Regulation of adipogenesis by ceramide 1-phosphate

1 Regulation of adipogenesis by ceramide 1-phosphate Marta Ordoñez, Natalia Presa, Asier Dominguez-Herrera, Miguel Trueba, and Antonio Gomez-Muñoz* Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), 48080 Bilbao, Spain. *Corresponding author: Telephone: 34-94-601 2455; FAX: 34-94-601 3500 E-mail: [email protected]s ABSTRACT We showed previously that ceramide kinase (CerK) expression increases during adipogenesis pointing to a relevant role of intracellular C1P in this process. In the present work we demonstrate that administration of exogenous C1P inhibits the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes through a mechanism involving activation of extracellularly regulated kinases (ERK) 1-2. Exogenous C1P reduced the accumulation of lipid droplets and the content of triacylglycerol in these cells, and potently inhibited the expression of the early and late adipogenic markers C/EBP and PPAR, respectively. C1P also reduced the secretion of leptin, which is a crucial regulator of energy balance and appetite in the organism, and is considered to be a late marker of adipogenesis. Interestingly, all of these C1P actions were reversed by pertussis toxin, suggesting the intervention of a Gi protein-coupled receptor previously identified for C1P, in this process. Also, exogenous C1P significantly reduced CerK activity. Altogether, the data presented in this work suggest that exogenous C1P may balance adipogenesis, and that targeting CerK may be a novel way for potential applications in the treatment of obesity or other inflammation-associated diseases. This is the accepted manuscript of the article that appeared in final form in Experimental Cell Research 372(2) : 150-157 (2018), which has been published in final form at https://doi.org/10.1016/j.yexcr.2018.09.021. © 2018 Elsevier under CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 Graphical abstract: ADIPOGENESIS C1P GPCR (+) (-) Ceramide Graphical Abstract Abbreviations: AIM, adipogenic induction medium; BSA, bovine serum albumin; CerK, ceramide kinase; C1P, ceramide 1-phosphate; EBP, enhancer binding protein ; FBS, fetal bovine serum; GAPDH, Glyceraldehide 3-phosphate dehydrogenase; GM, growth medium; IBMX, 3-isobutyl-1-methylxanthine; PPAR, peroxisome proliferatoractivated receptor gamma; Ptx, pertussis toxin; TG, triacylglycerol Keywords: Adipogenesis; ceramides; ceramide kinase; ceramide 1-phosphate; sphingolipids 1. Introduction Adipogenesis is the process by which undifferentiated pre-adipocytes are converted to differentiated adipocytes. The whole process is complex and is 3 accompanied by changes in cell morphology and gene expression of transcription factors such as CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor g (PPAR) [1]. Early stages of adipogenesis are controlled by members of the EBP transcription factors, such as C/EBP, whereas late stages of adipocyte differentiation are mainly controlled by PPAR, which is considered the master regulator of adipogenesis [2]. Hyperplasia caused by adipocyte proliferation and hypertrophy caused by adipogenesis are the main reasons for fat deposition in vivo, the majority of it being stored as triacylglycerol (TG). However, the adipose tissue is also an endocrine organ that regulates crucial pathophysiological processes through secretion of specific hormones such as leptin [3], or adiponectin [4]. Adipose cells can also secrete proand anti-inflammatory cytokines, actions that when uncontrolled, can lead to insulin resistance, type II diabetes and obesity. Hence, deciphering the mechanisms that are involved in adipocyte differentiation is essential for understanding the processes that are implicated in the onset and development of obesity and obesityassociated diseases. Several lines of evidence suggest that sphingolipids or the enzymes involved in their metabolism regulate vital cellular functions including cell proliferation, apoptosis, autophagy, or