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

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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.