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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 29, 2005 as doi:10.1096/fj.05-3953fje. |
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* Rowett Research Institute, Aberdeen, UK;
Department of Biochemistry, Veterinary School, University of Zaragoza, Zaragoza, Spain; and
Nutrigenomics Research Group, Department of Clinical Medicine, Institute of Molecular Medicine, St. James’s Hospital, Dublin, Ireland
1 Correspondence: Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, Scotland, UK. E-mail: b.deroos{at}rowett.ac.uk
SPECIFIC AIMS
Conjugated linoleic acids (CLA) are present as minor constituents of meat, milk, and dairy products or other foods derived from ruminant animals. These fatty acids affect atherosclerosis, but mechanisms are not well understood. The present study compares both the physiological and biochemical properties of cis9, trans11-CLA and trans10, cis12-CLA in a single well-validated animal model for atherosclerosis, the apolipoprotein E (apoE) knockout mice. Through a combination of physiological data obtained from plasma with hepatic protein expression profiles, and the application of biostatistical methods, we explored mechanisms through which these individual CLA isomers similarly or differently affect changes in pathways that are involved in atherosclerosis.
PRINCIPAL FINDINGS
1. Dietary treatment with cis9, trans11-CLA and trans10, cis12-CLA differentially altered lipid and glucose metabolism in apoE knockout mice
To assess the effects of the two CLA isomers on lipid and glucose metabolism, apoE knockout mice were fed a semisynthetic high fat (30% energy) and cholesterol-containing (0.15% w/w) diet supplemented with either 1% (w/w) cis9, trans11-CLA (n=9) or 1% (w/w) trans10, cis12-CLA (n=9) or 1% (w/w) linoleic acid (n=10, control group) for 12 wk.
Cis9, trans11-CLA and trans10, cis12-CLA instigated isomer-specific effects on lipid and glucose metabolism. Fasting plasma triglyceride concentrations were >50% lower and fasting plasma NEFA concentrations were almost 35% lower (both P<0.05) upon intervention with cis9, trans11-CLA compared with the control group. In contrast, fasting plasma triglyceride concentrations were >300% higher and fasting plasma NEFA concentrations were almost 170% higher (both P<0.01) upon intervention with trans10, cis12-CLA compared with the control group. Plasma glucose concentrations were 16% lower after intervention with cis9, trans11-CLA but >30% higher after intervention with trans10, cis12-CLA, compared with the control group (both P<0.05). Plasma insulin concentrations were 26% lower after intervention with cis9, trans11-CLA compared with the control group. Intervention with trans10, cis12-CLA did not significantly affect plasma insulin levels compared with the control group (Table 1
).
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2. Regulation of hepatic proteins by the dietary interventions in apoE knockout mice, identified by proteomics
Two-dimensional gel electrophoresis of individual liver cytoplasmic protein fractions revealed 113 proteins of which levels were significantly up- or down-regulated by cis9, trans11-CLA and/or trans10, cis12-CLA, compared with linoleic acid. 104 (92%) of these proteins were identified with MALDI-TOF mass spectrometry and LC-MS/MS. This approach provided a detailed overview of how the two individual CLA isomers very differently affected both novel and recognized alterations in lipid degradation and gluconeogenensis pathways, reflecting changes in triglyceride, NEFA, glucose and insulin concentrations. Principal component analysis revealed that the treatment effect of trans10, cis12-CLA was mainly explained by up-regulation of key enzymes in the gluconeogenic, ß-oxidation and ketogenesic pathways. In contrast, the treatment effect of cis9, trans11-CLA was mainly explained by the up-regulation of different post-translationally modified forms of heat shock protein 70 kDa.
Trans10, cis12-CLA provoked insulin resistance in the apoE knockout mice, as indicated by a simultaneous increase in the rate of fatty acid ß-oxidation (significant up-regulation of long chain acyl-CoA thioester hydrolase), the rate of gluconeogenesis (significant up-regulation of phosphoenolpyruvate carboxykinase, 
enolase, fructose 1,6-bisphosphatase, glycerol-3-phosphate dehydrogenase, and glycerol kinase), and the rate of ketone body formation (significant up-regulation of HMG-CoA synthase) in the liver.
For the first time, we showed that cis9, trans11-CLA significantly and specifically increased levels of five different post-translationally-modified forms of heat shock protein (HSP) 70 kDa, whereas levels of this protein were not altered by trans10, cis12-CLA. Increased plasma levels of this molecular chaperone molecule have been associated with a lowered coronary artery disease risk, independent of traditional risk factors, in humans. The protective mechanisms responsible for the inverse relationship between HSP 70 kDa and coronary artery disease might include anti-inflammatory pathways. HSP 70 kDa forms complexes with inhibitory 
B protein and attenuates nuclear factor-B activation. Since nuclear factor-B is a key transcription factor modulating the expression of proinflammatory genes, this could be a key anti-inflammatory activity of HSP 70 kDa. We found that only cis9, trans11-CLA enhanced protein levels of hepatic IKB, indicating an inhibited degradation of this NF-B pathway intermediate, which may lead to a decrease in NF-B activity.
