Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden.
Center for Hypothalamic Research, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA; Division of Endocrinology, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA; Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX, USA.
Mol Metab. 2021 Sep;51:101223. doi: 10.1016/j.molmet.2021.101223. Epub 2021 Mar 31.
The orexigenic hormone ghrelin exerts its physiological effects by binding to and activating the growth hormone secretagogue receptor (GHSR). The recent development of a Ghsr-IRES-Cre knock-in mouse line has enabled to genetically access GHSR-expressing neurons. Inserting a Cre construct using a knock-in strategy, even when following an upstream internal ribosome entry site (IRES) can, however, interfere with expression of a targeted gene, with consequences for the phenotype emerging. This study aimed to phenotype, both physically and metabolically, heterozygous and homozygous Ghsr-IRES-Cre mice, with a view to discovering the extent to which the ghrelin signalling system remains functional in these mice.
We assessed feeding and arcuate nucleus (Arc) Fos activation in wild-type, heterozygous and homozygous Ghsr-IRES-Cre mice in response to peripherally-administered ghrelin. We also characterised their developmental and growth phenotypes, as well as their metabolic responses upon an overnight fast.
Insertion of the IRES-Cre cassette into the 3'-untranslated region of the Ghsr gene led to a gene-dosage GHSR depletion in the Arc. Whereas heterozygotes remained ghrelin-responsive and more closely resembled wild-types, ghrelin had reduced orexigenic efficacy and failed to induce Arc Fos expression in homozygous littermates. Homozygotes had a lower body weight accompanied by a shorter body length, less fat tissue content, altered bone parameters, and lower insulin-like growth factor-1 levels compared to wild-type and heterozygous littermates. Moreover, both heterozygous and homozygous Ghsr-IRES-Cre mice lacked the usual fasting-induced rise in growth hormone (GH) and displayed an exaggerated drop in blood glucose and insulin compared to wild-types. Unexpectedly, fasting acyl-ghrelin levels were allele-dependently increased.
Our data suggest that (i) heterozygous but not homozygous Ghsr-IRES-Cre mice retain the usual responsiveness to administered ghrelin, (ii) the impact of fasting on GH release and glucose homeostasis is altered even when only one copy of the Ghsr gene is non-functional (as in heterozygous Ghsr-IRES-Cre mice) and (iii) homozygous Ghsr-IRES-Cre mice exhibit growth retardation. Of the many transgenic models of suppressed ghrelin signalling, Ghsr-IRES-Cre mice emerge as best representing the full breadth of the expected phenotype with respect to body weight, growth, and metabolic parameters.
促食欲激素胃饥饿素通过与生长激素促分泌素受体（GHSR）结合并激活来发挥其生理作用。最近开发的 Ghsr-IRES-Cre 敲入小鼠品系使人们能够在遗传上访问 GHSR 表达神经元。然而，使用敲入策略插入 Cre 构建体，即使是在内部核糖体进入位点（IRES）上游，也可能干扰靶向基因的表达，并对出现的表型产生影响。本研究旨在对杂合子和纯合子 Ghsr-IRES-Cre 小鼠进行身体和代谢表型分析，以期发现这些小鼠中胃饥饿素信号系统的功能在多大程度上仍然存在。
我们评估了野生型、杂合子和纯合子 Ghsr-IRES-Cre 小鼠对外周给予的胃饥饿素的进食和弓状核（Arc）Fos 激活反应。我们还描述了它们的发育和生长表型，以及它们在 overnight fast 后的代谢反应。
IRES-Cre 盒插入 Ghsr 基因的 3'-非翻译区导致 Arc 中 GHSR 基因剂量减少。杂合子仍然对胃饥饿素有反应，并且更接近野生型，而胃饥饿素的食欲促进作用降低，并且在纯合子同窝仔鼠中未能诱导 Arc Fos 表达。与野生型和杂合子同窝仔鼠相比，纯合子体重较低，体长较短，脂肪组织含量较少，骨骼参数改变，胰岛素样生长因子-1 水平较低。此外，杂合子和纯合子 Ghsr-IRES-Cre 小鼠均缺乏禁食诱导的生长激素（GH）升高，并显示出与野生型相比血糖和胰岛素水平的过度下降。出乎意料的是，禁食酰基胃饥饿素水平与等位基因依赖性增加。
我们的数据表明：（i）杂合子但不是纯合子 Ghsr-IRES-Cre 小鼠保留了对给予的胃饥饿素的通常反应性；（ii）即使只有一个 Ghsr 基因失活（如杂合子 Ghsr-IRES-Cre 小鼠），禁食对 GH 释放和葡萄糖稳态的影响也会发生改变；（iii）纯合子 Ghsr-IRES-Cre 小鼠表现出生长迟缓。在许多抑制胃饥饿素信号的转基因模型中，Ghsr-IRES-Cre 小鼠在体重、生长和代谢参数方面最能代表预期表型的全貌。
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