Fish oil and krill oil supplementations differentially regulate lipid catabolic and synthetic pathways in mice
© Tillander et al.; licensee BioMed Central Ltd. 2014
Received: 17 December 2013
Accepted: 11 April 2014
Published: 27 April 2014
Marine derived oils are rich in long-chain polyunsaturated omega-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have long been associated with health promoting effects such as reduced plasma lipid levels and anti-inflammatory effects. Krill oil (KO) is a novel marine oil on the market and is also rich in EPA and DHA, but the fatty acids are incorporated mainly into phospholipids (PLs) rather than triacylglycerols (TAG). This study compares the effects of fish oil (FO) and KO on gene regulation that influences plasma and liver lipids in a high fat diet mouse model.
Male C57BL/6J mice were fed either a high-fat diet (HF) containing 24% (wt/wt) fat (21.3% lard and 2.3% soy oil), or the HF diet supplemented with FO (15.7% lard, 2.3% soy oil and 5.8% FO) or KO (15.6% lard, 2.3% soy oil and 5.7% KO) for 6 weeks. Total levels of cholesterol, TAG, PLs, and fatty acid composition were measured in plasma and liver. Gene regulation was investigated using quantitative PCR in liver and intestinal epithelium.
Plasma cholesterol (esterified and unesterified), TAG and PLs were significantly decreased with FO. Analysis of the plasma lipoprotein particles indicated that the lipid lowering effect by FO is at least in part due to decreased very low density lipoprotein (VLDL) content in plasma with subsequent liver lipid accumulation. KO lowered plasma non-esterified fatty acids (NEFA) with a minor effect on fatty acid accumulation in the liver. In spite of a lower omega-3 fatty acid content in the KO supplemented diet, plasma and liver PLs omega-3 levels were similar in the two groups, indicating a higher bioavailability of omega-3 fatty acids from KO. KO more efficiently decreased arachidonic acid and its elongation/desaturation products in plasma and liver. FO mainly increased the expression of several genes involved in fatty acid metabolism, while KO specifically decreased the expression of genes involved in the early steps of isoprenoid/cholesterol and lipid synthesis.
The data show that both FO and KO promote lowering of plasma lipids and regulate lipid homeostasis, but with different efficiency and partially via different mechanisms.
KeywordsOmega-3 fatty acids Plasma lipids High-fat diet Gene regulation Krill oil
Omega-3 polyunsaturated fatty acids (PUFAs) such as eiocosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) are well known bioactive dietary compounds that are found particularly in marine-derived food sources such as e.g. fatty fish, seaweed, shellfish, microalgae and krill. Since 1970, regular consumption of fish (preferably fatty fish) has been stated to have several positive effects on cardiovascular health[1–5]. The American Heart Association dietary guidelines for healthy individuals proposes consumption of at least two servings of fish per week which should yield an intake of approximately 400-500 mg EPA and DHA, and they recommend an even higher intake of omega-3 fatty acids to patients with documented coronary heart disease[6, 7]. However, in large parts of the world the consumption of fish is considered to be inadequate and fish oil (FO) from anchovy, sprat, herring and salmon as a source of DHA and EPA has become widely used as a dietary supplement. The reported health benefits of FO have however led to an increased demand that may endanger natural resources of fish, and krill oil (KO) has recently emerged on the market as an alternative source of omega-3 PUFAs. Most FO on the market today have their omega-3 PUFAs incorporated into (triacylglycerols) TAG or in ethyl esters. However in KO are the majority of these omega-3 PUFAs esterified in phospholipids (PLs)[8–11]. KO has been stated to be a safe source of EPA and DHA that like other marine-based oils is able to efficiently raise the plasma levels of EPA and DHA[12–16]. However, the structural differences in the PUFA-rich lipid molecules may affect the distribution in cellular lipid fractions and tissue uptake and thereby promote different regulatory effects on lipid homeostasis. KO also contains astaxanthin, which due to its anti-oxidative effect, might enhance the stability of the omega-3 PUFAs in the oil and thereby preserve them from lipid oxidation.
Intake of EPA and DHA has been shown to improve cardiovascular health by regulating lipid and glucose metabolism by acting as ligands for several nuclear transcription factors (e.g. peroxisome proliferator-activated receptors (PPARs) -α, -β/δ, and -γ and sterol regulatory element-binding protein 1 (SREBP-1))[18–20]. EPA and DHA also have anti-inflammatory effects due to their conversion to less inflammatory signaling molecules at the expense of production of pro-inflammatory molecules from arachidonic acid (for reviews see[21, 22]).
The liver is a central metabolic organ that regulates both circulating blood lipids and glucose levels by catabolism as well as synthesis of lipids and carbohydrates. Marine-derived omega-3 PUFAs have previously been shown to modulate the gene transcription profile in liver to enhance lipid degradation and decrease VLDL secretion (for review, see). Recently, also KO was shown to modulate the transcriptional profile in mouse and rat liver and to affect plasma and liver lipids in mice[24–28].
Gene expression is regulated in the intestine in response to different metabolic conditions in order to cope with changes in nutrient load and content, to signal satiety and other stimuli to the rest of the body and to keep the intestinal defense barrier against pathogens intact. In addition, the intestine contributes to the plasma lipoprotein profile by absorbing lipids for chylomicron synthesis and being responsible for a significant part of the HDL production in the body[29, 30]. To the best of our knowledge, so far no study has addressed the effects of KO on regulation of gene expression in the small intestine, although KO was recently shown to attenuate inflammation and oxidative stress in colon in an experimental rat model of ulcerative colitis.
The aim of this study was to compare the effects of two of the major sources of omega-3 supplements on the market today, FO and KO, when supplemented to a Western-like high-fat diet.
Equal amount of FO and KO (6% by weight) were added to a high fat diet, and the effects on plasma and liver lipids and gene regulation in liver and intestine were measured. In spite of the markedly lower omega-3 PUFA content in KO, both dietary supplementations raised the content of omega-3 PUFAs in plasma as well as in liver phospholipids to a similar extent. However, FO more efficiently lowered plasma lipids and this decrease was associated with accumulation of lipids in liver. In contrast, KO was less efficient in lowering plasma lipids with less, if any, sign of TAG accumulation in the liver. These different effects by FO and KO can at least in part be ascribed to differential regulation of gene expression in liver and intestine and different effects on VLDL secretion.
