This is the submitted version of the journal article: Méndez Lara, Karen Alejandra; Rodríguez-Millán, Elisabeth; Sebastián, David; [et al.]. «Nicotinamide protects against diet-induced body weight gain, increases energy expenditure, and induces white adipose tissue beiging». Molecular Nutrition and Food Research, Vol. 65 Núm. 11 (2021), p. 2100111. DOI 10.1002/mnfr.202100111 This version is available at https://ddd.uab.cat/record/271568 under the terms of the license Nicotinamide protects against diet-induced body weight gain through increased energy expenditure and white adipose tissue beiging Short title: Anti-obesity effects of the amide form of vitamin B3 Authors: Karen Alejandra Méndez-Lara 1,2, Elisabeth Rodríguez-Millán 1, David Sebastián 3, 4, 5, Rosi Blanco-Soto 6, Mercedes Camacho 1, Madalina N. Nan 7, Elena M. G. Diarte-Añazco 1, Eugènia Mato 6, Silvia Lope-Piedrafita 6, 8, Núria Roglans 9, Juan Carlos Laguna 9, Núria Alonso 5, 10, Dídac Mauricio 3, 5, 11, Josep A. Villena 5, 12, Antonio Zorzano 3, 4, 5, Francesc Villarroya 3, 13, Francisco Blanco-Vaca 2, 5, 7, †, Josep Julve 1, 2, 5, † Author affiliation: 1 Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau i Institut d’Investigació Biomèdica de l'Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain. 2 Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain. 3 Departament de Bioquímica i Biomedicina. Facultat de Biologia, Universitat de Barcelona (UB), Barcelona, Spain. 4 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain 5 Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, Madrid, Spain. 6 Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBERBBN), CIBERDEM, Madrid, Spain. 7 Servei de Bioquímica. Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain. Page 1 of 30 Barcelona, Spain. 8 Servei de Ressonància Magnètica Nuclear. Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain. 9 Departament de Bioquímica i Biologia Molecular. Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Spain. 10 Servei d'Endocrinologia, Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain 11 Servei de Endocrinologia i Nutrició. Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain. 12 Laboratori de Metabolisme i Obesitat. Unitat de Diabetis i Metabolisme. Institut de Recerca del Vall d’Hebron. Barcelona, Spain. 13 Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición, CIBEROBN, Madrid, Spain. † Corresponding authors Highlights NAM supplementation enhances energy metabolism in diet-induced obese mice White adipose tissue beiging and oxidative metabolism is induced upon NAM administration NAM administration prevents white adipose tissue inflammation NAM supplementation ameliorates fatty liver In brief Méndez-Lara et al. show that NAM supplementation prevents weight gain by inducing NAD+ content and white adipose tissue beiging. An increased number Page 2 of 30 of brown and beige/brite adipocyte clusters, protein abundance of Ucp1, mitochondrial activity, adipose NAD+, EL ANALSISIS DE SENSIBILIDAD A INSULINA NO ES ESPECIFICO DE TAB and Ampk levels was observed in white adipose tissue of treated mice. Hepatic steatosis was prevented and inversely related to decreased gene expression of Cd36. The effect of NAM in diet-induced obese mice also ameliorated the inflammatory phenotype by increasing circulating levels and adipose gene expression of adiponectin and IL-10. Abstract Interventions that boost NAD+ availability are of potential therapeutic interest for obesity treatment. The potential of nicotinamide (NAM), the amide form of vitamin B3 and a physiological precursor of NAD+, in preventing weight gain has not previously been studied in vivo. Other NAD+ precursors have been shown to favorably decrease weight gain in vivo; however, their impact on adipose tissue was not described. Here we show that NAM supplementation protected against diet-induced obesity by augmenting global body energy expenditure. The manipulation markedly altered adipose morphology and metabolism, particularly in inguinal (i) white adipose tissue (iWAT). An increased number of brown and beige/brite adipocyte clusters, protein abundance of Ucp1, mitochondrial activity, adipose NAD+, insulin sensitivity, and induced Ampk levels was observed in iWAT of treated mice. Notably, a significant improvement in hepatic steatosis and inflammation was also observed in NAM high-dose (HD)-treated mice. These data indicate that NAM is a nutrient supplement that influences whole-body energy expenditure by driving changes in the adipose phenotype. Thus, NAM is Page 3 of 30 a novel and attractive dietary supplement for preventing obesity and associated complications. Keywords: adiposity, beiging, fatty liver, inflammation Address correspondence to: Dr. Josep Julve, Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau, C/ Sant Quintí 77-79, 08041 Barcelona, Spain; Dr. Francisco Blanco-Vaca, Hospital de la Santa Creu i Sant Pau, Servei de Bioquímica, C/ Sant Quintí 89 08041 Barcelona, Spain Funding This work was funded by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, grant FIS PI17-00232 (to J.J.), PI18/0164 (to F.B.