Discussion On Changes Of Hepatic Lipid Metabolism In Pregnant Mice Based On Endoplasmic Reticulum Stress And Increased FGF21 Secretion

Background:To meet the needs of fetal growth and development,the coordinated physiological adaptation of multiple organs occurs during pregnancy.These widespread maternal physiological changes are highly coordinated with the development and growth of the placenta and fetus,which are critical to the successful establishment and maintenance of pregnancy.Previous studies showed that the serum lipid profile increased gradually during pregnancy.The liver acts as a central organ for lipid metabolism,marker changes occur in lipid profiles during pregnancy.For instance,the lipid uptake and transport capacities of the liver cell and TG secretion levels were up-regulated throughout reproduction,and liver X receptor(LXR)targets promote lipogenesis during early pregnancy by increased lipogenic enzymes fatty acid synthase(FASN)and stearoyl-CoA desaturase 1(SCD1).Abnormal lipid metabolism is often accompanied by adverse pregnancy outcome,which seriously affects maternal and infant health.Endoplasmic reticulum stress is a protective stress response.Endoplasmic reticulum stress activates the unfolded protein response,the survival pathway.ER stress includes three pathways;inositol-requiring enzyme(IRE)-1α path ways,activating transcription factor(ATF)-6αpathways and protein kinase R-like ER kinase(PERK)pathways.When endoplasmic reticulum stress was induced by external factors,BIP was separated from transmembrane proteins in these three pathways and bound to unfolded proteins.After the induction of stress signals,the cells are protected.There was evidence that ER stress is a common feature of fatty liver.a greater expression of ER stress related protein in bovine liver before calving has been reported previously.Fibroblast growth factor-21(FGF21)was found and named based on its membership in the FGF family in 2000,which belongs to an FGF subfamily which has hormone-like actions.Serum FGF21,secreted mostly from liver,could be delivered to pancreas or adipose tissue and caused a series of effects about weight loss and improved insulin sensitivity.The relationship between FGF21 and hepatic lipid metabolism has been described in mice.Knockdown of FGF21 has been shown to contribute directly to liver steatosis through inhibition of β-oxidation.Recently,the novel metabolic hormone FGF21 was shown to increase suddenly at the onset of lactation in dairy cows or during late pregnancy in the mouse.In recent years,the study on the mechanism of liver lipid metabolism has been progressing,but the upstream regulatory targets of lipid metabolism in pregnancy have not been clearly reported.Further explanation of the mechanism of lipid metabolism during pregnancy is helpful to reduce the incidence of adverse pregnancy.Objective:This experiment is intended to clarify Lipid metabolism in mice liver during pregnancy and to explore whether ER stress or increased secretion of FGF21 in the liver of mice in late pregnancy is related to the changes of lipid metabolism in the liver.Methods:1.Pregnant mouse model was successfully constructed:8-week-old female and male C57BL/6 mice were fed and allowed to acclimatize for one week.Female rats were randomly divided into the virgin group and the pregnant group.Female and male rats were caged at 2:1 every evening,and pregnancy was confirmed by checking a vaginal plug in the morning after caged(the day of appearance of the vaginal plug was assumed as day I of pregnancy.).Subsets of mice were euthanized in the virgin state(V),on day 5 of pregnancy(P5),on day 12 of pregnancy(P12),on day 19 of pregnancy(P19)and on lactation day 2(L2).2.To assessme mouse body metabolism with metabolic chambers:On day 15 or 16 of pregnancy,mice were acclimated in the animal monitor chamber(PhenoMaster,TSE Systems,Germany)for 24h and then placed into individual metabolic chambers.Afterwards,Oxygen consumption(VO2),carbon dioxide production(VCO2),Respiratory exchange rate(RER)and Heat production were simultaneously measured for 2 days.3.The serum lipid profile and serum β-hydroxybutyrate were measured using a Beckman Chemistry Analyzer,and the level of serum FGF21 was measured using ELISA.Moreover,we used tissue triglyceride and cholesterol content analysis kit to detect the lipid content of mouse liver tissue.Lipid accumulation in liver tissues of mice was evaluated by oil red O staining.4.Quantitative real-time PCR and RT2 PCR array:genetic screening of related to glucose and lipid metabolism during pregnancy.In addition,FGF21 and its receptor and FGF21 secretion were detected.5.Protein extraction and Western blot were used to detect