VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice

RESEARCH ARTICLE VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice Filomena D’Amato1*, Barbara Noli1, Laura Angioni1, Efisio Cossu2, Michela Incani2, Irene Messana3, Barbara Manconi3, Paola Solinas1, Raffaella Isola1, Stefano Mariotti2, Gian-Luca Ferri1‡, Cristina Cocco1‡ 1 Department of Biomedical Sciences, University of Cagliari, 09042, Monserrato, Italy, 2 Department of Medical Sciences, University of Cagliari, 09042, Monserrato, Italy, 3 Department of Life and Environmental Sciences, University of Cagliari, 09042, Monserrato, Italy ‡ These authors are co-senior authors on this work. * Abstract OPEN ACCESS Citation: D’Amato F, Noli B, Angioni L, Cossu E, Incani M, Messana I, et al. (2015) VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice. PLoS ONE 10(11): e0142333. doi:10.1371/journal.pone.0142333 Editor: Juergen Eckel, GDC, GERMANY Received: July 4, 2015 Accepted: October 19, 2015 Published: November 12, 2015 Copyright: © 2015 D’Amato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and Supporting Information files. Funding: This study was sponsored by the ARS (Autonomous Region of Sardinia) through the ‘Sardinia PO FSE 2007-1013’ funds (the L.R. 7/2007 for the ‘Promotion of the Scientific Research and of the Technological Innovation in Sardinia’). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92 ±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0142333 November 12, 2015 1 / 16 VGF Peptides in Obesity and T2D Introduction Type 2 diabetes (T2D) is a chronic disorder of carbohydrate, fat and protein metabolism, steadily increasing worldwide. The global epidemic of T2D appears to be largely secondary to insulin resistance induced by obesity [1]. Of metabolically healthy adults, those with body mass index (BMI)  30 kg.m-2 show an increased risk of developing T2D, compared to normal weight individuals [2–4]. Adipose tissue appears to be a critical component in metabolic control, responding to nutritional, hormonal and neuronal signals [5, 6] by releasing hormones including adipocytokines, which in turn regulate insulin sensitivity [7]. The neurotrophin responsive gene vgf (nonacronymic name) has recently been shown to be involved in the regulation of energy balance [8, 9]. VGF mRNA is selectively expressed in neurons and neuroendocrine elements, and its primary translation product, the VGF protein, gives rise to several low molecular weight VGF peptides [10, 11]. These are stored in secretory vesicles and can be secreted upon stimuli [12, 13]. One such naturally occurring VGF peptide, TLQP-21, was shown to increase resting energy expenditure upon intracerebroventricular injection [14]. Such peptide appears to be present in sympathetic nerve fibres in WAT, to bind at high affinity to adipocyte membranes, and to increase lipolysis via activation of noradrenaline/β-adrenergic receptor pathways [15, 16]. Chronic administration of TLQP-21 delayed the onset of overt diabetes by preserving islet cell mass in Zucker Diabetic Fatty rats [17]. The Cterminally extended peptide named TLQP-62 distinctly stimulated basal insulin secretion in several insulinoma cell lines [18]. A further peptide derived from a different portion of VGF, named NERP-2, was found to increase glucose-stimulated insulin secretion from the β-cell line MIN6, as well as from isolated mouse pancreatic islets [19]. Further complexity, as well as possible diverging roles of yet uncharacterized VGF peptides are suggested by the remarkable phenotype of vgf knockout mice, which are hyperactive and hypermetabolic, with a deranged hypothalamic response to feeding [8, 20]. None the less, only a few studies have so far addressed human obesity and/or diabetes. In a group of patients treated for idiopathic intracranial hypertension, VGF immunoreactivity proved higher in cerebro-spinal fluid from obese, compared to non-obese subjects [21]. The number of neurons expressing both NPY and VGF was increased in the hypothalamic infundibular nucleus, but decreased in the nucleus of tractus solitarius of T2D patients, compared to non-diabetic controls [22]. To address the potential role of VGF in obesity and T2D, we studied a mouse model of high-fat diet induced obesity, in parallel with human newly diagnosed diabetics and agematched euglycemic controls, classified according to their body mass index (BMI). Four VGF peptides were investigated, including the TLQP peptides which exert known actions on metabolic regulations [14, 15, 18]. Material and Methods Human studies Patients (20–81 years, male, BMI 19 (...truncated)