Ghrelin⁺ cells originate from alpha-cells in the alpha-cell-specific <i>Pax6</i> KO islets.

<p>Double immunofluorescence staining of pancreatic cryosections from 1 month old mice. Ghrelin expression is not detected in the control islets (a-c). In alpha-cell-specific <i>Pax6</i> KO islets ghrelin expression is up regulated in YFP labeled cells (d-f) and rarely ghrelin expression co-localizes with glucagon expression (arrows d-f).</p

Co-expression of ghrelin with insulin, iAPP, and PC1/3 in the islets of beta-cell-specific <i>Pax6</i> KO mice.

<p>Double immunofluorescence staining of pancreatic cryosections from 2.5 month old mice at 2 weeks after tamoxifen induction. Ghrelin⁺ cells are not detected in the control islets (a-c). In the beta-cell-specific <i>Pax6</i> KO islets, ghrelin expression is up regulated (d-f) and some of the ghrelin⁺ cells co-express insulin (d). Additionally, these ghrelin⁺ cells co-express iAPP (e) and PC1/3 (f).</p

Development of hyperglycemia and resulting beta-cell regeneration in the beta-cell-specific <i>Pax6</i> KO mice.

<p>Measurement of blood glucose level in mice after tamoxifen induction at 3 weeks (a) or 1.5 month (b) (n = 5). Increase in blood glucose level is observed at either age but a slight decrease in the long term is only seen in young mice (a). Quantification of insulin⁺ YFP^-, YFP⁺ insulin^-, and insulin⁺ YFP⁺ cells in the islets of 13 week old mice at 10 weeks after tamoxifen induction (c) and 6 month old mice at 4.5 month after tamoxifen induction (d) (n = 3). Number of insulin⁺ YFP^- cells is increased at either age indicating some beta-cell regeneration. Due to the loss of insulin expression in YFP labeled <i>Pax6</i>-deficient cells, a significant increase in the number of YFP⁺ insulin^- and a decrease in the number of YFP⁺ insulin⁺ cells is also observed in the KO islets. (Control = <i>RIP-CreER;R26-YFP;Pax6</i>^<i>+/+</i>, Pax6 KO = <i>RIP-CreER;R26-YFP;Pax6</i>^<i>fl/fl</i>). Error bars represent SEM; *p<0.05.</p

Different experimental time lines used for the beta-cell-specific <i>Pax6</i> ablation.

<p>Age at tamoxifen induction and analysis time point post-induction is indicated. After each timeline, the respective figure number is also mentioned.</p

Alpha-cell-specific ablation of <i>Pax6</i>.

<p>Double immunofluorescence staining of pancreatic cryosections from 1 month old mice. In control islets, all the YFP⁺ cells express <i>Pax6</i> and glucagon (a-c). In alpha-cell-specific <i>Pax6</i> KO islets, most of the YFP⁺ cells are negative for <i>Pax6</i> and have lost the expression of glucagon as well (d-f). YFP^- glucagon⁺ cells in the KO islets express <i>Pax6</i> (arrowheads d-f). Rarely YFP⁺ glucagon⁺<i>Pax6^-</i> cells are also found in the KO islets (arrows d-f). Quantification of YFP⁺ glucagon⁺ cells in relation to total glucagon⁺ cells in 1 month old non-inducible alpha-cell-specific control (<i>Glu-Cre;R26-YFP;Pax6</i>^<i>+/+</i>) mice, and in Dox-inducible alpha-cell-specific control (<i>Glu-rtTA;TetO-Cre;R26-YFP;Pax6</i>^<i>+/+</i>) mice at 4 months of age following 6 weeks of Doxycycline treatment (g) (n = 3). Nearly 68% and 75% of the glucagon⁺ cells are labeled with YFP in non-inducible and Dox-inducible alpha-cell-specific control mice, respectively. Error bars represent SEM.</p

Ghrelin cells originate from beta-cells in the beta-cell-specific <i>Pax6</i> KO islets.

