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Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland and * Unit of Nutrition, Department of Clinical Medicine, Trinity Health Sciences Centre, St. James’s Hospital, Dublin, Ireland
2To whom correspondence should be addressed. E-mail: hmroche{at}tcd.ie.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.03) as a result of increased cellular de novo TAG levels. Chronic Caco-2 cell t10,c12-CLA supplementation, prior to the acute oleic acid (OA) treatment, significantly increased (P = 0.005) the ratio of cellular de novo TAG to de novo PL (cTAG/cPL), to a greater extent than that following chronic linoleic acid (LA) (P = 0.001) or c9,t11-CLA supplementation (P = 0.005). Again, this effect was attributed to increased cellular de novo TAG synthesis. Neither CLA isomer affected the ratio of secreted de novo TAG to de novo PL (sTAG/sPL). No effects on Caco-2 cell apoB synthesis and secretion were observed after acute or chronic CLA treatments. In conclusion, chronic t10,c12-CLA supplementation modulated intestinal TRL metabolism, by increasing cellular de novo TAG synthesis but had no effect on de novo TAG secretion in Caco-2 cells.
KEY WORDS: • conjugated linoleic acid (CLA) • lipid metabolism • triacylglycerol (TAG) • Caco-2 cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The two main CLA isomers are trans-10, cis-12 CLA (t10,c12-CLA) and cis-9, trans-11 CLA (c9,t11-CLA). CLA in animal foodstuffs consists of approximately 80% c9,t11-CLA. However, in commercial vegetable oils the isomers are 43% t10,c12-CLA:43% c9, t11-CLA with other isomers accounting for the additional 14% because of partial hydrogenation during the extraction process (2
).
Feeding diets containing CLA has been associated with marked changes in lipid metabolism. Mice fed diets containing 0.5–1.2% CLA have enhanced feed efficiency, reduced body fat accumulation and increased lean body mass, in a dose-responsive manner that is not due to reduced energy intake (3
–5
). The effects of CLA on body composition appear to be due to reduced fat deposition, increased lipolysis in adipocytes and enhanced fatty acid oxidation in muscle and adipose tissue (6
). The t10,c12-CLA isomer is responsible for the body compositional changes in mice whereby it inhibits lipoprotein lipase (LPL) activity and enhances lipolysis in murine 3T3-L1 adipocytes (2
). A number of animal studies have shown that CLA also improves plasma lipoprotein metabolism (4
,8
) and inhibits the progression of atherosclerosis (9
). However, contrary to the findings in animal studies, one study in humans (n = 10 females, 20–41 y) showed that 3.9 g/d (11.4% c9,t11-CLA and 14% t10,c12-CLA) over 8 wk did not alter body composition or energy expenditure (10
) while transiently reducing circulating leptin concentrations (11
). Fasting lipid and lipoprotein metabolism [cholesterol, LDL-cholesterol, HDL-cholesterol and triacylglycerol (TAG)] were also not altered in that human CLA intervention study (12
). Our group has shown that CLA supplementation [3 g/d, 50:50 t10, c12-CLA:c9,t11-CLA (n = 16; 31.5%:31%)] for 8 wk significantly reduced fasting plasma TAG concentrations compared to those of controls (13
). Plasma TAG metabolism is a dynamic system that reflects fatty acid metabolism in adipose tissue, liver and the small intestine. To date, in vitro and in vivo studies have shown that CLA affects TAG metabolism in adipocytes and hepatocytes. CLA inhibits cellular differentiation, TAG synthesis and accumulation in 3T3-L1 adipocytes (14
, 15
). In vivo, CLA reduces adipose tissue mass by promoting adipocyte fatty acid oxidation (6
) and apoptosis (16
). In the human hepatoma (HepG2) cell line, the t10, c12-CLA isomer suppresses TAG secretion (17
). In vivo, feeding a CLA-rich diet promotes hepatic peroxisome proliferator-activated receptor 
responsive gene expression, which explains the profound effect of CLA on hepatic lipid metabolism (18
).