Animals and diets
Nine to ten week old male C57BL/6J mice were fed either a high-fat diet (HF) containing 24% (wt/wt) fat (21.3% lard and 2.3% soy oil, n = 9), or the HF diet supplemented with FO (EPAX 6000 TG®, a generous gift of Epax A/S, Ålesund, Norway) (15.7% lard, 2.3% soy oil and 5.8% FO, n = 6) or the HF diet supplemented with KO (Superba™, a generous gift of Aker BioMarine, Oslo, Norway) (15.7% lard, 2.3% soy oil and 5.7% KO, n = 6) and water ad libitum for 6 weeks. Diets were packaged in airtight bags and freeze stored until use to prevent lipid oxidation. Mice were housed in groups of three per cage at a constant temperature of 22 ± 2°C and a light/dark cycle of 12/12 h. Body weights of the animals were measured approximately every seventh day and food intake was measured three times in the beginning of the 6-week study to optimize the food supply. Animals were fasted overnight, anesthetized with 2% isoflurane (Schering-Plough, Kent, UK) and blood was collected by heart puncture. The blood was centrifuged and EDTA-plasma was frozen until further analysis. Livers were collected and the intestines were removed, rinsed with ice-cold phosphate buffered saline, cut into four segments of equal length that were further cut open and the epithelial cell layers were scraped off to separate the epithelial cells from the smooth muscle. All tissue samples were immediately frozen in liquid nitrogen and stored at -80°C until further analysis. The animal experiments were carried out with ethical permission obtained from the Norwegian State Board for Biological Experiments and followed the Norwegian Research Councils ethical guidelines.
RNA isolation and cDNA synthesis
Total RNA from the intestinal epithelium and liver tissues was purified using the MagMax total RNA isolation system (Applied Biosystems, Carlsbad, CA, USA) after tissue homogenization. The quantity of the RNA was measured spectrophotometrically using a NanoDrop 1000 (NanoDrop Products, Wilmington, DE, USA) and the quality of the RNA was analyzed using the Experion Automated Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA). The quality limit for further use of RNA was set to a R/Q value of ≥7 (out of 10). cDNA was synthesized with 500 ng RNA per reaction, using High Capacity RNA-to cDNA Mastermix (Applied Biosystems).
Two types of TaqMan® Low Density Arrays in 96-well formats (format 96b, Applied Biosystems) were custom made to investigate the expression of genes related to peroxisomal and mitochondrial metabolic pathways, respectively (see Additional file1 for gene lists). The plates were run at the Bioinformatics and Expression Analysis core facility (BEA) at Karolinska Institutet and the run data were analyzed by RQ Manager (Applied Biosystems). Gene expression was calculated using the 2^ -ΔΔCt method according to Livak et al., using 18S as reference gene and one individual sample in the high fat group as a calibrator (n = 4 per group). Some additional genes of interest (not being on the TLDA-plates) were analyzed in individual samples from liver and intestine (HF n = 6, FO n = 5, KO n = 6) using TaqMan or SYBR Green gene expression assays (for primers and additional TaqMan expression assays, see Additional file1). SYBR Green primers were used at concentrations ranging from 100 to 200 nM and run with the Power SYBR Green Master Mix (Applied Biosystems). Again, gene expression data were calculated using the 2^ -ΔΔCt method (due to known efficiency of the primers used in the SYBR Green assay), however, the average Ct value of three different reference genes (18S, Hprt and Ppia) was used as control values and one individual sample in the high fat group as a calibrator.
Liver lipids were extracted according to Bligh and Dyer, solvents were evaporated under nitrogen and the samples were re-dissolved in isopropanol before analysis. Lipids from liver extracts or plasma were then measured enzymatically on a Hitachi 917 system (Roche Diagnostics, Mannheim, Germany) using kits for analyzing total TAG (GPO-PAP kit, Roche Diagnostics), cholesterol (CHOD-PAP kit, Roche Diagnostics), total PLs (bioMérieux SA, Marcy l'Etoile, France) and NEFA (FS kit, DiaSyS, Holzheim, Germany). Aliquots of extracted liver lipids were separated by thin layer chromatography using silica gel plates (Merck, Darmstadt, Germany) and hexane:diethylether (1:1) as the liquid phase. The absolute levels of fatty acids of the diets, plasma and the TAG and PL fractions from livers were analyzed using gas chromatography as described previously by Grimstad et al.. Lipoproteins were analyzed by size exclusion chromatography of plasma samples from individual mice (five mice in each group) according to Parini et al..
Hepatic enzyme activities
The liver tissue samples were homogenized and post-nuclear fractions were prepared as previously described. Carnitine palmitoyltransferase 1 (CPT-I) activity was measured in the absence and presence of malonyl-CoA (15 μM) essentially as described by Bremer. Peroxisomal acyl-CoA oxidase (ACOX) activity was determined by the coupled assay described by Small et al. and fatty acid synthase (FAS) activity was measured as described by Skorve et al..
Since normal distribution could not be assumed for the number of animal used in this study, the Kruskal-Wallis test was used for analysis of differences among the groups. If significance was obtained (p < 0.05) with the Kruskal-Wallis test, Dunn’s multiple comparison test was performed on all combinations, i.e. FO vs. KO, FO vs. high fat and KO vs. high fat. Significance indicated in tables and figures is shown for the Kruskal-Wallis test (as p-values in tables or as “K-W” in figures), and if significance was obtained in the Kruskal-Wallis test, also significance obtained by the post hoc test between the individual groups is indicated. A trend of difference between groups was set to 0.1 > p > 0.05 for the Kruskal-Wallis test. All values are presented as median and range. The statistics were calculated using GraphPad Prism 5.0d.
