Growth Hormone & IGF Research 25 (2015) 28–33 Contents lists available at ScienceDirect Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir Preliminary data on the relationship between circulating levels of Sirtuin 4, anthropometric and metabolic parameters in obese subjects according to growth hormone/insulin-like growth factor-1 status☆ Silvia Savastano a,⁎,CarolinaDiSommab, Annamaria Colao a, Luigi Barrea c, Francesco Orio d, Carmine Finelli e, Fabrizio Pasanisi a, Franco Contaldo a,GiovanniTarantinof a Dipartimento di Medicina Clinica e Chirurgia, Università Federico II di Napoli, Italy b IRCCS SDN, Napoli, Italy c Coleman & IOS srl, Naples, Italy d Dipartimento di Scienze Motorie e del Benessere Università Parthenope Napoli, Italy e Center of Obesity and Eating Disorders, Stella Maris Mediterraneum Foundation, C/da S. Lucia, Chiaromonte, 80035 Potenza, Italy f Centro Ricerche Oncologiche di Mercogliano, Istituto Nazionale Per Lo Studio e La Cura Dei Tumori “Fondazione Giovanni Pascale”,IRCCS,Italy article info abstract Article history: Background: The main components of GH/insulin-like growth factor (IGF)-1 axis and Sirtuin 4 (Sirt4), highly Received 28 July 2014 expressed in liver and skeletal muscle mitochondria, serve as active regulators of mitochondrial oxidative capac- Received in revised form 20 October 2014 ity with opposite functions. In obesity both GH/IGF-1 status and serum Sirt4 levels, likely mirroring its reduced Accepted 21 October 2014 mitochondrial expression, might be altered. Available online 28 October 2014 Objective: To evaluate the association between circulating levels of Sirt4, body composition, metabolic parame- ters and cardio-metabolic risk profile in obese patients according to their different GH/IGF-1 status. Keywords: Design: Cross-sectional study with measurement of serum Sirt4, GH after GH releasing hormone (GHRH) + GH/IGF-1 axis – Serum Sirtuin 4 Arginine test, IGF-1 and assessment of body composition, glucose and lipid metabolism in 50 class II III obese 2 Body composition subjects (BMI 35.6 to 62.1 kg/m ) and 15 normal weight subjects. Low GH secretion and IGF-1 were defined Cardio-metabolic risk using pre-determined cutoff-points. The Homeostatic Metabolic Assessment of insulin resistance index and Visceral adiposity index were also calculated. The association of Sirt4 with peak stimulated GH and IGF-1, body composition, metabolic parameters and cardio-metabolic risk profile was assessed. Results: Serum Sirt4 was inversely related to anthropometric and metabolic parameters and positively related to peak GH and IGF-1. After adjusting for peak GH and IGF-1, the relationships between Sirt4 and BMI became not significant. At multiple regression analysis IGF-1 (p b 0.001) was the independent predictor for Sirt4. Conclusion: There was a close relationship between low IGF-1 and low serum Sirt4. This observation suggested that in obese patients, low GH/IGF-1 status was likely associated with a major compensatory decrease in circu- lating levels of Sirt4 to oppose to its negative regulator effect on mitochondrial oxidative capacity. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction overall regulation of body composition across the feeding/fasting cycle, being adipose tissue and skeletal muscle as major targets of the Apart from their effects on linear growth before puberty, growth action of the GH/IGF-1 axis. Of interest, GH and IGF-1 receptors hormone (GH) and insulin-like growth factor (IGF)-1, the main compo- expressed on skeletal muscle have been reported to regulate basal skel- nents of the GH axis, have defined anabolic functions and act as key reg- etal muscle lipid oxidation [2] and to promote mitochondrial oxidative ulators of energy metabolism in adulthood [1]. Actually, the interaction capacity [3,4]. Albeit mainly reversible after calorie restriction and between the GH axis and body weight can be viewed as part of the sustained weight loss [5], the low GH/IGF-1 status in obese individuals might be one of the factors accounting for heterogeneity in metabolic phenotype among equally obese subjects, as this subset of obese sub- jects presented with higher prevalence of cardio-metabolic risk factors ☆ SS conceived the study and drafted the manuscript. GT contributed to drafting the [6] and worse body composition compared with obese individuals manuscript and carried out the statistical analysis of the data. CDS, LB,andCF performed with a normal GH/IGF-1 axis [7]. In particular, we previously reported laboratory analyses. FO and FP collected the data. AC and FC critically revised the text. that up to 1/3 of severely obese patients exhibited a low GH/IGF-I status ⁎ Corresponding author at: Dipartimento di Medicina Clinica e Chirurgia, Unità di fi Endocrinologia, Università