| Prediabetic state(or "impaired glucose regulation")is the one of the most common lifestyle diseases,and it poses a severe threat to global public health worldwide.Prediabetes typically defined as blood glucose levels above normal but below diabetes thresholds,acting as the intermediate state and process of the development of type 2 diabetes mellitus(T2DM).As a result,new treatment approaches be able to convert prediabetes and also improve glucose and lipid metabolism are of growing importance.The Qi-collateral disease theory of Traditional Chinese Medicine(TCM)constructed by Professor Wu Yiling for the prevention and treatment of' endocrine metabolism diseases has drown increasing attention,in particular,he has put forward the new viewpoint "the mutual transformation of qi,blood,body fluid and essence in Qi-collateral is closely related to the function of endocrine system in the regulation of internal metabolic activities".Therefore,prediabetes,T2DM and other related metabolic syndrome are classified into the category of TCM Qi-collateral diseases.Brown adipose tissue(BAT),a highly metabolically active organ of energy expenditure,maintaining core body temperature during cold acclimation via non-shivering thermogenesis.Mediated by the uniquely expression of the BAT-specific uncoupling protein 1(UCP1)within the mitochondrial inner membrane,BAT functions to promote the whole-body energy metabolism,or the uncoupling of ATP production to generate proton leakage and then substrate oxidation.Therefore,improving BAT activity and increasing BAT content have been proposed as promising therapeutic strategies for the prevention and treatment of obesity,prediabetes,T2DM and other associated metabolic diseases due to its increased capacity for promoting adaptive thermogenesis and energy expenditure.Traditional Chinese medicine compound Jinlida granules(JLD)is an effective prescription for the prevention and treatment of T2DM developed under the guidance of Qi-collateral theory"spleen regulation",which has been widely used in clinical practice for many years.Current knowledge of the effects of JLD are regulation of glucose and lipid metabolism,improvement of insulin sensitivity and insulin secretion(?-cell function)and so on.Research demonstrated that the clinical usage of JLD therapy could lead to an improvement in body weight,and converting prediabetes back to normoglycaem,instead of developing and evolving to T2DM.However,the effects of JLD on obesity-associated metabolism disorders and BAT function in high fat diet-induced C57BL/6J mice are remained unknown.Therefore,this study was carried out from the viewpoint of traditional Chinese medicine(TCM)Qi-collateral theory and combined with experimental analysis,in order to explore the potential regulatory effects of JLD on metabolic disorders and BAT function.Objective:(1)Theoretical research:Under the guidance of the Qi-collateral theory of TCM,the main aim of this study was to summarize and analysis the pathogenesis of prediabetes state from the viewpoint of the function of Qi-collateral and Qi-activity and Qi-transformation,and elaborate the theoretical basis of "spleen regulation"therapy.After a thorough theoretical analysis,thus further providing the theoretical guidance and support for prediabetes and other related metabolism disorders.At the same time,the study could be considered as a theoretical instruction for investigating the preventive effects of JLD on prediabetes and the potential mechanisms.