Exendin-4 Stimulation of Cyclin A2 in β-Cell Proliferation
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Figures
- FIG. 1.
Exendin-4 treatment and CBP-S436A+/− mutation result in increased β-cell proliferation and islet size. Representative fluorescence photomicrographs with pseudo-coloring of islet sections of C57Bl/6 littermates treated with either vehicle (PBS) (A) or exendin-4 (B) for 5 days and of wild-type (G) and CBP-S436A+/− (H) littermates. A, B, G, and H are partitioned as follows: top left, DAPI nuclear stain (blue); top right, fluorescein isothiocyanate (FITC) immunostain for insulin (green); bottom left, Cy3 immuno-stain for Ki67 (red); and bottom right, a merged image of the three preceding panels. Compared with controls (A), exendin-4–treated animals (B) exhibit increased islet area (C), β-cell proliferation as assessed by Ki67 in insulin-positive β-cells (D), and β-cells per islet (E) and unchanged calculated β-cell size (F). Similarly, compared with wild-type controls (G), CBP-S436A+/− mice (H) exhibit increased islet area (I), β-cell proliferation as assessed by Ki67 in insulin-positive β-cells (J), and β-cells per islet (K) and unchanged calculated β-cell size (L). Means ± SE in graphs are provided above the corresponding bars. *Significant difference from the corresponding control with P < 0.05. (Please see http://dx.doi.org/10.2337/db07-1541 for a high-quality digital representation of this figure.)
- FIG. 2.
A: Exendin-4 treatment increases PDX-1, cyclin A2, and Skp-2 and decreases p27 islet protein levels. Islets isolated from C57Bl/6 mice treated with either vehicle or exendin-4 for 5 days were analyzed for protein levels of cyclin A2 (A), cyclin D1 (B), cyclin D2 (C), PDX-1 (D), p27 (E), and Skp2 (F). B: CBP-S436A+/− islets exhibit increased cyclin A2 and decreased p27 but unchanged PDX-1 and Skp2 protein levels. Islets isolated from wild-type and CBP-S436A+/− littermates were analyzed for protein levels of cyclin A2 (G), cyclin D1 (H), cyclin D2 (I), PDX-1 (J), p27 (K), and Skp2 (L). Representative immunoblots are shown together with corresponding immunoblot for actin. Corresponding graphs below the immunoblots depict densitometric analysis of antigen band in relation to the corresponding actin band. Data are normalized to the first control densitometry, which is depicted as 1.0 in the first column of the graph. Numbers indicate absolute value of the corresponding column immediately below them.
- FIG. 3.
A: Exendin-4 stimulates cyclin A2 and PDX-1 transcript in ex vivo–cultured mouse islets. Cultured isolated C57Bl/6 mouse islets were exposed to PBS or 20 nmol/l exendin-4 for 0, 2, 4, 6, 8, and 12 h. Transcript levels of cyclin A2 (A), cyclin D2 (B), and PDX-1 (C) were measured by quantitative RT-PCR. Transcripts were normalized to housekeeping gene 36B4 at corresponding time point and are provided in Table 2 in fold change over baseline (time 0 h) as means ± SE of quadruplicate experiments. *P < 0.05 and statistical significant change. B: Exendin-4 stimulates cyclin A2 and PDX-1 protein in ex vivo–cultured mouse islets. Cultured isolated C57Bl/6 mouse islets were exposed to PBS or 20 nmol/l exendin-4 for 0, 2, 4, 6, 8, and 12 h. Representative immunoblots for cyclin A2, cyclin D2, and PDX-1 are shown. Densitometric analysis of relative protein levels normalized to actin at corresponding time points are provided in Table 2 and depicted as fold change over baseline (0 h) as means ± SE of triplicate studies. *P < 0.05 and statistical significant change.
- FIG. 4.
