原代培养中分化的小鼠脂肪细胞：胰岛素抵抗模型
Summary
该协议描述了从皮下脂肪中分离小鼠前脂肪细胞，它们分化为成熟脂肪细胞以及诱导胰岛素抵抗。通过蛋白质印迹的胰岛素信号通路成员的磷酸化/激活来评估胰岛素作用。该方法可以直接测定原代脂肪细胞的胰岛素抵抗/敏感性。
Abstract
胰岛素抵抗是胰岛素对其靶细胞的影响降低，通常源于胰岛素受体信号传导的减少。胰岛素抵抗导致2型糖尿病（T2D）和其他肥胖衍生疾病的发展，这些疾病在世界范围内患病率很高。因此，了解胰岛素抵抗的潜在机制非常重要。几种模型已被用于研究 体内 和 体外胰岛素抵抗;原代脂肪细胞是研究胰岛素抵抗机制和鉴定抵消这种情况的分子和胰岛素增敏药物分子靶标的一种有吸引力的选择。在这里，我们使用用肿瘤坏死因子-α（TNF-α）处理的培养物中的原代脂肪细胞建立了胰岛素抵抗模型。
通过磁细胞分离技术从胶原酶消化的小鼠皮下脂肪组织中分离出的脂肪细胞前体细胞（APC）分化为原代脂肪细胞。然后通过TNF-α治疗诱导胰岛素抵抗，TNF-是一种促炎细胞因子，可减少胰岛素信号级联反应成员的酪氨酸磷酸化/激活。通过蛋白质印迹定量胰岛素受体 （IR）、胰岛素受体底物 （IRS-1） 和蛋白激酶 B （AKT） 磷酸化降低。该方法为研究脂肪组织中介导胰岛素抵抗的机制提供了极好的工具。
Introduction
胰岛素是由胰岛β细胞产生的合成代谢激素，是葡萄糖和脂质代谢的关键调节因子。在其众多功能中，胰岛素调节葡萄糖摄取、糖原合成、糖异生、蛋白质合成、脂肪生成和脂肪分解1。胰岛素与其受体（IR）相互作用后的初始分子信号是IR2的内在酪氨酸蛋白激酶活性的激活，导致其自身磷酸化3和随后称为胰岛素受体底物（IRS）的蛋白质家族的激活，其与衔接蛋白结合导致级联蛋白激酶的激活4.胰岛素激活两种主要的信号通路：磷脂酰肌醇3激酶（PI3K）蛋白激酶B（AKT）和Ras-丝裂原活化蛋白激酶（MAPK）。前者构成一个主要的分支点或节点4，5，用于激活参与各种生理功能的众多下游效应子，包括调节燃料稳态，而后者调节细胞生长和分化4，6。胰岛素作用最终取决于细胞类型和生理环境7。
主要的胰岛素反应代谢组织之一是脂肪组织。白色脂肪组织是人类和啮齿动物中最丰富的脂肪类型，分布在皮下脂肪（皮肤和肌肉之间）和内脏脂肪（腹腔器官周围）中。鉴于其体积大，脂肪细胞或脂肪细胞是脂肪组织中最丰富的细胞类型。这些脂肪细胞可以是棕色/米色（产热）、粉红色（乳腺）和白色8，9。白色脂肪细胞以甘油三酯的形式保持体内的主要能量储备，这是一种胰岛素依赖性过程。胰岛素促进葡萄糖转运和脂肪生成，同时抑制脂肪分解或脂质分解7，10。它还有助于前脂肪细胞分化为脂肪细胞 – 成熟的脂肪储存细胞11。
当正常胰岛素水平产生减弱的生物反应时，就会发生胰岛素抵抗，从而导致代偿性高胰岛素血症12。胰岛素抵抗是一种与超重和肥胖相关的疾病5，当它们结合在一起时会导致2型糖尿病（T2D）和其他代谢性疾病13。高胰岛素血症补偿外周组织中的胰岛素抵抗，以维持正常的血糖水平14。然而，最终的β细胞丢失或衰竭，以及胰岛素抵抗加剧，导致血糖水平升高，符合T2D5。因此，胰岛素抵抗和高胰岛素血症可导致肥胖衍生代谢疾病的发展15。此外，肥胖可能导致慢性低度局部炎症，促进脂肪组织中的胰岛素抵抗15，16，17。此外，肥胖引起的脂肪组织改变，如纤维化、炎症以及血管生成和脂肪生成减少，导致脂联素血清水平（一种胰岛素增敏剂）降低，纤溶酶原激活剂抑制剂 1 （PAI-1）、游离脂肪酸和外泌体等因子分泌增加进入血液，加剧胰岛素抵抗17。
胰岛素抵抗的许多方面仍然未知。已经开发了体外和体内模型来研究介导主要靶组织（包括脂肪组织）中胰岛素抵抗的机制。体外模型的优点是研究人员可以更好地控制环境条件，并且可以评估特定细胞类型的胰岛素抵抗。特别是，脂肪细胞前体细胞（APC）具有供体组织的个体表型，这可能比脂肪细胞系更好地反映生理学。体外诱导胰岛素抵抗的一个主要因素是肿瘤坏死因子-α（TNF-α）。TNF-α是由脂肪组织中的脂肪细胞和巨噬细胞分泌的促炎细胞因子18。虽然适当的脂肪组织重塑和扩增是必需的19，但长期暴露于TNF-α会在体内脂肪组织和体外脂肪细胞中诱导胰岛素抵抗20。几种细胞类型的慢性TNF-α处理导致IR和IRS-1的丝氨酸磷酸化增加，从而促进酪氨酸磷酸化减少21。IRS-1在丝氨酸残基上的磷酸化增加抑制IR酪氨酸激酶活性，可能是慢性TNF-α治疗损害胰岛素作用的关键机制之一22，23。TNF-α激活涉及核因子ĸB激酶β（IKKβ）和c-Jun N末端激酶（JNK）的丝氨酸/苏氨酸激酶抑制剂的途径24。JNK诱导复杂的促炎转录程序，但也直接磷酸化IRS-16。
