The recent discovery that genetically modified α cells can regenerate and convert into β-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-β-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into β-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated β-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in β-like cell counts, suggestive of α-to-β-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated β-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.

The regenerative capacity of glucagon-producing cells and their potential to convert into β-like cells are of great interest in the context of T1D research. However, the aforementioned transgenic approaches cannot be translated to diabetes treatment in humans. We therefore performed a number of screens aiming to identify small molecules/chemical compounds mimicking the effects of the ectopic expression of Pax4/inhibition of Arx in α cells. Here, we report the identification of γ-aminobutyric acid (GABA) as an α-to-β-like cell inducer. Interestingly, previous studies have suggested a role for GABA in the endocrine pancreas and in diabetes mellitus. GABA, synthesized from glutamate by glutamic acid decarboxylase (GAD) in β cells, is an extracellular signaling molecule acting on the pancreatic islets (). Once released, GABA is thought to serve as a functional regulator of pancreatic hormone secretion or as a fast-acting paracrine signaling molecule for the communication between β cells and other islet cells (). In addition, other studies have demonstrated that GABA participates in maintaining the β cell mass and in protecting β cells from apoptosis in vitro (). Of note, a putative β cell proliferation was suggested upon short-term GABA treatment but, in the absence of lineage tracing, this remains to be ascertained (). Here, using lineage tracing, we provide evidence that long-term GABA treatment eventually induces the conversion of glucagon-expressing α cells into functional insulin-producing β-like cells. Interestingly, this α-to-β-like cell conversion leads to the reactivation of developmental processes resulting in a compensatory α cell neogenesis. Upon sustained GABA administration, these newly generated α cells are again converted into β-like cells, such cycle of neogenesis and conversion eventually leading to β-like cell hyperplasia. Importantly, upon GABA administration, the entire β cell mass can be regenerated at least twice following two cycles of streptozotocin-mediated β cell ablation. Lastly, we provide in vitro and ex vivo evidence that human α cells can also be converted into β-like cells upon maintained GABA exposure, such results opening the way for clinical trials with GABA or GABA-like compounds.

The mature pancreas includes three main cell subtypes: acinar, duct, and endocrine cells. Acinar cells produce and secrete digestive enzymes, which are routed to the intestine by a branched ductal network. Endocrine cells are involved in regulating nutrient metabolism and glucose homeostasis. Specifically, these are organized into cell clusters termed islets of Langerhans that are composed of five cell subtypes, each secreting a specific endocrine hormone: α-, β-, δ-, ε-, and PP-cells producing glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide (PP), respectively (). Both type 1 and 2 diabetes (T1D and T2D, respectively) ultimately result in pancreatic β cell loss and chronic hyperglycemia (). Importantly, despite the most recent advances in diabetes care, patients suffering from T1D still display, on average, a shortened life expectancy and a worsened quality of life as compared to healthy individuals. Aiming to develop alternative treatments, the replacement of lost β cells using different cell sources, such as stem, precursor, or differentiated cells, represents a promising strategy. However, despite major advances in mimicking embryonic β cell development in vitro (), a deeper understanding of the genetic program underlying human β cell genesis is still required to eventually generate fully differentiated β cells (). Toward this goal, and using the mouse as a model, a number of studies have demonstrated that during pancreas morphogenesis, several transcription factors are successively involved in the specification of pancreatic precursors toward the different endocrine cell fates. Among these, Pdx1 is required for pancreatic epithelium determination () whereas Ngn3 specifies the endocrine cell lineage (). Following Ngn3 induction, a complex network of transcription factors, including Arx and Pax4, drives endocrine precursor cells toward the different endocrine cell fates. These two factors were found to mutually inhibit each other’s transcription and to display antagonistic activities for proper endocrine cell lineage allocation (). Indeed, Pax4 promotes the β and δ cell lineages, whereas Arx is involved in specifying the α cell fate (). Importantly, it was previously demonstrated that embryonic glucagon-producing cells can continuously regenerate and convert into insulin-producing β-like cells through the sole ectopic expression of Pax4 (). It was subsequently shown that the misexpression of Pax4 in mouse adult α cells also induces their neogenesis and conversion into β-like cells, these being functional and allowing the reversion of multiple cycles of chemically induced hyperglycemia (). Finally, the downregulation of Arx activities in glucagoncells was recently found to represent the main trigger for α-to-β-like cell conversion ().

The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the alpha- and beta-cell lineages in the mouse endocrine pancreas.

The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the alpha- and beta-cell lineages in the mouse endocrine pancreas.

Aiming to determine whether human α cells could also be converted into insulincells in vitro, we first used 3D culture of human islets (). Importantly, following 14 days of culture in presence of GABA, we observed a significant 37% diminution in the glucagoncell counts and a concomitant 24% increase in the insulincell numbers, as compared to saline-supplemented islet cultures ( Figure 7 A). These results being suggestive of α-to-β-like cell conversion in human, the reprogramming process was investigated in human islet xenografts. Thus, 500 human islet equivalents were transplanted under the kidney capsule of immunodeficient mice that were subsequently treated daily with GABA (or saline) for 1 month before being sacrificed. A close examination of the transplants indicated a 1.21-fold increase in the total endocrine cell count (chromogranin Acells) and similar 1.19-fold augmentation in the (α+β) cell counts when comparing GABA-treated animals versus saline-administered counterparts ( Figures 7 B–7F). Importantly, upon GABA administration, the percentage of glucagoncells decreased 3.8-fold while the percentage of β cells increased 1.4-fold ( Figure 7 F). Despite the present lack of reliable methods for human α cell lineage tracing, these results suggest a conversion of human α cells into insulin-producing cells upon GABA exposure, thereby opening new and promising avenues in the context of diabetes research.

