Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro

    Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro Cristina Salmer´on, Marcus Johansson, Maryam Asaad, Anna R. Angotzi, Ivar Rønnestad, Sigurd O. Stefansson, Elisabeth J¨onsson, Bj¨orn Thrandur Bj¨ornsson, Joaquim Guti´errez, Isabel Navarro, Encarnaci´on Capilla PII: DOI: Reference:

S1095-6433(15)00170-1 doi: 10.1016/j.cbpa.2015.06.017 CBA 9909

To appear in:

Comparative Biochemistry and Physiology, Part A

Received date: Revised date: Accepted date:

1 April 2015 8 May 2015 12 June 2015

Please cite this article as: Salmer´on, Cristina, Johansson, Marcus, Asaad, Maryam, Angotzi, Anna R., Rønnestad, Ivar, Stefansson, Sigurd O., J¨ onsson, Elisabeth, Bj¨ornsson, Bj¨ orn Thrandur, Guti´errez, Joaquim, Navarro, Isabel, Capilla, Encarnaci´on, Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro, Comparative Biochemistry and Physiology, Part A (2015), doi: 10.1016/j.cbpa.2015.06.017

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro

1

2

1

3

3

Cristina Salmerón , Marcus Johansson , Maryam Asaad , Anna R. Angotzi , Ivar Rønnestad , 2

2

T

3

1

Sigurd O. Stefansson , Elisabeth Jönsson , Björn Thrandur Björnsson , Joaquim Gutiérrez , 1

1

SC R

IP

Isabel Navarro , Encarnación Capilla *

1

Department of Physiology and Immunology, Faculty of Biology, University of Barcelona,

Barcelona 08028, Spain. 2

NU

Fish Endocrinology Laboratory, Department of Biological and Environmental Sciences,

University of Gothenburg, Gothenburg 40590, Sweden. 3

MA

Department of Biology, University of Bergen, Bergen 5020, Norway.

D

Running title: Leptin and ghrelin effects on trout adipocytes

TE

*Corresponding author: Encarnación Capilla. Department of Physiology and Immunology, Faculty of Biology, University of Barcelona. Av. Diagonal 643, Barcelona 08028, Spain.

CE P

Phone +34 934039634, Fax +34 934110358

AC

E-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Leptin and ghrelin are important regulators of energy homeostasis in mammals, whereas their physiological roles in fish have not been fully elucidated. In the present study, the effects of leptin and ghrelin on adipogenesis, lipolysis and on expression of lipid metabolism-related

T

genes were examined in rainbow trout adipocytes in vitro. Leptin expression and release

IP

increased from preadipocytes to mature adipocytes in culture, but did not affect the process of adipogenesis. While ghrelin and its receptor were identified in cultured differentiated adipocytes,

SC R

ghrelin did not influence either preadipocyte proliferation or differentiation, indicating that it may have other adipose-related roles. Leptin and ghrelin increased lipolysis in mature freshly isolated adipocytes, but mRNA expression of lipolysis markers was not significantly modified. Leptin significantly suppressed the fatty acid transporter-1 expression, suggesting a decrease in

NU

fatty acid uptake and storage, but did not affect expression of any of the lipogenesis or βoxidation genes studied. Ghrelin significantly increased the mRNA levels of lipoprotein lipase,

MA

fatty acid synthase and peroxisome proliferator-activated receptor-β, and thus appears to stimulate synthesis of triglycerides as well as their mobilization. Overall, the study indicates that ghrelin, but not leptin seems to be an enhancer of lipid turn-over in adipose tissue of rainbow trout, and this regulation may at least partly be mediated through autocrine/paracrine

D

mechanisms. The mode of action of both hormones needs to be further explored to better

TE

understand their roles in regulating adiposity in fish.

CE P

Keywords: Leptin, ghrelin, insulin, adipocyte primary culture, isolated mature adipocytes,

AC

lipolysis, proliferation, differentiation, adipose tissue.

ACCEPTED MANUSCRIPT List of abbreviations adipose triglyceride lipase

CPT1

carnitine palmitoyltransferase-1

EF1α

elongation factor 1α

FAS

fatty acid synthase

FATP1

fatty acid transporter-1

GHS-R1a

growth hormone secretagogue receptor 1a, ghrelin receptor

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

HSL

hormone sensitive lipase

LepA1

leptin paralogue A1

LepA2

leptin paralogue A2

LepRL

leptin receptor long isoform

LPL

lipoprotein lipase

PPARs

peroxisome proliferator-activated receptors

qPCR

quantitative real-time polymerase chain reaction

NU MA

D

TE

CE P AC

SC R

IP

T

ATGL

ACCEPTED MANUSCRIPT 1. INTRODUCTION In addition to the classical functions exerted on lipid metabolism, the adipose tissue has more recently been recognized as an important endocrine organ through the secretion of a wide range of factors (Londraville et al., 2014; Won and Borski, 2013). Among these, leptin and

T

ghrelin together with their corresponding functional receptors, the long isoform leptin receptor

IP

(LepRL) and the growth hormone secretagogue receptor 1a (GHS-R1a), respectively have been identified in a number of fish species including salmonids (Angotzi et al., 2013; Gong et al.,

SC R

2013; Jönsson, 2013; Kaiya et al., 2009a; Rønnestad et al., 2010).

Most in vivo studies on the role of leptin in lipid metabolism of fish have used heterologous mammalian leptin, although the low degree of leptin sequence conservation among vertebrates

NU

(Londraville et al., 2014) makes difficult to gauge the relevance of the obtained data. In goldfish (Carassius auratus), for example, treatment with human leptin decreases hepatic lipid content (de Pedro et al., 2006), but it is not known if homologous leptin will have a similar effect. In

MA

terms of the modulatory effect of leptin on the expression of lipid-related genes, homologous leptin treatment decreases hepatic stearoyl CoA desaturase and lipoprotein lipase (LPL), as well as increases hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and

D

carnitine palmitoyltransferase-1 (CPT1) in grass carp (Ctenopharyngodon idellus) (Li et al., 2010a; Lu et al., 2012), supporting an anti-adipogenic role for leptin in fish. Moreover, the mutant medaka (Oryzias latipes) show large deposits of visceral fat not found in the

TE

-/-

LepRL

wild type indicating that leptin signaling is involved in lipid allocation in teleosts (Chisada et al.,

CE P

2014). In mammals, leptin treatment of primary preadipocytes and 3T3-L1 cells has inconsistent effects on proliferation and differentiation, in some cases depending on the hormone concentrations used (Kim et al., 2008; Wagoner et al., 2006). However, leptin consistently increases energy expenditure in vivo enhancing lipid oxidation while it inhibits insulin-induced

AC

lipogenesis and promotes lipolysis in adipocytes, hepatocytes and other cell types (Hwa et al., 1997; Reidy and Weber, 2000). Importantly, some of these effects appear to be modulated through autocrine or paracrine signaling (Frühbeck et al., 1997; Kim et al., 2008). In fish, in vivo ghrelin treatment increases lipid deposition in the liver and muscles of Mozambique tilapia (Oreochromis mossambicus) and goldfish (Kang et al., 2011; Riley et al., 2005), but not in rainbow trout (Oncorhynchus mykiss) (Jönsson et al., 2010). No data are available about ghrelin effects on adipogenesis or adipocyte lipid metabolism in piscine models. Ghrelin has been shown to promote growth and adiposity in rat (Castañeda et al., 2010; Choi et al., 2003; Tschöp et al., 2000), and stimulatory effects of ghrelin on preadipocyte proliferation have been well described in mammalian models such as the 3T3-L1 cells, with the proliferationpromoting effect showing time-dependence (Kim et al., 2004; Liu et al., 2009; Zhang et al., 2004; Zwirska-Korczala et al., 2007). In addition, ghrelin enhances adipocyte differentiation in vitro, with a concomitant increase in the mRNA expression of two key adipogenic transcription factors: the peroxisome proliferator-activated receptor-γ (PPARγ) and the CCAAT/enhancer binding protein-α (Liu et al., 2009; Pulkkinen et al., 2010). Further, ghrelin induces abdominal

ACCEPTED MANUSCRIPT obesity and hepatic steatosis in rodents by increasing the number of lipid droplets and their lipid content as well as decreasing fat utilization (Davies et al., 2009; Tschöp et al., 2000). In visceral adipose tissue, ghrelin also stimulates lipid accumulation by enhancing the expression of several genes including fatty acid synthase (FAS), LPL and perilipin among others (Pulkkinen et

T

al., 2010).

