Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involving activation of peroxisome proliferator-activated receptors (PPARs) and proliferation of mitochondria. This study aimed to penetrate the effect of TTA on the development of atherosclerotic lesions in apolipoprotein (apo)-E-/- mice fed a high-fat diet containing 0.3% TTA for 12 weeks. These mice displayed a significantly less atherosclerotic development vs control. Plasma cholesterol was increased by TTA administration and triacylglycerol (TAG) levels in plasma and liver were decreased by TTA supplementation, the latter, probably due to increased mitochondrial fatty acid oxidation and reduced lipogenesis. TTA administration also changed the fatty acid composition in the heart, and the amount of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) was reduced and increased, respectively. The heart mRNA expression of inducible nitric oxidase (NOS)-2 was decreased in TTA-treated mice, whereas the mRNA level of catalase was increased. Finally, reduced plasma levels of inflammatory mediators as IL-1α, IL-6, IL-17, TNF-α and IFN-γ were detected in TTA-treated mice. These data show that TTA reduces atherosclerosis in apoE-/- mice and modulates risk factors related to atherosclerotic disorders. TTA probably acts at both systemic and vascular levels in a manner independent of changes in plasma cholesterol, and triggers TAG catabolism through improved mitochondrial function.
Citation: Vik R, Busnelli M, Parolini C, Bjørndal B, Holm S, Bohov P, et al. (2013) An Immunomodulating Fatty Acid Analogue Targeting Mitochondria Exerts Anti-Atherosclerotic Effect beyond Plasma Cholesterol-Lowering Activity in apoE-/- Mice . PLoS ONE 8(12): e81963. https://doi.org/10.1371/journal.pone.0081963
Editor: Andrej A. Romanovsky, St. Joseph's Hospital and Medical Center, United States of America
Received: August 27, 2013; Accepted: October 18, 2013; Published: December 4, 2013
Copyright: © 2013 Vik et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from NordForsk, grant no. 070010, MitoHealth; the Research Council of Norway, grant no. 190287/110; and the European Community's Seventh Framework Programme (FP7/2007-2013) AtheroRemo, grant no. 201668. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interest exist.
Atherosclerosis is a complex vascular disease with a bidirectional interaction between lipids and inflammation as a major feature. These interactions involve monocytes/macrophages, T cells, vascular smooth muscle cell (SMC) and endothelial cells, and in addition to inflammation and lipid deposition, matrix remodelling is an important characteristic of the atherosclerotic lesion . The liver, which is a central regulator of fatty acid and glucose metabolism, is also involved in an atherosclerosis development and systemic low-grade inflammation characterizing this disorder, as demonstrated by the increased subclinical atherosclerosis in patients with non-alcoholic fatty liver disease (NAFLD) . Additionally, there is growing evidence supporting a role of mitochondrial dysfunction that also involves enhanced oxidative stress in the pathogenesis of atherosclerosis [3,4]. Despite state-of-the-art cardiovascular treatment including the use of statins, 38% of all deaths in North America is related to atherosclerosis, and it is the most common cause of death in European men over 45 and in woman over 65 years of age . The growing epidemic of obesity and diabetes associated with a metabolic phenotype characterized by dyslipidemia may further contribute to an increased risk of cardiovascular disease. These factors force us to consider new strategies for prevention and treatment of atherosclerotic disorders. Although statins have been shown to possess anti-inflammatory properties, modulation of the non-resolving inflammation that characterizes the atherosclerotic process is still a therapeutic challenge.
Tetradecylthioacetic acid (TTA) is a modified saturated fatty acid (SFA) analogue made of 16 carbons, plus a sulphur atom inserted in the 3-position from the carboxylic end. Following cellular uptake, TTA is converted to TTA-CoA , which is a good substrate for the carnitine palmitoyltransferase system (CPT1 and CPT2) , transporting acyl-CoA into the mitochondria. However, due to the sulphur atom at the 3 position TTA is blocked for β-oxidation , and the S-substitution also contribute to the antioxidant effect . TTA stimulates mitochondrial fatty acid oxidation of normal β-oxidizable fatty acids  and induces mitochondrial proliferation in rat hepatocytes . As a pan-ligand for members of the peroxisome proliferator-activated receptor (PPAR) family of nuclear hormone receptors, TTA regulates expression of fatty acid metabolizing enzymes, in particular those of the catabolic pathway [12–14]. TTA is reported to have both cholesterol- and triacylglycerol (TAG) lowering effect in in psoriasis patients . The increased mitochondrial fatty acid oxidation and mitochondrial proliferation seem to be important in regulating plasma TAG level [11,12]. Moreover, TTA is an antioxidant  with immunomodulating properties, able to lower plasma level of tumor necrosis factor (TNF)-α, vascular cell adhesion molecule (VCAM)-1 and interleukin (IL)-8 in vitro  as well as in vivo . The anti-inflammatory effects of TTA appear to involve both PPARα-dependent and –independent pathways . Finally, TTA is found to be present in the coronary wall , suggesting a potential for a direct effect on coronary atherosclerosis.
Based on its hypolipidemic properties as well as its anti-oxidative and anti-inflammatory potential involving mitochondrial related mechanisms, we examined the ability of TTA to modulate atherosclerotic lesion development through an intervention study in apolipoprotein E-deficient (apoE-/-) mice. ApoE-/- mice are a widely recognized model for hypercholesterolemia that spontaneously develops atherosclerotic lesions, even on a standard chow diet with low fat and no cholesterol  and have been used to study the effect of pharmacological or dietary treatments on atherosclerosis development .