(F) Quantitative immunohistochemical analyses were performed and demonstrated a slight increase in the total endocrine cell count and a similar augmentation in the combined (α+β) cell sum, upon GABA exposure. Interestingly, by comparing the proportion of α and β cells in the (α+β) cell sum between the saline or GABA-treated groups, we observed an almost 4-fold reduction in the α cell proportion and a concomitant 38% augmentation in the β cell amount GABA-treated animals. These results therefore support the notion of the conversion of human α cells into β-like cells upon GABA administration. (n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

(B–E) Immunodeficient mice were transplanted with 500 human islet equivalents under their kidney capsules. These mice were then treated daily either with saline (B and C) or with GABA (D and E) for 1 month before being sacrificed. Immunohistochemical assessment of GABA-treated pancreatic transplants outlined a clear increase in the number of insulin-producing cells and a concurrent decrease in the number of glucagon-expressing cells compared to their saline-treated counterparts.

(A) Human islets were subjected to 14 days of 3D culture in presence of saline or GABA. The relative proportions of glucagon + and insulin + populations were quantified following immunohistochemical detection. Importantly, a 37% decrease in glucagon + cells and a concomitant 24% increase in insulin + cells were observed when comparing GABA-treated islet versus their saline-supplemented counterparts.

The aforementioned studies established that GABA administration induces the neogenesis and conversion of α cells into functional β-like cells in mice. However, whether the same applies to other species remained to be tested. Similarly, whether GABA could protect β cells from apoptosis (in addition to inducing their neogenesis) remained to be determined. To address these issues, we first analyzed the effects of cytokine-induced apoptosis (using interleukin-1β [IL-1β] and interferon γ [IFNγ]) on GABA-treated rat INS-1E β cells and on primary fluorescence-activated cell sorting (FACS)-purified rat β cells. In both instances, the apoptosis rates were similar ( Figures S7 A and S7B), indicating that GABA does not prevent cytokine-induced apoptosis. Subsequently, we determined whether FACS-sorted cultured rat α cells could be reprogrammed into insulin-producing cells upon 14 days of GABA treatment ( Figures S7 C–S7M). Interestingly, we observed a GABA dose-dependent decrease in glucagoncell content (albeit not significant) and a significant diminution in Arx transcript contents ( Figures S7 K and S7L). Concomitantly, a significant increase in the number of insulincell numbers was outlined ( Figure S7 M). Additional analyses demonstrated no effect of GABA treatment on the proliferation of FACS-sorted α or β cells ( Figure S7 K, bottom, and data not shown). Thus, despite the lack of lineage tracing, these data strongly suggest a conversion of α cells into insulincells. Unfortunately, the duration of GABA exposure could not be extended beyond 14 days, which corresponds to the maximal duration of viability of primary rat α cells under the present culture conditions.

(K–M) Quantification of glucagon- (K) and insulin- (M) positive cells as well as Arx transcript contents (L) were made by two observers. The results obtained indicate a decrease in the numbers of glucagon + cells and Arx transcripts whereas the contents in insulin + cells are augmented. The number of proliferating KI67 + cells were also counted in FACS-sorted α- (K) or β-cells (data not shown) treated with GABA or saline: no significant change was detected in either cell population. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 treated versus untreated; ANOVA followed by Student’s t test with Bonferroni correction.

(B) FACS-purified rat β-cells (>90% purity) were pre-treated with vehicle or GABA (1mM) during 48 hr. Culture medium was changed every 24h and fresh GABA added. After this pre-treatment, cells were left untreated or exposed to IL-1β + IFNγ (50 and 500U/ml, respectively) for 24h in the presence or absence of GABA (1mM). Apoptosis was evaluated by staining with the nuclear dies Hoechst 33342 and PI. Results are means ± SEM (n = 3); ∗∗∗ p < 0.001 cytokine-treated versus untreated; ANOVA followed by Student’s t test with Bonferroni correction. (C-L) FACS-purified rat α-cells (>90% purity) were treated during 14 days with different concentrations of GABA (0.05-1mM). Culture medium was changed every 24h and fresh GABA added. Glucagon (green), insulin (red) positive cells and total amount of cells (blue) were evaluated by immunocytochemistry.

(A) INS-1E cells were pre-treated with vehicle or GABA (1mM) for 72h. Culture medium was changed every 24h and fresh GABA added. After this pre-treatment, cells were left untreated or exposed to IL-1β + IFNγ (10 and 100U/ml, respectively) for 24h in the presence or absence of GABA (1mM). Apoptosis was evaluated by staining with the nuclear dies Hoechst 33342 and PI. Results are means ± SEM (n = 6); ∗∗∗ p < 0.001 cytokine-treated versus untreated; ANOVA followed by Student’s t test with Bonferroni correction.

To determine whether GABA could induce more than one cycle of β cell regeneration, mice that went through a first round of β cell ablation and β-like cell regeneration were subjected to a second round of streptozotocin treatment (1 month after stopping GABA treatment). These mice were then administered GABA (or saline) when their glycemia reached 300 mg/dL. Importantly, and as observed previously, while saline-treated mice (survivors of a first ablation round) eventually died from severe hyperglycemia, their GABA-treated counterparts saw, following a transitory peak, a normalization of their blood glucose levels resulting again in an extended lifespan ( Figure 6 K). Taken together, these results demonstrate that GABA treatment counteracts the consequences of chemically induced diabetes, even when the animals are severely diabetic. Importantly, GABA administration can induce at least two cycles of complete β cell mass neogenesis by virtue of regenerating α cells. These findings also provide evidence that the arrest of GABA treatment halts these α cell-mediated β-like cell neogenesis processes, indicating that these can be tightly controlled.

