| Adipose tissues regulate whole-body energy homeostasis.Lipodystrophy and obesity are extreme and opposite aspects of adipose tissue dysfunction,which both contribute to metabolic disorder,including insulin resistance and hepatic steatosis.Lipodystrophy syndrome is a metabolic disorder-related disease mainly characterized by lipid atrophy and metabolic disorder,accompanied by hyperproteinemia,hyperlipidemia,hyperinsulinemia,insulin resistance,kidney damage,etc.,which can severely increases the risk of death.The low lipid content of livestock meat not only causes abnormal metabolism and threatens animal health,but also greatly reduces meat quality and flavor,reduces reproduction rate,and affects fur quality,which are difficult to meet people’s growing demand for meat product quality and dietary nutrition,and also affect the economic benefits of animal husbandry.It can be seen that normal development of adipose tissue is critical in humans and animals.More and more reports have demonstrated that cyclin-dependent kinases(CDKs)are not only directly involved in the regulation of the cell cycle,but may also participate in the normal function of adipose tissue and maintenance of metabolic homeostasis.However,the role of CDK7 in adipose tissue function and systemic metabolism remains unclear,which requires further research.In this project,the Cre-LoxP recombinase system was used to specifically knock out the CDK7 gene in mouse adipose tissue,combining with EchoMRI,indirect calorimetry,ELISA,Western Blotting,qPCR,RNA sequencing,histological staining,glucose tolerance test and insulin sensitivity test to study the effect of CDK7 deletion on mouse fat accumulation,function and systemic metabolism under different conditions including basal condition,cold stimulation,aging,high-fat diet-induced obesity,and β3-adrenoceptor agonist-induced white adipose tissue browning,providing a theoretical basis for the involvement of the cell cycle regulator CDK7 in regulating fat development and energy homeostasis.In addition,3T3-L1 adipocytes,C57BAT adipocytes and porcine primary adipocytes were treated with CDK7 inhibitor to study the effect of inhibiting CDK7 on lipid metabolism and proliferation and apoptosis of adipocytes.This thesis provides a theoretical basis for exploring the involvement of the cell cycle regulator CDK7 in regulating fat development and energy homeostasis.The main results in our experiments are as follows:1.The regulatory effect of CDK7 adipocyte-specific knockout on lipodystrophy in mice fed a normal diet.Firstly,CDK7 adipocyte-specific knockout mice(CDK7 aKO)were successfully constructed via the Cre-LoxP recombinase system.Compared with littermate control mice(CDK7 fl/fl),the mRNA levels of CDK7 were significant decreased in the subcutaneous white adipose tissue,perigonadal white adipose tissue,and the interscapular brown adipose tissue of CDK7 aKO mice(P<0.001).But there was no significant difference between the body weights of the two genotypes(P>0.05).The expression of fat-related genes were reduced in white adipose tissue of CDK7 aKO mice,such as FABP4,FAS,PPARy(P<0.05),and the serum free fatty acids and glycerol concentrations were also significantly reduced(P<0.05).The brown adipose tissue of CDK7 aKO mice was smaller,and the expression levels of thermogenic genes was decreased,while the expression levels of inflammation-related genes was increased,such as TNFa and MCPI(P<0.001).CDK7 aKO mice had impaired thermogenic function,which caused a lower survival rate in cold stimulation than control(P<0.001).There were no significantly differences in fasting blood glucose,glucose tolerance and insulin sensitivity between the two groups(P>0.05).No obvious difference was detected in oxygen consumption,respiratory exchange rate,and activity between the two groups(P>0.05).Under aging condition,CDK7 aKO mice also exhibited severe lipoatrophy,accompanied by marked hepatic hypertrophy.The brown adipose tissue was whitening,and the brown adipocytes were obviously enlarged.Serum free fatty acids,triglycerides,and glycerol