Roles Of TIPE2 In Regulating Hepatic Gluconeogenesis

BackgroundGlucose is important for energy metabolism in mammalian.Homeostasis of blood glucose is the basis of all physiological activities.The body regulates the input of glucose into peripheral organs as well as the output and release of endogenous glucose based on the diet intake,thus maintaining glucose concentration in the blood T2DM(type 2 diabetes mellitus)is a chronic metabolic disease,manifested by hyperglycemia and insulin resistance in clinic.T2DM causes many complications including vascular damage and glycolipid metabolic disorders,which endangers heart,brain,eyes and peripheral nerves.Non-sugar substances such as amino acid,lactic acid,glycerin can be transformed into glucose in vivo through a series of biotransformation.This physiological process is called gluconeogenesis Gluconeogenesis is an important form of glucose metabolism,which is responsible for maintaining the concentration of blood glucose during fasting in mammals Moreover,the increase of blood glucose in patients with T2DM is resulted from the continuous activation of hepatic gluconeogenesis.The process of gluconeogenesis is mainly regulated by hormones in vivo,including glucagon and insulin.Glucagon is an important hormone to enhance gluconeogenesis in liver.Glucagon increases the level of cAMP,which induces the phosphorylation of cAMP-dependent protein kinase(AMPK.or protein kinase A,PKA),resulting into phosphorylation of cAMP-response element binding(CREB)protein.Thus,phosphorylated CREB interact with CREB binding protein(CBP)and the related protein p300,which form a complex to bind to the promoter region of gluconeogenic genes G6Pase(encoding the catalytic subunit of glucose-6-phosphatase)and PEPCK(encoding phosphoenolpyruvate carboxykinase).Similarly,phosphorylated CREB increases expression of PGC-1?,which can form a complex with FOXO1.binding to the promoter region of key enzymes of gluconeogenesis,mediating activation of the gluconeogenic program.In addition,ERK promotes FOXO1 phosphorylation and acetylation through Ets-1,which interfering with the FOXO1-PGC-1? interaction in a glucagon-sensitive manner Glucagon promotes gluconeogenesis through inhibiting ERK signaling pathway in hepatocytes.Insulin is the most important hypoglycemic hormone that inhibits gluconeogenesis in the body.Insulin can activate PI3K in hepatocytes,causing the increase of PIP3 content?which promotes the phosphorylation and activation of AKT Phosphorylated AKT leads to the phosphorylation of FOXO1,and then phosphorylated FOXO1 is degraded in the cytoplasm.It has also been reported that insulin induces phosphorylation of PGC-1?,and the phosphorylated PGC-1?interfering with the FOXO1-PGC-1? interaction in an insulin-sensitive manner,thus decreasing the gluconeogenesis.The signaling pathways mediated by these hormones are disturbed in patients with T2DM.Especially,insulin resistance leads to disorders of blood glucose metabolism.Therefore,it is helpful to explore the molecular mechanisms that regulate gluconeogenesis and provide theoretical and experimental basis for reducing the incidence of T2DM.TIPE2,a member of the TIPE(TNFAIP8)family,is predominantly expressed in lymphoid tissues and immune cells.TIPE2 is involved in the regulation of immunity and inflammation,which maintain immune homeostasis.TIPE2 is mainly expressed in thymus,spleen,lymph nodes and small intestinal mucosa where immune cells and lymphocytes are enriched.Protein structure analysis revealed that TIPE2 contains a hydrophobic domain in which hydrophobic cofactors and cytokines are bound Functional studies have found that TIPE2 deficiency mice can spontaneously develop multiple organ inflammation and splenomegaly.TIPE2 negatively regulates immune and inflammatory responses mediated by macrophages,dendritic cells,T cells and other immune cells.Further studies showed that macrophages depleted TIPE2 gene had significant increases in phagocytosis of pathogen and secretion of TNF-?,IL-6 and other inflammatory cytokines induced by LPS.Recent studies have shown that neutrophils deleted TIPE2 gene shows incomplete remodeling of the cytoskeleton and decreases of migration capacity in the process of polarization into the inflammatory sites.TIPE2 is also involved in the occurrence and development of tumors,but with different functions in different tumors.In gastric cancer,lung cancer and hepatocellular carcinoma,the expression of TIPE2 was downregulated and negatively correlated with the growth and migration of the tumors.But in renal cell carcinoma and colon cancer tissues,the expression of TIPE2 was upregulated and positively correlated with the growth and migration of the tumors.It has