Rb and p107 are required for alpha cell survival, beta cell cycle control and glucagon-like peptide-1 action
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Diabetes mellitus is characterised by beta cell loss and alpha cell expansion. Analogues of glucagon-like peptide-1 (GLP-1) are used therapeutically to antagonise these processes; thus, we hypothesised that the related cell cycle regulators retinoblastoma protein (Rb) and p107 were involved in GLP-1 action.
We used small interfering RNA and adenoviruses to manipulate Rb and p107 expression in insulinoma and alpha-TC cell lines. In vivo we examined pancreas-specific Rb knockout, whole-body p107 knockout and Rb/p107 double-knockout mice.
Rb, but not p107, was downregulated in response to the GLP-1 analogue, exendin-4, in both alpha and beta cells. Intriguingly, this resulted in opposite outcomes of cell cycle arrest in alpha cells but proliferation in beta cells. Overexpression of Rb in alpha and beta cells abolished or attenuated the effects of exendin-4 supporting the important role of Rb in GLP-1 modulation of cell cycling. Similarly, in vivo, Rb, but not p107, deficiency was required for the beta cell proliferative response to exendin-4. Consistent with this finding, Rb, but not p107, was suppressed in islets from humans with diabetes, suggesting the importance of Rb regulation for the compensatory proliferation that occurs under insulin resistant conditions. Finally, while p107 alone did not have an essential role in islet homeostasis, when combined with Rb deletion, its absence potentiated apoptosis of both alpha and beta cells resulting in glucose intolerance and diminished islet mass with ageing.
We found a central role of Rb in the dual effects of GLP-1 in alpha and beta cells. Our findings highlight unique contributions of individual Rb family members to islet cell proliferation and survival.
KeywordsAlpha cell Beta cell Diabetes p107 Rb
Glucose-stimulated insulin secretion
Glucose tolerance test
Insulin tolerance test
Proliferating cell nuclear antigen
Whole-body p107 knockout
Pancreas-specific Rb knockout
Small interfering RNA
Glucose homeostasis is tightly regulated by insulin and glucagon secreted by pancreatic beta and alpha cells, respectively [1, 2, 3]. Accordingly, a proper ratio of beta and alpha cells is crucial to meet the challenges of metabolic stress. Reduced number and function of beta cells with alpha cell excess is a pathological hallmark in diabetes [4, 5, 6]. Beta cell regeneration, in principle, could be achieved by neogenesis of islet stem/progenitor cells  or self-replication of pre-existing beta cells . However, islet progenitors are rare and virtually absent after birth, when most mature islet cells are post-mitotic [8, 9]. Therefore, increasing research has focused on boosting beta cell mass while ideally suppressing alpha cell hyperplasia. Understanding the mechanisms that control alpha and beta cell proliferation may lead to the development of novel diabetes therapies.
Cell proliferation is a finely tuned process that transits cells through specific restriction points . Adult pancreatic islet cells are mostly in the quiescent/G1/0 state but can re-enter cell cycle following appropriate stimuli. For example, the glucagon-like peptide-1 (GLP-1) analogue, exendin-4, stimulates beta cell proliferation [11, 12] while preventing alpha cell expansion [13, 14]. However, the mechanisms by which GLP-1 mediates these divergent effects in alpha and beta cells are unknown.
Retinoblastoma protein (Rb) is a well-known tumour-suppressor and gatekeeper of cell cycle re-entry through inhibition of E2f transactivation and E2f target genes . Rb, therefore, is a viable target for controlling islet cell cycle. Previous studies showed that Rb plays a critical role during the transition from proliferative to differentiated states, but has a limited role once cells exit the cell cycle and differentiate [15, 16]. Thus, Rb plays a reduced essential role in mature pancreatic beta cells , whereas disruption of Rb in proliferating progenitors improves glucose tolerance through its divergent role in alpha and beta cells .
Here we showed the essential role of Rb in the effects of GLP-1 in alpha and beta cells. We also observed reduced Rb in islets of humans with diabetes. To better understand the physiological role of Rb family proteins we assessed Rb homologue p107. Unlike Rb, p107 did not have essential roles in GLP-1 action or in alpha or beta cell homeostasis. However, p107 potentiated the effects of Rb such that combined deficiency increased apoptosis in both alpha and beta cells, and decreased islet mass with ageing. Our results show that Rb is essential in mediating the divergent actions of GLP-1 on alpha and beta cells, and together with its homologue p107, is critical in governing islet cell mass.
Pancreas-specific Rb knockout mice, Pdx1-Cre:Rb fl/fl (p-RbKO) , were bred to p107 −/− whole-body knockout mice (p107KO) (E. Zacksenhaus, Toronto, ON, Canada)  to generate Pdx1-Cre:Rb fl/fl/p107 −/− mice (p-DKO) and control Pdx1-Cre:Rb +/+/p107 +/+ littermates. Mice were maintained on a mixed C57BL6;129/Sv background and housed as previously described . Exendin-4 (Sigma, St Louis, MO, USA) in PBS was administrated i.p. at 09:00 hours and 17:00 hours for 3 days at a dose of 24 nmol/kg . Glucose tolerance tests (GTT), insulin tolerance tests (ITT) and glucose-stimulated insulin secretion (GSIS) were performed as previously described . Animal protocols were approved by the Toronto General Research Institute Animal Care Committee.
Immunohistochemistry, immunofluorescence and immunoblotting
Ki67, insulin and glucagon immunostaining, TUNEL assay and immunoblotting were performed as described previously . Antibodies for Rb, p107, E2F1, p53, p21, p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclin D1, cyclin E, cleaved caspase 3, glyceraldehyde-3-phosphate dehydrogenase, p-Akt, and proliferating cell nuclear antigen (PCNA; Cell Signaling, Beverly, MA, USA) were used .
Cell culture, siRNA transfection, exendin-4 treatment and adenovirus infection
Insulinoma cells (INS-1) and simian virus 40 T-antigen induced glucagonoma cells (alpha-TC) were cultured as previously described . Cells were serum-starved for 2 h and then treated with 10 nmol/l exendin-4. Cells were transfected with Rb, p107 or Silencer Select negative control small interfering RNA (siRNA) (Ambion, Carlsbad, CA, USA) . Cells were infected with recombinant adenovirus-CMV-Rb (Vector Biolabs, Philadelphia, PA) at a multiplicity of infection of 100 for 24 h. Flow cytometry was performed as described previously .
Human islets were provided by the ABCC Human islet distribution programme (University of Alberta, Edmonton, AB, Canada) [23, 24]. Islet donation was approved by the local institutional ethical review board. Quantitative PCR was performed as previously described .
Data are presented as mean ± SEM and were analysed by two-tailed independent-sample Student’s t test or one-way ANOVA, as appropriate. A p value <0.05 was considered statistically significant.
Critical role of Rb in mediating exendin-4 action in alpha and beta cells
Remarkably, the levels of E2F1 effectors in these two cell types were significantly different. In alpha-TC cells, cell cycle inhibitors, p53, p27 and p21 were increased, and cell cycle promoters cyclin D1 and E decreased (Fig. 1a); whereas in INS-1 cells, cell cycle inhibitors decreased with an increase in cyclin D1 and E (Fig. 1b). Accordingly, exendin-4 decreased viability in alpha-TC but increased viability in INS-1 cells (ESM Fig. 1e). To further understand the mechanism of these divergent effects of Rb, we next examined the expression of Arf, the E2F1 target and upstream regulator of p53. Interestingly, Arf was induced by exendin-4 in both cell types after 6 h but this induction was transient in beta cells. In alpha cells, Arf remained elevated at 24 h, potentially contributing to opposing p53 levels in the two cell types (Fig. 1c).
In order to assess whether or not Rb suppression was the central mechanism through which exendin-4 exerted its effects, we overexpressed Rb in both cell lines. Indeed, the effects on E2F1, cyclin D1 and E observed in both cells lines in response to exendin-4 were abolished with sustained Rb expression. In INS-1 cells with Rb overexpression, decreases in p53 and p21 were abrogated, and in alpha-TC cells with Rb overexpression, induction of cell cycle inhibitors by exendin was significantly attenuated. Attenuation of Arf induction in response to exendin-4 was also seen after Rb overexpression, supporting the important role of ADP-ribosylation factor (ARF) in mediating effects of Rb regulation. Overall, these data indicate the essential role of Rb suppression in the opposing biological effects of exendin-4 in these cell types (Fig. 1d, e).
To determine whether or not Rb is essential for GLP-1 effects in vivo, we next administered exendin-4 to mice with Rb deficiency specifically in the pancreas. We recently showed that Pdx1-Cre + Rb fl/fl mice (henceforth referred to as p-RbKO) with Rb deletion in pancreatic progenitors exhibit increased beta cell proliferation under basal conditions (Fig. 1f) . As expected, exendin-4 treatment in control mice led to beta cell proliferation (Fig. 1f). By contrast, further induction of proliferation was not seen in p-RbKO mice (Fig. 1f), supporting the importance of Rb in GLP-1 action in vivo.
We next assessed the role of Rb homologue, p107, on exendin-4 response using whole-body p107KO mice (ESM Fig. 2a) given exendin-4. In contrast to p-RbKO, p107KO mice showed an increase in proliferation in response to exendin-4 similar to control mice (Fig. 1f). Furthermore, islets of mice with double deletion of Rb and p107 (p-DKO) showed similar lack of additional proliferative response to exendin-4, indicating that Rb but not p107 suppression was required for exendin-4 effects on islet cell proliferation (Fig. 1f).
Reduced Rb but not p107 expression in islets of humans with diabetes
Individuals with diabetes undergo adaptive islet cell proliferation to overcome insulin resistance . Accordingly, a recent report showed upregulation of cell cycle genes CCND1 (encoding cyclin D1) and CDK4 (encoding cyclin-dependent kinase 4) in islets of humans with type 2 diabetes . We, therefore, investigated whether Rb and p107 levels were altered with diabetes and found reduced RB expression in islets from individuals with diabetes compared with controls (Fig. 1g). Reduction of p107 was not statistically significant (Fig. 1g). Thus, Rb but not p107 is likely to be a critical negative regulator of adaptive beta cell proliferation.
Unique role of p107 in potentiating apoptosis in Rb-deficient islets
Disruption of Rb and p107 in alpha and beta cells leads to deregulation of E2F
The increased p53 levels seen in islets from the p-DKO mice but not from single knockout mice were associated with induction of its pro-death targets, Puma (also known as Bbc3) and Noxa (also known as Pmaip1) (Fig. 5c). Moreover, in contrast to the single knockout mice, p-DKO mouse islets had decreased expression of the anti-apoptotic Bcl-X L (also known as Bcl2l1) gene as well as a decrease in P16ink4a (also known as Cdkn2a), involved in beta cell regeneration . These findings suggest that combined Rb and p107 deficiency further alters cell survival and proliferation. Overall, both alpha and beta cell apoptosis were increased (Figs 2c and 3e), in addition to an increase in proliferation in p-DKO islets, resulting in similar islet mass as controls. By contrast, p-RbKO islets with lower levels of E2Fs compared with p-DKO islets showed reduced Puma and Noxa mRNA levels, and an increase in Bcl-X L mRNA, which encodes an anti-apoptotic protein (Fig. 5c).
In INS-1 cells, combined knockdown of Rb and p107 induced p53 to a much greater extent than with single-gene knockdown (Fig. 6c), substantially increasing apoptotic SubG1 fraction (Fig. 6d), while knockdown of Rb alone led to p53 suppression without significant changes in apoptosis (Fig. 6d). Interestingly, Arf expression increased in both alpha and beta cells after combined Rb and p107 knockdown (Fig. 6e). Consistent with this, we observed increased Arf expression and apoptosis in islets of p-DKO mice (Fig. 6f). We also observed increases in Kir6.2 (also known as Kcnj11) expression in p-Rb and p-DKO mice, which is well known for regulation in insulin secretion and has also been implicated in beta cell proliferation, consistent with its regulation by E2F1 (ESM Fig. 3c) [32, 33]. These results show intricate regulation of p53 by Rb family proteins and the fine-tuning of intracellular signalling that is potentiated by Rb and p107.
Rb is thought to be dispensable in mature beta cells, possibly due to compensation by p130 [17, 35]. However, deletion of Rb in proliferating pancreatic and duodenal homeobox 1 (PDX1)-positive pancreatic progenitors improved glucose tolerance by persistently increasing neurogenin 3-positive cells that were associated with enhanced beta cell proliferation and differentiation, but disrupted alpha cell differentiation and survival . We show that combined deletion of Rb and p107 in proliferating islets does not exert additional proliferative effects compared with loss of Rb alone in beta cells; however, ageing increases beta cell apoptosis, leading to loss of beta cell mass and abolition of enhanced glucose homeostasis. These results demonstrate a unique role of p107 deletion in potentiating islet cell apoptosis in Rb-deficient islets through induction of E2F members and p53, thereby resulting in a decline of both alpha and beta cell mass.
Rb and p107 may compensate for each other through two major mechanisms. First, in the absence of Rb, p107 may interact with activating E2Fs (E2F1–3) and suppress genes normally regulated by Rb. Thus, combined inactivation of both pocket proteins is required to deregulate E2F1–3-responsive genes and promote cell cycle progression. Second, the spacer domain of p107 (but not Rb) acts as a CDK2 inhibitor . Indeed, deletion of p107 has a similar effect as deletion of the CDK inhibitor p27 on retinoblastoma formation in murine models . Deletion of Rb and p107 in some contexts leads to enhanced proliferation and cancer such as retinoblastoma in mice, but may also lead to more severe differentiation defects in muscle  or, as shown here, to increased apoptosis in pancreatic islet cells.
The distinct biological consequences of the loss of specific Rb family members can be due to differential regulation of E2F family proteins [10, 39]. For example, in alpha cells, Rb knockdown led to an approximately fivefold increase in E2F1 levels, which was associated with p53 induction and an increase in apoptosis. By contrast, a less dramatic twofold induction of E2F1 after a similarly efficient knockdown of Rb in beta cells resulted in a decrease in p53 with an increase in cell proliferation . Similarly, a mild elevation of E2F1 levels in fibroblasts resulted in increased cell proliferation, but an excessive increase in E2F1 led to apoptosis through activation of p53-dependent caspase signalling . These findings suggest that E2F1 may have a dose-dependent effect on cell fate. Accordingly, we show that combined deletion of Rb and p107 in beta cells led to a greater increase in E2F1 expression compared with loss of Rb alone, which not only increased proliferation but also apoptosis. Indeed, E2F1 is considered unique from other E2F members because of its effect in promoting pro-apoptotic activity [29, 30]. Persistently increased apoptosis with a concomitant reduction in proliferation resulted in net loss of beta cell mass in p-DKO mice with ageing, leading to the loss of improved glucose homeostasis observed in young p-DKO mice. Moreover, E2F1 has previously been shown to be involved in early pancreas development , insulin secretion [33, 41] and proliferation [41, 42, 43, 44]. These data show the importance of the precise regulation of E2F in determining the various biological outcomes of islets.
