Systematic Study Of Lipotoxicity And Glucolipotoxicity Of Pancreatic ? Cell And The Function Of DDX1 In Pancreatic ? Cell

Type 2 diabetes mellitus(T2D)is a major public health issue characterized by pancreatic islet ?-cell failure in the presence of insulin resistance.Accumulating evidence suggest that progressive deterioration of pancreatic ?-cell function and gradual loss of ?-cell mass could be the core events in T2 D,regardless of the therapy.Recent genome-wide association(GWAS)and sequencing studies have identified multiple risk variants for T2 D,most of which appeared to have a primary role in ?-cell function rather than impacting insulin resistance,further suggesting the importance of ?-cell in the pathogenesis of T2 D.Therefore,understanding the process and mechanism of the progressive deterioration of pancreatic ?-cell may provide new clues for the treatment and prevention of diabetes.In this paper,we quantified the temporal transcriptome and proteome of pancreatic beta cell line INS1 in the process of gradual deterioration of the lipotoxicity and glucolipotoxicity.The bioinformatics analysis revealed that the main pathways affected by lipotoxicity and glucolipotoxicity were lipid metabolism,glucose metabolism,endoplasmic reticulum stress,oxidative stress,and vesicle related genes.The differences between lipotoxicity and glucolipotoxicity were TCA cycle and pro-apoptosis genes expression which inhibited by lipotoxicity and promoted by glucolipotoxicity.In addition,glucolipotoxicity can promote synthesis of cholesterol and phospholipid.This may be the reason that glucolipotoxicity is more serious than the lipotoxicity to the pancreatic ?-cell function.As the lipid metabolism is the main pathway affected by lipotoxicity and glucolipotoxicity,thus we further study lipid metabolism pathway.Depending on the difference of metabolism products,the lipid metabolism genes can be divided into five modules(fatty acids beta oxidation,unsaturated fatty acid synthesis,cholesterol synthesis,phospholipids and neutral lipids synthesis,fatty alcohol synthesis).Fatty alcohol synthesis is a novel pathway which affected by palmitate,and overexpression of fatty alcohol synthase FAR1 can aggravate the ER Stress induced by palmitate.Then we verified the function of PKC? kinase which is downregulated by palmitate treatment.We found that knockout PKC? can alleviate the ER Stress and impaired insulin secretion induced by palmitate.These results indicate that downregulation of PKC? is a protective mechanism for cellular response to lipotoxicity.Finally,we found that SETD8 is a novel target which is necessary for the proliferation of ?-cell induced by glucose.Analysis of the transcriptome and proteome revealed that palmitate was able to inhibit the translation of vesiclar genes.Therefore,the insulin gene was used as a target to study the molecular mechanisms of translation inhibited by palmitate.First of all,we verified that palmitate can inhibit proinsulin translation in INS1 cells and isolated rat islets in vitro.And we identified a novel insulin mRNA binding protein DDX1 by ChIRP-MS.Western-blot and RIP-PCR results verified the interaction between DDX1 and insulin mRNA.We further found that knockdown DDX1 could inhibit the translation of proinsulin,and overexpression of DDX1 could promote the translation of proinsulin.These results indicate that DDX1 is necessary for insulin translation.Furthermore,knockdown DDX1 could alleviate the inhibition of insulin translation by palmitate,suggesting that the RNA binding protein DDX1 is responsible for fatty acid mediated inhibition of insulin translation.Then we further studied the molecular mechanism whcih DDX1 mediated fatty acid inhibtion of insulin translation.Firstly,we found that expression of DDX1 was not changed after palmitate stimulation,but the palmitate stimulation could promote DDX1 into the nucleus and Stress Granules.RIP-PCR and ChIRP western-blot results showed that palmitate stimulation could result in dissociation of insulin mRNA and DDX1.Further analysis found that palmitate could induce DDX1 phosphorylation at S295,S436,and S617/T621,which the sites of S295 and S436 are responsible for insulin translation.And the interaction protein network of DDX1 showed that DDX1 could interact with translation initiation complex.Therefore,we speculated the molecular mechanism is that palmitate stimulation causes the phosphorylation of DDX1 and dissociation from the insulin mRNA,which decreases insulin translation.In addition,we identified 2632 genes directly bound to DDX1 using CLIP-seq and mapped the binding sites of DDX1.The most count enriched GO term of DDX1 targeting mRNA was vesicle,suggesting that DDX1 is important for the secretory function of pancreatic ?-cell.All these genes whose translation were inhibited by palmitate contained DDX1 binding signal in the Clip-seq data.RIP-PCR verified that palmitate stimulation could dissociation of the DDX1/RNA complex.So we speculate that the mechanism of palmitate inhibition of the vesiclar genes translation is the same with insulin.Further analysis of insulin mRNA binding sites of DDX1 revealed that DDX1 mainly binded to the CDS region of insulin mRNA,and the binding motif was CTTTT.In high fat diet mice,inhibition of insulin translation happens at an early prediabetic stage before the elevation of glucose.We speculate that DDX1-mediated repression of insulin translation worsens the situation of insulin resistance and contributes to the elevation of blood glucose in obese animals.GO analysis of DDX1 interacting proteins revealed that DDX1 interacted with RNA processing proteins.Furthmore,Clip-seq result showed that DDX1 was mainly binding to introns of RNA.These results suggested that DDX1 could regulate mRNA alternative splicing.Therefore,we identified 450 genes which are alternative splicing upon knockdown DDX1 by RNA-seq.GO enrichment analysis showed that these genes were vesiclar genes and ion channels.Furhermore,2697 genes were differential expressed upon knockdown DDX1.Among them,the upregulated genes were ribosome related genes,while the downregulated genes were related to cell adhesion proteins,ion channels and vesicles.These results suggested DDX1 could affect the secretion of insulin which was verified in shDDX1 cells.