been demonstrated that TIPE2 is involved in many signaling pathways such as Rac1,AKT,caspase-8,NF-?B and MAPK.The function of TIPE2 is rarely studied in the field of metabolism.Few studies have shown that TIPE2 in macrophages can reduce the responsiveness to ox-LDL and reduce the formation of foam cells by inhibiting the MAPK and AKT signaling pathways in atherosclerotic diseases.Clinical data showed that the expression of TIPE2 in PBMC of patients with T2DM is upregulated and positively correlated with the expression of TNF-?,IL-6 and other inflammatory factors.Our previous study found that TIPE2 inhibited the growth and migration of hepatocellular carcinoma by inhibiting the activity of Racl.However,the expression of TIPE2 in normal hepatocytes and its physiological functions in glucose metabolism remain unclear.In this study,we explored the functions and underlying mechanisms of TIPE2 in regulating hepatic gluconeogenesis using cell and animal models,so as to elucidate the physiological function and clinical application potential of TIPE2 in metabolismMethods1.Detection of TIPE2 expression in mouse livera.Mouse treatmentMice fed normal diet:wild type C57BL/6 male mice(6-8 weeks)were divided into three groups:normal feeding group,24 hour fasting group,6 hour refeeding follow 24 hour fasting group.Liver tissue from the mice were collectedObese type II diabetic model mice:wild type C57BL/6 male mice(8 weeks)were fed with high-fat diet(HFD)and normal diet(ND)for 32 weeks.Body weight,fasting blood glucose and pyruvate tolerance were recorded.After 1 6 hours of fasting,liver tissue from the mice were collected.b.Expression of TIPE2 and gluconeogenesis related genes in mouse liverThe mRNA levels of PEPCK,G6Pase.PGC-1? and TIPE2 were detected by real-time quantitative PCR.The protein expression of TIPE2 were detected by western-blot.c.Correlation analysis of TIPE2 and gluconeogenesis related genes in liverAnalyzes the correlation analysis of TIPE2 mRNA level with PEPCK,G6Pase and PGC-1? mRNA levels were performed,respectively2.Detection of gluconeogenesis in TIPE2 deficiency micea.Establishment of TIPE2 deficiency miceTIPE2 deficiency mice were established using CRISPR-Cas9,and raised in the experimental animal center of Shandong University.The mice were genotyped by PCR amplification of their genomic DNAb.Detection of blood glucose in TIPE2 knockout miceTIPE2 deficiency mice(6-8 weeks)and littermate wild-type mice were fasted for 6 hours.The glucose concentration in blood from tail vein was measuredc.Detection gluconeogenesis of TIPE2 deficiency micePyruvate tolerance test:TIPE2 deficiency mice(6-8 weeks)and littermate wild-type mice were fasted for 6 hours.Blood glucose at 0,1 5,30.45.60,90 and 120 minutes after injection of sodium pyruvate were detectedAlanine stimulation experiment:TIPE2 deficiency mice(6-8 weeks)and littermate wild-type mice were fasted for 6 hours.Blood glucose at 0.60 minutes after injection of alanine were detected3.Effects of TIPE2 on gluconeogenesis in hepatocytesa.Effect of overexpression of TIPE2 on PEPCK and G6PaseHepG2 cells(human liver carcinoma cells)were transfected with the pRK5-Flag-TIPE2.The cells were incubated in serum-free M199 medium for 16 hours before stimulation with 100?m pCPT-cAMP for 6 hours.The expression of TIPE2,PEPCK and G6Pase were detected by real-time quantitative PCRb.Effect of TIPE2 deletion on PEPCK and G6PaseThe primary hepatocytes from TIPE2 deficiency mice and littermate wild-type mice were incubated for 12 hours after adherent to the plates,respectively.The cells were incubated in serum-free M199 medium for 16 hours before stimulation with 100?m pCPT-cAMP for 6 hours.The expression of PEPCK and G6Pase were detected by real-time quantitative PCR4.Signaling pathways involved in TIPE2 regulation in gluconeogenesisa.Construction of mouse Flag-TIPE2 plasmidThe plasmid expressing mouse TIPE2 was constructed.Mouse TIPE2 gene was amplified using the cDNA from mouse hepatocytes as the template,and was inserted into pcDNA3.1 with a Flag tag.The Flag sequence and the restriction endonucleases EcoR land Hand ? sequences of enzyme were amplified by primers using PCR.After enzyme identification,linkage,transformation and screening,the plasmid was constructedb.The effect of TIPE2 deletion on AKT signaling pathwayIn the TIPE2 deficiency mice(6-8 weeks)and littermate wild-type mice,the liver tissues were collected,the protein lysate was extracted,and the expression levels of p-AKT,AKT and TIPE2 were detected by western-blotThe primary hepatocytes from TIPE2 deficiency mice and littermate wild-type mice were cultured in serum-free medium overnight and stimulated with 100nm insulin.The expression of p-AKT,AKT and TIPE2 