An Islet-Targeted Genome-Wide Association Scan Identifies Novel Genes Implicated in Cytokine-Mediated Islet Stress in Type 2 Diabetes

Abstract

Genome-wide association studies in human type 2 diabetes (T2D) have renewed interest in the pancreatic islet as a contributor to T2D risk. Chronic low-grade inflammation resulting from obesity is a risk factor for T2D and a possible trigger of β-cell failure. In this study, microarray data were collected from mouse islets after overnight treatment with cytokines at concentrations consistent with the chronic low-grade inflammation in T2D. Genes with a cytokine-induced change of >2-fold were then examined for associations between single nucleotide polymorphisms and the acute insulin response to glucose (AIRg) using data from the Genetics Underlying Diabetes in Hispanics (GUARDIAN) Consortium. Significant evidence of association was found between AIRg and single nucleotide polymorphisms in Arap3 (5q31.3), F13a1 (6p25.3), Klhl6 (3q27.1), Nid1 (1q42.3), Pamr1 (11p13), Ripk2 (8q21.3), and Steap4 (7q21.12). To assess the potential relevance to islet function, mouse islets were exposed to conditions modeling low-grade inflammation, mitochondrial stress, endoplasmic reticulum (ER) stress, glucotoxicity, and lipotoxicity. RT-PCR revealed that one or more forms of stress significantly altered expression levels of all genes except Arap3. Thapsigargin-induced ER stress up-regulated both Pamr1 and Klhl6. Three genes confirmed microarray predictions of significant cytokine sensitivity: F13a1 was down-regulated 3.3-fold by cytokines, Ripk2 was up-regulated 1.5- to 3-fold by all stressors, and Steap4 was profoundly cytokine sensitive (167-fold up-regulation). Three genes were thus closely associated with low-grade inflammation in murine islets and also with a marker for islet function (AIRg) in a diabetes-prone human population. This islet-targeted genome-wide association scan identified several previously unrecognized candidate genes related to islet dysfunction during the development of T2D.

Figure 1.

Effects of cytokine (cyto) treatment on islet function. A and B, Islets isolated from db/db mice (A) and heterozygous (het) control mice (B) were exposed overnight to 10 pg/mL IL-1β + 20 pg/mL IL-6, 10× that dose, or left untreated. After 3 minutes in 3 mM glucose, islets were stimulated with 11 mM glucose and observed for changes in [Ca²⁺]_i using the ratiometric dye fura-2 AM. C and D, Mean values for basal (C) and phase 1 (D, peak) response to glucose stimulation. #, P < .10; *, P < .05; **, P < .01; ***, P < .001; N.S., not significant.

Figure 2.

Schematic diagram of the experimental design related to the microarray study of islets (A) and analysis of GUARDIAN data to identify genes related to β-cell function (B). LogFC, log fold change.

Figure 3.

Stressor effects on islet viability and function. A, Islets exposed to various stressors for 48 hours produced similar effects on phase 1 [Ca²⁺]_i responses to 3 to 11 mM glucose stimulation. All stressors reduced [Ca²⁺]_i responses (***, P < .001) compared with untreated control islets, but none of the stressor effects significantly differed from one another. (Adapted from Qureshi et al [30] with permission of Elsevier, © 2015). B, None of the stressors altered cell death rates compared with those of untreated control islets in duplicate trials measuring PI fluorescence assessing 18 to 37 islets/condition. Cytos, cytokines (10 pg/mL IL-1β + 20 pg/mL IL-6); 28G, 28 mM glucose; FFA, free fatty acids (50 μM palmitate + 100 μM oleate + 50 μM linoleate); Thaps, 100 nM thapsigargin; Rote, 20 nM rotenone.

Figure 4.

Gene expression levels for F13a1 in mouse islets after a 48-hour treatment with various stressors as described in detail in the Results and Discussion. This and all subsequent figures use n = 4 replicates for each condition to determine statistical significance. *, P < .05. Cytos, cytokines (10 pg/mL IL-1β + 20 pg/mL IL-6); 28G, 28 mM glucose; FFA, free fatty acids (50 μM palmitate + 100 μM oleate + 50 μM linoleate); Thaps, 100 nM thapsigargin; Rote, 20 nM rotenone.

Figure 5.

Gene expression levels for Klhl6 in mouse islets after a 48-hour treatment with various stressors as described in detail in Results and Discussion. **, P < .01. Cytos, cytokines (10 pg/mL IL-1β + 20 pg/mL IL-6); 28G, 28 mM glucose; FFA, free fatty acids (50 μM palmitate + 100 μM oleate + 50 μM linoleate); Thaps, 100 nM thapsigargin; Rote, 20 nM rotenone.

Figure 6.

Gene expression levels for Pamr1 in mouse islets after a 48-hour treatment with various stressors as described in detail in Results and Discussion. *, P < .05. Cytos, cytokines (10 pg/mL IL-1β + 20 pg/mL IL-6); 28G, 28 mM glucose; FFA, free fatty acids (50 μM palmitate + 100 μM oleate + 50 μM linoleate); Thaps, 100 nM thapsigargin; Rote, 20 nM rotenone.

Figure 7.

Gene expression levels for Ripk2 in mouse islets after a 48-hour treatment with various stressors as described in detail in Results and Discussion. *, P < .05; ***, P < .001. Cytos, cytokines (10 pg/mL IL-1β + 20 pg/mL IL-6); 28G, 28 mM glucose; FFA, free fatty acids (50 μM palmitate + 100 μM oleate + 50 μM linoleate); Thaps, 100 nM thapsigargin; Rote, 20 nM rotenone.

Figure 8.

Gene expression levels for Steap4 in mouse islets after a 48-hour treatment with various stressors as described in detail in Results and Discussion. *, P < .05; **P < .01. Cytos, cytokines (10 pg/mL IL-1β + 20 pg/mL IL-6); 28G, 28 mM glucose; FFA, free fatty acids (50 μM palmitate + 100 μM oleate + 50 μM linoleate); Thaps, 100 nM thapsigargin; Rote, 20 nM rotenone.