Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Abstract

Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-βH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.

Keywords: alternative splicing; apoptosis; autoimmunity; beta cell (B-cell); diabetes; insulin secretion.

FIGURE 1.

Pancreatic beta cells express neuron-enriched RNA-binding proteins. A, heat map representing the expression of RBPs in human islets and in 16 other human tissues. Gene expression was assessed by RNA-sequencing using a previously published dataset consisting of five different human islets preparations (24) and the Illumina BodyMap 2.0. Expression values were hierarchically clustered using Gene Pattern modules. Blue and red colors indicate low and high expressed genes, respectively. RBPs showing high expression in brain and in human islets are highlighted by a yellow square. B–E, mRNA expression of four RBPs assessed by qRT-PCR in human islets (n = 3), insulin-producing EndoC-βH1 cells (n = 3), and in a panel of normal human tissues (n = 1). B, ELAVL4; C, NOVA2; D, RBFOX1; and E, RBFOX2.

FIGURE 2.

Expression of neuron-enriched RBPs after REST overexpression in INS-1E cells. INS-1E cells were infected with adenovirus overexpressing Renilla luciferase (AdLuc) or REST (AdREST) for 24 h at m.o.i. of 10 or left untreated (NT, non-treated). Expression of the following was measured by qRT-PCR and normalized by the housekeeping gene Gapdh: A, REST; B, Snap25; C, Elavl4; D, Nova2; E, Rbfox1; and F, Rbfox2. Results are mean ± S.E. of four to six independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus AdLuc; paired t test.

FIGURE 3.

Compensatory regulation within RBPs families. INS-1E cells were transfected with siCTR or siRNAs targeting different RBPs for 48 h. The expression of the different RBPs was measured by qRT-PCR and normalized by the housekeeping gene Gapdh. Expression of the following was evaluated after Nova2 KD: A, Elavl4; B, Elavl1. Expression of Nova2 (C) and Nova1 (D) is shown. Expression of Rbfox1 (E) and Rbfox2 (F) was evaluated after Rbfox1 KD. Expression of Rbfox2 (G) and Rbfox1 (H) was evaluated after Rbfox2 KD. mRNA expression values were normalized by the highest value of each experiment, considered as 1. Results are from 3 to 5 independent experiments. *, p < 0.05; **, p < 0.01 and ***, p < 0.001 versus siCTR; paired t test.

FIGURE 4.

Elavl4 modulates pancreatic beta cells death. INS-1E cells (A–E), FACS-purified primary rat beta cells (F and G), and EndoC-βH1 cells (H and I) were transfected with siCTR or independent siRNAs targeting Elavl4 for 48 h and then exposed to the pro-inflammatory cytokines IL-1β + IFN-γ. Cytokine exposure was 24 h for INS-1E cells and 48 h for primary rat beta cells and EndoC-βH1 cells. A, two representative Western blottings showing Elavl4, cleaved caspase-9 and -3, and α-tubulin (used as loading control) after Elavl4 knockdown in INS-1E cells. B, Western blotting densitometric measurements of Elavl4. C, apoptosis in INS-1E cells was evaluated by propidium iodide staining. D, Western blotting densitometric measurements of cleaved caspase-9; E, cleaved caspase-3. F, mRNA expression of Elavl4 in FACS-purified primary rat beta cells measured by qRT-PCR and normalized by the housekeeping gene Gapdh; G, apoptosis evaluated by propidium iodide staining. H, protein expression of ELAVL4 and α-tubulin (used as loading control) in EndoC-βH1 cells measured by Western blotting. One representative Western blotting and the densitometric measurements are shown. I, apoptosis in EndoC-βH1 cells evaluated by propidium iodide staining. mRNA and protein expression values were normalized by the highest value of each experiment, considered as 1. Results are mean ± S.E. of three to five independent experiments. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 versus untreated siCTR; #, p < 0.05 and ##, p < 0.001, versus cytokine-treated siCTR; paired t test.

FIGURE 5.

