| Objective:Brain gut peptide plays an important role in body weight regulation.Leptin is known to be involved in regulating energy balance and inhibiting food intake,resulting in weight loss,which plays an important role in the development of obesity in rats.As a feedback mechanism,the leptin signaling pathway regulates food intake,body weight,and energy homeostasis by signaling to the central nervous system.The aim of this study was to investigate the effects of leptin116-130 on feeding and energy metabolism in rats and its underlying mechanisms.Methods:1.Expression of leptin receptor immunoreactive neurons in the hypothalamic ventromedial nucleus?VMH?:Five rats were randomly selected and western blot and immunohistochemical staining techniques were used to observe the expression of VMH leptin immunoreactive neurons in rats;2.Effect of microinjection of leptin116-130 on the expression of mRNA encoding metabolic related hormones:10 rats were randomly divided into NS control group?n=5?and VMH leptin116-130 group?n=5?,using Real time-PCR method,injection of leptin116-130into VMH and observe the changes of mRNA expression of the encoding metabolic related hormones,such as adiponectin,resistin,ghrelin,cholecystokinin?CCK?,tyrosin?PYY?,uncoupling protein 1?UCP-1?,insulin and glucagon;3.Leptin116-130 response neurons in VMH:25 rats were randomly selected and recorded by single cell discharge to observe the effect of VMH on neuronal discharge activity of leptin116-130 response;4.Effects of microinjection of leptin116-130 on the feeding and energy metabolism of rats:12 rats were randomly divided into NS control group?n=6?and VMH leptin116-130 group?n=6?.Using the Comprehensive Experimental Animal Monitoring System?CLAMS?,microinjection of leptin116-130 into the VMH,to observe the effect of the food?12 h?and water intake?12 h?,oxygen consumption?VO2?,CO2 production?VCO2?,respiratory exchange rate?RER?,heat production?CV?and total energy consumption?EE?;5.Effects of microinjection of leptin116-130 on the spontaneous activity of rats:12 rats were randomly divided into NS control group?n=6?and VMH leptin116-130 group?n=6?.Microinjection of leptin116-130 into the VMH,to observe the effect on day and night spontaneous activity?vertical activity,horizontal activity and total activity?in rats.Results1.Western blot and immunohistochemistry showed that the expression of leptin receptor protein and leptin receptor immunopositive neurons in VMH;2.The results of VMH microinjection of leptin116-130 on the expression of mRNA encoding metabolic related hormones show that,microinjection of leptin116-130 into the VMH,the expression of adiponectin mRNA?P<0.05?and resistin mRNA?P<0.05?in rat white adipose tissue?WAT,epididymis?,uncoupling protein 1?UCP-1?mRNA?P<0.01?in brown adipose tissue?BAT,subcutaneous scapula?,ghrelin mRNA?P<0.05?and in gastric mucosal tissue and cholecystokinin?CCK?mRNA?P<0.05?in small intestinal mucosa were significantly up-regulated,while the expression of PYY mRNA?P<0.05?in the colorectal mucosa is down-regulated,and in the leptin116-130 injection group,the expression of UCP1 mRNA in brown adipose tissue was 6 times higher than that in the NS group?P<0.05?.Compare with NS group,there was no significant difference in pancreatic insulin mRNA and glucagon mRNA expression between the leptin116-130injection group and the NS group?P>0.05?;3.Electrophysiological studies further found that VMH microinjection of leptin116-130,85.71%of neurons responded to leptin116-130,identified as leptin116-130 responsive neurons(LR116-130-N),48.57%of the neurons were leptin116-130 excitatory neurons(LR116-130-E),and the frequency of discharge increased from 5.23±1.67 Hz to 9.17±2.14 Hz?P<0.05?,with an average increase of 75.33±12.33%;37.12%of neurons For leptin116-130 inhibitory neurons(LR116-130-I),the discharge frequency decreased from 5.14±0.89 Hz to 3.13±0.69Hz?P<0.05?,with an average decrease of 39.11%,suggesting that VMH has leptin116-130responsive neurons.If pre-injected the recombinant rat leptin triploid antagonist?r Leptin tA?into VMH,the excitation/inhibition effect of leptin116-130 on LR116-130-N can be completely blocked;but injection of the NS or rLeptin tA alone had no significant effect on leptin116-130 responsive neuronal firing activity?P>0.05?;4.Microinjection of leptin116-130 into the VMH of rats,the feeding and drinking water results showed that compared with NS control group,during light period?8:00 Am?or dark period?20:00 Pm?,microinjection of leptin116-130 into the VMH,the cumulative food intake of rats in 12h?8:00Am-20:00Pm;20:00Pm-8:00Am?was significantly reduced?P<0.05?.Compared with the NS group in the dark phase,there was no significant difference in the food intake of the leptin116-130 group during the light period?P<0.05?.However,compared with the dark-stage leptin116-130 group,the anti-feeding effect of the leptin116-130 group in the light period was significantly weaker than that in the dark stage leptin116-130 group?P<0.05?.Compared with the NS control group,microinjection of leptin116-130 into the VMH,there was no significant difference in the water intake in the dark or light period?P>0.05?.Compared with the dark-phase NS group,microinjection of leptin116-130 into the VMH during the light period,the water intake of the rats was significantly decrease?P<0.05?,compared with the dark phase leptin116-130 group,microinjection of leptin116-130 during the light period,the water intake of rats was also significantly decrease?P<0.05?;5.The effect of VMH microinjection of leptin116-130 on energy metabolism in rats showed that in the dark or light phase,compared with the NS control group,VMH microinjection of leptin116-130,rats produce calories per hour?P<0.05?,hourly total energy consumption?P<0.05?and fatty acid oxidation rate per hour?P<0.05?were significantly increased,but there was no significant difference between the NS control group or the medication group in the dark or light period?P>0.05?;In the dark or light phase,compared with the NS control group,VMH Microinjection of leptin116-130 showed no significant change in the rate of carbohydrate oxidation per hour in rats?P>0.05?,and there was no significant difference between the NS control groups or the medication groups,whether in the dark or light period?P>0.05?.Compared with the NS control group,in the dark or light phase,VMH microinjection of leptin116-130,rat hourly oxygen consumption,or hourly CO2 production increased significantly?P<0.05?,and whether in the dark or light period,NS There was no significant difference between the control groups or the medication groups?P>0.05?.6.The effect of VMH microinjection of leptin116-130 on spontaneous activity in rats showed that in the dark phase,compared with NS group,VMH microinjection of leptin116-130,spontaneous activity in the horizontal axis direction?X-axis direction?of rats?P<0.05?,the spontaneous activity in the vertical axis direction?Y-axis direction??P<0.05?,the spontaneous activity in the vertical axis direction?Z-axis direction??P<0.05?,and the total spontaneous activity?P<0.05?were significantly increased,but there was no significant change in the illumination period compared with the NS group?P>0.05?.Further statistical analysis showed that compared with the dark period,the NS group or the photoperiod leptin116-130 group in the photoperiod period.Spontaneous activity in the horizontal axis direction?X-axis direction??P<0.05?,spontaneous activity in the vertical axis direction?Y-axis direction??P<0.05?,spontaneous activity in the vertical axis direction?Z-axis direction??P<0.05?,and total Spontaneous activities are significantly reduced.ConclusionsThere is a leptin116-130 action site in VMH;leptin116-130 can participate in the regulation of body energy balance by reducing food intake and increasing limb movement in rats.The feeding-related peptides Ghrelin,CCK,PYY and UCP-1 may also be involved in the regulation of this process.Leptin116-130 can be used as a novel metabolic peptide in rats. |
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