Investigation On The Anti-diabetic Effect Of Tiliroside Derivative C45 And Its Mechanism

AimsDiabetes becomes worldwide health issue.International Diabetes Federation 2019 reported that there are around 0.46 billion diabetic individuals and 0.37 billion impaired glucose tolerance individuals worldwide.China has the most diabetes cases in the world.90% diabetes are type 2 diabetes which shows insulin resistance,namely impaired insulin signal pathway-induced glucose and lipid metabolism disorder,as well as low grade systemic inflammation.Obesity is the main reason.High fat diet induces obesity.Obese adipose tissue is hypoxic accompany macrophages infiltration.Adipocytes and macrophages in adipose tissue release factors which cause systemic insulin resistance and inflammation.Our team previously found that Chinese herb Potentilla chinensis Ser.anti diabetes component tiliroside.We synthesized and evaluated tiliroside derivative Fla-CN.After further optimized the structure we synthesized more tiliroside derivatives and selected the most active one,C45.In order to evaluate the druggability of C45,we explored the anti diabetes and inflammation of C45 and related mechanism.In the first part,cultured hepatocytes,adipocytes,macrophages and high fat diet-induced insulin resistant obese(DIO)mice were used to study the effect of C45 on glucose and lipid metabolism and inflammation in vivo and in vitro.In the second part,we explored the effect of hypoxic obese adipose on the release of inflammatory cytokines from adipocytes and macrophages and the mechanism of these cytokines dysregulated glucose and lipid metabolism in skeletal muscle cells and liver cells.Furthermore,the in vitro insulin resistance cell model was established with conditioned medium from co-cultured adipocytes and macrophages.These cell models were used to explore the effect and mechanism of C45 on glucose and lipid metabolism and inflammation.MethodsIn the first part,the effects of C45 on glucose uptake,glycogen synthesis,gluconeogenesis and glycolysis in hepatocytes were detected.The effect of C45 on differentiation and lipid metabolism of adipocytes and on the release of inflammatory cytokines from macrophages were measured.In vivo experiments,C57BL/6 male mice were randomly divided into lean mice and DIO mice groups.The lean mice and DIO mice were fed with normal chow diet and high fat diet for 12 weeks,respectively.Then the DIO mice were further randomly divided into without C45,with low dose C45,with middle dose C45,with high dose C45 and with metformin groups,respectively.C45 was administrated for 4 weeks along with high fat diet.Metformin was used as positive control.Mice fasting glucose level,glucose tolerance,markers of lipid metabolism,liver function,oxidative stress and inflammation were measured.Lipid deposition in the liver was detected by Oil red O staining and pathological examination.Macrophages in liver and liver fibrosis were detected by immunohistochemistry.Flow cytometry was used to evaluate systemic inflammation.ResultsIn the first part,in vitro results showed that C45 significantly increased glucose uptake,but decreased glycogen synthesis and gluconeogenesis without affect glycolysis in hepatocytes.C45 inhibited adipocytes differentiation and lipid metabolism.In addition,C45 significantly inhibited LPS-stimulated release of inflammatory cytokines from macrophages.In vivo results showed that C45 decreased body weight,liver weight and adipose tissue weight of DIO mice without affect food intake.C45 down-regulated fasting glucose level and improve glucose tolerance of DIO mice.DIO mice plasma TG,TC,NEFA,LDL,ALT,AST and lipid deposition were significantly reduced where as HLD was increased by C45.On the other hand,C45 significantly lowered TNF?,IL-1?,IL-6,MCP-1 levels and inflammatory neutrophils percentage in peripheral blood of DIO mice.Meanwhile,the amount of macrophages in DIO mice liver was reduced by C45.Liver fibrosis was relieved by C45.Moreover,plasma MDA and SOD levels were decreased by C45.All above effect of C45 was dose dependently.In the second part,primary adipocytes from lean mice and DIO mice were cultured under normoxia and hypoxia conditions,respectively.The conditioned medium(CM)were collected and used to incubated macrophages.Then the CMs from macrophages were collected.More CMs were collected from adipocytes,macropahges and co-cultured these two type cells under nomoxia and hypoxia conditions,respectively.All these CMs were used to incubate C2C12 skeletal muscle cells and AML12 hepatocytes,respectively.The levels of TNF? adn MCP-1 in CM were measured by ELISA.Glucose and lipid metabolism-related genes and HIF-1? gene expressions were measured by real-time quantitative PCR.The proteins related to glucose and lipid metabolism and inflammation,as well as AMPK phosphorylation were detected by Western blot.The results showed that obese and hypoxia increased the secretion of TNF? and MCP-1 from adipocytes.CM from these adipocytes promoted macrophages to secrete TNF? and MCP-1.Co-cultured 3T3-L1 adipocytes and RAW264.7 macrophages released more TNF? and MCP-1 than individual cultures.However,hypoxia did not affect macrophages release TNF? and MCP-1.CMs from macrophages incubated by adipocytes CMs and from co-cultured 2 type cells inhibited the phosphorylation of insulin signal molecules Akt,AS160,GSK-3? and Fox O1,but increased phosphorylation of IRS S306,IRS S636/639,JNK,S6 K and NF-?B.These CMs also increased lipid metabolism-related genes PPAR? and SREBP-1c expressions.C45 inhibited gene expressions of PPAR? and SREBP-1c in the liver and adipose tissue of DIO mice,as well as adipose tissue HIF-1? gene expression.C45 lowered the release of TNF? and MCP-1 from macrophages.C45 stimulated AMPK phosphorylation in DIO mice adipose tissue,liver,skeletal muscle and the in vitro cultured these cells.The effect of C45 on AMPK was inhibited by AMPK inhibitor Compound C.ConclusionTiliroside derivative C45 relieves glucose and lipid metabolism disorder and insulin resistance by ameliorates related gene expressions,kinase activities and ameliorates inflammation.AMPK is involved in this mechanism.