The Effects Of HIIT Versus MICT On Adipose Tissue UCP1 Expression By Macrophage Polarization In High-fat Diet Mice

Background:President Xi Jinping put forward the"Carrying out the Healthy China initiative"at the 19th CPC National Congress,and stressed that a healthy population is a key mark of a prosperous nation and a strong country.In 2019,for promoting people's health,the State Council issued a new guideline to“implement the country's Healthy China initiative”in which eating properly and taking exercise are two of most important actions.We are faced with serious health problems of obesity and related metabolic diseases that were caused by excessive or inappropriate diet.Regular physical activities will help to improve prevention and treatment of common chronic diseases,and improve people's quality of life and sense of well-being.How to choose exercise reasonably is the focus attention of research,and is also the core problem of physical exercise and sports prescription.High-intensity interval training?HIIT?involves alternating short bursts of high intensity exercise with recovery periods or light exercise.The moderate intensity continuous training?MICT?is performed in a continuous manner and at lower intensities than HIIT.HIIT is superior to MICT in improving vascular function,glucose regulation and health related physical fitness.According to previous reasearch and meta-analysis,HIIT is a time-efficient strategy to decrease fat-mass deposits,including those of abdominal and visceral fat mass.A new effective treatment for obesity that is to increase thermogenic in adipose tissue through UCP1?uncoupling protein 1?is getting more attention.Is there difference in UCP1expression between HIIT and MICT?Has differernce in effect between HIIT and MICT on macrophage polarization that induced to UCP1 expression?Objectives:UCP1 is a marker protein for brown adipocytes.The aim of the present study was to test the hypotheses that 1)The morphology and UCP1 protein expression in inguinal subcutaneous white adipose tissue?sWAT?,perigonadal visceral white adipose tissue?vWAT?and interscapular brown adipose tissue?iBAT?of mice induced by high fat diet;2)compare the effects of MICT and HIIT on morphology and UCP1 protein expression in different fat tissue of high fat diet mice;3)Does MICT and UCP1 HIIT regulate UCP1expression in macrophage polarization manner differently?The purpose of study is to supplement the theory of weight-loss exercise prescription and provide an optional exercise protocol for people.Methods:?1?100 female C57BL/6J mice?9.62±1.04g?were divided into high fat diet?HFD,n=90?and normal diet?CON,n=10?groups.The mice were housed individually in cages at room temperatures of 26±2?and with free access to standard chow.After 15?weeks,10 overweight mice were selected from the high-fat diet group and continued eating high fat diet for 12?weeks.After diet intervention,sWAT,vWAT,iBAT and liver were collected to be detected morphology and thermogenesis marker UCP1 protein expression using HE stain and Western blotting.?2?30 overweight mice were choosed from female C57BL/6J mice induced by high fat diet for 15 weeks,then were divided into three groups HFD?no exercise?,MICT?moderate intensity continuous training,45?min at 50⁷0%VO₂peak?,HIIT?high-intensity interval training,1?min at 90¹00%VO₂peak+2?min at 50⁷0%VO₂peak,the distance of HIIT same to MICT?.Trained animals ran on a treadmill?25°slope?,5 days/week for 12 weeks.Liver,sWAT,vWAT and iBAT were collected at 48 hours after the last bout of exercise,to detect the effects on morphology and UCP1 protein expression between HIIT and MICT.?3?Immunohistochemistry,real-time PCR,flow cytometry and Western blotting were used to observe the changes of macrophages phenotype and related protein expression in white adipose tissue of diet induced obesity mice.The purpose of that is to find out differences between MICT and HIIT in regulating UCP1 expression by macrophage polarization manner.Results:1.High fat diet affects the morphology and browning of fat tissue in female mice.The results showed that compared with CON,?1?the weight of inguinal sWAT and perigonadal vWAT was significantly higher in HFD?P<0.01?,no change was in the weight of interscapular BAT?P=0.20?;HE staining results showed that the adipocyte volume of inguinal sWAT and perigonadal vWAT increased more significantly in HFD mice?P<0.01?,and the large fat drops in brown adipose tissue increased obviously.?2?HFD significantly increased significantly levels of serum TC,LDL-C?P<0.05?,and TG,TC content in liver more significantly in mice?P<0.01?.HE staining results showed that the cytoplasm of hepatocytes was loose or vacuolized,and the number of lipid droplets in liver tissue increased obviously by oil red O staining.?3?The expression of UCP1 protein in inguinal sWAT and interscapular BAT decreased significantly in HFD mice?P<0.05?,but there was no change in perigonadal vWAT.It is suggested that high fat diet increased the fat storage ability of adipocytes and hepatocytes,and also decreased the UCP1 protein expression in fat tissue.2.Exercise training reversed adipose tissue dysfunction and heat production capacity of adipose tissue that decreased by high fat diet.?1?Compared with HFD,the weight of inguinal sWAT and perigonadal vWAT decreased more significantly in MICT group?P<0.05?,and that decreased most significantly in HIIT group?P<0.01?.The weight of perigonadal vWAT between MICT and HIIT was significantly different.The weight of interscapular BAT has no difference in three groups.HE staining results showed that the adipocyte volume of inguinal sWAT and perigonadal vWAT decreased significantly in HIIT and MICT group?P<0.01?,the decrease in HIIT was more significantly than MICT?P<0.05?.The decrease of large fat droplets in brown adipose tissue in HIIT group was better than in MICT.?2?Compared with HFD,there is a downward trend of serum LDL-C and TG content in liver in MICT,but they decreased more significantly in HIIT group?P<0.01?.HE staining results showed that exercise decreased vacuoles in hepatocytes,and HIIT was better than MICT.Oil red O staining results showed HIIT decreased the number of lipid drops in liver more obveriously than MICT.?3?Compared with HFD,the expression of UCP1 protein in perigonadal vWAT was tend to downward in MICT group,but increased significant in HIIT group?P<0.05?;The expression of UCP1 protein in inguinal sWAT has a tendency to increase in MICT and HIIT group,but there was no significance between MICT and HIIT;the expression of UCP1protein in brown adipose tissue increased in HIIT group?P<0.05?,but there was no significant difference between MICT and HIIT.It is suggested that HIIT is superior to MICT in reducing the fat storage of adipocytes and hepatocytes,and also increasing the protein expression of UCP1 in fat tissue.3.MICT and HIIT regulate UCP1 protein expression in macrophage polarization manner differently.?1?Macrophages changes in perigonadal visceral adipose tissue.Compared with HFD,?1?MICT and HIIT decreased the number of crown-like structure?CLS??P<0.01?,and HIIT decreased more significantly than MICT?P<0.05?.?2?M1 macrophages were labeled with CD11c and M2 macrophages were labeled with CD206.Immunohistochemical staining results showed that MICT and HIIT reduced the number of M1 macrophages significantly?P<0.01?,and HIIT decreased more significantly than MICT?P<0.05?;MICT and HIIT increased the number of M2 macrophages?P<0.01?,but there was no significant difference between the two groups.?3?Real-time PCR results showed that the relative expression of TNF-?mRNA decreased significantly in MICT group?P<0.05?,and more significantly decreased in HIIT group?P<0.01?that was different from MICT group?P<0.05?;The relative expression of iNOS mRNA decreased significantly in MICT and HIIT group?P<0.05?,and there was no difference between the two groups.The relative expressions of Arg1 mRNA and YM1 mRNA were significantly increased in MICT and HIIT groups,and HIIT increased more significantly than MICT?P<0.01?.?4?Macrophages labeled with F4/80-FITC,CD11C-PE was designated as M1-type macrophages,those labeled with F4/80-FITC,and CD206-PE was designated as M2-type macrophage.Flow cytometry results showed that the percentage of M1 macrophages decreased significantly?P<0.05?in MICT group while more significantlyin HIIT group?P<0.01?,and the decrease was more in HIIT group than MICT?P<0.05?;The percentage of M2 macrophages increased in MICT and HIIT group,but there was no difference between the two groups.All resluts suggested that HIIT and MICT can increase M2 macrophages and decrease M1 