蛋白酪氨酸磷酸酶1B抑制成脂并介导TNFα在肥胖中的作用
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摘要
蛋白酪氨酸磷酸酶1B（PTP1B）是机体广泛表达的非受体型酪氨酸磷酸酶，与肿瘤、代谢、心血管、自身免疫及神经功能紊乱等许多疾病相关，尤以其对葡萄糖及胰岛素稳态的负性调节而受到密切关注。PTP1B敲除小鼠呈现显著的低体重、低体脂及对饮食诱导肥胖的抵抗，然而这种现象似乎存在一定的组织特异性：脂肪特异性PTP1B敲减小鼠并未出现良性体重调节，而PTP1B缺乏的离体脂肪组织中亦未见胰岛素受体磷酸化水平及葡萄糖摄取功能的变化，提示PTP1B对脂肪组织中的胰岛素信号转导可能无主导作用。与此同时，大量证据显示肥胖脂肪组织中普遍存在PTP1B表达的上调，这让我们不禁思考其在调节胰岛素敏感性以外的重要意义。
肥胖的实质是能量摄入与消耗失衡所引起的病理性脂质累积。脂肪细胞为储存过剩的能量而肥大并继发分化功能障碍，肥胖个体无法产生新的储脂细胞，过载的脂质流入循环、骨骼肌、肝脏甚至胰岛β细胞，形成异位沉积。在这一过程中，促炎脂肪因子TNFα的活化发挥了重要作用。TNFα在各种肥胖动物模型及人群的脂肪组织中亦存在显著的过表达，并引发包括分化功能障碍在内的脂肪微环境病理改变，然而其分子机制尚未阐明。另一方面，PTP1B似乎与部分成脂相关基因的转录相关，但现有研究结果因实验条件和对象不同而模棱两可。
因此，本研究拟探讨PTP1B对脂肪分化功能的影响及作用位点，并在此基础上初步验证其对肥胖时炎症与脂肪功能障碍的贡献，以期揭示PTP1B在肥胖发生发展中的意义，并为其临床应用提供新的理论依据。
一、PTP1B对成脂的负性调节作用
我们分别构建了携带PTP1B cDNA序列、RNA干扰寡核苷酸及活性位点突变DNA序列的慢病毒并转染3T3-L1小鼠白色前脂肪细胞系。其中PTP1B双突变体（PTP1B-D/A-Y/F）替换了其分子催化域中两个关键氨基酸Tyr-46和Asp-181，使其保留与底物结合的能力而失去催化活性，在本研究中作为序列突变对照被引入。对病毒绿色荧光蛋白的检测显示整合基因不受细胞传代、分化甚至冻存的影响；过表达组细胞内PTP1B蛋白水平升高1.71±0.02倍，而敲减组则减少至13.14±0.04%，突变组亦降低至58.83±0.27%，提示PTP1B稳定过表达、敲减及突变脂肪细胞模型的建立。以经典“鸡尾酒”法诱导三组细胞分化，于诱导后第8天收集细胞并评估分化情况。结果显示：过表达组细胞分化明显延迟，成熟脂肪细胞比率不足60%，且形态不典型，油红O染色吸光度仅为对照的67.15±0.62%；敲减组分化显著增强，几乎均呈成熟脂肪细胞形态，细胞变圆变大，胞浆内脂滴丰富，油红O染色吸光度亦明显高于对照；突变组呈现与敲减模型相同的成脂促进作用，其成熟脂肪细胞比率及油红O染色吸光度亦显著高于对照。对成脂重要标志物的检测也得到相同结果：过表达组PPARγ2、SREBP-1c、FAS及LPL基因表达显著下调，而敲减及突变组则均增强；其中PPARγ2变化最大，在过表达组抑制至11.05±1.43%，而在敲减组则升高8.14±0.46倍，SREBP-1c也在敲减组升高12.52±1.41倍，C/EBPα的表达则在三组均无改变。
在脂肪细胞的终末分化中，PPARγ2与C/EBPα同在早期活化，并在成熟脂肪细胞中维持高水平；SREBP-1c在分化前期被激活，并继而活化包括FAS、LPL在内的众多脂肪酸合成基因。我们的数据显示，PTP1B主要通过下调PPARγ2而非C/EBPα的转录水平抑制成脂，同时伴有脂质合成基因SREBP-1c、FAS、LPL等的平行变化。PTP1B-D/A-Y/F突变体过表达对PTP1B有显著的负显性抑制作用，这在以往应用底物捕获突变体的研究中从未报道。这种负显性抑制作用主要源于突变体“捕获”底物的高效性：强有力地与底物结合而不催化，从而竞争性抑制了内源性PTP1B的功能；而PTP1B-D/A-Y/F慢病毒对宿主基因组的整合似乎也能下调内源性PTP1B的表达。本部分通过特异性PTP1B过表达、敲减及负显性突变三种细胞模型，一致证实了PTP1B对白色前脂肪细胞成脂的重要调节作用。
二、PTP1B介导肥胖时TNFα诱导的脂肪分化不良
