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

The GPR120 agonist TUG‐891 promotes metabolic health by stimulating mitochondrial respiration in brown fat

Brown adipose tissue (BAT) activation stimulates energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies demonstrated a role for G protein‐coupled receptor 120 (GPR120) in BAT thermogenesis. Here, we investigated the therapeutic potential of GPR120 agonism and addressed GPR120‐mediated signaling in BAT. We found that activation of GPR120 by the selective agonist TUG‐891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice. These effects coincided with decreased brown adipocyte lipid content and increased nutrient uptake by BAT, confirming increased BAT activity. Consistent with these observations, GPR120 deficiency reduced expression of genes involved in nutrient handling in BAT. Stimulation of brown adipocytes in vitro with TUG‐891 acutely induced O2 consumption, through GPR120‐dependent and GPR120‐independent mechanisms. TUG‐891 not only stimulated GPR120 signaling resulting in intracellular calcium release, mitochondrial depolarization, and mitochondrial fission, but also activated UCP1. Collectively, these data suggest that activation of brown adipocytes with the GPR120 agonist TUG‐891 is a promising strategy to increase lipid combustion and reduce obesity

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery