Subtotal pancreatectomy (Px) is a well defined model of beta cell regeneration in rats. Initially after surgery (from 12h until 72h post-Px) an increased ...
UPREGULATION OF CELL DIFFERENTIATIONDIFFERENTIATION-RELATED GENES IN 90% PANCREATECTOMIZED RATS TREATED WITH GASTRIN Noèlia Téllez1,2, Géraldine Joanny1,2, Jéssica Escoriza1,2 and Eduard Montanya1,2. 1Laboratory
of Diabetes and Experimental Endocrinology , IDIBELL-University of Barcelona, Hospital Universitari de Bellvitge. 2CIBERDEM
RESULTS
BACKGROUND
EARLY Pancreatic Regeneration Period
In a recent study, we have shown that gastrin treatment enhanced beta cell neogenesis in 95% pancreatectomized rats.
LATE Pancreatic Regeneration Period
Pancreatic remnants harvested initially after Px double-stained for BrdU-insulin (black/brown)
Subtotal pancreatectomy (Px) is a well defined model of beta cell regeneration in rats. Initially after surgery (from 12h until 72h post-Px) an increased proliferation is seen first in the common pancreatic duct (CPD) and sequentially in smaller ducts of the ductal tree and in focal areas of proliferating small ductules (1). During this early period of regeneration small clusters of newly formed endocrine cells are found within these foci of regeneration (1). These observations, together with the increased PDX-1 expression in proliferative ductal cells found in pancreatic remnants 2 - 3 days after Px (2), suggest that pancreatic regeneration after Px recapitulates the pancreas formation during the embryonic development.
1 day post-Px
Pancreatic remnants harvested on days 5 and 7 after Px double-stained for BrdU-insulin (black/brown) 5 days post-Px
3 days post-Px
A
7 days post-Px
B (A) Increased
ductal
cell
After 5-7 days from Px ductal cell
proliferation initially after Px.
proliferation
Gene expression of gastrin and its Receptors during the EARLY pancreatic regeneration period
300 200
100
50
100
0
0
0
day3
Sham
day1
day3
2. Sharma,A, Zangen,DH, Reitz,P, Taneja,M, Lissauer,ME, Miller,CP, Weir,GC, Habener,JF, Bonner-Weir,S: The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 48:507-513, 1999
day1
Sham
*p≤ 0.05 vs Sham
400
400
400
day1
Sham
day3
*
*
day1
*
300
*
ck20
*
*
100
0 Sham
day1
day1
Sham
day3
200 100
0 Sham
day3
ca2
day1
100
0 Sham
day5
day7
po st -P x
Px + Vehicle
day1
Sham
day3
day1
day3
day5
EMBEDDING. Late Pancreatic Regeneration qPCR and IHC Period
Sham
day1
day3
100 0
Sham
day5
day7
Sham
day1
day3
day5
day7
day7
*
*
Sham
day5
100 50
*
0
0
0 day5
mafA
*
150
50
50 Sham
nkx6,1 200
100 RQ
RQ Sham
100 50 0 Shaml
day1
day3
day7
Sham
day5
Sham
day7
day5
day7
250 200 150 100 50 0
*
Sham
day5
day7
Gene expression profile of islet hormones: Gastrin treatment prevents downregulation of insulin2 gene expression 160 140 120 100 80 60 40 20 0
Insulin2
glucagon
150 100 50
* Sham
*p≤ 0.05 vs Sham
During the EARLY Pancreatic Regeneration Period after 90%-Px: 1.- There was an upregulation of gastrin and cckbr gene expression which coincided with an increased expression of ductal and stem cell markers.
Px + Gastrin
day7
pdxpdx-1
100
150
Sham
day5
150
*
150
glucagon
day3
* *
0
day7
neuroD1 200
140 120 100 80 60 40 20 0
200
day1
100
*p≤ 0.05 vs Sham
Insulin2 160 140 120 100 80 60 40 20 0
150 100 0
Sham
Gene expression profile of islet hormones remains stable after Px
PARAFFIN
neurog3 *
50
TOTAL RNA EXTRACTION OR
* 200
50
0 Sham
RQ
250 200
* RQ
RQ
RQ Sham
600 500 400 300 200 100 0
*p≤ 0.05 vs Sham
day5
0
day7
Sham
day5
day7
*p≤ 0.05 vs Sham
During the LATE Pancreatic Regeneration Period after 90%-Px: There was a dramatic reduction of pdx-1, nkx6.1 and insulin2 gene expression in 90%-Px rats treated with vehicle, which was prevented by gastrin treatment.