various lipid metabolism and endoplasmic reticulum stress-related protein expression level.Results:1 Alterations in serum lipids,hepatic fat content during pregnancy.Gestation led to both body and liver weight gain especially during advance pregnancy.And female mice showed very rapid reduction of body weight after childbirth while liver weight was not significantly changed.Compared with V group,the level of TG was all obviously increased in P12 and P19 groups.However,TC and HDL-C reached the minimum levels in P12 group and then gradually rose.To further examine the potential alterations in liver,we measured the lipid content in the liver tissue.Compared to V group,significant increase in hepatic TG content was observed in P12 and P19 groups,while hepatic cholesterol content rose in P19 group.Consistently,more lipid droplet and ballooning degeneration in the liver during late pregnancy,as shown by HE and Oil Red O staining.In conclusion,serum TG content in mice increased in the early and middle trimester of pregnancy,liver lipid gradually accumulated,and tended to be stable in late pregnancy,while serum TG rapidly decreased and liver steatosis rapidly decreased after delivery.2 Energy expenditure increased significantly during late pregnancy.To evaluate the effect of gestation on the body energy metabolism,energy expenditure was measured using the metabolic cage analysis.Compared with mice in V group,mice in P1 7-19 group,showed increased Vo2,Vco2.heat production,while RER decreased which reminded β-oxidation enhanced.Fatty acids can be oxidized into ketone bodies in the maternal liver,and ketone bodies are the alternative energy source for the fetus when the glucose energy supply is insufficient.In other words,the energy metabolism level of the body of mice in late pregnancy is significantly increased,which meets the needs of fetal growth and development.3 Hepatic anabolic metabolism is enhanced in early pregnancy and suppressed in late pregnancy.Throughout pregnancy the expression of TG metabolism genes followed a distinct biphasic pattern.In early pregnancy,the transcript and protein levels of the lipogenic targets FASN and SREBP1C were markedly raised.In contrast,during late pregnancy the mRNA and protein abundance of SREBP1C,FASN and SCD1 was substantially diminished.And the β-oxidation related genes including CPT1α、hadha、ACOX1、Ech1、Ehhadh and Mlycd increased during late pregnancy,consistent with no increase in liver TG between P12 and P19.Microarray analysis was performed to identify changes in murine hepatic gene expression between late pregnancy groups and virgin group,which revealed that a set of lipid synthesis-related genes was down-regulated.The most striking impact of lipid synthesis-related genes was SCD1,which was markedly down-regulated 19-fold in late pregnancy group compared with V group.4 Hepatic ER stress is increased in early pregnancy.WB analysis exhibited increased BIP,phosphorylation level of IRE,PERK and eIF2a in P5 vs V or P1 9,indicating the possible activation of all three branch of ER stress.There was no significant change in the content of triglycerides and lipid droplets in the liver of mice in P5 vs V,until the occurrence of hepatic steatosis in P12.This suggests that endoplasmic reticulum stress may occur earlier than hepatic TG accumulation in our pregnant mouse model.5 Hepatic FGF21 production and secretion is increased in late pregnancy in ketotic states.In late pregnancy,we observed a significant raising in serum ketones,and serum FGF21 increased from a little over 100 pg/ml to nearly 6000 pg/ml between V and P19 and returned to V levels by L2,which was also apparent.Meanwhile,the mRNA expression of FGF21 genes significantly increases more than 30-fold in P19 and transfer to former level in L2.Surprisingly,Protein level of hepatic FGF21 was almost undetectable in late pregnancy,which may be related to increased FGF21 secretion.The synthesis process of FGF21 in hepatocytes belongs to the transport of intracellular intimal vesicles,so we measured genes expression of secretory vesicles between the ER and Golgi,including Yipf6、Yipf5、Sarla,Sec23a,Sec24a、Sec31a and Sec13.Sec23a is a small GTPase that initiates COPⅡ coat assembly and increased during late pregnancy,which suggested that FGF21 could be secreted into blood through COPII in late pregnancy,leading to a sharp increase in serum FGF21 content.Summary and conclusions:With activation of endoplasmic reticulum stress and increased FGF21 secretion during pregnancy,hepatic triglyceride content changed,suggesting that ER stress and FGF21 may play an vital part in balancing lipid homeostasis and meeting maternal and infant energy requirements in late pregnancy.