Materials and Methods
Animals and diets
The animal study was conducted according to the national (D.L. 116, G.U. Suppl. 40, February 18, 1992, Circolare No. 8, G.U July 1994) and international laws and policies (EEC Council Directive 86/609, OJL 358, 1, December 12, 1987: Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996). The Italian Ministry of Health approved the protocol. Mice were kept under standard laboratory conditions with temperature 22±1°C, dark/light cycles of 12/12 h, relative humidity 55±5% and 20 air changes per hour. Food and tap water was given ad libitum.
Twenty-four female apoE -/- mice (North Carolina, Charles River Laboratories, Italy), 8 weeks old, were randomly divided, based on body weight, into two groups of 12 animals. Mean cholesterol levels pre-treatment were not different between groups (8.16±0.80 vs 7.73±0.62 mmol/L, p=0.429). The control group was fed a high-fat diet (23.7% w/w) consisting of 21.3% lard (a generous gift from Ten Kate Vetten BV, Musselkanaal, Netherlands) and 2.4% soy oil (Dyets. Inc., Betlehem, PA, USA), with 25% w/w casein (Dyets Inc., Bethlehem, PA, USA) as protein source, while the intervention diet was supplemented with 0.3% w/w TTA, and TTA was synthesized as previously described . The energy contribution from fat, carbohydrates, and protein were 46.9%, 32.8% and 20.3%, respectively and the full composition of the diets is given in Table S1. Mice had free access to tap water and feed during the 12 weeks of feeding.
Blood and tissue harvesting
Day 0 and after 28, 56 and 77 days of dietary treatment, blood was collected from the retro-orbital plexus into tubes containing 0.1% (w/v) EDTA after an overnight fasting. Blood samples were chilled on ice for at least 15 minutes, centrifuged and stored at -80°C prior to analysis. After 84 days of treatment, mice were anesthetized with 2% isoflurane (Forane, from Abbot Laboratories Ltd, Illinois, USA) and blood was removed by perfusion with phosphate-buffered saline (PBS). Aorta was rapidly dissected from the aortic root to the iliac bifurcation, periadventitial fat was removed and aorta was pinned flat on a black wax surface in ice-cold PBS and photographed. After photographing, aorta was immediately put in a tissue-freezing medium, snap-frozen in liquid nitrogen and stored at -80°C. For histological/immunohistochemical analysis, six hearts from each group were removed, fixed in 10% formalin for 30 min and transferred into PBS containing 20% sucrose (w/v) overnight at 4°C before being embedded in OCT compound (Sakura Finetek Euope B.V., Alphen aan den Rijn, The Netherlands) and stored at -80°C. An equal subset of hearts was dedicated to qPCR analysis and immediately snap-frozen in liquid nitrogen. Livers from all animals were harvested, and a portion was embedded in OCT compound. The embedded portion and the remaining tissue were snap-frozen and stored at -80°C until further processing.
En face analysis
Images of the aorta were captured with a stereomicroscope-dedicated camera (IC80 HD camera, MZ6 microscope, Leica Microsystems, Germany), and analyzed with ImageJ image processing program (http://rsb.info.nih.gov/ij/). An operator blinded to dietary treatment quantified atherosclerosis.
Aortic sinus and liver histology/immunohistochemistry
Serial cryosections (7 μm thick) of the aortic sinus were cut and stained with hematoxylin and eosin (Bio-Optica, Milano, Italy) to evaluate plaque area and with Masson’s Trichrome (04-010802, Bio-Optica, Milano, Italy) to evaluate extracellular matrix deposition. Oil red O staining was used to detect intraplaque neutral lipids (Sigma-Aldrich, St. Louis, MO, USA). Seven μm thick sections were also cut from the liver and stained with Red Oil O.
Macrophages and lymphocytes were detected using an anti-F4/80 antibody (ab6640, Abcam, Cambridge, UK), and an anti-CD3 antibody (ab16669, Abcam, Cambridge, UK), respectively. A biotinylated secondary antibody was used for streptavidine-biotin-complex peroxidase staining (Vectastain Abc Kit, Vector Laboratories, Peterborough, UK). 3,3’-Diaminobenzidine (DAB) was used as chromogen (Sigma-Aldrich, St. Louis, MO, USA), and sections were counterstained with hematoxylin (Gill’s Hematoxylin, Bio-Optica, Milano, Italy).
The Aperio ScanScope GL Slide Scanner (Aperio Technologies, Vista, CA, USA), equipped with a Nikon 20x/0.75 Plan Apochromat objective producing a 0.25 μm/pixel scanning resolution and the Aperio ImageScope software (version 22.214.171.1243), was used to acquire and process digital images with a 40x magnification. An operator blinded to dietary treatment quantified the plaque area, extracellular matrix and lipid composition, as well as inflammatory cell infiltrate.
Plasma and liver lipids and fatty acid composition in heart and plasma
Liver lipids were extracted according to Bligh and Dyer , evaporated under nitrogen, and redissolved in isopropanol before analysis. Lipids from liver extracts or plasma were then measured enzymatically on a Hitachi 917 system (Roche Diagnostics GmbH, Mannheim, Germany) using the TAG (GPO-PAP), cholesterol (total, free, LDL and HDL) kit (CHOD-PAP) from Roche Diagnostics, phospholipid kit from bioMérieux SA (Marcy l'Etoile, France) and non-esterified fatty acid (NEFA) kit from DiaSys Diagnostic Systems GmbH (Holzheim, Germany). Total plasma fatty acid composition in heart and plasma was analyzed as described previously .
Hepatic enzyme activities
Livers were homogenized and a post-nuclear fraction made as described earlier . The activity of carnitine palmitoyltransferase (CPT)-1 was performed according to Bremer , but with some modifications: the reaction mix contained 17.5 mM HEPES pH 7.5, 52.5 mM KCl, 5 mM KCN, 100 mM palmitoyl-CoA, and 6.67 mg BSA/mL. 100 μM [methyl-14C]-L-carnitine (1100 cpm/ηmol) inititated the reaction, and 30 μg total protein was used. The assay conditions for CPT-2 were identical except that BSA and KCN was omitted and 0.01% Triton X-100 was included, and a total of 35 μg was used.
