Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Abstract

Background and purpose: Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca(2+)]i) in beta cells, in the absence of any co-stimulating factor.

Experimental approach: Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca(2+)]i were measured using the ratiometric fluorescent Ca(2+) indicator Fura-2. Ca(2+) channel currents were recorded with the whole-cell patch-clamp technique.

Key results: Quercetin concentration-dependently increased insulin secretion and elevated [Ca(2+)]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L(-1)), but were nearly abolished by the L-type Ca(2+) channel antagonist nifedipine (1 μmol·L(-1)). Similar to the L-type Ca(2+) channel agonist Bay K 8644, quercetin enhanced the L-type Ca(2+) current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca(2+)]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L(-1)), with the two drugs having cumulative effects on [Ca(2+)]i.

Conclusions and implications: Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca(2+) influx through an interaction with L-type Ca(2+) channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.

Figure 1

Quercetin stimulates insulin secretion and increases the intracellular calcium ([Ca²⁺]_i) in INS-1 cells. (A) Insulin secretion was stimulated either with 20 μmol·L⁻¹ quercetin or with 15 mmol·L⁻¹ KCl in the presence of glucose, at a concentration (1.4 mmol·L⁻¹) devoid of any stimulating effect. Values represent the means ± SEM from 10 separate experiments. ***P < 0.0001; multiple comparison analysis. (B) [Ca²⁺]_i variations revealed by false-colour imaging of Fura-2 loaded INS-1 cells: (a) typical cell images illustrating the maximal fluorescence ratio (F₃₄₀/F₃₈₀) under basal conditions and after the addition of 20 μmol·L⁻¹ quercetin or 15 mmol·L⁻¹ KCl. (b) Concentration–response curve of maximal variation in the fluorescence ratio in response to quercetin. Values represent the means ± SEM from five experiments. (c) Time course of variations in the fluorescence ratio in response to quercetin or KCl. Values represent the means ± SEM from 10 experiments.

Figure 2

The quercetin-induced increase in [Ca²⁺]_i and insulin secretion in INS-1 cells require extracellular calcium but are independent of SERCA activity. (A) Typical recordings of variations in the fluorescence ratio under the different conditions. Cells were stimulated with 20 μmol·L⁻¹ quercetin and washed with KRB before further treatment. (a) Cells were then subjected to Ca²⁺ removal by substituting Ca²⁺-free KRB medium supplemented with 5 mmol·L⁻¹ EGTA, the fluorescence was allowed to stabilize before the re-addition of quercetin, and extracellular Ca²⁺ restored. (b) Thapsigargin (1 μmol·L⁻¹) was added in the absence of quercetin, the fluorescence was allowed to stabilize before the re-addition of quercetin, and quercetin was washed out once again. Arrows indicate the starting point of each drug or change of medium. (c) Bar graph representing the maximal variation in the fluorescence ratio induced by quercetin under basal conditions, in the absence of extracellular Ca²⁺ (Ca²⁺-free) or in the presence of thapsigargin. Results are presented as means ± SEM of five separate experiments. (B) Effects of 20 μmol·L⁻¹ quercetin on insulin secretion in the presence or absence of 1 μmol·L⁻¹ thapsigargin. Results are presented as means ± SEM from 4–6 separate experiments. ***P < 0.0001; **P < 0.001; ns: P > 0.05; multiple comparison analysis for the different treatment conditions.

Figure 3

The quercetin-induced increase in [Ca²⁺]_i and insulin secretion in INS-1 cells are blocked by the L-type Ca²⁺ channel antagonist nifedipine. (A) Typical recordings of variations in the fluorescence ratio. Arrows indicate the time of application of each drug. (a) Cells were incubated with 20 μmol·L⁻¹ quercetin followed by 1 μmol·L⁻¹ nifedipine. (b) Cells were incubated with 1 μmol·L⁻¹ nifedipine for 3 min before the addition of 20 μmol·L⁻¹ quercetin. (c) Bar graph representing the maximal variation in the fluorescence ratio induced by quercetin, nifedipine and quercetin in the presence of nifedipine. Results are presented as means ± SEM of five separate experiments. (B) The effects of 20 μmol·L⁻¹ quercetin on insulin secretion in the presence or absence of 1 μmol·L⁻¹ nifedipine. Values represent means ± SEM from 4–6 separate experiments. ***P < 0.0001; ns: P > 0.05; multiple comparison analysis for the different treatment conditions.