<p>Double immunofluorescence staining of pancreatic cryosections from 4 month old mice at 6 weeks after tamoxifen induction (a-f) and 6 month old mice at 4.5 months after tamoxifen induction (g-i). Ghrelin expression is not detected in the control islets (a-c). In beta-cell-specific <i>Pax6</i> KO islets, ghrelin expression is up regulated and that of insulin down regulated in the YFP labeled <i>Pax6</i>-deficient cells (d-f). Most of the ghrelin⁺ cells have lost the expression of insulin but some are still insulin⁺ (arrows d-f). Some YFP⁺ cells in the KO islets are negative for both insulin and ghrelin (arrowhead d-f). At 4.5 months after tamoxifen induction many of the YFP⁺ cells are negative for both insulin and ghrelin (arrowheads g-i).</p

A genetic mouse model for progressive ablation and regeneration of insulin producing beta-cells

<div><p>The putative induction of adult β-cell regeneration represents a promising approach for the treatment of type 1 diabetes. Toward this ultimate goal, it is essential to develop an inducible model mimicking the long-lasting disease progression. In the current study, we have established a novel β-cell ablation mouse model, in which the β-cell mass progressively declines, as seen in type 1 diabetes. The model is based on the β-cell specific genetic ablation of the transcription initiation factor 1A, TIF-IA, essential for RNA Polymerase I activity (TIF-IA^Δ/Δ). Using this approach, we induced a slow apoptotic response that eventually leads to a protracted β-cell death. In this model, we observed β-cell regeneration that resulted in a complete recovery of the β-cell mass and normoglycemia. In addition, we showed that adaptive proliferation of remaining β-cells is the prominent mechanism acting to compensate for the massive β-cell loss in young but also aged mice. Interestingly, at any age, we also detected β-like cells expressing the glucagon hormone, suggesting a transition between α- and β-cell identities or <i>vice versa</i>. Taken together, the TIF-IA^Δ/Δ mouse model can be used to investigate the potential therapeutic approaches for type 1 diabetes targeting β-cell regeneration.</p></div

Variable effect of <i>Pax6</i> ablation in the islets of adult-ubiquitous <i>Pax6</i> KO mice.

<p>Double immunofluorescence staining of pancreatic cryosections from mice that were injected with tamoxifen at 2 months of age and analysed at 1 week, 2 weeks, and 3 weeks post tamoxifen induction. Ghrelin⁺ cells are not detected in the control islets (a-d). In the ubiquitous <i>Pax6</i> KO islets, ghrelin expression is up regulated (e-p). Ghrelin expression shows co-localization with that of glucagon (e, i, m) and insulin (f, j, n) but never with that of somatostatin (g, k, o) and PP (h, l, p). In ubiquitous <i>Pax6</i> KO islets, a loss of glucagon (e, i, m) and insulin expression (f, j, n) was observed but not that of somatostatin (g, k, o) and PP (h, l, p). Furthermore, glucagon⁺ cells were the first ones to be affected in relation to the loss of glucagon expression and upregulation of ghrelin expression (e, i, m). (TI = Tamoxifen induction).</p

Expression of Rfx6 and Pdx1 in the islets of beta-cell-specific <i>Pax6</i> KO mice.

<p>Immunofluorescence staining of pancreatic cryosections from 6 month old mice at 4.5 month after tamoxifen induction. Expression of Rfx6 (e, f) and Pdx1 (g, h) is maintained in the YFP⁺<i>Pax6</i>-deficient cells of the beta-cell-specific <i>Pax6</i> KO islets.</p

Loss of Glut2 expression due to direct and/or indirect effect of <i>Pax6</i> ablation in the islets of beta-cell-specific <i>Pax6</i> KO mice.

<p>Double immunofluorescence staining of pancreatic cryosections from 2 month old mice at 4 weeks after tamoxifen induction. In the control islets, expression of Glut2 is detected in both YFP⁺ and YFP^- insulin⁺ cells (a-c). In the beta-cell-specific <i>Pax6</i> KO islets, Glut2 expression is lost in the YFP labeled <i>Pax6</i>-deficient cells as well as in a majority of the YFP^- insulin⁺ cells (arrows d-f). Rarely, some YFP^- insulin⁺ cells do express Glut2 in the KO islets (arrowhead d-f).</p