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

β-Adrenergic Inhibition of Contractility in L6 Skeletal Muscle Cells

The β-adrenoceptors (β-ARs) control many cellular processes. Here, we show that β-ARs inhibit calcium depletion-induced cell contractility and subsequent cell detachment of L6 skeletal muscle cells. The mechanism underlying the cell detachment inhibition was studied by using a quantitative cell detachment assay. We demonstrate that cell detachment induced by depletion of extracellular calcium is due to myosin- and ROCK-dependent contractility. The β-AR inhibition of L6 skeletal muscle cell detachment was shown to be mediated by the β2-AR and increased cAMP but was surprisingly not dependent on the classical downstream effectors PKA or Epac, nor was it dependent on PKG, PI3K or PKC. However, inhibition of potassium channels blocks the β2-AR mediated effects. Furthermore, activation of potassium channels fully mimicked the results of β2-AR activation. In conclusion, we present a novel finding that β2-AR signaling inhibits contractility and thus cell detachment in L6 skeletal muscle cells by a cAMP and potassium channel dependent mechanism

β-adrenergic signalling and novel effects in skeletal muscle

Skeletal muscles have, due to their large mass, a big impact on the whole body metabolism. There are many signals that can regulate the functions of skeletal muscles and one such signal is activation of α- and β-adrenoceptors (α- and β-ARs) by epinephrine and norepinephrine. This activation leads to several effects which are examined in this thesis.   Stimulation of β-AR on muscle cells induces glucose uptake, an event that both provides the muscle with energy and lowers the blood glucose levels. We discovered two key components in the β-ARs signal to glucose uptake: the transporter protein GLUT4 and the kinase mTOR, a molecule involved in several metabolic processes but not previously known to be activated by β-ARs.   The classical second messenger downstream of β-ARs, cAMP, was surprisingly found to be only partly involved in the β-adrenergic glucose uptake. We also found that a molecule called GRK2 is very important for this glucose uptake.   A novel effect of β-AR stimulation presented in this thesis is the inhibition of myosin II-dependent contractility in skeletal muscle cells. The intracellular pathway regulating this event was different from that regulating glucose uptake and involved both classical and novel molecules in the β-AR pathway.   Another stimulus that we found to activate insulin-independent glucose uptake in skeletal muscle cells was the natural compound Shikonin. Shikonin increased glucose uptake in skeletal muscle cells via a calcium- and GLUT4-dependent mechanism and improved glucose homeostasis in diabetic rats.   Taken together, we have identified new key molecules in the adrenergic signaling pathway as well as novel downstream effects. We conclude that glucose uptake in muscles can be activated by β-adrenergic stimulation or by Shikonin and that both treatments improves glucose homeostasis in diabetic animals. This knowledge can hopefully be used in the search for new drugs to combat type II diabetes.At the time of doctoral defence the following papers were unpublished and had a status as follows. Paper 1: Manuscript; Paper 3: Manuscript</p

β3-Adrenergically induced glucose uptake in brown adipose tissue is independent of UCP1 presence or activity: Mediation through the mTOR pathway

Objective: Today, the presence and activity of brown adipose tissue (BAT) in adult humans is generally equated with the induced accumulation of [2-18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) in adipose tissues, as investigated by positron emission tomography (PET) scanning. In reality, PET-FDG is currently the only method available for in vivo quantification of BAT activity in adult humans. The underlying assumption is that the glucose uptake reflects the thermogenic activity of the tissue. Methods: To examine this basic assumption, we here followed [18F]FDG uptake by PET and by tissue [3H]-2-deoxy-d-glucose uptake in wildtype and UCP1(−/−) mice, i.e. in mice that do or do not possess the unique thermogenic and calorie-consuming ability of BAT. Results: Unexpectedly, we found that β3-adrenergically induced (by CL-316,243) glucose uptake was UCP1-independent. Thus, whereas PET-FDG scans adequately reflect glucose uptake, this acute glucose uptake is not secondary to thermogenesis but is governed by an independent cellular signalling, here demonstrated to be mediated via the previously described KU-0063794-sensitive mTOR pathway. Conclusions: Thus, PET-FDG scans do not exclusively reveal active BAT deposits but rather any tissue possessing an adrenergically-mediated glucose uptake pathway. In contrast, we found that the marked glucose uptake-ameliorating effect of prolonged β3-adrenergic treatment was UCP1 dependent. Thus, therapeutically, UCP1 activity is required for any anti-diabetic effect of BAT activation. Keywords: Brown adipose tissue, Uncoupling protein 1, Glucose uptake, Adrenergic signaling, Positron emission tomograph

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 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 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 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