Glibenclamide treatment preserves Cx36 expression in beta cells.

<p><b>a)</b> At the end of the 25 wk experiment, immunostaining reveals the spotted distribution of Cx36 (yellow) in the few residual islets of control, untreated mice and in the more numerous islets of the glibenclamide dose 2-treated mice. The enlargements of the field outlined in the pictures show that Cx36 formed larger and more numerous spots (some are circled) in the islets of the latter animals. The dot line outlines the limit between an islet and its surrounding insulitis mantle (L). Bar, 40 μm in the pictures, 20 μm in the enlargements. The red background staining is due to the Evans blue counterstain. <b>b)</b> The volume density of Cx36 was significantly lower in the whole pancreas of untreated controls than in that of the glibenclamide-treated mice, due to the much larger number of islets found in the latter animals. <b>c)</b> In contrast, no significant difference in the volume density of Cx36 was reached in the residual islets of the 2 groups of mice that we compared, possibly because few surviving islets could be scored in the pancreas of the untreated NOD mice. ** p< 0.01 vs control group, as tested by Mann-Whitney and median tests. Median values are shown by the red lines.</p

Glibenclamide preserves a sizable mass of beta cells in adult NOD mice.

<p><b>a)</b> HE staining of pancreas sections shows representative islets of the 3 groups of NOD mice, which we compared. At the end of the experiment, untreated control mice aged 28 wks showed few islets with many of the residual cells featuring a poorly stained cytoplasm. In contrast, mice treated with either dose 1 or dose 2 glibenclamide featured many more and larger islets, which mostly comprised healthy looking cells. Insulitis was prominent in the islets of all mice groups. The dot and dash lines outline the borders of islets and insulitis mantle (L), respectively. Bar, 100 μm. <b>b)</b> At the end of the experiment, the numerical density (Nv) of beta cells was significantly higher in the glibenclamide dose 2-treated NOD mice (solid bars), than in untreated controls (open bars). The mice receiving dose 1 of glibenclamide featured an intermediate phenotype. <b>c)</b> The volume density (Vv) of beta cells was similarly modified by the glibenclamide treatments. Data are mean + SEM values of the number of pancreas sections indicated within the bars. * p< 0.05, ** p< 0.01, *** p< 0.001 vs control group, as tested by ANOVA.</p

Glibenclamide Prevents Diabetes in NOD Mice

<div><p>Previous work has revealed that Cx36, the sole connexin expressed in the insulin-producing beta cells, enhances the secretion of insulin, and promotes the resistance of beta cells against pro-inflammatory cytokines. In parallel, the anti-diabetic sulphonylurea glibenclamide was shown to promote the assembly and function of Cx36 channels. Here, we assessed whether glibenclamide could protect the insulin-producing cells against conditions mimicking those expected at the onset of type 1 diabetes. We found that the drug 1) protected <i>in vitro</i> the mouse MIN6 cells from the apoptosis and loss of Cx36, which are induced by Th1 cytokines; 2) prevented the development of hyperglycemia as well as the loss of beta cells and Cx36, which rapidly develop with aging in untreated NOD mice; 3) modified the proportion of effector CD4⁺ and CD8⁺ T cells in pancreatic draining lymph nodes. The data imply that an early glibenclamide treatment may help protecting beta cells against the autoimmune attack, which triggers the development of type 1 diabetes.</p></div

Glibenclamide reduces the apoptosis and the loss of CVx36 in MIN6 cells exposed to Th1 cytokines.

<p><b>a)</b> Apoptosis of MIN6 cells was evaluated by the proportion of Annexin V-stained cells (green) out of the total number of cells screened (identified by the blue DAPI staining of nuclei). Bar, 25 μm. <b>b)</b> A 18h exposure of MIN6 cells to a mix of IL-1β, IFN γ and TNFα induced a significant increase in the proportion of Annexin V-labelled cells (hatched bar), compared to the level observed in controls cultured in the absence of cytokines (open bar). Such an increase was not observed in cells exposed to 10 μM glibenclamide (solid bar). <b>c)</b> Comparable effects were seen when MIN6 cells were exposed to 10 μM glibenclamide 6 h before starting the exposure to the cytokines. <b>d)</b> Expression of Cx36 was evaluated by immunostaining, using an antibody that detects the small gap junction plaques made by the concentration of Cx36 within the membrane (yellow-green). Bar, 5 μm. <b>e)</b> A 18h exposure of MIN6 cells to IL-1β, IFN γ and TNFα (hatched bar) reduced the volume density of immunolabeled Cx36 under the control levels (open bar). Such a decrease was not observed in cells exposed to 10 μM glibenclamide (solid bar). <b>f)</b>. Comparable effects were seen when MIN6 cells were exposed to 10 μM glibenclamide 6 h before starting the exposure to the cytokines. Data are mean + SEM of 3 independent experiments. Numbers within bars indicate the total number of cells scored in each group. *** p < 0.001 vs control group;. ^§§§ p < 0.001 vs control + cytokines group, as tested by one-way ANOVA.</p

Glibenclamide alters the subsets of T cells in both spleen and pancreatic-draining lymph nodes.

<p><b>a)</b> After 3 wks of treatment (mice 6 wk of age), FACS analysis showed that the treatment with glibenclamide dose 2 (solid symbols) increased the proportion of the effector subsets of CD44^hiCD62L^- T cells, and decreased that of the naïve subset of CD44^loCD62L⁺ T cells in CD4⁺ (top panel) and CD8⁺ T cells (bottom panel), as compared to control values (open symbols). These changes took place in both spleen (S) and pancreatic-draining lymph nodes (PLN). <b>b)</b> Comparable observations were made after 6 wks of glibenclamide treatment (mice of 9 wks of age). Data are shown as scatters of individual values for the untreated control (open symbols, 5–10 mice) and the glibenclamide dose 2-treated group (solid symbols, 5–9 mice). Mean values are shown by the red lines. * p<0.05, ** p< 0.01, **** p<0.001 as compared to the untreated control group, using Mann-Whitney t-test.</p

Islets of untreated control and glibenclamide-treated NOD mice display insulitis.

