Martin D. Reid

Divergent mechanisms of cis9, trans11-and trans10, cis12-conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach

Conjugated linoleic acids (CLA) affect atherogenesis, but mechanisms are not well understood. We explored how two isomers of CLA, cis9, trans11‐CLA and trans10, cis12‐CLA, affected lipid and glucose metabolism, as well as hepatic protein expression, in apolipoprotein E knockout mice. After 12 wk of intervention, plasma triglyceride, NEFA, and glucose concentrations were significantly higher in the trans10, cis12‐CLA group, whereas plasma triglyceride, NEFA, glucose, and insulin concentrations were significantly lower in the cis9, trans 11‐CLA group, compared with control mice consuming linoleic acid. Proteomics identified significant up‐ or down‐regulation of 113 liver cytosolic proteins by either CLA isomer. Principal component analysis revealed that the treatment effect of cis9, trans11‐CLA was mainly explained by the up‐regulation of different posttranslational forms of heat shock protein 70 kD. In contrast, the treatment effect of trans10, cis12‐CLA was mainly explained by up‐regulation of key enzymes in the gluconeogenic, β‐oxidation, and ketogenesic pathways. Correlation analysis again emphasized the divergent effects of both CLA isomers on different pathways, but also revealed a linkage between insulin resistance and increased levels of hepatic serotransferrin. Thus, our systems biology approach provided novel insights into the mechanisms by which individual CLA isomers differentially affect pathways related to atherogenesis, such as insulin resistance and inflammation. Baukje De Roos, Garry Rucklidge, Martin Reid, Karen Ross, Gary Duncan, Maria A. Navarro, Jose M. Arbones‐Mainar, Mario A. Guzman‐Garcia, Jesus Osada, John Browne, Christine E. Loscher, Helen M. Roche Divergent mechanisms of cis9, trans11‐ and trans10, cis12‐conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach. FASEB J. 19, 1–21 (2005)

Response of apolipoprotein E*3-Leiden transgenic mice to dietary fatty acids: combining liver proteomics with physiological data

Dietary fatty acids have a profound impact on atherosclerosis, but mechanisms are not fully understood. We studied the effects of a saturated fat diet supplemented with fish oil, trans10,cis12 conjugated linoleic acid (CLA), or elaidic acid on lipid and glucose metabolism and liver protein levels of APOE*3 Leiden transgenic mice, a model for lipid metabolism and atherosclerosis. Fish oil lowered plasma and liver cholesterol and triglycerides, plasma free fatty acids, and glucose but increased plasma insulin. CLA lowered plasma cholesterol but increased plasma and liver triglycerides, plasma β‐hydroxybutyrate, and insulin. Elaidic acid lowered plasma and liver cholesterol. Proteomics identified significant regulation of 65 cytosolic and 8‐membrane proteins. Many of these proteins were related to lipid and glucose metabolism, and to oxidative stress. Principal component analysis revealed that fish oil had a major impact on cytosolic proteins, and elaidic acid on membrane proteins. Correlation analysis between physiological and protein data revealed novel clusters of correlated variables, among which a metabolic syndrome cluster. The combination of proteomics and physiology gave new insights in mechanisms by which these dietary fatty acids regulate lipid metabolism and related pathways, for example, by altering protein levels of long‐chain acyl‐CoA thioester hydrolase and adipophilin in the liver.

Identification of potential serum biomarkers of inflammation and lipid modulation that are altered by fish oil supplementation in healthy volunteers.

