The Biological Function Of GDF11 On Transformation Of White Adipocytes To Brown Adipocytes And The Expression Of Inflammatory Cytokines In Macrophage

Background:There are two types of adipose tissue in the mammals’ body, white adipose tissue(WAT) and brown adipose tissue(BAT). The former one is the important organ of storing energy and secreting hormones, while the latter plays an important role in maintaining body temperature and regulating energy balance. Compared to WAT, BAT contains plenty of mitochondrion which are rich in uncoupling protein 1(UCP1). UCP1 can release the energy in the form of heat via transporting protons across the mitochondrial membrane and disturbing oxidative phosphorylation. It has been indicated that WAT can acquire the characteristics of BAT, such as smaller lipid droplets, increased mitochondria and higher expression level of UCP1, CIDEA and DIO2, after they were stimulated by cold exposure, some hormones(Irisin, for example) or drugs(such as PPAR γ agonist and β- adrenaline). This transformation process is known as white adipose-brown adipose transition. It has been found that PGC-1α could promote the expression of UCP-1 and the formation of mitochondria. Besides, PRDM16, FGF21 and Plac8 are also involved in regulating white adipose-brown adipose transition. However, the progress of this research is impeded by the difficulty in directionally inducting WAT. Because white adipose-brown adiposetransition reverses the storage of energy into release of energy, it is becoming the new therapeutic target of obesity and type 2 diabetes. So, it is necessary to develop new medications to promote the transition of WAT into BAT.Macrophage plays a great role in the development of atherosclerosis. There are two types of macrophage: MI and MII. MI(classically activated macrophages) express proinflammatory cytokines, such as IL-1, IL-6 and TNFa. MII(alternatively activated macrophages) express cytokines which can inhibit the progression of inflammation, such as IL-10, TGFb and CD68. The transition of MI to MII can inhibit the progression of atherosclerosis.As a member of BMP/TGF-β superfamily, GDF11(Growth and differentiation factor-11) produces bone morphogenetic proteins(BMPs) which has important role in the early embryonic development. BMPs regulate the metabolism and differentiation of adipose cells. BMP7 was identified to promote the differentiation of mesenchymal progenitor cells C3H10T1/2 into brown fat cell lineage. But it is still unclear whether GDF11 has some effects in the transition of white fat to brown fat and the tansition of MI to MII.The present study is aimed to illustrate the role GDF11 played in white adipose-brown adipose transition and the possible mechanism in this progress through observing the changes in C3H101/2 cells after they were incubated with GDF11 in vitro. And identify the role of GDF11 in the transition of MI to MII.Objective:Explore the effect and mechanism of exogenous GDF11 in the white adipose-brown adipose transition and the expression of inflammatory cytokines in macrophage.Methods:1. Culture the C3H10T1/2 cells with different concentration of GDF11 and then detect the content of lipid droplet by oil red O staining.2. To examine the expression of the brown adipose markers UCP1, CIDEA, COX7 A, DIO2 and El OVl3 by RT-PCR and Western-Blot.3. To detect the variation of mitochondrial DNA copy number after incubating with different concentration GDF11 by RT-PCR.4. To detect the expression of GDF11 in RAW264.7 and HUVEC by western blot.5. To identify the effect of ox LDL on GDF11 expression in RAW264.7 by RT-PCR.6. Treat RAW264.7 with GDF11 or ox LDL or in the present of both. Extract the total RNA and detect the expression of IL-1 and IL-6 by RT-PCR.Results:1. Oil Red O staining suggested that C3H10T1/2 cells can be induced into mature adipose cells by adipogenic liquid.2. We evaluated the expression of brown adipose markers UCP1 by Western-Blot. This result implied that the expression of UCP1 can be significantly induced by adding 100 ng/ml GDF11.3. We detect the variation of mitochondrial DNA copy number by RT-PCR, and found that the GDF11 can increase the copy number of mt DNA. The RT-PCR results showed that the GDF11 can obviously promote the expression of UCP1, CIDEA, COX7 A, DIO2 and El OVl3.4. RAW264.7 express more GDF11 than HUVEC.5. Ox LDL can inhibit the expression of GDF11 in RAW264.7 cell line.6. Ox LDL can promote the expression of IL-1 and IL-6, which are the markers of MI. While GDF11 can reverse the induction of ox LDL.Conclusion:1. GDF11 plays a positive role in the white adipose-brown adipose transition, and can promote the expression of brown adipose markers UCP1, CIDEA, COX7 A, DIO2 and El OVl3 and increase the copy number of mt DNA.2. GDF11 is mainly expressed in RAW264.7. Ox LDL can inhibit the expression of GDF11 and promote the expression of IL-1 and IL-6. GDF11 can inhibit the induction effect of ox LDL on the expression of IL-1 and IL-6.