IP

The aim of this study was to elucidate the role of leptin and ghrelin in the regulation of adipogenesis and lipid metabolism in rainbow trout by using well-established in vitro systems for

SC R

cultured preadipocytes and freshly isolated mature adipocytes (Albalat et al., 2005a; Bouraoui et al., 2008; Cruz-Garcia et al., 2015; Salmerón et al., 2013; Salmerón et al., 2015).

NU

2. MATERIALS AND METHODS

MA

2.1. Fish, holding conditions and sampling procedures Adult rainbow trout from the fish farm Viveros de los Pirineos S.A. (El Grado, Huesca, Spain) were held in a recirculation system at the University of Barcelona in round fiberglass tanks (80

D

cm ø) of 400 L (8-9 trout/tank), at 15±1ºC, with 12 h light: 12 h dark photoperiod, and fed to

TE

satiation twice daily with a commercial feed (Trout evolution, Dibaq Diproteg S.A., Segovia, Spain). Prior to sampling, the fish were fasted for 24 h to avoid regurgitation of food and to ensure clean intestines. The fish were anesthetized with MS-222 (0.1 g/L) and sacrificed by a

CE P

blow to the head and medullar section, after which they were weighed, externally sterilized with 70% ethanol and visceral adipose tissue was taken with sterile dissection material. The study was divided in two parts. The first part concerned the effects of leptin and ghrelin on

AC

the development of preadipocytes, in which the number and mean body weight of the fish used was: 45 fish of 430.5±18 g for the leptin expression and secretion experiment; and 50 fish of 181.1±10 g for the immunofluorescence, proliferation and differentiation experiments. The second part of the study was with mature isolated adipocytes and encompassed 49 fish of 264.5±25 g. All animal handling procedures were approved by the Ethics and Animal Care Committee of the University of Barcelona, following the established legislation of the European Union, Spanish and Catalan Governments (reference numbers CEEA 237/12 and DAAM 6755).

2.2. Peptides Recombinant rainbow trout leptin was produced at the Department of Biology, University of Bergen (Norway) following the procedure described in (Murashita et al., 2008). Synthetic 20 amino-acid octanoylated rainbow trout ghrelin was obtained from the Peptide Institute Inc., Osaka (Japan). Porcine insulin was obtained from Sigma (Tres Cantos, Spain) and used instead of piscine insulin as its bioactivity in fish cells has been shown previously (Albalat et al., 2005a; Albalat et al., 2005b; Bouraoui et al., 2012; Salmerón et al., 2015).

ACCEPTED MANUSCRIPT

2.3. Roles of leptin and ghrelin in adipogenesis Preadipocytes were obtained following the protocol of Bouraoui et al., (2008) with minor

T

modifications. For each experiment, a pool of adipose tissue of approximately 28 g obtained from 4-9 fish was kept in Krebs-Hepes buffer pre-gassed with a mixture of O2-CO2 to stabilize

IP

the pH (pH 7.3-7.4) and supplemented with 1% (v/v) antibiotic/antimycotic solution. The adipose

SC R

tissue was minced into small pieces and incubated for 60 min in Krebs-Hepes buffer containing collagenase type II (130 U/mL) and 1% bovine serum albumin at 18ºC with gentle agitation. Cell suspension was filtered through a 100 µm cell strainer and centrifuged at 2.000xg for 10 min. The pellet obtained was treated with erythrocyte lysing buffer for 10 min at room temperature.

NU

Cells were centrifuged again and resuspended in growth medium containing Leibovitz’s L-15, 10% fetal bovine serum and 1% antibiotic/antimycotic solution. Finally, cells were counted, diluted, and plated into 6- or 12-well plates treated with 1% gelatin at a density of 3.0 and 1.2 x 5

MA

10 cells per well, respectively and incubated at 18°C in growth medium. 2.3.1. Immunofluorescence

To detect the presence of leptin and ghrelin and their receptors in mature adipocytes, cells from

D

four independent experiments were plated on glass cover slips and incubated in growth medium

TE

up to day 7. When confluence was reached, differentiation into adipocytes was induced by using a differentiation medium composed of growth medium plus 10 mg/mL insulin, 0.5 mM 1methyl-3-isobutylxanthine and 0.25 mM dexamethasone; supplemented also with lipid mixture

CE P

(5 µL/mL), as lipids are required to induce complete maturation of fish adipocytes (Bouraoui et al., 2008; Salmerón et al., 2013; Vegusdal et al., 2003). After 3 days, the culture conditions were changed to an adipocyte medium consisting of growth medium plus lipid mixture (5 µL/mL)

AC

to keep the cells already differentiating until day 13, when the cells were fixed. Immunostaining was performed as described by Capilla et al., (2007) using 3T3-L1 adipocytes. The primary antibodies were tested at the following dilutions: leptin (1:100 and 1:500), LepRL (1:10000), ghrelin (1:10000) and GHS-R1a (1:20000). The anti-rat ghrelin antibody was kindly provided by Dr. Hiroshi Hosoda (Japan) and all other antibodies were developed against rainbow trout in rabbits by Agrisera (Vännäs, Sweden) as previously described (Einarsdóttir et al., 2011; Jönsson et al., 2010). The secondary Alexa Fluor 568-conjugated goat anti-rabbit antibody (A21069 LifeTechnologies, Alcobendas, Spain; 1:1000) was used in combination with a Hoechst 342 stain (LifeTechnologies, Alcobendas, Spain; 1:2000). The cells were mounted with Prolong (Invitrogen, Alcobendas, Spain) and immunofluorescence was captured with a confocal microscope (Leica TCS SP2). The images were analyzed with the image processing software ImageJ version 1.47 (National Institutes of Health, USA). 2.3.2. Expression and secretion of leptin during adipogenesis To study leptin mRNA expression and secretion during adipogenesis, plated cells were incubated up to day 7 in growth medium and then, it was changed to differentiation medium

ACCEPTED MANUSCRIPT including 5 µL/mL of lipid mixture to induce differentiation. At day 10, and then every 2 days, the medium was changed again to adipogenic medium to maintain differentiation. Media and cell samples were obtained from 6-8 independent experiments at days 7 (preadipocytes) and 16 (differentiated cells) of culture. Cell culture medium (0.5 mL) from individual wells of a 6-well

T

plate were collected to analyze leptin secretion by radioimmunoassay using the established

IP

homologous protocol (Kling et al. 2009) validated for adipocyte media samples (Salmerón et al., 2015). In this radioimmunoassay, the peptide used is based on rainbow trout LepA1

SC R

(AB354909), which shares 71% and 21% sequence identity with LepA2 (JX123129) and LepB1 (JX131306), respectively (Angotzi et al., 2013). Thus, the assay primarily measures LepA1, although based on sequence similarities some cross-reactivity might exist with LepA2, but not LepB1. After removing the media, cells from 3 wells together were collected with 1 mL of TRI

NU

Reagent solution (Ambion, Alcobendas, Spain) and kept at -80ºC until leptin (LepA1 and LepA2) gene expression analysis by quantitative real-time PCR (qPCR).

