The CDK4-pRB-E2F1 pathway controls insulin secretion

Abstract

CDK4-pRB-E2F1 cell-cycle regulators are robustly expressed in non-proliferating beta cells, suggesting that besides the control of beta-cell number the CDK4-pRB-E2F1 pathway has a role in beta-cell function. We show here that E2F1 directly regulates expression of Kir6.2, which is a key component of the K(ATP) channel involved in the regulation of glucose-induced insulin secretion. We demonstrate, through chromatin immunoprecipitation analysis from tissues, that Kir6.2 expression is regulated at the promoter level by the CDK4-pRB-E2F1 pathway. Consistently, inhibition of CDK4, or genetic inactivation of E2F1, results in decreased expression of Kir6.2, impaired insulin secretion and glucose intolerance in mice. Furthermore we show that rescue of Kir6.2 expression restores insulin secretion in E2f1(-/-) beta cells. Finally, we demonstrate that CDK4 is activated by glucose through the insulin pathway, ultimately resulting in E2F1 activation and, consequently, increased expression of Kir6.2. In summary we provide evidence that the CDK4-pRB-E2F1 regulatory pathway is involved in glucose homeostasis, defining a new link between cell proliferation and metabolism.

Figure 1. Decreased secretagogue-stimulated insulin secretion in E2f1 −/− mice

a, Immunofluorescence analysis of serial pancreatic sections showing co-expression of E2F1, CDK4 and pRB (green) in β-cells, as demonstrated by insulin staining (red). Nuclei are stained with Hoechst reagent. Scale bars represent 100 μm. b, The Proliferating Cell Nuclear Antigen (PCNA, black arrow) proliferation marker is detected in β-cells (insulin, pink) by immunohistochemistry. Cells were counterstained with methyl green. Scale bars represent 100 μm. c, d, IPGTT measuring the levels of glucose (c) and insulin (b) at the indicated times after an intra-peritoneal injection of glucose in E2f1 +/+ and −/− mice (means ± s.e.m., n=7). e, Insulin secretion of E2f1 +/+ and −/− isolated islets in the presence of 2.8 mM and 20 mM glucose. Results were normalized by total insulin content. A representative result of 5 independent experiments is shown (means ± s.e.m.). f, Serum glucose levels were determined before and 60 minutes after intra-peritoneal injection of glibenclamide (means ± s.e.m., n=5).

Figure 2. Kir6.2, a component of the K_ATP channels regulating insulin secretion, is a direct E2F1 target gene

a, Gene expression analysis in E2f1 +/+ and −/− isolated islets. Quantification of the expression by Q-PCR of E2f1 and relevant genes involved in insulin secretion. Kir6.2, inwardly rectifying potassium channel; Sur1, sulfonyl urea receptor 1; Pcx, pyruvate carboxylase; Ucp2, uncoupling protein-2 (means ± s.e.m., n=5). b, Western blot showing KIR6.2 expression in E2f1 +/+ and −/− pancreas. c, Quantification of E2f1 and Kir6.2 mRNA expression in E2f1 +/+ isolated islets transfected with siRNA-control or siRNA-E2F1. The experiment was performed in triplicate (means ± s.e.m., n=3). d, Western blot showing E2F1 expression in isolated islets transiently transfected with a siRNA control or a siRNA targeting E2f1 mRNA. Actin was used as a loading control. e, Insulin secretion of E2f1 +/+ and −/− isolated islets transfected with pCDNA3 or pCDNA3-mKir6.2 in the presence of 2.8 mM and 20 mM glucose. Results were normalized by total insulin and DNA content (means ± s.e.m., n=3). f, The E2F1/DP-1 heterodimer modulates Kir6.2 promoter activity. COS7 cells were transiently co-transfected with the pGL3 (empty vector, negative control), the Kir6.2 promoter (Kir6.2-luc) and the mutated E2F1 responsive element Kir6.2 promoter (mutKir6.2-luc) luciferase constructs in the presence or absence of E2F1/DP-1. Results were normalized to β-galactosidase activity and are expressed as relative luciferase units (R.L.U.). g, ChIP demonstrating specific binding of E2F1 to the Kir6.2 promoter. Cross-linked chromatin from E2f1 +/+ and −/− pancreata were incubated with two different antibodies against E2F1 or IgG. Immunoprecipitates were analyzed by PCR using specific primers for the E2F-RE present in the Kir6.2 promoter. As a control, a sample representing 10% of the total chromatin was included in the PCR (Input). h, ChIP demonstrating specific binding of E2F1 to the Kir6.2 promoter in isolated islets. Cross-linked chromatin from E2f1 +/+ and −/− isolated islets were incubated with antibodies against E2F1 or IgG. Immunoprecipitates were analyzed as in g. See Supplementary Information, Fig S6 for full scans of blots in b and d.

