Effects Of Milk Proteins On Risk Factors For Cardiovascular Disease And Aging-Related Muscle Loss

Objective: The present dissertation aimed to provide deeper insights into the potential beneficial role of milk protein in the prevention of two challenging global public health concerns,namely cardiovascular disease and aging-related muscle loss,using meta-analyses,cross-sectional study,and animal study.Cardiovascular disease(CVD)is the leading cause of mortality worldwide,with 80% of the burden in low-income and middle-income countries.Primordial and primary prevention is crucial for improving cardiovascular health and preventing cardiovascular disease.The American Heart Association has defined ideal cardiovascular health as free from CVD and meeting all seven cardiovascular health metrics,which encompasses four health behaviors(smoking,physical activity,bodyweight,and diet)and three health risk factors(total cholesterol,blood pressure,and fasting blood glucose).A cost-effective nutritional approach to improve cardiovascular health and to prevent CVD is attractive considering the burden of CVD throughout the world.It is known that fruit,vegetables,whole grains,nuts,legumes,and fish consumption are important dietary factors protecting against CVD,whereas a high intake of added sugar,sodium,trans fat,and refined carbohydrates may contribute to the development of CVD.While the contribution of each of these foods to cardiovascular health is widely recognized,the extent of contribution of other foods,such as milk and dairy products,to the overall nutritional quality of diets remains uncertain.Milk and dairy products are plentifully available and widely consumed around the world.Although milk and dairy consumption is recommended in most dietary guidelines around the world,the question of whether milk and dairy products should be included in human diet has remained under debate in the last few decades.While milk and dairy products consumption contributes to the intake of a complete protein and micronutrients(e.g.,calcium,magnesium,selenium,riboflavin,vitamin B12,and pantothenic acid),milk and dairy products consumption also contributes to saturated fat intake.Because milk and dairy products are high in saturated fat the public are often led to misconception that consuming milk and dairy products may adversely affect blood lipids and increases cardiovascular disease and mortality.However,there is growing evidence that milk and dairy products consumption may have at least a neutral,if not protective,effect on cardiovascular disease.For example,the findings from the Prospective Urban Rural Epidemiology(PURE)study,a large multinational cohort of 136,384 adults between aged 35 and 70 years from 21 countries in five continents,reported that dairy consumption was inversely associated with the risks of mortality and major cardiovascular disease events.The protective role of dairy is thought to be largely attributed to its nutrients,one of them being protein.Concordantly,the results from clinical trials showed that milk proteins may have favorable effects on various cardiometabolic parameters.However,most of those trials were focused on lipid profile and glucose levels.Therefore,the potential benefits of milk protein on other parameters related to CVD remain less defined.High blood pressure is a modifiable risk factor of CVD.Endothelial dysfunction plays an important role in the pathogenesis of CVD.Endothelial dysfunction can be evaluated by measuring the degree of flow-mediated dilation(FMD)of the brachial artery.Chronic inflammation is firmly established as central to the development and complications of cardiovascular diseases.Previous trials suggest that higher dose of milk protein may reduce the levels of C-reactive protein(CRP),a marker of inflammation.However,the extent by which milk protein influences other inflammatory markers is unknown.Given all the aforementioned considerations,we performed a series of meta-analyses to clarify the effects of milk protein on blood pressure,FMD,and inflammatory markers interleukin-6(IL-6)and tumor necrosis-alpha(TNF-?)).Furthermore,we also performed a cross-sectional study to investigate the association between milk consumption,as a proxy of milk protein intake,and metabolic syndrome,which is a risk factor for CVD,and its components.In addition,a meta-analysis of observational studies was also performed to quantify the available evidence on the association between milk and metabolic syndrome and its components.The life expectancy of older people has increased globally,particularly in developed countries.However,disability free years are not increasing at the same rate as life expectancy,and many older people are living longer with chronic conditions and multiple comorbidities.Aging is associated with declines in skeletal muscle mass and strength(sarcopenia)that reduces physical activity levels,diminishes quality of life,and can lead to fall-related injuries.Decreased muscle mass and decreased physical activity levels contribute to a reduction in total energy expenditure in older people,leading to an increase in fat accumulation,which contribute to various cardiometabolic conditions.Apart from its potential beneficial effects on cardiovascular health,milk proteins,particularly in combination with exercise,has been suggested to improve lean muscle mass in older adults.Therefore,we performed a meta-analysis to investigate the effects of milk proteins on body composition(fat-free mass,fat mass,body weight)in older adults undergoing resistance training.Furthermore,we