cell differentiation [5-11]. Noteworthy, some sphingolipids are implicated in inflammatory responses [12-14] and inflammation-associated diseases such as atherosclerosis, cancer, diabetes, or obesity. In particular, ceramides have been associated with insulin resistance and the development of type II diabetes, and sphingosine and sphingosine 1-phosphate (S1P) levels are altered in the obese state [15, 16]. Another important ceramide metabolite is ceramide 1-phosphate (C1P), which is formed by the action of ceramide kinase (CerK) on ceramide. Initially, C1P was demonstrated to be a relevant regulator of cell proliferation and survival [17-24], and to 4 actively participate in inflammatory responses [12-14, 25-28]. However, the mechanisms or signaling pathways by which C1P exerts its biological actions have only been partially described. It is known that intracellularly generated C1P can act directly on intracellular targets, such as acidic sphingomyelinase (A-SMase) [23], serine palmitoyltransferase (SPT) [29], or type IV phospholipase A2 [13, 14] to modulate cell survival or inflammation, whereas exogenous C1P, which is present in plasma, can interact with a putative Gi protein-coupled receptor to promote cell migration [30-35] and glucose uptake [36]. The present work was undertaken to elucidate whether C1P might be able to regulate adipogenesis, and to study the mechanisms involved in this action. 2. Materials and methods 2.1. Materials The Dulbecco´s modified Eagle´s culture medium (DMEM) used in experiments was purchased from Lonza. The triacylglycerol assay kit was obtained from Abnova. NBD-Ceramide (NBD-N-hexanoyl-D-erythro-sphingosine) was from Cayman Chemicals. Dexamethasone, pertussis toxin, rosiglitazone, insulin, 3-Isobutyl-1methylxanthine (IBMX) and the Oil Red O dye were purchased from Sigma-Aldrich. Nitrocellulose membranes, protein markers, and BCA assay reagents were obtained from Bio-Rad. Fetal bovine serum (FBS) and newborn calf serum (NCS) were from GIBCO. The ELISA kit for determination of leptin was purchased from Preprotech. The PPARγ and EBP antibodies were supplied by Cell Signaling. The GAPDH antibody, non-targeting (negative) siRNA and ceramide kinase (CerK) siRNA were purchased from Santa Cruz Biotechnology. The CerK antibody was from Calbiochem or Abgent. 5 The rest of chemicals and reagents used in this work were of the highest grade available. 2.2. Cell culture The mouse pre-adipocyte (fibroblast) 3T3-L1 cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and was cultured following the manufacturer’s indications. The cells were grown in 175 cm2 plastic flasks in DMEM supplemented with 10% heat-inactivated newborn calf serum (NCS), 50 mg/l gentamicin, 200 µM L-glutamine and 4.5 g/l of glucose. Cells were incubated in a humidified 5% CO2 incubator at 37 °C and were subcultured every 3-4 days. Cells were used in experiments at about 80-100% confluence. 2.3. 3T3-L1 pre-adipocyte differentiation protocol The 3T3-L1 pre-adipocytes were seeded in 6-well plates at 1.2 x 105cells/well, or in 24-well plates at 6 x 104 cells/well, or in 96-well plates at 9 x 103 cells/well depending on the kind of experiment to be performed. Cells were then cultured in DMEM supplemented with 10% NCS until they were about 90-100% confluent. The cells were further incubated for 2 days. Pre-adipocytes were treated with adipogenic induction medium (AIM), which is a medium consisting of DMEM 10% FBS supplemented with an adipogenic cocktail containing 0.5 mM 3-isobutyl-1methylxanthine (IBMX), 1 g/ml insulin, 0.25 M dexamethasone and 2 M rosiglitazone. Two days later, the medium was removed and cells were incubated further in maintenance medium (DMEM 10% FBS + 1 g/ml insulin) for two additional days. The cells were then fed every two days with DMEM supplemented with 10% FBS and 1 g/ml insulin. 