Compound 19e, a Novel Glucokinase Activator, Protects against Cytokine-Induced Beta-Cell Apoptosis in INS-1 Cells

Abstract

Previously, compound 19e, a novel heteroaryl-containing benzamide derivative, was identified as a potent glucokinase activator (GKA) and showed a glucose-lowering effect in diabetic mice. In this study, the anti-apoptotic actions of 19e were evaluated in INS-1 pancreatic beta-cells co-treated with TNF-α and IL-1β to induce cell death. Compound 19e protected INS-1 cells from cytokine-induced cell death, and the effect was similar to treatment with another GKA or exendin-4. Compound 19e reduced annexin-V stained cells and the expression of cleaved caspase-3 and poly (ADP-ribose) polymerase protein, as well as upregulated the expression of B-cell lymphoma-2 protein. Compound 19e inhibited apoptotic signaling via induction of the ATP content, and the effect was correlated with the downregulation of nuclear factor-κB p65 and inducible nitric oxide synthase. Further, 19e increased NAD-dependent protein deacetylase sirtuin-1 (SIRT1) deacetylase activity, and the anti-apoptotic effect of 19e was attenuated by SIRT1 inhibitor or SIRT1 siRNA treatment. Our results demonstrate that the novel GKA, 19e, prevents cytokine-induced beta-cell apoptosis via SIRT1 activation and has potential as a therapeutic drug for the preservation of pancreatic beta-cells.

Keywords: NAD-dependent protein deacetylase sirtuin -1; apoptosis; beta-cell; compound 19e; cytokine; glucokinase activator.

FIGURE 1

The proposed molecular mechanisms of the compound 19e-mediated anti-apoptotic effect in INS-1 cells treated with cytokines.

FIGURE 2

Effect of GKA compounds on cell viability. (A) Structure of compound 19 (C19) and compound 19e (C19E) (B) INS-1 cells were treated with various concentrations of C19 or C19E for 24 h, and viability was determined using a cell counting kit. (C) INS-1 cells were treated with 20 ng/ml of IL-1β and 20 ng/ml TNF-α (CM) in the absence or presence of various concentrations of C19 or C19E. After 24 h, cell viability was determined by using a cell counting kit. Results represent the mean ± SEM from triplicate experiments. ^∗P < 0.05 vs. 0, ^∗∗P < 0.05 vs. CON, ^†P < 0.05 vs. CM alone.

FIGURE 3

Anti-apoptotic effect of compound 19e on cytokine-induced toxicity. INS-1 cells were treated with 20 ng/ml of IL-1β and 20 ng/ml TNF-α (CM) with or without (-) 10 μM of PSN-GK-1 (GK-1), 5 nM of exendin-4 (EX-4) or 5 μM of compound 19e (C19E) for 24 h. (A) Cell viability was determined by using a cell counting kit. (B) Cells were harvested and stained with annexin-V-phycoerthyrin. Apoptotic cells were counted by flow cytometry. (C) Cell lysates were subjected to western blot analysis using specific antibodies. (D) Densities of western blot signals were measured using Image J software and relative expression was normalized to actin. The results represent the mean ± SEM from triplicate experiments. ^∗P < 0.05 vs. CON, ^†P < 0.05 vs. CM alone.

FIGURE 4

The protective effect of compound 19e against cytokine-induced mitochondrial dysfunction by suppression of NF-κB signaling. INS-1 cells were treated as described in Figure 2. (A) Cell lysates were fractionated and the nuclear fraction was subjected to western blot analysis using specific antibodies. (B) Densities of western blot signals were measured using Image J software and relative expression was normalized to lamin B. (C) iNOS mRNA levels were analyzed by qRT-PCR and cyclophilin was used as a loading control. N.D, non-detected. (D) Cell lysates were subjected to western blot analysis using specific antibodies. Actin was used as a loading control. (E) Intracellular concentrations of ATP were determined using an ATP-dependent luminescent cell viability assay. Mean ± SEM from triplicate experiments. ^∗P < 0.05 vs. CON, ^†P < 0.05 vs. CM alone.

FIGURE 5

Effect of compound 19e on SIRT1 deacetylase activity. INS-1 cells were treated as described in Figure 2. (A) Cell lysates were subjected to western blot using specific antibodies. (B) Densities of western blot signals were measured using Image J software and relative expression was normalized to actin. (C) Nuclear protein was extracted and SIRT1 deacetylase activity was measured using fluorogenic substrate. The results represent the mean ± SEM from triplicate experiments. ^∗P < 0.05 vs. CON, ^†P < 0.05 vs. CM alone.

FIGURE 6

Amelioration of cytokine-induced cell death by SIRT1 activity inhibitor or SIRT1 siRNA treatment. (A) INS-1 cells were pre-treated with 2.5 mM nicotinamide (NAM) for 12 h and incubated with GK-1, EX-4, or C19E in the presence of CM for 24 h. Cell viability was determined by using a cell counting kit. (B) SIRT1 siRNA (siSIRT1) or scrambled siRNA (siCON) was transfected into INS-1 cells and SIRT1 mRNA levels were analyzed by qRT-PCR. Cyclophilin was used as a loading control. (C) Cells were transfected with SIRT1 siRNA (siSIRT1) or scrambled siRNA (siCON) for 24 h and then incubated with GK-1, EX-4, or C19E in the presence of CM for 24 h. Cell viability was determined by using a cell counting kit. Results represent the mean ± SEM from triplicate experiments. ^∗P < 0.05 vs. CON, ^†P < 0.05 vs. CM alone.