Exposure of Embryonic Pancreas to Metformin Enhances the Number of Pancreatic Progenitors

Abstract

Aims

Developing beta cells are vulnerable to nutrient environmental signals. Early developmental processes that alter the number of pancreatic progenitors can determine the number of beta cells present at birth. Metformin, the most widely used oral agent for diabetes, alters intracellular energy status in part by increasing AMP-activated protein kinase (AMPK) signaling. This study examined the effect of metformin on the developing pancreas and beta cells.

Methods

Pancreatic rudiments at embryonic day 13.0 (E13.0) were cultured with metformin or AICAR (an AMPK activator) or vehicle control in vitro. In another set of studies, pregnant mice were treated with metformin throughout gestation. Embryonic (E14.0) and neonatal pancreata were then analyzed for their morphometry.

Results

In vitro metformin treatment led to an increase in the proliferation and number of PDX1⁺ progenitors. These results were reproduced by in vitro culture of embryonic pancreas rudiments with AICAR, suggesting that AMPK activation was involved. Similarly, Metformin administration to pregnant dams induced an increase in both PDX1⁺ and NGN3⁺ progenitors in the embryonic pancreas at E14.0 and these changes resulted in an increased beta cell fraction in neonates.

Conclusions

These results indicate that gestational metformin exposure modulates early steps of beta cell development (prior to E14.0) to increase the number of pancreatic and endocrine progenitors and these changes ultimately result in a higher beta cell fraction at birth. These findings are of clinical importance given that metformin is currently used for the treatment of gestational diabetes.

Introduction

Type 2 diabetes is one of the most prevalent conditions affecting human health today. It is understood that both genetic and environmental factors contribute to type 2 diabetes risk [1], and one important environmental factor is maternal nutrition during pregnancy [2]. Developing beta cells have been shown to be critically sensitive to nutrient status [3–6]. Experimental models of metabolic stress during pancreatic development show permanent impairments in offspring beta cell mass and function [7–10]. This phenomenon, termed beta cell programming, is also seen in human observational studies, [11].

Pancreas development begins at embryonic day (E)8.5 within a region of the endoderm [12]. Pancreatic duodenal homeobox 1 (PDX1) positive cells represent a population of progenitor cells for all mature pancreatic cells [13]. These undifferentiated precursor cells can be specified towards the endocrine lineage by the expression of Neurogenin 3 (NGN3) [14]. After the expression of a cascade of transcription factors, these cells differentiate into the five endocrine cell types: alpha cells (glucagon), beta cells (insulin), delta cells (somatostatin), PP cells (pancreatic polypeptide) and epsilon cells (ghrelin) (reviewed in [13, 15]).

The mechanisms by which nutrition-related changes influence beta cell development are unclear, but signaling pathways that respond to changes in energy status are prime candidates. Mammalian target of rapamycin (mTOR) is a nutrient sensor that has been shown to be important for beta cell mass and function in rodent models [16, 17]. The role of mTOR complex 1 (mTORC1) signaling in regulation of mature beta cell mass and proliferation has been established [18, 19]. However, an understanding of the role of this pathway in the developing beta cell is only indirect [20]. Metformin, the most widely used oral anti-diabetic agent, has been demonstrated to decrease mTORC1 activity through various mechanisms including AMP-activated kinase (AMPK) induction [21]. Metformin also acts on nutrient signaling pathways via AMPK-independent mechanisms [22, 23]. Metformin is being studied for use during pregnancy in polycystic ovary syndrome and gestational diabetes [24, 25]. However, the implications for alterations in pancreatic embryonic development induced by metformin have not been characterized. We sought to directly examine the impact of metformin on pancreatic development using both an in vitro and an in vivo approach to assess the resultant alterations in the embryonic and neonatal pancreas.

