这项工作由奥地利研究促进机构 (FFG; 项目编号 850681) 资助, 应用科学大学上部奥地利基本供资倡议 (项目 GlucoSTAR) 和医学技术创新中心 (定时中心)那接受基本的资金由上部奥地利的省。

根据 2010/63/欧盟的指令, 在胚胎发育的前2/3 阶段, 未孵化的禽胚试验不需要道德委员会的许可。
1. 鸡蛋的贮藏和繁殖
<ol><li>从当地育种者那里获得受精卵 (材料表)。 注: 在使用实验前, 鸡蛋可以在湿润的大气中储存14摄氏度, 最多10天。</li>
	<li>孵化鸡蛋在38°c, 平均湿度为 40-60% 在10或11天的孵化器, 不断打开鸡蛋。</li>
</ol>2. 蛋的选择
<ol><li>孵化11天后, 检查卵子受精。使用烛光进行此步骤。将烛灯的边缘蘸上印泥, 并在鸡蛋的尖面上点燃蜡烛。</li>
	<li>要识别空气中的膀胱, 检查鸡蛋顶部的亮度差异。 注: 气囊呈圆形、更透明的区域, 而蛋白在卵 candled 时呈深色。</li>
	<li>在检测到气囊后, 用以前使用过的墨水按下灯对着鸡蛋进行标记。</li>
	<li>在这个步骤中排除非受精的卵子, 这并不表明蛋白和气囊之间的亮度有差别。</li>
</ol>3. 注射物质
<ol><li>通过在汉克平衡盐溶液 (HBSS) 缓冲液中稀释所需浓度的物质, 制备感兴趣的物质 (例如, 草药提取物或胰岛素)。例如, 在 HBSS 的一个商业上可以使用的胰岛素模拟的 3.3 U/毫升。</li>
	<li>对于所选化合物的应用, 请在气囊的标记区域小心地啄上一双尖头镊子。不要形成比注射器针直径大的洞。注入缓冲液 (300 µL), 包含感兴趣的物质通过注射器的啄区。 注: 使用无菌, 1 毫升单用注射器, 吸管提示, 和血管。给实验戴上手套和实验室大衣。</li>
	<li>为了确定某种物质的降血糖效果, 分别将卵放回孵化器中60、120和180分钟。为确定基础血糖水平, 包括至少 10-15 未治疗的控制卵子。</li>
</ol>4. 毒性试验
<ol><li>在选择的化合物24小时后, 平衡蛋壳膜与磷酸盐缓冲盐水 (PBS) 缓冲 (足以覆盖整个蛋壳膜) 至少三十年代。</li>
	<li>把多余的 PBS 缓冲液倒掉。</li>
	<li>用尖头的镊子小心地拉出蛋壳膜。</li>
	<li>检查血管的病变和确认活力 (例如, 运动) 的小鸡胚。 注意: 请参阅图 1 , 以比较在使用有毒草药提取物后的重要与非重要胚胎。</li>
</ol>5. 血糖值的测定
<ol><li>在各自的时间点, 小心地取出蛋壳上面的气囊和平衡的蛋壳膜与 PBS 缓冲 (足以覆盖整个蛋壳膜)。</li>
	<li>把多余的 PBS 缓冲液倒掉。</li>
	<li>用尖头的镊子小心地拉出蛋壳膜。</li>
	<li>用微剪刀切割和取出胚膜, 使其能很好地接触到合适的血管。切膜至少 2-3 厘米。不要在膜内切开任何大血管, 以避免胚胎不必要的失血。</li>
	<li>找到源自胚胎腹部的大血管。用闭合的微剪刀将这个容器从蛋白中提起, 并将其放在塑料 pH 条上。将 pH 条移动到容器下, 用微剪刀握住, 然后小心地拉回微剪刀。</li>
	<li>用吸管去除胚膜下的 1-2 毫升液体, 以避免下一步稀释血液。将容器和 pH 带用滤纸擦干, 在容器被切开之前。</li>
	<li>在一侧用微剪刀将血管仔细切开。不要把船完全切开。 注: 该船约1毫米厚。不要削减超过一半的船只, 以避免完全通过船只切割, 因为它可能会滑出的条, 没有血液收集是可能的。</li>
	<li>用吸管收集 pH 条上的漏血。用血糖仪测定葡萄糖水平需要十微升的血液。</li>
</ol>6. 统计评价
<ol><li>计算每个时间点的至少10个单独卵的平均值, 并将这些值规范化为控制卵子 (非或缓冲处理的卵子) 之一。</li>
	<li>随后, 确定减少的百分比。此外, 使用胰岛素作为一个积极的控制。 注: 重复每个实验至少三次。</li>
</ol>