-V.), PI17-01362 (to N. A.), FEDER “Una manera de hacer Europa”, and by La Marató de TV3 2016 (201602.30.31) (to N. A. and J.J.). K.A.M.-L. was recipient of an AGAUR grant FI-DGR2014 (Generalitat de Catalunya). E.M.G.D.-A. was recipient of The National Program of Foreign Postgraduate Scholarships “DON CARLOS ANTONIO LOPEZ” (BECAL) (BCAL04-451 - 3824321) by Ministerio de Educación (Republic of Paraguay). J.J. was recipient of a Miguel Servet Type 2 contract (CPII18/00004; ISCIII). CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is a project of Instituto de Salud Carlos III. J.J. is member of Red de Investigación en "Enfermedades Metabólicas y Cáncer" (RED2018-102799-T), Ministerio de Economía y Competitividad (MINECO), Madrid, Spain. Institut de Recerca de l'Hospital de la Santa Creu i Page 4 of 30 Sant Pau is accredited by the Generalitat de Catalunya as Centre de Recerca de Catalunya (CERCA). Conflicts of Interest: The authors declare no conflict of interest. Page 5 of 30 1. Introduction Obesity is a complex, chronic disease [1] and a main public health concern worldwide [2], which is defined as an imbalance of energy between energy intake (i.e, food consumption) and energy expenditure (i.e., basal metabolism, physical activity and adaptive thermogenesis) [3]. In the last two decades there has been a remarkable increase in our understanding of adipose biology. The white adipose tissue (WAT) is designed as the main long-term, energy-reservoir organ and actively controls energy homeostasis [4]. Given its energy buffering capacity and endocrine function, healthy WAT manages excess energy storage by controlling both energy intake and expenditure [4]. However, in obesity, WAT becomes severely dysfunctional and generally undergoes pathological remodeling and metabolic derangements, which triggers uncontrolled pro-inflammatory responses [5]. Compelling evidence suggest that an altered management of excess free fatty acids (FFA) by WAT results in metabolic stress and toxicity in adipocytes (lipotoxicity) and other relevant cell types, including myocytes, hepatocytes, and immune cells [4]. Systemic chronic, low-grade inflammation is another hallmark of WAT in obesity [4]. It is frequently characterized by moderate elevations in proinflammatory cytokines with concomitant reductions in anti-inflammatory cytokines, as well as infiltration and proinflammatory responses of different immune cell types, including macrophages [4]. Notably, chronic inflammation in WAT during obesity has a major impact on adipocyte metabolism and function. Obesity is a largely preventable disease [10]. However, the effectiveness of existing pharmacological and/or dietary initiatives for long-treat treatment of Page 6 of 30 obesity failed, at least in part could be due to strategies which do not target adipose tissue, a situation that is beginning to change [10]. Pharmacological and nutritional interventions stimulating mitochondrial biogenesis and function confers protection against diet-induced obesity (DIO) [7]. In this context, the role of nicotinamide adenine dinucleotide (NAD)+ has emerged as a signaling molecule that plays a major regulatory role in many cell functions, including inflammation and energy metabolism, by modulating the action of protein such as sirtuins, which use NAD+ as a coenzyme [7, 8]. Different lines of evidence suggest that strategies leading to increasing adipose tissue NAD+ could be a target for treating obesity. For instance, cellular NAD+ decline is a DIO hallmark and induces the development of many of the ailments associated with this condition [9-15]. Conversely, tissue content of NAD+ raises in response to exercise [16-19] or calorie restriction interventions [20], both of which are interventions associated with decreasing body weight. Furthermore, supplementation with NAD+ precursors, such as nicotinamide riboside (NR) and mononucleotide nicotinamide (NMN), increased energy metabolism and prevented body weight gain by improving mitochondrial function, confering protection against metabolic