Discovery and Functional Characterization of AMPK Activators for the Regulation of Energy Metabolism

Abstract

AMP-activated protein kinase has been described as a key metabolic enzyme which can regulate whole body energy homeostasis. The use of AMPK activators for the treatment of metabolic diseases has been explored. For example, adiponectin or natural plant derivative metformin stimulate glucose uptake and inhibit hepatic glucose production, leading to decrease blood glucose through AMPK. Although many chemicals or ligand hormones which increase AMPK activity have been identified, most of these agents must be administrated at high concentrations and they function at only restricted organs.To identify novel endogenous and exogenous ligand for activating AMPK, I used two methods based on drug repositioning and LPI (ligand profiling and identification) technology. Drug repositioning strategy is highlighted to replace traditional drug discovery and development. It means an identifying and developingVnew uses with pre-existing drugs for reduction of development time and costs. Using this strategy, we aimed to characterize novel AMPK activating compounds that have a much lower effective concentration than known natural derivative activators. As a result, Emodin, a natural anthraquinone derivative was shown to stimulate AMPK activity in skeletal muscle and liver cell. Emodin enhanced GLUT4 translocation and [14C] glucose uptake into myotube in an AMPK dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species (ROS) and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activity. Finally, we confirmed that a single-dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for eight days using an insulin tolerance test and insulin stimulated PI-3K phosphorylation in wild type and HFD induced diabetic mouse models. This suggests that emodin regulates glucose homeostasis in vivo by AMPK activation.Ligand profiling by mass spectrometry is also an effective means of isolating functional molecules in active HPLC fractions. Using this method, Apolipoprotein A1 (Apo A1) was identified as an endogenous ligand to activate AMPK. Apolioprotein A1 is a major lipoprotein component of high-densityVIlipoprotein (HDL) cholesterol. Despite the important role of Apolioprotein A1 in the regulation of short-term glucose uptake into skeletal muscle, the mechanisms by which Apolioprotein A1 activates AMPK and effects on mitochondrial biogenesis after chronic treatment remain largely unexplored. In this study, I investigated whether Apolipoprotein A1 increase mitochondrial biogenesis and protect fatty acid induced insulin resistance. Treatment of Apolioprotein A1 at myotube for 24-48 hours increased mitochondrial DNA and oxygen consumption rate. The increment of mitochondria was mediated through induction of transcription factors of mitochondria such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear transcription factor 1 (NRF-1). The inhibition of Apolioprotein A1 receptor expression or AMPK by a pharmacological agent inhibited induction of mitochondrial biogenesis. Furthermore, free fatty acid induced insulin resistance was largely attenuated by Apolipoprotein A1 treatment. In summary, Apolioprotein A1 can alleviate obesity related metabolic disease by inducing AMPK dependent mitochondrial biogenesis.Physical inactivity can lead to obesity and fat accumulation in various tissues. Critical complications of obesity include type II diabetes and nonalcoholic fatty liver disease (NAFLD). Exercise has been reported to have ameliorating effects on obesity and NAFLD. However, the underlying mechanism is not fully understood. We showed that liver expression of macrophage migration inhibitory factor (MIF) was increased after 4 weeks of treadmill exercise. Phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase in primary humanVIIhepatocyte cell lines was enhanced after MIF treatment. These responses were accompanied by increases in lipid oxidation. Moreover, inhibition of either AMPK or CD74 resulted in inhibition of MIF-induced lipid oxidation. Furthermore, the administration of MIF to a human hepatocyte cell line reduced LXR agonist-induced lipid accumulation. Taken together, these results indicate that MIF is expressed and secreted in the liver during physical exercise and prevents hepatic steatosis by activating the AMPK pathway.In the present study, three factors are discovered as an AMPK activator. Emodin is a positive regulator of glucose uptake in skeletal muscle and lowers blood glucose levels in both wild type and high fat induced diabetic model mice. Apolipoprotein A1 can protect fatty acid induce insulin resistance status by increasing mitochondria biogenesis. Finally, MIF is a negative regulator of lipid synthesis in liver. Therefore, all of these exo-/endogenous AMPK activators may represent a novel therapeutic aspect in the treatment of Type 2 diabetic models.