为进一步阐明PTP1B对脂肪分化调节作用在肥胖中的作用，我们对高脂饮食诱导肥胖小鼠的附睾脂肪组织进行检测。PTP1B与促炎脂肪因子TNFα在肥胖脂肪组织中存在明显重叠的过表达，同时伴有脂肪分化关键因子PPARγ2表达的下调。TNFα持续干预使3T3-L1前脂肪细胞分化能力明显受损，诱导后第8天分化率仅50～60%；而PTP1B表达却得到增强，尤其在第6-8天其蛋白水平为对照的2倍以上。然而在PTP1B功能受到抑制的突变体过表达3T3-L1细胞上，TNFα对脂肪分化的干预也明显减弱，提示PTP1B在TNFα的成脂抑制作用中扮演重要角色。最后，我们回到肥胖小鼠上探讨PTP1B对成脂调节的体内变化。以PTP1B经典抑制剂钒酸钠处理6周的肥胖（ob/ob）小鼠，其饮水量受影响，摄食、总体重与对照比较无差异，增重呈下降趋势；葡萄糖耐量在60、120min时明显改善。附睾脂肪组织比率无改变，但脂肪组织局部SREBP-1c、FAS等脂质合成基因明显上调，PPARγ2表达略上升但无统计学意义；TNFα基因表达及其下游重要靶点NFκB活性下调。
脂肪细胞功能失调是肥胖等许多代谢疾病的中心病理环节。前脂肪细胞增殖及分化能力障碍是脂肪细胞参与胰岛素抵抗、代谢综合征和2型糖尿病发生的重要因素。我们的结果提示PTP1B可能是肥胖时连接炎症浸润与脂肪功能受损的重要桥梁，其在肥胖脂肪组织中被TNFα过度激活，并介导了成脂抑制作用；而PTP1B抑制剂治疗可能通过改善肥胖动物脂肪组织的成脂功能，减轻游离脂肪酸过载，而减轻局部炎症反应，最终为整体胰岛素抵抗的改善做贡献。NFκB可能是PTP1B与TNFα相互作用的中间环节。
综上所述，我们提出PTP1B参与肥胖脂肪组织病理变化的可能机制：随着肥胖的进展，增大的脂肪细胞储脂能力超负荷，脂肪组织内分泌功能受损，大量分泌的TNFα通过激活PTP1B的表达抑制前脂肪细胞内PPARγ2、SREBP-1c、FAS、LPL等重要成脂因子的转录，最终导致脂肪细胞分化不足；而新分化储脂细胞的减少又反过来加重脂质过载，形成恶性循环；因此，抑制PTP1B的表达可能削弱肥胖时炎症对脂肪细胞功能的损害。本研究首次阐明了PTP1B在脂肪组织中的作用，尤其是其参与肥胖发生发展的可能机制，为其抑制剂的临床应用提供了新的理论依据。
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 adipocytedifferentiation
Lentiviral 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 obesity
Based 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.
肥胖的实质是能量摄入与消耗失衡所引起的病理性脂质累积。脂肪细胞为储存过剩的能量而肥大并继发分化功能障碍，肥胖个体无法产生新的储脂细胞，过载的脂质流入循环、骨骼肌、肝脏甚至胰岛β细胞，形成异位沉积。在这一过程中，促炎脂肪因子TNFα的活化发挥了重要作用。TNFα在各种肥胖动物模型及人群的脂肪组织中亦存在显著的过表达，并引发包括分化功能障碍在内的脂肪微环境病理改变，然而其分子机制尚未阐明。另一方面，PTP1B似乎与部分成脂相关基因的转录相关，但现有研究结果因实验条件和对象不同而模棱两可。
因此，本研究拟探讨PTP1B对脂肪分化功能的影响及作用位点，并在此基础上初步验证其对肥胖时炎症与脂肪功能障碍的贡献，以期揭示PTP1B在肥胖发生发展中的意义，并为其临床应用提供新的理论依据。
一、PTP1B对成脂的负性调节作用
我们分别构建了携带PTP1B cDNA序列、RNA干扰寡核苷酸及活性位点突变DNA序列的慢病毒并转染3T3-L1小鼠白色前脂肪细胞系。其中PTP1B双突变体（PTP1B-D/A-Y/F）替换了其分子催化域中两个关键氨基酸Tyr-46和Asp-181，使其保留与底物结合的能力而失去催化活性，在本研究中作为序列突变对照被引入。对病毒绿色荧光蛋白的检测显示整合基因不受细胞传代、分化甚至冻存的影响；过表达组细胞内PTP1B蛋白水平升高1.71±0.02倍，而敲减组则减少至13.14±0.04%，突变组亦降低至58.83±0.27%，提示PTP1B稳定过表达、敲减及突变脂肪细胞模型的建立。以经典“鸡尾酒”法诱导三组细胞分化，于诱导后第8天收集细胞并评估分化情况。结果显示：过表达组细胞分化明显延迟，成熟脂肪细胞比率不足60%，且形态不典型，油红O染色吸光度仅为对照的67.15±0.62%；敲减组分化显著增强，几乎均呈成熟脂肪细胞形态，细胞变圆变大，胞浆内脂滴丰富，油红O染色吸光度亦明显高于对照；突变组呈现与敲减模型相同的成脂促进作用，其成熟脂肪细胞比率及油红O染色吸光度亦显著高于对照。对成脂重要标志物的检测也得到相同结果：过表达组PPARγ2、SREBP-1c、FAS及LPL基因表达显著下调，而敲减及突变组则均增强；其中PPARγ2变化最大，在过表达组抑制至11.05±1.43%，而在敲减组则升高8.14±0.46倍，SREBP-1c也在敲减组升高12.52±1.41倍，C/EBPα的表达则在三组均无改变。
