The Role And Mechanism Of DHA Glucose-dependently Stimulating Insulin Secretion In Rat

Objective:Docosahexaenoic acid（DHA）is an essential polyunsaturated fatty acid in human body,and it is abundant in fish oil.Previous studies have shown that DHA can regulate glucose metabolism,and this study focuses on the effect of DHA on insulin secretion in rat pancreatic islets and its mechanism of action in pancreaticβ-cells,in order to further study the specific regulatory mechanism of insulin secretion and find potential targets for the treatment and prevention of diabetes.Methods:Normal male Wistar rat pancreatic islet tissue and primary isletβ-cells were used as the main study subjects.After acute execution of the rats,a concentration of 1 mg/m L of collagenase P was injected into the pancreas via the common bile duct,followed by rapid isolation and digestion at 55 rpm/min for 16 min on a thermostatic shaker at 37°C.Digestion was terminated with pre-cooled complete medium.Rat islet tissue was obtained by gradient centrifugation using Histopaque 1077 and then digested into islet cells using Dispase II.（1）Insulin secretion experiment was used to investigate the effect of DHA on insulin secretion in rat islets and INS-1 cells under different conditions.（2）CCK-8 was used to detect the effect of different concentrations of DHA on INS-1 cells viability.ELISA was used to detect the effect of DHA on rat islets intracellular c AMP content under different conditions.（3）The effect of DHA on the action potential of pancreaticβ-cells was examined in current-clamp mode using the patch-clamp technique;the effects of DHA on voltage-dependent potassium（Kv）channel currents and voltage-dependent Ca2+（VDCC）channel currents in pancreaticβ-cells were investigated in whole-cell voltage-clamp mode under different conditions;patch clamp technique was also used to explore the effect of DHA on the Kv channel current of CHO-Kv2.1 cell in whole-cell voltage clamp mode.（4）To monitor the changes in free Ca²⁺concentration in pancreatic isletβ-cells under different intervention factors using a calcium imaging system.Result:（1）DHA had no effect on insulin secretion in rat islets under low glucose（2.8 m M）conditions,but promoted insulin secretion in a concentration-dependent manner under high glucose（11.1 m M）,and the different concentrations of DHA（1-100μM）used in the experiments had no effect on INS-1 cells viability.（2）The results of electrophysiological experiments showed that DHA significantly prolongedβ-cell action potential duration time and had no effect on VDCC current,but could significantly reduce Kv current.（3）The results of calcium imaging showed that DHA could lead to an increase in intracellular Ca²⁺concentration.（4）Application of DC260126（the GPR40 receptor blocker）revealed that GPR40 was involved in DHA-stimulated insulin secretion,prolongation ofβ-cell action potential duration time,elevation of intracellular free calcium ion concentration,and inhibition of Kv channel current.（5）The specific adenylyl cyclase inhibitor（AC）inhibitor SQ22536,the phospholipase C（PLC）inhibitor U73122 and the GPR40 receptor blocker DC260126can all inhibited the effect of DHA on increasing c AMP levels in rat pancreatic islet cells.（6）Both SQ22536 and U73122 can significantly attenuated the insulin secretion of rat pancreatic islets and INS-1 cells by DHA and the inhibitory effect of DHA on the Kv current of pancreaticβcells.Conclusion:DHA dose-dependently promotes insulin secretion from rat islets under high glucose conditions,but does not affect insulin secretion under low glucose conditions,so there is no risk of hypoglycemia.The main mechanism by which DHA promotes insulin secretion is that DHA acts on GPR40 receptors,activates AC to increase c AMP levels and stimulates PLC,which in turn inhibits Kv channels,and inhibition ofβ-cell Kv channels results in a prolongation of the action potential time course in pancreaticβ-cells,which increases the concentration of free Ca²⁺inβ-cells and ultimately leads to an increase in cellular insulin secretion.