Aiming to further confirm the origin of neo-generated β-like cells, similar β cell ablation experiments were performed in Glu-Cre::ROSA mice allowing α cell lineage tracing. Importantly, using both X-Gal staining and immunohistochemical analyses ( Figures 6 G–6J), the vast majority of newly formed insulin-producing cells were found to be labeled with β-galactosidase (permanently marking cells that once expressed glucagon), demonstrating that most of the regenerated β-like cells passed through a glucagon-expressing transitional phase. Accordingly, the repetition of this β cell ablation procedure in presence of GABA and of an antagonist of the α cell-specific GABAreceptor, SR-95531 (), prevented the recovery from the lethal hyperglycemia, indicating that GABA mainly acts on α cells via the GABAreceptor ( Figure S6 B compared to 6 A). It is important to mention that mouse GABAreceptors are indeed expressed in mouse α cells () while mostly lacking in β cells, as outlined by RNA sequencing (RNA-seq) analyses on purified β cells (RPKM: Gabra4, <0.1; Gabrb3, 8; Gabrg2, 0; Gabre, <0.1; other GABAsubunits, < 0.1; G. Rutter, personal communication).

To determine whether GABA-induced neo-formed insulincells were functionally equivalent to endogenous β cells, 2-month-old WT animals were injected with a high dose of streptozotocin (STZ) to obliterate the pancreatic β cell mass. Once these animals were overtly diabetic, with a glycemia of ∼300 mg/dL, they were treated daily with GABA or saline (controls). While saline-treated control mice saw their glycemia increase further and died from extreme hyperglycemia, a steady recovery was observed (following a transitory peak in glycemia) in their GABA-treated counterparts ( Figure 6 A). Quantitative immunohistochemical analyses were performed on sections of saline-treated ( Figures 6 B and 6C) and GABA-treated ( Figures 6 D–6F) pancreata isolated 5–85 days post STZ administration. While STZ treatment induced a loss of insulin-producing cells in all conditions (above 96%; Figure 6 C), animals that received GABA displayed a progressive regeneration of their β-like cell mass ( Figures 6 D–6F), resulting in reconstituted islets 70–95 days following β cell ablation. It is worth noting that weight (data not shown) and glycemia were normal in the surviving animals that displayed an extended lifespan compared to controls ( Figure 6 A). The glucose responsiveness of these surviving GABA-treated mice was examined 4.5 months post β cell ablation, that is, following regeneration and additional neogenesis of insulincells. Upon challenge with a high dose of glucose, these animals exhibited an improved response with a lower raise in glycemia and a faster return to euglycemia as compared to controls ( Figure S6 A). Accordingly, a 2.1-fold increase in maximal insulin secretion, reflecting the augmented content of β-like cells, was observed in GABA-treated mice as compared to saline-administered animals ( Figure S6 A and inset).

(B) WT mice were subjected to streptozotocin (STZ) treatment. Upon reaching a diabetic state (glycemia ≥ 300mg/dl), they were treated daily with GABA or saline as shown in Figure 6 A. Three days later, all animals were also administered daily with SR-95531, a competitive antagonist of the GABAreceptor. Importantly, and unlike the recovery observed in streptozotocin-treated animals supplemented with GABA ( Figure 6 A), all animals that received SR-95531, including those that administered with GABA, died from severe hyperglycemia. These results therefore demonstrate that antagonizing the GABAreceptor prevents GABA-induced α cell-mediated β-like cell neogenesis.

(A) WT mice were subjected to streptozotocin (STZ) treatment. Upon reaching a diabetic state (glycemia ≥ 300mg/dl), these were treated daily with GABA for 4.2 months to first induce β-like regeneration and, subsequently, a β-like cell hyperplasia. Control animals were administered solely with saline for 4.2 months. Upon intraperitoneal glucose tolerance test, GABA-treated animals that recovered from STZ-induced diabetes were found to perform better than controls with a lower peak in glycemia and a faster return to euglycemia, suggestive of an increased and functional β-cell mass.

(K) WT animals were subjected to a first round of β cell ablation and treated with GABA for 2.7 months to regenerate their β cell mass. GABA administration was subsequently halted for 1 month prior to a second round of STZ-mediated β cell ablation. Half of the animals were treated with GABA daily once their glycemia reached 300 mg/dL, the other half with saline. Importantly, while saline-treated animals died due to severe hyperglycemia, their GABA-treated counterparts survived and eventually saw a normalization of their glycemia. This indicates that a functional β cell mass can be reconstituted at least two times solely upon sustained GABA exposure.

(G–J) Similar β cell ablation experiments were performed in Glu-Cre::ROSA mice allowing α cell lineage tracing. Importantly, upon GABA treatment, these animals also survived and showed a majority of regenerated insulin-producing cells labeled with the α cell tracer β-galactosidase, as outlined by X-Gal staining (G and H) and immunohistochemistry (I and J). This indicates that most regenerated β-like cells went through a transitional phase of glucagon expression.

(B–F) Quantitative immunohistochemical analyses during the course of these experiments outlined a loss of β cells 5 days post-STZ (B and C). Interestingly, upon GABA treatment, a progressive increase in insulin + cell count was observed, this continued augmentation eventually resulting in the replenishment of the whole β cell mass, 65–85 days post β cell ablation (D–F). β cell mass quantification is provided in (B–F) (in red) using the “WT+saline” condition as control (n = 4 for each condition).

(A) Two-month-old WT mice were subjected to high dose streptozotocin (STZ) treatment to ablate β cells and then treated with GABA (or saline) once they were overtly diabetic (glycemia ≥300 mg/dL). While saline-treated animals died due to severe diabetes, their GABA-treated counterparts, following a peak in glycemia, saw a progressive normalization of their blood glucose levels.

