| BackgroundIn recent years,there are many researches about the relationship between adipocyte-secreted factors and obesity,type 2 diabetes(T2D)and other metabolic diseases,and the role of adiponectin(ADPN)has attracted much attention.The research progress of adiponectin and other adipokines suggest that adiponectin not only have the exact effect on promoting fatty acid oxidation,reducing blood lipid,improving insulin resistance,which have a direct impact on blood sugar,but also reduces in the patients who suffered from obesity and diabetes,which means there is no "adiponectin resistance".So we expect to achieve the beneficial effects by the replenishment of adiponectin.Excessive intake of fat or unreasonable fatty acid composition have intimate connection with obesity and related metabolic diseases.N-3 polyunsaturated fatty acids(n-3 PUFAs)especially ALA,EPA and DHA,has the function of improving insulin resistance,anti-inflammatory,anti-oxidative stress,reducing lipid and so on,which can reduce the incidence of cardiovascular disease.But the role of certain fatty acids in n-6 PUFAs are opposite.Therefore,how to maintain the balance of the two has become a hot research topic in recent years.The recommend ratio of n-6/n-3 PUFAs composition is not accordance.During to the lack of strong evidence,it is not possible to reach agreement on the recommended intake of n-6/n-3PUFAs.In the previous study,we found that dietary fatty acids can affect the expression of adiponectin.Vitro experiment show that in a certain range of concentration,linoleic acid,DHA and EPA,especial DHA can up regulate the expression of adiponectin in 3T3-L1 mature cells.Some foreign research show that EPA/DHA can increase the level of plasma adiponectin,but linoleic acid can inhibit the expression of adiponectin.The results of these studies showed that n-6/n-3PUFAs had different regulatory effects on the expression of adiponectin.Therefore,this study will systematically research the effect of n-6/n-3PUFAs on the expression and secretion of adiponectin,and exploring the potential mechanism of the effect of n-6/n-3PUFAs on the expression of adiponectin.The present study reveals that the regulation of adiponectin expression is related to the peroxisome proliferator activated receptor γ(PPARγ).The activation of PPARγcan increase the expression of adiponectin mRNA,so that plays a key role in regulation of adipocyte differentiation and metabolism of glucose and lipid.Antidiabetic drugs thiazolidinediones(TZDs)such as rosiglitazone and pioglitazone,which are PPAR gamma agonists,are widespread used in clinical to reduce blood sugar and improve insulin resistance through improving the level of serum adiponectin by activing PPAR gamma.N-6/n-3PUFAs and other fatty acids are natural ligands PPARγ,so it is possible to induce(or inhibit)adiponectin expression through a similar pathway with TZDs.In the early stage of this study,the results of cell culture in vitro showed that there was a correlation between the expression of adiponectin and PPAR in the mature adipocytes of 3T3-L1.In addition,Choi et al.found that,in obesity mice induced by high fat diet,Cyclin-Dependent Kinas 5,CDK5 was activated in adipose tissue,which leading to the 273 serine phosphorylation of PPAR gamma without affecting its transcriptional activity and decrese expression of adiponectin.The study also found that rosiglitazone could inhibit the phosphorylation of PPAR mediated by CDK5,thus increase the expression and secretion of adiponectin.Another drug MRL-24 can also inhibit the phosphorylation of PPARy mediated by CDK5,although the effect is weak,it can also present a good effect of reducing blood glucose.In addition,a high concentration of free fatty acids was found to activate CDK5,leading to ser-273 phosphorylation of PPARγ in 3T3-L1 adipocytes.It is concluded that the same as the PPARγ ligands,a variety of fatty acids are also likely to regulate the expression of adiponectin by activating or inhibiting the phosphorylation of PPARγ mediated by CDK5.So,it is worth to discuss that the regulation of n-6 and n-3PUFAs on adiponectin expression in the case of different fat for energy ratio and different n-6/n-3PUFAs constituent ratio.ObjectiveWe intend to discuss and research from the following aspects:1 To study the effect of different n-6/n-3 PUFAs on the expression and secretion of adiponectinThe effect of different n-6/n-3 PUFAs on the expression and secretion of adiponectin was studied under the condition of fixed total dietary fatty acids(normal or high-fat diet fed)and dietary S:M:P(by the Chinese Nutrition Society recommended 1:1:1 and our previous study found can increase adiponectin expression of 1:1:2),and found the optimal n-6/n-3 PUFAs ratio which was ascertained to increase the expression of adiponectin.2 To investigate the molecular mechanism of n-6/n-3 PUFAs regulating the expression of adiponectinFor reasons such as noted above,we assume that the n-6/n-3 PUFAs can regulate the expression of adiponectin by two ways:① Direct activation or inhibition of PPARγ to raise or lower the expression and secretion of adiponectin.