Acyl-CoA oxidase (ACOX)-1 activity was measured using 20 μg protein, as described by Madsen et al .. 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA) were measured in the post-nuclear fraction as described by Skorve et al. , and citrate synthase activity was measured as previously described .
Gene expression analysis
Total cellular RNA was extracted from liver, homogenates from the whole heart and from pooled aortic arch segments, and cDNA was produced as previously described . Quantitative real-time PCR was performed on an ABI prisme 7900 H sequence detection system from Applied Biosystems with Sarstedt 384 well multiply-PCR Plates (Sarstedt Inc., Newton, NC, USA) using probes and primers from Applied Biosystems. The primers used are listed in Table S2. Six different reference genes were included for liver and heart, and three for aorta: 18s (Kit-FAM-TAMRA (Reference RT-CKFT-18s)) from Eurogentec, Belgium, ribosomal protein, large, P0 (Rplp0, Gene ID 11837), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1, AX-045271-00), ribosomal protein, large, 32 (Rpl32, AX-055111-00), polymerase II (DNA directed) polypeptide A, (Polr2a, AX-046005-00) and TATA-box binding protein (Tbp, AX-041188-00) from Thermo Fisher Scientific. GeNorm were used to evaluate the reference genes , and data normalized to Rplp0 and Rpl32 are presented for liver, Hprt1 for heart and 18S, Rplp0 and Hprt1 for aortic arch segment.
Measurements of inflammatory markers
Levels of IL-1α, IL-1β, IL-6, IL-10, IL-17, interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1 and TNF-α were analyzed on plasma samples collected at day 77 after treatment by Multiplex suspension technology using a customized Bio-Plex Pro Mouse assay (Bio-Rad Laboratories, Hercules, CA).
Data sets were analyzed using Prism Software (GraphPad Prism version 5.0; GraphPad Prism, San Diego, CA, USA) to determine statistical significance. The results are shown as means of 3 to 12 animals per group with their standard deviations. Normal distribution was determined by the Kolmogorov-Smirnov test (with Dallal-Wilkinson-Lilliefors P value). Unpaired t-test was performed to evaluate statistical differences between two groups, or Mann Whitney test when values were not normally distributed. P-values <0.05 were considered significant.
TTA effects on atherosclerosis development
After treatment, mice fed TTA displayed the same body weight as controls (Figure 1A). TTA diet administration significantly inhibited the development of atherosclerotic lesions at the aortic arch compared with the control diet (Figure 1B). A lower atherosclerotic development was also observed in TTA-treated mice at the thoracic and abdominal aorta without reaching statistical significance (Figure 1B). However, considering the entire aortic surface, TTA-treatment significantly inhibited atherosclerosis development (0.39 ± 0.22 x 106 μm2 in TTA-mice vs 1.09 ± 0.50 x 106 μm2 in controls; p=0.016, Figure 1B). In addition, a tendency towards a decrease in lesion area was observed at the aortic sinus of TTA mice compared with control mice (1.49 ± 0.30 x 105 μm2 vs. 2.02 ± 0.31 x 105 μm2, respectively; p=0.068; Figure 2A-C). In contrast to the decrease in lesion size, the histological/immunohistochemical characterization of atherosclerotic lesions did not show any significant difference in plaque composition between controls and TTA mice, displaying a comparable percentage of extracellular matrix (30.31 ± 18.25 % vs. 29.75 ± 15.89 %; Figure 2D-F), lipids (79.68 ± 6.45 % vs. 71.17 ± 6.97 %; Figure 2G-I), macrophages (60.47 ± 3.71 % vs. 57.13 ± 2.75 %; Figure 2J-L) and lymphocytes (22.62 ± 7.24 % vs. 27.26 ± 9.05 %; Figure 2M-O).
After 12 weeks of diet induction, TTA-fed mice displayed the same growth curve as controls (A). Whole aorta was collected and en-face analysis was performed to quantify aortic surface covered by atherosclerotic plaques (B). Data are shown as means ± SD for 6-12 mice for each diet. Unpaired t-test was used to detect statistical significance. *P<0.05 vs. control.
Representative photomicrographs and quantification of maximum plaque area (panels A-C). Representative photomicrographs and quantification of extracellular matrix deposition (panels D-F), lipid deposition (panels G-I), macrophages (panels J-L) and T lymphocytes (panels M-O). Bar in panel A = 100 μm. Data are shown as means ± SD for 6 mice for each diet.
TTA effects on genes related to inflammation and oxidative stress in heart and aorta
Enhanced inflammation and oxidative stress seem to be recurring factors in atherogenesis. Therefore it was of interest to observe that the gene expression of inducible nitric oxidase (Nos2) was significantly decreased in the heart of TTA-treated apoE-/- mice compared to controls (Figure 3A), whereas gene expression of the key antioxidant enzyme, catalase (Cat) was significantly increased (Figure 3B). The mRNA expression of Vcam1 and intracellular adhesion molecule 1 (Icam1) in the heart tended to be decreased by TTA administration, but the differences did not reach statistical significance (Figure 3C, D). The gene expression of the inflammatory mediators Mcp1 and Tnfa, and the antioxidants soluble superoxide dismutase (Sod1) and mitochondrial superoxide dismutase (Sod2) were unchanged (data not shown). The mean mRNA level of Icam1, Vcam1 and Mcp1 decreased in pooled aortic arch from six animals, whereas mRNA Nos2 levels seemed to be unchanged by the treatment (Figure 3E).
A) Nos2, B) Catalase, C) Icam1, D) Vcam1 and E) Aortic arch. Bars represent means ± SD for 3-4 mice for each diet. Mann-Whitney test was used to detect statistical significance and results significantly different from control are indicated **P<0.01.
