Resveratrol improves palmitic acid‑induced insulin resistance via the DDIT4/mTOR pathway in C2C12 cells

Abstract

The present study aimed to establish a model of palmitic acid (PA)-induced insulin resistance (IR) in C2C12 cells and to determine the mechanism underlying how resveratrol (RSV) improves IR. C2C12 cells were divided into the control (CON), PA, PA + RSV, PA + RSV + DNA damage-inducible transcript 4 (DDIT4)-small interfering (si)RNA and PA + RSV + MHY1485 (mTOR agonist) groups. Glucose contents in culture medium and triglyceride contents in cells were determined. Oil red O staining was performed to observe the pathological changes in the cells. Reverse transcription-quantitative PCR and western blotting were conducted to evaluate the mRNA and protein expression levels, respectively, of DDIT4, mTOR, p70 ribosomal protein S6 kinase (p70S6K), insulin receptor substrate (IRS)-1, PI3K, AKT and glucose transporter 4 (GLUT4). Compared with in the CON group, glucose uptake was decreased, cellular lipid deposition was increased, phosphorylated (p)-IRS-1, p-mTOR and p-p70S6K protein expression levels were increased, and p-PI3K, p-AKT, GLUT4 and DDIT4 protein expression levels were decreased in the PA group. By contrast, compared with in the PA group, culture medium glucose content and cellular lipid deposition were decreased, p-PI3K, p-AKT, GLUT4 and DDIT4 protein expression levels were increased, p-IRS-1 protein expression levels were decreased, and mTOR and p70S6K mRNA and protein expression levels were decreased in the PA + RSV group. Compared with in the PA + RSV group, DDIT4 protein and mRNA expression levels were reduced in the PA + RSV + DDIT4-siRNA group, but showed no change in the PA + RSV + MHY1485 group. Following transfection with DDIT4-siRNA or treatment with MHY1485, the effects of RSV on improving IR and lipid metabolism were weakened, mTOR and p70S6K protein expression levels were upregulated, p-PI3K, p-AKT and GLUT4 protein expression levels were down-regulated, p-IRS-1 protein expression levels were upregulated, and culture medium glucose content and cellular lipid deposition were increased. In conclusion, RSV may improve PA-induced IR in C2C12 cells through the DDIT4/mTOR/IRS-1/PI3K/AKT/GLUT4 signaling pathway, as well as via improvements in glucose and lipid metabolism.

Effect of mTORC Agonism via MHY1485 with and without Rapamycin on C2C12 Myotube Metabolism

“…However, one important consideration in these experiments is that mTORC activation was achieved via leucine treatment. Given that leucine is metabolized predominantly in the mitochondria and has previously been associated with altered mitochondrial function, we assessed the effect of mTORC1 activation on indicators of BCAA metabolism using MHY1485, which does not resemble any BCAA in structure [44,45]. Consistent with our other metabolic findings, MHY1485 had no effect of BCAA catabolic enzyme expression at the mRNA or protein level.…”

“…Differentiation was accomplished by replacing growth media with DMEM with 2% horse serum and 100 U/mL penicillin and 100 µg/mL streptomycin for 6-9 days [47]. MHY1485 from Cayman Chemical was dissolved in 100% DMSO, and cells were treated with a final concentration of 10µM for MHY1485 based on previous observations demonstrating effective mTORC activation in C2C12 cells [45]. To identify if any responses were dependent on mTORC activation, rapamycin from Cayman Chemical was dissolved in DMSO, and cells were then treated with either DMSO or MHY1485 at 10µM either with DMSO or rapamycin (RAPA) at 100 nM (with final DMSO at 0.2% vol:vol for all cells) for 24 h. For insulin-sensitivity Western blot experiments, cells were treated as described above and stimulated with insulin at 100 nM for 30 min in serum-free media or negative control (serum-free media only).…”

“…Therefore, the purpose of these experiments was to assess the effect of the mTORC1 agonist on myotube metabolism and insulin sensitivity using an mTORC agonist that does not share chemical resemblance to any BCAA. Specifically, this report will assess the effects of MHY1485, which has been shown to activate mTORC [44,45], both with and without mTORC inhibition via rapamycin treatment.…”

Exploring the Anti-Diabetic Potential of Quercetagitrin through Dual Inhibition of PTPN6 and PTPN9

“…In summary, QG alleviated insulin resistance induced by palmitic acid in C2C12 myoblasts by enhancing the phosphorylation of Akt. The intake of free fatty acids, notably palmitic acid, has been closely linked to the development of insulin resistance and a reduction in glucose uptake in muscle cells [16,35]. Whether QG confers a preventive response against palmitic acid-induced insulin resistance in differentiated C2C12 myoblasts was investigated.…”

Syrah Grape Polyphenol Extracts Protect Human Skeletal Muscle Cells from Oxidative and Metabolic Stress Induced by Excess of Palmitic Acid: Effect of Skin/Seed Ripening Stage

“…Grape, undoubtedly one of the most widely consumed fruits globally, is a noteworthy source of polyphenols, offering an impressive array of both quantity and variety. Grape polyphenols (GPPs) possess diverse biological effects and have demonstrated positive impacts on obesity, oxidative stress, mitochondrial activity and insulin sensitivity in both in vitro and in vivo settings [ 20 , 21 , 22 ]. The Syrah grape, also known as Shiraz in some regions, holds significant importance in the world of wine thanks to its versatility and ability to deliver high quality wines.…”