# The transcription factor E2F1 controls the GLP-1 receptor pathway in pancreatic β cells

Cyril Bourouh, Emilie Courty, Laure Rolland, Gianni Pasquetti, Xavier Gromada, Nabil Rabhi, Charlène Carney, Maeva Moreno, Raphaël Boutry, Emilie Caron, et al.

► To cite this version:

Cyril Bourouh, Emilie Courty, Laure Rolland, Gianni Pasquetti, Xavier Gromada, et al. The tran-
scription factor E2F1 controls the GLP-1 receptor pathway in pancreatic β cells. Cell Reports, 2022,
40 (6), pp.111170. 10.1016/j CELREP.2022.111170. hal-03810743

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**Cell Reports**

# The transcription factor E2F1 controls the GLP-1 receptor pathway in pancreatic β cells

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Cyril Bourouh, Emilie Courty,
Laure Rolland, ..., Amélie Bonnefond,
Frédérik Oger, Jean-Sébastien Annicotte

## Correspondence

jean-sebastien.annicotte@inserm.fr

## In brief

The glucagon-like peptide-1 (GLP-1) is an enterohormone that binds the GLP-1 receptor (GLP1R) and modulates insulin secretion. Bourouh et al. show that β cell-specific genetic ablation of *E2f1* impairs glucose homeostasis associated with decreased *GLP1R* expression, highlighting molecular crosstalk between E2f1 and Glp-1 signaling pathways in pancreatic islets.

## Highlights

* Deletion of *E2f1* in β cells impairs glucose homeostasis and insulin secretion

* E2F1 regulates Glp-1 receptor gene expression in β cells

* The GLP-1R agonist exendin-4 increases E2F1/pRB transcriptional activity

* Crosstalk between E2F1 and GLP1R signaling pathways modulates β cell function

<figure><img src="image_3.png" /></figure>
Report

The transcription factor E2F1 controls
the GLP-1 receptor pathway in pancreatic β cells

Cyril Bourouh,<fnref n="1" />,<fnref n="8" /> Emilie Courty,<fnref n="1" />,<fnref n="2" />,<fnref n="8" /> Laure Rolland,<fnref n="1" />,<fnref n="2" /> Gianni Pasquetti,<fnref n="3" /> Xavier Gromada,<fnref n="1" /> Nabil Rabhi,<fnref n="4" /> Charlène Carney,<fnref n="1" /> Maeva Moreno,<fnref n="1" /> Raphaël Boutry,<fnref n="1" /> Emilie Caron,<fnref n="5" /> Zohra Benfodda,<fnref n="6" /> Patrick Meffre,<fnref n="6" /> Julie Kerr-Conte,<fnref n="3" /> François Pattou,<fnref n="3" /> Philippe Froguel,<fnref n="1" />,<fnref n="7" /> Amélie Bonnefond,<fnref n="1" />,<fnref n="7" /> Frédérik Oger,<fnref n="1" /> and Jean-Sébastien Annicotte<fnref n="1" />,<fnref n="2" />,<fnref n="9" />,*

SUMMARY

The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving β cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating β cell mass and function. Here, we report that β cell-specific genetic ablation of *E2f1* (*E2f1*β⁻/⁻) impairs glucose homeostasis associated with decreased expression of the Glp-1 receptor (*Glp1r*) in *E2f1*β⁻/⁻ pancreatic islets. Pharmacological inhibition of E2F1 transcriptional activity in nondiabetic human islets decreases *GLP1R* levels and blunts the inhibin effect of GLP1R agonist exendin-4 (ex-4) on insulin secretion. Overexpressing *E2f1* in pancreatic β cells increases *Glp1r* expression associated with enhanced insulin secretion mediated by ex-4. Interestingly, ex-4 induces retinoblastoma protein (pRb) phosphorylation and E2f1 transcriptional activity. Our findings reveal critical roles for E2f1 in β cell function and suggest molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways.

INTRODUCTION

Glucose homeostasis is finely tuned by the coordinated action of
the glucagon and insulin that are produced by pancreatic α and β
cells, respectively. Dysregulation of this homeostatic process
leads to type 2 diabetes (T2D), which is characterized by
impaired function and mass of pancreatic β cells (Weir and Bon-
ner-Weir, 2004). Since lifestyle modifications are usually ineffec-
tive to cure T2D, pharmacological treatments are necessary to
restore normoglycemia. Targeting the glucagon-like peptide-1
(Glp-1) pathway has demonstrated promising efficacy to treat
T2D (Drucker, 2018). Glp-1 is an enterohormone produced by in-
testinal L cells (Holst et al., 1987) and pancreatic α cells (Cham-
bers et al., 2017) that bind the Glp-1 receptor (GLP1R), a G pro-
tein-coupled receptor (GPCR) expressed by several cell types,
including pancreatic β cells (Thorens, 1992). The activation of
this GPCR by natural Glp-1 or synthetic Glp-1 analogs, such
as exendin-4 (ex-4) or liraglutide (Nauck et al., 2021) favors
glucose-stimulated insulin secretion (GSIS) by pancreatic β cells
(Holz et al., 1993; Kreymann et al., 1987), inhibits glucagon
secretion by pancreatic α cells (de Heer et al., 2008), but also in-

creases β cell proliferation (Cornu et al., 2010; Friedrichsen et al.,
2006; Liu and Habener, 2008), and prevents pancreatic β cells
from apoptosis (Buteau et al., 2004; Li et al., 2003b; Quoyer
et al., 2010).

The E2F1-pRb-CDK4 pathway plays a major role in controlling β cell mass and function (Annicotte et al., 2009; Fajas et al., 2004; Iglesias et al., 2004; Kim and Rane, 2011; Li et al., 2003a). The E2F1 transcriptional activity is finely regulated through its association with the dimerization partner-1 (DP-1) protein, members of the retinoblastoma protein family such as pRb (Mendoza and Grossniklaus, 2015), and cyclin and cyclin-dependent kinase (CDK) complexes, such as CDK4/cyclinD1, that phosphorylate pRb (Kato et al., 1993). This leads to pRb release from E2F1, promoting E2F1 transcriptional activity, cell-cycle progression, and CDK inhibitors, namely p16INK4A, that bind to CDK and avoid CDK4/cyclin D complex formation, and subsequently repress pRb phosphorylation and E2F1 transcriptional activity (Schulze et al., 1994). Although these mechanisms mainly control the progression of the cell cycle (Poppy Roworth et al., 2015), recent studies have reported important roles for the E2F1-pRb-CDK4 pathway beyond the sole regulation of cell

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<footnote n="1">¹Université de Lille, INSERM, CNRS, CHU Lille, Institut Pasteur de Lille, U1283 - UMR 8199 - EGID, 59000 Lille, France</footnote>

<footnote n="2">²Université de Lille, INSERM, CHU Lille, Institut Pasteur de Lille, U1167 - RID-AGE - Facteurs de Risque et Déterminants Moléculaires des Maladies Liées au Vieillissement, 59000 Lille, France</footnote>

<footnote n="3">³Université de Lille, INSERM, CHU Lille, Institut Pasteur de Lille, U1190 - EGID, 59000 Lille, France</footnote>

<footnote n="4">⁴Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA</footnote>

<footnote n="5">⁵Université de Lille, INSERM, CHU Lille, U1172-LilNCog - Lille Neuroscience & Cognition - EGID - DISTALZ, 59000 Lille, France</footnote>

<footnote n="6">⁶Université de Nimes, UPR CHROME, 30021 Nimes Cedex 1, France</footnote>

<footnote n="7">⁷Department of Metabolism, Imperial College London, Hammersmith Hospital, London W12 0NN, UK</footnote>

<footnote n="8">⁸These authors contributed equally</footnote>

<footnote n="9">⁹Lead contact</footnote>

<footnote>*Correspondence: jean-sebastien.annicotte@inserm.fr</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2022.111170</footnote>
<figure><img src="image_5.png" /><figcaption>Figure 1. β cell-specific loss of E2f1 expression impairs glucose tolerance</figcaption></figure>

(A) E2f1 mRNA expression levels from E2f1$^{β+/+}$ and E2f1$^{β−/−}$ pancreatic islets (n = 4).

