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The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic beta-cells.

Harb G, Vasavada RC, Cobrinik D, Stewart AF - Diabetes (2009)

Bottom Line: In vivo loss of either p107 or p130 did not affect beta-cell replication or function.Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death.Pancreas and beta-cell mass were significantly reduced in double mutants.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT

Objective: The retinoblastoma protein family (pRb, p130, p107) plays a central role in the regulation of cell cycle progression. Surprisingly, loss of pRb in the beta-cell has no discernible effect on cell cycle control. Therefore, we explored the effects of individual loss of either p130 or p107 in addition to the simultaneous loss of both pRb/p130 on the beta-cell.

Research design and methods: Adult mice deficient in either p130 or p107 or both pRb/p130 were examined for effects on beta-cell replication, function, and survival. The Cre-Lox system was also used to inactivate pRb in wild-type and p130-deficient beta-cells in vitro.

Results: In vivo loss of either p107 or p130 did not affect beta-cell replication or function. Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death. Pancreas and beta-cell mass were significantly reduced in double mutants. Despite this, overall glucose tolerance was normal, except for mild postprandial hyperglycemia. Ex vivo, acute deletion of pRb in p130-deficient beta-cells also caused a striking increase in proliferation. The combined deletion of pRb/p130 upregulated islet expression of E2F2 but not E2F1.

Conclusions: These studies define an essential role for the pocket proteins in controlling the G(1)/S transition in beta-cells. When deficient in both pRb and p130, beta-cells undergo unrestrained cell cycle reentry and activation of apoptosis. These studies underscore the central role of the pRb pathway in controlling beta-cell turnover and provide new cellular targets for beta-cell regeneration.

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Targeted disruption of the p130 gene and metabolic characterization of p130- mice. A: The p130 gene was disrupted by insertion of a targeting vector into exon 2 (17). Mutant alleles that contain the neomycin resistance gene (neo) were detected using PCR with previously described primers represented by arrows (17). B: (upper) PCR products for genomic DNA from p130 wild-type (WT), heterozygous (HT), and knockout (KO) animals. The mutant p130 gene was identified based on amplification of a 320-bp PCR fragment. Gapdh was used as an internal control. B: (lower) A representative immunoblot of islet protein extracts from p130 WT, HT, and KO animals probed with a p130 antibody. Actin was used to ensure equal protein loading in all immunoblots. C–F: Metabolic characterization of p130- mice. At the time of a glucose tolerance test, fasted animals were weighed (C), then injected intraperitoneally with a glucose bolus. D: Shows the blood glucose levels of animals at the indicated time points following glucose administration. Blood obtained from fasting and postprandial mice were analyzed for circulating glucose (E) and insulin (F). There were no statistically significant differences between the KO (n = 6–8) and WT (n = 7–9) or HT (n = 7–9) mice. PGK, phosphoglycerate kinase; neo, neomycin.
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Figure 1: Targeted disruption of the p130 gene and metabolic characterization of p130- mice. A: The p130 gene was disrupted by insertion of a targeting vector into exon 2 (17). Mutant alleles that contain the neomycin resistance gene (neo) were detected using PCR with previously described primers represented by arrows (17). B: (upper) PCR products for genomic DNA from p130 wild-type (WT), heterozygous (HT), and knockout (KO) animals. The mutant p130 gene was identified based on amplification of a 320-bp PCR fragment. Gapdh was used as an internal control. B: (lower) A representative immunoblot of islet protein extracts from p130 WT, HT, and KO animals probed with a p130 antibody. Actin was used to ensure equal protein loading in all immunoblots. C–F: Metabolic characterization of p130- mice. At the time of a glucose tolerance test, fasted animals were weighed (C), then injected intraperitoneally with a glucose bolus. D: Shows the blood glucose levels of animals at the indicated time points following glucose administration. Blood obtained from fasting and postprandial mice were analyzed for circulating glucose (E) and insulin (F). There were no statistically significant differences between the KO (n = 6–8) and WT (n = 7–9) or HT (n = 7–9) mice. PGK, phosphoglycerate kinase; neo, neomycin.

Mentions: Mice with inactivating mutations in p130 or p107 have previously been developed and characterized (16,17). These mice develop normally, are fertile, and apart from modest skeletal changes in p107 mutants, exhibit no obvious adult phenotypes. However, these previous studies did not include any assessment of β function or glucose homeostasis. The gene-targeting strategies used to disrupt the p130 or p107 genes (16,17) are shown in Fig. 1A and supplementary Fig. S1A (available in the online-only appendix). Mutant mice were identified by PCR analysis of genomic DNA and confirmed as s by immoblotting for p130 or p107 in isolated islets (Fig. 1B; supplementary Fig. S1B, available in the online-only appendix). Metabolic analysis of adult p130- or p107-deficient mice revealed a completely normal phenotype, similar to that seen in mice with Rb- islets. Specifically, body weight, fasting and postprandial blood glucose, and plasma insulin values were entirely normal compared with heterozygous and wild-type littermates (Fig. 1C–F; supplementary Fig. S1C–F, available in the online-only appendix). Glucose tolerance was also normal in mice lacking p130 or p107, compared with wild-type littermates (Fig. 1D; supplementary Fig. S1D, available in the online-only appendix).


The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic beta-cells.