(2)Experimental research:In vivo study,we aimed to investigate the regulatory effects of JLD on obesity-associated metabolism disorders in high-fat diet(HFD)induced C57BL/6J mice compared to those on a low-fat diet(LFD)and high-fat diet group(HFD).At the animal levels,we observed the morphologically alteration and the expression of BAT-specific protein UCP1 in the BAT in the HFD group and JLD-treated mice.On the other hand,our study biologically investigated the effects of JLD on the expression levels of the BAT-specific thermogenic genes and mitochondrial fatty acid oxidation-related genes to determine the brown fat function in obese mice induced by a high-fat diet(HFD).In the cellular levels,we aimed to investigate whether JLD has an effect on the formation and function of C3H10T1/2 mesenchymal stem cells(MSCs)differentiated into the mature brown adipocytes.Methods:(1)Theoretical research:After a thorough theoretical analysis of Qi-collateral theory relevant literature,the theoretical study explored the close correlation between spleen Qi-activity and Qi-transformation,and substance conversion,energy metabolism disorders.We also explored the pathological mechanism of prediabetes and proposed the intervention strategy of "spleen regulation",in order to restore the spleen Qi-collateral function.(2)Experimental research:Part 1:A total of 45 male C57BL/6J mice(5-6-week-old,18-22g)were acclimatized to laboratory conditions for one week and randomly assigned into three groups according to their body weights as follows(n=15):low-fat diet(10%fat,70%carbohydrates and 20%protein,D12450B,New Brunswick,NJ,USA)group(LFD),high-fat diet(60%fat,20%carbohydrates,and 20%protein,D12492,New Brunswick,NJ,USA)group(HFD)and HFD plus JLD intervention group(HFD+JLD).Mice were administrated with JLD(3.8g/kg/d)by oral gavage simultaneously fed on a HFD.JLD was dissolved in 0.5%sodium carboxymethyl cellulose solution(CMC)to the concentration of 1.9 mg/mL and intraperitoneally injected in a volume of 0.2 mL/lOg.bw once daily up to 15 weeks.LFD and HFD mice were administration with 0.5% CMC as the control treatment.We measured the body weight and food intake of mice once a week for consecutive 15-week.At the end of the experiment,the mice were tested for intraperitoneal glucose tolerance test(IPGTT)and insulin tolerance test(ITT)to investigate the effect of JLD on glucose homeostasis.In addition,the areas under the blood glucose concentration curve(AUC)of IPGTT and ITT were also calculated.At sacrifice,after fasted for 12 h,their blood,inguinal WAT(iWAT),epididymal WAT(eWAT)and liver tissues were dissected,weighted,frozen in liquid nitrogen.The iWAT and eWAT in each group were subjected to the morphological observation.The serum was separated to analysis lipid profiles,and the liver tissues were collected to analysis morphological alteration and expression of related inflammatory factors.?Serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),free fatty acid(NEFA),aspartate aminotransferase(AST),alanine aminotransferase(ALT)were determined by automatic biochemical analyzer.?Morphological changes of iWAT and eWAT were observed by hematoxylin-eosin(HE)staining.?HE staining and Oil red O staining were performed to observe the lipid deposition of the liver tissues in each group.?Real-Time Quantitative PCR(qRT-PCR)analysis was performed to explore the mRNA expression of several inflammatory factors,such as tumor necrosis factor-?(TNF-?),Interleukin-6(IL-6),Medium chain acyl-CoA(MCAD)and Monocyte chemotactic protein-1(MCP1)in liver tissues.Part 2:Male C57BL/6J mice(5-6-week-old,18-22 g)were fed adaptively for one week and randomly assigned into two groups(n=15):high-fat diet group(HFD)and high-fat diet supplemented with Jinlida granules group(HFD+JLD).Mice were administrated with JLD(3.8g/kg/d)by oral gavage simultaneously fed on a HFD.In the final week,mice were removed to a cold room(4?),and their rectal temperature were recorded.At sacrifice,interscapular BAT was collected to detect the morphology and molecular biology alterations.?The HE staining of BAT was used to observe the morphological alterations in both groups.The immunohistochemical analysis were processed with anti-UCP1 antibodies to analysis the protein expression of UCP1 in BAT.?qRT-PCR analysis was performed to observe the mRNA expression levels of BAT thermogenesis-related genes UCP1,PR-domain containing protein-16(PRDM16),Type II iodothyronine deiodinase(Dio2),ELOVL fatty acid elongase 3(ELOVL3)and fatty acid oxidation-related genes Carnitine palmitoyl transferase-1 beta(CPT1?)and Peroxisome proliferator-activated receptor alpha(PPAR?)in BAT.?qRT-PCR analysis was performed to measure the mitochondrial DNA copy number in BAT.?Western blot analysis was performed to explore the protein expression levels of UCP1,peroxisome proliferator-activated receptor-gamma coactivator-1 alpha(PGC-l?)and mitochondrial oxidative complex proteins(OXPHOS)in BAT.Part 3:In an effort to investigate whether JLD is involved in brown adipogenesis and function in vitro,C3H10T1/2 mesenchymal stem cells were cultured and differentiatied treated with a brown adipogenic induction cocktail into mature brown adipocytes.JLD was dissolved in Dulbecco's Modified Eagle Medium(DMEM)for cell treatment.C3H10T1/2 mesenchymal stem cells were treated with JLD for 6 days during brown adipogenesis,vehicle-treated cells served as a control(Copt).C3H10T1/2 cells were treated with brown adipogenic induction medium(DMEM containing 10%FBS,20 mM insulin,2?g/mL dexamethasone,0.5 mM isobutylmethylxanthine,0.125 mM indomethacin,and 1 nM 3,3',5-Triiodo-L-thyronine(T3))for the first two days.The differentiation medium was then replaced by medium supplemented with only insulin and T3,which was changed every other day for four days.C3H10T1/2 cells were treated with or without JLD for six days during brown adipogenesis.On the sixth day,the fully differentiated brown adipocytes were used for all experiments in this study.?MTS assay was used to measure the cell viability in C3H10T1/2 mesenchymal stem cells cultured with or without various concentrations of JLD.?Oil red O staining was used to observe the morphological alterations in C3H10T1/2 mesenchymal stem cells treated with or without JLD during the brown adipogenesis.? qRT-PCR analysis was used to explore the gene expression levels of thermogenesis-related genes such as UCP1,ELO VL3,PGC-1?,peroxisome proliferator-activated receptor-gamma coactivator-1 beta(PGC-1?)and adipogenesis-related genes such as fatty acid-binding protein-4(FABP4),PPAR?,CCAAT/enhancer binding protein beta(C/EBP?)as well as fatty acid oxidation-related genes including CPT? and PPAR?in C3H10T1/2 cells.?Western blot analysis was performed to explore the thermogenic protein expression of UCP 1,Peroxisome proliferator-activated receptor-gamma(PPARy),PGC-1? and OXPHOS.?Confocal immunofluorescence staining was used to observe the mitochondrial content and UCP 1 expression regulated by JLD in mature brown adipocytes.?qRT-PCR analysis was performed to detect mitochondrial copy number regulated by JLD in mature brown adipocytes during the brown adipogenesis.?Oxygen consumption rates(OCR)were measured with a Seahorse Bioscience XF24 extracellular flux analyzer to test the respiratory function of mature brown adipocytes at day six in both groups.Results:1.Theoretical research:? The theoretical study puts forward Qi-collateral theory and provides a new theoretical guidance for the prevention and treatment of prediabetes.? Qi-activity and Qi-transformation abnormality are the crucial pathological mechanisms of prediabetes.?The disorder and imbalance of the energy intake and utilization caused by spleen deficiency are the core pathological mechanisms.? "Spleen regulation"Chinese medicine JLD provides a new approach for clinical prevention and treatment of prediabetes.2.Experimental research:Part 1:?At week 15 of intervention,we observed statistically significant differences in mean body weight in the groups.Compared with the HFD group,in vivo administration of JLD could significantly prevent body weight gain(P<0.001)from the fourth week,and this effect was continued through JLD supplementation till the end of the experimental period.While,there were no significant differences in the food intake between JLD-treated group and HFD control group(P>0.05).?Compared with the HFD control group,JLD-treated mice were also associated with tissue weight loss of iWAT and eWAT(P<0.05,P<0.01).In addition,Histologic analysis demonstrated that the sizes of lipid droplets in iWAT and eWAT from JLD-treated mice were smaller than those from the HFD control mice.?Compared with the HFD control group,the serum lipid profiles showed that JLD dramatically reduced the serum concentrations of TG,LDL-C,and NEFA(P<0.05,P<0.01),without any