Simvastatin inhibits glucose metabolism and legumain activity in human myotubes

Abstract

Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the Musculus obliquus internus abdominis of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with [¹⁴C]glucose or [³H]deoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of legumain. Dysregulation of glucose metabolism and decreased legumain activity by simvastatin points out new knowledge about the effects of statins on skeletal muscle, and may contribute to the understanding of the myotoxicity observed by statins.

Figure 1. Reduced glucose uptake in myotubes after treatment with simvastatin.

A. and B. Differentiated myotubes were incubated for 48 h with simvastatin (S) with or without mevalonolactone (1 mM; ML) and compared to untreated control (C), prior to incubation with radiolabeled substrates at day 7. A. Dose-response of simvastatin on glucose uptake after 4 h incubation with [¹⁴C(U)]glucose (0.2 mM, 21.5 kBq/ml) using a multiwell trapping device. Radioactivity was measured in cell lysates and in trapped CO₂ and corrected for total proteins (n = 3–8, student t-test, *p<0.05 vs. untreated). B. Effects of 5 µM simvastatin with or without ML on uptake of [³H]deoxyglucose (10 µM, 37 kBq/ml, 15 min) (n = 3, student t-test, *p<0.05 vs. S). C. Differentiated myotubes were pre-incubated for 48 h with simvastatin (30 µM) before subcellular fractionation. Equal amount of total proteins from the membrane fraction were loaded to the gel and immunoblot analysis performed. LAMP-2 was used as loading control. Quantification of GLUT1 band intensity is shown, corrected for LAMP-2 and normalized to untreated control (C) (n = 4). D. Myotubes were treated with 0–40 µM simvastatin with or without 50 µM or 1 mM ML for 48 h before cell viability was analyzed at day 7. The cells were incubated with MTS-reagent for 2 h before absorbance at 490 nm was measured (n = 3, student t-test, *p<0.05 vs. untreated control (C)).

Figure 2. Effects of simvastatin on glucose oxidation and expression of proteins involved in oxidative phosphorylation.

A. and B. Differentiated myotubes were pre-incubated for 48 h with simvastatin (A: 0–20 µM; B: 5 µM) prior to incubation for 4 h with [¹⁴C(U)]glucose (0.2 mM, 21.5 kBq/ml) with or without different agents at day 7. A. Dose-response of simvastatin on glucose oxidation with or without addition of FCCP (1 µM; n = 3). B. Effects on glucose oxidation of 0.1 µM rotenone, 0.1 µM antimycin A, 1 µg/ml oligomycin A or 1 µM FCCP with (black bars) or without (open bars) simvastatin. Radiolabeled [¹⁴C]CO₂ was trapped and counted in a MicroBeta® scintillation counter and corrected for total protein (n = 4–8, student paired t-test, *p<0.05 vs. basal). C. One representative immunoblot of proteins involved in oxidative phosphorylation from differentiated myotubes incubated for 48 h with (S) or without (C) 10 µM simvastatin is shown. Twenty µg total proteins were loaded to the gel and immunoblot analysis using MitoProfile® Total OXPHOS Human WB Antibody Cocktail and GAPDH were performed (n = 3).

Figure 3. Legumain activity and expression after treatment with simvastatin, mevalonolactone, geranylgeranyl pyrophosphate and/or farnesyl pyrophosphate.

Differentiated myotubes were incubated for 48(0–40 µM) with or without mevalonolactone (50 or 1000 µM; ML), geranylgeranyl pyrophosphate (3 µM; GGPP) or farnesyl pyrophosphate (3 µM; FPP) prior to harvesting at day 7. A. Dose-dependent effects of simvastatin (0–40 µM) on legumain activity (n = 3–15, student t-test, *p<0.05 vs. 0 µM). B. Effects on legumain activity caused by 30 µM simvastatin (S alone) with or without ML, GGPP or FPP. The data are compared and normalized to untreated myotubes (n = 6–12, student t-test, *p<0.05 vs. S alone; n = 12, paired student t-test, #p<0.01 vs. untreated). C. Effects on legumain expression caused by 30 µM simvastatin (S alone) with or without ML, GGPP or FPP. Equal amounts of total proteins (10 µg) of cell lysates were separated and immunoblot analyzes were performed. One representative immunoblot is shown and band intensity analysis are normalized to 36 or 56 kDa legumain immunobands, respectively, (n = 4–7, student t-test, *p<0.05 vs. S alone).

Figure 4. Legumain, cathepsin B and L expressions in cytosolic and membrane fractions of myotubes.

Myotubes were treated with (+) or without (−) 30 µM simvastatin for 48 h before subcellular fractionation was performed. One representative immunoblot of legumain, cathepsin B and L in the cytosolic and membrane fractions is shown. All lanes were loaded with equal amount of total proteins and probed with antibodies as indicated. LAMP-2 and α-tubulin are shown as cell compartment controls (n = 3).