Effect of TTA on systemic inflammation
In addition to its potential anti-inflammatory effect within the aortic arch, TTA administration decreased plasma levels of several inflammatory cytokines and chemokines in apoE-/- mice. As shown in Figure 4, IL-1α, IL-6, IL-17, TNF-α and IFN-γ levels were significantly lower in TTA-treated mice compared to controls. IL-6, IL-17, TNF-α and IFN-γ levels significantly and directly correlated with mouse aortic plaque area (r = 0.70, p=0.011; r = 0.74, p=0.006; r = 0.68, p=0.015; r = 0.64, p=0,027, respectively). No significant changes of IL-1β, IL-10, and MCP-1 could be observed after TTA administration (data not shown).
Blood samples collected at day 77 of treatment were analysed A) IL-1α, B) IL-6, C) IL-17, D) TNF-α and E) IFN-γ. Bars represent means ± SD for 4 mice for each diet. Unpaired t-test was used to assess statistical significance and results significantly different from control are indicated *P<0.05, **P<0.01, ***P<0.001.
Effect of TTA on lipid concentrations in plasma
The plasma concentrations of TAGs and NEFAs were significantly reduced in the TTA-treated mice compared to the control group already after 28 days of treatment (Figure 5A, B). Interestingly, plasma total and free cholesterol levels were increased (Figure 5C, D), as well as levels of HDL- and LDL-cholesterol (Figure 5E, F) whereas the plasma phospholipid concentration and the ratio of HDL/LDL-cholesterol were unaffected by TTA administration compared to the controls (data not shown).
After 28, 56 and 77 days of diet induction, blood was collected, EDTA plasma was isolated and plasma lipids were determined by enzymatic assay. A) Triacylglycerols, B) Non-esterified fatty acids, C) Cholesterol, D) Free cholesterol, E) High-density lipoprotein cholesterol and F) Low-density lipoprotein cholesterol. Bars represent means ± SD for 4 mice for each diet. Unpaired t-test was used to detect statistical significance and results significantly different from control are indicated *P<0.05, **P<0.01, ***P<0.001.
Effect of TTA on fatty acid composition in heart and plasma
Dietary supplementation with 0.3% TTA promoted different effects on heart and plasma fatty acid composition in apoE-/- mice fed a high-fat diet (Table 1). While TTA had no effect in the relative amounts of saturated fatty acids (SFA) in heart, it reduced the weight % of SFA in plasma, mainly as a consequence of reduced due amount of stearic acid (C18:0) (Table 1). In contrast, TTA had no significant effect on the relative amount of monounsaturated fatty acid in heart or plasma (Table 1). Δ9 desaturated TTA in addition to TTA was found in heart and plasma (Table 1) demonstrating an activity of SCD1. TTA significantly increased the relative amounts of n-9 polyunsaturated fatty acids (PUFAs) in both heart and plasma, mainly because of increased relative amounts of mead acid (C20:3n-9) (Table 1). While TTA induced a decrease in n-3 PUFAs in plasma, due to decreased relative amounts of C18:3n-3, C18:4n-3, C20:5n-3 and C22:6n-3, the relative amounts of n-3 PUFAs tended to increase during TTA treatment in the heart, due to significantly increased amounts of EPA (C20:5n-3) and DPA (C22:5 n-3) (Table 1). TTA decreased the weight % of n-6 PUFAs in heart, mainly reflecting a reduction in the prostaglandin precursor arachidonic acid (20:4n-6). TTA increased n-6 PUFAs in plasma and this was due to increased relative amounts of C20:3n-6, C20:4n-6 and C22:4n-6 (Table 1). Altogether, TTA administration for 12 weeks influenced the ratio of n-3 to n-6 PUFAs in two different ways: in heart an increase was observed, whereas in plasma a reduction was found (Figure 6H).
|SFAs||31 ± 0.68||31 ± 0.84||32 ± 0.47||32 ± 0.16|
|C18:0||16 ± 0.59||16 ± 1.11||12 ± 0.20||8.8 ± 0.20***|
|C22:0||0.17 ± 0.01||0.20 ± 0.02||0.40 ± 0.02||0.40 ± 0.03**|
|C23:0||0.05 ± 0.00||0.08 ± 0.01**||0.16 ± 0.02||0.21 ± 0.02**|
|C24:0||0.09 ± 0.01||0.12 ± 0.01*||0.31 ± 0.02||0.36 ± 0.02*|
|TTA||<0.00||32 ± 0.69*||<0.00||33 ± 0.20|
|MUFAs||21 ± 1.9||18 ± 4.4||31 ± 0.42||29 ± 0.81***|
|C18:1n-7||2.0 ± 0.05||2.0 ± 0.04||1.3 ± 0.03||1.2 ± 0.04**|
|n9 PUFAs||0.13 ± 0.00||0.24 ± 0.04*||0.21 ± 0.31||0.41 ± 0.29|
|C18:1n-9||17 ± 1.7||14 ± 3.7||25 ± 0.38||24 ± 0.77*|
|C20:3n-9||0.13 ± 0.00||3.2 ± 0.04*||0.2 ± 0.01||0.4 ± 0.03***|
|TTA:1n-8||<0.00||37 ± 2.4*||<0.00||30 ± 0.81|
|n6 PUFAs||26 ± 0.61||23 ± 1.0*||28 ± 0.37||32 ± 0.82***|
|C20:3n-6||0.66 ± 0.02||1.2 ± 0.18**||0.5 ± 0.01||1.2 ± 0.04***|
|C20:4n-6||8.7 ± 0.37||7.0 ± 0.64*||12 ± 0.4||15 ± 0.9***|
|C22:4n-6||0.77 ± 0.03||0.80 ± 0.11||0.21 ± 0.01||0.28 ± 0.02**|
|n3 PUFAs||23 ± 1.4||26 ± 4.0||6.4 ± 0.01||5.3 ± 0.10***|
|C18:3n-3||0.23 ± 0.01||0.20 ± 0.06||0.60 ± 0.09||0.40 ± 0.02**|
|C18:4n-3||<0.00||<0.00||0.05 ± 0.00||0.01 ± 0.00***|
|C20:5n-3||0.06 ± 0.00||0.11 ± 0.02*||0.5 ± 0.04||0.3 ± 0.00**|
|C22:5n-3||1.7 ± 0.08||3.0 ± 0.57*||0.31 ± 0.02||0.35 ± 0.03*|
|C22:6n-3||21 ± 1.3||23 ± 3.6||5.0 ± 0.3||4.2 ± 0.1**|
