Section on Clinical Psychoneuroendocrinology and Neuropsychopharmacology, National Institute on Alcohol Abuse and Alcoholism Division of Intramural Clinical and Biological Research and National Institute on Drug Abuse Intramural Research Program, National Institutes of Health, Bethesda, MD, USA.
Neurobiology of Addiction Section, National Institute on Drug Abuse Intramural Research Program, National Institutes of Health, Baltimore, MD, USA.
Int J Obes (Lond). 2019 Feb;43(2):344-354. doi: 10.1038/s41366-018-0013-5. Epub 2018 Jan 30.
BACKGROUND/OBJECTIVES: Ghrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts by binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing.
Using CRISPR/Cas9, we developed a GHSR KO in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood.
The RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT; p = 0.0322).
We have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.
背景/目的：胃饥饿素是一种源于胃部的激素，与摄食、奖赏、应激和成瘾行为等多种行为有关，其作用是与生长激素促分泌素受体（GHSR）结合。在这里，我们介绍了一种使用 CRISPR 基因组编辑技术创建的新型 GHSR 基因敲除（KO）Wistar 大鼠模型的开发、验证和初步特征。
我们使用 CRISPR/Cas9 在 Wistar 背景下开发了 GHSR KO。使用 RNAscope 在野生型（WT；n=2）和 KO（n=2）大鼠中对 GHSR mRNA 表达缺失进行组织学验证。我们测试了腹腔内酰基-胃饥饿素给药对 WT（n=8）和 KO（n=8）大鼠的食物消耗和血浆生长激素（GH）浓度的影响。我们还分析了这些动物的运动、食物消耗和体脂组成。从早期发育到成年期，我们监测了体重。
RNAscope 分析显示，WT 大鼠的下丘脑、中脑和海马体中存在大量 GHSR mRNA 表达，而 KO 大鼠中则没有观察到探针结合。胃饥饿素给药增加了 WT 大鼠的血浆 GH 水平（p=0.0067）和食物消耗（p=0.0448），但对 KO 大鼠没有影响。KO 大鼠在基础条件下整体消耗的食物较少，在整个发育过程中体重明显低于 WT（p=0.0001）。与 WT 相比，KO 大鼠的棕色脂肪组织（BAT）浓度更高（p=0.0322）。
我们使用组织学、生理学、神经内分泌学和行为学测量方法验证了我们的 KO 模型中的 GHSR 缺失。我们的研究结果表明，大鼠 GHSR 缺失不仅与对胃饥饿素的反应缺失有关，还与每日食物消耗和生长减少以及 BAT 增加有关。这种 GHSR KO Wistar 大鼠模型为研究胃饥饿素系统在肥胖和广泛的医学和神经精神疾病中的作用提供了一种新的工具。