Fat source (% in diet)
Soy oil 2.3%
Soy oil 2.3%
Soy oil 2.3%
Fish oil 5.8%
Krill oil 5.7%
Fatty acids (% of total fatty acids in diet)
Total ω-6 PUFA
Total ω-3 PUFA
Body weight gain and liver weight
Final body and liver weights
Final body weight (g)
Liver weight (g)
Liver weight/body weight (%)
Plasma lipids and fatty acid composition
2.05 (2.00–2.46) a**
1.50 (1.36–1.80) a*
0.65 (0.50–0.66) a**
0.49 (0.26–0.68) a**
2.42 (2.17–2.52) a**
Nonesterified fatty acids
0.05 (0.00–0.18) b*
Cholesterol and TAG content of the lipoprotein fractions in plasma
0.04 (0.04–0.06) a*, c*
1.92 (1.71–2.21) a*
0.20 (0.11–0.30) a*
Total fatty acid composition of plasma lipids
μg FA/ml plasma
μg FA/ml plasma
μg FA/ml plasma
18.9 (14.6–24.5)a**, c*
0.5 (0.4–0.6)a*, c**
ω– 3 PUFA
ω- 3/ω- 6 PUFA
Liver lipids and fatty acid composition
6.9 (6.0–10.4)a *
7.0 (5.9–9.7)b *
21.7 (21.5–23.5)a **
Fatty acid composition of the PL fraction in liver
μg FA/g liver
μg FA/g liver
μg FA/g liver
1935 (1677–2052)a*, c**
Fatty acid composition of the TAG fraction in liver
μg FA/g liver
μg FA/g liver
μg FA/g liver
Quantitative fatty acid analysis confirmed the increased fatty acid content in liver PLs by FO and further revealed a significantly increased fatty acid content in the PL fraction also by KO. This was due to increases in SFA, MUFA and omega-3 PUFAs in spite of decreased omega-6 fatty acid species, especially C18:2 by FO and C20:4 by KO and FO compared to HF (Table 7). The increased amount of SFA (25-30%) was mainly due to increases in C16:0 and C18:0. Interestingly, the EPA, DHA and DPA contents were quite similar in the FO and KO groups in spite of the lower omega-3 content in the KO diet. For liver PL fatty acid composition given in wt %, see Additional file5.
Effect of marine oils on gene expression in the liver
Liver enzyme activities
nmol/mg/min - 15μM Malonyl-CoA
nmol/mg/min + 15μM Malonyl-CoA
Interestingly, KO decreased the expression of the mitochondrial-associated Acacb (acetyl-CoA carboxylase 2), which produces malonyl-CoA, compared to FO (Figure 2, “Mitochondrial pathways”). The decreased Acacb expression indicates a positive effect by KO on mitochondrial β-oxidation rate by reduced production of the CPT1-inhibitor malonyl-CoA.
Many of the genes that were upregulated by FO supplementation are well known targets for PPARα and involved in fatty acid degradation. Cytosolic acyl-CoA thioesterase 1 (Acot1) is one of the most strongly PPARα-regulated genes and a previously characterized target for PPARα. Acot1 expression was upregulated in the FO group and also Hmgcs2 (mitochondrial rate limiting enzyme in ketone body formation) was upregulated in the FO group but not appreciably by KO (Figure 2, see “Additional metabolic pathways” and “Mitochondrial pathways”), suggesting a more potent PPARα-activation by FO.
The expression of the first enzymes of the cholesterol/isoprenoid synthesis pathway (Mvd, Mvk, Pmvk, Fdps and Idi1) did differ in the total study set, but the post hoc test only confirmed significant downregulation of Mvd, Mvk and Hmgcr by KO compared to HF (Figure 2, “Mevalonate pathway” and, “Additional metabolic pathways”), while no, or less pronounced decreases were found with FO supplementation. Pcsk9, a well-known regulator of degradation of the LDL receptor, was significantly downregulated by both KO and FO. No changes were found in the expression of lipoproteins ApoB or ApoAII or the TAG-synthesizing enzyme Dgat2 by FO or KO. Fatty acid synthase (Fas) expression was significantly downregulated by KO, in line with the (non-significantly) lower activity of the enzyme (c.f. Table 9). The increased FAS-activity in the FO group was however not supported by the tendency to decreased Fas mRNA expression (see Table 9 and Figure 2, “Additional metabolic pathways”).
No major changes in the expression of genes coding for proteins in the citric acid cycle (Figure 2, “Mitochondrial pathways”), or oxidative phosphorylation (data not shown) were observed in either group compared to the HF group. However, Pdk4, a known PPARα responding gene, was strongly upregulated in the FO group but not in the KO group (Figure 2, “Mitochondrial pathways”). Soluble phosphoenolpyruvate carboxykinase 1 (Pck1) expression was increased in the FO group compared to the KO group, but not when compared to HF (Figure 2, “Additional metabolic pathways). The same pattern was seen for the expression of the mitochondrial gluconeogenic gene Pck2, as well as the citric acid synthase coding gene Cs (Figure 2, “Mitochondrial pathways”). No major changes were seen in the expression of selected genes coding for proteins involved in apoptosis or in the metabolism of reactive oxygen species, except for increased expression of the pro-apoptotic Bid and uncoupling protein 2 (Ucp2) (see “Mitochondrial pathway”), a well-known PPARα target gene, and an increased expression of epoxide hydrolase (Ephx2, see “Peroxisomal pathways”) by FO compared to KO.
Expression was also investigated for members of the PPAR family of nuclear receptors, that are known to bind lipids as ligands and thereby respond to changes in lipid homeostasis, and the PPARγ coactivators 1a and 1b. However, no significant differences in the expression of these selected genes were found due to large variation of expression levels between individuals (Figure 2, “Nuclear transcription factors”). Although many PPARα regulated genes were upregulated in the FO group, Pparα itself was not significantly increased by FO or KO (p = 0.15 by Kruskal-Wallis test). Ppargc1a showed a trend (p = 0.058 by Kruskal-Wallis test) to be different between the groups. Expression of Srebp1c (sterol regulatory element binding protein-1c), which is a regulator of e.g. Fas and Acaca, was also investigated, however, contradictory to expectation no downregulation of the gene by KO was found (Figure 2, “Additional metabolic pathways”).