|n3/n6||0.88 ± 0.07||1.1 ± 0.12*||0.23 ± 0.01||0.17 ± 0.01*|
In liver extracts, fatty acids were measured using gas-liquid chromotography, enzyme activities were measured spectrophotometrically and lipid deposition was assessed using Red Oil staining. A) Tetradecylthioacetic aicd and B) TTA:1n-8, C) Carnitine palmitoyltransferase 1a activity, D) carnitine palmitoyltransferase 2 E) 3-hydroxy-3-methylglutaryl coenzyme A synthase activity F) Citrate synthase activity, G) Acyl-coenzyme A oxidase activity, H) Lipids droplets and I) Triacylglycerols. Bars represent means ± SD in 4-6 mice for each diet. Unpaired t-test was used to detect statistical significance and results significantly different from control are indicated *P<0.05, **P<0.01, ***P<0.001.
Hepatic gene and enzyme activities
TTA and delta 9 desaturated TTA were also found in liver of TTA-treated mice (Figure 6A, B), indicating that TTA-CoA is a suitable substrate for SCD1. The mRNA level of Scd1, however, was unchanged by TTA administration (Table 2). Under normal condition, fatty acids to the liver can either be oxidized or secreted in the VLDL-particles. Gene expression of Cd36 was up-regulated in TTA-treated mice (Table 2) indicating higher influx of fatty acids to the liver. We found that the activities of both CPT1 and CPT2 were stimulated in the TTA-fed mice compared to controls (Figure 6C, D, respectively), in addition to increased HMG-CoA synthase activity (Figure 6E) demonstrating increased mitochondrial fatty acid oxidation and ketogenesis. This was also associated with higher levels of the mitochondrial biomarker citrate synthase (Figure 6F). Up-regulation of these enzymes, as well as of ACOX1 (Figure 6G) by TTA administration, supports PPARα activation. The mRNA expression of Ppara, however, was unchanged (Table 2). The lipid droplets and the hepatic level of total TAG were decreased in TTA-treated apoE-/- mice (Figure 6H, I). Hepatic TAG biosynthesis and VLDL secretion seemed to be unaffected by TTA treatment as the gene expression of Gpat and ApoB were similar in TTA-treated mice compared to controls (Table 2). The mRNA expression of VLDL receptor (VLDLr) was significantly increased (Table 2). Reduced hepatic TAG content may partly be due to reduced lipogenesis as the mRNA expression of acetyl carboxylase alpha (Acaca) was decreased by TTA administration (Table 2).
|Lipid transport and β-oxidation||Diet groups||P value|
|Apob||Fatty acid transport||1.00 ± 0.09||0.87 ± 0.17||0.501|
|Cd36||Fatty acid import||1.00 ± 0.20||2.23 ± 0.52*||0.045|
|Cpt1a||β-oxidation||1.00 ± 0.27||0.55 ± 0.15||0.252|
|Cpt2||β-oxidation||1.00 ± 0.04||1.18 ± 0.16||0.260|
|Vldlr||Fatty acid import||1.00 ± 0.11||2.46 ± 0.56*||0.029|
|Lipogenesis, desaturation and transcription||Diet groups||P value|
|Acaca||Fatty acid synthesis||1.00 ± 0.08||0.676 ± 0.03||0.203|
|Gpat||Glycerolipid synthesis||1.00 ± 0.13||1.44 ± 0.14||0.075|
|Scd1||Desaturation||1.00 ± 0.14||0.72 ± 0.02||0.161|
|Ppara||Energy homeostasis||1.00 ± 0.20||0.68 ± 0.33||0.463|
TTA is reported to be an important regulator of lipids through PPARs [12,16,31] and to have positive effects on plasma lipid risk factors related to cardiovascular diseases  and most probably to atherosclerosis . Therefore, the ability of TTA to inhibit the development of atherosclerosis in apoE-/- mice fed on a high-fat diet was investigated. Notably, TTA was found to reduce atherosclerotic plaque area in the aortic arch in these mice. In addition, a decrease in lesion area was observed at the aortic sinus of TTA-treated mice, but failed to be significant possibly due to small sample size. However, our results suggest a potential beneficial effect of TTA treatment on plague formation.
The atherosclerotic process is initiated by accumulation of plasma lipoproteins in the subendothelial space of the blood vessel and uptake of these lipoproteins by machrophages will result in their transformation into foam cells . Noteworthy, the plasma cholesterol, free cholesterol, HDL- and LDL-cholesterol levels were increased by TTA treatment, suggesting that effect of TTA on plaque reduction is not related to its cholesterol lowering properties. Inflammation has emerged as an important factor in the development of atherosclerosis characterized by a state of non-resolving inflammation. In contrast to the lack of effect on plasma cholesterol, TTA induced a marked decrease in plasma levels of several inflammatory mediators with relevance to atherogenesis such as IL-1α, IL-6, IL-17, TNF-α and IFN-γ. Moreover, TTA also induced a decrease in the gene expression of Icam1, Vcam1 and Mcp1 within the aortic arch, suggesting anti-inflammatory effects of TTA both systemically and within the lesions. Although we have no firm evidence, it is tempting to hypothesize that the atheroprotective effect of TTA in apoE-/- mice involves both anti-inflammatory and anti-oxidative effects of this fatty acid.
