The Effect And Mechanism Of Plin1 Knockout On Lipid Metabolism And Inflammation In Adipose Tissue Of Obese Mice

Objective:To provide an exclusive and specific animal model for the study of PLIN1 related functions,a homozygous Plin1 gene knockout mouse strain was constructed.In order to further verify and explore the PLIN1 protein on lipid metabolism and inflammation in adipose tissue and its molecular mechanism,we observed the related effects of Plin1gene knockout on high-fat diet induced obese mice.Methods:1.Use CRISPR/Cas9,micro-injection and embryo transfer technology to construct Plin1 gene knockout mouse animal model,and carry out strain purification.According to the sequence of exon 2 of Plin1 gene,design,screen and synthesize sgRNA to construct plasmid-type sgRNA expression vector;The success of vector construction was verified with the double enzyme digestion method and gene sequencing;Follow the sgRNA kit instructions for in vitro transcription of sgRNA,the size and integrity of the transcription product was measured with agarose gel electrophoresis,the concentration and purity of the transcription product was determined by ultra-micro spectrophotometer;Female C57BL/6J mice aged 8-10 weeks was induced to superovulate by HCG and PMSG,and cage them with male mice of the same age to obtain fertilized eggs;The sgRNA and Cas9protein mixture was delivered to the pronucleus of the fertilized egg using micro-injection technology;The fertilized eggs in good condition after micro-injection were transfered into the fallopian tubes of pseudo-pregnant female mice using embryo transfer technology to make the recipient female mice pregnant;After 19-21 days,the F₀generation mice are born;Tail-cut and extract the offspring mouse genomic DNA,PCR amplify the Plin1 gene;F₀chimeric mice were verified and screened with agarose gel electrophoresis and gene sequencing;F₀generation chimera mice were inbred with wild-type heterosexual mice in the same litter,and F₁generation mice were obtained from 19-21 days pregnant mice;Agarose gel electrophoresis was used for genotyping and screening of heterozygous positive mice;Male and female heterozygous mice in the same litter were inbred,and F₂mice were obtained after 19-21 days.Homozygous Plin1 gene knockout mice were screened out by genotype identification;Homozygous heterosexual mice in the same litter were inbred to expand the group,and the offspring were trimmed after birth for genotype identification.If the results were all homozygous,it indicated that the Plin1 gene knockout mouse strain was favourably constructed.The expression levels of PLIN1 protein and mRNA in adipose tissue,liver tissue and skeletal muscle of wild-type mice,heterozygous Plin1 gene knockout mice,and homozygous Plin1 gene knockout mice were detected by Western blot and RT-PCR.2.To investigate the effect and mechanism of Plin1 gene knockout on obesity of mice and lipid metabolism in adipose tissue.12 male wild-type C57BL/6j mice and 12Plin1 gene knockout mice were randomly divided into 4 groups（n=6）:control group（CON-C group）,6 wild-type C57BL/6j mice,knockout group（KO-C group）,6 Plin1gene knockout mice,all fed with D12450B normal mouse growth maintenance feed（10%fat calories,3.85 kcal/g）;high fat group（CON-H group）,6 wild-type C57BL/6j mice,knockout high-fat group（KO-H group）,6 Plin1 gene knockout mice,all fed D1245145%high fat diet（45%fat calories,4.7 kcal/g）for 12 weeks.During the experiment,the mice’s food intake,body length and body weight were monitored.One week before the end of the experiment,the glucose tolerance test measures the glucose tolerance level of mice,the insulin resistance test measures the level of insulin resistance in mice.The energy metabolism rate of mice was compared by oxygen consumption measurement.After the experiment,all mice were sacrificed,and the serum levels of TC,TG,NEFAs,glycerol,HDL-c and LDL-c in each group of mice were detected by enzymatic method;Observe and weigh the changes of various organs in mice;The pathological changes of white and brown adipose tissue was detected by HE staining;The difference of ATGL,HSL,p-HSL and SREBP1 protein expression in epididymal adipose tissue was measure with Western blot;The difference of hsl,fas,atgl and acc gene mRNA expression in epididymal fat tissue was measure with RT-PCR;By culturing mouse adipose tissue in vitro and applying insulin（INS）or isoproterenol（ISO）intervention,the levels of NEFAs and glycerol in the culture medium were detected.3.To investigate the effect and mechanism of Plin1 gene knockout on the inflammation of adipose tissue in obese mice.The serum and part of the epididymal adipose tissue in vitro culture medium of each group were collected,and the levels of TNF-α,IL-6 and MCP-1 in the adipose tissue culture medium and serum of the mice was measured with ELISA;The expression of F4/80 in white adipose tissue was measured with immunohistochemical staining;The expression and translocation of NF-κB P65 in white adipose tissue were detected by immunofluorescence staining;The difference of tnf-α,il-6 and mcp-1 mRNA expression in epididymal adipose tissue was measured with RT-PCR.;The difference of NF-κB p-P65 expression in epididymal adipose tissue was measured with Western