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The inactivation of Arx in pancreatic α-cells triggers their neogenesis and conversion into functional β-like cells.

Courtney M, Gjernes E, Druelle N, Ravaud C, Vieira A, Ben-Othman N, Pfeifer A, Avolio F, Leuckx G, Lacas-Gervais S, Burel-Vandenbos F, Ambrosetti D, Hecksher-Sorensen J, Ravassard P, Heimberg H, Mansouri A, Collombat P - PLoS Genet. (2013)

Bottom Line: Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis.Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion.Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

View Article: PubMed Central - PubMed

Affiliation: Université de Nice Sophia Antipolis, iBV, UMR 7277, Nice, France ; Inserm, iBV, U1091, Nice, France ; CNRS, iBV, UMR 7277, Nice, France.

ABSTRACT
Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon(+) cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

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Related in: MedlinePlus

Localized cellular proliferation in the pancreatic ductal lining upon α-cell-specific Arx loss.(A–I) A 10-day BrdU pulse-chase was performed in 3 month-old IndGlu-ArxKO animals subjected to a 2 month-long Dox treatment. An increase in the number of proliferating cells was thereby noted, most of these being detected in the lining of the duct close to the non-β-cell cluster (B–C, E–F, H–I compared to A, D, G). Of note, the ductal epithelium was found thickened in mutant pancreata, suggesting an increased ductal cell number (C, F, I). (J–L) The investigation of cell proliferation in 13 month-old Glu-ArxKO animals also revealed increased numbers of proliferating cells (K–L) compared to controls (J), as assayed by the expression of the proliferation marker KI67. These were again mainly found in the ductal lining adjacent to the islets (K–L). For the purpose of clarity, islets are outlined in dashed yellow lines in selected photographs.
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pgen-1003934-g006: Localized cellular proliferation in the pancreatic ductal lining upon α-cell-specific Arx loss.(A–I) A 10-day BrdU pulse-chase was performed in 3 month-old IndGlu-ArxKO animals subjected to a 2 month-long Dox treatment. An increase in the number of proliferating cells was thereby noted, most of these being detected in the lining of the duct close to the non-β-cell cluster (B–C, E–F, H–I compared to A, D, G). Of note, the ductal epithelium was found thickened in mutant pancreata, suggesting an increased ductal cell number (C, F, I). (J–L) The investigation of cell proliferation in 13 month-old Glu-ArxKO animals also revealed increased numbers of proliferating cells (K–L) compared to controls (J), as assayed by the expression of the proliferation marker KI67. These were again mainly found in the ductal lining adjacent to the islets (K–L). For the purpose of clarity, islets are outlined in dashed yellow lines in selected photographs.

