The Effects And Mechanisms Of Roflumilast On Insulin Secretion And Cell Survival In Pancreatic β Cells

Objectives:Roflumilast,an inhibitor of phosphodiesterase-4,inhibits the degradation of intracellular cAMP and increases the content of intracellular cAMP,and plays multiple physiology functions.Recent studies had been reported that roflumilast decreased the levels of fasting blood glucose and glycosylated hemoglobin in COPD patients with diabetes,and reduced body weight,which can improve glucose homeostasis in vivo.Other researches suggested that roflumilast promoted insulin secretion by increasing plasma GLP-1.Considering that pancreaticβ-cell possesses electrical excitability,and intracellular cAMP concentration also affects the insulin secretion of pancreaticβcells.Therefore,in this study,we mainly investigated the effect of roflumilast on the insulin secretion ofβ-cell and the underlying electrophysiological mechanisms,as well as on the survival of pancreaticisletβcells,and further explored the possible related cellular signal transduction mechanisms of these effects.Methods:1.Isolation and culture of rat pancreaticislets andβcells.After the rat pancreas was isolated,rat pancreas was digested by collagenase P at 37℃.Rat pancreaticislets were obtained after density gradient centrifugation with Histopaque-1077,and were further separated into pancreaticβcells for subsequent experiments.2.To investigate the effect of roflumilast on insulin secretion in vitro,insulin secretion experiment was used to explore the effects of glucose and roflumilast with different concentrations on insulin secretion.3.The whole-cell patch clamp technique was used to explore the effects of roflumilast and specificblockers of signal pathway on action potential duration,voltage-dependent potassium channel and voltage-dependent calcium channel in pancreaticβcells.4.After applying specificfluorescent dye,Fluo4-AM,microplate reader was used to observe the effects of roflumilast on the fluorescence intensity ofβcells,which indicated the changes of the intracellular calcium ion concentration.5.CCK-8 method was used to detect the effect and possible mechanism of roflumilast on the cell viability of INS-1 cells and CHO-Kv2.1 cells induced by high concentration glucose and palmiticacid.6.The C57BL/6 mice and db/db diabeticmice were used to investigate the effects of roflumilast on insulin secretion and voltage-dependent potassium channels under in vivo and unusual high glucose condition,respectively.Results:1.Roflumilast（100nmol/L,200nmol/L,300nmol/L）had no significant effect on insulin secretion under low glucose（2.8 mmol/L）condition,but at higher glucose（8.3 mmol/L）level,roflumilast（200nmol/L,300nmol/L）significantly promoted insulin secretion in rat islets,indicating that roflumilast stimulated insulin secretion with a dose-dependent manner in rat islets.2.Under low glucose（2.8 mmol/L）condition,roflumilast（200nmol/L）had no effect on insulin secretion,but at higher glucose levels（8.3mmol/L and 16.7mmol/L）,roflumilast（200nmol/L）significantly prompted insulin secretion in rat islets,indicating that roflumilast increased insulin secretion of rat islets in a glucose concentration-dependent manner.3.Roflumilast inhibited the voltage-dependent potassium（Kv）channel currents and prolonged the action potential durations of pancreaticβcells.4.Roflumilast increased intracellular calcium ion concentration inβcells,but had no effect on voltage-dependent calcium channels ofβcells.5.Kv channels play an important role in insulin secretion induced by roflumilast from rat islets.6.Roflumilast had no direct inhibitory effect on the Kv channels of CHO-Kv2.1 cells using the whole-cell patch clamp method.7.Roflumilast inhibited the Kv channels currents and stimulated insulin secretion ofβcells through the AC/cAMP/Epacsignaling pathway.8.Roflumilast increased the intracellular calcium concentration via promoting extracellular Ca²⁺influx by inhibiting the Kv channels and prolonging the action potential durations.9.Roflumilast improved the cell viability of INS-1 cells induced by high concentration glucose and palmiticacid,and exerted protective effect on INS-1 cells.10.Roflumilast improved the cell viability of INS-1 cells induced by high concentration glucose and palmiticacid or by thapsigargin via inhibiting Kv channels,and exerted protective effect on INS-1 cells.11.Roflumilast did not improve the cell viability of CHO-Kv2.1 cells induced by high concentration glucose and palmiticacid,and had no protective effect on CHO-Kv2.1cells.12.Roflumilast increased the plasm insulin secretion level,and further decreased the blood glucose level in C57BL/6 mice.13.Roflumilast inhibited voltage-dependent potassium channels ofβcells,and promoted insulin secretion of islets in db/db diabeticmice.Conclusions:1.Roflumilast promoted insulin secretion with a dose-and glucose-dependent manner in rats islets.2.Roflumilast inhibited the Kv channels ofβcells through the AC/cAMP/Epacsignaling pathway,and ultimately stimulated insulin secretion.3.Roflumilast prolonged the action potential durations,increased the intracellular calcium concentration,and ultimately promoted insulin secretion ofβcells by inhibiting the voltage-dependent potassium channels rather than directly affecting the voltage-dependent calcium channels.4.Roflumilast improved the cell viability of INS-1 cells induced by high concentration glucose and palmiticacid or thapsigargin,and had protective effect on isletβcells.5.Roflumilast improved glucose metabolism in vivo by increasing insulin secretion in C57BL/6 mice,and by augmenting insulin secretion fromβcells in db/db diabeticmice.