blot.Results:1.Successfully designed and constructed the plasmid-type sgRNA expression vector of Plin1 gene,and obtained intact sgRNA with a concentration of more than 5000 ng/L through in vitro transcription.The pronuclear injection and embryo transfer of fertilized eggs were completed.The size of wild mouse Plin1 gene fragment was 1236 bp,and the size of knockout mouse Plin1 gene fragment was 495 bp.Homozygous Plin1 knockout mice(Plin1^-/-)and strains were obtained.The levels of PLIN1 protein and mRNA in the liver,adipose tissue and skeletal muscle of heterozygous Plin1 knockout mice were significantly reduced（P<0.05）,and the homozygous Plin1 knockout mice have almost no expression of PLIN1 protein and mRNA in liver,adipose tissue and skeletal muscle.2.After 12 weeks of feeding,compared with the CON-C group,KO-C mice had no significant changes in body weight,body length,food intake,and GTT level（P>0.05）.The energy metabolism rate and ITT level increased,and the epididymis and groin were increased.The weight of adipose tissue decreased,the proportion of lipid droplets in white adipose tissue decreased,and the proportion of lipid droplets in brown adipose tissue increased（P<0.05）.The levels of NEFAs and HDL-c in the serum increased significantly（P<0.05）.The expression and mRNA levels of lipolytic enzymes HSL,p-HSL and ATGL increased,the expression of SREBP1c and the mRNA levels of fas and acc genes decreased（P<0.05）,and the level of glycerol and NEFAs in the medium increased in the basal state of adipose tissue,the level of glycerol and NEFAs in the culture dish decreased to a small extent after the intervention of INS（P<0.05）,and the level of glycerol and NEFAs in the medium did not change significantly after the intervention of ISO.Compared with the CON-C group,the CON-H group mice were significantly obese,the metabolic rate decreased,the GTT and ITT levels increased（P<0.05）,the liver and adipose tissue weight increased,and the lipid droplet area in the adipose tissue increased.Increased blood lipid levels（P<0.05）,decreased lipolytic enzyme expression and mRNA levels,increased SREBP1 expression and fas and acc gene mRNA levels（P<0.05）.The level of glycerol and NEFAs in the culture medium increased in the basal state of adipose tissue,and the level of glycerol and NEFAs in the culture dish decreased to a small extent after the intervention of INS（P<0.05）.The level of glycerol and NEFAs in the culture medium did not change significantly after the intervention of ISO.Compared with the CON-H group,the weight of the KO-H group was significantly reduced,the energy metabolism rate was significantly increased（P<0.05）,the weight of the epididymis and inguinal adipose tissue was reduced,the proportion of lipid droplets in the white adipose tissue was reduced,and the brown adipose tissue The proportion of medium lipid droplets further increased,serum TG,TC and NEFAs levels were significantly reduced,HDL-c,LDL-c and glycerol levels increased（P<0.05）,lipolytic enzyme expression and mRNA levels in adipose tissue increased,The expression of SREBP1c and mRNA levels of fas and acc genes decreased（P<0.05）,and the levels of glycerol and NEFAs in adipose tissue foundation,INS and ISO intervention medium increased（P<0.05）.3.Compared with the CON-C group,the levels of IL-6 and TNF-αin the adipose tissue culture medium and serum of the mice in the CON-H group increased significantly,and there was no significant change in MCP-1 in serum,but the level of MCP-1 in adipose tissue culture medium was significantly increased（P<0.05）.The infiltration of M1 macrophages in adipose tissue,the fluorescence intensity and nuclear translocation level of NF-κBP65,the expression of p-P65,and the mRNA expression levels of tnf-α,il-6 and mcp-1 genes increased significantly（P<0.05）;Except that the mRNA expression levels of tnf-αand il-6 genes did not change significantly,the serum and adipose tissue of mice in the KO-C group also had similar changes.Compared with CON-H,the serum TNF-αof mice in the KO-H group increased,IL-6 and MCP-1 did not change significantly,the levels of three pro-inflammatory factors in the adipose tissue culture medium were significantly increased.The expression of F4/80,P65 fluorescence intensity and nuclear translocation,p-P65 expression and pro-inflammatory gene mRNA in adipose tissue all increased significantly（P<0.05）.Conclusion:1.Using CRISPR/Cas9,microinjection and embryo transfer technology,successfully constructed Plin1 gene knockout mice,and obtained stable genetic homozygous Plin1 knockout mice(Plin1^-/-)and their strains,which provide an exclusive and specific animal model for studying the related functions of PLIN1.2.Knockout of Plin1 gene up-regulates lipase expression,down-regulates lipid synthesis transcription factor and downstream target gene expression,promotes lipolysis and inhibits lipid synthesis in WAT,improves high fat diet-induced obesity in mice.3.Knockout of Plin1 gene promotes the inflammatory response of adipose tissue in obese mice.The mechanism may be that lipolysis mediated by PLIN1 inactivation increases,promotes the polarization and infiltration of M1 macrophages,and activates the NF-κB pathway to cause pro-inflammatory factors synthesis and release.