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Biology

测量体外分化的离体小鼠脂肪组织和原代前脂肪细胞的脂肪分解率

Published: March 17, 2023
doi:
10.3791/65106
,
1Weill Center for Metabolic Health, Department of Medicine,Weill Cornell Medicine

Summary

脂肪细胞中的甘油三酯脂肪分解是导致游离脂肪酸和甘油释放的重要代谢过程。在这里，我们提供了一个详细的方案来测量小鼠脂肪细胞和 离体 脂肪组织中的基础和刺激脂肪分解。

Abstract

脂肪细胞以甘油三酯的形式在脂滴中储存能量。这种能量可以通过脂解 动员 ，其中脂肪酸侧链从甘油骨架上依次裂解，导致游离脂肪酸和甘油的释放。由于甘油激酶在白色脂肪细胞中的低表达，甘油再摄取率可以忽略不计，而脂肪酸再摄取由白蛋白等培养基成分的脂肪酸结合能力决定。甘油和脂肪酸释放到培养基中都可以通过比色测定法进行定量，以确定脂解速率。通过在多个时间点测量这些因素，可以高置信度地确定脂肪分解的线性速率。在这里，我们提供了一个详细的方案，用于测量来自小鼠的体 外 分化脂肪细胞和 离体 脂肪组织中的脂肪分解。该协议也可以针对其他前脂肪细胞系或来自其他生物体的脂肪组织进行优化;讨论了考虑因素和优化参数。该协议旨在用于确定和比较小鼠模型和治疗之间的脂肪细胞脂肪分解率。

Introduction

多余的营养物质以甘油三酯的形式储存在脂滴的中性脂质核心中的白色脂肪组织中。甘油三酯储存通过脂解动员，脂肪分解是脂肪酸侧链被脂肪组织甘油三酯脂肪酶（ATGL），激素敏感脂肪酶（HSL）和甘油单酯脂肪酶（MGL）顺序切割的过程，导致游离脂肪酸（FFA）和甘油骨架的释放1，2。脂肪分解由脂肪组织中的儿茶酚胺信号激活。交感神经末梢局部释放儿茶酚胺，儿茶酚胺与脂肪细胞质膜上的β-肾上腺素能受体结合。配体结合后，这些G蛋白偶联受体（GPCR）通过Gαs激活腺苷酸环化酶。随后通过cAMP激活蛋白激酶A（PKA）导致ATGL和HSL的上调。PKA对perilipin-1的磷酸化导致ABHD5（也称为CGI-58）的解离，其结合并共激活ATGL3。PKA直接磷酸化HSL，促进其从细胞质质到脂滴的易位，其中与磷酸化的perilipin-1相互作用进一步促进其脂肪酶活性4，5，6，7。参与脂解的第三种脂肪酶MGL似乎不受儿茶酚胺信号传导的调节8。重要的是，脂肪细胞中的甘油三酯合成是由甘油脂质合成途径介导的，其不涉及甘油单酯作为中间体的形成;相反，甘油-3-磷酸酰基转移酶催化溶血磷脂酸的形成，溶血磷脂酸与另一种脂肪酰基辅酶A结合形成磷脂酸，然后在最终合成甘油三酯之前异构化为甘油二酯（图1）9，10，11。

Figure 1
图1：脂解和甘油脂质合成途径。 上图：脂解通路;红色显示的酶：脂肪组织甘油三酯脂肪酶（ATGL），激素敏感脂肪酶（HSL）和甘油单酯脂肪酶（MGL）。下图：甘油脂质合成途径;绿色显示的酶：甘油二酰基转移酶 （DGAT）、磷脂酸磷酸酶 （PAP）、溶血磷脂酸酰基转移酶 （LPAT，也称为 LPAAT） 和甘油-3-磷酸酰基转移酶 （GPAT）。脂质：甘油三酯 （TG）、甘油二酯 （DG）、甘油单酯 （MG）、游离脂肪酸 （FFA）、脂肪酰辅酶 A （FA-CoA）、溶血磷脂酸 （LPA） 和磷脂酸 （PA）。其他代谢物：无机磷酸盐（Pi）和甘油3-磷酸（G3P）。 请点击此处查看此图的大图。

细胞外腺苷是脂肪分解的另一种重要调节因子，通过Gs和Gi偶联GPCR影响腺苷酸环化酶活性。脂肪细胞中的主要腺苷受体ADORA1抑制腺苷酸环化酶，从而通过激活Gi12来抑制脂肪分解。ADORA2A以较低水平表达，主要在棕色脂肪细胞中表达，通过Gs信号传导激活脂肪分解13。ADORA1影响基础脂肪分解和对肾上腺素能激动剂的反应。腺苷对脂肪分解的作用可以通过添加腺苷脱氨酶来中和腺苷，以及ADORA1特异性激动剂苯基异丙基腺苷14，15来控制。Gq偶联GPCR的激素激活也可以通过磷脂酶C和蛋白激酶C16，17，18，19的活化来影响脂肪分解。炎症信号也会影响脂解率。LPS（和其他内毒素）的TLR4激活通过激活ERK来增加脂解率，ERK磷酸化perilipin-1和HSL20。TNF-α还通过ERK和NF-κB活化激活脂肪分解，以及磷酸二酯酶PDE-3B和CIDEC21，22，23的转录下调。IL-6 也与脂肪细胞脂肪分解增加有关，特别是在肠系膜脂肪组织中，其 FFA 释放影响肝脂肪变性和糖异生24，25，26。

