Bergenin protects pancreatic beta cells against cytokine-induced apoptosis in INS-1E cells

Abstract

Beta cell apoptosis induced by proinflammatory cytokines is one of the hallmarks of diabetes. Small molecules which can inhibit the cytokine-induced apoptosis could lead to new drug candidates that can be used in combination with existing therapeutic interventions against diabetes. The current study evaluated several effects of bergenin, an isocoumarin derivative, in beta cells in the presence of cytokines. These included (i) increase in beta cell viability (by measuring cellular ATP levels) (ii) suppression of beta cell apoptosis (by measuring caspase activity), (iii) improvement in beta cell function (by measuring glucose-stimulated insulin secretion), and (iv) improvement of beta cells mitochondrial physiological functions. The experiments were carried out using rat beta INS-1E cell line in the presence or absence of bergenin and a cocktail of proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon- gamma) for 48 hr. Bergenin significantly inhibited beta cell apoptosis, as inferred from the reduction in the caspase-3 activity (IC50 = 7.29 ± 2.45 μM), and concurrently increased cellular ATP Levels (EC50 = 1.97 ± 0.47 μM). Bergenin also significantly enhanced insulin secretion (EC50 = 6.73 ± 2.15 μM) in INS-1E cells, presumably because of the decreased nitric oxide production (IC50 = 6.82 ± 2.83 μM). Bergenin restored mitochondrial membrane potential (EC50 = 2.27 ± 0.83 μM), decreased ROS production (IC50 = 14.63 ± 3.18 μM), and improved mitochondrial dehydrogenase activity (EC50 = 1.39 ± 0.62 μM). This study shows for the first time that bergenin protected beta cells from cytokine-induced apoptosis and restored insulin secretory function by virtue of its anti-inflammatory, antioxidant and anti-apoptotic properties. To sum up, the above mentioned data highlight bergenin as a promising anti-apoptotic agent in the context of diabetes.

Fig 1. Suppression of cytokine-induced beta cell damage by bergenin.

(A) The chemical structure of bergenin. (B) Cellular ATP levels of INS-1E cells treated for two days with bergenin in the absence of cytokine treatment. INS-1E cells were treated for two days with proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) in the presence or absence of bergenin (0.25–10 μM). (C) Dose-dependent effects of bergenin on cellular ATP levels after 48 hr treatment with cytokines. (D) Dose-dependent effects of bergenin on caspase-3 activity after 48 hr of treatment with cytokines. Data are represented as the mean ± standard deviation of 12 independent wells for A-B. * indicates p <0.05, ‡<0.01, and §<0.001 relative to cytokine-treated cells.

Fig 2. Inhibition of cellular nitrite production and restoration of glucose-stimulated insulin secretion.

INS-1E cells were treated for two days with proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) in the presence or absence of bergenin (0.25–10 μM). (A) Dose-dependent effects of bergenin on cellular nitrite production after 48 hr treatment with cytokines. Data are represented as the mean ± standard deviation of 12 independent wells for nitrite production. (B) The glucose-stimulated insulin secretion was measured in “low glucose” (2 mM), and “high glucose” (16 mM) conditions in the presence of bergenin. Data are represented as the mean ± standard deviation of six independent wells for insulin secretion. * indicates p <0.05, ‡<0.01, and §<0.001 relative to cytokine-treated cells.

Fig 3. Cellular effects of bergenin on mitochondrial physiological parameters of beta cells in the presence of cytokines.

Effects of bergenin on mitochondrial membrane potential (ΔΨm), intracellular ROS production, and mitochondrial dehydrogenase activity (MDA) after 48 hr treatment with IL-1β, INF-γ, and TNF-α. For dose-response studies, INS-1E cells were treated with various concentrations of bergenin (0.25–10 μM). The treated INS-1E cells were assessed for their ability to restore mitochondrial membrane potential (ΔΨm) (A); to reduce cellular ROS production (B); and to improve mitochondrial dehydrogenase activity (C). Data are represented as the mean ± standard deviation of 12 independent wells for A-F. * indicates p <0.05, ‡<0.01, and §<0.001 relative to cytokine-treated cells.

Fig 4. Bergenin suppresses cytokine-induced apoptosis in beta cells.

INS-1E cells were treated with cytokines (IL-1β, INF-γ, and TNF-α) in the presence or absence bergenin (2–10 μM) for 48 hr, and were assessed for apoptosis inhibition. (A) Represents the flow cytometric analysis of INS-1E cells treated with bergenin in the presence of cytokines cocktail. The viable cell populations are in the lower left quadrant (Annexin V^-/PI^-); the cells at the early apoptosis are in the lower right quadrant (Annexin V⁺/PI^-); and the ones at the late apoptosis are in the upper right quadrant (Annexin V⁺/PI⁺); and dead cells are in the upper left quadrant (Annexin V^-/PI⁺). (B) Represents the live and apoptotic beta cell population after treatment with cytokines and bergenin. Data are represented the mean ± standard deviation of 2 independent experiments. * indicates p <0.05, ‡<0.01 and § <0.001 relative to cytokine-treated cells.

Fig 5. Bergenin suppressed cytokine-induced beta cell apoptosis.

Exposure of beta cell to proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) lead to apoptosis. However, the addition of bergenin suppressed deleterious effects of cytokine cocktail and prevented apoptosis in INS-1E cells.

Fig 6. Potential mechanism of action of bergenin in suppressing cytokine-induced apoptosis in INS-1E cells.

Proinflammatory cytokines (IL-1β, INF-γ, and TNF-α) initiate a cascade of signaling pathways leading to beta cell apoptosis. These cytokines stimulate JAK-STAT, NFκB, and MAPK pathways, which later induce intrinsic apoptotic pathway in beta cells. IL-1β also induces nitric oxide (NO) production, which causes inhibition of electron transport chain, increase in ROS production, decrease in glucose oxidation rate resulting in reduced ATP generation, and insulin production. Bergenin protected pancreatic beta cells through multiple mechanisms simultaneously. Bergenin suppressed beta cell apoptosis by potentially influencing NF-κB, MAPK and JAK/STAT pathways (represented by blue dashed lines). This is evident from the inhibition of downstream effector molecules (caspse-3, NO, ROS, and apoptosis) targeted in cell-based assays (represented by red dashed lines). By inhibiting cytokine-induced NO production, bergenin was able to increase in cellular ATP levels, decreased ROS production, and increased insulin production.