Download Pancreatic polypeptide cells of rat pancreas after chronic

Indian Journal of Experimental Biology
Vol. 39, May 2001, pp. 416-424
Pancreatic polypeptide cells of rat pancreas after chronic ethanol feeding
Yesna Kokol.2, Vera Todorovicl.2··, Neda Drndarevic 1, Radmila Glisic3 , Miroslava Nedeljkovic 2 &
Anna Nikolic 4
'Institute for Medical Researc h, Belgrade, Yugoslavia
Institute of Zoology, Faculty of Biology, Belgrade, Yugoslavia
3
lnstitute of Zoology, Faculty of Science, Kragujevac, Yugoslavia
4
lnstitut for the Application of Nuclear Energy, Zemun, Yugoslavia
2
Received 15 October 1999; revised 23 January 200 I
Male Wistar rats, (2 months old) were randomly divided into two groups accordi ng to the diet offered (C-control and Ecthanol treated rats). Final body weight was significantly increased but pancreatic weight as a percentage of body weight
2
was decreased in ethanol treated rats. Volume density, number of pancreatic poly peptide (PP)-cells per islet and per ~lm of
islet were significant ly increased. PP-cells were abundant and occ upied the whole periphery of islets in the splenic part of
the pancreas. Those cells showed strong immuno posit ivity. At the ultrastructural level PP granules had predominantly less
electron density. The mean diameter of PP g ranules was signifi ca ntly increased and the number of granules of larger diameter was greater in theE g roup of rats, than in the controls.
Pancreatic polypeptide (PP) is a 36 amino acid hormone belonging to a family of structurally related
peptides including peptide YY and neuropeptide yu_
PP is synthesized in and released from endocrine PP
cells situated within the pancreatic islets and scattered
throughout the exocrine tissue 3 .
The relative proportions of different pancreatic islet
cells show regional and species differences. ]n both
the human and rat pancreas, two distinct types of islets have been described, differing from each other in
their cellular content: a PP-rich , glucagon-poor islet
(PP islet) characteristic of the ventral lobe (derived
from the ventral primordium) and glucagon-rich, PPpoor islet (glucagon islet) characteristic of the dorsal
primordium4.
Under physiological circumstances, PP is mainly
secreted in response to ingestion of a meal. The
physiological function of the peptide is poorly understood, although it has been demonstrated that under
experimental conditions, the peptide inhibits the secretion of insulin in rats, dogs and mice, and inhibits
exocrine pancreatic secretion in man and in dogs 5.
*Correspondent author.
Institute for Medi cal Research
DR Suboti a 4, PO BOX 102
11129 Belgrade, Yugoslavia
Tel: 381 II 685 788. 381 II 684 484
Fax: 381 II 643 691. e-mail: [email protected]
The secretion of PP is predominantly regulated by
the parasympathetic nervous system 6·7 • The basal level
of serum PP has become a field of interest mainly due
to the role of PP as a tumour marker8. 10 , and as a
marker of pancreatic endocrine function.
In comparison with general mucosal alteration of
the gastrointestinal tract, relatively little is known
about the effect of alcohol on the diffuse neuroendocrine system (ONES) and its neurohormonal response11.14. Alcohol-associated effects are either due
to direct local action or mediated by numerous other
substances 15.20 . Although controversy regarding the
definition of different histological types of alcoholic
chronic pancreatitis in humans and animal models
prevails in the literature 21 , the association between
ethanol and chronic pancreatitis is well established.
Functional studies have shown that exocrine deficiency has a significant effect on islet cell function,
and there are exocrine pancreas-insulin, glucagon,
pancreatic polypeptide and somatostatin interactions,
as well as enteropancreatic interaction in chronic pancreatitis22'23.
To the best of our knowledge, complex quantitative
analysis of the light microscopic and fine structure of
islet endocrine cells has not been carried out in
chron ic alcoholism, especially on the PP-cells. The
aim of this study is to examine PP-cells at the light
and ultrastuctural levels in rats after long term (4
months) alcohol ingestion.
