Function And Mechanism Of Adssl1 And Clstn3 In Energy Metabolism Of Adipocytes

Objective: Promoting white adipose tissue(WAT)browning and enhancing brown adipose tissue(BAT)activity are attractive therapeutic strategies for obesity and its complications.The aim of this study is to investigate the regulation roles and mechanism of Adssl1(a gene involved in purine nucleotide metabolism)and Clstn3(a gene mediating the neuro-adipose synaptic junctions)in the energy metabolism of beige adipocytes.Methods: 1)Q-PCR and immunoblotting methods were used to observe the expression profile of Adssl1.Mediating knockdown and overexpression of Adssl1 in beige adipocytes,the expression levels of differentiation marker gene,adipogenic marker gene and thermogenic marker gene were then detected.Seahorse was used to detect the oxygen consumption of beige adipocytes.The level of purine nucleotiderelated metabolites in cells was measured by capillary electrophoresis-mass spectrometry.RNA-seq was used to analysis the gene expression profiles changes between Adssl1 knock-down group(ADsi group)and control group.Mitotraker staining,mitochondrial DNA copy number detection and electron microscopy were used to detect intracellular mitochondrial levels.2)Differentially expressed predicted secreted genes(DEPSGs)during WAT browning and BAT activation were identified.Expression of the key gene in mature adipocytes of WAT and BAT after cold exposure was examined.Differentiated adipocytes were treated with ISO to detect the expression changes of the key genes.Results: 1)Adssl1 is highly expressed in metabolically active tissues such as brown adipose tissue and muscle tissue.Adssl1 does not affect the differentiation and adipogenesis of beige adipocytes.The incresed expression of UCP1 and higher oxygen consumption were observed in ADsi group.The concentration of inhibitory purine nucleotides(ATP,ADP,GTP,and GDP)in the ADsi group was decreased.RNA-seq results showed that the differentially expressed gene profiles in beige adipocytes of ADsi group and in browning WAT were similar,and the type ? interferon signaling pathway was both significantly down-regulated.Mitotraker staining,mitochondrial DNA copy number detection,and electron microscopy results suggest that the increase of energy metabolism in the ADsi group is mainly due to an increase of mitochondrial function rather than a significant increase of mitochondria number.2)The secreted genes expression profiles of WAT browning and BAT activation have a relatively small similarity.Clstn3 was identified as the top gene expressed enriched in mature adipocytes.Its expression level was increased during WAT browning and BAT activation.In vitro study found that ISO could induce Clstn3 expression in adipocytes.Conclusion: 1)ADSSL1 regulates the energy metabolism of beige adipocytes by affecting the concentration of cellular inhibitory purine nucleotides.The specific mechanism involves not only the increase of Ucp1 activity,but also the increase of Ucp1 expression levels.2)Clstn3 may be a key gene mediating the neuro-adipose junction formation or remodeling in WAT browning and BAT activation process.