Trout Adipocytes Research Articles

Mature adipocytes inhibit differentiation of myogenic cells but stimulate proliferation of fibro-adipogenic precursors derived from trout muscle in vitro

Interactions between tissues and cell types, mediated by cytokines or direct cell–cell exchanges, regulate growth. To determine whether mature adipocytes influence the in vitro growth of trout mononucleated muscle cells, we developed an indirect coculture system, and showed that adipocytes (5 × 106 cells/well) derived from perivisceral adipose tissue increased the proliferation (BrdU-positive cells) of the mononucleated muscle cells (26% vs. 39%; p < 0.001) while inhibiting myogenic differentiation (myosin+) (25% vs. 15%; p < 0.001). Similar effects were obtained with subcutaneous adipose tissue-derived adipocytes, although requiring more adipocytes (3 × 107 cells/well vs. 5 × 106 cells/well). Conditioned media recapitulated these effects, stimulating proliferation (31% vs. 39%; p < 0.001) and inhibiting myogenic differentiation (32 vs. 23%; p < 0.001). Adipocytes began to reduce differentiation after 24 h, whereas proliferation stimulation was observed after 48 h. While adipocytes did not change pax7+ and myoD1/2+ percentages, they reduced myogenin+ cells showing inhibition from early differentiation stage. Finally, adipocytes increased BrdU+ cells in the Pdgfrα+ population but not in the myoD+ one. Collectively, our results demonstrate that trout adipocytes promote fibro-adipocyte precursor proliferation while inhibiting myogenic cells differentiation in vitro, suggesting the key role of adipose tissue in regulating fish muscle growth.

The autophagy response during adipogenesis of primary cultured rainbow trout (Oncorhynchus mykiss) adipocytes.

Adipogenesis is a tightly regulated process, and the involvement of autophagy has been recently proposed in mammalian models. In rainbow trout, two well-defined phases describe the development of primary cultured adipocyte cells: proliferation and differentiation. Nevertheless, information on the transcriptional profile at the onset of differentiation and the potential role of autophagy in this process is scarce. In the present study, the cells showed an early and transient induction of several adipogenic transcription factors genes' expression (i.e., cebpa and cebpb) along with the morphological changes (round shape filled with small lipid droplets) typical of the onset of adipogenesis. Then, the expression of various lipid metabolism-related genes involving the synthesis (fas), uptake (fatp1 and cd36), accumulation (plin2) and mobilization (hsl) of lipids, characteristic of the mature adipocyte, increased. In parallel, several autophagy markers (i.e., atg4b, gabarapl1 and lc3b) mirrored the expression of those adipogenic-related genes, suggesting a role of autophagy during in vitro fish adipogenesis. In this regard, the incubation of preadipocytes with lysosomal inhibitors (Bafilomycin A1 or Chloroquine), described to prevent autophagy flux, delayed the process of adipogenesis (i.e., cell remodelling), thus suggesting a possible relationship between autophagy and adipocyte differentiation in trout. Moreover, the disruption of the autophagic flux altered the expression of some key adipogenic genes such as cebpa and pparg. Overall, this study contributes to improve our knowledge on the regulation of rainbow trout adipocyte differentiation, and highlights for the first time in fish the involvement of autophagy on adipogenesis, suggesting a close-fitting connection between both processes.

Is methylmercury a new candidate obesogen?