Exendin-4 stimulates cyclin A2 transcription via the cAMP-PKA-CREB-CBP pathway. Min6 cells transiently transfected with −800-bp 5′-untranslated region of the murine cyclin A2 gene linked to a luciferase cDNA (−800-bp cyclin A2-LUC) or with the same vector carrying a mutation in the CRE (−800-bp ΔCRE cyclin A2-LUC). Exendin-4 (20 nmol/l for 4 h) stimulates the intact reporter (A) but not the reporter with the mutant CRE (B). Co-transfected expression vector to overexpress active PKAcs stimulates the intact (C) but not the CRE mutant promoter-reporter (D). Short-term (for 20 min before exendin-4 treatment) pharmacological inhibition of cAMP (H89 and PKI), but not other intracellular pathways (U0126 and LY294002) stimulated by exendin-4, inhibits exendin-4 effects on cyclin A2-LUC activity (E and F). Co-transfected expression vector to overexpress CREB stimulates and dominant-negative A-CREB suppresses −800-bp cyclin A2-LUC activity (G). Co-transfected expression vectors to overexpress CREB and CBP-S4356A mutant stimulate cyclin A2-LUC activity more potently than CREB with wild-type CBP (CBPwt) (H). Means ± SE in graphs are provided above the corresponding bars. *Significant difference from the corresponding control with P < 0.05.
- FIG. 5.
Knockdown of cyclin A2 in Min6 cells reduces proliferation rate. siRNA-mediated knockdown of cyclin A2 in Min6 cells is ascertained by immunoblot for cyclin A2. A representative immunoblot (A, top) with densitometric analysis of cyclin A2 relative to actin levels is shown (A, bottom). Scrambled siRNA does not change cyclin A2 levels (far right column). Data are normalized to the first control densitometry, which is depicted as 1.0 in the first column of the graph. Numbers indicate absolute value of the corresponding column immediately below them. B: Proliferation rate of Min6 cells is reduced by siRNA knockdown of cyclin A2, whereas scrambled siRNA does not influence proliferation rates. Graph depicts cell number at 48 h normalized to start (0 h) after no siRNA (left column), cyclin A2-specific (middle column), or scrambled siRNA (right column), respectively. C: Proliferation rate of Min6 cells is increased by exendin-4 (4 h) treatment and cyclin A2-specific, but not scrambled siRNA, abolishes the exendin-4 stimulation of cell proliferation. Graph depicts cell number at 48 h normalized to start (0 h) after no exendin-4, exendin-4 only, exendin-4 plus cyclin A2-specific siRNA, or scrambled siRNA, respectively. Means ± SE in graphs are provided above the corresponding bars. *Significant difference from the corresponding control with P < 0.05.
- FIG. 6.
Lentiviral expression of cyclin A2 in mouse islets stimulates β-cell proliferation. A–D: Representative pseudo-colored digitally imaged sections of islets infected for 48 h with control (CMV-; E1fα-EGFP) (A and D) or cyclin A2 expressing (CMV-cyclin A2; E1fα-EGFP) (B and E) lentivirus. Sections (Nomarksi image shown in top left subpanels, respectively) are co-immunostained and pseudo-colored for nuclei (DAPI, blue; top right subpanel), virally expressed EGFP (cyan; middle left subpanel), insulin (Cy5, red; bottom left subpanel), proliferation markers Ki67 (A and B) or phospho-histone-3B (D and E) (yellow; middle right subpanel). The bottom right subpanels depict the corresponding merged image of the fluorescence channels within the respective panels. White arrows indicate proliferation marker positive and positive lentivirus infection. Yellow arrows indicate proliferation marker negative, lentivirus postive infection. Quantitaive analysis of proliferation markers Ki67 (C) or phospho-histone 3B (F) in β-cells of mouse islets infected for 48 h with control (CMV-; E1fα-EGFP) or cyclin A2–expressing (CMV-cyclin A2; E1fα-EGFP) lentiviridae. β-Cells infected with cyclin A2–expressing lentivirus (▪) show significantly more Ki67 (Ki67+/EGFP+) and phospho-histone 3B (Phospho-H3B+/EGFP+) expression than β-cells infected with control virus (□) or β-cells not infected at all (Ki67+/EGFP−). Means ± SE in graphs are provided above the corresponding bars. *Significant difference from the corresponding control with P < 0.05. (Please see http://dx.doi.org/10.2337/db07-1541 for a high-quality digital representation of this figure.)
