Effects Of Pre-sleep Protein Supplementation Combined With Resistance Exercise On Energy And Glycolipid Metabolism In Sedentary Healthy Adults

Introduction:As a common modern lifestyle,sedentary behavior has many adverse effects on human health,which can reduce the rate of muscle protein synthesis,and cause damage to lean tissue and muscle mass.Currently,protein intake and resistance exercise are the predominant forms of promoting muscle protein synthesis.However,some studies have found that even if a certain amount of protein is ingested during the day,muscle protein synthesis is in a negative balance during the longer nutrient window at night.In addition,it has been suggested that pre-sleep protein intake to maintain and/or reduce the loss of muscle mass may be a new nutritional worth exploring concept.Studies have also found that pre-sleep protein intake combined with resistance exercise can reduce muscle protein breakdown during sleep,promote muscle protein synthesis,and have a beneficial impact on muscle health.However,some researchers believe that eating close to sleep may increase the risk of obesity,diabetes,and other metabolisms.Therefore,although pre-sleep protein intake combined with resistance exercise can promote muscle protein synthesis,the impact on the body’s metabolic health is not clear,especially energy metabolism and glycolipid metabolism.On this basis,it remains to be clarified whether the possible effects of pre-sleep protein intake combined with resistance exercise on metabolism health are caused by the total protein intake in a day or by the "pre-sleep intake time".In summary,whether this intervention model can be used as a nutritional exercise strategy to be incorporated into future sedentary people interventions needs further research to confirm.Purposes:(1)To explore the effects of the acute pre-sleep protein supplementation combined with resistance exercise on energy metabolism and glycolipid metabolism at different stages of 24-h in sedentary healthy people,and whether the impact may be caused by the specific "pre-sleep intake time";(2)To explore the effects of 8-week pre-sleep protein supplementation combined with resistance exercise on population composition,resting energy metabolism,and glycolipid metabolism in sedentary healthy people,and whether the impact may be caused by the specific "pre-sleep intake time";(3)Using metabolomics technology to screen the possible different metabolites,and to explore the effects of pre-sleep protein supplementation combined with resistance exercise on other metabolites in sedentary healthy people.Research methods:Experiment 1:The required sample size was calculated by the G*Power,and a total of 31 sedentary healthy subjects were recruited according to the experimental screening conditions.Each subject completed the following three 24-h experimental protocols in the human energy metabolic chamber in a random cross sequence,with at least 7-14 days intervals between each experiment:(1)Daytime protein intake: 40 g protein supplementation immediately after dinner combined with resistance exercise,and presleep placebo supplementation(PRO-PLA);(2)Pre-sleep protein intake: placebo supplementation immediately after dinner combined with resistance exercise,and presleep 40 g protein supplementation(PLA-PRO);(3)Placebo: immediately after dinner and pre-sleep placebo supplementation combined with resistance exercise(PLA).Before the experiment,the subjects were monitored and prohibited from vigorous physical activity,kept diet records and ingested standard meals,and wore a 24-h continuous glucose monitor.During the experiment,energy metabolism tests were performed and blood was collected at baseline and on the next day after the intervention,immediately after a meal,30 min,60 min,and 120 min.The postprandial 2-h t AUC and i AUC of serum GLU,INS,TG and FFA was calculated using Graph Pad software(Prism 9: Graph Pad,San Diego,USA).Paired samples t-test and the nonparametric test was used to analyze the changes of outcome indexes before and after intervention in the three experimental conditions.One-way ANOVA and nonparametric Kruskal-Wallis H test were used to analyze the differences of baseline indicators among the three experimental conditions.The effects of the three experimental conditions on all outcome indicators were analyzed using linear mixed models(LMM)and generalized linear mixed models(GLMM),and using non-target metabolomics techniques and orthogonal partial least squares discriminant analysis(OPLS-DA)screening differential metabolites and their changing trends.Experiment 2:According to the required sample size calculated by G*Power,a total of 34 sedentary healthy subjects were recruited according to the experimental and screening conditions.They were randomly divided into 8-week 40 g protein supplementation immediately after dinner combined with resistance exercise group(daytime protein intake,PRO-RE)and 8-week pre-sleep protein supplementation combined with resistance exercise group(pre-sleep protein intake,RE-PRO)equally.Before the experiment,the subjects’ physical activity and dietary controls were controlled same with the acute study.During the experiment,subjects completed