Simvastatin, but not Pravastatin, Blocks L-Type Ca2+ Channels In Rat Islet -Cells, Inhibiting Glucose-Induced Cytosolic Ca2+ Signaling and Insulin Release

Research Article

Austin Pharmacol Pharm. 2023; 7(2): 1029.

“Simvastatin, but not Pravastatin, Blocks L-Type Ca2+ Channels In Rat Islet -Cells, Inhibiting Glucose-Induced Cytosolic Ca2+ Signaling and Insulin Release”

Hari Prasad Sonwani*; Pragya Sahu; Rashi Bandey; Yuvraj Chandrawanshi; Prashant Verma

Department of Pharmacy, Apollo Group of Institutions, Durg CG, India

*Corresponding author: Hari Prasad Sonwani Department of Pharmacy, Apollo Group of Institutions, Durg CG, India. Email: [email protected]

Received: October 09, 2023 Accepted: November 16, 2023 Published: November 23, 2023


Patients with type 2 diabetes who have hypercholesterolemia frequently need to take HMG-CoA reductase inhibitors. A key factor in the pathophysiology of type 2 diabetes is altered pancreatic beta-cell activity, which results in a reduced ability of the pancreas to secrete insulin in response to glucose. The impact of HMG-CoA reductase inhibitors on -cell function must therefore be investigated. The regulation of -cell function is greatly influenced by the cytosolic Ca2+ concentration ([Ca2+]i). The current work looked at how HMG-CoA reductase inhibitors affected the [Ca2+]i signaling and insulin release that glucose-induced in rat islet -cells. Simvastatin, a lipophilic HMG-CoA reductase inhibitor, suppressed the initial phase increase and oscillation of [Ca2+]i caused by 8.3 mM glucose in single -cells at concentrations ranging from 0.1 to 3 g ml—1. The fewer Simvastatin-acid, a lipophilic inhibitor, decreased the first [Ca2+]i rise but was two orders of magnitude less effective. Pravastatin (100g ml—1), a hydrophilic inhibitor, has little impact on [Ca2+]. Simvastatin (0.3g ml–1) reduced glucose-induced insulin production from islets more effectively than simvastatin-acid (30g ml–1), whereas pravastatin (100g ml–1) had no effect. Simvastatin, but not pravastatin, showed a reversible blockage of the L-type Ca2+ channels in -cells in whole-cell patch clamp recordings. Simvastatin also prevented the opening of L-type Ca2+ channels, which is how L-arginine and HCl enhance [Ca2+] Conclusion: By blocking L-type Ca2+ channels in -cells, lipophilic HMG-CoA reductase inhibitors can prevent glucose-induced [Ca2+]i signaling and insulin secretion, and their inhibitory potencies are inversely correlated with their lipophilicities. Precaution must be takenWhen HMG-CoA reductase inhibitors are utilized in clinical settings, attention should be given to these findings, especially in type 2 diabetic patients.

Keywords: HMG-CoA reductase inhibitor; Simvastatin; Pravastatin; Lipophilicity; Islet; β-cell; Cytosolic Ca2+; L-type Ca2+ channel; Insulin secretion; Diabetes


The rate-limiting enzyme in cholesterol production is inhibited specifically and competitively by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors [1]. HMG-CoA reductase inhibitors have been shown to be effective in lowering plasma cholesterol levels and lowering the incidence of cardiovascular disease in long-term clinical studies (2–5.3 years) [7,18,19,21]. As a result, HMG-CoA reductase inhibitors are frequently utilized to treat hypercholesterolemic individuals [15]. Patients with diabetes and atherosclerotic plaque frequently experience hypercholesterolemia. One of the main causes of death among diabetic people is cardiovascular disease. HMG-CoA reductase inhibitors are so frequently given to diabetic individuals. According to numerous studies (Hosaka et al., 1977) [16,17,51], type 2 diabetes (non-insulin-dependent diabetes mellitus) is thought to develop and progress as a result of altered pancreatic beta-cell function that results in an impaired insulin secretory response to glucose. Therefore, it is crucial to look into any potential inhibition of -cell functions by HMG-CoA reductase inhibitors. It is well known that the control of -cell processes, including insulin secretion, depends heavily on the cytosolic Ca2+ concentration ([Ca2+]i) [10,26]. Ammala and others 1996). Recent studies have shown that [Ca2+]i oscillations can control gene expression [8,38]. Therefore, the current investigation looked at how HMG-CoA reductase inhibitors affected the [Ca2+]i oscillations and insulin secretion in islet -cells caused by glucose. HMG-CoA reductase inhibitors come in two different varieties: pravastatin, a hydrophilic inhibitor, and simvastatin and lovastatin, lipophilic inhibitors. Simvastatin's lactone ring makes it lipophilic. Simvastatin-acid, an in vivo active metabolite, is partly hydrophilic in its open acid form [20]. The acid form of pravastatin is hydrophilic [20]. Both versions of simvastatin and pravastatin have equivalent HMG-CoA reductase inhibitory activity in decreasing serum cholesterol levels. Inhibitors of HMG-CoA reductase have Although the frequency is very low, it has been clinically and experimentally reported to produce myotonia and severe rhabdomyolysis [24,25,33]. It has been demonstrated that the lipophilicity of HMG-CoA reductase inhibitors plays a key role in their ability to cause fast cell damage in L-myocytes [42]. Therefore, the effects of simvastatin, simvastatin-acid, and pravastatin on glucose-stimulated -cells were compared in the present investigation.


