The Effect Of Growth Hormone And Signal Transducer And Activator Of Transcription 5B (STAT5B) On Adipocyte Differentiation

When the body is exposed to excess nutrients, the fat cells will over-growth and over-accumulate lipid, namely, hypertrophy, consequently, will result in obesity. Studies showed that obesity could increase the risk of type 2diabetes and cardiovascular disease. It is noticed that the incidence of obesity is positively related to age. Growth hormone (GH) is secreted by the anterior pituitary gland, during the development its secretion reaches its perk at some point and thereafter, decreases with the increase of age. Clinical research showed that there was a significant correlation between the secretion of human growth hormone and obesity. Growth hormone can regulate the metabolism of adipose tissue and reduce visceral fat, but the specific mechanism is not clear. Study on transgenetic mice found that lost of singal transducer and activator of transcription (STAT5B) activation fragment in GH receptor (GHR) lead to obesity. In our previous work we found that GH and STAT5B pathway could inhibit Fatty acid synthase (Fasn) and hinder adipocyte differentiation. This evidence indicates that STAT5B may play an important role in the GH pathway to impact obesity. To clarify the mechanism is to provide a good theoretical basis for the prevention and treatment of obesity. Our work is divided into two parts:Part â…  the effect of growth hormone on 3T3-L1 adipocyte differentiation and adipocyte tissue developmentAim:To investigate the effect of growth hormone on the expression of gene in the differentiation of 3T3-L1 cells and the effect on the expression of gene in adipose tissue from C57BL/6 High Fat Diet (HFD) mice.Methods:1. To observe the effect of GH on the early stage of 3T3-L1 preadipocytes during adipogenesis. Cultured 3T3-L1 pre-adipocytes were exploited for adipogenesis, GH (the final concentration was 125ng/ml) or PBS (control) was added into the medium during differentiation. Cells were harvested and marked as DO, D2, D3, D4, and D5 for different induction days. We would also detect the expression of genes which are relevant to lipid metabolism.2. To observe the effect of STAT5B knocking-down on adipocyte differentiation. LV3-STAT5B-shRNA was constructed and packaged, and a LV3-control-virus expression nonsense sequence was used as control.3T3-L1 cells were respectively infected with LV3-virus and LV3-STAT5B-shRNA virus. After puromycin drug selction, two kinds of cells were obtained and were induced; the samples were harvested at D0, D2, D3, D4, D5 and D10. Western blot and qPCR were used to detect the expression of genes which are relevant to lipid metabolism.3. To observe the effect of GH on adipocyte tissues of HFD mice:6 weeks old C57BL/6 mice were randomly divided into 3 groups, normal-feeding, HFD and HFD with intraperitoneal injection of growth hormone (sterilized phosphate buffered saline (PBS) were used as control to other two groups). After 12 weeks of feeding, the mice were anesthetized and the blood glucose and biochemical parameters of serum were detected. Western blot were used to test the effect of growth hormone on the gene expression in visceral adipose tissue.4. To observe the mechanism of GH on HSL expression.3T3-L1 pre-adipocyte were induced in four different inducers as follows:Dex+IBMX+insulin (DMI),DMI+H89, DMI+GH, DMI+GH+H89. After two days, Western blot was used to detect the phosphorylation levels of phosphorylation of cAMP responsive element-binding protein (CREB) and hormone-sensitive lipase (HSL).Results:1. After induction, the cells of the two groups were able to differentiate, but oil red 0 staining shown that the differentiation of the growth hormone treatment group was significantly lower than that of the control group during the differentiation of fifth days. Western blot analysis of two sets of samples from different days showed that the STAT5B gene in the growth hormone treatment group was activated and the fat accumulation related proteins such as Fasn expression decreased, but the expression level of p-HSL protein was increased.2. After selected with puromycin, STAT5B expression in knocking-down group was reduced to ten percent compared to control group. The oil red O staining shown that the differentiation of STAT5B knockdown group was significantly higher than that of the control group during the differentiation of fifth days. Cells harvested from different days were analyzed by Western blot, Fasn exprssion was increased and p-HSL was down-regulated in STAT5B knock-down cells compared with control on the fifth day. When we cultured the cells for ten days after induction, the RNA levels of PPARy, CCAAT/enhancer binding protein a (C/EBPa) and Fabp4 were increased in STAT5B knock-down cells compared to control cells.3. Animal body weights were measured once a week and the body weight 20% greater than the normal diet mouse was considered to be fat. In HFD with GH group the body weight was between normal-feeding group and HFD group and the daily food intake and physical fitness levels were lower than the other two groups. Oral glucose tolerance test (OGTT) showed that the glucose tolerance in the GH injection group was higher