Differential glucose-regulation of microRNAs in pancreatic islets of non-obese type 2 diabetes model Goto-Kakizaki rat - PubMed (original) (raw)

Differential glucose-regulation of microRNAs in pancreatic islets of non-obese type 2 diabetes model Goto-Kakizaki rat

Jonathan Lou S Esguerra et al. PLoS One. 2011.

Abstract

Background: The Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions.

Methodology/principal findings: Global profiling of 348 miRNAs in the islets of GK rats and Wistar controls (females, 60 days, N = 6 for both sets) using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3'UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay.

Conclusions/significance: The perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Phenotype of the GK rats at the time of islets collection.

A. Non-fasting intra-cardial blood glucose levels are elevated in the GK rats compared to non-diabetic Wistar controls (N = 10 in both groups). B. Plasma insulin levels are at comparable levels between the two groups of animals (N = 8 in both groups). C. Insulin secretion is reduced in the isolated pancreatic islets of GK rat at 8.3 mM and 16.7 mM glucose (N = 3 independent RIA in quadruplicate per assay). Data are average ± SEM; (***) P<0.001 GK vs Wistar.

Figure 2

Figure 2. Global miRNA profiles of the pancreatic islets of GK and Wistar rats and qPCR validation.

A. Hierarchical clustering of array signals from 348 rat miRNAs allowed for the separation of the individual animals into two groups, 6 GK vs 6 Wistar. Rat miR-375, miR-132 and miR-708 are indicated for reference, representing no significant change (dark tones), upregulated (yellow tones) and downregulated (blue tones) miRNAs in GK. B. Significant Analysis of Microarrays (SAM) identified 30 differentially-regulated miRNAs in the GK rat pancreatic islets, clustered into 6 downregulated and 24 upregulated miRNAs (median False Discovery Rate = 0%). C. Stem-loop qPCR validation of selected 12 miRNAs in GK and Wistar islets. Mir-375 was included as a non-regulated control. Each miRNA was normalized to the geometric mean of U6 snRNA and U87 rat expressions as implemented in GeNorm v3.5. The 2-ΔΔCt method was used for relative quantification using the Wistar expression level as calibrator. The presented data are the average of N = 3 biological replicates performed independently each in triplicate qPCR wells ± SEM; (*) P<0.05, (**) P<0.01, (***) P<0.001 GK vs Wistar.

Figure 3

Figure 3. Glucose-dependence of miRNA expression after 1 h incubation at 2.8 mM, 8.3 mM and 16.7 mM glucose.

More pronounced variation of miRNA expression trajectories (magnitude and direction of expression) were observed in the GK compared to Wistar islets across the different glucose concentrations. Each miRNA was normalized to the geometric mean of U6 snRNA and U87 rat. The 2-ΔΔCt method was used for relative quantification using Wistar expression level at 2.8G as calibrator. The presented data are the average of N = 3 biological replicates performed independently each in triplicate qPCR wells ± SEM. Intra-sample (within same animal group) significance denoted by (*) P<0.05, (**) P<0.01, (***) P<0.001 vs 2.8G of same animal group. Inter-sample (W vs GK) significance denoted by (†) P<0.05, (††) P<0.01, (†††) P<0.001, compares expression levels from different animal groups of the same incubating glucose concentration. Different y-axis scaling was used for each miRNA to allow easy comparison of expression levels across different conditions.

Figure 4

Figure 4. Glucose-dependence of miRNA levels after 24 h incubation at 2.8 mM, 8.3 mM and 16.7 mM glucose.

Three general trends of miRNA expression trajectories were observed for Wistar islets at 2.8G vs 16.7G: i) increased expression as exhibited by rno-miR-132, rno-miR-212 and rno-miR-409-3p, ii) decreased expression as in the case of rno-miR-124, rno-miR-142-3p, rno-miR-375, rno-miR-335, rno-miR-130a and rno-miR-708 and, iii) no change as seen in rno-miR-376a, rno-miR-142-5p and rno-miR-433. For GK islet expression, the miRNAs generally exhibited expression trajectories aimed at attaining Wistar islet levels. Data analysis and presentation are as described for Figure 3.

Figure 5

Figure 5. In silico strategy to analyze the miRNAs upregulated in the GK rat islets.

One of the highlights of the approach is the filtering of predicted targets with known glucose-regulated mRNA data set from a previous study, significantly reducing false positive targets. This also resulted to a more focused enrichment of genes already implicated in islet functions.

Figure 6

Figure 6. Interaction of rno-miR-335 with the predicted binding sites in the 3′UTR of rat Stxbp1.

A. The rat Stxbp1 3′UTR contains two putative miR-335 binding sites as predicted by TargetScan 5.1. The two sites are located in the proximal region and both could be included in a 200 bp fragment cloned into a dual luciferase reporter plasmid. Arrows indicate the sites in the seed sequences that were mutated into complementary bases to act as negative control in the luciferase assay B. Two inserts, one with the wildtype rat Stxbp1-3′UTR sequence (Wt) and the other with sequences mutated at both miR-335 predicted binding sites (Mut) were cloned into the pmiRGLO dual luciferase vector. The pmiRGLO dual luciferase vector alone (Empty) was included as positive controls. HeLa cells were co-transfected with the empty vector or plasmid constructs and pre-miR-335 or pre-miR-scr (control with scrambled sequence) and assayed after 48 hours. Transfection efficiency was normalized using the Renilla signal. Data represents two independent transfections ± S.E.M. with n = 3. (**) P<0.01.

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