Nova2 knockdown increases apoptosis in pancreatic beta cells. INS-1E cells (A–E), FACS-purified primary rat beta cells (F and G), and EndoC-βH1 cells (H and I) were transfected with siCTR or independent siRNAs targeting Nova2 for 48 h and then exposed to the pro-inflammatory cytokines IL-1β + IFN-γ. Cytokine exposure was 24 h for INS-1E cells and 48 h for primary rat beta cells and EndoC-βH1 cells. A, protein expression of Nova2 and α-tubulin (used as loading control) in INS-1E cells was measured by Western blotting. One representative blot and densitometric measurements are shown. Apoptosis in INS-1E cells was evaluated by propidium iodide staining (B) and by Western blotting for cleaved caspase-9 and cleaved caspase-3 (C). Densitometric measurements of cleaved caspase-9 (D) and 3 (E) are shown. mRNA expression of Nova2 in FACS-purified primary rat beta cells was measured by qRT-PCR and normalized by the housekeeping gene Gapdh (F), and apoptosis was evaluated by propidium iodide staining (G). Protein expression of NOVA2 and α-tubulin (used as loading control) in EndoC-βH1 was measured by Western blotting (H). One representative blot and the densitometric measurements are shown. Apoptosis in EndoC-βH1 cells was evaluated by propidium iodide staining (I). mRNA and protein expression values were normalized by the highest value of each experiment, considered as 1. Results are mean ± S.E. of three to four independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus untreated siCTR; #, p < 0.05; ##, p < 0.01; and ###, p < 0.001 versus cytokine-treated siCTR. A and E–H, paired t test. B, D, and E, paired t test with Bonferroni's correction.

FIGURE 6.

Rbfox1 knockdown increases insulin secretion. INS-1E was transfected with siCTR or three independent siRNAs against Rbfox1 (siFOX1#1, siFOX1#2, and siFOX1#3) for 48 h. A, mRNA expression of Rbfox1 measured by qRT-PCR and normalized by the housekeeping gene Gapdh. B, protein expression of Rbfox1 and α-tubulin (used as loading control) measured by Western blotting. One representative blot and the densitometric measurements are shown. C, insulin secretion following Rbfox1 KD evaluated by ELISA after 30 min of incubation with 1.7 mm glucose, 17 mm glucose, or 17 mm glucose plus forskolin (20 μm). D, scatter plots showing individual insulin secretion experiments shown in C. Individual paired experiments are indicated by the same color. E, insulin secretion after 30 min of incubation with 1.7 mm glucose or 1.7 mm glucose plus 30 mm KCl. Insulin secretion values are expressed as fold increase as compared with siCTR exposed to 1.7 mm glucose. F, insulin content fold increase as compared with siCTR. G, mRNA expression of Ins2 measured by qRT-PCR and normalized by the housekeeping gene Gapdh. H, glucose metabolism following exposure to 1.7 or 17 mm glucose after Rbfox1 KD. Values are expressed as fold increase as compared with siCTR exposed to 1.7 mm glucose. I, oxygen consumption rates (OCR) relative to basal (1.7 mm glucose) following sequential stimulation with glucose (17 mm), oligomycin (5 μm), FCCP (4 μm), and rotenone (1 μm). J and K, electrophysiological characterization of voltage-gated Ca²⁺ channels following Rbfox1 KD. J, example trace of currents evoked by a depolarization from −70 to 0 mV in a single siFOX1#2- (lower trace) or siCTR (upper trace)-transfected cells. K, charge (Q)-voltage (V) relationship in siFOX1#2- (black squares) and siCTR (white circles)-transfected cells. Charge is measured as the area enclosed by the curve in J. mRNA and protein expression values were normalized by the highest value of each experiment, considered as 1. Results are mean ± S.E. of three to eight independent experiments (A–I). K, results are mean ± S.E. of 17–20 cells. A, B, F, G, I, and K: *, p < 0.05; **, p < 0.01, and ***, p < 0.001 versus siCTR. C and D: ***, p < 0.001 versus siCTR exposed to 1.7 mm glucose; #, p < 0.05, and ###, p < 0.001 versus siCTR exposed to 17 mm glucose; &, p < 0.05; &&, p < 0.01, and &&&, p < 0.001 versus siCTR exposed to 17 mm glucose plus forskolin. F, ***, p < 0.001 versus siCTR exposed to 1.7 mm glucose; ###, p < 0.001 versus siCTR exposed to 30 mm KCl. A, B, F, G, I, and K: paired t test. C–E and H, paired t test with Bonferroni's correction.

