The Regulation Of IRS-1 Expression And Its Impacts On Insulin Resistance In Neurons

BackgroundEpidemiological and basic studies have revealed the close interactions between Alzheimer's disease(AD)and type 2 diabetes mellitus(T2DM).T2DM is considered to be a risk factor for AD.AD is proposed as "type 3 diabetes" for the extensive abnormalities of insulin signaling in the brain.However,the molecular mechanism underlying the interactions between these two prevalent diseases remains unclear.Brain insulin resistance is the common feature shared by AD and T2DM,which contributes to cognitive dysfunction.Furthermore,intranasal insulin administration improved cognitive performance in both AD and T2DM patients with cognitive dysfunction.Insulin receptor substrate 1(IRS-1)is a core molecule in insulin signaling and also plays a pivotal role in insulin resistance.Its abnormal phosphorylation is believed to be a molecular marker of insulin resistance in the brain.After binding with insulin,insulin receptors will phosphorylate the tyrosine sites of IRS-1 and activate downstream signaling pathways.By contrast,specific serine phosphorylation of IRS-1 will inhibit insulin-induced tyrosine phosphorylation and thus disturb insulin signaling.Autopsy results revealed that IRS-1 serine phosphorylation was abnormally increased and total IRS-1 protein expression was decreased in AD patients' brains.The level of abnormal phosphorylation in specific brain regions was significantly related to the degree of cognitive impairment.Therefore,clarifying the regulation mechanism of IRS-1 expression in the brain might provide new therapeutic methods for brain insulin resistance and related cognitive impairment.However,the specific regulation mechanism is still unclear.Dual-specificity tyrosine phosphorylation-regulated kinase 1A(DYRK1A)is a Ser/Thr protein kinase located in the Down syndrome critical region(DSCR)at chromosome 21.It is comprised of multiple domains,including a nuclear localization signal(NLS),a kinase domain,a prolineglutamic acid-serine-threonine rich(PEST)domain,a consecutive histidine repeat(His)and a serine/threonine rich domain.DYRK1A is abundantly expressed in the brain and interacts with numerous cytoskeletal,synaptic and nuclear proteins in neurons.Through phosphorylating various substrates,DYRK1A participates in a broad spectrum of brain biological functions including the regulation of neural proliferation/differentiation,neurogenesis,dendritogenesis,and synaptogenesis.Its aberrant expression contributes to the abnormal brain development in Down syndrome(DS),and favors the pathological hallmarks of neurodegenerative diseases such as AD.Moreover,inhibition of DYRK1A stimulates robust ?-cell proliferation in adult primary islets and increases?-cell mass and improves glycaemic control in mice.The Wnt/?-catenin pathway is another important signaling pathway in the brain.When activated,?-catenin enters the nucleus and interacts with T cell factor/lymphoid enhancer factor(TCF/LEF)transcription factors.Together,they regulate the transcription of Wnt target genes.Through the regulation of target genes,the Wnt/?-catenin pathway is involved in activities such as synapse formation and maintenance,hippocampal neurogenesis,and neural circuit formation.Decreased pathway activity is associated with cognitive dysfunction in AD.There is also a close interaction between the Wnt/?-catenin pathway and the insulin pathway.For instance,deletion of ?-catenin and mutation of T cell factor 4(TCF4,also known as TCF7L2)could result in reduced islet ? cell mass and attenuated glucose homeostasis.In addition,the Wnt/p-catenin pathway is also involved in the regulation of insulin sensitivity in peripheral tissues,but its effect on the insulin pathway in the brain is unclear.Our research focused on whether the expression of IRS-1 in neurons is regulated by DYRK1A and Wnt/?