Disruption of the Igf2 gene alters hepatic lipid homeostasis and gene expression in the newborn mouse.

Newborns with intrauterine growth-restriction are at increased risk of mortality and life-long comorbidities. Insulin-like growth factor-II (IGF2) deficiency in humans, as well as in mice, leads to intrauterine growth restriction and decreased neonatal glycogen stores. The present study aims to further characterize the metabolic and transcriptional consequences of Igf2 deficiency in the newborn. We found that, despite being born significantly smaller than their wild-type ( Igf2+/+) littermates, brain size was preserved in Igf2 knockout ( Igf2-/-), consistent with nutritional deficiency. Histological and triglyceride analyses of newborn livers revealed that Igf2-/- mice are born with hepatic steatosis. Gene expression analysis in Igf2-/- newborn livers showed an alteration of genes known to be dysregulated in chronic caloric restriction, including the most upregulated gene, serine dehydratase. Multiple genes connected with lipid metabolism and/or hepatic steatosis were also upregulated. Ingenuity Pathway Analysis confirmed that the biological functions most altered in livers of Igf2-/- newborns are related to lipid metabolism, with the top upstream regulator predicted to be the peroxisome proliferator-activated receptor alpha, a master regulator of hepatic lipid and carbohydrate homeostasis. Together, our data indicate that Igf2 deficiency leads to a newborn phenotype strongly reminiscent of nutritional deficiency, including growth retardation, increased brain/body weight ratio, hepatic steatosis, and characteristic changes in hepatic gene expression. We propose that in addition to its growth factor proliferating functions, Igf2 may also regulate growth by altering the expression of genes that control nutrient metabolism in the newborn.

Insulin-like growth factor 2 and the insulin receptor, but not insulin, regulate fetal hepatic glycogen synthesis.

Whether insulin or IGFs regulate glycogen synthesis in the fetal liver remains to be determined. In this study, we used several knockout mouse strains, including those lacking Pdx-1 (pancreatic duodenal homeobox-1), Insr (insulin receptor), and Igf2 (IGF-II) to determine the role of these genes in the regulation of fetal hepatic glycogen synthesis. Our data show that insulin deficiency does not alter hepatic glycogen stores, whereas Insr and Igf2 deficiency do. We found that both insulin receptor isoforms (IR-A and IR-B) are present in the fetal liver, and their expression is gestationally regulated. IR-B is highly expressed in the fetal liver; nonetheless, the percentage of hepatic IR-A isoform, which binds Igf2, was significantly higher in the fetus than the adult. In vitro experiments demonstrate that Igf2 increases phosphorylation of hepatic Insr, insulin receptor substrate-2, and Akt proteins and also the activity of glycogen synthase. Igf2 ultimately increased glycogen synthesis in fetal hepatocytes. This increase could be blocked by the phosphoinositide 3-kinase inhibitor LY294008. Taken together, we propose Igf2 as a major regulator of fetal hepatic glycogen metabolism, the insulin receptor as its target receptor, and phosphoinositide 3-kinase as the signaling pathway leading to glycogen formation in the fetal liver.

Decreased motoneuron survival in Igf2 null mice after sciatic nerve transection.

The search for therapeutic targets to prevent neurons from dying is ongoing and involves the exploration of a long list of neurotrophic factors. Insulin-like growth factor 2 (IGF2) is a member of the insulin family with known neurotrophic properties. In this study, we used Igf2 knockout (Igf2) neonate mice to determine whether Igf2 deficiency is detrimental to motor neuron survival after axonal injury. Results show that Igf2 neonatal mice are more susceptible to motor neuron damage than Igf2 mice, as they have a significantly lower percentage of motor neuron survival after a sciatic nerve transection. Neuronal survival was significantly improved in Igf2 mice when IGF2 was administered. These results support the role of IGF2 in neonatal motor neuron survival.

Placental glycogen stores are increased in mice with H19 null mutations but not in those with insulin or IGF type 1 receptor mutations.

