Shikonin increases glucose uptake in L6 myotubes.

<p>(<b>A</b>) Glucose uptake in differentiated L6 myotubes in response to 100 nM, 1 µM or 10 µM shikonin treatment (2 h). In (<b>B</b>) and (<b>C</b>), cells were treated with 1 µM shikonin (sh) or 1 µM insulin (ins) for 2 h or 20 h respectively). Graphs show mean ± SEM of four (A), seven (B) or three (C) experiments. Asterisks represent statistical difference as analyzed by one way ANOVA between basal and treated cells (** P&lt;0.01 *** P&lt;0.001).</p

Shikonin stimulates translocation of GLUT4.

<p>(<b>A</b>) Representative confocal image of GLUT4-translocation which was detected by myc-antibody in cells stable transfected with GLUT4myc after 2 h treatment with either 1 µM shikonin or 1 µM insulin (<b>B</b>) Quantification of confocal images obtained in (A) by using Image J and expressed as % of basal. Graph show mean ± SEM of 3 experiments performed. Asterisks represent statistical difference as analyzed by one way ANOVA between basal and treated cells (***p&lt;0.001, *p&lt;0.05).</p

Shikonin increases free calcium in L6-myotubes.

<p>Intracellular levels of calcium in L6 cells before and after acute exposure to 1 µM insulin or shikonin. Data are means ± SEM (n = 15).</p

Shikonin does not affect AMP-phosphorylation or ATP/ADP-ratio.

<p>(<b>A</b>) Immunoblot phosphorylated AMPK at threonine 172 (t172) following treatment of L6 myotubes with water as control (c) or stimulated with either 1 µM shikonin (sh) or 2 mM AICAR (ai) for 2 h. Figure is representative of six independent experiments performed.(<b>B</b>) Quantification of AMPK phosphorylation (t172) expressed as a percentage of the basal levels. Asterisks represent statistical difference as analyzed by one way ANOVAbetween basal and treated cells (*** P&lt;0.001). Graph show mean ± SEM (n = 6). (<b>C</b>) AMP to ATP-ratio in L6 myotubes 1 µM shikonin or 1 µM DPI treatment. Graph show mean ± SEM (n = 4).</p

Calcium is involved in shikonin-medaited glucose uptake.

<p>(<b>A</b>) Insulin and A23187 stimulated glucose uptake in L6 myotubes. Data are mean ± SEM of three experiments (<b>B</b>) Measurement of intracellular calcium levels by the fluorescent ratiometric Ca²⁺ indicator indo-1 following acute treatment with shikonin (1 µM) or insulin (1 µM). ***p&lt;0.001, *p&lt;0.05, one way ANOVA . Graph show mean delta value ± SEM of 15 experiments performed. Asterisks represent statistical difference as analyzed by one way ANOVA between basal and treated cells (*p&lt;0.05, *** P&lt;0.001). (<b>C</b>) Effect of BAPTA-AM on glucose uptake mediated by shikonin in L6 mytoubes. Cell were stimulated with shikonin for 2 h in the absence or presence of 5 µM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester), BAPTA (BA). Graph show mean ± SEM of 3 experiments performed. Asterisks represent statistical difference as analyzed by one way ANOVA (*p&lt;0.05, **&lt;0.01).</p

Shikonin injections lower plasma glucose levels in diabetic GK-rats.

<p>(<b>A</b>) Shikonin effect on plasma glucose in GK-rats treated with DMSO/oil (squares) or shikonin (10 mg/kg intraperitoneally) (triangles) for 4 days. Results are mean ±SEM of 6 rats.**p&lt; 0.01 for shikonin day 2 compared to day 1, ***p&lt;0.001 for shikonin day 4 compared to day 1 (Students t-test). (<b>B</b>) Shikonin effect on insulin sensitivity in GK-rats. Plasma glucose was measured in GK-rats after s.c. insulin in GK rats treated with DMSO/oil (squares) or shikonin (triangles) for 4 days. Results are mean ±SEM of 6 rats.</p