MYCT1 Alters Glycogen Shunt By Regulating RACK1-mediated Enzyme Selective Translation

Introduction: Glycogen metabolism,as the primary storage unit of glucose,has a vital influence on maintaining the homeostasis of glucose and energy.The abnormalities of glycogen synthesis or degradation can cause glycogen storage diseases,glycogenic hepatopathy and nonalcoholic fatty liver disease.Recent advances in metabolic reprogramming that glycogen metabolism may exert important biological effects on tumorgenesis has attracted attention: excessive accumulation of glycogen has been found in a variety of solid tumors,which is negatively correlated with the proliferation rate.Oncogenes(such as Myc,PI3 K / Akt,etc.)and tumor suppressor genes(such as p53,LKB1/ AMPK,etc.)can indirectly regulate glycogen metabolism by influencing other metabolic pathways.It has been reported that the hypoxic microenvironment in tumor cells induced transcriptional upregulations of glycogen metabolizing enzymes in a dependent-HIF1αmanner leading to the accumulation of glycogen in tumor.However,whether in normal tissues or tumor cells,the molecular mechanism of abnormal glycogen metabolism and its role in the occurrence and development of diseases are still unclear,which needs further study.Glycogen shunt,which is described in condition when glucose is shunted to glycogen and subsequently consumed through glycolysis and other biosynthesis pathway even though adequate glucose.glycogen metabolism plays an unneglectable role in the carbon source redistribution through multiple metabolic pathways beyond its role as a reservoir of glucose including glycolysis and pentose phosphate pathways(PPP).In tumor cells,glycogen shunt also plays a similar role as normal tissues.A large number of studies have shown that glycogen shunt can affect tumor growth and metastasis by changing the glycolysis homeostasis of key cells of tumor proliferation and invasion.However,the regulatory mechanism of glycogen shunt in tumor cells remains unclear.Favaro et al.found that hypoxia induced tumor cells can increase the expression level of hepatic glycogen phosphorylase after accelerating glycogen synthesis by activating HIF1αpathway,so as to promote glycogen shunt.However,HIF1α-induced turnover of glycogen metabolism has not yet given a persuasive explanation with glycogen shunt in brain tissue and tumor cells with adequate glucose.MYCT1,the direct c-Myc target gene,regulates numerous target genes leading to the alterations of proliferation,apoptosis,migration and invasion in various tumors.To explore regulation mechanics of MYCT1,we generated a Myct1 knockout mouse model(Myct1-KO).Unexpectedly,Myct1-KO mice showed progressive liver glycogen accumulation instead of tumor related phenotypes consistent with i TRAQ proteomics data enriching a series of glycogen synthesis enzymes in our tumor research.These results suggest that MYCT1 may be involved in the regulation of glycogen metabolism.Futhermore,in the studies on MYCT1-targeted coimmunoprecipitation followed by mass spectrometry,a large number of translation regulation related proteins on ribosomes were enriched.RACK1 is a scaffold protein on ribosomes,which can interact with a variety of signal transduction molecules,translation cofactors and RNA binding proteins to regulate protein translation level,and its enrichment on ribosome directly affects the translation efficiency of specific m RNA.In yeast model studies,we found that knockout of cqc2 /asc1,a homologous protein of Rack1,can not only significantly reduce the overall level of protein translation,but also regulate the translation level of metabolism related molecules.We hypothesis that MYCT1 allows the selective translation regulation of glycogen enzymes through the interaction with RACK1,thus affecting glycogen metabolism.Materials and methods:1.Materials: Myct1 knockout mouse and wild-type mouse(C57BL/6),human normal liver cell line LO2,human hepatoma