Effect of marine oils on gene expression in small intestine
Omega-3 PUFA supplementation and adequate intake of dietary omega-3 PUFAs is stated to have numerous beneficial health promoting effects including TAG lowering in plasma in humans, which have also been found in numerous animal studies. The main outcome was the demonstration of a different metabolic regulation by KO and FO. FO significantly decreased several plasma lipid parameters (TAG, PLs and cholesterol) compared to HF, which was associated with lipid accumulation in liver. However, the post hoc test did not demonstrate any significant changes in total plasma TAG, PL or cholesterol in the KO supplemented group compared to HF, while plasma NEFA was significantly reduced. In addition, there were no significant differences between the two marine oil groups except for significantly lower VLDL cholesterol content in the FO group.
FO and KO differently regulated expression of genes involved in lipid degradation and synthesis. While FO provoked a strong PPARα activation like response in liver and intestine, these effects were weak, or absent, in the KO group, which instead mainly decreased the expression of genes involved in cholesterol and fatty acid synthesis.
The major factors contributing to these differences are likely the different content and structure of the omega-3 PUFAs in FO and KO. In this study we intentionally supplemented the high fat diet with equal amounts of oil (6% w/w) to compare the effects of commercially available omega-3 supplements. Although the omega-3 content is about double in FO, the concentration of omega-3 PUFAs in plasma was very similar between the two marine oil groups suggesting a higher bioavailability of KO, or that the content of omega-3 PUFAs in KO in our study is enough to obtain maximal plasma concentrations of omega-3 fatty acids. Incorporation of omega-3 PUFAs in PLs has been suggested to give these lipids a higher bioavailability[14, 42, 43], in fact, PLs enriched from KO was shown to be even more efficient in lowering plasma cholesterol than KO itself when given to rats fed a high cholesterol diet.
Unfortunately, in the present study was not enough plasma to analyze the fatty acid composition in TAG and PLs separately so it is not clear whether the similar levels of omega-3 PUFAs measured in plasma of the two groups are due to a specific enrichment in the PL fraction of the KO feed mice, which may be the case. However, the ratio of EPA/DHA (EPA > DHA) in total plasma was very similar in the FO and KO groups and rather closely resembled the ratio in the diet, indicating that these fatty acids are not modified during uptake in the intestine and transport in plasma. It is however evident that the omega-3 fatty acids are redistributed between the TAG and PL fractions following uptake and incorporation into liver lipids and that EPA is further metabolized to DHA, thereby markedly increasing the DHA/EPA ratio. DHA is mainly esterified into PLs in liver of the HF mice (constituting >90% of the omega-3 fatty acids in this fraction) and surprisingly reaching almost 50% of the DHA levels in the FO and KO groups. FO decreased total omega-6 fatty acids in total plasma lipids and liver PLs, while KO mainly lowered longer chain omega-6 fatty acid species in plasma and liver PLs. Both treatments thereby significantly increased the omega-3/omega-6 ratio in plasma compared to HF alone. Since KO specifically lowered the arachidonic acid content in the liver PL fraction and in plasma more efficiently than FO, KO may have a more potent anti-inflammatory effect. Such an effect by KO was seen in another FO and KO comparing supplementation study on rheumatoid arthritis with balanced amounts of DHA and EPA. The decreased arachidonic acid levels are also likely to decrease the amounts of 2-arachidonoylglycerol (2-AG), a potent signaling lipid, as previously described. Krill powder, which is also rich in omega-3 containing PLs, was shown to decrease another arachidonic acid derived endocannabinoid, anandamide (N-arachidonoylethanolamide, AEA), and its related metabolites palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) in plasma of obese men. KO has further been shown to be protective in a rat model of inflammatory bowel disease in which KO seemed to both act as an anti-inflammatory as well as anti-oxidative agent in the colon of these animals.
The plasma TAG lowering effect of EPA and DHA have been proposed to be at least partly mediated via PPARα, similar to fibrates, by stimulating the β-oxidation systems that would drain the liver of fatty acids and thereby decrease the production of VLDL particles. Our data, however, show a 3-4 fold TAG accumulation in livers from FO supplemented mice in spite of an apparent PPARα activation in both liver and intestine. Similar, but less pronounced, changes were seen also for fatty acids in plasma and liver PLs. The apparent ‘transfer’ of PLs from plasma to liver may reflect increased membrane synthesis, e.g. proliferation of peroxisomes (at least for the FO group) as indicated by increased expression of peroxisomal enzymes and mitochondria as indicated by slightly increased citrate synthase expression (see Figure 2). Another possibility is that liver PLs increase as a result of decreased HDL production that may at least in part explain the decrease in plasma HDL. Since fatty acid composition and amounts are similar in plasma and liver PLs in FO and KO supplemented mice, it is likely that the stronger PPARα activation seen in liver and intestine is mediated via omega-3 PUFAs of the TAG fraction of FO supplemented mice.
The increase in the fatty acid content of liver TAG found in the FO group indicates that the observed upregulation of genes coding for catabolic pathways (e.g. the upregulation of genes coding for fatty acid oxidation and the increased ACOX activity and possibly elevated activity of CPT1) is apparently of minor importance in determining liver lipid levels in these animals. It should be noted that PPARα activation by e.g. fenofibrate also increases liver expression of lipogenic genes a with concomitant increase in fatty acid synthesis and TAG accumulation. Thus, our data are consistent with PPARα activation by FO, although weaker than fibrate treatment, and hence increased lipid synthesis and possibly decreased VLDL secretion may at least partially explain the observed TAG accumulation in liver. Also, a recent study demonstrated that DHA attenuates postprandial hyperlipidemia in a PPARα dependent manner by activation of fatty acid oxidation genes in the intestine leading to decreased TAG and apoB secretion. However, PPARα-independent pathways that reduce the assembly and secretion of VLDL particles also appear to be involved[23, 48]. Some data indicate that FO decreases VLDL production mainly due to decreased plasma NEFA, which is suggested to be the main source of fatty acids for VLDL synthesis. However only KO significantly decreased fasting plasma NEFA in our study but did not significantly change plasma TAG, suggesting additional mechanisms being involved. EPA is reported to inhibit DGAT activity in liver[49, 50], and also negatively influence the assembly and secretion of VLDL via incorporation into choline and ethanolamine PLs, which due to the higher total content of EPA in FO could be an additional possible explanation for the different effects on plasma TAG by FO and KO in our study. This may also explain why similar levels of omega-3 PUFAs were found in plasma, since the TAG pool entering the VLDL fraction might be affected by the high levels of EPA, and that EPA may be shunted into the cytosolic TAG-pool for storage rather than entering VLDL in the FO group, while the PL pool (which contained similar amounts of omega-3 PUFAs in both groups in liver) that enters the different lipoprotein fractions, e.g. HDL, may not be as affected. We did not assess the severity of the lipid accumulation in liver in this study and therefore we can not predict the health effects associated with these changes and effects of long-term feeding of these omega-3 supplements.