-catenin pathway,and the impacts of such regulation on insulin resistance in neurons.This study can provide new insights in the pathogenesis of AD and T2DM-related cognitive impairment,and potential directions for the prevention and treatment of these diseases.Section 1 Regulation of DYRK1A on IRS-1 and its effects on insulin resistance in neuronsObjectiveThis study aimed to investigate the regulation of IRS-1 expression by DYRK1A and the underlying molecular mechanisms,and the effects of such regulation on insulin resistance in neurons.Materials and methods1.Regulation of DYRK1A on IRS-1 protein expression.HEK293 and SH-SY5Y cells were transfected with DYRK1A overexpression or control plasmid,rat primary neurons were infected with DYRK1A overexpression or control virus,and the protein levels of DYRK1A and IRS-1 were detected by western blotting.These cells were stimulated with DYRK1A inhibitor Harmine or its solvent control DMSO,and the protein levels of DYRK1A and IRS-1 were detected by western blotting.2.Effect of DYRK1A on IRS-1 protein degradation HEK293 cells were transfected with DYRK1A overexpressing plasmid or treated with Harmine,and then the degradation rates of IRS-1 in these cells were detected by cycloheximide(CHX)chase assay.HEK293 cells were co-transfected with IRS-1 and DYRK1A overexpression or control plasmids,and then IRS-1 protein was immunoprecipitated and detected for its ubiquitination.3.Protein-protein interaction between DYRK1A and IRS-1 Co-immunoprecipitation assay was used to study the interaction between DYRK1A and IRS-1 in HEK293 cells.Immunofluorescence was performed to detect the intracellular localization of DYRK1A and IRS-1,images were captured using LSM 780 fluorescent microscope and analyzed with ZEN software.Proximity ligation assay(PLA)was used to detect DYRK1A-IRS-1 colocalization in neurons.4.Detection of the phosphorylation sites of IRS-1 by DYRK1A HEK293 cells were co-transfected with IRS-1 and DYRK1A overexpression plasmids.IRS-1 protein was then immunoprecipitated,and western blotting was used to detect its total serine phosphorylation.HEK293 cells were co-transfected with IRS-1 and DYRK1A overexpression plasmids,and western blotting was used to detect IRS-1 Ser312 and Ser616 phosphorylation levels.5.Expression of DYRK1A and IRS-1 in mouse brain Western blotting was used to detect the expression levels of DYRK1A and IRS-1 in the hippocampus and prefrontal cortex(PFC)of db/db mice.Their mRNA levels were detected by real-time quantitative PCR.6.Establishment of insulin resistant cell models The cells were stimulated with high concentration of insulin for 24 h,and then re-stimulated with 10 nM insulin for another 15 min.The protein levels of pAKT S473,AKT5 pGSK3? S9,GSK3? were detected by western blotting.Ratios of pAKT Ser473/AKT and pGSK3? Ser9/GSK3? were calculated and the ratio changes after acute insulin re-stimulation were used to reflect cell responsiveness to insulin.7.Effects of DYRK1A on insulin resistance in SH-SY5Y SH-SY5Y cells were transfected with DYRK1A overexpression or control plasmids,and the cellular insulin responsiveness were detected under both basal and insulin resistant conditions,respectively.SH-SY5Y cells were treated with Harmine or DMSO,and the cellular insulin responsiveness were detected under both basal and insulin resistant conditions,respectively.8.Effects of DYRK1A on insulin resistance in neurons Rat primary neurons were infected with DYRK1A overexpression or control virus,and the cellular insulin responsiveness were detected under both basal and insulin resistant conditions,respectively.Neurons were treated with Harmine or DMSO,and the cellular insulin responsiveness were detected under both basal and insulin resistant conditions,respectively.Results1.DYRK1A upregulated IRS-1 protein expression Overexpression of DYRK1A up-regulated the expression of IRS-1 in HEK293 cells,SH-SYSY cells and neurons.And treating these three kinds of cells with Harmine reduced the expression of IRS-1.In addition,overexpression of DYRK1A at different doses could increase the protein level of IRS-1 in a dose-dependent manner in HEK293 cells.2.DYRK1A stabilized IRS-1 by decreasing its ubiquitination Overexpression of DYRK1A in HEK293 cells slowed the degradation rate of IRS-1 protein;while treating HEK293 cells with Harmine accelerated the degradation rate of IRS-1 protein.Moreover,overexpression of DYRK1A decreased IRS-1 ubiquitination