MA

2.3.3. Effects of leptin and ghrelin on preadipocyte proliferation The effects of leptin, ghrelin and insulin (included as a positive control) on cell proliferation were studied using preadipocytes at day 3. Cells were starved for 5 h by changing the growth

D

medium to a medium consisting of Leibovitz’s L-15 with 1% antibiotic/antimycotic solution and only 0.02% fetal bovine serum. Then, the medium was changed again to a medium consisting

TE

of Leibovitz’s L-15 containing 1% antibiotic/antimycotic solution and 2% fetal bovine serum alone (control), or with the corresponding concentration of peptide. Leptin was tested at 100 nM;

CE P

ghrelin at 10 nM; and insulin at 1000 nM final concentrations in duplicate wells in a 12-well plate. The concentrations used for leptin and ghrelin were those found to be most effective in the isolated adipocytes experiment (see section 2.4), while that of insulin was based on literature data on fish cells (Bouraoui et al., 2012; Capilla et al., 2011; Salmerón et al., 2013).

AC

Proliferation was analyzed after 48 h incubation (day 5) with a methylthiazolyldiphenyltetrazolium bromide assay as described for previously in fish adipocytes (Salmerón et al., 2013). Proliferation data were obtained by subtracting the background reading at 650 nm from the absorbance measured at 570 nm. Cells with phosphate buffered saline instead of methylthiazolyldiphenyl-tetrazolium bromide were used as non-specific controls, and this reading was subtracted from all the other data. Data were obtained from 6 independent experiments and presented as the average fold change with the absorbance values normalized to the control condition. 2.3.4. Effects of leptin and ghrelin on preadipocyte differentiation To analyze the effects of leptin, ghrelin and insulin on cell differentiation, confluent and undifferentiated cells grown in growth medium up to day 7 after plating, were then incubated during 6 days with differentiation medium as a control condition, or with differentiation medium containing the different treatments: 5 µL/mL lipid mixture as a positive control, leptin 100 nM, ghrelin 10 nM or insulin 1000 nM, or were left with growth medium as a negative control condition. At day 13, cells from 2 wells from a 6-well plate were recovered together as described

ACCEPTED MANUSCRIPT in section 2.3.2 and stored at -80ºC until gene expression analyses of well-known adipocyte differentiation markers such as PPARγ, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), LPL and fatty acid transporter-1 (FATP1). Furthermore, in a parallel set of 12-well plates, cell differentiation was also evaluated in duplicate by measuring lipid accumulation using the

T

protocol of oil red O staining adapted for fish adipocytes (Capilla et al., 2011; Salmerón et al.,

IP

2013). Quantification of cell lipid content was measured at 490 nm and was divided by the cell protein content reading at 630 nm. Results, presented as the average of 6 independent

SC R

experiments, are expressed as fold change with the values normalized to the control condition.

2.4. Effects of leptin and ghrelin on lipid metabolism in mature isolated adipocytes

NU

Adipocytes were isolated following the general protocol by Albalat et al., (2005a), further outlined by Salmerón et al., (2015). For each experiment (n=8), a pool of visceral adipose tissue of 20-28 g obtained from 3-10 fish was minced and incubated in collagenase type II (130 U/mL)

MA

prepared in Krebs-Hepes buffer containing 1% bovine serum albumin at 18ºC in a shaking water bath. Cell suspension was filtered through a 100 µm cell strainer and washed by flotation. Approximately 2 million isolated adipocytes were incubated in triplicate tubes during 3 h in the

D

shaking bath at 18ºC, in the absence (control) or presence of 10 and 100 nM of leptin or 0.1, 1 and 10 nM of ghrelin. The concentrations of peptides tested were based on previous literature

TE

(Kim et al., 2004; Kim et al., 2008; Salmerón et al., 2015; Wagoner et al., 2006; Zhang et al., 2004; Zwirska-Korczala et al., 2007). After incubation, samples were centrifuged and cell-free

CE P

aliquots of each triplicate were immediately transferred to a new tube and kept at -80ºC until measurement of glycerol and free fatty acids. The cells from each triplicate recovered together with 1 mL of TRI Reagent solution (Ambion, Alcobendas, Spain) were transferred to a single tube and kept at -80ºC until expression analysis of leptin (LepA1 and LepA2) and genes

AC

involved in fatty acid metabolism and transport into the cell and mitochondria (HSL, ATGL, LPL, FAS and FATP1) and related transcription factors (PPARα and PPARβ). Glycerol and free fatty acids levels in the media were determined enzymatically using commercial kits (Free glycerol determination kit, Sigma, Tres Cantos, Spain for glycerol and NEFA-HR2, Wako Chemicals GmbH, Neuss, Germany for free fatty acids).

2.5. RNA extraction and cDNA synthesis Total RNA was extracted using TRI Reagent (Ambion, Alcobendas, Spain), quantified (NanoDrop 2000; Thermo Scientific, Alcobendas, Spain), and its integrity analyzed. Then, total RNA was treated with DNase I (Invitrogen, Alcobendas, Spain) and used to synthesize firststrand cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Sant Cugat del Valles, Spain) or Oligo(dT)12-18 Primer and Superscript III (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendations.

ACCEPTED MANUSCRIPT 2.6. Quantitative real-time PCR (qPCR) The relative expression of the different genes analyzed was quantified on a CFX-96 Real-Time PCR detection system platform (Bio-Rad, Hercules, CA, USA) or a MyiQ thermocycler (Bio-Rad, El Prat de Llobregat, Spain) following standard procedures. Negative controls, a dilution curve

T

with a pool of samples and an amplicon dissociation analysis was run to confirm primer

IP

efficiency, specificity of the reaction and absence of primer-dimers formation. Information on forward and reverse primers is given in Table 1. The primers for ATGL not reported previously

SC R

were designed using the DNAMAN software package and Net primer (Premier BioSoft). SYBR Green fluorescence was recorded during the annealing-extending phase of cycling. Raw data were normalized to elongation factor 1α (EF1α), which was used as a reference gene, and

NU

analyzed by the delta-delta-Ct method (Livak and Schmittgen, 2001).

MA

2.7. Statistical analyses

Statistical analyses of all parameters were carried out using IBM SPSS Statistics 20.0 (IBM, Chicago, USA). Normality was analyzed according to the Shapiro-Wilk test and homogeneity of variance according to Levene’s test. Statistical differences were assessed by Student’s t-test or

D

one-way ANOVA followed by the correct post hoc test Tukey’s or Dunnett's T3. Non-parametric

TE

tests, Kruskal-Wallis and Mann-Whitney U test were used if the data were not normally distributed after logarithmic (log10) transformation. A statistical significance was set at the level of p<0.05 for all the statistical tests performed. Data are presented as means ± standard error of

AC

3. RESULTS

CE P

the mean (SEM).

3.1. Presence of leptin and ghrelin in cultured adipocytes To identify the presence of leptin, ghrelin and their receptors (LepRL and GHS-R1a, respectively) in differentiated adipocytes, cells were stained after 13 days in culture. Cells used as negative controls were incubated with secondary antibody, and showed only non-specific autofluorescence (Figure 1A-C). Cells incubated either with leptin or LepRL antibodies did not give specific signals (Supplementary Figure 1) independently of the dilutions and conditions tested, suggesting that these antibodies are unsuitable for immunofluorescence assays. Cells incubated with ghrelin primary antibody, showed a clear specific signal in the cytoplasm, in the structures surrounding the nuclei and inside the nuclei, as revealed by Hoechst staining colocalization (Figure 1D-F). Furthermore, cells incubated with the GHS-R1a primary antibody, showed a clear specific signal in the cytoplasm, and especially in the plasma membrane, mainly in the junctions between cells (Figure 1G-I).

ACCEPTED MANUSCRIPT LepA1 expression was detected in both preadipocytes at day 7 and in mature adipocytes at day 16, with higher levels of expression, although not significant, in the latter (Figure 2A). LepA2 mRNA expression was very low, almost undetectable (data not shown). Measurable levels of leptin were found in culture media from both preadipocytes (day 7) and mature adipocytes (day

IP

T

16), with significantly higher levels in the latter (Figure 2B).

