Silymarin induces insulin resistance through an increase of phosphatase and tensin homolog in Wistar rats

Abstract

Background and aims: Phosphatase and tensin homolog (PTEN) is a phosphoinositide phosphatase that regulates crucial cellular functions, including insulin signaling, lipid and glucose metabolism, as well as survival and apoptosis. Silymarin is the active ingredient in milk thistle and exerts numerous effects through the activation of PTEN. However, the effect of silymarin on the development of insulin resistance remains unknown.

Methods: Wistar rats fed fructose-rich chow or normal chow were administered oral silymarin to identify the development of insulin resistance using the homeostasis model assessment of insulin resistance and hyperinsulinemic- euglycemic clamping. Changes in PTEN expression in skeletal muscle and liver were compared using western blotting analysis. Further investigation was performed in L6 cells to check the expression of PTEN and insulin-related signals. PTEN deletion in L6 cells was achieved by small interfering ribonucleic acid transfection.

Results: Oral administration of silymarin at a dose of 200 mg/kg once daily induced insulin resistance in normal rats and enhanced insulin resistance in fructose-rich chow-fed rats. An increase of PTEN expression was observed in the skeletal muscle and liver of rats with insulin resistance. A decrease in the phosphorylation of Akt in L6 myotube cells, which was maintained in a high-glucose condition, was also observed. Treatment with silymarin aggravated high-glucose-induced insulin resistance. Deletion of PTEN in L6 cells reversed silymarin-induced impaired insulin signaling and glucose uptake.

Conclusions: Silymarin has the ability to disrupt insulin signaling through increased PTEN expression. Therefore, silymarin should be used carefully in type-2 diabetic patients.

Figure 1. Mean values for the direct measure of insulin sensitivity, as assessed by the glucose infusion rate during the last 20-h hyperinsulinemic-euglycemic clamp.

Silymarin (200 mg/kg body weight) was orally administered once daily to Wistar rats for 2 weeks. A glucose clamp was performed 30 min after the oral intake of silymarin (200 mg/kg) in Wistar rats fed normal or fructose-rich chow. VO-OHpic (PTEN inhibitor, 10 µg/kg) was i.p. injected 30 min before administration of silymarin. The vehicle used to dissolve the testing drugs was given at the same volume. Values (mean ± SE) were obtained from each group of 8 animals. ***P<0.001 compared with vehicle-treated normal chow-fed group. ^### P<0.001 compared with the vehicle-treated fructose-rich chow-fed group.

Figure 2. The effect of silymarin on the expression of PTEN.

Silymarin (200 mg/kg body weight) was orally administered once daily to Wistar rats for 2 weeks. Skeletal muscle (A) and liver (B) were isolated for identification of PTEN expression using western blotting analysis. Data are obtained from 6 individual experiments and expressed as mean ± standard error of mean. *P<0.05; **P<0.01 compared with the vehicle-treated group (first column).

Figure 3. Silymarin disrupted insulin signaling to decrease glucose uptake in L6 myotube cells.

L6 cells were maintained in normal-glucose (5 mM) or high-glucose (25 mM) medium. Cells were treated with 1 µM silymarin, and each group was exposed or not to 0.1 µM insulin for 30 min. Equal amounts of the total lysates of each group were immunoblotted with anti-phospho-Akt (pAkt) or Akt (A). The L6 cells were then treated with 200 µM 2-NBDG for 10 min to evaluate the glucose uptake ability of each group (B). Values represent means ± standard error of mean of 3 independent experiments. *P<0.05; **P<0.01 compared with the control group.

Figure 4. PTEN deletion in L6 cells reversed the effect of silymarin on insulin sensitivity and glucose uptake.

Cells were transfected for 48(A). Cells treated with 1 µM silymarin for 6 h with or without specific PTEN siRNA were exposed or not to 0.1 µM insulin. Equal amounts of the total lysates of each group were immunoblotted with anti-phospho-Akt (pAkt) or Akt (B). Cells were transfected for 48 h with 50 pmol PTEN siRNA or scramble siRNA were treated with 1 µM silymarin 6 h with or without specific PTEN siRNA and exposed or not to 0.1 µM insulin for 30 min. The L6 cells were then treated with 200 µM 2-NBDG for 1 h to evaluate the glucose uptake ability of each group (C). Values represent means ± standard error of mean for 3 independent.