Effect Of Cholesterol Sulfate On The Function Of Islet β Cell

Background:Diabetes is a chronic metabolic disease characterized by hyperglycemia,the mechanism of the pathogenesis of diabetes has not yet been fully understood.Most studies believe that the cause of diabetes is a relative or absolute lack of plasma insulin levels.The plasma insulin level is mainly determined by the function and number of pancreaticβcells,that is,the decline in the function or number of pancreaticβcells will cause insufficient plasma insulin levels,leading to hyperglycemia and diabetes.In pancreaticβcells,mature insulin is produced from proinsulin through the folding and modification in the endoplasmic reticulum and the Golgi.The insulin is then transferred to theβ-cell membrane by transport vesicles in the trans-Golgi apparatus,waiting for the stimulation of secretory signals.After feeding,the blood glucose concentration increases,glucose is absorbed into pancreaticβcells by glucose transporter 2（GLUT2）,which undergoes oxidative phosphorylation in the mitochondria to produce ATP.The increase in ATP production closes the ATP-sensitive potassium ion(K_ATP)channel,which leads to an increase in the intracellular K⁺concentration,depolarization of the membrane,and Ca²⁺flow into the cell,triggering the pancreaticβ-cells to secrete insulin through exocytosis.Therefore,maintainingβ-cell mitochondrial function,ATP production,and Ca²⁺are essential for insulin secretion.The increased reactive oxygen species（ROS）is the main factor that induces mitochondrial dysfunction andβcells dysfunction.Since the expression of antioxidant enzymes such as superoxide dismutase isoenzyme and glutathione peroxidase in pancreaticβcells is low,the pancreaticβcells are extremely vulnerable to ROS.The proliferation of pancreaticβ-cells is regulated by growth factors and nutrients,such as insulin and insulin-like growth factors,prolactin,epidermal growth factor（EGF）,platelet-derived growth factor（PDGF）,glucose and so on,which promote the proliferation and survival of pancreaticβ-cells by binding to their receptors and activating the IRS located in the cell.The activated IRS in turn activates PI3K and its downstream factor m TORC2.At the same time,PI3K uses PIP2 as a substrate to produce PIP3 on the cell membrane,and PIP3 then activates PDK1.The activated m TORC2 and PDK1 phosphorylate AKT.The activated AKT regulates the proliferation ofβ-cells through a variety of ways:（1）AKT can phosphorylate and activate m TORC1,which in turn promotes cyclin expression andβcell proliferation.（2）AKT can phosphorylate and inactivation Foxo1,and then up-regulates the transcription of p27and inhibits the Cyclin/Cdk complex,thereby regulate cell proliferation.（3）AKT also can phosphorylate and activate CREB up-regulating the transcription of key CREB-dependentβ-cell genes（Ins1,Ins2,Pdx1,Irs2,cyclin D1）to promoteβ-cell proliferation and survival.Cholesterol sulfate（CS）is a kind of sterol sulfate with a structure similar to cholesterol,it is found in blood,urine,semen,bile,and other body fluids.CS is a constituent of a variety of human tissues including skin,adrenal,liver,and so on.CS has a variety of biological functions,and participates in the regulation of physiological reactions such as oxidative stress,inflammation,and immunity.Recent research found CS is closely related to glucolipid metabolism and diabetes-related phenotypes,such as:（1）the expression of SULT2B1b,an enzyme that synthesizes CS in the liver,of mice on a high-fat diet,obese mice,and mice that changed from a fasted state to a fed state significantly increased;（2）CS can significantly improve glucose tolerance and insulin sensitivity in DIO mice and ob/ob obese mice;（3）the insulin secretion and the number of pancreaticβ-cells in hyperlipidemia and obesity people increased significantly,and the concentration of CS in the blood also increased significantly;（4）the endogenous ligand of CS,RORα,can up-regulating the expression of insulin 1/2.In summary,the function and number of pancreaticβcells directly affect insulin secretion,which is closely related to the occurrence and development of diabetes.Mitochondrial dysfunction and oxidative stress are important factors that affect the function and number ofβcells.Therefore,focusing on improving mitochondrial function and reducing oxidative stress is still an important solution to protectβcell function.CS is closely related to glucolipid metabolism and diabetes-related phenotypes,it also has anti-oxidation and may have the function of promoting insulin synthesis and secretion,which suggests that CS may have a potential protective mechanism for pancreaticβcells that are susceptible to ROS damage.However,there is still a lack of research on the correlation between CS and pancreaticβ-cell function.Therefore,this study intends to construct a model of isletβ-cell dysfunction by STZ,and explore the effect of CS on the function and number of pancreaticβ-cells and its specific mechanisms,so as to lay a certain theoretical basis for whether CS can be further used in the treatment of diabetesObjective:Based on the destruction effect of STZ on pancreaticβ-cells,a diabetes model was