| ObjectPalmitic acid(PA)in serum increased under obesity status can promote the expression of inflammatory factor interleukin-6(IL-6)by up-regulating Kruppel-like factor 7.However,the specific molecular mechanism that PA regulates KLF7 remain unclear.In our study on the basis of culturing adipocytes-3T3-L1 and human liver cancer cells-HepG2 in vitro and constructing diet-induced obesity C57BL/6 mouse mode in vivo to clarify whether PA up-regulates the expression of KLF7 through activating GPR40/GPR120-NF-κB signaling pathway which in turn influence the expression of pro-inflammatory cytokines,glucose consumption ability and insulin sensitivity,search experimental and theoretical basis for the clinical search for new targets for the treatment of obesity,inflammation and related metabolic diseases.Method(1)Culturing 3T3-L1 adipocytes and HepG2 cells in vitro,adding 200 μM PA into culture medium respectively for 48 hours or 24 hours.mRNA and protein expression of of GPR40,GPR120,p-p65,KLF7 and the glucose consumption ability of cells were determined by qRT-PCR,Western blot and kit method.(2)Adding GW1100(50 μM,100 μM,150 μM),AH7614(50 μM,100 μM,150 μM)and Bay11-7082(10 μM)respectively to block GPR40,GPR120 and p-p65 specifically while treating cells with 200 μM PA,mRNA and protein expression of p-IKKβ,p-IκB,p-p65,KLF7 as well as inflammatory factors IL-6,MCP-1,TNF-α,glucose transporter GLUT4,and the ability of glucose consumption of cells were determined by qRT-PCR,Western blot and kit method.(3)Human p65 overexpressed plasmid was constructed and transfected into 293 T and HepG2 cells.The mRNA and protein expressions of KLF7 were detected.(4)C57BL/6(5-week-old,male)mice were determined as two sets: Normal Chow Diet(NCD,n = 8),High Fat Diet(HFD,n = 32).After 7 week,HFD mice were grouped into four sets: HFD with intraperitoneal injection of DMSO(n = 8),intraperitoneal injection of GW1100(2.5 mg/kg body weight,n = 8),intraperitoneal injection of AH7614(2.5 mg/kg body weight,n = 8)and intraperitoneal injection of Bay 11-7082(2.5 mg/kg body weight,n = 8)to block GPR40,GPR120 and p-p65 respectively in mice,the body weight of mice was dynamically measured once a week.(5)Glucose Tolerance Test(GTT)and Insulin Tolerance Test(ITT)were carried out after injection of the above blockers from 5th to 12 th weeks.Collecting and weighing different parts of adipose tissues and liver,p-p65,KLF7,IL-6 and GLUT4 protein and mRNA expressions in vis WAT and liver were detected.PA,blood glucose and blood lipid in serum were determined by kit assay(Peroxidase method).Inflammatory cytokines(IL-6,TNF-α,MCP-1)were determined by ELISA,hepatic steatosis in different groups was observed by HE staining.Results1.Differentiation of 3T3-L1 mouse fibroblasts and culture of HepG2 cells in vitro1.1 3T3-L1 cells are spindle-shaped with clear boundaries before adding induction medium.After differentiation the cell gradually became round,irregular,and the boundaries were blur.Observing cell morphology at high magnification after adipocyte differentiation is successfully induced,it was observed that lipid droplets of different sizes,regular shapes,round and full were distributed around the cells,forming a "circular" structure,and stained red by oil red O staining,showing that3T3-L1 adipocytes were successfully induced.1.2 HepG2 cells were observed to grow in an "islet" like aggregation under a microscope 48 hours later,cells could grow to about 95%.Subsequent treatment could be performed according to experimental needs.2.PA promotes the expression of KLF7 through activating GPRS/NF-κB signaling pathway2.1 After adding 200μM PA into 3T3-L1 and HepG2 cells culture medium for 48 h and24h,promoting the protein expressions of GPR40,GPR120,p-p65 and KLF7,the mRNA expression level of KLF7 were increased,and the ability of glucose consumption of cells was significantly inhibited,differences were statistically significant(P < 0.05).2.2 Different concentrations of GW1100 were added to block GPR40 specifically while 3T3-L1 adipocytes and HepG2 cells were treated with 200 μM PA,the mRNA expressions of KLF7,IL6,MCP-1 and TNF-α were significantly inhibited,the lower expression of GLUT4 induced by PA was significantly reversed by GW1100.The protein expression levels of p-IKKβ,p-IκB,p-p65,KLF7 and IL-6 were significantly decreased compared with 200 μM PA-only treatment group.Blocking GPR40 significantly improved the impaired glucose consumption induced by PA.Protein expressions of p-p65 and KLF7 in HepG2 nucleus was significantly inhibited after inhibiting GPR40,differences were statistically significant(P < 0.05).2.3 Different concentrations of AH7614 were added to block GPR120 specifically while 3T3-L1 adipocytes and HepG2 cells were treated with 200μM PA,the mRNA expressions of KLF7,IL6,MCP-1 and TNF-α were significantly inhibited,the lower expression of GLUT4 induced by PA was significantly reversed by AH7614.The protein expression levels of p-IKKβ,p-IκB,p-p65,KLF7 and IL-6 were significantly decreased compared with 200 μM PA-only treatment group.Blocking GPR120 significantly improved the impaired glucose consumption induced by PA.Protein expressions of p-p65 and KLF7 in HepG2 nucleus was significantly inhibited after inhibiting GPR120,differences were statistically