Abstract
The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH) minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5) and reduced expression of growth hormone releasing hormone (Ghrh). Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH) deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety.
Highlights
Mouse models have proven to be versatile tools in biomedical research
Similar RT-qPCR signals were found when a forward primer annealing to the Cre fragment and a reverse primer annealing to the junction between exon 2 and 3 of the human growth hormone (hGH) minigene were used, indicating a single mRNA (Fig 2C)
In this study we have shown that the previously reported physiological abnormalities associated with the Nestin-Cre driver line may, at least partially, be explained by the ectopic expression of hGH in hypothalamus
Summary
Mouse models have proven to be versatile tools in biomedical research. Numerous publications have used knockout and transgenic mouse models. The discovery of the Cre-Lox recombinase in bacteriophage P1 and its application in conditional knockout models has advanced the field enormously [1] This system allows the excision of a DNA fragment that is flanked by two loxP sites (referred to as floxed), using the enzyme Cre-recombinase. The majority of Cre-driver lines are generated by pronuclear microinjection, in which the genomic integration site of the Cre-recombinase transgene is not controlled [2] It can potentially disrupt the expression of endogenous genes. In vivo it has been shown that this can lead to brain damage [6, 7] For all those reasons it is essential to include the Cre-driver lines as control mice in Cre-LoxP studies. Nestin-Cre mice have higher adiposity and circulating leptin levels and they are less tolerant to glucose challenge and more sensitive to insulin administration [16]
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