Effect Of Protein Tyrosine Phosphatase 1B In The Differentiation Of 3T3-L1 Preadipocyte

Part I The Cultivation, Differentiation and Identification of 3T3-L1 Preadipocytes3T3-L1 preadipocyte cell line, one of the most used preadipocyte cell lines, was derived from immortalized 19 day-nonclonal Swiss 3T3 embryonic fibroblast cells of mesenchymal origin and is already committed to the adipocytic lineage. When treated with an empirically derived, prodifferentiation regimen that includes cAMP, insulin, and glucocorticoids, these cells undergo differentiation to mature fat cells over a 4- to 6-d period. Within 24–36 h of hormonal induction, cells re-enter the cell cycle, undergo one to two rounds of mitosis or clonal expansion, after which they permanently withdraw from the cell cycle, begin to accumulate lipid, and undergo terminal differentiation into mature adipocytes. As the good simulation of adipocyte differentiation and function of live adipocyte, 3T3-L1 preadipocyte cell line has been thought as one of the best characterized and widely used invitro models to study adipocyte differentiation. The cultural condition, optimal medium and inducer also make great sense to the livingness and potency of differentiated adipocyte. In this part of our study, with the observation of the resuscitation, growth, confluence and differentiation of 3T3-L1 preadipocytes, we make great efforts to get the optimal condition(DMEM with high glucose level and supplemented with 10%FBS, 100u/ml penicillin and streptomycin ) and inducing concerntration of differentiation(0.5 mmol/L MIX, 0.25 umol/ L DEX and 10ug/ ml INS), and we used oil red O staining to identify the fully differentiated adipocytes, all of which make good basis for the after work of our study. Part II The Expression of Protein Tyrosine Phosphatase 1B during The Differentiation of 3T3-L1 PreadipocyteAs one of the first purified protein tyrosine phophatases, PTP1B has been thought as the negative regulator of insulin signal transducting. The researches about the inhibition of PTP1B have been new hotspots for the treatment of metabolic diseases such as obesity and type 2 diabetes.It has already been confirmed that whether knockout PTP1B-/- mice or PTP1B antisense oligonucleotide(ASO) pretreated mice showed enhanced phosphorylation of insulin receptor(InsR) and insulin receptor substrate(IRS) in liver and skeletal muscles, with enhanced insulin signal transducting and improvement of insulin sensitivity. But in adipose tissue, these studies gave us so different results. There is no change in the phosphorylation of InsR and IRS-1 in adipose tissue, and both the highest level of phosphorylation in InsR and the time needed to get that highest level being unaltered. PTP1B ASO can decrease the weight of ob/ob mice and diminish the expression of many genes involved in adipogenesis such as sterol regulatory element-binding protein( SREBP-1) without any changes in phosphorylation of insulin related protein kinase B(PKB) and in insulin induced glucose transportion. Another study indicated that the mice specially knock out PTP1B-/- in adipose tissue got weight gain. So what is the exact effect of PTP1B on adipose tissue?This time we first use 3T3-L1 preadipocytes and investigate the expression level of PTP1B during the differentiation of adipocytes, to elucidate the effect of PTP1B in the differentiation of adipocytes and in the course of lipids accumulation.In this study we use 3T3-L1 preadipocytes cultured in vitro and induced them with regular inducer(MIX+DEX+INS). Oil Red O stain was used to clarified matural adipocytes. RT-PCR and Real Time FQ-PCR were used to assess the level of PTP1B mRNA, and western blotting was used to detect the protein level of PTP1B. Our study shows that with the relatively high level in 3T3-L1 preadipocytes, both the expression of mRNA and protein level of PTP1B went down with the differentiation of adipocytes, and reached the lowest level in matured adipocytes. So we can conclude that the declining of PTP1B level during the differentiation of adipocyte maybe a promoter for adipocyte maturation. Part III The Impacts of TNF-alpha And Rosiglitazone on The Expression of Protein Tyrosine Phosphatase 1B During The Differentiation of 3T3-L1 PreadipocytesAdipose tissue has been accepted as an important endocrinal organ which can secrete so many hormones and cytokines such as leptin, resistin, adiponectin and tumor necrosis factor participating in more and more metabolic regulation. It has been confirmed that it is the abnormal adipocyte differentiation, but not the adiposity, is the culprit of metabolic syndrome such as type 2 diabetes, hypertension and coronary heart disease. Central obesity which is closely related with diabetes and coronary heart disease It is due to the dysfunction of adipocyte differentiation, which can not produce enough adipocytes to storage more energy, that causes ectopic aggradation of lipids in liver or muscles, inducing so many metabolic disorders.PTP1B, that can dephosphorylate the tyrosine in InsR and IRS-1, has been thought as the negative regulator of insulin signal transducting. But there is obvious tissue specificity of the effect of PTP1B. In liver and skeletal muscles, the lower level of PTP1B, the more of phosphorylation in InsR and IRSs, and the higher level of insulin stimulated whole-body glucose disposal. But to adiopose tissue, the exact effect is still not so clear, just as the relationship between PTP1B and insulin sensitivity.So we use in vitro cultured 3T3-L1 adipocytes to investigate the expression of PTP1B protein level and the phosphorylation level of insulin receptor, and their changes under the influences of tumor necrosis factor-alpha and rosiglitazone during the differentiation of 3T3-L1 adipocytes. We hope to get out the real effect of PTP1B in adipogenensis and the possible mechanisms of rosiglitazone's and tumor necrosis factor-alpha's influence on insulin sensitivity adipocyte.3T3-L1 preadipocytes were cultured in vitro and induced with three types of inducers, regular inducer( MIX+DEX+INS), regular inducer plus 20 ug/l TNF-α(CT) and regular inducer plus 10-5mol/l rosiglitazone(CR). Western blotting was used to detect the level of PTP1B protein in each group during the differentiation of adipocytes.Immunoprecipitation and western blot were used to detect the expression and phosphorylation level of insulin receptor in matured adipocytes of the three groups.Our study showed that compared with regular inducers, CR could promote the differentiation of adipocytes, while CT could retard this programme. Each group showed the relatively high level of PTP1B in 3T3-L1 preadipocytes, going down with the differentiation of adipocytes, and reaching the bottom in fully-matured adipocytes. Comparing the late period of differentiation in these three groups, CT one were sluggishly differentiated with more PTP1B protein, and CR group showed active differentiation with the lowest level of PTP1B. The expression of insulin receptor showed no difference in the three groups, but the phosphorylation level of tyrosine in insulin receptor decreased obviously in CT group, and increased dramatically in CR group.So we can conclude that the declining of PTP1B level and its activity during the differentiation of adipocytes is in favor of lipid accumulation and maybe a promoter for adipocytes maturation. The effects of TNF-αand rosiglitazone on adipocytes'sensitivity to insulin perhaps partly depended on their influences on PTP1B level and its activity in adipocytes.