Effects And Mechanisms Of CASK-specific Deletion In Pancreatic Beta Cells On Glucose Homeostasis And And Islet Function In Mice

Part Ⅰ Effects and mechanism of CASK-specific deletion in pancreaticβ-cells on glucose homeostasis and β-cell function in normal diet-fed miceBackground: The dysfunction of pancreatic islet β-cells is central to the development and progression of type 2 diabetes mellitus(T2DM).Previous studies have found that calcium/calmodulin-dependent serine protein kinase CASK participates in insulin secretion in rat insulinoma INS-1 cells.Cask knockdown in INS-1 cells reduces insulin release,possibly through effecting on the anchoring process of insulin vesicles onto the β-cell membrane.However,the exact role of CASK on islet β-cell function and glucose homeostasis in vivo has not been elucidated.Objectives: To investigate the effect of CASK on glucose homeostasis and islet β-cell function in mice fed a normal dietMethods: The Cre-lox P recombination system was used to specifically knock out the Cask gene in mouse pancreatic β cells(βCASKKO).The knockdown efficiency of CASK was determined by Western blot,q RT-PCR and immunofluorescence staining.The mice were fed with a normal diet until 16 weeks of age.The body weight of the mice was monitored weekly,and the fasting and postprandial blood glucose of the mice were measured every two weeks.Mice were performed intraperitoneal glucose tolerance test(IPGTT)at 16 weeks of age,intraperitoneal insulin tolerance test(IPITT)was performed three days after the experiment,and in vivo glucose-stimulated insulin secretion test(GSIS)was performed one week later;The mouse islets were extracted and cultured in vitro,and the glucose-stimulated insulin secretion and islet perfusion experiments were performed in vitro.Histomorphological changes of mouse pancreas were detected by HE staining and immunofluorescence staining.The morphology,size and anchoring of insulin vesicles in mouse pancreatic islets were detected by transmission electron microscopy.Results: 1.CASK was effectively knocked out in Pancreatic β-Cells.The m RNA quantification showed that the Cask level was 60% lower in islets from βCASKKO than from WT mice.Western blotting and immunofluorescence staining further confirmed a significant reduction in CASK expression in β-cells but no distinct changes in other tissues.2.The body weight,fasting blood glucose and postprandial blood glucose of βCASKKO mice were not significantly different from those of the littermate control mice.3.At age 16 weeks,higher blood glucose levels in the βCASKKO mice were found at 15 min after glucose injection than in other groups.In IPITT,no significant difference was detected among the groups in blood glucose Levels.GSIS tests performed on 16-week-old mice revealed lower insulin-releasing levels in βCASKKO mice in comparison with other groups.4.GSIS tests performed on pancreatic islets isolated from the mice revealed lower insulin secretion by βCASKKO islets than by other groups.5.The islet perfusion assays of dynamic insulin secretion revealed a typical biphasic response of insulin secretion in the pancreatic islets from both mouse groups following high glucose stimulation.Noticeably,insulin secretion was significantly inhibited in the βCASKKO islets.High potassium-stimulated insulin secretion was also decreased inβCASKKO islets compared with those of other groups.6.q RT-PCR revealed that CASK knockout did not affect the expression of genes involving insulin synthesis or exocytosis.Histological analysis and islet-specific staining did not reveal any difference in islet shape,size,cell arrangements and compositions between βCASKKO and WT mice.7.Electron microscopy examination further confirmed no significant alterations in morphology,size,or number of insulin dense-core vesicles in βCASKKO versus WT islets.However,the number of vesicles at or near the cell membrane was significantly lower in the βCASKKO islets.Conclusion: Under normal diet feeding,knockout of CASK in mouse pancreatic isletβ-cells appears to interfere with the transport or anchoring of insulin granules to the β-cell membrane,thereby inhibiting glucose-stimulated insulin secretion and increasing blood glucose after glucose challenge.Part Ⅱ Effects and mechanism of CASK-specific deletion in pancreatic β cells on glucose homeostasis in high-fat diet-fed miceBackground: Type 2 diabetes is an expanding global health problem closely linked to the obesity epidemic.Previous studies found that CASK is also involved in glucotoxicity-,palmitate-,or inflammatory cytokine–induced impairment in β-cells.These in vitro results reveal that