Simvastatin induces insulin resistance in L6 skeletal muscle myotubes by suppressing insulin signaling, GLUT4 expression and GSK-3β phosphorylation

Highlights

• Simvastatin but not pravastatin inhibits glucose uptake in L6 myotubes.

• Mevalonate and GGPP but not FPP restore simvastatin-decreased glucose uptake.

• Simvastatin suppresses IR-IRS1-AKT signaling cascade.

• Simvastatin activates GSK-3β to inhibit glycogen synthesis.

• Simvastatin downregulates GLUT4.

Abstract

Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor widely used for the treatment of hypercholesterolemia. Recent data indicates that simvastatin increases the risk of new-onset diabetes by impairing both insulin secretion and insulin sensitivity. However, systematic evaluation of mechanistic pathways is lacking. We aimed to explore the effects of simvastatin on glucose uptake and underlying mechanisms using L6 skeletal muscle myotubes. We performed our experiments at basal and insulin-stimulated conditions, at normal (5.5 mM) and high (16.7 mM) glucose. We showed that simvastatin inhibited glucose uptake at all conditions. We also found out that pravastatin, another widely used statin with different physicochemical properties, did not inhibit glucose uptake. The effect of simvastatin was reversed with geranylgeranyl pyrophosphate but not with farnesyl pyrophosphate, implying that reduced protein geranylgeranylation has a role in simvastatin-induced insulin resistance. Simvastatin also decreased phosphorylation of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), AKT and glycogen synthase kinase 3β (GSK-3β), and downregulated GLUT4. In conclusion, our data indicate that simvastatin decreased both basal and insulin-stimulated glucose uptake through inhibiting the critical steps in IR/IRS-1/AKT signaling cascade, and by hindering GLUT4 function and normal regulation of glycogen synthesis, contributing to insulin resistance.

Introduction

Statins are the most effective drugs in lowering plasma cholesterol and prevention of cardiovascular disease [1]. They act by inhibiting 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthesis. Large scale meta-analyses showed that statins induce the new-onset type 2 diabetes (T2D) [2]. The risk varied between different statins, pravastatin having the lowest effect [3]. In a large population-based METSIM study, statin therapy was associated with a 46% increased risk of T2D, a reduction in insulin sensitivity and a decrease in insulin secretion during a 6 year follow-up [4]. Statins also affect glycemic control in patients with pre-existing T2D [5].

Although several studies have investigated the molecular mechanisms for increased risk of T2D with statin treatment, they are not fully understood. T2D is caused by impaired insulin secretion and insulin resistance [6]. Effects of statins on insulin sensitivity have been explored mainly in adipose cells. Skeletal muscle accounts for the majority of whole body glucose uptake (∼80%) and insulin sensitivity [7], therefore understanding of the mechanisms of statin-induced insulin resistance in skeletal muscle is crucial for the prevention of T2D in individuals requiring statin treatment.

Glucose uptake in skeletal muscle is mediated by glucose transporter 4 (GLUT4) in both basal and insulin-stimulated conditions [8]. Statins were shown to inhibit GLUT4 expression and glucose uptake in adipocytes [9], [10], [11], [12]. In muscle cells, simvastatin was also reported to decrease glucose uptake [13], [14], but no change in GLUT4 protein expression was observed [14]. Furthermore, inhibition of glucose uptake by simvastatin was observed in insulin-stimulated but not in basal conditions in L6 myotubes [14]. Some studies reported that the effect of statins on glucose uptake is mediated by cholesterol biosynthesis inhibition [9], [10] whereas others reported an independent mechanism [14]. Glucose uptake and GLUT4 translocation is stimulated through insulin receptor (IR) - insulin receptor substrate 1 (IRS1) - phosphoinositide 3-kinase (PI3K) - AKT kinase pathway [15]. Treatment with statins has been shown to suppress this pathway in adipocytes [9], [12], and in skeletal muscle cells under insulin-stimulated conditions [14]. There is still need for further investigation of insulin signaling pathway and its downstream targets.

We systematically inverstigated the effects of simvastatin and pravastatin, two widely used statins with different physicochemical properties, on basal and insulin-stimulated glucose uptake and insulin signaling in L6 skeletal muscle myotubes. We performed the experiments at normal (5.5 mM) and high (16.7 mM) glucose concentrations to investigate whether these effects are modulated by hyperglycemia.