Materials and Methods

Pancreatic bud culture in vitro

Pancreatic rudiments were dissected from E13.0 embryos (the morning of vaginal plug was E0.5) according to the University of Michigan School of Medicine-approved protocols. Pancreatic rudiments were cultured as described previously [26, 27], for 72 hours in DMSO with or without 2 mmol/l metformin or 1mmol/l AICAR (Sigma-Aldrich, St. Louis, MO, USA). After culture, embryonic rudiments were fixed in 3.7 % formalin in PBS and pre-embedded in Histogel (Thermo Scientific, Kalamazoo, MI, USA) for paraffin embedding. A schematic of the experimental protocol appears in Fig. 1A.

Figure 1. Metformin increases the number and proliferation of pancreatic progenitors in vitro.

Schematic of in vitro experiments (A) E13.0 buds cultured for 72 hours with DMSO (B) or metformin (C) and stained for PDX1 (red) and a nuclear marker, DAPI (blue). Quantification for the total cell number (D) PDX1⁺ (E), and mesenchyme cells (F). E13.0 pancreatic buds exposed to DMSO (G) or metformin (H) and stained for PDX1 (red), KI-67 (green) and DAPI (blue). Overall proliferation rates at the end of the culture (I), of PDX1⁺ (J), and mesenchyme cells (K). * p<0.05, scale bars=50 µm

In vivo metformin programming mouse model

8 week-old virgin C57Bl6 animals were purchased from Jackson Laboratories and adapted to control diet (D02041001B, Research Diets Inc., New Brunswick, NJ, USA) for 3 weeks. Upon vaginal plug detection females were given unadulterated water or water with metformin (Sigma-Aldrich, St Louis, MO, USA) at 5 mg/ml. Water was changed weekly until sacrifice. A schematic of the experimental protocol appears in Fig. 3A. Blood glucose levels were measured using an AlphaTRAK blood glucose meter (Abbott Laboratories, Abbott Park, IL, USA).

Figure 3. Dam and offspring characteristics after gestational metformin exposure.

Schematic of in vivo experiments (A). Gestational weight gain in control (open circles) or metformin-treated (black circles) dams from baseline (n=5) (B). Maternal blood glucose at G14.0 (C) litter sizes (D) and metformin levels in pregnant dams and offspring (E). Dashed lines indicate the metformin therapeutic window.

Metformin quantitation in mouse plasma

Metformin was quantified using HPLC with UV detection. 300 µl of calibrators, controls, and samples were mixed with 30 µl of 10 µg/ml phenformin (internal standard) and 1.0 ml MeOH. Samples were vortexed and centrifuged at 3,200g for 10 min. Supernatants were dried to residue in glass tubes, redissolved in 200 µL of mobile phase (35% ACN, 65% 40 mmol/l KH₂PO₄ (pH 4.0)), and filtered using a microfilterfuge tube. 100 µl was injected into the HPLC-UV system at room temperature with a flow rate of 1.0 ml/min and 234 nm wavelength of absorbance. The ratios of the peak area of metformin to the internal standard were compared against a linear regression of ratios of calibrators at concentrations of 0, 62.5, 125, 250, 1000, 2000, and 4000 ng/ml. The HPLC-UV system consisted of a Dionex Omnipac PCX 500 column (4.6 × 250 mm), and a Waters 2487 UV detector, 717 autosampler, and 515 HPLC pump.

Morphometric analysis and immunostaining

Pancreatic rudiments were dissected from C57Bl6 embryos at E14.0. Embryonic rudiments were fixed in 3.7 % formalin in PBS, then pre-embedded in Histogel (Thermo Scientific, Kalamazoo, MI, USA) for paraffin embedding. Newborn pancreata harvested on postnatal day 1 (P1) were fixed in 3.7 % formalin in PBS for six hours before embedding.

