| Objective:To evaluate the browning markers in subcutaneous(sWAT)and visceral adipose tissue(vWAT)in people with obesity.In addition,we established high fat-diet(HFD)induced obese model on SD rats,and observed the activity of intercapular brown adipose tissue(iBAT)and the browning markers in sWAT in rats.Methods:1 We collected the abdominal subcutaneous adipose tissue and omental adipose tissue from patients with obesity undergoing weight loss surgery,to detect the browning markers in these fats by RT-PCR.Additinally,we analyzed the relationship between UCP1 expression in adipose tissue and body mass index(BMI)as well as insulin resistance index(HOMR-IR).2 Six-week-old male Sprague Dawley rats were adaptive feeding for 1 week and then randomly divided into two groups:(1)normal chow group(NC),with a standard chow diet for 18 weeks;(2)high fat diet group(HF),with high fat diet for 18 weeks.3 Body weight of rats was recorded weekly,insulin resistance related indicators were measured at the 18th week:fasting blood glucose,fasting blood insulin,insulin resistance index(HOMR-IR),intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT),the cold tolerance test was also evaluated.4 The iBAT and sWAT of rats were stained with H&E.The expression of uncoupling protein-1(UCP1)was detected by immunohistochemical staining(IHC).The mRNA levels of browning marker in iBAT and sWAT were detected by RT-PCR:UCP1,cell death inducing DFFA like effector a(Cidea),PR domain containing 16(PRDM16),transmembrane protein 26(Tmem26),peroxisome proliferator activated receptor γ(PPARy),PPARG coactivator la(PGCla),cluster of differentiation 137(CD137).The protein levels of phosphorylated serine/threonine kinase 1(p-AKT),phosphorylated hormone-sensitive lipase(p-HSL),UCP1,Complex III and Complex V in iBAT and sWAT were detected by Western Blotting(WB).Results:1 Compared with normal weight people,the mRNA levels of UCP1,Cidea,Tmem26,PGCla,PPARy,adiponectin and CD 137 in sWAT of people with obesity were significantly decreased.What’s more,the mRNA level of UCP1 in sWAT was negatively correlated with BMI and HOMR-IR in people with obesity.2 Compared with normal weight people,the mRNA levels of UCP1,Cidea,Tmem26,PGCla,PPARy,adiponectin and CD 137 in vWAT of people with obesity were significantly decreased.What’s more,the mRNA level of UCP1 in vWAT was negatively correlated with BMI and HOMR-IR in people with obesity.3 Significant weight gain and systematic insulin resistance were induced in SD rats after HFD for 18 weeks.4 Rats in the HF group were less tolerance to cold than NC group.5 In the HF group,the sizes of brown adipocytes were increased,the insulin sensitivity and lipolysis in iBAT were decreased.The expression levels of browning markers in iBAT were decreased,which became "whitening".6 In the HF group,the sizes of subcutaneous white adipocytes were increased,the insulin sensitivity and lipolysis in sWAT were decreased,and the expression levels of browning markers in sWAT were also decreased.The "browning" level in sWAT was reduced by HFD.Conclusions:1 The "browning" levels in sWAT and vWAT were decreased in people with obesity.The expression levels of UCP1 in sWAT and vWAT were all negatively correlated with BMI and HOMR-IR in people with obesity.2 HFD-induced systematic insulin resistance in rats,decreased adaptive thermogenensis,decreased insulin sensitivity and lipolysis in iBAT and sWAT,decreased browning level in iBAT and sWATObjective:We treat HFD rats with abelmoschus esculentus(AE)and metformin,to study whether AE and metformin could exhibit effects on the metabolic disorders,the activity of iBAT and browning of sWAT in HFD rats.Methods:1 Six-week-old male Sprague Dawley rats were randomly divided into the following four groups after adaptive feeding for 1 week:(1)normal diet group(NC),with a standard chow diet for 18 weeks,equal volume of 0.9%saline was administered daily from the 11th week by oral gavage,the intervention time was 8 weeks;(2)high-fat diet group(HF),with HFD for 18 weeks,equal volume of 0.9%saline was administered daily from the 11th week by oral gavage,the intervention time was 8 weeks;3)high fat diet with AE group(HF-AE),fed with HFD for 18 weeks,800mg/kg okra was administered daily from the 11th week by oral gavage,the intervention time was 8 weeks;(4)high fat diet with metformin group(HF-Met),fed with HFD for 18 weeks,200mg/kg metformin was administered daily from the 11th week by oral gavage,the intervention time was 8 weeks.2 The body weight was recorded weekly,and insulin resistance related indicators were measured at the 18th week:fasting blood glucose,fasting blood insulin,HOMR-IR,IPGTT,IPITT and cold tolerance test.3 The iBAT and sWAT