Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation

Abstract

Compounds that increase the activity of the energy sensor AMP-activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [ H]-2-deoxy-d-glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post-hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0 μmol/L CA a response (226 ± 9.62% of control, P=.001), similar to maximum insulin (201 ± 7.86% of control, P=.001) and metformin (213 ± 10.74% of control, P=.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA-stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo.

Rosmarinic Acid, a Rosemary Extract Polyphenol, Increases Skeletal Muscle Cell Glucose Uptake and Activates AMPK

“…Importantly, the activation of AMPK seen with RA treatment was at the same level as with 2mM metformin treatment ( Figure 4 A). Treatment with RE also increased AMPK phosphorylation ( Figure 4 A) in agreement with our previous studies [ 27 , 31 ]. To examine the involvement of AMPK in glucose uptake, we used the AMPK inhibitor, compound C (CC).…”

“…It is important to note that the maximum stimulation of glucose uptake seen with RA treatment (186 ± 7.31% of control, p < 0.001) was comparable to the response seen with maximum insulin (100 nM, 0.5 h, 204 ± 10.73% of control, p < 0.001) and metformin (2 mM, 2 h, 202 ± 14.37% of control, p < 0.001) stimulation ( Figure 3 A). Exposure of myotubes to 5 μg/mL of rosemary extract (RE) also significantly increased glucose uptake (197 ± 3.60% of control, p < 0.001) in agreement with our previous studies [ 27 , 31 ].…”

“…In our previous studies we found significant increase in AMPK phosphorylation by RE [ 27 ] and carnosic acid [ 31 ], and therefore we hypothesize that RA may also increase AMPK phosphorylation. Treatment of L6 myotubes with RA resulted in a robust increase in AMPK phosphorylation on threonine 172 [ 32 ], an indicator of activation ( Figure 4 A).…”

“…We previously found that treatment of L6 muscle cells with 5 μg/mL of rosemary extract increased glucose uptake to a level comparable to maximum stimulation observed with insulin and metformin [ 27 ], and we hypothesized that specific polyphenols present in the RE may contribute to the increase in glucose uptake. We measured the direct effect of carnosic acid on muscle cells and found a significant increase in glucose uptake and activation of AMPK [ 31 ]. In the current study, we continued our investigation and focused on the rosemary polyphenol rosmarinic acid (RA).…”

“…It is therefore possible that the beneficial effects observed with RE administration may be due to the action of a specific polyphenol. We recently found a significant increase in muscle cell glucose uptake and activation of AMPK by CA [ 31 ].…”