macrophages,and HIIT can reduce M1phenotype more obviously than MICT in visceral adipose tissue.?2?Macrophages changes in inguinal subcutaneous adipose tissue.Compared with HFD,?1?MICT and HIIT decreased the number of CLS?P<0.01?,but there was no difference between the two groups.?2?M1 macrophages were labeled with CD11c and M2 macrophages were labeled with CD206.Immunohistochemical results showed that MICT and HIIT reduced the number of M1 macrophages?P<0.01?and increased the number of M2macrophages?P<0.01?significantly,but there was no significant difference between the two groups.?3?Real-time PCR results showed that the relative expression of TNF-?mRNA decreased significantly in MICT?P<0.05?,and the relative expression of iNOS mRNA decreased significantly not only in MICT but also in HIIT?P<0.01?.The change of iNOS mRNA in MICT was not different from HIIT.The relative expression of Arg1 mRNA increased significantly in MICT and HIIT?P<0.01?,but there was no difference between the two groups.The expression of Ym1 mRNA has not significant change in the two groups.All above resluts suggested HIIT and MICT can decrease M1 phenotype and increase M2phenotype in subcutaneous adipose tissue,but HIIT is not superior to MICT.?3?Macrophages regulate UCP1 protein expression by cytokines.?1?In perigonadal visceral adipose tissue,compared with HFD,the expression of TNF-?mRNA and protein decreased more significantly in MICT group?P<0.05?,and decreased most significantly in HIIT group?P<0.01?which was significantly different from that in MICT group?P<0.05?.Compared with HFD,the relative expression of IL10 mRNA increased more significantly in MICT group?P<0.05?,and increased most significantly in HIIT group?P<0.01?which was significantly different from that in MICT group?P<0.05?.The protein expression of IL-10 in MICT and HIIT group was just an increasing trend.The expression of IL-6 mRNA was reduced significantly in HIIT and MICT groups?P<0.01?,but there was no significant difference between the two groups;The protein expression of IL-6 in MICT was reduced significantly?P<0.05?,the change of HIIT was not significant,but the difference between the HIIT and MICT was significant?P<0.05?.All above results suggested HIIT is superior to MICT in reducing the protein expression of TNF-?which inhibit UCP1 protein expression in visceral adipose tissue,and contribute to browning of visceral white adipose tissue.?2?In inguinal subcutaneous adipose tissue,compared with HFD,the relative expression of TNF-?mRNA decreased significantly in MICT group?P<0.05?,but did not change in HIIT group;The protein expression of TNF-?has a tendency to decrease in MICT and HIIT group,but there was no significantly difference between the two groups.The relative expression of IL-10 mRNA increased significantly in HIIT and MICT group?P<0.01?,but there was no significant difference between the groups;The protein expression of IL-10 increased more significantly in MICT group?P<0.05?and most significantly in HIIT group?P<0.01?,but there was no differernce between the two groups.The expressions of IL-6 mRNA and protein have no change in MICT and HIIT group.All above results suggested that HIIT and MICT effect on IL-10 protein expression that inhibit UCP1 protein expression,and stunted browning of subcutaneous white adipose tissueConclusion:1.High fat diet increased fat storage in different parts of fat tissue and liver of female mice,and decreased the protein expression of UCP1 in subcutaneous white adipose tissue and brown adipose tissue.Compared with MICT that has the same exercise distance as HIIT,HIIT is superior to MICT in reducing fat storage in visceral fat tissue and liver,in increasing the UCP1 protein expression in visceral adipose tissue and brown adipose tissue2.Compared with MICT,HIIT more significantly reduced M1 macrophages and TNF-?protein expression in visceral adipose tissue,which reduced the inhibition of UCP1 protein expression;HIIT and MICT promoted M2 macrophages in subcutaneous adipose tissue and increased IL-10 protein expression.3.12 weeks MICT and HIIT regulate UCP1 expression in macrophage polarization manner differently.The expression of UCP1 in visceral adipose tissue was significantly improved by HIIT with high exercise intensity,which provides reference for weight-loss exercise prescription.