Tables
- TABLE 1
Relative changes of indicated proteins in total islet extracts in exendin-4–treated (5d) C57Bl/6 mice normalized to vehicle-treated controls and CBP-S436A normalized to wild-type littermate islet extracts
Protein Exendin-4 versus PBS P value CBP-S436A+/− versus wild type P value Cyclin A2 2.69 ± 0.17 <0.05 2.02 ± 0.20 <0.05 Cyclin B1 0.90 ± 0.20 NS 0.59 ± 0.16 NS Cyclin B2 0.87 ± 0.31 NS 1.05 ± 0.31 NS Cyclin D1 1.26 ± 0.06 NS 0.86 ± 0.15 NS Cyclin D2 1.00 ± 0.09 NS 0.98 ± 0.31 NS Cyclin D3 1.11 ± 0.12 NS 0.85 ± 0.03 NS FoxO1 0.79 ± 0.05 0.05 0.98 ± 0.20 NS p27 Not detected <0.05 Not detected <0.05 Skp2 5.01 ± 0.98 <0.05 0.80 ± 0.21 NS PDX-1 1.93 ± 0.20 <0.05 0.85 ± 0.03 NS -
Data are means ± SE; n = 4 for each parameter and group. A significant difference (P < 0.05) from the corresponding control is indicated. For details, see text.
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- TABLE 2
Exendin-4 stimulates cyclin A2 and PDX-1 in cultured C57Bl6 mouse islets
Treatment Time (h) 0 2 4 6 8 12 Transcript Cyclin A2 PBS 1 0.98 ± 0.11 1.09 ± 0.09 1.05 ± 0.04 1.06 ± 0.10 0.99 ± 0.07 Cyclin A2 Exendin-4 1 2.75 ± 0.47* 4.45 ± 0.65* 3.88 ± 0.37* 4.86 ± 0.54* 4.47 ± 0.63* Cyclin D2 PBS 1 1.09 ± 0.07 1.07 ± 0.05 1.06 ± 0.07 1.05 ± 0.09 0.93 ± 0.10 Cyclin D2 Exendin-4 1 0.99 ± 0.07 1.08 ± 0.09 1.02 ± 0.09 1.18 ± 0.11 1.0 ± 0.15 PDX-1 PBS 1 1.22 ± 0.09 0.99 ± 0.0.07 1.06 ± 0.07 1.05 ± 0.16 0.9 ± 0.16 PDX-1 Exendin-4 1 1.35 ± 0.26 1.06 ± 0.13 2.04 ± 0.67* 6.23 ± 2.88* 7.49 ± 2.97* Protein Cyclin A2 PBS 1 0.96 ± 0.38 1.13 ± 0.39 1.13 ± 0.39 1.14 ± 040 0.15 ± 0.40 Cyclin A2 Exendin-4 1 1.56 ± 0.40* 1.82 ± 0.40* 1.68 ± 0.39* 2.01 ± 0.39* 1.69 ± 0.39* Cyclin D2 PBS 1 1.09 ± 0.07 1.07 ± 0.05 1.06 ± 0.07 1.05 ± 0.09 0.93 ± 0.10 Cyclin D2 Exendin-4 1 0.99 ± 0.07 1.08 ± 0.09 1.02 ± 0.09 1.18 ± 0.11 1.0 ± 0.15 PDX-1 PBS 1 1.15 ± 0.35 1.02 ± 0.35 1.15 ± 0.35 1.14 ± 0.35 0.15 ± 0.36 PDX-1 Exendin-4 1 1.18 ± 0.37 1.55 ± 0.37 1.67 ± 0.36* 1.97 ± 0.36* 2.67 ± 0.35* -
Data are means ± SE. Relative changes of indicated transcripts and immunoblots of indicated proteins in cultured C57Bl/6 islet extracts treated with PBS or 20 nmol/l exendin-4 for 0, 2, 4, 6, 8, and 12 h (n = 4 for each parameter and group). Transcripts were normalized to housekeeping gene 36B4 transcript at corresponding time points and results are fold change over baseline (0 h). Exendin-4 stimulates an early (2–4 h) increase in cyclin A2, whereas PDX-1 transcript increases after 6–8 h. Densitometric analysis of relative protein levels normalized to actin at corresponding time points of three independent experiments are shown as fold change over baseline (0 h). Exendin-4 stimulates an early (2–4 h) increase in cyclin A2, whereas PDX-1 levels increase after 6–8 h.
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* Significant difference (P < 0.05) from the corresponding control. For details, see text and Fig. 3.
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