| Backgrounds:Worldwide growing epidemic of obesity has become one of the most significant contributors to global health.Lifestyle intervention against the obesogenic environment including unhealthy diet and sedentary behavior is deemed to be the most important strategy in tackling obesity,especially for the children.Exercise has been proven to be the most efficiency and healthy way to lose weight,thus elucidating which exercise regime improves the metabolic status of individuals with obesity is essential.The debate about the value of high-intensity interval training?HIIT?vs.moderate-intensity continuous training?MICT?has been long lasting.Accumulating studies have suggested HIIT yield more favorable results in weight loss,metabolic and cardiovascular status improvement than those with MICT.In contrast,some other evidences supported similar health benefits of HIIT and traditional endurance exercise,even argued that HIIT may not be safe and tolerable.Hitherto the effect of HIIT on systemic and tissue-specific metabolism in obesity remains largely unexamined.Diet intervention is another crucial strategy against obesity.Mediterranean diet has been supposed to reduce cardiovascular disease morbidity and mortality,metabolic syndrome morbidity.The beneficial effects of virgin olive oil,the major dietary fat in the Mediterranean diet,have been attributed to its high content of phenolic compounds,such as oleuropein.Hydroxytyrosol?HT?,the major hydrolysate of oleuropein,is a simple phenolic compound with marked antioxidant and anti-inflammatory activity.Considerable researches on obesity have revealed nutrient overload induces activation of cellular stress and inflammatory signaling,and that,in turn impairs insulin signaling and lipid metabolism in metabolically active tissues.Among the stressful conditions responsible for obesity,accumulating evidences focus on the critical role of endoplasmic reticulum?ER?stress.Thus,therapeutic strategy to manipulate levels of ER stress in obesity is distinctly significant.However,report suggesting amelioration of ER stress as a core mechanism involved in insulin-sensitizing and hypolipidemic effects of HT is limited.In the present study,firstly we aimed to clarify how HIIT would influence obesity-related physiological variables,and compare changes in these adaptations with traditional MICT in a mouse model of high fat diet?HFD?induced obesity;secondly,we evaluated the effect of HT on obesity complication in diet-induced obesity?DIO?mice,and sought to prove in cultured cell that HT could improve insulin action and lipid metabolism by moderating ER stress,for the sake of providing theoretical basis for development of new and improved options to prevent and treat obesity and its related complications.Purpose:1.To compare adaptations in anthropometry and glucolipid metabolism index after HIIT and MICT intervention in DIO mice model.To explore the mechanistic link between HIIT and advantageous metabolic homeostasis through detecting mRNA levels of genes involved in hepatic lipogenesis and?-oxidation,adaptions in inflammatory and insulin signaling,as well as beige adipocyte recruitment in inguinal subcutaneous adipose tissue?scWAT?.2.To demonstrate the mechanism of insulin-sensitizing and hypolipidemic effects after HT intervention in DIO mice,by means of detecting ER stress indicators,insulin receptor signaling in insulin sensitive tissues,analyzing ER ultrastructural damage and lipid deposit in the liver,as well as the hepatic transcription factor and limiting enzymes expression involved in lipogenesis.Method:1.Modeling of DIO phenotype:one hundred of 3-weeks old male ICR mice were fed with HFD,and another 10 mice were fed with a standard diet for 7 weeks.Then the HFD-fed mice which were 20 percent heavier than that fed with a standard diet were considered to be the DIO model.Then the DIO mice were randomly assigned to different interventions?n=10 in each group?:?1?HIIT group?2?MICT group?3?sedentary?SED?group?4?HFD group?daily oral gavage of distilled water??5?HFD+HT group?daily oral gavage of HT?,and continued to consume the HFD.Meanwhile,lean control mice?CON??n=10?fed a standard control diet over the entire period were also included.2.Exercise Intervention:?1?Exercise protocol:The trained mice were running on a motored mice treadmill at 25oinclination 5 days/week?Monday to Friday?for 8 weeks.Both groups started with a warm-up at 5 m/min,where after HIIT consisted 10 bouts of4 min high-intensity(8590%VO2max)treadmill running,interspersed by 2 min active rest?5 m/min?;whereas MICT consisted of distance-matched continuous running,corresponding to 6570%of VO2max;?2?Mice were weighed every week,and their intake of water and food were recorded every other day.After execution,fat pads were removed and weighed,adiposity index was calculated.Slices of visceral white adipose tissue?vWAT?were