The Molecular Mechanism And Function Of RNA-binding Protein QKI In Cold-induced Adipose Thermogenic Energy Consumption

Background The cold environment is closely related to the human health.Cold exposure induces hypothermia and increases the risk of cardiovascular and cerebrovascular diseases.Recent studies have shown that cold exposure improved the body’s metabolic status and effectively alleviated the metabolic disorders of obesity and diabetes.Thus,cold exposure is a doubleedged sword for human health.Upon cold exposure,adipose tissue plays an important metabolic regulatory role to maintain the body temperature.Adipose tissue is distributed throughout the body and mainly divided into white fat,brown-like fat and brown fat.Upon cold exposure,white fat with stored fat pad can resist heat loss and promoted the lipid droplets lipolysis to provide a metabolic substrate for peripheral organs.Furtherly,cold exposure efficiently induces the formation of brown-like fat,which plays a similar role to brown fat.Instead of energy storage,brown-like fat and brown fat comsume the energy to produce heat and participate in the maintenance of body temperature.More importantly,current research shows that cold exposure activates the function of brown-like fat and brown fat,and alleviate the symptoms of metabolic disorders.Therefore,to elucidate the complex mechanism of adipose metabolism in cold exposure,it will help us to figure out the critical regulatory genes and provide potential targets for the prevention and treatment of hypothermia and metabolic diseases.Currently,transcriptional factors and signaling pathways regulating adipose metabolism have been well studied.However,the significance of posttranscriptional regulatory in controlling adipose energy homeostasis still remains largely unknown.During changes in cold-induced a dynamic gene expression programs,the post-transcriptional mechanisms that promptly alter m RNA availability is critically implicated in modulating fat metabolism balance.As the center of post-transcription regulation,RNA binding protein(RBP)is involved in determining the timing and magnitude of protein expression.Based on our previous work,we implicate that RNA binding protein,Quaking(QKI),is the high potential critical regulator of adipose metabolism.In this study,genetic or AAV-mediated ablation of QKI was utilized to generate adipose-specific QKI deficient mice.Analysis of QKI function and mechanism was undertaken through an m RNA microarray,RNA-IP sequencing and QKI target motif prediction.The data demonstrated,for the first time,that QKI controlled adipose theromengesis.Furthermore,our findings displayed that QKI bounds to the QKI response element in 3’UTR of UCP1 and PGC1α,affecting their m RNAs location,stability and transcriptional efficacy.And inhibition of QKI facilitated energy dissipation,decreased lipid droplet accumulation,and improved cold tolerance.More importantly,QKI played a negative feedback to c AMP-c AMP response element-binding protein(CREB)axis induced thermogenesis.Together,these data indicate QKI as a master regulator in adipose metabolic homeostasis and show that it as a promising target to prevent obesity and hypothermia.Aims 1.To reveal the role of QKI in the regulaton on body metabolism status2.To elucidate the role of QKI on adipose energy metabolism;3.To explore the molecular mechism of QKI in regulating adipose energy metabolism;4.To demonstrate the post-transcriptional regulatory role of QKI in controlling thermogenic m RNAs avability;5.To clarify the role of QKI as a target in the prevention and treatment of hypothermia,obesity and its related diseases.Methods 1.We employed western blot,immunohistochemistry,promoter reportor assay to detect the expression of QKI in adipose tissue unpon cold exposure,and its regulated signaling pathways.QKI adipose-specific knockout mice was generated to detect the energy metabolism by using the Comprehensive Lab Animal Monitoring System.2.We used western Bolt,RT-PCR,HE staining,transmission electron microscopy and Clark electrode,to determine the role of QKI on adipose energy comsuption.3.We analyzed the data from RNA profiling array and RNA-IP Sequencing to reveal the molecular signaling pathway and key molecules regulated by QKI.In vitro study,lentivirus or si RNA was used to intervene QKI expression in adipocytes.And we verified the regulatory role of QKI on downstream target genes by molecular biology methods such as Western blot,RT-PCR and immunofluorescence staining.4.Based on the data from 3’UTR luciferase reporter assay,RNA half-life,RNA Subcellular fractionation,and ribosomes ananlysis,we determined the effect of QKI on RNA stability,transportion and translational efficiency.5.Using the high-fat diet-induced obesity mouse model,we clarified the role of QKI in obesity and its related diseases by monitoring body weight gain,insulin sensitivity and glucose tolerance.Results 1.Cold exposure increases the level of QKI in adipose tissue.Western blot and immunohistochemistry staining showed that the expression of QKI was gradually increased in brown adipose and subcutaneous adipose tissue during cold exposure.And QKI promoter luciferase reporter assay showed that c AMP signaling transcriptional up-regulated the level of QKI.QKI deficiency in mice increased energy expenditure and defended cold-induced hypothermia.The result of indirect calorimetry system detection displayed that QKI adipose-specific knockout mice had a significantly higher level of oxygen consumption rate and heat production.Cold exposure experiment observed that QKI adipose-specific knockout mice sustained a relative higher body temperature,effectively resisted coldinduced hypothermia,and improved the ability of cold tolerance.2.Loss of QKI promotes adipose tissue thermogenic energy consumption.Based on the data form Western Bolt,RT-PCR,HE staining,transmission electron microscopy,and oxygen consumption rate test,we found that inhibition of QKI significantly increased adipose oxygen consumption rate,increased the number of mitochondria,reduced the lipid droplet size,up-regulated the expression level of the thermogenic genes,and enhanced the mitochondrial oxidative uncoupling metabolic function.Upon cold exposure,more brownlike fat cells were enriched in the QKI-knockout subcutaneous adipose tissue.3.QKI affects thermogenic signaling genes.RNA profile array and KEGG pathway analysis presented that QKI was involved in metabolic signaling pathways such as AMPK,PPAR and insulin.GSEA analysis provided that QKI was involved in the regulation of functional genes related to PPARα,Phospholipid metabolism,TCA Cycle and Respiratory electron transport,and Fatty acid triacylglycerol and ketone body metabolism.RNA-IP Sequencing analysis suggested that PGC1α,a key factor of thermogenic metabolism,was a potential target for QKI.QKI negatively controls the thermogenic signaling genes.Western blot,RTPCR and immunofluorescence staining showed that knockdown of QKI promoted the expression of its potential target genes PGC1α and UCP1.Furtherly,our data clarified that QKI regulated adipose thermogenic energy consumption mainly through the PGC1α.By using Forskolin and IBMX to stimulate the cellular c AMP signaling pathway to mimic the thermogenic metabolism pathway in vivo,we figured out that inhibition of QKI rapidly and effectively increased the expression of thermogenic genes,especially upon cold exposure.Therefore,our data concluded that QKI played a “braking” mechanism on c AMP signaling induced thermogenesis.4.QKI post-transcriptionally regulates the expression of thermogenic genes.3’UTR luciferase reporter assay demonstrated that QKI binded to the QRE region on PGC1α,UCP1 3’UTR and inhibited their luciferase activity.RNA half-life,RNA subcellular fractionation,and ribosome analysis showed that inhibition of QKI significantly improved the stability of PGC1α RNA,promoted the cytoplasmic transportion of PGC1α /UCP1 RNA and increased thier translational efficiency.5.QKI deficiency protects the mice from high fat diet-induced obesity.Using a high-fatinduced mouse obesity model,we demonstrated that QKI transgenic knockout mice alleviated diet-induced insulin resistance and hepatosteatosis.Administration of QKIknockdown AAV in adipose tissue suppressed HFD-induced body-weight gain and improved glucose tolerance.These findings suggested that QKI was a potential therapeutic target for obesity.Conclusions 1.QKI controls the thermogenic metabolism of brown fat and subcutaneous fat,and is an effective target for preventing cold-induced hypothermia;2.QKI negatively regulates thermogenic genes and palys a "brake" role in cold induced energy dissipation;3.QKI binds to the 3’UTR region of the thermogenic genes(PGC1α and UCP1),precisely controlling their expression;4.QKI post-transcriptional affects their targets PGC1α and UCP1 RNA stability,transportation and translational efficiency;5.QKI-mediated adipose thermogenic energy consumption effect can effectively involed in the high-fat diet induced obesity,and QKI is a potential target for prevention and treatment of obesity and its related metabolic diseases.