Adaptive β-Cell Neogenesis in the Adult Mouse in Response to Glucocorticoid-Induced Insulin Resistance
Article Figures & Tables
Figures
- Figure 1
Metabolic parameters and pancreatic analysis of mice treated with CORT for 8 weeks. A: Insulin tolerance test was carried out in mice treated with CORT (n = 8) or VEH (n = 7) that were subjected to a 6-h fast and injected with insulin (1 IU/kg). Results are expressed as the percentage of basal glycemia. B: IPGTT was performed after an 18-h fast. Glucose was injected (2 g/kg) into mice treated with CORT (n = 7) or VEH (n = 10). AUCs are shown in the inset. C: Plasma insulin levels during IPGTT (at time 0 [T0] and T15 after glucose challenge) in mice treated with CORT (n = 7) or VEH (n = 6). D: Insulin secretion was tested in response to 2.8 mmol/L glucose, 16.7 mmol/L glucose, or 50 mmol/L KCl and measured in isolated pancreatic islets from VEH-treated (n = 4) and CORT-treated (n = 4) mice. E and F: Representative images of insulin immunostaining of pancreatic sections of mice treated with VEH or CORT for 8 weeks. Counterstaining was performed using hematoxylin. Magnification is ×2.5. G–L: Pancreatic β-cell fraction, β-cell mass, mean islet size, islet density, islet distribution, and individual β-cell area after insulin or glucagon immunostaining of eight pancreatic slides from each animal after 8 weeks of treatment with CORT (n = 7) or VEH (n = 7). M: Percentage of α- or β-cells within islets after insulin and glucagon immunofluorescence on pancreatic slides after 8 weeks of treatment with CORT (n = 5) or VEH (n = 5). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 CORT vs. VEH mice; ####P < 0.001 VEH 16.7 mmol/L glucose or VEH 50 mmol/L KCl vs. VEH 2.8 mmol/L glucose.
- Figure 2
Pancreatic adaptation throughout CORT administration. A–D: Pancreatic β-cell fraction, β-cell mass, mean islet size, and islet density was quantified after insulin immunostaining of eight pancreatic slides from each animal after 1 (n = 3 in VEH and CORT), 4 (n = 6 in VEH and n = 5 in CORT), 6 (n = 3 in VEH and CORT), or 8 (n = 7 in VEH and CORT) weeks of treatment. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 CORT vs. VEH; ##P < 0.01 comparing CORT-treated mice at various points of treatment.
- Figure 3
The β-cell mass adaptation is reversible and can be reinduced. A: Scheme of the protocol. During 4 weeks, mice were treated with CORT or VEH (stage I). Mice were then subjected to a 4-week washout period (stage II). Finally, mice were again treated with CORT or VEH for 4 weeks (stage III). At the end of each stage, pancreata were dissected and analyzed. B–E: Pancreatic β-cell fraction, β-cell mass, mean islet size, and islet density were quantified after insulin immunostaining of eight pancreatic slides from each animal. Number of mice treated with CORT were five after the first treatment, three after the washout, and three after the retreatment and of those treated with VEH, five after the first treatment, three after the washout, and three after the retreatment; values are fold increase relative to the corresponding group of mice treated with VEH. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 CORT vs. VEH; $P < 0.05, $$P < 0.01 CORT vs. CORT and then subjected to washout; ##P < 0.01 CORT retreatment vs. CORT during 4 weeks.
- Figure 4
Chronic CORT treatment is associated with signs of β-cell neogenesis. A–C: mRNA levels in isolated adult pancreatic islets from mice treated with VEH or CORT for 8 weeks, including transcription factors involved in pancreatic development and endocrine cell maturation (A), genes encoding proteins involved in hormone secretion (B), and Ki67 islets (C) from mice treated with VEH (n = 3) or CORT (n = 3). D: Percentage of cells positive for insulin and BrdU within insulin-positive cells in pancreas from mice treated with VEH (n = 5) or CORT (n = 5). E and F: Images of coimmunostaining for insulin (red) and pancytokeratin (brown) performed on pancreatic sections from mice treated with CORT for 8 weeks (arrow points to β-cells in the ductal epithelium). Magnification is ×20. G and H: Quantification of ducts containing insulin cells and pancreatic islets adjacent to ducts in pancreata from mice treated with VEH (n = 3 at 1 week and n = 4 at 8 weeks) or CORT (n = 3 at 1 week and n = 4 at 8 weeks). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 CORT vs. VEH mice; #P < 0.05 CORT at 8 weeks vs. CORT at 1 week. d, duct; UA, arbitrary unit.
- Figure 5
New β-cells did not derive from Sox9- or Ngn3-positive cells after CORT treatment. A: Schematic of Sox9CreERTM;ROSA26YFP mice generation (left) and the lineage-tracing experiment (right). TMX was administered by four daily injections during the 1st week. After 3 weeks, CORT or VEH was administrated during 3 weeks. B–D: Pancreatic β-cell fraction, β-cell mass, and islet density were calculated by morphometrical analysis in VEH-treated (n = 5) and CORT-treated (n = 8) mice. E and F: Representative graphs of flow cytometry on the basis of FITC-A and cellular granulation (forward-scattered light [FSC]-A) of trypsinized cells from islets isolated from a mouse treated with VEH or CORT. G: Quantification of YFP-positive cells in VEH TMX (n = 3) and CORT TMX (n = 4) mice. H: Schematic of Ngn3CreERTM;ROSA26YFP mice generation (left) and the lineage-tracing experiment (right). TMX was administered by injection twice a week during 3 weeks simultaneously with CORT or VEH treatment. Treatment was continued during 3 weeks after stopping TMX injections. I–K: Pancreatic β-cell fraction, β-cell mass, and islet density were calculated by morphometrical analysis in VEH-treated (n = 3) and CORT-treated (n = 3) mice. L and M: Representative graphs of flow cytometry on the basis of FITC-A and cellular granulation (FSC-A) of trypsinized cells from islets isolated from a mouse treated with VEH or CORT. N: Quantification of YFP-positive cells in VEH TMX (n = 3) and CORT TMX (n = 4) mice. Data are mean ± SD. *P < 0.05, **P < 0.01 CORT vs. VEH mice.
- Figure 6
β-Cell neogenesis did not involve the ERα or MR. A–D: Pancreatic β-cell fraction, β-cell mass, mean islet size, and islet density were calculated by morphometrical analysis after insulin immunostaining of pancreatic sections from ERKO mice treated with VEH (n = 3) or CORT (n = 3). E–H: Pancreatic β-cell fraction, β-cell mass, mean islet size, and islet density were calculated by morphometrical analysis after insulin immunostaining of pancreatic sections from mice treated with VEH and CANRE (n = 3) or CORT and CANRE (n = 3). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 CORT vs. VEH mice.
- Figure 7
CORT treatment led to partial β-cell regeneration after chemical depletion. A: Blood glucose levels in mice after STZ injection (day 0) and treatment with VEH (n = 10) or CORT (n = 10). The arrow indicates the start of CORT or VEH treatment. B–E: Pancreatic β-cell fraction, β-cell mass, islet density, and mean islet size were calculated by morphometrical analysis after insulin immunostaining of pancreatic sections from mice injected with STZ and treated with VEH (n = 3) or CORT (n = 4). F: Blood insulin levels at the end of the treatment in mice injected with STZ and treated with VEH (n = 10) or CORT (n = 11). Dashed lines represent mean value for mice without STZ injection (n = 3–10 per condition). Data are mean ± SD. **P < 0.01, ***P < 0.001 CORT vs. VEH mice.
- Figure 8
Serum from CORT-treated mice stimulates β-cell neogenesis in embryonic pancreatic buds. A–J: Immunofluorescence for insulin and DAPI nuclear staining on representative sections from pancreatic buds cultured 7 days with 10% of VEH serum (A and F), 10% of CORT serum (B and G), 10−7 mol/L CORT alone (C and H), 0.4 ng/mL insulin (D and I), or 10−7 mol/L CORT and 0.4 ng/mL insulin (E and J) supplemented in the serum of VEH mice. Magnification is ×10. K: β-Cell fraction in embryonic pancreatic buds after 7 days of culture in the same conditions with 10% of VEH serum (n = 6), 10% of CORT serum (n = 4), 10−7 mol/L CORT alone (n = 3), 0.4 ng/mL insulin (n = 3), or 10−7 mol/L CORT and 0.4 ng/mL insulin (n = 3) supplemented in the serum of VEH mice. L: mRNA levels coding proteins of endocrine cell differentiation and maturation in pancreatic buds cultured 7 days in the presence of 10% of VEH serum (n = 3) or CORT serum (n = 3). Data are mean ± SD. *P < 0.05 CORT serum–treated buds vs. VEH serum–treated buds; #P < 0.05 CORT-treated buds vs. VEH serum–treated buds; $P < 0.05 CORT + insulin–treated buds vs. VEH serum–treated buds. UA, arbitrary unit.
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