To further investigate a putative reactivation of endocrine developmental processes upon daily GABA administration, we analyzed the expression of a number of developmental marker genes involved in endocrine cell genesis. Interestingly, we observed a reactivation of the expression of the pro-endocrine gene Ngn3 in cells located within the ductal compartment of GABA-treated pancreata ( Figures 5 B and 5C ), while almost no expression of Ngn3 could be detected in control mice ( Figure 5 A). Lineage tracing experiments were subsequently performed to determine the putative contribution of these Ngn3-re-expressing cells to the supplementary endocrine cell mass. We used Ngn3-CreER™ mice (harboring a transgene composed of the Ngn3 promoter upstream of the phage P1 Cre recombinase fused to a mutant estrogen receptor) () crossed with the aforementioned ROSA animals. In GABA- and Tam-treated Ngn3-CreER::ROSA transgenic mice, a large number of β-Gal-labeled cells were observed near the ductal lining and, most importantly, within the islets ( Figures 5 E–5J), while controls appeared negative for β-galactosidase ( Figure 5 D). To further confirm the role of Ngn3 in the endocrine cell hyperplasia observed in GABA-treated animals, Ngn3 expression was inhibited in vivo by a knock-down approach. Following 2 days of GABA treatment, 2-month-old WT animals were injected, via the pancreatic duct, with GFP-encoding lentiviruses producing either a small hairpin RNA (shRNA) targeting Ngn3 transcripts (previously found to induce a 45% decrease in Ngn3 transcript contents) () or a scrambled shRNA. Subsequently, the animals were treated daily with GABA and examined 2 months later. While GABA-treated scrambled-infected pancreata displayed hypertrophic islets ( Figures 5 K, S5 G, and S5H), GABA-treated Ngn3 knock-down pancreata exhibited a 40.5% reduction in islet overgrowth and 69% decrease in islet counts ( Figures 5 L, 5M, S5 G, and S5H), indicating that Ngn3 re-expression is required for GABA-mediated endocrine cell neogenesis in adult mice. Importantly, we observed an accumulation of mesenchyme-like cells surrounding ducts in Ngn3 knock-down mice ( Figures 5 L–5P), suggestive of endocrine cell neogenesis that was stopped at the mesenchymal stage. Indeed, such cells were found to widely express vimentin ( Figures 5 N and 5O) or Snai2 ( Figure 5 P). Altogether, our data suggest that long-term GABA treatment eventually induces the reawakening of the Ngn3-controlled endocrine developmental program, such cells undergoing EMT prior to the acquisition of an endocrine cell identity.

(K–P) After 2 days of GABA treatment, 2-month-old WT mice were infected using GFP-encoding lentiviruses producing either a shRNA targeting Ngn3 transcripts or a scrambled shRNA. After infection, the animals were injected daily with GABA for 2 months before being sacrificed for examination. Importantly, while scrambled-infected pancreata display a GABA-mediated increase in islet size and β-like cell counts (K), Ngn3 knock-down pancreata do not exhibit such GABA-induced islet hypertrophy or β-like cell hyperplasia (L and M), demonstrating the requirement of Ngn3 re-expression for endocrine cell neogenesis. Note the massive accumulation of cells around ducts upon Ngn3 knock-down, such cells expressing the EMT labels Vimentin (N and O) or Snai2 (P), suggesting that, in the absence of Ngn3 re-expression, cells are neo-generated upon GABA treatment but fail to acquire an endocrine cell identity and remain in a mesenchymal state. For the purpose of clarity, dashed lines are used to surround the ductal lumen (in white), the islets (in yellow), and the ductal thickening (in green).

(D–J) Lineage tracing of cells re-expressing Ngn3 in Ngn3-CreER::ROSA mice using X-Gal staining (D–F) and immunohistochemistry (G–J). While tamoxifen- and saline-treated animals do not exhibit any β-galactosidase labeling (D), tamoxifen- and GABA-administered mice display numerous β-galactosidase + cells in the ductal lining, in the ductal epithelium and, most importantly, within the islet (E–J), with a labeling found in insulin + (G–I) and glucagon + (J) cells, suggesting that Ngn3-re-expressing ductal cells eventually adopt an endocrine cell identity.

(A–C) Ngn3 and the EMT marker, Vimentin, are re-expressed in the pancreatic ductal lining of GABA 3 m animals (B and C), while these are not detected in controls (A).

Glucagoncells were consistently detected in GABA-treated pancreata despite their conversion into β-like cells ( Figures 1 F, 1G, 1I–1K, and S2 J). In addition, the apoptosis rate was similar in GABA- versus saline-treated animals (data not shown). Together, these observations suggest a continued neogenesis of glucagoncells upon GABA treatment. Therefore, long-term (10 days) 5-bromo-2′-deoxyuridine (BrdU) labeling was used to detect cells undergoing or having undergone replication. As expected, very few BrdU-labeled cells (mostly insulin-expressing cells) were noted in control mice ( Figures 4 A, 4D, 4G, and 4J). However, in GABA-treated animals, a significant 7.0-fold ± 0.6-fold increase in the number of BrdUcells was observed ( Figures 4 A–4C), mainly within the epithelial cells in and near the lining of ducts and, to a lesser extent, within the endocrine compartment ( Figures 4 B, 4C, 4E, 4F, 4H, 4I, 4K, and 4L). In addition, long-term GABA-treated mice often demonstrated a thickening of the ductal lining in a cell nuclei-dense area reminiscent of mesenchyme ( Figures S5 B and S5C compared to S5 A). Indeed, closer examination revealed that most of these cells re-expressed the canonical mesenchymal marker, vimentin ( Figure S5 C). Similarly, Snai2cells were also detected at the same loci in the pancreata of GABA-treated animals ( Figures S5 D–S5F). Note that these markers were only expressed in scattered cells of control mice ( Figures S5 A and S5D and data not shown). Because vimentin and Snai2 are transcriptional regulators/hallmarks of the developmental epithelium to mesenchymal transition (EMT), these data suggest that the neo-generated endocrine cells undergo EMT as observed during embryogenesis ().