Although research has been carried out in adipocytes and hepatocytes, to date there is little understanding of the isomer-specific effects of CLA on intestinal TAG metabolism. To address this, we investigated acute and chronic effects of the two major CLA isomers, t10,c12-CLA and c9,t11-CLA, on TAG, phospholipid (PL) and apolipoprotein B (apoB) synthesis and secretion in the colon carcinoma (Caco-2) cell line, a model system for intestinal lipid metabolism. The Caco-2 cell line is an accepted cell model of the human small intestine because it spontaneously differentiates in culture and displays some morphological and physiological characteristics of the human small intestine (19
). In addition, this model has been used to investigate the effects of other fatty acids on human intestinal TAG rich lipoprotein (TRL) metabolism (20
, 21
). Therefore the Caco-2 cell line was used to investigate the effect of acute CLA isomer treatments on intestinal lipid and apoB synthesis and secretion. Additionally, the effect of chronic CLA isomer supplementation on intestinal lipid and apoB synthesis and secretion following acute fatty acid treatments with oleic [18:1(n-9)] or palmitic acid (16:0) were investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Because the Caco-2 cell line was originally derived from an adenocarcinoma (22
) and CLA inhibits proliferation of tumors (23
), caution was required when using this cell line. The acute effects of the CLA isomers t10,c12-CLA and c9,t11-CLA were determined in Caco-2 cells that had been grown for 19 d. Bovine serum albumin (BSA) was used as a general control and linoleic acid (LA) was used as a control for (n-6) PUFA effects. In the acute study, cells were incubated for 22 h (with BSA or BSA plus LA, t10,c12-CLA or c9,t11-CLA) before harvesting the cells and basolateral media. 14C-glycerol was used to assess de novo TAG and PL synthesis and 35S-methionine was used to measure de novo apolipoprotein B100 (apoB100) and apolipoprotein B48 (apoB48) synthesis.
To investigate the acute-after-chronic effect of fatty acid supplementation on Caco-2 cell lipoprotein metabolism, the cells received the chronic fatty acid supplementation, followed by the acute fatty acid test. The acute-after-chronic experiment was carried out in Caco-2 cells that had been grown without supplements (controls), or in the presence of 0.05 mmol/L LA, t10,c12-CLA or c9,t11-CLA for 19 d. Previous investigation by our group showed that t10,c12-CLA supplementation inhibited tight junction formation in Caco-2 cells for up to 14 d postseeding (24
). However, by 19 d postseeding, Caco-2 cell proliferation and development were not affected by CLA supplementation. Therefore, all indices of TRL metabolism were measured in cells 19 d postseeding. Then, acute fatty acid treatment with BSA (control) or BSA plus palmitic acid (PA) or oleic acid (OA) for 22 h was used to assess the effects of chronic fatty acid supplementation on de novo TAG, PL (14C-glycerol) and apoB (35S-methionine) metabolism following acute treatment. For simplicity, acute BSA or fatty acid doses are referred to as treatments and chronic fatty acid interventions are referred to as supplements.
Caco-2 cell culture
An adherent colon carcinoma cell line (Caco-2) was obtained from the European Collection of Animal Cell Cultures (ECACC), and the cells were grown from passages 33 to 47. Cells were grown in either 75 or 125-cm2 flasks (Falcon, Lincoln Park, NJ) in media consisting of Dulbecco’s modified Eagle’s medium (DMEM; high glucose, with glutimax and 4500 mg/L D-glucose; GibcoBRL, Paisley, UK), 10% fetal calf serum (FCS; GibcoBRL), 1x sodium pyruvate (GibcoBRL) and 0.1 g/L gentimicin (GibcoBRL), at 37°C in a humidified atmosphere of 5% CO2 until confluence was attained and were split by using trypsin–EDTA solution (GibcoBRL). Cells were seeded at a density of 3 x 105 cells per 24 mm polycarbonate transwells (growth area: 4.7 cm2; pore size: 0.4 µm; Costar, Bucks, UK) and grown for 19 d. Apical and basolateral media were changed every second day during the course of cell growth and development.