CASK not only plays a crucial role in the physiological insulin secretion of β-cells,but also possibly contributes to β-cell injury during the development of T2 DM.Therefore,clarifying the role of CASK on islet β-cell function and glucose homeostasis during the development of T2 DM may provide new therapeutic targets for T2 DM.Objective: To investigate the effect of CASK on glucose homeostasis and islet β-cell function in high-fat diet-fed miceMethods: WT and βCASKKO mice were fed a high-fat diet(HFD)or normal diet(ND)at 10 weeks of age,and their body weights were monitored weekly.Western blot and q RT-PCR to detect the expression of CASK in pancreatic islets of ND-WT and HFD-WT mice.Fasting and postprandial blood glucose,fasting serum insulin,IPGTT,IPITT and GSIS were measured of mice fed an HFD for either 8 or 16 weeks.The mouse islets were extracted and cultured and GSIS was carried out in vitro.Immunohistochemical staining and quantitative β-cell analysis were performed to calculate β-cell mass.The hyperinsulinemic-euglycemic clamp assays were performed after 16-week feeding of HFD.Subsequent Western blots of liver,skeletal muscle,and epididymal adipose tissues removed immediately after completion of the clamp assays to measure the degree of AKT phosphorylation as an insulin sensitivity assessment.HE staining was used to detect the morphological changes of liver,muscle and adipose tissue.The insulin target tissue was extracted 15 minutes after the mice were injected intraperitoneally with insulin.Western blot was used to detect the expressions of insulin signaling pathway-related proteins in the liver,muscle and adipose tissue of mice.Results: 1.Mice fed the HFD for 16 weeks had significantly increased body weight in comparison with ND-fed mice;as in ND-fed mice,βCASKKO did not alter the body weight of HFD-fed animals.2.Immunoblotting and q RT-PCR analysis confirmed CASK expression was higher in the islets from HFD-fed WT mice than those from ND-fed WT mice.3.After 8 weeks of HFD feeding,the mice showed fasting hyperglycemia,and hyperinsulinemia,as well as impaired glucose tolerance and systemic insulin resistance.Simultaneously,HFD-WT mice and HFD-βCASKKO mice did not show any significant differences in glucose homeostasis.4.Strikingly,after consuming the HFD for 16 weeks,HFD-βCASKKO mice showed reduced fasting blood glucose and plasma insulin levels compared with HFD-WT mice.Meanwhile,the HFD-βCASKKO mice showed a significant improvement in glucose tolerance and insulin sensitivity.5.In response to glucose challenge,the HFD-βCASKKO mice secreted lower levels of insulin compared with HFD-WT mice.In vitro GSIS tests performed in islets from HFD-fed mice also showed lower insulin secretion by islets from HFD-βCASKKO mice than by those from HFD-WT mice.6.Insulin-stained sections and quantitative β-cell mass indicated the compensatory increase of islet β-cell mass in HFD-fed βCASKKO mice was lower compared with that in HFD-fed WT mice.7.The hyperinsulinemic-euglycemic clamp assays suggested that the glucose infusion rate showed significant increases in the HFD-βCASKKO mice relative to the HFD-WT mice during the clamp.Subsequent Western blots of liver,skeletal muscle,and epididymal adipose tissues showed that although no difference was observed in AKT phosphorylation in the liver or skeletal muscle,AKT phosphorylation in adipose tissue of HFD-βCASKKO mice increased significantly compared with HFD-WT mice.8.Hematoxylin-eosin staining of insulin target tissue sections from HFD-fed mice showed lipid deposition in the liver and a significant enlargement of the sarcolemma space and the epididymal adipocyte size.9.The insulin target tissue was extracted 15 minutes after the mice were injected intraperitoneally with insulin.Western blot further showed that AKT phosphorylation in the liver and skeletal muscle showed no difference within the HFD group,but in the adipose tissue,it was higher in HFD-βCASKKO mice than in HFD-WT mice.Further research found the phosphorylation of IRS1 serine residue,m TOR,and p70S6K1 was also significantly lower in adipose tissue from HFD-fed βCASKKO mice than from HFD-fed WT mice.Conclusion: Under high-fat diet feeding,selective knockout of the Cask gene in pancreatic β-cells in mice alleviated hyperinsulinemia in obese mice by reducing insulin secretion,thereby improving hyperglycemia,glucose intolerance and insulin resistance in obese mice.Further studies suggest that the IRS1/PI3K/AKT signaling pathway was upregulated in the adipose tissue of HFD-βCASKKO mice.These findings provide new evidence for CASK as a potential target for the treatment of T2 DM.