Figure 4

Effects of the L-type Ca²⁺ channel agonist Bay K 8644 on quercetin-induced increase in [Ca²⁺]_i and insulin secretion in INS-1 cells. (A) Typical recordings of variations in the fluorescence ratio. Arrows indicate the time of application of each drug. (a) Cells were stimulated with 20 μmol·L⁻¹ quercetin and, after a wash-out period with KRB, 1 μmol·L⁻¹ Bay K 8644 was added. Fluorescence was allowed to stabilize before the addition of 20 μmol·L⁻¹ quercetin, which was subsequently washed out. (b) Bar graph representing the maximal variation in the fluorescence ratio induced by quercetin, Bay K 8644 and quercetin in the presence of Bay K 8644. Results are presented as means ± SEM from five separate experiments. (B) The effects 20 μmol·L⁻¹ quercetin on insulin secretion in the presence or absence of 1 μmol·L⁻¹ Bay K 8644. Results are presented as means ± SEM from 4–6 separate experiments. ***P < 0.0001; **P < 0.001; multiple comparison analysis for the different treatment conditions.

Figure 5

Quercetin affects high- but not low-voltage-activated (LVA) Ca²⁺ channel currents carried by Ba²⁺ in INS-1 cells. (A) Typical traces of (a) a HVA Ba²⁺ current recorded at a test pulse (tp) of −10 mV and characterized by slow inactivation; (b) a predominantly LVA (T-type) current recorded at low depolarization (tp of −30 mV) and (c) a mixed LVA (with a fast-inactivating component) and HVA current (with a slow-inactivating component). The holding potential (HP) was −85 mV. (B) The effects of 20 μmol·L⁻¹ quercetin on (a) the HVA Ba²⁺ current and (b) the LVA current. (C) (a) Concentration–response curve for the effects of quercitin on the HVA Ba²⁺ current recorded at −10 mV. (b) Bar graph representing the effects of 20 μmol·L⁻¹ quercetin on the HVA Ba²⁺ current, Ca²⁺ current and the Ba²⁺ current recorded at an HP of −60 mV (step depolarization at −10 mV, HP −80 mV or −60 mV). Results are presented as means ± SEM (n = 5–11 cells). ns: P > 0.05; multiple comparison analysis for the different conditions. (D) Time course of the effect of 20 μmol·L⁻¹ quercetin on a HVA Ba²⁺ current (−10 mV, HP = −80 mV; frequency of stimulation 0.1 Hz). Data points indicate means ± SEM (n = 5 cells).

Figure 6

Effects of quercetin and Bay K 8644 on the HVA Ca²⁺ channel current carried by Ba²⁺ in INS-1 cells. (A) Effect of quercetin on the HVA current at all voltages of the current-to-voltage (I/V) relationship; (a) peak current and (b) the sustained component measured after 150 ms of depolarization, under control conditions (black line) or in the presence of 20 μmol·L⁻¹ quercetin (blue line). Data points indicate means ± SEM (n = 6 cells). (B) Effect of 1 μmol·L⁻¹ Bay K 8644 on (a) the HVA Ba²⁺ current and (b) the peak HVA current at all voltages of the current-to-voltage relationship, under control conditions (black line) or in the presence of 1 μmol·L⁻¹ Bay K 8644 (green line). Data points indicate means ± SEM (n = 3 cells). (C) (a) Effect of 20 μmol·L⁻¹ quercetin on the HVA Ba²⁺ current in the presence of 1 μmol·L⁻¹ Bay K 8644. (b) Bar graph representing the effects of quercetin on the HVA Ba²⁺ current in the presence or absence of Bay K 8644. Results are presented as means ± SEM (n = 5 cells). ns: P > 0.05; Student's t-test for paired samples.

Figure 7

Quercetin increases [Ca²⁺]_i and stimulates insulin secretion in rat isolated pancreatic beta cells and islets respectively. (A) Effect of quercetin on the fluctuations in [Ca²⁺]_i in rat isolated pancreatic beta cells: (a) typical cell images illustrating the maximal fluorescence ratio (F₃₄₀/F₃₈₀) under basal conditions and after the addition of 20 μmol·L⁻¹ quercetin. (b) Typical recordings of fluctuations in the fluorescence ratio. Arrows indicate the time of application of quercetin. Cells were stimulated with 2 μmol·L⁻¹ quercetin (Q2) and fluorescence was allowed to stabilize before the addition of 20 μmol·L⁻¹ quercetin (Q20), which was subsequently washed out. (c) Bar graph representing the maximal variation in the fluorescence ratio induced by increasing concentrations of quercetin (2, 10 and 20 μmol·L⁻¹). Results are presented as means ± SEM from five separate experiments. (B) Effect of increasing concentrations of quercetin (2, 10 and 20 μmol·L⁻¹) on insulin secretion in rat pancreatic islets. Results are presented as means ± SEM from three separate experiments. ***P < 0.0001; **P < 0.001; ns: P > 0.05; multiple comparison analysis for the different treatment conditions.