Celastrol Protects against Antimycin A-Induced Insulin Resistance in Human Skeletal Muscle Cells

Abstract

Mitochondrial dysfunction and inflammation are widely accepted as key hallmarks of obesity-induced skeletal muscle insulin resistance. The aim of the present study was to evaluate the functional roles of an anti-inflammatory compound, celastrol, in mitochondrial dysfunction and insulin resistance induced by antimycin A (AMA) in human skeletal muscle cells. We found that celastrol treatment improved insulin-stimulated glucose uptake activity of AMA-treated cells, apparently via PI3K/Akt pathways, with significant enhancement of mitochondrial activities. Furthermore, celastrol prevented increased levels of cellular oxidative damage where the production of several pro-inflammatory cytokines in cultures cells was greatly reduced. Celastrol significantly increased protein phosphorylation of insulin signaling cascades with amplified expression of AMPK protein and attenuated NF-κB and PKC θ activation in human skeletal muscle treated with AMA. The improvement of insulin signaling pathways by celastrol was also accompanied by augmented GLUT4 protein expression. Taken together, these results suggest that celastrol may be advocated for use as a potential therapeutic molecule to protect against mitochondrial dysfunction-induced insulin resistance in human skeletal muscle cells.

Figure 1

MTT cell viability assay on human skeletal muscle after AMA and celastrol treatment. Cell viability of AMA (A) and celastrol (B) was performed in a dose-dependent manner. The time-course evaluation of the optimal dose for AMA (30 µM) and celastrol (15 nM) was determined (C). Figures of the untreated cells (DMSO) (D), 30 µM AMA-treated cells (E), 15 nM celastrol-treated cell (F) and AMA-treated cells with 15 nM celastrol treatment (G) were taken at 40X magnification using fluorescence inverted microscope (Carl Zeiss, Göttingen, Germany). Figures represent one of three independent experiments. * p < 0.05 and ** p < 0.01 vs. untreated control.

Figure 1

MTT cell viability assay on human skeletal muscle after AMA and celastrol treatment. Cell viability of AMA (A) and celastrol (B) was performed in a dose-dependent manner. The time-course evaluation of the optimal dose for AMA (30 µM) and celastrol (15 nM) was determined (C). Figures of the untreated cells (DMSO) (D), 30 µM AMA-treated cells (E), 15 nM celastrol-treated cell (F) and AMA-treated cells with 15 nM celastrol treatment (G) were taken at 40X magnification using fluorescence inverted microscope (Carl Zeiss, Göttingen, Germany). Figures represent one of three independent experiments. * p < 0.05 and ** p < 0.01 vs. untreated control.

Figure 2

The mechanistic effects of AMA and celastrol treatment on the glucose uptake activity in skeletal muscle cells. Co-treatment with celastrol on skeletal muscle cells with mitochondrial dysfunction for 48 h improved insulin-mediated glucose uptake activity. However, wortmannin blocked this beneficial effects of celastrol on AMA-treated cells. # p< 0.05 vs. basal rate; + p < 0.05 vs. AMA-treated cells.

Figure 3

AMA and celastrol treatment of mitochondrial functions of skeletal muscle cells. After incubation, cells were assayed to measure intracellular ATP concentration (A), mitochondrial membrane potential (B), mitochondrial superoxide production (C) and citrate synthase activity (D). Basal and insulin-stimulated levels were shown. Basal rate refers to the rate of glucose transport in the absence of insulin. * p < 0.05 and ** p < 0.01 vs. untreated control; # p < 0.05 and ## p < 0.01 vs. basal rate; + p < 0.05 vs. AMA-treated cells.

Figure 4

Effects of AMA and celastrol on the oxidative properties of human skeletal muscle. The quantification of 8-OHdG DNA (A), protein carbonyls (B) and lipid peroxidation levels (C) was determined after 30 µM AMA treatment with or without addition of 15 nM celastrol and celastrol treatment alone. Values are normalized against standard curves generated according to standard protocol provided by manufacturers. * p < 0.05 and ** p < 0.01 vs. untreated control; + p < 0.05 and ++ p < 0.01 vs. AMA-treated cells.