Acute effects of LA and CLA
The acute effects of LA, t10,c12-CLA and c9,t11-CLA on de novo TRL metabolism were determined in Caco-2 cells that had been grown in media for 19 d. Cells were washed in 1x PBS (GibcoBRL). To determine lipid concentrations, the acute fatty acid treatment was administered by adding 2.5 mL of FCS-free media to each basolateral compartment and 1.5 mL of FCS-free media containing one of four acute treatments [5 g/L BSA only (control) or 5 g/L BSA with 0.5 mmol/L LA, t10,c12-CLA or c9,t11-CLA] to the apical compartment. Immediately, 13 µL [400 ng, 1,443,000 disintegrations per minute (dpm)] of 14C-glycerol (140–168 mCi/mmol; Amersham Pharmacia, Bucks, UK) was added to each apical compartment, and cells were incubated for 22 h at 37°C. The basolateral media was harvested directly into 15-mL tubes (Falcon) and stored at -80°C until lipids were extracted. The cells were washed in 1x PBS (GibcoBRL), scraped into 1.5 mL tubes (Starstead, Wexford, UK), pelleted at 800 x g for 2 min and resuspended in 1x PBS (GibcoBRL) before being stored at -80°C. De novo lipid levels (cellular n = 8, basolateral n = 4) were determined by isolating (25
) and separating the cellular and basolateral TAG and PL moieties on a thin layer chromatography plate (Whatman Biosystems, Kent, UK) (26
). The separated TAG and PL fractions were scraped into scintillation vials containing 10 mL of scintillation fluid (Lumagel safe, Lumac–LSC; Packard, Groningen, The Netherlands) and each vial was counted for 10 min (TriCarb 2100Tr, liquid scintillation analyzer; Packard).
Protein concentrations after acute LA, t10,c12-CLA and c9, t11-CLA treatments were measured by adding 2.5 mL FCS-free media minus methionine (DMEM minus methionine; GibcoBRL) to the basolateral compartment and 1.5 mL of FCS-free media minus methionine to the apical compartment. Acute 5 g/L BSA, 0.5 mmol/L LA and 5 g/L BSA, 0.5 mmol/L t10, c12-CLA and 5 g/L BSA or 0.5 mmol/L c9,t11-CLA and 5 g/L BSA were added to the apical compartment, then 10 µl (2,220,000 dpm) of 35S-methionine (40–500 mCi/mmol; Amersham Pharmacia) was added to each apical compartment and the cells were incubated for 22 h at 37°C. The basolateral media was harvested into 15-mL tubes (Falcon) and protease inhibitors were added [leupeptin, 1 g/L; pepstatin, 1 g/L; aprotinin, 1 g/L; PMSF, 10 g/L; benzamidine, 1 g/L (Sigma, Poole, UK)]. The cells were washed in 1x PBS (GibcoBRL) (4°C), then 200 µL of Boehringer Mannheim wash 1 (protein A–agarose protocol; Boehringer Mannheim GmbH, Germany) with protease inhibitors were added to the cells, which were scraped into 1.5-mL tubes (Starstead). Cells were centrifuged to remove cellular debris and the supernatant was transferred to a new tube. Cellular and basolateral (n = 4) fractions were cleared of any other antibodies by use of the protein A–agarose protocol (Boehringer Mannheim GmbH), and anti-human apoB antibody (ICN Biochemicals, Aurora, OH) was added. The immunoprecipitation of apoB48 and apoB100 was carried out using the Protein A-agarose (Boehringer Mannheim GmbH) according to the manufacturer’s instructions. The apoB100 and apoB48 isoforms were separated on 4% running, 2% stacking SDS–PAGE gels. The prestained protein large molecular weight ladder (GibcoBRL) was loaded on the SDS–PAGE gels along with the samples. The gels were run in 1x running buffer (25 mmol/L Tris–Cl; 250 mmol/L glycine; 0.1% SDS, pH 8.3) for approximately 90 min at 100 mV. Gels were dried on a gel dryer before autoradiography film (Hyperfilm; Amersham Pharmacia) was put on the gels. Films were developed after 2 d. The quantity of both forms of apoB, apoB100 and apoB48, were measured using an instant imager (Instant Imager, Model A202401; Packard, Reading,UK). The apoB100 (500 kDa) and apoB48 (230 kDa) proteins were identified by comparison with the apoB100 (Sigma) and apoB48 (internal laboratory standard) standards and the prestained large molecular weight protein ladder (GibcoBRL).
