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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

The inactivation of Pax4 in Arx-deficient glucagon-expressing cells does not impact β-like cell neogenesis.Representative photographs of immunohistochemical analyses performed on 6 month-old Glu-ArxKO/Pax4KO pancreata using the indicated antibody combinations. A clear loss of Arx was evidenced in Glu-ArxKO/Pax4KO glucagon+ cells (A), such cells not ectopically expressing Pax4 (as seen in Figures S2-S3). Interestingly, a number of insulin+ cells appeared Pax4−, such cells most likely deriving from Arx−/Y Pax4−/− glucagon+ cells (A–C). As noted in Glu-ArxKO and Dox+ IndGlu-ArxKO pancreata, an increase in islet size compared to controls (Figure 1), caused by an insulin+ cell hyperplasia, was observed in Glu-ArxKO/Pax4KO pancreata (A–I). Non-β-cell endocrine hormone-expressing cells displayed a preferential localization at poles of the islets, adjacent to neighboring ducts (E–I), reminiscent of the phenotype of the sole inactivation of Arx in glucagon+ cells. (Each photograph is representative of at least three independent animals).
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pgen-1003934-g010: The inactivation of Pax4 in Arx-deficient glucagon-expressing cells does not impact β-like cell neogenesis.Representative photographs of immunohistochemical analyses performed on 6 month-old Glu-ArxKO/Pax4KO pancreata using the indicated antibody combinations. A clear loss of Arx was evidenced in Glu-ArxKO/Pax4KO glucagon+ cells (A), such cells not ectopically expressing Pax4 (as seen in Figures S2-S3). Interestingly, a number of insulin+ cells appeared Pax4−, such cells most likely deriving from Arx−/Y Pax4−/− glucagon+ cells (A–C). As noted in Glu-ArxKO and Dox+ IndGlu-ArxKO pancreata, an increase in islet size compared to controls (Figure 1), caused by an insulin+ cell hyperplasia, was observed in Glu-ArxKO/Pax4KO pancreata (A–I). Non-β-cell endocrine hormone-expressing cells displayed a preferential localization at poles of the islets, adjacent to neighboring ducts (E–I), reminiscent of the phenotype of the sole inactivation of Arx in glucagon+ cells. (Each photograph is representative of at least three independent animals).

Mentions: It was previously shown that the forced expression of Pax4 in glucagon+ cells was sufficient to induce their neogenesis and conversion into β-like cells [23], [24]. Here, we show that, in fact, the inactivation of Arx initiated in embryonic, but also in adult, α-cells is sufficient to induce a similar outcome. One may therefore conclude that the misexpression of Pax4 in α-cells could induce the down-regulation of Arx and thereby trigger α-cell mediated β-like cell neogenesis. However, the opposite could also be true, that is, that the deletion of Arx could promote such processes by up-regulating Pax4. To discriminate between these two possibilities, we generated double-mutant animals allowing the conditional deletion of Arx and Pax4 specifically in α-cells. To achieve this purpose, we crossed ArxcKO animals with Pax4cKO animals (generated by knock-in of two LoxP sites within the Pax4 locus [35]). The resulting double transgenic animals were subsequently crossed with Glu-Cre mice to generate Glu-Cre::ArxcKO::Pax4cKO animals (referred to as Glu-ArxKO/Pax4KO). Using immunohistochemistry on 6 month-old triple transgenic pancreata, most glucagon-producing cells were found to be negative for both Arx and Pax4 (Figure 10A). Importantly, a number of insulin-producing cells were also found to lack Pax4, such cells most likely corresponding to α-cells converted into Arx−/Y Pax4−/− β-like cells (Figure 10B–C). Further examination of these triple transgenic pancreata by immunohistochemistry outlined, yet again, a substantial increase in the islet number and a clear islet hypertrophy caused by an insulin+ cell hyperplasia that was found to be similar to the one observed in animals with Arx deletion (Figure 10Dcompared to 1E–H). Quantitative analyses confirmed this augmentation in insulin+ cell numbers (Figure 11A–E), but also in the content in somatostatin+ cells, non-β-cells being again found preferentially located close to ducts within the islets (Figure 10E–I). Of note was the observation that, despite the lack of Pax4 in a number of β-cells, no alteration in basal glycemia of 4 month-old double-mutants could be detected as compared to controls (127±7 mg/dl and 121±4 mg/dl, respectively). Interestingly, upon glucose challenge, Glu-ArxKO/Pax4KO animals displayed a significantly improved response as previously seen in Glu-ArxKO mice (Figure 11F), suggestive of an increased functional β-like cell mass. Altogether, our analyses indicate that the combined loss of Arx and Pax4 in glucagon-producing cells results in a phenotype similar to that of Arx mutants, sustaining the notion that Arx represents the main player involved in α-cell-mediated β-like cell neogenesis processes.


