测量基底和福斯柯林刺激的脂肪分解在腹股沟脂肪脂肪垫
概要
该方案描述了从正常食物饮食（NCD）或高脂肪饮食（HFD）±辣椒素喂养的野生型小鼠获得的腹股沟脂肪垫中测定基础和毛喉素刺激的脂肪分解的方法。作为脂肪分解的指标，从腹股沟脂肪脂肪垫测量甘油释放。
Abstract
脂解是脂肪组织中作为甘油三酯存储的脂质水解成甘油和脂肪酸的过程。本文介绍了从正常食物饮食（NCD），高脂肪饮食（HFD）或含有0.01的高脂肪饮食（HFD）的野生型小鼠分离的腹股沟脂肪垫中测量基础和毛喉素（FSK）刺激的脂肪分解的方法百分比的辣椒素（CAP;瞬时受体潜在的香草素亚科1（TRPV1）激动剂）32周。本文所述的用于进行离体脂解的方法采用Schweiger 等人 1我们提供了一个详细的方案，通过紫外 – 可见（UV / VIS）分光光度法测量甘油水平。这里描述的方法可用于成功分离腹股沟脂肪垫用于脂肪分解测量以获得一致的结果。腹股沟脂肪垫描述的方案可以容易地扩展到其他组织中的脂肪分解。
Introduction
脂肪组织为所需的产热3,4脂肪2和脂肪酸氧化存储能量。通过饮食摄取的脂肪酸与脱蛋白一起包装到乳糜微粒中，并通过血液循环递送到身体的不同组织。虽然大多数细胞在体内储存能量的储备，脂肪组织储存多余的能量为脂肪5,6。脂肪组织中的脂肪分解是由复杂过程调节的，脂肪分解的分子细节仍然模糊7 。
脂肪分解是通过脂肪甘油三酯脂肪酶（ATGL） 8将存储在脂肪组织中的甘油三酯（TGL）水解以产生甘油和脂肪酸（FA）的过程。基础和刺激脂肪分解的改变是肥胖的特征。 basal脂肪分解由ATGL活化9调节，其将TGL转化为二酰基甘油（DAG），随后水解成单酰甘油（MAG）。 通过腺苷酸环化酶激活激素敏感性脂肪酶（HSL）激活环磷酸腺苷（cAMP）依赖性蛋白激酶A（PKA）刺激并引起脂肪分解。因此，脂肪分解，基础和刺激的测量对于分析涉及该过程的蛋白质的活性是重要的。另外，解开脂肪分解的分子调控可能有利于开发针对肥胖的新型治疗策略10 。因为刺激脂肪分解和脂肪酸氧化的分子是降低储存在贮库中的脂肪的潜在候选者，所以重要的是采用鲁棒的重现性测定法。
以前发表的数据表明，CAP在白色脂肪组织中表达的TRPV1蛋白的激活增强了基础和FSK（腺苷酸环化酶激活剂） – 腹股沟脂肪垫中的刺激性脂肪分解11 。以前的研究还表明，通过CAP长期激活TRPV1的激活PKA 12。因为PKA的活化刺激脂解13，14，测量从NCD或HFD（±CAP）32周供给各自的饮食后喂食的小鼠分离的腹股沟脂肪垫基础和PKA依赖性刺激脂解作用将验证TRPV1的作用激活脂解。
本文介绍了确定基础和刺激的脂肪分解的有效方法。虽然这采用glycerol和繁琐的高效液相色谱法或气相色谱法/质谱法测量15，16是可用的放射性同位素等方法，这种方法提供了一种更直接的，简单且成本有效的确定脂肪组织脂肪分解的技术。
Protocol
所有协议均遵循怀俄明大学的动物保健指南。 动物饲养注意：根据机构动物护理和使用委员会（IACUC）批准的方案，在研究动物设施中培育成年雄性野生型小鼠（C57BL / 6）（12至24周）。 从第6周开始，将小鼠分为四组，分为笼子，随机将其分配到非传染性疾病或高血压（±0.01％CAP）的喂养组，直到第38周岁。 注：CAP是在脂肪组织<…
Representative Results
为了评估CAP对基础和受刺激的脂肪分解的影响，本研究测量了从NCD或HFD（±CAP）野生型小鼠分离的腹股沟脂肪脂肪垫中的脂肪分解。腹股沟脂肪垫的基础和FSK刺激的脂肪分解的代表性结果在表I中给出。在Triacsin C存在下，基础和FSK刺激的甘油释放，其抑制酰基辅酶A合成酶并阻止TGL的再生。 如图1所示，HFD抑制了FSK刺激的脂肪分解，CAP增加?…
Discussion
TGL的击穿过程为甘油和脂肪酸被by ATGL 9 basal脂解期间催化和由蛋白质的阵列编排包括腺苷酸环化酶/ PKA-依赖性途径的激脂解21，22，23在活化。脂解作用增强增加脂肪酸的血浆水平为运输和能源使用24。脂肪酸被线粒体吸收为乙酰CoA，用于产生能量。
脂肪分解的程度和程?…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作得到AHA Award 15BGIA23250030，NIH国家综合医学科学研究所的支持，获得了第8P20 GM103432-12的奖学金和怀俄明州大学授予BT援助。
Materials
| Capsaicin | Sigma, USA | M2028 | TRPV1 agonist |
| Forskolin | Sigma, USA | F6886 | Adenylyl cyclase activator |
| DMEM | GE healthcare and life sciences, UT, USA | SH30081.01 | |
| High fat diet | Research diets, New Brunswick, USA | D12492 | Abbreviated as HFD |
| Tris | Amresco, USA | O497 | |
| Sodium chloride | Thermofisher Scientific | BP358-212 | |
| Sodium deoxycholate | Sigma, USA | D6750 | |
| Dithiothreitol | Sigma, USA | D9163 | |