KO feeding reduced the expression and the activity of fatty acid synthase (Fas), which is in line with previous findings that KO supplementation decreases the mRNA expression of this protein more efficiently than FO[25, 27, 28]. Expression of Acacb (acetyl-CoA carboxylase 2) was also decreased in the KO group compared to the FO group, which rather showed an increased expression of this gene. Acetyl-CoA carboxylase 2 is associated with mitochondria and generates malonyl-CoA, which is a key regulator of energy homeostasis, and deletion of Acacb in mice promotes fatty acid oxidation and increased energy expenditure[53, 54]. Therefore, in conjunction with an apparent downregulation of Fas (and possibly Acaca) and thereby fatty acid synthesis in the KO group, this may provide a possible alternative mechanism by which KO maintains lower liver TAG levels compared to FO.
KO also reduced the mRNA expression of the first segments of the cholesterol/isoprenoid synthesis pathway, including the rate-limiting enzyme in cholesterol synthesis HMG-CoA reductase. However, the reduction in expression seen in our current study did not reflect in significant changes in plasma cholesterol (-16%), and liver cholesterol was even slightly increased as in the FO group. This was also seen in a low fat/omega-3 PUFA balanced fish and krill oil based study in which KO caused a similar downregulation in expression of genes involved in gluconeogenesis and cholesterol and fatty acid synthesis without changing plasma lipids. Similar amounts of KO supplementation have previously been shown to decrease plasma and liver cholesterol in C56BL/6 mice on a HF diet, however in this study the mice were fed a different high fat diet containing buttermilk and 0.15% cholesterol ± KO for 8 weeks which may explain the different results. More experiments are needed to elucidate the discrepancy between gene expression and plasma/liver cholesterol levels, e.g. measurement of total body cholesterol pool and bile acid and cholesterol synthesis and excretion.
In the present study we have also investigated the effects of FO and KO on gene expression of proteins involved in lipid metabolism in the small intestine. A similar trend in gene regulation as in the liver could be seen in the intestine of these animals. The expression of genes involved in fatty acid modulation and transport, such as Acot1, Cd36, I-fabp and L-fabp were all upregulated by the FO-containing diet, which is in line with previous findings of PPARα activation in the intestine during increased ligand availability. KO on the other hand did not promote the same response to these PPARα driven genes. The increased expression of fatty acid transporters in the intestine of the FO fed group would potentially lead to an enhanced chylomicron production due to increased uptake of fatty acids, however FO has previously been shown to cause PPARα activation and thereby increased β-oxidation in murine intestine, which may balance an increased uptake of fatty acids. In a recent study, fenofibrate was shown to reduce blood TAG content in the postprandial state in part due to decreased dietary fat absorption and increased β-oxidation of fatty acids in spite of upregulation of chylomicron synthesizing genes and fatty acid transporters. It is possible that the different effects of FO and KO may in part be due to differences in PPARα activation and thereby oxidative degradation of fatty acids in the small intestine, although the quantitative importance of fatty acid oxidation in the intestine is likely to be small. The only significant changes found in the KO group in genes coding for proteins in the chylomicron synthesis pathway was a downregulation of Mttp in the first intestinal segment by KO compared to HF diet, and a significant difference between FO and KO in the expression of Acat2 (in segment 3) and Dgat1 (in segments 2 and 3). Feces were not collected for absorption studies or lipid analysis in our study, therefore it is not clear whether omega-3 PUFAs per se, or omega-3 PUFAs in TAG versus PLs, affect dietary lipid absorption in the small intestine. Some data indicate that FO may reduce lipid uptake in the intestine, or at least delay efflux into the circulation by a transient accumulation of lipids in the enterocytes. Whether KO affects lipid uptake in the intestine remains to be studied.
Both FO and KO raised plasma levels of omega-3 PUFAs to the same extent in spite of a markedly lower omega-3 PUFA content in the KO diet. FO lowered plasma TAG, PL and cholesterol and KO lowered NEFA in comparison to the control group. The two omega-3 fatty acid supplementations also promoted different gene expression profiles in liver and intestine with FO causing an apparent PPARα response by increasing the expression of genes coding for proteins in the two β-oxidation systems and other lipid metabolizing genes. In contrast, KO supplementation rather acted as a negative regulator of endogenous cholesterol and fatty acid synthesis, at least at the mRNA level. The stronger plasma lipid lowering effect with FO can be partly explained by increased lipid accumulation, mainly as TAG, in liver in spite of increased PPARα activation that may not compensate for decreased VLDL secretion. The physiological/pathological implications of the liver lipid accumulation by FO are not clear and may depend on diet composition and dose of FO and should be evaluated.
High density lipoprotein
Low density lipoprotein
Monounsaturated fatty acid
Nonesterified fatty acids
Peroxisome proliferator-activated receptor
Polyunsaturated fatty acid
Very low density lipoprotein.