AbstractRecent studies suggest that environmental pollutants play a role in the continuous increase of the worldwide prevalence of obesity. To our knowledge, there is only one study investigating the impacts of methylmercury, a common and more toxic form of mercury, mainly present in fish and shellfish, on adipose tissue. It shows that methylmercury can cause changes in the structure and function of 3T3-L1 preadipocytes. In this context, we decided to study the effects of methylmercury on primary cultured adipocytes of rainbow trout. This species is a well-known model to study the impact of pollution. In addition, fish adipose tissue metabolism is close to the one of humans. Using an in vitro model allows us to focus on adipocyte differentiation and lipid metabolism.Preadipocytes were obtained after isolating precursor cells from perivisceral adipose tissue by enzymatic digestion. Cells were cultured at 19°C until confluence before being incubated for 6 days in growth medium supplemented or not with a hormonal cocktail (insulin, dexamethasone, ciglitizone) enriched with 0, 0.3, 1.7 or 3.8 mM of methylmercury and lipid mixture. Cytotoxicity was evaluated every two days via LDH assay. At day 6, quantification of total mercury in cells was performed with Direct Mercury Analyser-80. Phospholipid and neutral lipid fractions were analyzed by gas chromatography and expression of adipocyte-related genes was evaluated by RT-PCR. Three biological replicates were carried out.No cytotoxicity was observed during the methylmercury treatment. Methylmercury was partially accumulated but in a dose-dependent manner. At day 6, all fatty acids (FA) from neutral lipid fraction significantly increased at the highest methylmercury concentration. However, the rate of increase was different for each FA. In particular, n-3 polyunsaturated FA were preferentially accumulated. The total amount of phospholipids was not impacted by methylmercury but a decrease of n-6 polyunsaturated FA was observed. Surprisingly, methylmercury decreased the expression of three adipocyte markers, FA transport protein 1 and glycerol-3-phosphate dehydrogenase in presence or absence of hormonal cocktail, and CCAAT/enhancer binding protein delta in presence of hormonal cocktail. On the other hand, methylmercury increased the expression of FA synthase and perilipin.As a conclusion, methylmercury appears to be a candidate obesogen as it increased the cellular lipid content in primary cultured trout adipocytes. Additional genes involved in adipogenesis, FA de novo synthesis and FA intracellular transport are being investigated. Further experiments currently conducted in our lab should allow us to better clarify the mechanisms involved.

Caffeic acid and hydroxytyrosol have anti-obesogenic properties in zebrafish and rainbow trout models.

Some natural products, known sources of bioactive compounds with a wide range of properties, may have therapeutic values in human health and diseases, as well as agronomic applications. The effect of three compounds of plant origin with well-known dietary antioxidant properties, astaxanthin (ATX), caffeic acid (CA) and hydroxytyrosol (HT), on zebrafish (Danio rerio) larval adiposity and rainbow trout (Onchorynchus mykiss) adipocytes was assessed. The zebrafish obesogenic test (ZOT) demonstrated the anti-obesogenic activity of CA and HT. These compounds were able to counteract the obesogenic effect produced by the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (RGZ). CA and HT suppressed RGZ-increased PPARγ protein expression and lipid accumulation in primary-cultured rainbow trout adipocytes. HT also significantly reduced plasma triacylglycerol concentrations, as well as mRNA levels of the fasn adipogenic gene in the adipose tissue of HT-injected rainbow trout. In conclusion, in vitro and in vivo approaches demonstrated the anti-obesogenic potential of CA and HT on teleost fish models that may be relevant for studying their molecular mode of action. Further studies are required to evaluate the effect of these bioactive components as food supplements for modulating adiposity in farmed fish.

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

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 genes were examined in rainbow trout adipocytes in vitro. Leptin expression and release 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, 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 fatty acid uptake and storage, but did not affect expression of any of the lipogenesis or β-oxidation genes studied. Ghrelin significantly increased the mRNA levels of lipoprotein lipase, 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 mechanisms. The mode of action of both hormones needs to be further explored to better understand their roles in regulating adiposity in fish.

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

The aim of this study was to determine the effects of growth hormone (GH) and insulin-like growth factor (IGF)-I on glycerol release and the regulation of IGF-I and IGF-II expression by GH in isolated rainbow trout adipocytes. Cells were also incubated with GH, tumor necrosis factor α (TNFα), or insulin to analyze the gene expression of peroxisome proliferator-activated receptors (PPARs) and lipid metabolism markers: hormone sensitive lipase, fatty acid synthase (FAS), and lipoprotein lipase. Complimentary in vivo experiments were performed by intraperitoneally administering insulin, TNFα, or lipopolysaccharide and subjecting the animals to fasting and refeeding periods. The results showed that IGF-I had an antilipolytic effect and GH had a lipolytic effect; the latter occurred independently of IGF modulation and in conjunction with a reduction in PPARα expression in adipocytes. The anabolic action of insulin was demonstrated through its upregulation of lipogenic genes such as lipoprotein lipase, FAS, and PPARγ, whereas GH, by contrast, inhibited FAS expression in adipose tissue. The gene transcription levels of PPARs changed differentially during fasting and refeeding, and the TNFα and/or lipopolysaccharide administration suggested that the regulation of PPARs helps maintain metabolic adipose tissue homeostasis in rainbow trout.

