| Objective:Obesity related diseases(such as NAFLD,diabetes)have been a growing threat to human life and health,which has caused wide public concern all over the world.These metabolic syndromes tend to be companied with fat metabolism disorders and abnormal accumulation of lipids in the cells of various organs.PAT protein family is one of the most studied lipid droplet proteins,while PLIN5 is a new comer,which has high expression in metabolically active tissues such as skeletal muscle,liver,and brown adipose tissue.Some studies have shown that the expression of plin5 in the subcutaneous white adipose tissue of mice is significantly increased after cold stimulation After patients of abnormal glucose tolerance(IGT)took pioglitazone(it could induce browning of white adipose)for 10 weeks,the mRNA level of PLIN5 in the subcutaneous adipose tissue was significantly higher than that before the treatment.Hence,it is speculated that PLIN5 might play an important role in regulating beige fat formation.This study will explore the effect of plin5 on browning of white fat and its molecular mechanism.Methods: Part ?: Investigate the effect of PLIN5 on browning of white adipose.The browning of white fat was induced by intraperitoneal injection of cl316243 into C57 BL /6 mice for 10 days,and the expression of plin5 and UCP1 in subcutaneous white adipose tissue was detected;Purchase 15 db/db mice and 5 wt(BKS)mice.10 db/db mice were randomly divided into two groups,which are insulin group and non-insulin group according to whether they were injected with insulin.After that,PLIN5 overexpressed lentivirus and negative control virus are injected into the caudal vein in each group.Fasting blood glucose was measured in each group,and insulin content was measured in serum of each group.Perform Real-Time PCR and Western Blot experiments on subcutaneous white adipose tissue in the insulin group,testing the expressions of PLIN5 and UCP1.Part ?: Perform RNA seq high-throughput sequencing on PLIN5 overexpressed lentivirus and negative control virus in transfected C3H10T1/2 cells,after analyzing the results,we found 6 up-regulated mitochondrial genes and 4 down-regulated mitochondrial genes.After Real-Time PCR verification of these genes was performed,the positive expression of BNIP3 was found and verified at the cellular and tissue levels;take C3H10T1/2 cells,transfect PLIN5 overexpressed plasmid +BNIP3 1000 bp promoter plasmid and negative control plasmid +BNIP3 1000 bp promoter plasmid,then perform Dual-Luciferase test to verify the expression quantity.Results: 1.After browning of white adipose was induced through CL316243,mRNA expressions of PLIN5 and UCP1 in subcutaneous white adipose and epididymal fat significantly enhanced in mice(P<0.05).2.In the subcutaneous white adipose tissue of mice injected with PLIN5 overexpressed lentiviruses through caudal vein,the serum insulin concentration and fasting blood glucose were lower than those in the control group(P<0.05).3.In the subcutaneous white adipose tissue of mice injected with PLIN5 overexpressed lentiviruses through caudal vein,mRNA expression of PLIN5 and UCP1 was obviously enhanced,and further Western Blot analysis showed that protein expression of PLIN5 and UCP1 also increased(P<0.05).4.According to RNA seq high-throughput sequencing results and subsequent validation,in C3H10T1/2 cells transfected with plin5-overexpressed lentivirus,the expression of BNIP3 mRNA increased simultaneously,which suggested that BNIP3 may also be involved in the browning of white fat(P<0.05).5.mRNA expression of BNIP3 improved significantly in subcutaneous fat of mice with chronic disease overexpressed by PLIN5 injected through the caudal vein(P <0.05).6.The double luciferase report indicated that the luciferase activity in the overexpression group of plin5 was 2 times higher than that in the control group(P<0.01).Conclusions: 1.PLIN5 was involved in the browning of white adipose.2.PLIN5 regulates brown adipose tissue cell differentiation through BNIP3. |
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