This work was supported by NordForsk under the Nordic Centers of Excellence programme in Food, Nutrition, and Health, Project (070010) “MitoHealth” and The Swedish Research Council. Krill oil (Superba™) was supplied by Aker BioMarine ASA, Oslo, Norway, and fish oil (EPAX 6000 TG™) by Epax A/S, Ålesund, Norway. We are grateful to Professor Paolo Parini and Lilian Larsson (Karolinska Institutet, Stockholm) for plasma lipid profiling analysis. Thanks to Liv-Kristine Øysæd for plasma and hepatic lipid analysis and to Eline Milde, Elin Strand, Natalya Vigerust, Thomas Lundåsen and the staff at the UiB animal facility, University of Bergen, Bergen, for assistance during the animal study. We also thank Kjetil Berge (Aker BioMarine, ASA) for valuably comments to the manuscript. We thank Dr. Claes Carneheim for advice on statistical analysis and comments on the manuscript. We also would like to thank the core facility BEA (Bioinformatics and Expression Analysis) at Karolinska Institutet, which is supported by the board of research at the Karolinska Institute and the research committee at the Karolinska hospital.
- de Oliveira Otto MC, Wu JH, Baylin A, Vaidya D, Rich SS, Tsai MY, Jacobs DR, Mozaffarian D: Circulating and dietary omega-3 and omega-6 polyunsaturated fatty acids and incidence of CVD in the Multi-Ethnic Study of Atherosclerosis. J Am Heart Assoc. 2013, 2 (6): e000506-10.1161/JAHA.113.000506.View ArticleGoogle Scholar
- Dyerberg J, Bang HO, Stoffersen E, Moncada S, Vane JR: Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis?. Lancet. 1978, 2 (8081): 117-119.View ArticleGoogle Scholar
- Konig A, Bouzan C, Cohen JT, Connor WE, Kris-Etherton PM, Gray GM, Lawrence RS, Savitz DA, Teutsch SM: A quantitative analysis of fish consumption and coronary heart disease mortality. Am J Prev Med. 2005, 29 (4): 335-346. 10.1016/j.amepre.2005.07.001.View ArticleGoogle Scholar
- Kromhout D, de Goede J: Update on cardiometabolic health effects of omega-3 fatty acids. Curr Opin Lipidol. 2014, 25 (1): 85-90. 10.1097/MOL.0000000000000041.View ArticleGoogle Scholar
- Lorente-Cebrian S, Costa AG, Navas-Carretero S, Zabala M, Martinez JA, Moreno-Aliaga MJ: Role of omega-3 fatty acids in obesity, metabolic syndrome, and cardiovascular diseases: a review of the evidence. J Physiol Biochem. 2013, 69 (3): 633-651. 10.1007/s13105-013-0265-4.View ArticleGoogle Scholar
- Kris-Etherton PM, Harris WS, Appel LJ: Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Arterioscler Thromb Vasc Biol. 2003, 23 (2): e20-e30. 10.1161/01.ATV.0000038493.65177.94.View ArticleGoogle Scholar
- Kris-Etherton PM, Harris WS, Appel LJ: Omega-3 fatty acids and cardiovascular disease: new recommendations from the American Heart Association. Arterioscler Thromb Vasc Biol. 2003, 23 (2): 151-152. 10.1161/01.ATV.0000057393.97337.AE.View ArticleGoogle Scholar
- Burri L, Hoem N, Banni S, Berge K: Marine omega-3 phospholipids: metabolism and biological activities. Int J Mol Sci. 2012, 13 (11): 15401-15419.View ArticleGoogle Scholar
- Phleger CF, Nelson MM, Mooney BD, Nichols PD: Interannual and between species comparison of the lipids, fatty acids and sterols of Antarctic krill from the US AMLR Elephant Island survey area. Comp Biochem Physiol B: Biochem Mol Biol. 2002, 131 (4): 733-747. 10.1016/S1096-4959(02)00021-0.View ArticleGoogle Scholar
- Tou JC, Jaczynski J, Chen YC: Krill for human consumption: nutritional value and potential health benefits. Nutr Rev. 2007, 65 (2): 63-77.View ArticleGoogle Scholar
- Winther B, Hoem N, Berge K, Reubsaet L: Elucidation of phosphatidylcholine composition in krill oil extracted from Euphausia superba. Lipids. 2011, 46 (1): 25-36. 10.1007/s11745-010-3472-6.View ArticleGoogle Scholar
- Maki KC, Reeves MS, Farmer M, Griinari M, Berge K, Vik H, Hubacher R, Rains TM: Krill oil supplementation increases plasma concentrations of eicosapentaenoic and docosahexaenoic acids in overweight and obese men and women. Nutr Res. 2009, 29 (9): 609-615. 10.1016/j.nutres.2009.09.004.View ArticleGoogle Scholar
- Ulven SM, Kirkhus B, Lamglait A, Basu S, Elind E, Haider T, Berge K, Vik H, Pedersen JI: Metabolic effects of krill oil are essentially similar to those of fish oil but at lower dose of EPA and DHA, in healthy volunteers. Lipids. 2011, 46 (1): 37-46. 10.1007/s11745-010-3490-4.View ArticleGoogle Scholar
- Schuchardt JP, Schneider I, Meyer H, Neubronner J, Von Schacky C, Hahn A: Incorporation of EPA and DHA into plasma phospholipids in response to different omega-3 fatty acid formulations--a comparative bioavailability study of fish oil vs. krill oil. Lipids Health Dis. 2011, 10: 145-10.1186/1476-511X-10-145.View ArticleGoogle Scholar
- Berge K, Musa-Veloso K, Harwood M, Hoem N, Burri L: Krill oil supplementation lowers serum triglycerides without increasing low-density lipoprotein cholesterol in adults with borderline high or high triglyceride levels. Nutr Res. 2014, 34 (2): 126-133. 10.1016/j.nutres.2013.12.003.View ArticleGoogle Scholar
- Ramprasath VR, Eyal I, Zchut S, Jones PJ: Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil. Lipids Health Dis. 2013, 12: 178-10.1186/1476-511X-12-178.View ArticleGoogle Scholar