SC R

3.2. Effects of leptin and ghrelin on proliferation and differentiation of cultured preadipocytes

Proliferation was significantly stimulated by insulin, but not by leptin or ghrelin (data not shown). Adipocyte differentiation was significantly stimulated by the lipid mixture, but neither leptin,

NU

ghrelin nor insulin increased lipid accumulation in these cells (data not shown). Transcript levels of the adipogenesis-associated transcription factor PPARγ and of the marker

MA

of adipocyte differentiation GAPDH were not significantly affected by any of the treatments (Figure 3A and 3B). Lipid mixture significantly increased the expression of LPL (Figure 3C) and FATP1 (Figure 3D), whereas treatment with leptin or growth medium, significantly decreased

TE

D

the expression of LPL (Figure 3C).

3.3. Effects of leptin and ghrelin on lipid metabolism in freshly isolated mature adipocytes

CE P

Both leptin concentrations tested as well as ghrelin at 1 and 10 nM increased glycerol release from adipocytes to a similar level (Table 2). Neither leptin nor ghrelin affected free fatty acids levels in the media.

AC

LepA2 expression was almost undetectable in mature adipocytes and, the mRNA expression of LepA1 was not significantly affected neither with leptin nor ghrelin (Table 2). The gene expression of HSL, ATGL, LPL and FAS in adipocytes treated with leptin was not significantly affected. However, LPL and FAS expression increased after incubation with ghrelin being significantly different respect to the control at the concentration of 10 nM (Table 2). FATP1 expression decreased significantly with leptin at 100 nM concentration but not with ghrelin, whereas CPT1b expression was unaffected upon leptin or ghrelin treatments (Table 2). Adipocyte gene expression of PPARα was not affected by any treatment. PPARβ expression increased significantly after incubation with ghrelin at 1 nM compared to the control, while it was unaffected by leptin (Table 2).

4. DISCUSSION This study shows that both leptin and ghrelin have direct regulatory effects on rainbow trout adipocyte lipid metabolism, although neither hormone appears to control adipogenesis in this species. The study demonstrates that the leptin gene is expressed in adipocytes and that the

ACCEPTED MANUSCRIPT peptide hormone is released from adipocytes in vitro. Further, the presence of ghrelin and its receptor in adipocytes suggest that some of the regulatory actions of ghrelin may occur in an

T

autocrine or paracrine manner in rainbow trout.

IP

4.1. Presence of leptin and ghrelin in cultured adipocytes

The present study shows that LepA1 is expressed in rainbow trout adipocytes in vitro, and that

SC R

leptin is produced and released by these cells during adipogenesis. This is in agreement with observations on mature adipocytes of rainbow trout (Salmerón et al., 2015), Atlantic salmon, Salmo salar (Vegusdal et al., 2003), mammals (van Harmelen et al., 2002) and 3T3-L1 cells (Sheng et al., 2014). Further, the present study clearly shows leptin production in rainbow trout

NU

preadipocytes, whereas in mammals leptin is almost exclusively produced in mature adipocytes since its gene expression seems to be activated with differentiation (Melzner et al., 2002; Schultz et al., 2014). Although the presence of leptin or its receptor at protein level could not be

MA

detected by immunofluorescence in the present study, their gene expression has previously been demonstrated in rainbow trout adipose tissue (Gong et al., 2013; Pfundt et al., 2009). Ghrelin and its functional receptor GHS-R1a were detected in differentiated adipocytes of

D

rainbow trout in culture by immunofluorescence. Although ghrelin is mainly produced by the

TE

stomach and its receptor is mostly present in the pituitary of vertebrates (Castañeda et al., 2010; Jönsson, 2013), our results are in line with the detection of both transcripts in visceral adipose tissue of Atlantic salmon and Mozambique tilapia (Kaiya et al., 2009b; Murashita et al.,

CE P

2009). Together, these data indicate that leptin and ghrelin are produced, and can act through their respective receptors at the level of the adipose tissue, thus suggesting autocrine/paracrine

AC

roles for both hormones.

4.2. Effects of leptin and ghrelin on preadipocyte proliferation and differentiation Proliferation of rainbow trout preadipocytes was unaffected by leptin or ghrelin treatments. In mammals, the effects of leptin on adipocyte development are contradictory. In rat preadipocyte primary culture, leptin increases proliferation at a low concentration, whereas a 10-fold higher concentration has the opposite effect (Wagoner et al., 2006). In 3T3-L1 cells, leptin either reduces proliferation (Zwirska-Korczala et al., 2007), or has no effects over a wide concentration range (Kim et al., 2008). These observations suggest that more studies are necessary to elucidate if leptin in fish may also show differential effects depending on the time and dosage of hormone used as found for mammalian preadipocytes. In contrast to our data in rainbow trout, ghrelin is known to stimulate preadipocyte proliferation in mammals (Kim et al., 2004; Zhang et al., 2004; Zwirska-Korczala et al., 2007) suggesting that it has evolved other/new functions in mammals such as on preadipocyte proliferation. The significant stimulation of preadipocytes by insulin is in agreement with published data on preadipocytes of fish (Oku et al., 2006; Salmerón et al., 2013; Wang et al., 2012) and mammals (Gélöen et al.,

ACCEPTED MANUSCRIPT 2006), strengthening the notion that the role of insulin as a growth factor is conserved among vertebrates. While neither leptin nor ghrelin were able to induce differentiation of rainbow trout preadipocytes, addition of lipids significantly induced adipocyte lipid accumulation. These

T

results are in agreement with previous studies where lipids stimulate adipocyte differentiation to

IP

a greater extent than hormones, including insulin, and suggest that one or more components of the lipid mixture act as adipogenic factor(s) (Bouraoui et al., 2008; Salmerón et al., 2013;

SC R

Vegusdal et al., 2003). In vivo and in vitro studies show that free fatty acids bind to PPARγ, the master regulator of adipogenesis, to stimulate transcription of target genes, a metabolic control that seems to be highly conserved between fish and mammals (Carmona-Antoñanzas et al., 2014). However in the present study, PPARγ expression was not affected upon treatments.

NU

Leptin has no effect on triglyceride accumulation or GAPDH activity in rodent primary preadipocytes and undifferentiated 3T3-L1 cells (Kim et al., 2008; Wagoner et al., 2006),

MA

whereas leptin inhibits the rosiglitazone-induced PPARγ expression (Rhee et al., 2008). Similar mechanisms may be at work in the present study with leptin suppressing the differentiating effect of the differentiation medium. The majority of ghrelin studies in rodents show stimulation of adipocyte differentiation determined by oil red O staining and PPARγ mRNA expression

D

(Choi et al., 2003; Liu et al., 2009), although in 3T3-L1 cells, ghrelin inhibits differentiation

(Zhang et al., 2004).

TE

suppressing PPARγ mRNA and protein expression, stimulating in parallel cell proliferation

CE P

Notwithstanding, genes involved in fatty acid metabolism such as FATP1 and LPL (Gregoire et al., 1998) were up-regulated with lipid mixture incubation, whereas addition of leptin only, decreased LPL expression. Thus, our results confirm that these two genes are good markers of adipocyte differentiation in fish (Bouraoui et al., 2012; Sánchez-Gurmaches et al., 2012) and

AC

also suggest that leptin might have an inhibitory role during adipocyte differentiation in rainbow trout consistent with the view that this hormone pushes the animal towards a negative energy balance. Ghrelin on the other hand, did not modify the expression of either FATP1 or LPL, contrary to previous studies on human adipocytes where it increases the expression of fat storage-related genes, including LPL (Rodríguez et al., 2009). The fact that ghrelin did not affect LPL expression in fish, suggests that its role in adipocyte development may depend on interaction with other factors, as it occurs for example with troglitazone (a PPARγ activator) or triiodothyronine that require the presence of insulin to modulate adipogenesis in rainbow trout and red sea bream (Pagrus major), respectively (Bouraoui et al., 2012.; Oku et al., 2006).

