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Age-dependent decline in beta-cell proliferation restricts the capacity of beta-cell regeneration in mice.

Tschen SI, Dhawan S, Gurlo T, Bhushan A - Diabetes (2009)

Bottom Line: The aim of this study was to elucidate whether age plays a role in the expansion or regeneration of beta-cell mass.The capacity to expand beta-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age.The ability of beta-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16(Ink4a)expression in the beta-cells.

View Article: PubMed Central - PubMed

Affiliation: Larry L. Hillblom Islet Research Center, Department of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

ABSTRACT

Objective: The aim of this study was to elucidate whether age plays a role in the expansion or regeneration of beta-cell mass.

Research design and methods: We analyzed the capacity of beta-cell expansion in 1.5- and 8-month-old mice in response to a high-fat diet, after short-term treatment with the glucagon-like peptide 1 (GLP-1) analog exendin-4, or after streptozotocin (STZ) administration.

Results: Young mice responded to high-fat diet by increasing beta-cell mass and beta-cell proliferation and maintaining normoglycemia. Old mice, by contrast, did not display any increases in beta-cell mass or beta-cell proliferation in response to high-fat diet and became diabetic. To further assess the plasticity of beta-cell mass with respect to age, young and old mice were injected with a single dose of STZ, and beta-cell proliferation was analyzed to assess the regeneration of beta-cells. We observed a fourfold increase in beta-cell proliferation in young mice after STZ administration, whereas no changes in beta-cell proliferation were observed in older mice. The capacity to expand beta-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age. The ability of beta-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16(Ink4a)expression in the beta-cells. Young Bmi1(-/-) mice that prematurely upregulate p16(Ink4a)failed to expand beta-cell mass in response to exendin-4, indicating that p16(Ink4a)levels are a critical determinant of beta-cell mass expansion.

Conclusions: beta-Cell proliferation and the capacity of beta-cells to regenerate declines with age and is regulated by the Bmi1/p16(Ink4a)pathway.

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Expression patterns of p16, p27, and cyclin D2 in wild-type and Bmi1−/− mice. Pancreatic sections from 2-week-old wild-type and Bmi1−/− mice were immunostained with antibodies to insulin (green) and p16, p27, or cyclin D2 (red) as indicated in the figure. KO, knockout; WT, wild type. (A high-quality digital representation of this figure is available in the online issue).
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Figure 4: Expression patterns of p16, p27, and cyclin D2 in wild-type and Bmi1−/− mice. Pancreatic sections from 2-week-old wild-type and Bmi1−/− mice were immunostained with antibodies to insulin (green) and p16, p27, or cyclin D2 (red) as indicated in the figure. KO, knockout; WT, wild type. (A high-quality digital representation of this figure is available in the online issue).

Mentions: We first assessed whether p16Ink4a was a specific cell cycle target of Bmi1 by analyzing expression levels of p16Ink4a along with other cell cycle regulators involved in β-cell proliferation. Immunohistochemistry analysis of pancreatic sections from 14-day-old Bmi1−/− mice and wild-type littermates showed p16Ink4a was upregulated in islets of Bmi1−/− mice (Fig. 4). Other cell cycle targets cyclin D2 and p27 were unaffected by the absence of Bmi1 (Fig. 4). To directly test whether Bmi1 regulates p16Ink4a, we measured the levels of p16Ink4a in islets isolated from 4-week-old wild-type and Bmi1−/− mice. Immunoblots showed increased levels of p16Ink4a in islets isolated from Bmi1−/− mice (Fig. 5A). To further confirm whether Bmi1 represses p16Ink4a, we generated an expression vector that allowed expression of Bmi1 fused with an NH2-terminal myc tag. The expression of myc-tagged Bmi1 in the Min6 cells resulted in reduced levels of p16Ink4a protein, thus confirming the repressive effect of Bmi1 on p16Ink4a expression (Fig. 5B). We next assessed whether Bmi1 regulated p16Ink4a expression by affecting the chromatin structure of the Ink4a/Arf locus that encodes p16Ink4a. We determined whether histone acetylation at the Ink4a/Arf locus, which results in the loosening of chromatin and lends itself to transcription, was regulated by Bmi1. We examined the levels of H3K9 acetylation associated with the Ink4a/Arf locus in islets isolated from 6-week-old Bmi1−/− mice and their wild-type littermates. In the absence of Bmi1, the levels of H3K9 acetylation were increased, suggesting that Bmi1 mediated repression of p16Ink4a via modulation of chromatin structure (Fig. 5C).


Age-dependent decline in beta-cell proliferation restricts the capacity of beta-cell regeneration in mice.

Tschen SI, Dhawan S, Gurlo T, Bhushan A - Diabetes (2009)

Expression patterns of p16, p27, and cyclin D2 in wild-type and Bmi1−/− mice. Pancreatic sections from 2-week-old wild-type and Bmi1−/− mice were immunostained with antibodies to insulin (green) and p16, p27, or cyclin D2 (red) as indicated in the figure. KO, knockout; WT, wild type. (A high-quality digital representation of this figure is available in the online issue).
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Related In: Results  -  Collection

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Figure 4: Expression patterns of p16, p27, and cyclin D2 in wild-type and Bmi1−/− mice. Pancreatic sections from 2-week-old wild-type and Bmi1−/− mice were immunostained with antibodies to insulin (green) and p16, p27, or cyclin D2 (red) as indicated in the figure. KO, knockout; WT, wild type. (A high-quality digital representation of this figure is available in the online issue).
Mentions: We first assessed whether p16Ink4a was a specific cell cycle target of Bmi1 by analyzing expression levels of p16Ink4a along with other cell cycle regulators involved in β-cell proliferation. Immunohistochemistry analysis of pancreatic sections from 14-day-old Bmi1−/− mice and wild-type littermates showed p16Ink4a was upregulated in islets of Bmi1−/− mice (Fig. 4). Other cell cycle targets cyclin D2 and p27 were unaffected by the absence of Bmi1 (Fig. 4). To directly test whether Bmi1 regulates p16Ink4a, we measured the levels of p16Ink4a in islets isolated from 4-week-old wild-type and Bmi1−/− mice. Immunoblots showed increased levels of p16Ink4a in islets isolated from Bmi1−/− mice (Fig. 5A). To further confirm whether Bmi1 represses p16Ink4a, we generated an expression vector that allowed expression of Bmi1 fused with an NH2-terminal myc tag. The expression of myc-tagged Bmi1 in the Min6 cells resulted in reduced levels of p16Ink4a protein, thus confirming the repressive effect of Bmi1 on p16Ink4a expression (Fig. 5B). We next assessed whether Bmi1 regulated p16Ink4a expression by affecting the chromatin structure of the Ink4a/Arf locus that encodes p16Ink4a. We determined whether histone acetylation at the Ink4a/Arf locus, which results in the loosening of chromatin and lends itself to transcription, was regulated by Bmi1. We examined the levels of H3K9 acetylation associated with the Ink4a/Arf locus in islets isolated from 6-week-old Bmi1−/− mice and their wild-type littermates. In the absence of Bmi1, the levels of H3K9 acetylation were increased, suggesting that Bmi1 mediated repression of p16Ink4a via modulation of chromatin structure (Fig. 5C).

Bottom Line: The aim of this study was to elucidate whether age plays a role in the expansion or regeneration of beta-cell mass.The capacity to expand beta-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age.The ability of beta-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16(Ink4a)expression in the beta-cells.

View Article: PubMed Central - PubMed

Affiliation: Larry L. Hillblom Islet Research Center, Department of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

ABSTRACT

Objective: The aim of this study was to elucidate whether age plays a role in the expansion or regeneration of beta-cell mass.

Research design and methods: We analyzed the capacity of beta-cell expansion in 1.5- and 8-month-old mice in response to a high-fat diet, after short-term treatment with the glucagon-like peptide 1 (GLP-1) analog exendin-4, or after streptozotocin (STZ) administration.

Results: Young mice responded to high-fat diet by increasing beta-cell mass and beta-cell proliferation and maintaining normoglycemia. Old mice, by contrast, did not display any increases in beta-cell mass or beta-cell proliferation in response to high-fat diet and became diabetic. To further assess the plasticity of beta-cell mass with respect to age, young and old mice were injected with a single dose of STZ, and beta-cell proliferation was analyzed to assess the regeneration of beta-cells. We observed a fourfold increase in beta-cell proliferation in young mice after STZ administration, whereas no changes in beta-cell proliferation were observed in older mice. The capacity to expand beta-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age. The ability of beta-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16(Ink4a)expression in the beta-cells. Young Bmi1(-/-) mice that prematurely upregulate p16(Ink4a)failed to expand beta-cell mass in response to exendin-4, indicating that p16(Ink4a)levels are a critical determinant of beta-cell mass expansion.

Conclusions: beta-Cell proliferation and the capacity of beta-cells to regenerate declines with age and is regulated by the Bmi1/p16(Ink4a)pathway.

Show MeSH