- Rossmeisl M, Jilkova ZM, Kuda O, Jelenik T, Medrikova D, Stankova B, Kristinsson B, Haraldsson GG, Svensen H, Stoknes I, Sjovall P, Magnusson Y, Balvers MG, Verhoeckx KC, Tvrzicka E, Bryhn M, Kopecky J: Metabolic effects of n-3 PUFA as phospholipids are superior to triglycerides in mice fed a high-fat diet: possible role of endocannabinoids. PLoS One. 2012, 7 (6): e38834-10.1371/journal.pone.0038834.View ArticleGoogle Scholar
- Hostetler HA, Kier AB, Schroeder F: Very-long-chain and branched-chain fatty acyl-CoAs are high affinity ligands for the peroxisome proliferator-activated receptor alpha (PPARalpha). Biochemistry. 2006, 45 (24): 7669-7681. 10.1021/bi060198l.View ArticleGoogle Scholar
- Takeuchi Y, Yahagi N, Izumida Y, Nishi M, Kubota M, Teraoka Y, Yamamoto T, Matsuzaka T, Nakagawa Y, Sekiya M, Sekiya M, Iizuka Y, Ohashi K, Osuga J, Gotoda T, Ishibashi S, Itaka K, Kataoka K, Nagai R, Yamada N, Kadowaki T, Shimano H: Polyunsaturated fatty acids selectively suppress sterol regulatory element-binding protein-1 through proteolytic processing and autoloop regulatory circuit. J Biol Chem. 2010, 285 (15): 11681-11691. 10.1074/jbc.M109.096107.View ArticleGoogle Scholar
- Sanderson LM, de Groot PJ, Hooiveld GJ, Koppen A, Kalkhoven E, Muller M, Kersten S: Effect of synthetic dietary triglycerides: a novel research paradigm for nutrigenomics. PLoS One. 2008, 3 (2): e1681-10.1371/journal.pone.0001681.View ArticleGoogle Scholar
- Larsen R, Eilertsen KE, Elvevoll EO: Health benefits of marine foods and ingredients. Biotechnol Adv. 2011, 29 (5): 508-518. 10.1016/j.biotechadv.2011.05.017.View ArticleGoogle Scholar
- Serhan CN, Petasis NA: Resolvins and protectins in inflammation resolution. Chem Rev. 2011, 111 (10): 5922-5943. 10.1021/cr100396c.View ArticleGoogle Scholar
- Shearer GC, Savinova OV, Harris WS: Fish oil - How does it reduce plasma triglycerides?. Biochim Biophys Acta. 2012, 1821 (5): 843-851. 10.1016/j.bbalip.2011.10.011.View ArticleGoogle Scholar
- Burri L, Berge K, Wibrand K, Berge RK, Barger JL: Differential effects of krill oil and fish oil on the hepatic transcriptome in mice. Front Genet. 2011, 2: 45-View ArticleGoogle Scholar
- Ferramosca A, Conte L, Zara V: A krill oil supplemented diet reduces the activities of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes in rats. J Anim Physiol Anim Nutr. 2012, 96 (2): 295-306. 10.1111/j.1439-0396.2011.01135.x.View ArticleGoogle Scholar
- Tandy S, Chung RW, Wat E, Kamili A, Berge K, Griinari M, Cohn JS: Dietary krill oil supplementation reduces hepatic steatosis, glycemia, and hypercholesterolemia in high-fat-fed mice. J Agric Food Chem. 2009, 57 (19): 9339-9345. 10.1021/jf9016042.View ArticleGoogle Scholar
- Ferramosca A, Conte A, Burri L, Berge K, De Nuccio F, Giudetti AM, Zara V: A krill oil supplemented diet suppresses hepatic steatosis in high-fat fed rats. PLoS One. 2012, 7 (6): e38797-10.1371/journal.pone.0038797.View ArticleGoogle Scholar
- Vigerust NF, Bjorndal B, Bohov P, Brattelid T, Svardal A, Berge RK: Krill oil versus fish oil in modulation of inflammation and lipid metabolism in mice transgenic for TNF-alpha. Eur J Nutr. 2013, 52 (4): 1315-1325. 10.1007/s00394-012-0441-2.View ArticleGoogle Scholar
- Green PH, Tall AR, Glickman RM: Rat intestine secretes discoid high density lipoprotein. J Clin Invest. 1978, 61 (2): 528-534. 10.1172/JCI108963.View ArticleGoogle Scholar
- Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, Coburn BA, Bissada N, Staels B, Groen AK, Hussain MM, Parks JS, Kuipers F, Hayden MR: Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest. 2006, 116 (4): 1052-1062. 10.1172/JCI27352.View ArticleGoogle Scholar
- Grimstad T, Bjorndal B, Cacabelos D, Aasprong OG, Janssen EA, Omdal R, Svardal A, Hausken T, Bohov P, Portero-Otin M, Pamplona R, Berge RK: Dietary supplementation of krill oil attenuates inflammation and oxidative stress in experimental ulcerative colitis in rats. Scand J Gastroenterol. 2012, 47 (1): 49-58.View ArticleGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25 (4): 402-408. 10.1006/meth.2001.1262.View ArticleGoogle Scholar
- Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959, 37 (8): 911-917. 10.1139/o59-099.View ArticleGoogle Scholar
- Parini P, Johansson L, Broijersen A, Angelin B, Rudling M: Lipoprotein profiles in plasma and interstitial fluid analyzed with an automated gel-filtration system. Eur J Clin Invest. 2006, 36 (2): 98-104. 10.1111/j.1365-2362.2006.01597.x.View ArticleGoogle Scholar
- Berge RK, Flatmark T, Osmundsen H: Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators. Eur J Biochem. 1984, 141 (3): 637-644. 10.1111/j.1432-1033.1984.tb08239.x.View ArticleGoogle Scholar
- Bremer J: The effect of fasting on the activity of liver carnitine palmitoyltransferase and its inhibition by malonyl-CoA. Biochim Biophys Acta. 1981, 665 (3): 628-631. 10.1016/0005-2760(81)90282-4.View ArticleGoogle Scholar
- Small GM, Burdett K, Connock MJ: A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase. Biochem J. 1985, 227 (1): 205-210.View ArticleGoogle Scholar
