Name: evaluation of hepatic glucose production in a polycystic ovary syndrome mouse model
Uploaded: 2022-03-05T13:30:00
Description: Watch this Scientific Journal Video about Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model at JoVE.com

这项工作得到了贝勒医学院（ALG）妇产科系的培训资助以及美国国立卫生研究院为CSB，SC和JM提供的R-01研究补助金（补助金编号DK114689）的支持。

所有动物程序均由贝勒医学院的机构动物护理和使用委员会（IACUC）批准。
1. [6，6-2H2]葡萄糖的制备
<ol><li>手术前一天，在生理盐水中制备稳定的同位素葡萄糖示踪剂。对于该实验，使用[6，6-2H2]葡萄糖作为示踪剂来测量血浆葡萄糖出现率。 注意：在本实验中，测量了空腹和富葡萄糖条件下的葡萄糖产生，因此在两种不同的制?...

<ol>
	<li>March, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+polycystic+ovary+syndrome+in+a+community+sample+assessed+under+contrasting+diagnostic+criteria.">The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria.</a> Human Reproduction. 25 (2), 544-551 (2009).</li><li>Yildiz, B. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+phenotype+and+cardiometabolic+risk+of+polycystic+ovary+syndrome+under+different+diagnostic+criteria.">Prevalence, phenotype and cardiometabolic risk of polycystic ovary syndrome under different diagnostic criteria.</a> Human Reproduction. 27 (10), 3067-3073 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+2003+consensus+on+diagnostic+criteria+and+long-term+health+risks+related+to+polycystic+ovary+syndrome.">Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome.</a> Fertility and Sterility. 81 (1), 19-25 (2004).</li><li>Goodarzi, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+ovary+syndrome:+etiology,+pathogenesis+and+diagnosis.">Polycystic ovary syndrome: etiology, pathogenesis and diagnosis.</a> Nature Reviews. Endocrinology. 7 (4), 219-231 (2011).</li><li>Azziz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction:+Determinants+of+polycystic+ovary+syndrome.">Introduction: Determinants of polycystic ovary syndrome.</a> Fertility and Sterility. 106 (1), 4-5 (2016).</li><li>Baskind, N. E., Balen, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic-pituitary,+ovarian+and+adrenal+contributions+to+polycystic+ovary+syndrome.">Hypothalamic-pituitary, ovarian and adrenal contributions to polycystic ovary syndrome.</a> Best Practice and Research. Clinical Obstetrics &amp; Gynaecology. 37, 80-97 (2016).</li><li>Burghen, G. A., Givens, J. R., Kitabchi, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+hyperandrogenism+with+hyperinsulinism+in+polycystic+ovarian+disease.">Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease.</a> The Journal of Clinical Endocrinology and Metabolism. 50 (1), 113-116 (1980).</li><li>Bremer, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+ovary+syndrome+in+the+pediatric+population.">Polycystic ovary syndrome in the pediatric population.</a> Metabolic Syndrome and Related Disorders. 8 (5), 375-394 (2010).</li><li>Dunaif, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+insulin+receptor+serine+phosphorylation+in+cultured+fibroblasts+and+in+skeletal+muscle.+A+potential+mechanism+for+insulin+resistance+in+the+polycystic+ovary+syndrome.">Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. A potential mechanism for insulin resistance in the polycystic ovary syndrome.</a> The Journal of Clinical Investigation. 96 (2), 801-810 (1995).</li><li>H&#248;jlund, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin-stimulated+phosphorylation+of+Akt+and+AS160+in+skeletal+muscle+of+women+with+polycystic+ovary+syndrome+is+reversed+by+pioglitazone+treatment.">Impaired insulin-stimulated phosphorylation of Akt and AS160 in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment.</a> Diabetes. 57 (2), 357-366 (2008).</li><li>Moghetti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergences+in+insulin+resistance+between+the+different+phenotypes+of+the+polycystic+ovary+syndrome.">Divergences in insulin resistance between the different phenotypes of the polycystic ovary syndrome.</a> The Journal of Clinical Endocrinology and Metabolism. 98 (4), 628-637 (2013).</li><li>Ovalle, F., Azziz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance,+polycystic+ovary+syndrome,+and+type+2+diabetes+mellitus.">Insulin resistance, polycystic ovary syndrome, and type 2 diabetes mellitus.</a> Fertility and Sterility. 77 (6), 1095-1105 (2002).</li><li>Dunaif, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+peripheral+insulin+resistance,+independent+of+obesity,+in+polycystic+ovary+syndrome.">Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome.</a> Diabetes. 38 (9), 1165-1174 (1989).</li><li>Hutchison, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+on+insulin+resistance+and+body+composition+in+overweight+and+obese+women+with+and+without+polycystic+ovary+syndrome.">Effects of exercise on insulin resistance and body composition in overweight and obese women with and without polycystic ovary syndrome.</a> The Journal of Clinical Endocrinology Metabolism. 96 (1), 48-56 (2011).</li><li>Stepto, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Women+with+polycystic+ovary+syndrome+have+intrinsic+insulin+resistance+on+euglycaemic-hyperinsulaemic+clamp.">Women with polycystic ovary syndrome have intrinsic insulin resistance on euglycaemic-hyperinsulaemic clamp.</a> Human Reproduction. 28 (3), 777-784 (2013).</li><li>Basu, R., Schwenk, W. F., Rizza, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+fasting+glucose+production+and+disappearance+are+abnormal+in+people+with+&amp;#34;mild&amp;#34;+and+&amp;#34;severe&amp;#34;+type+2+diabetes.">Both fasting glucose production and disappearance are abnormal in people with &amp;#34;mild&amp;#34; and &amp;#34;severe&amp;#34; type 2 diabetes.</a> American Journal of Physiology, Endocrinology and Metabolism. 287 (1), 55-62 (2004).</li><li>Blesson, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+dependent+dysregulation+of+hepatic+glucose+production+in+lean+Type+2+diabetic+rats.">Sex dependent dysregulation of hepatic glucose production in lean Type 2 diabetic rats.</a> Frontiers in Endocrinology. 10, 538 (2019).</li><li>Caldwell, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+reproductive,+metabolic,+and+endocrine+features+of+polycystic+ovary+syndrome+in+female+hyperandrogenic+mouse+models.">Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models.</a> Endocrinology. 155 (8), 3146-3159 (2014).</li><li>Chappell, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+androgen+induced+lean+PCOS+impairs+mitochondria+and+mRNA+profiles+in+oocytes.">Prenatal androgen induced lean PCOS impairs mitochondria and mRNA profiles in oocytes.</a> Endocrine Connections. 9 (3), 261-270 (2020).</li><li>Chacko, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+gluconeogenesis+using+glucose+fragments+and+mass+spectrometry+after+ingestion+of+deuterium+oxide.">Measurement of gluconeogenesis using glucose fragments and mass spectrometry after ingestion of deuterium oxide.</a> Journal of Applied Physiology. 104 (4), 944-951 (2008).</li><li>Bier, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+&amp;#34;true&amp;#34;+glucose+production+rates+in+infancy+and+childhood+with+6,6-dideuteroglucose.">Measurement of &amp;#34;true&amp;#34; glucose production rates in infancy and childhood with 6,6-dideuteroglucose.</a> Diabetes. 26 (11), 1016-1023 (1977).</li><li>Chacko, S. K., Sunehag, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gluconeogenesis+continues+in+premature+infants+receiving+total+parenteral+nutrition.">Gluconeogenesis continues in premature infants receiving total parenteral nutrition.</a> Archives of Disease in Childhood. Fetal and Neonatal Edition. 95 (6), 413-418 (2010).</li><li>Chacko, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ghrelin+on+glucose+regulation+in+mice.">Effect of ghrelin on glucose regulation in mice.</a> American Journal of Physiology, Endocrinology and Metabolism. 302 (9), 1055-1062 (2012).</li><li>Marini, J. C., Lee, B., Garlick, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-surgical+alternatives+to+invasive+procedures+in+mice.">Non-surgical alternatives to invasive procedures in mice.</a> Laboratory Animals. 40 (3), 275-281 (2006).</li><li>Jacobs, J. D., Hopper-Borge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+artery+infusions+for+pharmacokinetic+and+pharmacodynamic+analysis+of+taxanes+in+mice.">Carotid artery infusions for pharmacokinetic and pharmacodynamic analysis of taxanes in mice.</a> Journal of Visualized Experiments: JoVE. (92), e51917 (2014).</li><li>Ayala, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperinsulinemic-euglycemic+clamps+in+conscious,+unrestrained+mice.">Hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice.</a> Journal of Visualized Experiments: JoVE. (57), e3188 (2011).</li><li>Kmiotek, E. K., Baimel, C., Gill, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Intravenous+Self+Administration+in+a+Mouse+Model.">Methods for Intravenous Self Administration in a Mouse Model.</a> Journal of Visualized Experiments: JoVE. (70), e3739 (2012).</li><li>Marini, J. C., Lee, B., Garlick, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+urea+kinetic+studies+in+conscious+mice.">In vivo urea kinetic studies in conscious mice.</a> The Journal of Nutrition. 136 (1), 202-206 (2006).</li><li>Choukem, S. -. P., Gautier, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+hepatic+insulin+resistance.">How to measure hepatic insulin resistance.</a> Diabetes Metabolism. 34 (6), 664-673 (2008).</li></ol>

高血糖和葡萄糖代谢/稳态异常是 PCOS 的特征。血糖水平通过饮食中的葡萄糖和葡萄糖 通过 糖原分解和糖异生和糖原生成的组合来维持，在激素和酶的控制下。肝葡萄糖的产生因循环葡萄糖水平升高而受到抑制。在葡萄糖代谢异常的疾病中，葡萄糖产生的抑制调节受到损害，导致高血糖。虽然许多研究表明，在PCOS动物模型中间接测量了肝葡萄糖的产生，但很少有人直接测量肝葡萄糖的产生...

多囊卵巢综合征 （PCOS） 是一种常见疾病，见于 12%-20% 的育龄妇女1，2。它是一种复杂的疾病，导致表型多发性变异，包括多囊卵巢、月经不调和高雄激素血症的临床或实验室证据，通常在女性符合三个标准中的两个时诊断出来3。PCOS的一个主要方面，也是其发病机制的一个关键因素，是在患有该疾病的女性中发现的代谢紊乱。患有PCOS的女性胰岛素抵抗，葡萄糖不耐受，肥胖和代谢综合征的发生率较高3，4，5，6。胰岛素抵抗不仅是该疾病的一种表现，而且被认为通过增强卵巢中黄体生成素的作用来促进其发病机制，从而导致雄激素产生增加7，8。胰岛素抵抗被认为有几种可能的起源，但研究表明这可能是由于胰岛素受体信号传导的异常模式9，10。研究已经使用高胰岛素血症 - 正常血糖钳夹的金标准技术评估了PCOS患者的胰岛素抵抗11，12，13，14，15。与对照组相比，无论BMI如何，患有PCOS的女性具有更高水平的胰岛素抵抗。胰岛素对葡萄糖产生的控制在导致葡萄糖产生过量的胰岛素抵抗性疾病中受损。例如，糖尿病患者的糖异生率增加，糖原溶解抑制受损16。此外，在糖尿病大鼠中观察到葡萄糖产生的抑制受损17。虽然钳夹研究可以测量胰岛素抵抗，但PCOS中很少有研究专注于直接测量空腹和进食状态下的葡萄糖产生。这需要使用非放射性同位素葡萄糖示踪剂输注和 通过 质谱法进行测量。
动物模型已被广泛用于PCOS研究。瘦型和肥胖型 PCOS 小鼠模型都是通过产前、青春期前或青春期后施用雄激素来创建的18。啮齿动物PCOS模型也显示出与各自对照组相比的代谢差异。我们实验室先前的数据显示PCOS小鼠模型（瘦和肥胖）中的葡萄糖耐量测试异常（GTT），与人类PCOS文献一致19。使用瘦肉和肥胖的动物模型可以进一步研究代谢差异。具体而言，该模型允许使用同位素葡萄糖示踪剂直接评估葡萄糖产生速率。最常用的稳定同位素葡萄糖示踪剂之一是[6，6-2H2]葡萄糖。[6，6-2H2]葡萄糖富集可以使用前面描述的五乙酸衍生物进行测量20。
在这项研究中，我们的目标是使用同位素葡萄糖输注测量PCOS小鼠在空腹和富葡萄糖状态下肝葡萄糖的产生率。这些技术可以应用于涉及葡萄糖动力学的广泛实验。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sodium chloride solution</td>
			<td>McKesson</td>
			<td>275595</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL BD Luer-Lok tip syringe</td>
			<td>VWR</td>
			<td>75846-756</td>
			<td>Two syringes per animal (one for isotopic glucose solution, one for glucose-rich isotopic solution)</td>
		</tr>
		<tr>
			<td>1-inch clear transpore tape</td>
			<td>3M</td>
			<td>70200400169</td>
			<td></td>
		</tr>
		<tr>
			<td>1-inch Labeling tape</td>
			<td>Fisher</td>
			<td>GS07F161BA</td>
			<td>Brand is example</td>
		</tr>
		<tr>
			<td>5 mL syringe containing heparanized saline flush</td>
			<td>McKesson</td>
			<td>191-MIH-2235</td>
			<td>One can also prepare a heparin flush solution (10 units/mL heparin in 0.9% sodium chloride)</td>
		</tr>
		<tr>
			<td>5 mm Medipoint Goldenrod animal lancets</td>
			<td>Fisher Scientific</td>
			<td>NC9891620</td>
			<td>5 mm if animal is between 2 and 6 months</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced hot plate stirrer</td>
			<td>VWR</td>
			<td>97042-602</td>
			<td>Brand is example</td>
		</tr>
		<tr>
			<td>BD 27 gauge 0.5 inch needles</td>
			<td>Health Warehouse</td>
			<td>A283952</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 30 gauge 0.5 inch needles</td>
			<td>Medvet</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic Polyethylene (PE) tubing 0.28 mm ID x 0.61 mm</td>
			<td>VWR</td>
			<td>63019-004</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic Polyethylene (PE) tubing 0.28 mm ID x 0.61 mm</td>
			<td>VWR</td>
			<td>63019-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker, 1000 mL</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Caging pellets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clear VOA glass vials with closed-top cap</td>
			<td>Fisher Scientific</td>
			<td>05-719-120</td>
			<td>For storage of acetone and blood draw samples</td>
		</tr>
		<tr>
			<td>Copper toothless alligator clamp for tourniquet</td>
			<td>Amazon</td>
			<td></td>
			<td>Any Brand; smooth toothless alligator clips made of solid copper</td>
		</tr>
		<tr>
			<td>D-(+)-glucose &#62;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose (6,6-D2, 99%)</td>
			<td>Cambridge Isotope Laboratories, Inc.</td>
			<td>DLM-349-PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning silastic tubing 0.3 mm ID x 0.64 mm OD</td>
			<td>VWR</td>
			<td>62999-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifying glass</td>
			<td>Amazon</td>
			<td></td>
			<td>Any brand; similar to LANCOSC Magnifying Glass with Light and Stand</td>
		</tr>
		<tr>
			<td>Microbalance</td>
			<td>Ohaus Adventurer Pro</td>
			<td>AV264C</td>
			<td>Any similar model with 0.0001g accuracy can be used</td>
		</tr>
		<tr>
			<td>Nalgene bottle, 500 mL</td>
			<td>Sigma-Aldrich</td>
			<td>B0158-12EA</td>
			<td>Or any Similar brand; saw in half (including lid) and cut tail-sized notch in the bottom</td>
		</tr>
		<tr>
			<td>PHD Ultra multi-syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-3024A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plexiglass sheet</td>
			<td></td>
			<td></td>
			<td>Any brand; to stabalize mouse during catheter insertion</td>
		</tr>
		<tr>
			<td>Plexiglass sheets and dividers</td>
			<td></td>
			<td></td>
			<td>Any brand; used to cage mice during infusion</td>
		</tr>
	</tbody>
</table>

evaluation of hepatic glucose production in a polycystic ovary syndrome mouse model

多囊卵巢综合征（PCOS）是一种常见疾病，可导致葡萄糖代谢紊乱，如胰岛素抵抗和葡萄糖耐受。葡萄糖代谢失调是该疾病的重要表现，是其发病机制的关键。因此，涉及评估PCOS中葡萄糖代谢的研究至关重要。很少有研究使用非放射性葡萄糖示踪剂直接在PCOS模型中量化肝葡萄糖的产生。在本研究中，我们讨论了通过气相色谱 - 质谱法（GCMS）测量[6，6-2H2]葡萄糖（一种稳定的同位素葡萄糖示踪剂）的M + 2富集来量化PCOS小鼠模型中肝葡萄糖产生速率的分步说明。该过程涉及创建稳定的同位素葡萄糖示踪剂溶液，使用尾静脉导管放置和在同一小鼠中同时在空腹和富葡萄糖状态下输注葡萄糖示踪剂。在GCMS中使用五乙酸衍生物测量[6，6-2H2]葡萄糖的富集。该技术可应用于涉及直接测量肝葡萄糖产生速率的各种研究。

使用先前描述的同位素稀释方程，使用五乙酸衍生物从空腹和富葡萄糖条件下的M + 2富集[6，6-2H2]葡萄糖计算总血浆葡萄糖率（葡萄糖Ra）21。在稳态条件下，假设葡萄糖的出现速率等于葡萄糖的消失速率。对照组，空腹6 h后总葡萄糖Ra为19.98±2.53 mg/（kg·min），富糖条件下总葡萄糖Ra为25.80±1.76 mg/（kg·min）。使用上面列出的计算，空腹6小时后GPR为19...

Watch this Scientific Journal Video about Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model at JoVE.com

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

本研究描述了在多囊卵巢综合征小鼠模型中直接测量肝葡萄糖产生的方法是使用稳定的同位素葡萄糖示踪剂 通过 尾静脉在空腹和富葡萄糖状态中串联。

多囊卵巢综合征小鼠模型中肝葡萄糖产生的评价

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

该程序的目标是确定多囊卵巢综合征或PCOS小鼠模型中的肝葡萄糖产生。葡萄糖代谢失调是PCOS的重要表现，也是其发病机制的关键。因此，涉及评估PCOS中葡萄糖代谢的研究至关重要。在本研究中，我们讨论了通过气相色谱法，质谱法或GCMS测量M加两个六氘代葡萄糖的富集，稳定的同位素葡萄糖示踪剂，在PCOS小鼠模型中定量肝葡萄糖产生速率的分步说明。该过程涉及创建稳定的同位素葡萄糖示踪剂溶液，使用尾静脉导管放置和在同一小鼠中同时在空腹和富葡萄糖状态下输注葡萄糖示踪剂。手术前一天，在生理盐水中制备稳定的同位素葡萄糖示踪剂。对于该实验，在富含葡萄糖的条件下测量空腹期间的葡萄糖产生，因此在两种不同的制剂中制备葡萄糖同位素。示踪剂在无菌条件下通过溶解无菌和无热原的六六氘化葡萄糖和无菌0.9%氯化钠溶液与有或不含葡萄糖来制备。第一次注入仅用示踪剂制备。第二种输液含有示踪剂和非同位素葡萄糖。一旦溶液被制备出来，它们就用0.22微米过滤器进行无菌过滤，并在四摄氏度下储存。溶液的制备方式是，最终输注剂量分别接近每公斤和每分钟一毫克和两毫克。在富含葡萄糖的条件下，在基础条件下测量葡萄糖外观速率时，分别以每公斤1.08毫克和分钟1.9毫克/公斤的速度注注6种氘代葡萄糖的起始平均恒定速率输注。为了模拟喂养状态，我们在富含葡萄糖的条件下采用平均D-葡萄糖输注速率为每公斤和每分钟18.8毫克。将小鼠从家笼中取出，并在实验开始前三小时将它们放入禁食笼中。在这项实验中，我们使用了来自C57BL6J菌株的四个月大的雌性小鼠。通过将小鼠分组到每个胰岛素泵的设定数量的小鼠中来组装笼中设备。将隔板放在平坦，稳定的底座上，为小鼠制作单独的摊位。笼子是透明的，由有机玻璃制成。有一个平坦，稳定的基地，隔板位于该基础上。笼子的门滑动关闭，底部有一个缺口，允许尾巴通过。组装含有一毫升基底输液的注射器，然后使用聚乙烯管连接到输液泵管。通过将速率设置为每小时150微升（这是基本速率）来准备输液泵。将水浴加热至48摄氏度。准备靠近水浴的导管插入站，其中包含30号半英寸针头，硅胶管和一英寸透明运输胶带。选择一个鼠标并将其放在可以进入尾部的安全支架中。在尾部的近端部分放置一块胶带，以便为更远端的导管插入留出空间。将鼠标带到水浴槽中，将尾巴插入水浴中约30至45秒。这有助于扩张尾部脉管系统以进行导管放置。导管插入必须在无菌条件下进行。一旦尾巴变暖，清洁尾巴，并在尾巴的近端放置一个小的铜无齿鳄鱼夹作为止血带。在放大镜下可视化侧尾静脉。然后，小心地将导管插入尾静脉，并轻轻冲洗溶液以确保导管的通畅性。在插入部位周围缠绕一块运输胶带以固定导管，并从尾部取出小止血带。将鼠标放在单独的笼子中并关闭滑动门，确保尾巴突出穿过凹口并保持在笼子外面。在整个导管和尾部上再放一块胶带，将其固定在笼子的底板上。断开冲洗与尾静脉导管的连接，并将小夹子放在导管的硅胶管上，以防止在连接泵的输液管路时回流。牢固连接后，取下夹具并冲洗由输液组成的注油溶液。确保溶液在管中是透明的，没有血迹。一旦注输液管线正常工作，请从鼠标上取下盖子。将标准床上用品放在鼠标周围。用含有示踪剂的输液开始输注，并连续运行三个小时。在输注期间，继续检查小鼠的健康状况以及输液管路。确保输液管已正确固定，并且线路连接点没有泄漏。第一次输注完成后，停止输注并在导管管管上放置一个夹子以防止回流。轻轻地将小鼠从其围栏中取出而不干扰导管以收集血液。对于这个实验，小鼠使用四毫米的柳叶刀进行了脸颊静脉穿刺。将大约75微升的血液收集到您想要的小瓶中。为了在制备质谱分析中使样品脱蛋白，将约15微升血液加入500微升丙酮中。剩余的血液可用于通过血糖仪和/或离心机检查血糖水平，以分离血浆以备将来的激素测定。通过断开管道与注射器的连接，从注射器泵中取出输液，并将其替换为含有示踪剂和葡萄糖的第二次输液。使用富含葡萄糖的同位素葡萄糖输注重复先前的输注步骤。要达到稳定状态，请以每小时600微升的速度推注第二滴液，持续15分钟。记下每组笼子的开始时间。将输注速率降低到每小时150微升，以完成三小时的总输注时间。如前所述重复血液采样。断开输液，在导管部位施加压力，直到出血停止，然后将小鼠送回家笼。接下来，将样本发送到GCMS。使用五乙酸衍生物通过GCCMS计算葡萄糖峰下葡萄糖同位素的同位素富集。简而言之，该方法涉及葡萄糖的五乙酸衍生物的制备，然后在正化学电离模式下使用GCMS进行样品分析。对质荷比（171~169）进行选择性离子监测，以确定葡萄糖同位素的M加2富集。所有动力学测量均在假设稳态条件下进行。使用已建立的同位素稀释方程计算M的总血浆葡萄糖出现率加上葡萄糖同位素和血浆的两次富集。在稳态条件下，假设葡萄糖的出现速率等于葡萄糖的消失速率。内源性葡萄糖产生速率等于总血浆葡萄糖速率减去外源性葡萄糖。表一包括该研究的代表性结果。所有单位均以毫克/千克和分钟表示。使用先前描述的同位素稀释方程，计算总血浆葡萄糖率。在对照组中，空腹状态下的平均葡萄糖率为19.98。在富含葡萄糖的条件下，葡萄糖的出现率为25.8。空腹状态下的葡萄糖产生率为19.08，富葡萄糖状态下的葡萄糖产生率为8.56。葡萄糖代谢异常和体内平衡在 PCOS 中很常见。在葡萄糖代谢异常的疾病中，葡萄糖产生的抑制调节受到损害，导致高血糖。在这项研究中，我们描述了一种直接的方法来同时测量多只小鼠的肝葡萄糖产生速率。该实验的关键组成部分是插入导管并定义葡萄糖同位素和天然葡萄糖的精确测量，以便充分执行GCMS。该研究的优点是由于尾静脉导管插入以及使用易于获得的稳定葡萄糖示踪剂，以微创方式完成输注。该研究存在一些局限性，包括程序的技术需求和对可能的额外技能的需求。总体而言，我们描述了一种在PCOS小鼠模型中测量总肝葡萄糖产生速率的简单，准确的方法。该技术可以作为涉及小鼠模型葡萄糖代谢的多项研究的基础。

Research

JoVE Journal

Medicine

Murine Bioluminescent Hepatic Tumour Model

发光法小鼠肝肿瘤模型

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

科研

医学

A Preclinical Murine Model of Hepatic Metastases

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

肝硬化大鼠门脉高压综合征的侵袭性血流动力学特征

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...