6 2.4. Western blotting Pre-adipocytes were harvested and lysed in ice-cold homogenization buffer to analyze proteins by Western-blotting, essentially as described in [37]. Specifically, 3T3L1 cells were seeded at 1.2 x 105 cells/well in 6-well plates and were differentiated following the above described pre-adipocyte differentiation protocol (section 2.3). Western-blotting was performed as detailed in [33]. About 20-40 μg of protein from each sample was loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), using 12% separating gels. Proteins were transferred onto nitrocellulose membranes and blocked with 5% skim milk for 1 h in Tris-buffered saline (TBS) containing 0.1% Tween 20, and then incubated overnight with the primary antibody in TBS-0.1% Tween 20 at 4 ºC. After three washes with TBS-0.1% Tween 20, membranes were incubated with horseradish peroxidase-conjugated secondary antibody at 1:4000 dilution for 1 h. Bands were visualized by enhanced chemiluminescence and exposed films were analyzed with an ImageJ software. 2.5. Oil Red O staining protocol 3T3-L1 pre-adipocytes were seeded at 6 x 104 cells/well in 24-well plates and differentiated following the above described pre-adipocyte differentiation protocol (section 2.3). Intracellular lipid accumulation was determined using a solution of Oil Red O. Briefly, cells were washed with PBS and fixed in 3.8% formaldehyde for 10 min. They were then washed and stained with a solution containing Oil Red O (3 mg/ml) and isopropanol in water (60/40, v/v) for 20 min at room temperature. Stained cells were washed twice with water and photographed with a Nikon Eclipse TS100 microscope. To assess the degree of differentiation, 200 μl isopropanol was added and incubated for 30 min in a plate shaker. Then, 50 μl of Oil Red O extracted dye was 7 transferred into 96-well plates and quantified by reading the absorbance at a wavelength of 510 nm. The dye extracted from the empty wells represented the non-specific binding of the dye to the plate. The non-specific binding value was subtracted from the absorbance of each experimental condition to obtain accurate measurements of specific staining. 2.6. Measurement of triacylglycerol concentration 3T3-L1 pre-adipocytes were seeded at 9 x 103 cells/well in 96-well plates and differentiated following the above described pre-adipocyte differentiation protocol (section 2.3). Triacylglycerol (TG) content in cells was determined using an ELISA kit (from Abnova) following the manufacturer’s instructions. Briefly, cells were washed with PBS and 100 μl of the lipid extraction solution was added to each well. Then, the plates were incubated in a heating block at 90-100ºC for 30 min. After this time, 50 l/well of standard dilutions of TG or 5-50 μl of the lipid extracts were transferred into 96-well plates. Assay buffer was added where necessary to bring the volume up to 50 μl in all wells. Then, 2 μl of lipase solution was added to each well containing either sample or standard, and the whole plate was incubated for 10 min at room temperature. Subsequently, 50 μl of the reaction mixture (46 μl adipogenesis assay buffer + 2 μl Probe + 2 μl Enzyme mix) was added to each well and incubated at 37 ºC for 30 min in the dark. The absorbance was read at 570 nm in a plate reader. The protein concentration of the lipid extracts was determined using a commercial kit containing bicinchonic acid (BCA), from BioRad, and the values were used as internal controls to normalize the lipid concentration in the samples. 2.7. Determination of ceramide kinase activity 8 Ceramide kinase (CerK) activity was essentially determined as described by Don and Rosen [38]. 