| Sodium orthovanadate | Sigma, USA | S6508 | |
| Protease inhibitor cocktail | Sigma, USA | P8340 | |
| Free Glycerol reagent | Sigma USA | F6428 | |
| DMSO | Sigma, USA | D8779 | |
| Triacsin C | Sigma, USA | T4540 | Acyl CoA transferase inhibitor |
| Bovine serum albumin | Sigma, USA | A7030 | |
| Chloroform | Sigma Aldrich | 31998-8 | |
| Methanol | Thermofisher Scientific, USA | A412-1 | |
| Sodium hydroxide | Amresco, USA | O583 | |
| Sodium dodecyl sulfate | Sigma, USA | L3371 | |
| Bicinchoninic acid reagent | Sigma, USA | BCA1-1KT | |
| UV-VIS Spectrophotometer | Pharmacia Biotech, NJ, USA | Ultrospec 2000 | |
| Normal chow diet | Labdiet.com | 500I | abreviated as NCD |
| C57BL/6 mice | Jackson Laboratory, CT, USA | Stock number000664 | wild type mice |
| Parafilm | Heathrow Scientific, USA | HS 234526A | |
| Glycerol standard | Sigma, USA | G7793 |
参考文献
- Schweiger, M., et al. Measurement of lipolysis. Methods Enzymol. 538, 171-193 (2014).
- Coelho, M., Oliveira, T., Fernandes, R. Biochemistry of adipose tissue: an endocrine organ. Arch Med Sci. 9 (2), 191-200 (2013).
- Richard, D., Picard, F. Brown fat biology and thermogenesis. Front Biosci (Landmark Ed). 16, 1233-1260 (2011).
- Barquissau, V., et al. White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways. Mol Metab. 5 (5), 352-365 (2016).
- Rodriguez, A., Ezquerro, S., Mendez-Gimenez, L., Becerril, S., Fruhbeck, G. Revisiting the adipocyte: a model for integration of cytokine signaling in the regulation of energy metabolism. Am J Physiol Endocrinol Metab. 309 (8), E691-E714 (2015).
- Giordano, A., Smorlesi, A., Frontini, A., Barbatelli, G., Cinti, S. White, brown and pink adipocytes: the extraordinary plasticity of the adipose organ. Eur J Endocrinol. 170 (5), R159-R171 (2014).
- Langin, D. Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome. Pharmacol Res. 53 (6), 482-491 (2006).
- Zimmermann, R., Lass, A., Haemmerle, G., Zechner, R. Fate of fat: the role of adipose triglyceride lipase in lipolysis. Biochim Biophys Acta. 1791 (6), 494-500 (2009).
- Miyoshi, H., Perfield, J. W., Obin, M. S., Greenberg, A. S. Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes. J Cell Biochem. 105 (6), 1430-1436 (2008).
- Kolditz, C. I., Langin, D. Adipose tissue lipolysis. Curr Opin Clin Nutr Metab Care. 13 (4), 377-381 (2010).
