells is not known. The SA of this precursor pool
represents the integration of several dynamic processes
including amino acid uptake, amino-acylation of tRNA
molecules, and turnover of amino-acylated tRNA molecules.
Any one or all of these rates could change in cultured
acinar cells resulting in the reported decrease in SA.
in freshly isolated and cultured rat acinar cells. Despite
a decrease with time, cellular amylase activity remained
higher in cells from HC-fed rats compared to cells from HF-
fed rats during the culture period. These differences in
cellular amylase activity appeared to be at least partly
determined by changes in amylase relative synthesis in
response to antecedent diet. Rates of relative amylase
synthesis were higher in cells from rats fed HC diets
compared to those in cells from rats fed HF diets.
Although relative amylase synthesis slightly decreased in
both diet groups during 48 h in culture, the effects of
antecedent diet were preserved throughout the entire culture
period. These studies clearly demonstrated persistent
94
dietary adaptation of amylase in the cultured pancreatic
acinar cell.
Future studies should consider the mechanism of this
adaptation. One component of this work should examine the
level of amylase regulation - is it at the level of mRNA
synthesis or mRNA stabilization or mRNA translation?
Additionally, the possible effectors of this regulation at
the level of the acinar cell need to be investigated. That
is, are the effectors of this adaptation hormones, dietary
metabolites or a combination of both? The culture system
used herein would be a useful system in which to pursue
these investigations because these cells maintain the
synthesis of amylase in culture and demonstrate its dietary
adaptation.
APPENDIX A
UNIVERSITY OF ARIZONA
Tucson, Arizona @5721
96
VERIFICATION OF APPROVAL OF ANIMAL CARE AND USE BY THE INSTITUTIONAL COMMITTEE
Title: Ii'.!Cnanisms of Dinlary A<iapUi;ion of i'.nicrcatic Fi.n;t;i.ion
Principal Investigator: lliyimon, IVtsy II.
DwHUrtniHiit* 111J L'f 1 Li Oil n i'OU'.l j (JHLO Department:
Submission Date: ^G'>* '-'J> ^
Agency:
The University of Arizona University Leboratory Animal Care Committee reviews all sections of proposals to PHS which concern animal cere and use. The above proposal has:
»r v (•" ) Been reviewed and approved by ULACC and verification of review
iB attached. i-oiiunnaUon - ilu ciinngu in r.ninmi r.iutiiorlolot ilu ciinngu in r.nimal r.ifit-IiorioIo«.jv
] Been reviewed and approval withheld. Verification of review attached.
t ) Will be reviewed within the next 60 days and verification of review will be submitted.
Laurel L. Wilkening Vice President for Research
Date: April 13, 1907
Aasurence of Compliance Pending. Submitted for Approval to DHHS S2/30/B5.
97
REFERENCES
1. Williams, J.A. & Goldfine, I.D. (1986) The insulin-acinar relationship. In: The Exocrine Pancreas (Go, V.L.W., Gardner, J.D., Brooks, F.P., Lebenthal, E., DiMagno, E.P. & Scheele, G., eds.)/ P» 347, Raven Press, New York.
2. Boguist, P. (1981) The endocrine cells. In: The Pancreas (Keynes, W.M. & Keith, R.G., eds.), p.39, Appleton-Century-Crofts, New York.
3. Korc, M., Iwamoto, Y., Sankaran, H., Williams, J.A. & Goldfine, I.D. (1981) Insulin action in pancreatic acini from streptozotocin-treated rats. I. Stimulation of protein synthesis. Am. J. Phvsiol. 240: G56-G62.
4. Williams, J.A., Bailey, A.C., Preissler, M. 6 Goldfine, I.D. (1982) Insulin regulation of sugar transport in isolated pancreatic acini from diabetic mice. Diabetes 31: 674-682.
5. Lahaie, R.G. (1984) Translational control of protein synthesis in isolated rat pancreatic acini. Gastroenterology 86: 1149.
6. Kanno, T. & Saito, A. (1976) The potentiating influence of insulin on pancreozymin-induced hyperpolarization of amylase release in the pancreatic acinar cell. J. Phvsiol.(London) 261: 505-521.
7. Saito, A., William's, J.A. & Kanno, T. (1980) Potentiation of cholecystokinin-induced exocrine secretion by both exogenous and endogenous insulin in isolated and perfused rat pancreata. J. Clin. Invest. 65: 777-782.
8. Otsuki, M. & Williams, J.A. (1983) Direct modulation of pancreatic CCK receptors and enzyme secretion by insulin in isolated pancreatic acini from diabetic rats. Diabetes 32: 241-246.
9. Farese, R.V., Larson, R.E. & Subir, M. A. (1981) Insulin and its secretagogues activate Ca++ dependent phosphatidyl inositol breakdown and amylase secretion in rat pancreas in vitro. Diabetes 30: 396-401.
10. Dyck, W.P., Rudic, J., Hoexter, B. & Janowitz, H.D. (1969) Influence of glucagon on pancreatic exocrine secretion. Gastroenterology 56: 531-537.
14. Mortimer, C.H., Carr, D., Lind, T., Bloom, S.R., Mallinson, C.N., Schally, A.V., Tunbridge, W.M.G., Yeomans, L., Coy, D.H., Kastin, A., Besser, G.M. & Hall, R. (1974) Effects of growth hormone release-inhibiting hormone on circulation glucagon, insulin and growth hormone in normal, diabetic, acromegalic and hypopituitary patients. Lancet 1: 697-701.
