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RNA Isolation and Real-Time PCR Analysis Pleiotrophin deletion alters glucose homeostasis, energy metabolism and brown fat thermogenic function. Sevillano, Julio1#; Sánchez-Alonso, María Gracia1#; Zapatería, Begoña1; Calderón, María1; Alcalá, Martín1, Limones, María1; Pita, Jimena1; Gramage, Esther2; Vicente- Rodríguez, Marta2; Horrillo, Daniel4; Medina-Gómez, Gema4; Obregón, María Jesús5; Viana, Marta1; Valladolid-Acebes, Ismael3, Herradón, Gonzalo2, Ramos, María del Pilar1*. Affiliations 1Department of Chemistry and Biochemistry, Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain. 2Department of Pharmaceutical and Health Sciences, Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain. 3The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76, Stockholm, Sweden 4Department of Basic Sciences of Health. Universidad Rey Juan Carlos. Alcorcón. Madrid. Spain. 5Department of Endocrine and Nervous System Pathophysiology, Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Madrid, Spain. Keywords: Glucose homeostasis; Metabolism; Adipose tissue; Insulin resistance, Thermogenesis. *To whom correspondence should be addressed: Mª del Pilar Ramos Álvarez, PhD. Department of Chemistry and Biochemistry Facultad de Farmacia, Universidad CEU San Pablo Ctra. Boadilla del Monte km 5,3 28668, Madrid 1 +34-91-3724760 [email protected] Additional Title Page Footnotes #Co-first authors This study was supported by Spanish Ministry of Economy and Competitiveness (SAF2010-19603 and SAF2014-56671-R, SAF2012-32491, BFU2013-47384-R and BFU2016-78951-R) and Community of Madrid (S2010/BMD-2423, S2017/BMD-3864). Running Title: PLEITROPHIN AND ENERGY METABOLISM Aims/hypothesis: Pleiotrophin, a developmentally regulated and highly conserved cytokine, exerts different functions including regulation of cell growth, migration and survival. Here we hypothesize that this cytokine can play a regulatory role for glucose and lipid homeostasis. Methods: To test our hypothesis, we used gene targeted Ptn deficient female mice and their corresponding wild-type counterparts, and performed a longitudinal study characterizing the metabolic profile of our animal model at different ages from the young adulthood (3 months) to older ages (15 months). Results: Our results reveal for the first time that pleiotrophin is a key player to preserve insulin sensitivity, driving the dynamics of adipose tissue lipid turnover and plasticity and regulating energy metabolism and thermogenesis. Conclusions/interpretation This study opens therapeutic avenues for the treatment of metabolic disorders by targeting pleiotrophin in the white-brown adipose tissue crosstalk. RESEARCH IN CONTEXT 2 What is already known about this subject? Pleiotrophin signaling may be involved in the inhibition of white preadipocytes differentiation in vitro. What is the key question? Is pleiotrophin a modulator of lipid and glucose homeostasis regulating fat accumulation, body composition and brown fat thermogenic function? What are the new findings? Pleiotrophin deficiency ameliorates insulin sensitivity in later life that is associated with an altered expansibility of periovaric adipose fat as well as with an enhanced cell differentiation and thermogenesis of brown adipose tissue. The impaired lipid and glucose homeostasis in Ptn deficient mice can be attributed to a defective PPAR- activation. How might this impact on clinical practice in the foreseeable future? This study opens therapeutic avenues for the treatment of metabolic disorders by targeting pleiotrophin in the white-brown adipose tissue crosstalk. 3 INTRODUCTION Pleiotrophin (PTN) is a highly conserved cytokine that belongs to a family of heparin- binding growth factors [1, 2]. PTN is found in cells in early differentiation stages, especially during embryonic development [3, 4]. PTN contributes to the epithelial- mesenchymal interactions in organs undergoing branching morphogenesis [5-7] and participates in bone formation [8]. In adult rodents there is a residual Ptn expression restricted to uterine cells, and discrete populations of the nervous system [9]. An almost identical and limited expression profile was found in human adult tissues [10]. On the contrary, PTN levels are upregulated in the uterus and placenta during pregnancy [9], in early stages of differentiation of cell types involved in repair [11, 12], and in inflammatory processes [13]. Besides its role in tumor growth, PTN functions include regulation of cell growth, migration and survival [14]. Importantly, PTN signaling might have an inhibitory role in preadipocytes differentiation in vitro [15], that is attributable to the cross-talk between the PTN/PI3K/Akt/GSK-3/-catenin and the Wnt/β-catenin