The Effect Of Metabolic Stress On Pancreatic β-cell Function And The Mechanism Of Compensatory β-cell Hyperplasia

Background and Objectives Development of type 2 diabetes is associated with insulin resistance and pancreaticβ-cell dysfunction.Metabolic stress leads to insulin resistance and β-cell failure.Pancreatic β-cell can compensate for insulin resistance by increasing insulin secretion;however insufficient compensation leads to the onset of type 2 diabetes.Therefore,preventing progressive pancreatic β-cell dysfunction and increasing β-cell compensatory proliferation are very crucial factors for the treatment of type 2 diabetes.Excess caloric intake and obesity increase metabolic stress on β-cell,cause β-cell dysfunction and lead to the onset of type 2 diabetes diabetes.Caloric restriction can reduce metabolic stress and potentially prevent type 2 diabetes.However,the molecular mechanisms of protect β-cell function via caloric restriction have not been fully clarified.In the present study,we aim to explore whether caloric restriction protects the β-cell function in db/db mice.Furthermore,we investigate the mechanisms of compensatoryβ-cell hyperplasia under metabolic stress.Glucose is a crucial nutrient and can cause metabolic stress,which results in β-cell compensatory hyperplasia.Sfrp5 is an inhitior of WNT signaling that is involved in beta cell proliferation during obesity.However,the role of Sfrp5 in glucose-induced proliferation of pancreatic β-cells is still unknown.We seek to clarify whether Sfrp5 is involved in beta cell proliferation in response to glucose and to further explore the underlying molecular mechanisms both in vitro and in vivo with rat models of short-term glucose infusion.Understanding the underlying mechanisms ofβ-cell compensatory hyperplasia in metabolic stress will provide new insights for developing therapies to treat diabetes.Materials and Methods In the calorie restriction study,3 week old male C57 wild type mice(n=12)given free access to standard food were used as controls(wt-al).3 week old male db/db mice were divided into two groups with or without caloric restriction(n=12).Mice on calorie restriction were allowed to consume a meal amount which was adjusted to be equal in quantity to that of wild type mice.Body weight and food consumption were measured everyday,random plasma glucose was measured every other day,and fasting plasma glucose was measured every week.Intraperitoneal glucose tolerances test were performed at ages of 5,6,7,and 9 weeks.At the end of the study,serum was obtained to detect insulin levels.Pancreas insulin content was also measured.Pancreases from the 3 groups were harvested,fixed in 4% paraformaldehyde,embedded in paraffin,and consecutively sliced into 5μm sections.Morphometric analysis was performed by immunohistochemical staining.Immunostaining for insulin,Ki67,Glut2,Pdx-1,and Maf A was performed using immunofluorescence methods.To study Sfrp5 in mediating glucose induced β-cell proliferation,we used a rat model of short-term glucose infusion.INS-1 cells and primary rat islets were incubated in medium with different glucose levels and assessed for proliferation rate by Ed U incorporation.Sfrp5 m RNA and protein levels were also measured.Additionally,we overexpressed Sfrp5 by adenovirus in INS-1 cells and primary rat islets,measured glucose induced proliferation rate,and examined WNT/β-catenin signaling activity using luciferase gene reporter assay and western blot.We also measured the expression of cyclin D1 and cyclin D2.We further studied the mechanism of glucose induced downregulation of Sfrp5 expression by treating INS-1 cells with a PI3 K inhibitor and an AKT activator.Results1.Body weight,fasting plasma glucose,random plasma glucose,and glucose intolerance were significantly reduced in db/db mice on a caloric restriction diet as compared to db/db mice on an unrestricted diet.2.Pancreas insulin content was increased and the expression of Glut2,Pdx-1,and Maf A were increased in mice under calorie restriction.3.The number of β-cell with positive Ki-67 staining was increased in mice under calorie restriction.4.Short-term glucose infusion induced β-cell proliferation and reduced Sfrp5 expression.5.Ed U incorporation was further increased in INS-1 cells and in primary rat islets when incubated with 16.7m M glucose than with 5.6m M glucose,and Sfrp5 was downregulated.6.Overexpression of Sfrp5 reduced glucose-induced proliferation in INS-1 cells and primary islets,repressed WNT/β-catenin signaling and the expression of cyclin D2.7.Pancreatic β-cell proliferation associated with Sfrp5 downregulation is dependent on the PI3k/AKT signaling pathway.Conclusion1.Caloric restriction improves pancreatic β-cell function by reducing metabolic stress and upregulating Glut2,Pdx1,and Maf A expression.2.Downregulation of Sfrp5 is associated with compensatory β-cell hyperplasia by upregulating the expression of Cyclin D2.