DIO complications [10, 12, 21]. Despite data showing the efficacy of NAD+ precursors on adipose metabolism [15], the mechanisms accounting for the broad spectrum of their health benefits are still poorly understood. Indeed, the impact of these molecules on the biology/physiology of different adipose tissues, i.e., brown (BAT) and white adipose tissues (WAT), has not been addressed. Nicotinamide (NAM) is also a physiological precursor of NAD+. However, its contribution in boosting energy metabolism in adipose tissue and body weight gain remains largely unknown. Although a growing body of evidences supports a Page 7 of 30 role for NAM as an anti-diabetic agent [22][14, 23] which ameliorates non- alcoholic fatty liver disease [24], its potential contribution to body weight reduction remained to be characterized. We therefore tested the hypothesis that administration of NAM in vivo protects against diet-induced obesity. 2. Materials and methods All animal procedures were reviewed and approved by the Institutional Animal Care Committee and Use Committee of the Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau (Procedure id 10434), and the methods were conducted in accordance with the approved guidelines. The effect of NAM supplementation was conducted in male (strain C57BL/6J) mice. Two doses of NAM (high dose 1%; and low dose 0.25%) were given via drinking water to mice, starting at the same time as the high-fat diet feeding. The biochemical measures were determined using commercial kits adapted to a COBAS c501 autoanalyzer as described [25]. NAM was analyzed using high-performance liquid chromatography (HPLC) with mass spectrometry (MS). NAD+/NADH and adenosine monophosphate (AMP) in adipose tissues were determined using a commercial assay (from Abcam and Buffalo University, respectively) according to the manufacturer’s instructions. The design of the intervention is shown in Supplementary Figure 1. Systemic concentrations of cytokines (IL10, IL6, IL4 and TNF-a) were analyzed on a Luminex using xMAP® technology (Millipore Corporation, Billerica, MA). Adipokines were measured using enzyme-linked immunosorbant assay kits (ELISA; Invitrogen). RNA samples from tissues were extracted and reverse transcribed and relative mRNA levels of each gene determined by real time qPCR. Western blots were performed following standard procedures using specific antibodies against Ucp1 and total and phosphorylated Page 8 of 30 AMP‐activated protein kinase (p‐AMPK), respectively. Palmitate β-oxidation measurement in isolated mitochondria was performed as described [26]. White and brown adipose tissue and hepatic biopsies were taken for histological evaluation (hematoxylin and eosin staining) in paraffin embedded sections using a light field microscope. Measurements of oxygen consumption (VO2) and CO2 production (VCO2) were performed using an indirect calorimetry system (Oxymax/CLAMS, Columbus Instruments). Magnetic resonance imaging (MRI) and spectroscopy (MRS) analysis was performed using a 7T Bruker BioSpec 70/30 USR (Bruker BioSpin GmbH, Ettlingen, Germany) system. Data are mean ± SEM. Two-tailed student’s-tests were used for single comparisons and analysis of variance (ANOVA) with Newman-Keuls post-hoc tests for multiple comparisons. The relationships between different variables were determined by calculating Pearson’s or Spearman’s correlation coefficients, as appropriate. Statistical significance was assumed if P<0.05. Additional details Methods are available in the online version of the paper (Supplementary Methods). 3. Results NAM supplementation causes body weight gain prevention In order to determine the effects of NAM on preventing diet-induced obesity, mice were first fed a high-fat diet and treated with two different concentrations of NAM for 12 consecutive weeks. NAM was well-tolerated by mice as shown by similar water intake rates among the different groups (Table 1). Plasma levels of NAM were steadily elevated in NAM treated groups (NAM LD: 6.5-fold, P<0.05, NAM HD: 18-fold, P<0.05) compared with untreated mice (Figure 1, panel A; Table 1). Blood chemistry panels, including tests of liver and kidney function, and urine Page 9 of 30 did not detect any sign of NAM toxicity (Table 1, Supplementary Tables 1 and 2). Final body weight or volume (ME REFIERO A FIG 1 PANEL C) was significantly reduced only in mice receiving the highest dose of NAM compared with untreated mice (Table 1 and Figure 1, panel B and C). The average percentage of body weight prevention normalized to untreated mice was 10.2% (REVISALO, A MI ME SALE UN 