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)

The inactivation of Pax4 in Arx-deficient glucagon-expressing cells does not impact β-like cell neogenesis.Representative photographs of immunohistochemical analyses performed on 6 month-old Glu-ArxKO/Pax4KO pancreata using the indicated antibody combinations. A clear loss of Arx was evidenced in Glu-ArxKO/Pax4KO glucagon+ cells (A), such cells not ectopically expressing Pax4 (as seen in Figures S2-S3). Interestingly, a number of insulin+ cells appeared Pax4−, such cells most likely deriving from Arx−/Y Pax4−/− glucagon+ cells (A–C). As noted in Glu-ArxKO and Dox+ IndGlu-ArxKO pancreata, an increase in islet size compared to controls (Figure 1), caused by an insulin+ cell hyperplasia, was observed in Glu-ArxKO/Pax4KO pancreata (A–I). Non-β-cell endocrine hormone-expressing cells displayed a preferential localization at poles of the islets, adjacent to neighboring ducts (E–I), reminiscent of the phenotype of the sole inactivation of Arx in glucagon+ cells. (Each photograph is representative of at least three independent animals).
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pgen-1003934-g010: The inactivation of Pax4 in Arx-deficient glucagon-expressing cells does not impact β-like cell neogenesis.Representative photographs of immunohistochemical analyses performed on 6 month-old Glu-ArxKO/Pax4KO pancreata using the indicated antibody combinations. A clear loss of Arx was evidenced in Glu-ArxKO/Pax4KO glucagon+ cells (A), such cells not ectopically expressing Pax4 (as seen in Figures S2-S3). Interestingly, a number of insulin+ cells appeared Pax4−, such cells most likely deriving from Arx−/Y Pax4−/− glucagon+ cells (A–C). As noted in Glu-ArxKO and Dox+ IndGlu-ArxKO pancreata, an increase in islet size compared to controls (Figure 1), caused by an insulin+ cell hyperplasia, was observed in Glu-ArxKO/Pax4KO pancreata (A–I). Non-β-cell endocrine hormone-expressing cells displayed a preferential localization at poles of the islets, adjacent to neighboring ducts (E–I), reminiscent of the phenotype of the sole inactivation of Arx in glucagon+ cells. (Each photograph is representative of at least three independent animals).
Mentions: It was previously shown that the forced expression of Pax4 in glucagon+ cells was sufficient to induce their neogenesis and conversion into β-like cells [23], [24]. Here, we show that, in fact, the inactivation of Arx initiated in embryonic, but also in adult, α-cells is sufficient to induce a similar outcome. One may therefore conclude that the misexpression of Pax4 in α-cells could induce the down-regulation of Arx and thereby trigger α-cell mediated β-like cell neogenesis. However, the opposite could also be true, that is, that the deletion of Arx could promote such processes by up-regulating Pax4. To discriminate between these two possibilities, we generated double-mutant animals allowing the conditional deletion of Arx and Pax4 specifically in α-cells. To achieve this purpose, we crossed ArxcKO animals with Pax4cKO animals (generated by knock-in of two LoxP sites within the Pax4 locus [35]). The resulting double transgenic animals were subsequently crossed with Glu-Cre mice to generate Glu-Cre::ArxcKO::Pax4cKO animals (referred to as Glu-ArxKO/Pax4KO). Using immunohistochemistry on 6 month-old triple transgenic pancreata, most glucagon-producing cells were found to be negative for both Arx and Pax4 (Figure 10A). Importantly, a number of insulin-producing cells were also found to lack Pax4, such cells most likely corresponding to α-cells converted into Arx−/Y Pax4−/− β-like cells (Figure 10B–C). Further examination of these triple transgenic pancreata by immunohistochemistry outlined, yet again, a substantial increase in the islet number and a clear islet hypertrophy caused by an insulin+ cell hyperplasia that was found to be similar to the one observed in animals with Arx deletion (Figure 10Dcompared to 1E–H). Quantitative analyses confirmed this augmentation in insulin+ cell numbers (Figure 11A–E), but also in the content in somatostatin+ cells, non-β-cells being again found preferentially located close to ducts within the islets (Figure 10E–I). Of note was the observation that, despite the lack of Pax4 in a number of β-cells, no alteration in basal glycemia of 4 month-old double-mutants could be detected as compared to controls (127±7 mg/dl and 121±4 mg/dl, respectively). Interestingly, upon glucose challenge, Glu-ArxKO/Pax4KO animals displayed a significantly improved response as previously seen in Glu-ArxKO mice (Figure 11F), suggestive of an increased functional β-like cell mass. Altogether, our analyses indicate that the combined loss of Arx and Pax4 in glucagon-producing cells results in a phenotype similar to that of Arx mutants, sustaining the notion that Arx represents the main player involved in α-cell-mediated β-like cell neogenesis processes.

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