Browning Of 3T3-L1 Preadipocytes Induced By Fermented Barley Protein And Its Mechanism

Obesity caused by unhealthy lifestyles and dietary imbalances has become a enormous burden for society.An imbalance between energy intake and energy consumption leads to an abnormal increase in body fat.However,there is a kind of brown fat containing high mitochondria content in mammals,which leads to a direct conversion of nutrient energy into heat via the uncoupling protein 1(UCP1).Recent studies have found that white fat can undergo browning with certain environmental and material stimulations,thus having certain characteristics of brown fat.Our previous study found that obese rats that were ingested with lactic acid bacteria fermented barley extract had significantly lower serum triglycerides and cholesterol,and brown fat also increased.Cell tests showed that the major component inhibiting fat synthesis was the protein in the fermentation extract,but it is still unclear whether it can cause browning of white fat.In this study,barley was fermented by Lactobacillus plantarum dy-1 to isolate and purify the protein(LFBE-P)from the fermented barley extract and its effect on the browning of 3T3-L1 preadipocytes was studied.Then we select trait evaluation indicators,screen the active ingredients in LFBE-P,and explore the mechanisms that induce the browning of 3T3-L1 preadipocytes.The main research contents and results are as follows:1.The separation method of LFBE-P and the difference before and after fermentation were studied.Coomassie brilliant blue method and Kjeldahl method were used to determine the protein content of the extracts before and after barley fermentation.Proteins were separated by ammonium sulfate fractionation.Proteins were analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE)before and after barley fermentation.The results showed that the recovery of LFBE-P and unfermented barley protein extract(RBE-P)by ammonium sulfate fractionation was 84.6% and 73.2%,respectively;the relative molecular mass of RBE-P was between 20 and 120 kDa,and for LFBE is mainly below 45 kDa.2.The effect of LFBE-P on the browning of 3T3-L1 preadipocytes was studied.Cell viability was measured by CCK-8;glucose consumption of cells was measured by glucose oxidase method;relative gene expression was detected by real-time fluorescence quantitative PCR(RT-PCR).The results showed that the survival rate(at a concentration of 40?g/mL for LFBE-P)of 3T3-L1 preadipocytes at 24 h and 48 h were 81% and 69%,respectively.The survival rates(at a concentration of 80?g/m L for RBE-P)of 3T3-L1 preadipocytes at 24 h and 48 h were 93.3% and 69.7%,respectively.LFBE-P could increase the glucose consumption of 3T3-L1 preadipocytes,and the glucose consumption of cells was increased by 80% compared with the control group.At the same time,LFBE-P could up-regulate the expression of the brown specific genes and cause the browning of 3T3-L1 preadipocytes,but RBE-P had no obvious effect.3.LFBE-P was isolated and purified,and the high activity component that induced the browning of 3T3-L1 preadipocytes was screened and its effect on the browning of 3T3-L1 preadipocytes was examined.We isolated and purified proteins by ultrafiltration,anion exchange chromatography and gel chromatography to obtain the most active LFBE-P4 fraction.The relative gene expression was detected by real-time fluorescence quantitative PCR(RT-PCR).The effect of LFBE-P4 on the differentiation of 3T3-L1 preadipocytes was examined by oil red O staining;triglyceride content was detected by the TG assay kit;and glucose was measured by the glucose oxidase method.The mitochondria conten was studied by real-time fluorescent quantitative PCR to investigate the effect of Cytochrome B regulation.The results showed that the protein with a relative molecular mass of 3-20 kDa has the most obvious effect inducing the browning of 3T3-L1 preadipocytes;the activity of P2 and P3 in the elution peak of anion exchange was the largest;after the gel column chromatography,the protein peak SP4 with the highest activity component is named LFBE-P4;LFBE-P4 can inhibit the differentiation of 3T3-L1 preadipocytes with a differentiation rate of only 53.2%.It also inhibits the anabolism of cellular fat and reduces intracellular triglyceride content;LFBE-P4 can significantly promote cell catabolism and increase cell glucose consumption;LFBE-P4 can significantly increase the relative content of mitochondria,thereby inducing browning of 3T3-L1 preadipocytes.4.The molecular mechanism of LFBE-P4 inducing white fat browning.The effect of LFBE-P4 on gene expression at different stages of differentiation of 3T3-L1 preadipocytes was analyzed by real-time fluorescence quantitative PCR;Western blot was used to analyze the expression of UCP1 protein in the cells.The results showed that LFBE-P4 could promote the expression of Pgc-1? and Ucp1 genes in 3T3-L1 preadipocytes.During the differentiation of 3T3-L1 preadipocytes,LFBE-P4 had a significant up-regulation effect on most brown genes.It can significantly down-regulate the expression of white fat adipocyte genes including Cidea and C/ebp?.For 3T3-L1 mature adipocytes,LFBE-P4 can significantly up-regulate browning-related genes,and the expression of UCP1 is increased about 5 times(the concentration of LFBE-P4 is at 15 ?g/mL).