changes in serum TC(P>0.05).?IPGTT test revealed that JLD could significantly enhance glucose metabolic activity after the glucose injection compared with the HFD control group(P<0.05,P<0.01).?ITT test indicated that the JLD-treated mice were more sensitive to insulin challenge compared with the HFD control group(P<0.01,P<0.001).?Compared with the HFD-induced control mice,JLD treatment could notably ameliorated liver lipid contents in HFD-induced C57BL/6J mice,as evidenced by the H&E and Oil Red O staining of liver tissue samples.?Compared with the HFD control group,JLD treatment significantly downregulated the mRNA levels of the inflammatory cytokines in liver tissue induced by HFD treatment,including TNF-a,IL-6,MCAD,and MCP1(P<0.05,P<0.01).Part 2:?Compared with the HFD control group,cold challenge experiment indicated that JLD treatment significantly upregulated core body temperature when mice were exposed to a cold environment(4?)(P<0.01).?Histology and immunohistochemistry analysis of BAT revealed that JLD treatment could significantly improve the morphology of brown fat cells and increase the expression of BAT-specific thermogenic protein UCP1.?qRT-PCR analysis demonstrated that the mRNA levels of BAT-specific thermogenic genes including UCP1,PRDM16,Dio2 and ELOVL3 and other genes related to fatty acid oxidation including CPT1? and PPARa in the BAT were significantly upregulated by the JLD treatment(P<0.05,P<0.01)..?qRT-PCR analysis demonstrated that JLD treatment could significantly elevate the DNA copy number of mitochondria in the BAT(P<0.05).?Western blot analysis demonstrated that JLD treatment could also significantly increase the protein levels of UCP1,PGC,1?(P<0.05,P<0.01).Compared with the HFD control group,the protein levels of the mitochondrial oxidative phosphorylation OXPHOS proteins were significantly elevated in BAT of the mice treated with JLD,which included DADH dehydrogenase ubiquinone Subcomplex 5(NDUFB8),Succinate dehydrogenase complex and subunit B(SDHB),Ubiquinol-cytochrome C reductase core protein?(UQCRC2),Mitochondrially Encoded Cytochrome C Oxidase Subunit ?(MTCO1),ATP synthase subunit CV alpha(ATP5A)(P<0.05,P<0.01).Part 3:?MTS assay demonstrated that administration of JLD at the concentration of 400?g/mL has no obvious cytotoxicity.Therefore,in the subsequent cellular experiment,400p.g/mL concentration of JLD was used as the optimal dosage.?Oil Red O staining showed that JLD-treated cells accumulated much more small lipid droplets as compared with the control cells,thus displaying appearance of mature brown-fat-like adipocytes.In consistent with Oil-Red O staining,JLD also significantly increased the mRNA levels of several adipogenic transcription factors,such as PPARy2,CEBP/?,and Fabp4(P<0.05,P<0.01)·?Compared to the control cells,qRT-PCR analysis confirmed that JLD could significantly upregulate the mRNA levels of BAT-related thermogenic genes such as UCP1,PGC-1? and ELOVL3 during brown adipogenesis(P<0.05,P<0.01,P<0.001).In addition,JLD also dramatically increased the expression levels of several genes related to fatty acid oxidation such as CPT1? and PPARa during in vitro brown adipogenesis(P<0.01).?In comparison to the control group,Western blot analysis demonstrated that JLD treatment could significantly elevate the expression levels of key thermogenic proteins such as UCP1,PGC-la and mitochondrial oxidative complex proteins OXPHOS in C3H10T1/2 cells(P<0.05).The expression level of maker adipogenic protein PPARy was also significantly increased after the JLD treatment(P<0.05).?In comparison to the control group,JLD significantly increased mitochondrial DNA copy number by qRT-PCR.Compared with the control cells,the expression levels of BAT-specific protein UCP1 and mitochondrial content were dramatically elevated after the JLD treatment,as evidenced by the immunofluorescence imaging results.?Seahorse oxygen consumption analysis indicated that the basal oxygen consumption,uncoupled oxygen consumption,and maximal oxygen consumption rates were significantly higher in the JLD-treated cells as compared with the control