Effects And Regulatory Mechanisms Of GADD45α On Differentiation And Metabolism Of Animal Adipocyte

Fat deposition and muscle development are the crucial factors affecting meat quality.The important point of improving meat quality is to increase intramuscular fat(IMF)deposition.Jinhua pig,an excellent local variety in our country,has higher intramuscular fat content and exhibits better meat quality traits than Landrace.In our previous study,differential expression gene GADD45α(Growth arrest and DNA damage 45 alpha)was screened out by transcriptome profiling between the two breeds.The expression of GADD45α is higher in longissimus dorsi muscles of Jinhua pig than landrace.However,whether GADD45α can regulate fat deposition especially intramuscular fat deposition remains largely unknown.Up to now,the gene sequence of porcine GADD45α has been cloned,but the specific structure and function of GADD45α have not been reported.It has been reported that GADD45α is highly expressed in white adipose tissue,but the role and regulatory mechanism of GADD45α in different types of adipose tissue are still unclear.Here,in this study,pigs and mice were used as experimental animal model.By analyzing the correlation between GADD45α expression and fat deposition,constructing overexpression and knockdown vector,and using the GADD45α knockout mouse model to study the effect and regulatory mechanism of GADD45α on differentiation and adipogenesis,glucose and lipid metabolism of different types of adipocytes at the cellular,tissue and animal levels.The main results are as follows:1.Analysis of the relationship between GADD45α expression and intramuscular fat depositionIn this experiment,the differentially expressed genes related to intramuscular fat deposition were screened based on the transcriptome analysis.Our results indicted that GADD45α expression in the dorsal longest muscle(LDM)of Jinhua pig was markedly higher than Landrace.Further verification revealed that the expression of GADD45α in muscle of high IMF content breeds(Jinhua pig and Laiwu pig)was significantly higher than that of low IMF content breeds(Landrace and DYL).By analyzing the gene expression profiles of lean and marbled beef with different IMF content,we also found that the expression of GADD45α in muscle of cattle with high IMF content(HF and HER)was significantly higher than that of lean cattle with low IMF content(LIM),suggesting that GADD45α may be related to beef marbling.Likewise,the expression of GADD45α in muscle with high IMF content(psoas and soleus)was markedly higher than that with low IMF content(longus dorsi and extensor digitalis).Taken together,the results suggest that GADD45α expression is positively correlated with IMF deposition,which implies that GADD45α may be involved in regulating IMF deposition.2.The effect of GADD45α on differentiation and lipogenesis of adipocyteIn order to explore the function of GADD45α in lipid deposition and metabolism,we successfully isolated intramuscular,subcutaneous and brown precursor adipocytes,and explored GADD45α expression and lipid droplets(LDs)deposition in undifferentiated and differentiated adipocytes.Furthermore,we constructed GADD45α overexpression and interference vectors to study the effect of GADD45αon the preadipocytes differentiation.The results indicated that GADD45α was highly expressed in differentiated porcine intramuscular and subcutaneous adipocyte.Similar results were observated in intramuscular and subcutaneous fat cells,as well as brown adipocyte in mice.GADD45α overexpression significantly increased lipid accumulation in white adipocyte,promoting brown adipocyte adipogenic differentiation and upregulating the expression of Pparg,Leptin,Adipoq and Fabp4.Oppositely,GADD45α knockdown markedly inhibited intracellular lipid accumulation,suppressing brown adipocyte lipogenesis and downregulating the expression of Pparg,Leptin,Adipoq and Fabp4.These results collectively suggest that GADD45α plays important roles in regulating the differentiation and lipogenesis of different types of adipocyte.3.Effect of GADD45α deletion on adipose tissue deposition and glucose and lipid metabolismIn this experiment,we use the Gadd45a-/-mouse model to explore the function of GADD45α on adipose tissue development and glucose and lipid metabolism.Our data showed that GADD45α knockout significantly reduced the mass of white adipose tissue(WAT).All other non-adipose tissue masses were not affected by GADD45αdeficiency.The KO WAT had the smaller lipid droplets and volume of adipocyte,contained numerous brown adipocyte-like cells filled with multilocular lipid droplets.GADD45α deficiency induced WAT browning and upregulated the mRNA levels of browning-specific genes such as Ucpl and Cidea.By contrast,the mRNA levels of pan-adipocyte