KOKO eta/. : PANCREATIC POLYPEPTIDE CELLS AFTER CHRONIC ETHANOL FEEDING
Materials and Methods
Animals and f eeding protocol - Male Wi star
rats (2 month s old) , weighing approximately 240g
were randomly allocated to two groups (C-control;
E-ethanol treated animals). The control group of 15
rats was fed a commercial diet. The E group of 14
rats was given free access to the diet and a solution
of 25% sucrose-32% ethanol as recommended by
Hartroft 24 . The quantity of food offered to the
control animal s was adjusted to the energy intake of
anim als receiving alcohol (i.e. control rats were
pair fed). The amount of tap water given to the
eth anol-treated rats was 25 ml , and the mean
co nsumption per rat per day of th e alcohol solution
was 12 ml per ani mal. Ethanol treated rats therefore
cons umed 23% of th e total calories as alcohol
(mean 8.42 g/kg/day).
Blood ethanol determination - Samples for blood
eth anol determin ation were obtained from the tail
vein, and blood ethanol concentration was determined
using the Sigma diagnostic alcohol procedure (No.
322-UY, Si gma Chemical Co., St. Loui s, Mo) . Mean
blood ethanol concentration was calculated from
multiple blood determinations (at 1000 hrs on day 1,
15, 30, 45 , 60, 75, 90, 105 and 120) throughout the
exposure period of 120 days.
Blood glucose and plasma insulin determination Fasting blood glucose level was measured by the
glucose oxidase method 25 . Plasma insulin was
measured by radioimmunoassay usi ng commerci al
kits in accordance with the instructions (INEPDiagnostics, Ze mun ) and rat insulin standard (Novo
Industry, Bagsvaerd, Denmark) .
Preparation of histological samples - After 4
months, rats were starved for 24 hr and given water
ad libitum before being killed in the morning (0700
hrs) by a blow on the neck. The pancreas was removed, weighed in air, and the splenic part of the
pancreas was cut and used for light and electron microscopic investigation. Samples were immersed for
24 hr in Bouin fixative and embedded in paraffin according to the standard procedure. Immunohistochemistry was performed in 5 J..lm serial section s using polyclonal antibodies agai nst somatostatin, insulin, glucagon and pancreatic polypeptide. Sections
were immunostained employing the streptavidinbiotin or PAP technique. Convention al morphometry
and standard stereological equations were used to calculate the volu me density and frequency of pancreatic
endocrine cells26 .
417
Electron microscopy - Several pieces of the splenic part of the pancreas of each animal were fixed in
2.5% glutaraldehyde in 0.1 M caccodylate buffer and
postfixed in 2% osmium tetroxide in the same buffer.
After dehydration the specimens were cut on an LKB
ultratome, double stained with uranyl acetate and lead
citrate and examjned under Philips CM 12 electron
microscope.
Statistics- Data were subjected to Student's t-test
and the results expressed as mean± SE.
Results
There was no significant difference between the
eth anol-treated and control rats with regard to dail y
energy intake and energy from protein and fat
(Fig. 1). The average ethanol intake of group E was
3.09 g/day which was 23 % of the daily energy intake.
Mean blood ethanol concentration was 125 ± 13
mg/ml and ranged from 107 to 138 mg/100 mi.
Animals in the examined groups had similar weight
gains (data not shown) and body mass increased du ring the experiment continually . Total pancreatic
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Fig. !- Daily energy intake and percentage of particular nutri tional ingredients in the rats of groups C and E.
INDIAN J EXP BIOL, MAY 2001
418
weight and volume were similar in both groups, but
relative pancreatic weight in rats of group E was decreased (Table I). The results for fasting blood glucose and insulin level were similar in the two groups
of animals. There was significant a difference between the ethanol-treated rats and controls with regard
to fin al body weight.
Volume density, number of PP-cells per islet and
per Jlm 2 of islet were significantly increased (Table 2). Mean profile area of the PP-cell was similar in
both groups of rats. The number of PP-cells in the
exocrine part of the pancreas was reduced, but statistically significant differences were not found.
