Investigation Of Drak2 Function And Discovery Of Natural Molecule Diphyllin During The Differentiation And Thermogenesis Process Of Brown/Beige Adipocytes

Long-term energy imbalance can induce obesity and other metabolic diseases.Obesity has become a global disease and one of the risk factors threatening human health.Adipose tissue is an important organ that regulates energy balance in the body.It is divided into white,brown and beige fat according to its function.White fat is used to store energy,while brown and beige fat increase energy loss and release heat mainly through mitochondrial uncoupled proteins.The formation and activity of brown and beige adipocytes can be induced by different ways,including signalling pathways,epigenetic factors,transcription factors,endogenous factors and exogenous small molecules.A number of recent studies have shown that promoting energy consumption through brown or beige fat may be a new strategy for the treatment of metabolic diseases.This thesis is dedicated to discovering new mechanisms and new chemical molecule regulating the functions of brown and beige adipocytes.The research content mainly includes investigating the roles of the serine/threonine kinase Drak2 and small molecule diphyllin in the differentiation and thermogenesis of brown and beige fats,respectively.The first part of the work concerns the role of diphyllin in brown and beige adipocytes.A screening method for promoting brown fat differentiation was established by using mesenchymal stem C3H10-T1/2 cells.The natural products of Li Jinlong’s research group in Nantong University were screened and diphyllin was found to promote the formation of brown adipocytes and the expression of UCP1.At the cellular level,diphyllin promoted the formation of brown and beige adipocytes and also had a significant increase in thermogenesis.At the animal level,diphyllin promoted the thermogenesis of brown/beige fat in diet-induced obese mice,resisted diet-induced obesity,and improved the glycolipid metabolic dysfunction.Mechanism studies showed that diphyllin may be involved in regulating the formation and function of brown and beige fat cells by inhibiting V-ATPase activity and reducing intracellular autophagy.The second part of the work concerns the role of Drak2 in brown and beige adipocytes.Drak2 is a member of the death-related protein DAPK family.Previous studies reported that Drak2 was mostly associated with apoptosis,and also played important roles in immunity and type 1 diabetes.Drak2 is widely expressed in immune cells and expressed in fat tissues which provides theoretical basis and possibility for exploring the role of Drak2 in adipocytes.At the cellular level,we used RNA interference technology to knockdown Drak2 and investigated its effects on the differentiation and thermogenesis of brown and beige adipocytes.The results showed that knockdown of Drak2 could significantly promote the thermogenesis of brown/beige adipocytes.At the animal level,we used the Lox P/Cre recombination enzyme system to construct the brown and beige adipocytes specific Drak2 knockout mice and further investigated its phenotypes,we found Drak2 knockout mice showed resistance to diet induced obesity,increased energy consumption,and resistance to cold stimulation.These results suggest that knockout of Drak2 may increase the thermogenesis of brown and beige fat,and thus increase the wholebody energy consumption in mice.We further explored the role of Drak2 by MC385,a selective molecule inhibitor our lab discovered on the differentiation and thermogenic function of brown and beige adipocytes.We found that MC385 can increase expression of thermogenic related genes and UCP1,and increased oxygen consumption,which further indicate Drak2 inhibition may promote the thermogenesis.At the animal level,administration of MC385 in UCP1 reporter mice increased UCP1 contents in the interscapular brown adipose tissue and subcutaneous white adipose tissue.Long-term administration of MC385 promoted energy consumption and heat production of adipose tissue,reduced fat weight and cell size in obese mice,and had a therapeutic effect on diet-induced obesity and metabolic syndrome.Adipose tissue specific Drak2 knockout mice and self-developed inhibitors demonstrated the negative regulatory effect of Drak2 on the thermogenesis of brown and beige fat and energy consumption.To sum up,this paper focuses on the function and mechanism studies of Drak2 protein and compound diphyllin in fat browning and energy metabolism,which provides new potential protein and drug candidate for targeting brown and beige fat in the treatment of obesity and related metabolic diseases.