in HEK293 cells.3.DYRK1A interacted with IRS-1 in cells Co-IP assay showed that DYRK1A efficiently precipitated IRS-1.Reciprocally,IRS-1 precipitated DYRK1A.Immunofluorescent assay showed that DYRK1A and IRS-1 co-localized in both HEK293 cells and T98G cells.PLA assay showed that DYRK1A and IRS-1 co-localized in neurons.4.DYRK1A phosphorylated IRS-1 at Ser312 and Ser616 Overexpression of DYRK1A in HEK293 increased the total serine phosphorylation of IRS-1;DYRK1A increased the phosphorylation of Ser312 and Ser616 sites of IRS-1 protein.5.DYRK1A and IRS-1 were coordinately up-regulated in PFC of db/db mice The mRNA levels of IRS-1 and DYRK1A in the brain of db/db mice were not significantly changed compared to m/m mice,while the protein levels of DYRK1A and IRS-1 were increased in the PFC.Moreover,the expression levels of DYRK1A and IRS-1 proteins in hippocampus and PFC showed significant positive correlations.6.Establishment of insulin resistant cell models After chronic high insulin treatment,the expression of IRS-1 in the cells was significantly reduced,and the increased expression of pAKT S473 and pGSK3?S9 induced by acute insulin stimulation disappeared,indicating that insulin resistance occurred.7.DYRK1A ameliorated chronic high insulin-induced insulin resistance in SH-SY5Y cells Overexpression of DYRK1A increased IRS-1 expression in both basal and insulin resistant states.In the case of insulin resistance,the insulin sensitivity was partially recovered in cells overexpressing DYRK1A compared with control cells,indicating that DYRK1A could ameliorate insulin resistance.Moreover,Stimulating SH-SY5Y with Harmine reduced the insulin responsiveness at basal state.8.DYRK1A ameliorated chronic high insulin-induced insulin resistance in neurons Overexpression of DYRK1A increased IRS-1 expression in the basal state and insulin resistant state.And DYRK1A overexpression partially restored the insulin sensitivity of the neurons in the state of insulin resistance,which means that DYRK1A could alleviate the insulin resistance of neurons.In contrast.stimulating neurons with Harmine reduced the insulin response at basal stateConclusions1.DYRK1A up-regulates IRS-1 protein expression by repressing its degradation.2.DYRK1A physically interacts with IRS-1 and phosphorylates IRS-1 at Ser312 and Ser616.3.DYRK1A upregulation of IRS-1 reinforces insulin sensitivity and ameliorates insulin resistance in SH-SY5Y cells and rat primary neuronsSignificanceIRS-1 is a pivotal molecule in the insulin pathway,and its abnormal expression plays a key role in brain insulin resistance,but its expression regulation mechanism is still unclear.In this study,for the first time,DYRK1A was identified to be a regulatory factor of IRS-1,and it was demonstrated that DYRK1A could protect against the insulin resistance in neurons by regulating the expression of IRS-1.These findings further enrich our understanding of the regulatory mechanism of IRS-1 and provide potential therapeutic strategies for brain insulin resistance and the related cognitive impairment.Section 2Regulation of Wnt/?-catenin signaling on IRS-1 and its effects on neuronal insulin resistanceObjectiveThe Wnt/p-catenin signaling is known to regulate insulin sensitivity in peripheral tissues,but its effect on brain insulin action is unknown.In this section,we mainly studied the regulation of IRS-1 expression in neurons by the Wnt/?-catenin signaling and the effects of this regulation on downstream insulin signaling pathways,so as to explore the role of the Wnt/?-catenin signaling in the regulation of insulin sensitivity in the brain.Materials and methods1.Regulation of Wnt/?