(B) Body weight of 16-week-old E2f1$^{β+/+}$ and E2f1$^{β−/−}$ mice fed with chow diet (CD, n = 12–15).

(C and D) IPGTT in E2f1$^{β+/+}$ and E2f1$^{β−/−}$ mice fed with CD (n = 5–7) and the corresponding area under curve (AUC, D).

(E) Insulin plasma levels during IPGTT measured 0 and 15 min after glucose injection in CD-fed E2f1$^{β+/+}$ and E2f1$^{β−/−}$ mice (n = 5–7).

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proliferation (Denechaud et al., 2017; Kahoul et al., 2020). Indeed, several reports have demonstrated the role of the E2F1 pathway in the control of metabolic functions in non-proliferative cells, including adipocytes (Chen et al., 2020; Fajas et al., 2002), hepatocytes (Denechaud et al., 2016; Giralt et al., 2018; Lai et al., 2017), muscle and brown adipose tissue (Blanchet et al., 2011, 2012), and pancreatic β cells (Annicotte et al., 2009; Fajas et al., 2004; Grouwels et al., 2010; Kim and Rane, 2011). Interestingly, germline-deficient E2f1 mice are resistant to diet-induced obesity (Fajas et al., 2002) and have enhanced energy metabolism in muscle (Blanchet et al., 2011), as observed in global Glp1r knockout (KO) mice (Ayala et al., 2010).

While E2F1 and Glp-1 pathways are both involved in the con-
trol of β cell function and mass, whether they were physiologi-
cally connected remains unknown. Here, we demonstrate a mo-
lecular crosstalk between the GLP1R and E2F1/pRb pathways in
pancreatic β cells. We show that E2F1 modulates the Glp-1
signaling pathway through the transcriptional control of Glp1r
expression by pancreatic β cells. Importantly, we found that
a treatment of human islets with the pan E2F inhibitor
HLM006474 decreases E2F1 expression, as observed in T2D
human islets (Lupi et al., 2008), as well as GLP1R expression.

RESULTS

Loss of β cell-specific E2f1 expression impairs glucose
tolerance under a chow diet and during metabolic stress

To evaluate the role of E2f1 in the control of glucose homeostasis, we generated pancreatic β cell-specific E2f1-deficient mice, called hereafter E2f1<sup>β−/−</sup>. The recombination efficiency at the E2f1 locus was confirmed in pancreatic islets isolated from E2f1<sup>β−/−</sup> mice compared to E2f1<sup>β+/+</sup> control littermates (Figure 1A). E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice displayed comparable body weight (Figure 1B), fasting glycemia, pancreatic islet morphology, islet number, islet density, the percentage of endocrine cells per pancreatic section and insulin-positive cells per islet, and the insulin content (Figures S1A–S1G, respectively). Intraperitoneal glucose tolerance tests (IPGTTs) in 3-month-old E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice fed with chow diet (CD) revealed that glucose clearance was impaired in E2f1<sup>β−/−</sup> mice (Figures 1C and 1D), associated with defects in secreting insulin in response to glucose (Figure 1E). Plasma Glp-1 levels were not affected in control mice before and 10 min after the intraperitoneal (i.p.) injection of glucose (Figure 1F). Intriguingly, circulating Glp-1 levels were increased by the deletion of E2f1 in β cells after glucose injection (Figure 1F). Similarly, 4-month-old E2f1<sup>β−/−</sup> mice demonstrated impaired glucose tolerance when chal-

lenged with an oral bolus of glucose compared to age-matched
E2f1<sup>β+/+</sup> mice (Figures 1G and 1H), associated with a significant
alteration of insulin secretion only upon glucose stimulation but
not in fasting conditions (Figure 1l). Strikingly, although insulin
levels were dampened in E2f1<sup>β−/−</sup> mice upon glucose stimula-
tion, glucose-induced plasma Glp-1 levels during an oral GTT
(OGTT) were not affected by the deletion of E2f1 in β cells
(Figure 1J).

Metabolic stress, such as high-fat diet (HFD) feeding, pro-
foundly affects β cell function and mass. Using public datasets,
we monitored E2f1 transcript levels in pancreatic islets from
mice fed CD or HFD (Gao et al., 2021). E2f1 transcripts were
significantly increased in pancreatic islets isolated from mice af-
ter 12 weeks of HFD compared to age-matched CD-fed controls
(Figure S1H). We next evaluated whether such metabolic dysho-
meostasis promoted by β cell-selective E2f1 knockdown could
be exacerbated upon an HFD. Accordingly, we challenged
E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice with an HFD for 16 weeks. Despite
body weight (Figures 1K and S1l), glucose tolerance during
IPGTT (Figures 1L and 1M), and insulin sensitivity (Figure S1J)
being similar between both genotypes, insulin secretion was
impaired in E2f1<sup>β−/−</sup> mice as compared to controls fed an HFD
(Figure 1N). Again, HFD-fed E2f1<sup>β−/−</sup> mice demonstrated
impaired glucose tolerance when challenged with an oral
bolus of glucose compared to E2f1<sup>β+/+</sup> mice (Figures 1O and
1P). Although insulinemia was similar between controls and
E2f1<sup>β−/−</sup> mice in fasting conditions, insulin secretion was damp-
ened 30 min after oral glucose intake in E2f1-deficient mice (Fig-
ure 1Q), whereas circulating Glp-1 levels before and 10 min after
an oral glucose bolus were not significantly different between
control and E2f1<sup>β−/−</sup> mice either when fed an HFD (Figure 1R).
Our data demonstrate that the loss of E2f1 expression within β
cells impairs glucose homeostasis and insulin secretion upon
physiologic conditions and during metabolic stress.

Overexpression of human E2F1 in murine β cells has a
limited impact on glucose homeostasis and insulin
secretion

We next investigated whether E2F1 overexpression in pancre-
atic β cells of wild-type animals could mirror the phenotype
observed in E2f1<sup>β−/−</sup> (i.e., increased insulin secretion in
response to glucose). Therefore, we generated a mouse model
that overexpresses the human form of E2F1 (hE2F1) specifically
in β cells (E2f1<sup>βover</sup>). hE2F1 overexpression was confirmed at the
mRNA levels (Figure 2A). The β cell-specific expression of hE2F1
proteins was confirmed using co-immunostaining with anti-insu-
lin and anti-hemagglutinin (HA) antibodies, an HA tag being fused

(F) Blood Glp-1 levels before and 10 min after an i.p. glucose injection (n = 5).

(G and H) OGTT in E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice fed with CD (G) (n = 5–7) and the corresponding AUC (H).

(I) Insulin plasma levels during OGTT measured 0 and 30 min after glucose gavage in CD-fed E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice (n = 5–7).

