Electrophysiological Properties Of Pancreatic Islets And Hypothalamus In Response To Body Mebabolic Regulatory Factors

Endocrine and neuroendocrine cells are typical excitable cells in our body.Their main physiological function is releasing relevant hormones, neuro-hormones,or neurotransmitters.As excitable cells,membrane electrophysiological properties,such as cross-membrane potential and resistance,have been changed by stimulatory or inhibitory factors before hormone secretion is changed.It is therefore vital to clarify modification of ion channels and signaling systems by regulatory factors in these cells. Endocrine and neuroendocrine systems are under constant influence of body metabolic factors,such as levels of carbohydrates,amino acids,and fatty acids,and also under regulation by metabolic regulatory hormones, such as ghrelin,obestatin,leptin,adiponectin,etc.In order to clarify the regulatory mechanisms in these cells,current study selected a typical and important endocrine cell-pancreaticβcell,and a neuroendocrine cell-hypothalamic GHRH neuron as experimental cell models.Effect of metabolic factor,free fatty acids on beta cells have been investigated to search for endocrine cell function changing mechanism;and effects of key metabolic regulatory hormones,ghrelin and obestatin,on GHRH neurons to search for a better understanding of GH level control by metabolic regulatory hormones.This study is divided into three chapters.In the fist and second chapters,for further understanding of mechanisms underlying the effect of FFAs on pancreaticβ-cell functions,we focus on the clarification of effect of FFAs on ion channels in pancreaticβ-cells.In chapter 3,we investigated the effects of Ghrelin and Obestatin on glutamatergic and GABAergic synaptic responses recorded from mouse GHRH neurons expressing eGFP under the control of the GHRH promoter.Chapter 1 The effect of long chain FFAs on the voltage-gated K⁺ currents in rat pancreaticβ- CellsIn this study,we investigated the action of linoleic acid on voltage-gated K⁺ currents and voltage-dependent Ca²⁺ currents(VDCC). A long-chain unsaturated FFA,linoleic acid(10μM),reversibly decreased the amplitude of K⁺ currents(down to less than 10%).This reduction was significantly reduced by the treatment of GPR40-specific small interfering RNA,whereas the cAMP-induced reduction of K⁺ current was not affected.We conclude that linoleic acid reduces the voltage-gated K⁺ current in ratβ-cells through GPR40,leading to an increase in[Ca²⁺]_i and insulin secretion.Chapter 2 The effect of lioleic acid on the voltage-dependent Ca⁺ currents in rat pancreaticβ- CellsIn this chapter,we examined the effect of linoleic acid on the VDCC in primary cultured ratβ-cells.The VDCC was identified as high-voltage-gated Ca²⁺ channels due to there being no-difference in current amplitudes under holding potential of-70 mV or -40 mV. Linoleic acid(10μM) significantly inhibited VDCC currents inβ-cells, an effect which was fully reversible upon washout.Methyl-linoleic acid, which does not activate GPR40,neither did alter VDCC currents in ratβ-cells nor did influence linoleic acid-induced inhibition of VDCC currents.Linoleic acid-induced inhibition of VDCC currents was not blocked by pretreatment ofβ-cells with either the specific protein kinase A inhibitor,H89,or the PKC inhibitor,chelerythrine.However, pre-incubation ofβ-cells with thapsigargin,which depletes intracellular Ca²⁺ stores,completely abolished linoleic acid-induced decrease in VDCC currents.Measurement of intracellular Ca²⁺ concentration([Ca²⁺]_i) illustrated that linoleic acid induced an increase in[Ca²⁺]i and that thapsigargin pre-incubation inhibited this increase.Methyl-linoleic acid neither induced increase in[Ca²⁺]_i nor blocked linoleic acid-induced increase in[Ca²⁺]_i.These results suggest that linoleic acid stimulates Ca²⁺ release from intracellular Ca²⁺ stores and inhibits VDCC currents in rat pancreaticβ-cells via Ca²⁺ -induced inactivation of VDCC.Chapter 3 Glrenlin and Obestatin Modulate Glutamatergic and GABAergic Synaptic Inputs onto GHRH Neurons in eGFP-GHRH Transgenic MiceIn this study,we found Both Ghrelin and Obestatin had no effect on glutamatergic synaptic transmission in the recorded 16 and 19 neurons respectively.Ghrelin dramatically decreased GABAergic synaptic transmission in 53%of the recorded GHRH neurons:26 neurons displayed a significantly decreased GABA response during ghrelin application while the response was not modified in other 23 neurons.This effect was dose dependent between 10 nM and 1μM.Ghrelin-induced inhibition of GABAergic synaptic currents in GHRH neurons was abolished by pretreatment with the specific GHSR-1A inhibitor, BIM28163.Obestatin had no effect by itself on GABAergic synaptic transmission but it blocked Ghrelin-induced decrease in GABA responses in 75%of tested neurons.In 4 neurons,Ghrelin was applied first and inhibition on GABA response was obtained with full recovery after wash; the combination of the Ghrelin and Obestatin peptides were then applied showing a total abolishment of Ghrelin-induced inhibition on GABA responses.This result indicates that Obestatin-induced inhibition occurs in Ghrelin-responding cells.Effect of Obestatin on Ghrelin-induced inhibition was not due to desensitization of the GHS-R since,in another 4 neurons,two successive applications of ghrelin led to similar extend of decreases in the GABA responses.GHRH assay results showed that Ghrelin had no effect on basal GHRH release from hypothalamic explants but significantly increased the GHRH response to 28 mM KCl.In summary,the marked inhibitory effect of Ghrelin on GABAergic transmission is likely to result in an increased excitability of GHRH neurons by act on GHSR-1A,thereby driving an increased secretion of GH from pituitary somatotropes.In parallel,Obestatin counteracts Ghrelin action by directly antagonizing Ghrelin effects on GABAergic transmission on hypothalamic GHRH neurons.In present study,we first investigated the direct effect of FFAs on ion channels in pancreaticβcells by activating on the specific receptor, GPR40,which may provide new therapeutic targets for the treatment of type 2 diabetes.We also revealed the modulatory effects of Ghrelin and Obestatin on the neurotransmitter synaptic inputs to GHRH neurons in ARC nucleus,which contributed to the direct efference of Ghrelin on GHRH neurons,stimulating GH release.Last,the concentration of GHRH from static incubated mouse hypothalamic explants was at the first time detected by radio-immunoassay in incubation medium and showed an enhancement of secretion by ghrelin.