The sheer amplitude of biological actions of insulin-like growth factor I (IGF-1) affecting all types of cells in all tissues suggests a vast signaling landscape for this ubiquitous humoral signal. While the canonical signaling pathways primarily involve the Ras/MAPK and PI3K/AKT cascades, the evolutionary conservation of insulin-like peptides (ILPs) and their pathways hints at the potential for novel functions to emerge over time. Indeed, the evolutionary trajectory of ILPs opens the possibility of either novel functions for these two pathways, novel downstream routes, or both. Evidence supporting this notion includes observations of neofunctionalization in bony fishes or crustaceans, and the involvement of ILPs pathways in invertebrate eusociality or in vertebrate bone physiology, respectively. Such evolutionary processes likely contribute to the rich diversity of ILPs signaling observed today. Moreover, the interplay between conserved signaling pathways, such as those implicated in aging (predominantly involving the PI3K-AKT route), and lesser known pathways, such as those mediated by biased G-protein coupled receptors and others even less known, may underpin the context-dependent actions characteristic of ILPs signaling. While canonical IGF-1 signaling is often assumed to account for the intracellular pathways utilized by this growth factor, a comprehensive analysis of all the pathways mediated by the IGF-1 receptor (IGF-1R) remains lacking. This review aims to explore both canonical and non-canonical routes of IGF-1R action across various cell types, offering a detailed examination of the mechanisms underlying IGF-1 signaling and highlighting the significant gaps in our current understanding.

Insulin is known mainly for its effects in peripheral tissues, such as the liver, skeletal muscles and adipose tissue, where the activation of the insulin receptor (IR) has both short-term and long-term effects. Insulin and the IR are also present in the brain, and since there is evidence that neuronal insulin signaling regulates synaptic plasticity and that it is impaired in disease, this pathway might be the key to protection or reversal of symptoms, especially in Alzheimer's disease. However, there are controversies about the importance of the neuronal IR, partly because biophysical data on its activation and signaling are much less complete than for the peripheral IR. This review briefly summarizes the neuronal IR signaling in health and disease, and then focuses on known differences between the neuronal and peripheral IR with regard to alternative splicing and glycosylation, and lack of data with respect to phosphorylation and membrane subdomain localization. Particularities in the neuronal IR itself and its environment may have consequences for downstream signaling and impact synaptic plasticity. Furthermore, establishing the relative importance of insulin signaling through IR or through hybrids with its homolog, the insulin-like growth factor 1 receptor, is crucial for evaluating the consequences of brain IR activation. An improved biophysical understanding of the neuronal IR may help predict the consequences of insulin-targeted interventions.

GH signaling yields multiple anabolic and metabolic effects. GH binds the transmembrane GH receptor (GHR) to activate the intracellular GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signals, including signal transducer and activator of transcription 5 (STAT5) activation and IGF-1 gene expression. Some GH effects are partly mediated by GH-induced IGF-1 via IGF-1 receptor (IGF-1R), a tyrosine kinase receptor. We previously demonstrated in non-human cells that GH causes formation of a GHR-JAK2-IGF-1R complex and that presence of IGF-1R (even without IGF-1 binding) augments proximal GH signaling. In this study, we use human LNCaP prostate cancer cells as a model system to further study the IGF-1R's role in GH signaling. GH promoted JAK2 and GHR tyrosine phosphorylation and STAT5 activation in LNCaP cells. By coimmunoprecipitation and a new split luciferase complementation assay, we find that GH augments GHR/IGF-1R complex formation, which is inhibited by a Fab of an antagonistic anti-GHR monoclonal antibody. Short hairpin RNA-mediated IGF-1R silencing in LNCaP cells reduced GH-induced GHR, JAK2, and STAT5 phosphorylation. Similarly, a soluble IGF-1R extracellular domain fragment (sol IGF-1R) interacts with GHR in response to GH and blunts GH signaling. Sol IGF-1R also markedly inhibits GH-induced IGF-1 gene expression in both LNCaP cells and mouse primary osteoblast cells. On the basis of these and other findings, we propose a model in which IGF-1R augments GH signaling by allowing a putative IGF-1R-associated molecule that regulates GH signaling to access the activated GHR/JAK2 complex and envision sol IGF-1R as a dominant-negative inhibitor of this IGF-1R-mediated augmentation. Physiological implications of this new model are discussed.