signaling pathways to repress adipogenesis [16]. In fact, after induction of white adipocyte differentiation endogenous PTN decreases [17], and the in vitro administration of rPTN inhibits white adipocyte differentiation, including a decreased expression of white adipocyte markers, like Ppar2 [16, 17]. Our hypothesis points out a novel role of pleiotrophin as a modulator of lipid and glucose homeostasis that might be involved in fat accumulation and body composition, due to adipose tissue-specific actions. To address this question, we monitored and phenotyped a whole-body constitutive pleiotrophin knockout-out mouse model (Ptn-/-) at different stages of adult life. 4 METHODS Animals PTN genetically deficient (Ptn-/-) mice on a C57BL/6J background were generated as described [18, 19]. Female Ptn-/- and wild-type (Ptn+/+) mice were housed at 22–24°C with 12-h/12-h light/dark cycles, from 08:00 to 20:00 h, and free access to water and chow diet. All the animals were maintained in accordance with European Union Laboratory Animal Care Rules (86/609/ECC directive) and the protocols approved by the Animal Research Committee of CEU-San Pablo University. Fasted mice of 3, 6, 12 and 15- months of age from each genotype were exposed to CO2 and killed by decapitation. Plasma and organs were collected and preserved at -80ºC. Determination of body fat. Total body fat was extracted of the dried mummies of 6-months old mice in a soxhlet using chloroform-methanol (3:1). Plasma analysis, estimation of insulin resistance and glucose tolerance tests (GTT) Glucose (GOD-PAP, Roche Diagnostics), triacylglycerides (LPL/GPO-Trinder, Roche Diagnostics), and nonesterified fatty acids (NEFA) (ACS-ACOD; Wako Chemicals GmbH, Germany) were determined by enzymatic colorimetric tests. Plasma insulin (Mercodia), and free T4 (DRG) were determined using immunoassay kits. Plasma leptin was measured with a Bioplex pro-Mouse diabetes-Immunoassay kit (Bio-Rad). Insulin sensitivity was estimated as previously described [20]. GTT were performed in 6-h-fasted mice after intraperitoneal glucose administration (2g/kg) and the areas under the curve for glucose were calculated. 5 q-PCR in white adipose tissue. RNA was isolated with Rneasy Mini Kits (Qiagen). First-strand cDNA were synthesized using the iScript cDNA-Synthesis kit (Bio-Rad), and were used for real-time qPCR analysis in a CFX96 Real-Time System (Bio-Rad). See Table1 for primer sequences. Histological studies Periovaric adipose tissue (AT) was stained with hematoxylin-eosin and analyzed in an optical microscope. Lipolysis analysis in isolated adipocytes. Adipocytes isolation from periovaric AT of 15-months old mice was performed as described [21]. To test catecholamine-stimulated lipolysis, isolated adipocytes were incubated with 1 U/ml of adenosine deaminase (Sigma-Aldrich, Spain) in the absence or presence of isoproterenol (Sigma-Aldrich). Lipolysis was quantified as liberation of glycerol into the media, determined by the GPO-Trinder method (Sigma-Aldrich). Basal lipolysis, half-maximal effective agonist concentration (EC50) and maximum lipolytic effect (Emax) values were estimated from the concentration-effect curves of glycerol. To measure the effect of insulin on lipolysis, isolated adipocytes were incubated with 0, 1, 10 or 100 nM insulin (Sigma, Madrid) prior to addition of isoproterenol (100 nM), and incubated for 90 min. Half-maximal effective agonist concentration (IC50) and maximum effect (Imax) values were estimated. Indirect calorimetry. 6 month-old Ptn+/+ and Ptn-/- mice were individually housed in chambers maintained at 24±2°C with free access to chow and water, and adapted to the new environment for 72h. After adaptation mice were housed three days at 24ºC, and seven days at 30ºC. Oxygen consumption (VO2) and CO2 production (VCO2) of each individual mouse were measured 6 every 30 min during 24h in labMaster metabolic cages. The respiratory exchange ratio (RER) was calculated as VCO2/ VO2. Data of total energy expenditure (EE) were used for calculation of cold-induced thermogenesis as a fraction of total daily EE as described [22]. Analysis in brown adipose tissue (BAT). Total T3 and T4 concentrations were determined in BAT from 6-months old mice by radioimmunoassays, as described [23]. The Dio2 activity was assayed in BAT homogenates as previously described [24]. Mouse Dio2 mRNA was quantified by qRT- PCR using specific Taqman probes for mouse Dio2. Results were normalized to cyclophilin. Ucp-1 was determined by western blot, using a specific anti-UCP1 antibody (Chemicom) followed by corresponding secondary HRP-antibody (Sigma-Aldrich), and visualization by ECL (Amersham Biosciences); values were normalized
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