(G–H) Quantitative analyses of islet number and surface upon Ngn3 knock-down in GABA-treated pancreata. Quantitative comparison of the number (G) and surface (H) of islets between GABA-treated Ngn3 knock-down (Ngn3KD) mice and control animals (n = 3 for each group). Note the strong decrease in both islet size (40%) and number (69%) upon inhibition of Ngn3 re-expression. All data depicted as mean ± SEM in (G) and (H). Related to Figure 5

(A–F) EMT re-awakening in GABA-treated mice. Immunohistochemical analyses reveal that, in GABA-treated animals, the mesenchymal marker vimentin is strongly re-expressed in the thickened region surrounding ducts and close to islets (B–C). Accordingly, the transcriptional regulator of EMT, Snai2, is also found re-expressed in the ductal lining (E–F). Little to no vimentin or Snai2 labeling was detected in control mice (A), (D).

A 10-day BrdU pulse was performed in 2-month-old saline 2 m (A, D, G, and J) and GABA 2 m (B, C, E, F, H, I, K, and L) animals. While few proliferating cells were detected in controls, seven times more proliferating cells were observed in GABA-treated animals (B and C versus A; n = 4, p < 0.001). Interestingly, this increase in proliferating cell numbers was preferentially detected in the pancreatic ductal lining and epithelium, adjacent to islets (B, C, E, F, H, I, K, and L). Note also the presence of insulin- (B and C), glucagon- (E and F), or somatostatin- (H and I) expressing cells labeled with BrdU, suggesting that such cells have proliferated prior to hormone expression. For the purpose of clarity, the ductal lumen is outlined with dashed white lines, whereas the islets are outlined with dashed yellow lines.

To determine the origin of the hyperplastic endocrine cells observed in GABA-treated pancreata, further immunohistochemical analyses were performed. In very rare instances, we observed cells co-expressing both insulin and glucagon ( Figures 3 A and 3B ). Aiming to determine whether these may correspond to cells transitioning from an α- to a β-like cell phenotype, we treated Glu-Cre::ROSA mice (harboring the following transgenes: the rat glucagon promoter upstream of the phage P1 Cre recombinase [] and the Rosa26 promoter upstream of a loxP-Stop-loxP-β-galactosidase cassette (]) with GABA (or saline) for 2 months. Using X-gal staining to visualize β-galactosidase activity, we followed the outcome of cells having expressed or still expressing glucagon. β-galcells were detected solely in the mantle zone of islets from control mice treated with saline where glucagon-expressing cells are located ( Figures 3 C–3E). Importantly, in GABA-treated animals, β-galcells were located not only in the islet mantle zone but also in the islet core where insulincells are classically detected ( Figures 3 F–3H). Accordingly, immunofluorescent staining confirmed the presence of numerous insulincells that contained the α cell label β-galactosidase ( Figures 3 I–3K and S4 D–S4I ) in the pancreas of GABA-treated but not of control mice ( Figures S4 A–S4C). Quantitative analyses further validated these findings, with an initial labeling of 65%–68% of α cells with β-galactosidase in Glu-Cre::ROSA animals and the subsequent detection of this label in ∼60% of supplementary β-like cells in GABA 2 m pancreata ( Figure S4 J). These results therefore provide conclusive evidence that long-term GABA administration converts glucagon-expressing cells into β-like cells.

(J) Quantitative analyses confirmed such α-to-β-like cell conversion. Indeed, 65%–68% of α-cells were found to be labeled by β-galactosidase in Glu-Cre::ROSA pancreata. Interestingly, 31.1 ± 4.4% of β-like cells were found positive for β-galactosidase upon GABA administration, the β-like cell mass being concomitantly 2.1 ± 0.4-fold increased compared to controls. Thus, labeled β-cells represent 59.9 ± 8.5% of supplementary β-like cells (calculated as follow: (31.09x2.08)/1.08 = 59.88), a proportion in line with the initial 65%–68% labeling of α-cells. All data depicted as mean ± SEM. Related to Figure 3

(A–I) Glu-Cre::ROSA animals were used to label glucagon–producing cells and follow their progeny. These animals were treated with saline (A–C) or GABA (D-I) for 2 months. Pancreatic sections were labeled using a combination of anti-insulin and anti-β-galactosidase antibodies. Note, in GABA-treated animals (D–I), the detection of numerous cells that once expressed glucagon (β-galactosidase + ) and now produce the insulin hormone, such co-expression being expectedly not observed in their saline-administered counterparts (A–C).

(C–H) Lineage tracing of α cells in the pancreas of Glu-Cre::ROSA mice treated for 2 months with saline (C–E) or GABA (F–H) using X-Gal staining. While β-galactosidase labeling is expectedly detected in the islet mantle (where α cells reside) in both saline- (C–E) and GABA-treated conditions (F–H), such labeling is also observed within the islet core (where insulin + cells are normally located) in the pancreas of GABA-treated animals (F–H).