② To validate the presumption whether n-6/n-3 PUFAs can regulate the expression and secretion of adiponectin by the pathway of inhibition or activation of phosphorylated PPARγmediated by CDK5.3 Dynamic observing relevant parameters,analyzing the relationship between these parameters,understanding the role of adiponectin on the occurrence and development of obesity,insulin resistance,dyslipidemia.This animal experiment emphatically research the role of different dietary n-6/n-3 polyunsaturated fatty acids constitute on adiponectin expression and secretion,looking to the appropriateratio of n-6/n-3 polyunsaturated fatty acids which can raise adiponectin expression and secretion,and conducive to the prevention and treatment of obesity and related metabolic diseases,and then,exploring the underlying molecular mechanism.MethodsPart Ⅰ Comparing effects of different composition of n-6/n-3 polyunsaturated fatty acid on the expression of adiponectin under the fat energy supply ratio of 25%1 Rat feed formula,animal selection and groupingIn this study,we selected SPF adult male Wistar rats as the research object,which were purchased from the medical animal experimental center of Southern Medical University.Randomization according to the weight,each group has 12 rats.Detecting 26 kinds of fatty acid content of 6 kinds of edible oil,which are butter,peanut oil,corn oil,tea oil,deep sea fish oil and sunflower seeds oil,and adjusting fat energy supply for 25%,feed S:M:P composition for 1:1:1 and 1:1:2,and ratio of n-6/n-3 for 1,5,10,20 according to the detection results,then testing various fatty acid content in feeds.Control group:fed with basic feed(reference AIN-93G recommended animal feed formula)Experimental group A-E as follows:group A:S:M:P=1:1:1,n-6/n-3=1;group B:S:M:P=1:1:1,n-6/n-3=5;group C:S:M:P=1:1:1,n-6/n-3=10;group D:S:M:P=1:1:1,n-6/n-3=20;group E:S:M:P=1:1:2,n-6/n-3=5.Anesthetizing rats and drawing ocular blood about 1.5 mL respectively in the experiment of 0,4th and 8th week,separating the serum,and frozening storage in-80℃ to be measured;At the 12th week,Anesthetizing rats and drawing abdominal aortic blood approximately 5 mL,separating the serum.At same time,separating heart,liver,kidney,testis,spleen,perinephric and perine-testis adipose tissue and skeletal muscle,weighing organ wet weight to calculate organ coefficient and fat accumulation.Liver and kidney peripheric adipose tissue were prepared to do pathological section,and the rest of the organs will storage to be measured in liquid nitrogen after freezing,-80℃.2 Index detection①Histological observationMaking Liver and visceral adipose tissue HE staining pathological sections②Blood biochemical indexTriglyceride(triacylglycerol TG),total cholesterol(TC),adiponectin(ADPN),fasting blood glucose(FBG),glutathione peroxidase(GSH-Px),superoxide enzyme(SOD)and malondialdehyde(MDA).③Molecular biological index of visceral adipose tissueThe mRNA and protein expression levels of adiponectin,PPAR,p-PPAR,273Ser and CDK5 in visceral adipose tissue.Part Ⅱ Comparing effects of different composition of n-6/n-3 polyunsaturated fatty acid on the expression of adiponectin under the fat energy supply ratio of 40%In addition to formula feed fat for the ratio of 40%,the fatty acid composition is same as the first part.Control group:fed with basic feed(reference AIN-93G recommended animal feed formula)Experimental group F-J as follows:group F:S:M:P=1:1:1,n-6/n-3=1;group G:S:M:P=1:1:1,n-6/n-3=5;group H:S:M:P=1:1:1,n-6/n-3=10;group I:S:M:P=1:1:1,n-6/n-3=20;group J:S:M:P=1:1:2,n-6/n-3=5.Serum and tissue sample collection methods,detection indicators and detection methods are the same as part Ⅰ.ResultsChapter Ⅰ Comparing effects of different composition of n-6/n-3 polyunsaturated fatty acid on the expression of adiponectin under the fat energy supply ratio of 25%.)1 Thechanges of rats’ weight,fat accumulation,serum TG,TC and FBG levelsWhen S:M:P is 1:1:1,the levels of serum triglyceride(TG),TC were increased with the raise of the ratio of n-6/n-3PUFA composition in group A(n-6/n-3=1:1),group B(n-6/n-3=5:1),group C(n-6/n-3=10:1)and group D(n-6/n-3=20:1)The level of serum TG,TC were decreased in group A and B and was the highest in group D,but there were no significant difference when it were compared with control group.When S:M:P was 1:1:2,there was no significant change on