A genetic strategy to measure insulin signaling regulation and physiology in Drosophila.
A genetic strategy to measure insulin signaling regulation and physiology in Drosophila.
复制标题
DOI：
10.1371/journal.pgen.1010619
复制
发表时间：
2023-02
期刊：
影响因子：
4.5
通讯作者：
中科院分区：
文献类型：
--
作者：
胰岛素调节是健康的标志，而受损的胰岛素信号会促进糖尿病等代谢疾病。然而，目前用于测量所有动物中的胰岛素信号传导的测定仍然是半定量的，并且缺乏定量体内生理信号传导动力学所需的灵敏度、组织特异性或时间分辨率。胰岛素信号转导在后生动物中是非常保守的，包括胰岛素依赖性磷酸化和Akt/蛋白激酶B的调节。在这里，我们产生了转基因果蝇允许组织特异性表达的免疫表位标记的Akt（AktHF）。我们开发了酶联免疫吸附试验（ELISA），以定量皮摩尔水平的磷酸化（pAktHF）和总AktHF在单个苍蝇，揭示动态组织特异性生理调节pAktHF响应禁食和再喂养，外源性胰岛素，或有针对性的基因抑制建立胰岛素信号转导调节。基因筛选显示Pp 1 - 87 B是Akt和胰岛素信号传导的未被识别的调节因子。这里的工具和概念提供了发现体内胰岛素信号应答的组织特异性调节剂的机会。胰岛素是一种必需的激素，通过调节能量的使用和靶组织的生长来控制所有动物的代谢。人类中受损的胰岛素信号传导（“抗性”）是2型糖尿病发展的基础，2型糖尿病是一种引起显著发病率和死亡率的流行性疾病。胰岛素抵抗的遗传风险是复杂的，糖尿病的研究受到缺乏以敏感、定量和组织特异性方式测量胰岛素抵抗的工具的限制。在这里，我们描述了一种新的技术来测量果蝇中胰岛素信号的强度。通过将果蝇遗传学与基于抗体的测定相结合，我们可以量化成年果蝇特定组织中的磷酸化Akt，这是一种进化上保守的胰岛素信号转导靶点。我们表明，这项技术可以检测空腹和再喂养后，添加外源性胰岛素，或胰岛素信号通路的遗传破坏胰岛素信号的变化。我们用这种方法发现了一种新的胰岛素信号调节剂，一种由Pp 1 - 87 B编码的磷酸酶。这一令人兴奋的新工具将提高我们研究和发现胰岛素信号传导和抵抗的其他调节因子的能力。
Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. Here, we generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). We developed enzyme-linked immunosorption assays (ELISA) to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts here provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses. Insulin is an essential hormone that controls metabolism in all animals by regulating energy use and growth of target tissues. Impaired insulin signaling (“resistance”) in humans underlies development of type 2 diabetes, a pandemic disease causing significant morbidity and mortality. The genetic risk for insulin resistance is complex, and studies of diabetes are limited by a lack of tools to measure insulin resistance in a sensitive, quantitative, and tissue-specific way. Here, we describe a new technique to measure the strength of insulin signaling in fruit flies. By combining fruit fly genetics with antibody-based assays, we can quantify phosphorylated Akt, an evolutionarily conserved target of insulin signaling, in specific tissues of the adult fly. We show this technique can detect changes in insulin signaling after fasting and refeeding, addition of exogenous insulin, or genetic disruption of the insulin signaling pathway. We used this method to discover a new regulator of insulin signaling, a phosphatase enzyme encoded by Pp1-87B. This exciting new tool should advance our ability to study and discover additional regulators of insulin signaling and resistance.
登录
查看更多内容
影响因子：
7.8
作者：
Bai H;Kang P;Tatar M
通讯作者：
Tatar M
影响因子：
3.3
作者：
Gramates LS;Agapite J;Attrill H;Calvi BR;Crosby MA;Dos Santos G;Goodman JL;Goutte-Gattat D;Jenkins VK;Kaufman T;Larkin A;Matthews BB;Millburn G;Strelets VB;the FlyBase Consortium
通讯作者：
the FlyBase Consortium
影响因子：
29
作者：
Kim SK;Tsao DD;Suh GSB;Miguel-Aliaga I
通讯作者：
Miguel-Aliaga I
影响因子：
64.8
作者：
Kahn, Steven E.;Hull, Rebecca L.;Utzschneider, Kristina M.
通讯作者：
Utzschneider, Kristina M.
影响因子：
3.7
作者：
Gajewski, Kathleen M.;Schulz, Robert A.
通讯作者：
Schulz, Robert A.