PACAP Glucose-dependently Stimulates Insulin Secretion In Rat Pancreatic ?-cells Via Relevant Mechanism

Objective:Pituitary adenylate cyclase-activating polypeptide?PACAP?is a neuropeptide that belongs to the secretin-glucagon superfamily.The present study aimed to explore the effect of PACAP and the underlying mechanism of this action in primary rat pancreatic?-cells.Methods:Islets and?-cells of Adult male Wistar rats were employed to the experiment.After rat was sacrificed,collagenase P was injected into the pancreas through the common bile duct for digesting pancreas.And scattered rat islet particles were obtained by Histopaque 1077 gradient centrifugation.Islet cells were separated from islets by Dispase?.?1?Insulin secretion test was used for probing the relationship between PACAP and insulin secretion in different environments.?2?Patch clamp technique was to explore the effect of PACAP on the action potential of islet?-cells.Voltage-dependent potassium channel currents and voltage-dependent calcium channel currents were recorded in whole-cell voltage clamp mode.?3?Calcium imaging technique was used to monitor the change of Ca²⁺concentration in?-cells under different interventions.Results:?1?PACAP dose-dependently regulated insulin secretion at 8.3 mmol/L glucose concentrations.And PACAP stimulated insulin secretion at high glucose concentrations?8.3 or 16.7 mmol/L?instead of at at low glucose?2.8 mmol/L?concentrations.?2?Whole-cell patch-clamp recordings illustrated that PACAP lengthened action potential duration and inhibited voltage-dependent potassium?Kv?channels.?3?Insulin secretion results indicated that PACAP stimulated insulin release not only by voltage-dependent potassium channels,but also by voltage-dependent Ca²⁺channels.And PACAP38 enhanced voltage-dependent Ca²⁺channel currents.?4?Calcium imaging techniques showed that PACAP increased intracellular Ca²⁺concentration,which could be mediated by voltage-dependent Ca²⁺channels.?5?The effect of PACAP on insulin secretion could be attenuated by PAC₁ receptor?PAC1-R?antagonist,but not VPAC₁ and VPAC₂ receptor antagonist.?6?The inhibitive effect of voltage-dependent potassium channels were antagonized by PAC₁ receptor blocker.?7?PACAP mediated insulin secretion via adenylate cyclase?AC?and protein kinase A?PKA?.Meanwhile,PACAP-inhibited voltage-dependent potassium channels were reversed by inhibitor of AC or PKA.?8?PACAP-increased voltage-dependent Ca²⁺channel currents and intracellular Ca²⁺concentration were weaken by PAC₁ receptor blocker.Conclusions:PACAP dose and glucose-dependently stimulates insulin secretion in rat pancreatic islets.Furthermore,PACAP has no effect on insulin secretion at low glucose concentrations,which suggests that PACAP could be potentially applied to treat type 2 diabetes mellitus without the risk of hypoglycemia.The main mechanism of PACAP-induced insulin secretion is that PACAP acts on PAC₁ receptor,stimulating AC and its downstream effector PKA,inhibiting Kv channels,prolonging action potential duration,potentiating opening time of voltage-dependent Ca²⁺channels,increasing intracellular Ca²⁺concentration and finally stimulating insulin secretion.PACAP also binds to PAC₁ receptor,enhancing voltage-dependent Ca²⁺channels currents,augmenting intracellular Ca²⁺concentration and ultimately stimulating insulin secretion.These findings provide a new opinion on the mechanism underlying PACAP-regulated insulin secretion.Moreover,our research demonstrates that the PAC₁ receptor could be a potential target for the therapeutic action of type 2 diabetes,which provides a useful clue to further investigate PAC₁ receptor agonists as a novel agent for the treatment of type 2 Diabetes.