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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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Related in: MedlinePlus

Assessment of β-cell replication by quantitative staining of nuclear DNA and multicolor flow cytometry.Purified islets from naïve and diabetic EAD and NOD mice, and insulinoma-developing Rip-Tag2 Tg mice were dissociated into a single cell suspension, then were fixed and stained for insulin (Ins) and glucagon (Gcg). Nuclei were stained using a quantitative nuclear dye (Vybrant DyeCycle violet). A: Nuclear DNA staining profiles of gated Ins+ β-cells. The range of different frequencies of DNAhi β-cells from groups of mice is shown. These were non-diabetic, naïve (range 0.8–1.9), diabetic EAD (range 2.5–5.7), and Rip-Tag2 transgenic mice (range 7.4–14.6). The lower panel represents a magnified y-axis range to better illustrate the DNAhi β-cells. B: Statistical analysis of increased β-cell DNA content displayed as percentage of overall Ins+ cells (mean±SE): EAD (naïve, 1.5±0.2, n = 15; and CTL-induced pre-diabetic, 2.1±0.2, n = 12; and diabetic, 3.9±0.3, n = 20); NOD (pre-diabetic, 2.9±0.2, n = 13; diabetic, 6.2±0.5, n = 6), and Rip-Tag2 (WT, 1.6±0.1, n = 25; transgenic, 11.2±0.5, n = 16). Significance levels: (*) p = 0.026, and (***) p<0.0001. The inset depicts a similar DNA content analysis on Gcg+ α-cells of EAD and Rip-Tag2 mice, showing no detectable difference in α-cell DNA content among mice.
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pone-0004827-g001: Assessment of β-cell replication by quantitative staining of nuclear DNA and multicolor flow cytometry.Purified islets from naïve and diabetic EAD and NOD mice, and insulinoma-developing Rip-Tag2 Tg mice were dissociated into a single cell suspension, then were fixed and stained for insulin (Ins) and glucagon (Gcg). Nuclei were stained using a quantitative nuclear dye (Vybrant DyeCycle violet). A: Nuclear DNA staining profiles of gated Ins+ β-cells. The range of different frequencies of DNAhi β-cells from groups of mice is shown. These were non-diabetic, naïve (range 0.8–1.9), diabetic EAD (range 2.5–5.7), and Rip-Tag2 transgenic mice (range 7.4–14.6). The lower panel represents a magnified y-axis range to better illustrate the DNAhi β-cells. B: Statistical analysis of increased β-cell DNA content displayed as percentage of overall Ins+ cells (mean±SE): EAD (naïve, 1.5±0.2, n = 15; and CTL-induced pre-diabetic, 2.1±0.2, n = 12; and diabetic, 3.9±0.3, n = 20); NOD (pre-diabetic, 2.9±0.2, n = 13; diabetic, 6.2±0.5, n = 6), and Rip-Tag2 (WT, 1.6±0.1, n = 25; transgenic, 11.2±0.5, n = 16). Significance levels: (*) p = 0.026, and (***) p<0.0001. The inset depicts a similar DNA content analysis on Gcg+ α-cells of EAD and Rip-Tag2 mice, showing no detectable difference in α-cell DNA content among mice.

