Discovery And Pharmacological Investigation Of New Natural Modulators Of AMPK And LXR

This dissertation is grouped into two parts. One is about the discovery of AMP-activated protein kinase (AMPK) agonists and related pharmacological research, while the other involves the study of the regulation of prostaglandin F2α (PGF2α) as a selective endogenous antagonist of LXR/RXR heterodimer against β-amyloid in microglia.Physical inactivity lifestyle and over-intake high-energy food are considered as the major causes of metabolic syndrome, like type2diabetes. The dysfunction of energy metabolism finally results in the occurrence of type2diabetes and related cardiovascular diseases. Exercise training leads to fiber type transformation, mitochondrial biogenesis and angiogenesis, thus further enhancing fat oxidation which is associated with improvement of insulin sensitivity. AMPK as an energy sensor plays pivotal roles in the regulation of energy homeostasis. Activated AMPK switches on ATP-generating, catabolic pathways and switches off ATP-consuming, anabolic pathways, while long-time activation of AMPK could mimic exercise training to enhance exercise endurance. AMPK is thought to be an important target to prevent and treat type2diabetes and to explore AMPK agonist has significant value.We screened our lab in-house natural product library in HEK293T cells and finally determined that arctigenin (ATG) efficiently activated AMPK phosphorylation. We next investigated the effect of ATG on AMPK activation in muscle cells and the data indicated that ATG could also enhance AMPK phosphroylation and promote the transcription of of its downstream gene, pGC-1α, in both H9C2and C2C12cells. Futher study demonstrated that ATG phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase11(LKB Independent pathways rather than direct activation of AMPK. Administration of ATG efficiently improved mice endurance by enhancing AMPK phosphorylation and increasing the expression of uncoupling protein3(fatty acid oxidation) and cytochrome C (mitochondrial biogenesis) in muscle tissues. Besides, we screened ATG derivatives designed for modification and optimization against ATG and finally discovered several compounds with better activity. Our work discovered the natural product ATG could mimic exercise traning to enhance mice treadmill endurance as an AMPK agonist and provided valuable structure information for the development of new drugs to treat metabolic diseases.As reported, crude mogrosides isolated from the fruit of Siraitia grosvenorii Swingle, traditional Chinese medicine, could regulate mice blood glucose level by stimulating insulin secretion, however, the molecular mechanism and active ingredients were unknown. We screened nine compounds isolated from crude mogrosides and found that two of them could activate AMPK in HepG2cells. This work suggested AMPK activation was the potential mechanism by which crude mogrosides exerted its anti-hyperglycemic effects. In the second part, we investigated the regulation of prostaglandin F2α (PGF2α) as a selective endogenous antagonist of LXR/RXR heterodimer against β-amyloid (Ap) in microglia.Alzheimer’s disease (AD) is a neurodegenerative disease featured by impairment of memory and cognition. The pathological features of AD are the extracellular deposit of Aβ and accumulation of intracellular neurofibrillary tangles in the brain. AD ranks the fourth-leading cause of death among elderly individuals and threatens the health of the elderly seriously. AD is probably caused by complex interactions among multiple genetic, epigenetic and environmental factors and no cure has been found against this disease to date. PGF2α as one of the cyclooxygenase metabolites of arachidonic acid plays essential roles in regulation of varied key physiological processes, such as endometrial cyclic changes and ovarian function. Current studies have revealed that PGF2a is associated with the regulation of chronic and acute inflammation, however, the mechanism underlying the function of PGF2Q in this inflammation regulation is not clear.We identified that PGF2α was an endogenous antagonist of LXR and RXR and could selectively antagonize LXR/RXR and RXR/RXR dimers through mammalian one-hybrid and transactivation assays. It has been reported that LXR could alleviate AD by promoting apoE expression to enhance Aβ clearance and negatively regulating inflammation response. Our data showed that PGF2a could antagonize t0901317-stimulated promotion of AP clearance in microglia by antagonizing the expression of apoE. Besides, we investigated the association of PGF2a as an antagonist of LXR with neuroinflammtion. We found that t0901317potently inhibited the inflammatory response to LPS and Aβ, meanwhile, PGF2a antagonized t0901317-induced decrease of inflammatory response. Furthermore, cell apoptosis assay indicated that PGF2α antagonized t0901317-induced protection against LPS and Aβ.Our results demonstrated that PGF2α might be involved in the progression of AD through LXR antagonism, which may help better understand the pathogenesis of AD.