- Baskaran, P., Krishnan, V., Ren, J., Thyagarajan, B. Capsaicin induces browning of white adipose tissue and counters obesity by activating TRPV1 channel-dependent mechanisms. Br J Pharmacol. 173 (15), 2369-2389 (2016).
- Yang, D., et al. Activation of TRPV1 by dietary capsaicin improves endothelium-dependent vasorelaxation and prevents hypertension. Cell Metab. 12 (2), 130-141 (2010).
- Ding, L., et al. Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity. Sci Rep. 6, 34374 (2016).
- Ohyama, K., et al. A combination of exercise and capsinoid supplementation additively suppresses diet-induced obesity by increasing energy expenditure in mice. Am J Physiol Endocrinol Metab. 308 (4), E315-E323 (2015).
- Beylot, M., Martin, C., Beaufrere, B., Riou, J. P., Mornex, R. Determination of steady state and nonsteady-state glycerol kinetics in humans using deuterium-labeled tracer. J Lipid Res. 28 (4), 414-422 (1987).
- Gilker, C. D., Pesola, G. R., Matthews, D. E. A mass spectrometric method for measuring glycerol levels and enrichments in plasma using 13C and 2H stable isotopic tracers. Anal Biochem. 205 (1), 172-178 (1992).
- Baskaran, P., et al. TRPV1 activation counters diet-induced obesity through sirtuin-1 activation and PRDM-16 deacetylation in brown adipose tissue. Int J Obes (Lond). , (2017).
- Smith, N. C., Fairbridge, N. A., Pallegar, N. K., Christian, S. L. Dynamic upregulation of CD24 in pre-adipocytes promotes adipogenesis. Adipocyte. 4 (2), 89-100 (2015).
- Schweiger, M., et al. Measurement of lipolysis. Methods Enzymol. 538, 171-193 (2014).
- Duncan, R. E., Ahmadian, M., Jaworski, K., Sarkadi-Nagy, E., Sul, H. S. Regulation of lipolysis in adipocytes. Annu Rev Nutr. 27, 79-101 (2007).
- Ahmadian, M., Duncan, R. E., Sul, H. S. The skinny on fat: lipolysis and fatty acid utilization in adipocytes. Trends Endocrinol Metab. 20 (9), 424-428 (2009).
- Jaworski, K., Sarkadi-Nagy, E., Duncan, R. E., Ahmadian, M., Sul, H. S. Regulation of triglyceride metabolism. IV. Hormonal regulation of lipolysis in adipose tissue. Am J Physiol Gastrointest Liver Physiol. 293 (1), G1-G4 (2007).
- Jeppesen, J., Kiens, B. Regulation and limitations to fatty acid oxidation during exercise. J Physiol. 590 (5), 1059-1068 (2012).
- Nakamura, M. T., Yudell, B. E., Loor, J. J. Regulation of energy metabolism by long-chain fatty acids. Prog Lipid Res. 53, 124-144 (2014).
- Murray, A. J., Panagia, M., Hauton, D., Gibbons, G. F., Clarke, K. Plasma free fatty acids and peroxisome proliferator-activated receptor alpha in the control of myocardial uncoupling protein levels. Diabetes. 54 (12), 3496-3502 (2005).
- Barbera, M. J., et al. Peroxisome proliferator-activated receptor alpha activates transcription of the brown fat uncoupling protein-1 gene. A link between regulation of the thermogenic and lipid oxidation pathways in the brown fat cell. J Biol Chem. 276 (2), 1486-1493 (2001).
- Leung, F. W. Capsaicin as an anti-obesity drug. Prog Drug Res. 68, 171-179 (2014).
- Hursel, R., Westerterp-Plantenga, M. S. Thermogenic ingredients and body weight regulation. Int J Obes (Lond). 34 (4), 659-669 (2010).
- Kang, J. H., et al. Dietary capsaicin reduces obesity-induced insulin resistance and hepatic steatosis in obese mice fed a high-fat diet. Obesity (Silver Spring). 18 (4), 780-787 (2010).
- Winkler, B., Steele, R., Altszuler, N. Relationship of glycerol uptake to plasma glycerol concentration in the normal dog. Am J Physiol. 216 (1), 191-196 (1969).
- Dugan, C. E., Cawthorn, W. P., MacDougald, O. A., Kennedy, R. T. Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes. Anal Bioanal Chem. 406 (20), 4851-4859 (2014).