The effects of TTA on fatty acid composition could also be of relevance for the effect of TTA on plaque size. Thus, while TTA treatment resulted in significant increased plasma level of arachidonic acid (C20:4n-6), the precursor for prostaglandins , and a tendency to reduced amount of the anti-inflammatory n-3 PUFAs in plasma, TTA increased the amount of the n-3 PUFAs EPA (C20:5n-3) and DHA (C22:6n-3), and decreased the amount C20:4n-6 within the heart. We have previously shown that TTA is taken up in endothelial cells  and macrophages , and after local delivery reduces the local inflammatory responses and collagen accumulation . TTA inhibits proliferation of smooth muscle cells and has the ability to attenuate TNFα-mediated endothelial cell activity involving both PPARα-dependent and –independent pathways . As previously reported, TTA was taken up in the arterial wall . Thus, its anti-atherosclerotic effect could be a direct action on the vascular cells with anti-inflammatory and antioxidative effects, at least partly related to its effect of the local fatty acid composition.
In normolipidemic rats  and chronic inflammatory mice  the plasma levels of TAGs and NEFAs, as well as the hepatic concentration of TAGs, are regulated by the hepatic mitochondrial activity after TTA treatment. In the present study, the increased mRNA level of Cd36, which may be involved in fatty acid translocase, could result in an increased fatty acid uptake by the liver. The activation of fatty acids to their CoA -esters, catalyzed by long-chain acyl-CoA synthetase, is probably stimulated by TTA administration . Plasma TAG levels depend on the balance between TAG synthesis, degradation and catabolism by the liver, as well as on its intravascular catabolism. We have previously reported that thia fatty acid treatment resulted in a more pronounced decrease in plasma TAGs than the decrease VLDL-secretion, indicating that changes in clearance of VLDL might contribute to the reduction in plasma TAGs . Indeed, the lipoprotein lipase (LPL) mRNA in 3T3-L1 preadipocytes was increased by TTA . Thus, this LPL action may well result in a decrease of plasma TAGs in TTA-treated apoE(-/-) mice. The apoE-/- mice exhibited increased hepatic activities of both CPT1 and CPT2 and a slightly up-regulation of CPT2 could be confirmed at the mRNA level. Moreover, proliferation of mitochondria and peroxisomes seemed to take place in the liver of apoE-/- mice as activities of both citrate synthase and ACOX1 were increased by TTA supplementation. TTA significantly reduced the hepatic TAG level in apoE-/- mice and this is most probably due to increased fatty acid catabolism and decreased lipogenesis as the gene expression of Acaca was reduced. Both TTA-treated mice and control mice displayed similar weight gain during the study, showing that our beneficial effects of TTA are not simply due to suppression of appetite, lack of palatability or blockage of intestinal uptake of dietary fatty acids. Overall, we hypothesize that the triggered TAG metabolism accompanied with increased mitochondrial function in liver may contribute to the beneficial action of TTA in apoE-/- mice.
The present study has some limitations such as the lack of protein data and lack of data on fatty acid composition within the lesion. However, our observations suggest that the modified fatty acid analogue, TTA, inhibits atherosclerosis development in vivo, an effect most likely independent of changes in plasma cholesterol levels, but related to an inflammatory response both at vascular and systemic levels, in addition to an antioxidant effect at the vascular level. This could be related to the effect of TTA on local fatty acid composition as well as its effect on mitochondrial function in the liver. Forthcoming studies should further explore the mechanism by which TTA exerts its anti-atherosclerotic effects. If its athero-protective effects are confirmed in additional studies on experimental atherosclerosis, TTA should be explored as treatment modality in clinical atherosclerosis.
Composition of the diets shown as g/kg diet. The diets were isocaloric and isonitrogenous.
We thank Kari Williams, Liv Kristine Øysæd, Randi Sandvik and Svein Krüger for excellent technical assistance.
Conceived and designed the experiments: BB MB TB. Performed the experiments: RV MB CP TB PB SM GSG. Analyzed the data: RV MB CP BB SH SM GSG FD OKN PA CRS GC RKB. Contributed reagents/materials/analysis tools: SH BH. Wrote the manuscript: RV BB RKB.
- 1. Luis AJ, Mar R, Pajukanta P (2004) Genetics of atherosclerosis. Genomics-- Human Genetics 5: 189-218. doi:https://doi.org/10.1146/annurev.genom.5.061903.175930.
- 2. Thakur ML, Sharma S, Kumar A, Bhatt SP, Luthra K et al. (2012) Nonalcoholic fatty liver disease is associated with subclinical atherosclerosis independent of obesity and metabolic syndrome in asian indians. Atherosclerosis 223; No. 2: 507-511. doi:https://doi.org/10.1016/j.atherosclerosis.2012.06.005. PubMed: 22748277.
- 3. Madamanchi NR, Runge MS (2007) Mitochondrial dysfunction in atherosclerosis. Circ Res 100: No. 4: 460-473. doi:https://doi.org/10.1161/01.RES.0000258450.44413.96. PubMed: 17332437.
- 4. Lakshmi SV, Padmaja G, Kuppusamy P, Kutala VK et al. (2009) Oxidative stress in cardiovascular disease. Indian J Biochem Biophys 46; No. 6: 421-440. PubMed: 20361706.
