A. Joseph D’Ercole

The role of the insulin-like growth factors in the central nervous system

Increasing evidence strongly supports a role for insulin-like growth factor-I (IGF-I) in central nervous system (CNS) development. IGF-I, IGF-II, the type IIGF receptor (the cell surface tyrosine kinase receptor that mediates IGF signals), and some IGF binding proteins (IGFBPs; secreted proteins that modulate IGF actions) are expressed in many regions of the CNS beginningin utero. The expression pattern of IGF system proteins during brain growth suggests highly regulated and developmentally timed IGF actions on specific neural cell populations. IGF-I expression is predominantly in neurons and, in many brain regions, peaks in a fashion temporally coincident with periods in development when neuron progenitor proliferation and/or neuritic outgrowth occurs. In contrast, IGF-II expression is confined mainly to cells of mesenchymal and neural crest origin. While expression of type I IGF receptors appears ubiquitous, that of IGFBPs is characterized by regional and developmental specificity, and often occurs coordinately with peaks of IGF expression.In vitro IGF-I has been shown to stimulate the proliferation of neuron progenitors and/or the survival of neurons and oligodendrocytes, and in some cultured neurons, to stimulate function. Transgenic (Tg) mice that overexpress IGF-I in the brain exhibit postnatal brain overgrowth without anatomic abnormality (20–85% increases in weight, depending on the magnitude of expression). In contrast, Tg mice that exhibit ectopic brain expression of IGFBP-1, an inhibitor of IGF action when present in molar excess, manifest postnatal brain growth retardation, and mice with ablated IGF-I gene expression, accomplished by homologous recombination, have brains that are 60% of normal size as adults. Taken together, these in vivo studies indicate that IGF-I can influence the development of most, if not all, brain regions, and suggest that the cerebral cortex and cerebellum are especially sensitive to IGF-I actions. IGF-I’s growth-promoting in vivo actions result from its capacity to increase neuron number, at least in certain populations, and from its potent stimulation of myelination. These IGF-I actions, taken together with its neuroprotective effects following CNS and peripheral nerve injury, suggest that it may be of therapeutic benefit in a wide variety of disorders affecting the nervous system.

Postnatal growth responses to insulin-like growth factor I in insulin receptor substrate-1-deficient mice.

Organ weight was compared in adult mice with deletion of one (IRS-1−/+) or both (IRS-1−/−) copies of the insulin receptor substrate-1 (IRS-1) gene and IRS-1+/+ littermates. IRS-1−/+ mice showed modest reductions in weight of most organs in proportion to a decrease in body weight. IRS-1−/− mice showed major reductions in weight of heart, liver, and spleen that were directly proportional to a decrease in body weight. In IRS-1−/− mice, kidney and particularly small intestine and brain exhibited proportionately smaller weight reductions, and gastrocnemius muscle showed a proportionately greater weight reduction than the decrease in body weight. Growth deficits in IRS-1−/− mice could reflect impaired actions of multiple hormones or cytokines that activate IRS-1. To assess the requirement for IRS-1 in insulin-like growth factor I (IGF-I)-dependent postnatal growth, IRS-1−/+ mice were cross-bred with mice that widely overexpress a human IGF-I transgene (IGF+) to generate IGF+ and wild-type mice on an IRS-1+/+, I...

Insulin-like growth factor-I (IGF-I) inhibits neuronal apoptosis in the developing cerebral cortex in vivo

