Limits...
Ectopic expression of E2F1 stimulates beta-cell proliferation and function.

Grouwels G, Cai Y, Hoebeke I, Leuckx G, Heremans Y, Ziebold U, Stangé G, Chintinne M, Ling Z, Pipeleers D, Heimberg H, Van de Casteele M - Diabetes (2010)

Bottom Line: At 48 h after infection, the total beta-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated.Conditional overexpression of E2F1 in mice resulted in a twofold increase of beta-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase beta-cell mass.In vivo E2F1 expression significantly increases the insulin content and function of adult beta-cells, making it a strategic target for therapeutic manipulation of beta-cell function.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium.

ABSTRACT

Objective: Generating functional beta-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G(1)- to S-phase transition during the cycling of many cell types and is required for pancreatic beta-cell growth and function. However, the consequences of overexpression of E2F1 in beta-cells are unknown.

Research design and methods: The effects of E2F1 overexpression on beta-cell proliferation and function were analyzed in isolated rat beta-cells and in transgenic mice.

Results: Adenovirus AdE2F1-mediated overexpression of E2F1 increased the proliferation of isolated primary rat beta-cells 20-fold but also enhanced beta-cell death. Coinfection with adenovirus AdAkt expressing a constitutively active form of Akt (protein kinase B) suppressed beta-cell death to control levels. At 48 h after infection, the total beta-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated. Conditional overexpression of E2F1 in mice resulted in a twofold increase of beta-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase beta-cell mass. Glucose-challenged insulin release was increased, and the mice showed protection against toxin-induced diabetes.

Conclusions: Overexpression of E2F1, either in vitro or in vivo, can stimulate beta-cell proliferation activity. In vivo E2F1 expression significantly increases the insulin content and function of adult beta-cells, making it a strategic target for therapeutic manipulation of beta-cell function.

Show MeSH

Related in: MedlinePlus

Effect of Akt expression on cell death and proliferation in isolated β-cells overexpressing E2F1. Data are presented as means ± SEM. A: Immunocytochemical detection of constitutively active (hemagglutinin [HA] tagged) Akt using a HA-specific antibody. Akt was localized at the cell membrane of rat β-cells after their infection with adenovirus AdAkt, suggesting myristoylation of Akt. B: BrdU incorporation (n = 3) and cell survival (n = 4) after co infection of β-cell cultures with both AdAkt and AdE2F1 at the indicated MOI, and as studied in Fig. 2. C: Quantitation of P-HH3+ cells in β-cell cultures infected with AdAkt and/or AdE2F as indicated (n = 4, *P < 0.05, ***P < 0.001 vs. AdNull). Results for MIN6 cell cultures are shown for reference. D: Total β-cell numbers determined by CyQuant assay at 48 and 96 h after viral infection with AdNull or AdE2F (MOI 20) or with AdE2F+AdAkt (both MOI 10). Assays were performed with ∼13,000 cells as starting β-cell number (n = 6, **P < 0.01, ***P < 0.001 vs. no virus). (A high-quality digital color representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874704&req=5

Figure 3: Effect of Akt expression on cell death and proliferation in isolated β-cells overexpressing E2F1. Data are presented as means ± SEM. A: Immunocytochemical detection of constitutively active (hemagglutinin [HA] tagged) Akt using a HA-specific antibody. Akt was localized at the cell membrane of rat β-cells after their infection with adenovirus AdAkt, suggesting myristoylation of Akt. B: BrdU incorporation (n = 3) and cell survival (n = 4) after co infection of β-cell cultures with both AdAkt and AdE2F1 at the indicated MOI, and as studied in Fig. 2. C: Quantitation of P-HH3+ cells in β-cell cultures infected with AdAkt and/or AdE2F as indicated (n = 4, *P < 0.05, ***P < 0.001 vs. AdNull). Results for MIN6 cell cultures are shown for reference. D: Total β-cell numbers determined by CyQuant assay at 48 and 96 h after viral infection with AdNull or AdE2F (MOI 20) or with AdE2F+AdAkt (both MOI 10). Assays were performed with ∼13,000 cells as starting β-cell number (n = 6, **P < 0.01, ***P < 0.001 vs. no virus). (A high-quality digital color representation of this figure is available in the online issue.)

Mentions: Purified rat β-cells were transduced with AdAkt to express constitutively active Akt/protein kinase B (Fig. 3A). In coinfection experiments with AdE2F1 and AdAkt, the E2F1-induced β-cell death at 48 h was nearly completely prevented (Fig. 3B). Infection with AdAkt alone did not increase β-cell proliferation (Fig. 3B), but its coinfection with AdE2F1 increased the E2F1-induced BrdU incorporation (Figs. 2A and 3B). The fraction of P-HH3+ β-cells also enlarged upon combined expression of E2F1 and Akt, compared with E2F1 alone (Fig. 3C). Expression of active E2F1 and Akt resulted in a net increase of β-cell numbers in vitro. The total cell number increased by 38 and 46% at 48 h, and by 39 and 72% at 96 h after infection with AdE2F1 and AdE2F1+AdAkt, respectively (Fig. 3D). Thus, in contrast to expression of E2F1 alone, combined expression of E2F1 and Akt continued to increase the absolute number of β-cells in vitro for more than 48 h.


