Pharmacological polysulfide suppresses glucose-stimulated insulin secretion in an ATP-sensitive potassium channel-dependent manner

Abstract

Hydrogen sulfide (H₂S) is an endogenous gaseous transmitter synthesized in various cell types. It is well established that H₂S functions in many physiological processes, including the relaxation of vascular smooth muscle, mediation of neurotransmission, regulation of inflammation, and modulation of insulin signaling. In recent years, it has been revealed that polysulfides, substances with a varying number of sulfur atoms (H₂Sn), are generated endogenously from H₂S in the presence of oxygen. A series of studies describes that sulfane sulfur has the unique ability to bind reversibly to other sulfur atoms to form hydropersulfides and polysulfides, and that polysulfides activate ion channels and promote calcium influx. Furthermore, polysulfides regulate tumor suppressor activity, promote the activation of transcription factors targeting antioxidant genes and regulate blood pressure by vascular smooth muscle relaxation. Insulin secretion from pancreatic β cells plays a critical role in response to increased blood glucose concentration. H₂S has emerged as an important regulator of glycemic control and exhibits characteristic regulation of glucose homeostasis. However, the effects of polysulfides on glucose-stimulated insulin secretion (GSIS) are largely unknown. In this study, we demonstrated that pharmacological polysulfide salts including Na₂S₂, Na₂S₃, and Na₂S₄ considerably inhibit GSIS in mouse and rat pancreatic β-cell-derived MIN6 and INS-1 cell lines, and that the effect is dependent on the activation of ATP-sensitive potassium channels. In addition, we demonstrated that a mixture of Na₂S and diethylamine NONOate inhibits GSIS in a similar way to the pharmacological administration of polysulfide salts.

Figure 1

Dose- and time-dependent effects of polysulfide salt, sodium tetrasulfide (Na₂S₄) on glucose-stimulated insulin secretion in MIN6 cells. (a,b) Mouse MIN6 cells were exposed to Na₂S₄ (0, 1.5, 3.1, 6.2, 12.5, 25 and 50 µM) for 4 h under 2 mM glucose conditions, and insulin secretion was determined in the presence of 2 mM (a) or 20 mM glucose (b) (n = 5). (c) MIN6 cells were treated with 20 mM glucose (glu), sucrose (suc) or maltose (mal) for 1 h (n = 8). (d) MIN6 cells were exposed to Na₂S₄ (0, 1.5, 3.1, 6.2, 25, and 50 µM) for 1 h or 4 h and then insulin secretion was determined under 20 mM glucose conditions. (n = 5) (e) MIN6 cells were exposed to Na₂S₄ for 4 h and then to 20 mM glucose or incubated for 6 h without Na₂S₄ and then insulin secretion was determined in 20 mM glucose conditions (n = 3). Data are presented as mean ± SD. Differences between treatments were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ^#P < 0.05, as compared with the control: (Na₂S₄ 0 µM: a and b). *P < 0.05 for comparison of the indicated groups (c, d, and e).

Figure 2

Effects of hydrogen sulfide donor and polysulfide salts on glucose-stimulated insulin secretion in MIN6 cells and its dependency of S-S bonds. (a) Mouse MIN6 cells were exposed to Na₂S, Na₂S₂, or Na₂S₃ (0, 6.2,12.5, 25 and 50 µM) for 1 h in the presence of 2 mM glucose and then insulin secretion was determined in 20 mM glucose. (n = 3) (b) MIN6 cells were exposed to Na₂S or DEA/NO in 2 mM or 20 mM glucose. (n = 3) (c) MIN6 cells were exposed to Na₂S or Na₂S₄ in 20 mM glucose. (n = 3) (d,e) MIN6 cells were exposed to 50 µM Na₂S or 50 µM Na₂S₄ with 250 µM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (d) or Na₂S or Na₂S₄ treated by immobilized-TCEP (iTCEP). (n = 3) (e) for 1 hour and then insulin secretion was determined under 20 mM glucose conditions. (n = 3) Data are presented as mean ± SD (n = 3). Differences between treatments were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ^#P < 0.05, as compared with the control (0 µM Na₂S₄: 20 mM or 10 mM glucose)

