The Impact Of Growth Hormone And STAT5B Signaling On Adipocyte Differentiation And The Associated Crosstalk With Insulin

Obesity is a chronic metabolic disease owing to fat over-storage or accumulation. The development of obesity will further result in higher rate of vascular diseases, metabolic syndrome and type2diabetes. The adipocytes of our body are differentiated from pre-adipocytes, therefore, pre-adipocyte over-differentiation or over-enlargement is closely related to obesity formation.Growth hormone (GH), a22-kDa peptide, is secreted in anterior pituitary, plays important roles in regulation of growth and metabolism. The GH association with pre-dimmer of GH receptor activates JAK2and triggers a series of signal pathways. Signal transducer and activatorof transcript5(STAT5) is the downstream signaling molecule. There are two types of STAT5molecules, STAT5A and STAT5B, from different chromosomes, but their sequences are highly homologous (96%identity). Transgenic animal studies show that GH receptor lack of STAT5activation sequence will result in diminished-sized animal and obesity. STAT5B has important role in GH signaling, while its role in modulating adipocyte differentiation and adipocyte accumulation is far from being clarified. Understanding the mechanism of GH modulating adipocytes maturation will provide the theoretical base for people to tackle the problem of obesity and will also provide clues for new anti-obesity drug exploiting. GH and insulin play important roles in regulation body development and metabolism. These two hormones work coordinately to maintain the metabolic homeostasis. Both insulin and GH signaling share PI3K and ERK pathways, while GH has an additional signal pathway that is STAT5pathway. It is worth exploring that if these two hormones (GH and insulin) have some synergic effect or inhibitory effect on the fate of adipocyte. Our works includes three parts:Part I The impact of growth hormone signaling on gene transcription in differentiated3T3-F442A adipocytesAims:To investigate the effect of GH on gene expression in mature adipocytes and on pre-adipocytes differentiation.Methods:1.3T3-F442A pre-adipocytes were induced to differentiate as described in literature. The morphology change was observed under inverted microscope; Oil red O staining was applied; absorbance values at OD49onm were measured to further verify the pre-adipocyte differentiation;2. To further verify the pre-adipocyte differentiation, the expression levels of the essential regulators and markers for adipocyte differentiation, such as PPARy, C/EBPβ and FAS, were analyzed by RT-PCR;3. DNA microarray assay was exploited to screen the target genes enhanced by GH stimulation in mature3T3-F442A adipocytes; the differentially expressed genes were verified by RT-PCR;4. Differentiated3T3-F442A adipocytes were treated with GH at125ng/mL (equal volume of PBS was used to as control) for24h and48h and the mRNA levels of SOCS family, adiponectin and its receptors in these adipocytes were determined;5. Differentiated3T3-F442A adipocytes were treated with GH at125ng/mL (equal volume of PBS was used to as control) for24h and48h and the mRNA levels of genes related to fatty synthesis and lipolysis were analyzed;6. To investigate the role of GH in F442A differentiation, the pre-adipocytes cells were cultured in differentiation medium, or in differentiation medium plus GH (125ng/mL); cells were harvested at day0,2,4,6,8,10and12. The adipocyte differentiation marker gene (C/EBPP) and fatty acid synthesis (FAS) were determined by RT-PCR.Results:1. After differentiation, the cytoplasmic lipid droplets could be detected by Oil-red-O staining as early as day4post differentiation induction, and reached its peak on day12; OD49onm value of Oil-red-O staining extract increased with time on. Adipocytes differentiation successfully reaches.2. The mRNA and protein levels of PPARy were significantly increased from day4post differentiation induction, compared with control cells (DO)(p<0.05). RNA expressions of C/EBPβ and FAS increased significantly after induction.3. DNA Micoroarray analysis showed that Adipor2and SOCS2up-regulated under GH-treatment in mature3T3-F442A adipocytes.4. Compared with negative control cells (treated with PBS), under125ng/mL GH-treatment, IGF-I expression increased; gene expression of SOCS family, such as CIS, SOCS1, SOCS2and SOCS3, increased at24h; the expression of Adiporl and2significantly increased. These data indicated that GH modulates the expression of some proteins in mature3T3-F442A cells.5. Compared with control cells (treated with PBS), differentiated mature3T3-F442A cells treated with125ng/mL GH resulted in FAS, FABP and PPARy increased expression while no expression changes of C/EBPp, HSL, ATGL were observed.6. GH interference in pre-adipocyte differentiation experiment showed that mRNA levels of C/EBPβ and FAS decreased at different time points of adipogenesisunder125ng/mL GH treatment. But GH did not stop the adipocytes development progress. Conclusions:1. The Model of3T3-F442A adipocytes differentiation was successfully established.2. Micoroarray analysis showed that GH-treatment resulted in lipolysis in mature3T3-F442A adipocytes and promoted metabolism.3. In pre-adipocytes differentiation progress, GH hadsome inhibitory effect, but did not stop the differentiation progress. Part Ⅱ Possible role of STAT5B in adipocytes differentiationAim:To investigate the probable role of STAT5B in adipogenesis of3T3-L1pre-adipocytes in vitro.Methods:1. STAT5A and STAT5B mRNA levels during the differentiation of3T3-L1pre-adipocytes were analyzed;2. Lentivirus encoding shRNA targeting to STAT5B was designed, generated and described as LV-STAT5B-shRNA, the control lentivirus marked as LV-NC.3T3-L1pre-adipocytes were infected with these two viruses separately and stable cell polls were selected