group(P<0.05,P<0.01,P<0.001).Conclusions:1.Under the guidance of Qi-collateral theory of TCM,the theoretical study provides us a new theoretical guidance for the prevention and treatment of prediabetes and related metabolic diseases.Qi-activity and Qi-transformation abnormality are the crucial pathological mechanisms of prediabetes based on a close relationship between spleen Qi-collateral and substance conversion,energy metabolism.The metabolic disorders and imbalance of the energy intake and utilization caused by spleen deficiency has been revealed as the core pathogenesis of prediabetes.Based on the principle of "Spleen regulation",Chinese medicine JLD provides us a new approach for clinical prevention and treatment of prediabetes.Taken together,the theoretical study provided us new ideas and approaches for the prevention and treatment of prediabetes and related metabolic diseases.2.In the animal study,we showed that JLD could effectively improve the disorders of glucose and lipid metabolism induced by a HFD in mice,which might be,at least in part,due to the activation of BAT thermogenesis.Part 1 confirmed that JLD could effectively decrease body weight gain,whole-body adipose tissue mass and the size of the adipocytes,improve lipid metabolism disorders,enhance glucose tolerance and insulin sensitivity,and dramatically alleviate lipid deposition and inflammation of liver tissue in HFD-induced C57BL/6J mice.Part 2 indicated that JLD could significantly upregulate the mRNA expression of several brown fat-specific genes such as UCP1,PRDM16,Dio2 and ELOVL3.At the same time,several thermogenic proteins such as UCP1,PGC-la and mitochondrial oxidative phosphorylation-related protein OXPHOS were notably raised in BAT from JLD-treated mice.In addition,mitochondria DNA copy number and fatty acid oxidation related genes such as CPT1?and PPARa were significantly elevated in the BAT after the JLD treatment.Our in vivo results confirmed that JLD treatment could effectively resisted to high-fat diet(HFD)-induced body weight gain,improved glucose homeostasis and ameliorated hepatic steatosis and inflammation in C57BL/6J mice fed with a high-fat diet.Data of Part 2 also showed that JLD treatment markedly enhance the thermogenesis activity of BAT,as evidenced by the improvement of BAT morphology and molecular biology.These results revealed for the first time that JLD treatment effectively reduces adiposity and related metabolic disorders by significantly enhancement of BAT activity in HFD-induced C57BL/6J mice.Taken together,our results demonstrate that JLD as a promising BAT activator,may have great and beneficial potential for the prevention and treatment of obesity,prediabetes and related metabolic disorders.3.JLD could significantly promote the adipogenic differentiation of C3H10T1/2 cells into mature brown adipocytes and also improve the thermogenic function of brown adipocytes.JLD could significantly upregulate the gene expression levels of key adipogenic transcription factors such as FABP4,PPARy2 and CEBP/?,and BAT-specific thermogenic genes such as UCP1,ELOVL3,PGC-la and PGC-1?p and also increase the expression levels of BAT thermogenesis-related proteins such as UCP1,PGC-la and mitochondrial oxidative phosphorylation-related protein OXPHOS.Our data suggested that JLD could regulate the activity of BAT and improve the function of mitochondrial during brown adipogenesis.In addition,JLD treatment markedly increased mitochondria DNA copy number and fatty acid oxidation,related genes such as CPT1? and PPARa in mature brown adipocytes.At the same time,mitochondria content and the expression of UCP1 were both dramatically up-regulated after the JLD treatment in mature brown adipocytes.Oxygen consumption analysis suggested that JLD could effectively improve the respiratory function of brown fat cells.Taken together,our in vitro results suggested that JLD could significantly promote the formation of brown fat cells,up-regulate the activity of brown adipocytes and mitochondrial function during brown adipogenesis. |
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