| Objective: The incidence rate of diabetes and its related complications has increased year by year and has become a serious health problem facing the whole world.Nesfatin-1 is a brain gut peptide with negative energy balance found in recent years.It can reduce blood sugar,inhibit food intake,reduce weight,slow heart rate,increase basic metabolism,etc.The level of plasma nesfatin-1 is related to body mass index(BMI),body weight and fat content.It is involved in the regulation of peripheral lipid accumulation and liver lipid metabolism.Peripheral administration of nesfatin-1 can reduce the plasma glucose level.It is suggested that nesfatin-1 may play an important role in the development of diabetes or metabolic syndrome.Is central nesfatin-1 involved in glucose or energy balance regulation? Is it involved in the regulation of food intake and energy metabolism in diabetic rats? What is the mechanism? It's still unclear.The purpose of this study was to compare and analyze the effects of microinjection of nesfatin-1 into ventral dorsal tegmental area(VTA)on food intake,excitability and energy metabolism of glucose sensitive neurons in normal and diabetic rats.To provide reliable experimental basis for the prevention and treatment of metabolic diseases such as diabetes and obesity.Methods: 1.To prepare the model of type I diabetic rats: SD rats were randomly selected(220-250 g),fasted for 12 hours,and injected with 65 mg / kg streptozotocin(STZ)intraperitoneally.72 hours later,the tail vein was taken and the fasting blood glucose was measured.The blood glucose was higher than 7.0 mmol / L and the postprandial blood glucose was higher than 11.1 mmol / L;2.The activity of VTA glucose(GS)sensitive neurons in normal or diabetic rats was observed by using single extracellular recording and microinjection of nesfatin-1,melanocortin 3 / 4 receptor antagonist SHU9119 or normal saline(NS);3.The food intake of normal and diabetic rats for 0-4 h was observed by VTA microinjection of nesfatin-1,SHU9119 or ns.In this part,28 normal and diabetic rats were randomly divided into 4 groups(n = 7):(1)NS group(0.5 ?l NS in VTA);(2)Nesfatin-1 group(0.5 ?l 0.5 ?g nesfatin-1 in VTA);(3)SHU9119 group(0.5 ?l 0.25 ?g SHU9119 in VTA);(4)Nesfatin-1 + SHU9119 group(0.5 ?l 0.5 ?g nesfatin-1 in VTA and 0.5 ?l 0.25 ?g SHU9119 in VTA).In the experiment of the above groups,the drug was slowly injected into VTA through a tube,and then put into a feeding measuring cage to measure the food intake of rats 0-4 h after administration.;4.VTA was microinjected with nesfatin-1,SHU9119 or ns to observe the effect on energy metabolism of normal and diabetic rats.In this part,28 normal and diabetic rats were randomly divided into 4 groups(n = 7):(1)NS group(0.5 ?l NS in VTA);(2)Nesfatin-1 group(0.5 ?l 0.5 ?g nesfatin-1 in VTA);(3)SHU9119 group(0.5 ?l 0.25 ?g SHU9119 in VTA);(4)Nesfatin-1 + SHU9119 group(0.5 ?l 0.5 ?g nesfatin-1 in VTA and 0.5 ?l 0.25 ?g SHU9119 in VTA).The drug was slowly injected into VTA through catheterization,and the heat production per hour,total energy consumption per hour,oxygen consumption per hour and CO2 production per hour were detected by the crams system.Results: 1.Effects of microinjection of nestin-1 by VTA on excitability of GS neurons in normal and diabetic rats:(1)88 spontaneous firing neurons were recorded in VTA of 30 normal rats,64 of which(64 / 88,72.73%)responded to glucose injection and were identified as GS responsive neurons.Of the 64 GS responsive neurons,35(35 / 64,54.69%)were identified as GS-E neurons with significantly increased firing frequency(4.67 ± 0.53 Hz vs.6.92 ± 1.03 Hz,P < 0.05),while 29(29 / 64,45.31%)were identified as GS-I neurons with significantly decreased firing frequency(4.98 ± 0.76 Hz vs.3.29 ± 0.44 Hz,P < 0.05).It is suggested that there are glucose regulated neurons in VTA;Among the 35 GS-E neurons,23 of them(23 / 35,65.71%)were significantly higher(P < 0.05,4.32 ± 0.75 Hz vs.7.99 ± 0.74 Hz),with an average increase of 84.95 ± 7.59 %If the VTA was microinjected with the melanocortin 3 / 4 receptor antagonist SHU9119,the excitatory effect induced by nesfatin-1 could be partially blocked(P < 0.05);but the firing activity of the VTA GS-E neurons did not change significantly(P > 0.05);Among the 29 GS-I neurons,17 of them(17 / 29,58.62%)were treated with VTA microinjection of nesfatin-1,and the discharge frequency was significantly reduced(P < 0.05,5.97 ± 1.23 Hz vs.3.06 ± 0.75 Hz),with an average decrease of 48.74 ± 2.19%(P < 0.05).The inhibitory effect of nesfatin-1 on GS-I neurons was partially blocked by the injection of SHU9119 by VTA(P < 0.05).There was no significant change in the firing activity of GS-E response neurons(P > 0.05).It is suggested that nesfatin-1 may be involved in the regulation of excitability of VTA GS-E or GS-I neurons through melanocortin 3 / 4 receptor pathway.