- 5. Aukrust PI, Halvorsen B, Yndestad A, Ueland T, Øie E (2008) Chemokines and cardiovascular risk. Atherosclerosis, Thrombosis and Vascular Biology 28: 1909-1919. doi:https://doi.org/10.1161/ATVBAHA.107.161240.
- 6. Elholm M, Garras A, Neve S, Tornehave D, Lund TB et al. (2000) Long-chain acyl-coa esters and acyl-coa binding protein are present in the nucleus of rat liver cells. J Lipid Res 41; No. 4: 538-545. PubMed: 10744774.
- 7. Skrede S, Wu P, Osmundsen H (1995) Effects of tetradecylthiopropionic acid and tetradecylthioacrylic acid on rat liver lipid metabolism. Biochem J 305 ( 2): 591-597. PubMed: 7832778.
- 8. Morken T, Bohov P, Skorve J, Ulvik R, Aukrust P et al. (2011) Anti-inflammatory and hypolipidemic effects of the modified fatty acid tetradecylthioacetic acid in psoriasis-a pilot study. Scand J Clin Lab Invest 71; No. 4: 269-273. doi:https://doi.org/10.3109/00365513.2011.559552. PubMed: 21338276.
- 9. Wergedahl H, Berge K, Bohov P, Tronstad KJ, Madsen L et al. (2008) Long term treatment with tetradecylthioacetic acid improves the antioxidant status in obese zucker (fa/fa) rats. Drug Metab Lett, 2: 138-145. PubMed: 19356083.
- 10. Gudbrandsen OA, Dyrøy E, Bohov P, Skorve J, Berge RK (2005) The metabolic effects of thia fatty acids in rat liver depend on the position of the sulfur atom. Chem Biol Interact 155; No. 1-2: 71-81. doi:https://doi.org/10.1016/j.cbi.2005.04.007. PubMed: 15949791.
- 11. Kryvi H, Aarsland A, Berge RK (1990) Morphologic effects of sulfur-substituted fatty acids on rat hepatocytes with special reference to proliferation of peroxisomes and mitochondria. J Struct Biol 103; No. 3: 257-265. doi:https://doi.org/10.1016/1047-8477(90)90044-D. PubMed: 2261310.
- 12. Berge RK, Tronstad KJ, Berge K, Rost TH, Wergedahl H et al. (2005) The metabolic syndrome and the hepatic fatty acid drainage hypothesis. Biochimie 87; No. 1: 15-20. doi:https://doi.org/10.1016/j.biochi.2004.11.011. PubMed: 15733731.
- 13. Madsen L, Garras A, Asins G, Serra D, Hegardt FG et al. (1999) Mitochondrial 3-hydroxy-3-methylglutaryl coenzyme a synthase and carnitine palmitoyltransferase ii as potential control sites for ketogenesis during mitochondrion and peroxisome proliferation. Biochem Pharmacol 57; No. 9: 1011-1019. doi:https://doi.org/10.1016/S0006-2952(99)00004-0. PubMed: 10796071.
- 14. Vaagenes H, Madsen L, Asiedu DK, Lillehaug JR, Berge RK (1998) Early modulation of genes encoding peroxisomal and mitochondrial beta-oxidation enzymes by 3-thia fatty acids. Biochem Pharmacol 56; No. 12: 1571-1582. doi:https://doi.org/10.1016/S0006-2952(98)00193-2. PubMed: 9973177.
- 15. Muna ZA, Gudbrandsen OA, Wergedahl H, Bohov P, Skorve J et al. (2002) Inhibition of rat lipoprotein oxidation after tetradecylthioacetic acid feeding. Biochem Pharmacol 63; No. 6: 1127-1135. doi:https://doi.org/10.1016/S0006-2952(01)00934-0. PubMed: 11931845.
- 16. Dyrøy E, Yndestad A, Ueland T, Halvorsen B, Damås JK et al. (2005) Antiinflammatory effects of tetradecylthioacetic acid involve both peroxisome proliferator-activated receptor alpha-dependent and -independent pathways. Arterioscler Thromb Vasc Biol 25; No. 7: 1364-1369. doi:https://doi.org/10.1161/01.ATV.0000171982.57713.96. PubMed: 15920037.
- 17. Helledie T, Antonius M, Sorensen RV, Hertzel AV, Bernlohr DA, Kølvraa S et al. (2000) Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm. J Lipid Res 41; No. 11: 1740-1751. PubMed: 11060343.
- 18. Pettersen RJ, Muna ZA, Kuiper KK, Svendsen E, Müller F et al. (2001) Sustained retention of tetradecylthioacetic acid after local delivery reduces angioplasty-induced coronary stenosis in the minipig. Cardiovasc Res 52; No. 2: 306-313. doi:https://doi.org/10.1016/S0008-6363(01)00404-7. PubMed: 11684079.
- 19. Smith JD, Breslow JL (1997) The emergence of mouse models of atherosclerosis and their relevance to clinical research. Journal of Internal Medicine 24; No. 2: 99-109.
- 20. Kolovou G, Anagnostopoulou K, Mikhailidis DP, Cokkinos DV (2008) Apolipoprotein e knockout models. Curr Pharm Des 14; No. 4: 338-351. doi:https://doi.org/10.2174/138161208783497769. PubMed: 18289060.
- 21. Spydevold O, Bremer J (1989) Induction of peroxisomal beta-oxidation in 7800 c1 morris hepatoma cells in steady state by fatty acids and fatty acid analogues. Biochim Biophys Acta 1003; No. 1: 72-79. doi:https://doi.org/10.1016/0005-2760(89)90101-X. PubMed: 2713397.
- 22. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37; No. 8: 911-917. doi:https://doi.org/10.1139/o59-099. PubMed: 13671378.
- 23. Bjørndal B, Vik R, Brattelid T, Vigerust NF, Burri L et al. (2012) Krill powder increases liver lipid catabolism and reduces glucose mobilization in tumor necrosis factor-alpha transgenic mice fed a high-fat diet. Metabolism, 61: 1461–72. PubMed: 22538117.