19,8%) (P<0.05) in NAM HD-treated mice. The final body weight of mice correlated inversely with plasma NAM (Spearman r = 0.76, P<0.05) (Supplementary Figure 2, panel A). Body weight gain prevention was 69.5%, P<0.05) in NAM HD-treated mice and 35,8% in NAM LD respect to untreated mice (Table 1). This was mainly attributed to body fat gain prevention and, to a lesser extent, also to decreased total lean mass (Table 1). Consistently, NMR and fat mass depot analysis (Figure 1, panels D, E and F) showed similar information and total fat pad was directly associated with body weight in these mice (Spearman r = 0.9522, P<0.05 (Supplementary Figure 2, panel B),. In contrast, body fat fraction in NAM LD-treated mice often did not differ from untreated mice. Fat pad reduction was accompanied by a concomitant reduction (59.5%, P<0.05) in the cross-sectional area of adipocyte from NAM HD-treated mice (Figure 1, panel G). Although plasma glucose or insulin levels in NAM-treated mice did not significantly differ from untreated mice (Table 1), NAM treatment improved glucose tolerance in mice treated with the highest dose of NAM (21%, P<0.05) compared with untreated mice (Figure 1, panels H and I). No changes were observed in insulin sensitivity (Figure 1, panels J and K). Plasma concentrations of FFA were significantly decreased in NAM-treated mice compared with Page 10 of 30 untreated mice, whereas total cholesterol and triglycerides did not differ among groups (Table 1). The decreased adiposity shown by HFD-fed animals was accompanied of an elevation in circulating adiponectin in NAM HD mice (1.4-fold, P<0.05) compared with untreated mice (Table 1). In contrast, plasma leptin concentrations were reduced in NAM HD mice (79.1%, P<0.05) compared with untreated mice (Table 1). Histological analysis of WAT revealed the presence of characteristic crown-like structures (CLS) in untreated mice (Figure 1, panel L). Of note, the number of CLS was significantly diminished (85%, P<0.05) in WAT from NAM HD mice diminished (300  30 CLS/10,000 adipocytes) compared with untreated mice (45  8 CLS) (Figure 1, panel L). Consistently, two gene markers of macrophage infiltration were significantly reduced in WAT of NAM HD mice (Cd68: ~40%, P<0.05) compared with untreated mice (Figure 1, panel M). Additionally, relative mRNA levels of Il10 in WAT of NAM-HD mice presented significant elevation (~2.7-fold, P<0.05) whereas the mRNA levels of Tnfa did not differ among groups (Figure 1, panel M). Plasma levels of several pro- and anti-inflammatory cytokines were also measured (Figure 1, panel N). Plasma IL-10 showed a close-to-significant trend to be increased, while plasma IL-6 was marginally decreased in NAM HD mice. NAM administration ameliorates fatty liver NAM did not alter liver weight (Table 1, Figure 1, panel F). Plasma levels of ALT and AST in NAM-treated mice did not differ either from those of untreated mice (Supplementary Table 2). Interestingly, NAM supplementation prevented HFD- induced hepatic steatosis (Figure 2, panels A and B) as seen by the decreased intracellular lipid droplets (39.9%, P<0.05) in histological preparations in NAM HD Page 11 of 30 mice ) and MRS analysis -(69.5%, P<0.05), respect untreated mice. Furthermore, the liver fat fraction directly correlated to both whole-body fat volume (Spearman r = 0.9301, P<0.05) and whole-body volume (Spearman r = 0.7552, P<0.05) (Figure 2, panels C and D). Notably, the hepatic mRNA levels of Cd36 and Fgf21 and the plasma concentration of Fgf21 were reduced, especially in NAM HD mice (Cd36: -51%, P<0.05; Fgf21: -39%, P<0.05; Fgf21: (-83%, P<0.05, respectively) (Figure 2, panel E and F). Effects of NAM administration on energy expenditure Body weight gain prevention by NAM was not accompanied by significant changes in the daily food consumption of NAM-treated mice (Table 1, Figure 3, panel A). It is worthy to note that food intake was measured before body weights diverged to eliminate confusions by body-weight differences [11]. Additionally, fecal triglycerides did not differ among groups (NAM HD: 1.74 ± 0.43 µmol/g vs. Un: 1.52 ± 0.36 µmol/g, P=0.98. Therefore, increased energy expenditure rather than decreased food intake or steatorrhea may explain the fatless in NAM HD mice. Indeed, feed efficiency (body weight change per kcal of food eaten) was significantly reduced (84.6%, P<0.05) only in NAM HD mice (Figure 3, panel B). Increased energy expenditure was directly demonstrated by indirect calorimetry The 24-h VO2 and the respiratory exchange ratio (RER) of NAM HD mice were significantly higher (VO2: 20% during day and night (Figure 3, panel C). RER in NAM HD mice was increased 2.4% during day and 3.6% during night (P<0.05) respect untreated mice (Figure 3, panel D; Supplementary Figure 2, panels C and D), NAM-LD treated mice only showed a significant VO2 increase during night (VO2: 10%, P<0.05; RER: 2.3%, P<0.05) (Figure 3, panels C and D). RER was inversely related to either body weight (day: Spearman r = -0.6731, P<0.05; Page 12 of 30 night: Spearman r = -0.6082, P<0.05) and fat pad (day: Spearman r = -0.5985, P<0.05; night: Spearman r = -0.5857, P<0.05) (Supplementary Figure 2, panels E and F). Data from energy expenditure analysis in different groups of mice were Ancova corrected prior to comparison among different group pairs (Figure 3, panel E). Energy expenditure in NAM LD mice did not differ from untreated mice but was significantly increased in NAM HD mice (1.4-fold, P<0.05) compared with untreated mice (Figure 3, panel E). Locomotor activity did not differ among groups (Supplementary Figure 2, panel G). NAM administration induces WAT beiging Since we observed an increase in energy expenditure, NAM might promote increased iBAT activity. NAM manipulation did not influence iBAT mass (Figure 4, panel A). Analysis of iBAT from NAM HD showed that brown adipocytes had much less accumulation of lipid vesicles (0.6-fold, P<0.05) than untreated mice (Figure 4, panel B). Notably, mitochondrial fatty acid -oxidation was elevated (1.3-fold, P<0.05) in iBAT of NAM HD mice compared with untreated mice (Figure 4, panel C). This finding was not associated with changes in the expression of mitochondrial genes (Figure 4, panel D). Similarly, the relative protein abundance of Ucp1 in iBAT of NAM HD mice did not differ from untreated mice (Figure 4, panel E, left bar chart subpanel). However, corrected levels of Ucp1 by total iBAT protein were significantly increased in these mice (1.9-fold, P<0.05) (Figure 4, panel E, right bar chart subpanel), suggesting iBAT hyperplasia. Under certain conditions adipose thermogenesis may be activated and contribute to energy expenditure. Beige adipocytes, which may be found in WAT, can functionally mimic the metabolic actions of classical brown adipocytes, including Page 13 of 30 thermogenesis [27]. As inguinal (i)WAT is the subcutaneous fat depot most readily beiged in C57BL/6 mice [28, 29], we investigated whether NAM-mediated increased energy expenditure was associated with WAT beiging. NAM HD administration in mice markedly altered adipose morphology (Figure 5, panel A), promoting the appearance of small-sized adipocytes. The number of clusters of small-sized adipocytes containing multiple lipid vesicles was abundantly present in iWAT of NAM HD mice compared with untreated mice, suggesting a shift into a brown-like phenotype. Indeed, NAM administration stimulated a brown/beige fat thermogenic gene program of this inguinal fat depot. In addition to the upregulation of iWAT Pgc1a and Pgc1b genes, relative Ucp1 mRNA levels (10.4- fold, P<0.05) and Ucp1 protein abundance were significantly increased in NAM HD mice (:2.5-fold, P<0.05) compared with untreated mice (Figure 5, panels B and C). Mitochondrial -oxidation was significantly elevated in iWAT (NAM LD: 1.9-fold, P<0.05; NAM HD: 1.4-fold, P<0.05) of NAM-treated mice (Figure 5, panel D), suggesting that this tissue was metabolically more active in treated mice. Consistent with this view, the expression of genes involved in mitochondrial homeostasis, Pgc1a, Ppargc1b, Mfn2 were either elevated (Pgc1a: 3-fold, P<0.05; Pgc1b: 2.5-fold, P<0.05; Mfn2: 4.3-fold, P<0.05) or marginally elevated (Cpt1b: 2.6-fold, P<0.07) upon NAM HD treatment (Figure 5, panel). Given that NAM may be a main substrate for NAD+ synthesis, we evaluated the in vivo efficacy of NAM to increase NAD+ in iWAT. NAM treatment dose- dependently increased both total NAD and NAD+ content in iWAT. The NAD+/NADH ratio was higher in iWAT from HD NAM-treated mice (Figure 5, panel E). NAM treatment did not influence the relative mRNA levels of adipose Page 14 of 30 nicotinamide phosphoribosyltransferase (Nampt) or NAM riboside kinase (Nmrk)1, but augmented (2.9-fold, P<0.05) the expression of nicotinamide N- methyltransferase (Nnmt) (Figure 5, panel G). Thus, NAD+ elevations in adipose tissue could be due to an elevated bioavailability of NAM in this tissue due to an increased synthesis from NAD. Because AMP-activated protein kinase (AMPK) cascade is a critical regulator of brown and beige adipose tissue [30, 31], we examined the protein abundance of phosphorylated (active) AMPK in iWAT. Adipose content of phosphorylated AMPK was indeed increased (2.2-fold, P<0.05) in iWAT of NAM HD mice (Figure 5, panel H). Interestingly, the adipose content of AMP was significantly augmented (3.2-fold, P<0.05) in NAM HD mice compared with untreated mice (Figure 5, panel I (CAMBIAR ORDEN DE LAS LETRAS DE PANELES EN LA FIGURA) ), consistently with its known allosteric regulation on AMPK. Phosphorylated AMPK was correlated to Ucp-1 (Pearson r = 0.59, P<0.05) (Figure 5, panel K). 