The Preventive Effect Of Mussel Oil On Gestational Diabetes Mellitus And Its Mechanism In Mice

ObjectiveThe present study investigated the preventive effect and mechanism of mussel oil(MO)on gestational diabetes mellitus(GDM)in mice induced by a high-fat and high-sucrose(HFHS)diet compared with fish oil(FO)and corn oil(CO).Method1.Establishment and intervention of the animal model: One hundred and thirty 5-week-old C57BL/6J mice(100 females and 30 males)were given free access to water and standard diet.After 1 week of adaptation,the mice were randomly divided into 2 groups.The healthy control group was fed an AIN-93 G diet(contains 64% carbohydrate,20%protein and 16% fat)and the HFHS groups were fed a HFHS diet(contains 35%carbohydrate,20% protein and 45% fat)for 4 weeks and weighed once a week.Female mice were mated with males at a female/male ratio of 3/1,and the day when a vaginal plug was found was recorded as the 0.5th day of pregnancy(E0.5d).Pregnant mice were randomly divided into four groups: healthy control group(Normal diet+CO,n=7),corn oil group(HFHS+CO,n=8),fish oil group(HFHS+FO,n=7)and mussel oil group(HFHS+MO,n=7).The Normal diet+CO and HFHS groups were fed the same diet during pregnancy as before pregnancy.The Normal diet+CO and HFHS+CO groups were supplemented with CO,and HFHS+FO and HFHS+MO groups were supplemented with FO and MO by gavage at 7 g/kg/d.The mice were weighed every 3 days.Insulin tolerance test(ITT)was performed intraperitoneally on E15.5 d and oral glucose tolerance test(OGTT)was performed on E17.5 d.Fasting blood glucose(FBG)was measured on E18.5d and the pregnant mice were sacrificed.Blood was collected and liver,fetuses and placentas were collected and weighed.Crown-rump length of fetuses was measured using vernier calipers.Blood samples and fresh tissues were stored at-80 °C for further analyses.2.Laboratory analysis: Serum triglyceride(TG),total cholesterol(TC),low density lipoprotein-cholesterol(LDL-C)and high density lipoprotein-cholesterol(HDL-C)were measured by biochemical kits.Fasting serum insulin(FINS),glycosylated hemoglobin(Hb A1c),interleukin 6(IL-6),interleukin 10(IL-10)and tumor necrosis factor α(TNF-α)were determined using ELISA kits.The homeostasis model insulin resistance index(HOMA-IR)was calculated.The fatty acid composition in the liver of pregnant rats was measured by gas chromatography.The protein expression levels of sphingosine kinase 1(Sph K1),sphingosine kinase 2(Sph K2),Akt and phosphorylation of Akt(p-Akt,Ser473)in the liver were detected by western-blot analysis.Ultra performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-QTOFMS)was used for the determination of liver metabolites,and liver sphingosine(So),sphinganine(Sa),sphingosine-1-phosphate(So1P)and sphinganine-1-phosphate(Sa1P)content.Result1.There was no significant difference in body weight among the four groups before and during pregnancy(P<0.05).The weight ratio of fetus to placenta in the HFHS groups was significantly lower than that in the Normal diet+CO group(P<0.05).There were no significant differences in fetus and placenta weight among groups(P>0.05).2.The area under the curve(AUC)of OGTT and ITT in the HFHS+CO group was significantly higher than that in the Normal diet+CO group(P<0.05).The AUC of OGTT in HFHS+MO and HFHS+FO groups was significantly lower than that in HFHS+CO group(P<0.05).The 30-min and 60-min blood glucose levels of OGTT in HFHS+CO group were significantly higher than HFHS+MO group(P<0.05),and was comparable with that in the HFHS+FO group(P>0.05).The 90-min glucose level of OGTT in HFHS+MO group was significantly lower than HFHS+FO and HFHS+CO groups(P<0.05).The AUC of ITT in HFHS+CO group was significantly higher than that in HFHS+MO group(P<0.05),and was comparable with that in the HFHS+FO group(P>0.05).3.Serum FINS and HOMA-IR levels in HFHS+MO group were significantly lower than that in HFHS+CO and HFHS+FO groups(P<0.05).MO improved liver fat deposition induced by the HFHS diet.4.HFHS+MO and HFHS+FO groups had significantly higher liver levels of C20:5n-3,C22:6n-3,and total n-3 polyunsaturated fatty acids(PUFA)than Normal diet+CO and HFHS+CO groups(P<0.05);and lower levels of C18:2n-6,n-6 PUFA and ratio of n-6PUFA/n-3 PUFA than Normal diet+CO and HFHS+CO groups(P<0.05).Liver C22:6n-3and total n-3 PUFA levels were negatively correlated with the AUC of OGTT and ITT in pregnant mice(P<0.05).5.Untargeted metabolomic analysis of the liver revealed that HFHS+MO group significantly decreased metabolites of fatty acyls and glycerolipids(P<0.05),and increased ceramide(Cer)16:0 compared to HFHS+CO group(P<0.05).The AUC of OGTT,FINS,and HOMA-IR were positively correlated with lysophosphatidylethanolamine(LPE)(18:1)(P<0.05),and were inversely associated with Cer 16:0 level(P<0.05).6.The liver Sphk1 protein level in the HFHS+CO group was significantly higher than Normal diet+CO group(P<0.05),while the protein levels of Sphk2,Akt,and P-Akt were significantly lower than Normal diet+CO group(P<0.05).The HFHS+MO group had higher Sphk2,Akt and P-Akt levels and lower Sphk1 level than the HFHS +CO group(P<0.05).7.The HFHS+CO group had lower So and higher So1 P and Sa1 P content than the Normal diet+CO group in the liver(P<0.05),and no significant difference in Sa content between HFHS+CO group and Normal diet+CO group(P>0.05).The HFHS+MO group had significantly lower Sa1 P content than the HFHS+CO group(P<0.05).The Sa content in the HFHS+FO group was significantly higher than HFHS+CO group(P<0.05).ConclusionMO improved HFHS diet-induced glucose intolerance and insulin resistance during pregnancy better than FO.The mechanism of MO may be related to the regulation of liver lipid metabolism,sphingolipid metabolic pathway and insulin signaling pathway.These findings provide us with a new thought for the nutritional intervention to prevent GDM.