Bioactives of Artemisia dracunculus L. mitigate the role of ceramides in attenuating insulin signaling in rat skeletal muscle cells

Abstract

Ectopic lipids in peripheral tissues have been implicated in attenuating insulin action in vivo. The botanical extract of Artemisia dracunculus L. (PMI 5011) improves insulin action, yet the precise mechanism is not known. We sought to determine whether the mechanism by which PMI 5011 improves insulin signaling is through regulation of lipid metabolism. After differentiation, cells were separately preincubated with free fatty acids (FFAs) and ceramide C2, and the effects on glycogen content, insulin signaling, and ceramide profiles were determined. The effect of PMI 5011 on ceramide accumulation and ceramide-induced inhibition of insulin signaling was evaluated. FFAs resulted in increased levels of total ceramides and ceramide species in L6 myotubes. Saturated FFAs and ceramide C2 inhibited insulin-stimulated phosphorylation of protein kinase B/Akt and reduced glycogen content. PMI 5011 had no effect on ceramide formation or accumulation but increased insulin sensitivity via restoration of Akt phosphorylation. PMI 5011 also attenuated the FFA-induced upregulation of a negative inhibitor of insulin signaling, i.e., protein tyrosine phosphatase 1B (PTP1B), and increased phosphorylation of PTP1B. PMI 5011 attenuates the reduction in insulin signaling induced by ceramide accumulation, but the mechanism of improved insulin signaling is independent of ceramide formation.

FIG. 1.

Glycogen content in both basal and insulin-stimulated states was measured as a marker of insulin action in control cells and in those treated for 24 h with FFA or ceramide (CER) with or without PMI 5011. As shown, glycogen content was significantly reduced with FFA and ceramide C2 incubation. PMI 5011 was observed to mitigate the effect of ceramide C2 and palmitic acid on reducing insulin-stimulated glycogen content. Data are presented as mean ± SEM. *P < 0.05 vs. FFA or C2 treatment. All experiments for each condition were conducted in triplicate.

FIG. 2.

Effect of PMI 5011 and myriocin on the ceramide profile and total ceramides formed in L6 cells pretreated with palmitic acid as determined by liquid chromatography/tandem mass spectrometry (LCMS/MS). As demonstrated, myriocin significantly inhibited ceramide formation, whereas PMI 5011 had no effect on ceramide formation or accumulation. There was no statistically significant difference in total ceramide levels between FFA-treated cells and those treated with FFA and PMI 5011. Data are presented as mean ± SEM. *P < 0.05 vs. control; n = 3 assessments for each treatment condition.

FIG. 3.

Ceramide profiles in L6 cells pretreated with different saturated and unsaturated FFAs as determined by liquid chromatography/tandem mass spectrometry (LCMS/MS). C16, C18, and C24:1 were the most prominent ceramide species formed regardless of the FFA treatment used (i.e., C16:0, C16:1, C18:0, and C18:1). Total ceramides as compared with the control treatment increased only in cells treated with saturated FFAs. Unsaturated FFAs had no effect on total ceramide content. n = 3 experiments for each experimental condition.

FIG. 4.

Demonstration of a comparison of effect of FFA type on glycogen content (A) and total ceramides formed (B) in L6 cells. A: Glycogen content was used as a marker of insulin sensitivity in cells treated with FFAs for 24 h. B: Another set of cells also treated for 24 h was used to measure total ceramides by liquid chromatography/tandem mass spectrometry (LCMS/MS) after extraction of the lipid fraction by methanol/chloroform partition. As demonstrated, palmitic acid and stearic acid were significantly different from the control for both total ceramides and glycogen content. Elevated ceramide levels were associated with significantly lower glycogen content. *P < 0.05 vs. control; n = 3 experiments for each treatment condition.

FIG. 5.

Effect of FFA, ceramide (CER), and PMI 5011 on Akt-1, Akt-2, and PTP1B expression and phosphorylation (phos). L6 cells were pretreated with palmitic acid (200 μM) or ceramide C2 (20 ng/mL) for 16 h before insulin stimulation. The bar graphs summarize the results of quantification of gel plots from three experiments. *P < 0.05 indicates a significant difference for each parameter compared with the control. As shown, palmitic acid and ceramide C2 attenuated the increase in phosphorylation of Akt-1 and -2 postinsulin stimulation. PMI 5011 incubation with either C2 or with FFA restored AKT1 phosphorylation and downregulated PTP1B expression compared with incubation with either C2 or FFA alone (see lanes 5 and 6 vs. lanes 3 and 4, and lanes 9 and 10 vs. lanes 8 and 9). Increased Akt-1 and Akt-2 phosphorylation resulted in increased PTP1B phosphorylation.

FIG. 6.

Effect of pretreatment with fatty acids and ceramide (CER) on PTP1B activity in L6 cells. Cells were treated with 200 μM palmitic acid or 20 ng/mL C2 with or without PMI 5011 for 24 h. For comparison, cells were also incubated in the presence of an inhibitor of PTP1B. Palmitic acid and C2 significantly increased PTP1B activity. PMI 5011 was observed to significantly reduce PTP1B activity in the presence of FFA or ceramide (C2) exposure. *P < 0.05 vs. control; n = 3 experiments for each treatment condition.

FIG. 7.

A and B: Demonstration of use of RNAi to downregulate PTP1B expression in FFA and C2-treated cells. L6 control and transfected cells were pretreated with palmitic acid (200 μM) or ceramide C2 (20 ng/mL) for 16 h before insulin stimulation (10 nmol/L for 10 min). Akt-1, Akt-2, PTP1B levels, and phosphorylation were assessed. The bar graphs summarize the results of quantification of gel plots from three experiments. When compared with the negative vector, use of RNAi reduced PTP1B levels in L6 cells treated with C2 and FFA only (lanes 5 and 6 vs. lanes 9 and 10). Use of small interfering RNA (SiRNA) in the presence of C2 and FFA treatment increased insulin-stimulated Akt-1 and Akt-2 phosphorylation modestly compared with the C2 and FFA treatment (lanes 9 and 10 vs. lanes 5 and 6). Data are means ± SEM. *P < 0.05 indicates a significant difference compared with the control.

FIG. 8.

A and B: AKT 1, AKT 2, and PTP1B gene expression shown as fold induction compared with the control. The effects of 200 μM palmitic acid and 20 ng ceramide (Cer) C2 with or without PMI 5011 on mRNA expression in differentiated L6 cells were determined. As shown, both FFA and ceramide C2 increased PTP1B, but not Akt-1 or -2 gene expression. RNAi and PMI 5011 reduced PTP1B gene expression in the presence of FFA or ceramide C2. Data are means ± SEM. *P < 0.01 vs. control; n = 3 experiments for each treatment condition. siRNA, small interfering RNA.