在脂肪细胞的终末分化中，PPARγ2与C/EBPα同在早期活化，并在成熟脂肪细胞中维持高水平；SREBP-1c在分化前期被激活，并继而活化包括FAS、LPL在内的众多脂肪酸合成基因。我们的数据显示，PTP1B主要通过下调PPARγ2而非C/EBPα的转录水平抑制成脂，同时伴有脂质合成基因SREBP-1c、FAS、LPL等的平行变化。PTP1B-D/A-Y/F突变体过表达对PTP1B有显著的负显性抑制作用，这在以往应用底物捕获突变体的研究中从未报道。这种负显性抑制作用主要源于突变体“捕获”底物的高效性：强有力地与底物结合而不催化，从而竞争性抑制了内源性PTP1B的功能；而PTP1B-D/A-Y/F慢病毒对宿主基因组的整合似乎也能下调内源性PTP1B的表达。本部分通过特异性PTP1B过表达、敲减及负显性突变三种细胞模型，一致证实了PTP1B对白色前脂肪细胞成脂的重要调节作用。
二、PTP1B介导肥胖时TNFα诱导的脂肪分化不良
为进一步阐明PTP1B对脂肪分化调节作用在肥胖中的作用，我们对高脂饮食诱导肥胖小鼠的附睾脂肪组织进行检测。PTP1B与促炎脂肪因子TNFα在肥胖脂肪组织中存在明显重叠的过表达，同时伴有脂肪分化关键因子PPARγ2表达的下调。TNFα持续干预使3T3-L1前脂肪细胞分化能力明显受损，诱导后第8天分化率仅50～60%；而PTP1B表达却得到增强，尤其在第6-8天其蛋白水平为对照的2倍以上。然而在PTP1B功能受到抑制的突变体过表达3T3-L1细胞上，TNFα对脂肪分化的干预也明显减弱，提示PTP1B在TNFα的成脂抑制作用中扮演重要角色。最后，我们回到肥胖小鼠上探讨PTP1B对成脂调节的体内变化。以PTP1B经典抑制剂钒酸钠处理6周的肥胖（ob/ob）小鼠，其饮水量受影响，摄食、总体重与对照比较无差异，增重呈下降趋势；葡萄糖耐量在60、120min时明显改善。附睾脂肪组织比率无改变，但脂肪组织局部SREBP-1c、FAS等脂质合成基因明显上调，PPARγ2表达略上升但无统计学意义；TNFα基因表达及其下游重要靶点NFκB活性下调。
脂肪细胞功能失调是肥胖等许多代谢疾病的中心病理环节。前脂肪细胞增殖及分化能力障碍是脂肪细胞参与胰岛素抵抗、代谢综合征和2型糖尿病发生的重要因素。我们的结果提示PTP1B可能是肥胖时连接炎症浸润与脂肪功能受损的重要桥梁，其在肥胖脂肪组织中被TNFα过度激活，并介导了成脂抑制作用；而PTP1B抑制剂治疗可能通过改善肥胖动物脂肪组织的成脂功能，减轻游离脂肪酸过载，而减轻局部炎症反应，最终为整体胰岛素抵抗的改善做贡献。NFκB可能是PTP1B与TNFα相互作用的中间环节。
综上所述，我们提出PTP1B参与肥胖脂肪组织病理变化的可能机制：随着肥胖的进展，增大的脂肪细胞储脂能力超负荷，脂肪组织内分泌功能受损，大量分泌的TNFα通过激活PTP1B的表达抑制前脂肪细胞内PPARγ2、SREBP-1c、FAS、LPL等重要成脂因子的转录，最终导致脂肪细胞分化不足；而新分化储脂细胞的减少又反过来加重脂质过载，形成恶性循环；因此，抑制PTP1B的表达可能削弱肥胖时炎症对脂肪细胞功能的损害。本研究首次阐明了PTP1B在脂肪组织中的作用，尤其是其参与肥胖发生发展的可能机制，为其抑制剂的临床应用提供了新的理论依据。
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 adipocytedifferentiation
Lentiviral 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 obesity
Based 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.
引文
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