- 24. Berge RK, Flatmark T, Osmundsen H (1984) Enhancement of long-chain acyl-coa hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators. Eur J Biochem 141; No. 3: 637-644. doi:https://doi.org/10.1111/j.1432-1033.1984.tb08239.x. PubMed: 6146524.
- 25. Bremer J (1981) The effect of fasting on the activity of liver carnitine palmitoyltransferase and its inhibition by malonyl-coa. Biochim Biophys Acta; no. 665: 628-631. doi:https://doi.org/10.1016/0005-2760(81)90282-4. PubMed: 7295757.
- 26. Madsen L, Rustan AC, Vaagenes H, Berge K, Dyrøy E et al. (1999) Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference. Lipids 34; No. 9: 951-963. doi:https://doi.org/10.1007/s11745-999-0445-x. PubMed: 10574660.
- 27. Skorve J, al-Shurbaji A, Asiedu D, Björkhem I, Berglund L et al. (1993) On the mechanism of the hypolipidemic effect of sulfur-substituted hexadecanedioic acid (3-thiadicarboxylic acid) in normolipidemic rats. J Lipid Res 34; No. 7: 1177-1185. PubMed: 8371065.
- 28. Rose-Kahn G, Bar-Tana J (1985) Inhibition of lipid synthesis by beta beta'-tetramethyl-substituted, c14-c22, alpha, omega-dicarboxylic acids in cultured rat hepatocytes. J Biol Chem 260; No. 14: 8411-8415. PubMed: 4008497.
- 29. Vigerust NF, Bjorndal B, Bohov P, Brattelid T, Svardal A et al. (2012) Krill oil versus fish oil in modulation of inflammation and lipid metabolism in mice transgenic for tnf-alpha. Eur J Nutr.
- 30. Rekawiecki R, Rutkowska J, Kotwica J (2012) Identification of optimal housekeeping genes for examination of gene expression in bovine corpus luteum. Reprod Biol 12; No. 4: 362-367. doi:https://doi.org/10.1016/j.repbio.2012.10.010. PubMed: 23229008.
- 31. Løvås K, Røst TH, Skorve J, Ulvik RJ, Gudbrandsen OA et al. (2009) Tetradecylthioacetic acid attenuates dyslipidaemia in male patients with type 2 diabetes mellitus, possibly by dual ppar-alpha/delta activation and increased mitochondrial fatty acid oxidation. Diabetes Obes Metab 11; No. 4: 304-314. doi:https://doi.org/10.1111/j.1463-1326.2008.00958.x. PubMed: 19267708.
- 32. Hafstad AD, Khalid AM, Hagve M, Lund T, Larsen TS et al. (2009) Cardiac peroxisome proliferator-activated receptor-alpha activation causes increased fatty acid oxidation, reducing efficiency and post-ischaemic functional loss. Cardiovasc Res 83; No. 3: 519-526. doi:https://doi.org/10.1093/cvr/cvp132. PubMed: 19398469.
- 33. Muna ZA, Doudin K, Songstad J, Ulvik RJ, Berge RK (1997) Tetradecylthioacetic acid inhibits the oxidative modification of low density lipoprotein and 8-hydroxydeoxyguanosine formation in vitro. Arterioscler Thromb Vasc Biol 17; No. 11: 3255-3262. doi:https://doi.org/10.1161/01.ATV.17.11.3255. PubMed: 9409320.
- 34. Stocker R, Keaney JF Jr (2004) Role of oxidative modifications in atherosclerosis. Physiol Rev 84; No. 4: 1381-1478. doi:https://doi.org/10.1152/physrev.00047.2003. PubMed: 15383655.
- 35. Mamalakis G, Kafatos A, Tornaritis M, Alevizos B (1998) Anxiety and adipose essential fatty acid precursors for prostaglandin e1 and e2. J Am Coll Nutr 17; No. 3: 239-243. doi:https://doi.org/10.1080/07315724.1998.10718753. PubMed: 9627909.
- 36. Gjøen T, Kleveland EJ, Moya-Falcón C, Frøystad MK, Vegusdal A et al. (2007) Effects of dietary thia fatty acids on lipid composition, morphology and macrophage function of atlantic salmon (salmo salar l.) kidney. Comp Biochem Physiol B Biochem Mol Biol 148; No. 1: 103-111. doi:https://doi.org/10.1016/j.cbpb.2007.04.021. PubMed: 17572126.
- 37. Pettersen RJ, Salem M, Rotevatn S, Kuiper KK, Larsen TH et al. (2012) Effects of local delivery of tetradecylthioacetic acid within the injured coronary vessel wall. Scand Cardiovasc J 46; No. 6: 366-373. doi:https://doi.org/10.3109/14017431.2012.725477. PubMed: 22934973.
- 38. Madsen L, Guerre-Millo M, Flindt EN, Berge K, Tronstad KJ, et al. (2002) Klimes, B. Staels and R. K. Berge, Tetradecylthioacetic acid prevents high fat diet induced adiposity and insulin resistance. J Lipid Res 43; no. 5, 742-750.
- 39. Burri L, Bjørndal B, Wergedahl H, Berge K, Bohov P et al. (2011) Tetradecylthioacetic acid increases hepatic mitochondrial beta-oxidation and alters fatty acid composition in a mouse model of chronic inflammation. Lipids 46; No. 8: 679-689. doi:https://doi.org/10.1007/s11745-011-3536-2. PubMed: 21479675.
- 40. Raspé E, Madsen L, Lefebvre AM, Leitersdorf I, Gelman L et al. (1999) Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (tta) via pparalpha activation. J Lipid Res 40; No. 11: 2099-2110. PubMed: 10553013.