Increased expression of insulin‐like growth factor‐I (IGF‐I) in embryonic neural progenitors in vivo has been shown to accelerate neuron proliferation in the neocortex. In the present study, the in vivo actions of (IGF‐I) on naturally occurring neuron death in the cerebral cortex were investigated during embryonic and early postnatal development in a line of transgenic (Tg) mice that overexpress IGF‐I in the brain, directed by nestin genomic regulatory elements, beginning at least as early as embryonic day (E) 13. The areal density of apoptotic cells (NA, cells/mm2) at E16 in the telencephalic wall of Tg and littermate control embryos was determined by immunostaining with an antibody specific for activated caspase‐3. Stereological analyses were conducted to measure the numerical density (NV, cells/mm3) and total number of immunoreactive apoptotic cells in the cerebral cortex of nestin/IGF‐I Tg and control mice at postnatal days (P) 0 and 5. The volume of cerebral cortex and both the NV and total number of all cortical neurons also were determined in both cerebral hemispheres at P0, P5 and P270. Apoptotic cells were rare in the embryonic telencephalic wall at E16. However, the overall NA of apoptotic cells was found to be significantly less by 46% in Tg embryos. The volume of the cerebral cortex was significantly greater in Tg mice at P0 (30%), P5 (13%) and P270 (26%). The total number of cortical neurons in Tg mice was significantly increased at P0 (29%), P5 (29%) and P270 (31%), although the NV of cortical neurons did not differ significantly between Tg and control mice at any age. Transgenic mice at P0 and P5 exhibited significant decreases in the NV of apoptotic cells in the cerebral cortex (31% and 39%, respectively). The vast majority of these apoptotic cells (>90%) were judged to be neurons by their morphological appearance. Increased expression of IGF‐I inhibits naturally occurring (i.e. apoptotic) neuron death during early postnatal development of the cerebral cortex to a degree that sustains a persistent increase in total neuron number even in the adult animal.

Astrocyte-Specific Overexpression of Insulin-Like Growth Factor-1 Protects Hippocampal Neurons and Reduces Behavioral Deficits following Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) survivors often suffer from long-lasting cognitive impairment that stems from hippocampal injury. Systemic administration of insulin-like growth factor-1 (IGF-1), a polypeptide growth factor known to play vital roles in neuronal survival, has been shown to attenuate posttraumatic cognitive and motor dysfunction. However, its neuroprotective effects in TBI have not been examined. To this end, moderate or severe contusion brain injury was induced in mice with conditional (postnatal) overexpression of IGF-1 using the controlled cortical impact (CCI) injury model. CCI brain injury produces robust reactive astrocytosis in regions of neuronal damage such as the hippocampus. We exploited this regional astrocytosis by linking expression of hIGF-1 to the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, effectively targeting IGF-1 delivery to vulnerable neurons. Following brain injury, IGF-1Tg mice exhibited a progressive increase in hippocampal IGF-1 levels which was coupled with enhanced hippocampal reactive astrocytosis and significantly greater GFAP levels relative to WT mice. IGF-1 overexpression stimulated Akt phosphorylation and reduced acute (1 and 3d) hippocampal neurodegeneration, culminating in greater neuron survival at 10d after CCI injury. Hippocampal neuroprotection achieved by IGF-1 overexpression was accompanied by improved motor and cognitive function in brain-injured mice. These data provide strong support for the therapeutic efficacy of increased brain levels of IGF-1 in the setting of TBI.

Deficient expression of insulin receptor substrate-1 (IRS-1) fails to block insulin-like growth factor-I (IGF-I) stimulation of brain growth and myelination.

To determine whether insulin receptor substrate-1 (IRS-1) is essential in mediating insulin-like growth factor-I (IGF-I) stimulation of brain growth and myelination in vivo, we cross-bred IGF-I transgenic (Tg) mice with IRS-1 null mutant (IRS-1(-/-)) mice and examined brain growth and expression of myelin-specific proteins in mice that overexpress IGF-I with or without IRS-1 expression. We found that while IGF-I overexpression stimulates a dramatic increase in brain weight (43%) by 7-8 weeks of age in the absence of IRS-1, it stimulates a greater increase (50%) with intact IRS-1 expression. To evaluate myelination we investigated IGF-I-stimulated expression of myelin basic protein (MBP) and proteolipid protein (PLP) in the cerebral cortex CTX and brainstem, and found similar increases in each region in IRS-1(-/-) and wild type mice. In studies using mixed glial cultures derived from IRS-1(-/-) mice, IGF-I also increased the abundance of MBP and PLP mRNA. To assess possible alternate mediators of IGF-I actions, we examined IRS-2 and IRS-4 and found that the abundance of each is increased in the CTX of IRS-1(-/-) mice and IGF-I Tg mice. Our results suggest that IRS-1 is not essential in IGF-I promotion of oligodendrocyte development and myelination, and that IRS-2 and IRS-4 may compensate for the loss of IRS-1 expression and function in the cells of oligodendrocyte lineage. Nonetheless, the finding that IGF-I stimulates brain growth less well in the absence of IRS-1 suggests that IRS-1-mediated signaling may be more central to IGF-I action in cells other than glia and oligodendrocytes.

