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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 8, 2005 as doi:10.1096/fj.04-2974fje. |
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,1
,
,||,¶,#
* Rowett Research Institute, Aberdeen, Scotland, UK;
TNO Prevention and Health, Gaubius Laboratory, Leiden, The Netherlands;
TNO Nutrition and Food Research, Zeist, The Netherlands;
Departments of Endocrinology,
|| Cardiology and
¶ Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands,
# Wageningen Centre for Food Sciences/NIZO Food Research, Ede, The Netherlands
2 Correspondence: Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK. E-mail: b.deroos{at}rowett.ac.uk
SPECIFIC AIMS
Dietary fatty acids have a profound impact on the development of atherosclerosis largely because of their detrimental or beneficial effects on the lipoprotein profile. Mechanisms by which dietary fatty acids exert these effects are still not fully understood. In this study we compared the physiological effects of three classes of dietary fatty acids that affect atherosclerosis (fish oil, t10,c12-conjugated linoleic acid (CLA), and elaidic acid) on lipid and glucose metabolism in apoE*3-Leiden transgenic mice, a sensitive and validated animal model for lipid metabolism, insulin resistance, and atherosclerosis. We explored the mechanisms of dietary fish oil, CLA, and elaidic acid by identification of proteins that regulate changes in lipid and glucose metabolism as well as other biochemical pathways.
PRINCIPAL FINDINGS
1. Dietary treatment with fish oil, t10,c12 CLA, and elaidic acid altered lipid and glucose metabolism in APOE*3-Leiden mice
To assess the effects of different dietary fatty acids on lipid and glucose metabolism, APOE*3-Leiden transgenic mice were fed a high saturated fat, high-cholesterol diet (control diet) or this diet supplemented with 3% (w/w) fish oil (n=8), 1% t10,c12 CLA (n=8), or 3% (w/w) elaidic acid (n=8) for 3 wk. Another intervention group received the high saturated fat, high-cholesterol diet supplemented with 0.04% (w/w) fenofibrate (n=8); like fish oil and CLA, fibrates are PPAR agonists that stimulate cellular fatty acid uptake, conversion of fatty acids to acyl-CoA derivatives, and catabolism of fatty acids via ß-oxidation pathways.
Dietary fish oil and fenofibrate instigated similar effects on lipid and glucose metabolism (Table 1
). Both treatments significantly lowered plasma cholesterol and triglyceride levels vs. the control treatment. These changes were reflected in liver composition, where both treatments significantly decreased the amount of triglycerides, free cholesterol, and cholesteryl esters compared with control treatment. Fish oil and fenofibrate treatment significantly decreased the amount of free fatty acids in plasma to almost half the concentrations in the control group. These effects are likely caused by an enhanced fatty acid oxidation rate, which is consistent with the significant increase in ß-hydroxybutyrate levels in plasma of fish oil or fenofibrate treatment, although the increase was significant only in the fenofibrate-treated animals. Plasma glucose levels were significantly lowered by fish oil and fenofibrate treatments whereas plasma insulin levels were slightly increased in fish oil-fed animals compared with control animals.
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The t10,c12 isomer of CLA, like fish oil and fenofibrate, caused a significant decrease in plasma cholesterol levels (Table 1)
. However, this dietary fatty acid significantly increased levels of plasma triglycerides compared with control treatment. Again, changes in plasma lipids were reflected in liver composition: liver cells contained significantly more triglycerides and fewer cholesteryl esters than the control group. t10,c12 CLA caused a 2-fold increase in liver weight compared with the control group (P<0.05). Plasma ß-hydroxybutyrate levels were significantly increased by t10,c12 CLA treatment, indicative of an increased ß-oxidation rate. We observed hyperinsulinemia as evidenced by a 3-fold induction in plasma insulin levels in t10,c12 CLA-fed animals.
Consumption of elaidic acid decreased levels of plasma cholesterol and significantly increased levels of plasma ß-hydroxybutyrate (Table 1)
. Dietary elaidic acid had no effect on plasma levels of triglycerides, glucose, or insulin in our mouse model, unlike the effects in humans where trans fat in the diet increases levels of LDL cholesterol and triglycerides. A high intake of trans fat has been associated with the development of insulin resistance and type 2 diabetes in humans. Whether this differential effect is the result of species difference, a difference in the administered dose, or trans fatty acid isomer remains to be established.
2. Proteomics identified significant regulation of liver proteins by the dietary interventions in APOE*3-Leiden mice
When comparing the 2-dimensional gel electrophoresis gels, we found significant changes in levels of 74 liver cytosolic proteins and 14 liver membrane proteins that were up- or down-regulated by at least one of the dietary treatments compared with the control group. We identified 65 liver cytosolic proteins and 8 liver membrane proteins using MALDI-TOF mass spectrometry. Clear effects could be seen on pathways involved with glucose and lipid metabolism and in oxidation and aging. Regulation of these pathways is consistent with the physiological effects observed.
We found, for example, that not only fish oil and fenofibrate, but also t10,c12 CLA, increased liver protein levels of catalase and long chain acyl-CoA thioester hydrolase (cytosolic and mitochondrial forms), indicative of increased ß-oxidation rate. Until now, long chain acyl-CoA thioester hydrolase has not been linked to specific dietary fatty acid treatments. Acyl-CoA thioesterases hydrolyse CoA esters to free fatty acids and CoA-SH and are likely to play important roles in maintaining appropriate CoA-SH levels during periods of increased ß-oxidation and fatty acid overload. The existence of selective acyl-CoA thioesterases could provide important control points in the oxidation of many peroxisomal substrates; they may regulate intracellular levels of CoA esters and CoA-SH.