In an effort to potentially discriminate newly formed from pre-existing insulincells in GABA-treated pancreata, we assayed the expression of several endocrine cell marker genes ( Figures 2 C, 2D, 2G, 2H, 2K, 2L, 2O, 2P, 2S, 2T, 2W, 2X, 2Z1, 2Z2, 2Z5, and 2Z6) compared to saline-treated controls ( Figures 2 A, 2B, 2E, 2F, 2I, 2J, 2M, 2N, 2Q, 2R, 2U, 2V, 2Y, 2Z, 2Z3, and 2Z4) by immunohistochemistry. These analyses revealed that all insulincells in GABA-treated islets uniformly expressed the bona fide β cell markers, such as the prohormone convertase 1/3 (PC1/3) ( Figures 2 A–2D), the transcription factors Nkx6.1 ( Figures 2 E–2H), and Pdx1 ( Figures 2 I–2L), the β cell-specific glucose transporter Glut 2 ( Figure 2 M–P), as well as the pan-endocrine markers Pax6 ( Figures 2 Q–2T) and NeuroD1 ( Figures 2 U–2X). These cells also lacked the non-β cell labels, such as glucagon ( Figures 1 E–1J) and somatostatin ( Figures 1 H–1J). Interestingly, as expected from the in vitro data ( Figure S1 B), a number of glucagoncells were found negative for Arx expression ( Figures 2 Z3–2Z6) or misexpressing Pax4 ( Figures 2 Y–2Z2), suggesting a loss of the α cell identity. Due to technical limitations, cells could not be co-labeled simultaneously for Arx and Pax4 ( Figures 2 Y–2Z6). In addition, pancreas tissue sections from GABA 2 m mice were analyzed by electron microscopy following insulin immunogold labeling: a thorough examination of more than 200 photographs per pancreas (n = 3) indicated that all cells displaying a β cell ultrastructure expressed insulin and that all insulin-labeled cells exhibited a β cell phenotype in GABA-treated pancreata ( Figures S3 A–S3D). Interestingly, functional tests performed in GABA 3 m animals confirmed improved glucose tolerance, these animals demonstrating a lower peak in glycemia, a faster return to normoglycemia, and a 3.1-fold increase in insulin secretion as compared to saline-treated animals ( Figure S3 E and inset), all suggestive of an augmented functional β-like cell mass. Nevertheless, despite a significant β-like cell hyperplasia, GABA treatment did not cause any resistance to insulin as outlined by insulin tolerance tests carried out in GABA 6 m animals ( Figure S3 F). Taken together, our data suggest that long-term GABA administration induces a downregulation of Arx expression/ectopic expression of Pax4 in α cells and a concomitant increase in β-like cell mass. Furthermore, daily administration of GABA to WT mice leads to islet neogenesis accompanied by progressive islet hypertrophy, the latter resulting from hyperplasia of functional β-like cells, as well as an increase in glucagonand somatostatincell numbers.

(E and F) Intraperitoneal glucose tolerance (E) and insulin tolerance (F) tests were performed in GABA- (or saline-) treated WT animals. Importantly, GABA 3 m animals displayed a clear improvement in response to a glucose bolus, with a reduction in the glycemic peak and a faster return to euglycemia (E). Measurements of circulating insulin levels confirmed an increased capability to secrete insulin upon glucose challenge, as expected from an increased β-like cell mass (whereas basal levels were found to be normal (E). As important was the finding that GABA 6 m animals did not develop resistance to insulin despite the β-like cell hyperplasia (F). All data depicted as mean ± SEM in (E) and (F). Related to Figure 2

(A–D) Electron microscopy examination was combined with insulin detection using immuno-gold staining to examine islets of animals treated for 2 months with saline or GABA. In GABA-treated islets, all cells displaying a β-cell ultrastructure express insulin. Similarly, all cells labeled with insulin exhibit a typical β-cell ultrastructure (n = 3, 200 photographs analyzed per sample).

Representative pictures of immunohistochemical analyses performed on pancreas sections from controls (A, B, E, F, I, J, M, N, Q, R, U, V, Y, Z, Z3, and Z4) and from age-/sex-matched GABA-treated mice (C, D, G, H, K, L, O, P, S, T, W, X, Z1, Z2, Z5, and Z6) using the indicated antibody combinations. When compared to controls, GABA-treated pancreata exhibit an islet hypertrophy as a result of an insulin + cell hyperplasia, the latter uniformly expressing the bona fide β cell markers PC1/3 (A–D), Nkx6.1 (E–H), Pdx1 (I–L), Glut-2 (M–P), but also Pax6 (Q–T), NeuroD1 (U–X), and Pax4 (Y–Z2). Interestingly, Pax4 was found misexpressed in few glucagon + cells (Z2 and inset), while Arx expression was lost in a number of glucagon + cells (Z3–Z6 and inset in Z6), suggestive of a loss of the α cell phenotype (each photograph is representative of four independent animals); a minimum of 50 islets were examined per animal. Average islet size increase in GABA 2 m versus controls: 2.1 ± 0.5, p < 0.05 (n = 6).

Such alterations being suggestive of an activation of GABA pathways, we initially assayed its short-term role in vitro using the α-TC1-6 α cell line (). These cells were treated (or not) daily with concentrations of GABA ranging from 5 μM to 1 mM and kept in culture for up to 6 weeks ( Figure S1 B). Insulin expression appeared unchanged upon GABA treatment (data not shown). However, the α-TC1-6 cell line, derived from an induced adenoma and passaged over time, may not fully recapitulate the endogenous inherent α cell plasticity. We therefore focused on Arx expression whose downregulation triggers α-to-β-like cell conversion in mice (). Interestingly, even at low GABA concentrations, a comparative analysis of Arx expression by qRT-PCR outlined a rapid decrease in Arx transcript contents, with a maximum of 31% as compared to controls ( Figure S1 B). These encouraging results prompted us to test GABA activities in vivo by combining long-term GABA administration and immunohistochemical analyses. Thus, 2.5- to 10-month-old wild-type (WT) mice were subjected to a 1- to 6-month (1–6 m) GABA treatment (250 μg/kg) by daily intraperitoneal injections. These animals were viable, healthy, and fertile, their lifespan, weight, and basal glycemia remaining within normal range ( Figure 1 A and data not shown). Importantly, the examination of GABA-treated pancreata ( Figures 1 C, 1D, 1F, 1G, 1I, and 1J) outlined a dramatic islet hypertrophy mostly resulting from a massive increase in the insulincell numbers (while the size of insulincells was found unchanged; data not shown), as compared to control mice ( Figures 1 B, 1E, and 1H). This insulincell hyperplasia further increased with the duration of GABA treatment, aging not seeming to impact these processes ( Figures 1 A and 1K). Furthermore, the number of islets in GABA-treated mice was found to be doubled compared to controls, independently of the duration of GABA exposure ( Figures 1 A and 1K). In addition, GABA-treated mice also displayed higher numbers of glucagonand somatostatincells as compared to their non-treated counterparts ( Figures 1 D–1K). It is also important to note the atypical positioning of these non-β cells within the islets, most of these being detected in clusters close to ducts ( Figures 1 F, 1G, 1I, and 1J) and not uniformly distributed within the islet mantle zone, as seen in controls ( Figures 1 E and 1H). Additional experiments using increasing concentrations of GABA (1–5 mg/kg) demonstrated a dose-dependent augmentation in the number of insulincells and islets ( Figures S2 A–S2J). In all subsequent experiments, we used 250 μg/kg of GABA, corresponding to the minimal concentration that caused a significant increase in insulincell numbers.