3T3-L1 cells were seeded in 6-well plates at 1.2 x 105 cells/well and differentiated in the absence or in the presence of CerK siRNA, following the preadipocyte differentiation protocol described above (section 2.3). Cell lysates (50-100 μg of protein) were mixed with reaction buffer (100 μl, 20 mM Hepes (pH 7.4), 10 mM KCl, 15 mM MgCl2, 15 mM CaCl2, 10% glycerol, 1 mM DTT, 1 mM ATP) containing 10 μM of NBD-C6-ceramide (N-hexanoyl-D-erythro-sphingosine). The reactions were allowed to proceed for 20 min in the dark at 35ºC. Then, 250 μl aliquots of chloroform:methanol (2:1) were added to terminate the reactions. Samples were then centrifuged at 21,800 g for 30 s and 100 μl aliquots of the upper aqueous phase were transferred into 96-well plates. Subsequently, 100 µl aliquots of dimethylformamide (DMF) were added before reading the NBD fluorescence in an appropriate plate reader. Fluorescence was quantified with a 495 nm excitation filter and a 520 nm emission filter with a Synergy HT (Biotek) plate reader equipped with Gen5 software. 2.8. Determination of leptin secretion Leptin concentration in the culture medium was determined using a “Mouse Standard ELISA Development Kit” (Peprotech) following the manufacturer’s indications. 2.9. Statistical analyses Results are expressed as means ± SEM of three to six independent experiments performed in duplicate unless indicated otherwise. Statistical analyses were performed using the two-tailed, paired Student’s t-test, where p<0.05 was considered to be significant (GraphPad Prism software, San Diego, CA) [33]. 9 3. Results 3.1. Exogenous C1P inhibits pre-adipocyte differentiation into mature adipocytes. We have recently shown that CerK is upregulated during adipogenesis [39], suggesting a relevant role of intracellular C1P in this process. However, as mentioned above, C1P is present in plasma and can act through interaction with a putative plasma membrane receptor to modulate a variety of cell functions [30, 31, 35]. To examine the effects of exogenous C1P on pre-adipocyte differentiation, confluent 3T3-L1 cells were grown in growth medium (GM) or adipogenic induction medium (AIM) for ten days. Administration of C1P at concentrations that are found in vivo (0.5 – 20 µM) [40-42] decreased the accumulation of lipid droplets, which were visualized using Oil Red O staining, in a time dependent manner in these cells (Fig. 1). In addition, the content of TG, which was increased in differentiated cells compared to pre-adipocytes, was substantially decreased by administration of C1P (Fig. 2), also indicating that this phosphosphingolipid inhibits adipocyte differentiation. 3.2 C1P inhibits the expression of early and late adipogenic markers. The process of adipocyte differentiation is divided into early, intermediate, and late stages. Early stages of adipogenesis are controlled by members of the EBP transcription factors, such as C/EBP, whereas late stages of adipocyte differentiation are mainly controlled by PPAR. Fig. 3 shows that treatment with C1P caused a significant reduction in the levels of C/EBP phosphorylation (panels A, B) as well as a significant decrease in PPAR expression (panels C, D), suggesting that C1P promotes its anti-adipogenic activity by downregulating these transcription factors. C1P did not 16 concentration was measured 10 days after inducing cell differentiation as described in section 2.5. Results are normalized to protein concentration of each sample and are the mean ± SEM of 4 independent experiments performed in triplicate (cells incubated in GM vs cells incubated in AIM in the absence of C1P, ***p<0.001; cells incubated in AIM in the absence of C1P vs cells incubated in AIM in the presence of 20 µM C1P, ##p<0.01). Figure 3. C1P decreases EBP and PPAR expression. 