Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta -cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation

Abstract

Context: Elevated glucose levels impair islet function and survival, and it has been proposed that intraislet expression of IL-1beta contributes to glucotoxicity.

Objective: The objective was to investigate IL-1beta mRNA expression in near-pure beta-cells of patients with type 2 diabetes (T2DM) and study the regulation of IL-1beta by glucose in isolated human islets.

Methods: Laser capture microdissection was performed to isolate beta-cells from pancreas sections of 10 type 2 diabetic donors and nine controls, and IL-1beta mRNA expression was analyzed using gene arrays and PCR. Cultured human islets and fluorescence-activated cell sorter-purified human beta-cells were used to study the regulation of IL-1beta expression by glucose and IL-1beta.

Results: Gene array analysis of RNA from beta-cells of individuals with T2DM revealed increased expression of IL-1beta mRNA. Real-time PCR confirmed increased IL-1beta expression in six of 10 T2DM samples, with minimal or no expression in nine control samples. In cultured human islets, IL-1beta mRNA and protein expression was induced by high glucose and IL-1beta autostimulation and decreased by the IL-1 receptor antagonist IL-1Ra. The glucose response was negatively correlated with basal IL-1beta expression levels. Autostimulation was transient and nuclear factor-kappaB dependent. Glucose-induced IL-1beta was biologically active and stimulated IL-8 release. Low picogram per milliliter concentrations of IL-1beta up-regulated inflammatory factors IL-8 and IL-6.

Conclusion: Evidence that IL-1beta mRNA expression is up-regulated in beta-cells of patients with T2DM is presented, and glucose-promoted IL-1beta autostimulation may be a possible contributor.

Figure 1

Responsiveness of human islets to glucose-induced IL-1β expression and IL-1Ra inhibition depends on baseline IL-1β expression levels. Human islets were cultured for 4 d at 5.5 (basal) or 33.3 mm glucose. IL-1β and IL-1Ra mRNA levels were quantified using 18s RNA as an internal standard. The mean of triplicate determinations for each preparation is shown. A, Basal IL-1β mRNA levels expressed relative to a human islet preparation arbitrarily designated as H0 and defined as 1. B Effect of 33.3 vs. 5.5 mm glucose on IL-1β mRNA in different human islet preparations expressed as fold of basal levels (5.5 mm glucose). C, Effect of 33.3 mm glucose on IL-1Ra mRNA expressed as fold of basal levels, D, Variation of basal expression of different gene products in 11–12 human islet preparations. For each separate gene transcript, we defined the sample with the lowest expression as 1. E, Linear regression analysis of basal IL-1β mRNA vs. fold stimulation of IL-1β mRNA by glucose. F, Fold glucose-stimulated IL1β mRNA vs. fold glucose-inhibited IL-1Ra mRNA.

Figure 2

IL-1β mRNA is induced by IL-1β autostimulation and glucose. A, Effect of 33.3 mm glucose (n = 12 separate donors), 0.2 ng/ml IL-1β (n = 11 separate donors), and a combination of 33.3 mm glucose and 0.2 ng/ml IL-1β (n = 8 separate donors) on IL-1β mRNA levels in whole-islet cultures relative to basal levels (5.5 mm glucose). Means ± se, significance vs. 5.5 mm glucose was determined with the Wilcoxon rank test. *, P < 0.05; **, P < 0.01. B, Effect of 0.2 ng/ml IL-1β or 33.3 mm glucose on IL-1β mRNA levels in purified β-cells (mean ± se, n = 3) relative to 5.5 mm glucose (Mann Whitney test). C, Effect of 33.3 mm glucose (n = 12), 0.2 ng/ml IL-1β (n = 10), or 1 μg/ml IL-1Ra (n = 6) on IL-1Ra mRNA levels relative to untreated islet control cultures. Means ± se, significance vs. untreated controls was determined with the Wilcoxon rank test, **, P < 0.01.