Long chain n‐3 polyunsaturated fatty acids (n‐3 LCPUFA) lower risk of coronary heart disease (CHD), but mechanisms are not well understood. We used proteomics to identify human serum proteins that are altered by n‐3 LCPUFA. Such proteins could identify pathways whereby they affect CHD. Eighty‐one healthy volunteers entered a double blind randomised trial to receive 3.5 g of fish oil or 3.5 g of high oleic sunflower oil daily. Serum was collected before and after 6 wk of intervention. Serum was analysed by proteomics using 2‐DE. Proteins that were differentially regulated were identified by MS. We also analysed serum apolipoprotein A1 (apo A1), high‐density lipoprotein (HDL) particle size and haptoglobin. Serum levels of apo A1, apo L1, zinc‐α‐2‐glycoprotein, haptoglobin precursor, α‐1‐antitrypsin precursor, antithrombin III‐like protein, serum amyloid P component and haemopexin were significantly downregulated (all p<0.05) by fish oil compared with high oleic sunflower oil supplementation. Fish oil supplementation caused a significant shift towards the larger, more cholesterol‐rich HDL2 particle. The alterations in serum proteins and HDL size imply that fish oil activates anti‐inflammatory and lipid modulating mechanisms believed to impede the early onset of CHD. These proteins are potential diagnostic biomarkers to assess the mechanisms whereby fish oil protects against CHD in humans.

ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

Attenuation of inflammation and cellular stress-related pathways maintains insulin sensitivity in obese type I interleukin-1 receptor knockout mice on a high-fat diet.

The development of insulin resistance in the obese is associated with chronic, low‐grade inflammation. We aimed to identify novel links between obesity, insulin resistance and the inflammatory response by comparing C57BL/6 with type I interleukin‐1 receptor knockout (IL‐1RI−/−) mice, which are protected against diet‐induced insulin resistance. Mice were fed a high‐fat diet for 16 wk. Insulin sensitivity was measured and proteomic analysis was performed on adipose, hepatic and skeletal muscle tissues. Despite an equal weight gain, IL‐1RI−/− mice had lower plasma glucose, insulin and triacylglycerol concentrations, compared with controls, following dietary treatment. The higher insulin sensitivity in IL‐1RI−/− mice was associated with down‐regulation of antioxidant proteins and proteasomes in adipose tissue and hepatic soluble epoxide hydrolase, consistent with a compromised inflammatory response as well as increased glycolysis and decreased fatty acid β‐oxidation in their muscle. Their lower hepatic triacylglycerol concentrations may reflect decreased flux of free fatty acids to the liver, decreased hepatic fatty acid‐binding protein expression and decreased lipogenesis. Correlation analysis revealed down‐regulation of classical biomarkers of ER stress in their adipose tissue, suggesting that disruption of the IL‐1RI‐mediated inflammatory response may attenuate cellular stress, which was associated with significant protection from diet‐induced insulin resistance, independent of obesity.

Blood Folate Status and Expression of Proteins Involved in Immune Function, Inflammation, and Coagulation: Biochemical and Proteomic Changes in the Plasma of Humans in Response to Long-Term Synthetic Folic Acid Supplementation

We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

The response of human colonocytes to folate deficiency in vitro: functional and proteomic analyses.

Low folate intake is associated with colon cancer. We combined a proteomics and biochemical approach to identify proteins and pathways affected by folate deficiency in human colonocytes. Folate differentially altered activity and expression of proteins involved in proliferation [e.g., PCNA], DNA repair [e.g., XRCC5, MSH2], apoptosis [e.g., BAG family chaperone protein, DIABLO and porin], cytoskeletal organization [e.g., actin, ezrin, elfin], and expression of proteins implicated in malignant transformation [COMT, Nit2].

Disruption of lipid metabolism in the liver of the pregnant rat fed folate-deficient and methyl donor-deficient diets.

