Atlantic salmon (Salmo salar) muscle precursor cells differentiate into osteoblasts in vitro: polyunsaturated fatty acids and hyperthermia influence gene expression and differentiation.

The formation and mineralisation of bone are two critical processes in fast-growing Atlantic salmon (Salmo salar). The mechanisms of these processes, however, have not been described in detail. Thus, in vitro systems that allow the study of factors that influence bone formation in farmed Atlantic salmon are highly warranted. We describe here a method by which unspecialized primary cells from salmon white muscle can differentiate to osteoblasts in vitro. We have subsequently used the differentiated cells as a model system to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions, namely polyunsaturated fatty acids, PUFAs, and temperature. Muscle precursor cells changed their morphology from triangular or spindle-shaped cells to polygonal or cubical cells after 3 weeks in osteogenic medium. In addition, gene expression studies showed that marker genes for osteoblastogenesis; alp, col1a1, osteocalcin, bmp2 and bmp4 increased after 3 weeks of incubation in osteogenic media showing that these cells have differentiated to osteoblasts at this stage. Adding CLA or DHA to the osteoblast media resulted in a reduced PGE(2) production and increased expression of osteocalcin. Further, temperature studies showed that differentiating osteoblasts are highly sensitive to increased incubation temperature at early stages of differentiation. Our studies show that unspecialized precursor cells isolated from salmon muscle tissue can be caused to differentiate to osteoblasts in vitro. Furthermore, this model system appears to be suitable for the study of osteoblast biology in vitro.

Socializing makes thick-skinned individuals: on the density of epidermal alarm substance cells in cyprinid fish, the crucian carp (Carassius carassius).

In cyprinid fish, density of epidermal club cells (i.e. alarm substance cells) has been found to vary between lakes with different predator fauna. Because predators can be labelled with chemical cues from prey, we questioned if club cell density could be controlled indirectly by predators releasing prey cues. In particular, we suspected a possible feedback mechanism between chemical alarm signals and their cellular source. We raised crucian carp singly and in groups of four. For both rearing types, fish were exposed to skin extracts of either conspecifics or brown trout (without club cells), and provided either low or high food rations. Independent of rearing type, condition factor and club cell density increased with food ration size, but no change was found in club cell density following exposure to conspecific alarm signals. However, the density of club cells was found significantly higher for fish raised in groups than for fish raised alone. We conclude that an increased condition factor results in more club cells, but crucian carp may also possess an awareness of conspecific presence, given by higher club cell densities when raised in groups. This increase in club cell density may be induced by unknown chemical factors released by conspecifics.

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; effects of n-3 and n-9 fatty acids.

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.

Effects of dietary thia fatty acids on lipid composition, morphology and macrophage function of Atlantic salmon (Salmo salar L.) kidney.

High lipid levels are being used in modern salmonid diets to promote rapid growth; however there is a limiting supply of the traditional fish oils as the fish farming industry expands. One way to utilize the lipid sources better, could be to find ways to stimulate fatty acid (FA) oxidation so that Atlantic salmon use more energy for muscle growth and less for storage in perivisceral adipose tissue. We have previously shown that dietary inclusion of the thia FA tetradecylthioacetic acid (TTA) promoted hepatic beta-oxidation and reduced total body lipid levels. However, dietary TTA also had some negative effects, leading to accumulation of sulfone and sulfoxide metabolites of TTA in the kidney and increasing mortality rates, particularly at low water temperatures. Therefore we also wish to investigate the effects of TTA on kidney function at high and low temperatures, including some immune system parameters. The production of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) immunoreactive material from exogenously added arachidonic acid in isolated head kidney macrophages was affected by both diet and temperature. The phagocytic activity in these cells was reduced by DTA in the 12 degrees C group and there was significantly higher protein degradation in head kidney macrophages at 12 degrees C compared to 5 degrees C in all dietary groups. Interestingly, the incorporation of thia FAs in the kidney was higher at 5 degrees C (0.3% TTA and 0.6% DTA) than at 12 degrees C (0.1% TTA and 0.5% DTA). Additionally, there were lower levels of saturated FAs, while higher levels of polyunsaturated FAs (PUFAs) in the kidney of TTA fed fish at 5 degrees C. We also observed temperature-independent tubular dilatation and a reduction in the density of melanomacrophages of the kidney in salmon fed TTA. Nevertheless, the mRNA expression of some immune-relevant genes in head kidney tissue was not affected by TTA-inclusion in salmon diets. In conclusion, it is clear that 0.6% TTA-inclusion in the feed leads to changes in the kidney function particularly at low water temperatures.

Effects of 3-thia fatty acids on expression of some lipid related genes in Atlantic salmon (Salmo salar L.).

