The Neural Mechanism Of Selective Activation Of Kisspeptin Neurons In The ARC Of The Hypothalamus Regulate Bown Adipose Thermogenesis In Female Mice

Objectives:Overweight and obesity currently burden our public health,and especially with the aging process in China,the incidence of obesity is significantly higher in women than that in men partly because of the changes of hypothalamus-pituitary-sex glands axis.Kisspeptin neurons in the arcuate nucleus involved in the regulation of the HPG axis play an important role in the regulation of central energy metabolism.To explore the neural circuits of Kisspeptin neurons regulate brown fat thermogenesis and improve metabolism provides new molecular targets and drug research targets for the treatment of obesity and metabolic complications in female.Methods:1.Eight-week-old female KISS1-CreGFP mice were randomly divided into two groups and fed with adaptive high-fat diet,and addno assciated virus（AAV-DIO-hM3D（Gq）-mCherry）was injected into the bilateral ARC of hypothalamus.Three weeks later,daily injections of chlorazepine N-oxide（CNO）or saline（NS）,and the body weight of the mice was measured daily.After 2 weeks of continuous intraperitoneal injection,fasting was performed for intraperitoneal glucose tolerance test、Metabolic cage analysis.The mice were sacrificed the next day for sampling,including（1）rapid separation of brown adipose tissue（BAT）,subcutaneous white adipose tissue（sWAT）,perigonadal white adipose tissue（eWAT）and renal white adipose tissue（rWAT）,weighing statistics,pathology,IF,RT-PCR,WB,HPLC,etc.（2）Cerebral perfusion immunofluorescence,（3）adipose tissue sympathetic activity2.Female mice were randomly divided into four groups,a HHdaptability to high fat feed.Two clusters of sympathetic nerves in brown fat（iBAT）in the interscapular region mice were surgically denervated and 3 weeks postoperatively,by stereotaxic injecting adeno-associated virus vector of the ARC to kiss1-CreGFP mice,CNO activation kisspeptin^ARC neurons after 3 weeks,at the same time set up wild type control,sham control,saline control.Histological analysis of fat,sympathetic nerve and brain was performed as above.3.Three-dimensional brain reconstruction and immunofluorescence labeling experiments were used to construct the central neural network of Kisspeptin^ARCneurons regulating brown fat thermogenesis.Results:1.The hypothalamus of Kiss1-CreGFP mice was injected with AAV-DIO-HM3D（Gq）-mCherry virus in ARC.Three weeks later,AAV was successfully transfected and expressed the corresponding receptor.（1）Selective activation of Kisspeptin neurons in ARC resulted in decreased body weight,improved glucose metabolism,increased energy expenditure,increased BAT,decreased sWAT,eWAT、rWAT in female mice（P<0.05）;（2）Kisspeptin neurons of ARC were successfully transfected with AAV-DIO-hM3D-mCherry and were greatly activated by CNO,and the expression of neuronal activation index c-fos was significantly enhanced.（3）The norepinephrine（NE）concentration,sympathetic specific indicator tyrosine hydroxylase（TH）,the number of brown adipose cells and the expression of thermogenic related genes were significantly increased in female iBAT activated group（P<0.05）.2.In the Kissppetin neuron activation experiment after sympathetic nerve denervation,the SHAM operation group（SHAM）of beneficial effects of CNO activation on weight reduction,improved glucose metabolism and activation of brown fat thermogenesis still existed（P<0.05）.While the sympathetic denervation,the metabolic improvement effect of activating kisspeptin^ARC neurons disappeared,and female mice gained weight,impaired glucose metabolism,failed activation of brown fat,and significantly decreased thermogenesis（P>0.05）.3.Kisspeptin neurons in ARC of female mice had a large number of neural projection and synaptic connections with nuclei and neurons that regulate fat Browning and activate brown fat.Conclusions:1.Chemogenetics can relatively specifically activate Kisspeptin neurons in the ARC of the hypothalamus in female,and the activated Kisspeptin has an effect on the body’s energy metabolism,mainly weight loss,improved glucose metabolism,increased energy expenditure,increased thermogenic and dissipative,decreased white adipose tissue,increased sympathetic activity innervating iBAT,and significantly improved obesity.2.iBAT sympathetic denervation experiments confirmed that Kisspeptin ^ARC neurons regulate energy metabolism in female mice regulate iBAT activation through sympathetic nerve.3.Kisspeptin neurons in ARC have projection and synaptic connections with a large number of activated sympathetic regulatory iBAT thermogenic nuclei and neurons（POMC,NPYAg RP,DMH,VMH）,which may be an important neural mechanism to improve energy metabolism in female mice.