stained with hematoxylin and eosin?H&E?for histopathology and adipocyte size was calculated;?3?Serological examination:the serum alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,total cholesterol?TC?,triglyceride?TG?,high-density-lipoprotein cholesterol?HDL-c?,and low-density lipoprotein cholesterol?LDL-c?were measured by auto chemistry analyzer and high-sensitivity C-reactive protein?hsCRP?by automatic special protein analyzer;IL-6and adiponectin were determined by chemiluminescence,fasting insulin?FINS?level was analyzed using ELISA kit;?4?Sections of liver were stained with H&E for histopathology;TG levels in liver tissues were analyzed by glycerol lipase oxidase?GPO-PAP?method;mRNA levels of lipolysis-related genes CPT-I,HAD and PPAR?,lipogenesis-related genes ACC,FAS,SCD-1 and SREBP-1c were analyzed by real time quantitative PCR?RT-q PCR?,protein levels of PPAR?and SREBP-1 were analyzed by Western blotting?WB?;ultrastructure of mitochondria in the liver tissues was evaluated by transmission electron microscopy?TEM?analysis;?5?Glucose tolerance tests?GTTs?and insulin tolerance tests?ITTs?were manipulated?48h after treadmill running?to evaluate glucose tolerance and insulin sensitivity,homeostasis model assessment of insulin resistance?HOMA-IR?was calculated;sections of vWAT were for immunofluorescence?IF?analysis to assess macrophage infiltration?F4/80+?;markers of inflammatory?p-c-Jun/JNK1,IKK?,TNF?,IL-1??and insulin receptor?ps-IRS-1/IRS-1,p-Akt/Akt,GLUT4?pathways were detected by WB;?6?Thermogenic protein uncoupling protein-1?UCP1?of interscapular brown adipose tissue?iBAT?were analyzed by immunohistochemistry?IHC?analysis,“browning”switch in vWAT and scWAT were examined through IHC analysis?UCP1?,WB?UCP1 and PRDM16?and TEM?mitochondrial level?.3.Diet intervention:?1?HFD group:gavage with a daily oral of distilled water?0.1mL/10g bw?,HT group:gavage with a daily oral of HT?20 mg/kg bw?for 10 weeks;?2?After execution,fat pads were removed and weighed,adiposity index was calculated;?3?Fasting blood glucose?FBG?level was measured every week,GTTs and ITTs were manipulated after intervention;?4?ALT,AST,TC,TG,HDL-c,and LDL-c were measured by auto chemistry analyzer and hsCRP by automatic special protein analyzer;IL-6 and adiponectin were determined by chemiluminescence,FINS levels were analyzed using ELISA kit;?5?Markers of ER stress?p-PERK/PERK,p-IRE-1?/IRE-1?,ATF6,GRP78?,inflammatory?p-c-Jun/JNK1,TNF?,IL-1??and insulin receptor?ps-IRS-1/IRS-1,p-Akt/Akt,GLUT4/2?pathways were detected by WB;sections of liver tissues were stained with Oil Red O for evaluation of lipid droplets deposition,TG levels in liver tissues were analyzed by GPO-PAP method;ultrastructure of ER in liver tissues were evaluated by TEM analysis;Lipolysis-and lipogenesis-related genes and protein levels were detected by qRT-PCR and WB,respectively;?6?Experiments in vitro:ER stress was induced by palmitate?PA?in HepG2 cell pretreated by HT or4-PBA?ER stress inhibitor?,specific pharmacological JNK inhibitor SP600125?SP?was further used to verify the specific pathway?IRE1?/JNK/IRS1?that HT worked on the PA stimulated cel.Result:Part One:Exercise Intervention1.Weight gain of the training mice was significantly less than sedentary counterparts,in spite of higher total caloric intake.In relative terms,HIIT induced weight loss more notable than MICT?P=0.001?.HIIT induced a striking reduction of fat mass compared with SED,while MICT exerted a smaller extent of reduction.On average,the size of the adipocytes of HIIT mice was smaller than that of the SED and MICT mice?both P=0.000?.2.Serum ALT and TG content of two training groups were significantly lower than that observed in SED group.TC and LDL-c level of HIIT mice were significantly lower than that of SED mice,but did not differ between MICT and SED mice.SED group exhibited a substantial amount of lipid deposition,which was markedly alleviated in the HIIT and MICT groups.Moreover,HIIT almost restored the morphology to the same as normal controls.Biochemical analysis of TG content in liver tissues showed consistent results with morphological evaluation.TEM showed higher density?P=0.000 vs.MICT?and size?P=0.035 vs.MICT?of mitochondria in liver cell of HIIT mice,with the morphology of mitochondria restored into elliptical or circular bursa.Declined expressions of oxidative transcription PPAR?and its downstream targets,CPT1a and HAD levels in SED were totally prevented by HIIT but not MICT.At the protein level,PPAR?expression showed a similar profile to its transcriptional level;moreover,SREBP1 was down-regulated effectively by training,and still the effect of HIIT was more remarkable in comparison with MICT?P=0.013?.3.FBG,FINS,HOMA-IR,and AUCITT were significantly lower in two training groups,nevertheless,HIIT played a more profound impact than MICT.Serum pro-inflammatory IL-6 and hsCRP level were