Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver

Abstract

ObjectiveLaron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized.MethodsCRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue.ResultsGHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2.ConclusionGHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials.

Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy

“…Monogenic disorder is a promising target for developing model pigs for cardiomyopathy by using currently available technologies [18][19][20][21]. Here, we focused on δ-sarcoglycan (δ-SG) gene that causes genetic cardiomyopathy in hamsters [22,23] and mice [24,25].…”

Multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus

“…Concentrations and phosphorylation levels of insulin receptor (INSR)-related signaling molecules in the liver were evaluated by western blot analyses as described previously [25], [26]. Briefly, liver tissue samples were homogenized in Laemmli extraction buffer, and the protein content was determined by the bicinchoninic acid protein assay.…”

A decade of experience with genetically tailored pig models for diabetes and metabolic research

“…Moreover, a reduced incidence of diabetes mellitus in LS patients compared to controls was reported (Guevara-Aguirre et al, 2011), making this condition an interesting model for diabetes and metabolic research. For studies of the underlying mechanisms, we developed GHR deficient (GHR-KO) pigs as a large animal model for the human LS (Hinrichs et al, 2018). Frameshift mutations in the GHR gene were introduced by CRISPR-Cas9 in porcine zygotes.…”

“…Frameshift mutations in the GHR gene were introduced by CRISPR-Cas9 in porcine zygotes. GHR-KO pigs show important hallmarks of the human disease, including reduced levels of insulin-like growth factor 1 (IGF1) and IGF-binding protein 3 (IGFBP3), increased serum GH concentrations, postnatal growth retardation, juvenile hypoglycemia, and a progressive increase in total body fat (Hinrichs et al, 2018).…”