3T3-L1 cells were seeded in 6-well plates (1.2 x 105 cells/well) and grown in DMEM containing 10% NCS until they were about 90-100% confluent. The cells were further incubated for 2 days before inducing cell differentiation. After this time, cells were cultured in growth medium (GM) or adipogenic induction medium (AIM), with or without C1P. The medium was changed every 2 days and C1P or vehicle were added each time. A. On day 4, cell lysates were prepared and C/EBPβ phosphorylation was detected by Western blotting using a specific antibody against p-C/EBPβ. Equal loading of protein was assessed with an antibody against GAPDH. Similar results were obtained in each of 3 independent experiments. B. Results of the scanning densitometry of exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 3 independent experiments (cells incubated in GM vs cell incubated in AIM in the absence of C1P, *p<0.05; cells incubated in AIM in the absence of C1P vs cells incubated in AIM in the presence of 20 µM C1P, ##p<0.01). C. Cell lysates were prepared from cells that were differentiated for 7 days and PPARγ expression was detected by Western blotting using a specific antibody against PPARγ. Equal loading of protein was assessed with an antibody against GAPDH. Similar results were obtained in each of 5 replicate experiments. D. Results of the scanning densitometry of exposed 17 film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 5 independent experiments (cells incubated in GM vs cell incubated in AIM in the absence of C1P, ***p<0.001; cells incubated in AIM in the absence of C1P vs cells incubated in AIM in the presence of 20 µM C1P, #p<0.05). Figure 4. C1P reduces leptin secretion. 3T3-L1 cells were seeded in 6-well plates (1.2 x 105 cells/well) and they were grown in DMEM containing 10% NCS until they were about 90-100% confluent. Cells were further incubated for 2 days before inducing cell differentiation. A. After this time, cells were incubated in adipogenic induction media (AIM) in order to induce cell differentiation, as described in section 2.5. The culture medium was collected at the indicated time points, and centrifuged. Leptin concentration in supernatants was measured using an ELISA kit, as indicated in section 2.5. The cells were harvested in lysis buffer in order to measure protein concentration. Results are normalized to the protein concentration and are the mean ± SEM of 4 independent experiments performed in duplicate (*p<0.05). B. Cells were incubated in adipogenic induction media (AIM) for two days in the absence or in the presence of 20 μM of C1P. The medium was changed every 2 days and C1P or vehicle were added each time. On day 10, the culture medium was collected, and centrifuged. Leptin concentration in supernatants were measured using an ELISA kit, as indicated in section 2.5. Cells were harvested in lysis buffer in order to measure protein concentration. Results are normalized to the protein concentration and are the mean ± SEM of 5 independent experiments performed in duplicate (#p<0.05). Figure 5. C1P induces ERK 1-2 phosphorylation in 3T3-L1 cells. Pre-adipocytes were seeded in 6-well plates (1.2 X 105 cells/well) and grown in DMEM containing 18 10% NCS until they were about 90-100% confluent. Cells were further incubated for 2 days before inducing cell differentiation. After this time, cells were incubated in adipogenic induction medium (AIM) with or without C1P, as indicated. A. Cells were harvested after the indicated periods of time, and ERK phosphorylation was detected by Western-blotting using a specific antibody against phosphorylated (p) ERK1-2. Cells from day 0 were collected after 1 h of incubation in AIM. Equal loading of protein was assessed with antibodies to GAPDH or total ERK, as indicated. Similar results were obtained in each of 3 replicate experiments. B. Results of the scanning densitometry of the exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 3 independent experiments (*p<0.05). C. ERK phosphorylation, total ERK and GAPDH were detected as in B. D. Results of the scanning densitometry of the exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 5 independent experiments (*p<0.05; **p<0.01). Figure 6. Inhibition of ERK 1-2 phosphorylation decreases TG levels in 3T3-L1 cells. Pre-adipocytes were seeded in 6-well plates (1.2 x 104 cells/well) to quantify lipid accumulation, or in 96-well plates (9 x 103 cells/well) for measurement of TG concentration. Cells were incubated as indicated in figure 5, and were pretreated with vehicle or with the