serum TG and TC in group E(n-6/n-3=5).There was no difference on the level of FBG in different n-6/n-3PUFAs groups.The rats in(of)each experimental group had no significant change in FBG content at different time,and there was no significant difference between these groups and control group,P>0.05.It can be seen that the different n-6/n-3PUFA composition ratio has little effect on body weight and blood glucose change,but has different effects on blood lipid,which serum TG and TC levels were increased with the increase of n-6/n-3PUFA composition ratio.2 The level of oxidative stress in each groupAt twelfth weeks,the serum GSH,PX and SOD activity of each group were increased,and the activity of serum GSH-PX and SOD activity in group A was significantly higher than that in control group(P<0.05).In addition to group A,the serum GSH-PX and SOD activity in the rest of experimental group was not significantly different(P>0.05)before and after the experiment.The serum MDA level in each group were decreased,and the serum MDA in group A was significantly lower than that in control group.In addition to the D group,the serum MDA content in the rest experimental group,was significantly decreased compared with that of before experiment(P<0.05).Therefore,appropriately reducing dietary n-6/n-3PUFA(such as 5:1)can decrease the oxidative damage on rats.3 Changes of adiponectin mRNA and protein expression in rats visceral adipose tissue of each groupWhen S:M:P was 1:1:1,the expression level of adiponectin mRNA in visceral adipose tissue of group B was higher than that in control group(P<0.05)and was the highest in all group.The expression levels of adiponectin mRNA in group B,C and D,were decreased with the increase of n-6/n-3 composition ratio.The expression level of mRNA in group D was the lowest,but there was no significant difference between these groups(P>0.05).When S:M:P was 1:1:2,the expression level of adiponectin mRNA in group E was higher than that in control group(P<0.05),and was higher than that in group D.The expression level of adiponectin protein in visceral adipose tissue in each group was basically consistent with that of RT-PCR.The expression level of adiponectin protein in visceral adipose tissue of group B,higher than that in control group(P<0.05),and was the highest in all groups.The expression level of adiponectin protein in D group was the lowest,which was lower than that in control group(P<0.05),and the expression level was down regulated in group A,B,C(P<0.05).There was no difference in the expression level of adiponectin protein in the group E and control group.The expression of adiponectin in adipose tissue of rats was synchronous with different n-6/n-3 composition.When the ratio of n-6/n-3 was 5:1,the expression level of adiponectin in adipose tissue was the highest,and was decreased with the increase of n-6/n-3PUFA ratio.4 Changes of PPARy mRNA and protein expression in visceral adipose tissue of rats in each groupWhen S:M:P was 1:1:1,the expression level of PPARy in visceral adipose tissue of group B was increased compared with group A,C and D,but there was no significant difference when compared with control group(P<0.05).When S:M:P was 1:1:2,the expression level of PPARy in visceral adipose tissue of group E was up-regulated,but there was no significant difference when compared with control group(P>0.05).The expression level of PPARγ in visceral adipose tissue of group A was statistical higher than that in control group(P<0.05).The expression level of PPARγ in visceral adipose tissue of group B was higher than that in control group(P<0.05).The expression level of adiponectin in group D was the lowest,which was lower than that in control group(P<0.05).Compared with the control group,the expression level of visceral fat PPARγ in group E was higher than that in control group(P<0.05).The expression level of PPAR gamma protein was positively correlated with adiponectin protein in visceral adipose tissue of rats(r=0.479,P<0.05).The expression level of PPARγ in visceral adipose tissue of rats was decreased with the increase of n-6/n-3 ratio,which was positively correlated with the expression of adiponectin in adipose tissue(P<0.05).When S/M/P was 1:1:1,and the ratio of n-6/n-3 was 5:1,the effect of PPARγ on adiponectin expression was the strongest.5 The changes of CDK5mRNA and protein expression and p-PPARγ(273Ser)protein expression in visceral adipose tissue in each groupThe expression level of CDK5 mRNA in visceral adipose tissue in each group was slightly raised,but there was no significant difference compared with control group(P>0.05).When S:M:P was 1:1:1,CDK5 protein expression in visceral adipose tissue of group A,B,C and D,was lower than that in control group(P<0.05).When