FIGURE 7.

Rbfox2 knockdown increases insulin secretion and content. INS-1E cells were transfected with siCTR or siFOX2#1 siRNA for 48 h. A, mRNA expression of Rbfox2 measured by qRT-PCR and normalized by the housekeeping gene Gapdh. mRNA expression values were normalized by the highest value of each experiment, considered as 1. B, insulin secretion evaluated by ELISA after 30 min of incubation with 1.7 mm glucose, 17 mm glucose, or 17 mm glucose plus forskolin (20 μm) following Rbfox2 KD. Values are expressed as fold increase as compared with siCTR exposed to 1.7 mm glucose. Individual paired experiments are indicated by the same color. C, insulin content was evaluated by ELISA. A and C: *, p < 0.05, and ***, p < 0.001 versus siCTR; paired t test. B: ###, p < 0.001 versus siCTR exposed to 17 mm glucose, and &&, p < 0.01 versus siCTR exposed to 17 mm glucose plus forskolin; paired t test with Bonferroni's correction.

FIGURE 8.

Rbfox1 regulates the alternative splicing of key genes related to pancreatic beta cell function. A–C, representative images of agarose gels showing that Rbfox1 controls alternative splicing of gelsolin (Gsn) (A), insulin receptor (Insr) (B), and voltage-gated calcium channel 1C (Cacna1c) (C) in INS-1E cells. The percentage of inclusion/exclusion of each exon was quantified by densitometry and is shown under the respective gels. Results are mean ± S.E. of five to ten independent experiments. *, p < 0.05, and **, p < 0.01 versus siCTR; paired t test.

FIGURE 9.

Gelsolin silencing prevents the insulin secretion increase produced by Rbfox1 knockdown. INS-1E cells were transfected with siCTR, siFOX1#2, siGSN, or siFOX1#2 + siGSN for 48 h. mRNA expression of Rbfox1 (A) and gelsolin (B) was measured by qRT-PCR and normalized by the housekeeping gene Gapdh. mRNA expression values were normalized by the highest value of each experiment, considered as 1. C, insulin secretion was evaluated by ELISA after 30 min of incubation with 1.7 mm glucose, 17 mm glucose, or 17 mm glucose plus forskolin (20 μm). Values are expressed as fold increase as compared with siCTR exposed to 1.7 mm glucose. Results are mean ± S.E. of six independent experiments. A and B, *, p < 0.05; **, p < 0.01, and ***, p < 0.001 versus siCTR; paired t test. C, ###, p < 0.001 versus siCTR exposed to 17 mm glucose; &&, p < 0.01 versus siCTR exposed to 17 mm glucose plus forskolin; and **, p < 0.01 as indicated by bars. Paired t test with Bonferroni's correction.

FIGURE 10.

Rbfox1 knockdown accelerates actin depolymerization dynamics after glucose stimulation, a phenomenon prevented by gelsolin silencing. A, confocal microscopy images of actin cytoskeleton in INS-1E cells following incubation at 0 or 17 mm glucose during 10 min. Cells were transfected with siCTR, siFOX1#1, siFOX1#2, or siFOX1#2 + siGSN for 48 h. B and C, quantification of actin depolymerization following glucose stimulation by the F/G ratio in INS-1E cells. Atto 550-phalloidin (red) staining actin filaments and DNase I-Alexa 488 (green) staining globular actin were used to quantify the emitted fluorescence, and calculation of the ratio is shown in C. ###, p < 0.001 versus siCTR exposed to 17 mm glucose during 5 min; &&&, p < 0.001 versus siCTR exposed to 17 mm glucose during 10 min; @@@, p < 0.001 versus siCTR exposed to 17 mm glucose during 15 min; *, p < 0.05; **, p < 0.01, and ***, p < 0.001 as indicated by bars. Paired t test with Bonferroni's correction. Scale bars, 10 μm (A) and 20 μm (B).