also performed an experimental animal study to confirm whether milk protein,whey protein,and its derived peptides can preserve muscle mass and reduce fat mass in elderly with obesity.In addition,we also investigated the effects of whey protein and its derived peptides on lipid and glucose metabolism and oxidative stress.Methods: 1)Meta-analyses Pub Med,Web of Science,and Cochrane databases were searched for relevant randomized controlled trials on the effects of milk proteins on blood pressure,body composition in older adults undergoing resistance training,FMD,IL-6,and TNF-?.The study characteristics were abstracted from each study using a standardized data-collection form.Two investigators independently performed the literature search,data extraction,and quality assessment.Any discrepancies regarding inclusion were resolved by consensus.2)Cross-sectional study A cross-sectional study was performed to investigate the association between milk consumption and metabolic syndrome and its components among the residents of Suzhou Industrial Park,Suzhou,China.All participants were interviewed by trained interviewers using a standardized questionnaire to collect the information about age,sex,education level,physical activity,alcohol intake,smoking status,sleep duration,television watching duration,and the use of medications for diabetes,dyslipidemia,or hypertension.Interviewer-administered food frequency questionnaires were used to collect the information about dietary intake.The overnight 10-12 hours fasting blood samples were drawn by venipuncture to measure serum glucose,total cholesterol(TC),triglycerides,high-density lipoproteins cholesterol(HDL-C),and low-density lipoproteins cholesterol(LDL-C)using an autoanalyzer.Metabolic syndrome was defined according to the joint interim statement(JIS)issued by several internationally renowned institutions.A total of 5149 participants were included in the final analysis.3)Animal study Old male C57BL/6J mice aged 10 to 12 months were fed on a normal diet(control group),a high-fat diet(HFD),HFD supplemented with whey protein(WP)and HFD with Whey protein-peptides(WPP)for 20 weeks.Body weight and food intake were monitored weekly.Body composition by nuclear magnetic resonance(NMR)was analyzed at baseline and week 20.Glucose tolerance test was conducted at the end of the study.After 20 weeks,mice were killed and blood samples were centrifuged to collect serum.Liver,fat tissue and muscle were immediately collected.The levels of TC,triglyceride,LDL-C,HDL-C and free fatty acids(FFA)in serum were analyzed by automatic biochemical meter.Fresh gastrocnemius(GAS)and fat tissues were fixed for Hematoxylin and Eosin(HE)staining.The cross-section average area of muscle fibers and fat tissues were measured using Image-Pro Plus.Ultrathin sections of GAS were and observed under a transmission electron microscope.The antioxidant capacity in the muscle was determined by measuring the total antioxidant capacity(T-AOC)and activity of glutathione peroxidase(GPx)using corresponding assay kits.Western blot was used to determine the expressions of protein related to muscle synthesis and decomposition.4)Statistical analyses For meta-analysis,a random-effects model was used to calculate the pooled estimates and 95% confidence intervals(CIs)of effect sizes or relative risks(RRs).The degree of heterogeneity across trials was assessed using Q and I2 statistics.Both Begg's rank correlation test and Egger's linear regression test were performed to investigate potential publication bias.If evidence of publication bias was observed,the trim and fill method was applied to correct the bias.A random-effects model was used to calculate the pooled estimates and 95% CIs of effect sizes.To explore the possible influences of study and participant characteristics on combined effect sizes,subgroup and meta-regression analyses were performed.In addition,we conducted sensitivity analysis to investigate the influence of a single trial on the overall effect estimated by omitting one trial in each turn.All analyses were performed using STATA version 11.0(Stata Corp,College Station,TX,USA).For cross-sectional study,a logistic regression model was applied to estimate the odds ratios(ORs)and 95% CIs for the prevalence of metabolic syndrome and its components according to milk consumption(milk consumers vs non-milk consumers).Multivariable model was adjusted for age,sex,education,smoking,alcohol,physical activity,sleep duration,television watching duration,and consumption of red meat,poultry,fish,fruits,vegetables,nuts,soy,and salted vegetable.For animal studies,variables from the four groups(control,HFD,WP and WPP)were compared using ANOVA followed by multiple comparisons in which least significant difference(LSD)or Tamhane's T2 was used.A P value < 0.05 was considered to be statistically significant,unless otherwise specified.Results: 1)Effects of milk proteins on blood pressure: a meta-analysis of randomized control trials Seven RCTs were eligible for analyses of systolic blood pressure and diastolic blood pressure.Milk proteins interventions significantly lowered systolic blood pressure by-3.33 mm Hg(95% CI:-5.62,-1.03)and diastolic blood pressure by-1.80 mm Hg(95% CI:-3.38,-0.22).No evidence of heterogeneity was observed in analyses of systolic blood pressure(I2 = 0%,P = 0.754)and diastolic blood pressure(I2 = 0%,P = 0.959).More pronounced reduction in blood pressure was observed in trials that included fewer participants(systolic blood pressure:-4.96 mm Hg(95 % CI:-7.99,-1.93),diastolic