Chronic LA and CLA isomer supplementation
For the chronic supplementation study, Caco-2 cells were grown under the same conditions as described in the cell culture section, only they were either grown in media supplemented with fatty acids (0.05mmol/L LA, t10,c12-CLA or c9,t11-CLA) or in unsupplemented media (control). LA (cis-9, cis-12-octadecanoic acid, Sigma), t10,c12-CLA (trans-10, cis-12-octadecanoic acid; Loders Croklaan BV, Wormerveer, The Netherlands) and c9,t11-CLA (cis-9, trans-11-octadecanoic acid; Loders Croklaan BV) were stored in ethanol at -20°C. Before use, the fatty acids had sodium ions added to make them soluble in media (27
) and Na+LA, Na+ t10,c12-CLA and Na+c9,t11-CLA were dried. Before addition to the cells, a 30 mmol/L solution of each fatty acid was prepared in deionized H2O. This was incubated in boiling water for 2 min to solubilize the fatty acids before adding them to the apical cell culture media. The fatty acid supplements were delivered to the apical compartment of the transwells; the basolateral compartment did not receive fatty acid supplementation. This was done to mimic the intestine where the apical surface of enterocytes is exposed to dietary fats. Gas chromatography demonstrated alterations in Caco-2 cell fatty acid composition dependent on supplementary fatty acid. Supplementation with t10,c12-CLA increased TAG-t10,c12-CLA levels by 1.5% and PL-t10,c12-CLA levels by 1.9% relative to control cells, whereas c9,t11-CLA supplementation increased TAG-c9,t11-CLA levels by 3% and PL-c9,t11-CLA levels by 2.2% relative to controls.
Caco-2 Cell Characterization
Characterization assays of cell growth and proliferation of the control and supplemented Caco-2 cells were conducted (23
). Transepithelial electrical resistance (TER) of Caco-2 cells growing in the transwells was measured with an EVOM epithelial voltohmmeter and STX2 electrode (World Precision Instruments, Sarasota, FL), as described (23
). Translocation assays with a paracellular (mannitol) and intracellular (propranolol) markers were carried out at 6d and 14d postseeding. The transwells were washed in 1x PBS (GibcoBRL) before being equilibrated in Hanks’ balanced salt solution (HBSS; GibcoBRL) for 30 min at 37°C. Six-well plates were set up with 2.5 mL of HBSS (GibcoBRL). Samples were taken at 15, 30, 45, 60, 90 and 120 min were taken after addition of 150,000 dpm of both 14C mannitol (50–62 mCi/mmol; Amersham Pharmacia) and 3H-propranolol (15–30 Ci/mmol; Amersham Pharmacia) to the apical compartment of the transwell in a volume of 1.5 mL of HBSS (GibcoBRL). To assess the translocation of the intracellular and paracellular markers, 1 mL of basolateral HBSS was removed from each well of the 6-well plates to vials containing 10 mL of scintillation fluid (Lumagel safe, Lumac–LSC; Packard). Vials were counted for 10 min at radioactivity windows of 0.0 to 12.0 for 3H and 12.0 to 156 for 14C in a liquid scintillation counter (TriCarb 2100Tr, liquid scintillation analyzer; Packard).