Effects of nutritional status on plasma leptin levels and in vitro regulation of adipocyte leptin expression and secretion in rainbow trout

As leptin has a key role on appetite, knowledge about leptin regulation is important in order to understand the control of energy balance. We aimed to explore the modulatory effects of adiposity on plasma leptin levels in vivo and the role of potential regulators on leptin expression and secretion in rainbow trout adipocytes in vitro. Fish were fed a regular diet twice daily ad libitum or a high-energy diet once daily at two ration levels; satiation (SA group) or restricted (RE group) to 25% of satiation, for 8weeks. RE fish had significantly reduced growth (p<0.001) and adipose tissue weight (p<0.001), and higher plasma leptin levels (p=0.022) compared with SA fish. Moreover, plasma leptin levels negatively correlated with mesenteric fat index (p=0.009). Adipocytes isolated from the different fish were treated with insulin, ghrelin, leucine, eicosapentaenoic acid or left untreated (control). In adipocytes from fish fed regular diet, insulin and ghrelin increased leptin secretion dose-dependently (p=0.002; p=0.033, respectively). Leptin secretion in control adipocytes was significantly higher in RE than in SA fish (p=0.022) in agreement with the in vivo findings, indicating that adipose tissue may contribute to the circulating leptin levels. No treatment effects were observed in adipocytes from the high-energy diet groups, neither in leptin expression nor secretion, except that leptin secretion was significantly reduced by leucine in RE fish adipocytes (p=0.025). Overall, these data show that the regulation of leptin in rainbow trout adipocytes by hormones and nutrients seems to be on secretion, rather than at the transcriptional level.

Interplay of adiponectin, TNFα and insulin on gene expression, glucose uptake and PPARγ, AKT and TOR pathways in rainbow trout cultured adipocytes

Adipose tissue is being increasingly recognized as an important endocrine organ that produces and releases a variety of factors. In the present study we have evaluated in primary cultures of rainbow trout adipocytes, obtained from visceral adipose tissue, the interplay of the adiponectin system, TNFα and insulin at a transcriptional level and, their effects on the adipogenic transcription factor PPARγ, as well as on the activation of main insulin signaling pathways. Likewise, the implication of these adipokines in the regulation of glucose uptake in the adipocyte and their interactions with insulin or IGF-I were also evaluated. Similarly to the mammalian model, insulin enhanced adiponectin gene expression, while it exerted a negative modulation on adiponectin receptors. TNFα increased the mRNA levels of adiponectin receptor 1, but neither adiponectin nor TNFα modulated each other expression. Therefore, the reciprocal suppressive effect of both adipokines previously reported in mammals was not present in this model. Furthermore, the anti-adipogenic effect of TNFα was revealed by the down-regulation of PPARγ at a protein level, meanwhile adiponectin increased PPARγ expression in insulin-stimulated adipocytes, supporting its insulin-sensitizing role. Both adipokines stimulated glucose uptake without modifying AKT or TOR phosphorylation; however, glucose uptake in insulin-treated adipocytes was enhanced by TNFα but not by adiponectin. All in all, these results contribute to gain knowledge on the role of adipokines in rainbow trout adipose tissue and, to better understand the mechanisms that regulate glucose metabolism in this species.