4.3. Effects of leptin and ghrelin on lipid metabolism in isolated adipocytes In the present study, leptin and ghrelin increase glycerol release from isolated adipocytes without a proportional release of free fatty acids. The leptin effects are in agreement with studies in mammals, where leptin stimulates lipolysis as shown by increased glycerol without a concomitant release of free fatty acids both in vivo and in vitro (Li et al., 2010b; Tajima et al.,

ACCEPTED MANUSCRIPT 2005; Wang et al., 1999). The present data are also in line with a study on fatty degenerated hepatocytes of grass carp (Lu et al., 2012), supporting the idea that free fatty acids are preferentially oxidized or re-esterified inside the adipocyte rather than exported. Moreover, the ghrelin data obtained here are contrary to that generally observed in rat adipocytes, where

T

ghrelin has anti-lipolytic effects by inhibiting isoproterenol-induced lipolysis (Choi et al., 2003;

IP

Muccioli et al., 2004). Nevertheless, it is known that ghrelin stimulates both in vivo and in vitro pituitary release of growth hormone (Won and Borski, 2013). This suggests that in rainbow

SC R

trout, ghrelin may stimulate lipolysis directly and indirectly, as growth hormone has been proved to be lipolytic in fish as in mammals (Albalat et al., 2005b; Cruz-Garcia et al., 2015). In terms of gene expression regulation, HSL mRNA levels in isolated adipocytes were not affected by leptin treatment, but tended to increase after incubation with ghrelin, suggesting that

NU

ghrelin may stimulate lipolysis directly through HSL, whereas leptin may act by activating other lipases. For this reason, the expression of ATGL was also measured. This is a novel, poorly

MA

studied lipase, which interacts with HSL in the lipolytic process, and is modulated by leptin in mammalian adipocytes (Lampidonis et al., 2011; Li et al., 2010b). The present study is the first report on the expression of ATGL in rainbow trout adipose tissue and indicates that this lipase is not regulated by leptin or ghrelin. As leptin up-regulates HSL and ATGL expression in fatty

D

degenerated cultured hepatocytes of grass carp (Lu et al., 2012), it appears likely that tissue or

TE

species-specific differences may exist among teleosts. FATP1 mRNA expression decreases in response to leptin, suggesting that leptin reduces the

CE P

transport of free fatty acids into the adipocyte. Moreover, the expression of FAS and LPL significantly increase in response to ghrelin, supporting a lipogenic role for ghrelin in rainbow trout adipocytes. This is in agreement with in vivo data on rat where the mRNA expression levels of various fat storage-promoting enzymes such as LPL, acetyl-CoA carboxylase, FAS,

AC

and stearoyl-CoA desaturase increase in adipose tissue following central ghrelin infusion (Theander-Carrillo et al., 2006). PPARα and PPARβ activate transcription of genes involved in lipid utilization pathways, including fatty acid uptake and oxidation, and have been characterized as central regulators of mitochondrial lipid catabolism (Huss and Kelly, 2004; Leaver et al., 2008). In the present study, while neither leptin nor ghrelin affects CPT1b or PPARα expression, ghrelin significantly increases PPARβ mRNA expression, suggesting that ghrelin regulates fat metabolism and oxidation through the activation of this transcription factor. In conclusion, the present study demonstrates that leptin stimulates lipolysis, and decreases fatty acid uptake in adipocytes, supporting an anti-adipogenic role for leptin in rainbow trout, and that both cultured preadipocytes and mature adipocytes secrete leptin. Moreover, the present study shows for the first time the presence of ghrelin and its receptor in the adipose tissue of rainbow trout, and a direct effect of ghrelin enhancing lipolysis as well as the expression of lipogenesis and fatty acid oxidation genes in adipocytes. These data suggests that ghrelin may induce a general activation of lipid metabolism by stimulating anabolic and catabolic pathways, although the final balance on adipose tissue metabolism remains unclear. Overall, this study

ACCEPTED MANUSCRIPT supports the notion that both leptin and ghrelin regulate adipocyte energy homeostasis in fish. However, further studies are required to better understand the final outcome and the mechanistic basis for their actions. Such knowledge may provide future methods to prevent

T

excessive fat accumulation in fish and help improve product quality for aquaculture.

IP

ACKNOWLEDGEMENTS

SC R

The authors thank Manel Bosch from the “Servei de Microscopia de la Universitat de Barcelona” (CCiTUB) for his help with the acquisition and analyses of confocal images and Dr. Hiroshi Hosoda for the kind gift of the ghrelin antibody. This work was supported by funds from the “Ministerio de Ciencia e Innovación” (grants AGL2010-17324 to E.C. and AGL2011-24961 to

NU

I.N.), the Catalonian Government (grant 2009SGR-00402 to J.G.) and through the “Xarxa de Referència d’R+D+I en Aqüicultura”, a grant from Signhild Engkvists foundation to E.J., the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning th

MA

(Formas) grant 223-2011-1356 to Th.B., and by funds from the 7 Framework Program of the

D

European Union (project LIFECYCLE FP7-222719).

TE

REFERENCES

Albalat, A., Gutiérrez, J., Navarro, I. 2005a. Regulation of lipolysis in isolated adipocytes of rainbow trout (Oncorhynchus mykiss): the role of insulin and glucagon. Comp. Biochem.

CE P

Physiol. A. Mol. Integr. Physiol. 142, 347–354. Albalat, A., Gómez-Requeni, P., Rojas, P., Médale, F., Kaushik, S., Vianen, G.J., Van den Thillart, G., Gutiérrez, J., Pérez-Sánchez, J., Navarro, I. 2005b. Nutritional and hormonal control of lipolysis in isolated gilthead seabream (Sparus aurata) adipocytes. Am. J.

AC

Physiol. Reg. Integr. Comp. Physiol. 289, R259–R265. Angotzi, A.R., Stefansson, S., Nilsen, T.O., Rathore, R.M., Rønnestad, I. 2013. Molecular cloning and genomic characterization of novel Leptin-like genes in salmonids provide new insight into the evolution of the Leptin gene family. Gen. Comp. Endocrinol. 187, 48-59. Bouraoui, L., Gutiérrez, J., Navarro, I. 2008. Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss). J. Endocrinol. 198, 459–469. Bouraoui, L., Cruz-Garcia, L., Gutiérrez, J., Capilla, E., Navarro, I. 2012. Regulation of lipoprotein lipase gene expression by insulin and troglitazone in rainbow trout (Oncorhynchus mykiss) adipocyte cells in culture. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 161, 83–88. Capilla, E., Suzuki, N., Pessin, J.E., Hou, J.C. 2007. The glucose transporter 4 FQQI motif is necessary for Akt Substrate of 160-Kilodalton-dependent plasma membrane translocation but not Golgi-localized γ-ear-containing Arf-binding protein-dependent entry into the insulin-responsive storage compartment. Mol. Endocrinol. 21, 3087–3099.

ACCEPTED MANUSCRIPT Capilla, E., Teles-García, A., Acerete, L., Navarro, I., Gutiérrez, J. 2011. Insulin and IGF-I effects on the proliferation of an osteoblast primary culture from sea bream (Sparus aurata). Gen. Comp. Endocrinol. 172, 107–114. Carmona-Antoñanzas, G., Tocher, D.R, Martinez-Rubio, L., Leaver, M.J. 2014. Conservation of

T

lipid metabolic gene transcriptional regulatory networks in fish and mammals. Gene 534,

IP

1–9.

Castañeda, T.R., Tong, J., Datta, R., Culler, M., Tschöp, M.H. 2010. Ghrelin in the regulation of

SC R

body weight and metabolism. Front. Neuroendocrinol. 31, 44–60.

Chisada, S.-i., Kurokawa, T., Murashita, K., Rønnestad, I., Taniguchi, Y., Toyoda, A., Sakaki, Y., Takeda, S., Yoshiura, Y. 2014. Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food

NU

intake and stage specific effects on growth and fat allocation. Gen. Comp. Endocrinol. 195, 9-20.

