Shikonin Increases Glucose Uptake in Skeletal Muscle Cells and Improves Plasma Glucose Levels in Diabetic Goto-Kakizaki Rats

Glucose is the most common substrate for energy metabolism. Despite the varying demands for glucose, the body needs to regulate its internal environment and maintain a constant and stable condition. Glucose homeostasis requires harmonized interaction between several tissues, achieving equilibrium between glucose output and uptake. In this thesis we aimed to investigate factors modulating glucose homeostasis in a rat model of type 2 diabetes, the Goto-Kakizaki (GK) rat. In addition, we investigated sex differences in hepatic carbohydrate and lipid metabolism in healthy rats. In Paper I, three-week but not three-day treatment with a Southeast Asian herb, Gynostemma pentaphyllum (GP), significantly reduced plasma glucose (PG) levels in GK rats. An intra-peritoneal glucose tolerance test (IPGTT) was significantly improved in GP-treated compared to placebo-treated group. In the GP treated rats, the glucose response in an intra-peritoneal pyruvate tolerance test was significantly lower, indicating decreased gluconeogenesis, and hepatic glucose output (HGO) was reduced. GP-treatment significantly reduced hepatic glycogen content, but not glycogen synthase activity. The study provides evidence that the GP extract exerted anti-diabetic effect in GK rats, reducing PG levels and HGO, suggesting that GP improves the hepatic insulin sensitivity by suppressing gluconeogenesis. In Paper II, shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increased glucose uptake in L6 myotubes, but did not phosphorylate Akt. Furthermore we found no evidence for the involvement of AMP activated protein kinase (AMPK) in shikonin induced glucose uptake. Shikonin increased the intracellular levels of calcium in these cells and stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myotubes. In GK rats treated with shikonin once daily for 4 days, PG levels were significantly decreased. In an insulin sensitivity test, the absolute PG levels were significantly lower in the shikonin-treated rats. These findings suggest that shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. In Paper III, GK and control Wistar rats were injected daily for up to 4 weeks with either a non-hematopoietic erythropoietin analog ARA290 or with placebo. PG levels in GK but not Wistar rats were significantly lower in ARA290-treated compared to placebo. After 2 and 4 weeks, the IPGTT was significantly improved in ARA290 treated GK rats. In insulin and pyruvate tolerance tests, glucose responses were similar in ARA290 and placebo groups. In isolated GK rat islets, glucose-stimulated insulin release was two-fold higher and islet intracellular calcium concentrations in response to several secretagogues were significantly higher in ARA290-treated than in placebo-treated GK rats. These findings indicate that treatment with ARA290 significantly improved glucose tolerance in diabetic GK rats, most likely due to improvement of insulin release. In Paper IV, sex differences in hepatic carbohydrate and lipid metabolism were characterized in healthy rats. No sex-differences were observed regarding hepatic triglyceride content, fatty acid oxidation rates or insulin sensitivity. Male rats had higher ratios of insulin to glucagon levels, increased hepatic glycogen content, a lower degree of AMPK phosphorylation, a higher rate of glucose production and higher expression levels of gluconeogenic genes, as compared to female rats. A sex-dependent response to mild starvation was observed with males being more sensitive. In conclusion, sex-differences reflect a higher capacity of the healthy male rat liver to respond to increased energy demands. Key words: glucose homeostasis, type 2 diabetes, GK rats, L6 myotubes, hepatic glucose output, insulin sensitivity, sex differences

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 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

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

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