constructed to explore the effects of CS on pancreaticβ-cell function（insulin synthesis and secretion）,survival and proliferation ofβ-cells,and clarify the mechanism of action.Materials and Methods:1.8-week male C57BL6J mice were injected intraperitoneally with DMSO solution,CS solution,CS+STZ solution,or STZ solution to establish mouse diabetes models and CS treatment mouse model.2.The LC-MS/MS method was used to detect plasma CS content in human and mice.3.Diabetes phenotype analysis of mice:The body weight and random blood glucose of the mice were continuously monitored,and after the 6th week of administration,the food intake,water intake,glucose tolerance（GTT）and blood lipids of the mice were measured.Small animal CT was used to detect the live fat volume of the mice.After the mice were sacrificed,the epididymis and subcutaneous white adipose tissue of the groin and the subscapular brown adipose tissue were weighed,and the size and weight of the mouse adipose cells were detected by HE staining.4.Use Immunofluorescence or immunohistochemistry for insulin and glucagon staining,and electron microscope to observed the ultrastructure of isletβ-cells.5.Isolate mouse pancreatic islets to detect the effect of CS on insulin secretion;study the effect of CS onβ-cell insulin secretion signaling pathway through the dynamic changes of calcium ion fluorescence.6.The expressions of Pdx1,Mafa,Ins1/2 were detected by Western blot or q PCR method in NIT-1 cells.7.In mouse pancreatic islets and NIT-1 cells,confocal was used to detect the effect of CS on the uptake of glucose analogues（2-NBDG）;in NIT-1 cells,Western blotting was used to detect the effect of CS on the expression of GLUT2.8.Detection of the effect of CS on the mitochondrial function of pancreatic islet cells:Use luciferase activity to detect the effect of CS on the ATP content of NIT-1 cells;use q PCR to detect the transcription of respiratory chain enzymes;use JC-1,fluorescent probes,and mitotracker to detect the influence of CS on cell membrane potential,ROS and mitochondria distribution and morphology after treatment with different oxidants.9.Detect the effect of CS on the proliferation,viability and apoptosis of pancreatic islet cells,and its molecular mechanism:CCK8 and cell flow cytometry were used to detect the influence of CS onβcell survival and apoptosis;the protein expression of AKT signaling pathway was detected by Western blot.Results:1.High blood glucose is accompanied by an increase in blood CS content.The plasma CS concentration of patients with hyperlipidemia and T2D was significantly higher than that of normal controls;The blood CS content of high-fat diet mice and STZ model mice was also significantly higher than that of control mice.2.CS can alleviate diabetes and related phenotypes caused by STZ destruction of mouse pancreatic isletβcells.CS can improve the symptoms of polydipsia,polyphagia,weight loss and body fat loss in STZ diabetic mice,and increase glucose tolerance,lower blood glucose and blood lipids.3.CS promotes insulin synthesis gene expression and insulin synthesis.The insulin content in the pancreatic islets of mice treated with CS increased significantly;CS treatment significantly increased the transcription level and protein expression of insulin synthesis genes Ins1/Ins2 and its key regulatory factors Pdx1 and Mafa in mouse pancreatic islets and NIT-1 cells.4.CS can alleviate the damage ofβ-cell mitochondria by STZ and promote insulin secretion.In mice and isolated pancreatic islets,CS can significantly promote glucose-stimulated insulin secretion,and CS can promote the release of Ca²⁺from pancreatic islet cells under the action of high glucose,but does not affect the release of Ca²⁺stimulated by high KCl.Mechanism studies have found that CS can promote the absorption of glucose analogue 2-NBDG in NIT-1 cells and pancreatic islets,promote the expression of GLUT2 in NIT-1 cells,and promote mitochondrial synthesis of ATP and the transcription of respiratory chain-related enzymes.CS can alleviate the infiltration of murineαcells induced by STZ.Transmission electron microscopy showed that CS can alleviate the damage of STZ toβ-cell mitochondria.5.CS inhibits cell apoptosis.CS can increase the activity of NIT-1 cells and inhibit the reduction of pancreatic islet and NIT-1 mitochondrial membrane potential caused by STZ,PA or H₂O₂,inhibit the production of cellular ROS,and inhibit cell apoptosis.6.CS can activateβ-cell AKT signaling pathway.CS promote the phosphorylation of AKT,CREB and m TOR,and promote the expression of Bcl-2.p-AKT inhibitors（MK）cannot inhibit the activation of CREB by CS and the expression of anti-apoptotic factor Bcl-2.Conclusion:1.CS can significantly improve the diabetic phenotype of mice with pancreaticβ-cell injury induced by STZ2.CS can promote the expression of Ins1/2,promote the synthesis of insulin.3.CS can alleviate the damage of STZ toβ-cell mitochondria and maintain the function of insulin secretion.4.CS activates CREB in an AKT-dependent or AKT-independent manner,up-regulates the expression of anti-apoptotic factor Bcl-2,promotes cell survival and growth,and inhibits pancreaticβ-cell apoptosis.