Effects Of Regulation By Medium-chain Fatty Acids On Lipid Metabolism And Their Mechanisms In C57BL/6J Obese Mice

Objective1.To confirm that medium-chain fatty acids (MCFA) as a natural source of small moleculefatty acids can effectively reduce body weight of obese mice, and improve lipid metabolism.2.To investigate the mechanism and the possible effective pathways and targets of MCFA inbody and offer the incidence for theory of MCFA regulating lipid metabolism.3.To observe the effect of caprylic acid and capric acid on body weight, body fat and lipidmetabolism of obese mice and to confirm the influence of MCFA on triglyceride andcholesterol metabolism.Methods1. Mouse obesity model100C57BL/6J male mice, aged4-5weeks, were used and were fed normal diet to adaptcircumstance one week. According to their fasting weight,15mice were randomly chosen tofeed normal diet (AIN-96G), which was used to be a control group. The other mice were fedhigh fat diet (HFD). The diet contains19.42%fat from the total weight and fat carloie is40.5%from total carloie. After four weeks feeding, the mice fed high-fat, weight higher than10%of individuals in the average body weight of the control group, weight distributionanalysis, randomly selected15mice in the distribution of peak frequency,as the obese group(HFD-1), weight less than control mice10%of individuals at the same time, after weightdistribution analysis, randomly selected15mice in the distribution of peak frequency range,set to the obesity resistance group (HFD-2). The body length, Lee's index, BMI, and weightgain during the study of mice as well as serum glucose, TG, TC and HDL-C and LDL-C,HDL-C/LDL-C ratio were measured and calculated. The liver, mesenteric fat fads,epididymal fat pads and perirenal fat fads were taken out and weighed. Another slice ofepididymal adipose tissue was HE stained. During the study, the body weights of the miceand diet consumption were measured, and food efficiency ratio (KJ/d) were calculated.2. Regulations of lipid metabolism of MCFA on the C57BL/6J obese miceThe obese model of mice was estabolished according to the methods of the first part ofthe experiment, and the obese mice were divided into three parts to carry out threeexperiments: acute gavage experiment, short-term gavage experiments and long-term feedingexperiments.(1) Acute gavage experiment36obese C57BL/6J male mice were randomly divided into2groups (n=18) according to the fasting weight, and were orally administered the MCT containing MCFA and LCTcontaining LCFA, which was a dose of2mg/kg. After1hour,2hours,4hours, respectively,6mice of each group were sacrificed, then, blood dsampling were taken from the abdominalaortic ateroia and serum TG, TC, HDL-C and LDL-C, and HDL-C/LDL-C ratio weremeasured.(2) short-term gavage experiment24C57BL/6J obese male mice were randomly divided into two groups (n=12) accordingto the fasting weight, and were orally administered MCT and LCT, which was a dose of2mg/kg daily for two weeks. During the study, mice were fed high fat diet, diet consumptionwas recorded and food efficiency ratio (KJ/d) were calculated. After two weeks, body weightwas weighed, and the body length, Lee's index, BMI, and weight gain were measured. SerumTG, TC and HDL-C and LDL-C, HDL-C/LDL-C ratio were measured and calculate, and liver,mesenteric fat pads, epididymal pads, perirenal fat pads were excised weighed. Another sliceof liver tissue was made into homogenates, and concentration of protein, TG, TC, ApoA1andApoB were determined and ApoA1/ApoB ratio was calculated.