Figure 5

Effects of AMA and celastrol treatments on mitochondrial fusion and fission of human myotubes. Representative images of western blot analysis (A) on the relative expression of (B) mfn1, (C) mfn2 and (D) drp1 proteins were quantified with the corresponding antibodies using a densitometer. β-actin was used as loading control. Protein levels calculated by densitometry were normalized relative to β-actin signals. * p < 0.05 and ** p < 0.01 vs. untreated control; + p < 0.05 and ++ p < 0.01 vs. AMA-treated cells.

Figure 6

Effects of AMA and celastrol on the production of pro-inflammatory cytokines with regard to mitochondrial-induced insulin resistance in human myotubes. Cells were incubated for 48 h with DMSO (control), AMA, celastrol-AMA and celastrol only. The quantification of IL-6 (A), TNF-α (B) and IL-1β (C) was determined as briefly described in materials and methods. Values are normalized against standard curves generated according to standard protocols provided by manufacturers. * p < 0.05 and ** p < 0.01 vs. untreated control; + p < 0.05 and ++ p < 0.01 vs. AMA-treated cells.

Figure 7

AMA treatment of human myotubes in the absence and presence of celastrol. Cells were grown in 6-well plates and treated with AMA and celastrol for 48 h. The representative images of western blot analysis (A) of the relative expression level of NF-κB (B) and IκBα (C) activity was measured and quantified. β-actin was used as a loading control. Protein levels calculated by densitometry were normalized relative to β-actin signals. * p < 0.05 vs. untreated control; + p < 0.05 vs. AMA-treated cells.

Figure 7

AMA treatment of human myotubes in the absence and presence of celastrol. Cells were grown in 6-well plates and treated with AMA and celastrol for 48 h. The representative images of western blot analysis (A) of the relative expression level of NF-κB (B) and IκBα (C) activity was measured and quantified. β-actin was used as a loading control. Protein levels calculated by densitometry were normalized relative to β-actin signals. * p < 0.05 vs. untreated control; + p < 0.05 vs. AMA-treated cells.

Figure 8

Effects of AMA and celastrol treatment on insulin signaling pathways and glucose transporters (GLUT4) of human myotubes. Cells were treated with AMA (30 µM) for 48 h before incubation with celastrol (15 nM). β-actin was used as an internal protein loading control. Protein levels for each corresponding antibody obtained from densitometry were normalized to the β-actin signal (A–F). * p < 0.05 and ** p < 0.01 vs. untreated control; # p< 0.05 vs. basal rate; + p < 0.05 and ++ p < 0.01 vs. AMA-treated cells.

Figure 8

Effects of AMA and celastrol treatment on insulin signaling pathways and glucose transporters (GLUT4) of human myotubes. Cells were treated with AMA (30 µM) for 48 h before incubation with celastrol (15 nM). β-actin was used as an internal protein loading control. Protein levels for each corresponding antibody obtained from densitometry were normalized to the β-actin signal (A–F). * p < 0.05 and ** p < 0.01 vs. untreated control; # p< 0.05 vs. basal rate; + p < 0.05 and ++ p < 0.01 vs. AMA-treated cells.

Figure 9

Effects of AMA and celastrol treatments on the protein expression of AMPK (Thr172) and PKC θ (Ser643/676) in human skeletal muscle-derived myoblast. Cells were cultured in media containing 30 µM AMA in the absence and presence of 15 nM celastrol for 48 h. Thereafter, cell lysates were subjected to western blot analysis (A). The percentage of specific and total protein phosphorylation was calculated to determine the relative level of exact amino acid residue phosphorylation (A–C). β-actin was used as internal protein loading control. Protein levels for each corresponding antibody obtained from densitometry were normalized to the β-actin signal. * p < 0.05 and ** p < 0.01 vs. untreated control; + p < 0.05 and ++ p < 0.01 vs. AMA-treated cells.

Figure 10

Schematic representation of the mechanistic action of celastrol on mitochondrial dysfunction-induced insulin resistance in human skeletal muscle cells.