specific MEK inhibitor PD98059 for 1 h prior to induction of adipogenesis. When present, C1P was at 20 µM. A. Cells were stained with Oil Red O, and lipid accumulation in the cells was determined as indicated in section 2.5. For quantitative analysis, the dye was dissolved in isopropanol. Results are expressed relative control (AIM) values and the absorbance of empty wells (without cells) was subtracted from the absorbance of experimental wells. Results are the mean ± SEM of 5 19 independent experiments performed in triplicate (cells incubated in GM vs cells incubated in AIM in the absence of C1P, ***p<0.001; cells incubated in adipogenic induction medium (AIM) in the absence of C1P vs cells incubated in AIM in the presence of 20 µM C1P, ###p<0.001; cells incubated in AIM in the presence of 20 µM C1P vs cells incubated in AIM in the presence of 20 µM C1P and 10 or 20 µM of PD98059, as indicated, *P<0.05, **P<0.01). B. TG concentration was measured 10 days after inducing cell differentiation as described in section 2.5. Results are normalized to protein concentration of each sample and are the mean ± SEM of 3 independent experiments performed in triplicate (cells incubated in GM vs cells incubated in AIM in the absence of C1P, *p<0.05; cells incubated in adipogenic induction medium (AIM) in the absence of C1P vs cells incubated in AIM in the presence of 20 µM C1P, #p<0.05; cells incubated in AIM in the presence of 20 µM C1P vs cells incubated in AIM in the presence of 20 µM C1P and 10 or 20 µM of PD98059, as indicated, *P<0.05, **P<0.01). There was no statistically significant differences between cells treated with C1P and PD98059 in cells grown in GM. Figure 7. Effect of pertussis toxin (Ptx) on C1P-induced reduction of lipid accumulation, TG concentration and ERK phosphorylation. Cells were seeded and treated as in figures 1, 2 and 5 for determination of lipid accumulation, TG concentration, or ERK phosphorylation, respectively. When present, C1P was at 20 µM. In experimental conditions containing Ptx (0.1 µg/ml), the cells were pre-incubated with the toxin for 16 h prior to addition of C1P or vehicle. A. Oil Red O dye was dissolved in isopropanol and absorbance was measured to quantify neutral lipid accumulation. The absorbance of empty wells (without cells) was subtracted from the absorbance of experimental wells. Results are the mean ± SEM of 5 independent experiments 20 performed in triplicate (cells incubated in GM vs cells incubated in AIM in the absence of C1P and Ptx, ***p<0.001; cells incubated in AIM in the absence of C1P vs cells incubated in AIM in the presence of 20 µM C1P, ###p<0.001; cells incubated in AIM in the presence of 20 µM C1P and 0.1 µg/ml Ptx, *p<0.05; differences between cells incubated in AIM in the absence of C1P and in the presence of 0.1 µg/ml Ptx were not statistically significant, n.s.). B. TG concentration was measured 10 days after inducing cell differentiation as described in section 2.5. Results are normalized to protein concentration of each sample and are the mean ± SEM of 3 independent experiments performed in triplicate (cells incubated in GM vs cells incubated in AIM in the absence of C1P and Ptx, *p<0.05; cells incubated in AIM in the absence of C1P vs cells incubated in AIM in the presence of 20 µM C1P, #p<0.05; cells incubated in AIM in the presence of 20 µM C1P vs cells incubated in AIM in the presence of 20 µM C1P and 0.1 µg/ml Ptx, *p<0.05. The effect of Ptx on TG concentration was not statistically significant). C. ERK phosphorylation was detected as in figure 5. Similar results were obtained in each of 5 replicate experiments. D. Results of scanning densitometry of exposed films. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of five independent experiments (cells incubated in AIM in the absence of C1P and Ptx vs cells incubated in AIM in the presence of 20 µM C1P, **p<0.05; cells incubated in AIM in the presence of 20 µM C1P vs cells incubated in the presence of 20 µM C1P and 0.1 µg/ml Ptx, ##p<0.05). 