(J) Blood Glp-1 levels before and 10 min after glucose gavage of E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice (n = 5).

(K) Body weight of E2f1<sup>β−/−</sup> and E2f1<sup>β+/+</sup> mice fed an HFD for 16 weeks (n = 7).

(L and M) IPGTT in E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice fed with HFD (L) (n = 7) and the corresponding AUC (M).

(N) Insulin plasma levels during IPGTT in HFD-fed E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice (n = 7).

(O and P) OGTT (O, n = 7) and its respective AUC (P, n = 7).

(Q) Insulin plasma levels during OGTT in HFD-fed E2f1<sup>β−/−</sup> and E2f1<sup>β+/+</sup> mice (n = 6).

(R) Total Glp-1 levels before and 10 min after an oral bolus of glucose in E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> mice after 20 weeks of HFD (n = 7). All of the values are expressed as means ± SEMs. \*p < 0.05, \*\*p < 0.01.
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<figure><img src="image_17.png" /><figcaption>Figure 2. The overexpression of E2f1 in pancreatic β cells slightly increases insulin secretion but does not improve glucose homeostasis</figcaption></figure>

(A) mRNA expression of *hE2F1* in *E2f1βctrl* and *E2f1βover* pancreatic islets (n = 6).

(B) Body weight of CD-fed *E2f1βctrl* and *E2f1βover* mice (n = 10).

(C) IPGTT in *E2f1βctrl* and *E2f1βover* mice (n = 11–15).

(D) AUC of IPGTT from *E2f1βctrl* and *E2f1βover* mice.

(E) Insulin plasma levels during IPGTT before (0 min) and 30 min following glucose injection (n = 11–15).

(F) Blood glucose levels during OGTT of *E2f1βctrl* and *E2f1βover* mice under CD (n = 5).

(G) AUC of OGTT from *E2f1βctrl* and *E2f1βover* mice (n = 5).

(H) Insulin plasma levels during OGTT measured 0 and 30 min after glucose gavage in *E2f1βctrl* and *E2f1βover* mice under CD (n = 5).

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with the *hE2F1* transgene (Scheijen et al., 2003) (Figure S2A). Overexpressing human E2F1 did not affect mouse *E2f1* transcript levels (Figure S2B). No particular phenotype was observed in these mice when fed CD, including islet morphology (Figures S2C and S2D), insulin content (Figure S2E), body weight (Figure 2B), fasting glycemia (Figure S2F), insulin sensitivity (Figure S2G), and i.p. glucose tolerance (Figures 2C and 2D). Intriguingly, the insulin levels were increased in *E2f1<sup>βover</sup>* mice in response to *in vivo* glucose injection when compared to littermate controls (*E2f1<sup>βCtrl</sup>*; Figure 2E). Finally, *E2f1<sup>βover</sup>* mice demonstrated a weak but not significant (p = 0.07) improvement in glucose tolerance when challenged with an oral bolus of glucose (Figures 2F and 2G), associated with comparable insulin levels in basal conditions and after glucose gavage in controls and *E2f1<sup>βover</sup>* mice (Figure 2H).

We next sought to evaluate the effect of *hE2F1* overexpression in pancreatic β cells on glucose homeostasis when challenged with HFD for 16 weeks. The body weight and the weight gain of *E2f1<sup>βover</sup>* mice remained comparable to control mice (Figures 2I and S2H, respectively). The overexpression of *hE2F1* in pancreatic β cells did not alter the insulin sensitivity (Figure S2I) or the glucose tolerance after an i.p. injection of glucose (Figures S2J and S2K). OGTT revealed similar glucose tolerance in *E2f1<sup>βover</sup>* mice after glucose gavage (Figures 2J and 2K). Interestingly, *E2f1<sup>βover</sup>* mice secreted more insulin 30 min after an oral glucose load compared to control mice (Figure 2L). Our data demonstrate that *E2f1<sup>βover</sup>* mice exhibit limited improvement of glucose homeostasis and insulin secretion upon physiologic conditions and during metabolic stress.

GSIS potentiation by the Glp-1 agonist exendin-4 is impaired in the absence of E2f1

To evaluate whether Glp-1 agonists modulate E2f1-dependent insulin secretion in a cell-autonomous manner, we performed static GSIS experiments in the *E2f1* knockdown mouse Min6 β cell line and their controls. Strikingly, a 55% decrease in *E2f1* mRNA levels was sufficient to blunt GSIS (Figures 3A and 3B). While ex-4 potentiated glucose effect on insulin secretion in control Min6 cells, that was not the case following *E2f1* knockdown (Figure 3B). To corroborate these findings, we performed a similar GSIS experiment using pancreatic islets isolated from *E2f1<sup>β+/+</sup>* and *E2f1<sup>β−/−</sup>* mice. Whereas *E2f1<sup>β+/+</sup>* pancreatic islets responded well to glucose, with an outcome potentiated in the presence of ex-4, *E2f1<sup>β−/−</sup>* pancreatic islets failed to exhibit both enhanced response to glucose and potentiation by ex-4 (Figure 3C). The treatment of non-diabetic human islets with the E2F pan inhibitor HLM006474 (Rosales-Hurtado et al., 2019) provoked a marked decrease in insulin secretion in response to ex-4 treatment, compared to DMSO-treated human islets (Figure 3D). Interestingly, GSIS assays on pancreatic islets isolated from control and *E2f1<sup>βover</sup>* mice showed that the overexpression of *E2F1* increased the potentiation impact of ex-4 on

GSIS (Figure 3E). To further demonstrate a specific loss of ex-4 capacity to potentiate insulin secretion in the absence of *E2f1*, we treated pancreatic islets with forskolin to evaluate whether activating the glp-1 pathway downstream of its receptor is functional in *E2f1<sup>β+/+</sup>* and *E2f1<sup>β−/−</sup>* pancreatic islets. We observed that treating pancreatic islets with forskolin potentiated more efficiently GSIS in *E2f1<sup>β−/−</sup>* pancreatic islets than in controls (Figure 3F). These data suggest that E2f1, both in mouse and human pancreatic islets, affects ex-4 effects on GSIS, a defect that can be rescued by forskolin treatment.

