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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

Quantitative analyses upon the dual inactivation of Arx and Pax4 in glucagon-producing cells.(A–E) Quantitative comparison of the number of insulin- (A), glucagon- (B) and somatostatin- (C) expressing cells between 4 month-old Glu-ArxKO/Pax4KO animals and age-/sex-matched WT controls. A significant increase in the numbers of insulin- and somatostatin-expressing cells was observed in Glu-ArxKO/Pax4KO animals, whilst no significant variation in glucagon+ cells was noted. Interestingly, both islet count (D) and size (E) were significantly increased in these animals compared to their WT counterparts, suggesting a process of islet neogenesis in addition to an increased insulin+ cell mass. (F) 4 month-old Glu-ArxKO/Pax4KO animals (and age/sex-matched WT controls) were challenged with glucose. Double-mutant animals displayed an increased capacity to counteract the glucose bolus with a lower peak in glycemia, suggestive of a functional increased β-cell mass. n≥3 in all experiments, ** p<0.01, * p<0.05 using ANOVA. (G) Schematic detailing the consequences of Arx (and Pax4) inactivation triggered in α-cells. Following the inactivation of Arx (and Pax4), α-cells are converted into β-like cells (1–2). The resulting shortage in glucagon (and/or putative additional signals - 3) promotes the proliferation of duct-lining cells, some of which re-express the developmental factor Ngn3. Our results indicate that such Ngn3+ cells adopt an endocrine cell identity, the glucagon+ cell fate being clearly favored (4). Whether neo-generated somatostatin+ cells contribute to the supplementary β-like cell mass remains to be determined (“?”). Similarly, one could assume that Ngn3+ cells could directly give rise to β-like cells, but additional experiments and mouse lines would be required to address this question (“?”). Subsequently, neo-formed glucagon+ cells are, yet again, turned into β-like cells upon the inactivation of Arx (and Pax4) (5). Such repeated cycles of neogenesis/double conversion (3 to 5) eventually result in an islet hypertrophy caused by a β-like cell hyperplasia (6).
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pgen-1003934-g011: Quantitative analyses upon the dual inactivation of Arx and Pax4 in glucagon-producing cells.(A–E) Quantitative comparison of the number of insulin- (A), glucagon- (B) and somatostatin- (C) expressing cells between 4 month-old Glu-ArxKO/Pax4KO animals and age-/sex-matched WT controls. A significant increase in the numbers of insulin- and somatostatin-expressing cells was observed in Glu-ArxKO/Pax4KO animals, whilst no significant variation in glucagon+ cells was noted. Interestingly, both islet count (D) and size (E) were significantly increased in these animals compared to their WT counterparts, suggesting a process of islet neogenesis in addition to an increased insulin+ cell mass. (F) 4 month-old Glu-ArxKO/Pax4KO animals (and age/sex-matched WT controls) were challenged with glucose. Double-mutant animals displayed an increased capacity to counteract the glucose bolus with a lower peak in glycemia, suggestive of a functional increased β-cell mass. n≥3 in all experiments, ** p<0.01, * p<0.05 using ANOVA. (G) Schematic detailing the consequences of Arx (and Pax4) inactivation triggered in α-cells. Following the inactivation of Arx (and Pax4), α-cells are converted into β-like cells (1–2). The resulting shortage in glucagon (and/or putative additional signals - 3) promotes the proliferation of duct-lining cells, some of which re-express the developmental factor Ngn3. Our results indicate that such Ngn3+ cells adopt an endocrine cell identity, the glucagon+ cell fate being clearly favored (4). Whether neo-generated somatostatin+ cells contribute to the supplementary β-like cell mass remains to be determined (“?”). Similarly, one could assume that Ngn3+ cells could directly give rise to β-like cells, but additional experiments and mouse lines would be required to address this question (“?”). Subsequently, neo-formed glucagon+ cells are, yet again, turned into β-like cells upon the inactivation of Arx (and Pax4) (5). Such repeated cycles of neogenesis/double conversion (3 to 5) eventually result in an islet hypertrophy caused by a β-like cell hyperplasia (6).