Selection of-Cells and the Populations of Islets and Individual-Cells

As described before [30], islets and single -cells were produced. In a nutshell, islets of Langerhans were extracted by collagenase digestion from Wistar rats aged 8–12 weeks. Pentobarbital was administered intraperitoneally to the animals to make them unconscious.80 mg/kg-1. After ligating the common bile duct proximal to the pancreas, the abdomen was opened, and collagenase (3 mg ml—1) was injected into the distal end of the duct. This was done in a solution containing 5 mM Ca2+. Cervical dislocation caused the rats to die. After being removed, the pancreas was incubated for 17 minutes at 37°C. Hand-collected islets were either divided into single cells in Ca2+-free HRB for insulin release tests or used in insulin release experiments. Eagle's minimal essential medium, which contains 5. mM glucose, 10% Foetal Bovine Serum (FBS), 100 g ml—1 streptomycin, and 100 U ml—1 penicillin, was used to maintain the single cells in short-term culture for up to 3 days after plating them on coverslips. 5% CO2 and 95% air make up the atmosphere. Throughout this period of culture, the cells reacted to the test chemicals in a predictable way. Physiological and morphological parameters were used to select -cells, as previously described [29].


NaCl 121.7, HCl 4.4, HH2PO4, CaCl 2, MgSO4, NaHCO3, and 4-(2-hydro- xyethyl) were the main components of HRB (in mM).HEPES, a -1-piperazineethanesulphonic acid with a pH of 7.4, with 0.1% bovine serum albumin. The following ingredients made up the standard pipette solution for whole-cell patch clamp recording of Ca2+ channel current (in mM): aspartate 75, tetraethylammonium (TEA) 30, HEPES 11, EGTA 11, MgCl2 3, CaCl2, ATP-Na2 3, and GTP 0.1 (at pH 7.2) (Boehringer Mannheim, Indianapolis, IN). To create a whole-cell clamp, an external solution containing Tris-HCl 100, TEA-Cl 30, HEPES 10, and CaCl2 (at pH 7.3) was utilized. 2.

Calculations of [Ca2+Ji As previously described [28,30], [Ca2+]i was quantified using dual-wavelength fura-2 microfluorometry in conjunction with imaging. In a nutshell, 1 M fura-2 acetoxymethylester was incubated with cells on coverslips for 30 min at 37°C in HRB containing 2.8 mM glucose. Then, with the cells mounted in a chamber, HRB was superfused at a rate of 1 ml min—1 at 37°C. An Intensified Charge-Coupled Device (ICDD) camera was used to detect emission signals at 510 nm, and an Argus-50 system created ratio images while the cells were activated at 340 and 380 nm in alternate intervals every 2.5 s. Japan's Hamamatsu is home to Hamamatsu Photonics. Calibration curves were used to convert ratio results to [Ca2+]i [30].

Electrical Physiological Signals

Using the conventional whole-cell patch-clamp arrangement, voltage-dependent Ca2+ channel currents in single -cells were captured under superfusion conditions at 1 ml min—1 at 30°C [11]. Pipettes were made of borosilicate glass (Sutter Instruments Co., Novata, CA, USA), fire polished, and at their tips were coated with Sylgard 184 (Dow Corning, Midland, MI, USA). When the usual pipette solution was used to fill them, their resistance was 2 7 MM. An Axopatch-200B amplifier and the program pCLAMP (Axon Instruments, Inc., Foster City, CA, U.S.A.) were used to monitor membrane currents. The potential was maintained at —70 mV and pulsed with depolarizing voltage to 0 mV. Applied at a frequency of 0.2 Hz for a period of 100 ms. The data were digitalized at 10 kHz, filtered at 5 kHz, and saved in a computer (IBM; Tokyo, Japan). The Clampfit tool was used to analyze the data. The reference potential was the pipette's zero-current potential, which was acquired right after the junction potential had been corrected but before the seal had been established.