E2f1 controls Glp1r expression in pancreatic β cells

As *E2f1* deficiency did not affect Glp-1 circulating levels during OGTT (Figures 1J and 1R) but inhibits insulin secretion in response to glucose stimulation and incretins, we hypothesized that E2f1 modulates Glp-1 signaling in β cells rather than Glp-1 production. Since Glp-1 agonists modulate insulin secretion through the Glp1r (Muller et al., 2019), we analyzed *Glp1r* expression in our different mouse and human models of *E2f1* modulation. Knocking down *E2f1* in Min6 cells resulted in a significant decrease in *Glp1r* mRNA levels (approximately 65% of decrease, p = 0.0022; Figure 3G). We confirmed these results in *E2f1<sup>β−/−</sup>* pancreatic islets (Figure 3H) and human islets treated with the E2F inhibitor HLM006474 (Figure 3I). Interestingly, this E2F inhibitor decreased not only *GLP1R* expression in islets from nondiabetic humans but also *E2F1* mRNA levels (Figure 3J). Notably, we observed a mirrored rise in *Glp1r* mRNA levels in *E2f1<sup>βover</sup>* isolated islets (Figure 3J). *In silico* analysis revealed the presence of E2f1 binding sites in the promoter region of the *Glp1r* gene (Figures 3K, S3A, and S3B). To further demonstrate the ability of the E2f1-dependent pathway to control *Glp1r* expression, we performed co-transfection experiments in Min6 cells using a plasmid construct combining the mouse *Glp1r* promoter DNA sequence and the luciferase reporter gene. To activate or repress E2F1 pathways, we used expression vectors encoding E2F1 and its dimerization partner DP-1, as well as pRb, the bona fide repressor of E2F1 (Mendoza and Grossniklaus, 2015). Our data demonstrate that the activation of the E2F pathway increased *Glp1r*-dependent luciferase activity, while pRb repressed the increased luciferase activity mediated by E2F1-DP-1 (Figure 3L). Finally, we performed chromatin immunoprecipitation (ChIP)-qPCR experiments to determine whether E2F1 binds directly to the *Glp1r* promoter at the chromatin level. In the absence of reliable antibodies to ChIP E2F1, Min6 cells were transfected with plasmid containing the cytomegalovirus promoter (pCMV) or pCMV-hE2F1-FLAG to immunoprecipitate E2F1 using a FLAG antibody. Enrichment at the *Glp1r* promoter was observed when cells were transfected with pCMV-hE2F1-FLAG, demonstrating that E2F1 directly binds to the *Glp1r* promoter (Figure 3M). Our data highlight that E2F1 modulates *Glp1r* expression both in mouse and human islets, likely through the control of its promoter region. Strikingly, when E2F1-DP-1

<footnote>(I) Body weight of *E2f1<sup>βctrl</sup>* and *E2f1<sup>βover</sup>* mice after 16 weeks of HFD (n = 5).</footnote>

<footnote>(J) OGTT in *E2f1<sup>βctrl</sup>* and *E2f1<sup>βover</sup>* mice after 16 weeks of HFD (n = 5).</footnote>

<footnote>(K) AUC of OGTT from *E2f1<sup>βctrl</sup>* and *E2f1<sup>βover</sup>* mice (n = 5).</footnote>

<footnote>(L) Insulin plasma levels during OGTT.</footnote>

<footnote>All of the values are expressed as means ± SEMs. \*p < 0.05, \*\*p < 0.01.</footnote>
<figure><img src="image_18.png" /><figcaption>**Figure 3.** *E2f1 modulates Glp-1-mediated insulin secretion associated to Glp1r expression*</figcaption></figure>

(A) E2f1 mRNA expression levels from control (siCtrl) or E2f1 silencing (siE2f1) in Min6 cells (n = 5).

(B) GSIS measured in siCtrl or siE2f1 Min6 cells treated as indicated (n = 7).

(C) GSIS in E2f1<sup>β+/+</sup> and E2f1<sup>β−/−</sup> pancreatic islets (n = 3–9).

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are co-transfected with pRb, the activity of the *Glp1r* promoter is dampened (Figure 3L), showing that the activity of the E2F1-DP-1 complex on the *Glp1r* promoter is regulated by pRb.

## Glp-1 increases the phosphorylation of pRb through the activation of the CDK signaling pathway in mouse pancreatic islets

Since the molecular mechanisms of serine and threonine kinase activation controlled by the Glp-1 pathway are not fully understood, a kinome analysis using the Pamgene technology was performed on pancreatic islets isolated from C57Bl6/J mice treated with 20 mM glucose compared to 20 mM glucose plus 50 nM ex-4 for 30 min (Figure 4A). Following these treatments, an increase in 43 phosphorylation sites of 41 peptides was observed when pancreatic islets were treated with glucose-ex-4 compared to glucose alone (Figure 4B; Table S3). It is noteworthy that treatment of mouse islets with 20 mM glucose plus 50 nM ex-4 increased Creb phosphorylation at serine 133 (Creb<sup>S133</sup>, Figure 4B, log2 fold change = 0.45, p = 0.003), as previously shown (Jhala et al., 2003). Strikingly, the co-treatment of mouse pancreatic islets with 20 mM glucose plus 50 nM ex-4 for 30 min increased the phosphorylation level of the pRb on serine 807/811 (pRb<sup>S807/811</sup>; log2 fold change = 0.63, p = 0.043), the bona fide repressor of E2F1 transcriptional activity. The ex-4-mediated induction of pRb<sup>S807/811</sup> phosphorylation was further confirmed in Min6 β cells (Figures S4A-S4C). Integrating the phosphorylation data using Bionavigator software revealed the specific activation of kinases upon ex-4 treatment, including several CDKs (Figure 4C). Finally, Ingenuity Pathway Analysis further demonstrated that several canonical pathways were controlled by ex-4 treatment, such as the opioid, AMP kinase (AMPK) or protein kinase A (PKA) signaling pathways (Figure 4D). These results demonstrate that the activation of the Glp-1 pathway by its agonist ex-4 in mouse pancreatic islets activates several kinases, including CDKs, leading to the phosphorylation of pRb.

## Ex-4 increases E2F1 transcriptional activity

These data led us to postulate that GLP1R activation through an ex-4 treatment could increase E2F1 transcriptional activity, since the *E2f1* target gene expression is strongly controlled by the phosphorylation status of pRb (Mendoza and Grossniklaus, 2015). To test this hypothesis, we performed luciferase assays using a plasmid containing E2F-responsive elements (E2F-RE) cloned in front of the thymidine kinase (Tk) promoter and the luciferase gene (E2F-RE-Tk-Luc). Upon treatment with 2.8 mM glucose,

co-transfection with the E2F1 and DP-1 expression vectors induced luciferase activity compared to control conditions, validating increased E2F1 transcriptional activity in the presence of E2F1 and DP-1 (Figure 5A). The treatment of Min6 cells with 20 mM glucose further increased E2f1 transcriptional activity when compared to 2.8 mM glucose treatment (Figure 5A). Interestingly, this effect was even more potentiated with a 50-nM ex-4 treatment (Figure 5A), suggesting that ex-4 enhanced the E2f1 transcriptional activity on the promoter of its target genes. However, the co-transfection with a pRb expression vector nearly abolished E2f1 transcriptional activity (Figure 5A), thus demonstrating, as expected, that pRb inhibits E2f1 transcriptional activity. However, when Min6 cells were treated with 50 nM of ex-4, the pRb repressive effect was partially alleviated, thus allowing a weak transcriptional activity of the E2F1-DP1 heterodimer on the E2F-RE-Tk promoter (Figure 5A). These data suggest the loss of the pRb repressive activity following the treatment with the GLP1R agonist (Figure 5A). Since ex-4 increased E2f1 transcriptional activity, we next wondered whether the expression of genes involved in β cell mass and/or function was modulated upon ex-4 treatment. Since PDX-1, a master regulator of β cell identity that controls Insulin gene expression (Fujitani, 2017), is regulated by ex-4 treatment (Kodama et al., 2005; Wang et al., 2001), we checked whether short-term treatment with 50 nM of ex-4 (1-4 h) modulated its expression in Min6 cells or mouse pancreatic islets in which *E2f1* expression level is downregulated. Our results showed that treatment with 50 nM of ex-4 increased the mRNA levels of *E2f1* (Figure 5B) and *Pdx1* (Figure 5C) in Min6 cells transfected with a non-targeting, control small interfering RNA (siRNA). Knocking down *E2f1* in Min6 cells completely blocked the ex-4 mediated increase in *E2f1* (Figure 5B) and *Pdx1* (Figure 5C) mRNA levels. These results were also observed when C57Bl6J pancreatic islets were treated with glucose and ex-4 (Figures 5D and 5E). Interestingly, treating *E2f1*<sup>β/-</sup> pancreatic islets with ex-4 failed to increase *E2f1*, confirming the reproducibility and robustness of our model, but most interestingly failed to increase *Pdx1* expression levels (Figures 5F and 5G, respectively). Altogether, these results suggest that ex-4 modulates pRb phosphorylation and E2F1 transcriptional activity, which in turn may modulate insulin secretion through the upregulation of E2f1-dependent genes such as *E2f1* itself or *Pdx1* mRNA levels.

