GPAT3 Knockout Improves The Abnormal Metabolic Phenotype Of Seipin Defects

Adipose tissue is a very important lipid storage and endocrine organ that regulates systemic metabolic homeostasis and is closely related to atherosclerotic cardiovascular disease.Lipodystrophy is a rare,heterogeneous disease that is divided into hereditary and acquired disease characterized by varying degrees of body fat loss and related to metabolic complications,including insulin resistance,dyslipidemia,hepatic steatosis,andincreased susceptibility to atherosclerotic cardiovascular disease（ASCVD）.Seipin is a causative gene of congenital lipodystrophy type 2（CGL2）that regulates lipid droplet expansion and affects adipose tissue regeneration.Defects in Seipin lead to metabolic abnormalities such as systemic fat loss,fatty liver and insulin resistance.Our previous study found that Seipin could interact with 3-phosphoglyceryl acyltransferases（GPATs）and affect the activity of GPATs.Knocking down GPAT3 improved lipid droplet morphological abnormalities and adipocyte dysfunction in Seipin deficiency cells.It is suggested that the abnormal phenotype caused by Seipin deficiency may be triggered by GPAT3,but there is still no evidence of mammalian experimentation.The main objective of the part one of this study was to investigate the role of GPAT3 knockout in the metabolic abnormal phenotype of Seipin-deficient mice at the global animal level using a genetically engineered mice model,and to elucidate the role of GPAT in the development of lipodystrophy caused by Seipin deficiency.A large number of experiments in the past have confirmed that obesity can aggravate the progression of atherosclerosis（As）,while reports of adipose tissue reduction and As are rare.Our previous study found that Seipin deficiency could aggravate atherosclerosis（As）in LDLR knockout（LDLR-KO）mice;Seipin^-/-LDLR^-/-mice also exhibit extreme hypertriglyceridemia and hypercholesterolemia.Seipin deficiency aggravates As because of the loss of adipose tissue or the genetic defect itself,which is still inconclusive.The main purpose of part two of this study was to remove the adipose tissue of LDLR-KO mice by surgery to observe the systemic lipid homeostasis,fatty liver and insulin sensitivity,and the pathological changes of As in LDLR-KO mice and to demonstrate the role of maintenance of systemic adipose tissue on metabolic homeostasis and As.Part one GPAT3 knockout improves the abnormal metabolic phenotype of Seipin defectsObjective:The main objective of this part was to investigate the role of GPAT3 knockout in the metabolic abnormal phenotype such as hypertriglyceridemia,insulin resistance,fatty liver,adipose tissue atrophy of Seipin-deficient mice at the global animal level using a genetically engineered mice model,and to elucidate the role of GPAT knockoutin lipodystrophy caused by Seipin deficiency.Methods:The CRISPR/Cas9 system was used to simultaneously knock out Seipin and GPAT3（DKO）in mice,and all the results were compared with Seipin single knockout（SKO）mice.The genotypes of mice were detected by PCR and sequencing genomic DNA.The blood glucose and blood lipid levels were determined by enzyme reaction colorimetry.The phenotypic changes of adipose tissue were determined by immunohistochemistry and H&E staining.The lipid droplets morphology were determined by incubation of oleic acid in MEF cells.The MEF was induced to differentiate into adipocyte.Rosiglitazone feeding determines the effect of Pparg agonist on fat regeneration and metabolic phenotype in mice.Results:Seipin and GPAT3 were simultaneously knocked out（DKO）in mice using the CRISPR/Cas system,and the results were compared with Seipin single knockout（SKO）mice.1.Successfully constructed Seipin-GPAT3 double knocking miceGenotypes of mice were identified by PCR and sequencing of genomic DNA.The expression of Seipin and/or GPAT3 in knockout mice was confirmed by Real time PCR and Western Blot at m RNA and protein levels.2.GPAT3 knockout can improve hyperlipidemia in SKO miceDetection of plasma total cholesterol（TC）,triglyceride（TG）,and non-esterified fatty acids（NEFA）showed that GPAT3 knockout improved mild hypercholesterolemia in SKO mice,but did not correct plasma low TG and low NEFA levels after long time fasting in SKO mice.3.GPAT3 knockout can partially correct fat loss in SKO miceWe analyzed the subcutaneous fat,epididymal fat and brown fat of the four groups of mice to evaluate the effect of GPAT3 knockout on the adipose atrophy phenotype of SKO mice.Compared with SKO mice,the weight of subcutaneous fat and