-catenin signaling on IRS-1 expression The Wnt signaling was activated in SH-SY5Y cells and rat primary neurons in various ways,and then the mRNA level of IRS-1 was detected by real-time quantitative PCR and the protein level of IRS-1 was detected by western blotting.SH-SY5Y cells were transfected with dominant-negative TCF4(TCF4DN),and IRS-1 protein and mRNA levels were detected.2.Identify the Wnt-responsive element(WRE)on the IRS-1 promoter First,a series of IRS-1 promoter truncations were constructed by subcloning IRS-1 promoter deletions into the pGL3-Basic vector.Dual-luciferase assay was used to detect the transcriptional regulation of these truncations by Wnt activators and inhibitors.Then the JASPAR database was used to predict the possible binding site of the Wnt pathway transcription factor TCF4 on the IRS-1 promoter.And the corresponding mutant versions of IRS-1 promoter truncations were also constructed with the predicted WRE sequence mutated.Finally,the electrophoretic mobility shift assay(EMSA)and chromatin immunoprecipitation(ChIP)experiments were used to verily the predicted WRE sequence on the IRS-1 promoter.3.Effects of Wnt ligand Wnt3a on insulin sensitivity in neurons Rat primary neurons were treated with Wnt3a or not and then cells were stimulated with insulin for a short period of time.Western blotting was used to detect changes in pAKT S473 and pGSK3? S9 protein levels caused by acute insulin stimulation.4.Effects of Wnt3a on insulin resistance in neurons First,a neuronal insulin resistant cell model was constructed by chronic incubation of neurons with high concentration of insulin.Based on this model,neurons were treated with Wnt3a or not,and then pAKT S473,pGSK3? S9 protein levels induced by acute insulin stimulation were detected by western blotting.5.Effects of Wnt3a on glucose uptake induced by insulin stimulation in neurons Neurons were treated with or without Wnt3a,and then stimulated with low concentration of insulin for a short time at both basal state and the insulin resistant state.The glucose uptake capacity of the neurons induced by the acute insulin stimulation was measured using the glucose uptake kitResults1.Wnt/?-catenin signaling up-regulated IRS-1 expression The activation of Wnt/?-catenin pathway increased the mRNA and protein expression of IRS-1 in SH-SY5Y cells and rat primary neurons.However,over-expression of TCF4DN down-regulated the mRNA and protein expression of IRS-1.2.-529?-516 bp of IRS-1 promoter was a WRE site The dual luciferase assay showed that the regulation of IRS-1 by Wnt agonists or inhibitors is closely related to the-529?-516 bp of the IRS-1 promoter.EMSA and ChIP experiments further confirmed that TCF4 can bind to-529?-516 bp of IRS-1.3.Wnt3a improved neuronal insulin sensitivity After incubation with Wnt3a,the expression of IRS-1 was increased in neurons.Moreover,the protein level changes of pAKT S473,pGSK3? S9 induced by acute insulin stimulation were all increased,which indicated enhanced insulin sensitivity of neurons.4.Wnt3a ameliorated neuronal insulin resistance Prolonged stimulation with high concentrations of insulin successfully induced insulin resistance in neurons.Based on this model,the neurons were treated with or without Wnt3a,and then the cells were acutely stimulated with insulin.The results showed that pAKT S473,pGSK3? S9 protein levels were almost unchanged by acute insulin stimulation in control group.However,the pAKT S473,pGSK3? S9 protein levels were significantly elevated by acute insulin stimulation in the Wnt3a treatment group.5.Wnt3a enhanced insulin-induced glucose uptake in neurons After acute insulin stimulation,the Wnt3 a-treated neurons could take more glucose in the same period of time than the control neurons at both basal state and the insulin resistant state,that is,Wnt3a enhanced the insulin-induced glucose uptake capacity of neurons.Conclusions1.Activation of Wnt/?-catenin signaling upregulates IRS-1 expression.2.TCF4 could combine with the WRE(-529--516 bp)site of IRS-1 promoter and stimulate IRS-1 transcription.3.Wnt3a could promote insulin signaling and effectively ameliorate insulin resistance in neurons.4.Wnt3a could enhance insulin-mediated glucose uptake in neurons.SignificanceThis study demonstrated for the first time that Wnt/?-catenin could up-regulate the expression of IRS-1 in neurons,and this up-regulation could activate the insulin signaling pathway and alleviate insulin resistance in neurons.These findings further deepen our understanding of the regulation of IRS-1 expression in neurons and also emphasize the important role of Wnt/p-catenin signaling in the treatment of neurodegenerative diseases such as AD.