## DISCUSSION

Following previous studies demonstrating that the E2F1-pRb pathway controls pancreatic β cell mass (Cai et al., 2013,

<footnote>(D) GSIS experiments on human isolated islets treated for 48 h with 0.1% DMSO or 10 µM HLM006474 (n = 3).</footnote>

<footnote>(E) GSIS in *E2f1βctrl* and *E2f1βover* isolated islets (n = 3–4).</footnote>

<footnote>(F) GSIS in *E2f1β+/+* and *E2f1β−/−* pancreatic islets exposed to 2.8 mM glucose, 16.7 mM glucose, treated or not with 2.5 µM forskolin (n = 8–10).</footnote>

<footnote>(G) Glp1r mRNA expression levels in *siCtrl* or *siE2f1* Min6 cells (n = 6).</footnote>

<footnote>(H) Glp1r mRNA expression levels in *E2f1β+/+* and *E2f1β−/−* isolated islets (n = 4).</footnote>

<footnote>(I) E2F1 and GLP1R mRNA expression levels in human islets treated for 48 h with 0.1% DMSO or 10 µM HLM006474 (n = 3).</footnote>

<footnote>(J) Glp1r mRNA expression levels in *E2f1βctrl* and *E2f1βover* isolated islets (n = 4–5).</footnote>

<footnote>(K) Sequence of the E2F1 responsive element (E2F RE) in the mouse Glp1r promoter region.</footnote>

<footnote>(L) Transient transfection experiment in Min6 cells with a mGlp1r-promoter-luciferase construct (Glp1r-Luc vector) and pcDNA3 vector (empty vector, negative control), E2F1-DP-1, pRb, E2F1-DP-1-pRb.</footnote>

<footnote>(M) ChIP-qPCR demonstrating E2F1 binding to the mouse Glp1r promoter from Min6 cells transfected with pCMV or pCMV-hE2F1-FLAG plasmids (n = 2).
All of the values are expressed as means ± SEMs. *p* < 0.05, **p* < 0.01, ***p* < 0.001, ****p* < 0.0001.</footnote>
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>**Figure 4.** Short-term Glp1r activation induces pRb phosphorylation in pancreatic islets</figcaption></figure>

(A) Schematic representation of the kinome profiling strategy following ex-4 treatment of mouse pancreatic islets.

(B) Volcano plot exhibiting differential peptide phosphorylation in mouse pancreatic isolated islets treated with 20 mM glucose (n = 3) or 20 mM glucose + 50 nM ex-4 (n = 3) for 30 min.

(C) Analysis of upstream kinases involved in differential peptide phosphorylation.

(D) Canonical pathways controlled by ex-4 treatment.
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /><figcaption>**Figure 5. Ex-4 modulates E2f1 transcriptional activity**</figcaption></figure>

(A) Min6 cells transiently co-transfected with an E2F-RE-Tk-luciferase construct (E2F-RE-Tk-Luc vector), an empty pcDNA3 vector (negative control), or E2F1-DP-1, or pRb or E2F1-DP-1-pRb, and treated for 30 min with 2.8 mM glucose, 20 mM glucose, or 20 mM glucose + 50 nM ex-4 (n = 3).

(B) E2f1 mRNA expression from siCtrl or siE2f1 Min6 cells treated with 20 mM glucose or 20 mM glucose and 50nM ex-4 (ex-4) during 1, 2, or 4 h (n = 3).

(C) Pdx1 mRNA expression from siCtrl or siE2f1-Min6 cells treated as in (B) (n = 3).

(D) E2f1 mRNA expression from 12-week-old C57Bl6J pancreatic islets treated with 20 mM glucose (vehicle) or 20 mM glucose and 50 nM ex-4 (n = 3).

(E) Pdx1 mRNA expression from pancreatic islets as in (D) (n = 3).

(F) E2f1 mRNA expression in E2f1<sup>β−/−</sup> and E2f1<sup>β+/+</sup> pancreatic islets treated for 4 h with 20 mM glucose (DMSO) or 20 mM glucose and 50 nM ex-4 (ex-4) (n = 3–4).

(G) mRNA expression of Pdx1 in E2f1<sup>β−/−</sup> and E2f1<sup>β+/+</sup> pancreatic islets treated as in (F) (n = 3–4).

All of the values are expressed as means ± SEMs. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001.
2014; Fajas et al., 2004; Grouwels et al., 2010) and function (An-
nicotte et al., 2009; Boni-Schnetzler et al., 2018), we identify here
a molecular link between this transcription factor and the drug-
gable Glp-1 signaling.

In the last 2 decades, a particular emphasis has been placed
on the development of specific GLP1R agonists to treat T2D
(Hinnen, 2017). Indeed, GLP1R agonists improve glycemia in pa-
tients with T2D by increasing insulin synthesis and secretion and
by inducing β cell proliferation (Baggio and Drucker, 2007;
Campbell and Drucker, 2013). Although targeting the GLP1R
signaling pathway is a promising therapeutic strategy, it has
been reported that the incretin effects decrease in humans
with the onset of obesity and T2D, thus urging us to uncover
the underlying mechanisms involved in the loss of GLP1R
agonist sensitivity in patients with T2D (Ahrén, 2013; Knop
et al., 2007). Several studies suggest that part of this deleterious
mechanism could rely on the downregulation of the Glp1r
expression in different tissues involved in metabolic homeosta-
sis, including pancreatic β cells (Pan et al., 2009; Xu et al.,
2007). Other studies have demonstrated that hyperglycemia
not only downregulates both Glp-1 et Gip receptors expression
in pancreatic islets but it also impairs the Glp-1 signaling
pathway through the loss of Glp1r at the β cell cytoplasmic mem-
brane (Rajan et al., 2015). Preserving Glp1r expression in meta-
bolic and non-metabolic tissues appears to be crucial to main-
tain glucose homeostasis. In addition, the identification of the
signaling pathways that regulate Glp1r expression could repre-
sent potential targets toward T2D treatment. For instance, the
Pax6 (Gosmain et al., 2012) or the farnesoid X receptor (Kong
et al., 2021) directly modulate the Glp1r gene expression at the
chromatin level; therefore, targeting these pathways may be
promising for the improvement of glucose homeostasis and in-
sulin secretion in a metabolic stress context. We also observe
that overexpressing E2F1 in pancreatic β cells increases Glp1r
expression, suggesting that targeting this pathway would also
represent an alternative strategy, with caution regarding the pro-
liferative functions displayed by the E2F1/pRb pathway. More-
over, since E2F1 expression is decreased in T2D islets (Lupi
et al., 2008), our study suggests a putative association between
the loss of GLP1R expression in patients with T2D and the loss of
E2F1 expression. Although single-cell RNA sequencing analyses
are required to unequivocally demonstrate concomitantly
decreased levels of E2F1 and GLP1R within the same cell, we
can speculate that the long-term loss of efficacy of Glp1r agonist
treatment to restore glucose homeostasis in patients with T2D
may be related, at least partially, to impaired E2F1-dependent
modulation of GLP1R expression.