epididymal fat in DKO mice were increased by about 2times.The H&E staining of adipose tissue showed that the number of adipocytes in DKO mice increased and the lipid droplet size was more uniform.The weight recovery of brown adipose tissue is more pronounced.Compared with SKO mice,gene expression such as fat differentiation and lipid synthesis in subcutaneous fat of DKO mice was restored.The plasma adipokines Leptin of four groups of mice were detected by ELISA.It was found that the DKO group was increased by about 2 times compared with the SKO group,suggesting that the secretory function of adipose tissue in DKO mice was significantly improved.F4/80 immunohistochemistry and Masson staining showed that the macrophage infiltration and fibrosis levels of adipose tissue in DKO mice were significantly reduced.It can be seen that GPAT3knockout can partially improve the lipoatrophic phenotype of SKO mice.4.Seipin-GPAT3 mice develop a white fat browning phenotypeWe also found that the m RNA levels of the brown adipose tissue marker genes Ucp1,Cidea,Elovl3 and Cox8b in the subcutaneous fat and epididymal fat of the DKO group were significantly up-regulated,suggesting a white fat browning change.Western Blot and immunohistochemistry of Ucp1 further confirmed the brown fat-like changes in white fat of DKO mice,which was beneficial to improve the abnormal phenotype of systemic metabolism.5.GPAT3 knockout can improve insulin resistance and fatty liver in SKO miceSevere fatty liver and systemic insulin resistance occured in Seipin-deficient mice.H&E and oil red O staining showed that lipid deposition in the liver was significantly reduced in DKO mice;TG content in liver of DKO mice was significantly decreased,indicating that GPAT3 knockout can significantly improve the fatty liverof SKO mice.Plasma insulin levels were significantly down-regulated in DKO mice,suggesting that GPAT3 knockout significantly improved insulin resistance in SKO mice.6.GPAT3 knockout can correct morphological abnormalities of lipiddrops and adipocyte dysfunction caused by Seipin defectsTo determine the effect of GPAT3 knockout on lipiddrops（LDs）morphological abnormalities and adipocyte neonatal disorders caused by Seipin deficiency,we extracted embryonic fibroblasts（MEF）of four groups mice.After oleic acid incubation,the phenotype of lipid droplets was observed.It was found that the SKO mice showed heterogeneity of lipid droplet morphology,many small lipid droplets dispersed with large lipid droplets,while lipid droplet diameter in DKO mice exhibited good uniformity,suggesting significantly improvement the abnormal morphology of lipid droplets compared with SKO mice.The MEF was induced to differentiate into adipocyte,and then stained with oil red O.The results showed that the SKO mice had a adipose-stasis disorder,and no mature adipocytes were observed,while a small amount of mature adipocytes were observed in the DKO group.It can be seen that GPAT3 knockout can partially improve the adipose regeneration disorder in SKO mice.7.Rosiglitazone feeding improves insulin sensitivity in four groups of miceWe also administered rosiglitazone（Rosi）,a agonist of the adipose proliferator-activated receptor gamma（Pparg）,a key nuclear receptor for adipose differentiation,in four groups of mice.It was found that Rosi could increase the weight of subcutaneous fat and improve insulin sensitivity in four groups of mice.Summary:Our study showed that GPAT3 knockout could increase the weight of adipose tissue in SKO mice,partially improve adipose tissue morphological abnormalities and secretory dysfunction,and have a white-brown-like change.GPAT3 knockout could significantly improve systemic metabolic disorders such as fatty liver and insulin resistance in SKO mice.GPAT3 knockout could also improve the abnormality of lipid droplets and differentiation of MEF cells in SKO mice.It can be seen that at the global animal level,GPAT3 knockout can partially improve the adipose atrophy and metabolic abnormal phenotype caused by Seipin deficiency.Part two Effects of fat loss on metabolic phenotype and atherosclerosis in LDLR^-/-miceObjective:White adipose tissue（WAT）and/or brown adipose tissue（BAT）in LDLR-KO mice were removed by surgery to observe systemic lipid homeostasis,fatty liver and insulin sensitivity andthe pathological condition of As in LDLR-KO mice with partial fat loss.In order to demonstrate the role of maintenance of systemic adipose tissue on metabolic homeostasis and As.Methods:The LDLR-KO mice were removed brown fat,subcutaneous fat,or epididymal fat of the