Martin L. Adamo

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Regulation by fasting of rat insulin-like growth factor I and its receptor. Effects on gene expression and binding.

We have examined, in liver and extrahepatic tissues, the effects of fasting on total insulin-like growth factor I (IGF-I) mRNA levels, on levels of different IGF-I mRNAs generated by alternative splicing of the primary IGF-I transcript, and on IGF-I receptor binding and mRNA levels. A 48-h fast decreased total IGF-I mRNA levels by approximately 80% in lung and liver, approximately 60% in kidney and muscle, and only approximately 30-40% in stomach, brain, and testes. In heart, IGF-I mRNA levels did not change. The levels of the different splicing variants, however, were essentially coordinately regulated within a given tissue. Specific 125I-IGF-I binding in lung, testes, stomach, kidney, and heart was increased by fasting by approximately 30-100%, whereas in brain 125I-IGF-I binding did not change in response to fasting. In tissues in which fasting increased IGF-I receptor number, receptor mRNA levels increased approximately 1.6- to 2.5-fold, whereas when IGF-I receptor number was unchanged in response to fasting, receptor mRNA levels did not change. These data demonstrate that the change in IGF-I and IGF-I receptor mRNA levels during fasting is quantitatively different in different tissues and suggest that regulation of IGF-I and IGF-I receptor gene expression by fasting is discoordinate.

Insulin and insulin-like growth factor receptors in the nervous system.

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 reuptake 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.

Minireview: Mechano-Growth Factor: A Putative Product of IGF-I Gene Expression Involved in Tissue Repair and Regeneration

The discovery that IGF-I mRNAs encoding isoforms of the pro-IGF-I molecule are differentially regulated in response to mechanical stress in skeletal muscle has been the impetus for a number of studies designed to demonstrate that alternative splicing of IGF-I pre-mRNA involving exons 4, 5, and 6 gives rise to a unique peptide derived from pro-IGF-I that plays a novel role in myoblast proliferation. Research suggests that after injury to skeletal muscle, the IGF-IEb mRNA splice variant is up-regulated initially, followed by up-regulation of the IGF-IEa splice variant at later time points. Up-regulation of IGF-IEb mRNA correlates with markers of satellite cell and myoblast proliferation, whereas up-regulation of IGF-IEa mRNA is correlated with differentiation to mature myofibers. Due to the apparent role of IGF-IEb up-regulation in muscle remodeling, IGF-IEb mRNA was also named mechano-growth factor (MGF). A synthetically manufactured peptide (also termed MGF) corresponding to the 24 most C-terminal residues of IGF-IEb has been shown to promote cellular proliferation and survival. However, no analogous peptide product of the Igf1 gene has been identified in or isolated from cultured cells, their conditioned medium, or in vivo animal tissues or biological fluids. This review will discuss the relationship of the Igf1 gene to MGF and will differentiate actions of synthetic MGF from any known product of Igf1. Additionally, the role of MGF in satellite cell activation, aging, neuroprotection, and signaling will be discussed. A survey of outstanding questions relating to MGF will also be provided.

Insulinlike Growth Factors and Their Receptors as Growth Regulators in Normal Physiology and Pathologic States

Abstract Insulinlike growth factors (IGFs), their binding proteins, and receptors are expressed by many different tissues, suggesting that they may act as parts of an autocrine paracrine system in addition to having a classic endocrine role. Since these growth factors are essential for the normal growth and development of the organism, their altered rate of production in a number of important disease states results in severe growth alterations. These include nutritional deprivation, growth hormone deficiency, diabetes, and malignancy.

The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor.

We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).

A circadian-regulated gene, Nocturnin, promotes adipogenesis by stimulating PPAR-γ nuclear translocation

Nocturnin (NOC) is a circadian-regulated protein related to the yeast family of transcription factors involved in the cellular response to nutrient status. In mammals, NOC functions as a deadenylase but lacks a transcriptional activation domain. It is highly expressed in bone-marrow stromal cells (BMSCs), hepatocytes, and adipocytes. In BMSCs exposed to the PPAR-γ (peroxisome proliferator-activated receptor-γ) agonist rosiglitazone, Noc expression was enhanced 30-fold. Previously, we reported that Noc−/− mice had low body temperature, were protected from diet-induced obesity, and most importantly exhibited absence of Pparg circadian rhythmicity on a high-fat diet. Consistent with its role in influencing BMSCs allocation, Noc−/− mice have reduced bone marrow adiposity and high bone mass. In that same vein, NOC overexpression enhances adipogenesis in 3T3-L1 cells but negatively regulates osteogenesis in MC3T3-E1 cells. NOC and a mutated form, which lacks deadenylase activity, bind to PPAR-γ and markedly enhance PPAR-γ transcriptional activity. Both WT and mutant NOC facilitate nuclear translocation of PPAR-γ. Importantly, NOC-mediated nuclear translocation of PPAR-γ is blocked by a short peptide fragment of NOC that inhibits its physical interaction with PPAR-γ. The inhibitory effect of this NOC-peptide was partially reversed by rosiglitazone, suggesting that effect of NOC on PPAR-γ nuclear translocation may be independent of ligand-mediated PPAR-γ activation. In sum, Noc plays a unique role in the regulation of mesenchymal stem-cell lineage allocation by modulating PPAR-γ activity through nuclear translocation. These data illustrate a unique mechanism whereby a nutrient-responsive gene influences BMSCs differentiation, adipogenesis, and ultimately body composition.

Does reduced IGF-1R signaling in Igf1r+/- mice alter aging?