Following pioneer observations that the E2F1-CDK4-pRb
pathway is regulated by glucose and insulin in pancreatic islets
(Annicotte et al., 2009), we show here that the treatment of
pancreatic islets with a Glp1r agonist modulates the E2F1
pathway through the phosphorylation of pRb and the activation
of the E2F1 transcriptional activity. Our data are in line with pre-
vious studies showing that the treatment of Ins1 cells with ex-4
decreased both mRNA and protein levels of pRb and increased
E2f1 protein levels (Cai et al., 2014). It is well established that
ex-4 and Glp1r agonists increase cell-cycle progression through
the increased expression of cyclins A2 and D1, stimulating

pancreatic β cell proliferation (Kim et al., 2006; Song et al.,
2008). Since the E2F1-pRb-CDK4 pathway positively modulates
the expression of *Ccna2* and *Ccnd1* (Stanelle et al., 2002), our
data suggest that the cell-cycle progression of pancreatic β
cell through the induction of *Ccna2* and *Ccnd1* with ex-4 treat-
ment could be associated with an increase in E2f1 expression
and/or activity. Moreover, in human islets, the treatment with
ex-4 also increases the expression of several cyclins, leading
to increased pancreatic β cell proliferation (Dai et al., 2017).
Recent data obtained from hypophosphorylated pRb knockin
mice demonstrated that pRb phosphorylation may affect
glucose homeostasis at the β cell level and mediates β cell pro-
liferation upon ex-4 treatment (Jiang et al., 2022). Although islet
number and β cell morphology were preserved in *E2f1<sup>β−/−</sup>* in
basal conditions, further investigations are warranted to un-
equivocally demonstrate a functional link between E2f1 and β
cell proliferation upon ex-4 treatment during metabolic stress
or β cell regeneration.

In this study, through the use of β cellspecific E2f1 KO mice, we further confirm a cell-autonomous function for E2f1 in controlling insulin secretion. More important is the fact that in E2f1<sup>β−/−</sup> mice, E2f1 controls insulin secretion and, contrary to global E2f1 KO mice (Fajas et al., 2004; Iglesias et al., 2004; Li et al., 2003a), without modifying β cell mass.

In conclusion, our study reveals a crosstalk between E2f1 and
Glp-1 signaling pathways in both mice and human pancreatic is-
lets. It opens the possibility of understanding the differential ef-
fects of Glp-1 in the control of β cell mass and function and their
relevant molecular mechanisms. How this crosstalk modulates β
cell proliferation, β cell dedifferentiation, or loss of identity; which
downstream signaling molecules are involved; and how these
mechanisms are actionable in T2D pathophysiology remain
open questions that require further investigation.

Limitations of the study

A limitation of this study is the observation that E2f1<sup>β−/−</sup> mice display glucose intolerance, pointing to a more general dysfunction of the E2f1<sup>β−/−</sup> pancreatic islets to secrete insulin in response to glucose and, de facto, to Glp1-R agonists. In addition, although we demonstrate that ex-4 increases pRb phosphorylation and E2F1 activity, we do not provide evidence that this mechanistic link between the E2f1/pRb/Glp-1 directly modulates insulin secretion and contributes to the phenotype we observe in E2f1<sup>β−/−</sup> mice. Future work is needed to precisely assess how lack of glucose responsiveness in the absence of E2f1 influences the Glp-1 pathway and contributes per se to glucose homeostasis impairments.

STAR★METHODS

Detailed methods are provided in the online version of this paper
and include the following:

● KEY RESOURCES TABLE

● RESOURCE AVAILABILITY

○ Lead contact

○ Materials availability

○ Data and code availability
* EXPERIMENTAL MODEL AND SUBJECT DETAILS

* METHOD DETAILS

* Pancreatic islets studies

* Cell culture, transfections, and siRNA knock-down

* Protein extracts and western blot experiments

* *In silico* analysis of the mouse *Glp1r* promoter region

* Chromatin immunoprecipitation

* RNA extraction, measurements, and profiling

* Kinome profiling

* Immunohistochemistry, immunofluorescence, and
morphometry

* QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2022.111170.

ACKNOWLEDGMENTS

We thank Drs. David Blum, Stéphane Dalle, and Benoit Pourcet for scientific discussions. Human islets were provided through JDRF award 31-2008-416 (ECIT Islet for Basic Research program). The authors thank UMS2014-US41 and the experimental resources platform from Université de Lille. This work was supported by Agence Nationale de la Recherche (ANR) grants (EGID ANR-10-LABX-46; LIGAN-PM Equipex 2010 ANR-10-EQPX-07-01; ANR-16-IDEX-0004 ULNE; ANR BETAPLASTICITY, ANR-17-CE14-0034), EFSD, INSERM, CNRS, Université de Lille, Institut Pasteur de Lille (CTRL Melodie), Fonderation pour la Recherche Médicale (EQU202103012732), I-SITE ULNE (EpiRNAdiab Sustain grant), Conseil Régional Hauts de France, Métropole Européenne de Lille, and Société Francophone du Diabète.

AUTHOR CONTRIBUTIONS

Conceptualization, C.B., E. Courty, and J.-S.A.; methodology, C.B., E. Courty, and J.-S.A.; investigation, C.B., E. Courty, L.R., G.P., X.G., N.R., C.C., M.M., R.B., and E. Caron; resources, Z.B., P.M., J.K.-C., F.P., P.F., A.B., and F.O.; writing – original draft, C.B. and J.-S.A.; writing – review & editing, C.B., E. Courty, and J.-S.A.; visualization, C.B., E. Courty, and J.-S.A.; supervision, J.-S.A.; funding acquisition, J.-S.A.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: April 28, 2021