21 References [1] Y.J. Jang, H.J. Koo, E.H. Sohn, S.C. Kang, D.K. Rhee, S. Pyo, Theobromine inhibits differentiation of 3T3-L1 cells during the early stage of adipogenesis via AMPK and MAPK signaling pathways, Food Funct 6 (2015) 2365-2374. [2] K. Sarjeant, J.M. Stephens, Adipogenesis, Cold Spring Harb Perspect Biol 4 (2012) a008417. [3] Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold, J.M. Friedman, Positional cloning of the mouse obese gene and its human homologue, Nature 372 (1994) 425-432. [4] P.E. Scherer, S. Williams, M. Fogliano, G. Baldini, H.F. Lodish, A novel serum protein similar to C1q, produced exclusively in adipocytes, J Biol Chem 270 (1995) 26746-26749. [5] A. Gomez-Munoz, N. Presa, A. Gomez-Larrauri, I.G. Rivera, M. Trueba, M. Ordonez, Control of inflammatory responses by ceramide, sphingosine 1phosphate and ceramide 1-phosphate, Prog Lipid Res 61 (2016) 51-62. [6] Y.A. Hannun, L.M. Obeid, Principles of bioactive lipid signalling: lessons from sphingolipids, Nat Rev Mol Cell Biol 9 (2008) 139-150. [7] A.H. Merrill, E.M. Schmeltz, D.L. Dillehay, S. Spiegel, J.A. Shayman, J.J. Schroeder, R.T. Riley, K.A. Voss, E. Wang, Sphingolipids--the enigmatic lipid class: biochemistry, physiology, and pathophysiology, Toxycology and Applied Pharmacology 142 (1997) 208-225. 22 [8] A.H. Merrill, M.C. Sullards, W. E., K.A. Voss, R.T. Riley, Sphingolipid metabolism: roles in signal transduction and disruption by fumonisins, Environ. Health Perspect. 109 (2001) 283-289. [9] N. Presa, A. Gomez-Larrauri, I.G. Rivera, M. Ordonez, M. Trueba, A. GomezMunoz, Regulation of cell migration and inflammation by ceramide 1phosphate, Biochim Biophys Acta 1861 (2016) 402-409. [10] S. Spiegel, A.H. Merrill, Sphingolipid metabolism and cell growth regulation, FASEB J. 10 (1996) 1388-1397. [11] J. Stiban, R. Tidhar, A.H. Futerman, Ceramide synthases: roles in cell physiology and signaling, Adv Exp Med Biol 688 (2010) 60-71. [12] C. Chalfant, S. Spiegel, Sphingosine-1-phosphate and ceramide-1-phosphate: expanding roles in cell signaling, J. Cell Sci. 118 (2005) 4605-4612. [13] B.J. Pettus, A. Bielawska, S. Spiegel, P. Roddy, Y.A. Hannun, C.E. Chalfant, Ceramide kinase mediates cytokineand calcium ionophore-induced arachidonic acid release, J. Biol. Chem. 278 (2003) 38206-38213. [14] B.J. Pettus, A. Bielawska, P. Subramanian, D.S. Wijesinghe, M. Mayceka, C.C. Leslie, J. Freiberg, P. Roddy, Y.A. Hannun, C.E. Chalfant, Ceramide-1phosphate is a direct activator of cytosolic phospholipase A2., J. Biol. Chem. 279 (2004) 11320-11326. [15] P.J. Larsen, N. Tennagels, On ceramides, other sphingolipids and impaired glucose homeostasis, Mol Metab 3 (2014) 252-260. [16] M.H. Moon, J.K. Jeong, S.Y. Park, Activation of S1P2 receptor, a possible mechanism of inhibition of adipogenic differentiation by sphingosine 1phosphate, Mol Med Rep 11 (2015) 1031-1036. 23 [17] P. Gangoiti, L. Arana, A. Ouro, M.H. Granado, M. Trueba, A. Gomez-Munoz, Activation of mTOR and RhoA is a major mechanism by which Ceramide 1phosphate stimulates macrophage proliferation, Cell Signal 23 (2011) 27-34. [18] P. Gangoiti, C. Bernacchioni, C. Donati, F. Cencetti, A. Ouro, A. GomezMunoz, P. Bruni, Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts, Biochimie 94, 597-607 (2012). [19] P. Gangoiti, M.H. Granado, L. Arana, A. Ouro, A. Gomez-Munoz, Activation of protein kinase C-alpha is essential for stimulation of cell proliferation by ceramide 1-phosphate, FEBS Lett 584 (2010) 517-524. [20] P. Gangoiti, M.H. Granado, S.W. Wang, J.Y. Kong, U.P. Steinbrecher, A. Gomez-Munoz, Ceramide 1-phosphate stimulates macrophage proliferation through activation of the PI3-kinase/PKB, JNK and ERK1/2 pathways, Cell Signal 20 (2008) 726-736. [21] A. Gómez-Muñoz, P.A. Duffy, A. Martin, L. O'Brien, H.S. Byun, R. Bittman, D.N. Brindley, Short-chain ceramide 1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides, Mol. Pharmacol. 