- Skorve J, Al Shurbaji A, Asiedu D, Bjorkhem I, Berglund L, Berge RK: On the mechanism of the hypolipidemic effect of sulfur-substituted hexadecanedioic acid (3-thiadicarboxylic acid) in normolipidemic rats. J Lipid Res. 1993, 34 (7): 1177-1185.Google Scholar
- Dongol B, Shah Y, Kim I, Gonzalez FJ, Hunt MC: The acyl-CoA thioesterase I is regulated by PPARalpha and HNF4alpha via a distal response element in the promoter. J Lipid Res. 2007, 48 (8): 1781-1791. 10.1194/jlr.M700119-JLR200.View ArticleGoogle Scholar
- Lambert G, Charlton F, Rye KA, Piper DE: Molecular basis of PCSK9 function. Atherosclerosis. 2009, 203 (1): 1-7. 10.1016/j.atherosclerosis.2008.06.010.View ArticleGoogle Scholar
- Rakhshandehroo M, Knoch B, Muller M, Kersten S: Peroxisome proliferator-activated receptor alpha target genes. PPAR Res. 2010Google Scholar
- Cansell M, Nacka F, Combe N: Marine lipid-based liposomes increase in vivo FA bioavailability. Lipids. 2003, 38 (5): 551-559. 10.1007/s11745-003-1341-0.View ArticleGoogle Scholar
- Ramirez M, Amate L, Gil A: Absorption and distribution of dietary fatty acids from different sources. Early Hum Dev. 2001, 65 (Suppl): S95-S101.View ArticleGoogle Scholar
- Li DM, Zhou DY, Zhu BW, Chi YL, Sun LM, Dong XP, Qin L, Qiao WZ, Murata Y: Effects of krill oil intake on plasma cholesterol and glucose levels in rats fed a high-cholesterol diet. J Sci Food Agric. 2013, Epub ahead of printGoogle Scholar
- Ierna M, Kerr A, Scales H, Berge K, Griinari M: Supplementation of diet with krill oil protects against experimental rheumatoid arthritis. BMC Musculoskelet Disord. 2010, 11: 136-10.1186/1471-2474-11-136.View ArticleGoogle Scholar
- Berge K, Piscitelli F, Hoem N, Silvestri C, Meyer I, Banni S, Di Marzo V: Chronic treatment with krill powder reduces plasma triglyceride and anandamide levels in mildly obese men. Lipids Health Dis. 2013, 12: 78-10.1186/1476-511X-12-78.View ArticleGoogle Scholar
- Oosterveer MH, Grefhorst A, van Dijk TH, Havinga R, Staels B, Kuipers F, Groen AK, Reijngoud DJ: Fenofibrate simultaneously induces hepatic fatty acid oxidation, synthesis, and elongation in mice. J Biol Chem. 2009, 284 (49): 34036-34044. 10.1074/jbc.M109.051052.View ArticleGoogle Scholar
- Lu Y, Boekschoten MV, Wopereis S, Muller M, Kersten S: Comparative transcriptomic and metabolomic analysis of fenofibrate and fish oil treatments in mice. Physiol Genomics. 2011, 43 (23): 1307-1318. 10.1152/physiolgenomics.00100.2011.View ArticleGoogle Scholar
- Rustan AC, Nossen JO, Christiansen EN, Drevon CA: Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A:1,2-diacylglycerol acyltransferase. J Lipid Res. 1988, 29 (11): 1417-1426.Google Scholar
- Berge RK, Madsen L, Vaagenes H, Tronstad KJ, Gottlicher M, Rustan AC: In contrast with docosahexaenoic acid, eicosapentaenoic acid and hypolipidaemic derivatives decrease hepatic synthesis and secretion of triacylglycerol by decreased diacylglycerol acyltransferase activity and stimulation of fatty acid oxidation. Biochem J. 1999, 343 (Pt 1): 191-197.View ArticleGoogle Scholar
- Tran K, Sun F, Cui Z, Thorne-Tjomsland G, St Germain C, Lapierre LR, McLeod RS, Jamieson JC, Yao Z: Attenuated secretion of very low density lipoproteins from McA-RH7777 cells treated with eicosapentaenoic acid is associated with impaired utilization of triacylglycerol synthesized via phospholipid remodeling. Biochim Biophys Acta. 2006, 1761 (4): 463-473. 10.1016/j.bbalip.2006.03.018.View ArticleGoogle Scholar
- Choi CS, Savage DB, Abu-Elheiga L, Liu ZX, Kim S, Kulkarni A, Distefano A, Hwang YJ, Reznick RM, Codella R, Zhang D, Cline GW, Wakil SJ, Shulman GI: Continuous fat oxidation in acetyl-CoA carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity. Proc Natl Acad Sci U S A. 2007, 104 (42): 16480-16485. 10.1073/pnas.0706794104.View ArticleGoogle Scholar
- Abu-Elheiga L, Brinkley WR, Zhong L, Chirala SS, Woldegiorgis G, Wakil SJ: The subcellular localization of acetyl-CoA carboxylase 2. Proc Natl Acad Sci U S A. 2000, 97 (4): 1444-1449. 10.1073/pnas.97.4.1444.View ArticleGoogle Scholar
- Abu-Elheiga L, Matzuk MM, Abo-Hashema KA, Wakil SJ: Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Science. 2001, 291 (5513): 2613-2616. 10.1126/science.1056843.View ArticleGoogle Scholar
- Bunger M, van den Bosch HM, van der Meijde J, Kersten S, Hooiveld GJ, Muller M: Genome-wide analysis of PPARalpha activation in murine small intestine. Physiol Genomics. 2007, 30 (2): 192-204. 10.1152/physiolgenomics.00198.2006.View ArticleGoogle Scholar
- Mori T, Kondo H, Hase T, Tokimitsu I, Murase T: Dietary fish oil upregulates intestinal lipid metabolism and reduces body weight gain in C57BL/6J mice. J Nutr. 2007, 137 (12): 2629-2634.Google Scholar
- Uchida A, Slipchenko MN, Cheng JX, Buhman KK: Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, alters triglyceride metabolism in enterocytes of mice. Biochim Biophys Acta. 2011, 1811 (3): 170-176. 10.1016/j.bbalip.2010.12.011.View ArticleGoogle Scholar
- Larsen LF, Marckmann P, Kornerup Hansen A, Bukhave K: Fish oil feeding is associated with an increased accumulation of dietary lipids in enterocytes: results from an in vivo study in rats. Scand J Gastroenterol. 2003, 38 (7): 712-718.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.