Glucagon-producing
A-cells,
somatostatinproducing 0-cells and pancreatic polypeptideproducing PP-cells were increased in number, while
insulin-producing B-cells were decreased (Table 3).
Results obtained for individual endocrine cells per
islet showed changes in the percentage of those cells
in the E group of rats (Fig. 2). The percentage of Aand PP-cells was increased and that of B-decreased in
ethanol-treated rats. The percentage of 0-cells was
unchanged. In comparison with the control, the increase in number of PP-cells amounted to about 42%
2
per islet and 83 % per Jlm of islet.
It was observed that PP-cells were situated peripherally on the islets singly or in small clusters in the C
group of rats while in some islets of Langerhans there
were no PP cells. In the exocrine part of the pancreas,
pancreatic
polypeptide-producing-PP-cells
were
found both in the acini and in the small ducts.
In ethanol-treated rats PP-cells were abundant and
occupied the whole periphery of the islets (Fig. 3c).
There were no islets without PP-cells unlike in the
control (Fig. 3b). In the exocrine tissue PP-cells were
Table 1-
found in the acini and in the small ducts (Figs 4a,b). A
great number of PP-cells had very strong immunoreactivity suggesting accumulation of pancreatic polypeptide granules in the cytoplasm.
Electron microscopic examination showed that PPcells in alcohol-treated rats con tained granules in the
whole cytoplasmic area. PP granules had less dense
cores and closely fitting limiting membranes
Table 2-Volume density, number or PP cell s and mean profile
areas of PP cells
[Values are mean± SE]
GroupE
Group C
Volume density (m m2)
0.!3±0.015
0.24±0.030**
Npplprolile area of islet
11.4 ± 1.64
16.7 ± 1.76
Nppl!ln/ of islet
0.0012 ± 0.0001
0.0022 ± 0.0002**
NPP in the exocrine pan of pancreas
25.6 ± 4.91
19.8 ± 2.92"'
Npplmm 2 of ex.ocrine pancreas
1.36 ± 0.24
1. 155 ± 0. 18"'
Mean profile area of PP cell
88.96±3.67
P values:** <0.00 1; "' not significant
87. ! 3 ±4.95'"
Table 3-Number of part' cuiar re llslislet
Cell type
c
E
a
21.2± 1.95
27. 1 ± 3.75"'
~
D
30.8 ± 1.48
25.5 ± 2.73"'
3.7 ± 0.38
4.2 ± 0.55"'
11.0 ± 1.52
16.7 ± 1.75*
pp
P values: * < 0.05; "'not signifi cant
Body and pancreatic weight, total pancreatic vo lume and fasting blood glucose and insulin level in the rats
[Values are mean± SE fro m 15 animals]
Group C
GroupE
Initi al body weight (g)
232±1.27
240±2.37'"
Final body weight (g)
359±5.61
426±!8.00**
Pancreati c weight (g)
1.30±0.039
1.33±0.073"'
Pancreatic weight
(% of body weight)
Total pancreatic volume (cm 3 )
0.36±0.012
0.32±0.014*
1.24±0.037
1.26±0.070"'
Glucose (mmol/L)
5.28±0.26
5.36±0.13"'
Insulin (mlU/L)
22.0±1.34
2!.5±3.06"'
P values:** <0.001; * <0.02; "' not significant
KOKO eta/.: PANCREATIC POLYPEPTIDE CELLS AFTER CHRONIC ETHANOL FEEDING
419
(Figs 5a,b ). The mean diameter of PP granules was
significantly increased (209 ± 3.8 in the control and
224 ± 4.0 in theE group of rats). Although, the largest
number of PP granules was found in the same diameter group in both groups of rats (222 nm), in the ethanol treated group the number of granules of larger
diameter was greater (Fig. 6).
Discussion
In a series of experiments, started in 1989, we have
been investigating the diffuse neuroendocrine system
(ONES) in the upper part of the digestive tract and
pancreas after long-term (4 or 6 months) ethanol consumption with or without adequate protein in the diet.
