Proinsulin C-Peptide Enhances Cell Survival and Protects against Simvastatin-Induced Myotoxicity in L6 Rat Myoblasts

Abstract

The repair capacity of progenitor skeletal muscle satellite cells (SC) in Type 1 diabetes mellitus (T1DM) is decreased. This is associated with the loss of skeletal muscle function. In T1DM, the deficiency of C-peptide along with insulin is associated with an impairment of skeletal muscle functions such as growth, and repair, and is thought to be an important contributor to increased morbidity and mortality. Recently, cholesterol-lowering drugs (statins) have also been reported to increase the risk of skeletal muscle dysfunction. We hypothesised that C-peptide activates key signaling pathways in myoblasts, thus promoting cell survival and protecting against simvastatin-induced myotoxicity. This was tested by investigating the effects of C-peptide on the L6 rat myoblast cell line under serum-starved conditions. Results: C-peptide at concentrations as low as 0.03 nM exerted stimulatory effects on intracellular signaling pathways-MAP kinase (ERK1/2) and Akt. When apoptosis was induced by simvastatin, 3 nM C-peptide potently suppressed the apoptotic effect through a pertussis toxin-sensitive pathway. Simvastatin strongly impaired Akt signaling and stimulated the reactive oxygen species (ROS) production; suggesting that Akt signaling and oxidative stress are important factors in statin-induced apoptosis in L6 myoblasts. The findings indicate that C-peptide exerts an important protective effect against death signaling in myoblasts. Therefore, in T1DM, the deficiency of C-peptide may contribute to myopathy by rendering myoblast-like progenitor cells (involved in muscle regeneration) more susceptible to the toxic effects of insults such as simvastatin.

Keywords: Type 1 diabetes; myotoxicity; proinsulin C-peptide; simvastatin.

Figure 1

Rat C-peptide activates ERK1/2 in rat skeletal muscle cell line L6. L6 myoblasts cells were stimulated with rat C-peptide for 5 min in Dulbecco’s Modified Eagle’s Medium (DMEM). (A) Phosphorylation of ERK1/2 was determined by the Western blot using specific anti-phospho ERK1/2 antibody. 2% dialysed foetal bovine serum (DFBS) was used as a positive control. As a loading control, membranes were reprobed with an antibody against ERK2. Quantification by densitometry of data from three such experiments is presented in histograms; (B) as mean ± SEM. * p < 0.05 versus the unstimulated control.

Figure 2

Dose response study of Akt activation by rat C-peptide in L6 cells. Cells were stimulated with rat C-peptide for 5 min in DMEM. Pertussis toxin (PTX) denotes the effect of co-incubation with 100 ng/mL Pertussis toxin. Phosphorylation of Akt was determined by the Western blot (A) using specific anti-phospho-Akt antibody. DFBS (2%) was used as a positive control. As a loading control, membranes were reprobed with antibody against total Akt; (B) quantification by densitometry of data from three such experiments: mean ± SEM * p < 0.05 versus the unstimulated control.

Figure 3

Simvastatin decreases L6 myoblast viability in a time- and dose-dependent manner (A). Cytotoxicity was assessed by methylthiazoletetrazolium (MTT) assay in cells treated with simvastatin at different concentrations and at different time points indicated above. Data are mean ± SEM of (n = 4) independent experiments performed in triplicate, * p < 0.05 (compared to untreated cells at the same time point); (B) C-peptide protects against simvastatin-induced cytotoxicity assessed using MTT assay. Cells were incubated with varying concentrations of simvastatin (with or without 3 nM C-peptide) for 72 h. Data are presented as mean ± SEM of four independent experiments with each condition performed in triplicate, * p < 0.05; (C) pertussis toxin, PTX (100 ng/mL) inhibits the protective effect of C-peptide against simvastatin-induced cell death. Cytotoxicity was assessed using the MTT assay. Cells were treated for 72 h as indicated in the key on the figure. 3 nM scrambled human C-peptide was used as a negative control. Data are presented as % of the untreated control value (Cont) in each experiment. Values are mean ± SEM of three independent experiments performed in duplicate, * p < 0.05.

Figure 4

(A) C-peptide protects against simvastatin-induced cell toxicity. L6 myoblasts were serum starved in DMEM (i); treated with 3 nM C-peptide alone (ii); treated with simvastatin 10 μM (iii); or treated with simvastatin in the presence of 3 nM rat C-peptide (iv) for 72 h. Morphological visualization was assessed by using the Wright stain and light microscopy. Black arrows indicate shrunken cells (consistent with apoptosis). Magnification ×200; (B) C-peptide blunts the inhibitory effect of simvastatin on phospho-Akt activation in L6 myoblasts. Cells were treated with 10 µM simvastatin and co-incubated with 3 nM rat C-peptide for 72 h. Phosphorylation of Akt was determined by the Western blot using specific anti-phospho Akt antibody. As a loading control, membranes were reprobed with antibody against total Akt (shown in the lower blot); (C) densitometric analysis of four experiments. Data are presented as mean ± SEM * p < 0.05.

Figure 5

C-peptide blunts the effect of simvastatin–induced caspase-3 cleavage in L6 myoblasts. Cells were treated with 10 and 30 µM simvastatin and co-treated with 3 nM rat C-peptide for 72 h. (A) Cleavage of caspase-3 was determined by the Western blot using specific anti-caspase-3 antibody. As a loading control, membranes were reprobed with antibody against β actin; (B) densitometry analysis of four experiments was performed at 72 h and data are presented as mean ± SEM.* p < 0.05.

Figure 6

Simvastatin-induced ROS generation in L6 myoblasts, assessed by colorimetric detection using Nitro Blue Tetrazolium (NBT). (A) Cells were serum starved overnight in DMEM and then treated with a range of simvastatin concentrations for the times shown. H₂O₂ (100 μM) and (M) menadione (30 μM) were used as positive controls. Data are pooled from three independent experiments and presented as mean ± SEM. * p < 0.05; ** p < 0.01 compared to control at the same time point. (B–E) C-peptide blunting of simvastatin-induced ROS generation in L6 myoblasts. Cells were serum starved overnight in DMEM and treated with simvastatin alone or in-combination with 3 nM C-peptide for 30–360 min. H₂O₂ (100 μM) and menadione (M) (30 μM) were used as positive controls. Data are pooled from three independent experiments and presented as mean ± SEM. * p < 0.05.