To study insulin-like growth factor 1 (IGF1)-independent effects of permanent growth hormone (GH) excess on body and organ growth and pathology in vivo, hemizygous bovine GH transgenic mice with homozygous disruption of the Igf1 gene (Igf1(-/-)/GH) were generated, and examined in comparison to Igf1(-/-), Igf1(+/-), wild-type (WT), Igf1(+/-)/GH, and GH mice. GH mice and Igf1(+/-)/GH mice showed increased serum IGF1 levels and the well-known giant-phenotype of GH transgenic mice. In contrast, the typical dwarf-phenotype of Igf1(-/-) mice was only slightly ameliorated in Igf1(-/-)/GH mice. Similar to GH mice, Igf1(-/-)/GH mice displayed hepatocellular hypertrophy, glomerulosclerosis, and reduced volumes of acidophilic cells in the pituitary gland. However, GH excess associated skin lesions of male GH mice were not observed in Igf1(-/-)/GH mice. Therefore, development of GH excess induced liver-, kidney-, and pituitary gland-alterations in GH transgenic mice is independent of IGF1 whereas GH stimulated body growth depends on IGF1.

Insulin receptors, as well as IGF-1 receptors and their postreceptor signaling partners, are distributed throughout the brain. Insulin acts on these receptors to modulate peripheral metabolism, including regulation of appetite, reproductive function, body temperature, white fat mass, hepatic glucose output, and response to hypoglycemia. Insulin signaling also modulates neurotransmitter channel activity, brain cholesterol synthesis, and mitochondrial function. Disruption of insulin action in the brain leads to impairment of neuronal function and synaptogenesis. In addition, insulin signaling modulates phosphorylation of tau protein, an early component in the development of Alzheimer disease. Thus, alterations in insulin action in the brain can contribute to metabolic syndrome, and the development of mood disorders and neurodegenerative diseases.

A steadily growing number of studies have begun to establish that the brain and insulin, while traditionally viewed as separate, do indeed have a relationship. The uptake of pancreatic insulin, along with neuronal biosynthesis, provides neural tissue with the hormone. As well, insulin acts upon a neuronal receptor that, although a close reflection of its peripheral counterpart, is characterized by unique structural and functional properties. One distinction is that the neural variant plays only a limited part in neuronal glucose transport. However, a number of other roles for neural insulin are gradually emerging; most significant among these is the modulation of ligand-gated ion channel (LGIC) trafficking. Notably, insulin has been shown to affect the tone of synaptic transmission by regulating cell-surface expression of inhibitory and excitatory receptors. The manner in which insulin regulates receptor movement may provide a cellular mechanism for insulin-mediated neuroprotection in the absence of hypoglycemia and stimulate the exploration of new therapeutic opportunities.

Growth hormone (GH) function is mediated through multiple endocrine pathways. In the liver, GH also transcriptionally activates hepatocyte nuclear factor-6 (HNF-6; OC-1), a liver-enriched transcription factor that regulates the expression of genes essential to hepatic function. We hypothesize that GH modulates hepatic function in the normal and injured liver through HNF-6 and HNF-6 target genes. CD1 mice received PBS or GH for the 1-, 7-, and 28-day course of Sham operation or bile duct ligation (BDL). Proliferation-, metabolic-, and profibrotic-specific hepatic functions were assessed with a focus on candidate HNF-6 transcriptional target genes. Confirmation of HNF-6 regulation was done by analysis of target gene expression in liver infected with recombinant adenovirus AdHNF-6 expression vectors. GH administration upregulated HNF-6 expression throughout the course of liver injury. This was associated with increased expression of HNF-6 proliferative target genes cyclin D1 and metabolic gene Cyp7A1 and downregulation of profibrogenic TGFb2R. Hepatic function improved such as enhanced hepatocyte proliferation, higher cholesterol clearance throughout the course of injury, and attenuated fibrogenic response at day 28 of BDL. GH treatment also transcriptionally increased albumin expression in an HNF-6-independent manner. This was associated with enhanced serum albumin levels. In conclusion, the GH/HNF-6 axis is a potential in vivo mechanism underlying GH diverse function in the liver to modulate the liver repair response to BDL.