[21] Estimation of tissue concentrations of somatomedin C/insulin-like growth factor I

Publisher Summary The somatomedins, somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II), act on their cells of origin or on the cells near their origin. When hypophysectomized rats are injected with a single dose of growth hormone (GH), Sm-C/IGF-I concentrations in tissues increase before a rise in blood is apparent. Therefore, a more precise understanding of somatomedin regulation might accrue from the studies of tissue concentrations of these growth factors. The somatomedins in blood are complexed to binding proteins, and they can be dissociated from these proteins under acid conditions (pH optimum 3.6). This chapter discusses the method of extraction of somatomedin from its binding proteins. The chapter also describes the assays and calculations of Sm-C/IGF-I radioimmunoassay (RIA).

Hepatic mRNAs up-regulated by starvation: an expression profile determined by suppression subtractive hybridization

Delineating the molecular basis for the metabolic switch from the well‐fed state to starvation is crucial to understanding nutritionally regulated metabolic abnormalities. We have examined the molecular events associated with nutrient deprivation, using suppression subtractive hybridization to define the transcriptional programs up‐regulated in rat liver by starvation. Of the genes that displayed significant increases in their hepatic mRNA levels following 48‐h starvation, most could be assigned to one of five major functional classes. We found up‐regulation of genes involved in energy and protein metabolism, genes that respond to stress, and genes encoding nutrient transporters or signaling transducers. The genes with functions in energy and protein metabolism have roles in initiating gluconeogenesis, switching fuel sources from carbohydrates to fatty acids, and protein turnover. A variety of stress response genes, including acute‐phase reactants, exhibited a marked increased in expression, indicating an attempt to restore homeostasis. The expression of several integrated membrane nutrient transporters that supply essential metabolic substrates was increased dramatically. Some known cytosolic signal transducers, likely involved in the metabolic shift from an anabolic to a catabolic state and in the stress response, were significantly enhanced as well. We also observed increased expression of a variety of other known and novel genes. Collectively, our findings indicate that starvation stimulates multiple signaling pathways, which likely lead to extensive metabolic alterations in the liver. These data should serve to enhance our understanding of the molecular mechanisms underlying energy and nitrogen expenditure in the starved state.

A non-transformed oligodendrocyte precursor cell line, OL-1, facilitates studies of insulin-like growth factor-I signaling during oligodendrocyte development

The process by which oligodendrocyte progenitors differentiate into mature oligodendrocytes is complex and incompletely understood in part because of the paucity of oligodendrocyte precursors cell lines that can be studied in culture. We have developed a non‐immortalized rat oligodendrocyte precursor line, called OL‐1, which behaves in a fashion consistent with developing oligodendrocytes in vivo. This OL‐1 line provides a model for the study of oligodendrocyte development and offers an alternative to the CG‐4 cell line. When OL‐1 cells are propagated in conditioned growth media, they have morphology consistent with immature oligodendrocytes and exhibit A2B5 antigen positive and myelin basic protein‐negative immunoreactivity. Withdrawal of conditioned growth media and culture in serum‐free medium results in OL‐1 cell maturation, manifested by a shift to myelin basic protein‐positive immunoreactivity, A2B5 antigen‐negative immunoreactivity, decreased NG2 mRNA expression, increased expression of proteolipid protein mRNA, and increased expression of CNP protein. In addition, the expression of proteolipid protein and its splicing variant DM‐20 exhibit a pattern that is similar to brain proteolipid protein expression during development. When OL‐1 cells are exposed to Insulin‐like growth factor‐I, there are significant increases in proteolipid protein mRNA expression (p < 0.05), the number of cell processes (p < 0.05), and cell number (p < 0.05). Treatment with the caspase inhibitors Z‐DEVD‐FMK and Z‐VAD‐FMK (inhibitors of caspases 3, 6, 7, 8, 10 and 1, 3, 4, respectively), Insulin‐like growth factor‐I, or both, results in a similar increase in cell number. Because Insulin‐like growth factor‐I does not substantially increase the BrdU labeling of OL‐1 cells, these data collectively indicate that Insulin‐like growth factor‐I increases OL‐1 cell number predominately by promoting survival, rather than stimulating proliferation. This non‐immortalized oligodendrocyte precursor cell line, therefore, exhibits behavior consistent with the in vivo development of oligodendrocytes and provides an excellent model for the study of developing oligodendrocytes.