levels tended to decrease in CDK7 aKO mice(P>0.05).No difference in glucose tolerance between two groups(P>0.05),but CDK7 aKO mice had insulin resistance(P<0.05)and increased serum insulin concentrations(P<0.05).This experiment shows that CDK7 ablation causes adipose atrophy in mice fed a normal diet,especially in aged mice.2.The regulatory effect of CDK7 adipocyte-specific knockout on lipodystrophy in obese mice fed a high-fat diet.Under the high-fat diet-induced obesity,we found that CDK7 aKO mice were resistant to obesity,because the significantly lower body weight than control group(P<0.001).CDK7 aKO mice had severe lipoatrophy in subcutaneous white adipose tissue,perigonadal white adipose tissue,and retroperitoneal white adipose tissue.And the interscapular brown adipose tissue was whitening.Fat-related genes were reduced in the white and brown fat,such as Adiponectin(P<0.01),C/ebpα(P<0.001),Fabp4(P<0.01),ATGL(P<0.01),HSL(P<0.01),and Perilipin(P<0.01).The concentrations of leptin(P<0.05)and resistin(P<0.001)were decreased.The expression of macrophage related markers were increased in brown adipose tissue,such as F4/80(P<0.05),IL6(P<0.05)and TNFα(P<0.01).In addition,CDK7 aKO mice exhibited increased liver lipid deposition and insulin resistance(P<0.001).These results suggest that adipocyte-specific CDK7 ablation causes lipodystrophy and metabolic disorders in mice under high-fat diet-induced obesity.3.The effect of CDK7 adipocyte-specific knockout on white adipose tissue browning in mice.After injecting two groups of mice with the β3-adrenoceptor agonist CL-316,243 for 7 days to induce white adipose tissue browning,the different phenotypes of the two genotypes mice in response to CL stimulation were observed.Our data showed that CL can normally induce the browning of white adipose tissue in CDK fl/fl control group,which was characterized by the decomposition of single-chambered large lipid droplets into multi-chambered small lipid droplets in white adipocytes,and increased expression of thermogenic genes(P<0.001).But CDK7 aKO mice had less response to CL-stimulated browning,and had glucose intolerance compared with CL-treated control mice(P<0.05).RNA-seq data indicated that lipid metabolism-related genes were down-regulated in white adipose tissues of CDK7 aKO mice,such as Fasn(P<0.05),Dgat2(P<0.001),and Lipe(P<0.01).In addition,we found obvious immune cell infiltration in pgWAT of CL-treated knockout group,and PPARy-related pathways were down-regulated in our RNA-seq data,which were similar to the phenotype of PPARy knockout lipodystrophy mice model.These results showed that CDK7 ablation resulted in a reduced response to white adipose tissue browning in mice mediated by the β3-AR pathway.4.Effects of CDK7 inhibition on lipid metabolism and proliferation in adipocytes.In order to explore the effects of CDK7 inhibition on adipogenesis,lipolysis,energy metabolism,proliferation,and apoptosis,we treated 3T3-L1 preadipocytes,C57BAT brown adipocytes,and primary porcine adipocytes with the CDK7-specific inhibitor THZ1 in in vitro experiments.The data indicated that THZ1 could inhibit the formation lipid droplet in 3T3-L1 preadipocytes,while no significant effect on lipolysis and energy metabolism.The respiratory function of C57BAT adipocytes with THZ1 treatment had a tendency to be reduced(P>0.05).THZ1 was toxic to primary porcine adipocytes(P<0.01)and could restrict cell proliferation.These results suggest that CDK7 is required for normal adipogenesis,metabolism and proliferation of adipocytes.Taken together,we demonstrated that loss of CDK7 in adipose tissue leaded to lipodystrophy,insulin resistance,impaired adipokines secretion,and down-regulation of fat-specific genes,which phenotypes were exacerbated on high-fat diet-induced obesity and aging.In addition,CDK7 was required for β3-adrenoceptor agonist-induced white adipose tissue browning.Our study demonstrated that CDK7 is a key regulator in maintaining adipose tissue healthy and whole-body energy homeostasis. |
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