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The pro-Forms of Insulin-Like Growth Factor I (IGF-I) Are Predominant in Skeletal Muscle and Alter IGF-I Receptor Activation

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Endocrinology (Impact Factor: 4.64). 03/2013; 154(3):1215-24. DOI: 10.1210/en.2012-1992
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ABSTRACT IGF-I is a key regulator of muscle development and growth. The pre-pro-peptide produced by the Igf1gene undergoes several posttranslational processing steps to result in a secreted mature protein, which is thought to be the obligate ligand for the IGF-I receptor (IGF-IR). The goals of this study were to determine what forms of IGF-I exist in skeletal muscle, and whether the mature IGF-I protein was the only form able to activate the IGF-IR. We measured the proportion of IGF-I species in murine skeletal muscle and found that the predominant forms were nonglycosylated pro-IGF-I and glycosylated pro-IGF-I, which retained the C-terminal E peptide extension, instead of mature IGF-I. These forms were validated using samples subjected to viral expression of IGF-I combined with furin and glycosidase digestion. To determine whether the larger molecular weight IGF-I forms were also ligands for the IGF-IR, we generated each specific form through transient transfection of 3T3 cells and used the enriched media to perform kinase receptor activation assays. Compared with mature IGF-I, nonglycosylated pro-IGF-I had similar ability to activate the IGF-IR, whereas glycosylation of pro-IGF-I significantly reduced receptor activation. Thus, it is important to understand not only the quantity, but also the proportion of IGF-I forms produced, to evaluate the true biological activity of this growth factor.

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Available from: Anastassios Philippou, Aug 29, 2015
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    • "In contrast to the activity of mature and pro-IGF-I, we found that glycosylated pro-IGF-I was inefficient at receptor activation in vitro [23]. How, then, can we explain the multipronged benefits to muscle mass, strength and regenerative capacity in mice expressing IGF-IA, in which the predominant form that is stored is glycosylated pro-IGF-I [33] [35]? "
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    ABSTRACT: It is virtually undisputed that IGF-I promotes cell growth and survival. However, the presence of several IGF-I isoforms, vast numbers of intracellular signaling components, and multiple receptors results in a complex and highly regulated system by which IGF-I actions are mediated. IGF-I has long been recognized as one of the critical factors for coordinating muscle growth, enhancing muscle repair, and increasing muscle mass and strength. How to optimize this panoply of pathways to drive anabolic processes in muscle as opposed to aberrant growth in other tissues is an area that deserves focus. This review will address how advances in the bioavailability, potency, and tissue response of IGF-I can provide new potential directions for skeletal muscle therapeutics.
    Growth Hormone & IGF Research 10/2014; 24(5). DOI:10.1016/j.ghir.2014.06.003 · 1.33 Impact Factor
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    • "These E-peptides are either cleaved by proteases to release mature IGF or stay attached and together with ‘mature IGF sequence’ to form pro-IGF-I (A, B or C). It has been recently demonstrated that pro-IGF-1A form is as potent as mature IGF-1 to activate IGF-1R and is a predominant form present in muscle (12). Another level of complexity in the IGF-1 activity is glycosylation of IGF-1A isoform. "
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    ABSTRACT: The human Igf-1 gene not only produces insulin‑like growth factor-I (IGF-I), but also different carboxy‑terminal extensions, known as E peptides, through alternative splicing. We and others have shown that human Eb peptide (hEb) derived from Igf-1 has intrinsic biological activity and is localized to nuclei of transfected cells. Since hEb actions can complement the activity of IGF-I itself, the aim of the present study was to compare IGF-I isoforms at the endogenous protein and transcript level in cancer cell lines, including HeLa, U2OS, HepG2 and K562 cells. Quantitative real-time PCR (qRT‑PCR) using Igf-1 isoform specific primers was performed to determine expression patterns, using β-actin as a reference gene. The overall relative Igf-1 transcript level was different across the cell lines, with ~80-fold higher expression in K562 (130.2±31.2) than in U2OS cells (1.7±1.1). The relative copy number of Igf-1b was the highest in HepG2 (69.9±28.6) and K562 cells (28.3±6.7), whereas the relative copy numbers of Igf-1a and Igf-1c were significantly higher in K562 cells compared to all other cell lines. Immunoblotting using total cell lysates, cytoplasmic and nuclear fractions were carried out to determine the level and distribution of IGF-I proteins. K562 cells exhibited the highest level of hEb in total cell lysates and nuclear fractions and no cell lines displayed hEb in the cytoplasmic fractions. In contrast, IGF-IA was the highest in HeLa cells and was enriched only in the cytoplasmic fraction. Since relatively low IGF-1A transcript level but relatively high pro‑IGF-1A protein level is plausible, we hypothesized that these transcripts could be processed with higher efficiency and/or the protein product may be stabilized by viral HPV oncogenes in HeLa cells. We assert that while it is important to analyze Igf-1 transcript level, it may be more relevant to determine the IGF isoforms at the protein level.
    Oncology Reports 07/2014; DOI:10.3892/or.2014.3329 · 2.19 Impact Factor
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    • "Subtilisin-related proprotein convertases like furin can cleave polypeptides that include this motif, resulting in free mature IGF-I and an E-peptide (Duguay et al., 1995, 1997; Duguay, 1999). Intriguingly, uncleaved pro-IGF-I is detectable in conditioned media and in vivo in serum (Powell et al., 1987; Conover et al., 1989, 1993; Wilson et al., 2001; Barton et al., 2012; Durzynska et al., 2013a). To date, however, it is unclear if pro-IGF-I is bioactive or simply an inactive precursor or source for mature IGF-I and/or E-peptides. "
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    ABSTRACT: Insulin-like growth factor I (IGF-I) is a key regulator of muscle development and growth. The pre-pro-peptide produced by the Igf1 gene undergoes several post-translational processing steps to result in a secreted mature protein, which is thought to be the obligate ligand for the IGF-I receptor (IGF-IR). However, the significance of the additional forms and peptides produced from Igf1 is not clear. For instance, the C-terminal extensions called the E-peptides that are part of pro-IGF-I, have been implicated in playing roles in cell growth, including cell proliferation and migration and muscle hypertrophy in an IGF-IR independent manner. However, the activity of these peptides has been controversial. IGF-IR independent actions suggest the existence of an E-peptide receptor, yet such a protein has not been discovered. We propose a new concept: there is no E-peptide receptor, rather the E-peptides coordinate with IGF-I to modulate activity of the IGF-IR. Growing evidence reveals that the presence of an E-peptide alters IGF-I activity, whether as part of pro-IGF-I, or as a separate peptide. In this review, we will examine the past literature on IGF-I processing and E-peptide actions in skeletal muscle, address the previous attempts to separate IGF-I and E-peptide effects, propose a new model for IGF-I/E-peptide synergy, and suggest future experiments to test if the E-peptides truly modulate IGF-I activity.
    Frontiers in Endocrinology 03/2013; 4:42. DOI:10.3389/fendo.2013.00042
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