了解胰岛素抵抗的发病机制对于指导未来针对T2D的疗法的开发变得越来越重要。APC已被证明是研究脂肪细胞生物学的优秀模型，包括对胰岛素的敏感性和抵抗力，以及独立于全身环境鉴定脂肪细胞的内在特性。APC可以很容易地从不同的脂肪库获得，并在适当的条件下分化成成熟的脂肪细胞。使用这种方法，可以评估对脂肪细胞中胰岛素抵抗/敏感性的直接影响。
Protocol
Representative Results
Discussion
本文提供了一种研究胰岛素抵抗的方法，该方法使用用TNF-α处理的培养物中的原代脂肪细胞。该模型的优点是原代脂肪细胞可以在规定的条件下长时间培养，并严格控制细胞环境因素26。测定持续时间为15-20天，尽管实验之间可能发生分化脂肪细胞百分比的变化。原代脂肪细胞比细胞系具有优势，因为它们在培养中没有持续扩增，并且更紧密地捕获了它们来源的组织多样性<sup clas…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢丹尼尔·蒙德拉贡、安东尼奥·普拉多、费尔南多·洛佩斯-巴雷拉、马丁·加西亚-塞尔文、亚历杭德拉·卡斯蒂利亚和玛丽亚·安东尼埃塔·卡尔巴霍的技术援助，感谢杰西卡·冈萨雷斯·诺里斯对手稿的批判性编辑。该议定书得到了墨西哥国家科学和技术委员会（CONACYT）、教育部门研究基金赠款284771（致Y.M.）的支持。
Materials
| 1. Isolation mouse adipocyte precursor cells | |||
| ACK lysing buffer | LONZA | 10-548E | |
| Anti-Biotin Microbeads | Miltenyi | 130-090-485 | |
| Anti-CD31 | eBioscience | 13-0311-85 | |
| AutoMACS Pro Separator | Miltenyi | ||
| Basement membrane matrix (matrigel) | Corning | 354234 | |
| bFGF | Sigma | F0291 | Growth factor |
| BSA | Equitech-Bio, Inc. | BAC63-1000 | |
| CD45 Monoclonal Antibody (30-F11) – Biotin | eBioscience | 13-0451-85 | |
| Collagenase, Type 1 | Worthington Biochem | LS004197 | |
| Dexamethasone | Sigma | D1756 | |
| DMEM | GIBCO | 12800017 | |
| DMEM low glucose | GIBCO | 31600-034 | |
| EGF | Peprotech | 315-09 | Growth factor |
| FBS | GIBCO | 26140-079 | |
| ITS mix | Sigma | I3146 | |
| L-ascorbic acid 2-phosphate | Sigma | A8960 | |
| LIF | Millipore | ESG1107 | Growth factor |
| Linoleic acid-albumin | Sigma | L9530 | |
| MCDB 201 medium | Sigma | M6770 | |
| Normocin | InvivoGen | ant-nr-2 | |
| PDGF-BB | Peprotech | 315-18 | Growth factor |
| Peniciline-Streptomycine | BioWest | L0022-100 | |
| Pre-Separation Filters (70 µm) | Miltenyi | 130-095-823 | |
| Purified Rat Anti-Mouse CD16 / CD32 | BD Pharmingen | 553142 | |
| Trypsin-EDTA | GIBCO | 25300062 | |
| 2. Adipocyte differentiation and insulin resistance induction | |||
