| BackgroundObesity and its relative diseases have become a serious health threat to human beings and urgently need to be solved by doctors and researchers all over the world.Human and other species of mammal have two kinds of adipose tissue, white adipose tissue(WAT) can reserve energy, and brown adipose tissue(BAT) can improve the nonshivering thermogenesis, insulin sensitivity and insulin resistance.BAT has been found in interscapular region, while WAT has been found in inguinal adipose tissue, epididymal adipose tissue and visceral adipose tissue. PRDM16( PRD1-BF1-RIZ1 homologous domain containing 16) is one of transcription factors which can be strongly expressed on BAT and shows a positive regulation in the procedure of the differentiation of brown adipose cell. It improves the number of mitochondrion and enhances the function of mitochondrion. Then the procedure of the oxidative phosphorylation is promoted and causes the expenditure of energy and calorie production. Type Ⅱ iodothyronine deiodinase(Dio-2) is one of the marker of BAT, it promotes energy metabolism via the binding of special site in the gene of uncoupling protein 1(UCP1) and then activates transcription. Hypothalamus plays an important role in energy homeostasis. Neuropeptide Y(NPY) is an important peptide which can promote the appetite. The regulation almost relies on the NPY neuron in hypothalamus. ARC, DMH and many other areas of hypothalamus can express NPY, but it is expressed at the top level on ARC. The research has shown that knocking down the expression of NPY gene in the ARC of high-fat diet induced obese rat can improve the energy metabolism.Fibroblast growth factor-21(FGF21) shows its kinds of effects via the complex of FGF21/ b-Klotho/FGFR, improving the glucose and lipid metabolism. FGF21 induces the change from WAT to BAT via SNS. Now it is not clear that the change of expression of FGF21, PRDM16 and Dio-2 in the adipose tissue when knockdown of NPY expression in the DMH. ObjectiveTo investigate the rats weight, FGF21, PRDM16 and Dio-2 in the adipose tissue by creating the model of rat knockdown of NPY in the DMH and feeding high-fat diets. MethodsThe method of modelling as follows, the animal were 4-5 weeks of 80, SPF, male Sprague Dawley rats, including 20 rats(weight:270-300g) and 60 rats(weight:130-150g). After one week for adaption, creating the AAV-mediated RNAi vector can cause the low level expression of NPY in the DMH, and it was the recombinant viral vector AAVsh NPY. At the same time, creating the recombinant viral vector AAVshCTL was as control. The rats(weight:270-300g) were used for examining whether the modelling was successful or not. Finding the DMH nuclei three-dimensional coordinates by the rats’ brain atlas of Paxinos and Watson.15 rats were randomly received bilateral DMH injections of AAVshNPY and 5 rats randomly received bilateral DMH injections of AAVshCTL. 15 rats(received AAVshNPY) were randomly sacrificed(n=5, each week) at 1,2,4 weeks post- viral injection, and another 5 rats received AAVshCTL were sacrificed after 4 weeks of viral injection. All of the 20 sacrificed rats were quickly taken their hypothalamus. Half of their hypothalamus tissue was kept in liquid nitrogen, and then made as frozen section for finding the areas of viral vector. The remain was made as paraffin section for testing the level of DMH NPYmRNA expression by fluorescence in situ hybridisation. These steps were to make sure that knockdown of the NPY expression in the dorsomedial hypothalamus was accomplished. And then, put the remain 60 rats into two groups randomly(n=30, each group) and found the DMH nuclei three-dimensional coordinates by the rats’ brain atlas of Paxinos and Watson. Then did the same step above, the two groups respectively received AAVshNPY and AAVshCTL.The weight of rats was recorded at the same time once a week. All rats were feed with regular chow(RC) at the first 4 weeks of experiment. Half of AAVsh NPY and half of AAVshCTL were chosen randomly and their food was changed from RC to HF, and others were still feed with RC. Thus now all rats were divided into 4 groups, they were AAVshNPY HF, AAVshNPY RC, AAVshCTL HF and AAVshCTL RC.All the rats were sacrificed at the end of 16 weeks, and were quickly taken the epididymal and inguinal adipose tissue which was kept in liquid nitrogen. These tissue was used to measure the expression of FGF21 mRNA, PRDM16 mRNA and Dio-2mRNA by quantitative RT-PCR, and the level of PRDM16 expression by western blot. ResultsThe animal models were successfully established. The viral vector had located in DMH. Compared with AAVshCTL, the level of NPYmRNA significantly decreased in 1,2,4 weeks after injection, the difference was significant(P<0.05).In the 5th week, compared AAVshNPY group and AAVshCTL group, it showed a significant difference(P<0.05), this showed knockdown of DMH NPY expression had reduced the speed of weight increase. In the 16 th week, compared with the RC groups, the HF groups’ weight had a significant increase, the difference was significant(P<0.01), this indicated that high-fat diet can cause weight increase quickly; compared AAVshNPY groups with AAVshCTL groups, it showed a significant difference(P<0.01), this showed that knockdown of DMH NPY expression can reduce the speed of weight increase.In the inguinal and epididymal adipose tissue, compared with the RC groups, the HF groups’ FGF21 mRNA had a low level expression, which showed a significant difference(P<0.01), this indicated that high-fat diet can reduce the FGF21 mRNA expression in the adipose tissue. While compared AAVshNPY groups with AAVshCTL groups, there were no statistically significant differences between the groups(P(29)0.05), which indicated that knockdown of DMH NPY expression had no influence on FGF21 mRNA expression in the adipose tissue.Both PRDM16 and Dio-2mRNA showed the low level of expression in the epididymal adipose tissue, there was no significant difference between any two groups(P(29)0.05), this indicated that knockdown of neither DMH NPY expression nor high-fat diet had an effect on PRDM16 and Dio-2mRNA expression in the epididymal adipose tissue.In the inguinal adipose tissue, compared with AAVshCTL groups, AAVshNPY groups’ PRDM16 and Dio-2mRNA were strongly expression, which showed a significant difference(P<0.01), this indicated that knockdown of DMH NPY expression can induce the significant expression of PRDM16 and Dio-2mRNA in the inguinal adipose tissue; compared with RC groups, the expression showed down regulation in the HF groups, and the difference is significant(P<0.01), it indicated that HF can reduce the expression of PRDM16 and Dio-2 mRNA in the inguinal adipose tissue.In the inguinal adipose tissue, compared AAVshNPY HF group and AAVshCTL HF group, PRDM16 protein of AAVshNPY HF group showed strongly expression and the difference was significant(P<0.01), compared AAVshCTL RC group and AAVshCTL HF group, PRDM16 protein of AAVshCTL HF group showed low level expression and the difference was significant(P<0.01). But there is no significant difference between any two groups in the epididymal adipose tissue(P(29)0.05). These result were consistent with qRT-PCR.ConclusionsKnockdown of NPY expression in the DMH can upgrade the expression of PRDM16 and Dio-2 in the inguinal adipose tissue of rats and improve lipid metabolism. And then high-fat diet induced obesity can be improved.High-fat diet can reduce the expression of FGF21 in the adipose tissue of rats. And knockdown of NPY expression in the DMH can’t regulate the expression of FGF21 in the adipose tissue. |
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