Study On Long-chain Fatty Acid Receptors:Agonists Screening And Pharmacological Investigations Of The Anti-diabetic Effect

Due to the aged population and changed lifestyles,incidence of type 2 diabetes(T2DM)and its complications is rising.As T2 DM has now become a seriously global health problem,it is of great practical importance to investigate new targets and drugs for diabetes therapy.T2 DM is one of the most common endocrine diseases and characterized with pancreatic ? cell dysfunction and insulin sensitivity impairment.Recent evidence demonstrated that the processes of free fatty acids(FFA)sensing and metabolism were tightly associated with insulin secretion as well as peripheral tissue sensitivity to insulin.GPR40 mainly expresses on pancreatic ? cell and promotes glucose-stimulated insulin secretion,while GPR120 distributes in various tissues and is tightly associated with appetite,glucolipid metabolism and bone formation.GPR120 signaling in macrophage and adipocyte suppresses inflammatory response in peripheral tissue,resulting in a improved insulin sensitivity.Moreover,both GPR40 and GPR120 in enteroendocrine cells mediate secretion of incretins,whch could directly increase insulin secretion and satiety.To date,clinical trials had confirmed the efficacy of GPR40 agonists on T2 DM therapy,and GPR120 is also considered to be a potential target of anti-diabetic drug discovery.Part ?(including chapter 2 and 3): GPR120 agonists screening and pharmacological investigations of the anti-diabetic effect.By using a cell based GPR120 agonist screening system,we found that a potential GPR120 agonist L34(EC50=100 nM)could not only improve oral glucose tolerance in mice by augmenting glucose-stimulated insulin release,but also markedly repress the LPS-induced inflammatory response.Orally administration of L34(50 mg/kg)for 4 weeks improved the hyperinsulinmia,fatty liver and insulin resistance.In the further investigation of molecular mechanism,we found that L34 significantly repressed the inflammatory pathway and reduced the expression of inflammatory genes in the liver and adipose tissue of db/db mice.Results showed that GPR120 agonists possessed anti-diabetic effect by mutiple mechanisms,highlitghting their promising application on T2 DM treatment.On the other hand,we found that Rb2 could upregulated GPR120 expression and subsequently enhanced the anti-inflammatory effect of ?-3 fatty acid in LPSstimulated mouse macrophage cell line RAW264.7 cells.Co-treatment with Rb2 and ?-linolenic acid(ALA)dramatically reduced lipopolysaccharide(LPS)-induced inflammatory pathways activation as well as inflammatory genes expression and subsequently led to significantly lower production of pro-inflammatory cytokines than ALA treatment alone.Our study provides a new mechanism to explain the suppressing effect of ginsenosides on inflammation.In addition,our results also suggest that the decreased response efficacy caused by receptor desensitization can be overcome by increasing GPR120 expression,indicating that GPR120 remains a promising target for treating chronic inflammation and T2 DM.Part ?(chapter 4): GPR40 full agonist identification and its anti-diabetic effects evaluation.Similar method was employed to evaluated the activity of GPR40 agonist,and we obtained a series of specific GPR40 agonists with novel structure,among which compound(R)-7k(EC50=81 nM)was a full agonist and represented stronger positive effect on glucose-stimulated insulin secretion in pancreatic ? cells.Moreover,compound(R)-7k(10 mg/kg)improved oral glucose tolerance in normal mice.We demonstrated the advantages of GPR40 full agonist on T2 DM therapy through the pharmacology study on compound(R)-7k.Part ?(chapter 5): we present a discovery that ginsenoside Rb2 could improved nonalcoholic fatty liver disease(NAFLD)and insulin resistance by restoring autophagy.Although drug therapies with sufficient efficacy and safety for NAFLD have not been fully established,numerous evidence indicates that lipid metabolism plays a key role in NAFLD.Autophagy is a self-renewal pathway that mediates the degradation of cytoplasmic contents,thus keeping cellular energy homeostasis.Recent researchs demonstrate that hepatic autophagy is closely associated with NAFLD.However,it is controversial that autophagy promotion could improve NAFLD.Rb2 treatment significantly improved insulin intolerance,hepatic lipid accumulation and hepatic autophagy in db/db mice.In vitro,Rb2 obviously increased autophagic flux in hepG2 cell and mouse primary hepatocytes,and consequently reduced the lipid accumulation induced by high FFA plus high glucose.Further study of the mechanism showed that the effect of Rb2 on autophagy induction relied on promotion of both AMPK-mTOR and sirt1-Foxo1 pathways.Results together revealed the underlying mechanism of Rb2 improving NAFLD and insulin resistance,providing evidence for autophagy-related pathways to be new targets of NAFLD therapy.