A porcine model for investigating the role of an insulin signaling regulator in development and disease

摘要 在人类和家畜中，发育中的胎儿经历新陈代谢器官和细胞器的编程 以适应子宫内营养供应不足和预计出生后类似的营养短缺。这 被称为“健康和疾病的发育起源”或“节俭表型”的现象是 在人类和家畜中观察到，表现为“小于胎龄儿”(SGA)或“低出生体重” (体重)出生时的后代。在人类中，SGA后代患肥胖症、糖尿病的风险很高 以及出生后生活中的心血管疾病。另一方面，LBW仔猪表现出饲料效率降低， 生长性能和屠体性状发生变化，造成巨大的经济损失。因此，这项研究 在猪模型中，特别是具有自然产生的节俭表型的Ossabaw猪，DOHaD的 提高畜牧业的动物健康、产品质量和盈利能力的潜在优势，同时 同时作为人类的翻译模式，使其非常适合 福利“拨款计划。利用这个模型，我们的主要目标是研究GRB10(生长激素)的作用 受体结合蛋白10)，是介导DOHaD的胰岛素信号转导的适配器蛋白和抑制物。在人类身上， Grb10与10%患有严重SGA的Silver-Russell综合征(SRS)病例有关，从Gwas研究到 II型糖尿病。在小鼠中，消融和过度表达实验强调Grb10是一种拮抗剂 胰岛素信号，生长抑制因子，代谢综合征的介体。 我们的中心假设是，改变的胰岛素信号是调节节俭表型的关键，并调节 因此，Grb10的表达将是克服DOHaD的关键。在本研究中，使用CRISPR/CAS系统，GRB10 获得KO和转基因敲打蛋白(KI)Ossabaw胎儿成纤维细胞。以及前体野生型(WT) 体细胞核移植将产生KO、KI和WT仔猪的细胞、克隆系。使用这个 实验管道，AIM-1将研究GRB10的消融和过表达在产前和 出生后的生长。将仔猪在足月或出生后一段时间内处死，以评估 GRB10基因缺失或过表达对生长速度的影响。AIM-2将研究改变的胰岛素信号的影响 由GRB10介导的代谢健康和胎儿规划。我们将调查GRB10的损失或 肥胖饮食对肥胖和糖尿病发展的过度表达。展出的动物 将培育代谢综合征，并调查后代的跨代遗传 表型。我们预计，该项目的结果将验证GRB10作为增长调节器-A 具有重要的农业特性，可作为糖尿病和代谢综合征治疗干预的候选药物。

ABSTRACT In humans and domestic animals, the growing fetus undergoes programming of metabolic organs and organelles to adapt for poor nutrient supply in utero and in anticipation of similar shortage of nutrients postnatally. This phenomenon known as “Developmental Origins of Health and Disease (DOHaD)” or “thrifty phenotype” is observed in humans and domestic animals, and manifests as “small for gestation age” (SGA) or “low birth weight” (LBW) offspring at the time of birth. In humans, the SGA offspring are at high risk for developing obesity, diabetes and cardiovascular diseases in postnatal life. The LBW piglets on the other hand exhibit reduced feed efficiency, growth performance and altered carcass characteristics, resulting in great economic losses. Therefore, the study of DOHaD in a pig model, specifically the Ossabaw pigs that have a naturally occurring thrifty phenotype has the potential advantage of improving animal health, product quality and profitability in animal agriculture, while simultaneously serving as a translational model for humans, making it an ideal fit for “Dual-purpose with dual- benefit” grant program. Using this model, our main goal is to investigate the role of GRB10 (growth hormone receptor binding protein 10), an adaptor protein and inhibitor of insulin signaling in mediating DOHaD. In humans, GRB10 is linked to 10% of Silver–Russell syndrome (SRS) cases with severe SGA, and from GWAS studies to Type II diabetes. In mice, ablation and overexpression experiments have highlighted Grb10 as an antagonist of insulin signaling, a growth inhibitor, and mediator of metabolic syndrome. Our Central hypothesis is that altered insulin signaling is key to mediating thrifty phenotype, and modulation of GRB10 expression will therefore be key to overcoming DOHaD. In this study, using CRISPR/Cas system, GRB10 KO and transgene knockin (KI) Ossabaw fetal fibroblasts were generated. Along with precursor wildtype (WT) cells, clonal lines of KO, KI, and WT piglets will be generated by somatic cell nuclear transfer. Using this experimental pipeline, Aim-1 will investigate the effect of ablation and overexpression of GRB10 in prenatal and postnatal growth. Piglets will be sacrificed at term or after a period of postnatal growth to evaluate the effect of loss or overexpression of GRB10 on growth rate. Aim-2 will investigate the effect of altered insulin signaling mediated by GRB10 on metabolic health and fetal programming. We will investigate the loss of GRB10 or overexpression on the development of obesity and diabetes when fed obesogenic diet. Animals that exhibit metabolic syndrome will be bred and the offspring investigated for transgenerational inheritance of the phenotype. We anticipate that the results from the project will validate GRB10 as a regulator of growth-an agriculturally important trait, and as a candidate for therapeutic intervention of diabetes and metabolic syndrome.