(3) Long-term feeding experiment30C57BL/6J obese mice were randomly divided into two groups (n=15) according tothe fasting weight, and were fed high fat diet with2%concentration of MCT or LCT. After12weeks, the same indicators used in the short-term experiment were determinated. At thesame time, the epididymal adipose tissue was kept for HE staining3. Study of mechanism of MCFA regulating lipid metabolism in C57BL/6J obese miceIn the long-term feeding experiment, blood samples were collected after12weeks, anda part of the liver tissue and epididymal adipose tissue were frozen, then, were made of10%concentration of tissue homogenates with0.9%sodium chloride injection. Theconcentrations of HSL, cAMP, PKA, FFA, GLY, NADR and T3in the serum, the levels ofHSL, ATGL, cAMP, PKA, LPL, FAS, ACC, Leptin, APN, PPAR-γ, TNF-α in adipose tissue,and the levels of LPL, FAS, the ACC, ME, G6PD, Leptin, APN, PPAR-γ, TNF-α in livertissue, were measured by ELISA methods. The protein concentration of the adipose tissueand liver tissue homogenates were determinated by BCA method. mRNA expression ofHSL, ATGL, UCP2, β3-AR, leptin, PPAR-γ, SREBP-1and C/EBP-α in adipose tissue weretested by Real-time PCR assay. The protein expression of β3-AR in adipose tissue weretested using Western blotting analysis. In addition, the HSL activities in serum and adiposetissue were determinated according to the reports of Fredrikson G and Belfrage P.4. Regulations of lipid metabolism by different MCFA on C57BL/6J obese miceThe same experiment was done as the long-term feeding experiment, which was usinghigh fat diet containing2%concentration of octanoic acid (C8), decanoic acid (C10) andoleic acid (C18). After eight weeks, according to above methods, index as followed weremeasured. They were fasting body weight weekly, consumption of diets, food efficiency ratio,body length of mice, Lee's index, BMI and weight gain, the weight of liver and adiposetissue, fat cell morphology, the concentrations of TG, TC, HDL-C, LDL-C, HDL-C/LDL-C in serum, the levels of HSL, ATGL, cAMP, PKA and Leptin, TNF-α, PPAR-γ, and mRNAexpression of HSL, ATGL and β3-AR in adipose tissue, the levels of ApoA1, ApoB, LPL,FAS, CYP7A1, HMGCoA, TNF-α, and mRNA expression of CYP7A1and HMGCoA inliver tissue.Results1. Mouse obesity modelAt the end of study, the body weight of mice, the body length, Lee's index, BMI, andweight gain, the weight of liver and adipose tissue, the concentrations of serum glucose, TC,LDL-C in the HFD-1group were significantly higher than that of in the HFD-2group and thecontrol group (P<0.05). The ratio HDL-C/LDL-C in the HFD-1group was significantlylower than that of in the HFD-2group and the control group (P<0.05). Results of fat cellmorphology analysis showed that cell diameter, short diameter in the HFD-1group weresignificantly greater, and the number of fat cells of a single field of vision was less, than thatof in the HFD-2group and the control group (P<0.05). Obese mice model in the HFD-1group was successfully established.2. Regulations of lipid metabolism of MCFA on the C57BL/6J obese miceThe results of acute gavage experiment showed that mice fed the MCT or LCT after twohours and four hours, the concentration of serum TG in the MCT group was significantlylower than that of in the LCT group (P<0.05), and other blood lipid-related indicators werenot shown significant differences (P>0.05). The results of short-term gavage experimentshowed that the body weight of mice, the index of blood lipids, the indicators of liverhomogenates were not shown significant differences between MCT group and LCT groupafter two weeks (P>0.05). The results of long-term feeding experiment showed that the bodyweight of mice, body length, Lee's index, BMI, and weight gain, liver weight, the weight ofperirenal and epididymal adipose tissue, the concentrations of serum TG, TC and LDL-C inthe MCT diet group were significantly lower, while the levels of serum HDL-C andHDL-C/LDL-C ratio, the concentrations of ApoA1of liver homogenates, and ApoA1/ApoBratio, were significantly higher than that of in the LCT diet group (P<0.05). The results of fatcell morphology analysis showed that cell diameter and short diameter of adipose tissue weresignificantly lower, and the number of cells of a single field of vision was significantly higherin the MCT group than that of in the LCT group (P<0.05).3. Study of mechanism of MCFA regulating lipid metabolism in C57BL/6J obese miceThe results of ELISA analysis showed that the levels of serum HSL and NADR, LPLlevel of liver tissue, ATGL, HSL and cAMP levels of adipose tissue of C57BL/6J obese micein MCT group were significantly higher, and serum FFA concentration, the levels of FAS,leptin and TNF-α of adipose tissue in MCT group were