The entire pancreatic bud and neonatal pancreata were sectioned at 5 µm thickness. For the in vitro study every other section of the bud was stained for PDX1 and KI-67 (8–16 sections counted per bud). Alternate sections were stained for NGN3 and KI-67. For the in vivo studies at E14.0, 4 sections were taken from each quartile of the organ. For the neonatal studies 5 sections were taken at equal intervals [28]. Sections were deparaffinized, rehydrated and incubated overnight at 4°C with primary antibodies as previously described [29]. Specific primary antibodies used were insulin (Guinea pig, Dako, Denmark), KI-67 (Rabbit, Vector Laboratories, Burlingame, CA, USA), E-Cadherin (Mouse, BD Biosciences, San Jose, CA, USA), PDX1 (Rabbit, Millipore, Temecula, CA, USA), NGN3 (Mouse, Beta Cell Biology Consortium), Phospho-S6 (Rabbit, Ser240, Cell Signaling, Danvers, MA, USA) followed by secondary antibodies conjugated to FITC, AMCA or Cy3 (Jackson Immunoresearch, West Grove, PA, USA). TUNEL staining was performed using the ApopTag kit (Millipore, Billerica, MA, USA). Images were acquired using a Leica DM5500B fluorescent microscope (Leica Microsystems, Wetziar, Germany).

For neonates, the areas of the pancreatic section and of insulin-positive tissue were assessed on 5 independent sections. For proliferation analysis at E14.0, KI-67⁺ nuclei were hand-counted from 4 sections. The total number of nuclei was counted using ImageJ software [30]. In neonates (n=4–5 per group), a total of 1000–3000 beta cells were manually counted from 3 sections. Mesenchyme cells were calculated by subtracting the total number of PDX1⁺ cells from the total number of cells (DAPI⁺). For determination of cell size at E14.0 the area of each E-Cadherin⁺ cell was directly measured on 25 cells per bud from pancreatic sections using the magic wand tool in Photoshop CS4 (Adobe Inc., San Jose, CA) to assess the area of the cytoplasm delimited by the E-Cadherin staining.

Immunoblotting

Immunoblotting was performed as previously described [20, 31]. Briefly, whole embryonic pancreata at E13.0 pooled from 3 dams were cultured in collagen for three days, then removed from collagen gel and immediately suspended in RIPA lysis buffer and sonicated. 30 µg of protein lysates were used for the western blots using Phospho-S6 (Ser 240) and phosphorylated Acetyl-CoA Carboxylase (ACC) (Cell Signaling, Danvers, MA, USA) antibodies. Mouse Cyclophilin B (Fisher Scientific, Pittsburgh, PA, USA) was a loading control.

Statistical analysis

Statistical significance was assessed through the Mann-Whitney test (u-test) or t-test where appropriate using GraphPad Prism (version 6.0c, GraphPad Software, La Jolla, CA, USA). Results were considered significant with a p-value < 0.05.

Results

Metformin increases the number of pancreatic progenitors in vitro

To assess the effect of metformin during embryonic development, E13.0 pancreatic rudiments were cultured with metformin (2 mol/l) or vehicle (DMSO) for 72 hours (Fig. 1A). Metformin-treated buds were strikingly larger than controls (Fig. 1B–C). The total number of cells counted throughout the bud was higher in the metformin-treated group (Fig. 1D). To determine the cause of this size difference, we examined the number of PDX1⁺ progenitors and found an increase in the metformin-treated buds (Fig. 1E). Exposure to metformin also increased the number of mesenchyme cells (Fig. 1F).

We next assessed the contribution of proliferation to the increased cell populations observed. Metformin-exposed pancreatic buds displayed an increased proliferation rate compared to controls (Fig. 1G–I). The metformin-treated rudiments also showed an increase in PDX1⁺ (Fig. 1J) and mesenchyme cell proliferation (Fig. 1K). These studies suggest that metformin increases the size of the pancreatic bud by increasing the number and proliferation of mesenchyme and epithelial cells.

Metformin decreases the number of endocrine progenitors in vitro

We then examined the effect of metformin on the number and proliferation of the endocrine progenitors (NGN3⁺ cells). The fraction of NGN3⁺ cells was decreased in the metformin-exposed rudiments compared to controls (Fig. 2A–C). Proliferation of the NGN3⁺ progenitors at the end of the culture, however, was unchanged (Fig. 2D–F). This suggests that the increase in proliferation of PDX1⁺ progenitors delays the differentiation to NGN3⁺ cells.