脂肪分解在进食状态下被胰岛素抑制。AKT 磷酸化并激活 PDE-3B 以抑制 cAMP 信号传导并阻止 PKA 活化27。胰岛素也转录下调ATGL28。肥胖通过多种机制促进儿茶酚胺耐药性，包括下调脂肪细胞29，30，31，32，33中的β-肾上腺素能受体。脂肪细胞表达所有三种β-肾上腺素能受体（β-1、β-2 和 β-3）。虽然β-1和β-2肾上腺素能受体普遍表达，但β-3肾上腺素能受体主要在小鼠34，35的脂肪细胞中表达。Adrb3 表达由 C/EBPα 在脂肪生成36 期间诱导。β-3肾上腺素能受体在成熟脂肪细胞中高表达。由于β-arrestin37的反馈抑制，β-1和β-2肾上腺素能受体的激活是自限性的。β-3肾上腺素能受体的反馈抑制由其他信号通路介导，其降低Adrb3表达33，38，39。

许多化合物可用于激活脂肪细胞脂质分解。儿茶酚胺是脂解的主要生理激活剂。去甲肾上腺素（或去甲肾上腺素）和肾上腺素（或肾上腺素）激活所有三种β肾上腺素能受体40。去甲肾上腺素和肾上腺素也通过激活α-肾上腺素能受体信号传导 来 影响脂肪分解41。常用的β-肾上腺素能受体激动剂包括异丙肾上腺素能激动剂，它是一种非选择性β-肾上腺素能受体激动剂，以及β-3肾上腺素能受体激动剂CL-316，243和米拉贝隆42。鉴于脂肪细胞主要表达β-3肾上腺素能受体，我们在这里使用CL-316，243作为一个例子。它对β-3肾上腺素能受体的特异性也使其成为脂肪细胞儿茶酚胺信号传导的相对特异性激活剂，也可以安全地用于 体内。请注意，细胞培养物中常用的10μM CL-316，243浓度比达到最大反应所需的~0.1μM剂量高几个数量级33。佛司可林绕过肾上腺素能受体， 直接激活腺苷酸环化酶和下游脂解信号.还有更多的激活剂，以及脂肪分解的抑制剂。在选择刺激脂解的化合物时，应在实验设计中仔细考虑受体特异性和下游信号通路。

白色脂肪组织中脂肪分解的速度是影响禁食或运动期间耐寒性和营养可用性的重要代谢因素43，44，45，46。该协议的目的是测量脂肪细胞和脂肪组织中脂肪分解的速率，这将有助于了解脂肪细胞代谢以及它如何影响各种小鼠模型的代谢表型。为了量化脂解率，我们测量了脂解产物在培养基中的外观（即FFA和甘油）。该方法依赖于脂解产物从脂肪细胞释放到培养基中。由于白色脂肪细胞表达低水平的甘油激酶，因此甘油再摄取率较低47。相反，还应考虑通过脂肪分解以外的代谢途径产生FFA和甘油。脂肪细胞似乎表达一种具有抗甘油-3磷酸活性的磷酸酶，使得能够从葡萄糖48，49，50衍生的甘油-3-磷酸产生甘油。糖酵解是用于白色脂肪细胞中FFA再酯化的甘油-3-磷酸的来源。当葡萄糖水平有限时，甘油新生需要其他3碳来源，如乳酸和丙酮酸51。细胞内脂肪分解释放的FFA的通道及其代谢命运知之甚少;脂肪分解释放的FFA必须在再酯化或经历β氧化之前转化为脂肪酰基辅酶A。看来脂肪分解释放的FFAs可能会在被带回并转化为脂肪酰辅酶A 52，53，54，55，56，57，58，59，60，61，62之前离开细胞.FFA可以通过白蛋白隔离在细胞外。重要的是，已知长链FFAs如果不被白蛋白63，64，65，66，67隔离，则反馈抑制脂肪分解。因此，在脂解测定过程中优化培养基的FFA缓冲能力至关重要。这里描述的程序类似于先前发表的测量小鼠和人类脂肪细胞和离体脂肪组织中脂解率的方法15，68，69，70，71。该协议通过使用串行采样而有所不同;通过执行连续采样，我们可以在内部验证线性阶段测量脂肪分解，并利用多次测量来计算脂肪分解速率，从而减少测量误差，以提高最终计算值的置信度。连续取样的缺点是检测需要更多的时间和试剂;然而，较长的时间范围减少了测量误差对速率估计标准误差的影响。此外，该协议测量FFA和甘油释放，并考虑FFA：甘油释放的比例，目标是实现3：1的比例，正如完全脂解和脂解产物释放到培养基中的预期一样72。