Measuring Insulin Ejection

The procedures for measuring insulin release were followed exactly [9,28]. In a nutshell, groups of five islets that were extracted from Wistar rats between 8 and 12 weeks of age were first stabilized for 30 min in HRB containing 2.8 mM glucose. Islets were then treated in 1 ml of HRB for 30 minutes at 37°C. Amount of insulin was discovered by the use of an enzyme immunoassay kit from Morinaga in Yokohama, Japan.


Sankyo Co. (Tokyo, Japan) synthesized simvastatin, simvastatin-Na (simvastatin-acid), and pravastatin and graciously contributed them for our study. Pravastatin and simvastatin-acid were dissolved in distilled water, whereas simvastatin was dissolved in 100% ethanol. For the experiments, HRB was treated with tiny aliquots of the stock solutions of HMG-CoA reductase inhibitors. At a final ethanol concentration of less than 0.1%, [Ca2+]i in -cells remained unaffected. Dojin Chemical (Humamoto, Japan) provided the fura-2 and fura-2 acetoxymethylester. Life Technologies Inc. (New York, NY, USA) provided the FBS. We bought mevalonic acid lactone from Nacalai-Tesque Chemical Co. in Tokyo, Japan. Other compounds came from either Nacalai-Tesque or Sigma Chemical Co. (St. Louis, Missouri, United States).

Statistic Evaluation

The mean and s.e. mean were used to express the calculated values (n = number of observations). The Student's t-test was used for the statistical analysis. When P 0.05, differences were deemed statistically significant. Inhibition was therefore visible at the single cell level if the experiments revealed that the response to the initial glucose stimulation obtained in the presence of HMG-CoA reductase inhibitors was consistently lower than the response to the subsequent glucose stimulation obtained after washing out the inhibitors. It was established at the conclusion of the studies that the cells responded to 300 M tolbutamide with significant increases in [Ca2+]i, a feature of healthy -cells [9]. Simvastatin, simvastatin-acid, and pavastatin effects on the glucose-induced first phase [Ca2+Ji induction in single -cells] The peak of the first phase [Ca2+]i increase in response to 8.3 mM glucose was diminished in the presence of 0.3 g ml—1 simvastatin (Figure 1b). Simvastatin at a greater dosage of The first phase [Ca2+]i rise was severely reduced or even completely stopped by 3 g ml—1 (Figure 1c). Simvastatin is removed after washing, and the first phase [Ca2+]i rises in reaction to the second 8.3 mM glucose causes an increase in [Ca2+]i during the first phase. However, simvastatin-acid marginally decreased the first phase [Ca2+]i rise at 30 g ml—1 (Figure 3a) and severely inhibited it at 100 g ml—1 (Figure 3b) in a reversible way. a mean Simvastatin inhibits glucose-induced first phase [Ca2+]i rises in individual -cells. (a) In superfusion conditions, a rise in glucose concentration from 2.8 to 8.3 M induced the first phase [Ca2+]i increase in individual rat pancreatic - cells. Most -cells reacted to two consecutive challenges.

2+ Using 8.3 mM glucose (G8.3)gains in [Ca] with a comparable The restoration of stimulation with 8.3 mM glucose showed that the inhibition was reversible (Figure 1b and c). Simvastatin at concentrations of 0.1–3 g ml–1 also decreased the mean amplitude of the first phase [Ca2+]i increase for the single -cells under study (P 0.05, P 0.02, P 0.001, and P 0.0001 for the three g ml–1 simvastatin concentrations, respectively) (Figure 2). Simvastatin inhibits the first phase [Ca2+]i response to glucose as a result, and this inhibition is concentration-dependent. Simvastatin-acid, which has inhibitory effects at doses of 1 and 10 g ml—1, had no effect on it.


Temporal profile of the [Ca2+Ji-induced incuease in single uat pancueatic -cells due to glucose At a basal glucose content of 2.8 mM, [Ca2+]i in individual -cells varied between 30 and 150 nM (7.8–3.7 nM for 58 cells). As previously reported, an increase in glucose concentration from 2.8 mM to 8.3 or 1.07 mM resulted in a biphasic increase in [Ca2+]i in -cells: a first peak at about 400–500 nM (Figure 1), followed by a moderate elevation at about 100–200 nM that was occasionally superimposed with an oscillation of [Ca2+]i (second phase). Unless otherwise stated, the effects of HMG-CoA reductase inhibitors on the initial [Ca2+]i increase were investigated in the current investigation. Glucose stimulation was performed twice: once with inhibitors present and once without to assess the impact of the inhibitors at the single cell level. In control studies, 8.3 mM glucose was used to stimulate -cells twice. Twenty of the 29 cells responded to the first stimulation by increasing [Ca2+]i, while 24 of the 29 cells responded to the second stimulation. When it came to the amplitude of the first phase [Ca2+]i increase, the response to the first stimulation was either greater than or equivalent to the response to the second stimulation in 22 of these 24 responding cells (92%; Figure 1a).