Figure 2. Metformin decreases endocrine progenitors in vitro and AICAR increases PDX1⁺ progenitor proliferation in vitro.

E13.0 control (A) or metformin (B) buds stained for NGN3 (red) and DAPI (blue). The fraction of NGN3⁺ cells (C). Staining for NGN3 (red) and KI-67 (green) in the control (D) and metformin group (E). The proliferation of NGN3⁺ cells (F). E13.0 control buds (G) or exposed to AICAR (H) stained for PDX1 (red) KI-67 (green) and DAPI (blue). Proliferation rates at the end of the culture (I), of PDX1⁺ (J), and NGN3⁺ cells (K).* p<0.05, scale bars=50 µm.

AICAR increases the bud size and rate of proliferation in vitro

Metformin regulates multiple intracellular processes by inducing changes in signaling pathways including AMPK and mitochondrial function. To understand the mechanisms responsible for the metformin effects on pancreas development, we examined the contribution of AMPK by culturing pancreatic buds with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an AMPK activator. Buds exposed to AICAR were larger than control buds (Fig. 2G,H), similar to the findings with metformin. The overall proliferation rate of bud cells was increased compared to controls (Fig. 2I). PDX1⁺ cell proliferation was also increased as seen with metformin (Fig. 2J). Thus, treatment of embryonic pancreas rudiments with an AMPK activator has a similar effect on bud size, PDX1⁺ cell number and proliferation as metformin. Also similar to the results with metformin there was no change in the proliferation rate of the NGN3⁺ cells (Fig. 2K).

Metformin exposure in utero does not alter birth parameters

The previous studies showed a significant effect of metformin on growth of the embryonic pancreas in vitro. We next sought to determine if metformin administration in vivo recapitulated these changes. Metformin was given to pregnant dams in drinking water at a dose that has been demonstrated to yield a blood level in the human therapeutic range (equivalent to 0.03 mmol/l) [32, 33]. Pregnant dams were exposed to metformin throughout gestation from E0.5 (day of the vaginal plug) to delivery of the pups (Fig. 3A). Metformin-treated dams gained an equal amount of weight compared to controls and had no change in blood glucose at G14.0 (Fig. 3B and C). There were no changes in litter size (Fig. 3D). Plasma analysis revealed metformin levels in the upper therapeutic range in dams at delivery, and at the lower therapeutic limit in neonates (Fig. 3E).

Gestational metformin exposure increases the number of pancreatic progenitors in vivo

Given the augmentation in pancreatic bud size and proliferation of pancreatic progenitors seen in vitro, we then looked for alterations in pancreatic development at E14.0. E14.0 was selected as a time when there is a high number of undifferentiated PDX1⁺ cells and NGN3⁺ cells begin appearing in increasing numbers, just before the secondary transition for endocrine differentiation [34]. Metformin was administered to pregnant dams from E0.5 to E14.0, at which point embryonic pancreata were harvested. Upon microscopic examination of the E14.0 embryonic pancreata, metformin-treated buds were again noted to be larger than controls (Fig. 4A, B). When the total number of cells across the bud was counted, it was significantly increased in the metformin-exposed pancreata (Fig. 4C). Assessment of the total number of PDX1⁺ cells showed a significant increase in the metformin-exposed group (Fig. 4D). The total number of mesenchyme cells, however, was not altered (Fig. 4E).

Figure 4. Metformin increases bud size and number of pancreatic progenitors in vivo.