Mentions: Reasoning that adapting flow cytometry-based techniques to pancreas research would greatly facilitate a more quantitative analysis of β-cell proliferation in islets undergoing autoimmune destruction, we have developed protocols to isolate islets from naïve and diabetic mice, dissociate them into single cells, and identify their endocrine cell lineage by co-staining for intracytoplasmic insulin, glucagon, and somatostatin (data not shown). Supporting Information Figure S3 illustrates electronic gating strategies to identify islet β-cells, based sequentially on CD45+ hematopoetic-lineage cell exclusion, insulin-staining, and eliminating not fully dissociated islet cell clusters using the hydrodynamic focusing-based, electronic doublet exclusion gating strategy on nuclear fluorescence. As illustrated in Figure 1A, we determined the fraction of cells with increased DNA content (ranging from less than 1% to more than 10%) using a quantitative DNA-intercalating dye, and electronically-gating on β-cells from healthy, naive (left), and recently diabetic mice (middle), and among Rip-Tag2 transgenic animals that develop insulinomas (right) [46]. Quantitative analysis (Figure 1B) demonstrates that islet cells isolated from newly diabetic EAD mice had a higher proportion of β-cells with increased DNA content (3.9%, range 2.5–5.7, n = 20) compared to naïve mice (1.6%, range 0.8–2.4%, n = 25), suggesting that diabetic mice have a greater proportion of β-cells with ongoing DNA synthesis and cell cycle progression. Expectedly, tumor-prone Rip-Tag2 transgenic mice, when tested well before they developed clinical symptoms of (transgene-mediated) insulinomas, regularly showed a substantially increased proportion of β-cells with the increased DNA content (11.2%, range 7.4–14.5, n = 16). Evidence supporting increased β-cell cycle progression at diabetes onset was not unique to our CTL-induced EAD model; we found similar results using islets isolated from spontaneously diabetic NOD mice (6.2%, range 4.0–7.3, Figure 1B). Pre- or non-diabetic NOD mice of a comparable age but with incompletely characterized glucose tolerance had a somewhat increased proportion of β-cells with elevated DNA content (2.9%, range 1.8–4.4). Importantly, in rodent models of both autoimmunity and ß cell tumor development, we found increased DNA content only in β-cells and not in α-cells (Figure 1B inset), based on glucagon staining and a comparable gating strategy. While the DNA content analysis for determining proliferation enjoys the important advantage of not having to label tissue in vivo, the technique does suffer from technical and/or biological limitations. These potentially confounding factors include that: (1) DNA is partially degraded in the early stages of islet cell apoptosis, which may occur during islet inflammation or the islet isolation process, (2) the S-Phase cell DNA quantification requires establishing a somewhat arbitrary cutoff, (3) dysfunctional islet cells may be arrested in the G2/M phase after completing S-phase but before they undergo cell division, and (4) doublet exclusion efficacy may vary. Cellular uptake of the nucleoside analog BrdU during S-Phase transition would, unlike DNA content, likely be more stringent in labeling proliferating cells, but the technique does not label G2/M-Phase cells, and has further limitations. For instance, if BrdU is given some time before islet isolation, the amount incorporated into DNA reflects the cumulative history of cell cycles completed during the BrdU exposure. Consequently, BrdU-labeling experiments (especially those in which the BrdU was dosed some time before euthanasia) cannot distinguish between endocrine cell replication and progenitor cell replication followed by later differentiation into an endocrine phenotype. Hence, we have used the term “proliferation” or “replication” more broadly to accommodate both possibilities.


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)