The importance of folic acid and the methionine cycle in fetal development is well recognised even though the mechanism has not been established. Since the cycle is active in the maternal liver, poor folate status may modify hepatic metabolism. Pregnant rats were fed diets deficient in folic acid (-F) or in three key methyl donors, folic acid, choline and methionine (-FLMLC) and the maternal liver was analysed on day 21 of gestation. Two-dimensional gel electrophoresis of soluble proteins identified differentially abundant proteins, which could be allocated into nine functional groups. Five involved in metabolic processes, namely, folate/methionine cycle, tyrosine metabolism, protein metabolism, energy metabolism and lipid metabolism, and three in cellular processes, namely, endoplasmic reticulum function, bile production and antioxidant defence. The mRNA for sterol regulatory element-binding protein-1c and acetyl-CoA carboxylase-1 (fatty acid synthesis) were decreased by both -F and -FLMLC diets. The mRNA for PPARalpha and PPARgamma and carnitine palmitoyl transferase (fatty acid oxidation) were increased in the animals fed the -FLMLC diets. Changes in the abundance of proteins associated with intracellular lipid transport suggest that folate deficiency interferes with lipid export. Reduced fatty acid synthesis appeared to prevent steatosis in animals fed the -F diet. Even with increased oxidation, TAG concentrations were approximately three-fold higher in animals fed the -FLMLC diet and were associated with an increase in the relative abundance of proteins associated with oxidative stress. Fetal development may be indirectly affected by these changes in hepatic lipid metabolism.

Intervention with fish oil, but not with docosahexaenoic acid, results in lower levels of hepatic soluble epoxide hydrolase with time in apoE knockout mice.

Long-chain n-3 PUFA from fish oil protect against death from CHD but mechanisms are not well understood. Preliminary results indicate that fish oil may affect the enzyme soluble epoxide hydrolase (sEH) and influence inflammatory pathways in a time-dependent manner. In the present study male apoE knockout (Apoe-/-) mice were randomised to three dietary groups receiving a high-fat high-cholesterol diet supplemented with 2 % (w/w) high-oleic acid sunflower-seed (HOSF) oil, DHA oil or fish oil. Livers and proximal aortas were collected on day 2 and on weeks 1, 2, 4 and 10 to determine hepatic sEH levels, hepatic fatty acid composition, hepatic proteome and atherosclerotic plaque size in the aortic root. Intervention with fish oil, but not with DHA, resulted in significantly lower levels of hepatic sEH levels with time compared with HOSF oil. DHA and fish oil caused differential regulation of thirty-five hepatic proteins which were mainly involved in lipoprotein metabolism and oxidative stress. All mice developed atherosclerosis without differences in plaque size between the three groups. Thus EPA may be responsible for lowering levels of hepatic sEH and both fish oil and DHA could beneficially affect lipoprotein metabolism and oxidative stress.

Alperujo extract, hydroxytyrosol, and 3,4‐dihydroxyphenylglycol are bioavailable and have antioxidant properties in vitamin E‐deficient rats—a proteomics and network analysis approach

SCOPE Olive products are rich in phenolic compounds, which are natural antioxidants in vitro. We tested the in vivo effects of alperujo, an olive production by-product, as well as hydroxytyrosol and 3,4-dihydroxyphenylglycol (DHPG) isolated from alperujo, on indices and pathways of oxidative and metabolic stress in a vitamin E-deficient rat model. METHODS AND RESULTS Rats were fed a vitamin E-deficient diet for 10 weeks, followed by this diet supplemented with either 100 mg/kg diet dα-tocopherol, alperujo extract, hydroxytyrosol, or 10 mg/kg diet DHPG, for a further 2 weeks. We detected alperujo phenolics in tissues and blood, indicating they are bioavailable. Alperujo extract partially ameliorated elevated plasma levels of thiobarbituric acid reactive substances and also lowered plasma cholesterol levels, whereas hydroxytyrosol increased plasma triglyceride levels. Proteomics and subsequent network analysis revealed that hepatic mitochondrial aldehyde dehydrogenase (ALDH2), of which protein and activity levels were regulated by dα-tocopherol and olive phenolics, represents a novel central regulatory protein hub affected by the dietary interventions. CONCLUSION The in vivo free radical scavenging properties of olive phenolics appear relatively modest in our model. But alternative mechanisms, including regulation of ALDH2, may represent relevant antioxidant mechanisms by which dietary olive phenolics could have beneficial impact on cardiovascular health.