Mentions: While the conversion of α-cells into β-like cells is of interest, it cannot account for the dramatic β-like cell hyperplasia noted in our animal models, nor for the continuous detection of glucagon+ cells despite their conversion. Hence, to gain further insight into the mechanisms underlying these processes, we assayed proliferating cells combining BrdU labeling and immunohistochemical detection of the KI67 marker. Importantly, in 2mDox+ IndGlu-ArxKO pancreata of animals subjected to a long-term 10-day BrdU pulse-chase, we observed a thickened ductal epithelium (insets in Figure 6B–C, E–F, H–I), most of the BrdU+ cells being detected in some epithelial cells and in the adjacent ductal lining while a few appeared located at a pole of the islet where non-β-cells are detected (Figure 6A–I). Similar observations were made using KI67 labeling in Glu-ArxKO animals, even in mice as old as 13 months of age (Figure 6J–L). We therefore investigated whether Ngn3 could be re-expressed in animals lacking Arx in α-cells. Indeed, Ngn3 was previously found to be re-expressed in duct-lining cells in animals that underwent pancreatic duct ligation [31], [32] or with the forced misexpression of Pax4 in α-cells [23], [24]. Additional work suggested that Ngn3+ cells could be continuously generated and converted into endocrine cells [23], [24], [32]. Hence, we assayed Glu-ArxKO pancreata for Ngn3 using immunohistochemistry. Ngn3 has been previously detected in endocrine cells being expressed at very low levels [33]: we confirmed these observations (Figure 7A, D). However, a much stronger expression of Ngn3 was noted mainly in cells located in the pancreatic ductal lining of animals lacking Arx in α-cells (Figure 7B–C, E–F). A time course analysis of Ngn3+ cells in pancreata of IndGlu-ArxKO animals subjected to increasing durations of Dox treatment outlined Ngn3+ cells within the ductal epithelium and lining, as well as in islet cells located close to ducts, as early as 10 days post-Dox treatment initiation, a similar trend being detected 11 days later (Figure 7G–I). Importantly, the downstream target of Ngn3, Rfx6 [34], was also found ectopically expressed in the ductal lining (Figure 7J–L) of Glu-ArxKO animals. These results suggesting a reactivation of, at least, a part of the endocrine differentiation program, we queried the mechanisms triggering such processes. One possibility could be the loss of α-cells, and the resulting shortage in glucagon, provoked by their conversion into β-like cells. To verify this hypothesis, we initiated long-term glucagon supplementation experiments in IndGlu-ArxKO animals. Interestingly, while Dox+ IndGlu-ArxKO pancreata showed a significant augmentation of the insulin+ or somatostatin+ cells mass following 3 weeks of treatment with saline and Dox (as compared to controls – Figure 8A), animals treated with glucagon and Dox exhibited a diminished increase in insulin+, glucagon+, and somatostatin+ cell counts (Figure 8A). Taken together, these results provide evidence that, upon α-cell-mediated Arx deficiency, α-cells are converted into β-like cells. This, in turn, induces glucagon shortage-dependent regeneration processes characterized by the continuous proliferation of duct-lining cells, the re-expression of the developmental factors Ngn3 and Rfx6, and the compensatory neogenesis of endocrine cells, neo-formed glucagon+ cells acquiring a β-like cell identity upon glucagon expression and subsequent Arx inactivation.


The inactivation of Arx in pancreatic α-cells triggers their neogenesis and conversion into functional β-like cells.

Courtney M, Gjernes E, Druelle N, Ravaud C, Vieira A, Ben-Othman N, Pfeifer A, Avolio F, Leuckx G, Lacas-Gervais S, Burel-Vandenbos F, Ambrosetti D, Hecksher-Sorensen J, Ravassard P, Heimberg H, Mansouri A, Collombat P - PLoS Genet. (2013)

Localized cellular proliferation in the pancreatic ductal lining upon α-cell-specific Arx loss.(A–I) A 10-day BrdU pulse-chase was performed in 3 month-old IndGlu-ArxKO animals subjected to a 2 month-long Dox treatment. An increase in the number of proliferating cells was thereby noted, most of these being detected in the lining of the duct close to the non-β-cell cluster (B–C, E–F, H–I compared to A, D, G). Of note, the ductal epithelium was found thickened in mutant pancreata, suggesting an increased ductal cell number (C, F, I). (J–L) The investigation of cell proliferation in 13 month-old Glu-ArxKO animals also revealed increased numbers of proliferating cells (K–L) compared to controls (J), as assayed by the expression of the proliferation marker KI67. These were again mainly found in the ductal lining adjacent to the islets (K–L). For the purpose of clarity, islets are outlined in dashed yellow lines in selected photographs.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814322&req=5