(2)A total of 94 spontaneous firing neurons were recorded in VTA of 30 diabetic rats,67 of which(67 / 94,71.28%)responded to glucose and were identified as GS responsive neurons.Of the 67 GS responsive neurons,39(39 / 67,58.21%)were identified as GS-E neurons(3.96 ± 0.78 Hz vs.6.07 ± 0.97 Hz,P < 0.05),while 28(28 / 67,41.79%)were identified as GS-I neurons(4.23 ± 0.64 Hz vs.2.65 ± 0.31 Hz,P < 0.05).Among 39 GS-E neurons,26 of them(26 / 39,66.67%)discharge frequency increased significantly(P < 0.05,3.15 ± 0.69 Hz vs.6.24 ± 0.90 Hz),with an average increase of 98.10 ± 9.14%(P < 0.05);if VTA was injected with SHU9119 in advance,the excitatory effect induced by nesfatin-1 could be partially blocked(P < 0.05);if SHU9119 was injected alone,the discharge activity of VTA GS-E neurons had no significant change(P > 0.05).Among the 28 GS-I neurons,15(15 / 28,53.57%)of them were treated with VTA microinjection of nesfatin-1,and the discharge frequency was significantly reduced(P < 0.05,5.86 ± 0.78 Hz vs.2.35 ± 0.91 Hz),with an average decrease of 59.90 ± 3.09%(P < 0.05).The inhibitory effect of nesfatin-1 on GS-I neurons in diabetic rats was partially blocked by the injection of SHU9119 in advance by VTA(P < 0.05).There was no significant change in the firing activity of VTA GS-E neurons after injection of SHU9119 alone(P > 0.05).The results suggest that,similar to normal rats,nesfatin-1 can also participate in the regulation of excitability of VTA GS neurons in diabetic rats through melanocortin 3 / 4 receptor signaling pathway.(3)Compared with normal rats,the excitatory effect of nesfatin-1 on GS-E neurons in diabetic rats was significantly increased(85.14 ± 7.59% vs.98.10 ± 9.14%,P < 0.05),and the inhibitory effect of nesfatin-1 on GS-I neurons in diabetic rats was significantly stronger than that in normal rats(48.74 ± 2.19% vs.59.90 ± 3.09%,P < 0.05).It is suggested that nesfatin-1 is more sensitive to the regulation of glucose responsive neurons in the pathological state of diabetic glucose imbalance,and may play an important role in the pathogenesis of diabetes.2.Effect of microinjection of nesfatin-1 into VTA on food intake of normal and diabetic rats:(1)In normal rats,compared with NS group,the amount of food consumed per hour in 0-4h was significantly reduced after the VTA microinjection of nesfatin-1(P < 0.05).If VTA was injected with SHU9119,the inhibition effect of nesfatin-1 could be partially weakened(P < 0.05),that is to say,the inhibition effect of nesfatin-1 might be partly realized by activating the signal pathway of melanocortin 3 / 4 receptor.However,SHU9119 alone had no significant effect on Rats' food intake(P > 0.05);(2)In diabetic rats,compared with NS group,VTA microinjection of nesfatin-1 significantly reduced the food intake per hour for 0-4 hours(P < 0.05).If VTA was injected with SHU9119,the inhibitory effect of nesfatin-1 could be partially blocked(P < 0.05).However,SHU9119 alone had no significant effect on food intake(P > 0.05);(3)Compared with the normal or diabetic rats,the consumption per hour of 0-2 h in diabetic rats decreased significantly(P < 0.05).It is suggested that Nesfatin-1 may be more sensitive to the inhibition and regulation of food intake under the condition of blood glucose imbalance.3.The effect of VTA microinjection of nesfatin-1 on energy metabolism in normal and diabetic rats:(1)In the normal rats,compared with the NS group,the amount of heat production per hour(P < 0.05),the total energy consumption per hour(P < 0.05),the oxygen consumption per hour(VO2/ml/Kg/h,P<0.05)and the amount of CO2 production per hour(VCO2/ml/Kg/h,P<0.05)in the VTA microinjection group were significantly increased;if the VTA was injected with SHU9119 in advance,the effect of promoting energy consumption in the VTA microinjection group partially disappeared(P < 0.05),That is to say,the activation of melanocortin 3 / 4 receptor signal may be involved in the process of promoting energy consumption by nesfatin-1.There was no significant effect on the energy consumption of normal rats(P > 0.05);(2)In diabetic rats,the energy production per hour(P < 0.05),total energy consumption per hour(P < 0.05),oxygen consumption per hour(VO2 / ml / kg / h,P < 0.05)and CO2 production per hour(VCO2 / ml / kg / h,P < 0.05)were significantly higher than those in NS group when VTA was microinjected with SHU9119;if VTA was pre injected with SHU9119,the energy consumption promoting effect of nesfatin-1 could be weakened(P < 0.05).However,there was no significant effect on the energy consumption of the rats by injection of SHU9119 alone(P > 0.05);(3)Compared with the normal and diabetic rats,the energy metabolism of the above-mentioned nesfatin-1 group was more significantly increased by VTA microinjection of nesfatin-1,the caloric production per hour(P < 0.05),total energy consumption per hour(P < 0.05),oxygen consumption per hour(P < 0.05),and CO2 production per hour(P < 0.05).It is suggested that nesfatin-1 may be more sensitive to the regulation of energy metabolism in diabetic rats.Conclusions: VTA nesfatin-1 is involved in the regulation of the excitability of VTA glucose responsive neurons.VTA nesfatin-1 can inhibit food intake and promote energy consumption,which may be related to the activation of melanocortin 3 / 4 receptor pathway.Nesfatin-1 is more effective in the above regulation of diabetic rats.This study can provide a new research strategy for the prevention and treatment of diabetes and energy metabolism disorders,such as obesity. |
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