1. Volume 128, Issue 5, Pages 1391-1404 (May 2005)
Aging Is Associated With Decreased Pancreatic Acinar Cell Regeneration and Phosphatidylinositol 3-Kinase/Akt Activation 
Hiroaki Watanabe, Hiroshi Saito, Piotr G. Rychahou, Tatsuo Uchida, B. Mark Evers 
Gastroenterology 
Volume 128, Issue 5, Pages (May 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Aging is associated with decreased pancreatic regeneration after partial Px. (A) The wet weight of remnant pancreas from young (•) and aged (■) mice was measured 0, 3, 7, and 14 days after partial Px. Data are shown as fold increase compared with day 0. All data were normalized by body weight (BW). Values are mean ± SEM, n = 3–9. *P < .05 vs day 0 group. Pancreatic weight (B) and contents of DNA (C) and protein (D) of remnant pancreas after partial Px were measured in young and aged mice. Pancreatic wet weight and contents of DNA and protein in remnant pancreas 7 days after partial Px were measured. All data were normalized by BW (g). Values are mean ± SEM, n = 3–5. *P < .05 comparing sham vs Px in young mice, †P < .05 comparing young and aged mice treated by partial Px.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
BrdU incorporation and BrdU-labeling index of remnant pancreas after partial Px. BrdU incorporation in remnant pancreas was immunohistochemically estimated on 0, 3, and 7 days after partial Px (A) and BrdU-labeling index was counted as described in the Materials and Methods section (B). Values are mean ± SEM, n = 5. *P < .05 compared with other groups. Arrowheads represents BrdU-positive nuclei. (Original magnification 100×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Expression of phosphorylated Akt (pAkt) and phosphorylated ERK (pERK) in remnant pancreas. The expression of pAkt in remnant pancreas was determined on 0, 3, and 7 days after partial Px by immunohistochemical staining (A). Typical positive areas are indicated as follows: arrowhead, pAkt-positive islet cells; arrows, pAkt-positive acini; I, islet, D, ductal cell; A, acinar cell (original magnification, ×200). The quantitative pAkt expression in remnant pancreas was measured by Western blot and densitometric analysis (B). Each lane represents pooled samples from the pancreas of 3 mice at the indicated time points. pAkt was normalized by the corresponding total Akt density. The expression of pERK in remnant pancreas was immunohistochemically determined on 0, 1, 3, and 7 days after partial Px (C). Typical positive areas are indicated as follows: arrow, pERK-positive duct cell; I, islet; D, ductal cell; A, acinar cell (original magnification 200×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Wortmannin, a pharmacologic PI3K inhibitor, completely blocks pancreatic regeneration in young mice. The effects of wortmannin (Wort) on pancreatic regeneration after partial Px was assessed. Pancreatic weight (A), DNA (B), and protein content (C) in remnant pancreas from young mice undergoing partial Px were assessed and compared with sham-operated mice. All data were normalized by body weight (BW) (g). Values are mean ± SEM, n = 3–7. *P < .05 comparing sham with Px in vehicle injection group, †P < .05 comparing vehicle with wortmannin treatment in Px group. Immunohistochemistry of pAkt in remnant pancreas was assessed (D). The level of pAkt was immunohistochemically analyzed in remnant pancreas from young mice following vehicle or wortmannin treatment at 7 days after partial Px. Arrowhead indicates pAkt-positive islet cells; arrows indicate pAkt-positive acini (original magnification, ×400).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
PI3K p85α regulatory subunit siRNA significantly attenuates pancreatic regeneration in young mice. Young mice were injected with p85α siSTABLE (Dharmacon) or scrambled control siRNA (10 μg/mouse) by hydrodynamic tail vein injection using TransIT In Vivo Gene Delivery System (Mirus). Total injection volume (mL) per mouse was calculated by mouse body weight (g)/10. To assess the siRNA delivery efficiency, CX-Rhodamine-labeled siRNA or control vehicle (ie, transfection reagent) was injected into mice, and frozen sections of pancreas from mice killed 24 hours after siRNA injection were prepared; CX-Rhodamine (red) in the sections was assessed by fluorescent microscopy (587 nm) (original magnification, ×400) (A) Blue: DAPI. The effects of p85α siRNA on pancreatic regeneration after partial Px was determined. Pancreatic weight (B), DNA (C), and protein content (D) in remnant pancreas from young mice undergoing partial Px were assessed and compared with sham-operated mice. All data were normalized by body weight (BW) (g). Two independent experiments performed with similar results were obtained; the data were combined from the 2 separate studies. Values are mean ± SEM, n = 5–7. *P < .05 comparing sham with Px in control siRNA treatment group, †P < .05 comparing control with p85α siRNA treatment in Px group. Western blot analysis of p85α, pAkt, and Akt expression in remnant pancreas was assessed 3 and 7 days after partial Px (E). Each lane represents pooled samples from 3 mice at the indicated times.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