15. Efendic, S., Claro, A, & Luft, R. (1976) Studies on the mechanism of somatostatin on insulin release. Acta Endocrinology 81: 753-761.
16. Wilson, P.M., Boden, G. & Owen, D.E. (1978) Pancreatic polypeptide responses to a meal and to intra duodenal amino acids and sodium oleate. Endocrinology 102: 859-863.
17. Hellman, B., Wallgreen, A. & Peterson, B. (1962) Cytological characteristics of the exocrine pancreatic cells with regard to their position in relation to the islets of Langerhans. Acta Endocrinology 465-473.
18. Bendayan, M. & Ito, S. (1979) Immunohistochemical localization of exocrine enzymes in normal rat pancreas. J. Histochem. Cvtochem. 27: 1029-1034.
19. Desnuelle, P. & Figarella, C. (1979) Biochemistry In: The Exocrine Pancreas (Howat. H.T. & Sarles, H., eds.), p. 87, W.B. Saunders Co., Ltd., Philadelphia
20. Blobel, G. & Dobberstein, B. (1975) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane bound ribosomes of murine myeloma. J. Cell Biol. 67: 835-851.
21. Blobel, G. & Dobberstein, B. (1975) Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous compounds. J. cell Biol. 67: 852-862.
99
22. Jackson, R.C. & Blobel, G. (1977) Post-translational cleavage of pre-secretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc. Nat. Acad. Sci. USA 74: 5598-5602.
23. Caro, L.G< & Palade, G.E. (1964) Protein synthesis storage and discharge in the pancreatic exocrine cell. J. Cell Biol. 20: 473-495.
24. Jamieson, J.D. & Palade, G.E. (1967) Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J. Cell Biol. 34: 577-596.
25. Jamieson, J.D. & Palade, G.E. (1967) Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J. Cell Biol. 34: 597-615.
26. Jamieson, J.D. & Palade, G.E. (1968) Intracellular transport of secretory proteins in the pancreatic exocrine cell. III. Dissociation of intracellular transport from protein synthesis. J. Cell Biol. 39: 580-588.
27. Jamieson, J.D. & Palade, G.E. (1968) Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J. Cell Biol. 39: 589-603.
28. Reggio, H.A. & Palade, G.E. (1978) Sulfated compounds in the zymogen granules of the guinea pig pancreas. J. Cell Biol. 77: 288-314.
29. Hand, A.R. & Oliver, C. (1977) Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells. Histochem. J. 9: 375-392.
30. Novikoff, A.B., Mori, M., Quintana, N. & Yam, A. (1977) Studies of the secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles. J. Cell Biol. 75: 148-165.
31. Rothman, S.S. (1975) Protein transport by the pancreas. The current paradigm is analyzed and an alternative hypothesis is proposed. Science 190: 747-753.
32. Palade, G. (1975) Intracellular aspects of the process of protein secretion. Science 184: 347-358.
100
33. DeCamilli, P., Peluchetti, D. & Meldolesi, J. (1974) Structural difference between lumenal and lateral plasmalemma in the pancreatic acinar cells. Nature 248: 245-246.
34. Jamieson, J.D. & Palade, G.E. (1971) Synthesis, intracellular transport and discharge of secretory proteins in stimulated pancreatic exocrine cells. J. Cell Biol. 50: 135-158.
35. Rothman, S.S. & Isenman, L.D. (1974) Secretion of digestive enzymes derived from two parallel intracellular pools. Am. J. Phvsiol. 226: 1082-1087.
36. Robberecht, P., Cremer, M. & Christophe, J. (1977) Discharge of newly synthesized proteins in pure juice collected from the human pancreas. Gastroenterology 72: 417-420.
37. Roberge, M. & Beaudoin, A.R. (1982) Newly synthesized secretory proteins from pig pancreas are not released from a homogeneous granule compartment. Biochim. Biophvs. Acta 716: 331-336.
38. Dean, P.M. & Matthews, E.K. (1972) Pancreatic acinar cells: Measurement of membrane potential and miniature depolarization potentials. J. Phvsiol. 225: 1-13.
39. Hoist, J.J., Fahrenkrug, J., Jensen, S.L., Lundberg, J. & Nielson, O.V. (1984) PHI/VIP in the pig pancreas: cotranslation, coexistence and co-secretion and co-operative effects. Dig. Pis. Sci. 29 (suppl.): 37S.
40. Gardner, J. D. & Jensen, R. T. (1981) Regulation of pancreatic exocrine secretion in vitro: The action of secretagogues. Phil. Trans. R. Soc. Lond. B. 296: 17-26.
41. Gardner, J.D. & Jensen, R.T. (1983) Gastrointestinal peptides: The basis of action at the cellular level. Rec. Prog. Horm. Res. 39: 211-244.
42. Hoist, J.J., Knuhtsen, S., Jensen, S.L., Nielsen, O.V. (1983) Role of gastrin releasing peptide (mammalian bombesin) nerves in pancreatic endocrine and exocrine secretion. Digestion 28: 35.
43. Vaysse, N., Bastic, M.J., Pascal, J.P., Roux, P., Martinick, C., Lacroix, A. & Riber, A. (1975) Role of cholinergic mechanisms in the response to secretin of isolated canine pancreas. Gastroenterology 69: 1269-1277.
101
44. Schultz, I. (1980) Messenger role of calcium in function of pancreatic acinar cells. Am. J. Phvslol. 239: G335-G347.
45. Williams, J.A. (1980) Regulation of pancreatic acinar cell function by intracellular calcium. Am. J. Phvsiol. 238: G269-G279.
