Inhibition by simvastatin, but not pravastatin, of glucose-induced cytosolic Ca2+ signalling and insulin secretion due to blockade of L-type Ca2+ channels in rat islet beta-cells

Abstract

1. Hypercholesterolaemia often occurs in patients with type 2 diabetes, who therefore encounter administration of HMG-CoA reductase inhibitors. Alteration of pancreatic beta-cell function leading to an impaired insulin secretory response to glucose plays a crucial role in the pathogenesis of type 2 diabetes. Therefore, it is important to examine the effects of HMG-CoA reductase inhibitors on beta-cell function. 2. Cytosolic Ca2+ concentration ([Ca2+]i) plays a central role in the regulation of beta-cell function. The present study examined the effects of HMG-CoA reductase inhibitors on the glucose-induced [Ca2+]i signalling and insulin secretion in rat islet beta-cells. 3. Simvastatin, a lipophilic HMG-CoA reductase inhibitor, at 0.1-3 microg ml(-1) concentration-dependently inhibited the first phase increase and oscillation of [Ca2+]i induced by 8.3 mM glucose in single beta-cells. The less lipophilic inhibitor, simvastatin-acid, inhibited the first phase [Ca2+]i increase but was two orders of magnitude less potent. The hydrophilic inhibitor, pravastatin (100 microg ml(-1), was without effect on [Ca2+]i. 4. Simvastatin (0.3 microg ml(-1)), more potently than simvastatin-acid (30 microg ml(-1)), inhibited glucose-induced insulin secretion from islets, whereas pravastatin (100 microg ml(-1)) had no effect. 5. Whole-cell patch clamp recordings demonstrated a reversible inhibition of the beta-cell L-type Ca2+ channels by simvastatin, but not by pravastatin. Simvastatin also inhibited the [Ca2+]i increases by L-arginine and KCl, agents that act via opening of L-type Ca2+ channels. 6. In conclusion, lipophilic HMG-CoA reductase inhibitors can inhibit glucose-induced [Ca2+]i signalling and insulin secretion by blocking L-type Ca2+ channels in beta-cells, and their inhibitory potencies parallel their lipophilicities. Precaution should be paid to these findings when HMG-CoA reductase inhibitors are used clinically, particularly in patients with type 2 diabetes.

Figure 1

Glucose-induced first phase [Ca²⁺]_i increases and their inhibition by simvastatin in single β-cells. (a) A rise in glucose concentration from 2.8 to 8.3 M, under superfusion conditions, evoked the first phase [Ca²⁺]_i increase in single rat pancreatic β-cells. The majority of β-cells responded to two sequential challenges by 8.3 mM glucose (G8.3) with increases in [Ca²⁺]_i with a similar pattern. The amplitude of the first phase [Ca²⁺]_i increase in response to the first challenge was either larger than or comparable to that in response to the second challenge. In the presence of simvastatin (Simva) at 0.3 μg ml⁻¹ (b) and 3 μg ml⁻¹ (c), the first phase [Ca²⁺]_i increase was reduced and abolished, respectively. After washing out simvastatin, the first phase [Ca²⁺]_i increase was restored in response to the second challenge with 8.3 mM glucose. Tolbutamide at 300 μM (Tolb) induced rapid increases in [Ca²⁺]_i as seen in (a) and (b). The bars above the tracing indicate the periods of exposure to the agents specified. Dotted lines indicate beginning of exposure. The basal glucose concentration was 2.8 mM throughout measurements when not indicated otherwise. The results shown are representative of 14 cells in (a), six in (b) and ten in (c).

Figure 2

Concentration-response relationship for the inhibition of glucose-induced first phase [Ca²⁺]_i increases by HMG-CoA reductase inhibitors in single β-cells. The mean amplitude of the first phase [Ca²⁺]_i increase in response to 8.3 mM glucose was averaged and expressed as the mean±s.e.mean. The number of single β-cells examined was 127 for the control and 18–50 for each experimental condition. The mean amplitude of the first phase [Ca²⁺]_i increase was reduced by simvastatin at 0.1–3 μg ml⁻¹ and simvastatin-acid at 30–100 μg ml⁻¹. }P<0.05 for 0.1 μg ml⁻¹ simvastatin, #P<0.02 for 0.3 μg ml⁻¹ simvastatin and 30 μg ml⁻¹ simvastatin-acid, ##P<0.002 for 100 μg ml⁻¹ simvastatin-acid, ^*P<0.001 for 1 μg ml⁻¹ simvastatin, and ^**P<0.0001 for 3 μg ml⁻¹ simvastatin vs the control.