IGF-1 significantly increases BrdU incorporation of isolated pancreatic acinar cells. BrdU incorporation of isolated pancreatic acinar cells in response to IGF-1 was measured (A). Pancreatic acinar cell cultures were treated with various concentrations of IGF-1 for 24 hours. BrdU (10 μmol/L) was added to the culture 6 hours after the initiation of IGF-1 treatment. Incorporation of BrdU was determined by a BrdU ELISA. Values are mean ± SEM, n = 5. *P < .05 compared with control (vehicle treatment). Phosphorylation of IGF-1R, Akt, and ERK in cultured pancreatic acinar cells in response to IGF-1 (B) was measured by Western blot analysis. Cultured acinar cells were treated with IGF-1 (10 nmol/L) and harvested at designated time points for expression of phosphorylated IGF-1R (pIGF-1R), phosphorylated Akt (pAkt), total Akt (Akt), and phosphorylated ERK (pERK). Histogram represents the densitometric analysis of the bands in Figure 6B after IGF-1 stimulation (C). pIGF-1R and total pERK (phosphorylated ERK1 and ERK2) were normalized by the corresponding β-actin density, and pAkt was normalized by the corresponding total Akt density. Similar results were obtained in 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
Wortmannin, but not PD98059, blocks cell proliferation in pancreatic acinar cells. The effect of wortmannin or PD98059 on BrdU incorporation of isolated acinar cell was assessed (A). Isolated pancreatic acinar cells were preincubated with wortmannin (100 nmol/L) or PD98059 (30 μmol/L) for 30 minutes followed by addition of IGF-1 (10 nmol/L). BrdU was added 6 hours after IGF-1 administration, and cells were harvested 18 hours later. Values are mean ± SEM, n = 5. *Compared with no IGF-1 treatment; †P < .05 compared with IGF-1 treatment alone. The effect of wortmannin on PI3K/Akt activation after IGF-1 administration was measured by Western blot analysis (B). Isolated pancreatic acinar cells were preincubated with wortmannin (100 nmol/L) or PD98059 (30 μmol/L) for 30 minutes prior to IGF-1 (10 nmol/L) treatment and then harvested 1 hour later for protein extraction and determination of phosphorylated Akt (pAkt), total Akt (Akt), and phosphorylated ERK (pERK) expression. Similar results were obtained in 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
p85α siRNA inhibits cell proliferation in pancreatic acinar cells. Isolated pancreatic acinar cells were transfected with either p85α or control siRNA. To assess the transfection efficiency, CX-Rhodamine-labeled siRNA was transfected into the acinar cells and CX-Rhodamine in the cells was estimated by fluorescent microscopy (587 nm) 48 hours later (original magnification: upper panel, ×100; lower panel, ×200) (A). The effect of p85α siRNA on BrdU incorporation of isolated acinar cells was measured (B). One day after transfection of p85α siRNA, cells were treated with IGF-1 (10 nmol/L); BrdU was added to the culture medium 6 hours after IGF-1 administration, and cells were harvested 18 hours later. Values are mean ± SEM, n = 5. *P < .05 compared with vehicle treatment of cells transfected with control siRNA; †P < .05 compared with IGF-1 treatment of cells transfected with control siRNA. The effect of p85α siRNA on PI3K activation in isolated acinar cells after IGF-1 administration was assessed by Western blot and densitometric analysis (C and D). p85α siRNA was transfected 1 day after isolation. Twenty-four hours after transfection, cells were stimulated with IGF-1 (10 nmol/L) for 1 hour and harvested, and protein was extracted for determination of p85α, phosphorylated Akt (pAkt), and phosphorylated ERK (pERK) expression (C and D, upper panels). p85α and total pERK (phosphorylated ERK1 and ERK2) were normalized by the corresponding β-actin density, and pAkt was normalized by the corresponding total Akt density (C and D, bottom panels). Similar results were obtained in 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions