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Blood glucose levels regulate pancreatic beta-cell proliferation during experimentally-induced and spontaneous autoimmune diabetes in mice.

Pechhold K, Koczwara K, Zhu X, Harrison VS, Walker G, Lee J, Harlan DM - PLoS ONE (2009)

Bottom Line: For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs.Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells).We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD, USA. klausp@intra.niddk.nih.gov

ABSTRACT

Background: Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic beta-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of beta-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ss-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation.

Methodology and principal findings: We adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5'-bromo-2'-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that beta-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ss endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells).

Conclusion: We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient beta-cell mass to restore normal glucose homeostasis.

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Detection specificity of multiparameter flow cytometry of continuously in vivo BrdU-labeled islet cells.Mice aged 7–9 weeks were given BrdU at 1 mg/ml in their drinking water continuously for 7 days. Islets were isolated, dissociated, and 4-color stained for insulin (red), glucagon (blue), somatostatin (green), and for incorporated BrdU. BrdU incorporation (x-axis) was plotted for the endocrine islet cell subsets from naïve, C57BL/6 (top panel), and insulinoma-developing yet non-symptomatic Rip-Tag2 Tg mice. Percentages indicate the relative frequency of BrdU-stained, single cells among the particular endocrine subset identified by hormone staining. Note that depending of the stage of insulinoma development, the frequency of glucagon+ α-cells and somatostatin+ δ cells are correspondingly reduced among all islet cells acquired from purified islets of insulinoma carrying mice.
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pone-0004827-g002: Detection specificity of multiparameter flow cytometry of continuously in vivo BrdU-labeled islet cells.Mice aged 7–9 weeks were given BrdU at 1 mg/ml in their drinking water continuously for 7 days. Islets were isolated, dissociated, and 4-color stained for insulin (red), glucagon (blue), somatostatin (green), and for incorporated BrdU. BrdU incorporation (x-axis) was plotted for the endocrine islet cell subsets from naïve, C57BL/6 (top panel), and insulinoma-developing yet non-symptomatic Rip-Tag2 Tg mice. Percentages indicate the relative frequency of BrdU-stained, single cells among the particular endocrine subset identified by hormone staining. Note that depending of the stage of insulinoma development, the frequency of glucagon+ α-cells and somatostatin+ δ cells are correspondingly reduced among all islet cells acquired from purified islets of insulinoma carrying mice.

Mentions: In accordance with numerous studies using flow cytometry to detect BrdU-labeled hematopoetic cells, Figure 2 demonstrates the feasibility of this approach using dissociated islet cells (see Supporting Information Figure S2 illustrating the gating strategy applied to islet cell BrdU staining). Analysis of 8–10 week-old WT mice given continuous BrdU for 7 days in their drinking water labeled just 8.7% of their β-cells (Figure 2). In contrast, age-matched Rip-Tag2 transgenic mice incorporated BrdU in more than half their β-cells during the same labeling period. The BrdU uptake in insulinoma-prone mice was β-cell specific; i.e. both WT and insulinoma-prone mouse islet α- and δ-cells incorporated very little BrdU. However, we found that endothelial cells, and perhaps other islet cell subsets, markedly expanded during insulinoma formation (data not shown), which accounts for the increased BrdU incorporation present in non-endocrine islet cells (Figure 2, lower panel, lower right quadrants). We also noted the 2-fold increase in proliferating somatostatin-positive δ cells in the Rip-Tag2 transgenic mice, but we did not further explore the significance of this finding.


Blood glucose levels regulate pancreatic beta-cell proliferation during experimentally-induced and spontaneous autoimmune diabetes in mice.

Pechhold K, Koczwara K, Zhu X, Harrison VS, Walker G, Lee J, Harlan DM - PLoS ONE (2009)

Detection specificity of multiparameter flow cytometry of continuously in vivo BrdU-labeled islet cells.Mice aged 7–9 weeks were given BrdU at 1 mg/ml in their drinking water continuously for 7 days. Islets were isolated, dissociated, and 4-color stained for insulin (red), glucagon (blue), somatostatin (green), and for incorporated BrdU. BrdU incorporation (x-axis) was plotted for the endocrine islet cell subsets from naïve, C57BL/6 (top panel), and insulinoma-developing yet non-symptomatic Rip-Tag2 Tg mice. Percentages indicate the relative frequency of BrdU-stained, single cells among the particular endocrine subset identified by hormone staining. Note that depending of the stage of insulinoma development, the frequency of glucagon+ α-cells and somatostatin+ δ cells are correspondingly reduced among all islet cells acquired from purified islets of insulinoma carrying mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654100&req=5

pone-0004827-g002: Detection specificity of multiparameter flow cytometry of continuously in vivo BrdU-labeled islet cells.Mice aged 7–9 weeks were given BrdU at 1 mg/ml in their drinking water continuously for 7 days. Islets were isolated, dissociated, and 4-color stained for insulin (red), glucagon (blue), somatostatin (green), and for incorporated BrdU. BrdU incorporation (x-axis) was plotted for the endocrine islet cell subsets from naïve, C57BL/6 (top panel), and insulinoma-developing yet non-symptomatic Rip-Tag2 Tg mice. Percentages indicate the relative frequency of BrdU-stained, single cells among the particular endocrine subset identified by hormone staining. Note that depending of the stage of insulinoma development, the frequency of glucagon+ α-cells and somatostatin+ δ cells are correspondingly reduced among all islet cells acquired from purified islets of insulinoma carrying mice.
Mentions: In accordance with numerous studies using flow cytometry to detect BrdU-labeled hematopoetic cells, Figure 2 demonstrates the feasibility of this approach using dissociated islet cells (see Supporting Information Figure S2 illustrating the gating strategy applied to islet cell BrdU staining). Analysis of 8–10 week-old WT mice given continuous BrdU for 7 days in their drinking water labeled just 8.7% of their β-cells (Figure 2). In contrast, age-matched Rip-Tag2 transgenic mice incorporated BrdU in more than half their β-cells during the same labeling period. The BrdU uptake in insulinoma-prone mice was β-cell specific; i.e. both WT and insulinoma-prone mouse islet α- and δ-cells incorporated very little BrdU. However, we found that endothelial cells, and perhaps other islet cell subsets, markedly expanded during insulinoma formation (data not shown), which accounts for the increased BrdU incorporation present in non-endocrine islet cells (Figure 2, lower panel, lower right quadrants). We also noted the 2-fold increase in proliferating somatostatin-positive δ cells in the Rip-Tag2 transgenic mice, but we did not further explore the significance of this finding.

Bottom Line: For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs.Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells).We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD, USA. klausp@intra.niddk.nih.gov

ABSTRACT

Background: Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic beta-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of beta-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ss-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation.

Methodology and principal findings: We adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5'-bromo-2'-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that beta-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ss endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells).

Conclusion: We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient beta-cell mass to restore normal glucose homeostasis.

Show MeSH
Related in: MedlinePlus