Representative pictures of immunohistochemical analyses performed on islets of animals treated for 2 months with saline (A and B), GABA 1mg/kg (C and D), GABA 2.5mg/kg (E and F) and GABA 5mg/kg (G and H). Increasing concentrations of GABA induce a dose-dependent increase in the number of islets (I) and of hormone-producing cell contents (J).p < 0.05,p < 0.01,p < 0.001; n ≥ 3; data statistically analyzed by a one-way ANOVA by comparing all data points to saline-treated controls, all data depicted as mean ± SEM. Related to Figure 1

(K) Quantitative comparison of the numbers of insulin + , glucagon + , and somatostatin + pixels in GABA-treated islets versus age-/sex-matched controls. Note the GABA duration-dependent increase in all endocrine cell populations. ∗∗ p < 0.01, ∗ p < 0.05 using an unpaired ANOVA test; all data depicted as mean ± SEM (n ≥ 3).

(B–J) Immunohistochemical staining using different hormone combinations of pancreatic sections from controls (B, E, and H) and GABA-treated (C, D, F, G, I, and J) animals. A massive increase in islet size and numbers is observed. Interestingly, glucagon + and somatostatin + cell counts are also found augmented. For the purpose of clarity, the ductal lumen is outlined with dashed white lines, whereas the islets are outlined with dashed yellow lines.

(A) Wild-type mice were treated with GABA at the indicated ages and for the specified durations. Note that while life expectancy and basal glycemia are within normal ranges, an increase in islet count and size is observed in all conditions, such increases being dependent on the duration of treatment. Interestingly, aging does not impact on these neogenesis processes. Indeed, 10-month-old and 2.5-month-old animals treated with GABA for 3 months exhibit a similar augmentation in islet size and number. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 using a Mann-Whitney test; all data depicted as mean ± SEM (n ≥ 6).

Through the generation and analysis of Glu-rtTA::TetO-Pax4 double transgenic animals (harboring transgenes encompassing the rat glucagon promoter upstream of the reverse tetracycline-dependent transactivator and the Tet operator upstream of Pax4 cDNA), we previously demonstrated that the misexpression of Pax4 triggered in adult α cells induces their continuous neogenesis and subsequent conversion into β-like cells (). Subsequent work provided evidence that the downregulation of Arx expression in α cells is in fact the main trigger of their conversion into β-like cells (). Aiming to gain further insight into the molecular mechanisms underlying these processes, we performed a number of screens (, [this issue of Cell]), including transcriptomic analyses using islets isolated from Glu-rtTA::TetO-Pax4 animals treated for increasing durations with doxycycline compared to saline-treated controls. Our results, sorted by fold change, outlined strong alterations in the expression of several genes involved in GABA signaling (unpublished data; Figure S1 A). For instance, when comparing 3-month doxycycline-treated versus saline-treated pancreata, the expression of the β cell-specific GABAreceptor was increased >15 times. Similarly, a 7-fold augmentation in GABAreceptor transcripts, a 10.5-fold rise in GAD transcripts, and an 8.5-fold increase in the expression of the GABAreceptor target, gephyrin, were observed ( Figure S1 A).

(B) Arx transcript content quantification in α-TC1-6 cells treated with GABA. α-TC1-6 cells were treated with saline or increasing GABA concentrations for the indicated periods of time. Relative Arx transcript contents were quantified using quantitative RT-PCR. Note the progressive decrease in Arx expression (a hallmark of α-to-β-cell conversion) () in all GABA treatment conditions, such decrease progressing with GABA exposure duration.p < 0.05,p < 0.01,p < 0.001; n ≥ 3; data statistically analyzed by a one-way ANOVA by comparing all data points to matching control treatment durations, all data depicted as mean ± SEM.

(A) Transcriptomics analyses of the pancreata from Glu-rtTA::TetO-Pax4 transgenic mice. Glu-rtTA::TetO-Pax4 animals were treated for 3 months with Dox or Saline prior to sacrifice for pancreatic islet isolation. Transcriptomics analyses were subsequently performed based on RNaseq. By focusing on genes displaying a significant 5-fold change in expression, our analyses outlined 48 genes comparing Dox- versus Saline-treated pancreata. Importantly, 22 of these appeared potentially linked to the GABA signaling pathway.

Discussion

Here, we report an unsuspected role of GABA in inducing a continuous conversion of α cells into β-like cells, accompanied by α cell regeneration. Prolonged GABA exposure results in a significant increase in islet size and number due to a β-like cell hyperplasia. This hyperplasia closely correlates with the dose and duration of GABA treatment. Specifically, our results suggest that GABA induces the conversion of α cells into β-like cells through the downregulation of Arx expression, GABA acting via the GABA A receptor located on α cells. This triggers α cell replacement mechanisms involving the mobilization of ductal precursor cells that re-enact the endocrine differentiation program as seen during pancreas development, such α cells being subsequently converted into β-like cells upon sustained GABA exposure. Importantly, the regenerated β-like cells are functional and can repeatedly reverse the consequences of chemically induced β cell ablation, even in severely diabetic animals. As important is the demonstration that the treatment of transplanted human islets with GABA results in a decrease in α cell numbers accompanied by a concomitant increase in the β-like cell counts, suggestive of an α-to-β-like cell conversion capacity in humans. These findings could therefore pave the way toward targeted therapies to restore β cell mass in types 1 and 2 diabetes, both conditions eventually resulting in the loss of β cells.

