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Mörth, Corinna (2005): Consequences of postnatal insulin-like growth factor II overexpression in insulin-like growth factor I deficient mice. Dissertation, LMU München: Tierärztliche Fakultät



Insulin-like growth factor I (IGF-I) and -II (IGF-II) are single chain peptides produced by many tissues, functioning in an endocrine, autocrine or paracrine fashion to regulate cellular proliferation, survival and differentiation. IGF actions are initiated upon binding to the insulin-like growth factor I receptor (IGF-IR) and are modulated through interactions with a family of six secreted IGF-binding proteins (IGFBP-1 to -6). IGF-I is necessary for normal growth and differentiation during both, embryonic and postnatal development. IGF-II is a stimulator of fetal growth but its functions in the postnatal period are still unclear. Notably, expression of IGF-II is shut down shortly after birth in rodents (but not in humans). Previous studies in phosphoenolpyruvate-carboxykinase (PEPCK)-IGF-II transgenic mice demonstrated that overexpression of IGF-II resulted in disproportionate growth of specific organs but a significant increase in body size was not observed. Homozygous IGF-I deficient mice were shown to be severely retarded in growth. The aim of this study was to test whether elevated levels of circulating IGF-II can rescue the dwarfism in IGF-I deficient mice and thereby function as a stimulator of postnatal growth in the absence of IGF-I. For this purpose, we crossed heterozygous IGF-I deficient mice [I+/- IIwt] with heterozygous IGF-I deficient mice carrying PEPCK-IGF-II transgenes [I+/- IItg]. The resulting offspring comprised six different groups: homozygous IGF-I knockout and PEPCK-IGF-II wildtype mice [I-/- IIwt], homozygous IGF-I knockout and PEPCK-IGF-II transgenic mice [I-/- IItg], animals lacking one IGF-I allele and wildtype for the PEPCK-IGF-II transgene [I+/- IIwt], lacking one IGF-I allele and harbouring the PEPCK-IGF-II transgene [I+/- IItg], wildtype for the IGF-I mutation and carrying the PEPCK-IGF-II transgene [I+/+ IItg], and completely wildtype [I+/+ IIwt]. The genotype of all mice was determined by PCR. Body weight of mice was recorded daily until the age of 8 weeks. The nose-rump length (NRL) and the weights of individual organs and of the carcass were recorded and the femurs and lumbar vertebras prepared for further investigations. At an age of 8 weeks, mean serum concentrations of IGF-I were beyond detection level in [I-/- IIwt] and [I-/- IItg] mice, intermediate in [I+/- IIwt] and [I+/- IItg] animals and highest in [I+/+ IIwt] and [I+/+ IItg] mice. IGF-II levels were significantly increased in animals harbouring the PEPCK-IGF-II transgene ([I-/- IItg], [I+/- IItg], and [I+/+ IItg]) when compared to their wildtype counterparts ([I-/- IIwt], [I+/- IIwt], and [I+/+ IIwt]). This reflected the genotype, demonstrating the appropriateness of our experimental model. Analysis of body weight data from day 3-4 after birth until day 60 revealed that in the absence of IGF-I, elevated levels of IGF-II have no effect on body weight gain. The same was found for the nose-rump length and the carcass. The weight of specific organs, however, was altered. Compared to the wildtype counterparts ([I-/- IIwt]), the relative kidney weight in [I-/- IItg] mice was significantly increased. IGF-I is known to play an important role in bone growth and in cancellous bone homeostasis. Investigations of geometric and structural bone parameters showed that in the presence or absence of IGF-I, an increase in the circulating levels of IGF-II was without effect on the skeleton and could not substitute for the skeletal functions of IGF-I in IGF-I-ablated mice. Homozygous IGF-I deficient mice are known to have elevated levels of growth hormone (GH). To demonstrate that the lack of effect on growth in our [I-/- IItg] animals was not due to a loss of these elevated GH-levels, a GH-Western immunoblot was performed, revealing that, despite elevated levels of IGF-II, increased levels of GH were still present in [I-/- IItg] animals. Evaluation of the serum levels of IGFBPs by Western ligand blot analysis demonstrated that IGFBP-1 and IGFBP-4 levels were similar in all groups, whereas the levels of IGFBP-2 and IGFBP-3 were strongly reduced in [I-/- IIwt] animals. In the presence of IGF-II ([I-/- IItg]), they were partially restored but the amounts were still smaller than in the IGF-I wildtype animals ([I+/+ IIwt] and [I+/+ IItg]). In summary, these results show that under our experimental conditions, IGF-II is not able to rescue the postnatal growth deficit of IGF-I knockout mice and apparently does not exert a negative feedback on the secretion of growth hormone. However, it could be demonstrated, that the IGFs have differentiated effects on the regulation of the expression/stability of individual IGFBPs.