Hep G2 cell line and human hepatoma Huh7 cell line.2.Methods: Paraffin embedded sections,HE staining,PAS staining,transmission electron microscopy,quantitative real-time PCR,western blot,cell culture,gene transient transfection,lentivirus stable transformation,immunoprecipitation,immunofluorescence and polyribosome analysis.Results:1.Using CRISPR/Cas9 to generate Myct1 knockout mice,DNA,RNA and protein were extracted from the tissues of Myct1 knockout mice.Sanger sequencing was performed confirming 8 bases deletions in the domains of the second exon of Myct1,resulting in frameshift mutation and no complete MYCT1 protein production.Bioinformatics predicted that the mutated protein had lost its original functional domain.Western blot was conducted indicating no functional MYCT1 expression in the Myct1 knockout mouse livers.2.The results showed that the weight of the Myct1-KO mice was gradually higher than that of the wild-type(n = 38).The liver visceral index increased at 200 days significantly(P<0.05)and decreased at 300-400 days significantly(P<0.05).Compared with the wildtype mice,the fasting blood glucose decreased significantly(P<0.05),and the glucose tolerance test showed the increased 2-hour-blood-glucose(P<0.05).3.Compared with the wild-type mice,pathological examination and transmission electron microscopy(TEM)of the livers of the mice were performed.HE staining and PAS staining of paraffin embedded sections exhibited the cell damage and glycogen accumulation increased with age in Myct1-KO mice;TEM results showed that a large amount of glycogen accumulation and endoplasmic reticulum rupture occurred in the liver cells of Myct1-KO mice.4.The transcription and protein levels of glycogen metabolizing enzymes in liver tissues of mice were detected showing that compared with WT littermates,the transcription levels of glycogen metabolic enzymes Pgm1,Ugp2 and Gsk3 a in Myct1 KO mice were upregulated less than 10% or had no statistical significance.Western blotting results showed that compared with wild-type littermates,PGM1,UGP2 and GSK3 A of Myct1 KO mice decreased by 34%(P<0.05),42%(P<0.05)and 43%(P<0.05),30%(P<0.05),39%(P<0.05)and 42%(P<0.05),27%(P<0.05),38%(P<0.05)and 50%(P<0.05)respectively.5.The ribosomal m RNA complexes of Myct1-KO and WT mice were isolated by sucrose density ultracentrifugation.The translation efficiency of glycogen metabolic enzymes was detected by quantitative real-time PCR.The results showed that the translation efficiencies of glycogen metabolic enzymes in Myct1 KO mice were significantly lower than those in WT mice.Pgm1 decreased by 51%(P<0.05),Ugp2 decreased by 39%(P<0.05),and Gsk3 a decreased by 27%(P<0.05).6.Glycogen metabolic intermediates were detected by colorimetric assay kit and ELISA in Myct1-KO and WT mice.The results showed that the contents of glycogen and glucose-1-phosphate in liver tissues of Myct1-KO mice were significantly increased with age(P<0.05),while the contents of glucose-6-phosphate and UDPG were significantly decreased(P<0.05).7.Three types of liver cells with different expression levels of MYCT1,namely human hepatoma cell line Hep G2,Huh7 and human normal liver cell line LO2,were selected and analyzed by Western blot.The results showed that MYCT1 enhanced the expression of glycogen metabolic enzymes.8.Glycogen metabolic intermediates were detected by colorimetric assay kit and ELISA in MYCT1 overexpressing and knockdown cells.The results showed that MYCT1 decreased the contents of glycogen and glucose-1-phosphate and increased glucose-6-phosphate and UDPG remarkably.9.For lentivirus-MYCT1 infected Hep G2,the transcription,translation and protein levels of glycogen metabolizing enzymes were detected by RT-q PCR and Western blot.The results showed that MYCT1 had no effect on the transcription level of glycogen metabolic enzymes but increased the translation efficiencies(P<0.05)and protein levels of PGM1,UGP2 and GSK3 A significantly(P<0.05).10.Under