Protocol

所有动物的使用都得到了康奈尔大学威尔康奈尔医学院机构动物护理和使用委员会（IACUC）的批准。 1. 缓冲液和收集板的制备 通过将 5 g BSA 溶解在 100 mL 不含酚红的 Dulbecco 改性鹰培养基 （DMEM） 中，制成 5% 牛血清白蛋白 （BSA）。轻轻搅拌BSA溶解（摇晃适得其反）。BSA完全溶解后，用0.2μm过滤器对培养基进行过滤灭菌。将BSA培养基在4°C下储存长达1个月。<…

Representative Results

我们测量了体外分化脂肪细胞的基础和刺激脂解率。通过用5μM地塞米松、0.5mM IBMX、1μg/mL胰岛素和1μM曲格列酮处理融合细胞4天，将来自腹股沟白色脂肪组织的原代前脂肪细胞分化为脂肪细胞，然后用1μg/mL胰岛素再处理3天。在脂肪分解测定之前，将细胞在没有胰岛素的培养基中孵育24小时。在时间= 0h时，用PBS洗涤细胞一次，然后将含有10μM CL-316，243或载体对照的2%BSA的无酚红DMEM加入到12孔?…

Discussion

在这里，我们提供了一种用于测量脂肪细胞和离体脂肪组织中脂肪分解速率的基本方案。为了量化脂解，重要的是测量线性相中的脂解速率。我们使用连续采样技术，其中收集大部分培养基并定期更换为新鲜培养基。这种半保守的方法允许添加具有FFA缓冲能力的新鲜BSA，并延迟反馈抑制，延长线性脂解的持续时间。该实验设计试图概括体内脂肪组织的血管化，其提供新鲜白蛋白以结?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国立卫生研究院向S.M.R.拨款R01DK126944的支持。

Materials

24-Well tissue culture treated plate Corning Inc 3527 Must be tissue culture treated for adipocyte differntiation
48-Well flat bottom plate with lid Corning Inc 353078 Can be tissue culture treated
6-Well flat bottom plate with lid Corning Inc 353046 Can be tissue culture treated
96-Well PCR Plate USA sceintific 1402-9100 Any conical 0.2 mL PCR plate will be convenient 
Bovine Serum Albumin Sigma Aldrich A9418 FFA free BSA such as A8806, is also commonly used. The BSA should not have detectable FFA, also lot to lot variations in BSA can impact the observed rate of lipolysis
CL-316,243 Sigma Aldrich C5976 CAS #: 138908-40-4 availaible from other suppliers
CO2 incubator PHCBI MCO-170AICUVH CO2 should ideally be set to 10% for adipose tissue, however 5% CO2 will also work
DMEM, low glucose, no phenol red Thermofischer 11054020 Any phenol red free media should work, DMEM/F12, RPMI, but should contain volatile buffering capacity, i.e. biocarbonate
FFA-free Bovine serum albumin Equitech-Bio, Inc,  BAH66
Free Glycerol Reagent Sigma Aldrich F6428
Glycerol Standard Solution Sigma Aldrich G7793  This can also be made by diluting glycerol to the desired concentration
HR Series NEFA Standard Solution Fujifilm 276-76491
HR Series NEFA-HR (2) Color Reagent A Fujifilm 999-34691
HR Series NEFA-HR (2) Color Reagent B Fujifilm 991-34891
HR Series NEFA-HR (2) Solvent A  Fujifilm 995-34791
HR Series NEFA-HR (2) Solvent B  Fujifilm 993-35191
Microbiological Incubator Fischer Scientific S28668 Any incubator at 37C can be used
Nunc MicroWell 96-Well Plates Thermo Scientific 269620 Any optically clear, flat bottom 96-well plate works
Silicone Laboratory Benchtop Mat VWR 76045-300 Glass plate can also be used. Absorbant surfaces are not recommended
Spectrophotometer/Microplate Reader Molecular devices SpectraMax i3x  Any plate reader that can read at 540, 550 and 660 mm will work
V Bovine serum albumin Sigma-Aldrich 810531
WypAll X70 Wipers Kimberly-Clark 41200 Any high quality paper towel will work
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Keywords: LipolysisPreadipocytesMurineEx VivoIn VitroFree Fatty AcidsBSADMEMInsulinDifferentiationDPBSStimulationControlSerial SamplingMeasurementOptimization
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Bridge-Comer, P. E., Reilly, S. M. Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro. J. Vis. Exp. (193), e65106, doi:10.3791/65106 (2023).
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