The apparent permeability coefficient (Papp) was calculated from the following equation: Papp (cm/s) = {[dQ/dt (s)]/A (cm2)} x C0, where dQ/dt is the slope of the graph of the dpm vs. time (s), A is the area of the transwell (4.7 cm2) and C0 is the initial dpm (150,000 dpm) added to the transwell. A proliferation assay was carried out using the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). Caco-2 cells were seeded at a density of 105 cells/well for 24 h in a 96-well microtiter plate (Falcon) or at 4 x 104 cells/well (same density as in transwells) for 6 d with the media being changed every second day. The proliferation assay was conducted according to the manufacturer’s protocol and the cells were incubated for 4 h. Absorbance was read at 490 nm on an ELISA plate reader (Dynatech MR5000 microplate reader; Dynex, Middlesex, UK).
Fatty acid supplementation with 0.05 mmol/L LA and c9,t11-CLA in the apical compartment of the transwell did not alter cell TER compared to control cells. In a previous study, 0.05 mmol/L t10,c12-CLA supplementation inhibited tight junction formation up to 14 d postseeding (23
). However, by 17 d postseeding the TER of Caco-2 cells supplemented with 0.05 mmol/L t10,c12-CLA isomer were not significantly different from control cells (Fig. 1
). Supplementation of Caco-2 cells with 0.05 mmol/L LA, t10,c12-CLA or c9,t11-CLA did not alter cell proliferation compared to control cells (data not shown). Therefore, all studies to investigate the effects of CLA on TRL metabolism in the Caco-2 cell model were conducted in cells grown for 19 d.
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The acute-after-chronic effect of fatty acid supplementation on de novo lipid and apoB (cellular, n = 7; basolateral, n = 7) synthesis and secretion was determined in Caco-2 cells following the 19-d chronic fatty acid supplementation phase with control, 0.05 mmol/L LA, 0.05 mmol/L t10,c12-CLA or 0.05 mmol/L c9, t11-CLA. This concentration was chosen because it had been shown by characterization not to alter Caco-2 cell growth compared to Caco-2 cells grown in media alone. Acute treatments were with 5 g/L BSA, 0.5 mmol/L PA and 5 g/L BSA or 0.5 mmol/L OA and 5 g/L BSA for 22 h, given that the literature primarily discusses these fatty acids. De novo TAG and PL cellular (n = 14) and secreted (n = 7) levels were measured using 14C-glycerol. The lipids were isolated, separated and counted on a scintillation counter, as described above. The cellular apoB100, apoB48 (n = 7) and secreted (n = 7) apoB100 levels were measured using 35S-methionine. The apoB isoforms were isolated, separated and measured using an instant imager, as described above.
Statistical analysis
To investigate de novo TAG, PL and apoB synthesis and secretion the following ratios were used as metabolic indicators of intestinal lipid and apoB metabolism: ratio of nonsecreted TAG to secreted TAG (cTAG/sTAG), ratio of nonsecreted TAG to nonsecreted PL (cTAG/cPL), ratio of secreted TAG to secreted PL (sTAG/sPL), ratio of nonsecreted apoB100 to secreted apoB100 (cB100/sB100) and ratio of nonsecreted apoB100 to nonsecreted apoB48 (cB100/cB48). The effects of acute treatment with 5 g/L BSA or 5 g/L BSA and 0.5 mmol/L LA, 0.5 mmol/L t10,c12-CLA or 0.5 mmol/L c9,t11-CLA were analyzed by ANOVA (Data Desk 6.0, Data Descriptions, New York, NY), and post hoc protected least-significant difference (LSD) tests were used to identify different means. The data obtained from Caco-2 cells that had been chronically supplemented followed by acute treatment were analyzed by ANOVA with interactions (Data Desk 6.0), where the main factors were the acute treatment, the chronic supplementation and their interaction. Post hoc comparisons were done using protected LSD tests. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Acute fatty acid treatment affected the sTAG/sPL ratio (P = 0.0151) (Table 1
), which was attributed to the acute LA treatment increasing TAG secretion compared to all other acute treatments, BSA (P = 0.003), t10, c12-CLA (P = 0.012) and c9,t11-CLA (P = 0.05). However, the cTAG/sTAG, cTAG/cPL, cB100/sB100 and cB100/cB48 ratios were not affected.
