Proteomic analysis allows for early detection of potential markers of metabolic impairment in very young obese children

International Journal of Pediatric Endocrinology Proteomic analysis allows for early detection of potential markers of metabolic impairment in very young obese children Gabriel Martos-Moreno 0 1 2 3 Lucila Sackmann-Sala 1 5 6 Vicente Barrios 0 2 3 Darlene E Berrymann 1 Shigeru Okada 1 4 Jess Argente 0 2 3 John J Kopchick 1 4 5 0 Hospital Infantil Universitario Nino Jesus, Department of Endocrinology, Instituto de Investigacion la Princesa, Universidad Autonoma de Madrid, Department of Pediatrics , Av. Menendez Pelayo, 65, E-28009 Madrid , Spain 1 Edison Biotechnology Institute, Ohio University, Konneker Research Laboratories , 1 Water Tower Drive, The Ridges, 45701 Athens, Ohio , USA 2 CIBER Fisiopatologia Obesidad y Nutricion (CIBERobn), Instituto de Salud Carlos III , Madrid , Spain 3 Hospital Infantil Universitario Nino Jesus, Department of Endocrinology, Instituto de Investigacion la Princesa, Universidad Autonoma de Madrid, Department of Pediatrics , Av. Menendez Pelayo, 65, E-28009 Madrid , Spain 4 Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University , Athens, Ohio , USA 5 Molecular and Cellular Biology Program, Ohio University , Athens, Ohio , USA 6 Department of Biological Sciences, College of Arts and Sciences, Ohio University , Athens, Ohio , USA Background: Early diagnosis of initial metabolic derangements in young obese children could influence their management; however, this impairment is frequently not overt, but subtle and undetectable by routinely used clinical assays. Our aim was to evaluate the ability of serum proteomic analysis to detect these incipient metabolic alterations in comparison to standard clinical methods and to identify new candidate biomarkers. Methods: A cross-sectional study of fasting serum samples from twenty-two prepubertal, Caucasian obese (OB; 9.22 1.93 years; 3.43 1.08 BMI-SDS) and twenty-one lean controls (C; 8.50 1.98 years; -0.48 0.81 BMI-SDS) and a prospective study of fasting serum samples from twenty prepubertal, Caucasian obese children (11 insulin resistant [IR]) before (4.77 1.30 BMI-SDS) and after weight reduction (2.57 1.29 BMI-SDS) by conservative treatment in a reference hospital (Pros-OB) was performed. Proteomic analysis (two-dimension-eletrophoresis + mass spectrometry analysis) of serum and comparative evaluation of the sensitivity of routinely used assays in the clinics to detect the observed differences in protein expression level, as well as their relationship with anthropometric features, insulin resistance indexes, lipid profile and adipokine levels were carried out. Results: Study of the intensity data from proteomic analysis showed a decrease of several isoforms of apolipoprotein-A1, apo-J/clusterin, vitamin D binding protein, transthyretin in OB vs. C, with some changes in these proteins being enhanced by IR and partially reversed after weight loss. Expression of low molecular weight isoforms of haptoglobin was increased in OB, enhanced in IR and again decreased after weight loss, being positively correlated with serum interleukin-6 and NAMPT/visfatin levels. After statistical correction for multiple comparisons, significance remained for a single isoform of low MW haptoglobin (OB vs. C and IR vs. non-IR) and Apo A1 (IR vs. non-IR). Assays routinely used in the clinical setting (ELISA/kinetic nephelometry), only partially confirmed the changes observed by proteomic analysis (ApoA1 and haptoglobin). Conclusion: Proteomic analysis can allow for the identification of potential new candidate biomarkers as a complement to routinely used assays to detect initial changes in serum markers of inflammation and lipid metabolism impairment in young obese children. Proteomic; Two dimension electrophoresis; Childhood obesity; Insulin resistance - Background Early-onset obesity is a definite risk factor for adult obesity, its associated comorbidities and reduced life expectancy [1,2]. Most of the metabolic comorbidities overtly observed in obese adults are usually subtle or even undetectable in most young (prepubertal) obese children when the currently available biomarkers, including the growing number of adipokines, are used. Adipokines play a preeminent role in carbohydrate metabolism, the derangement of which is the most important metabolic comorbidity of obesity, which can ultimately lead to type 2 diabetes mellitus ([T2DM] [3,4]). Impaired adipokine synthesis and secretion, which can be detected even in young obese children, contributes to the generation of insulin resistance (IR) in a dual fashion. Obesity induces changes in serum levels of insulin sensitizing adipokines [4-7] and also generates a generalized proinflammatory environment by increasing the circulating levels of resistin, interleukin 6 (IL-6) and tumoral necrosis factor alpha (TNF-) [6,8]. However, these changes in circulating adipokines related with carbohydrate metabolism have not been shown to be of value for the detection of early stages of homeostatic derangement in young obese children. Hence, there is a lack of sensitive markers for the initial stages of carbohydrate metabolism impairment in childhood obesity. A similar situation is observed with regard to the obesity related impairment of lipid metabolism, mainly represented by the decrease in high density lipoprotein (HDL) levels in this age range and definitely influenced by the existence of IR. Consequently, analysis of the serum proteome of young children in different weight-related conditions (normal, excess and later reduction), with or without IR, could be useful for the detection of new more sensitive diagnostic biomarkers. An advantageous tool for the study of the circulating proteome in serum under different conditions is the use of two dimension electrophoresis (2DE). This technique allows for the isolation and identification of proteins in a given sample and has previously been applied to human serum samples for different purposes, with a limited amount (below 0.5 ml) of serum needed to identify up to 300 protein spots [9-12]. Therefore, the aims of this study were: 1) To investigate the influence of obesity and weight loss on the serum proteomic profile of young prepubertal obese children. 2) To evaluate the differences in the serum proteome between young obese children according to the presence or lack of estimated IR and 3) To identify new candidate biomarkers of early metabolic impairment in young obese children. Subjects and methods Subjects Three groups of children were enrolled for this study: obese patients (OB), controls (C) and a second cohort of prospectively followed obese patients (Pros-OB). Obese patients (OB) This group was made up of twenty-two prepubertal (Tanner stage I) obese (BMI > +2 SDS according to Spanish standards [13]) Caucasian children (13 males / 9 females). Their mean age at recruitment was 9.22 1.93 years (range 5.53-12.89) and their mean BMI 3.43 1.08 SDS. Samples from these patients were obtained (...truncated)