Absorption, utilisation and storage of lipids in Atlantic salmon. A focus on the gastro-intestinal tract, hepatocytes, muscle cells and adipocytes / Opptak, utnyttelse og lagring av lipider i Atlantisk laks. Med fokus på mage-tarmkanal, hepatocytter, muskelceller og adipocytter
The overall aim of the work described in this thesis was to gain more insight into the absorption, utilisation and storage of lipids in Atlantic salmon (Salmo salar L.) , with focus on the main lipid metabolising tissues: the gastro-intestinal (GI) tract, the liver (hepatocytes), muscle (muscle cells) and adipose tissue (adipocytes).
Publication I describes lipid absorption in different segments of the GI-tract of Atlantic salmon. The levels of radioactivity in stomach, anterior, mid, and distal pyloric ceca (PC), mid intestine (MI), and distal intestine (DI) were measured after force-feeding of isotope-labelled free fatty acid (FFA) [14C]-10:0 and triacylglycerol (TAG) [9,10-3H(N)]-triolein (trioleic acid; [3H]-18:1n-9). Radioactivity from both fatty acid (FA) substrates was highest in tissue from PC 12 to 18 hours after force-feeding. The level of radioactivity from [14C]-10:0 was significantly higher in the anterior part of PC than it was in other parts of PC, whereas the [3H]-18:1n-9 substrate was distributed more evenly along the PC and the MI. However, when taking into consideration the total weight of the different segments of the intestine, the PC was the most important site for absorption of both substrates. Only negligible amounts of radioactivity were present in the DI. Morphometric analysis of the total lipid accumulation in tissue from the mid PC, MI and DI confirmed the findings from the isotope study.
Publication II describes accumulation and secretion of lipids in cultivated hepatocytes isolated from Atlantic salmon fed diets supplemented with either 100% soybean oil (SO) or 100% fish oil (FO). Hepatocyte cultures were incubated with [3H]-glycerol and an FA-free medium or a medium supplemented with 0.75 mM of either 18:1n-9, 20:5n-3, 22:6n-3 or the non-β-oxidizable sulphur-substitued FA analogue tetradecylthioacetic acid (TTA). The total secretion of [3H]-glycerolipids was significantly lower in fish fed the FO diet than it was in fish fed the SO diet. Addition of 18:1n-9 to hepatocyte cultures markedly stimulated, whereas 20:5n-3 and TTA reduced, both the total secretion of [3H]-glycerolipids and the secretion of TAG. 22:6n-3 increased the total secretion of [3H]-glycerolipids, but had no effect on TAG secretion. Morphometric measurements showed that significantly more lipid was accumulated in cells incubated with 18:1n-9, 20:5n-3 or 22:6n-3, than in cells incubted with TTA or an FA-free medium. The total area occupied by mitochondria was highest in hepatocytes incubated with 20:5n-3.
Publication III describes how salmon mucle cells differentiated in vitro metabolise FAs with different chain-lengths. Almost all (98%) [14C]-8:0 substrate was oxidised to acid-soluble products (ASPs), while only approximately 50% of the [14C]-18:1n-9 and [14C]-20:5n-3 substrated was oxidised. The main ASPs for all three substrates were acetate and a combined fraction of the tricarboxcylic acid (TCA)-cycle products oxaloacetate and malate. There was nearly twice as much radioactivity from [14C]-20:5n-3 substrate in cell-associated lipids than there was from [14C]-18:1n-9, whereas there was approximately three times more [14C]-18:1n-9 than [14C]-20:5n-3 in the form of FFAs in the media, indicating that [14C]-20:5n-3 was taken up more rapidly into the muscle cells than [14C]-18:1n-9. More [14C]-20:5n-3 than [14C]-18:1n-9 was recovered as FFAs, TAG and mono- and diacylglycerols (MDG) in cell-associated lipids, whereas almost the same amounts of the two FAs were found in the phospholipid (PL) fraction of the muscle cells. Significant amounts of radioactivity from both [14C]-18:1n-9 and [14C]-20:5n-3 were secreted as esterified lipids into the culture media. Differentiated muscle cells expressed the peroxisome proliferator-activated receptor α (PPARα), PPARβ and apolipoprotein (apo)A-I.
Publication IV describes an in vitro method for studying the proliferation and differentiation of preadipocytes isolated from Atlantic salmon visceral adipose tissue. The cells started to proliferate within 48 hours after seeding, and continued to proliferate throughout the culture period of two weeks. After one week in culture, the preadipocytes showed a fibroblast-like morphology and reached confluence. At this stage, the differentiation was triggered by the addition of a lipid mixture to the cell cultures. The cells gradually accumulated more lipid during the subsequent days. Glycerol-3-phosphate dehydrogenase (GPDH) activity was used as a marker of adipocyte differentiation. Immunocytochemical staining revealed that differentiated adipocytes expressed the three regulatory proteins associated with adipocyte differentiation: PPARγ, CCAAT/enhancer binding protein α (C/EBPα) and leptin. The results show that salmon adipose tissue contains preadipocytes that are similar to preadipocytes in mammals. Exogenous lipids induced the differentiation of salmon preadipocytes to mature adipocytes in vitro.
Pr. desember 2004: Anne Vegusdal, Akvaforsk, Postboks 5010, 1432 Ås