significantly lower in HIIT group compared with SED and MICT groups,the content of anti-inflammatory adiponectin was significantly higher in HIIT group compared with MICT group?P=0.05?.IF staining of vWAT showed that F4/80+cells were down-regulated in two training groups,especially in the HIIT group?P=0.026 vs.MICT?.Consistently,a training-related decline in TNF?and IL-1?protein expressions compared to sedentary counterparts was observed,and still the decline in HIIT group was more pronounced.JNK phosphorylation decreased in both HIIT and MICT group,when compared with SED mice?both P=0.000?,but was comparable between two training groups.Both HIIT and MICT demonstrated significant suppression of p-IRS?Ser307?,activation of p-Akt?Ser473?and GLUT4 in adipose tissue and skeletal muscle compared with SED group,and the value was significantly different between HIIT and MICT mice.4.Gross appearance of iBAT showed a striking deeper color in two training group,in consistent with the emergence of more abundant small,multilocular UCP1+adipocytes.The quantification analysis further revealed HIIT was a stronger inducer of UCP1 expression in iBAT compared with MICT?P=0.016?.IHC staining reveals an evident reduced volume of lipid droplets in inguinal scWAT of HIIT mice than SED and MICT mice.This was accompanied by presence of more multilocular adipocytes and a marked increase in UCP1 content in this fat depot,while MICT mice showed no change in appearance relative to SED animals.Concomitant with morphology analysis,only HIIT significantly increased protein expression of UCP1,as well as PRDM16 in sc WAT.Accordingly,HIIT group showed the highest value of mitochondrial population density as evidenced by TEM.Part Two:Diet intervention1.FBG,FINS and HOMA-IR levels were significantly lower in HT-treated mice compared with vehicle-treated mice.Accordingly,AUCs of ITTs and GTTs significantly reduced after 10 weeks of HT intervention.HT treatment effectively prevented HFD-induced upregulation of F4/80+cells in adipose tissue,as well as protein expressions of TNF?and IL-1?in adipose and liver tissue.Similarly,the anti-inflammatory effect of HT was confirmed by the reduction of circulating hsCRP and IL-6 levels.In HT-treated mice,PERK,IRE-1?and JNK phosphorylation,ATF6and GRP78 was markedly reduced in adipose and liver tissue,in comparison with vehicle-treated mice.Consistently,HFD-induced serine phosphorylation of IRS-1?Ser307?and suppressed Akt phosphorylation?Ser473?were both restored by HT administration in liver,adipose tissue and skeletal muscle.GLUT4 significantly increased in respond to HT treatment in adipose tissue and skeletal muscle,though the alterations of GLUT2 in liver tissue were not statistically significant.In vitro,HT treatment at 100?M inhibited PERK,IRE1?and JNK phosphorylation stimulated by PA,and the efficacy was comparable with the ER stress inhibitor 4-PBA.Expression of GRP78 also confirmed the effect of HT on ER stress modulation.Moreover,JNK inhibitor SP600125 effectively reversed the activation of IRS?tyrosine phosphorylation?and Akt?Ser473 phosphorylation?under HT interference in the cell stimulated by PA.2.HT treatment showed no significant changes in weight gain and adiposity.Circulating levels of ALT,AST,TC,TG and LDL-c were not affected by HT,except for content of HDL-c.However,lipid deposition in the liver and hepatic TG content was relieved by HT.TEM analysis revealed that HFD caused dramatic swelling and dilatation of the ER in hepatocytes,which was alleviated by HT supplementation.HFD feeding and PA stimulation led to significantly increased SREBP1 protein expression,in the liver and Hep G2 cells,respectively.Oral administration of HT and pretreatment with HT significantly reduced SREBP1 expression.Meanwhile,the altered mRNA level of SREBP1 in the liver,as well as of its downstream targets,ACC1,FAS and SCD1caused by HFD was recovered by HT replenishment.ConclusionPart One:Exercise Intervention1.HIIT protocol exhibited prominent beneficial effects in weight and fat mass loss,adipocyte size decrease.2.HIIT is more effective than MICT in treatment of hyperlipidemia and hepatic steatosis,principally through alleviating ultrastructural damage to hepatocytes caused by HFD,and altering expression of molecules related to hepatic lipid metabolism.3.HIIT ameliorated whole-body glucose homeostasis and inflammation,in accordance with modification of inflammatory and insulin signaling in adipose tissues.4.HIIT was particularly prone to enhancing thermogenic activity of BAT and inducing“browning”of subcutaneous adipose tissue.Part Two:Diet intervention1.HT ameliorated glucose homeostasis,chronic inflammation and decreased hepatic steatosis in DIO mice.2.HT rescued insulin receptor signaling and suppressed hepatic lipogenesis pathway through mitigating ER stress. |
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