Choi, K., Roh, S.-G., Hong, Y.-H., Shrestha, Y.B., Hishikawa, D., Chen, C., Kojima, M.,

MA

Kangawa, K., Sasaki, S.-I. 2003. The role of ghrelin and growth hormone secretagogues receptor on rat adipogenesis. Endocrinology 144, 754–759. Cruz-Garcia, L., Sánchez-Gurmaches, J., Monroy, M., Gutiérrez, J., Navarro, I. 2015.

D

Regulation of lipid metabolism and peroxisome proliferator-activated receptors in rainbow trout adipose tissue by lipolytic and antilipolytic endocrine factors. Domest Anim

TE

Endocrinol. 51, 86-95.

Davies, J.S., Kotokorpi, P., Eccles, S.R., Barnes, S.K., Tokarczuk, P.F., Allen, S.K., Whitworth,

CE P

H.S., Guschina, I.A., Evans, B.A.J., Mode, A., et al. 2009. Ghrelin induces abdominal obesity via GHS-R-dependent lipid retention. Mol. Endocrinol. 23, 914–924. De Pedro, N., Martínez-Alvarez, R., Delgado, M.J. 2006. Acute and chronic leptin reduces food intake and body weight in goldfish (Carassius auratus). J. Endocrinol. 188, 513–520.

AC

Einarsdóttir, I.E., Power, D.M., Jönsson, E., Björnsson, B.T. 2011. Occurrence of ghrelinproducing cells, the ghrelin receptor and Na+,K + -ATPase in tissues of Atlantic halibut (Hippoglossus hippoglossus) during early development. Cell Tissue Res. 344, 481–498. Frühbeck, G., Aguado, M., Martínez, J.A. 1997. In vitro lipolytic effect of leptin on mouse adipocytes: evidence for a possible autocrine/paracrine role of leptin. Biochem. Biophys. Res. Commun. 240, 590–594. Géloën, A., Collet, A. J., Guay, G., Bukowiecki, L.J. 1989. Insulin stimulates in vivo cell proliferation in white adipose tissue. Am. J. Physiol. Cell Physiol. 256, C190–C196. Gong, N., Einarsdottir, I.E., Johansson, M., Björnsson, B.T. 2013. Alternative splice variants of the rainbow trout leptin receptor encode multiple circulating leptin-binding proteins. Endocrinology 154, 2331–2340. Gregoire, F.M., Smas, C.M., Sul, H.S. 1998. Understanding adipocyte differentiation. Physiol. Rev. 78, 783–809. Huss, J.M., Kelly, D.P. 2004. Nuclear receptor signaling and cardiac energetics. Circ. Res. 95, 568–578.

ACCEPTED MANUSCRIPT Hwa, J.J., Fawzi, A.B., Graziano, M.P., Ghibaudi, L., Williams, P., Van Heek, M., Davis, H., Rudinski, M., Sybertz, E., Strader, C.D. 1997. Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice. Am. J. Physiol. Reg. Integr. Comp. Physiol. 272, R1204–R1209.

T

Jönsson, E. 2013. The role of ghrelin in energy balance regulation in fish. Gen. Comp.

IP

Endocrinol. 187, 79–85.

Jönsson, E., Kaiya, H., Björnsson, B.T. 2010. Ghrelin decreases food intake in juvenile rainbow

SC R

trout (Oncorhynchus mykiss) through the central anorexigenic corticotropin-releasing factor system. Gen. Comp. Endocrinol. 166, 39–46.

Kaiya, H., Mori, T., Miyazato, M., Kangawa, K. 2009a. Ghrelin receptor (GHS-R)-like receptor and its genomic organisation in rainbow trout, Oncorhynchus mykiss. Comp. Biochem.

NU

Physiol. A: Mol. Integr. Physiol. 153, 438–450.

Kaiya, H., Riley, L.G., Janzen, W., Hirano, T., Grau, E.G., Miyazato, M., Kangawa, K. 2009b. Identification and genomic sequence of a ghrelin receptor (GHS-R)-like receptor in the

MA

Mozambique tilapia, Oreochromis mossambicus. Zoolog. Sci. 26, 330–337. Kang, K.S., Yahashi, S., Matsuda, K. 2011. The effects of ghrelin on energy balance and psychomotor activity in a goldfish model: an overview. Int. J. Pept. 2011, 171034.

D

Kim, M.S., Yoon, C.Y., Jang, P.G., Park, Y.J., Shin, C.S., Park, H.S., Ryu, J.W., Pak, Y.K., Park, J.Y., Lee, K.U., et al. 2004. The mitogenic and antiapoptotic actions of ghrelin in

TE

3T3-L1 adipocytes. Mol. Endocrinol. 18, 2291–2301. Kim, W.K., Lee, C.Y., Kang, M.S., Kim, M.H., Ryu, Y.H., Bae, K.-H., Shin, S.J., Lee, S.C., Ko, Y.

CE P

2008. Effects of leptin on lipid metabolism and gene expression of differentiationassociated growth factors and transcription factors during differentiation and maturation of 3T3-L1 preadipocytes. Endocr. J. 55, 827–837. Kling, P., Rønnestad, I., Stefansson, S.O., Murashita, K., Kurokawa, T., Björnsson, B.T. 2009. A

AC

homologous salmonid leptin radioimmunoassay indicates elevated plasma leptin levels during fasting of rainbow trout. Gen. Comp. Endocrinol. 162, 307–312. Lampidonis, A.D., Rogdakis, E., Voutsinas, G.E., Stravopodis, D.J. 2011. The resurgence of Hormone-Sensitive Lipase (HSL) in mammalian lipolysis. Gene 477, 1–11. Leaver, M.J., Bautista, J.M., Björnsson, B.T., Jönsson, E., Krey, G., Tocher, D.R., Torstensen, B.E. 2008. Towards fish lipid nutrigenomics: Current state and prospects for fin-fish aquaculture. Rev. Fish. Sci. 16, 73–94. Li, G.-G., Liang, X.-F., Xie, Q., Li, G., Yu, Y., Lai, K. 2010a. Gene structure, recombinant expression and functional characterization of grass carp leptin. Gen. Comp. Endocrinol. 166, 117–127. Li, Y., Zheng, X., Liu, B., Yang, G. 2010b. Regulation of ATGL expression mediated by leptin in vitro in porcine adipocyte lipolysis. Mol. Cell. Biochem. 333, 121–128. Liu, J., Lin, H., Cheng, P., Hu, X., Lu, H. 2009. Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes. J. Huazhong Univ. Sci. Technolog. Med. Sci. 29, 227–230.

ACCEPTED MANUSCRIPT Livak, K.J., Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2

−ΔΔCT

Method. Methods 25, 402–408.

Londraville, R.L., Macotela, Y., Duff, R.J., Easterling, M.R., Liu, Q., Crespi, E.J. 2014. Comparative endocrinology of leptin: Assessing function in a phylogenetic context. Gen.

T

Comp. Endocrinol. 203, 146-157.

IP

Lu, R.H., Liang, X.F., Wang, M., Zhou, Y., Bai, X.L., He, Y. 2012. The role of leptin in lipid metabolism in fatty degenerated hepatocytes of the grass carp Ctenopharyngodon idellus.

SC R

Fish Physiol. Biochem. 38, 1759–1774.

Melzner, I., Scott, V., Dorsch, K., Fischer, P., Wabitsch, M., Brüderlein, S., Hasel, C., Möller, P. 2002. Leptin gene expression in human preadipocytes is switched on by maturationinduced demethylation of distinct CpGs in its proximal promoter. J. Biol. Chem. 277,

NU

45420-45427.