Control E14.0 embryonic pancreas (A) or metformin-exposed (B) and stained for PDX1 (green) and DAPI (blue). Results for: total number of bud cells (C), PDX1⁺ (D), and mesenchyme (E). E14.0 control (F) and metformin-exposed buds (G) stained for E-Cadherin (red), KI-67 (green) and DAPI (blue) and with higher magnification views. Proliferation rates for: total bud cells (H), epithelial cells (I) and mesenchyme (J). Apoptosis rate for PDX1⁺ cells (K) * p<0.05, scale bars=50 µm.

The rate of proliferation was then quantified in the different compartments of the embryonic pancreas. Overall, the percentage of proliferating cells in the embryonic rudiment in both conditions was the same (Fig. 4F–H). There was also no significant difference in the proliferation rates of the epithelium (E-Cadherin⁺ cells, Fig. 4I) or the mesenchyme cells between the groups (Fig. 4J). We also assessed the contribution of apoptosis during development to the changes induced by metformin but found no difference in apoptosis of the PDX1⁺ progenitors (Fig. 4K). Thus, exposure of the developing pancreas to metformin in vivo does not alter the rate of proliferation or apoptosis when assessed at E14.0.

Gestational metformin exposure increases the number of endocrine progenitors in vivo

We next analyzed the effect of metformin on the endocrine precursors. We examined the total number of NGN3⁺ endocrine progenitors across the bud, and this was significantly increased in the metformin group (Fig. 5A–C). NGN3⁺ cells did not display a higher proliferation rate at this stage when the mother received metformin (Fig. 5D–F).

Figure 5. Metformin increases the number of endocrine progenitors in vivo.

E14.0 control buds (A) or metformin-exposed (B) and stained for PDX1 (green) and NGN3 (red). Results for the number of NGN3⁺ cells (C). Staining for NGN3 (red) and KI-67 (green) in control (D) and metformin-exposed pancreata (E) at low power and high power. Proliferation of NGN3⁺ cells (F). * p<0.05, scale bars=50 µm.

Gestational metformin exposure alters intracellular signals downstream of mTORC1

In order to assess whether the changes observed in the developing pancreata could be due to altered mTOR signaling, we assessed the expression of phosphorylated ribosomal protein S6 (p-S6) at E14.0 on embryonic tissue sections from metformin-exposed animals. While this approach is not quantitative, pancreata from the metformin-exposed embryos displayed increased staining intensity of p-S6 (Fig. 6A,B). The average cell area of the epithelium (E-Cadherin⁺) cells was also increased (Fig. 6C–E), as would be expected from the increased S6 activity. Finally embryonic pancreata were dissected at E13.0 and cultured with or without metformin for 72 hours. At the end of the experiments, western blots indicated an increase of phospho-ACC (known target of metformin, used as a control) and p-S6 serine 240 in the metformin-treated rudiments (Fig. 6F).

Figure 6. Metformin exposure increases mTORC1 signaling.

Immunostaining from E14.0 control (A) or metformin buds (B) stained for E-Cadherin (red) and p-S6 (green). High power images from control (C) and metformin-exposed buds (D) stained for E-Cadherin (red). Results are quantitated for average cell area (E). Representative immunoblotting for p-ACC and p-S6 from E13.0 pancreatic buds cultured with or without metformin for 72 hours (F). * p<0.05, scale bars=50 µm.

Gestational metformin exposure leads to an increase in neonatal beta cell fraction

Both in vivo and in vitro experiments indicated that the developing pancreatic rudiment grows larger under metformin treatment. The in vitro experiments also lead us to hypothesize that acute metformin treatment could delay the differentiation of the endocrine progenitors while significantly increasing the number of undifferentiated PDX1⁺ progenitors. Given these findings, we sought to determine the extent to which these changes might result in altered beta cell fraction at birth. Offspring of mothers characterized above were sacrificed on the morning of birth (P1). Both metformin-exposed and control dams delivered pups with no difference in birth length or weight (Fig. 7A, B). The blood glucose was significantly decreased in metformin-exposed neonates (Fig. 7C).

Figure 7. Metformin exposure in utero increases beta cell fraction at birth.