Role of LXR in trout adipocytes: Target genes, hormonal regulation, adipocyte differentiation and relation to lipolysis

In the present study, we describe an initial approach to investigate the role of LXR in fish adipose tissue. Rainbow trout (Oncorhynchus mykiss) isolated adipocytes were incubated with LXR agonists, unsaturated fatty acids, tumour necrosis factor-α (TNFα), insulin or growth hormone (GH) for 6h and LXR expression was analyzed. Lipolysis was measured after incubation with one of the LXR agonists and LXR expression was compared with levels of lipolysis. LXR expression was also analyzed during the differentiation of adipocytes in culture. The incubations with agonists in isolated adipocytes indicated that ATP-binding cassette transporter A1 (ABCA1) is an LXR target gene, but lipoprotein lipase (LPL), fatty acid synthase (FAS), hormone-sensitive lipase (HSL) and peroxisome proliferator-activated receptor (PPARs) are not. LXR agonists also induced LXR expression and raised lipolysis levels. Besides, LXR expression was upregulated in parallel with basal lipolysis. LXR mRNA expression was regulated by unsaturated fatty acids, insulin, TNFα and GH in isolated adipocytes. Besides, LXR showed an upregulation during adipocyte differentiation. All these data indicate that LXR is involved in orchestrating the transcriptional regulatory network in trout adipocyte lipid metabolism, specifically, in cholesterol transport, adipocyte differentiation and lipolysis.

Insulin and insulin-like growth factor I signaling pathways in rainbow trout (Oncorhynchus mykiss) during adipogenesis and their implication in glucose uptake

Primary cultures of rainbow trout (Oncorhynchus mykiss) adipocytes were used to examine the main signaling pathways of insulin and insulin-like growth factor I (IGF-I) during adipogenesis. We first determined the presence of IGF-I receptors (IGF-IR) and insulin receptors (IR) in trout preadipocytes (day 5) and adipocytes (day 14). IGF-IRs were more abundant and appeared to be in higher levels in differentiated cells than in preadipocytes, whereas IRs were detected in lower but constant levels throughout the culture. The cells were immunoreactive against ERK1/2 MAPK, and AKT/PI3K, components of the two main signal transduction pathways for insulin and IGF-I receptors. Stimulation of MAPK phosphorylation by IGF-I was higher in preadipocytes than in adipocytes, while no effects were observed in MAPK phosphorylation after incubation of cells with insulin. AKT phosphorylation increased in the presence of both insulin and IGF-I, with higher levels of stimulation in adipocytes than in preadipocytes. Activation of both pathways was blocked by the use of specific inhibitors of MAPK (PD98059) and AKT (wortmannin). We describe here, for the first time, the effects of IGF-I and insulin on 2-deoxyglucose uptake in primary culture of trout adipocytes. IGF-I was more potent in stimulating glucose uptake than insulin, and PD98059 and wortmannin inhibited the stimulation of glucose uptake by this growth factor, suggesting that IGF-I plays an important metabolic role in trout adipocytes. Our results suggest that differential activation of the MAPK and AKT pathways are involved in the IGF-I- and insulin-induced effects of trout adipocytes during the various stages of adipogenesis.

Differentially expressed proteins in rainbow trout adipocytes isolated from visceral and subcutaneous tissues

In rainbow trout, subcutaneous (in dorsal and ventral positions) and visceral fat deposits are known to influence the yield of edible flesh, whilst their respective roles in metabolism, storage and release of fatty acids have not, so far, been directly studied. The present work aimed to identify, by using 2D electrophoresis, proteins differentially expressed in isolated mature adipocytes originating from these various localizations in prepubescent females. A total of nine proteins were estimated to be differentially expressed according to the localisation of the adipocytes. Seven protein spots were considered to be present in the three fat deposits at differing abundances, and among them only six were estimated as being specific to fat tissues. Among these, five were more abundant in subcutaneous adipocytes of both sites compared to perivisceral adipocytes. Four were identified: three as H-FABP, ATP synthase, serum deprivation-response protein, indicating higher metabolic activity in subcutaneous adipocytes, while the latter, annexin, indicative of a higher proportion of less mature adipocytes, as also suggested by their smaller mean diameter. The more abundant protein in visceral isolated adipocytes is actin, known to be involved in cytoskeleton structure and to increase during adipogenesis. This allows us to suggest their more mature stage of development, in relation with their higher mean diameter.