4. Discussion The results presented provide proof of the concept that NAM can prevent a diet- induced obesity and related complications. NAM administration very notably prevented both body weight and adiposity gain after a HFD. The mechanisms implicated involve increases in whole-body energy expenditure, at by stimulating WAT beiging, and eliciting the metabolic activity in iWAT, while reducing adipose inflammation. Importantly, NAM administration also ameliorated fatty liver and, modestly, glucose tolerance in treated mice. Effect of NAM on adipose beiging Page 15 of 30 Adaptive thermogenesis is the main iBAT function, especially in rodents [32]. Our data did not reveal changes in the relative gene expression or protein abundance of Ucp1 in this tissue. Nevertheless, Ucp1 corrected by the total iBAT deposit was indeed increased in HD NAM-treated mice SEGUN EL PANEL DE LA FIG 4 NO HAY HIERPTROFIA DEL TEJIDO. Histological examination showed that brown adipocytes from iBAT of NAM HD mice were much less loaded with lipids than the untreated mice, suggesting that this tissue would be more active in treated mice. Also, mitochondria in iBAT might be more somewhat more metabolically active in NAM HD treated mice than in untreated mice, as revealed by the enhanced -oxidation of fatty acids, but this does not seem to require increased gene expression of mitochondrial markers of lipolysis or biogenesis. NAM administration prevented HFD-induced iWAT hypertrophy, as revealed by the reduced cross-sectional size of white adipocytes, and induced beiging, as shown by an increased abundance of multilocular adipocyte clusters embedded in iWAT, with an a concomitant upregulation of Ucp1, a known marker of BAT adipocytes [33]. Supporting to this finding, the relative mRNA levels of some molecular targets commonly involved in the WAT beiging (i.e., Pgc1a, Pparg) [34] were significantly upregulated or showed a trend to be induced in the inguinal fat depot of NAM HD mice. It is of note, that increased Pgc1a also leads to increased Comentat [LJ1]: Josep Villena suggereix citar papers que relacionin beiging i BAT activation en reduir el pes corporal i adipositat. oxidative metabolism in WAT protecting from HFD-induced obesity in mice [35, 36]. Consistently, our data revealed enhanced WAT mitochondrial -oxidation of fatty acids in NAM HD-treated mice. NAD+ plays a key role in cellular energy metabolism, as it can be reduced to NADH, which provides reducing equivalents to the mitochondrial electron transport chain to fuel oxidative phosphorylation [7]. Additionally, NAD+ also acts Page 16 of 30 as a coenzyme for a wide range of enzymes, such as the sirtuins, particularly Sirt1, involved in the regulation of energy metabolism [7]. Significantly, AMPK is expressed in many tissues, including WAT [37]. Phosphorylated (at Thr172) levels of the α1 isoform of AMPK [37] were increased in iWAT of HD NAM treated mice. Activated AMPK also leads to enhanced oxidation of fatty acids by enhancing mitochondrial functions [38]. Consistently, mitochondrial fatty acid oxidation in WAT of NAM HD mice in correlated with a dose-dependent increase of NAD+ in this tissue. In relation to the potential action of Sirt1 in explaining AMPK activation, it is worth to note that LKB1, which phosphorylates AMPK, is also a target of Sirt1 [39]. Accumulating evidence suggests that the thermogenic activities of iBAT are activated by AMPK [40]. Indeed, cold-induced BAT activation results in the phosphorylation of AMPK to begin to increase thermogenesis. AMPK also upregulates Ucp1 and induces beiging in WAT [41] and, conceivably, could enhance iWAT beiging in NAM HD mice. I. Our data shows that the relative protein abundance of Ucp-1 was directly related to that of p-AMPK, thereby suggesting that thermogenesis could be rather induced by Ucp-1-dependent mechanisms in our treated mice. AMPK activation may be driven by Sirt1-dependent and independent mechanisms may be involved in AMPK activation in vivo [43]. Intriguingly, adipose tissue NAD+ elevations were accompanied by a rise of adipose