Neonatal length (A) weight (B) and blood glucose of control or metformin-treated offspring (C). Staining for insulin (green) and DAPI (blue) for control (D) and metformin-exposed neonates (E). Beta cell fraction (F) neonatal pancreas area (G) beta cell proliferation (H) and acinar proliferation (I) are quantitated. * p<0.05, scale bars=50 µm.

Offspring pancreata were analyzed to establish the beta cell fraction and beta cell and acinar proliferation rates. Metformin exposure throughout gestation led to a significant increase in the beta cell fraction at birth in metformin versus control pups (Fig. 7D–F) but there was no increase in the overall size of the pancreas estimated by the total cross-sectional area of the representative images (Fig. 7G). Assessment of beta cell and acinar proliferation rates at birth also indicated no differences between groups (Fig. 7H–I). A summary of the findings in each set of experiments is presented in Table 1.

Table 1. Summary of gestational metformin exposure on the developing pancreas.

A summary of the key findings in each experiment. The groups examined were embryonic pancreas in vitro at E13.0, in vivo at 14.0 and neonatal pancreas in vivo. Parameters examined were epithelium size and proliferation, and endocrine progenitor number and proliferation.

Stage of Development	Epithelium size (PDX1 cells)	Epithelium proliferation (E-cadherin, KI-67+)	Number of endocrine progenitors (NGN3+)	Endocrine progenitor proliferation (NGN3+, KI-67+)	Beta cell fraction (insulin+)	Beta cell proliferation (insulin+, KI-67+)
In vitro (E13.0)
In vivo (E14.0)
In vivo neonatal

Discussion

The studies presented here provide surprising evidence of a previously unreported effect of metformin to modulate the developmental program of the pancreas. The studies using an in vitro culture system demonstrate that direct treatment of embryonic pancreas with metformin enhances the proliferation and number of PDX1⁺ and mesenchyme cells. In the in vivo model, maternal exposure to metformin also induced the number of PDX1⁺ and NGN3⁺ cells at E14.0. Continuing the treatment throughout gestation led to an increase in the beta cell fraction at birth. These findings are summarized in Table 1 and suggest that a commonly used anti-diabetic agent alters the developmental program of the embryonic pancreas in favor of increasing beta cell fraction at birth, which may then confer protection from type 2 diabetes in adulthood. These observations are clinically relevant because of the ongoing studies of metformin for use in pregnancy [25] and suggest that metformin could have beneficial effects on beta cell mass in humans.

In the in vitro culture system exposure of the developing pancreatic buds to a high dose of metformin increased the pool of PDX1⁺ progenitors and augmented PDX1⁺ cell proliferation. The associated decrease in NGN3⁺ cells may result from a delay in differentiation following the proliferation of the PDX1⁺ progenitors, as cell cycle exit is generally linked with differentiation [35]. It is possible that a longer incubation could have been necessary to observe an effect on the NGN3⁺ cell number, as pancreatic development does not occur at a typical pace in culture. Interestingly, a positive effect of metformin on the number of NGN3⁺ cells was observed in vivo at E14.0. The discrepancy of these findings could reflect a longer exposure to metformin, since the developing pancreas in the in vivo model was exposed to metformin from the very early stages of pancreas development (E9.5). Alternatively, it is also possible that the differences could reflect the different concentrations of metformin used in vivo and in vitro.