were detected by western-blotc.Effect of TIPE2 overexpression on AKT signaling pathwayHepG2 cells were transfected with the pRK5-Flag-TIPE2.The cells were incubated in serum-free medium for 16 hours before stimulation with 100nM insulin.The expression of p-AKT,AKT and Flag was detected by western-blotAML 12 cells(mouse hepatocyte cells)were transfected with the pcDNA3.1-Flag-TIPE2.The cells were incubated in serum-free medium for 16 hours before stimulation with 100nM insulin.The expression of p-AKT,AKT and Flag was detected by western-blot.d.The effect of TIPE2 deletion on ERK signaling pathwayTIPE2 deficiency mice(6-8 weeks)and littermate wild type mice were fasted 24 hours,liver tissues were collected.Protein lysate was extracted,and expression of p-ERK,ERK and TIPE2 were detected by western-blotThe primary hepatocytes from TIPE2 deficiency mice and littermate wild-type mice were cultured in serum-free medium overnight and stimulation with 100?M pCPT-cAMP for 60 minutes.The expression of p-ERK,ERK and TIPE2 were detected by western-blote.Effect of overexpression of TIPE2 on ERK signaling pathwayHepG2 cells were transfected with the pRK5-Flag-TIPE2.The cells were incubated in serum-free medium for 16 hours before stimulation with 100?M pCPT-cAMP.The expression of p-ERK.ERK and Flag was detected by western-blot.AML 12 cells were transfected with the pcDNA3.1-Flag-TIPE2 plasmid.The cells were incubated in serum-free medium for 16 hours before stimulation with 100?M pCPT-cAMP.The expression of p-ERK.ERK and Flag was detected by western-blot.Results1.The expression of TIPE2 is positively correlated with the expression of gluconeogenesis key genes in liver tissuea.TIPE2 expression is upregulated by fasting in liver tissuesThe livers of C57BL/6 mice were collected after fasting and fasting follow by refeeding.Compared with normal feeding mice,fasting significantly increased the transcriptional expression of TIPE2 and PEPCK,G6Pase,PGC-1?.But refeeding significantly decreased the expression of the above genes.Western-blot results confirmed the increase of TIPE2 in protein levels in liver tissues from fasting mice compared with those from normal feeding mice,while refeeding caused a significant decrease TIPE2 in protein levels in the liver tissues of the mice.These results suggested that TIPE2 may play a role in the process of gluconeogenesis induced by fasting.b.TIPE2 expression is positively correlated with the expression of PEPCK,G6Pase,PGC-1? in liver tissues from the miceAfter analyzing the correlation between the mRNA levels of TIPE2 and the mRNA levels of gluconeogenesis related genes in the liver tissues of the mouse,we showed that there were positive correlations between the TIPE2 mRNA expression and the mRNA expression of PEPCK,G6Pase,PGC-1?.c.High-fat diet induces the upregulation of TIPE2 expression in liver tissuesThe glucose levels in blood from fasting HFD-fed mice were significantly higher than those from ND-fed mice.Pyruvate tolerance test showed that the gluconeogenesis ability of HFD-fed mice was significantly higher than that of ND-fed mice.After 16 hours of fasting,the expression levels of TIPE2 in liver tissues from HFD-fed mice was significantly higher than those from ND-fed mice.These results suggested that TIPE2 may be involved in the hepatic gluconeogenesis in mice with T2DM.2.TIPE2 deficiency impairs gluconeogenesis in micea.Establishment of TIPE2 deficiency miceCRISPR/Cas9 technology was used to establish TIPE2 deficiency mice,in which the second exon of TIPE2 was depleted.The results from mouse genotyping showed that only 5' 1820bp sequence was identified in wild type mice,both 5' 1820bp and 3'695bp sequences were identified in heterozygous mice,while only 3' 695bp sequencewas identified in TIPE2 deficiency mice.b.TIPE2 deficiency reduces plasma glucose levels in fasting miceAfter 6 hours of fasting,littermate wild-type mice maintained a relatively normal levels of blood glucose,but for TIPE2 deficiency mice,the levels of blood glucose decreased significantly by fasting,suggesting that TIPE2 is essential for maintaining blood glucose in fasting mice.c.TIPE2 deficiency reduces gluconeogenesis in micePvruvate tolerance test and alanine stimulation test showed that the blood glucose levels of TIPE2 deficiency mice were significantly lower than that of wild-type mice.These data indicated that TIPE2 deficiency mice failed to maintain normal gluconeogenesis3.TIPE2 promotes gluconeogenesis in hepatocytesa.TIPE2 overexpression promotes the expression of PEPCK and G6Pase induced by pCPT-cAMP in hepatocytesIn response to pCPT-cAMP stimulation,HepG2 cells transfected with pRK5-Flag showed significantly higher levels of TIPE2 mRNA and protein