46. Williams, J.A. (1984) Regulatory mechanisms in pancreas and salivary acini. Annu. Rev. Phvsiol. 46: 361-375.
47. Moghimzadeh, E., Ekman, R., Hakanson, R., Yanaihara, N. & Sundler, F. (1983) Neuronal gastrin-releasing peptide in the mammalian gut and pancreas. Neuroscience 10: 553-563.
48. Larsson, L.I. (1979) Innervation of the pancreas by substance P, enkephalin, vasoactive intestinal peptide, and gastrin/CCK immunoreactive nerves. J. Histochem. Cvtochem. 27: 1283-1284.
49. Bishop, A.E., Polak, J.M., Green, I.C., Bryant, M.G. & Bloom, S.R. (1980) The location of VIP in the pancreas of man and rat. Diabetoloaie 18: 73-78.
50. Larsson, I., Fahrenkrug, J., Hoist, J.J. & Schaffalitzky de Muckadell, O.B. (1978) Innervation of the pancreas by vasoactive intestinal peptide (VIP) immunoreactive nerves. Life Sci. 22: 773-780.
51. Sundler, F., Alumets, J., Hakanson, R., Fahrenkrug, J. & Schaffalitzky de Muckadell, O.B. (1978) Peptidergic (VIP) nerves in the pancreas. Histochemistry 55: 173-176.
52. Dooley, C.P. & Valenzuela,. J.E. (1984) Duodenal volume and osmoreceptors in the stimulation of human pancreatic secretion. Gastroenterology 86: 23-27.
53. Robberecht, P., Deschodt-Lanckman, M., Lammens, M., DeNeef, P. & Christophe, J. (1977) Iq vitro effects of secretin and vasoactive intestinal peptide on hydrolase secretion and cyclic AMP levels in the pancreas of five animal species. A comparison with caerulein. Gastroenterol. Clin. Biol. 1: 519-529.
54. Folsch, U.R., Fischer, H., Soling, H.D. & Creutzfeldt, W. (1980) Effects of gastrointestinal hormones and carbamyl-choline on cAMP accumulation in isolated pancreatic duct fragments from the rat. Digestion 20: 277-292.
55. Chey, W.Y. & Konturek, S.J. (1982) Plasma secretin and pancreatic secretion in response to liver extract meals with
102
varied pH and exogenous secretin in the dog. J. Phvsiol. (London! 324: 263-272.
56. Rominger, J.M., Chey, W.Y. & Chang, T.M. (1981) Plasma secretin concentration and gastric pH in healthy subjects and patients with digestive diseases. Dig. Pis. Sci. 26: 591-597.
57. Chey, W.Y., Kim, M.S. & Lee, K.Y. (1979) Influence of the vagus nerve on the release and action of secretin in dog. J. Phvsiol. (London) 293: 435-446.
58. Schaffalitzky de Muckadell, O.B., Fahrenkrug, J., Watt-Boolsen, S. & Woraing, H. (1978) Pancreatic response and plasma secretin concentration during infusion of low dose secretin in man. Scand. J. Gastroenterol. 13: 305-311.
59. You, C.H., Rominger, J.M. & Chey, N.Y. (1983) Potentiation effect of cholecystokinin-octapeptide on pancreatic bicarbonate secretion stimulated by a physiological dose of secretin in humans. Gastroenterology 85: 40-45.
60. Chey, W.Y., Kim, M.S., Lee, K.Y. & Chang, T.M. (1979) Effect of rabbit antisecretin serum on postprandial pancreatic secretion in dogs. Gastroenterologv 77: 1268-1275.
61. Schaffalitzky de Muckadell, O.B., Fahrenkrug, J. & Rune, J. (1979) Physiological significance of secretin in the pancreatic bicarbonate response. I. Responsiveness of the secretin-releasing system in the upper duodenum. Scand. J. Gastroenterol. 14: 79-83.
62. Kim, M.S., Lee, K.Y., and Chey, W.Y. (1979) Plasma secretin concentrations in fasting and postprandial states in dog. Am. J. Phvsiol. 236: E539-E544.
63. Folsch, U.R. & Wormsley, K.G. (1973) Pancreatic enzyme response to secretin and cholecystokinin-pancreozymin in the rat. J. Phvsiol. (London! 234: 79-94.
64. Granger, D.N., Barrowman, J.A. & Kvietys, P.R. (1985) The pancreas In: Clinical Gastrointestinal Physiology, pp. 97-115, H.B.Saunders Co., Philidelphia.
66. Erspamer, V., Improta, v . , Melchiorri, P., et al. (1977) Evidence of cholecystokinin release by bombesin in the dog. Br. J. Pharmacol. 52: 227-232.
67. Gardner, J.D. (1979) Regulation of pancreatic exocrine function in vitro: Initial steps in the action of secretagogues. Annu. Rev. Phvsiol. 41: 55-66.
69. Gregory, R.A. & Tracy, H.J. (1964) Constitution and properties of two gastrins extracted from the hog antral mucosa. Gut 5: 103-117.
70. Baca, I., Feule, G.E., Hass, N. & Mernitz, T. (1983) Interaction of neurotensin, cholecystokinin, and secretin in the stimulation of the exocrine pancreas in the dog. Gastroenterology 84: 556-561.
71. Konturek, S.J. & Tasler, V. (1974) Characteristics of inhibition of pancreatic secretion by glucagon. Digestion 10:138-149.