the baseline test(body composition,resting metabolism,and the collection of fasting blood,immediately after meals,30 min,60min,and 120 min blood),and then started the 8-week intervention.After the intervention,the same tests were performed with the baseline indicators.In addition,Data processing and statistical analysis were consistent with the acute study.Results:Experiment 1:(1)For energy metabolism and appetite,the 24-h TEE of PLA-PRO and PROPLA were significantly higher than PLA by 3.0% and 3.3% respectively.But,the significance disappeared after adjusting for total 24-h energy intake;In addition,the SEE of PLA-PRO was higher than PLA and PRO-PLA by 11.2% and 3.6%respectively,which has the “pre-sleep intake time effect”;Compared with PLA,appetite the next morning after PLA-PRO intervention was significantly reduced,and satiety was significantly enhanced with " pre-sleep intake time effect".(2)For serum glucose and insulin,the In GLU i AUC of PLA-PRO was significantly reduced by 4.89 mmol·L-1·h compared with PLA,but the significance disappeared after the adjustment of the total 24-h energy intake;In addition,the reduction in fasting GLU was significantly higher for PRO-PLA(Δ-0.11mmol/L,p=0.017)and PLA-PRO(Δ-0.21mmol/l,p=0.003)than for PLA(Δ-0.05mmol/L);Compared with baseline fasting INS,PLA and PLA-PRO were significantly reduced by 23.2%(p=0.010)and 19.9%(p=0.012)respectively.(3)For blood lipid indexes,the 2-h TG t AUC of PLA-PRO was significantly higher than PLA by 2.5%(p=0.044)independent of 24-h total energy intake,but the postprandial 2-h TG increasement(i AUC)was significantly lower than that of PLA by 56.3%(p=0.046);The postprandial 2-h FFA t AUC for PLA-PRO was significantly higher than PLA by 7%(p=0.036),and the postprandial 2-h FFA reduction(i AUC)was 7.3% higher than PLA(p=0.036)with the "pre-sleep intake time effect";Compared with the baseline value,other blood lipid indexes in the 3 experimental conditions were significantly reduced.(4)For the non-target metabolomics,it was found that 13 small-molecularweight differential metabolites all had favorable trends and no other unfavorable metabolic changes.Experiment 2:(1)For the body composition,the body fat percentage in the RE-PRO group was significantly decreased after 8-week intervention(p=0.047),while there was no significant change in the PRO-RE group.(2)For the resting energy metabolism,the fat oxidation rate in RE-PRO and PRO-RE in resting state was significantly increased after an 8-week intervention,and the increase in fat oxidation rate in the RE-PRO(Δ+25%)was significantly higher than PRO-RE(Δ+14.3%)(p=0.045),with an " pre-sleep intake time effect".(3)For serum glucose and insulin: the fasting GLU and postprandial 2-h GLU t AUC and i AUC in the PRO-RE group were significantly increased after the 8-week intervention,while there were no significant changes in the RE-PRO group;In addition,postprandial 2-h INS i AUC in PRO-RE and RE-PRO groups were significantly increased by 24.7%(p=0.000)and 22.5%(p=0.001)after intervention respectively,with no significant effect on HOMA-IR.(4)For blood lipid indexes,the postprandial 2-h FFA t AUC and reduction(i AUC)in the RE-PRO group were significantly decreased by 33.6%(p=0.039)and37.1%(p=0.023)after the 8-week intervention respectively.(5)For the non-target metabolomics,8 small-molecular-weight differential metabolites related to intervention methods were found,and no other adverse metabolic changes were found.Conclusions:(1)The acute pre-sleep protein supplementation combined with resistance exercise can significantly increase SEE and satiety in the next morning with an "presleep intake time effect",and increase the 24-h TEE and CHO oxidation rate during sleep and reduce the appetite in the next morning not independent of 24-h total energy intake,but had no significant effect on next morning BMR;In addition,reduced fasting GLU and improved postprandial TG and FFA responses had the " pre-sleep intake time effect" and partial effect influenced by 24-h total energy intake.(2)Although 8-week pre-sleep protein supplementation combined with resistance exercise had no significant effect on REE,it can significantly improve the choice of metabolic substrates for energy supply in the resting state,and increase fat utilization and reduce CHO utilization,which had the "pre-sleep intake time effect".In addition,there is no adverse effect on fasting GLU and HOMA-IR and the 8-week intervention can increase postprandial insulin response and reduce postprandial FFA response.(3)The results of metabolomics showed that the acute pre-sleep protein supplementation combined with resistance exercise can cause changes in various metabolites that are beneficial to metabolic regulation,while 8-week intervention only caused changes in a small number metabolites,suggest that long-term intervention may produce metabolic adaptations.Thus,it also has beneficial effects on the energy metabolism,glycolipid metabolism and other metabolic changes based on the beneficial effects of pre-sleep protein intake combined with resistance exercise on muscle health,which can provide a reference for the formulation of nutrition and exercise strategies for sedentary healthy people.