were stained with H&E,and the expression of UCP1 was detected by IHC.The mRNA levels of browning marker in iBAT and sWAT were detected by RT-PCR:UCP1,Cidea,PRDM16,Tmem26,PGCla,PPARy,CD137.In addition,the inflammatory factors were quatified by RT-PCR in iBAT and sWAT.The protein levels of p-AKT,p-HSL,UCP1,complex III and complex V in iBAT and SWAT were also detected by WB.Results:1 Compared with HF group,both AE and metformin significantly improved the weight gain,fat deposition and systematic insulin resistance.2 Rats in HF-AE and HF-Met group exhibited more tolerance to cold than HF group.3 AE and metformin reduced the sizes of brown adipocytes induced by HFD,improved insulin sensitivity and lipolysis ability in iBAT,up-regulated the expressions of UCP1 and other browning markers in iBAT,as well as improving the inflammation in iBAT.4 AE and metformin can decrease the sizes of subcutaneous adipocytes induced by HFD,increase insulin sensitivity and lipolysis in sWAT,the expressions of UCP1 and other browning markers in sWAT were also up-regulated.The inflammatory response caused by HFD was improved in sWAT by AE and metformin.Conclusions:1 AE and metformin could improve the metabolic disorders induced by HFD in rats.2 AE and metformin can increase the activity of iBAT in HFD,up-regulating the expressions of UCP1 as well as other browning markers in iBAT.The adaptative thermogenesis was potentiated by AE and metformin.The inflammation in iBAT induced by HFD was also attenuated by AE and metformin.3 AE and metformin can increase the "browning" of sWAT,increasing the expression of browning markers in sWAT.The inflammation induced by HFD was reduced by AE and metformin.Objective:To explore the specific molecular mechanism of AE and metformin in increasing the activity of iBAT and promoting "browning" of sWAT both in vivo and in vitro.Methods:1.Isolate and culture primary stromal vascular fraction(SVF)from rat iBAT,sWAT and human sWAT,separately.Then differented the SVF into mature adipocytes.AE and metformin were added into the differented medium.At the end of differentiaon,the protein and mRNA levels of the browning markers in the adipocytes were detected by WB and RT-PCR,the level of citrate synthase activity was also measured.2.In the animal model established in Part 2,the level of p-AMPK in iBAT and sWAT of rats were detected by WB;The p-AMPK in the primary adipocytes after AE and metformin stimulation were also evaluated.3.When start the differentiation,the AMPK inhibitor was added into the primary SVF isolated from rats iBAT and sWAT,and then stimulated with AE and metformin,respectively.After the cells were differentiated into mature adipocytes,the relative mRNA expressions of browning genes were detected by RT-PCR.The protein levels of UCP1,Complex Ⅲ,and Complex Ⅴ were measured by WB.4.When started the differentiation,the AMPK activator was added into the primary SVF isolated from rats iBAT and sWAT,and then stimulated with AE and metformin,respectively.After the cells were differentiated into mature adipocytes,the relative mRNA expressions of browning genes were detected by RT-PCR.The protein levels of UCP1,Complex Ⅲ,and Complex Ⅴ were measured by WB.Results:1.AE and metformin increased the mRNA levels of browning genes in primary adipocytes isolated from rats iBAT,sWAT and human sWAT,respectively.Consistently,the protein levels of UCP1,Complex III and Complex V were also significantly increased in rat brown and white adipocytes.Additionally,the activity of citrate synthase was also up-reuglated in primary adipocytes from rat iBAT and sWAT.2.The p-AMPK level was reduced in iBAT and sWAT of rats from HF group.While AE and metformin could up-regulate the p-AMPK levels in HF-AE and HF-Met group.In addition,AE and metformin increased p-AMPK level in primary adipocytes from rats iBAT and sWAT,respectively.3.After adding the AMPK inhibitor-Compound C into the primary SVF from rats iBAT and sWAT in vitro,the up-regulated browning genes by AE and metformin were abolished,no signigicant increase of the protein levels of UCP1,Complex III and Complex V were observed in AE and metformin intervention.4.After adding AMPK activator-AICAR into the primary SVF from rats iBAT and sWAT in vitro,AE and metformin induced a more significant increase of browing genes and protein levels of UCP1,Complex III and Complex VConclusions:1.AE and metformin also increase browning and mitochondrial function in both brown and white adipocytes,which proves that AE and metformin indeed increased iBAT activity and promoted "browning" in WAT.2.AE and metformin promote iBAT activity and "browning" of WAT by activating the AMPK. |
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