72. Shaw, H.M. & Heath T.J. (1973) The effect of glucagon on the formation of pancreatic juice and bile in the rat. Can. J. Phvsiol. Pharmacol. 51: 1-5.
73. Konturek, S.J., Tasler, J., Obtulowicz, W., Coy, D.H. & Schally, A.V. (1976) Effect of growth hormone-releasing inhibiting hormones on hormones stimulating exocrine pancreatic secretion. J. Clin. Invest. 58: 1-6.
74. Douglas, W.W. (1968) Stimulus-secretion coupling. The concept and clues from chromaffin and other cells. Br. J. Pharmacol. 34: 451-474.
75. Halenda, S.P. & Rubin, R.P. (1982) Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phosholipase C. Biochem J. 208: 713-721.
76. Orchard, J.L., Davis, J.S., Larson, R.E. & Farese, R.V. (1984) Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro. Biochem. J. 217: 281-287.
on [32P] phosphoinositol-4,5-biphosphate metabolism in the exocrine pancreas. Biochem. J. 212: 483-488.
78. Broeckman, M.J. (1984) Phosphatidylinositol-4,5-biphosphate may represent the site of release of plasma membrane bound calcium upon stimulation of human platelets. Biochem. Biophvs. Res. Comm. 120: 226-231.
79. Ohsaka, S. & Deguchi, T. (1983) Phosphatidic acid mimics the muscarinic action of acetylcholine in cultured bovine chromaffin cells. Eur. J. Biochem. 152: 62-66.
80. Nishizuka, Y. (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308: 693-698.
81. Long, B.W. & Gardner, J.D. (1977) Effects of cholecystokinin on adenylate cyclase activity in dispersed pancreatic acinar cells. Gastroenterology 73: 1008-1014.
82. Burham, D.B. & Willians, J.A. (1984) Activation of protein kinase activity in pancteatic acini by calcium and cyclic AMP. Am. J. Phvsiol. 246: G500-G508.
83. Scheele, G.A. (1980) Biosynthesis, segregation and secretion of exportable proteins by the exocrine pancreas. Am. J. Phvsiol. 238: G467-G477.
84. Rinderknecht, H., Renner, I.G., Douglas, A.P. & Adham, A.F. (1978) Profiles of pure pancreatic secretions obtained by direct pancreatic duct cannulation in normal healthy human subjects. Gastroenterology 75: 1083-1089.
85. Grendell, J.H. (1985) Nonparallel secretion of digestive enzymes by the pancreas-implications for models of protein secretion. In: Nonvesicular Transport (Rothman, S.S. & Ho, J.J.L. eds.) pp. 347-57, John Wiley and Sons, New York
86. Goldberig, D.M., Sale, J.K. & Worms ley, K.G. (1973) Ratio of chymotrypsin to trypsin in human duodenal aspirate. Digestion 8: 101-109.
87. Dagorn, J.C. (1978) Nonparallel enzyme secretion from rat pancreas: in vivo studies. J. Phvsiol. 280: 435-448.
88. Steer, M.L. & Manabe, T. (1979) Choleycystokinin-pancreozymin induces the parallel discharge of digestive enzymes from the in vitro rabbit pancreas. J. Biol. Chem. 254: 7228-7229.
105
89. Scheele, 6.H. & Palade, G.E. (1975) Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. J. Biol. Chem. 250: 2660-2670.
90. Gumbiner, B. & Kelly, R.B. (1982) Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell 28: 51-59.
91. Mainz, D.L., Black, 0. & Webster P.D. (1973) Hormonal control of pancreatic growth. J. Clin. Invest. 52: 2300-2304.
92. Renaud, W., Giorgi, 0., Iovanna, J. & Dagorn, J.C. (1986) Regulation of concentration of mRNA for amylase, trypsinogen I and chymotrypsinogen B in rat pancrease by secretagogues. Biochem. J. 235: 305-308.
9 3 . Peakall, D.B. (1967) Incorporation of 14C-orotic acid and 14C-amino acid into pigeon pancreas slices following cholinergic stimulation. Proc. Soc. EXP. Biol. Med. 126: 198-201.
94. Dagorn, J.C. & Mongeau, R. (1977) Different action of hormonal stimulation on the biosynthesis of three pancreatic enzymes. Biochim. Biophvs. Acta 498: 76-82.
95. Mongeau, R., Dagorn, J.C. & Morisset, J. (1976) Further evidence that protein synthesis can be decreased in vivo following hormonal stimulation in the rat pancreas. Can. J. Phvsiol. Pharmacol. 54: 305-313.
96. Reggio, H. & Cailla, H.L. (1974) Effect of actinomycin D, pancreozymin and secretin on RNA synthesis and protein synthesis measured in vivo in rat pancreas. Biochim. Biophvs. Acta 338: 37-42.
97. Leroy, J., Morisset, J. & Webster, P.D. (1971) Dose-related response of pancreatic synthesis and secretion to cholecystokinin pancreozymin. J. Lab. Clin. Med. 78: 149-157.
98. Mongeau, R., Couture, Y., Dunnigan, J. & Morisset, J. (1974) Early dissociation of protein synthesis and amylase secretion following hormonal stimulation of the pancreas. Can. J. Phvsiol. Pharmacol. 52: 198-205.
99. Wicker, C.A., Puigserver, A. & Scheele, G. (1984) Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas. Eur. J. Biochem. 139: 381-387.
