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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

Reduced islet β-cell BrdU uptake in diabetic mice treated to restore normoglycemia.Diabetic Rip-CD80+GP+ mice were randomly assigned for treatment groups to normalize BG levels, followed by BrdU pulse labeling (3 doses indicated by arrows of 1.5, 1.0, 1.0 mg/mouse i.p. over the final 3 days of the experiment), and islet cell analysis by flow cytometry. A: Treatment regimen, expected and exemplified results are illustrated in columns. β-cell BrdU incorporation was increased in the presence of untreated hyperglycemia (lower panel). Note that both the abundance and BrdU incorporation of islet infiltrating CD45+ leucocytes was essentially unchanged among treatment groups (middle panel). B: BrdU-uptake of islet β-cells is controlled by BG levels. Groups of treated diabetic mice (no℞, n = 8; anti-CD8 mAb, n = 6; s.c. insulin pellet, n = 4; ins-pellet removed, n = 3; islet-Tx, n = 7) were compared to age-matched, naïve mice (n = 7). Results are shown as mean±SE, with the following significance levels: (*) p<0.05, (**) p<0.01, and (***) p<0.001. C: Correlation plot between the final 3-day average random BG readings (x-axis) and the frequency of BrdU+ islet β-cells (y-axis). Each symbol represents a single mouse, and all mice, regardless of treatment group are included. Correlation coefficient r = 0.70; 95% confidence interval (dashed lines).
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pone-0004827-g004: Reduced islet β-cell BrdU uptake in diabetic mice treated to restore normoglycemia.Diabetic Rip-CD80+GP+ mice were randomly assigned for treatment groups to normalize BG levels, followed by BrdU pulse labeling (3 doses indicated by arrows of 1.5, 1.0, 1.0 mg/mouse i.p. over the final 3 days of the experiment), and islet cell analysis by flow cytometry. A: Treatment regimen, expected and exemplified results are illustrated in columns. β-cell BrdU incorporation was increased in the presence of untreated hyperglycemia (lower panel). Note that both the abundance and BrdU incorporation of islet infiltrating CD45+ leucocytes was essentially unchanged among treatment groups (middle panel). B: BrdU-uptake of islet β-cells is controlled by BG levels. Groups of treated diabetic mice (no℞, n = 8; anti-CD8 mAb, n = 6; s.c. insulin pellet, n = 4; ins-pellet removed, n = 3; islet-Tx, n = 7) were compared to age-matched, naïve mice (n = 7). Results are shown as mean±SE, with the following significance levels: (*) p<0.05, (**) p<0.01, and (***) p<0.001. C: Correlation plot between the final 3-day average random BG readings (x-axis) and the frequency of BrdU+ islet β-cells (y-axis). Each symbol represents a single mouse, and all mice, regardless of treatment group are included. Correlation coefficient r = 0.70; 95% confidence interval (dashed lines).

Mentions: Figure 4A illustrates how hyperglycemia resolved as a result of the different treatments (upper panel, Supporting Information Table S1 provides the treatment group's average BG concentrations recorded during the 3-day BrdU labeling period), and exemplifies the concomitant detection of BrdU uptake by infiltrating leucocytes (middle panel) or non-lymphocytic islet cell subsets (lower panel). Figure 4B quantifies the various treatments' impact on endogenous β-cell proliferation. Exogenous insulin (s.c. pellets or islet-transplantation) restored normoglycemia in every mouse, whereas depleting CD8+ T cells transiently halted direct CTL-mediated β-cell killing so gradually restored normoglycemia in the majority of diabetic mice (responders), with remissions typically lasting for more than one week. Regardless the treatment (T cell depletion by anti-CD8 mAb, exogenous insulin by s.c. insulin pellets, or islet transplantation), restoring normoglycemia to diabetic mice profoundly blunted the increased β-cell proliferation observed in newly diabetic, hyperglycemic mice. In fact, using our CTL-induced EAD model, we found a robust correlation (r = 0.70) between β-cell proliferation (BrdU pulse-labeled for 3 days prior to islet isolation) and the average random BG level during the same period prior to euthanasia (Figure 4C). The importance played by the BG level was further emphasized in mice that were not restored to normal BG levels (non-responders) by the anti-CD8 antibody treatment; in such mice with persistent hyperglycemia, β-cell proliferation remained elevated (8.0±2.3%, n = 3, data not shown). This also argues against the anti-CD8 antibody causing an inhibitory or otherwise toxic effect on β-cell proliferation. Most importantly, residual β-cells remained responsive to changes in BG levels. For instance, if we restored normoglycemia using insulin pellets and thereby decreased β-cell proliferation, removing the pellets rapidly resulted in both hyperglycemia and increased β-cell proliferation (Figure 4B, “pellet-rem”).


