Arsenic is more potent than cadmium or manganese in disrupting the INS-1 beta cell microRNA landscape

Abstract

Diabetes is a metabolic disorder characterized by fasting hyperglycemia and impaired glucose tolerance. Laboratory and population studies have shown that inorganic arsenic (iAs) can impair these pathways. Other metals including cadmium (Cd) and manganese (Mn) have also been linked to diabetes phenotypes. MicroRNAs, short non-coding RNAs that regulate gene expression, have emerged as potential drivers of metabolic dysfunction. MicroRNAs responsive to metal exposures in vitro have also been reported in independent studies to regulate insulin secretion in vivo. We hypothesize that microRNA dysregulation may associate with and possibly contribute to insulin secretion impairment upon exposure to iAs, Cd, or Mn. We exposed insulin secreting rat insulinoma cells to non-cytotoxic concentrations of iAs (1 µM), Cd (5 µM), and Mn (25 µM) for 24 h followed by small RNA sequencing to identify dysregulated microRNAs. RNA sequencing was then performed to further investigate changes in gene expression caused by iAs exposure. While all three metals significantly inhibited glucose-stimulated insulin secretion, high-throughput sequencing revealed distinct microRNA profiles specific to each exposure. One of the most significantly upregulated microRNAs post-iAs treatment is miR-146a (~ + 2-fold), which is known to be activated by nuclear factor κB (NF-κB) signaling. Accordingly, we found by RNA-seq analysis that genes upregulated by iAs exposure are enriched in the NF-κB signaling pathway and genes down-regulated by iAs exposure are enriched in miR-146a binding sites and are involved in regulating beta cell function. Notably, iAs exposure caused a significant decrease in the expression of Camk2a, a calcium-dependent protein kinase that regulates insulin secretion, has been implicated in type 2 diabetes, and is a likely target of miR-146a. Further studies are needed to elucidate potential interactions among NF-kB, miR-146a, and Camk2a in the context of iAs exposure.

Keywords: Arsenic; Cadmium; Diabetes; Manganese; RNA; miRNA.

Figure 1.

Read length distribution from small RNA-seq in INS-1 832/13 cells after 24-hour exposure to 1 μM iAs, 5 μM Cd, or 25 μM Mn.

Figure 2.

Glucose stimulated insulin secretion by INS-1 832/13 cells (a) and cell viability (b) after 24-hour exposure to 1 μM iAs, 5 μM Cd, or 25 μM Mn. Data are represented as mean of 6–8 technical replicates + SE for 3 or more biological replicates. **p < 0.01; ***p < 0.001 treatment versus control high glucose stimulation or treatment versus control fluorescence.

Figure 3.

(a) PCA plot of the top 50 most variable miRNAs across all samples and (b) volcano plot of miRNA expression changes after exposure to iAsIII, Cd, or Mn. Vertical lines represent a fold change of +/− 1.5, and the horizontal line is an adjusted p-value of 0.05; N=3 per treatment group (c) RT-qPCR validation of sequencing results for two robustly expressed miRNAs altered by metal exposure, miR-146a and miR-708. Data are represented as mean of technical replicates (N=12 for each exposure) + SE for 3 or more biological replicates. *p <0.05; **p < 0.01; ***p < 0.001 treatment versus control relative expression.

Figure 4.

GSIS (a) and miR-146a (b) expression (as determined by RT-qPCR) after 24-hour exposure to 0.5 or 1 μM iAs (miR-146a, 0 μM iAs N=27, 0.5 μM iAs N=15, 1 μM iAs N=22). Also, GSIS (c) and miR-146a (d) expression after 24-hour exposure to 1 μM iAs or 0.5 μM MAs (miR-146a, 0 μM iAs N=12, 0.5 μM iAs N=9, 1 μM iAs N=12). Data are represented as mean of technical replicates + SE for 3 or more biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001 treatment versus control high glucose stimulation or treatment versus control relative expression.

Figure 5.

(a) A heatmap of Euclidean distance between samples based on DESeq2 normalized gene expression. (b) Volcano plot displaying genes altered by 24-hr iAs exposure. The vertical lines represent a fold change of +/− 1.5, and the horizontal line shows adjusted p=0.05. Control, N = 5; iAs, N=4.

Figure 6.

(a) Results of pathway enrichment analysis for upregulated genes (n=678) using the CORUM database. (b) Results of ChIP-X enrichment analysis for upregulated genes. (c) Results of protein-protein interaction enrichment analysis for upregulated genes. Dashed vertical line represents adjusted p=0.05.