106
100. Schick, J., Kern, H. & Scheele, G. (1984) Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis. J. Cell Biol. 99: 1569-1574.
101. Rothman, S.S. & Wells, H. (1967) Enhancement of pancreatic enzyme synthesis by pancreozymin. Am. J. Phvsiol. 213: 215-218.
102. Folsch, U.R., Winckler, K. & Wormsley, K.G. (1978) Influence of repeated administration of cholecystokinin and secretin on the pancreas of the rat. Scand. J. Gastroenterol. 13: 663-671.
103. Solomon, T.E., Peterson, H., Elashoff, J. & Grossman, M.I. (1978) Interaction of caerulein and secretin on pancreatic size and composition in the rat. Am. J. Phvsiol. 235:E714—E719.
104. Solomon, T.E., Peterson, H., Elashoff, J. & Grossman, M.I. (1979) Effects of chemical messenger peptides on pancreatic growth in rats. In: Gut Peptides (Miyoshi, A., ed.), pp.213-219, Kodansha, Tokyo.
105. Rausch, U., Vasiloudes, P., Rudiger, K. & Kern, H.F. (1985) In vivo stimulation of rat pancreatic acinar cells by infusion of secretin. I. Changes in enzyme content, pancreatic fine structure and total rate of protein synthesis. Cell and Tissue Research 242:633-639.
106. Rausch, U., Vasiloudes, P., Rudiger, K. & Kern, H.F. (1985) In vivo stimulation of rat pancreatic acinar cells by infusion of secretin. II. Changes in individual rates of enzyme and isoenzyme biosynthesis. Cell and Tissue Research 242:641-644.
107. Burham, D.B. & Williams, J.A. (1982) Effects of carbachol, cholecystokinin and insulin on protein phosphorylation in isolated pancreatic acini. J. Biol. Chem. 257:10523-10528.
108. Mossner, J., Logsdon, C.D., Williams, J.A. & Goldfine, I.D. (1985) Insulin, via its own receptor, regulates growth and amylase synthesis in pancreatic acinar AR42J cells. Diabetes 34:891-897.
109. Morisset, J.A. & Webster, P.D. (1972) Effects of fasting and feeding on protein synthesis by the rat pancreas. J. Clin. Invest. 51:1-8.
107
110. Webster, P.D., Singh, M., Tucker, P.C. & Black, O. (1972) Effect of fasting and feeding on the pancreas. Gastroenterology 62:600-605.
111. Meldolesi, J. (1970) Effect of caerulein on protein synthesis and secretion in the guinea pig pancreas. Br. J. Pharmacol. 40:721-731.
112. Viera-Matos, A.N. & Tenenhouse, A. (1977) The effect of fasting on the in vitro synthesis of amylase in rat exocrine pancreas. Can. J. Phvsiol. Pharmacol. 55:90-97.
113. Black, O. & Webster, P.D. (1974) Nutritional and hormonal effects on RNA polymerase enzyme activities in pancreas. Am. J. Phvsiol. 227:1276-1280.
114. Webster, P.D. & Tvor, M.P. (1967) Effects of fasting and feeding on uridine-^H incorporation into RNA by pancreas slices. Am. J. Phvsiol. 212:203-206.
115. Webster P.D. (1969) Effect of stimulation on pancreatic amylase secretion and nuclear RNA synthesis. Proc. Soc. EXP. Biol. Med. 132:1072-1076.
116. Morisset, J.A., Black, 0. & Webster, P.D. (1972) Effects of fasting, feeding and bethanecol chloride on pancreatic microsomal protein synthesis in vitro. Proc. Soc. EXP. Biol. Med. 140:1308-1314.
117. Black, O. & Webster, P.D. (1973) Protein synthesis in pancreas of fasted pigeons. J. Cell Biol. 57:1-8.
118. Pavlov, J.P. (1902) The Work of the Digestive Gland. Charles Griffin & Co., Ltd, London.
119. Corring, T. (1977) Possible role of hydrolysis porducts of the dietary components in the mechanisms of the exocrine pancreatic adaptation to the diet. Wld Rev. Nutr. Diet. 27:132-144.
120. Grossman, M.I., Greengard, H. & Ivy, A.C. (1943) The effect of dietary composition on pancreatic enzymes. Am. J. Phvsiol. 138:676-682.
121. Ben Abdeljlil, A. & Desnuelle, P. (1964) Sur 1'adaptation des enzymes exocrines du pancreas a la composition du regime. Biochim. Biophvs. Acta 81:136-149.
122. Robberecht, P. Deschodt-Lanckman, M., Camus, J., Bruylands, J. & Christophe, J. (1971) Rat pancreatic
108
hydrolases from birth to weaning and dietary adaptation after weaning. Am. J. Phvsiol. 221:376-381.
123. Snook, J.T. (1971) Dietary regulation of pancreatic enzymes in the rat with emphasis on carbohydrate. Am. J. Phvsiol. 221:1383-1387.
124. Reboud, J.P., Marchis-Mouren, G., Pasero, L., Cozzone, A. & Desnuelle, P. (1966) Adaptation de la Vitesse de biosynthese de 1'amylase pancreatique et du chymotrypsinogene a des regimes riches en amidase ou en proteines. Biochim. Biophvs. Acta 117:351-367.
125. Johnson, A., Hurwitz, R. & Kretchmer, N. (1977) Adaptation of rat pancreatic amylase and chymotrypsinogen to changes in diet. J. Nutr. 107:87-96.
