Effects Of Whey Protein On Combating Obesity And Potential Mechanisms

Overweight and obesity are strong predictors of the development of chronic metabolic diseases.Excessive accumulation of fat contributes to the activation of the immune response and inflammation,which leads to insulin resistance.Recently,whey protein(WP)with bioactive properties has been suggested to play an important role in combating obesity.Therefore,it is of great clinical significance to investigate the effect of whey protein on lipid metabolism,inflammation and insulin sensitivity in overweight and obese subjects as well as the potential mechanisms.Objective:The aim of this study was to determine the effect of whey protein on body composition,lipid metabolism,insulin sensitivity and inflammation in Chinese overweight/obesity subjects.Then through animal experiments,to explore the underlying mechanisms,including regulatory effects of whey protein on adipose tissue lipolysis and the modulation on hepatic lipid metabolism,immune response and inflammation related signaling pathways in high-fat diet fed mice,and to further address mechanisms about whey protein effects on insulin sensitivity.Methods:1.Randomized controlled trialTwenty-nine Overweight/obesity patients were randomly divided into whey protein intervention group(consuming 2×15g whey protein/d)and control group(consuming 2×15g maltodextrin/d)for 12 wk.During intervention,we examined following indexes: body weight,body composition,fasting blood glucose and lipid profiles,serum insulin,HOMA-IR,and the inflammatory status,as well as blood pressure and dietary survey.2.Animal experimentsExperiment 1: The preventive effect of whey protein on high-fat diet fed mice: sixty 5-6 week-old Male C57BL/6J mice were randomly divided into four groups that were fed either a standard diet(Control group)or a high-fat diet(HFD group)and that were supplemented with either 12.5% WP(HFD+12.5%WP group)or 25% WP(HFD+25%WP group)for 12 weeks.Experiment 2: The treatment effect of whey protein on obese model mice: forty-five 5-6 week-old Male C57BL/6J mice were fed a high-fat diet for 12 weeks to establish the obesity mice.Then obesity mice were randomly divided into three groups that were fed either a standard diet(Control group)or a standard diet supplemented with either 12.5% WP(12.5%WP group)or 25% WP(25%WP group)for 12 weeks.At the end of these two experiment,IPGTT,ITT,blood biochemical index and chronic inflammation factors were measured.After comparing the preventive and therapeutic effects of whey protein on high-fat diet fed mice,we selected the animal experiment with obvious interventional effect to carry out the further study.Further mechanism study included:(1)Effect of whey protein on adipose tissue lipolysis: Adipose tissue explants was incubated in vitro,and isoproterenol stimulated lipolysis were determined;(2)We conducted an isobaric tags for relative and absolute quantification(iTRAQ)proteomic analysis to identify whey protein regulated differential proteins and metabolic pathways that control the lipid metabolism and inflammatory response in liver tissue;(3)Western blot was used to detect the regulation of whey protein on HMGB1-TLR4 signaling pathway;(4)Western blot was used to detect the regulation of whey protein on insulin signaling pathway by modulating Gal3.Results:1.Randomized control trial(1)After 12 weeks,mean changes of waist circumference,body fat,percentage of body fat and visceral fat area of overweight/obesity patients were clinically significant between whey protein group and control group.(2)The within group mean changes of TC and LDL-R of whey protein group were significant;However,none of the lipid profile was significantly different between the groups.(3)After 12 weeks,the mean changes of serum insulin was significant between whey protein group and control group.(4)After whey protein intervention,the increase of Gal3 was significantly inhibited in whey protein group.(5)Before whey protein intervention,a positive correlation was found between visceral fat area and serum insulin,and between visceral fat area and Gal3.After whey protein intervention,no significant correlation was found.2.Animal experiment(1)Comparison of the preventive effect of whey protein on high-fat diet fed mice and the treatment effect of whey protein on obese model mice,the preventive effect of WP on HFD-fed mice was more effective.Therefore,subsequent mechanism studies will be conducted based on Experiment 1.(2)In epididymal adipose tissue(EPI),non-esterified fatty acid(NEFA)secretion was significantly increased in HFD+12.5%WP group and HFD+25%WP group after isoproterenol stimulated;In subcutaneous adipose tissue(SC),glycerol and NEFA were both significantly increased in HFD+25%WP group after isoproterenol stimulated.(3)In iTRAQ proteomic analysis,differential proteins of PKA and LDLR were up-regulated significantly while aPKC,IKKα and IκB were down-regulated significantly by WP,which participated in the regulation of lipogenesis,lipolysis,cholesterol metabolism and inflammatory response related signaling pathway.(4)The regulation of whey protein on HMGB1-TLR4 signaling pathway: Whey protein significantly reduced the HFD-associated increase in the liver tissue protein expression of HMGB1,TLR4,IL-1β and TNF-α.(5)The regulation of whey protein on insulin signaling pathway by inhibiting Gal3: There was a marked increased expression of Gal3 protein in HFD group and a corresponding decrease expression in insulin receptor substrate 1(IRS-1),insulin receptor substrate 2(IRS-2)and AKT phosphorylation.After WP intervention,a significant decrease in Gal3 was detected,causing increased expressions in IRS-1,IRS-2 and AKT phosphorylation.Conclusions:1.In the randomized control trial,whey protein regulates the waist circumference and body composition effectively in overweight/obese people,and there is also a beneficial effect on insulin levels and inflammatory status.2.Potential mechanisms: WP intervention improves lipolysis disorder in adipose tissue caused by HFD.3.In iTRAQ proteomic analysis,WP intervention inhibits the hepatic lipid accumulation by regulating differential proteins of PKA,aPKC and LDLR,and inhibits the inflammatory response via HMGB1-TLR4-NF-κB signaling pathway.In addition,WP improves insulin sensitivity by downregulating Gal3.