LDLR-KO mice to mimics systemic lipodystrophy.The blood glucose and blood lipid levels were determined by enzyme reaction colorimetry.The phenotypic changes of adipose tissue were determined by immunohistochemistry and H&E staining.The effect of lipodystrophy on As was determined by immunohistochemistry and oil red O staining.Results:1 The results of removal of subcutaneous fat,epididymal fat and brown fat of LDLR-KO mice.1.1 Mild hypercholesterolemia in the three-part fat removal miceThe plasma total cholesterol（TC）,triglyceride（TG）,and non-esterified fatty acid（NEFA）levels of the three-part fat removal mice increased moderately,suggesting mild hypercholesterolemia in fat removal mice.1.2 Aggravated insulin resistance and fatty liver in the three-part fat removal miceSevere fatty liver and systemic insulin resistance occur in mice with systemic lipodystrophy.H&E and oil red O staining showed that the lipid deposition in the liver of the three-part fat removal mice was significantly aggravated;the TG and TC contents in the liver of the three-part fat removal mice were significantly increased.It is indicated that the fat removal of the 3parts can significantly aggravate the fatty liver of LDLR-KO mice.We also found that the expression of CHOP protein level in the liver of the three-part fat removal group was significantly up-regulated,suggesting that endoplasmic reticulum stress might occur.Plasma insulin levels,glucose tolerance and insulin tolerance results showed that plasma insulin levels were significantly elevated in the three-part fat removal mice;glucose tolerance was impaired,and insulin sensitivity was decreased,suggesting that significantly aggravated insulin resistance in the three-part fat removal LDLR-KO mice.1.3 Residual fat compensatory increase in the three-part fat removal miceWe analyzed the retroperitoneal fat and mesenteric fat in the three-part fat removal group and evaluated the effect of fat removal on residual fat.Compared with sham-operated mice,the weight of retroperitoneal fat and mesenteric fat in the three-part fat removal mice were significantly increased.The H&E staining of adipose tissue showed that the adipocytes size in the three-part fat removal mice was bigger.We also found that the expression of genes related to lipid synthesis in retroperitoneal fat and mesenteric fat was up-regulated in the three-part fat removal group,the expression of p-AKT in adipose tissue was also up-regulated;and the expression of HSL and ATGL related to lipid hydrolysis genes was down-regulated.It can be seen that the remained fat in the three-part fat removal mice has a moderate compensatory increase,and the insulin sensitivity in adipose tissue is enhanced.1.4 The area of As lesions did not change in the three-part fat removal miceIn order to determine the effect of fat removal on As,we performed oil red O staining on the aorta and the aorta roots,and stained the aortic roots with Sirius red,H&E,CD68 and SM22α.Compared with the sham group,the area of As lesions did not change after the removal fat of 3 parts;the expression of CD68 protein in As lesions,the expression of SM22αprotein in vascular smooth muscle cells,and the content of vascular collagen were not changed,but the vascular area and the vascular cavity area were reduced,suggesting an abnormal vascular remodeling.2 The results of removal of subcutaneous fat and/or gonadal fat of LDLR-KO mice.After HFD,the plasma cholesterol level,liver lipid deposition,liver triglyceride and cholesterol content,and As lesion area were not changed in the subcutaneous fat and/or gonadal fat removal group compared with the control group.Compared with the control group,the weight of subcutaneous fat in the gonadal fat removal group increased significantly,which may be related to the increased synthesis of triglycerides.Summary:The three-part fat removal group can have hypercholesterolemia,aggravate fatty liver and insulin resistance,and the area of As lesion has no effect.There were no significant metabolic abnormalities and changes in the area of As lesions in the subcutaneous and/or gonadal fat removal group.It can be seen that removal of part of the adipose tissue did not aggravate As in LDLR-KO mice.Conclusion:GPAT3 knockout can partially improve fat loss,lipid droplet morphology and fat differentiation disorder caused by Seipin defect,and significantly improve fatty liver and insulin resistance;multi-site adipose tissue loss can aggravate fatty liver and insulin resistance in LDLR-KO mice,and partial fat tissue removal did not aggravate As in LDLR-KO mice.