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A significant acute effect (P = 0.001) and an acute x chronic interaction (P = 0.0188) were observed for the ratio of cTAG/sTAG (Table 2
). The acute effect indicated that, irrespective of the chronic fatty acid supplement, acute OA treatment reduced the cTAG/sTAG ratio because OA increased de novo TAG secretion, compared to that of the acute BSA treatment (P < 0.001). The acute x chronic interaction showed that the acute fatty acid treatments had different effects on the cTAG/sTAG ratio, depending on the chronic fatty acid supplement received by the cells. The fatty acid composition of the chronic supplement did not affect the cTAG/sTAG ratio after the acute BSA (control) and acute OA treatments. However, the response to the acute PA treatment was affected by the composition of the chronic fatty acid supplement. Chronic t10, c12-CLA supplementation, before the acute PA treatment, increased in cTAG/sTAG ratio compared to the chronic control (P < 0.001) and chronic LA supplement (P = 0.003). The t10,c12-CLA isomer significantly increased cellular de novo TAG levels. Chronic t10,c12-CLA supplementation before acute PA treatment increased the cTAG/sTAG ratio, albeit to a lesser extent, compared to the control supplement (P = 0.03), again by increasing cellular de novo TAG levels.
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Fatty acids had significant acute (P = 0.0001) and chronic effects (P = 0.0001) and an acute x chronic interaction (P = 0.0002) on the cTAG/cPL ratio (Table 3
). Acute PA treatments appeared to be highly lipogenic, as indicated by the greater cTAG/cPL ratio compared to that of acute BSA treatments, irrespective of chronic fatty acid intervention (P < 0.001). Acute PA treatment preferentially increased de novo TAG synthesis (> 2-fold) over PL synthesis (< 1.5-fold). The significant acute x chronic interaction demonstrated that the chronic fatty acid supplement determined the response to the acute fatty acid treatment. The acute OA treatment increased the cTAG/cPL ratio only after chronic fatty acid supplementation (P < 0.001) albeit to a less extent than acute PA treatments. When the cells received chronic t10, c12-CLA supplementation, before the acute OA treatment, the increase in the cTAG/cPL ratio was particularly augmented, and greater than that following chronic LA (P = 0.001) and c9,t11-CLA supplementation (P = 0.005). This was attributed to the t10,c12-CLA isomer preferentially increasing de novo TAG synthesis. In Caco-2 cells that received chronic c9,t11-CLA supplementation, the cTAG/cPL ratio was increased by both PA (P = 0.002) and OA (P < 0.05) compared to acute BSA treatment, which was attributed to increased de novo TAG synthesis (> 2-fold). The increase in the cTAG/cPL ratio was greater after acute PA treatment compared to that after acute OA treatment.
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The sTAG/sPL ratio was significantly affected by acute fatty acid treatment (P = 0.0001) only (Table 4
). Acute PA treatments increased the cTAG/cPL ratio compared to acute BSA treatment (P < 0.001). The acute OA treatments further augmented the increase in the sTAG/sPL ratio compared to acute BSA (P < 0.001) and acute PA treatments (P = 0.001), irrespective of the fatty acid composition of the chronic intervention. Acute OA and PA treatments increased de novo TAG secretion, with OA causing a greater increase.