genes including Pparg and Adipoq were significantly lower in the KO WAT.GADD45α deficiency promoted iWAT mitochondrial function.Meanwhile,GADD45α knockout promoted proliferation and upregulated the expression of BAT-specific genes(Ucpl,Pgcla and Prdm16)and mitochondrial marker genes(Uqcr10,Cox5b,Cox7a and Ppara)in brown adipocyte.Mitochondrial staining,transmission electron microscopy and mitochondrial complex protein detection demonstrated that GADD45α knockout affected mitochondrial biogenesis and increase heat production through upregulating the expression of UCP1 and PGC1α.This observation indicated that GADD45α deficiency affected BAT development and increased energy metabolism.GTT(Glucose tolerance test)showed that Gadd45a-/-mice have improved glucose tolerance.ITT(Insulin sensitivity test)indicated that Gadd45a-/-mice have higher insulin sensitivity.To gain further insight into the effect of GADD45α deficiency on whole-body metabolism,metabolic cages were used for the simultaneous measurement of food intake,energy expenditure,heat production and physical activity in the mice.Food intake was significantly higher in the KO mice compared to WT mice.In addition,the Gadd45a-/-mice had an increased general activity,higher rates of O2 consumption and CO2 production.We administered high-fat-diet(HFD)to both WT and KO mice.Although body masses remained similar in KO and WT mice before and after HFD feeding,fat mass in KO mice was consistently lower than in the WT mice,and the volume of fat cells was significantly smaller in KO mice.Notably,the KO mice had higher food intake and maintained better glucose tolerance and lower insulin resistance after HFD feeding.Further GTT and ITT experiments found that KO mice have better glucose tolerance and lower insulin sensitivity after HFD feeding,indicating that GADD45α knockout can resist high-fat induced obesity and insulin resistance.The above results suggest that GADD45α knockout significantly inhibited the development of WAT such as subcutaneous adipose tissue(SAT),induced browning of WAT,and promoted brown adipocyte proliferation and increased systemic glucose and lipid metabolism.4.Molecular mechanism of GADD45α regulating adipocyte proliferation and differentiationIn this experiment,RNA-seq,immunofluorescence,co-immunoprecipitation,luciferase assay and chromatin precipitation were used to reveal the molecular mechanism by which GADD45a regulates adipocyte differentiation and lipogenesis.In this experiment,shRNA lentivirus was used to establish a stable Gadd45a knockdown cell line,which was used in the following experiments.Ki67 immunofluorescence staining,crystal violet staining and cell growth detection assay indicated that GADD45α knockout significantly promoted brown adipocyte proliferation,while GADD45α overexpression had the opposite affect.Gene ontology(GO)enrichment analysis revealed pronounced changes in genes involved in biological processes such as negative cell growth regulation and negative cell cycle regulation,and down-regulated the expression of cell cycle related genes(Cdk5,Ccnb1,Cdknlb,Cdkn1c,Cdkn2a,Cdkn2b,Cdkn2c and Cdk5rap2)and cell proliferation marker gene(mKi67).These results suggest that deletion of Gadd45a promotes brown adipocyte proliferation through upregulating cell cycle related genes.In brown adipocytes,Gadd45a knockdown dramatically decreased the expression of PPARy,while Gadd45a overexpression had the opposite effects.Rosiglitazone(a PPARγ agonist)treatment rescued the differentiation of the Gadd45a-deficient adipocytes.Co-IP assay indicated that both exogenous and endogenous interactions between GADD45α and PPARy were found in differentiated adipocyte.Luciferase and ChIP assay further confirmed that GADD45α promoted brown adipogenesis via interacting with PPARy and upregulating its transcriptional activity,then promoting the expression of downstream Fabp4.These results suggest that GADD45α regulates cell proliferation through regulation of cell cycle-related gene expression.GADD45αregulates adipocyte differentiation by interacting with PPARy and upregulating the transcriptional activity of PPARγ to upregulate the expression of Fabp4 and adipocyte differentiation.In summary,the above results charified the relationship between GADD45α and intramuscular fat deposition.Our study elucidated the important role of GADD45α in regulating adipocyte proliferation and differentiation,tissue development,lipid metabolism and energy metabolism,and revealed the mechanism of GADD45α in regulating adipocyte differentiation.Our data provides new insights into the mechanistic role of GADD45α in target-regulating fat deposition and regulation of meat quality,as well as provides a certain reference for the prevention and treatment of human metabolic diseases such as obesity.