Harb G, Vasavada RC, Cobrinik D, Stewart AF - Diabetes (2009)

Targeted disruption of the p130 gene and metabolic characterization of p130- mice. A: The p130 gene was disrupted by insertion of a targeting vector into exon 2 (17). Mutant alleles that contain the neomycin resistance gene (neo) were detected using PCR with previously described primers represented by arrows (17). B: (upper) PCR products for genomic DNA from p130 wild-type (WT), heterozygous (HT), and knockout (KO) animals. The mutant p130 gene was identified based on amplification of a 320-bp PCR fragment. Gapdh was used as an internal control. B: (lower) A representative immunoblot of islet protein extracts from p130 WT, HT, and KO animals probed with a p130 antibody. Actin was used to ensure equal protein loading in all immunoblots. C–F: Metabolic characterization of p130- mice. At the time of a glucose tolerance test, fasted animals were weighed (C), then injected intraperitoneally with a glucose bolus. D: Shows the blood glucose levels of animals at the indicated time points following glucose administration. Blood obtained from fasting and postprandial mice were analyzed for circulating glucose (E) and insulin (F). There were no statistically significant differences between the KO (n = 6–8) and WT (n = 7–9) or HT (n = 7–9) mice. PGK, phosphoglycerate kinase; neo, neomycin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2712776&req=5

Figure 1: Targeted disruption of the p130 gene and metabolic characterization of p130- mice. A: The p130 gene was disrupted by insertion of a targeting vector into exon 2 (17). Mutant alleles that contain the neomycin resistance gene (neo) were detected using PCR with previously described primers represented by arrows (17). B: (upper) PCR products for genomic DNA from p130 wild-type (WT), heterozygous (HT), and knockout (KO) animals. The mutant p130 gene was identified based on amplification of a 320-bp PCR fragment. Gapdh was used as an internal control. B: (lower) A representative immunoblot of islet protein extracts from p130 WT, HT, and KO animals probed with a p130 antibody. Actin was used to ensure equal protein loading in all immunoblots. C–F: Metabolic characterization of p130- mice. At the time of a glucose tolerance test, fasted animals were weighed (C), then injected intraperitoneally with a glucose bolus. D: Shows the blood glucose levels of animals at the indicated time points following glucose administration. Blood obtained from fasting and postprandial mice were analyzed for circulating glucose (E) and insulin (F). There were no statistically significant differences between the KO (n = 6–8) and WT (n = 7–9) or HT (n = 7–9) mice. PGK, phosphoglycerate kinase; neo, neomycin.
Mentions: Mice with inactivating mutations in p130 or p107 have previously been developed and characterized (16,17). These mice develop normally, are fertile, and apart from modest skeletal changes in p107 mutants, exhibit no obvious adult phenotypes. However, these previous studies did not include any assessment of β function or glucose homeostasis. The gene-targeting strategies used to disrupt the p130 or p107 genes (16,17) are shown in Fig. 1A and supplementary Fig. S1A (available in the online-only appendix). Mutant mice were identified by PCR analysis of genomic DNA and confirmed as s by immoblotting for p130 or p107 in isolated islets (Fig. 1B; supplementary Fig. S1B, available in the online-only appendix). Metabolic analysis of adult p130- or p107-deficient mice revealed a completely normal phenotype, similar to that seen in mice with Rb- islets. Specifically, body weight, fasting and postprandial blood glucose, and plasma insulin values were entirely normal compared with heterozygous and wild-type littermates (Fig. 1C–F; supplementary Fig. S1C–F, available in the online-only appendix). Glucose tolerance was also normal in mice lacking p130 or p107, compared with wild-type littermates (Fig. 1D; supplementary Fig. S1D, available in the online-only appendix).

Bottom Line: In vivo loss of either p107 or p130 did not affect beta-cell replication or function.Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death.Pancreas and beta-cell mass were significantly reduced in double mutants.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT

Objective: The retinoblastoma protein family (pRb, p130, p107) plays a central role in the regulation of cell cycle progression. Surprisingly, loss of pRb in the beta-cell has no discernible effect on cell cycle control. Therefore, we explored the effects of individual loss of either p130 or p107 in addition to the simultaneous loss of both pRb/p130 on the beta-cell.

Research design and methods: Adult mice deficient in either p130 or p107 or both pRb/p130 were examined for effects on beta-cell replication, function, and survival. The Cre-Lox system was also used to inactivate pRb in wild-type and p130-deficient beta-cells in vitro.

Results: In vivo loss of either p107 or p130 did not affect beta-cell replication or function. Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death. Pancreas and beta-cell mass were significantly reduced in double mutants. Despite this, overall glucose tolerance was normal, except for mild postprandial hyperglycemia. Ex vivo, acute deletion of pRb in p130-deficient beta-cells also caused a striking increase in proliferation. The combined deletion of pRb/p130 upregulated islet expression of E2F2 but not E2F1.

Conclusions: These studies define an essential role for the pocket proteins in controlling the G(1)/S transition in beta-cells. When deficient in both pRb and p130, beta-cells undergo unrestrained cell cycle reentry and activation of apoptosis. These studies underscore the central role of the pRb pathway in controlling beta-cell turnover and provide new cellular targets for beta-cell regeneration.

Show MeSH
Related in: MedlinePlus