| 3-Isobutyl-1-methylxanthine [IBMX] | Sigma | I5879 | Differentiation cocktail |
| BMP4 | R&D Systems | 5020-BP-010 | Differentiation cocktail |
| Dexamethasone | Sigma | D1756 | Differentiation cocktail |
| Insulin | Sigma | I9278 | |
| Rosiglitazone | Cayman | 71742 | Differentiation cocktail |
| TNFα | R&D Systems | 210-TA-005 | |
| 3. Evaluation of insulin signaling pathway by western blot | |||
| Anti-beta tubulin antibody | Abcam | ab6046 | |
| Bromophenol blue | BioRad | 161-0404 | Laemmli buffer |
| EDTA | Sigma | E5134 | RIPA buffer |
| EGTA | Sigma | E4378 | RIPA buffer |
| FluorChem E system | ProteinSimple | ||
| Glycerol | Sigma | G6279 | Laemmli buffer |
| Glycine | Sigma | G7126 | Running and Transfer buffer |
| Igepal | Sigma | I3021 | RIPA buffer |
| 2- mercaptoethanol | Sigma | M3148 | Laemmli buffer |
| Methanol | JT Baker | 907007 | Transfer buffer |
| NaCl | JT Baker | 3624-05 | TBS-T |
| NaF | Sigma | 77F-0379 | RIPA buffer |
| NaOH | JT Baker | 3722-19 | |
| Na4P2O7 | Sigma | 114F-0762 | RIPA buffer |
| Na3VO4 | Sigma | S6508 | RIPA buffer |
| Nitrocellulose membrane | BioRad | 1620112 | |
| Nonfat dry milk | BioRad | 1706404 | Blocking solution |
| Prestained protein standard | BioRad | 1610395 | |
| Protease inhibitor cocktail | Sigma | P8340-5ML | |
| Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-035-132 | |
| Phospho- Insulin Receptor β | Cell signaling | 3024 | |
| Phospho-Akt (Ser473) Antibody | Cell signaling | 9271 | |
| Phospho-IRS1 (Tyr608) antibody | Millipore | 9432 | |
| Saccharose | JT Baker | 407205 | RIPA buffer |
| SDS | BioRad | 1610302 | Running and laemmli buffer |
| SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Scientific | 34577 | |
| Tris-base | Promega | H5135 | Running, transfer and laemmli buffer |
| Tris-HCl | JT Baker | 4103-02 | RIPA buffer – TBS |
| Tween 20 | Sigma | P1379 | TBS-T |
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