+ cell hyperplasia progresses with the dose and duration of GABA treatment. Interestingly, this insulin+ cell hyperplasia does not lead to insulin resistance or augmented circulating insulin levels, such results being in line with previous observations indicating that an increased β cell mass does not impair glucose homeostasis ( Rahier et al., 2008 Rahier J.

Guiot Y.

Goebbels R.M.

Sempoux C.

Henquin J.C. Pancreatic beta-cell mass in European subjects with type 2 diabetes. Topp et al., 1998 Topp J.

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Jansson L. Evidence of a negative feedback system regulating the total beta-cell volume in nondiabetic rats that received pancreas transplants. + cell neogenesis, as 10-month-old animals respond in a similar fashion to their 2.5-month-old counterparts. Interestingly, an increase in islet number is observed under all conditions tested. However, it appears to plateau at approximately twice the number of islets found in control pancreata. This suggests that GABA can also trigger islet neogenesis but these processes appear to be limited. Gaining further insight into the molecular mechanisms controlling this limitation and determining ways to overcome it would be of obvious interest in the context of β cell regeneration research. Our analyses reveal that long-term GABA treatment results in a significant, but controllable, increase in insulin-producing cell numbers. Such insulincell hyperplasia progresses with the dose and duration of GABA treatment. Interestingly, this insulincell hyperplasia does not lead to insulin resistance or augmented circulating insulin levels, such results being in line with previous observations indicating that an increased β cell mass does not impair glucose homeostasis (). In addition, our findings indicate that aging does not impact GABA-mediated insulincell neogenesis, as 10-month-old animals respond in a similar fashion to their 2.5-month-old counterparts. Interestingly, an increase in islet number is observed under all conditions tested. However, it appears to plateau at approximately twice the number of islets found in control pancreata. This suggests that GABA can also trigger islet neogenesis but these processes appear to be limited. Gaining further insight into the molecular mechanisms controlling this limitation and determining ways to overcome it would be of obvious interest in the context of β cell regeneration research.

Soltani et al., 2011 Soltani N.

Qiu H.

Aleksic M.

Glinka Y.

Zhao F.

Liu R.

Li Y.

Zhang N.

Chakrabarti R.

Ng T.

et al. GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes. + cells incorporated the nucleotide analog BrdU upon GABA exposure. However, such GABA-induced insulin+ cell proliferation does not appear to represent the main mechanism underlying GABA activities upon longer exposure. Indeed, our analyses demonstrate that regenerating α cells represent the main source of newly formed β-like cells. This is further evidenced following β cell ablation in animals allowing α cell lineage tracing: most regenerated β-like cells clearly display an α cell ontogeny. Previous reports have suggested a putative role of GABA in inducing β cell proliferation but showed no lineage tracing (). In addition, much shorter durations of treatment were used compared to our current report. Our results partly support these findings as we observed that a number of insulincells incorporated the nucleotide analog BrdU upon GABA exposure. However, such GABA-induced insulincell proliferation does not appear to represent the main mechanism underlying GABA activities upon longer exposure. Indeed, our analyses demonstrate that regenerating α cells represent the main source of newly formed β-like cells. This is further evidenced following β cell ablation in animals allowing α cell lineage tracing: most regenerated β-like cells clearly display an α cell ontogeny.

The demonstration that neo-generated β cells can functionally replace all endogenous β cells at least twice following in vivo chemical ablation is of importance for type 1 and 2 diabetes research, both conditions eventually resulting in loss or decrease of β cells. As mentioned above, GABA was hitherto thought to induce β cell proliferation. While this feature is of interest, a putative application to type 1 diabetic patients would not have been possible due to the loss/lack of pre-existing β cells. However, the present demonstration that, upon long-term GABA administration, regenerating α cells can be used to replenish the β cell mass appears extremely appealing. Similarly, the finding that exposure to GABA could potentially turn human α cells into β-like cells is also exciting. Although the results obtained by transplanting human islets into immunodeficient mice are suggestive, the definitive demonstration of such human α-to-β-like cell conversion will await the development of a cell lineage tracing system allowing the labeling of human α cells and the monitoring of their outcome. Another limitation to these transplantation experiments is the lack/very small number of functional human ductal cells to determine whether these would contribute to the α cell-mediated β-like cell neogenesis.

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Eizirik D.L. Differential cell autonomous responses determine the outcome of coxsackievirus infections in murine pancreatic α and β cells. Marroqui et al., 2015c Marroqui L.

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Greenbaum C.J. Disease modifying therapies in type 1 diabetes: Where have we been, and where are we going?. Should one consider a putative application of these findings to type 1 diabetic patients, one would need to take into account the autoimmune nature of this condition. Indeed, putatively regenerated β-like cells could be targeted by the immune system, which retains memory of β cell antigens. Four possibilities could be envisaged to alleviate this issue. First, GABA could promote the neogenesis and conversion of α cells into β-like cells, the latter retaining some α cell features rendering them “immune/more resistant” to an autoimmune attack. In line with this possibility, we have recently shown that α cells are more resistant than β cells to apoptosis induced by both “diabetogenic” viruses () and metabolic stress (). Second, β-like cell regeneration could be faster than the autoimmune-mediated β cell loss. Indeed, it has been shown that type 1 diabetes is a slow progressing condition, as outlined by the progressive decline in insulin secretion in antibody-positive individuals, long before the development of overt diabetes (). Third, GABA could potentially harbor some immune-protective activities as previously suggested (). Fourth, and in line with the prevailing view that preventive or curative therapies for T1D must be multifactorial (), GABA therapy could be coupled to a mild immunomodulatory approach, aiming to both restore β cell mass and to tolerize the immune system to these neo-formed β cells. Clearly, further work is necessary to test these hypotheses, but these paths of research could lead to exciting new findings.