Protein Tyrosine Phosphatase1B Inhibits Adipogenesis And Mediates TNFα Action In Obesity

Protein tyrosine phosphatase1B (PTP1B) is a ubiquitously expressednon-transmembrane protein tyrosine phosphatase. It is associated with numerous diseases,including cancer, metabolic and cardiovascular diseases, autoimmune and neurologicaldisorders. PTP1B becomes the target of intensive investigation because of its importancein glucose and insulin homeostasis. Global PTP1B knockout mice exhibit remarkablylower adiposity and are protected against diet-induced obesity, but several studies haveshown that this effect seems tissue-specific. Adipose-specific PTP1B deletion do notcontribute to the beneficial effect on body fat; furthermore, the insulin-stimulated receptorphosphorylation or glucose transport in isolated adipose tissue from total andadipocyte-specific PTP1B knockout mice have no difference compared with controls,suggesting an inessential role of PTP1B on insulin signaling in adipocytes. However,accumulating evidence shows that PTP1B is up-regulated in adipose tissue of obesity. Thispromotes us to explore the possibility that PTP1B is involved in other important functionsrather than insulin sensitivity in adipose.As the essence of obesity, the imbalance between energy intake and expenditure leadsto a pathologic accumulation of lipid. Adipose cells become hypertrophy in response toexcess energy, which is accompanied with a failure of adipocyte differentiation. Henceobese individuals are difficult to produce new fat cells, resulting in a lipid overload and theflow of fatty acids into circulation, muscle, liver and even β-cell. During this progression,the actions of pro-inflammatory adipokine TNFα plays an important role. The expressionof TNFα in adipose tissue is induced in variety of obese rodents and humans, whichimpairs the differentiation of preadipocytes and finally contributes to an alteration ofadipose microenvironment. But the underlying molecular mechanism has not beenunderstood currently. On the other hand, several studies have shown that PTP1B may beinvolved in the transcription of adipogenic markers, with ambiguous results derived fromdifferent experimental methods and subjects.Here, we explore the role of PTP1B in adipocyte differentiation, and its action inTNFα inhibiting adipocyte differentiation in obesity, to reveal the significance of PTP1B inthe obesity and provide new theory for its clinical application. Part Ⅰ. PTP1B acts as a negative regulator of adipocytedifferentiationLentiviral vector encoding mouse PTP1B cDNA sequence, RNA interference oligoand active sites mutant cDNA sequence were constructed and transfected into mouse3T3-L1white preadipocytes respectively. Among them, the mutant (PTP1B-D/A-Y/F) wasintroduced as sequence mutant control with two key residues within catalytic domain,Tyr-46and Asp-181, replaced by Phe and Ala respectively. Therefore it retains the abilityto bind substrates without catalysis. The gene intergration did not weaken withdifferentiation, passage or freeze determined by EGFP fluorescence. Intracellular PTP1Bexpression was raised by70%in cells with PTP1B overexpression, reduced by87%and41%in cells with PTP1B knockdown and mutant respectively, indicating a successfulconstruction of three cell models. Transfected cells were induced to differentiation byclassic “cocktail” and harvested at eight days after to evaluation experiments. PTP1Boverexpression delayed the differentiation of adipocytes, since the maturation efficiencywas less than60%, the morphology of adipocytes was indistinctive, and the absorbance ofOil Red O staining was only58.8%of the corresponding control. In contrast, PTP1Bknockdown accelerated the differentiation, since almost all cells demonstrated a maturemorphology, and absorbance of Oil Red O staining was higher than control. Mutantoverexpression also showed observed enhancement in adipocyte differentiation, withhigher level in both the maturation efficiency and Oil Red O absorbance. Several importantadipogenic markers, PPARγ2, SREBP-1c, FAS and LPL, were markedly down-regulatedby overexpression of PTP1B, and up-regulated by knockdown and mutant of PTP1B,consistent with the morphology of the cells. In particular, PPARγ2was