Molecular mechanisms of simvastatin-induced myopathy and insulin resistance

Statins or 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors are the most prescribed lipid-lowering drugs worldwide, used to treat hypercholesterolemia and efficient to reduce mortality and morbidity associated to cardiovascular diseases. They act primarily in the liver, where they inhibit the biosynthesis of cholesterol, alongside with other non-sterol intermediates, such as mevalonate, dolichol, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, leading to the impairment of several cellular processes for example protein post-translational modifications and proliferation.
Statins have several beneficial effects on the cardiovascular system and are in general well tolerated. However, inhibition of cholesterol synthesis pathway can induce adverse effects, mainly towards the skeletal muscle. These adverse effects range from muscle pain to rhabdomyolysis in rare cases, which can ultimately lead to death. Moreover, recently, an increased occurrence of insulin resistance and new-onset diabetes have been reported in patients treated with statins.
Considering the huge proportion of people under statin therapy worldwide and the prevalence of cardiovascular diseases and type 2 diabetes, it is urgent to elucidate molecular mechanisms leading to myopathy and new-onset diabetes.
This thesis includes four papers, two that are published and two that are in preparation.
The first paper presents the effects of insulin on simvastatin-induced toxicity as well as on the impairments induced by simvastatin on the insulin receptor (IR) signaling in C2C12 myotubes. Simvastatin strongly reduced membrane integrity and depleted the intracellular ATP in C2C12 cells. Additionally, simvastatin induced endoplasmic reticulum (ER) stress. Insulin was potent to not only prevent, but also rescue partially and time-dependently simvastatin toxicity. Simvastatin significantly reduced Akt phosphorylation on the serine 473 residue, done by mTORC2, while inhibiting only by trend the phosphorylation on the threonine 308 residue (done by PDK1). In like manner, downstream effectors of Akt were affected, inducing a reduced mTORC1 activity, atrophy and apoptosis. Insulin prevented these effects in a dose-dependent fashion. These data demonstrate that impaired Akt activation is a consequence of impaired mTORC2 activity and that insulin can prevent deleterious effects of simvastatin on the insulin receptor transduction pathway.
Our second paper correlates potentiality of insulin to prevent cell death and maintaining insulin receptor signaling in simvastatin-treated C2C12 myotubes, to the reported new-onset diabetes and insulin resistance concomitant to statin therapy. We demonstrated the effects of simvastatin on glucose metabolism in mice treated orally with simvastatin and elucidated the mechanisms leading to insulin resistance using C2C12 myotubes. Simvastatin-treated mice had higher plasmatic glucose during ip glucose tolerance test (IGTT) and a reduced glucose uptake in skeletal muscle compared to water-treated mice. A reduced glucose uptake was also observed in C2C12 myotubes treated with the statin as well as an impaired expression and phosphorylation of the insulin receptor β chain. Akt (Ser473) phosphorylation was significantly decreased in treated myocytes, which was explained and demonstrated with a decreased mTOR phosphorylation. Cells displayed also an impaired phosphorylation of GSK3β, leading to a reduced glucose transporter 4 (GLUT4) translocation to the cell surface. These data provide the evidence that simvastatin can cause insulin resistance in mice, and highlights new potential molecular targets for the management of insulin resistance during statin therapy, with the identification of a defect of mTORC2 activity and of GLUT4 translocation to the cell membrane for glucose absorption.
The third paper integrates the knowledge we acquired with simvastatin-treated myotubes to perform a comparison between C2C12 myoblasts and myotubes, the precursor and mature muscle cells respectively, in order to evaluate how post-natal myogenesis was affected by simvastatin. We observed that myoblasts were more sensitive to toxic effects of simvastatin in comparison to their differentiated form. We identified that geranylgeraniol was strongly potent in rescuing simvastatin toxicity. We assessed mitochondrial respiration and superoxide generation and found out that only myoblasts were disturbed by simvastatin at this level, whereas the mitochondrial function was not affected in myotubes, probably due to a higher expression of superoxide dismutase 2 (SOD2). We then characterized proliferation, differentiation and fusion processes in simvastatin-treated myoblasts. Proliferation of myoblasts was strongly inhibited, as well as the expression of differentiation and fusion markers. Mevalonate could prevent these effects in co-treatment. Last, upon simvastatin treatment, both cell models underwent apoptosis, which was prevented by insulin. This study demonstrates differences in sensitivity between C2C12 myoblasts and differentiated myotubes treated with simvastatin and might represent a good start point in the understanding of why statin-treated patients experience muscle pain or weakness during exercise or muscular stress.
Our fourth paper evaluates the contribution of mTORC1 and mTORC2 in simvastatin-induced myopathy, and confirms for the first time that mTORC2 inhibition is the key event in statin-induced myotoxicity. We showed that mTORC1 inhibition was cytoprotective in C2C12 myotubes and did not recapitulate simvastatin myotoxicity and impaired insulin receptor signaling. Inhibiting mTORC2 by knocking down Rictor displayed a similar toxicity pattern to simvastatin treatment in control cells and led to a reduced Akt (Ser473) and downstream effectors phosphorylation, thus recapitulating simvastatin-induced impairments. The mechanisms leading to mTORC2 and subsequently Akt inactivation in myocytes treated with statins were unprenylation of cellular GTPases and induction of mitochondrial reactive oxygen species (ROS) production. These findings highlight the primary molecular events occurring with simvastatin therapy, giving future opportunities for solutions to better manage and prevent statin-induced myopathy.