tissue content of AMP. The increase of AMP in WAT of treated mice is unknown, but it could be at least partly explained by different mechanisms. First, NAD+, which may be a main intracellular source of adenosine [44],[45], can conceivably increase endogenous production of adenosine, and hence contribute to the Page 17 of 30 intracellular AMP pool [46] in this tissue. Additionally, adenosine may be also formed by the hydrolysis of S-adenosylhomocysteine (SAH), which could be augmented by Nnmt. Nnmt catalyzes the transfer of a methyl group of S- adenosylmethionine (SAM) to NAM and produces SAH and me-NAM [47, 48]. In this regard, plasma concentrations of me-NAM and mRNA levels of Nnmt were upregulated in WAT. Although Nnmt activity was notdetermined , previous reports indicate that Nnmt function parallel mRNA levels in adipose tissue [11]. ADP- ribosyl produced by the cluster of differentiation (CD)38, which also uses NAD+ as a substrate [49], can subsequently be degraded to AMP [50, 51]. The effect of NAM in augmenting the NAD+ content of WAT differed from that observed in independent studies using other NAD+ intermediates, NMN and NR [10, 12]. Although we do not know the basis of such differences, they could be lattributed to the type of NAD+ precursor used and dose-related issues. Importantly, in these other studies, NAD+ precursors did not induce beiging in WAT of treated mice [10, 12])) NAM induces an anti-inflammatory adipose phenotype Inflammatory signalling has only recently been recognized as playing a role in adipose beiging [52], even though it is a well-known, relevant component of the adipose alterations in obesity [53]. IIt is noteworthy that chronic exposure to β- adrenergic agonists not only induces adipose beiging [54-56], but also inhibit the production of proinflammatory cytokines, such as Tnfa, and stimulate the production of anti-inflammatory cytokines, such as IL-10 [57]. The proliferation of (M2) activated macrophages is also a key feature related to WAT beiging [52, 58]. In agreement with this view, it would be feasible that the induction of beiging by NAM in iWAT could be related with the well-known anti-inflammatory NAM Page 18 of 30 effects [59, 60]. Indeed, the gene expression of Il10, an anti-inflammatory target and a key molecular marker of M2 macrophages in mice [61]e. Also, the relative mRNA levels of Cd68, a marker of macrophage infiltration, was downregulated in WAT from NAM HD mice. Consistently, the number of CLS, which commonly accumulate in fat depots from obese mice [62], were also diminished in WAT from NAM HD mice. Several evidence support a role for adiponectin in alleviating chronic adipose tissue inflammation [63, 64]. Indeed, adiponectin induces the production of the anti-inflammatory IL-10, display M2 polarization under the influence of adiponectin. and reduces obesity-induced macrophage infiltration and inflammation [71]. Additionally, macrophages [68] In agreement with a previous report [14],.Plasma concentrations of adiponectin were elevated in response to NAM manipulation in NAM HD mice. It would be thus, attractive to suggest that plasma NAM-mediated elevations of this adipokine might contribute to the polarization of macrophages towards an M2 (i.e. anti-inflammatory) phenotype and hence elicit adaptive thermogenesis in WAT of NAM HD mice [58] [65-68] . Supporting the potential favorable impact of adiponectin in thermogenesis, AMPK also appears activated by adiponectin [72]. Our NAM HD mice also displayed a modest improvement of glucose tolerance compared with untreated mice. Adiponectin is also a widely recognized insulin signaling sensitizer [73]. Our data are consistent with a previous report [14], whereby NAM-mediated improved glucose tolerance and increased circulating levels of adiponectin in treated obese rats. Intriguingly, in this case [14], NAM treatment did not induce reduction in body weight. A possible explanation of this finding could be the differences in the experimental model and design used. Page 19 of 30 Taken together, our data, i.e., NAD+ elevations, enhanced energy expenditure as a result of increased beiging of iWAT and oxidative capacity of iBAT support combined mechanisms to explain adipose gain prevention in NAM HD mice. Reduced WAT accumulation together with tissue and systemic alleviated inflammation as a result of NAM treatment may also contribute to improve glucose homeostasis. Limitations of the study Supplemented water was replaced weekly. Although we did not confirmed whether NAM remained intact in drinking water at room temperature for a 