significant(P < 0.05).3.PA up-regulates the expression of KLF7 by promoting p65 phosphorylation3.1 Adding Bay 11-7082 to repress phosphorylation of p65 while treating 3T3-L1 adipocytes with 200 μM PA the expression of KLF7 mRNA and protein were significantly inhibited and the glucose consumption capacity of cells was significantly increased,differences were statistically significant(P < 0.05).3.2 Adding Bay 11-7082 to repress phosphorylation of p65 while treating HepG2 cells with 200 μM PA,the mRNA and protein expression of KLF7 were repressed,as well as nucleoprotein expression of p-p65 and KLF7 compared with 200 μM PA-only treatment group.Blocking GPR120 significantly improved the impaired glucose consumption induced by PA,differences were statistically significant(P < 0.05).4.Upregulation of p65 significantly promoted KLF7 mRNA and protein expression4.1 After transfect p65 overexpressed plasmid into 293 T cells and HepG2 cells for48 h,the mRNA and protein expression levels of KLF7 were significantly increased;While p65 was overexpressed in HepG2 cells,the highly expressed KLF7 due to p65 transfection was significantly reversed after the phosphorylation of p65 was specifically blocked by the addition of Bay 11-7082,differences were statistically significant(P < 0.05).5.Construction of diet-induced obesity mouse model5.1 After feeding with HFD for 7 weeks,the body weight(30.9±0.32g)was higher than NCD mice(25.8±0.3g),and exceeded 20% of the body weight of NCD group,differences were statistically significant(P < 0.05).HE staining showed that the lipid accumulation in hepatocyte of HFD mice increased,results above suggest that the diet-induced obesity mouse model was successfully constructed.5.2 After feeding mice with HFD for 12 weeks,the weight(34.9±1.2g)was higher than NCD mice(28.6±0.53g).Serum PA,TG,and glucose levels were significantly higher than those in NCD group,differences were statistically significant(P < 0.05).5.3 Feeding HFD mice for 12 weeks,subcutaneous adipose tissue(sub WAT),visceral adipose tissue(vis WAT),epididymal adipose tissue(epi WAT),perirenal adipose tissue(pr WAT),brown adipose tissue(BAT)and Liver weight were significantly higher than those of NCD mice,differences were statistically significant(P < 0.05).6.Effects of GPRs and p-p65 blocker on fat accumulation,blood glucose and lipid levels in diet-induced obese mice6.1 After feeding mice with HFD for 7 weeks,obese mice were grouped into four sets:HFD group,HFD group with intraperitoneal injection of GW1100(blocking GPR40 in vivo),intraperitoneal injection of AH7614(blocking GPR120 in vivo),and intraperitoneal injection of Bay 11-7082(blocking p-p65 in vivo)for 5 weeks.6.2 Compared with HFD mice,the body weight of mice injected with GPR40 blocker GW1100 was significantly decreased,the weight of sub WAT,vis WAT,epi WAT and BAT were decreased,blood glucose was significantly decreased.After the injection of GPR120 blocker AH7614,the body weight and adipose tissue weight showed no difference,blood glucose was reduced.Body weight,weight of sub WAT,vis WAT,epi WAT and BAT in mice were significantly reduced after injection of p-p65 blocker Bay 11-7082,and the blood glucose was reduced.GTT and ITT in obesity-induced mice were improved by blocking p-p65,differences were statistically significant(P <0.05).Blocking GPR40,GPR120 and p-p65 did not affect the blood lipids of mice.7.The effects of GPRs and p-p65 blockers on the expression of p-p65,KLF7,IL-6,GLUT4 in adipose tissue and liver of diet-induced obese mice7.1 Compared with HFD mice,after feeding with high fat diet and intraperitoneal injection of GW1100 to specifically block GPR40,the mRNA expression of KLF7,IL-6 in vis WAT were reduced,the mRNA expression of GLUT4 was increased.Protein expression levels of p-p65,KLF7 were reduced,as well as the content of inflammatory factors IL-6,TNF-α,MCP-1,differences were statistically significant(P < 0.05).7.2 Compared with HFD mice,after feeding with high fat diet and intraperitoneal injection of AH7614 to specifically block GPR120,the mRNA expression of KLF7,IL-6 in vis WAT were reduced,the mRNA expression of GLUT4 was increased.Protein expression levels of p-p65,KLF7 were reduced,the release of inflammatory factors(IL-6,TNF-α,and MCP-1)were inhibited,differences were statistically significant(P < 0.05).7.3 Compared with HFD mice,after feeding with high fat diet and intraperitoneal injection of Bay 11-7082 to specifically block p-p65,the mRNA expression levels of KLF7,IL-6 in vis WAT were reduced,the mRNA expression of GLUT4 was increased.Protein expression levels of p-p65,KLF7 were reduced,differences were statistically significant(P < 0.05).Conclusion1.Palmitic acid promotes phosphorylation of p65 and KLF7 expression by activating GPR40/GPR120,induce inflammatory response of adipocytes,inhibit the expression of GLUT4 and glucose consumption ability of adipocytes.2.Palmitic acid content in serum of mice increased after obesity,which promoted phosphorylation of p65 and up-regulated KLF7 expression through activating GPR40/GPR120 in adipose tissue,leading to inflammatory response,resulting in decreased GLUT4 expression in adipose tissue,decreased insulin sensitivity and increased blood glucose in mice. |
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