Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; Effects of n-3 and n-9 fatty acids
Introduction
The use of high-energy feeds has been of major importance for the development of cost-effective fish farming. By increasing the energy levels in salmonid diets, growth and feed utilisation are improved [1]. However, increased dietary energy also increases fat deposition in the fish's fat storage organs and thereby reduces harvest yields [2]. It is of major importance to develop strategies to prevent excessive fat deposition in cultivated fish in order to strengthen the sustainability of the aquaculture industry. An improved knowledge of the underlying molecular events that regulate the differentiation process of preadipocytes to adipocytes in Atlantic salmon (Salmo salar) may open new avenues for the prevention of excessive storage of lipids in this important aquaculture species.
The primary sites for triacylglycerol (TAG) deposition in Atlantic salmon are the visceral adipose tissue [3], [4], [5] and myosepta surrounding the muscle [6], [7]. Adiposity may arise from both an increased size of individual adipose cells due to lipid accumulation, and from an increased number of adipocytes arising from the proliferation of precursor cells. It has been suggested that in fish, as in mammals, this process occurs not only during early life stages [8] but also throughout life [9]. When energy intake is excessive, both the number and size of fish adipocytes increase [9], [10]. The underlying molecular processes that control adipocyte differentiation in fish are poorly known. We have, however, previously shown that primary preadipocytes from Atlantic salmon differentiate to mature adipocytes in vitro and that these cells may be used as a model system for studies of adipose tissue development in fish [11]. In contrast to fish, the differentiation process in several mammalian species has been relatively well described: it is regulated by a complex network of molecular events controlled by signalling from hormones, growth factors and components of the extracellular matrix. The transcription factors peroxisome proliferator activated receptor gamma (PPAR) γ and CCAAT binding proteins (C/EBPs), are key regulators involved in initiating differentiation and inducing the expression of adipose-associated genes during differentiation [12], [13].
Nutritional studies in humans and rats have demonstrated that energy balance and body fat content can be affected by changing the dietary long-chain polyunsaturated fatty acid (PUFA) level [14], [15], [16]. Diets enriched in n-3 PUFAs decrease adipose tissue mass and suppress the development of obesity in rats [17]. De Vos et al. [18] demonstrated that n-3 PUFAs limit the development of visceral adipose tissue by suppressing the late phase of adipocyte differentiation through modifications of PPARγ. Fish oil (FO), being a very rich source of n-3 highly unsaturated fatty acids (HUFAs), has been traditionally used as the dominating lipid component in feed for salmonids. Due to a general shortage of marine feed sources, vegetable oils (VOs) are being included to an ever-increasing degree in Atlantic salmon diets. However, to date it is more or less unknown how changing from dietary FOs to VOs affects visceral fat deposition in Atlantic salmon. In order to avoid an increase in the amount of visceral adipose tissue when replacing a diet rich in n-3 HUFAs with one rich in 18-carbon fatty acids (FAs) from VOs, the different dietary FAs from VOs should preferably be easily β-oxidized rather than being primarily deposited. The role of n-3 HUFAs in promoting FA oxidation and repressing lipid deposition has not been reported in fish so far. This study was therefore conducted in order to investigate firstly, the molecular events regulating adipocyte differentiation in Atlantic salmon; secondly, how oleic acid (18:1n-9, OA), a FA highly present in VOs, affects preadipocyte differentiation, cell morphology, FA deposition and utilisation in comparison to two typical marine FAs, namely eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA); and thirdly, how different FAs are deposited and utilised at different stages of adipocyte differentiation.
Section snippets
Materials
Atlantic salmon were obtained from AKVAFORSK (Averøy, Norway). Fetal bovine serum (FBS), essential FA free bovine serum albumin (BSA), antibiotics (mixture of pencillin, streptomycin and amphotericin B), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), l-glutamin, lipid mixture, laminin, Thermanox cover slips, Hank's balanced salt solution (HBSS), oil red O, phosphate buffered saline solution (PBS), Leibowitz-15 (L-15), albumin-solution, diethylether, 2′,7′dichlorofluorescein,
Preadipocyte isolation and culture conditions
Atlantic salmon were reared in sea water (average temperature 12–13 °C) on a commercial diet to an average weight of 2–3 kg. Random fish were sampled and anaesthetized in metacain. After the anaesthesia, arch bows of the gills were cut. After bleeding for a couple of minutes, the fish were killed by a blow to the head and the abdomen was cut open to expose the visceral adipose depot. Visceral fat was carefully excised in order to avoid contamination with intestinal contents. Salmon
Morphology of preadipocytes at different stages during the differentiation process
After seeding, the isolated preadipocytes were small with a cytoplasm devoid of lipid droplets and morphologically very similar to fibroblasts. Electron microscopy revealed that preadipocytes at confluence (day 7) showed an extensive cytoplasm with nucleus located in a central region of the cell. The cells at this stage were relatively rich in mitochondria and were almost devoid of lipid droplets (Fig. 1a).
At confluence, the growth medium was replaced by a differentiation-inducing medium
Discussion
Atlantic salmon primary preadipocytes differentiate to mature adipocytes when a growth medium containing lipid mixture as the only factor inducing differentiation is added [11]. In this study, however, we show that the hormones insulin, dexamethasone, triiodothyronine and isobutylmethylxanthine also improve the differentiation capacity of Atlantic salmon preadipocytes towards mature adipocytes. Our results agree with results from human preadipocytes [31], [32], [33].
The transcription factor
Acknowledgments
We wish to thank Inger Ø. Kristiansen, Målfrid T. Bjerke (Akvaforsk), Anne-Lise Rishovd (Department of Pharmaceutical Biosciences, School of Pharmacy at University of Oslo) and Betty Irgens (NIFES) for skilful technical assistance. This work was carried out with support from the Norwegian Research Council, project number 158930.
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2023, AquacultureCitation Excerpt :In the study of blunt snout bream (M. amblycephala), dietary DHA addition led to decreased expression of srebp-1 and acc, which further inhibited lipogenesis (Cong-cong et al., 2020). Liu et al. (2014) pointed out that the expression level of hsl was significantly increased when grass carp (Ctenopharyngodon idellus) mature adipocytes were incubated with EPA and DHA for 6 h. Similarly, Wang et al. (2010) found that the expression of adipogenic genes decreased and the expression of lipolytic genes increased after incubating adipocytes with DHA for 24 h. Todorcević et al. (2008) demonstrated that EPA and DHA increased the β-oxidation in Atlantic salmon (S. salar), thereby reducing the content of triacylglycerol in adipocytes. In the study of Kjær et al. (2016), it was also proved that DHA can promote the β-oxidation in Atlantic salmon (S. salar).