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No significant effects of fatty acid supplementation or treatment on the ratios of cB100/sB100 or cB100/cB48 were observed (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In contrast, another human intervention study showed no effect of a lower dose of CLA in a smaller cohort (12
). Adipose tissue, the liver and the intestine are key organs for determining TAG metabolism. In vitro and in vivo animal feeding trials showed that CLA could mediate its ameliorative effect on TAG metabolism through altered hepatic and adipocyte TAG metabolism (6
,17
,18
). Thus, the objective of the present study was to investigate the isomer-specific effects of CLA on intestinal TRL metabolism. Caco-2 cells are often used as a model to study the effect of dietary fatty acid composition on intestinal TRL metabolism (20
,21
). Within this context, much of the literature on effects of fatty acid composition on Caco-2 cell TRL metabolism has focused on short-term (or acute treatments) for up to 24 h. In humans, postprandial TAG metabolism is affected by both acute and chronic fat composition (28
); therefore, we investigated the acute and chronic effects of CLA on Caco-2 cell TRL metabolism. Because the Caco-2 cell line was originally derived from an adenocarcinoma and CLA inhibits proliferation of tumors (22
), we completed all of our studies in confluent cells (19 d postseeding) because at this stage, neither CLA isomer affected Caco-2 cell development. In essence, our study showed that chronic CLA supplementation, rather than acute CLA treatment, modulated Caco-2 cell TRL metabolism.
We compared the effects of the t10,c12-CLA and c9, t11-CLA isomers with LA and BSA on indices of TRL metabolism. Only the acute LA treatment increased the ratio of secreted TAG to secreted PL (sTAG/sPL) compared to all other acute treatments by increasing de novo TAG secretion. This concurs with previous studies that showed that acute LA treatments increased TAG synthesis and de novo TAG secretion (29
), and the de novo TAG were secreted in the TRL fraction (27
). Although it was not our objective to determine the molecular basis of these effects, it may be that LA was a better substrate than the CLA isomers for microsomal triglyceride transfer protein, which may promote TRL secretion (30
,31
). A previous study investigating intestinal lipoprotein production in Caco-2 cells showed that fatty acid carbon chain length, double-bond positional and geometric configuration were important determinants of de novo TAG secretion (32
). The study investigated the effect of an acute (18 h) dose (0.5 mmol/L) of a variety of fatty acid isomers (16:0, cis-16:1
9, trans-16:19, 18:0, cis-18:19, trans-18:19, cis-18:111 or trans-18:111 bound to BSA) on Caco-2 cell TAG secretion. Oleic acid (cis-18:19) doubled de novo TAG secretion, compared to vaccenic acid (cis-18:111), irrespective of the cis or trans configuration the double bond (32
). However, for 16:19 monounsaturated fatty acids, de novo TAG secretion was dependent on the geometric configuration of the double bond; cis-16:19 increased de novo TAG secretion compared to 16:0, but trans-16:19 did not (32
). In our study, acute t10,c12-CLA treatment increased cellular TAG levels as indicated by the greater cTAG/sTAG and cTAG/cPL ratios. Even though these changes were not significant (P = 0.07), our results suggest that the geometric configuration of CLA may effect TAG synthesis, whereby the t10,c12-CLA isomer may be the preferred substrate for TAG synthesis compared to LA or c9,t11-CLA.
Chronic CLA supplementation had the greatest effect on indices of Caco-2 cell TRL metabolism. In particular, significant acute by chronic interactions were observed for the ratio of cellular to secreted de novo TAG (cTAG/sTAG) and cellular de novo TAG to PL (cTAG/cPL). Therefore, the composition of the chronic fatty acid supplement determined the effect of the acute fatty acid treatment. For the cTAG/sTAG ratio, the response to the acute PA treatment was determined by the composition of the chronic fatty acid supplement. The t10,c12-CLA and c9,t11-CLA isomers increased de novo cellular TAG levels compared to those of unsupplemented control cells. Indeed, the increase in the cTAG/sTAG ratio was greater after chronic t10,c12-CLA supplementation, almost twice that after chronic LA supplementation. LA is the most potent stimulator of de novo TAG secretion, whereas t10,c12-CLA had the least effect on TAG secretion. Our results concur with those in HepG2 cells, whereby acute treatment with the t10,c12-CLA isomer simulated TAG synthesis but suppressed de novo TAG secretion compared to acute BSA treatment (17
).