In the in vivo studies when a pharmacologically relevant dose of metformin was delivered to pregnant dams the effect was an increase in the number of PDX1⁺ and NGN3⁺ cells in the bud. The increase in the number of PDX1⁺ cells was not accompanied by increased proliferation suggesting that metformin induces proliferation of the PDX1⁺ progenitors prior to E14.0, as demonstrated in vitro. In addition, there was no evidence that apoptosis contributed to the changes in PDX1⁺ progenitors seen with metformin treatment. The increase in both the number of PDX1⁺ and NGN3⁺ is likely responsible for the increased beta cell fraction in neonates. This is consistent with previous evidence demonstrating that the initial pool of PDX1⁺ and NGN3⁺ progenitors are major determinants of the final beta cell number [36, 37]. Interestingly, the proliferation of PDX1⁺ cells in vitro was in marked contrast to the lack of alteration in NGN3⁺ cell proliferation. These intriguing findings suggest that PDX1⁺ progenitors are more susceptible to proliferative signals induced by metformin than NGN3⁺ cells. Another possibility may be that a rise in the proliferation of NGN3⁺ cells occurs at a later time point than was examined in this study, as the peak of NGN3⁺ proliferation typically occurs at E14.5 [38]. Finally, it is possible, that fetal administration of metformin induces long-lasting consequences that could alter early postnatal beta cell remodeling and the responses of beta cells to diabetogenic conditions. This could be tested by assessing beta cell proliferation and apoptosis at during the first four weeks of life and by exposing these mice to high fat diet.

The in vitro experiments demonstrated that metformin increased mesenchyme cell number and proliferation. These results are particularly interesting, as the mesenchyme has been shown to secrete growth factors and mitogenic signals that regulate proliferation and differentiation of PDX1⁺ progenitors, and this may have contributed to the effects seen in PDX1⁺ cells [36, 39, 40]. Culturing the pancreatic epithelium with metformin could assess the direct role of metformin on PDX1⁺ proliferation in the absence of mesenchyme.

Metformin has been shown to induce AMPK activity, but the role of AMPK signaling on the developing endocrine pancreas has not been evaluated in detail [41, 42]. Adult animals with AMPK a1 and 2 double knock out have no change in beta cell mass when compared to controls [43]. However no evaluation of embryonic stages or neonatal beta cell fraction was done in this model. Here we demonstrate that modulation of these signaling pathways by metformin during embryogenesis has a beneficial effect on endocrine pancreas development. How activation metformin induces proliferation during developmental stages is not completely understood, but it is possible that these effects could be mediated by inducing the secretion of mitogenic factors by the mesenchyme. Metformin has been shown to negatively impact mTOR signaling through several pathways and most often demonstrates an anti-proliferative effect on cells [23, 44–48]. In these experiments we present surprising evidence suggesting that metformin increases mTORC1 signaling in embryonic pancreata. This positive effect of metformin has been recently demonstrated in hypothalamic tissue and suggests that the responses to metformin could be tissue and developmental stage specific [49]. The effect on PDX1⁺ proliferation induced by metformin was reproduced after treatment with AICAR in vitro, providing strong evidence that this is an AMPK-dependent effect. The ability of AICAR to induce cell proliferation has also been demonstrated previously in an embryonic cell line [50]. Whether the effects of metformin on the embryonic pancreas are AMPK and mTOR-dependent may be elucidated with further studies using genetic mouse models.

The results of these studies uncovered that in vivo metformin exposure during pancreatic development leads to an increase in the pancreatic beta cell population at birth. It remains to be demonstrated if this change persists into adult life. These findings provide important clinical information, because metformin may be used during pregnancy depending on the outcome of ongoing clinical trials. The relevance of this study to the clinical use of metformin in pregnancy is currently unknown. One important difference is that administration of metformin throughout pregnancy, as in the studies described here, is not often used in pregnant women. However, a potential relevant clinical scenario is the treatment of women with PCOS during the first trimester to delivery as described in some studies [24, 51–55]. Future studies will be required to explore the effect of dosing and timing of metformin treatment in diabetic women. The findings described here also underscore the importance of possible programming phenomena resulting from altering cellular energy status in both the mother and developing fetus. Further examination of these programming phenomena is warranted and may provide a strategy to enhance beta cell mass at birth in populations at risk for type 2 diabetes later in life.

Abbrevations

AICAR

5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside

AMPK

AMP-activated protein kinase

mTOR

mammalian target of rapamycin

mTORC1

mTOR complex 1

NGN3

neurogenin 3

PDX1

pancreatic duodenal homeobox 1