Study On The Action And Mechanism Of Fatty Acid Binding Protein 4 And Its Inhibitors

The occurrence and development of type 2 diabetes mellitus(T2DM)is closely related to the abnormal lipid metabolism and non-infectious low-grade inflammation of peripheral tissues.Fatty acid binding protein 4(FABP4)has been proved to play a vital role in lipid metabolism and inflammation.FABP4 can sense fatty acid signals by reversibly binding fatty acids and other hydrophobic ligands to influence their intracellular localization and by direct interaction with proteins,thus becoming a key factor in associating inflammation and lipid metabolism.FABP4 can also be secreted into blood,clinical and epidemiological studies have shown that serum FABP4 concentration is closely related to obesity-related diseases such as T2 DM and cardiovascular diseases,acting as an important biomarker in serum.Many studies have demonstrated that FABP4 deficiency or inhibition can bring a series of metabolic improvements to genetically deficient or high fat-induced obese diabetic mice,making FABP4 a drug target for prevention and treatment of T2 DM with great potential.Hundreds of FABP4 inhibitors have been reported,targeting obesity,hyperlipidemia,T2 DM,atherosclerosis and so on,but no drugs have yet entered the clinical stage.In view of the important role of FABP4 in the metabolic process,the development of safe and effective FABP4 inhibitors has become a research hotspot in recent years.Firstly,we screened a series of FABP4 inhibitors with good activity and selectivity by using the in vitro activity and selective screening platform of FABP4 in our laboratory.The inhibitory activity of some compounds on FABP4 was stronger than that of the preclinical compound BMS309403.We selected two compounds 16 dk and 16 do for further study,and found they could significantly inhibit the lipolysis of mature adipocytes and promote lipogenesis during the differentiation of preadipocytes,which is consistent with the phenotype of adipocytes in FABP4-deficient mice.We also conducted a preliminary pharmacodynamic evaluation of the compounds in spontaneously obese diabetic db/db mice,and the results showed that the two compounds could reduce fasting blood glucose,increase insulin sensitivity,and significantly reduce serum triglyceride and free fatty acid levels.The staining results showed that the two compounds reduced the lipid and inflammatory infiltration of liver and inflammatory infiltration of adipose tissue.Further detection of insulin signaling pathway showed that the two compounds also reduced insulin resistance in the liver.To sum up,we discovered a class of active and selective FABP4 inhibitors that could inhibit adipocytes lipolysis and improve the disorders of glycolipid metabolism in obese diabetic mice.It has been demonstrated that FABP4 deficiency will lead to the compensatory elevation of FABP5 in adipose tissue,FABP4 and FABP5 double-knockout mice displayed more significant protection from diet-induced obesity,insulin resistance,type 2 diabetes,and fatty liver disease than FABP4 or FABP5 single knockout mice.The research and development of FABP4/5 double target inhibitors has gradually become the mainstream.Therefore,we focused on the research and development of FABP4/5 double-target inhibitors.Our pharmacochemical cooperation group continued to optimize the lead compound 16 dk to obtain hundreds of compounds.Through in vitro activity screening,we found that some compounds maintained or even increased the inhibitory activity of FABP4,but also showed inhibitory effect towards FABP5.We selected two compounds A16 and B8 with good FABP4/5 activity for preliminary evaluation at the cellular level,and found that the two compounds significantly inhibited adipocyte lipolysis,with a stronger effect than the leading compound 16dk;The two compounds also significantly reduced the expression of inflammatory genes MCP-1,TNF-?,IL-6 and COX-2 on the macrophage inflammation model induced by lipopolysaccharide(LPS),and their effects were also stronger than that of the leading compound 16 dk.Preliminary studies on inflammatory pathways showed that the two compounds exerted anti-inflammatory effects through IKK/NF-?B pathway.These results suggest that compounds with dual inhibitory activity of FABP4/5 have better effect than pure FABP4 inhibitors,which deserves further study on pharmacodynamics and mechanism in vivo after improving pharmacokinetic stability.In the last part of the thesis,we focused on exploring the mechanism of secreted FABP4,which is a research hotspot in recent years.Previous laboratory studies showed that the serum FABP4 level in db/db obese diabetic mice was higher than that in their lean littermates.FABP4 inhibitor I-9 showed beneficial effects on improving glycolipid metabolism and hyperinsulinemia in db/db mice after 4 weeks of administration,I-9 also significantly reduced serum FABP4 content.In my follow-up study,we found that I-9 increased insulin sensitivity and decreased inflammation in peripheral tissues.Moreover,I-9 enhanced autophagy levels in the liver and adipose tissue and reduced lipid infiltration in the liver of db/db mice.Combined with the latest research,we speculated that FABP4 inhibitors acted at least partially by affecting secreted FABP4,which might exert a direct impact on the function of peripheral tissues and glycolipid metabolism besides its function on transporting fatty acids.On the one hand,FABP4 inhibitors might inhibit the secretion of FABP4 and thus reduce the content of FABP4 in blood and tissues;On the other hand,they might block the direct effect of secreted FABP4 on tissues and cells.In order to confirm our hypothesis,firstly we used differentiated 3T3-L1 adipocytes to detect the effect of FABP4 inhibitor on FABP4 secretion,the results showed that I-9 significantly inhibited the secretion of FABP4.Then we used the recombinant FABP4 protein as secreted FABP4 to construct in vitro pathological models on hepatocytes and adipocytes,respectively.The results showed that secreted FABP4 induced lipid accumulation and insulin resistance of primary hepatocytes,with autophagy played an important regulatory role in this process.We also found that secreted FABP4 inhibited the differentiation of 3T3-L1 preadipocytes by negative regulation of PPAR? and promoted p38 MAPK mediated lipolysis and inflammation of mature adipocytes.Finally,we validated the in vivo effects of secreted FABP4 by short-term injection of recombinant FABP4 into C57BL/KsJ mice.It was found that lipid accumulation and insulin resistance of liver deteriorated,insulin secretion increased,inflammation of adipose tissue aggravated by FABP4 treatment,which were consistent with the cellular results.FABP4 inhibitor I-9 could partially block the metabolic disorders caused by secreted FABP4.Our study partially elucidate the role and mechanism of secreted FABP4 in the pathophysiological changes of metabolic disorders,which is of great significance for its feasibility as a drug target for metabolic disease prediction or treatment.