pgen-1003934-g006: Localized cellular proliferation in the pancreatic ductal lining upon α-cell-specific Arx loss.(A–I) A 10-day BrdU pulse-chase was performed in 3 month-old IndGlu-ArxKO animals subjected to a 2 month-long Dox treatment. An increase in the number of proliferating cells was thereby noted, most of these being detected in the lining of the duct close to the non-β-cell cluster (B–C, E–F, H–I compared to A, D, G). Of note, the ductal epithelium was found thickened in mutant pancreata, suggesting an increased ductal cell number (C, F, I). (J–L) The investigation of cell proliferation in 13 month-old Glu-ArxKO animals also revealed increased numbers of proliferating cells (K–L) compared to controls (J), as assayed by the expression of the proliferation marker KI67. These were again mainly found in the ductal lining adjacent to the islets (K–L). For the purpose of clarity, islets are outlined in dashed yellow lines in selected photographs.
Mentions: While the conversion of α-cells into β-like cells is of interest, it cannot account for the dramatic β-like cell hyperplasia noted in our animal models, nor for the continuous detection of glucagon+ cells despite their conversion. Hence, to gain further insight into the mechanisms underlying these processes, we assayed proliferating cells combining BrdU labeling and immunohistochemical detection of the KI67 marker. Importantly, in 2mDox+ IndGlu-ArxKO pancreata of animals subjected to a long-term 10-day BrdU pulse-chase, we observed a thickened ductal epithelium (insets in Figure 6B–C, E–F, H–I), most of the BrdU+ cells being detected in some epithelial cells and in the adjacent ductal lining while a few appeared located at a pole of the islet where non-β-cells are detected (Figure 6A–I). Similar observations were made using KI67 labeling in Glu-ArxKO animals, even in mice as old as 13 months of age (Figure 6J–L). We therefore investigated whether Ngn3 could be re-expressed in animals lacking Arx in α-cells. Indeed, Ngn3 was previously found to be re-expressed in duct-lining cells in animals that underwent pancreatic duct ligation [31], [32] or with the forced misexpression of Pax4 in α-cells [23], [24]. Additional work suggested that Ngn3+ cells could be continuously generated and converted into endocrine cells [23], [24], [32]. Hence, we assayed Glu-ArxKO pancreata for Ngn3 using immunohistochemistry. Ngn3 has been previously detected in endocrine cells being expressed at very low levels [33]: we confirmed these observations (Figure 7A, D). However, a much stronger expression of Ngn3 was noted mainly in cells located in the pancreatic ductal lining of animals lacking Arx in α-cells (Figure 7B–C, E–F). A time course analysis of Ngn3+ cells in pancreata of IndGlu-ArxKO animals subjected to increasing durations of Dox treatment outlined Ngn3+ cells within the ductal epithelium and lining, as well as in islet cells located close to ducts, as early as 10 days post-Dox treatment initiation, a similar trend being detected 11 days later (Figure 7G–I). Importantly, the downstream target of Ngn3, Rfx6 [34], was also found ectopically expressed in the ductal lining (Figure 7J–L) of Glu-ArxKO animals. These results suggesting a reactivation of, at least, a part of the endocrine differentiation program, we queried the mechanisms triggering such processes. One possibility could be the loss of α-cells, and the resulting shortage in glucagon, provoked by their conversion into β-like cells. To verify this hypothesis, we initiated long-term glucagon supplementation experiments in IndGlu-ArxKO animals. Interestingly, while Dox+ IndGlu-ArxKO pancreata showed a significant augmentation of the insulin+ or somatostatin+ cells mass following 3 weeks of treatment with saline and Dox (as compared to controls – Figure 8A), animals treated with glucagon and Dox exhibited a diminished increase in insulin+, glucagon+, and somatostatin+ cell counts (Figure 8A). Taken together, these results provide evidence that, upon α-cell-mediated Arx deficiency, α-cells are converted into β-like cells. This, in turn, induces glucagon shortage-dependent regeneration processes characterized by the continuous proliferation of duct-lining cells, the re-expression of the developmental factors Ngn3 and Rfx6, and the compensatory neogenesis of endocrine cells, neo-formed glucagon+ cells acquiring a β-like cell identity upon glucagon expression and subsequent Arx inactivation.

Bottom Line: Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis.Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion.Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

View Article: PubMed Central - PubMed

Affiliation: Université de Nice Sophia Antipolis, iBV, UMR 7277, Nice, France ; Inserm, iBV, U1091, Nice, France ; CNRS, iBV, UMR 7277, Nice, France.

ABSTRACT
Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon(+) cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

Show MeSH
Related in: MedlinePlus