126. Poort, S.R. & Poort, C. (1980) Effect of diet composition on the protein synthetic pattern of the rat pancreas after a feeding period of five days. Biochim. Biophvs. Acta 606:138-147.
127. Wicker, C. & Puigserver, A. (1987) Effects of inverse changes in dietary lipid and carbohydrate on the synthesis of some pancreatic secretory proteins. Eur. J. Biochem. 162:25-30.
128. Lahaie, R.G. & Dagorn, J.C. (1981) Dietary regulation of pancreatic protein synthesis. II. Kinetics of adaptation of protein synthesis and its effect on enzyme content. Biochim. Biophvs. Acta 654:119-123.
129. Dagorn, J.C. & Lahaie, R.G. (1981) Dietary regulation of pancreatic protein synthesis. I. Rapid and specific modulation of enzyme synthesis by changes in dietary composition. Biochim. Biophvs. Acta 654:111-118.
130. Giorgi, D., Renaud, W., Bernard, J.P. & Dagorn, J.C. (1985) Regulation of proteolytic enzyme activities and mRNA concentrations in rat pancreas by food content. Biochem. Biophvs. Res. Comm. 127:937-942.
131. Brants, F. & Morisset, J. (1976) Trophic effect of cholecystokinin-pancreozymin on pancreatic acinar cells from rats of different ages. Proc. Soc. EXP. Biol. Med. 153:523-527.
132. Grossman, M.I., Greengard, H. & Ivy, A.C.(1944) On the mechanism of the adaptation of pancreatic enzymes to dietary composition. Am. J. Phvsiol. 141:38-41.
109
133. Green, G.M., Olds, B.A. Matthews, G. & Lyman, R.L. (1973) Protein, as a regulator of pancreatic enzyme secretion in the rat. Proc. Soc. EXP. Biol. Med. 142:1162-1167.
134. Dagorn, J.C. (1986) Mechanism of pancreatic adaptation to diet. Biochimie 68:329-331.
135. Gidez, L.I. (1973) Effect of dietary fat on pancreatic lipase levels in the rat. J. Lipid Research 14:169-177.
136. Bazin, R. & Lavau, M. (1979) Diet composition and insulin effect on amylase to lipase ratio of diabetic rats. Digestion 19:386-391.
137. Deschodt-Lanckman, M., Robberecht, P., Camus, J. & Christophe, J. (1971) Short-term adaptation of pancreatic hydrolases to nutritional and physiological stimuli in adult rats. Biochimie 53:789-796.
138. Christophe, J., Camus, J. Deschodt-Lanckman, M., Rothe, J., Robberecht, P., Vandermeers-Piret, M.C. & Vandermeers, A. (1971) Factors regulation biosynthesis, intracellular transport and secretion of amylase and lipase in the rat exocrine pancreas. Horm. Metab. Res. 3:393-403. 139. Saraux, B., Girard-Globa, A., Ouagued, M. & Vacher, D. (1982) Response of the exocrine pancreas to quantitative and qualitative variations in dietary lipids. Am. J. Physiol. 243:G10-G15.
140. Sabb, J.E., Godfrey, P.M. & Brannon, P.M. (1986) Adaptive response of rat pancreatic lipase to dietary fat: Effects of amount and type of fat. J. Nutr. 116:892.
141. Bazin, R. & Lavau, M. (1978) Pancreatic lipase and ketogenic conditions. Biomedicine 28:160-165.
142. Cozzone, P., Pasero, L., Beaupoil, B. & Marchis-Mouren, G. (1970) Characterization of porcine pancreatic isoamylases. Chemical and physical studies. Biochim. Biophvs. Acta 207: 490-504.
143. Pommier, G., Cozzone, P. & Marchis-Mouren, G. (1974) The sulfhydryl groups of porcine pancreatic ot-amylase: Unmasking reactivity and function. Biochim. Biophvs. Acta 350:71-83.
144. Granger, M., Abadie, B., Mazzei, Y. & Marchis-Mouren, G. (1975) Enzymatic activity of TNB blocked porcine pancreatic amylase. Eur. J. Biochem. 50:276-285.
110
145. Steifel, D.J. & Keller, P.J. (1973) Preparation and some properties or human pancreatic amylase including a comparison with human parotid amylase. Biochim. Biophvs. Acta 302:345-361.
146. Fischer, E.H. & Stein, E.A. (1960) a-Amylases. In: The Enzvmes (Boyer, P.D., Lordy, H. & Hyrback, K., eds.), pp.313-343, Academic Press, Hew York.
147. Hsiu, J., Fischer, E.H. & Stein, E.A. (1964) Alpha-amylases as calcium-metalloenzymes. II. Calcium and the catalytic activity. Biochemistry 3:61-66.
148. Michaelis, L. & Pechstein, H. (1914) Die wirkungsbedingungen der speicheldiastase. Biochem. Z. 59:77-99.
149. Thomas, J.A., Spradline, J.E. & Dygert, S. (1971) Plant and animal amylases. In: The Enzvmes (Boyer, P.D., ed.), pp.115-119, Academic Press, New York.
150. Phillips, D.C. (1966) The three dimensional structure of an enzyme molecule. Scientific American 215:78-90.
151. Loyter, A. & Schramm, M. (1962) The glycogen-amylase complex as a means of obtaining highly purified a-amylases. Biochim. Biophvs. Acta 65:200-206.