Federico II di Napoli Via Sergio Pansini, 5, 80131 Naples, Italy. associated with signi cantly higher fat mass and waist circumference E-mail address: [email protected] (S. Savastano). [8] or metabolic syndrome prevalence [9]. http://dx.doi.org/10.1016/j.ghir.2014.10.006 1096-6374/© 2014 Elsevier Ltd. All rights reserved. S. Savastano et al. / Growth Hormone & IGF Research 25 (2015) 28–33 29 Sirtuins are NAD+-dependent enzymes that couple cellular energy conducted without support from the pharmaceutical industry, after production to metabolic demand [10]. Sirtuin 1 and Sirtuin 4 (Sirt4) approval by the institutional review board of the University of Naples, are involved in insulin secretion [11] and in maintaining the balance Italy (n. 5/14). As control group 15 Caucasian normal weight subjects of glucose/lipid metabolism in type 2 diabetes mellitus (T2DM) [12]. (5 males, mean age 33.4 ± 7.8 years) were chosen between volunteers Sirt4, ubiquitously expressed in many organs including skeletal muscle and employees of our Institution, with comparable education, income, and the liver, functions within the mitochondria as negative regulator of marital status, smoking habits, or participation in physical activity. The mitochondrial oxidative capacity [13]. Evidence now indicates that the purpose of the protocol was explained to the patients and an informed increased oxidative stress, due to excess visceral obesity, as the main written consent was obtained from each patient at the beginning of source of cytokines and adipokines [14], is one of the factors involved the study. in mitochondrial dysfunction [15]. Very recently, we evidenced that cir- culating levels of Sirt4, likely mirroring its reduced mitochondrial ex- 2.2. Assay methods pression, are low in obese patients, likely as an attempt to preserve in obesity the mitochondrial oxidative capacity, and are associated with Samples were collected in the morning between 8 and 10 a.m., after cardiovascular risk factors [16]. Aside from Sirt4, also GH has been re- an overnight fast of at least 8 h and stored at −80 °C until being proc- ported to actively regulate mitochondrial oxidative capacity. Actually, essed. All biochemical analyses including fasting plasma glucose, total both low GH/IGF-1 status and low serum Sirt4 levels could occur con- cholesterol, HDL cholesterol, LDL cholesterol, triglycerides and transam- comitantly in obesity. However, the possible relationship between inases were performed with a Roche Modular Analytics System in the Sirt4 and anthropometric and metabolic parameters among obese sub- Central Biochemistry Laboratory of our Institution. LDL cholesterol and jects according to GH/IGF-1 status has not been investigated formerly. HDL cholesterol were determined by a direct method (homogeneous The aim of the present study was to evaluate the association be- enzymatic assay for the direct quantitative determination of LDL and tween circulating levels of Sirt4, body composition, metabolic parame- HDL cholesterol). Glycated hemoglobin (HbA1C) was measured by ters and cardio-metabolic risk profile in obese patients according to HPLC. In premenopausal women hormonal investigations were per- their different GH/IFG-1 status. formed during the early follicular phase, 5–7 days after spontaneous menses. Serum GH levels were measured by immunoradiometric 2. Materials and methods assay (IRMA) using commercially available kits (HGH-CTK-IRMA, Sorin, Saluggia, Italy). The sensitivity of the assay was 0.2 μg/l. The 2.1. Study design and population intra- and interassay coefficients of variations (CVs) were 4.5 and 7.9%, respectively. Plasma IGF-1 was measured by IRMA after ethanol In order to calculate the minimum required sample the means of extraction. The sensitivity of the assay was 0.8 μg/l. According to our Sirt4 were evaluated. Out of the 97 consecutive morbidly obese subjects population reference values [18], the normal IGF-1 range in 20–40, referred to the out-patient Endocrinology Unit, from January 2013 to 41–60, and over 60-year-old subjects was 110–494, 100–300, and 78– December 2013, 50 obese subjects were enrolled in this cross- 260 μg/l, respectively. The intra-assay CVs were 3.4, 3.0, and 1.5% for sectional study. All subjects underwent complete medical history and low, medium, and high points on the standard curve, respectively. The clinical examination and the selection was based on GH/IGF-1 status inter-assay CVs were 8.2, 1.5, and 3.7% for low, medium, and high points as assessed during their screening procedure for bariatric surgery by on the standard curve, respectively. Total insulin levels were measured evaluating peak GH after the GHRH + Arginine (ARG) stimulation test by a solid-phase chemiluminescent enzyme immunoassay