The differential induction of E2F1 can in turn lead to specific regulation of Arf, which could contribute to divergent cell fate. Arf was transiently induced in exendin-4-treated beta cells, returning to baseline levels after 24 h, whereas high Arf levels persisted in alpha cells following treatment, and these inductions were attenuated with Rb overexpression. These results show a distinct dynamic response of Arf in alpha and beta cells, which may contribute to dose-dependent effects of E2F1 on islet cell fate. While E2F1 is perhaps best recognised as a major target of Rb regulation in cell cycle, further work is required to determine the potential roles of other E2Fs, which have increasingly been shown to play a role in cell survival and cycle regulation , including E2F3a and E2F3b which have been implicated in cell survival and Arf regulation [46, 47].
In addition, we found that combined loss of Rb and p107 in alpha cells led to an even greater increase in apoptosis, as evidenced by an approximately twofold increase in alpha cell apoptosis in p-DKO vs p-RbKO mice. We showed previously that E2F1 can bind directly to the promoter regions of Arx, a key alpha cell transcription factor, and likely represses gene transcription. As such, Rb-deficient mice exhibit low alpha cell mass . The more significant loss in alpha cell mass in p-DKO mice suggests that the combined loss of Rb family proteins potentiates Arx repression and alpha cell death. The profound reduction in alpha cell mass in p-DKO mice likely contributed to improved glucose homeostasis in young mice with normal beta cell mass. Moreover, the reduced alpha cell mass is also likely to have contributed to normal glucose tolerance in aged p-DKO mice with decreased beta cell mass.
As the gatekeeper of cell cycle entry, Rb proteins play a critical role in determining the transition between proliferation, differentiation and apoptosis [16, 48]. A recent report has demonstrated that another typically non-proliferative and post-mitotic cell type, muscle cells, can become regenerative by inactivating Rb and ARF . Moreover, expansion of stem cell populations have also been shown to be restricted by the Rb pathway . Combined with our results in human and mouse islets, these lines of evidence further support the notion that differentiated mammalian cells still retain the capacity to re-enter cell cycle and this regenerative ability may be largely controlled by the activities of Rb proteins.
The results of the present study demonstrate that Rb family proteins have unique and opposing effects on alpha and beta cells that provide a unifying mechanism for the divergent role of GLP-1 in these islet cell types. In contrast to the opposing effects of Rb loss alone, the Rb homologue p107, while not essential by itself, augments apoptosis in both alpha and beta cells in the setting of combined Rb and p107 loss. Given that a decline in beta cells and concomitant increase in alpha cells are core pathologies in diabetes, our findings on the dichotomous effects of Rb in alpha and beta cells provide valuable insight towards novel therapeutic strategies.
We thank T. Jin (Department of Physiology, University of Toronto, Toronto, ON, Canada) for providing INS-1 and alpha-TC cell lines. We thank M. Wheeler (Department of Physiology, University of Toronto, Toronto, ON, Canada) for providing human islet cDNA and K. J. Prentice for human islet cDNA preparation.
This work was supported by Canadian Institute of Health Research (CIHR) operating grants MOP-81148 and CCI-125690 to MW. EPC is supported by the Canadian Diabetes Association (CDA) Doctoral Student Research Award. CTL is supported by the Eliot Phillipson Clinician Scientist Training Program and postdoctoral fellowships from the Banting and Best Diabetes Centre (BBDC) and the CDA. SYS is supported by the Canadian Liver Foundation Graduate Studentship, CDA Doctoral Student Research Award and CIHR–Frederick Banting and Charles Best Canada Graduate Scholarship. TS is supported by a BBDC Novo Nordisk Studentship, CDA Doctoral Student Research Award and CIHR – Frederick Banting and Charles Best Canada Graduate Scholarship. JJB was supported by a BBDC Novo Nordisk Studentship. MW is supported by the Canada Research Chair in Signal Transduction in Diabetes Pathogenesis.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
All authors contributed to the study conception and data, and approved the final version of the manuscript. EPC and CTL contributed to the generation and analyses of research data, and preparation of the manuscript. XW, SAS, SYS, TS and JJB contributed to the generation of research data and reviewed the manuscript. EZ and MW designed experiments, supervised students, contributed to discussion and interpretation of the data, and reviewed and edited the manuscript. MW is the guarantor of this work.