Figure 3

Simvastatin-acid, but not pravastatin, inhibits glucose-induced first phase [Ca²⁺]_i increases in single β-cells. Simvastatin-acid (Simva-acid) at 30 μg ml⁻¹ reduced (a) and at 100 μg ml⁻¹ abolished (b) the first phase [Ca²⁺]_i increase in response to 8.3 mM glucose (G8.3). After washing out simvastatin-acid, the first phase [Ca²⁺]_i increase was restored in response to the second challenge with 8.3 mM glucose. (c) Pravastatin (Prava) at 100 μg ml⁻¹ had no effects on the first phase [Ca²⁺]_i increase in response to 8.3 mM glucose. The results shown are representative of eight cells in (a), six in (b) and 17 in (c).

Figure 4

Simvastatin, but not pravastatin, inhibits glucose-induced [Ca²⁺]_i oscillations in single β-cells. (a) Simvastatin (Simva) at 0.3 μg ml⁻¹ partially and at 3 μg ml⁻¹ almost completely inhibited [Ca²⁺]_i oscillations induced by 8.3 mM glucose in a reversible manner. (b) Pravastatin (Prava) at 100 μg ml⁻¹ had no effects on [Ca²⁺]_i oscillations. The results shown are representative of five cells in (a) and 15 in (b). (c) Effects of simvastatin and pravastatin on the incidence of [Ca²⁺]_i oscillations. Among the 14 cells exhibiting [Ca²⁺]_i oscillations in response to 8.3 mM glucose, two cells oscillated during treatment with 1 μg ml⁻¹ simvastatin for about 15 min, and 13 cells oscillated after washing out this drug. In contrast, among the 18 oscillating cells, 17 and 15 cells oscillated during treatment with and after washing out 100 μg ml⁻¹ pravastatin, respectively.

Figure 5

Simvastatin and simvastatin-acid, but not pravastatin, inhibit glucose-induced insulin secretion in islets. Insulin release from islets stimulated with 8.3 mM glucose, under static incubation conditions, was inhibited in the presence of 0.3 μg ml⁻¹ simvastatin (Simva). Glucose-stimulated insulin release was mildly reduced by 30 μg ml⁻¹ simvastatin-acid (Simva-a), but not significantly. Pravastatin (Prava) at 100 μg ml⁻¹ was without effect on glucose-stimulated insulin release. The results are expressed as the mean±s.e.mean of six experiments. #, Significant difference (P<0.05) vs the 8.3 mM glucose (G8.3) group and the pravastatin group.

Figure 6

Simvastatin inhibits Ca²⁺ currents in single β-cells. (A) Whole-cell Ca²⁺ currents in a single β-cell depolarized to 0 mV from the holding potential of −70 mV before (a), during (b) and after exposure to 3 μg ml⁻¹ simvastatin (c) under superfusion conditions. (B) Recording of the temporal change of the whole-cell Ca²⁺ currents. a, b and c, specify the time points at which the current traces in Aa, Ab and Ac were taken. (C) The current-voltage relationship of the peak Ca²⁺ current amplitude in the control and in the presence of 3 μg ml⁻¹ simvastatin. The results shown are representative of five cells in (A) and (B) and three in (C).

Figure 7

Simvastatin (Simva) inhibits [Ca²⁺]_i increases in response to L-arginine (Arg) and KCl in single β-cells. (a) An increase in [Ca²⁺]_i induced by 10 mM L-arginine was inhibited in the presence of 3 μg ml⁻¹ simvastatin, but not 60 μg ml⁻¹ pravastatin (Prava). (b) A sustained increase in [Ca²⁺]_i induced by 25 mM KCl was almost instantaneously inhibited by the addition of 3 μg ml⁻¹ simvastatin, and it was restored upon washing out the drug. The glucose concentration was 8.3 mM in (a) and 2.8 mM in (b). G16.7: change to 16.7 mM glucose. The results shown are representative of five cells in (a) and 14 in (b).