Muccioli, G., Pons, N., Ghè, C., Catapano, F., Granata, R., Ghigo, E. 2004. Ghrelin and desacyl ghrelin both inhibit isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a

MA

growth hormone secretagogue receptor. Eur. J. Pharmacol. 498, 27–35. Murashita, K., Uji, S., Yamamoto, T., Rønnestad, I., Kurokawa, T. 2008. Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss).

D

Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 150, 377–384. Murashita, K., Kurokawa, T., Nilsen, T.O., Rønnestad, I. 2009. Ghrelin, cholecystokinin, and

TE

peptide YY in Atlantic salmon (Salmo salar): molecular cloning and tissue expression. Gen. Comp. Endocrinol. 160, 223–235.

CE P

Oku, H., Tokuda, M., Okumura, T., Umino, T. 2006. Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromalvascular cells of red sea bream, Pagrus major. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 144, 326–333.

AC

Pfundt, B., Sauerwein, H., Mielenz, M. 2009. Leptin mRNA and protein immunoreactivity in adipose

tissue

and

liver

of

rainbow

trout

(Oncorhynchus

mykiss)

and

immunohistochemical localization in liver. Anat. Histol. Embryol. 38, 406–410. Pulkkinen, L., Ukkola, O., Kolehmainen, M., Uusitupa, M. 2010. Ghrelin in diabetes and metabolic syndrome. Int. J. Pept. 2010, 248948. Reidy, S.P., Weber, J. 2000. Leptin: an essential regulator of lipid metabolism. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 125, 285–298. Rhee, S.D., Sung, Y.-Y., Jung, W.H., Cheon, H.G. 2008. Leptin inhibits rosiglitazone-induced adipogenesis in murine primary adipocytes. Mol. Cell. Endocrinol. 294, 61–69. Riley, L.G., Fox, B.K., Kaiya, H., Hirano, T., Grau, E.G. 2005. Long-term treatment of ghrelin stimulates feeding, fat deposition, and alters the GH/IGF-I axis in the tilapia, Oreochromis mossambicus. Gen. Comp. Endocrinol. 142, 234–240. Rodríguez, A., Gómez-Ambrosi, J., Catalán, V., Gil, M.J., Becerril, S., Sáinz, N., Silva, C., Salvador, J., Colina, I., Frühbeck, G. 2009. Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes. Int. J. Obes. (Lond) 33, 541–552.

ACCEPTED MANUSCRIPT Rønnestad, I., Nilsen, T.O., Murashita, K., Angotzi, A.R., Gamst Moen, A.-G., Stefansson, S.O., Kling, P., Björnsson, B.T., Kurokawa, T. 2010. Leptin and leptin receptor genes in Atlantic salmon: cloning, phylogeny, tissue distribution and expression correlated to long-term feeding status. Gen. Comp. Endocrinol. 168, 55–70.

T

Salmerón, C., Acerete, L., Gutiérrez, J., Navarro, I., Capilla, E. 2013. Characterization and

IP

endocrine regulation of proliferation and differentiation of primary cultured preadipocytes from gilthead sea bream (Sparus aurata). Domest. Anim. Endocrinol. 45, 1–10.

SC R

Salmerón, C., Johansson, M., Angotzi, A.R., Rønnestad, I., Jönsson, E., Björnsson; B.Th., Gutiérrez, J., Navarro, I., Capilla, E. 2015. Effects of nutritional status on plasma leptin levels and in vitro regulation of adipocyte leptin expression and secretion in rainbow trout. Gen. Comp. Endocrinol. 210, 114-123.

NU

Sánchez-Gurmaches, J., Cruz-Garcia, L., Gutiérrez, J., Navarro, I. 2012. mRNA expression of fatty acid transporters in rainbow trout: in vivo and in vitro regulation by insulin, fasting and inflammation and infection mediators. Comp. Biochem. Physiol. A. Mol. Integr. Physiol.

MA

163, 177–188.

Schultz, N.S., Broholm, C., Gillberg, L., Mortensen, B., Jørgensen, S.W., Schultz, H.S., Scheele, C., Wojtaszewski, J.F., Pedersen, B.K., Vaag, A. 2014. Impaired leptin gene

D

expression and release in cultured preadipocytes isolated from individuals born with low birth weight. Diabetes 63, 111-121.

TE

Sheng, X., Tucci, J., Malvar, J., Mittelman, S.D. 2014. Adipocyte differentiation is affected by media height above the cell layer. Int. J. Obes. (Lond) 38, 315-320.

CE P

Tajima, D., Masaki, T., Hidaka, S., Kakuma, T., Sakata, T. and Yoshimatsu, H. 2005. Acute central infusion of leptin modulates fatty acid mobilization by affecting lipolysis and mRNA expression for uncoupling proteins. Exp. Biol. Med. (Maywood). 230, 200–206. Theander-Carrillo, C., Wiedmer, P., Cettour-Rose, P., Nogueiras, R., Perez-Tilve, D., Pfluger,

AC

P., Castaneda, T.R., Muzzin, P., Schürmann, A., Szanto, I., et al. 2006. Ghrelin action in the brain controls adipocyte metabolism. J. Clin. Invest. 116, 1983–1993. Tschöp, M., Smiley, D.L., Heiman, M.L. 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913.

Van Harmelen, V., Dicker, A., Rydén, M., Hauner, H., Lönnqvist, F., Näslund, E., Arner, P. 2002. Increased lipolysis and decreased leptin production by human omental as compared with subcutaneous preadipocytes. Diabetes 51, 2029–2036. Vegusdal, A., Sundvold, H., Gjøen, T., Ruyter, B. 2003. An in vitro method for studying the proliferation and differentiation of Atlantic salmon preadipocytes. Lipids 38, 289–296. Wagoner, B., Hausman, D.B., Harris, R.B.S. 2006. Direct and indirect effects of leptin on preadipocyte proliferation and differentiation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1557–R1564. Wang, M. Y., Lee, Y. and Unger, R. H. 1999. Novel form of lipolysis induced by leptin. J. Biol. Chem. 274, 17541–17544.

ACCEPTED MANUSCRIPT Wang, X., Huang, M. and Wang, Y. 2012. The effect of insulin, TNFα and DHA on the proliferation, differentiation and lipolysis of preadipocytes isolated from large yellow croaker (Pseudosciaena Crocea R.). PLoS One 7, e48069. Won, E.T. and Borski, R.J. 2013. Endocrine regulation of compensatory growth in fish. Frontiers

T

Endocrinol. 4, 74.

IP

Zhang, W., Zhao, L., Lin, T. R., Chai, B., Fan, Y., Gantz, I. and Mulholland, M. W. 2004. Inhibition of adipogenesis by ghrelin. Mol. Biol. Cell 15, 2484–2491.

SC R

Zwirska-Korczala, K., Adamczyk-Sowa, M., Sowa, P., Pilc, K., Suchanek, R., Pierzchala, K., Namyslowski, G., Misiolek, M., Sodowski, K., Kato, I., et al. 2007. Role of leptin, ghrelin, angiotensin II and orexins in 3T3 L1 preadipocyte cells proliferation and oxidative

AC

CE P

TE

D

MA

NU

metabolism. J. Physiol. Pharmacol. 58, 53–64.

ACCEPTED MANUSCRIPT TABLES

Table 1. Nucleotide sequences of the primers used to evaluate mRNA abundance by

T

quantitative real time PCR (qPCR) in rainbow trout adipocytes. HSL: hormone sensitive lipase; ATGL: adipose triglyceride lipase; LPL: lipoprotein lipase; FAS: fatty acid synthase;

IP

FATP1: fatty acid transporter 1; CPT1b: carnitine palmitoyltransferase 1b; PPARα: peroxisome

peroxisome

proliferator-activated

receptor

γ;

SC R

proliferator-activated receptor α; PPARβ: peroxisome proliferator-activated receptor β; PPARγ: GAPDH:

glyceraldehyde

3-phosphate

dehydrogenase; LepA1: leptin A1; LepA2: leptin A2; and EF1α: elongation factor 1α. (1) LepA1 and EF1α primers used for leptin expression analyses during adipogenesis and (2) LepA1 and

NU

EF1α primers used in the remaining experiments. FW: forward primer; RV: reverse primer; Ta:

MA

annealing temperature; bp: base pair; DFCI: Dana Farber Gene Indices.

