Development and functions of adipose tissue in Atlantic salmon

Abstract

The trend in salmon aquaculture is to use feed with high lipid content. In current feeds, energy from lipids can comprise as much as 50% of the total energy, compared to 10% approximately 20 years ago. This change was undertaken in order to increase growth and reduce expensive and scarce marine proteins in the feed [1]. However, these lipid-rich diets lead to high lipid deposition in salmonid fishes both in the fillet and around the internal organs [2]. High levels of visceral adipose tissue result in production losses when the salmon is gutted at harvest [3]. The relationship between excess lipid deposition in viscera and health is not well known for fish, but the occurrence of high mortality in slaughter-sized salmonids due to poor heart health and stress has been observed by Tørud and Hillestad [4]. The overall aim of the work described in this thesis was to gain more insight into Atlantic salmon visceral adipose tissue development and functions as well as the effects of n-3 highly unsaturated fatty acids (HUFAs) on adipocytes by combining in vivo fish trials with an in vitro cell culture model. Article I: describes an in vitro method for studying the differentiation of preadipocytes isolated from Atlantic salmon visceral adipose tissue. Isolated preadipocytes differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. After one week in culture, preadipocytes reach confluence. At this stage, the differentiation process is triggered with hormones. Several adipogenic gene markers were measured in order to follow the differentiation process. The expression of the adipogenic gene markers; peroxisome proliferated activated receptor (PPAR) α, lipoprotein lipase, microsomal triglyceride transfer protein, fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during the maturation of adipocytes. In this article, we further describe a novel alternatively spliced form of PPAR γ (PPAR γ short). The expression of PPAR γ short increased during differentiation, while the expression of PPAR γ long was down regulated. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are known to lower the triacylglycerol (TAG) accumulation in human adipocytes both in vivo and in vitro. We show that this is also the situation in fish, both EPA and DHA significantly lower TAG accumulation and increase fatty acid (FA) -oxidation in salmon adipocytes compared to OA. Article II: describes how increasing dietary levels of n-3 HUFAs affects lipid storage and mitochondrial β-oxidation in Atlantic salmon white adipose tissue in vivo. Further, it investigates how EPA and DHA influence the susceptibility to oxidative stress. Increased dietary levels of n-3 HUFAs resulted in lower fat percentage in white adipose tissue; in agreement with the in vitro observations in Article I. Mitochondrial FA β-oxidation activity was higher in the fish oil group than it was in the rapeseed oil group. The relative levels of EPA and DHA in phospholipids from white adipose tissue and mitochondrial membranes increased with the increasing dietary levels of these HUFAs. Together with reduced cytochrome c oxidase activity and increased superoxide dismutase activity in the high HUFA groups, these data show an increased incidence of oxidative stress resulting in non-functional mitochondria with no detectable mitochondrial FA β-oxidation activity. The increased activity of caspase 3, in the high n-3 HUFA groups, further indicated some degree of apoptosis occurring in white adipose tissue of these groups. Decreased fat cell number due to apoptosis, may be one factor explaining the lower TAGs percentage found in the high HUFA groups. Article III: describes development-associated changes in gene expression during determination and terminal differentiation of Atlantic salmon adipose-derived stromo-vascular fraction (aSVF). The determination phase was characterised with cellular heterogeneity. After confluence, however, the cellular heterogeneity decreased as evidenced by the down-regulation of markers of osteo/chondrogenic, myogenic, immune and vasculature cell lineages. Genes involved in nucleotide metabolism and DNA replication, essential processes for cellular division, implied attenuation of proliferation after day 9, in agreement with the number of cells staining positive for proliferating cell nuclear antigen. The terminal differentiation phase was characterised by high lipid accumulation and decreased recruitment of new adipocytes. This was accompanied with increased expressions of several genes involved in lipid and glucose metabolism, including markers of the adipocyte lineage. The gene expression of glyceraldehyde 3-phosphate dehydrogenase and transaldolase, suggested interplay between glycolysis and pentose phosphate pathways in order to secure the production of the glycerol backbone for TAG synthesis. The coordinated-expression of several genes in different antioxidant producing pathways, including the glutathione-based system, pointed to the importance of maintaining a reduced intracellular environment in cells after confluence in order to be able to safely store large amounts of lipids. Signs of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occurred at the later stages of adipocyte differentiation, in parallel with increased lipid droplet formation and increased gene expression of the secretory proteins adipsin and visfatin. The UPR markers, Xbox binding protein 1 and activating transcription factor 6, were induced together with genes involved in ubiquitin-proteasome and lysosomal proteolysis. Notably, changes in expression of a panel of genes belonging to different immune pathways were observed throughout adipogenesis. Article IV: describes how HUFAs may influence oxidative stress responses in salmon adipocytes in vitro. Terminally differentiating adipocytes were cultivated on HUFA-rich media and treated with two agents that affected their oxidative status: buthionine sulfoximine depleted stores of the intracellular antioxidant glutathione and exacerbated oxidative stress, while α-tochopherol protected cells from oxidative stress. Lipid accumulation and the expression of adipogenic gene markers were lower in cells with high HUFA levels and no antioxidant supplemented than in cells added the antioxidant α-tocopherol. Depletion of glutathione with buthionine sulfoximine was associated with the highest activity of superoxide dismutase and the highest levels of reactive oxygen species (ROS) as measured by increased level of thiobarbituric acid reactive substances. α-tocopherol supplementation mediated a reduction in the oxidative stress response in the glutathione-depleted cells, independent of glutathione peroxidases and superoxide dismutase. α-tocopherol seem to have a strong pro-adipogenic effect, while oxidative stress induced by HUFAs and buthionine sulfoximine have anti-adipogenic effects. In addition, α-tocopherol had anti-apoptotic and anti-inflammatory effects and induced the expression of activating transcription factor 6, a marker of ER-stress. The high expression of transcription factor 6 in the α-tocopherol groups may be explained by the higher lipid level found in these groups, since high lipid level is known to induce ER stress.