Calcineurin/NFATc2 and PI3K/AKT signaling maintains β-cell identity and function during metabolic and inflammatory stress

Abstract

Pancreatic islets respond to metabolic and inflammatory stress by producing hormones and other factors that induce adaptive cellular and systemic responses. Here we show that intracellular Ca²⁺ ([Ca²⁺]_i) and ROS signals generated by high glucose and cytokine-induced ER stress activate calcineurin (CN)/NFATc2 and PI3K/AKT to maintain β-cell identity and function. This was attributed in part by direct induction of the endocrine differentiation gene RFX6 and suppression of several β-cell "disallowed" genes, including MCT1. CN/NFATc2 targeted p300 and HDAC1 to RFX6 and MCT1 promoters to induce and suppress gene transcription, respectively. In contrast, prolonged exposure to stress, hyperstimulated [Ca²⁺]_i, or perturbation of CN/NFATc2 resulted in downregulation of RFX6 and induction of MCT1. These findings reveal that CN/NFATc2 and PI3K/AKT maintain β-cell function during acute stress, but β-cells dedifferentiate to a dysfunctional state upon loss or exhaustion of Ca²⁺/CN/NFATc2 signaling. They further demonstrate the utility of targeting CN/NFATc2 to restore β-cell function.

Keywords: Cell biology; Diabetology; Endocrinology; Molecular biology; Molecular interaction.

Graphical abstract

Figure 1

Identification of genes and transcription factors that regulate β-cell differentiation and disallowed genes in human islets exposed to metabolic and inflammatory stress (A) Relative abundance of RNA-Seq differentially expressed mRNA transcripts determined by DESeq2 average normalized count values for β-cell differentiation and disallowed genes in human islets exposed to 16.7 mM glucose (G16.7) and IL-1β. (B and C) Enrichr enrichment analysis visualization grid, network, and bar graph and (C) table of results ranked by combined score of odds ratio and p value (p < 0.05) as determined by TRANSFAC and JASPAR software analyses of position weight matrices of transcription factors for gene targets identified by RNA-Seq (padj <0.05). The grid represents enriched terms that cluster with highest significance. Network nodes represent enriched terms and links represent gene content similarity among enriched terms. Brighter colors represent higher significance.

Figure 2

Requirements of CN and NFATc2 to regulate genes controlling β-cell identity and function (A) Scatterplot of gene sets clustered by relatedness to integrated pathways affected by FK506 inhibition of CN in human islets exposed to 16.7 mM glucose (G16.7) and IL-1β. (B) Volcano plot of p values of associated integrated pathway gene sets with corresponding odds ratio. (C) Top integrated pathways affected by FK506 in human islets determined by cumulative p value and odds ratio as shown in the table. (A-C) Darker blue shading represents decreasing p value and gray points are not significant (p > 0.05) where lighter shades represent increasing p values as determined by Enrichr software analyses of position weight matrices of transcription factors for gene targets identified by RNA-Seq. (Enrichr/NIH BioPlanet, padj <0.01). (D) Top β-cell development and functional genes affected by FK506 in human islets (Enrichr/NIH BioPlanet, padj <0.05). (E) Heatmap comparing effects of 16.7mM glucose and cytokines, CN inhibitor FK506, or NFATc2βKO on fold change expression of β-cell differentiation genes and disallowed genes in human and mouse islets. (F) Venn diagram representing overlapping CN-dependent and NFATc2-dependent effects on upregulation (red arrows) and downregulation (blue arrows) of β-cell differentiation and disallowed genes in human islets and NFATc2βKO mouse islets, respectively (padj <0.05).

Figure 3

Effect of metabolic and inflammatory stress on RFX6 and MCT1 gene transcription in MIN6 β cells (A). Flow cytometry analysis of RFX6, INS, and GCG in human islets treated with 24 h 16.7 mM glucose (G16.7) and IL-1β (B). Time-course analysis of effects of G16.7 and IL-1β on RFX6 gene expression in human islets, mouse islets, and MIN6 cell culture (C) RFX6 promoter activity in MIN6 β cells. Time-course analysis of effects of G16.7 and IL-1β on (D) RFX6 and (E) MCT1 mRNA expression by qPCR in MIN6 β cells. Effects of FK506, RAPA, and WM on (F) RFX6 and (G) MCT1 mRNA expression and (H) RFX6 and (I) MCT1 by promoter-reporter assay in G16.7 and IL-1β treated MIN6. Effects of FK506, RAPA, and WM on (J) RFX6 and (K) MCT1 mRNA expression and (L) RFX6 and (M) MCT1 by promoter-reporter assay in Thapsigargin (Tg) treated MIN6. (N) CHOP protein expression by immunoblot in MIN6 treated with Tg or IL-1β. Effects of inhibitors on association of NFATc2 with (O) RFX6 and (P) MCT1 gene promoters by ChIP assay in MIN6. Data shown are representative of results from at least three independent experiments (n = 3) using replicate assays. Asterisks denote statistical significance (^#p < 0.05, ^##p < 0.01, ^###p < 0.001 as compared to control, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as compared to no drug) in mean values based on the two-tailed Student t test.