strongly affected byPTP1B since it was suppressed to10.9%in overexpression group and induced to8-fold inknockdown group. SREBP-1c was also markedly induced to more than12-fold inknockdown group. Of note, C/EBPα expression was not significantly affected by bothoverexpression and knockdown of PTP1B.During terminal differentiation, PPARγ2and C/EBPα are induced before transcriptionof most adipocyte genes and sustain high level in mature adipocyte. Besides, SREBP-1c isalso stimulated early and subsequently activates numerous fatty acid biosynthetic genessuch as FAS and LPL. Our data suggested that PTP1B regulates white preadipocytedifferentiation and lipogenesis probably through the modulation of PPARγ2expressioninstead of C/EBPα. SREBP-1c and its target genes changed in parallel with differentiation.In addition, we demonstrated a dominant-negative inhibition of PTP1B function by PTP1B-D/A-Y/F double mutant, which has not been reported before. This effect may becaused by the powerful ability of PTP1B-D/A-Y/F to interact with numerous substrateswithout catalysis, leading to a competitive inhibition of endogenous PTP1B. Moreover, theintegration of PTP1B-D/A-Y/F gene into host genome seemed to downregulate theexpression of wide-type PTP1B. Summarily, this part comprehensively demonstratesPTP1B is an important regulator of adiponesis by three cell model that elevate, knockdown and dominant-negative inhibit PTP1B expression respectively.Part Ⅱ. PTP1B mediates TNFα-induced insufficiency of adipocytedifferentiation in obesityBased on the established regulation of PTP1B in adipogenesis, we next examined thepossibility that PTP1B participated in TNFα action in obesity. In high fat diet-inducedobesity mice, PTP1B overexpression in epididymal adipose tissue coincides with increasedexpression of pro-inflammatory adipokine TNFα and down-regulation of PPARγ2. Thecontinued treatment of TNFα enhanced PTP1B expression but inhibited differentiation of3T3-L1preadipocytes, whereas PTP1B knockdown significantly prevented the inhibitoryeffect of TNFα on adipocyte differentiation. These results indicate an essential role ofPTP1B in TNFα-induced inhibition on adipocyte differentiation. Finally, to investigate thein vivo action of PTP1B in adipocytes, obese (ob/ob) mice were treated with PTP1Binhibitor vanadate for6weeks. There was no significant change in adipose mass besides areduction trend in weight gain in vanadate-treated group. The expression of adipogenicgenes including SREBP-1c and FAS was induced by vanadate in adipose tissue, althoughthe increase of PPARγ2was not significant. TNFα was down-regulated by vanadate inadipose tissue of obese mice, accompanied with a decreased phosphorylation level ofNF-κB p65subunit.Adipocyte dysfunction is the central pathological change of obesity. The lowproliferation and differentiation capability of preadipocyte is an important factor of insulinresistance, metabolism syndrome and type2diabetes. Our study supposes that PTP1B maybuild a bridge between inflammatory infiltration and adipocyte dysfunction in obesity, bymediating TNFα action in adipocyte differentiation; PTP1B inhibitor treatment mayremodel adipocyte function and further alleviate inflammatory lesion induced by excessivefatty acids in obesity. Furthermore, NF-κB p65subunit may serve as the intermediatebetween TNFα and PTP1B, which is also supported by our previous study. In summary, we propose the implication of PTP1B in obese adipose tissue: with theprogress of obesity, enlarged adipocytes reach an overload in excess energy storage and theendocrine function of adipose tissue is affected; numerous TNFα derived from adipocytesand macrophages suppresses the transcription of PPARγ2, SREBP-1c, FAS and LPL inpreadipocytes largely through enhancing PTP1B expression, finally leading to aninsufficient differentiation of adipocytes. In turn, the loss of ability to differentiate newadipocytes aggravates the overload of lipid accumulation in adipose tissue, resulting in avicious circle. Our study provides novel evidence for the important role of PTP1B inobesity, that PTP1B inhibition in obese adipose may remodel adipocyte function andfurther alleviate inflammatory lesion induced by excessive fatty acids.