Effect And Mechanism Of Five Coptis Monomer On Glucose Uptake And Fatty Acid Oxidation In3T3-LI Adipocytes

Background and ObjectiveDiabetes accompanied by hyperlipidemia is an endocrine metabolic disease which isdue to absolute or relative insulin deficiency,In traditional Chinese medicine, diabetes is ascope of Diabetes, the ancient medical books recorded a large number of traditionalChinese medicine and the prescription of the treatment of Diabetes, the current studiesfound that most of these Chinese herbal formula containing Coptis or its main ingredientCoptis alkaloid..Coptis is the Ranunculaceae perennial herb, Its roots has the efficacy oflowering blood glucose and lipid regulating. Modern medical researches shown that theeffect of the Coptis’s hypoglycemic and lipid regulating is related to its roots whichcontained berberine,jatrorrhizine table berberine,.palmatine, coptisine and so on. Berberineis the role of the most studied Coptis alkaloid, It can improve hyperlipidemia and insulinresistance. Chemical analysis of the medicine shown that jatrorrhizine,table berberine,palmatine,and coptisine are isoquinoline alkaloids, which are homologue with berberine,Ithas been reported that their chemical structure are similar to berberine,aiso they havesimilar biological functions with berberine, but its specific mechanism of action is unclear.Peroxisome proliferators-activated receptors (PPARs) are members of theligand-activated nuclear transcription factor superfamily,If they binding to the ligand theirtranscriptional activity will change, and then the PPARs target gene expression change.thereby causing lipogenesis, lipid metabolism, insulin resistance, impaired glucosetolerance, obesity and a series of physiological activities. The liver X receptor а (LXRа)have been found a dozen target genes, most of which are closely related to glucosemetabolism, inflammation, cell proliferation and lipid metabolism, It suggesting that theLXRа may be extensively involved in lipid quality of metabolic disorders, type2diabetesand atherosclerosis and other diseases’s occur.In this study we aimed to investigate the effect and mechanism of Coptis monomers on glucose uptaking as well as fatty acid oxidation in the3T3-L1adipocytes and theexpression changes of PPASs、LXRα’s mRNA and protein. Furthermore, to clear thepossible mechanisms of glucose and lipid metabolism, In order to provide the experimentalbasis for the clinical application of the Alkaloids of Coptis chinensis.Methods1.3T3-L1preadipocytes is cultured according to recommended method on ATCC anddifferentiated into adipocytes induced by IBMX (with final concentration of0.5mM),dexamethasone (with final concentration of1μM) and insulin (with final concentration of10mg/L). Differentiation of preadipocytes to mature adipocytes was confirmed byobservation via microscope and by Oil Red O staining of lipid vesicles.2. The differentiated cells were cultured in96well plates and induced for12h,24h,48h and72h,including five concentrations of Coptis monomer(265.65μmol/L、53.75μmol/L、10.75μmol/L、2.15μmol/L、0.45μmol/L). In addition, blank and PBS wereused as negative controls. Finally, OD values were detected after phagocytosis MTT andeach group were repeated at least three times.3. The glucose uptaking was detected by [3H]-labeled deoxyglucose,, and fatty acidoxidation was measured using14C-labected palmitic with optimal concentration of Coptismonomer in3T3-L1adipocytes after cocultured for48h. Please note that all theassumptions of experiments were the same except the PBS solution was set to be thebaseline4. The3T3-L1adipocytes were cultured with Coptis monomers for48h with optimalconcentration And then The genetic expression of PPARs and LXRα were described byreal-time PCR, and the proteins expression were presented by Western Blot. Please notethat all the assumptions of experiments were the same except the PBS solution was set to bethe baselineResults1. The culture, differentiation and identification of3T3-L1preadipocyte: During theinduction and differentiation process of3T3-L1preadipocytes into mature adipocytes, Itwas found that cytomorphology get round with cytoplasmic lipid droplets turning to largerand round. Finally the fat drips were stained by oil red O after induction of differentiationby9-10days. 2. The preferable reaction time for coculture with3T3-L1adipocytes of Coptismonomers are48hours, while the optimal concentration of berberine and table berberineare2.15μmol/L,.palmatine and coptisine are10.75μmol/L,jatrorrhizine is0.45μmol/L3. Glucose uptake in3T3-L1adipocytes is promoted by insulin. Insulin-stimulatingglucose uptake in3T3-L1adipocytes in vitro was promoted by berberine and tableberberine and palmatine but not by coptisine and jatrorrhizine4. Fatty acid oxidation in3T3-L1adipocytes wasn’t significantly promoted by tableberberine、palmatine and coptisine.. On the contrary, it was promoted by berberine andjatrorrhizine5. The results of the genes expression and protein expression of PPARs,LXRα in3T3-L1adipocytes coculture with Coptis monomers for48h:(1) The results of real-time PCR： Compared with PBS control group,the geneexpression of PPARα and β were significantly up-regulated in3T3-L1adipocytes by fiveCoptis monomers,but LXRα is up-regulated by berberine and jatrorrhizine. The geneexpression of PPARγ was significantly up-regulated by berberine、table berberine andpalmatine(2) The results of protein expression：Compared with PBS control group,berberine andtable berberine can significantly promote the protein expression of PPARs and LXRα in3T3-L1adipocytes. While jatrorrhizine can obviously promote the protein expression ofPPARα, PPARβ and LXRα. However, palmatine can up-regulate the protein expression ofPPARβ and PPARγ.Conclusion:1. The preferable reaction time for3T3-L1adipocytes of five Coptis monomers are48hours,while the optimal concentration of berberine and table berberine are2.15μmol/L,.palmatine and coptisine are10.75μmol/L,jatrorrhizine is0.45μmol/L.2. Berberine、table berberine and palmatine can promote the glucose uptaking in3T3-L1adipocytes, the main mechanism of berberine may be attribute to upregulationmRNA and protin of PPARs and LXRα.While table berberine and palmatine play an criticalrole on uptake of glucose in3T3-L1adipocytes in vitro, by way of upregulating the mRNAand protein expression of PPARα, PPARβ and PPARγ.3. Berberine and jatrorrhizine can promote the fatty acid oxidation in3T3-L1 adipocytes. Berberine may be associated with the up-regulation of PPARs and LXRα..While jatrorrhizine by way of enhancing the expression of PPARα, PPARγ and LXRα.