Marte Avranden Kjær defended her PhD thesis on February 20th 2009

Ane Gro Siri Skjelfjord

In the work described in this thesis, several aspects of lipid metabolism were investigated in two commercially important aquacultured Norwegian fish species, Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua)

In humans, dietary highly unsaturated fatty acids (HUFAs) are known to exert a hypolipidemic effect, which may have implications for problems related to coronary heart diseases. Whether this is the case for fish is largely unknown. The goal of the first paper was therefore to investigate the effects of different HUFA levels on the accumulation and secretion of lipids by salmon hepatocytes. Both high n-3 eicosapentaenoic acid (EPA) and high n-3 docosahexaenoic acid (DHA) dietary groups had a significantly lower lipid secretion from the hepatocytes than the low n-3 HUFA (rapeseed oil (RO)) group. Our results further show that EPA and DHA possess different hypolipidemic properties. Both EPA and DHA inhibit triacylglycerol (TAG) synthesis and secretion, but only EPA induces mitochondrial proliferation and reduces intracellular lipid level. Our data indicate that the underlying mechanisms for the hypolipidemic effect observed with high n-3 HUFA diets include inhibition of TAG synthesis. This is seen by a higher mono- and diacylglycerol (MDG) to TAG ratio in the high n-3 HUFA groups than in the groups fed with lower n-3 HUFA levels. The phospholipid (PL) to TAG ratio was higher in the high n-3 EPA group, indicating that an increase in PL synthesis may also be involved. An increase in β-oxidation is another possible factor, indicated by the increased mitochondrial proliferation in the high n-3 EPA group. It is also probable that factors involved in the assembly of the very low density lipoprotein (VLDL) particles are inhibited, and therefore may be important factors regulating the hypolipidemic response.

The HUFAs, EPA and DHA, are vital constituents of cell membranes, being essential for membrane structure and function. These fatty acids (FAs) are, however, highly susceptible to attack by oxygen and organic radicals. Little is known about n-3 HUFAs' potential unfavourable effects. The major aims of the second paper were, hence, to investigate how increasing dietary levels of n-3 HUFAs affect mitochondrial function and expression of lipid regulatory genes in Atlantic salmon. Further, to investigate how high levels of EPA and DHA may influence the susceptibility to oxidative stress. The expressions of Δ5-desaturase and Δ6-desaturase genes decreased with increasing dietary levels of EPA and DHA, suggesting that high dietary HUFA levels decrease the capacities to elongate and desaturate essential FAs (EFAs). The group fed the highest level of DHA had higher expressions of peroxisome proliferator-activated receptor (PPAR)-β and the FA β-oxidation genes acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase (CPT)-II, compared to the low n-3 groups, indicating that n-3 HUFAs increase the capacity for β-oxidation. A decreased percentage of major PLs in the mitochondrial and microsomal membranes of the liver, including cardiolipin,.were apparent in the group fed the highest level of n-3 HUFAs. This suggests an increase in the permeability of the membranes, which can cause an inactivation of membrane-bound enzymes. The high n-3 HUFA groups also showed reduced activity of mitochondrial βoxidation. Together with reduced mitochondrial cytochrome c oxidase activity and increased activities of superoxide dismutase (SOD) in the high n-3 HUFA groups, these data show an increased incidence of oxidative stress resulting in non-functional mitochondria. Increased caspase-3 activity in the liver of fish fed the two high n-3 HUFA diets further indicates a subsequent induction of apoptosis.

Whole muscle and muscle mitochondrial fraction were also analyzed for possible effects of lipid peroxidation when fed different levels of dietary n-3 HUFAs in paper three. Salmon whole muscle contained a significantly different PL composition than the mitochondrial fraction, being richer in n-3 HUFAs. The mitochondria had a higher content of C18 FAs compared to total muscle. That the mitochondria were less polyunsaturated than the whole tissue may be a protection mechanism against oxidative damage. Nevertheless, feeding high n-3 HUFA diets led to increased incorporation of n-3 HUFAs in mitochondrial lipids. The PLs, cardiolipin and sphingomyelin, are important components in mitochondrial membranes. These PLs are particularly sensitive to lipid peroxidation. There were lower relative levels of cardiolipin and sphingomyelin in both the high n-3 EPA and high n-3 DHA dietary groups, and reduced cytochrome c oxidase. activity, compared to the intermediate n-3 HUFA group, indicating a more damaged mitochondrial membrane when fed high HUFA levels. SOD assay, thiobarbituric acid reactive substances (TBARS) level, caspase-3 activity and gene expression of casp3, casp7 and bax, also point to a higher degree of oxidative stress and induced apoptosis in the high n-3 HUFA groups, especially in the high n-3 DHA group. Electron microscopy revealed myofibre-myofibre detachments in all groups, however, there was observed a tendency to higher degree of muscle structure deterioration in both high n-3 EPA and high n-3 DHA dietary groups.

Atlantic cod deposits dietary lipid mainly in the liver. High-energy diets contain high levels of lipids which is one of the factors causing development of large fatty livers. Underlying mechanisms, however, for lipid transport and liver lipid deposition in cod are scarcely studied. In the fourth paper we therefore investigated how high (30.5 % of dry matter) and low (11.4 % of dry matter) lipid levels in the diet, in addition to fasting affected lipid transport and mobilisation in Atlantic cod. The lipid level in the diet did not significantly affect fish growth. The hepatosomatic index (HSI), however, increased by a factor of 1.5, and the liver lipid index by a factor of 1.7, when fish were fed the high-fat diet. The increase in HSI was mainly due to a larger average size of liver cells. The cell size increased in the high-fat fed cod, owing to higher levels of lipid deposition than in the livers of fish fed the low-fat diet. The low-fat fed cod had more and smaller liver cells with less lipid. Vesicles with the size of chylomicrons were found in intestinal enterocytes of cod fed the high-fat diet. The high lipid levels found in cod livers have led to speculation that lipoproteins and transport out of the liver do not exist in this species. In fish from the high-fat dietary group, however, vesicles with the size of VLDLs were found in liver cells. When the fish had been fasted, these particles were not present in the tissues. High density lipoprotein (HDL) was the predominant lipoprotein class in cod serum, with low density lipoprotein (LDL) as second most common class. Some VLDLs and chylomicrons were also present in the serum. The apolipoprotein apoB was the major protein in chylomicrons, VLDLs and LDLs, while apoA predominated in HDLs, showing a similar pattern as found in other species. There was a tendency to reduced liver lipid index in fasted cod, indicating lipid mobilisation from the liver. Mobilised lipids were mainly transported esterified in TAG, primarily in HDLs. An increased degree of blood capillary vascularisation (angiogenesis) was seen after fasting, this is likely a mechanism to improve the supply of nutrients and FAs from the liver via serum to other organs and tissues.

Updated: 11.06.09
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