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)

Reduced islet β-cell BrdU uptake in diabetic mice treated to restore normoglycemia.Diabetic Rip-CD80+GP+ mice were randomly assigned for treatment groups to normalize BG levels, followed by BrdU pulse labeling (3 doses indicated by arrows of 1.5, 1.0, 1.0 mg/mouse i.p. over the final 3 days of the experiment), and islet cell analysis by flow cytometry. A: Treatment regimen, expected and exemplified results are illustrated in columns. β-cell BrdU incorporation was increased in the presence of untreated hyperglycemia (lower panel). Note that both the abundance and BrdU incorporation of islet infiltrating CD45+ leucocytes was essentially unchanged among treatment groups (middle panel). B: BrdU-uptake of islet β-cells is controlled by BG levels. Groups of treated diabetic mice (no℞, n = 8; anti-CD8 mAb, n = 6; s.c. insulin pellet, n = 4; ins-pellet removed, n = 3; islet-Tx, n = 7) were compared to age-matched, naïve mice (n = 7). Results are shown as mean±SE, with the following significance levels: (*) p<0.05, (**) p<0.01, and (***) p<0.001. C: Correlation plot between the final 3-day average random BG readings (x-axis) and the frequency of BrdU+ islet β-cells (y-axis). Each symbol represents a single mouse, and all mice, regardless of treatment group are included. Correlation coefficient r = 0.70; 95% confidence interval (dashed lines).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654100&req=5

pone-0004827-g004: Reduced islet β-cell BrdU uptake in diabetic mice treated to restore normoglycemia.Diabetic Rip-CD80+GP+ mice were randomly assigned for treatment groups to normalize BG levels, followed by BrdU pulse labeling (3 doses indicated by arrows of 1.5, 1.0, 1.0 mg/mouse i.p. over the final 3 days of the experiment), and islet cell analysis by flow cytometry. A: Treatment regimen, expected and exemplified results are illustrated in columns. β-cell BrdU incorporation was increased in the presence of untreated hyperglycemia (lower panel). Note that both the abundance and BrdU incorporation of islet infiltrating CD45+ leucocytes was essentially unchanged among treatment groups (middle panel). B: BrdU-uptake of islet β-cells is controlled by BG levels. Groups of treated diabetic mice (no℞, n = 8; anti-CD8 mAb, n = 6; s.c. insulin pellet, n = 4; ins-pellet removed, n = 3; islet-Tx, n = 7) were compared to age-matched, naïve mice (n = 7). Results are shown as mean±SE, with the following significance levels: (*) p<0.05, (**) p<0.01, and (***) p<0.001. C: Correlation plot between the final 3-day average random BG readings (x-axis) and the frequency of BrdU+ islet β-cells (y-axis). Each symbol represents a single mouse, and all mice, regardless of treatment group are included. Correlation coefficient r = 0.70; 95% confidence interval (dashed lines).
Mentions: Figure 4A illustrates how hyperglycemia resolved as a result of the different treatments (upper panel, Supporting Information Table S1 provides the treatment group's average BG concentrations recorded during the 3-day BrdU labeling period), and exemplifies the concomitant detection of BrdU uptake by infiltrating leucocytes (middle panel) or non-lymphocytic islet cell subsets (lower panel). Figure 4B quantifies the various treatments' impact on endogenous β-cell proliferation. Exogenous insulin (s.c. pellets or islet-transplantation) restored normoglycemia in every mouse, whereas depleting CD8+ T cells transiently halted direct CTL-mediated β-cell killing so gradually restored normoglycemia in the majority of diabetic mice (responders), with remissions typically lasting for more than one week. Regardless the treatment (T cell depletion by anti-CD8 mAb, exogenous insulin by s.c. insulin pellets, or islet transplantation), restoring normoglycemia to diabetic mice profoundly blunted the increased β-cell proliferation observed in newly diabetic, hyperglycemic mice. In fact, using our CTL-induced EAD model, we found a robust correlation (r = 0.70) between β-cell proliferation (BrdU pulse-labeled for 3 days prior to islet isolation) and the average random BG level during the same period prior to euthanasia (Figure 4C). The importance played by the BG level was further emphasized in mice that were not restored to normal BG levels (non-responders) by the anti-CD8 antibody treatment; in such mice with persistent hyperglycemia, β-cell proliferation remained elevated (8.0±2.3%, n = 3, data not shown). This also argues against the anti-CD8 antibody causing an inhibitory or otherwise toxic effect on β-cell proliferation. Most importantly, residual β-cells remained responsive to changes in BG levels. For instance, if we restored normoglycemia using insulin pellets and thereby decreased β-cell proliferation, removing the pellets rapidly resulted in both hyperglycemia and increased β-cell proliferation (Figure 4B, “pellet-rem”).

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