than that in the high fat diet group (blood glucose level after 2 hours of oral glucose normal diet:8.6mmol/L, HFD with GH: 10.4 mmol/L, HFD:14.6 mmol/L), which was similar to that in the normal diet group. Analysis of biochemical parameters of serum showed that HDF with GH group had lower Triglycerides (TG), Cholesterol (CHO) levels than HFD group. We also detected Fasn, Fabp4 and p-HSL expression in adipocyte tissues but only p-HSL levels was changed between HFD with GH injection group and HFD group.4. The inducer DMI could active HSL. H89 was an inhibitor of PKA, could down-regulate p-CREB and p-HSL level. When treated with GH+DMI+H89, the p-CREB and p-HSL levels were higher than that treated with MDI+H89.Conclusions:1. Growth hormone can effect the differentiation of fat cells and attenuate the lipid accumulation caused by HFD in C57BL/6 mice WAT.2. STAT5B knockdown changes the expression of genes in the process of adipogenesis to the opposite of GH treatment such as Fasn and p-HSL. That suggests GH may regulate the fat cell lipid metabolism by STAT5B molecules.3. Growth hormone affects adipogenesis in vitro or in vivo by regulating fat accumulation by down-regulating Fasn expression and up-regulating lipolysis expression.4. GH regulats p-HSL through PKA.Part II Growth hormone signaling molecule STAT5B impacts adipocyte differentiationAim:To investigate the mechanism of STAT5B in regulating adipocytes differentiation.Method:1.3T3-L1 cells were cultured for adipogenesis and oil red O staining was applied to detect differentiation efficiency. Protein samples were collected to test the difference of STAT5B and other genes expression between the pre-adipocytes and adipocytes.2. Protein extract samples were collected in different days (day 0,2,4,6,8,10,); MOF and STAT5B proteins level were detected by western blot.3. Western blot was performed to detect the changes of MOF and STAT5B in the fat tissues of animals in the Part â… .4. The expression level of MOF was detected in the STAT5B knocking down 3T3-L1 cell.5. Dual luciferase assay were applied to detect effect of STAT5B on MOF promoter transactivation. Cells were co-transfected with 400ng luciferase reporter plasmid (pGL3-Full-length MOF promoter or pGL3-MOF promoter deletion or mutation) or PGL3-basic (control),400ng protein expression plasmid (pcDNA-STAT5B) or 400ng pcDNA (vector) and 40ng pRL-TK(a Renilla luciferase plasmid). We also did Chromatin immunoprecipitation (ChIP) in pre-adipocyte and adipocye cells to detect the physical association between STAT5B and MOF promoter.6. The MOF expression in 3T3-L1 cell was knocked down by using shRNA. After induction, samples at day 0 and day 10 were collected to see if knock-down MOF expression would effect adipocyte differentiation.7. MOF expression vector containing FLAG tags and C/EBPa promoter with reporter genes were used to detect if over-expression MOF could impact the transcription activity of C/EBPa promoter. To figure out the important C/EBPa promoter fragment related to MOF activation, we generated sires C/EBPa promoter reporters with different lenght and detect luciferase activity.Results:1. Compared with pre-adipocyte, MOF, STAT5B and H4K16ac proteins level were increased in adipocyte cells.2. The protein level of MOF was gradually elevated in the process of adipogenesis accompanied with the increase of STAT5B protein level. PPARy and oil red O staining were the critical markers for adipogenesis, which were exploited to monitor the differentiation level of preadipocytes.3. The protein levels of MOF and STAT5B were significantly increased in HFD mice adipocyte tissue compared with normal diet mice, and the expression of MOF and STAT5B was consistent with that of the cells cultured in vitro.4. MOF expression declined accompanied with the decrease of STAT5B in STAT5B knock-down 3T3-L1 cells.5. Luciferase assay showed that over-expression STAT5B and GH treatment both could improved MOF promoter trans-activity. We mutated the putative STAT5B binding site in the full-length promoter, and found that GH treatment could not activate promoter trans-activity. ChIP assay suggested that STAT5B could bind to MOF promoter at the (GAS) sequence in adipocyte cell.6. MOF expression was knocked-down to 30 percent compared to control group by shRNA. Cells harvested were analyzed for ten days after initial adipogenesis, Western blot and qPCR results shown that Fasn, PPARy and Fabp4 exprssion were increased in MOF knock-down cells compared to control cells.7. Over-expression MOF in HeLa cell could inhibit C/EBPa promoter trans-activity, but had no effect on PPARy promoter. Further analysis showed that there was no inhibitory effect of MOF on C/EBPa trans-activity when the upstream of the C/EBPa promoter from 800bp to 200bp were deleted.Conclusion:1. STAT5B and MOF expression are different between 3T3-L1 pre-adipocyte and 3T3-Lladipocyte cells.2. STAT5B regulates adipocyte differentiation partially through modulating MOF expression.3. Knocking-down MOF expression in 3T3-L1 pre-adipocyte resulted in increased expression of adipogenesis activators during differentiation.4. Over-expression MOF attenuates C/EBPa promoter trans-activation.5. STAT5B and MOF feedback regulates adipocyte differentiation.