blood pressure:-2.54 mm Hg(95% CI:-4.72,-0.37)),in trials with younger participants(systolic blood pressure:-4.45 mm Hg(95% CI:-7.28,-1.61),diastolic blood pressure:-2.14 mm Hg(95% CI:-4.12,-0.15)),in trials with a higher dose of protein(systolic blood pressure:-4.96 mm Hg(95% CI:-7.99,-1.93),diastolic blood pressure:-2.54 mm Hg(95% CI:-4.72,-0.37))and in trials that used milk protein supplements(systolic blood pressure:-4.36 mm Hg(95% CI:-7.36,-1.35),diastolic blood pressure:-2.33 mm Hg(95% CI:-3.31,-1.34)).2)Effects of milk proteins on flow-mediated dilatation,interleukin-6 and tumor necrosis factor-alpha: meta-analysis of randomized controlled trials Six RCT were included for analysis of flow-mediated dilation(FMD),12 for analysis of IL-6,and 10 for analysis of TNF-?.Furthermore,milk proteins supplementation did not significantly affect IL-6(-0.50 pg/m L;95% CI:-0.50,0.16 pg/m L),but milk proteins supplementation increased FMD(0.94%;95% CI: 0.51,1.38%)and reduced TNF-?(-0.44 pg/m L;95% CI:-0.80,-0.08 pg/m L).High heterogeneity was observed in the analyses of IL-6(I2= 52.1%,P = 0.01)and TNF-?(I2= 90.1%,P <0.01),whereas no evidence of heterogeneity was observed in the analysis of flow-mediated dilation(I2= 0%,P = 0.713).There was no evidence of publication bias in the analyses of systolic blood pressure,diastolic blood pressure,fat-free mass,body weight,fat mass,flow mediated dilation,IL-6,and TNF-?.3)Effects of milk proteins supplementation in older adults undergoing resistance training: a meta-analysis of randomized control trials Nine RCTs were included for fat-free mass,eight for analysis of body weight,and seven RCTs for analysis of fat mass.Regarding body composition in older adults undergoing resistance training,the combination of milk proteins supplementation and resistance training did not have significant effect on fat mass(0.30,95% CI:-0.25,0.86 kg)or body weight 1.02,95% CI:-0.01,2.04 kg).However,a positive effect of milk proteins supplementation paired with resistance training on fat-free mass was observed(0.74,95% CI: 0.30,1.17 kg).Moderate heterogeneity was observed in the analysis of body weight(I2 = 35.3%,P = 0.147),whereas no noteworthy heterogeneity was observed in the analyses of fat mass(I2 = 0%,P = 0.43)and fat-free mass(I2 = 12.4%,P = 0.326).Greater fat-free mass gains were observed in studies that included more participants(0.73,95% CI: 0.30,1.16 kg),and in studies that enrolled participants with aging-related medical conditions(1.60,95% CI: 0.92,2.28 kg).4)The association between milk consumption and metabolic syndrome: a cross-sectional study of the residents of Suzhou Industral Park and a meta-analysis In the cross-sectional studies including 5149 residents of Suzhou Industrial park,there was no significant difference in the odds of having metabolic syndrome between milk consumers and non-milk consumers(OR 0.86,95% CI: 0.73,1.01).Compared with non-milk consumers,milk consumers had lower odds of having elevated waist circumference(OR 0.78,95% CI: 0.67,0.92),elevated triglyceride(OR 0.83,95% CI: 0.70,0.99),and elevated blood pressure(OR 0.85,95% CI: 0.73,0.99).However,the odds of having reduced HDL cholesterol(OR 1.14,95% CI: 0.96,1.36)and elevated fasting blood glucose(OR 0.99,95% CI: 0.82,1.20)were not significantly different between milk consumers and non-milk consumers.In the meta-analysis that included our cross-sectional study and other published observational studies,high milk consumption was inversely associated with metabolic syndrome(RR 0.80,95% CI: 0.72,0.88)and its components(except elevated fasting blood glucose).5)Whey protein-peptides ameliorated elderly obesity in mice induced by a high-fat diet with the preservation of muscle mass Compared to the HFD group,the WPP group had a 19.7% drop in final bodyweight(P<0.05)and a 10.1% drop in energy intake(P<0.05).NMR scan showed that WPP significantly reduced HFD-induced fat gain by 39.4% for fat mass and by 9.7% for relative fat mass(both P<0.05).Compared to the HFD group,subcutaneous fat mass and perirenal fat mass were decreased by 56.2%(P<0.05)and 53.3%(P<0.05)in the WPP group,which is consistent with the sizes of adipocytes in HE staining.Compared to the HFD group,gastrocnemius muscle weight was increased by 12.5%(P<0.05)in the WP group and by 9.8%(P<0.05)in the WPP group.However,no significant difference in the expressions of protein related to muscle synthesis and decomposition in any intervention group.Transmission electron microscopy showed both WP and WPP supplementation improved the myofibrillar arrangement and decreased amounts of abnormal mitochondria.WP and WPP supplementation alleviated oxidative stress in muscle with increased T-AOC by 35.5% and GPx by 18.0% in the WP grouyp and increased T-AOCby 47.2% and GPx by 31.1% in the WPP group(all P<0.05).Conclusions: In summary,milk proteins slightly but significantly lower both systolic and diastolic blood pressure.Moreover,milk proteins supplementation improves FMD and reduces TNF-?.The potential cardiometabolic benefit of milk protein is further corroborated by the findings of population-based studies showing that high milk consumption is inversely associated with metabolic syndrome and nearly all of its components.Regarding aging-related muscle loss,milk protein,in combination with resistance training,is effective to elicit fat-free mass gain in older adults.Consistently,WPP reduces fat mass in high-fat diet-induced obesity in mice model of elderly obesity.WP and WPP both ameliorated mitochondrial abnormality and oxidative stress in muscle.