The Role Of CUL4B In Regulating Brown Fat And White Fat Browning

Background and Objective:Obesity and its complications are important factors threatening global public health.The main reason for their occurrence is that the body’s energy intake is far greater than its energy consumption,leading to the accumulation of excess energy in the form of triglycerides in adipocytes.The body’s adipose tissues include white fat,brown fat,and beige adipose tissue.The main function of white adipose tissue is storage energy,while brown adipose tissue cells release energy in the form of heat due to their abundance of mitochondria and high expression of uncoupling protein 1(UCP1);Beige adipose tissue is transformed from white adipose tissue under conditions such as cold stimulation,and its morphological and functional characteristics are similar to brown adipose tissue.Therefore,increasing the proportion and activity of brown and beige fat in the body’s adipose tissue can increase energy consumption and prevent the occurrence of obesity,and is also considered a potential means of treating obesity related metabolic diseases.Therefore,it is crucial to identify key factors that regulate the brown fat and white fat browning.Cullin 4B(CUL4B)is the scaffold protein of E3 ubiquitin ligase complex(CRL4B),which participates in many life processes through ubiquitination modification of substrate proteins.Loss of function mutations in CUL4B lead to Xlinked mental retardation syndrome.In addition to intellectual disability,patients also have centripetal obesity.Moreover,the expression level of CUL4B is negatively correlated with BMI.Adipocytes-specific Cul4b knockout(AKO)mice are predisposed to obesity induced by a high-fat diet.However,it is unclear whether CUL4B regulates brown and white fat browning.Therefore,we aim to explore the role of CUL4B in regulating brown fat and white fat browning.Methods and results:1)First,we analyzed the correlation between mitochondrial function related genes(MFN1,MFN2,FIS1,OPA1,DRP1)and the RNA expression level of CUL4B,and found that CUL4B was positively correlated with MFN1,MFN2,and FIS1.Western blotting showed that the expression level of CUL4B in brown adipose tissue was higher than that in white adipose tissue.The expression of CUL4B was significantly upregulated during brown fat activation and white fat browning stimulated by cold orβ-Adrenaline agonists.2)An adipocyte-specific Cul4b knockout mouse(AKO)was generated.The metabolic cage assay showed that there was no significant difference in metabolic levels between control mice and AKO mice in a thermoneutral environment.After transient stimulation at 4℃,the oxygen consumption and thermogenesis of AKO mice tended to decrease,but there was no significant difference.When short-term feeding at 4℃,AKO mice had no significant changes in oxygen consumption and thermogenesis,but their food intake significantly increased.Under fasting conditions,both the oxygen consumption and thermogenesis of AKO mice were significantly decreased whether they were given transient stimulation at 8℃ or short-term feeding at 8℃.In addition,under cold fasting conditions,the temperature of AKO mice decreased more significantly,and the survival rate was significantly lower than control group.3)Western Blotting and qRT-PCR were used to analyze UCP1,PRDM16,PGC1α,CIDEA and DIO2 in BAT and ingWAT of AKO mice and their controls induced by cold exposure.The results showed that the expression level of thermogenic factors such as UCP1 in BAT and ingWAT of AKO mice was significantly lower than control mice.Analysis of mature adipocytes derived from SVF of mouse adipose tissue confirmed that deletion of CUL4B resulted in the expression levels of thermogenic factors such as UCP1,PRDM16,PGC-1α，CIDEA and DIO2 were significantly lower in the AKO group than that in the control group.Knocking down Cul4b in the brown lipoma cell HIB-1b cell line also significantly inhibited the expression of thermogenic genes.Analysis of cell energy metabolism showed that knocking down Cul4b reduced the rate of cell oxygen consumption.The above results suggest that CUL4B deficiency inhibits their thermogenic function.Conclusion:The analyses of mouse models and in vitro cell models showed that the loss of CUL4B resulted in a reduced ability to activate brown fat and browning white fat,suggesting that CUL4B plays a role in activating brown adipose tissue and white adipose browning.