In human preadipocytes, the t10,c12-CLA isomer decreases TAG concentrations and may inhibit lipogenesis (33
). Moreover, CLA interferes with TAG metabolism in murine 3T3-L1 adipocytes, whereby both the t10,c12-CLA and c9,t11-CLA isomers reduced heparin-releasable-LPL activity, and in the case of the t10,c12-CLA isomer, this effect was independent of insulin (34
). Acute OA treatment increased the cTAG/cPL ratio in a manner that was dependent on the composition of the chronic fatty acid supplement. Chronic LA and c9, t11-CLA supplementation significantly increased the cTAG/cPL ratio compared to nonsupplemented cells, attributed to increased de novo TAG synthesis. The cTAG/cPL ratio was further increased by chronic the t10,c12-CLA supplementation, also attributed to de novo TAG synthesis. Similarly, in HepG2 cells, t10,c12-CLA stimulated TAG synthesis compared to c9,t11-CLA and LA, which promoted TAG synthesis to a similar extent (17
). Of note is that acute PA treatment was associated with greater concentrations of nonsecreted TAG than acute OA treatment, and that no difference was found between acute OA and BSA treatments in the unsupplemented controls.
Previous cell studies have demonstrated that acute PA treatments are associated with high cellular TAG levels (20
,35
). HepG2 cells given acute PA treatments (0.4 mmol/L) had more cellular TAG than that of HepG2 cells treated with acute OA doses (35
). In Caco-2 cells, acute OA (0.5 mmol/L) treatment was associated with higher cellular TAG concentrations than those in PA-treated cells (20
). However, acute OA treatment resulted in the cells having a lower cTAG/bTAG ratio than that in PA-treated cells (20
), which also was observed in the present study. Therefore, acute PA treatment may be associated with reduced de novo TAG secretion, resulting in higher de novo cellular TAG concentrations compared to those in acute OA treatment.
The ratio of sTAG/sPL was not affected by chronic fatty acid supplementation, nor did we observe an acute x chronic interaction, which shows that CLA had no effect on either de novo TAG or PL secretion. The significant acute effect indicates that both the acute PA and OA treatments increased the ratio attributed to preferential TAG secretion over PL secretion, irrespective of the composition of the chronic fatty acid supplement. The present study suggests that neither acute nor chronic LA, c9,t11-CLA or t10,c12-CLA treatments affects Caco-2 cell apoB metabolism.
In conclusion, results from this study show that CLA has the greatest effect on Caco-2 cell TRL metabolism when provided as a chronic supplement rather that as an acute treatment. Chronic CLA supplementation promotes de novo TAG synthesis and does not affect apoB metabolism in Caco-2 cells. Hypothetically, if we were to extrapolate these data to intestinal lipoprotein metabolism, the results imply that t10,c12-CLA supplementation might delay the secretion of dietary TAG from the intestinal enterocytes and attenuate postprandial lipemia. However, further research is required to confirm this.
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FOOTNOTES |
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3 Abbreviations used: apo, apolipoprotein; BSA, bovine serum albumin; Caco-2, human colon carcinoma cell line; cB100/cB48, cellular apolipoprotein B100 to cellular apolipoprotein B48; cB100/sB100, cellular to secreted apolipoprotein B100; CLA, conjugated linoleic acid; cTAG/cPL, cellular triacylglycerol to cellular phospholipid; cTAG/sTAG, cellular to secreted triacylglycerol; c9,t11-CLA, cis-9, trans-11 conjugated linoleic acid; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; HepG2, human hepatoma cell line; LA, linoleic acid; LPL, lipoprotein lipase; OA, oleic acid; PA, palmitic acid; PL, phospholipid; sTAG/sPL, secreted triacylglycerol to secreted phospholipid; TAG, triacylglycerol; TER, transepithelial electrical resistance; TRL, triacylglycerol-rich lipoprotein; t10,c12-CLA, trans-10, cis-12 conjugated linoleic acid.
Manuscript received 19 December 2001. Initial review completed 14 February 2002. Revision accepted 16 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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