Revised: April 11, 2022

Accepted: July 15, 2022

Published: August 9, 2022

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Anti-phospho-Rb<sup>S807/811</sup></td><td>Cell signaling</td><td>CS#9308; RRID: AB_331472</td></tr><tr><td>anti-Rb 4H1</td><td>Cell signaling</td><td>CS#9309; RRID: AB_823629</td></tr><tr><td>Anti-tubulin</td><td>Sigma-Aldrich</td><td>T5168; RRID: AB_477579</td></tr><tr><td>Anti-glucagon</td><td>Sigma-Aldrich</td><td>G2654; RRID: AB_259852</td></tr><tr><td>Anti-HA</td><td>Sigma-Aldrich</td><td>H3663; RRID: AB_262051</td></tr><tr><td>Anti-insulin</td><td>Agilent technologies</td><td>A0564; RRID: AB_10013624</td></tr><tr><td>Anti-insulin</td><td>Agilent technologies</td><td>IR00261-2; RRID: AB_2800361</td></tr><tr><td>Anti-mouse alexa fluor 488 donkey</td><td>Thermo Scientific</td><td>A21202; RRID: AB_141607</td></tr><tr><td>Anti-rabbit alexa fluor 488 donkey</td><td>Thermo Scientific</td><td>A21206; RRID: AB_2535792</td></tr><tr><td>Anti-guinea pig alexa fluor 594 goat</td><td>Thermo Scientific</td><td>A11076; RRID: AB_2534120</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>DH5α Chemically Competent Cells</td><td>Thermo Scientific</td><td>18265017</td></tr><tr><td colspan="3"><strong>Biological samples</strong></td></tr><tr><td>Human islets</td><td>ECIT Islet for Basic Research program</td><td>https://ecit.dri-sanraffaele.org</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Glucose 45%</td><td>Sigma-Aldrich</td><td>G8769</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I9278</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>83264</td></tr><tr><td>Histopaque 1119</td><td>Sigma-Aldrich</td><td>11191</td></tr><tr><td>RPMI 1640</td><td>Fisher scientific</td><td>12027599</td></tr><tr><td>Exendin-4</td><td>Sigma-Aldrich</td><td>E7144</td></tr><tr><td>HLM006474</td><td>Sigma-Aldrich</td><td>SML1260</td></tr><tr><td>DMEM high glucose glutamax</td><td>Fisher scientific</td><td>12077549</td></tr><tr><td>β-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td></tr><tr><td>Fatty-acid free BSA</td><td>Sigma-Aldrich</td><td>A7030</td></tr><tr><td>Dharmafect1</td><td>Horizon discovery</td><td>T-2001-03</td></tr><tr><td>Lipofectamine 2000</td><td>Fisher scientific</td><td>10696153</td></tr><tr><td>Protease Inhibitor cocktail</td><td>Sigma-Aldrich</td><td>05892791001</td></tr><tr><td>Fetal bovine serum</td><td>Fisher scientific</td><td>10270106</td></tr><tr><td>Penicillin-streptomycin</td><td>Fisher scientific</td><td>15-140-122</td></tr><tr><td>Phosphatase inhibitors</td><td>Fisher scientific</td><td>10668304</td></tr><tr><td>Type V collagenase</td><td>Sigma-Aldrich</td><td>C9263</td></tr><tr><td>SuperSignal West Dura</td><td>Fisher scientific</td><td>10220294</td></tr><tr><td>Trizol reagent</td><td>Fisher scientific</td><td>15596-018</td></tr><tr><td>FastStart SYBR Green master mix</td><td>Sigma-Aldrich</td><td>4913914001</td></tr><tr><td>Pierce ECL Western blotting substrate</td><td>Fisher scientific</td><td>A38555</td></tr><tr><td>RIPA Lysis Buffer</td><td>Fisher scientific</td><td>89901</td></tr><tr><td>M-PER protein extraction reagent</td><td>Fisher scientific</td><td>78501</td></tr><tr><td>Diprotein A (DPP4 inhibitor)</td><td>Sigma-Aldrich</td><td>I9759</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>Mouse insulin ELISA kit</td><td>Mercodia</td><td>10-1247-01</td></tr><tr><td>Total GLP-1 NL-ELISA kit</td><td>Mercodia</td><td>10-278-01</td></tr><tr><td>Mini Elute PCR purification kit</td><td>Qiagen</td><td>28004</td></tr><tr><td>RNeasy Plus Microkit</td><td>Qiagen</td><td>74034</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>RNA-seq data from pancreatic islets isolated from HFD-fed C57Bl6N mice</td><td>Gao et al., 2021</td><td>GSE153222</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>Min6 cells</td><td>AddexBio</td><td>C0018008</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>C57Bl6/J homozygous E2f1 floxed mice (E2f1fl/fl)</td><td>This study</td><td>N/A</td></tr><tr><td>Rip-Cre Mouse - Tg(Ins2-cre)23Herr</td><td>Laboratory of Pr Pedro Herrera, University of Geneva, Switzerland</td><td>RRID: MGI:6195021</td></tr><tr><td>RIPCre<sup>+/+</sup>/E2f1fl/fl</td><td>This study</td><td>N/A</td></tr><tr><td>RIPCre<sup>Tg/+</sup>/E2f1fl/fl</td><td>This study</td><td>N/A</td></tr><tr><td>Rosa-26-loxP-LacZ-loxP-hE2F1 mice (RFC<sup>Tg/+</sup>)</td><td>Laboratory of Dr Ulrike Ziebold (MDC, Berlin, Germany)</td><td>N/A</td></tr><tr><td>RIPCre<sup>+/+</sup>/RFC<sup>Tg/+</sup> mice</td><td>This study</td><td>N/A</td></tr><tr><td>RIPCre<sup>Tg/+</sup>/RFC<sup>Tg/+</sup> mice</td><td>This study</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>Primers for RT-qPCR and ChIP-qPCR, see Table S1</td><td>This study</td><td>N/A</td></tr><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>pCMV10</td><td>This study</td><td>N/A</td></tr><tr><td>pCMV10-hE2F1-Flag</td><td>This study</td><td>N/A</td></tr><tr><td>pCMV-E2F1</td><td>Fajas et al., 2002</td><td>N/A</td></tr><tr><td>pCMV-DP-1</td><td>Fajas et al., 2002</td><td>N/A</td></tr><tr><td>pECE-pRB</td><td>Fajas et al., 2000</td><td>N/A</td></tr><tr><td>pE2fRE-Tk-Luc</td><td>Laboratory of Pr L. Fajas, CIG, Lausanne, Switzerland</td><td>N/A</td></tr><tr><td>pCMV-Glp1r-Luc</td><td>E-zyvec</td><td>This study</td></tr><tr><td>siE2f1</td><td>Dharmacon</td><td>#13555</td></tr><tr><td>siControl</td><td>Dharmacon</td><td>D-001810-01-20</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>GraphPad Prism 9.0 software</td><td>GraphPad Software</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr><tr><td>Image J</td><td>Schneider et al., 2012</td><td>https://imagej.nih.gov/ij/index.html</td></tr><tr><td>Ingenuity Pathway Analysis</td><td>Qiagen</td><td>https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/</td></tr><tr><td>LASAGNA-Search tool</td><td>Lee and Huang, 2013</td><td>N/A</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>High-fat diet</td><td>Research Diet</td><td>D12492i</td></tr><tr><td>AccuCheck Performa</td><td>Roche Diagnostics</td><td>N/A</td></tr></tbody></table>

RESOURCE AVAILABILITY

Lead contact

Further information and request for reagents and resources should be directed to and will be fulfilled by the lead contact, Dr Jean Sébastien Annicotte (jean-sebastien.annicotte@inserm.fr).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

* This paper analyzes existing, publicly available data. These accession numbers for the datasets are listed in the key resources table. All original data reported in this paper will be shared by the lead contact upon request.

* This paper does not report original code.
● Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice were maintained according to European Union guidelines for the use of laboratory animals. In vivo experiments were performed in compliance with the French ethical guidelines for studies on experimental animals (animal house agreement no. 59-350294, Authorization for Animal Experimentation, project approval by our local ethical committee no. APAFIS#2915-201511300923025v4). All experiments were performed with male mice. Mice were housed under a 12-hr light/dark cycle and given a regular chow. For high-fat diet (HFD) studies, 6-week-old mice were placed on a HFD (60% of calories from fat) for 16 to 20 weeks. IPGTT and OGTT was performed after 16h of fasting and glucose (2g/kg) was administrated by intraperitoneal injection or oral gavage respectively. For mice under HFD, IPGTT and OGTT was performed after 16h of fasting and glucose (1.5g/kg) was administrated by intraperitoneal injection or oral gavage respectively. Glycemia was measured using the AccuCheck Performa. Circulating insulin levels were measured using the insulin ELISA kit. Circulating GLP-1 levels were measured using Total GLP-1 NL-ELISA kit. C57Bl6/J homozygous E2f1 floxed (E2f1 flox/flox) mice and heterozygous rat insulin 2 promoter (RIP)-Cre mice (RIPcreTg/+ ) were previously described (Denechaud et al., 2016; Herrera et al., 1998) and then further intercrossed to generate RIPcre+/+/E2f1 flox/flox (E2f1β+/+) and RIPcreTg+/E2f1 flox/flox (E2f1β-/-) mice. A PCR genotyping strategy was subsequently used to identify E2f1β+/+ and E2f1β-/- littermates. C57Bl6/J mice harboring the Rosa-26-loxP-LacZ-loxP-hE2F1 conditional expression cassette (RFC mice) were obtained from Dr Ulrike Ziebold (MDC, Berlin, Germany; (Scheijen et al., 2003)). Heterozygous RFC/+ mice were crossed with RIPcreTg/+ mice to obtain RIPcreTg+/RFCTg+/ (E2f1βover) and control RIPcre+/+/RFCTg+/ (E2f1βCtrl) mice.