7-day period, plasma concentration of NAM exhibited a dose-dependent elevation in treated mice. Plasma concentration of NAM was not measured at the beginning of the study; however, genetically identical mice in the untreated group of DIO mice had significantly lower plasma concentration of NAM than NAM-treated mice. Although NAM content was not determined in adipose tissue, our data strongly supports the notion that NAM administration favorably influenced NAD+ biosynthesis in iWAT. Core body temperature was not directly evaluated in this work, but iBAT histology, and protein abundance of Ucp1 suggested enhanced metabolic activity and adaptive thermogenesis. This work provides quantifiable evidence that NAM may exerts beneficial effects on weight gain prevention. However, the contribution of other tissues also important in metabolic homeostasis, such as skeletal muscle, were not evaluated Page 20 of 30 [10, 21]. In a preliminary analysis, we found increased evaluated muscle fatty acid β-oxidation and, also there are previous studies on NAM effects in this tissue. 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Effect of NAM administration on gross parameters and plasma Untreated NAM LD NAM HD P Gross parameters 35.04 28.11 Body weight [g] 32.86 (4.06)† <0.05 (1.47) (1.28)* 11.40 Final weight gain [g] 7.32 (0.75)*† 3.48 (1.01)* <0.05 (2.17) Fat Pad [g] 4.55 (1.55) 2.55 (0.50)*† 1.31 (0.19)* <0.05 31.17 27.11 Lean Weight [g] 28.54 (1.36)* <0.05 (1.26) (1.14)* Liver weight [g] 1.21 (0.26) 1.08 (0.28) 1.06 (0.22)* <0.05 Liver-to-body weight 0.03 (0.00) 0.03 (0.00) 0.04 (0.00) <0.05 ratio Diet intake [g/day] 2.74 (0.63) 2.92 (0.22) 2.99 (0.56) 0.34 Water intake [g/day] 4.95 (1.58) 4.17 (0.77) 4.39 (1.26) 0.31 Calculated dose 0.32 (0.08) 1.40 (0.26) [g/kg/day] Biochemical parameters 13.18 Glucose [mM] 12.92 (2.16) 15.19 (1.47) 0.13 (1.85) Insulin [ng/mL] 0.48 (0.07) 0.39 (0.05) 0.39 (0.07) 0.63 Total cholesterol [mM] 3.97 (1.07) 3.71 (0.53) 3.88 (0.10) 0.30 Page 27 of 30 Triglycerides [mM] 0.83 (0.31) 1.01 (0.40) 1.44 (0.57) 0.16 FFA [mM] 0.85 (0.11) 0.67 (0.05)* 0.54 (0.10)* <0.05 75.96 105.40 Adiponectin [ng/mL] n. d. <0.05 (11.13) (15.80) 30,809 6,540 Leptin [ng/mL] n. d. <0.05 (16,913) (4,311) biochemistry. Results are expressed as the means (standard deviation) (n = 8 mice per group). All analyses were made at three months of age. At the age of two months the mice were challenged to high-fat diet and NAM manipulation for three months. Food intake was measured at the end of the study as described in the Materials and Methods section. Differences between the mean values were assessed by the nonparametric a Kruskal Wallis followed by Dunn’s posttest or ANOVA followed a Newman-Keuls posttest, as appropriate; differences were considered significant when P<0.05. Specifically, # P<0.05 vs. non-obese group, * P<0.05 vs. untreated group; or † P<0.05 vs. NAM LD-treated mice. Abbreviations used: NAM LD, low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice, FFA, free fatty acids; HDL, high-density lipoprotein; n. d., not determined. Page 28 of 30 Supplementary Table 1. Transitions used in QqQ shown by different metabolites. 1st Transition (CE 2nd Transition (CE Metabolite RT (min) (V)) (V)) N-Me-Nicotinamide 0.76 137 → 80 (24) 137 → 108 (16) d4-Nicotinamide (IS) 0.91 127 → 84 (24) 127 → 81 (28) Nicotinamide 0.93 123 → 80 (20) 123 → 53 (32) DIO untreated NAM LD NAM HD P NAM [µM] 17.80 (30.32) 177.50 568.40 <0.05 (84.94)*† (51.45)* Me-NAM [ µM ] 2.2e-004 2.5e-004 (9.2e- 7.1e-0.004 <0.05 (1.6e-004) 005)† (2.3e-004)* Creatinine [mM] 0.03 (0.00) 0.03 (0.00) 0.03 (0.00) 0.23 BUN [M] 7.58 (2.00) 6.94 (1.60) 8.69 (1.93) 0.23 AST [U/L] 152.8 (61.04) 173.1 (66.15) 124.6 (58.06) 0.56 ALT [U/L] 59.18 (46.07) 39.17 (17.77) 21.98 (6.49) 0.27 Page 29 of 30 Urine Creatinine 3.33 (0.54) 3.14 (0.86) 3.35 (1.83) 0.85 [mM] Supplementary Table 2. Effect of NAM on hepatic and renal parameters in DIO mice. Results are expressed as the means (standard deviation) (n = 8 mice per group). At the age of two months the mice were challenged to high-fat diet and NAM manipulation for three months. Plasma concentration of NAM and me- NAM were given in relative units (n = 5-6 mice per group). Differences between the mean values were assessed by the nonparametric a Kruskal Wallis followed by Dunn’s posttest; differences were considered significant when P<0.05. Specifically, *P<0.05 vs. untreated group. Abbreviations used: AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; NAM LD, low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice. Page 30 of 30