Figure 3

Dose- and time-dependent effects of polysulfide salts on glucose-stimulated insulin secretion in INS-1 cells and mouse pancreatic β-cells/islets. (a,b) Rat insulinoma INS-1 cells were exposed to Na₂S₄ (0, 0.05, 0.5, 5, or 50 µM) for 1 h in the presence of 2 mM glucose and then insulin secretion was determined under 2 mM (a) or 10 mM (b) glucose conditions. (n = 3) (c) INS-1 cells were exposed to 50 µM Na₂S, Na₂S₂, Na₂S₃, or Na₂S₄ for 1 h in 2 mM glucose and then insulin secretion was determined under 20 mM glucose conditions (n = 3). (d) Mouse pancreatic β-cells/islets were exposed to 50 µM Na₂S₄ for 1 h in 2 mM or 20 mM glucose (n = 3). In the case of β-cells/islets insulin concentrations were compensated with total protein weight. Data are presented as mean ± SD (n = 3). Differences between treatments were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ^#P < 0.05, as compared with the control (Na₂S₃ or Na₂S₄ 0 µM).

Figure 4

Impact of Na₂S₄ on death of mouse MIN6 cells. (a,b) MIN6 cells were exposed to Na₂S₄ at doses from 0 µM to 100 µM and to lidocaine as positive control at 10 mM and cultured for periods ranging from 0 h to 4 h prior to cell viability evaluation. Cell death was also assayed by flow cytometry (a) and trypan blue exclusion method (b). Differences between treatments were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ^#P < 0.05, as compared to the control cell population (Na₂S_{4 ;}0 µM, lidocaine; 0 mM treatment).

Figure 5

Effects of Na₂S₄ on cellular energy metabolism. (a) Mouse MIN6 cells were cultured from 1 h to 4 h with 25 µM Na₂S₄ prior to determination of the cellular ATP level (n = 3) under 2 mM or 20 mM glucose conditions. (b,c) Mouse MIN6 cells were exposed to Na₂S₄ (0, 1.5, 3.1, 6.2, 12.5, 25 and 50 µM) for a period of 4 h, followed by oxygen consumption rate (OCR) assay (b) and extracellular acidification rate (ECAR) assay (c) (n = 9). Differences between treatments were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ^#P < 0.05, as compared to the control cell population (Na₂S₄ 0 µM, glucose 20 mM). *P < 0.05 for comparison of the indicated groups.

Figure 6

Effects of polysulfide salts on insulin secretion induced by glibenclamide. (a,b) Mouse MIN6 cells were exposed to Na₂S₄ (a) or Na₂S₃ (b) (0, 6.2, 12.5, 25, or 50 µM) for 4 h without or with glibenclamide (10 µM) under 2 mM or 20 mM glucose conditions (n = 3). (c) MIN6 cells were exposed to 60 mM K⁺ with Na₂S₄ (n = 3). Insulin secretion was determined as described in Materials and Methods. Data are presented as the mean ± SD (n = 4). Differences between treatments were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ^#P < 0.05, as compared with the control (glucose = 2 mM, without glibenclamide or without diazoxide treatment or without high K⁺ treatment).

Figure 7

Effects of Na₂S₄ on the membrane potential of mouse MIN6 cells. (a) Mouse MIN6 cells were exposed to 50 µM Na₂S with 20 mM glucose and harvested. Then the mRNA levels of Kir6.2, and SUR1 were assayed by qRT-PCR. Data are presented as mean ± SD (n = 3). ^#P < 0.05, as compared with the control (Na₂S₄ 0 µM). (b) Na₂S₄ (50 μM) induced hyperpolarization of membrane potential with gramicidin-perforated patch. Glibenclamide (Glb; 100 μM) induced depolarization. These traces are representative of six glucose-responsive cells. (c) Representative current–voltage relationships for the whole-cell currents with gramicidin-perforated patch. The current was elicited by a voltage ramp from −123 to −43 mV with a rate of 0.2 V/s. Na₂S₄ (50 μM) increased the current in the KRBH buffer contained 10 mM glucose (C; control). Thereafter, the current induced by Na₂S₄ decreased by glibenclamide (Glb, 10 μM). (d) Summary of the effects of Na₂S₄ and glibenclamide on the conductance (n = 9). *P < 0.05 for comparison of the indicated groups.

Figure 8

Effect of Na₂S₄ on voltage-dependent outward potassium currents in mouse MIN6 cells. (a) Current–voltage relationships of whole-cell currents with varying concentrations of Na₂S₄ (in 0, 5, 25, 50, or 500 μM). Na₂S₄ increased inwardly rectifying potassium currents in a dose-dependent manner (n = 5). (b) Semi-logarithmic plot of the conductance between −108 and −68 mV by concentration of Na₂S₄ with the standard K⁺ pipette solution included ATP at 0.01 mM (closed circles) and 1 mM (open circles).