Identification of the IGF-1 processing product human Ec/rodent Eb peptide in various tissues: Evidence for its differential regulation after exercise-induced muscle damage in humans George Vassilakos, Anastassios Philippou, Michael Koutsilieris ⁎ Department of Experimental Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece abstract article info Article history: Received 13 June 2016 Received in revised form 16 August 2016 Accepted 2 November 2016 Available online 4 November 2016 Objective: Insulin-like growth factor-1 (IGF-1) is a pleiotropic factor expressed in various tissues and plays a critical role in skeletal muscle physiology. Alternative splicing of the IGF-1 gene gives rise to different precursor polypep- tides (isoforms) which could undergo post-translational cleavage, generating the common mature IGF-1 peptide and different carboxyl terminal extension (E-) peptides, with the fate of the latter being, so far, unknown. The ob- jective if this study was to identify the IGF-1Ec forms or processing product(s), other than mature IGF-1, generated in different human and rodent tissues and particularly in human skeletal muscle after exercise-induced damage. Design: Protein lysates from a wide range of human and rodent tissues were immunoblotted with a rabbit anti- human Ec polyclonal antibody raised against the last 24 amino acids of the C-terminal of the Ec peptide. This anti- body can recognize the Ec peptide, both as part of IGF-1Ec and alone, and also the corresponding rodent forms, due to the high homology that the human Ec shares with the rodent Eb. Results: We were able to confirm, for the first time, that the human Ec peptide and its rodent homologous Eb peptide are produced simultaneously with their precursor protein (pro-IGF-1Ec/Eb) in vivo, in a wide range of tissues (e.g. muscle, liver, heart). Proprotein convertase furin digestion of human muscle and liver protein lysates confirmed that the higher molecular form, pro-IGF-1Ec, can be cleaved to produce the free Ec peptide. Furthermore, initial evidence is provided that Ec peptide is differentially regulated during the process of muscle regeneration after exercise- induced damage in humans. Conclusions: The findings of this study possibly imply that the post-translational modification of the IGF-1Ec pro- peptide may regulate the bioavailability and activity of the processing product(s). © 2016 Published by Elsevier Ltd. Keywords: Ec/Eb peptide IGF-1Ec forms Post-translational processing Pro-protein cleavage Muscle damage 1. Introduction Insulin-like growth factor (IGF-1) is a pleiotropic factor regulating homeostasis in multiple tissues via its endocrine, autocrine and para- crine actions [1]. The induction of variable cellular responses via IGF-1, such as proliferation [2], differentiation [3] and protein synthesis [4] has placed it as a key player in the physiological processes of body growth and development [5], and metabolism [6]. IGF-1 is expressed lo- cally in various tissues (e.g. heart, lung, placenta, and kidney) [7], never- theless liver [8] and skeletal muscle [9] are considered as the primary pools of its production and secretion in the circulation. IGF-1 gene is comprised of 6 exons in humans and rodents and spans a DNA region of approximately 80 kb [10]. Its regulation is complex and involves the usage of multiple promoters [11], different transcription initiation sites [12], alternative splicing and multiple polyadenylation sites [13–15]. Specifically, transcription initiation from either exon 1 or exon 2 generates transcripts referred as class I or II, respectively [16], while alternative splicing at the 3′-end gives rise to three mRNA transcripts in humans (IGF-1Ea, IGF-1Eb and IGF-1Ec) and two in ro- dents (IGF-1Ea and IGF-1Eb) [16,17]. The various IGF-1 transcripts encode several precursor (pre-pro- IGF-1) peptides, thus different IGF-1 products can be derived from var- ious tissues and under different conditions or stimuli [16]. IGF-1 precur- sor polypeptides can undergo post-translational modifications, to produce the mature IGF-1 peptide and variable amino- and carboxyl- terminal peptides, termed as signal and extension (E-) peptides, respec- tively [18]. It is believed that the signal peptide is removed after facilitat- ing the passage of the polypeptide into the endoplasmic reticulum and the secretory pathway [16]. Conversion of pro-IGF-1 to mature peptide cleaves the E-peptides and this cleavage can occur by pro-protein convertases, such as furin, which processes pro-proteins at highly con- served, unique pentabasic motif [16,19,20],(Fig. 1A). Growth Hormone & IGF Research 32 (2017) 22–28 ⁎ Corresponding author at: Department of Experimental Physiology, Medical School, National and Kapodistrian University of Athens, 75 Micras Asias, Goudi, Athens 115 27, Greece. E-mail address: mkoutsil@med.uoa.gr (M. Koutsilieris). http://dx.doi.org/10.1016/j.ghir.2016.11.001 1096-6374/© 2016 Published by Elsevier Ltd. Contents lists available at ScienceDirect Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir