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Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes
Biochimica et Biophysica Acta 1737 (2005) 36 – 43 http://www.elsevier.com/locate/bba
Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes Shizuko Tachibana a, Kan Sato a,*, Yoshitake Cho a, Tomoyuki Chiba a, Wolfgang J. Schneider b, Yukio Akiba a b
a Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria
Received 11 May 2005; received in revised form 28 August 2005; accepted 6 September 2005 Available online 26 September 2005
Abstract Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucylnorleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids. D 2005 Elsevier B.V. All rights reserved. Keywords: Apolipoprotein B; Chicken hepatocyte primary culture; Octanoate; Very low density lipoprotein
1. Introduction The involvement of increased plasma lipid levels in the development of atherosclerosis has been demonstrated in many studies [1]. Hypertriglyceridaemia often accompanies an increase in small and dense low-density lipoprotein (LDL) and is now generally accepted as a risk factor for coronary heart disease [2]. From a biochemical point of view, regulation
Abbreviations: ALLN, N-acetyl-leucyl-leucyl-norleucinal; apoA, apolipoprotein A; apoB, apolipoprotein B; ER, endoplasmic reticulum; FAS, fatty acid synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; HNF-4, hepatic nuclear factor 4; MTP, microsomal triglyceride transfer protein; TG, triacylglycerol; VLDL, very low-density lipoprotein * Corresponding author. Tel.: +81 22 717 8689; fax: +81 22 717 8691. E-mail address: [email protected] (K. Sato). 1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2005.09.001
of the secretion of apolipoprotein B (apoB), the major protein of atherogenic lipoproteins, from hepatic and intestinal cells has attracted much attention to date [3,4]. Most studies have focused on apoB metabolism in the liver, given the greater contribution to the plasma apoB pool made by that organ and the availability of convenient primary and transformed hepatic cell models [5]. Medium-chain fatty acids with 6 to 12 carbon atoms have been shown to possess several specific biological properties that distinguish them from long chain fatty acids [6]. It has been shown that medium-chain triacylglycerols are potential agents for the prevention of obesity [7]. There is considerable evidence to show that the anti-obesity effect of dietary medium-chain fatty acids is caused by the fast rate of oxidation of these fatty acids, an effect that is not observed with dietary long-chain fatty acids [8]. In addition, we previously reported
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that addition of octanoate (C8:0) to chicken hepatocyte cultures in vitro decreased the secretion of triacylglycerol (TG) and apoB from cells [9] and suggested that the decrease in fat deposition in chickens fed on medium chain fatty acids may be ascribed to impairment of very low density lipoprotein (VLDL)-apoB secretion from the liver. It is, therefore, likely that medium-chain fatty acids, at least octanoate, may be a new nutrient involved in the control of fat deposition and hyperlipidemia. However, the mechanisms by which octanoate inhibits VLDL secretion from hepatocytes are still to be elucidated. The present study was undertaken to clarify the mechanisms underlying the effects of octanoate on VLDL secretion in primary cultures of chicken hepatocytes. We hypothesized that octanoate inhibits VLDL secretion by regulating intracellular apoB metabolism and then investigated the effect of octanoate on three aspects of apoB metabolism: (1) VLDL secretion (TG, cholesterol and apoB secretion); (2) VLDL assembly (apoB synthesis, apoB degradation and microsomal triglyceride transfer protein (MTP) level); and (3) apoB gene expression (apoB mRNA expression). 2. Materials and methods 2.1. Materials Fatty acid sodium salts, bovine serum albumin (BSA, essentially fatty acidfree), glucagon and aprotinin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Basal Medium Eagle, Minimum Medium Eagle without methionine and cysteine and antibiotics were obtained from Gibco BRL (Rockville, MD, USA). Rooster serum was prepared from 6-week-old male broiler chickens. l-[35S]methionine, [a-32P]dCTP, enhance solution and protein A were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL, USA). A monoclonal antibody to chicken apoB was developed in our laboratories [10]. Rabbit anti-chicken MTP antiserum was developed by immunization with synthetic peptide. Other reagents were obtained from Wako Pure Chemical Co. Ltd. (Osaka, Japan).
2.2. Animals and diets Male broiler chickens (Ross, provided from Matsumoto Hatchery, Zao, Japan) were fed ad libitum on a commercial grower’s diet (crude protein 22 g/kg diet, metabolisable energy 13.0 MJ/kg diet) and housed in wire cages under controlled temperature (25 T 3 -C) conditions. The birds were used for experiments when their body weights were within the range of 1000 to 1200 g at about 4 weeks of age.