Lowering of liver lipids in fish oil and fenofibrate-treated animals and the significant increase in liver triglycerides in CLA-treated animals matched a large decrease and increase, respectively, in adipophilin in the liver. Adipophilin is a protein associated with lipid storage droplets, dynamic structures that function as storage deposits for triglycerides and cholesterol esters. Increased expression of adipophilin has been associated with liver steatosis, and CLA-mediated liver steatosis has occurred in different strains of mice. Liver steatosis is often associated with obesity, diabetes, hyperinsulinemia, and VLDL overproduction; we saw some but not all these associations in the t10,c12 CLA-fed mice.
3. Principal component analysis revealed fish oil having a major effect on changes in cytosolic protein levels and elaidic acid on membrane proteins
Principal component analysis (PCA) was used to analyze the effects of the various treatments on protein levels in the complete dataset. Each protein set per treatment is reduced to a single point, and these are projected in space so that the variation in original variables, protein levels, is expressed to a maximum. This approach visualizes the extent to which different treatments have similar or very different effects on protein expression.
Although fish oil and fenofibrate are believed to share common mechanisms that affect lipoprotein metabolism, our PCA analysis revealed diverse effects of both dietary interventions. Fish oil treatment had the most prominent effect on the first principal component, the one that explains most of the variation in a dataset. Fenofibrate, on the other hand, showed a notable treatment effect on the second principal component. The proteins responsible for the treatment effect of fish oil were involved in a range of metabolic functions whereas proteins responsible for the treatment effect of fenofibrate were dominated by those involved in ß-oxidation of fatty acids. This indicates that fish oil, unlike fenofibrate, triggers a more diverse range of mechanisms that could affect the physiological outcome.
The greatest treatment effect on changes in membrane protein levels was produced by elaidic acid on the first and the second principal component. Proteins that provided the largest contribution to the differences between the elaidc acid treatment and the other dietary treatments were aldehyde dehydrogenase and CTP synthase.
4. Correlation analysis between physiological and protein data revealed novel clusters of correlated variables, among which was a metabolic syndrome cluster
This dataset with relatively mild perturbations, representing animals of equal body weights, revealed a cluster of associated variables containing plasma and liver triglycerides, plasma glucose, plasma free fatty acids, and protein levels of hepatic fructokinase and fructose 1,6-bisphosphatase. These variables are related to dyslipidemia and glucose intolerance, both related to the metabolic syndrome or syndrome X. The position of sepiapterin reductase in the middle of this cluster was unexpected, as there does not seem to be a known link between this enzyme and metabolic syndrome. However, sepiapterin reductase is involved in the biosynthe-sis of tetrahydrobiopterin, an essential cofactor for eNOS activity. Therefore, this enzyme could potentially play a role in the relationship that has been described among dyslipidemia, insulin resistance, and endothelial dysfunction.
A second example of a recognized association is the cluster that highlights associations between catalase and two different forms of long chain acyl CoA thioester hydrolases, all of which are related to the ß-oxidation of fatty acids. The association of cysteine sulfinic acid decarboxylase to this cluster, however, has not been described before. Cysteine sulfinic acid decarboxylase is a rate-limiting enzyme for taurine biosynthesis, and taurine can be tissue-protective in many models of oxidant-induced injury. Therefore, cysteine sulfinic acid decarboxylase, like catalase, might be involved in protecting cells against oxidative stress generated by fatty acid oxidation.
CONCLUSIONS
Fish oil (or EPA and DHA), t10,c12 CLA, and elaidic acid each have unique influences on the development of heart disease. Fish oils are believed to have beneficial effects in delaying or preventing the development of CHD, whereas the opposite is true for trans fatty acids. Current evidence from the literature is ambiguous as to whether consumption of CLA would protect against CHD. Consumption of each of these fatty acids revealed distinct effects on lipoprotein metabolism and glucose/insulin levels in APOE*3-Leiden mice consistent with previous studies, reinforcing the suitability of this transgenic model.
That proteins represent the functional output of a cell reinforces the usefulness of proteomics in elucidating pathways that affect physiological outcome parameters upon intervention with dietary fatty acids in our mouse model. Proteomics of diet-induced changes in the liver of APOE*3-Leiden mice revealed a wide array of proteins that were affected by the various dietary interventions. Our approach visualized the 500–800 most abundant proteins from a liver cell on a 2-dimensional electrophoresis gel, and this provided a detailed overview of novel as well as recognized alterations in lipid degradation and glycolysis pathways, reflecting changes in lipoprotein and glucose metabolism upon dietary treatment. We found that consumption of specific dietary fatty acids induced a differential expression of long chain acyl-CoA thioester hydrolase protein (as an indicator of ß-oxidation) and adipophilin (as an indicator of liver lipid content). Our integrative approach combining physiological and proteomics outcomes (Fig. 1
)revealed many associations, some well known (such as metabolic syndrome), reassuring the validity of our approach; others provide the basis of intriguing new leads for further studies.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2974fje;
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