Fig 1.
PTH1 receptor levels increase in islets of PPx mice.
(A) PPx experimental design for analysis of glucose and beta cell homeostasis. Eight week-old male Balb/c mice underwent PPx or sham-operation at day 0, and were injected s.c. for 5 days/week with vehicle (veh) or PTHrP(1–36) (P36) at 160μg/kg body weight, resulting in four groups of mice: sham-veh, sham-PTHrP, PPx-veh, and PPx-PTHrP. The four groups of mice were treated for 7 days (n = 5 mice/group), 30 days (n = 4 mice/group), or 90 days (n = 6 mice/group). IPGTT was performed at days 25 and 79 (solid arrows) and ITT at days 16 and 74 (dotted arrows) on the four groups of mice. Representative western blot analysis of (B) PTHrP, and (D) PTH1R, with tubulin as loading control, in islets isolated from mice treated for 7 days. Western blot analysis was performed on a separate additional group of mice from those shown in Fig 1A. Quantification of the expression of (C) PTHrP/tubulin, and (E) PTH1R/tubulin ratios in the four groups of mice: sham-veh (black bars), sham-PTHrP (stippled bars), PPx-veh (grey bars) and PPx-PTHrP (horizontally-stippled bars) (n = 9–12 mice/group). Significance of * p<0.02 by ANOVA versus corresponding sham controls.
Fig 2.
Body weight and glucose homeostasis.
Average (A) body weight and (B) blood glucose values at 30-day intervals in the sham (solid line) -veh (diamond), -PTHrP (square), and PPx (dotted line) -veh (triangle), -PTHrP (circle) mice, treated for 90 days (n = 6 mice/group). Fasting plasma glucose at (C) day 25 and (D) day 79, plasma insulin at (E) day 30 and (F) day 90 in the four groups of mice treated for 30 days (C, E, n = 4 mice/group) or 90 days (D, F, n = 5 mice/group), as represented in Fig 1.
Fig 3.
(A,C) IPGTT, (B,D) and area under the curve (AUC) for IPGTT performed on (A,B) day 25 and (C,D) day 79. (E,G) ITT, (F,H) and AUCs for ITT performed on (E,F) day16 and (G,H) day 74, in the four groups of mice, as represented in Fig 2 for the line graphs, and in Fig 1 for the bar graphs. The day 16 and day 25 procedures were performed on the 30 day treatment groups (n = 4 mice/group), and the day 74 and day 79 procedures were performed on the 90 day treatment groups (n = 6 mice/group). Y-axis depicts fold-change in plasma glucose relative to basal (time 0) taken as 100%. There was no significant difference in the basal plasma glucose among the four groups in all experiments. Significance of * p≤0.02 versus sham-Veh and sham-PTHrP groups by ANOVA.
Fig 4.
PTHrP(1–36) further increases beta cell proliferation induced by PPx.
(A) Pancreatic sections from sham or PPx mice treated for 7 days with vehicle (veh) or PTHrP(1–36) (P36) and stained for insulin (green) and BrdU (red), with BrdU-positive beta cells marked by arrows. Quantification of the percentage of BrdU-positive beta cells after (B) 7 days (n = 5 mice/group), (C) 30 days (n = 4 mice/group) and 90 days (n = 6 mice/group) of treatment in the four groups of mice, as represented in Fig 1. Average of 1032±61 beta cells/pancreas was counted. Significance of * p<0.01 versus corresponding sham groups, and # p<0.05 versus corresponding Veh-treated groups, by ANOVA.
Fig 5.
Exocrine cell proliferation, islet number and size distribution.
(A) Quantification of the percentage of pHH3-positive exocrine cells in sham and PPx mice treated with vehicle (Veh) or PTHrP(1–36) (P36) for 7 days (D7, n = 5 mice/group), 30 days (D30, n = 4 mice/group) and 90 days (D90, n = 6 mice/group), as represented in Fig 1. Average of 3921±133 exocrine cells/pancreas was counted. (B) Total islet number/pancreatic area, (C) islet size distribution, counting average number of islets in size ranges from <25 to >200μm diameter/ pancreatic area, and (D) average number of singlet and doublet beta cell-containing islets/ pancreatic area, in the four groups of mice treated for 90 days, as represented in Fig 1 (n = 6 mice/group). Significance of * p<0.05 versus sham-veh group by ANOVA.
Fig 6.
PTHrP(1–36) significantly enhances beta cell mass at 90 days in PPx mice.
Histomorphometric analysis of pancreata in sham-veh (black bars) mice treated for 7 (D7) and 90 days (D90), and PPx-veh (grey bars) and PPx-PTHrP (P36) (horizontally-stippled bars) mice treated for 7 (D7, n = 5 mice/group), 30 (D30, n = 4 mice/group), and 90 (D90, n = 6 mice/group) days, measuring (A) pancreatic weight, (B) ratio of beta cell area/pancreatic area, and (C) beta cell mass. Significance of * p<0.05 versus sham-veh mice at D7, # p<0.05 versus sham-veh mice at D90, and ^ p≤0.05 versus PPx-PTHrP mice at D90, by ANOVA.