Mutations in insulin/IGF-1 signaling pathway have been shown to lead to increased longevity in various invertebrate models. Therefore, the effect of the haplo- insufficiency of the IGF-1 receptor (Igf1r+/−) on longevity/aging was evaluated in C57Bl/6 mice using rigorous criteria where lifespan and end-of-life pathology were measured under optimal husbandry conditions using large sample sizes. Igf1r+/− mice exhibited reductions in IGF-1 receptor levels and the activation of Akt by IGF-1, with no compensatory increases in serum IGF-1 or tissue IGF-1 mRNA levels, indicating that the Igf1r+/− mice show reduced IGF-1 signaling. Aged male, but not female Igf1r+/− mice were glucose intolerant, and both genders developed insulin resistance as they aged. Female, but not male Igf1r+/− mice survived longer than wild type mice after lethal paraquat and diquat exposure, and female Igf1r+/− mice also exhibited less diquat-induced liver damage. However, no significant difference between the lifespans of the male Igf1r+/− and wild type mice was observed; and the mean lifespan of the Igf1r+/− females was increased only slightly (less than 5%) compared to wild type mice. A comprehensive pathological analysis showed no significant difference in end-of-life pathological lesions between the Igf1r+/− and wild type mice. These data show that the Igf1r+/− mouse is not a model of increased longevity and delayed aging as predicted by invertebrate models with mutations in the insulin/IGF-1 signaling pathway.

Generation of a New Congenic Mouse Strain to Test the Relationships Among Serum Insulin-like Growth Factor I, Bone Mineral Density, and Skeletal Morphology In Vivo†

Insulin‐like growth factor (IGF) I is a critical peptide for skeletal growth and consolidation. However, its regulation is complex and, in part, heritable. We previously indicated that changes in both serum and skeletal IGF‐I were related to strain‐specific differences in total femoral bone mineral density (BMD) in mice. In addition, we defined four quantitative trait loci (QTLs) that contribute to the heritable determinants of the serum IGF‐I phenotype in F2 mice derived from progenitor crosses between C3H/HeJ (C3H; high total femoral BMD and high IGF‐I) and C57BL/6J (B6; low total femoral BMD and low IGF‐I) strains. The strongest QTL, IGF‐I serum level 1 (Igflsl‐1; log10 of the odds ratio [LOD] score, ∼9.0), is located on the middle portion of chromosome (Chr) 6. For this locus, C3H alleles are associated with a significant reduction in serum IGF‐I. To test the effect of this QTL in vivo, we generated a new congenic strain (B6.C3H‐6T [6T]) by placing the Chr 6 QTL region (D6Mit93 to D6Mit150) from C3H onto the B6 background. We then compared serum and skeletal IGF‐I levels, body weight, and several skeletal phenotypes from the N9 generation of 6T congenic mice against B6 control mice. Female 6T congenic mice had 11‐21% lower serum IGF‐I levels at 6, 8, and 16 weeks of age compared with B6 (p < 0.05 for all). In males, serum IGF‐I levels were similar in 6T congenics and B6 controls at 6 weeks and 8 weeks but were lower in 6T congenic mice at 16 weeks (p < 0.02). In vitro, there was a 40% reduction in secreted IGF‐I in the conditioned media (CMs) from 6T calvaria osteoblasts compared with B6 cells (p < 0.01). Total femoral BMD as measured by peripheral quantitative computed tomography (pQCT) was lower in both 6T male (−4.8%, p < 0.01) and 6T female (−2.3%, p = 0.06) congenic mice. Geometric features of middiaphyseal cortical bone were reduced in 6T congenic mice compared with control mice. Femoral cancellous bone volume (BV) density and trabecular number (Tb.N) were 50% lower, whereas trabecular separation (Tb.Sp) was 90% higher in 8‐week‐old female 6T congenic mice compared with B6 control mice (p < 0.01 for all). Similarly, vertebral cancellous BV density and Tb.N were lower (−29% and −19%, respectively), whereas Tb.Sp was higher (+29%) in 16‐week‐old female 6T congenic mice compared with B6 control mice (p < 0.001 for all). Histomorphometric evaluation of the proximal tibia indicated that 6T congenics had reduced BV fraction, labeled surface, and bone formation rates compared with B6 congenic mice. In summary, we have developed a new congenic mouse strain that confirms the Chr 6 QTL as a major genetic regulatory determinant for serum IGF‐I. This locus also influences bone density and morphology, with more dramatic effects in cancellous bone than in cortical bone.

Molecular and Cellular Aspects of Insulin-like Growth Factor Action

Publisher Summary This chapter discusses the molecular and cellular aspects of insulin-like growth factor (IGF) action. The IGF-I and IGF-II are pluripotent factors that regulate growth, differentiation, and the maintenance of differentiated function in numerous tissues and in specific cell types. Both IGFs are produced in largest amounts by the liver and are secreted into the circulation, where they function as classical endocrine agents by interacting with specific cell-surface receptors present on target tissues. The widespread distribution of IGF receptors, and the production and secretion of the IGFs themselves by almost all extrahepatic tissues, suggests that these factors employ autocrine and paracrine modes of action. The actions of the IGFs are also influenced by a family of IGF binding proteins that are found in the circulation and in extracellular fluids; these proteins may have positive or negative effects on IGF action through different mechanisms. The overall actions of these important molecules are governed by a complex interplay between ligands, receptors, and binding proteins. The levels and the regulation of each component contribute significantly to the ultimate biological effects manifested in a given situation.