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)

Quantitative analyses upon the dual inactivation of Arx and Pax4 in glucagon-producing cells.(A–E) Quantitative comparison of the number of insulin- (A), glucagon- (B) and somatostatin- (C) expressing cells between 4 month-old Glu-ArxKO/Pax4KO animals and age-/sex-matched WT controls. A significant increase in the numbers of insulin- and somatostatin-expressing cells was observed in Glu-ArxKO/Pax4KO animals, whilst no significant variation in glucagon+ cells was noted. Interestingly, both islet count (D) and size (E) were significantly increased in these animals compared to their WT counterparts, suggesting a process of islet neogenesis in addition to an increased insulin+ cell mass. (F) 4 month-old Glu-ArxKO/Pax4KO animals (and age/sex-matched WT controls) were challenged with glucose. Double-mutant animals displayed an increased capacity to counteract the glucose bolus with a lower peak in glycemia, suggestive of a functional increased β-cell mass. n≥3 in all experiments, ** p<0.01, * p<0.05 using ANOVA. (G) Schematic detailing the consequences of Arx (and Pax4) inactivation triggered in α-cells. Following the inactivation of Arx (and Pax4), α-cells are converted into β-like cells (1–2). The resulting shortage in glucagon (and/or putative additional signals - 3) promotes the proliferation of duct-lining cells, some of which re-express the developmental factor Ngn3. Our results indicate that such Ngn3+ cells adopt an endocrine cell identity, the glucagon+ cell fate being clearly favored (4). Whether neo-generated somatostatin+ cells contribute to the supplementary β-like cell mass remains to be determined (“?”). Similarly, one could assume that Ngn3+ cells could directly give rise to β-like cells, but additional experiments and mouse lines would be required to address this question (“?”). Subsequently, neo-formed glucagon+ cells are, yet again, turned into β-like cells upon the inactivation of Arx (and Pax4) (5). Such repeated cycles of neogenesis/double conversion (3 to 5) eventually result in an islet hypertrophy caused by a β-like cell hyperplasia (6).
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pgen-1003934-g011: Quantitative analyses upon the dual inactivation of Arx and Pax4 in glucagon-producing cells.(A–E) Quantitative comparison of the number of insulin- (A), glucagon- (B) and somatostatin- (C) expressing cells between 4 month-old Glu-ArxKO/Pax4KO animals and age-/sex-matched WT controls. A significant increase in the numbers of insulin- and somatostatin-expressing cells was observed in Glu-ArxKO/Pax4KO animals, whilst no significant variation in glucagon+ cells was noted. Interestingly, both islet count (D) and size (E) were significantly increased in these animals compared to their WT counterparts, suggesting a process of islet neogenesis in addition to an increased insulin+ cell mass. (F) 4 month-old Glu-ArxKO/Pax4KO animals (and age/sex-matched WT controls) were challenged with glucose. Double-mutant animals displayed an increased capacity to counteract the glucose bolus with a lower peak in glycemia, suggestive of a functional increased β-cell mass. n≥3 in all experiments, ** p<0.01, * p<0.05 using ANOVA. (G) Schematic detailing the consequences of Arx (and Pax4) inactivation triggered in α-cells. Following the inactivation of Arx (and Pax4), α-cells are converted into β-like cells (1–2). The resulting shortage in glucagon (and/or putative additional signals - 3) promotes the proliferation of duct-lining cells, some of which re-express the developmental factor Ngn3. Our results indicate that such Ngn3+ cells adopt an endocrine cell identity, the glucagon+ cell fate being clearly favored (4). Whether neo-generated somatostatin+ cells contribute to the supplementary β-like cell mass remains to be determined (“?”). Similarly, one could assume that Ngn3+ cells could directly give rise to β-like cells, but additional experiments and mouse lines would be required to address this question (“?”). Subsequently, neo-formed glucagon+ cells are, yet again, turned into β-like cells upon the inactivation of Arx (and Pax4) (5). Such repeated cycles of neogenesis/double conversion (3 to 5) eventually result in an islet hypertrophy caused by a β-like cell hyperplasia (6).
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