Figure 4

Cell-signaling requirements for NFATc2 co-occupation with p300 and HDAC1 on RFX6 and MCT1 gene promoters (A–L). Effects of 16.7 mM glucose (G16.7) and IL-1β in the presence of (A-F) FK506, RAPA, and WM and (G-L) Nif, Dant, and NAC on association of NFATc2 (A, D, G, and J), p300 (B, E, H, and (K), and HDAC1 (C, F, I, and L) on RFX6 and MCT1 gene promoters by ChIP-Re-ChIP assay in MIN6. Data shown are representative of results from at least three independent experiments (n = 3) using replicate assays. Asterisks denote statistical significance (^#p < 0.05, ^##p < 0.01, ^###p < 0.001 as compared to control, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as compared to no drug) in mean values based on the two-tailed Student t test.

Figure 5

Signal-selective enrichment of NFAT-p300 and NFAT-HDAC1 on the RFX6 and MCT1 gene promoters (A–I). Effects of 16.7 mM glucose (G16.7) and IL-1β, K⁺-induced depolarization and H₂O₂ on association of NFATc2 (A and D), p300 (B and E), and HDAC1 (C and F) on RFX6 and MCT1 gene promoters by ChIP-Re-ChIP assay in MIN6. Effect of (G) FK506 and WM on G16.7 and IL-1β–induced activation of AKT by phosphorylated-AKT (pAKT) immunoblot. Effect of (H) WM and (I) NAC on H₂O₂-induced activation of AKT. Data shown are representative of results from at least three independent experiments (n = 3) using replicate assays. Blots were performed in duplicates (n = 2) or are representative of at least three independent experiments performed in MIN6 (n = 3). Asterisks denote statistical significance (^#p < 0.05, ^##p < 0.01,^###p < 0.001 as compared to control, ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001 as compared to no drug) in mean values based on the two-tailed Student t test.

Figure 6

Effect of metabolic and inflammatory stress on intracellular Ca²⁺, NFAT activation, and NFATc2-mediated RFX6 gene expression (A) Time course of acute (2 h) and long-term (24 h) effects of 16.7 mM glucose (G16.7) and IL-1β and ISX9 on basal and stimulatory intracellular Ca²⁺ (KCl stimulation indicated by arrow) in MIN6 (n = 3). (B) Area under the curve of Ca²⁺ measurements expressed as mean ± SD (n = 3). (C) Effect of G16.7 and IL-1β and ISX9 on NFAT-luciferase promoter-reporter activity in MIN6 (n = 3). (D) Effect of G16.7 and IL-1β and ISX9 on association of NFATc2 with the RFX6 gene promoter in MIN6 (n = 6). (E) Effect of G16.7 and IL-1β and ISX9 on RFX6 gene expression determined by qPCR in non-treated and FK506 treated human islets (n = 6). (F) Effect of G16.7 and IL-1β and ISX9 on RFX6 gene expression determined by qPCR in WT and NFATc2βKO mouse islets (n = 6). (G) Stimulation Indexes calculated from GSIS experiments expressed as mean ± SD (n = 3). Asterisks denote statistical significance (^#p < 0.05, ^##p < 0.01,^###p < 0.001 as compared to control, ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001 as compared to no drug) in mean values based on the two-tailed Student t test.

Figure 7

Requirement of CN/NFATc2 signaling to preserve β-cell function during metabolic and inflammatory stress (A–H) Effect of long-term (24 h) 16.7 mM glucose (G16.7) and IL-1β exposure with (A and D) NFATc2βKO mouse islets and FK506-treated MIN6 (B, C, E, and (F) on (A-C) G (glucose)-stimulated insulin secretion (GSIS) and (D-F) pyruvate-stimulated insulin secretion (PSIS). (C and F) Effect of ISX9 on GSIS and PSIS after 24 h exposure to G16.7 and IL-1β. Effect of ISX9 on marginal doses of human islets transplanted to nude mouse recipients on (G) blood glucose up to 30 days posttransplant and (H) intraperitoneal glucose tolerance test performed 30 days posttransplant in recipients of pre-graft and post-graft removal by nephrectomy (PN). Data shown are representative of results from three to five mice (n = 3-5) per treatment group experiments and assays with three replicates. Asterisks denote statistical significance (^#p < 0.05, ^##p < 0.01,^###p < 0.001 as compared to control, ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001 as compared to no drug) in mean values based on the two-tailed Student t test.