Figure 3

Glucose-induced IL-1β and IL-8 expression is antagonized with IL-1Ra. A, IL-1β mRNA expression in human islet cell cultures maintained at 5.5 mm glucose (control) or 33.3 mm glucose or treated with 0.2 ng/ml IL-1β in the presence (black bars) or absence (open bars) of 1 μg/ml IL-1Ra. Data are expressed relative to the untreated controls (means ± se, n = 7). Significance of the effect of IL-1Ra and glucose was determined with ANOVA and Bonferroni’s post hoc test. *, Control vs. IL-1Ra, P < 0.05 ; **, 33.3 mm glucose vs. IL-1Ra, P < 0.01 ; γ, control vs. 33.3 mm glucose or IL-1β P < 0.05. B, IL-8 mRNA and statistics determined as described for A (means ± se, n = 6). *, 33.3 mm glucose vs. IL-1Ra, P < 0.05; **, IL-1β vs. IL-1Ra, P < 0.01; γ, control vs. 33.3 mm glucose, P < 0.05; γγ, control vs. IL-1β, P < 0.01. C. Human islet cell cultures were treated as described under A, and islet cell extracts were subjected to Western blotting with an anti-IL-1β and an antiactin antibody. D, Secreted IL-8 in culture supernatants of cultures treated for 4 d with 33.3 mm glucose or 5.5 mm glucose in the presence (black bars) or absence (open bars) of 1 μg/ml IL-1Ra. Results from each islet preparation were expressed as percent of the untreated control (mean IL-8 concentration ± sd: 12.5 ± 4.4 ng/ml, n = 5). *, 33.3 mm glucose vs. IL-1Ra, P < 0.05 ; γ, control vs. 33.3 mm glucose, P < 0.05.

Figure 4

Glucose and IL-1β dose dependence of IL-1β expression and kinetics and NF-κB dependence of IL-1β autostimulation. A, IL-1β mRNA levels of human islet cultures treated for 4 d in the presence of increasing concentrations of glucose. Each data point shows the mean (± se) of quadruplicate cultures from two donors analyzed in triplicate. Significance relative to the lowest glucose value was determined with Mann Whitney. *, P < 0.05; **, P < 0.01. B, Human islet cell cultures were treated for 4 d without (0) or with increasing concentrations of recombinant human IL-1β. Results were expressed as fold of the untreated control and represent the mean of three islet preparations ± se. Significance relative to the value without IL-1β treatment was determined with Mann Whitney. *, P < 0.05. C, Kinetics of IL-1β mRNA stimulation. Islet cultures were treated with (dashed lines) or without (solid lines) 0.2 ng/ml IL-1β; results were expressed as fold of the 0 time point, and the mean ± se of three different islet preparations is shown. Significance was determined for control vs. IL-1β-treated cultures for each individual time point with Mann Whitney. *, P < 0.05. D, Cultured human islets were treated for 2 h with 5 μm BAY 11–7082 or 0.01% DMSO (solvent) alone. After washing, the islet cultures were incubated for 24 h with or without 0.2 ng/ml IL-1β. Data were expressed as percent of the cultures stimulated with 0.2 ng/ml IL-1β together with 0.01% DMSO and are means ± se of four different islet preparations. Statistical significance was determined using the Mann Whitney test. **, P < 0.01 for BAY 11–7082/ IL-1β vs. IL-1β alone.

Figure 5

IL-1β induces IL-8 and IL-6 expression and release from human islets. A and B, Human islet cell cultures were treated for 4 d without (control) or with 0.2 ng/ml IL-β, or IL-1β and 33.3 mm glucose in the presence (black bars) or absence (open bars) of 1 μg/ml IL-1Ra. IL-8 (A) and IL-6 (B) concentrations in culture supernatants (200 islets per 2 ml media) were determined and expressed as percent of the untreated controls (100%). Mean ± se for four different islet preparations are shown in A and for six islet preparations in B. Significance was determined with Mann Whitney. *, P < 0.05; **, P < 0.01 of cultures with vs. without IL-1Ra. C and D, Kinetics of IL-8 (C) and IL-6 (D) mRNA induction by 0.2 ng/ml IL-1β. Results were expressed as fold of the 0 time point, the mean ± se of three different islet preparations is shown, and significance was determined with Mann Whitney. *, P < 0.05; **, P < 0.01 of control vs. IL-1β-treated cultures for each time point. E, IL-8 mRNA levels of FACS-purified β-cells treated with or without 0.2 ng/ml IL-1β (n = 3 separate β-cell purifications; *, P < 0.05, Mann Whitney). F and G, Dose responses of IL-1β-induced IL-8 and IL-6 released into culture media from human islets treated for 4 d with various concentrations of IL-1β. For IL-8 (F), the mean ± se of three islet preparations is shown. Significance vs. the data point without IL-1β was determined by ANOVA with the Dunnett’s posttest. *, P < 0.05; **, P < 0.01. The dose response of IL-1β-induced IL-6 release (G) is expressed as fold of untreated control (0.7–5.4 ng/ml) and the mean ± sd of two islet preparations is shown.