Insulin-like growth factor type I receptor (IGF-IR) is frequently overexpressed in human hepatocellular carcinoma cells (HCC), and this overexpression has been correlated with increased tumor growth. The protective response of HCC to reactive oxygen species (ROS) produced by chemotherapeutic agents is mediated with the induction of phase II detoxifying genes including glutathione transferase (GST). To understand the roles of IGF-IR overexpression in HCC in terms of its detoxifying effect on ROS and conferred resistance to chemotherapy, we analyzed whether IGF-IR overexpressions affect IGF-1-inducible GST expression. GSTalpha was induced by exposure to IGF-1 in IGF-IR cells but not in cells expressing normal levels of IGF-IR. Furthermore, IGF-IR-overexpressed HCCs (IR-HCC) are more resistant to doxorubicin than control HCC cells, which was associated with the increased GST induction by IGF-1. Molecular analyses using GSTA2 promoter supported the involvement of xenobiotic response element (XRE) in GSTalpha induction. IGF-1 caused the nuclear translocation of CCAAT/enhancer-binding protein beta (C/EBPbeta), which might be responsible for XRE activation. In addition, IGF-1 increased the activities of phosphatidylinositol 3-kinase (PI3-kinase) and extracellular signal-regulated kinase in IR-HCCs. Moreover, the inhibition of PI3-kinase completely abolished the nuclear translocation of C/EBPbeta and the up-regulation of GSTalpha protein in IR-HCC treated with IGF-1. However, specific inhibitors against extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase did not alter IGF-1-inducible GSTalpha expression. These results provide evidence that one of the pathological consequences of IGF-IR overexpression in HCCs is the potentiation of GSTalpha inducibility by IGF-1. Moreover, this potentiation of GST may be associated with decreased susceptibility to chemotherapeutic agents such as doxorubicin.

Growth hormone (GH), acting through its receptor (GHR), is essential for somatic growth and development and maintaining metabolic homeostasis. GHR gene-deficient (GHR(-/-)) mice exhibit drastically diminished insulin-like growth factor-I (IGF-I) levels, proportional growth retardation, elevated insulin sensitivity, and reduced islet beta-cell mass. Unlike the liver, which is mostly unaffected by changes in IGF-I level, skeletal muscles express high levels of IGF-I receptor (IGF-IR). The net result of a concurrent deficiency in the actions of both GH and IGF-I, which exert opposite influences on insulin responsiveness, has not been evaluated. We studied insulin-stimulated early responses in the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and p85 subunit of phosphatidylinositol 3-kinase. Upon in vivo insulin stimulation, skeletal muscles of GHR(-/-) mice exhibit transient delayed responses in IR and IRS-1 phosphorylation but normal levels of p85 association with IRS-1. This is in contrast to normal/elevated insulin responses in hepatocytes and indicates tissue-specific effects of GHR gene deficiency. In addition to stimulating normal islet cell growth, GH may participate in islet cell overgrowth, which compensates for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of male GHR(-/-) mice to high-fat diet (HFD)-induced obesity. After 17 wk on a HFD, GHR(-/-) mice became more significantly obese than wild-type mice and exhibited increased beta-cell mass to a slightly higher extent. These data demonstrate that GH signaling is not required for compensatory islet growth. Thus, in both muscle insulin responsiveness and islet growth compensation, normal levels of GH signals do not seem to play a dominant role.