significantly lower than that of inLCT group (P<0.05). The indicators of PPAR-γ of adipose tissue, FAS, ACC, Leptin, APNand TNF-α of liver were not shown significant differences between the two groups (P>0.05).mRNA expression of ATGL, HSL, UCP2, β3-AR in adipose tissue were significantly higher, and mRNA expression of leptin, SREBP-1and C/EBP-α in adipose tissue were significantlylower in MCT group than that of in LCT group(P<0.05), and no significant differenceswere shown in PPAR-γ mRNA expression between MCT and LCT group. β3-AR proteinexpression in adipose tissue was significantly higher in MCT group than in LCT group(P<0.01). In addition, the results of HSL activities assay showed that HSL activities inadipose tissue in the MCT group were significantly higher than in the LCT group (P<0.01),while HSL activities in the serum, were not shown significant differences between the twogroups (P>0.05).4. Regulations of lipid metabolism by different MCFA on C57BL/6J obese miceC57BL/6J mice were fed high fat diet containing C8, C10or C18fatty acids after8weeks, in the C10group, the body weight of mice, Lee's index, BMI, and weight gain, theweight of epididymal adipose tissue were significantly reduced, fat cell volume wassignificantly reduced by analysis of morphology, such as cell length and short diameter weresignificantly decreased, while the average number of cells of a single field of visionsignificantly increased, moreover, the concentrations of serum TG, the levels of ATGL, HSL,cAMP in adipose tissue were significantly increased, the levels of leptin and TNF-α inadipase tissue and FAS level in liver were significantly decreased, compared with the C18group (P<0.05). mRNA expression of ATGL, HSL, and β3-AR of adipose tissue in the C10group were significantly higher than that of in the C18group(P<0.05).In the C8group, the concentrations of serum TC and LDL-C were significantly lower,and HDL-C/LDL-C ratio of serum, ApoA1concentration and ApoA1/ApoB ratio, the level ofCYP7A1in liver tissue were significantly higher than that of in the C18group (P<0.05). Inaddition, mRNA expression of CYP7A1in liver tissue in the C8group were significantlyincreased compared with the C10and C18groups (P<0.05). mRNA expression ofHMGCoA of liver was not shown any significant differences between the three groups(P>0.05).Conclusions1. Medium-chain fatty acids (MCFA) as a natural source of small molecule nutrients caneffectively reduce body weight, reduce body fat accumulation, and improve blood lipids,blood cholesterol levels of obese mice.2. Its mechanism may be:(1) MCFAcould increase body energy consumption due to its unique metabolic pathways,then, it could increase the activities of the sympathetic nervous system, and thus increasedrelease of the peripheral NADR, which activated the expression of β3-AR on the fat cellmembrane, and leaded to increasing of expression of UCP2and enhancing the activities andlevels of enzymes related triglyceride metabolism, such as of ATGL and HSL. These couldaccelerate fat mobilization of body, and contribute to the decomposition of adipose tissue, asresults.(2) The role of activation energy metabolism was not achieved by stimulating leptin. (3) MCFA may inhibit fat cell differentiation pathway, reduce the aggregation of fat cellsby down-regulating the levels and mRNA expression of TNF-α, SREBP-1and C/EBP-α inadipose tissue, thus improve the body's lipid metabolism disorders.(4) It was uncertain that MCFA should regulate the pathway of fatty acid synthesis andexpression of adipokines in the liver, and regulatory role of leptin, APN, and PPAR-γpathway.3. Octanoic acid (C8) and decanoic acid (C10) both could be effective in improving thelevels of blood lipids and blood cholesterol of obese mice, but octanoic acid was mainlyregulation of cholesterol metabolism in mice by increasing hepatic CYP7A1expression,while, decanoic acid mainly regulated triglyceride metabolism in mice by and increasing theexpression levels of β3-AR, ATGL and HSL in adipose tissue. The synergistic effect ofoctanoic acid and decanoic acid may be one of the mechanisms of the MCFA reducing bodyweight and improving blood lipids.