152. Vandermeers, A. & Christophe, J. (1968) a-Amylase et lipase du pancreas de rat purification chromatographique, recherche du poids moleculaire et composition en acides amines. Biochim. Biophvs. Acta 154:110-129.
153. Buonocore, V. & Poerio, E. (1975) Affinity column purification of amylases on protein inhibitors from wheat kernel. J. Chromatography 114:109-114.
154. Ettalibi, M., Ben Abdeljlil, A. & Marchis-Mouren, G. (1975) Purification and characterization of ovine pancreatic a-amylase. Biochimie 57:995-999.
155. Takeuchi, T. (1979) Human amylase isoenzymes separated on Concanavalin A-Sepharose. Clin. Chem. 25:1406-1410.
156. Burrill, P.H., Brannon, P.M. & Kretchmer, N. (1981) A single-step purification of rat pancreatic and salivary amylase by affinity chromatography. Analytical Biochemistry 117:402-405.
Ill
157. Schmidt, D.D., Frommer, W., Zunge, B., Muller, L., Wingender, W., Truschert, E. & Schafer, D. (1977) a-Glucosidase inhibitors. Naturwissenschaften 64:535-536.
158. Bergami, M. & Cacace, M. (1978) Direct coupling of reducing oligosaccharides to aminohexyl-sepharose: Purification of a,a-trehalase from Artemia salina. In: Affinity Chromatography (Hoffman-Ostenhof, O., Breitanbach, M., Roller, F., Kraft, D. & Scheiner, 0., eds.), pp.111-114, Pergamon Press, New York.
159. Wicker, C., Puigserver, A., Rausch, U., Scheele, G. & Kern, H. (1985) Multiple-level caerulein control of the gene expression of secretory proteins in the rat pancreas. Eur. J. Biochem. 151:461-466.
160. Logsdon, C.D., Mossner, J., Williams, J.A. & Goldfine, I.D. (1985) Glucocorticoids increase amylase mRNA levels, secretory organelles & secretion in pancreatic acinar AR42J cells. J. Cell Biology 100:1200-1208.
161. Ben Abdeljlil, A., Palla, J.C. & Desnuelle, P. (1965) Effect of insulin on pancreatic amylase and chymotrypsinogen. Biochem. Biophvs. Res. Comm. 18:71-75.
162. Soling, H.D. & Unger, K.O. (1972) The role of insulin in the regulation of a-amylase synthesis in the rat pancreas. Eur. J. Clin. Invest. 2:199-212.
163. Korc, M., Owerbach, D., Quinto, C. & Rutter, W.J. (1981) Pancreatic islet-acinar cell interaction: Amylase messenger RNA levels are determined by insulin. Science 213:351-353.
164. Morisset, J. & Dunnigan, J. (1971) Effects of glucose, amino acids, and insulin on adaptation of exocrine pancreas to diet. Proc. Soc. EXP. Biol. Med. 136:231-234.
165. Palla, J.C., Ben Abdeljlil, A. & Desnuelle, P. (1968) Action de l'insuline sur la biosynthese de 1'amylase et de quelques autres enzymes du pancreas de rat. Biochim. Biophvs. Acta 158:25-35.
166. Malaisse-Lagae, F., Ravazzola, M., Robberecht, P., Vandermeers, A., Malaisse, W.J. & Orci, L. (1976) Hydrolases content in peri-insular and tele-insular exocrine pancreas. In: Endocrine Gut and Pancreas (Fujita, T., ed.), pp.313-320, Elsevier, Amsterdam.
112
167. Korc, M., Sankaran, H., Wong, K.Y., Williams, J.A. & Goldfine, I.D. (1978) Insulin receptors in isolated mouse pancreatic acini. Biochem. Biophvs. Res- comm. 84:293-299.
168. Bazin, R. & Lavau, M. (1982) Effects of high-fat diet on glucose metabolism in isolated pancreatic acini of rats. Am. J. Phvsiol„ 243:G448-G454.
169. Reboud, J.P., Ben Abdeljlil, A. & Desnuelle, P. (1962) Variations de la teneur en enzymes du pancreas de rat en fonction de la composition des regimes. Biochim. Biophvs. Acta 58:326-337.
170. Ben Abdeljlil, A., Visani, A.M. & Desnuelle, P. (1963) Adaptation of the exocrine secretion of rat pancreas to the composition of the diet. Biochem. Biophvs. Res. Comm. 2:112-116.
171. Ben Abdeljlil, A. & Desnuelle, P. (1964) Sur 1'adaptation des enzymes exocrines du pancreas a la composition du regimes. Biochim. Biophvs. Acta 81:136-149.
172. Marchis-Mouren, G., Pasero, L. & Desnuelle, P. (1963) Further studies on amylase biosynthesis by pancreas of rats fed on a starch-rich or a casein-rich diet. Biochem. Biophvs. Res. Comm. 13:262-266.
173. Poort, S.R. & Poort, C. (1981) Effect of feeding diets of different composition on the protein synthetic pattern of the rat pancreas. J. Nutr. 111:1475-1479.
174. Schick, J., Verspohl, R., Kern, H. & Scheele, G. (1984) Two distinct adaptive responses in the synthesis of exocrine pancreatic enzymes to inverse changes in protein and carbohydrate in the diet. Am. J. Phvsiol. 247:G611-G616.
175. Giorgi, D., Bernard, J.P., LaPointe, R. & Dagorn, J.C. (1984) Regulation of amylase messenger RNA concentration in rat pancreas by food content. The EMBO Journal 3:1521-1524.
