Studies On The Alleviation Of Casein Hydrolysates And VPP/IPP Analogues In Insulin Resistance And Inflammation In High-fat Diet-induced Obesity Mice

In recent years,due to the change of modern lifestyle and dietary habits,obesity has become a major health risk factor.Obesity induced chronic inflammation and insulin resistance are mainly attributed to excessive and ectopic accumulation of fat in the body,especially in the viscera,caused by abnormal body energy metabolism.The major obesity-associated complications include metabolic syndrome,T2 DM,cardiovascular disease,hypertension and hyperlipidemia,etc.At present,traditional methods to improve inflammation and insulin resistance all have some side effects or certain limitations.Bovine casein as one of the important sources of high-quality natural protein has been widely used to obtain and produce various bioactive peptides.VPP and IPP,two bioactive tripeptides identified from casein hydrolysate(CH)showed significant ACE inhibitory activity in vitro and antihypertensive effects in vivo,as well as improving adipocyte inflammation and insulin resistance.In addition,the structureactivity relationship of peptides has been widely applied to discover more new peptides with strong activity such as ACE inhibitory activity from foodborne proteins.Therefore,it is of great importance to study the effect of CH containing VPP and IPP on alleviating and improving obesity-induced chronic inflammation and insulin resistance,and to try to obtain potent bioactive peptides with better anti-inflammatory and anti-insulin resistant activities through structure and activity relationship.At the beginning of the project,Casein was used as the object of study.VPP and IPP were firstly released from bovine casein in vitro by enzymatic hydrolysis and their concentrations were determined.The effects of CH containing VPP and IPP on impaired glucose tolerance,insulin resistance and chronic inflammation in high-fat diet-induced obese mice were studied.Next,we established an inflammatory model of preadipocyte differentiation by stimulating 3T3-L1 cells with TNF-α,and studied the amelioration mechanism of CH on impaired differentiation and chronic inflammation in 3T3-L1 cells.Then,in order to find tripeptide analogues with potential hypoglycemic activity,the structure-activity relationship of VPP/IPP analogs in promoting glucose uptake in L6 myotube was studied.Finally,high-fat diet induced obese mice model was established again to further study the intervention effects of VPP/IPP analogues on insulin resistance and chronic inflammation in mice;and by exploring the effects of dietary intervention on intestinal flora,the possible mechanisms of VPP/IPP analogues on improving insulin resistance and chronic inflammation induced by obesity were studied.The major conclusions of this research are as follows:1.By hydrolyzing casein with Aspergillus oryza-derived protease A2 SD,LCMS/MS technique was used for VPP and IPP identification and content determination in CH.It was found that the content of IPP in CH was higher than VPP,the contents were 926.69 ± 1.58 ng/mg dry base(VPP)and 184.93 ± 1.32 ng/mg dry base(IPP).CH containing VPP and IPP did not reduce the body weight of obese mice and significantly improve the dyslipidemia.CH did not play a hypoglycemic role and improve the impaired glucose tolerance in obese mice induced by high fat diet.However,CH could significantly alleviate macrophage infiltration in adipose tissue and reduce the contents of pro-inflammatory cytokines IL-6,IL-1β,MCP-1 and TNFα in serum of obese mice,moreover,CH could increase the insulin sensitivity but could not alleviate the insulin resistance in HFD-induced obese mice.2.CH containing IPP and VPP could significantly promote the growth and proliferation of 3T3-L1 preadipocytes.CH attenuated TNF-α-induced 3T3-L1 preadipocyte dysdifferentiation by activating C/EBPα rather than PPARγ,and attenuated TNF-α-induced 3T3-L1 preadipocyte inflammation by inhibiting NF-κB p65 phosphorylation and JNK phosphorylation.In addition,CH promoted the normal differentiation of 3T3-L1 preadipocytes through activation of MAPK-Erk signaling pathway in a dose-dependent manner.3.CH and VPP/IPP analogues had no significant toxic effect on the L6 myoblasts.The structure-activity relationship of peptide analogues supported that replacement of either the middle amino acid or the C-terminal amino acid of VPP and IPP with any other amino acids,especially negatively charged amino acids and their respective amides,could substantially improve the activity;the presence of P either in the middle position or the C-terminal position impaired the glucose uptake activity of peptide analogues.Four tripeptide analogues IQP,VEP,IPQ and VPE showed relatively high activity in promoting glucose uptake,and mainly existed in cereal storage proteins.A2 SD and pepsin hydrolysis could release these tripeptide analogs from rice protein and wheat protein successfully.In addition,the glucose uptake activities of these peptides are via phosphorylation of AMPK,independent of insulin signaling pathways.4.VPP/IPP analogs IQP,VEP and IPQ could not reduce the weight of obese mice induced by high fat diet,but IQP,VEP and IPQ could all reduce the levels of glucose and insulin in serum of obese mice.IQP promoted glucose uptake of obese mice via restoring Akt and AMPK signaling pathway,and VEP promoted glucose uptake of obese mice via restoring AMPK signaling pathway,and improved the insulin tolerance of obese mice,and significantly reduced the insulin resistance index of obese mice.Both VEP and IPQ could improve the dyslipidemia and alleviate the adipose tissue hypertrophy and liver steatosis,and IPQ was more effective.IQP,VEP and IPQ could all significantly alleviate macrophage infiltration in adipose tissue and reduce the contents of pro-inflammatory cytokines IL-6,IL-1β,MCP-1 and TNFα in serum of obese mice,the anti-inflammatory effect was IPQ > VEP > IQP.5.Both the formation of obesity in mice and the positive role of VPP/IPP analogues IQP,VEP and IPQ in the improvement of insulin resistance and chronic inflammation were associated with the change of gut microbiota.IQP,VEP and IPQ could significantly reverse the gut microecological environment,increase the diversity of gut microbiota,reduce the Firmicutes/Bacteroidota ratio and increase the abundance of Bacteroidota as well as other beneficial bacteria.IPQ may improve chronic inflammation and insulin resistance in obese mice mainly by increasing the abundance of Clostridia and Desulfovibrionales,respectively.