Recent evidence indicates that growth hormone (GH) is involved in liver regeneration. To test whether insulin-like growth factor I (IGF-I) mediates this effect, we studied liver regeneration induced by partial hepatectomy in liver-specific IGF type 1 receptor knockout (LIGFREKO) mice. The absence of IGF-1R caused a significant decrease in hepatocyte proliferation in males (-52%), but not in females, as assessed by Ki67 immunohistochemistry. Cyclin D1 and cyclin A protein levels in the livers of LIGFREKO males were only half those in controls, indicating that cyclin induction during liver regeneration is dependent on IGF-1R signaling. Analyzing the signaling cascade initiated by IGF-1R, we observed a lack of IRS-1 induction in LIGFREKO livers. In contrast, the induction of IRS-2 synthesis was similar in LIGFREKO and control groups, suggesting the existence of differential regulation of IRS synthesis during liver regeneration. Regenerating livers from LIGFREKO animals also showed significantly less activated ERKs than controls. Our findings demonstrate that IGF-1R makes a significant contribution to liver regeneration. Using the LIGFREKO model, we provide new evidence that IGF-1R/IRS-1/ERK signaling may be the intracellular pathway controlling the cell cycle via cyclin D1 and cyclin A in the regenerating liver.

Liver regeneration is a fundamental mechanism by which the liver responds to injury. This process is regulated by endogenous growth factors and cytokines, and it involves proliferation of all mature cells that exist within the intact organ. To understand the role of the GH/IGF-I axis in liver regeneration, we performed partial hepatectomies in three groups of mice: GH antagonist (GHa) transgenic mice, in which the action of GH is blocked; liver IGF-I-deficient mice that lack IGF-I specifically in the liver and also lack the acid-labile subunit (ALS; LID+ALSKO mice), in which IGF-I levels are very low and GH secretion is increased; and control mice. Interestingly, the survival rate of GHa transgenic mice was dramatically reduced after partial hepatectomy (57%) compared with the survival rate of controls (100%) or LID+ALSKO mice (88%). In control mice, the liver was completely regenerated after 4 d, whereas liver regeneration required 7 d in LID+ALSKO mice. In contrast, in GHa mice, liver regeneration reached only 70% of the original liver mass after 4 d and did not improve thereafter. Strikingly, 36 and 48 h after hepatectomy, the livers of control and LID+ALSKO mice, respectively, exhibited intense 5-bromo-2'-deoxyuridine (BrdU) staining, whereas BrdU staining was dramatically decreased in the livers of GHa-treated mice. These results suggest that GH plays a critical role in liver regeneration, although whether it acts directly or indirectly remains to be determined.

Deregulation of the insulin-like growth factor (IGF) axis, including the autocrine production of IGFs, IGF binding proteins (IGFBPs), IGFBP proteases, and the expression of the IGF receptors, has been identified in the development of hepatocellular carcinoma (HCC). Characteristic alterations detected in HCC and hepatoma cell lines comprise the increased expression of IGF-II and the IGF-I receptor (IGF-IR), which have emerged as crucial events in malignant transformation and the growth of tumours. Alterations of IGFBP production and the proteolytic degradation of IGFBPs resulting in an excess of bioactive IGFs, as well as the defective function of the IGF degrading IGF-II/mannose 6-phosphate receptor (IGF-II/M6PR), may further potentiate the mitogenic effects of IGFs in the development of HCC.

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.

Recent evidence that insulin-like growth factor-1 (IGF-1) influences certain properties of H4IIE hepatoma cells independent of insulin led us to examine whether H4IIE cells express IGF-1 receptors. Competitive binding experiments demonstrated IGF-1, but not insulin or IGF-II, could compete with [125I]IGF-1. Chemical crosslinking detected a protein with an apparent mass of 175 kDa and its identity as the IGF-1 receptor alpha-subunit was confirmed by Western blotting. The apparent molecular mass of this protein decreased to 135 kDa following deglycosylation. Immunofluorescence microscopy verified that both insulin and IGF-1 receptors were present, although measurement of IGF-1 receptor quantity revealed they were less abundant than insulin receptors. Binding of IGF-1 was low in growing cells and higher in a quiescent cell population. Scatchard analysis confirmed that receptor density was increased in non-growing H4IIE cells while there was no apparent difference in receptor affinity. Western blot analysis and RT-PCR revealed that both protein and mRNA levels were elevated as cell growth ceased. Interestingly, addition of insulin to quiescent H4IIE cells, which stimulates cell proliferation, further increased IGF-1 receptor protein levels with a peak at 12-24 h. Distinct modes of regulating IGF-1 receptor expression are indicated.

It is commonly believed that the insulin receptor mainly mediates the metabolic effects of insulin, whereas the closely related IGF-I receptor is considered a major factor for the regulation of cell proliferation. Experimental and epidemiological evidence indicates, however, that insulin and insulin receptors may play an important role in breast cancer. This article reviews evidence indicating that (a) insulin receptors are overexpressed in human breast cancer, (b) insulin stimulates growth in breast cancer cells, (c) cells transfected with human insulin receptor may acquire a ligand-dependent transformed phenotype, and (d) breast cancer is associated with insulin resistance and hyperinsulinemia. These findings may open new possibilities in breast cancer prevention, prognosis assessment, and therapy. (Trends Endocrinol Metab 1997; 8:306-312). (c) 1997, Elsevier Science Inc.

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 beginning in 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.

Insulin-like growth factor I (IGF I) is an autocrine/paracrine growth factor that is produced in multiple tissues and is essential for normal developmental growth. Its effects are mediated by activation of a membrane-bound tyrosine kinase receptor, IGF IR. On the basis of the partial rat IGF IR alpha-chain cDNA sequence previously reported, we cloned cDNA encoding the full-length rat IGF IR. The deduced amino acid sequence predicts a 1370-amino acid receptor precursor, which includes signal sequence, a 707-amino acid alpha-chain, a 4-Arg cleavage site, and a 629-amino acid beta-chain. Overall, similarity to human IGF IR is 89% and 98% at the nucleotide and amino acid levels, respectively. Antisense IGF IR expression constructs in vectors incorporating Epstein-Barr virus replicative signals and the cytomegalovirus promoter/enhancer or the inducible human metallothionein IIa promoter/enhancer were assembled and stably transfected into cultured rat aortic smooth muscle cells. Clone CA9 (constitutively expressing abundant antisense IGF IR transcripts), clones MA5 and MA7 (expressing antisense IGF IR transcripts inducibly), and clones ME8 and ME10 (expressing vector alone) were characterized. There was a 57% reduction in IGF IR mRNA levels in clone CA9 after confluence compared with clone ME10. This resulted in a 51% decrease in IGF I binding sites in clone CA9, without a change in binding affinity (Kd), and a 55% and 57% reduction in DNA synthesis rates, basally and in response to 10 ng/mL IGF I, respectively. Clones MA5/MA7 similarly showed a 54% reduction in IGF IR number after confluence following exposure to 100 mumol/L ZnSO4 and a 44% and 58% reduction in DNA synthesis, basally and in response to 10 ng/mL IGF I, respectively. Growth curves indicated that proliferation of clone CA9 in the presence of 10% serum was reduced by 60% compared with clone ME10. Thus, cloning of cDNA encoding the full-length rat IGF IR indicates that this receptor is highly conserved. Antisense targeting of this receptor in vascular smooth muscle cells (VSMCs) demonstrates that a decrease in IGF IR density results in marked inhibition of VSMC proliferation. These findings indicate an important role for this ligand-receptor system in regulating VSMC growth. Specifically, they suggest that modulation of VSMC IGF IR density may be an important mechanism whereby growth of these cells is controlled.