IGF-I improved bone mineral density and body composition of weaver mutant mice

Our recent report on a parallel decrease in the body weights and serum IGF-I levels of weaver mice suggests that IGF-Is endocrine function may be impaired in neurodegenerative diseases. To further understand the overall effects of IGF-I deficiency on the postnatal growth, we measured bone mineral density (BMD), bone mineral content (BMC), lean body mass (LBM) and fat mass in male and female weaver mice and wild-type littermates on D21 (prepuberty), D45 (puberty), and D60 (postpuberty) using dual-energy X-ray absorptiometry (DEXA). In both male and female weaver mice, we found that the levels of circulating IGF-I paralleled those of BMD, BMC, and LBM, but not the fat mass. Male weaver mice have normal fat mass at all three ages studied, whereas female weaver mice showed a trend to increase their fat mass as they mature. To determine whether circulating IGF-I is a determinant of body composition, we crossbred IGF-I transgenic mice with homozygous weaver mice, which resulted in a significant increase in circulating IGF-I levels in both male and female weaver mice and normalization of their BMD, BMC and body weights. In summary, our results demonstrated that normal circulating IGF-I levels are important in maintaining BMD, BMC, and body composition in neurodegenerative diseases, such as hereditary cerebellar ataxia.

Down-regulation of 14-3-3 η gene expression by IGF-I in mouse cerebellum during postnatal development

Insulin-like growth factor I (IGF-I) overexpression in the postnatal cerebellum of transgenic (Tg) mice results in remarkable cerebellar overgrowth characterized by a near doubling of granule cell number that is predominantly due to inhibition of apoptosis. Using this Tg model we set out to investigate IGF-I anti-apoptotic mechanisms by defining the influence of IGF-I on gene expression. Using a cDNA array technique, we screened a total of 243 mouse apoptosis-related genes, and found that 14-3-3 eta gene expression was significantly reduced in the cerebella of Tg mice compared with their wild-type (Wt) littermates. Using Northern blot analysis to corroborate our microarray finding, we showed that 14-3-3 eta mRNA abundance was decreased from postnatal day P5 through P17. Nonetheless, the expression pattern of 14-3-3 eta in Tg mice followed the same pattern observed in Wt mice, and was indistinguishable from that in Wt mice at P20 and P23. 14-3-3 eta protein abundance, as determined by Western immunoblot analyses, showed similar decreases in the cerebella of Tg mice. In situ hybridization demonstrated that 14-3-3 eta was predominantly, if not exclusively, expressed and regulated in Purkinje cells. 14-3-3 proteins have multiple functions, including participation in pathways that favor cell survival. Our finding of IGF-I-induced down-regulation of 14-3-3 eta expression in Purkinje cell at a time when IGF-I promotes granule cell survival leads us to speculate that down-regulation of 14-3-3 eta may: (a) serve a negative feedback role to modulate Purkinje cell survival, i.e. limit Purkinje cell number, and/or (b) function as part of a distinct signaling mechanism, perhaps one that augments the capacity of Purkinje cells to promote granule cell survival.