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作者没有什么可透露的。

在工业12中, 该方法是一种广泛应用于动物试验的替代测试系统。此处描述的蛋系统表示的是一种修改后的多式凸轮检测方法。在其原始形式下, 对化合物的刺激性特性和配方进行了研究, 并对血管生成进行了分析14,15,16, 我们已经适应了测试方法。具有假定的胰岛素模拟特性的化合物12。根据 2010/63/欧盟的指令, 在胚胎发育的前2/3 阶段, 未经孵化的禽胚实验不需要道德委员会的许可, 因为这些实验不被认为是动物实验。最近的研究已经证明了蛋系统的适用性, 以描述在 GLUT4-translocation-based 主屏幕10中确定的草药提取物的功效, 以降低血糖水平缺少胰岛素12,13。对蛋系统的良好性能的关键点包括: 首先, 一个可靠的育种者提供受精卵。罗曼经典棕鸡是一种很好的选择品种。其次, 实施毒性试验, 排除对所研究化合物的毒性作用需要做的。另外, 制备适合血液收集的血管是很重要的。重要的是要避免切割大血管在胚膜本身, 因为这可能导致非最好的失血。此外, 在将容器切割到 pH 条之前, 必须用过滤纸拍干, 以防止收集到的血液稀释。最后, 有足够数量的实验似乎很重要。我们建议每一个时间点使用至少10个鸡蛋, 和三倍的重复实验。
当然, 此蛋方法有一些限制。因为它需要30分钟, 直到物质通过蛋壳膜吸收, 并与胚膜紧密接触, 不可能量化快速反应的胚胎对应用化合物。此外, 一些化合物可能是有毒的应用浓度, 这往往导致血管病变的胚膜。因此, 基于长期孵化的化合物的细胞毒性试验大约24小时似乎是合理的。最后, 我们发现用于化合物的缓冲系统对该方法的性能有显著影响。12当前使用的是 HBSS 缓冲区, 因为它会对胚胎的血糖水平产生最小的影响。
对于将来的应用程序, 可以测试其他缓冲系统作为 HBSS 的替代方法。此外, 草药提取物对其他血液参数如胆固醇、甘油三酯或脂蛋白的影响可能是一个有吸引力的问题。
我们的新的蛋系统是一个有希望和重要的工具, 以测试具有胰岛素模仿特征的物质在一个活的有机体, 而不需要动物实验。因此, 它填补了体外和体内方法之间的差距。与现有的蛋模型系统相比,17没有必要通过链脲佐菌素 (STZ) 治疗来诱发糖尿病, 因为我们使用胚胎在孵化的10天或11。在这个阶段, 胰岛素的生产尚未开始, 但胚胎已经是胰岛素敏感。此外, 如果使用了 14-17 天的胚胎17, 则可能需要道德委员会的许可。此外, 与备选策略18相比, 此处描述的复合应用程序的危害性和费力程度更低, 增加了实验性的通过投入率。
结合起来, 这种蛋方法是一个有吸引力的系统, 如果一个胰岛素模拟物质的减血作用需要在生物体中测试。

糖尿病是一种代谢性疾病的特点是高血糖, 是由缺陷的胰岛素分泌或胰岛素作用的1。90% 的糖尿病病例均为2型糖尿病 (T2DM), 其中个体表现为胰岛素抵抗, 主要为胰岛素缺乏症2。众所周知, 有几个因素会增加 T2DM 的全球发病率, 包括生活方式的改变, 特别是与营养过剩、衰老和身体不活动有关的变化。根据国际糖尿病联合会 (IDF), 全世界约有4亿人患有糖尿病。此数字预计在未来20年3中达到6亿。
高血糖和胰岛素抵抗引起的糖尿病并发症, 如高血压、血脂异常、葡萄糖不耐受、冠心病和脑血管疾病, 导致预期寿命显著降低, 质量为4。受影响的个体需要降糖药物治疗;但是, 药物的使用, 如二甲双胍5通常与戏剧性的副作用6,7,8相关。因此, 进一步的抗糖尿病的方法, 是安全的, 广泛的可用和廉价的是必要的。
不同的草药化合物、提取物、植物和保健品被归因于通过调节各种细胞功能来特征胰岛素模仿的特性。一个重要的特点是刺激葡萄糖转运体 4 (GLUT4) 到血浆膜从细胞内储存室, 从而增加摄取葡萄糖的肌肉和脂肪组织在没有胰岛素, 被称为胰岛素模仿属性9。以前, 我们实施了一种基于荧光显微镜的方法来量化 GLUT410的易位过程。从提取库中分析大量植物提取物11使用这一化验结果, 确定了有前途的候选者。然而, 需要对生物体进行进一步的试验。本文详细描述的方法是一种改进的鸡胚模型, 已被证明是一种很有前景的识别胰岛素样物质的模型。它代表了一个吸引人的工具, 填补了体外和体内研究之间的巨大差距, 并与现有系统 (如可接受的成本、简单的处理过程和适当的吞吐量率) 相比提供了一些优势。此外, 不需要由道德委员会批准。

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Biochrom</td><td>L1825</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution (HBSS)</td><td>Thermo Scientific</td><td>SH30268.02</td><td></td></tr><tr><td>NovoRapid</td><td>Novo Nordisk</td><td>8-0905-82-304-6</td><td></td></tr><tr><td>Hens eggs (Lohmann classic brown chicken)</td><td>Local Breeder</td><td></td><td></td></tr><tr><td>Accu-check performa</td><td>Roche</td><td>6454011</td><td></td></tr><tr><td>Accu-check Inform II Teststreifen</td><td>Roche&amp;nbsp;</td><td>5942861</td><td></td></tr><tr><td>Incubator&amp;nbsp; HEKA- Turbo 288</td><td>HEKA Brutger&amp;auml;te</td><td></td><td></td></tr><tr><td>Syringe Omnican F 1 mL</td><td>Braun</td><td>09161502S&amp;nbsp;</td><td></td></tr><tr><td>Extracts</td><td>PEKISH extract library</td><td></td><td></td></tr><tr><td>Lamp Tempo Nr. 119</td><td>ORBAN</td><td></td><td></td></tr></tbody></table>

an in ovo model for testing insulin-mimetic compounds

2型糖尿病 (T2DM) 高血糖水平是一种复杂的多因素代谢疾病, 是由胰岛素抵抗和β细胞衰竭引起的。采取各种策略, 包括注射胰岛素或使用胰岛素增敏药物, 以治疗 T2DM 或至少减少症状。此外, 中草药化合物的应用也引起了越来越多的关注。因此, 有必要找到有效的测试系统来识别和表征胰岛素模拟化合物。在这里, 我们开发了一个改良的小鸡胚模型, 它可以测试合成化合物和草药提取物的胰岛素模仿的性质。使用基于荧光显微镜的主屏幕, 将葡萄糖转运体 4 (Glut4) 的易位量化为等离子膜, 我们能够识别出化合物, 主要是草药提取物, 从而导致细胞内葡萄糖的增加。脂肪细胞中的浓度。然而, 这些物质的功效需要在生物体中进一步验证。因此, 我们使用了一个蛋方法来确定它们的降血糖性能。由于在胚胎发育的头2/3 中使用鸡胚不被认为是动物实验, 所以不需要伦理委员会的批准。在此, 详细介绍了该模型的应用。

Gluc 方法的描述:
孵化后与感兴趣的物质, 受精卵被打开, 并准备通过删除蛋壳的血液收集 (图 2)。进一步的准备工作包括平衡和去除蛋壳膜, 以获得胚膜。然后仔细切开这膜, 为血液收集提供合适的血管。优选地, 起源于胚胎腹部的大血管放置在塑料 pH 带上。为了避免在收集过程中稀释血液, 容器和 pH 带被用滤纸拍干。然后将血管切开, 用吸管将血液泄漏到 pH 带上进行分析。
精选中草药提取物对血糖水平的影响:
通过最近建立的 GLUT4-translocation 定量的主屏幕, 我们能够识别出具有胰岛素模拟特性10的物质。对数以百计的水溶性草药提取物进行筛选, 结果发现了几起命中。这些提取物在我们的 in 蛋模型中进行了测试. 从Combretum 菊花(仰光爬虫) 和非公开提取物 (称为萃取物 0845) 中提取的萃取物用于这些实验。我们溶解在 HBSS 缓冲区中的提取物在300毫克/升, 一个浓度, 结果是适当的以前的研究12,13。将物质应用于孵化11天的胚胎。如图 3所示, 仰光爬虫提取物没有导致显著的血糖降低蛋。然而, 血糖浓度已成功减少, 提取物 0845, 轻微下降后60分钟, 这是可比的效果观察使用商业上可用的胰岛素模拟。当卵孵化时间延长时, 观察到的效果显著。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57237/57237fig1.jpg"> 图 1: 有毒化合物对胚胎活力的影响.卵孵化11天(A 和 C)或治疗与有毒草药提取物在10天为另 24 h (B 和 D).应用提取物导致严重的血管病变的胚膜(D)和胚胎(B)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57237/57237fig2.jpg"> 图 2:蛋模型的说明.从左向右: 蛋壳的打开和移除(1-2);胚膜对蛋壳膜的平衡和去除(3);在 pH 条上制备的容器(4);使用葡萄糖表(5)收集血液和葡萄糖浓度的测定。自 Haselgruebler et al(201712) 改编而成。<a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57237/57237fig3.jpg"> 图 3: 草药提取物对血糖水平的影响与未经治疗或胰岛素治疗的卵子相比.卵孵化了11天, 并与所表明的物质 (商用胰岛素模拟: 3.3 U/毫升; 提取物: 300 毫克/升) 溶解在 HBSS 缓冲 (300 µL 体积), 高达 3 h. 用血糖仪测定血糖水平。结果显示没有缓冲区的影响。误差线基于平均值的标准误差。* p < 0.05 和 ** p < 0.0001, 在相同的孵化时间 HBSS 处理的卵显著减少。自 Haselgruebler et al(201712) 改编而成。<a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An In Ovo Model for Testing Insulin-mimetic Compounds at JoVE.com

An In Ovo Model for Testing Insulin-mimetic Compounds

在本研究中, 我们描述了一个蛋系统, 作为一个有希望的工具来测试在生物体中的胰岛素模拟化合物。主要的优势包括适当的吞吐量和可接受的成本, 这使植物化学物质的识别与胰岛素模仿的性质。

用于测试胰岛素模拟化合物的蛋模型

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

an-in-ovo-model-for-testing-insulin-mimetic-compounds

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Unit 1

An In Ovo Model for Testing Insulin-mimetic Compounds

Insulin and Hypoglycemic Drugs

SCIENTISTS IN ACTION

9.7K 次观看.  University of Applied Sciences Upper Austria. 在本研究中, 我们描述了一个蛋系统, 作为一个有希望的工具来测试在生物体中的胰岛素模拟化合物。主要的优势包括适当的吞吐量和可接受的成本, 这使植物化学物质的识别与胰岛素模仿的性质。

视频: An In Ovo Model for Testing Insulin-mimetic Compounds

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

β细胞的有丝分裂原持续管理局完整小鼠胰岛<em&gt;离体</em&gt;使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

科研

生物工程

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

生物学

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

医学

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

使用共同分泌的Luciferase代理物测量相对胰岛素分泌

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

用于测试胰岛素模拟化合物的蛋模型

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β细胞的有丝分裂原持续管理局完整小鼠胰岛

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