Regulation of lipolysis in isolated adipocytes of rainbow trout ( Oncorhynchus mykiss): The role of insulin and glucagon

In the present study, we have examined the effects of insulin and glucagon on the lipolysis of rainbow trout ( Oncorhynchus mykiss). To this end, adipocytes were isolated from mesenteric fat and incubated in the absence (basal lipolysis) or presence of different concentrations of insulin and glucagon. In addition, to further elucidate the effects of these hormones in vivo on adipocyte lipolysis, both fasting and intraperitoneal glucagon injection experiments were performed. Basal lipolysis, measured as the glycerol released in the adipocyte medium, increased proportionally with cell concentration and incubation time. Cell viability was verified by measuring the release of lactate dehydrogenase (LDH) activity in the medium. Insulin (at doses of 35 and 350 nM) decreased lipolysis in isolated adipocytes of rainbow trout in vitro, while glucagon was clearly lipolytic at concentrations of 10 and 100 nM. Furthermore, hypoinsulinemia induced by fasting, as well as glucagon injection, significantly increased lipolysis in isolated adipocytes approximately 1.5- and 1.4-fold, respectively, when compared with adipocytes from control fish. Our data demonstrate that lipolysis, as measured in isolated adipocytes of rainbow trout, can be regulated by both insulin and glucagon. These results not only indicate that insulin is an important hormone in lipid deposition via its anti-lipolytic effects on rainbow trout adipocytes, but also reveal glucagon as a lipolytic hormone, as shown by both in vitro and in vivo experiments.

Control of adipose tissue lipid metabolism by tumor necrosis factor-α in rainbow trout (Oncorhynchus mykiss)

Tumor necrosis factor-alpha (TNF alpha) is a cytokine with multiple biological functions which, in mammals, has been shown to modulate muscle and adipose tissue metabolism. In fish, TNF alpha has been identified in several species. However, few studies have examined the role of TNF alpha in fish outside the immune system. In this study, we assessed the effects of human recombinant TNF alpha and conditioned media from rainbow trout lipopolysaccharide (LPS)-stimulated macrophages (LPS-MCM) on lipolysis in isolated rainbow trout adipocytes. Furthermore, we studied the effects of an LPS injection in vivo on lipid metabolism. In our study, human recombinant TNF alpha stimulated lipolysis in trout adipocytes in a time- and dose-dependent manner. Similarly, LPS-MCM stimulated lipolysis in trout adipocytes when compared with control conditioned medium. Experiments using specific inhibitors of the MAP kinase pathway showed that p44/42 and p38 are partially involved in the lipolytic effects of TNF alpha. On the other hand, adipocytes from LPS-injected rainbow trout showed higher basal lipolysis than adipocytes from control fish after 24 h, while this effect was not seen at 72 h. Furthermore, lipoprotein lipase (LPL) activity in adipose tissue of LPS-injected fish was lower than in the controls at 24 h. These data suggest that TNF alpha plays an important role in the control of lipid metabolism in rainbow trout by stimulating lipolysis in vitro and in vivo and by down-regulating LPL activity of adipose tissue in vivo.

Uptake studies in adipocytes isolated from rainbow trout ( Salmo gairdnerii). A comparison with adipocytes from rat and cat

1. 1. Adipocytes were isolated from mesenteric adipose tissue of rainbow trout ( Salmo gairdnerii) by incubation of tissue slices at 20°C in a buffer containing 3 mg collagenase per ml. These cells were compared to adipocytes from the cat and the rat, isolated by conventional technique (1 mg collagenase per ml buffer, incubation temperature 37°C). 2. 2. Uptake studies of some metabolites were performed with fish, rat and in some cases cat adipocytes. At a glucose concentration of 0.33 mM, the glucose uptake into rat cells was more than twice as fast as in cells from the cat, and more than five times as fast as in trout cells. 2-Amino butyrate resembled glucose in relative uptake rates between species. 3. 3. Metabolite uptake into rat cells was specific, with different uptake rates for different metabolites. The uptake into trout adipocytes proceeded at similar rates for all metabolites tested, provided the concentrations were the same. 4. 4. The uptake rate of glucose into rat cells was stimulated by insulin. Insulin had no effect on glucose uptake into adipocytes from trout.