The function of glycogen in the placenta remains controversial. Whether it is used as a source of fuel for placental consumption or by the fetus in times of need has yet to be determined. Two imprinted genes, insulin-like growth factor 2 (Igf2) and H19 are highly expressed in the placenta. We have previously demonstrated that mice with Igf2 deficiency have lower levels of placental glycogen. In this study, we used mice with targeted disruption of the H19 gene (H19(-/-)) to determine the importance of Igf2 over-expression in placental growth and glycogen stores. In addition, since Igf2 mediates most of its functions by signaling through the insulin and/or IGF Type 1 receptors, we determined whether gene deletions to these receptors could affect placental glycogen stores. Our data demonstrate that placentas from H19(-/-) mice are heavier, have higher number of glycogen cells, and contain larger glycogen concentrations than those of H19(+/+) mice. No differences in GSK-3, ERK, or total Akt expression or phosphorylation were found between genotypes; however, Akt1 protein expression levels were significantly increased in H19(-/-) placentas. Results obtained from insulin receptor or IGF Type 1 receptor mutant mice did not show differences in placental glycogen content compared to their wild-type littermates, supporting the notion of a specific placental Igf2 receptor. Taken together, these results support a role for Igf2 and Akt1, but not the insulin nor the IGF Type 1 receptors, in the regulation of placental growth and glycogen metabolism.

IGF2 knockout mice are resistant to kainic acid-induced seizures and neurodegeneration.

Insulin-like growth factor 2 (Igf2), a member of the insulin gene family, is important for brain development and has known neurotrophic properties. Though Igf2, its receptors, and binding proteins, are expressed in the adult CNS, their role in the adult brain is less well-understood. Here we studied how Igf2 deficiency affects brains of adult Igf2 knockout (Igf2(-/-)) mice following neurotoxic insult produced by the glutamate analog kainic acid (KA). Igf2(-/-) mice exhibited attenuated epileptiform activity in response to KA and were less susceptible to hippocampal neurodegeneration compared with Igf2(+/+) mice. Other brain areas protected by the lack of Igf2 included the amygdala complex, septal nuclei, and thalamic region. Apoptosis, as determined by TUNEL and Hoechst 33342 staining, was accordingly less for Igf2(-/-) mice. Hippocampal slices from Igf2(-/-) mice also were protected against the effects epileptogenic effects of KA compared to Igf2(+/+) mice suggesting that neuroprotection afforded by a lack of Igf2 may be developmental in origin and experiments demonstrating enhanced synaptic inhibition in slices taken from Igf2(-/-) mice support this hypothesis. Taken together, these results suggest that Igf2 may be important for mechanisms and circuits that contribute to neurodegeneration and epilepsy.

Igf2 deficiency results in delayed lung development at the end of gestation.

IGF-II is a polypeptide hormone with structural homology to insulin and IGF-I. IGF-II plays an important role in fetal growth as mice with targeted disruption of the IGF-II gene (Igf2) exhibit severe growth retardation. The role of IGFs in the fetal lung has been suggested by several studies, including those that have identified IGF mRNA expression, and that of their receptors and binding proteins in the lungs at different stages of development. In this study, we used mice carrying a null mutation of Igf2 (Igf2-/- mice) to determine whether the absence of IGF-II had any effect in fetal lung maturation. Our results showed that the lungs of Igf2-/- fetuses had thicker alveolar septae and poorly organized alveoli when compared with those of Igf2+/+ on d 17.5 and 18.5 of gestation. These morphological alterations may be the result of exposure to lower levels of glucocorticoids because plasma corticosterone levels were significantly lower in Igf2-/- mothers compared with wild-type controls. In support of this, fetuses from homozygous knockout matings, where mothers were treated with 15 microg/ml corticosterone, and Igf2-/- fetuses obtained from heterozygous matings had similar lung histology to those of wild-type fetuses. Finally, we found that IGF-I and SP-B mRNA levels were up-regulated in the lungs of Igf2-/- fetuses at the end of gestation. This study suggests that Igf2 plays an important role in the development of the fetal lung and may affect fetal lung maturation by regulating maternal factors, such as corticosterone levels, during pregnancy.