under drug pressure. Selected cell pool with STAT5B knock-down was designated as KD and cell pool selected from control virus infected cells was designated as NC;3. The Roles of STAT5B in adipogenesis in3T3-L1pre-adipocytes were evaluated. KD and NC cells were induced for adipogenesis, at different time points samples were collected for RNA purification, some differentiation associated genes were analyzed by RT-PCR;4. Effect of GH-interference during adipogenesis was investigated in both KD cells and NC cells.125ng/mL GH was applied to interfere the differentiation of both KD and NC cells and the expression levels of genes related to fatty synthesis and lipolysis were analyzed at different time points during differentiation.Results:1. Compared with un-induced3T3-L1pre-adipocytes, STAT5B expression level was significantly increased at day2.2. Lentivirus-mediated STAT5B gene silencing was detected in3T3-L1pre-adipocytes. The mRNA and protein expression levels of STAT5B were reduced to30%and10%in KD cells, respectively, in comparison with NC cells.3. During adipogenesis induction, the regulators and markers for adipocyte differentiation, such as PPARa, PPARy, C/EBPa and C/EBPβ were reduced in KD cells, in comparison with NC cells.4. At the same differentiation phase, the expressions of FAS and FABP were reduced in KD cells. No significantly difference between KD cells and NC cells as regard to Acetyl-CoA carboxylase (ACC) expression was detected.5. GH inhibited expression of adipogenesis regulators(PPARa,PPARy, C/EBPaand C/EBPβ) and lipid synthesis related genes (FAS and FABP) at Day6both in KD and NC cells, and the inhibition effects of GH in NC cells were more remarkable.6. During adipogenesis in culture, GH treatment up-regulated ATGL (adipose triglyceride lipase), a lipolysis related gene. GH markedly enhanced UCP1expression during adipogenesis in culture in NC cells not in KD cells, which showed the enhanced expression of UCP1is in a STAT5B dependent manner.Conclusions:1. During the process of adipogenesis of3T3-L1preadipocyte, the expression of STAT5B increased to some point.2. shRNA mediated STAT5B knockdown affected the expression of some important genes for adipogenesis, such as PPARa,PPARy, C/EBPaand C/EBPβ, FAS and FABP. 3. GH interference resulted in down-regulation of adipogenesis regulators and lipid synthesis genes during adipogenesis inculture both in NC and KD cells, especially in NC cells.4. During adipogenesis in culture, GH treatment enhanced the expression of ATGL, GH treatment markedly enhanced the expression of UCP1in NC cells, not in KD cells, which means the enhancement of expression of UCP1is in a STAT5B dependent manner. Part Ⅲ Effects of insulin and IGF-I on growth hormone-induced STAT5activation in3T3-F442A adipocytesAim:To investigate the role of insulin and IGF-I on GH-induced STAT5activation in3T3-F442A adipocytes in vitro and in vivo.Methods:1. Differentiated F442A adipocytes were starved in culture medium containing0.5%(w/v) bovine serum albumin (BSA) for16h before stimulation. For dose-response experiments, serum-starved cells were treated with:GH (0,5,25,50,125, or500ng/mL) for10min;2. For time course experiments, serum-starved cells were treated with:GH (125ng/mL) for different periods (0,1,3,5,7,10or15min), IGF-I (100ng/mL) or insulin (200nM) for different periods (0,1,5,10,20or30min);3. GH (0,5,25,50, or125ng/mL) plus200nM insulin for10min; GH in the presence of insulin (10,100or200nM) for30min (added20min prior to GH); GH plus100ng/mL IGF-I for10min; or GH plus IGF-I for30min (100ng/mL IGF-I was added20min prior to GH). Stimulation was terminated by washing cells twice with an ice-cold phosphate-buffered saline (PBS) in presence of0.4mM sodium orthovanadate;4. Female C57/BL6mice, aged12-weeks, weighing20-24g, were randomly divided into three groups:the "control group","GH group", and "GH+insulin group". GH group mice were injected intraperitoneally (i.p.) with50μg of GH per kg of body weight (BW) in0.2ml saline for10min before sacrifice. GH+insulin group mice were first injected i.p. with2μmol insulin/kg.BW in0.1mL saline and20min later with50μg/kg.BW GH in0.1mL saline before sacrifice10min thereafter. The control mice were injected with saline. Prior to injection, all mice were fasted overnight. After treatment, mice were sacrificed by decapitation under anesthesia with10%chloral hydrate. Visceral adipose tissues around the kidneys were collected for western blot analysis.Results:1. Results of western blot showed that GH induced STAT5tyrosine phosphorylation in differentiated3T3-F442A cells in dose-and time-dependent manners;2. Insulin (200nM) or IGF-I (100ng/mL) induced maximum MAPK activation at10min or5min respectively, but did not induce detectable STAT5tyrosine phosphorylation;3. Results showed that GH and insulin co-treatment significantly increased STAT5phosphorylation compared to GH alone; insulin pretreatment for20min further increased GH-induced STAT5phosphorylation compared to GH and insulin co-treatment;4. Insulin pretreatment before GH treatment increased STAT5phosphorylation in adipose tissues by60%as compared to mice treated with GH alone;5. IGF-I co-treatment with GH enhanced GH (5ng/mL) induced STAT5and had an additive effect on GH induced MAPK activation; IGF-I pretreatment for20min enhanced STAT5activation induced by GH of varying concentrations (GH5,25,50ng/mL); but did not change the profile of MAPK. Conclusions:1. GH specifically induced phosphorylation of STAT5but not insulin or IGF-I.2. Insulin specifically potentiates GH-induced STAT5activation in mature adipocytes both in vitro and in vivo. Insulin and GH might act synergistically to regulate some specific functions in mature adipocytes.3. As IGF and insulin have similarities in their mode of signaling and functions, IGF-Ialso promoted GH-induced activation of STAT5in our findings.