## METHOD DETAILS

### Pancreatic islets studies

For mouse islets studies, pancreas was digested by type V collagenase (1.5mg/mL) for 10 minutes at 37°C as previously described (Annicotte et al., 2009; Rabhi et al., 2016). After pancreas digestion and separation of pancreatic islets in a polysucrose density gradient medium, islets were purified by handpicking under a macroscope and were cultured during 16 hours before experiments. For GSIS experiments, approximately 30 islets were exposed to 2.8 mM glucose, 16.7 mM glucose, 20 mM glucose, 20 mM glucose and 50 nM exendin-4 or 16.7 mM glucose and 2.5 µM forskolin in Krebs-Ringer buffer supplemented with HEPES and 0.5% fatty-acid free BSA. Insulin released in the medium was measured using the mouse insulin ELISA kit. Insulin content was measured after lysis in a buffer containing 75% of ethanol and 1.5% HCl using mouse insulin ELISA kit. Data are expressed as a ratio of total insulin content. Human pancreatic tissue was harvested from human, non-diabetic, adult donors. Isolation and pancreatic islet culture were performed as previously described (Kerr-Conte et al., 2010). Human islets were treated for 48 hours with the pan-E2F inhibitor HLM006474 at 10µM (Rosales-Hurtado et al., 2019) and data were expressed as a ratio of total insulin content. For mRNA quantification, human islets were isolated as described above and snap-frozen for further processing.

### Cell culture, transfections, and siRNA knock-down

Min6 cells were cultured in DMEM with 15% fetal bovine serum, 100 mg/mL penicillin-streptomycin and 55 mM β-mercaptoethanol. Cells were transfected with non-targeting siRNA mouse negative controls (siCont) and siE2f1 (#13555, SMARTpool, Dharmacon) using Dharmafect1 and experiments were performed 48 hours later. Transient transfection experiments were performed in Min6 cells using Lipofectamine 2000 following the manufacturer's instructions. Luciferase assays were performed 48 hours post-transfection and normalized to β-galactosidase activity as previously described (Annicotte et al., 2009). For pharmacological treatments, Min6 cells and pancreatic islets were treated with HLM006474 (Rosales-Hurtado et al., 2019) at 10µM for 48h. Exendin-4 was used at 50 nM at different time as indicated.

### Protein extracts and western blot experiments

Western blot was performed as previously described (Rabhi et al., 2018). Min6 cells were washed twice with cold PBS 1X and lysed with cell lysis buffer (50 mM Tris-HCl pH 8, 137 mM NaCl, 10% glycerol, 1% NP-40) supplemented with Protease Inhibitor cocktail and phosphatase inhibitors. Immunoblotting experiments were performed using 40 µg of total proteins and loaded on Precast SDS gel (Biorad). After electromigration, proteins were transferred on nitrocellulose membrane during 1h at 110V that were further incubated in TBS-Tween 0.05% (TBS-T) supplemented with 5% of milk. Membranes were incubated 16 h at 4°C with primary antibodies as indicated in blocking buffer supplemented with 3% of bovine serum albumin or milk. After washing, membranes were incubated 1 h with the secondary antibody conjugated with horseradish peroxidase. The revelation of luminescent bands was performed using Pierce ECL Western blotting substrate or SuperSignal West Dura Extended duration substrate with Chemidoc Xrs+ (Biorad).

### In silico analysis of the mouse Glp1r promoter region

The DNA sequence of the promoter region of the Glp1r gene was obtained from Ensembl (https://www.ensembl.org/index.html). Motif search was performed using LASAGNA-Search tool (Lee and Huang, 2013).
## Chromatin immunoprecipitation

Min6 cells were transfected with the empty vector pCMV10 or pCMV10-hE2F1-Flag. 48 hours after transfection, DNA-protein complexes from Min6 cells were formaldehyde-crosslinked to DNA at a final concentration of 1% for 10 minutes. The reaction was stopped by adding glycine at a concentration of 0.125M during 5 minutes. After cell lysis and sonication with the Bioruptor Pico (Dialgenode, ref B01060010) for 8 minutes, proteins were immunoprecipitated with either the non-specific Ig G or anti-Flag 2 antibodies. After washing, protein-DNA complexes were decrosslinked by heating the samples for 16 hours at 65°C. DNA was purified using Mini Elute PCR purification kit and qPCR were performed using promoter-specific primers.

## RNA extraction, measurements, and profiling

Total RNA was extracted from Min6 cells using trizol reagent. For mouse and human islets, total RNA was extracted with the RNeasy Plus Microkit following manufacturer's instructions. mRNA expression levels were measured after reverse transcription by quantitative real-time PCR (qRT-PCR) with FastStart SYBR Green master mix using a LC480 instrument (Roche). qRT-PCR were normalized to cyclophilin mRNA levels. The results are expressed as the relative mRNA level of a specific gene expression using the formula $2^{-ΔΔCt}$.

## Kinome profiling

Serine-threonine kinase (STK) microarrays were purchased from PamGene International BV. Each array contained 140 phosphorylable peptides as well as 4 control peptides. Sample incubation, detection, and analysis were performed in a PamStation 12 according to the manufacturer's instructions. The experiments were performed on mouse pancreatic islets as previously described (Rabhi et al., 2018).

## Immunohistochemistry, immunofluorescence, and morphometry

Immunofluorescence and immunohistochemistry were performed exactly as described previously (Rabhi et al., 2016). Pancreatic tissues were fixed in 10% formalin, embedded in paraffin and sectioned at 5 µm. For immunofluorescence microscopy analyses, after antigen retrieval using citrate buffer, 5-µm formalin-fixed paraffin embedded (FFPE) pancreatic sections were incubated with the indicated antibodies. Immunofluorescence staining was revealed by using a fluorescein-isothiocyanate-conjugated anti-mouse (for Glucagon and HA), fluorescein-isothiocyanate-conjugated anti-rabbit (for pRb$^{S807/811}$), anti-guinea pig (for Insulin) secondary antibodies. Nuclei were stained with Hoechst. Morphometrical parameters (percentage of endocrine cells and insulin positive cells, islet number, and density) were determined as previously described (Courty et al., 2019; Rabhi et al., 2016).

## QUANTIFICATION AND STATISTICAL ANALYSIS

Data are presented as mean ± s.e.m. Statistical analyses were performed using a two-tailed unpaired Student's t-test, one-way analysis of variance (ANOVA) followed by Dunnett's *post hoc* test or two-way ANOVA with Tukey's *post hoc* tests comparing all groups to each other, using GraphPad Prism 9.0 software. Differences were considered statistically significant at $p < 0.05$ (*p* < 0.05, **p* < 0.01, ***p* < 0.001 and ****p* < 0.0001).