than those in cells transfected with mock plasmid.Special attention should be paid to HepG2 cells transfected with mock plasmid,pCPT-cAMP could obviously induce the upregulation of TIPE2 mRNA levelsb.TIPE2 deficiency reduces the expression of PEPCK and G6Pase in hepatocytesThe primary hepatocytes from TIPE2 deficiency mice and littermate wild-type mice were incubated in serum-free medium for 16 hours,and then were stimulated with or without pCPT-cAMP for 6 hours.The results showed that there was no significant difference in the mRNA levels of PEPCK and G6Pase between the wild-type and TIPE2 deficiency hepatocytes in the absence of pCPT-cAMP Treatment with pCPT-cAMP caused significant upregulation in the mRNA levels of PEPCK and G6Pase in both wild-type and TIPE2 deficiency hepatocytes,but the mRNA levels of PEPCK-and G6Pase were significantly lower in TIPE2 deficiency hepatocytes than those in wild-type hepatocytes.The results indicated that TIPE2 deletion in hepatocytes reduced the expression of gluconeogenesis key enzyme4.TIPE2 inhibits the phosphorylation of AKT and ERK during gluconeogenesisa.Construction of plasmid expressing mouse TIPE2Genetic sequencing results showed that the sequence of TIPE2 gene in the plasmid pcDNA3.1-Flag-TIPE2 was correct.Western blot showed that Flag protein was expressed in HepG2 cells transfected with pcDNA3.1-Flag-TIPE2.b.TIPE2 deletion promotes insulin-induced AKT phosphorylationIn order to determine the relationship between TIPE2 and AKT during gluconeogenesis.we detected AKT phosphorylation in liver tissues from TIPE2 deficiency mice and wild-type mice,respectively.The results showed that the phosphorylation levels of AKT in liver tissues from TIPE2 deficiency mice were higher than those from wild-type mice.The results showed that TIPE2 deletion promotes AKT phosphorylation in liver that may inhibit hepatic gluconeogenesisPrimary hepatocytes from TIPE2 deficiency mice and wild-type mice were incubated in medium without serum for 16 hours,and then stimulated with insulin.Western-blot showed that insulin could significantly induce AKT phosphorylation in primary hepatocytes,while AKT phosphorylation in TIPE2 deficiency hepatocytes was higher than that in wild-type hepatocytes,suggesting that TIPE2 deletion in hepatocytes could promote AKT phosphorylation induced by insulinc.TIPE2 overexpression reduces insulin-induced AKT phosphorylationThe human HepG2 cells and mouse AML 12 cells overexpressed Flag-TIPE2.After stimulated with insulin,both HepG2 cells and AML 12 cells showed significant increases in AKT phosphorylation,but AKT phosphorylation in TIPE2-overexpressed cells was significantly lower than that in cells transfected with mock plasmid.These results suggested that TIPE2 overexpression in hepatocytes could reduce AKT phosphorylation induced by insulin.d.TIPE2 deletion promotes ERK phosphorylationIn order to determine the relationship between TIPE2 and ERK during gluconeogenesis,we detected ERK phosphorylation in liver tissues of TIPE2 deficiency mice and wild-type mice after 24 hours of fasting,respectively Western-blot showed that the levels of p-ERK in liver tissues from TIPE2 deficiency mice was significantly higher than those from wild-type mice,suggesting that TIPE2 deletion could promote AKT phosphorylation in liver tissues.The primary hepatocytes from TIPE2 deficiency mice and wild-type mice were incubated in serum-free medium for 16 hours,and then were stimulated with pCPT-cAMP for 60 minutes.Western-blot showed that pCPT-cAMP could significantly reduce ERK phosphorylation in primary hepatocytes,while ERK phosphorylation in TIPE2 deficiency hepatocytes was higher than that in wild-type hepatocytes.suggesting that TIPE2 deletion could promote ERKphosphorylation in hepatocytes.e.TIPE2 overexpression reduces ERK phosphorylationHepG2 cells and AML 12 cells overexpressing Flag-TIPE2 were stimulated with pCPT-cAMP after incubation with serum-free medium.Western blot showed that the levels of ERK phosphorylation were decreased by pCPT-cAMP stimulation in both HepG2 cells and AML12 cells,but the levels of ERK phosphorylation in TIPE2 overexpressed cells was significantly lower than that in cells transfected with mock plasmid.These results suggested that TIPE2 overexpression in hepatocytes could inhibit ERK phosphorylation.Conclusions1.Fasting induces the upregulation of TIPE2 expression in the mice liver.2.High fat diet induces the upregulation of TIPE2 expression in the mice liver.3.TIPE2 deletion reduces gluconeogenesis and plasma glucose levels in fasting mice.4.TIPE2 promotes the expression of gluconeogenesis key enzymes in hepatocytes.5.TIPE2 inhibits the phosphorylation of AKT and ERK during gluconeogenesis.