Our study mainly consisted of morphofunctional and
stereological investigations at the light and electron
microscopic level because there are few data about
this problem. In the present study, as we11 as in previous investigations we applied a nutritional schedule
that made it possible to obtain similar mean daily energy intakes in both groups of rats. Rats given a
chronic dose of ethanol (about 23 % of total calories)
consumed an adequate amount of other nutrients and
had a normal growth rate when compared to pair fed
controls. In this respect, the ethanol diet was nutritionally adequate, and possible effects of alcohol were
not confounded with the effects of malnutrition 12.24.27.28 .
There are no data in the available literature about
the detailed morphometric parameters of the PP-ce11s
after long-term of ethanol ingestion in rats and our
results are the first in this area of investigation.
Owiang et al. 23 showed that the number of PP-ce11s
so
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A cells
B cells
D cells
PP cells
Fig. 2-Percentage of each endocrine cell expresed per islet
Fig. 3-(a) Splenic part of pancreas in control rat. Small
number of PP cells situated peripherally and islet without PP
cells; PAP; x 20; (b) Splenic part of pancreas in ethanol treated
rat. Abundant PP cells occupied all periphery of islet; PAP;
X 40
420
INDIAN J EXP BIOL, MAY 200I
23
appear to increase in chronic alcoholic pancreatitis .
Islets were composed largely of PP-cells, and a large
number of isolated PP-cells were frequently found in
29
close contact with duct epithelium . This observation
suggests that the increase in PP-cells may represent a
regenerative phenomenon after pancreatic injury.
There was morphological evidence for an alcoholinduced selective intrapancreatic nerve degeneration
in mice after 4 months of alcohol consumption. This
affected mainly the nerve fibers that are inhibitory to
the exocrine pancreas, and may represent the morphological background of the hypersecretory state of
the pancreas in chronic alcoholism. A sli ght decrease
was found in the intensity of VIP and SP immunoreactivity and the PP fibers almost di sappeared. Majority of periacinar nerve terminals showed degenerative changes whereas the perivascular nerve fibers
were relatively well preserved 30 .
Fig. 4-(a) PP cells in the acini. PAP; x 40; (b) PP cells in the
small duct: PAP; x 40.
The present results showed a significant increase in
number of PP-cells in the islets of Langerhans after 4
months consumption of ethanol. It is not clear what is
the function al significance of this increase. Other results obtained in the present experiment showed that
the number of A-and D-cells was slightly increased
while that of B-cells decreased. In addition, some
morphological abnormality of A-and B-cells was recorded. T he profile area of A-cells was small er, as
well as the profil e area and volume densi ty of th eir
31
nuclei . The B-cells appear more affected and sensitive than the other endocrine cells in the islets 32 . Both
B-and A-cells showed degenerative processes suggestive of apoptotic death. The D-and PP-cells in the
islets of Langerh ans did not express simil ar morphological changes.
Previous results for other endocrine cells belonging
to DNES also showed changes in their number12"14"31J2.33. Thus all endocrine cells in th e fundic
and pyloric part of the sto mach were significantly decreased in number and some cytological abnormality
was also observed.
In the rat pancreas glucagon and pancreatic polypeptide are present in di sti nct cell types, but they also
coexist in one population of islet cells termed glucagon/PP-cells. The glucagon/PP-cells were found in
significant numbers in all pancreatic regions, being
more numerous than the other two types of PP-and Acells34.
Many of the PP granules in ethanol treated rats
were electron pale. It seems th at these PP-cells pro35
duced predominantly pancreatic polypeptide . In this
context present results showed that there was proliferation of these cells, although no mitoses was observed in the PP-cells. A possible explanation may be
that the population of cells with coex istence of both
peptides changed their genetic programme and began
to produce predominantly pancreatic polypeptide. The
increased diameter of PP granules in the E group of
rats probably results fro m accumu lation of pancreatic
polypeptide.