2.- Gastrin treatment resulted in a stimulation of neurogenin3, neuroD1, pdx-1 and nkx6.1 gene expression.
250 Blood glucose (mg/dl)
day3
300
Downregulation of pdxpdx-1 and nkx6.1 gene-expression is prevented by Gastrin treatment in Px rats
mafA
nkx6,1 *
HARVESTING,
RESULTS Sham
day1
hnf1β / tcf2
hnf6
RQ
7 da ys
Sham
day7
200
50
RQ
5 days postpost-Px
REMNANT
500 400 300 200 100 0
day5
ND: Non Detectable
*p≤ 0.05 vs Sham
pdxpdx-1
*
RQ
3 day postpost-Px
Early Pancreatic Regeneration Period
300 250 200 150 100 50 0
RQ
or Vehicle (Px+V (Px+V))
x t-P stpo ay 1d
neuroD1
* RQ
RQ
neurog3 300 250 200 150 100 50 0
Sham
150
50
0
day3
Increased gene-expression of neurogenin 3, neuroD1, pdx-1 and nkx6.1 in Gastrin-treated Px rats mafA is unaffected
METHODS
ND
0
day7
prominin 1/ CD133
150 100
*p≤ 0.05 vs Sham
SD Rats 4weeks of age
day5
150
200
100
day3
100 Sham
250
300
200
200
0
0 Sham
300
400
200
100
200 0
200
hnf1β / TCF2
hnf6 500
* 600 RQ
600
RQ
RQ
To define a temporal gene expression profile of beta cell neogenesis-related genes induced by gastrin in pancreatectomized rats at different stages of the regenerative process.
prominin 1/ CD133 800
*
300
day7
Normal gene expression pattern of ductal and pluripotent stem cell markers during the late pancreatic regeneration period
RQ
ca2
*
800
RQ
ck20 1000
AIM
200
0 day5
ND: Non Detectable
RQ
.
300
*p≤ 0.05 vs Sham
The increased gene-expression of ductal and pluripotent stem cell markers early after Px is ameliorated by gastrin
3. Wilson,ME, Scheel,D, German,MS: Gene expression cascades in pancreatic development. Mech.Dev. 120:65-80, 2003
400
100 50
Sham
day3
150
RQ
day1
ND
500
RQ
400
150 100
Sham
1. Bonner-Weir,S, Baxter,LA, Schuppin,GT, Smith,FE: A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42:1715-1720, 1993
200
cckbr
200
RQ
*
600 500 400 300 200 100 0
RQ
*
200
500
Px + Gastrin
cckar
RQ
References:
300
RQ
RQ
400
250
Px + Vehicle
RQ
*
Sham gastrin
cckbr
RQ
500
We aimed to identify whether the endocrine regeneration induced by gastrin treatment in Px rats involved modifications on the gene expression pattern related to beta cell differentiation in pancreatic remnants.
cckar
RQ
gastrin
Gene expression of gastrin and its Receptors during the LATE pancreatic regeneration period
Px + Gastrin
RQ
Px + Vehicle
RQ
Sham
Gastrin (150µ (150µg/Kg· g/Kg·12h)(Px+G)
focal
regeneration after Px.
On the other hand, the proper expression of these transcription factors in mature beta cells is essential for the maintenance of the functional phenotype.
Treatment:
and
found during the initial period of
increased cell replication.
Studies from the developmental biology field have shown the hierarchical expression of key transcription factors required for beta cell formation in pancreatic development (3), among them, the master-regulator neurogenin3 and pdx-1.
90%-Px
declined
areas were smaller than those
(B) Areas of regeneration with
90%-Px rats developed mild hyperglycemia after surgery with similar blood glucose concentrations between Px+V and Px+Gast groups.
200 150 100
CONCLUSION. Gastrin treatment stimulated the expression of the crucial transcription factors ngn3, neuroD1, pdx-1 and nkx6.1 required for beta cell differentiation during
50
the early phase of regeneration after 90%-Px. Moreover, gastrin administration resulted in the maintenance of the mature/functional beta cell phenotype preventing pdx-1,
0 Px
2
4 Time (days)
6
8
nkx6.1 and insulin2 gene expression downregulation found in late Px+V rats.