S:M:P was 1:1:2,the CDK5 expression level of visceral fat in group E was higher than that in control group(P<0.05),and the expression level CDK5 was higher in group A,B,C and D,and the difference was statistically significant(P<0.05).There was a negative correlation between the expression level of CDK5 protein and the level of adiponectin protein expression in visceral adipose tissue of rats,but there was no statistical difference(r=-0.029,P>0.05).The expression of p-PPARγ(273 Ser)in visceral adipose tissue in group A,B,C,D were lower than that of the control group(P<0.05).The expression of p-PPARγ(273Ser)in visceral fat in group E was higher than that in control group,but there was no significant difference between the two groups(P>0.05).The expression level of p-PPARγ(273Ser)protein was negatively correlated with adiponectin protein in visceral adipose tissue of rats(r=-0.249,P<0.05).Chapter Ⅱ Effects of different n-6/n-3 polyunsaturated fatty acid composition on the expression of adiponectin in rats with high fat diet1 The changes of body weight,fat accumulation,serum TG,TC and FBG levels in ratsRats in each experimental group were heavier than control group.From the fourth weeks,the weight gain of group H(S/M/P=1:1:1,n-6/n-3=10:1)was significantly higher than that of control group(P<0.05).When S/M/P was 1:1:1,the body fat of rats in group F(n-6/n-3=1:1),group G(n-6/n-3=5:1),group Ⅰ(n-6/n-3=20:1)were significantly higher than control group(P<0.05).When S/M/P was 1:1:2,the body fat of group J(S/M/P=1:1:2,n-6/n-3=10:1)were significantly higher than control group(P<0.05).From the eighth weeks,the serum levels of TG and TC in the experimental group were higher than that in control group(P<0.05).At twelfth weeks,the serum levels of TG and TC in group 1 were higher than that in control group(P<0.05),and higher than that in group G,group F,group H and group J rats(P<0.05).FBG in group H and group Ⅰ were significantly higher than that in the control group(P<0.05).It can be seen that long-term excessive intake of fat can increase the weight of rats,with excessive accumulation of visceral adipose tissue,and even the occurrence of fatty liver disease.High fat diet can induce the increase of serum TG,TC and FBG levels,and raised with the increase of n-6/n-3PUFA composition.2 The level of oxidative stress in each groupCompared with control group,the serum GSH-PX and SOD activity were significantly decreased and MDA content were significantly increased in group G,H,I,J at the 12th weeks.(P<0.05).The serum GSH-PX and SOD were significantly decreased in the group Ⅰ.Compared with pre-experiment,the serum GSH-PX and SOD were significantly decreased(P<0.05),and the content of MDA was significantly increased(P<0.05).So,we can conclude that excessive intake of total amount of fat can reduce the ability of anti-oxidative damage in rats,with serum GSH-PX,SOD activity were decreased and MDA was increased.3 The effects on adiponectin expression in visceral adipose tissue of each groupWhen S:M:P was 1:1:1,the expression of adiponectin mRNA in visceral adipose tissue was decreased with the increase of the n-6 1 n-3 composition ratio,and adiponectin mRNA expression in group Ⅰ was significantly down regulated,which was lower than that of the control group(P<0.05),and group F and G.When S:M:P was 1:1:2,the adiponectin mRNA expression level of group J was slightly lower,but there was no significant difference compared with the control group(P>0.05).The expression levels of adiponectin in visceral adipose tissue in each group were lower than that in control group(P<0.05).When S:M:P was 1:1:1 and n-6/n-3 was greater than 5,the adiponectin protein expression in group G H,I were decreased with the increase of the proportion of n-6/n-3PUFA.The expression level of adiponectin in group Ⅰ was lowest,and lower than that in control group and group F,G,H,J(P<0.05),So it can be seen that excessive intake of fat can make the expression of adiponectin mRNA and protein reduced in visceral adipose tissue of rats.4 The change of PPARγ expression level in visceral adipose tissue of ratsWhen S:M:P was 1:1:1,the expression of PPARγ mRNA in group F,H,I were significantly lower than that of control group(P<0.05),and PPARγ mRNA was the lowest in group Ⅰ.When S:M:P was 1:1:2,the expression level of PPARγ in group J was slightly lower than that in control group,but there was no significant difference compared with control group(P>0.05).The expression level of PPARγ in visceral adipose tissue of rats in each group was significantly lower than that in control group(P<0.05),and were decreased with the increase of n-6/n-3 composition.The expression level of PPARγ in group Ⅰ was the lowest,which was lower than that in control