Study On The Regulatory Mechanism Of Raspberry Ketone On Obesity In Mice Based On Brown Adipose Tissue Thermogenesis And Gut Microecology

The role of plant compounds in controlling obesity has received widespread attention in the fields of food,nutrition,and medicine.Red raspberry is a dual-use plant widely grown in China for medicine and food.One of its functional ingredients,raspberry ketone（RK）,can be used as essence and spice in China.In the United States,it is a "GRAS" certified dietary supplement.At present,the products containing raspberry ketone on the market include perfume,drinks and dietary supplements.The weight control effect of RK first attracted scientists’attention in 2005.On one hand,it has been reported that the addition of RK（2%,10 weeks）in mouse diet significantly decreased mouse body weight,compared to high-fat diet（HFD）-fed mice.In line with this study,two other studies used HFD induced obesity model,and results indicated that RK（55 or 200 mg/kg,gavage daily for 4 or 8 weeks,respectively）reduced body wight as well as inhibiting oxidative stress,or increasing fat oxidation.However,some other studies suggested that the weight control effect of RK was mainly due to the reduction of food intake,and RK（1.74%,w/w）could not reduce the body weight of mice on a high-fat diet.Therefore,the weight control effect of RK is still controversial,and the underlying mechanism is very limited.In order to explore the mechanism of RK in weight control,this study first detect the main metabolites and their distribution of RK in mice using UPLC-QqQ/MS.Then,a HFD-induced obesity C57BL/6J mouse model was used to explore the role of the main metabolites of RK in weight control,aimed to clarify the material basis of RK in weight control.Further experimental methods such as metabolic cages,infrared thermography,and subcutaneous implantation of temperature sensors were used to investigate the role of RK in promoting thermogenesis in mice.By constructing wild-type and mutant PPARα plasmids,combined with computer simulations and cell models,this study investigated the effects of RK on PPARα/FGF21 signal axis,and its effects on thermogenesis.Finally,the FGF21 whole body gene knockout mouse model was used to verify this signal axis.The main results are as follows:（1）The main metabolites of RK in mice include tyrosol,zingerone,rhododendrol,etc.RK and its main metabolites can be detected in liver,brain,white adipose tissue and brown adipose tissue.After a single oral administration of 200 mg/kg RK,the peak plasma concentration was 276.91±42.95 nmol/mL,and among the metabolites,rhododendrol had the highest plasma concentration of 100.88±19.54 nmol/mL.（2）Tyrosol,zingerone and rhododendrol can effectively control body weight of obese mice.Mechanistically,we found that tyrosol and zingerone can promote brown adipose tissue thermogenesis and increase inguinal white adipose tissue browning to control body weight.Rhododendrol can change the composition of liver metabolites in mice,including enhancing the abundance of metabolites related to alanine,aspartate and glutamate metabolism,and arginine and proline metabolism to alleviate nonalcoholic fatty liver disease（NAFLD）in obese mice.In addition,the three metabolites can change the gut microbiota structure of mice,including increasing the abundance of Bacteroides etc.Therefore,these results showed that the molecular basis for raspberry ketone exerting weight loss effects includes itself and these three major metabolites.（3）Raspberry ketone can reduce the body weight of obese mice by increasing the expression of thermogenic genes and proteins and increasing energy expenditure.After 16 weeks of dietary intervention,RK（0.2%,w/w）significantly reduced body weight in obese mice（31.32±0.45 g vs.HFD35.14±0.62 g,p<0.05）,decreased the epididymal adipose tissue weight（RK 0.78±0.25g vs.HFD 1.19±0.25g,p<0.05）,reduced the cell area of BAT,iWAT and epididymal adipose tissue（eWAT）,reduced the lipid content in liver（RK41.61±6.85%vs.HFD81.29±5.26%,p<0.05）,improved blood glucose and lipid indexes,and did not affect the food intake of mice.Raspberry ketone increased energy expenditure（RK 14.56±1.04 kcal/h/kg vs.HFD11.62±1.26 kcal/h/kg,p<0.05）,BAT temperature and core body temperature in mice.RT-qPCR results showed that the UCP1 gene expression levels were increased by 1.4 times（p<0.05）in BAT and 5.0 times（p<0.05）in iWAT respectively,after gavage of RK（200 mg/kg/d）for 3 consecutive days.Meanwhile,through RT-qPCR and Western Blotting experiments,it was found that after 16 weeks of RK diet（0.2%,w/w）intervention,the expressions levels of thermogenic genes and proteins in BAT and iWAT,such as UCP1,PGC-1α,DIO2 and PRDM16 were significantly higher than those in HFD group（p<0.05）.