<p><b>a)</b> Few beta cells are immunostained for insulin (green) in the islets of untreated controls, used for morphometry, whereas many more beta cells are seen in the islets of the animals treated with glibenclamide. The blue DAPI staining outlines the insulitis mantle (L). Lower panels are enlargements of the fields outlined in the pictures to visualize the dense packing of the immune cells, mostly lymphocytes that formed the insulitis mantle. Bar, 100 μm in the pictures, 40 μm in the enlargements. <b>b)</b> All mice featured a similar percentage of islets with insulitis. <b>c)</b> However, a higher volume density of insulitis per unit pancreas volume was found after the glibenclamide treatments, due to the much larger number of islets surviving under these conditions. Data are mean + SEM values of the number of islets (b) or pancreas sections (c) indicated within the bars. * p< 0.05, ** p< 0.01, *** p< 0.001 vs control group, as tested by ANOVA.</p

Glibenclamide treatment prevents the development of diabetes in NOD mice.

<p>a) All mice had normal blood glucose levels at week 5. Thereafter, the proportion of untreated animals, which remained normoglycaemic, rapidly dropped with time (open symbols). In contrast, all littermates that received the high dose of glibenclamide (glibenclamide dose 2, black symbols) remained normoglycaemic till the end of the experiment. Mice that received the lower dose of glibenclamide (glibenclamide dose 1, grey symbols) featured an intermediate evolution. ** p< 0.01,**** p< 0.0001 vs control group, as tested by the Mantel-Cox log-rank test. n are the numbers of animals tested in 1 (glibenclamide dose 1 group)-2 (control and glibenclamide dose 2 groups) experiments. <b>b)</b> Bars show the percentage of NOD mice featuring normoglycaemia (open bars), glucose intolerance (grey bars) and overt diabetes (solid bars) at the end of the 25 wk experiment. n are numbers of mice investigated in 1 (glibenclamide dose 1 group)-2 (control and glibenclamide dose 2 groups) experiments. No error bars are given in panels a) and b) given that these graphs show a single percentage value calculated for the entire mouse population tested. ***p< 0.001 vs control group, ^§§§p < 0.001 vs glibenclamide dose 1 group, as tested by the Chi square test. <b>c)</b> After a glucose challenge untreated mice surviving till the week 25 (open symbols) displayed a sustained hyperglycaemia indicating glucose intolerance, whereas the glibenclamide-treated mice rapidly returned to normoglycaemia. Data are mean + SEM values of glycaemias from an experiment that tested in parallel the 3 groups of NOD mice. *** p< 0.001 compared to the glibenclamide-treated groups, as tested by ANOVA. n are numbers of mice investigated.</p

1,25(OH)₂D₃ and TX527 reduce IFN-γ, IL-4, and IL-17 but increase IL-10 in expanded human CD4⁺CD25^highCD127^low T cells.

<p>Peripheral blood CD3⁺ T cells from control donors (n = 5) or type 1 diabetes patients (n = ) were cultured for 8 days in the presence of 10^-8 M 1,25(OH)₂D₃ (1,25D₃) or TX527 or corresponding concentration of vehicle (CTR). CD4⁺CD25^highCD127^low T cells were sort-purified and mRNA expression of IFN-γ, IL-4, IL-17, and IL-10 was quantified by real-time RT-PCR using B2M and RPL27 as normalization genes. Bar graphs represent the mean ± SEM. Significance was tested using a two-tailed Mann-Whitney test. *<i>P</i><0.05; **<i>P</i><0.01. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃- or TX527-exposed human T cells from control donors and type 1 diabetes patients can suppress autologous CD4 and CD8 T cell responses.

<p><b>A, B</b>: CFSE-labeled responder cells from control (Control, n = 5–7) and type 1 diabetes (T1D, n = 7–10) donors were stimulated for 4 days with anti-CD3/CD28 mAbs and co-cultured with autologous unsorted CD4⁺ (<b>A</b>) or sorted CD4⁺CD25^highCD127^low (<b>B</b>) T cell populations (day 8) from control-, 1,25(OH)₂D₃- or TX527-treated cultures, as indicated. Shown are bar graphs summarizing the percentage suppression of proliferation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109194#s2" target="_blank">Methods</a> section) of CD4⁺ (top) and CD8⁺ (bottom) T cells without or with Tregs at a 2∶1 (in case of unsorted CD4⁺ T cells, <b>A</b>) or 1∶1 (in case of sorted CD4⁺CD25^highCD127^low T cells, <b>B</b>) Treg:Tresponder ratio. Significance was tested using a two-tailed Mann-Whitney test, all not significantly different. * <i>P</i><0.05. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃ and TX527 reduce T helper cytokines in human T cell cultures.

<p>Human peripheral blood CD3⁺ T cells, isolated from control subjects (n = 19) and type 1 diabetes patients (n = 20), were activated using anti-CD3/CD28 and treated with vehicle (CTR), 10⁻⁸ M 1,25(OH)₂D₃ (1,25D₃) or 10⁻⁸ M TX527. Concentrations of indicated cytokines were determined in the supernatant of day 8 cultures. Results are shown as bar graphs of mean ± SEM, data are grouped per treatment and donor type. Significance was calculated using a two-tailed Mann-Whitney test. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001. All other comparisons were not statistically significant.</p