Ectopic expression of E2F1 stimulates beta-cell proliferation and function.

Grouwels G, Cai Y, Hoebeke I, Leuckx G, Heremans Y, Ziebold U, Stangé G, Chintinne M, Ling Z, Pipeleers D, Heimberg H, Van de Casteele M - Diabetes (2010)

Effect of Akt expression on cell death and proliferation in isolated β-cells overexpressing E2F1. Data are presented as means ± SEM. A: Immunocytochemical detection of constitutively active (hemagglutinin [HA] tagged) Akt using a HA-specific antibody. Akt was localized at the cell membrane of rat β-cells after their infection with adenovirus AdAkt, suggesting myristoylation of Akt. B: BrdU incorporation (n = 3) and cell survival (n = 4) after co infection of β-cell cultures with both AdAkt and AdE2F1 at the indicated MOI, and as studied in Fig. 2. C: Quantitation of P-HH3+ cells in β-cell cultures infected with AdAkt and/or AdE2F as indicated (n = 4, *P < 0.05, ***P < 0.001 vs. AdNull). Results for MIN6 cell cultures are shown for reference. D: Total β-cell numbers determined by CyQuant assay at 48 and 96 h after viral infection with AdNull or AdE2F (MOI 20) or with AdE2F+AdAkt (both MOI 10). Assays were performed with ∼13,000 cells as starting β-cell number (n = 6, **P < 0.01, ***P < 0.001 vs. no virus). (A high-quality digital color representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874704&req=5

Figure 3: Effect of Akt expression on cell death and proliferation in isolated β-cells overexpressing E2F1. Data are presented as means ± SEM. A: Immunocytochemical detection of constitutively active (hemagglutinin [HA] tagged) Akt using a HA-specific antibody. Akt was localized at the cell membrane of rat β-cells after their infection with adenovirus AdAkt, suggesting myristoylation of Akt. B: BrdU incorporation (n = 3) and cell survival (n = 4) after co infection of β-cell cultures with both AdAkt and AdE2F1 at the indicated MOI, and as studied in Fig. 2. C: Quantitation of P-HH3+ cells in β-cell cultures infected with AdAkt and/or AdE2F as indicated (n = 4, *P < 0.05, ***P < 0.001 vs. AdNull). Results for MIN6 cell cultures are shown for reference. D: Total β-cell numbers determined by CyQuant assay at 48 and 96 h after viral infection with AdNull or AdE2F (MOI 20) or with AdE2F+AdAkt (both MOI 10). Assays were performed with ∼13,000 cells as starting β-cell number (n = 6, **P < 0.01, ***P < 0.001 vs. no virus). (A high-quality digital color representation of this figure is available in the online issue.)
Mentions: Purified rat β-cells were transduced with AdAkt to express constitutively active Akt/protein kinase B (Fig. 3A). In coinfection experiments with AdE2F1 and AdAkt, the E2F1-induced β-cell death at 48 h was nearly completely prevented (Fig. 3B). Infection with AdAkt alone did not increase β-cell proliferation (Fig. 3B), but its coinfection with AdE2F1 increased the E2F1-induced BrdU incorporation (Figs. 2A and 3B). The fraction of P-HH3+ β-cells also enlarged upon combined expression of E2F1 and Akt, compared with E2F1 alone (Fig. 3C). Expression of active E2F1 and Akt resulted in a net increase of β-cell numbers in vitro. The total cell number increased by 38 and 46% at 48 h, and by 39 and 72% at 96 h after infection with AdE2F1 and AdE2F1+AdAkt, respectively (Fig. 3D). Thus, in contrast to expression of E2F1 alone, combined expression of E2F1 and Akt continued to increase the absolute number of β-cells in vitro for more than 48 h.

Bottom Line: At 48 h after infection, the total beta-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated.Conditional overexpression of E2F1 in mice resulted in a twofold increase of beta-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase beta-cell mass.In vivo E2F1 expression significantly increases the insulin content and function of adult beta-cells, making it a strategic target for therapeutic manipulation of beta-cell function.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium.

ABSTRACT

Objective: Generating functional beta-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G(1)- to S-phase transition during the cycling of many cell types and is required for pancreatic beta-cell growth and function. However, the consequences of overexpression of E2F1 in beta-cells are unknown.

Research design and methods: The effects of E2F1 overexpression on beta-cell proliferation and function were analyzed in isolated rat beta-cells and in transgenic mice.

Results: Adenovirus AdE2F1-mediated overexpression of E2F1 increased the proliferation of isolated primary rat beta-cells 20-fold but also enhanced beta-cell death. Coinfection with adenovirus AdAkt expressing a constitutively active form of Akt (protein kinase B) suppressed beta-cell death to control levels. At 48 h after infection, the total beta-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated. Conditional overexpression of E2F1 in mice resulted in a twofold increase of beta-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase beta-cell mass. Glucose-challenged insulin release was increased, and the mice showed protection against toxin-induced diabetes.

Conclusions: Overexpression of E2F1, either in vitro or in vivo, can stimulate beta-cell proliferation activity. In vivo E2F1 expression significantly increases the insulin content and function of adult beta-cells, making it a strategic target for therapeutic manipulation of beta-cell function.

Show MeSH
Related in: MedlinePlus