176. Desnuelle, P., Reboud, J.P. & Ben Abdeljlil, A. (1962) Influence of the composition of the diet on the enzyme content of rat pancreas. In: Ciba Foundation Symposium on the Exocrine Pancreas (de Reuck, A.V.S. & Cameron, M.P., eds.), pp.90-114, Little, Brown, Boston.
177. Howard, F. & Yudkin, J. (1963) Effect of dietary change upon the amylase and trypsin activities of the rat pancreas. Br. J. Nutr. 17:281-294.
113
178. Bucko, A., Simko, V. & Kopec, Z. (1969) The effect of various kinds of carbohydrates on amylase formation in the pancreas. Nutr. Diet. 11:203-213.
179. Lavau, M., Bazin, R. & Herzog, J. (1974) Comparative effects of oral and parenteral feeding on pancreatic enzymes in the rat. J. Nutr. 104:1432-1437.
180. Oliver, C. (1980) Isolation and maintenance of differentiated exocrine gland acinar cells in vitro. In Vitro 16:297-305.
181. Brannon, P.M., Orrison, B.M. & Kretchmer, N. (1985) Primary cultures of rat pancreatic acinar cells in serum-free medium. In Vitro Cell. Dev. Biol. 21:6-14.
182. Orly, J., Sato, G., & Erickson, G.F (1980) Serum suppresses the expression of hormonally induced functions in cultured granulosa cells. Cell 20:817-827.
183. Nimrod, A., Tsafriri, A., & Linder, H.R. (1977) In vitro induction of binding sites for hCG in rat granulosa cells by FSH. Nature 267:632-633.
184. Hiller, S.G., Selenik, A.J. & Ross, G.T. (1968) Independence of steroidogenic capacity and LH receptor induction in developing granulosa cells. Endocrinology 102:937-946.
185. Yaffe, D. & Saxel, O. (1977) A myogenic cell line with altered serum requirements for differentiation. Differentiation 7:159-166.
186. Logsdon, C.D. & Williams, J.A. (1986) Pancreatic acinar cells in monolayer culture: direct trophic effects of caerulein in vitro. Am. J. Phvsiol. 250:G440-G447.
187. Bendayan, M., Duhr, H-A. & Gingras, D. (1986) Studies on pancreatic acinar cells in tissue culture: basal lamina (basement membrane) matrix promotes three-dimensional reorganization. Eur. J. Cell Biol.42:60-67.
188. Fujita, M., Spray, D.C., Choi, H., Saez, J., Jefferson, D.M., Hertzberg, E., Rosenberg, L.C. & Reid, L.M. (1986) Extracellular matrix regulation of cell-cell communication and tissue-specific gene expression in primary liver cultures. Progress in Clinical and Biological Research 226:333-352.
114
189. Logsdon, C.D. & Williams, J.A. (1983) Pancreatic acini in short-term culture: regulation by EGF, carbachol, insulin, and corticosterone. Am. J. Phvsiol. 244:G675-G682.
190. Vander, A.J., Sherman, J.H. & Luciano, D.S. (1980) The digestion and absorption of food. In: Human Physiology: The Mechanisms of Body Function (Vastyan, J.E. & Wagley, S., eds.), p.429, McGraw-Hill, Inc., New York.
191. Gorelick, F.S. & Jamieson, J.D. (1981) Structure function relationships of the pancreas. In: Physiology of the Gastrointestinal Tract (Johnson, L.R., ed.), pp.773-794, Raven Press, New York.
192. Scheele, G. (1986) Two-dimensional electrophoresis in the analysis of exocrine pancreatic proteins. In: The Exocrine Pancreas (Go, V.L.W., Gardner, J.D., Brooks, F.P., Lebenthal, E., DiMagno, E.P. & Scheele, G., eds.), pp.185-192. Raven Press, New York.
193. Brannon, P.M., Demerest, A.S., Sabb, J.E. & Korc, M. (1986) Dietary modulation of epidermal growth factor action in cultured pancreatic acinar cells of the rat. J. Nutr. 116:1306-1315.
194. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
195. Ceska, M., Hultman, E. & Ingelman, B.G.-A. (1969) A new method for determination of a-amylase. Experientia 25:555-556.
196. Ceska, M., Birath, K. & Brown, R. (1969) A new and rapid method for the clinical determination of a-amylase activities in human serum and urine. Optimal conditions. Clin. Chim. Acta 26:437-444.
197. Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London1 227:680-685.
198. Steel, R.G.O. & Torie, J.H. (1960) Principles and Procedures of Statistics. McGraw-Hill Book Co., New York.
199. Meda, P., Bruzzone, R., Knodel, S. & Orci, L. (1986) Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase. J. Cell Biol. 103:476-483.
115
200. Bruzzone, R., Trimble, E.R., Gjinovci, A., Traub, O., Willecke, K. & Meda, P. (1987) Regulation of pancreatic exocrine function: A role for cell-to-cell communication? Pancreas 2:262-271.
201. Iovanna, J., Giorgi, D. & Dagorn, J.C. (1986) Newly synthesized amylase, lipase and serine proteases are transported at different rates in rat pancreas. Digestion 34:178-184.
202. Schimke, R.T. & Doyle, D. (1970) Control of enzyme levels in animal tissues. Annu. Rev. Biochem. 39:929-976.
203. Brannon, P.M. & Scott, D. (1987) Impairment of pancreatic acinar function by reserpine in vivo and in vitro. In Vitro Cell. Dev. Biol. 23:429-435.