Figure 3. Glucose regulates CDK4 activity, pRB phosphorylation and E2F1 transcriptional activity both in vitro and in vivo

a, Kir6.2 mRNA levels following glucose treatment of isolated islets (means ± s.e.m, n=3). b, CDK4 activity in vivo. SDS-PAGE autoradiography showing phosphorylated purified pRB by immunoprecipitated CDK4 from pancreata after an intra-peritoneal injection of glucose (lane 2) or a saline solution (lane 1). As a positive control for pRb phosphorylation, purified CyclinD3/CDK4 proteins were used (lane 3). c, Quantification of phosphorylated pRB in pancreatic islets after in vivo injection of glucose. Mice were treated with (n=5) or without glucose (n=5), killed one hour post-injection. Pancreata were then collected, fixed in 4% PFA and subsequently processed for paraffin embedding. IHC was performed on pancreas sections using an anti-phospho pRb antibody. Total and positively-stained cells were counted. Around 50 islets per condition were counted (means ± s.e.m., n=5). d, Glucose treatment modulates the Kir6.2 promoter activity in the presence of E2F1/DP-1 heterodimer. Min6 cells were treated as indicated, and results are presented in fold induction. e, Activity generated by the Kir6.2-Luc reporter cotransfected with the E2F1/DP-1 expression vector in the absence or presence of increasing amount of wild-type pRB and the CDK4 inhibitor IDCX (1, 5 and 10 μM). Experiments were performed in COS7 cells in the presence of glucose. f, ChIP demonstrating binding of E2F1 and pRB to the Kir6.2 promoter. As a control, a sample representing 10% of the total chromatin was included in the PCR (Input). g, Re-ChIP assays demonstrating interaction between E2F1 and pRB on the Kir6.2 promoter. Immunoprecipitates were analyzed as in f. h, Western blot analysis of pRb phosphorylation status in Min6 cells upon glucose treatment transiently transfected with wild-type pRb or a phosphorylation-defective constitutive active pRB mutant construct (pRB Δp34). i, Quantification of Kir6.2 mRNA expression in Min6 cells transfected with wild-type pRb (pRb wt) or pRB Δp34 treated or not with 20mM glucose (means ± s.e.m., n=3). j, Insulin secretion of Min6 cells transfected with pRb wt or pRBΔp34 in the presence of 2.8 mM and 20 mM glucose. Results are presented as fold induction compared to 2.8 mM glucose (means ± s.e.m, n=3). See Supplementary Information, Fig S6 for full scans of blots in b and h.

Figure 4. Insulin regulates CDK4 activity, pRB phosphorylation and E2F1 transcriptional activity in vivo through an autocrine effect

a, Kir6.2 mRNA levels in E2f1 +/+ and −/− isolated islets 1 hour after glucose, insulin, diazoxide and glucose/diazoxide treatment. The experiment was done using ~20 islets per condition in triplicate (means ± s.e.m.). b, CDK4 activity in E2f1 +/+ pancreata. SDS-PAGE autoradiography showing radiolabelled phosphorylated purified pRB (^33P pRb) by immunoprecipitated CDK4 from pancreatic tissue of mice treated 1 hour as indicated (lane 1 to 4). As a positive control for pRb phosphorylation, purified Cyclin D3/CDK6 proteins were used (lane 5). c, ChIP showing differential binding of E2F1 and pRb to the Kir6.2 promoter in the pancreas of mice treated as indicated. d, Re-ChIP assays demonstrating differential interaction between E2F1 and pRB on the Kir6.2 promoter. Chromatin was prepared from pancreatic tissues obtained after i.p. injection of a saline, a glucose, an insulin or glucose/diazoxide solution and subjected to the ChIP procedure with the antibody against E2F1 and re-immunoprecipitated using IgG or anti-pRB antibody. Immunoprecipitates were analyzed as described above. e, Representative immunoprecipitation (IP) assays showing interaction between CCND2 and CDK4. Immunoblot (IB) analysis revealed an increased interaction between CDK4 and CCND2 in Min6 cells after glucose and insulin treatment. f, Densitometry analysis of results shown in 4e, and in two independent experiments. Results are expressed as the ratio of the signal reported to the input signal (means ± s.e.m.). Images were analyzed by ImageJ software. g, Quantification of Cyclin D2 (CcnD2) and Cdk4 mRNA levels in Min6 cells treated or not with 5 nM EGF or 10 nM IGF (triplicate, means ± s.e.m.). h, Kir6.2 mRNA levels in Min6 cells treated as described (triplicate, means ± s.e.m.). See Supplementary Information, Fig S6 for full scans of blots in b and e.

Figure 5. Glucose intolerance, decreased insulin secretion and KIR6.2 levels in C57Bl/6 treated with CDK4 inhibitor

a, b, IPGTT measuring the levels of glucose (a) and insulin (b) at the indicated times after an intra-peritoneal injection of glucose in C57/Bl6J mice treated for 3 days with vehicle or IDCX solutions (means ± s.e.m., n=4). c, Kir6.2, Sur1 and Pcx mRNA levels in E2f1 +/+ islets isolated from mice treated with IDCX as described in a. The experiment was done using ~40 islets per condition in triplicate (means ± s.e.m.). d, Western blot analysis of KIR6.2 protein levels in E2f1 +/+ pancreas isolated from mice treated without (control, n=4) or with IDCX (n=4) as described in a. A representative radiography of pancreas from 2 non-treated and treated animals is shown. Levels of induction (n-fold) are indicated. Tubulin was used as a loading control. See Supplementary Information, Fig S6 for full scans of blots in d.