the stimulation of high glucose,content of glycogen metabolites was measured by colorimetry and ELISA at 0,1,3,6,12 and 24 hours in lentivirus-MYCT1 infected Hep G2 cells.The results showed that the rate and content of glycogen synthesis were significantly decreased,while the rate and content of UGPD and G6 P were significantly increased with the overexpression of MYCT1.11.TCGA database was used to analyze the expression of MYCT1 in various tumors,and the expression analysis of MYCT1 in glycogen-rich tumors and non-glycogen-rich tumors were compared.The results showed the high expression of MYCT1 represented in glycogen-rich tumors.12.The biological binding of MYCT1 and RACK1 in mouse liver tissues and Hep G2 cells was identified by coimmunoprecipitation and immunofluorescence.13.Polyribosome analysis and Western blot was used to detect the enrichment of ribosomal RACK1 in mouse liver tissues and Hep G2 cells.The results showed that the enrichment of RACK1 on ribosomal small subunit,ribosomal monomer and polyribosomes in liver tissues of Myct1 KO mice was decreased.The enrichment of RACK1 on ribosomal small subunit,ribosomal monomer and polyribosomes in lentivirus-MYCT1 infected Hep G2 was increased.14.The interaction of RACK1 to RPS3 in lentivirus-MYCT1-infected Hep G2 cells was detected by immunoprecipitation and immunofluorescence.The results showed that the binding rate of RACK1 to RPS3 increased significantly with the overexpression of MYCT1.15.Western blot were used to detect the protein levels of glycogen metabolizing enzymes in MYCT1-stable Hep G2 cells,RACK1-knockdown Hep G2 cells and MYCT1-stable Hep G2 cells with RACK1-knockdown.The results showed that MYCT1-stable Hep G2 cells had higher protein levels of glycogen metabolizing enzymes compared with control.RACK1 downregulated the protein levels of glycogen metabolizing enzymes.RACK1 knockdown blocked the upregulation of MYCT1 on glycogen metabolizing enzymes.16.The ribosomal m RNA complexes of MYCT1-stable Hep G2 cells,RACK1-knockdown Hep G2 cells and MYCT1-stable Hep G2 cells with RACK1-knockdown were isolated by sucrose density ultracentrifugation.The translation efficiency of glycogen metabolic enzymes was detected by quantitative real-time PCR.The results showed that: MYCT1 could significantly improve the translation efficiency of glycogen metabolizing enzymes(P<0.05);knockdown of RACK1 could reduce the translation efficiencies of glycogen metabolizing enzymes(P<0.05);knockdown of RACK1 could block the improvement of the translation efficiencies of glycogen metabolizing enzymes by MYCT1(P<0.05),and MYCT1 could weaken the negative regulation of RACK1 on the translation efficiencies of glycogen metabolizing enzymes(P<0.05).17.Glycogen metabolic intermediates were detected by colorimetric assay kit and ELISA in MYCT1-stable Hep G2 cells,RACK1-knockdown Hep G2 cells and MYCT1-stable Hep G2 cells with RACK1-knockdown.The results showed that RACK1 significantly increased the contents of glycogen and G1 P and decreased the contents of G6 P and UDPG(P<0.05).RACK1 knockdown could block the regulation of glycogen metabolites by MYCT1(P<0.05).Conclusion:1.Myct1 KO mice were successfully constructed,and Myct1 deletion was verified at the genome level,transcription level and protein level.2.Myct1 can regulate glycogen metabolism and maintain the homeostasis of glycogen metabolism by regulating the expression levels of glycogen metabolism enzymes Pgm1,Ugp2 and Gsk3 a.It plays an important role as a "glycogen shunt switch".3.MYCT1 can regulate the glycogen shunt of tumor cells and play different roles in different types of tumor glycogen metabolism,which is of great significance for tumor metabolic reprogramming.4.MYCT1 can interact with RACK1 and regulate the translation efficiencies of glycogen metabolizing enzymes PGM1,UGP2 and GSK3 A by affecting the enrichment of RACK1 on ribosome,thus collectively regulating tumor glycogen shunt.