The Metabolic End And Associated Molecular Mechanisms Of Hypothalamic Endoplasmic Reticulum Stress In Offspring Mice Born To Obese Dams During The Postnatal Development

Objective: To investigate metabolic effect of early activated hypothalamic ER stress on offspring exposed to maternal obesogenic environment and the underlying mechanism.Methods: In vivo study,we established pre-pregnant maternal obese model with 5-week-old ICR female mice fed with high-fat high sucrose(HFHS)diet for 5 weeks,while those fed with low fat diet(LFD)as healthy control.Body weight(BW)gain was examined weekly,then female mice from each group were bred with male of the same age.Diets of each group were maintained with the same one of pre-pregnant during pregnancy.On the day of delivery,number of each litter was adjusted to 10.The pups from each group were randomly selected for the postnatal(P)4–16 daily intervention,receiving either phosphate buffer saline(PBS)or4-phenylbutyric acid(4-PBA)through intraperitoneal injection.The daily BW of the pups was recorded from P4 to 21.At weaning,only male offspring of each group were selected to given LFD or HFHS until the age of 10 weeks,respectively.During this period,BW of each group was measured weekly.Glucose tolerance test,fasting-refeeding test and norepinephrine thermogenesis test were performed to determine the changes in glucose tolerance,feeding behavior and thermogenic function of adipose tissue.Besides,immunofluorescence staining was conducted to determine the neuropeptidergic axon projection in paraventricular nucleus of hypothalamus(PVH)of the adult offspring.Hematoxylin-eosin staining was performed to assess morphological feature of adipose tissue.Furthermore,we determined the levels of effectors mediated by unfolded protein response(UPR),heat shock response(HSR)and leptin signal in offspring hypothalamus at weaning and adult with western blot(WB).At the same time,hypothalamic RNA extracted from the weaning offspring were used to perform transcriptomic sequence.R package cluster Profiler were used for the downstream data analysis.In vitro study,palmitic acid(PA)and thapsigargin(Tg)were used for establishing ER stress model in GT1-7 cell.Real-time polymerase chain reaction(RT-PCR)and WB were used to measure the levels of key effectors in the UPR and HSR,with the optimal concentration of 4-PBA for relieving ER stress screened.The expression of key molecules regulating leptin signal was assessed through leptin sensitivity test in the ER stress model.Furthermore,Heat shock protein70(HSP70)knockdown model was constructed by transfecting the sh-Hspa1 b lentivirus,and the knockdown efficiency was confirmed by RTq-PCR and WB.Then the above experiments were performed again in the knock-down model.Results: Maternal obesity significantly increased the BW gain of the offspring both in the early postnatal and post-weaning environment,especially exposed to HFHS.Offspring born to obese dams exhibited hyperphagia and impaired adipose thermogenesis.Further,defective neuropeptidergic axon projections in the PVH were markedly presented in those born to obese dams.There was significant hypothalamic ER stress in the weaning offspring born to obese dams,accompanied with occluded leptin signal transduction.Besides,the activated hypothalamic ER stress persisted into adulthood in both the LFD and HFHS group.Surprisingly,those who received early postnatal 4-PBA intervention showed more severe BW gain and glucose intolerance,whereas the structural effects of axon projection in PVH were more severe.Although early 4-PBA intervention could inhibit hypothalamic ER stress during the early postnatal period,the inhibitory effect was temporary,and failed to permanently inhibit the further activation of hypothalamic ER stress in the adult offspring.We found that the expression of HSP70,a member of the HSP family regulating HSR,was significantly up-regulated in the weaning hypothalamus of those born to obese dams,while early 4-PBA intervention significantly inhibited its expression and even persisted into adulthood.In subsequent in vitro experiments,we established an ER stress model induced by PA or Tg in the hypothalamic leptin receptor expressing cell,GT1-7.We confirmed the HSP70 knock-down efficiency at m RNA and protein levels.We found the stress response both from HSR and UPR was significantly inhibited once knock down HSP70 in the PA indued ER stress model.Further,we detected that the level of UPR effector was significantly increased in 4-PBA pretreated HSP70 knock-down cells when exposed to PA or Tg,resembling the postnatal 4-PBA intervened adult offspring born to obese dams.In addition,down-regulation of HSP70 expression significantly inhibited leptin signal transduction,especially exposed to ER stress induced by PA.Conclusion: Maternal obesity may induce supraphysiological levels of stress response both from HSR and UPR and interfere with the leptin signal in the hypothalamus of offspring during early postnatal period,leading to the hypothalamic feeding-circuit insults,which might underlie the origin of obesity.Furthermore,the coordinated balance between HSR and UPR is necessary for the postnatal hypothalamic feeding-circuit development.