Unipolar depression, alcoholism and suicide have become more common over the past decades. Genetic studies have attempted to link (bipolar) affective disorder to the short arm of chromosome 11 (where the loci for insulin, insulin growth factor (IGF), tyrosine hydroxylase (TH) and h-ras-oncogene are located) but these have failed. Since TH and the insulin receptor require phosphorylation by protein kinases, then a defect of the h-ras-oncogene or its products (p21) could disorder both these systems and compromise catecholaminergic transmission in neurones and energy flow in glial cells. This could lead not only to a predisposition to depression ('trait markers') but to neurotoxic damage, predisposed by inadequate cytosol Mg2+ levels of hypometabolism. Tyrosine, tryptophan and phenylalanine hydroxylases all require tetrahydrobiopterin (BH4) which allosterically regulates its own activity as well as that of these enzymes. Anything which impairs this cofactor could lead to overt depression in predisposed individuals, and the heterocyclic amines are being increasingly implicated. These substances are derived from fried and broiled meats, azo food dyes, soft drinks and hard candies, but particularly from cigarette and petroleum fumes. The heterocyclic amines can inhibit aromatic-l-amino-acid-decarboxylase (AADC) as well as the hydroxylases reversibly, but BH4 is inhibited noncompetitively. Thus, susceptible individuals (those with inherited defective protein kinase phosphorylation) might be 'tipped over' by chronic exposure to these neurotoxins. The rising incidence of unipolar depression-associated morbidity could be significantly linked to increasing levels of heterocyclic amines in the developed nations.

Insulin is a peptide hormone involved in the regulation of glucose homeostasis. Its synthesis and function in the peripheral tissues have been extensively studied and well understood. In contrast, demonstration of insulin in the brain has raised questions concerning its origin and physiological significance. In spite of extensive studies, the source of insulin present in the brain has not yet been conclusively identified. Evidence exists in support of both peripheral and central origins of this hormone in the brain. Recognized physiological effects of insulin in the central nervous system (CNS) include regulation of food intake, control of glucose uptake and trophic actions on neuronal and glial cells. These actions of insulin are mediated by insulin receptor resembling closely that in peripheral tissues and coupled with tyrosine kinase signal transduction pathway. In this review we will discuss theories concerning the origin of insulin in the CNS. In addition, we will present current information on both cellular and physiological effects of this hormone in the brain.

We studied how stimulation of protein kinase C and cAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differentiation while others degenerated. IGF-I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF-I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and cAMP-dependent protein kinases were not involved in mediating the effects of bFGF and IGF-I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular cAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF-I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl cAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1-methyl-4-phenyl-pyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results suggest that increased intracellular levels of cAMP protect dopaminergic neurons in situations of stress like the process of dissociation and plating or the exposure to neurotoxic compounds. Our results reveal novel possibilities for the treatment of Parkinson's disease.

1. The distribution and relative insulin-like growth factor (IGF) binding capacities of membranes derived from 14 tissues of the turkey were examined. 2. Affinity cross-linking analyses using [125I]IGF-I and [125I]IGF-II with membranes derived from the liver, pectoralis major muscle, gizzard, heart and brain indicated that both IGFs interact with only type-I IGF receptors on these tissues. 3. There was no evidence for the existence of a type-II IGF receptor in any tissue. 4. Although considerable variation was detected in the molecular weights of the IGF receptor alpha subunits between tissues (112.2-132.9 kDa), these differences did not appear to influence receptor-ligand affinities.

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.

1. The interaction of insulin-like growth factors (IGFs) with receptors on clonal-derived turkey satellite cells and embryonic myoblasts was compared using competitive binding assays and affinity cross-linking analysis. 2. Although [125I]IGF-I and [125I]IGF-II were displaced similarly by IGF-I and IGF-II within cell lines (P greater than 0.05), displacement, and therefore dissociation constants, differed between cell lines (P less than 0.0001). 3. Receptor cross-linking analysis using iodinated IGFs suggests that both IGF-I and IGF-II interact with the type I receptor on turkey embryonic and posthatch myogenic cells.