In this study, the effects of in vivo administration of 3-thia fatty acids (FAs) on lipid metabolism in muscle and liver of Atlantic salmon were investigated. Prior to analysis, the fish were kept in tanks supplied with 5 degrees C seawater for 20 weeks. The fish were fed fish meal and fish oil (FO)-based diets supplemented with either nothing (FO), or 0.3% and 0.6% of the 3-thia FAs dodecylthioacetic acid (DTA) and tetradecylthioacetic acid (TTA) respectively. The fish grew from an initial weight of 110 g to 220 g in the FO group and to approximately 160 g in the 3-thia FA groups. There was a significant higher mortality (66%) in fish fed 0.6% TTA than in fish fed the 0.3% DTA (15%) and FO diets (15%). None of the 3-thia FA diets affected the lipid content of the salmon muscle. The liver index, however, was significantly higher and the total liver fat content lower in the TTA group than in the FO group. Both DTA and TTA were incorporated into the lipid fraction of muscle and liver (0.4% to 0.9%). There were no major differences in the total FA composition of liver and muscle between the dietary groups; except for a small increase of n-3 polyunsaturated FAs (PUFAs) in liver of the DTA group. The mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, apolipoprotein AI (ApoAI), apolipoprotein CII (ApoCII) and low-density lipoprotein receptor (LDL-R) was down-regulated in liver of the salmon fed 0.3% DTA. PPARalpha and ApoAI transcripts were also reduced in liver of salmon fed 0.6% TTA. Additionally, the hepatic lipoprotein lipase (LPL) mRNA level was 3.8 fold increased in TTA fish relative to the FO group. In muscle there were no significant changes in gene expression pattern of any of the genes investigated. This is the first report on the effects of 3-thia FAs on gene expression in Atlantic salmon.

An in vitro method for studying the proliferation and differentiation of Atlantic salmon preadipocytes.

The aim of the present study was to develop a cell culture system for studying the proliferation and differentiation of preadipocytes isolated from Atlantic salmon adipose tissue. The expression of proliferating cell nuclear antigen (PCNA) was used as a marker for cell proliferation. The cells started to proliferate within 48 h after seeding and continued to proliferate throughout the culture period of 2 wk. Undifferentiated preadipocytes showed a fibroblast-like morphology with a homogeneous cytoplasm devoid of lipid droplets. At confluence, an exogenous lipid mixture was added to the cell cultures. The preadipocytes became larger and rounder during the subsequent days, and the cytoplasm gradually filled with lipid-rich droplets. These droplets were revealed by oil red O staining. Immunocytochemical staining showed that differentiated adipocytes expressed detectable levels of the three regulatory proteins associated with adipocyte differentiation: peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), and leptin. The cells also showed activity of glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), a biochemical marker of adipocyte differentiation. The morphological and biochemical data presented here show that fish preadipocytes have properties that are similar to those of preadipocytes in mammals. We conclude therefore that salmon adipose tissue contains a sizable population of preadipocytes. Exogenous lipids promote the activation of adipose-related genes and induce the differentiation of fish preadipocytes in vitro.

N-3 HUFAs affect fat deposition, susceptibility to oxidative stress, and apoptosis in Atlantic salmon visceral adipose tissue.

We have investigated how n-3 highly unsaturated fatty acids (HUFAs) in the diet affect fatty acid (FA) utilization, fat storage and oxidative stress (OS) in Atlantic salmon (Salmo salar) white adipose tissue (WAT). Four groups of Atlantic salmon were fed for 21 weeks on one of the four diets supplemented with 23% (of dry matter) lipid. Docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) levels increased from 10% of total FAs in the rapeseed oil (RO) diet, to 20% in the fish oil (FO) diet, and to 50% and 55% in the DHA-enriched and EPA-enriched diets, respectively. Increased dietary levels of n-3 HUFAs resulted in lower fat percentage in WAT. Furthermore, mitochondrial FA beta-oxidation activity was higher in the FO group than it was in the RO group. The relative levels of DHA and EPA in phospholipids (PLs) from WAT and mitochondrial membranes increased with the increasing dietary levels of these HUFAs. In general, the mitochondrial membrane PLs were characterised by lower relative levels of n-3 HUFAs and higher relative levels of linoleic acid (LA; 18:2 n-6) than WAT membrane PLs. The predominance of LA relative to n-3 HUFAs in mitochondrial membrane PLs may help to protect these PLs from peroxidation. Cytochrome c oxidase measurements revealed higher incidence of disrupted mitochondrial membranes in the DHA and EPA dietary groups than in the FO and RO dietary groups. This disruption further affected the mitochondrial function, resulting in a marked reduction in FA beta-oxidation capacities. The reduction in mitochondrial function and the increase in the activity of superoxide dismutase (SOD) in the DHA and EPA groups showed that high dietary dose of DHA and EPA resulted in oxidative stress (OS). The increased activity of caspase 3 in the high n-3 HUFA groups suggested the induction of apoptosis and increased incidence of cell death in WAT, which may be one of the factors explaining the lower fat percentage found in these groups.

Molecular cloning of the Atlantic salmon activin receptor IIB cDNA - Localization of the receptor and myostatin in vivo and in vitro in muscle cells.

In mammals, the activin receptor type IIB (ActRIIB) binds with high affinity several members of the transforming growth factor-beta (TGF-beta) superfamily, including the negative muscle regulator myostatin (MSTN). In this study, an actRIIB cDNA of 1443 bp was isolated by reverse transcription (RT)-PCR from the liver of Atlantic salmon (Salmo salar) encoding almost the complete receptor. The deduced salmon ActRIIB of 481 amino acids (aa) contained the conserved catalytic domain of serine/threonine protein kinases, and showed the highest sequence identity (83-87%) to the zebrafish, chicken and goldfish ActRIIB. Salmon actRIIB mRNA was identified by RT-PCR in all the examined tissues of juvenile fish that was confirmed by in situ hybridization. In comparison, the salmon MSTN signal was less widespread, and co-expression of the receptor and this putative ligand was only demonstrated in skeletal muscle. Consistently, both ActRIIB and MSTN were immunocytologically identified in salmon myoblasts and differentiated myotubes in culture.