Connective Tissue Growth Factor Modulates Adult β-Cell Maturity and Proliferation to Promote β-Cell Regeneration in Mice

CTGF treatment after 50% β-cell destruction elicits improved α-/β-cell ratios. A and B: Example of actual raw data obtained: the numbers of ins⁺ (green) and glucagon⁺ (red) cells were quantified. Under normal conditions, β-cells constitute ∼75% of the total number of counted cells (Control). After DT injection, 50% of β-cells are ablated, while α-cells remained unchanged (Ablation). This results in β-cells accounting for ∼60% of counted cells. In Ablation+CTGF animals, β-cells account for ∼70% of counted cells. C: Quantification of α-/β-cell ratios after 2 (left) and 4 (right) weeks of Dox administration. n = 8. **P < 0.01, ***P < 0.001, ****P < 0.0001.

CTGF does not mediate β-cell regeneration via hypertrophy, neogenesis, increased vascularization, or E-cadherin. 1. Control; 2. CTGF; 3. Ablation; and 4. Ablation+CTGF. β-Cell size after either 2 (A) or 4 (A′) weeks of CTGF treatment. Average number of islets per animal after 2 (B) or 4 (B′) weeks of CTGF induction. Number of small ins⁺ clusters after CTGF induction for 2 (C) or 4 (C′) weeks. (D and E) Islet vascularization quantification, as assessed by immunolabeling for blood vessels (PECAM; red) within islets (insulin; green). n = 8. (F) No change in E-cadherin mRNA expression as assessed by qRT-PCR. Real-time reactions were carried out in technical duplicates on a CFX Real-Time PCR Detection system (Bio-Rad). (G) No alteration in the percentage of proliferating β-cells either completely (gray bars) or incompletely surrounded (white bars) by E-cadherin. (H) Representative images of Ablation+CTGF islets at 2 days Dox. Immunolabeling for insulin (green), E-cadherin (red), and Ki67 (yellow). Yellow arrowheads indicate a proliferating β-cell surrounded by E-cadherin localized to the membrane. White arrowheads indicate a proliferating β-cell with incomplete E-cadherin membrane localization. A cell was considered E-cadherin positive if >75% of the cell membrane displayed E-cadherin immunolabeling. For qRT-PCR, n = 3 for CTGF and n = 4 for Control, Ablation, and Ablation+CTGF. For proliferation analysis, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001.

Priming islets with CTGF does not improve β-cell survival or enhance β-cell proliferation and mass. A: Experimental outline. Mice were administered 2 mg/mL of Dox in 2% Splenda in drinking water. DT (126 ng) was given intraperitoneally three times at 8 weeks of age. Cohorts are as follows: 1. Control; 2. CTGF; 3. Ablation; and 4. Ablation+CTGF. B and B′: β-Cell survival as assessed by quantifying the percentage of TUNEL⁺ β-cells. The percentage of apoptotic or necrotic β-cells was determined by dividing the number of TUNEL/insulin double-positive cells by the total number of insulin cells. A minimum of 4,000 cells were counted. C: Representative images of β-cell death in prophylactic- (left) and therapeutic-treated (right) islets (green, insulin; red, TUNEL; blue, DAPI; red arrowheads, proliferating ins⁺ cells). D: β-Cell mass. E: β-Cell proliferation. For priming time point, n = 5. For 2-day time point, n = 6. **P = 0.0027. N.S., not significant.

β-Cell proliferation characteristics in response to ablation and CTGF. Cohorts: 1. Control; 2. CTGF; 3. Ablation; and 4. Ablation+CTGF. β-Cell maturation (A) and proliferative state (B). Mature β-cells (red bars) and immature β-cells (yellow bars). Detection of Ki67 required direct conjugation to a Cy5 fluorophore using the Zenon conjugation kit according to the manufacturer’s instructions (Life Technologies). A minimum of 4,000 cells were counted. (C–C″) Representative images of Ablation+CTGF islets at 2 days' CTGF. Insulin (green), Ki67 (yellow), and MafA (red). (C) Yellow arrowheads, proliferating β-cells. (C′) Red arrowheads, mature β-cell; white arrowhead, immature β-cell. (C″) Orange arrowhead, mature proliferating β-cell; yellow arrowhead, immature proliferating β-cell. (D) Experimental outline for double uridine analog labeling. Mice were administered 2 mg/mL of Dox in 2% Splenda in drinking water. DT (126 ng) was given intraperitoneally three times at 8 weeks of age. Uridine analogs (CldU or IdU; both from Sigma-Aldrich) were administered at 1 mg/mL in Dox-treated drinking water. β-Cell replication during the first 2 days (red bars), last 5 days (green bars), and both labeling periods (yellow bars) at 2 (E) and 4 (F) weeks was determined. A minimum of 4,000 cells were counted. The percentage of β-cells undergoing replication during both labeling periods was obtained by dividing the number of CldU/IdU/Insulin triple-positive cells by the total number of ins⁺ cells. The percentage of dual-labeled β-cells at 4 weeks was significantly higher than at 2 weeks (demarked by #). Ratios between subgroups (i.e., CldU⁺:IdU⁺, CldU:CldU⁺;IdU⁺, and IdU:CldU⁺; IdU⁺) were determined. (G–G″) Representative images at 4 weeks. Replicating β-cells in the first 2 days incorporated CldU (red arrowheads) and in the last 5 days incorporated IdU (green arrowheads). Replicating cells in both periods incorporated CldU and IdU (yellow arrowheads). n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #P = 0.0414. N.S., not significant.

CTGF induces β-cell proliferation ex vivo and expression of genes involved in key signaling pathways and cell-cycle regulators. Dispersed islets labeled for insulin and Ki67, with representative images of the vehicle-treated (0) (A) and 250 ng/mL rhCTGF-treated (B) islets. Pink arrowheads: proliferating β-cells. (C) Quantification of β-cell proliferation (dual insulin/Ki67-positive cells) for various concentrations of rhCTGF. (D–F) Gene expression analysis on whole islets using TaqMan Universal PCR Master Mix (with UNG; Applied Biosystems). Islets isolated from animals with/without β-cell ablation ± CTGF treatment for 2 days. (E′ and F′) Islets from wild-type animals treated ex vivo with rhCTGF for 4 days. Islet cell markers (D), cell-cycle regulators (E and E′), and signaling pathways and growth factors (F and F′). All samples were run in duplicate. n = 4 for D–F and n = 3 for E′ and F′. *Compared with Control, #compared with CTGF, ^compared with ablation. *,^,#P < 0.5; **,##P < 0.01; ***,###P < 0.001; ****,^^^^P < 0.0001.