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Mol Metab. 2018 May;11:113-128. doi: 10.1016/j.molmet.2018.03.006. Epub 2018 Mar 15.

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

Author information

1
Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany; Center for Innovative Medical Models (CiMM), Department of Veterinary Sciences, LMU Munich, Hackerstr. 27, 85764 Oberschleißheim, Germany.
2
Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany; Center for Innovative Medical Models (CiMM), Department of Veterinary Sciences, LMU Munich, Hackerstr. 27, 85764 Oberschleißheim, Germany; Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama, Kawasaki, 214-8571, Japan.
3
Institute of Veterinary Pathology, Center for Clinical Veterinary Medicine, LMU Munich, Veterinärstr. 13, 80539 Munich, Germany.
4
Livestock Center of the Veterinary Faculty, LMU Munich, St.-Hubertus-Str. 12, 85764 Oberschleißheim, Germany.
5
Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany; German Center for Diabetes Research (DZD), Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
6
Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany; Center for Innovative Medical Models (CiMM), Department of Veterinary Sciences, LMU Munich, Hackerstr. 27, 85764 Oberschleißheim, Germany; German Center for Diabetes Research (DZD), Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
7
Human Biology and Bioimaging, Faculty of Biology, Biocenter, LMU Munich, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany.
8
German Center for Diabetes Research (DZD), Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Institute of Experimental Genetics, Helmholtz Zentrum München, and Chair of Experimental Genetics, Technical University of Munich, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
9
Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama, Kawasaki, 214-8571, Japan.
10
Cell Signaling Unit, Institute of Genome Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.
11
University Children`s Hospital, University of Giessen, Feulgenstr.12, 35392 Gießen, Germany.
12
Endocrine Laboratory, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstr. 1, 80336 Munich, Germany.
13
Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany; Center for Innovative Medical Models (CiMM), Department of Veterinary Sciences, LMU Munich, Hackerstr. 27, 85764 Oberschleißheim, Germany; Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama, Kawasaki, 214-8571, Japan; German Center for Diabetes Research (DZD), Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany. Electronic address: ewolf@lmu.de.

Abstract

OBJECTIVE:

Laron 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.

METHODS:

CRISPR/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.

RESULTS:

GHR-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.

CONCLUSION:

GHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials.

KEYWORDS:

Dwarfism; Growth hormone receptor; Hypoglycemia; Insulin-like growth factor 1; Laron syndrome; Pig model; Signaling

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