We also showed a mainly cis9, trans11- CLA-, but also a trans10, cis12-CLA-related decrease in hepatic levels of macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine that is expressed by vascular cells. In vivo evidence supports direct participation of MIF in atherogenesis as deficiency of MIF reduces atherogenesis in LDLr–/– mice. Therefore, the decreased hepatic protein levels of MIF, and also the increased protein levels of HSP 70 kDa, could potentially play a role in the decreased development of atheroslerotic lesions observed upon intervention with cis9, trans11-CLA in our study.
3. Pairwise correlation analysis between physiological and protein data revealed novel clusters of correlated variables
Across treatments, correlation analysis revealed both predictable and novel clusters of associated variables. Most noticeable were two clusters that were related to glucose metabolism. The first cluster contained all the plasma-derived parameters relating to glucose metabolism and insulin resistance (plasma triglycerides, plasma NEFA, plasma glucose, plasma insulin), as well as three derived plasma indices of insulin resistance: HOMA, QUICKI and the revised QUICKI. This cluster was plausibly linked to three post-translational forms of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme in the gluconeogenesis pathway. However, this link was unexpectedly also associated with the enzyme glutathione-S-transferase zeta, also known as maleylacetoacetate isomerase, catalyzing the fifth step in phenylalanine catabolism (Fig. 1
). The second cluster contained enzymes involved in glucose metabolism (e.g., glucose phosphomutase, fructose 1,6-bisphosphatase and glycerol-3-phosphate dehydrogenase) as well as serotransferrin, an iron binding transport protein (Fig. 1)
. Both glucose and iron metabolism were affected upon consumption of trans10, cis12-CLA, as we observed a simultaneous up-regulation of these gluconeogenic enzymes as well as significantly higher levels of hepatic serotransferrin and serotransferrin precursor, indicative for increased levels of hepatic iron. Insulin resistance-associated hepatic iron overload combined with liver steatosis has been observed before, although mechanisms underlying this association have not yet been identified. Hepatic iron overload with hepatic steatosis may down-regulate insulin receptors and hence, insulin action. Alternatively, insulin resistance may lead to the redistribution of transferrin receptors to the cell surface, where they could mediate the uptake of extracellular iron. Another explanation involves inflammation: since the insulin resistance syndrome is an atherogenic state, widespread activation of inflammatory cytokines in the subendothelial space increases transcription of ferritin mRNA in macrophages, and these cells may subsequently transfer ferritin to hepatocytes. This is, as far as we know, the first dietary intervention study that links insulin resistance to a hepatic iron overload.
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CONCLUSIONS AND SIGNIFICANCE
Many studies assessing the mechanism by which CLA affects pathways involved in atherosclerosis have so far used a mixture of CLA. However, recent research suggests that the two most important CLA isomers, cis9, trans11-CLA and trans10, cis12-CLA, although structurally very similar, may act on different biochemical pathways. To our knowledge, this study is the first that explores the mechanisms of the individual effects of both CLA isomers in a well-validated model for atherosclerosis using statistical analysis of interactions between proteome and physiological outcome parameters.
In this study, proteomics of liver proteins provided insight into novel and recognized mechanisms relating to lipid degradation, gluconeogenesic pathways and ketone body formation. Altered expression of acyl CoA thioester hydrolase, enolase, pyruvate dehydrogenase, glycerol-3-phosphate dehydrogenase, fructose 1,6-bisphophatase, phosphoenolpyruvate carboxykinase and hydroxymethylglutaryl CoA synthase proteins explained most of the anti-diabetic treatment effects of cis9, trans11-CLA and the pro-diabetic effects of trans10, cis12-CLA in the apoE knockout mice. For the first time, we showed that cis9, trans11-CLA specifically induces an increased expression of the anti-inflammatory HSP 70 kDa, as well as a decreased expression of the proinflammatory macrophage migration inhibitory factor. Both effects could potentially contribute to a less severe inflammatory response, or protection against the development of atherosclerosis upon consumption of cis9, trans11 CLA.
Correlation analysis again emphasized the divergent effects of both CLA isomers on specific metabolic pathways, but also revealed a novel linkage between insulin resistance and increased hepatic serotransferrin protein. Thus, our integrative approach combining physiological and proteomics outcomes (Fig. 1)
revealed many associations, some of which are predictable, confirming the validity of our approach, while others provide intriguing new leads for further studies into the mechanisms by which individual CLA isomers differentially affect pathways related to atherogenesis.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3953fje; doi: 10.1096/fj.05-3953fje.
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  J. M. Arbones-Mainar, M. A. Navarro, S. Acin, M. A. Guzman, C. Arnal, J. C. Surra, R. Carnicer, H. M. Roche, and J. Osada Trans-10, cis-12- and cis-9, trans-11-Conjugated Linoleic Acid Isomers Selectively Modify HDL-Apolipoprotein Composition in Apolipoprotein E Knockout Mice J. Nutr., February 1, 2006; 136(2): 353 - 359. [Abstract] [Full Text] [PDF]
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