Assessment of β-cell replication by quantitative staining of nuclear DNA and multicolor flow cytometry.Purified islets from naïve and diabetic EAD and NOD mice, and insulinoma-developing Rip-Tag2 Tg mice were dissociated into a single cell suspension, then were fixed and stained for insulin (Ins) and glucagon (Gcg). Nuclei were stained using a quantitative nuclear dye (Vybrant DyeCycle violet). A: Nuclear DNA staining profiles of gated Ins+ β-cells. The range of different frequencies of DNAhi β-cells from groups of mice is shown. These were non-diabetic, naïve (range 0.8–1.9), diabetic EAD (range 2.5–5.7), and Rip-Tag2 transgenic mice (range 7.4–14.6). The lower panel represents a magnified y-axis range to better illustrate the DNAhi β-cells. B: Statistical analysis of increased β-cell DNA content displayed as percentage of overall Ins+ cells (mean±SE): EAD (naïve, 1.5±0.2, n = 15; and CTL-induced pre-diabetic, 2.1±0.2, n = 12; and diabetic, 3.9±0.3, n = 20); NOD (pre-diabetic, 2.9±0.2, n = 13; diabetic, 6.2±0.5, n = 6), and Rip-Tag2 (WT, 1.6±0.1, n = 25; transgenic, 11.2±0.5, n = 16). Significance levels: (*) p = 0.026, and (***) p<0.0001. The inset depicts a similar DNA content analysis on Gcg+ α-cells of EAD and Rip-Tag2 mice, showing no detectable difference in α-cell DNA content among mice.
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pone-0004827-g001: Assessment of β-cell replication by quantitative staining of nuclear DNA and multicolor flow cytometry.Purified islets from naïve and diabetic EAD and NOD mice, and insulinoma-developing Rip-Tag2 Tg mice were dissociated into a single cell suspension, then were fixed and stained for insulin (Ins) and glucagon (Gcg). Nuclei were stained using a quantitative nuclear dye (Vybrant DyeCycle violet). A: Nuclear DNA staining profiles of gated Ins+ β-cells. The range of different frequencies of DNAhi β-cells from groups of mice is shown. These were non-diabetic, naïve (range 0.8–1.9), diabetic EAD (range 2.5–5.7), and Rip-Tag2 transgenic mice (range 7.4–14.6). The lower panel represents a magnified y-axis range to better illustrate the DNAhi β-cells. B: Statistical analysis of increased β-cell DNA content displayed as percentage of overall Ins+ cells (mean±SE): EAD (naïve, 1.5±0.2, n = 15; and CTL-induced pre-diabetic, 2.1±0.2, n = 12; and diabetic, 3.9±0.3, n = 20); NOD (pre-diabetic, 2.9±0.2, n = 13; diabetic, 6.2±0.5, n = 6), and Rip-Tag2 (WT, 1.6±0.1, n = 25; transgenic, 11.2±0.5, n = 16). Significance levels: (*) p = 0.026, and (***) p<0.0001. The inset depicts a similar DNA content analysis on Gcg+ α-cells of EAD and Rip-Tag2 mice, showing no detectable difference in α-cell DNA content among mice.
Mentions: Reasoning that adapting flow cytometry-based techniques to pancreas research would greatly facilitate a more quantitative analysis of β-cell proliferation in islets undergoing autoimmune destruction, we have developed protocols to isolate islets from naïve and diabetic mice, dissociate them into single cells, and identify their endocrine cell lineage by co-staining for intracytoplasmic insulin, glucagon, and somatostatin (data not shown). Supporting Information Figure S3 illustrates electronic gating strategies to identify islet β-cells, based sequentially on CD45+ hematopoetic-lineage cell exclusion, insulin-staining, and eliminating not fully dissociated islet cell clusters using the hydrodynamic focusing-based, electronic doublet exclusion gating strategy on nuclear fluorescence. As illustrated in Figure 1A, we determined the fraction of cells with increased DNA content (ranging from less than 1% to more than 10%) using a quantitative DNA-intercalating dye, and electronically-gating on β-cells from healthy, naive (left), and recently diabetic mice (middle), and among Rip-Tag2 transgenic animals that develop insulinomas (right) [46]. Quantitative analysis (Figure 1B) demonstrates that islet cells isolated from newly diabetic EAD mice had a higher proportion of β-cells with increased DNA content (3.9%, range 2.5–5.7, n = 20) compared to naïve mice (1.6%, range 0.8–2.4%, n = 25), suggesting that diabetic mice have a greater proportion of β-cells with ongoing DNA synthesis and cell cycle progression. Expectedly, tumor-prone Rip-Tag2 transgenic mice, when tested well before they developed clinical symptoms of (transgene-mediated) insulinomas, regularly showed a substantially increased proportion of β-cells with the increased DNA content (11.2%, range 7.4–14.5, n = 16). Evidence supporting increased β-cell cycle progression at diabetes onset was not unique to our CTL-induced EAD model; we found similar results using islets isolated from spontaneously diabetic NOD mice (6.2%, range 4.0–7.3, Figure 1B). Pre- or non-diabetic NOD mice of a comparable age but with incompletely characterized glucose tolerance had a somewhat increased proportion of β-cells with elevated DNA content (2.9%, range 1.8–4.4). Importantly, in rodent models of both autoimmunity and ß cell tumor development, we found increased DNA content only in β-cells and not in α-cells (Figure 1B inset), based on glucagon staining and a comparable gating strategy. While the DNA content analysis for determining proliferation enjoys the important advantage of not having to label tissue in vivo, the technique does suffer from technical and/or biological limitations. These potentially confounding factors include that: (1) DNA is partially degraded in the early stages of islet cell apoptosis, which may occur during islet inflammation or the islet isolation process, (2) the S-Phase cell DNA quantification requires establishing a somewhat arbitrary cutoff, (3) dysfunctional islet cells may be arrested in the G2/M phase after completing S-phase but before they undergo cell division, and (4) doublet exclusion efficacy may vary. Cellular uptake of the nucleoside analog BrdU during S-Phase transition would, unlike DNA content, likely be more stringent in labeling proliferating cells, but the technique does not label G2/M-Phase cells, and has further limitations. For instance, if BrdU is given some time before islet isolation, the amount incorporated into DNA reflects the cumulative history of cell cycles completed during the BrdU exposure. Consequently, BrdU-labeling experiments (especially those in which the BrdU was dosed some time before euthanasia) cannot distinguish between endocrine cell replication and progenitor cell replication followed by later differentiation into an endocrine phenotype. Hence, we have used the term “proliferation” or “replication” more broadly to accommodate both possibilities.

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