This study demonstrated species differences in IGF-1 and IGF-2 receptor binding and localization in the hippocampus of the rat and mouse. Competition binding studies indicated that there were no differences in the relative binding affinities for the type 1 or type 2 receptors between the brains of these animals. These results suggested that the observed species differences were not attributable to alterations in IGF receptor kinetics. Receptor autoradiographic analyses demonstrated that IGF-1 binding differed in both the localization and overall receptor densities observed, with the rat demonstrating more specific localization and greater receptor density in the hippocampus than the mouse. The rat also exhibited a greater density of IGF-2 receptors in the hippocampus than the mouse. Despite differences in IGF receptor populations, both species exhibit similar hippocampal structure and lamination. Therefore, these results demonstrate a disparity in the localization of IGF receptor binding in the rat and mouse, suggesting that IGFs in these species are differentially regulated, with distinct neuromodulatory, neurotrophic, and/or developmental roles in this region of the brain. Previous comparative anatomical studies of the hippocampal formation of rats and mice fail to offer an explanation for the absence or reduction of binding of IGF-1 in the mouse. Although the mouse has a greater cell density in the s. granulosum than the rat, and both species exhibit similar glia and synaptic contact densities in the s. moleculare of the dentate gyrus, the mouse exhibits a complete absence of IGF-1 binding in this region. The lack of anatomical differences in the hippocampal formation of these species suggests that the patterns observed in IGF binding result from alterations in either neurochemical modulation of these neurons or specific neurotrophic requirements of the cells in this region. Differences have been reported on the concentrations and binding of various neurotransmitters in the hippocampus of these species, however these differences do not easily account for the variations observed in IGF binding in this study. IGFs are known to influence acetylcholine neurotransmission in the hippocampus as well as other brain areas in the rat. Recently, a truncated form of IGF-1, in which a tripeptide is cleaved from the N-terminus of the peptide, has been reported in brain. The cleaved tripeptide has been shown to activate glutamate receptors, which may dramatically influence excitatory neurotransmission in this region. Therefore, in addition to the possible neurotrophic actions of the peptide itself, subsequent processing of IGF-1 may be an important aspect of IGF-1 activity in the brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Interactions of insulin-like growth factors (IGFs) from recombinant human and natural ovine sources with sheep liver plasma membranes have been studied. Total specific binding of 125I-hIGF-II (40%) to liver plasma membranes greatly exceeded that of 125I-hIGF-I (1.5%) after incubation at 20 C for 90 min. Binding of 125I-hIGF-II to the plasma membranes was dependent upon time, temperature and membrane concentration of the incubation. Binding of 125I-hIGF-II was only partially reversed by addition of 100 nM IGF-II (18%) or by dilution with excess buffer (36%). Competitive inhibition studies of 125I-hIGF-II binding demonstrated that IGF-II from ovine or recombinant human sources was more effective at inhibiting binding than ovine or human IGF-I. Insulin did not affect binding of 125I-hIGF-II. Plasma membranes were affinity cross-linked to 125I-IGF-II followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Following autoradiography, radioactive bands were localized at 274,000 Mr and 210,000-215,000 Mr in the presence and absence of reducing agent, respectively. This pattern was unaffected by 100 nM human or ovine IGF-I or 1,000 nM insulin, but coincubation with 100 nM human or ovine IGF-II eliminated the radioactive band. These data indicate that an IGF-II specific receptor is present in sheep liver plasma membranes which has characteristics similar to those of nonruminant Type II receptors.

