Mechanism Study And Drug Screening Of PKA/PKC Regulating Islet ? Cell Function Through TXNIP

BACKGROUND:Type 2 diabetes(T2D)is currently the most common chronic metabolic disease and has become the third major cause of illness and death in the elderly.However,according to statistical studies,almost half(50.3%)of all patients with diabetes are diagnosed and treated,and nearly half of them are not diagnosed and treated in a timely manner.Although there are many anti-diabetic drugs in the clinic,it can only improve the symptoms of patients,delay the course of the disease,and the obvious toxic and side effects of drugs have always been the bottleneck of the treatment of this disease.Therefore,research on new mechanisms and new target drugs for the onset of T2D is imminent.Hyperglycemia is a typical feature of T2D.In 2002,it was found that thioredoxin interacting protein(TXNIP)was up-regulated most obviously under the stimulation of glucose,and TXNIP level was significantly increased in T2D mouse model,which revealed the important role of TXNIP in the pathological process of T2D.In T2D patients,high glucose induced up-regulation of TXNIP expression through gene transcription process,while TXNIP inhibited insulin secretion through miR-204,leading to further increase in blood glucose and causing glycotoxicity and apoptosis of islet ? cells.In addition,up regulation of TXNIP also mediated oxidative stress,endoplasmic reticulum stress,cell inflammatory response and autophagy of islet ? cells.Due to the lack of antioxidant defense system,islet ? cells apoptosis induced by long-term stress.The decrease in the number of islet ? cells leads to a severe shortage of insulin secretion,which aggravates the pathological process of T2D.Therefore,the drug development targeting TXNIP has a better therapeutic prospect in the treatment of T2D.At present,many natural compounds and their derivatives have been reported to reduce TXNIP level,and their hypoglycemic effects have been verified in animals.However,these compounds have a wide range of mechanisms,low specificity and low clinical application prospects.To solve this problem,on the one hand,we studied the degradation mechanism of TXNIP,in order to obtain a new mechanism of TXNIP;at the same time,we use TXNIP as the target to carry out a series of drug screening experiments to obtain better lead compounds for clinical use.METHODS:1.CRISPR-Cas9 technology for PKA Ca knockout of ? cell.2.The effects of PKA Ca knockout on TXNIP protein and inflammatory factor expression,effects of PKCs inhibitors on endoplasmic reticulum(ER)stress gene and inflammatory factor expression induced by high glucose and thapsigargin(THAP),and effects of luteolin on endoplasmic reticulum stress gene and inflammatory factor expression induced by THAP were detected by real-time quantitative PCR and western blotting.3.The ability of insulin secretion of islet ? cells under various treatments were detected by glucose stimulated insulin secretion(GSIS).4.The interaction between PKA Ca and TXNIP was detected by bimolecular fluorescence complementary(BIFC)and co-immunoprecipitation(Co-IP)experiments.The interaction between PKC? and TXNIP and PP2A was detected by Co-IP.5.The effects of PKC inhibitors on apoptosis induced by THAP and high glucose were measured by hoechst nuclear staining assay and apoptosis kit method;the effects of luteolin on apoptosis induced by THAP were analyzed by flow cytometry(FACS).RESULTS:1.Study on the degradation mechanism of TXNIP mediated by PKA Ca.In the ?cell knocking out PKA Ca,the insulin secretion ability of ? cell was greatly decreased,and the expression of TXNIP and inflammatory factors were up-regulated.Also exendin-4 had no effect on the level of TXNIP and inflammatory factors.In the ? cell overexpression of PKA C?,insulin secretion ability of ? cell was enhanced,and the expression of TXNIP and inflammatory factors were decreased.The results of CO-IP experiment showed that PKA Ca interacts with TXNIP.2.Study on the degradation mechanism of TXNIP mediated by PKC?.In PKC?-overexpressed ? cells,cell nuclear was shrinkage and the number of cells are greatly reduced.Also the expression of ER stress-related genes and inflammatory factors increase greatly.However,blocking PKC can inhibit the increase of apoptosis,ER stress genes and inflammatory factors induced by THAP and high glucose.Inhibition of PKC can also improve the impairment of?-cell insulin secretion caused by THAP and high glucose.Co-IP experiment results show the interaction of PKC?,TXNIP and PP2A.3.Drug screening studies for TXNIP.? cell apoptosis,ER stress genes,HNF4?signaling pathway genes and inflammatory factors induced by THAP were inhibited by Luteolin.Luteolin can also maintain the homeostasis of calcium in ?cells.However,knocking down HNF4? would reduce the effect of Luteolin.CONCLUSIONS:Our research found for the first time that Exendin-4 mainly promotes the phosphorylation of TXNIP through PKA Ca and promotes its degradation through the protease pathway,thereby protecting ? cell function;at the same time,we also found that PKC? interacts with TXNIP by recruiting PP2A to maintain its stability,leading to impaired ? cell function,while inhibition of PKC? significantly improved the function of ? cell.In the process of TXNIP drug screening,we found that luteolin,a natural drug,can inhibit the expression of TXNIP induced by THAP and high glucose,while it can maintain the calcium homeostasis and inhibit apoptosis by inhibiting the HNF4? signal pathway.Moreover,luteolin has little toxicity,strong effect and wide source,so it is likely to be developed into a safe and efficient anti-T2D drug.