group and group F,G H(P<0.05).The expression level of PPARγ in group J was lower than that in control group and group F(P<0.05),it was higher than that in group Ⅰ(P<0.05).The correlation analysis showed that there was a positive correlation between the two proteins(r=0.331,P<0.05),suggesting that PPARγ might be the key molecule to regulate the expression of adiponectin.Based on these results,we can conclude that excessive intake of fat can also down-regulate PPARγ mRNA and protein expression,and it were decreased with the increase of the n-6/n-3 proportion.5 Changes in the expression level of CDK5 and p-PPARγ(273Ser)in visceral adipose tissue of ratsThe expression level of CDK5mRNA in visceral adipose tissue of each experimental group was up·regulated.The expression of CDK5mRNA in group F,I was significantly higher than that in control group(P<0.05).The expression level of CDK5mRNA in group J was not significant,and there was no difference between the control group and group J(P>0.05),but it lower than that in group F and Ⅰ(P<0.05).When S:M:P was 1:1:1,the CDK5 protein of visceral adipose tissue in group F,G,H,I were significantly higher than that in control group(P<0.05).The expression level of CDK5 protein in group I was highest,and it was)higher than that in group F,GqH(P<0.05).When S:M:P was 1:1:2,the expression of CDK5 protein in visceral adipose tissue of group J was higher than that in control group(P<0.05),but lower than that in I group(P<0.05).(The expression level of CDK5 protein was negatively correlated with adiponectin protein in visceral adipose tissue of rats(r=-0.750,P<0.05).The level of p-PPARγ(273Ser)protein in group F,G H,I was significantly higher than that in the control group(P<0.05).The expression level of p-PPARγ(273Ser)protein in visceral adipose tissue of group I was slightly higher than that in G group(P<0.05).The expression level of p-PPARy(273Ser)protein in group J was higher than that in control group and group F,G,H,I(P<0.05).The expression level of p-PPARγ(273Ser)protein was negatively correlated with adiponectin protein in visceral adipose tissue of rats(r=-0.483,P<0.05).It can be seen that the total amount of dietary fat intake can up-regulate the expression level of CDK5mRNA and protein and p-PPARy(273Ser)protein in visceral adipose tissue of rats.The regulation effects is different with the n-6/n-3 composition,and it was the lowest when n-6/n-3 is 5:1.Conclusion1 The body weight,blood lipid and blood glucose of rats was not affected by the dietary which the fat energy supply ratio is 25%.When n-6/n-3 PUFA ratio was 1:1,the ability of averting oxidant damage was significantly enhanced.Long term intake of the dietary which fat for energy ratio is 40%can make the body weight of the rats increased,visceral fat accumulated,lipid metabolic disorder,reduce and the antioxidative ability of the rats.The levels of blood lipid and blood glucose of rats was significantly increased with PUFA composition ratio increasing,and the antioxidant capacity of rats was decreased with PUFA composition ratio increasing.2 The expression level of adiponectin in adipose tissue of rats was significantly increased when the ratio of fat supply to energy was 25%and the ratio of n-6/n-3PUFA was 5:1.The expression level of adiponectin in adipose tissue of rats can be reduced by long-term intake of fat for 40%feed.When the ratio of n-6/n-3 was greater than 5:1,the expression level of adiponectin was decreased with the increase of the proportion of the n-6/n-3PUFA.3 When the fat supply ratio was 25%,and the the ratio of n-6/n-3PUFA was 5:1,the expression level of PPARγ in adipose tissue of rats was significantly increased.Long term intake of fat for 40%feed can reduce the expression level of PPARγ in adipose tissue of rats.When the ratio of n-6/n-3 was 5:1,the expression level of PPARγ was decreased with the increase of the composition ratio of the n-6/n-3PUFA.The expression level of PPARγ in adipose tissue of rats was positively correlated with the expression of adiponectin,and high expression of PPARγ can promote the expression and secretion of adiponectin.4 The expression of CDK5 and p-PPARγ in adipose tissue of rats was down regulated by intake the dietary which fat supply energy ratio is 25%.CDK5 protein and p-PPARγ protein expression in adipose tissue of rats can be increased with the dietary which fat supply energy ratio is 40%,and it was increasd with the increase of n-6/n-3PUFA composition.The expression level of CDK5 and p-PPARγ in adipose tissue was negatively correlated with the expression level of adiponectin,and the expression level of CDK5 and p-PPARγ could inhibit the expression and secretion of adiponectin. |
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