Functional studies using various tests for insulin
secretion have been performed both in patients and
animals in chronic pancreatitis. Fasting insulin levels
in chronic pancreatitis were found to be normal or
22
moderately raised 36·37 or diminished and the re38
36 38
sponse to oral glucose was variable · . Folch et at.
reported that blood glucose concen trations were
higher in patients with pancreatitis when compared to
controls 38 . On the other hand, serum immunoreactive
KOKO eta/.: PANCREATIC POLYPEPTIDE CELLS AFfER CHRONIC ETHANOL FEEDING
l:
Fig. 5-(a) PP granules in control rat; urany l acetate; lead citrate; bar I Jlm; (b) PP granules with less dense cores in
alcoholized rat; uranyl acetate; lead citrate; bar= I Jlm.
421
INDIAN 1 EXP BIOL, MAY 2001
422
___.__co ntrol
· ··•··· ethanol
(/)
~
30
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25
....
0'1
.....
20
s:::
C'll
0
Cll
0'1
-...
C'll
s:::
15
10
Cll
(J
Cll
c..
5
0
.
130 148 166 185 203 222 241 259 278 296 315
PP granules diameter (nm)
Fig. 6--Size- frequency distribution of the profile diameter of
granules inC and E groups of rats
insulin (IRI) concentrations were diminished in
chronic human alcoholics 39 , while ethanol had no effect on IRI in ethanol-treated rats 40.4 1• Infusion of
ethanol at increasing concentrations caused progres42 43
sive inhibition of insulin and glucagon secretion · .
44
However, Jauhonen and Hassinen observed that
ethanol itself produced no marked changes in blood
glucose, IRI and glucagon after intravenous infusion.
The present results showed that there was no hypoglycaemia which is a strong stimulus for production
of pancreatic polypeptide.
The plasma level of PP increased significantly after
intravenous ethanol administration 45 . Ingestion of alcohol by healthy volunteers did not stimulate the release of cholecystokinin (CCK), which is the chief
hormonal stimulant of pancreatic enzyme secretion,
nor did it significantly alter fasting levels of pancreatic polypeptide46. These data suggest that, in patients
who do not have chronic pancreatitis, alcohol does not
induce acute pancreatitis, either by stimulating CCK
release or by stimulating enzyme secretion directly.
On the contrary, in chronic alcoholic pancreatitis,
fasting and postprandial levels of pancreatic polypeptide were significantly increased and the link between
CCK and pancreatic polypeptide was disrupted 47 . In
addition, Hajnal et al. 48 tested the effect of a 12%
(v/v) alcoho l solution added to a meal on basal and
postprandial levels of PP in both nonalcoholic volunteers and chronic alcoholics 48 . Basal plasma PP levels
were similar in both groups, but the postprandial increments in PP levels observed in nonalcoholics were
not observed in alcoholics. However, basal and postprandial levels of gastrin and CCK were similar in
alcoholics and nonalcoholics. They concluded that
postprandial hypersecretion of pancreatic enzymes in
alcoholics is not related to increased plasma levels of
CCK or gastrin but it is possible that the impaired release of PP may participate in the mechanism for increased pancreatic enzyme secretion in chronic alcoholics48.
Hyperplasia of PP-cells in the PP-rich region of the
pancreas (ventral embryonic origin) was observed in
patients previously treated surgicall y for functioning
duodenal or pancreatic gastrinoma not associated with
multiple endocrine neoplasia syndrome49 . PP hyperplasia was observed in the pancreas of j uvenile diabetes50. Although hyperplasia of PP-cells took place
predominantly in altered exocrine pancreases, in the
present study all rats (with or without histological
disturbances of the exocrine pancreas) showed hyperplasia of PP-cells only in the islets of Langerhans.
Proliferation of PP-cells was also observed in ethanol
treated rats which were fed with a low protein di et
(data not shown). It may be concluded that ethanol
had a direct effect on this proliferation , al though the
exact mechanisms of this phenomenon can not be explained at present.
Acknowledgement
This work was supported by grant 13M 13 from the
Serbian Ministry of Science and Technology, Yugoslavia.
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