（4）RT-qPCR assay showed that raspberry ketone significantly increased FGF21 gene expression level in Huh7 cells（3.9 times,p<0.05）.At the same time,the expression level of FGF21 gene in liver of mice was increased by 7.1 times（p<0.05）after single administration of raspberry ketone（200 mg/kg）.The expression level of FGF21 gene in the liver of mice was also increased by 2.5 times（p<0.05）after 16 weeks of diet containing raspberry ketone（0.2%,w/w）.ELISA showed that single gavage（200 mg/kg）or 16 weeks of dietary intervention with raspberry ketone（0.2%,w/w）could increase the plasma FGF21 concentration of mice（8.0 and 1.78 times,respectively,p<0.05）.Using FGF21 knockout mice model,we found that feeding with high-fat diet or high-fat plus RK diet 16 weeks have no influence on whole body weight,liver weight,epididymal adipose tissue weight and glucose tolerance of mice.There was no significant difference in energy expenditure and oxygen consumption neither between the two groups.Further RT-qPCR and Western Blotting results indicates that,no significant difference was found between two groups in genes and proteins expression levels,such as UCPland PGC-1α.These results indicate that FGF21 plays central role in raspberry ketone combating obesity.（5）Raspberry ketone activates the PPARα/FGF21 signaling pathway.FGF21 is mainly regulated by PPARα.The cellular thermal shift assay was conducted on the liver of mice given RK for 3 consecutive days.It was found that RK could enhance the thermal stability of PPARα protein,and the cellular thermal curve shifted to the right,indicating that RK could bind to PPARα protein.Meanwhile,luciferase assay was performed on HepG2 cells transfected with PPARα plasmid,and the fluorescence value of RK group was 3.10 times higher than that of control group（p<0.05）,which also confirmed that RK can bind to PPARα and promote the transcription of its downstream genes.The results of molecular docking showed that RK could interfere with the amino acid residues of ASN219,MET220,THR279 and TYR334 of PPARα.After specific deletion of ASN219 and MET220 amino acids in PPARα plasmid,it was found that the transcriptional promoting effect of RK was significantly decreased.These results indicate that RK can act as a ligand to bind PPARα and affect downstream signaling molecule transcription.（6）Through 16S rDNA sequencing,it was found that RK could change the structure of gut microbiota in mice.At the phylum level,RK significantly reduced the relative abundance of Firmicutes（RK 47.86±7.5%vs.HFD 80.1±12.2%）,increased the abundance of Bacteroides（RK 18.71±5.9 vs.2.8±2.0%）and Verrucomicrobiota（RK 9.45±4.52%vs.HFD 2.30±1.22%）（p<0.05）compared with the high-fat diet group.At the species level,RK significantly increased Lachnospiraceae_bacterium₂8-4（RK 5.05±3.74%vs.HFD 0.15±0.17%）and Bacteroides_sartorii（RK 3.55±2.75%vs.HFD 0.48±0.50%）and reduced the relative abundance of Lachnospiraceae_bacterium_DW59（RK 0.68±0.31%vs.HFD 4.24±2.23%）（p<0.05）.Through the fecal microbiota transplantation（FMT,rectal perfusion）,it was found that,after 2 or 3 weeks of transplantation,the body weight of the recipient mice receiving the RK diet decreased significantly,but at the 4th to 7th week of the experiment,the body weight was unchanged by FMT.Through T-test analysis of the relative abundance of gut microbiota,it was found that Bacteroides and Lachnospiraceae bacterium 28-4 increased significantly in the third week of fecal bacteria transplantation,suggesting that these two microbiomes may be involved in the regulation of RK on the weight of obese mice.In summary,this paper systematically studies the molecular mechanism of RK on weight control.In terms of material basis,the main metabolites of RK can effectively control the weight gain of mice caused by high-fat diet.Among them,tyrosol and zingerone reduced the body weight through increasing the expression levels of genes and proteins related to thermogenesis in BAT and iWAT.While rhododendrol mainly changes the composition of liver metabolites in mice,including increased the abundance of metabolites related to alanine,aspartate and glutamate metabolism.Meanwhile,all the three metabolites can change the gut microbiota structure of mice.Further,we investigated the upstream molecular mechanism of RK on weight control,and found that RK can act as a ligand for PPARα,and promote BAT thermogenesis,increase energy expenditure,and thus reduce weight through PPARα/FGF21 signaling axis.Through fecal transplantation experiments,we found that Bacteroides and Lachnospiraceae bacterium 28-4 may contribute to the weight control effect of RK.