47 (1995) 883-889. [22] A. Gómez-Muñoz, J.Y. Kong, K. Parhar, S.W. Wang, P. Gangoiti, M. González, S. Eivemark, B. Salh, V. Duronio, U.P. Steinbrecher, Ceramide-1-phosphate promotes cell survival through activation of the phosphatidylinositol 3kinase/protein kinase B pathway, FEBS Lett. 579 (2005) 3744-3750. [23] A. Gómez-Muñoz, J.Y. Kong, B. Salh, U.P. Steinbrecher, Ceramide-1phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages, J. Lipid Res. 45 (2004) 99-105. 24 [24] A. Ouro, L. Arana, M. Riazy, P. Zhang, A. Gomez-Larrauri, U. Steinbrecher, V. Duronio, A. Gomez-Munoz, Vascular endothelial growth factor mediates ceramide 1-phosphate-stimulated macrophage proliferation, Exp Cell Res 361 (2017) 277-283. [25] K. Baudiss, C.K. Ayata, Z. Lazar, S. Cicko, J. Beckert, A. Meyer, A. Zech, R.P. Vieira, R. Bittman, A. Gomez-Munoz, I. Merfort, M. Idzko, Ceramide-1phosphate inhibits cigarette smoke-induced airway inflammation, Eur Respir J 45 (2015) 1669-1680. [26] K. Baudiss, R. de Paula Vieira, S. Cicko, K. Ayata, M. Hossfeld, N. Ehrat, A. Gomez-Munoz, H.K. Eltzschig, M. Idzko, C1P Attenuates LipopolysaccharideInduced Acute Lung Injury by Preventing NF-kappaB Activation in Neutrophils, J Immunol 196 (2016) 2319-2326. [27] D.S. Wijesinghe, M. Brentnall, J.A. Mietla, L.A. Hoeferlin, R.F. Diegelmann, L.H. Boise, C.E. Chalfant, Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts, J Lipid Res 55 (2014) 1298-1309. [28] D.S. Wijesinghe, P. Subramanian, N.F. Lamour, L.B. Gentile, M.H. Granado, A. Bielawska, Z. Szulc, A. Gomez-Munoz, C.E. Chalfant, Chain length specificity for activation of cPLA2alpha by C1P: use of the dodecane delivery system to determine lipid-specific effects, J Lipid Res 50 (2009) 1986-1995. [29] M.H. Granado, P. Gangoiti, A. Ouro, L. Arana, A. Gomez-Munoz, Ceramide 1phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages, Biochim Biophys Acta 1791 (2009) 263-272. [30] L. Arana, M. Ordonez, A. Ouro, I.G. Rivera, P. Gangoiti, M. Trueba, A. GomezMunoz, Ceramide 1-phosphate induces macrophage chemoattractant protein-1 25 release: involvement in ceramide 1-phosphate-stimulated cell migration, Am J Physiol Endocrinol Metab 304 (2013) E1213-1226. [31] M.H. Granado, P. Gangoiti, A. Ouro, L. Arana, M. Gonzalez, M. Trueba, A. Gomez-Munoz, Ceramide 1-phosphate (C1P) promotes cell migration Involvement of a specific C1P receptor, Cell Signal 21 (2009) 405-412. [32] J.L. Hankins, K.E. Ward, S.S. Linton, B.M. Barth, R.V. Stahelin, T.E. Fox, M. Kester, Ceramide 1-phosphate mediates endothelial cell invasion via the annexin a2-p11 heterotetrameric protein complex, J Biol Chem 288 (2013) 19726-19738. [33] M. Ordonez, I.G. Rivera, N. Presa, A. Gomez-Munoz, Implication of matrix metalloproteinases 2 and 9 in ceramide 1-phosphate-stimulated macrophage migration, Cell Signal 28 (2016) 1066-1074. [34] M.Z. Ratajczak, M. Suszynska, S. Borkowska, J. Ratajczak, G. Schneider, The role of sphingosine-1 phosphate and ceramide-1 phosphate in trafficking of normal stem cells and cancer cells, Expert Opin Ther Targets 18 (2014) 95-107. [35] I.G. Rivera, M. Ordonez, N. Presa, P. Gangoiti, A. Gomez-Larrauri, M. Trueba, T. Fox, M. Kester, A. Gomez-Munoz, Ceramide 1-phosphate regulates cell migration and invasion of human pancreatic cancer cells, Biochem Pharmacol 102 (2016) 107-119. [36] A. Ouro, L. Arana, P. Gangoiti, I.G. Rivera, M. Ordonez, M. Trueba, R.S. Lankalapalli, R. Bittman, A. Gomez-Munoz, Ceramide 1-phosphate stimulates glucose uptake in macrophages, Cell Signal 25 (2013) 786-795. [37] J.A. Hamilton, D. Myers, W. Jessup, F. Cochrane, R. Byrne, G. Whitty, S. Moss, Oxidized LDL can induce macrophage survival, DNA synthesis, and enhanced proliferative response to CSF-1 and GM-CSF, Arterioscler Thromb Vasc Biol 19 (1999) 98-105. 32 33 34 HIGHLIGHTS Exogenous C1P inhibits adipogenesis ERK1-2 are implicated in the inhibition of adipogenesis by C1P C1P inhibition of adipogenesis is blocked by Gi protein inhibitor pertussis toxin