Although the presence of significant amounts of insulin-like growth factor (IGF)-I receptors in fetal tissues is well documented, adult liver has been reported to contain little or no IGF-I binding activity. In the present investigation, substantial amounts of specific IGF-I receptors were detected in crude membrane fractions and in partially purified receptor preparations of female adult rat liver. Insulin was 100 times less potent than IGF-I in competing for 125I-IGF-I binding. IGF-I binding activity was much less than that of insulin binding in both the microsomal fraction and partially purified receptor preparations. Affinity cross-linking of 125I-IGF-I to purified receptors and microsomal fractions followed by electrophoretic analysis under nonreducing conditions revealed labeling of proteins with relative molecular weight (Mr) of 350,000 and 210,000-220,000, corresponding to the molecular mass of the intact tetramer and alpha-beta dimers, respectively. Under reducing conditions, the labeling of proteins with Mr of 130,000 and 260,000, corresponding to the alpha-subunit of IGF-I receptor and its dimer, respectively, was observed. Treatment of microsomes as well as partially purified receptors with 0.5-1 mM dithiothreitol resulted in decreased IGF-I binding, and this correlated with structural changes in the receptor as detected by affinity labeling and electrophoretic analysis. Hepatic IGF-I binding activity was significantly diminished in female rats exposed to chronic growth hormone excess, suggesting down-regulation of IGF-I receptors by the enhanced circulating levels of IGF-I in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive binding studies indicated that PC12 cells have receptors for insulin-like growth factor-I (IGF-I). There are approximately 11,000 +/- 1,500 IGF-I receptors/cell; these receptors have an apparent KD for IGF-I of 7.2 +/- 0.6 nM. Covalent cross-linking of 125I-IGF-I to PC12 cells labeled a 125,000-130,000-Mr protein, presumably the alpha-subunit of the IGF-I receptor. Although PC12 cells also have insulin receptors, the 125I-IGF-I appeared to be cross-linked to IGF-I receptors, because 100 nM IGF-I competed for labeling but 100 nM insulin did not. Bovine chromaffin cells also have IGF-I receptors. The protein tyrosyl kinase activity of IGF-I receptors from bovine adrenal medulla and PC12 cells was examined after purification of the receptors by wheat germ agglutinin-Sepharose chromatography. IGF-I (10 nM) stimulated autophosphorylation of the beta-subunits of the IGF-I receptors from both preparations; the beta-subunits from both sources had Mr values of approximately 97,000. IGF-I also stimulated phosphorylation of the synthetic substrate poly(Glu:Tyr)4:1 by both receptor preparations. IGF-I (IC50 of approximately 0.2 nM) was much more potent than insulin at stimulating phosphorylation of poly(Glu:Tyr) by the bovine adrenal medulla preparation. A maximal concentration of IGF-I (10 nM) increased phosphorylation approximately threefold. IGF-I was slightly more effective than insulin at stimulating the phosphorylation of poly(Glu:Tyr) by the PC12 cell receptor preparation, but neither ligand produced a maximal effect at concentrations up to 100 nM. This result probably reflects the presence of comparable numbers of IGF-I and insulin receptors on PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin and the insulin-like growth factors (I and II) are homologous peptides essential to normal metabolism as well as growth. These peptide hormones are present in the brain, and, based on biosynthetic labeling studies as well as evidence for local gene expression, they are synthesized by nervous tissue as well as being taken up by the brain from the peripheral circulation. Furthermore, the presence of insulin and IGF receptors in the brain, on both neuronal and glial cells, also suggests a role for these peptides in the nervous system. Thus, these ligands affect brain electrical activity, either as neurotransmitters or as neuromodulators, altering the release and re-uptake of other neurotransmitters. The insulin and IGF-I and -II receptors found in the brain exhibit a lower molecular weight than corresponding receptors on peripheral tissues, primarily caused by alterations in glycosylation. Despite these alterations, both brain insulin and IGF-I receptors exhibit tyrosine kinase activity in cell-free systems, as do their peripheral counterparts. Brain insulin and IGF-I receptors are developmentally regulated, with the highest levels appearing in fetal or perinatal life. However, the altered glycosylation of brain receptors does not appear until late in fetal development. The receptors are widely distributed in the brain, but especially enriched in the circumventricular organs, choroid plexus, hypothalamus, cerebellum, and olfactory bulb. These studies on the insulin and IGF receptor in brain, add strong support to the suggestion that insulin and IGFs are important neuroactive substances, regulating growth, development, and metabolism in the brain.