Amplicon Database size (bp)

Gene

Primer Sequence (5’-3’)

HSL1

FW: AGGGTCATGGTCATCGTCTC RV: CTTGACGGAGGGACAGCTAC FW: CGTGTCCGAGTTCAAGTC RV: GGAGAGATGCTGATGGTG FW: TAATTGGCTGCAGAAAACAC RV: CGTCAGCAAACTCAAAGGT FW: GAGACCTAGTGGAGGCTGTC RV: TCTTGTTGATGGTGAGCTGT FW: AGGAGAGAACGTCTCCACCA RV: CGCATCACAGTCAAATGTCC FW: CCCTAAGCAAAAAGGGTCTTCA RV: CATGATGTCACTCCCGACAG FW: CTGGAGCTGGATGACAGTGA RV: GGCAAGTTTTTGCAGCAGAT FW: CTGGAGCTGGATGACAGTGA RV: GTCAGCCATCTTGTTGAGCA FW: GACGGCGGGTCAGTACTTTA RV: ATGCTCTTGGCGAACTCTGT FW: TCTGGAAAGCTGTGGAGGGATGGA RV: AACCTTCTTGATGGCATCATAGC

58

175

DFCI

TC172767

56

174

GenBank

BX318925

59

164

GenBank

AJ224693

54

186

Sigenae

tcaa0001c.m.06_5.1.om.4

60

157

DFCI

CA373015

60

149

GenBank

AF606076

54

195

GenBank

AY494835

60

195

GenBank

AY356399.1

60

171

GenBank

HM536192.1

61

210

GenBank

NM_001123561

FW: TTGCTCAAACCATGGTGATTAGGA

60

68

GenBank

AB354909

54

148

GenBank

AB354909

60

95

GenBank

JX123129

60

125

GenBank

AF498320

59

159

GenBank

AF498320

FATP1 CPT1b PPARα PPARβ PPARγ GAPDH LepA1 (1)

D

TE

FAS

CE P

LPL

AC

ATGL

Ta (ºC)

Accession number

RV: GTCCATGCCCTCGATCAGGTTA LepA1 (2)

FW: GGTGATTAGGATCAAAAAGCTGGA RV: GACGAGCAGTAGGTCCTGGTAGAA

LepA2

FW: TGGGAATCAAAAAGCTCCCTTCCTCTT RV: GCCTCCTATAGGCTGGTCTCCTGCA

EF1α (1)

FW: ATTAACATTGTGGTCATTGGCCATGTC RV: ATCTCAGCTGCTTCCTTCTCGAACTTTT

EF1α (2)

FW: TCCTCTTGGTCGTTTCGCTG RV: ACCCGAGGGACATCCTGTG

ACCEPTED MANUSCRIPT Table 2. Effects of leptin and ghrelin in lypolisis and lipid metabolism-related genes expression in rainbow trout isolated adipocytes. Glycerol and free fatty acids secreted into the media and, quantitative relative expression of several lipid metabolism-related genes normalized to EF1α of isolated adipocytes untreated (control, C) or treated during 3 h with leptin

T

at 10 and 100 nM (L10 and L100) or ghrelin at 0.1, 1 and 10 nM (G0.1, G1 and G10). Data are

IP

normalized to control adipocytes and shown as means ± SEM (n=4-13). Different upper or lower case letters indicate significant differences between treatments for leptin and ghrelin,

SC R

respectively at p<0.05. FFAs: free fatty acids; LepA1: leptin A1; HSL: hormone sensitive lipase; ATGL: adipose triglyceride lipase; LPL: lipoprotein lipase; FAS: fatty acid synthase; FATP1: fatty acid transporter 1; CPT1b: carnitine palmitoyltransferase 1b; PPARα: peroxisome proliferator-activated receptor α; PPARβ: peroxisome proliferator-activated receptor β; EF1α:

C

L10

Fold change Aa

L100

MA

Treatment

NU

elongation factor 1α.

B

B

ab

1.01±0.03

0.98±0.01

0.98±0.02

1.02±0.04

1.02±0.02

1.00±0.23

0.95±0.32

0.91±0.28

0.92±0.31

1.00±0.27

0.91±0.29

HSL

1.00±0.08

0.92±0.06

0.97±0.07

1.23±0.15

1.28±0.13

1.43±0.44

ATGL

1.00±0.17

0.66±0.10

0.74±0.15

1.03±0.17

0.90±0.17

TE

FAS

1.00±0.15

PPARβ

1.00±0.25

A

1.00±0.16 1.00±0.17 1.00±0.10

AC

PPARα

a

CE P

1.00±0.13

CPT1b

D

1.03±0.02

LepA1

a

0.76±0.12

1.10±0.31

1.45±0.25

0.60±0.22

1.07±0.42

1.76±0.40

0.57±0.15

AB

B

ab

1.99±0.71 2.40±0.95

1.37±0.04

ab

2.38±0.78 3.32±0.80

0.51±0.05

1.10±0.50

0.93±0.36

1.11±0.21

0.75±0.13

1.00±0.25

0.87±0.28

1.05±0.26

0.83±0.19

1.40±0.32

0.97±0.08

1.36±0.13

1.29±0.30 ab

1.55±0.12

b

0.98±0.16 ab

1.16±0.17 0.87±0.06

0.40±0.03

ab

1.27±0.04

b

FFAs

LPL

1.21±0.09

G10

1.06±0.03

a

1.29±0.07

G1

Glycerol

FATP1

1.26±0.09

G0.1

b b

1.50±0.32 b

1.13±0.14

ab

ACCEPTED MANUSCRIPT FIGURES LEGENDS

Figure 1. Immunofluorescence of ghrelin and ghrelin receptor (GHS-R1a) in rainbow trout

T

primary cultured adipocytes. Primary and secondary antibodies incubations and nuclei counterstain (visualized in blue) were performed as described in Materials and Methods. In the

IP

left column images acquired in the red channel (target signal) are shown; in the middle column

SC R

are the images acquired in the green channel (unspecific signal); and in the right column the final images, product of the subtraction of the signal from the green channel (autofluorescence) to the corresponding image from the red channel to obtain the specific target signal are shown. Cells incubated with secondary antibody only (negative control) are in panels A-C, and cells

NU

incubated with ghrelin or GHS-R1a primary antibodies are in panels D-F and G-I, respectively. All images were captured with the 63x objective in immersion oil. Representative images from

MA

n=4 independent experiments are shown.

Figure 2. Leptin expression and release in rainbow trout primary cultured cells during adipogenesis. (A) Quantitative relative expression of LepA1 normalized to EF1α and (B) leptin

D

concentration in the media of primary cultured preadipocytes at day 7 and mature adipocytes at

TE

day 16. Data are shown as means ± SEM (n=6-8). Asterisks indicate significant differences

CE P

between culture days at p<0.05. LepA1: leptin A1; EF1α: elongation factor 1α.

Figure 3. Expression of genes related to adipocyte differentiation in rainbow trout primary cultured adipocytes under different treatments. Quantitative relative expression of PPARγ (A), GAPDH (B), LPL (C) and FATP1 (D) normalized to EF1α in adipocytes at day 15

AC

growing in differentiation medium (C), growth medium (GM), or differentiation medium with lipid mixture (LIP), leptin 100 nM (L100), ghrelin 10 nM (G10) or insulin 1000 nM (I1000). Data are shown as means ± SEM (n=5-6). Different letters indicate significant differences between treatments at p<0.05. PPARγ: peroxisome proliferator-activated receptor γ; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; LPL: lipoprotein lipase; FATP1: fatty acid transporter 1; EF1α: elongation factor 1α.

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT