Deacetylation Of RNA Binding Protein QKI By SIRT1under Fasting Conditions Controls The Adaptive Hepatic Energy Metabolism

Liver plays a critical role on regulation of the energy balance in the state of starvation. Inthe early stage of starvation, the rapid hepatic glycogenolysis maintains the normalconcentration of blood sugar, and the hepatic gluconeogenesis starts meantime. At thesame time, some nonsugar convert into glucose in liver, which can maintain bloodglucose level and provide energy after release into blood. Lipolysis provide glycerinumand fatty acids as the starvation continues. Oxidation of fatty acids provide energy to thewhole body. Liver produces ketone body in the procedure of fatty acid oxidation, whichcan be transported to several tissues and organs with blood to provide energy. So Liverplays a central role in the energy balance of starvation, and the underlying mechanism ofthe energy balance refers to several complex molecular signals. In other words, theessence of energy supply effects such as glycogenolysis, gluconeogenesis, fatty acidoxidation and ketogenesis is cell and molecular signaling pathways in hunger environmentadaptability of control process in essence in the state of starvation. Obstacles of signaltransduction related to the energy balance of liver and disorders of molecular regulationoccurred in many pathological conditions, such as diabetes, fatty liver, that leads to the occurrence and development of the disease. Therefore, further study of the livermetabolism molecular mechanisms can enrich theory of molecular metabolism andprovide the new method for the diagnosis and treatment of disease.Signaling molecules change their own expression and activity under the multipleregulation of, epigenetic, transcription, post-transcription, translation, post-translation inorder to adapt to environmental changes. In the state of starvation, NAD dependentprotein acetylation enzyme Sirt1in the liver promote the activity of metabolism relatedtranscription factors (PGC1α, FOXO1,etc.)by modification after translation (toacetylation). However, through transcriptional regulation mechanism, activatedtranscription factors increase the expression of downstream metabolism-related enzymes,which promotes the sugar dysplasia and fatty acid oxidation of liver under chronic hungerand regulate blood sugar balance and energy supply. In conclusion, to maintain energybalance, metabolism related signaling molecule in liver cells multiple are regulated bydifferent levels in the state of hunger.RNA binding protein mediated post-transcription regulation in the metabolism of liveris rarely researched. QKI (quaking) encoded by qki gene as a member of the family ofSTAR is an RNA-binding protein, which can produce multiple isomer and express in avariety of tissues and cells. QKI5mainly located in the nucleus, which can also move backand forth to the cytoplasm, while QKI6and QKI7are mainly distributed in the cytoplasm.Combining with the specific recognition elements （QRE）of3’UTR in mRNA, QKImediate orientation of cytoplasm/cell nucleus, stability and translation efficiency of targetmRNA. QKI can enhance the expression of MBP in nervous system, thus increasing theformation of the myelin sheath. QKI plays the anti-oncogene effect in colon cancer byinhibiting the translation efficiency of β-catenin. We found that QKI also expressed in theliver, and studies in liver hasn’t been reported.Objective（1）To observe the expression level of QKI in hepatic energy balance of the hungerand analyze the relationship between gluconeogenesis, fatty acid oxidation, ketoplasia andQKI in vivo and in vitro; （2）To observe whether QKI was regulated by acetylation, then illustrate the functionof acetylation in hepatic energy balance of the hunger in vivo and in vitro;（3）To observe the upstream regulatory mechanisms of QKI acetylation, investigatehow the upstream regulatory molecular effect QKI and its role in starvation metabolism ofliver;（4）Find out downstream molecules that were accommodated by acetylated-QKI, theneffect the energetic metabolism of starvation liver. In addition, to clarify the furthermechanism of downstream regulation of QKI molecules.Methods and Results(1) After starvation, in mice liver tissue, low carbohydrates and Forskolin/dexamethasone treated-HepG2cells starvation models, immunochemistry, qRT-PCR andwestern-blotting were performed to test the expression changes of QKI and itsrelated-metabolism molecules. The results showed that the level of QKI5was obviouslyincreased in hungry situation, which consistent with gluconeogenesis, fatty acid oxidationand ketoplasia related enzymes.(2) In the above starvation models, we used acetylation antibody to conductco-immunoprecipitation and western-blotting to detect the acetylation level of QKI5;Biological technique was used to predict the possible acetylation sties of QKI5;Constructed QKI5acetylation sites mutant vector, discussed the effect of acetylatedchange on energetic metabolism of starvation liver. The results showed that QKI presenteddeacetylation in starvation liver. Moreover, we found that when lysine located in QKI5KH domain mutant to arginine (MTR5, acetylation sites deficiency) after interfered bymutation, which promoted the production of gluconeogenesis, fatty acid oxidation andketoplasia; However, when lysine located in QKI5KH domain mutant to glutamine(MTR5, equal to acetylated amino acid), it suppressed the production of gluconeogenesis,fatty acid oxidation and ketoplasia.(3) The acetylation level of QKI5was tested by using IP analysis when liver cellswere treated with inhibitor or activator of histone deacetylase Sirt1; testing the acetylationlevel of QKI5by inhibiting expression of Sirt1; testing acetylation level of QKI5in Sirt1 knockdown mice liver. Our study revealed that inhibiting Sirt1activity of Sirt1increasedQKI5acetylation level. In contrast, increased Sirt1activity resulted in QKI5deacetylation.The acetylation level of QKI5were increased no matter in the liver cells of Sirt1genesilenced or liver tissue of Sirt1knockdown mice.(4) qRT-PCR was used to test the expression of key molecular involved in livermetabolism in the hungry model. Interferometric QKI5or overexpression of MTQ5caninhibit the expression of FOXO1and PPARα. Over expression of QKI5and MTR5increased the expression of FOXO1and PPARα. QKI5interacted with mRNA of FOXO1and PPARα. MTR5have stronger interaction with mRNA of FOXO1and PPARα. MTQ5have no interaction with mRNAof FOXO1and PPARα.(5) We verified the posttranscriptional regulation mechanism of targeting FOXO1orPPARα by QKI5、MTR5and MTQ5. Our study revealed that over expression of QKI5andMTR5could increase the luciferase activity of FOXO1and PPARα. In contrast, MTQinhibited the luciferase activity of FOXO1and PPARα.Conclusions:In summary, our data demonstrates that QKI, RNA binding protein, play an importantrole in energy metabolism in hunger liver. Sirt1promoted QKI deacetylation in liver.Deacetylation of QKI increased the expression of FOXO1and PPARα by posttranscriptional regulatory mechanism, and thus promoted gluconeogenesis, fatty acidoxidation and ketogenesis and exerted profound influence in energy balance of liver at thesame time.