2.3. Primary culture of chicken hepatocytes Chicken liver cells were prepared and maintained in a monolayer culture as described previously [11]. In brief, male broiler chickens were starved for 3 h and hepatocytes then isolated by perfusion of the liver with 0.05% collagenase. The hepatocytes were plated (5.0 105 cells//60 mm collagen type I-coated dish) with incubation medium for 24 h at 37 -C in 5% CO2 in order to achieve monolayer culture. After a 24-h incubation, the monolayer cultures of hepatocytes were used in experiments.
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Intracellular TG and VLDL-TG were extracted according to the method of Folch and Sloan-Stanly [12]. The TG concentrations were quantified by the method of Fletcher [13]. VLDL-cholesterol was measured using the methods of Allain et al. [14]. Intracellular protein content was determined by the method of Lowry et al. [15] using BSA as the standard.
2.5. Western blot analysis The hepatocyte culture media or cell lysates were separated by 6% SDSpolyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad Laboratories, Hercules, CA, USA). Western blotting experiments utilized phosphate-buffered saline solution (PBS) containing 7% non-fat dry milk to block the membrane. The membranes were then incubated with 5 Ag/ml of specific anti-chicken apoB monoclonal antibody or chicken MTP antisera followed by corresponding antimouse or -rabbit IgG conjugated to horseradish peroxidase. After rinsing five times in PBS containing 0.3% Tween 20, the membranes were incubated in enhanced chemiluminescence solution (Super Signal, Pierce Biotechnology, Inc., Rockford, IL, USA) for 5 to 10 min, and exposed to Kodak XAR-5 film for 1 to 10 min. ApoB proteins were detected semi-quantitatively with densitometer tracing using a Molecular Imager FX (Bio-Rad Laboratories).
2.6. Pulse-chase analysis of apoB metabolism Primary chicken hepatocytes were pre-incubated in methionine-free minimum essential medium supplemented with essential amino acids at 37 -C for 1 h and pulsed with 100 ACi/ml of [35S] methionine/cysteine (redivue Promix l-[35S] in vitro cell labeling mix, Amersham Pharmacia Biotech, Arlington Heights, IL, USA) for 60 min. After the pulse, the cells were washed twice and chased in hepatocyte attachment media supplemented with 10 mM methionine. At various chase times (0.5, 1, 2, 3 h), the cells and media were harvested and the cells were lysed in solubilization buffer (5 mM EDTA) supplemented with a cocktail of protease inhibitors (Complete Protease inhibitor, Roche Diagnostics, Rotkreuz, Switzerland) and homogenized. The homogenate was added to carbonate extraction buffer (100 mM Na2CO3 pH 11.5) and incubated for 30 min on ice. The lysates were centrifuged at 15,000 rpm for 10 min at 4 -C and the supernatants mixed with Tris buffer (500 mM Tris-HCl, pH 6.8) and then subjected to immunoprecipitation. All buffers were supplemented with a cocktail of protease inhibitors. In some experiments, the proteasomal inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) was added to the pulse and chase medium at a concentration of 40 Ag/ml. The pulse and chase times were as described above.
2.7. Immunoprecipitation, SDS-PAGE, fluorography and measurement of labeled apoB The supernatants of hepatocyte lysates and culture media were diluted with IP buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 1% Triton-X-100) supplemented with a protease inhibitor cocktail (Complete Protease inhibitor, Roche Diagnostics). Chicken anti-apoB monoclonal antibody was added to cell lysates and incubated for 4 h at 4 -C. Protein A (Protein A CL-4B, Amersham Pharmacia Biotech, Arlington Heights, IL) was then added to the lysates and the incubation continued for over 15 h at 4 -C with the tube rotating. Protein A – antibody – apoB complexes were collected by centrifugation at 15,000 rpm for 20 min. The immunoprecipitates were washed three times with IP buffer and twice with PBS. To detect the radiolabeled apoB, the pellet was resuspended in sample buffer (62.5 mM Tris-HCl, pH 7.4, 2% SDS, 5% mercaptoethanol) and boiled for 5 min. After centrifugation, supernatants were analyzed by 6% SDS-PAGE. The gels were fixed, and then fluorographed by incubating in enhancer (Amplify, Amersham Pharmacia Biotech). The gels were dried and exposed to X-ray film at 80 -C and the radioactivity of apoB measured using a liquid scintillation counter.
2.4. VLDL isolation and measurements of lipid and protein content 2.8. RNA isolation and Northern blot analysis VLDL in the culture media was quantitatively collected by ultracentrifugation (d < 1.065 g/ml) for 3 h with KONTRON ultracentrifuges (Kontron Instrument K.K., Zurich, Switzerland) fitted with a TFT65.13 rotor.
All probes were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) from chicken hepatocyte RNA. All PCR primers were
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designed based on published sequences from Genbank as follows; apoB (M18421), apolipoprotein A-I (apoA-I, M18746), fatty acid synthase (FAS, J03860), 3-hydroxy-3-metylglutaryl-CoA reductase (HMGR, AB109635) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AF047874). Total RNA was isolated from the culture dishes using Trizol-Reagent (Invitrogen, San Diego, CA, USA). For Northern blot analysis, 20 Ag of total RNA were resolved using glyoxal solution and electrophoresed on a 1.0% agarose gel, and transferred to membrane (Zeta-Probe Blotting Membranes, Bio-Rad Laboratories, Hercules, CA). Hybridized RNA blots and quantification of gene expression were carried out as described by our previous report [16]. The blots were subsequently hybridized with a GAPDH cDNA probe to correct for differences in the amounts of RNA loaded onto the gel.
2.9. Statistical analysis A computer-generated SAS applications package was used for statistical calculations (Statistical Analysis System Version 6.03, SAS Institute Inc., Cary, NC). Group data for multiple comparisons were analyzed by ANOVA using a general linear model procedure followed by Duncan’s multiple range tests.
3. Results 3.1. Effect of octanoate on intracellular TG accumulation and VLDL-TG, VLDL-cholesterol and apoB secretion No changes in cellular protein concentrations, cell number, and results of the trypan blue exclusion test were observed in cultures incubated with up to 2.0 mM of fatty acids for 5 days incubation (data not shown). Intracellular TG accumulation in hepatocytes was not affected by the addition of fatty acids (Fig.
Fig. 2. Effect of octanoate on microsomal triglyceride transfer protein expression in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. MTP expression was determined by Western blotting analysis. Representative illustration of the Western blot is documented in the upper panel. Results are expressed as mean T S.D. (n = 3).
1A). In contrast, VLDL-TG secretion from hepatocytes incubated with 2.0 mM octanoate was significantly lower than both control and palmitate-treated cultures (Fig. 1B). In addition, both VLDL-cholesterol and apoB secretion from hepatocytes incubated with 2.0 mM octanoate were significantly reduced compared to both control and palmitate-treated cells (Fig. 1C and D). These results confirm that octanoate
Fig. 1. Effect of octanoate on intracellular TG accumulation and VLDL-TG, VLDL-cholesterol and apoB secretion. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing various concentrations of octanoate (0, 0.2, 1.0, 2.0 mM) or 0.2 mM palmitate complexed with 1.88% BSA and the cells incubated for 24 h. Intracellular TG accumulation (A), VLDL-TG secretion (B), VLDL-cholesterol secretion (C) were measured as described in Section 2. ApoB secretion (D) was determined by Western blotting analysis. Representative illustration of the Western blot is documented in the upper panel (D). Results are expressed as mean T S.D. (n = 4). Different letters indicate significantly difference, P < 0.05.
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reduces VLDL secretion in the aspects of TG, cholesterol and apoB from chicken hepatocytes. 3.2. Effect of octanoate on MTP in hepatocytes Fig. 2 shows that MTP levels in the hepatocytes were not changed by any of the treatments. No differences in cellular protein concentrations were observed in the cultures supplemented with saturated fatty acids. 3.3. Effect of octanoate on apoB synthesis, degradation and secretion in hepatocytes The level of radiolabeled apoB following a 1 h pulse of [35S] methionine/cysteine was increased by palmitate addition and reduced by octanoate addition, relative to the control. Octanoate treatment significantly reduced intracellular apoB accumulation, compared to both control and palmitate
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treatments, 1 h into the chase period. (Fig. 3A, B). In culture media, the apoB concentrations in cultures supplemented with octanoate were also reduced. Three hours into the chase period, intracellular apoB and apoB accumulation in the media (apoB secretion) in the octanoate-treated group were reduced compared to the control and palmitate groups (Fig. 3C). The degradation of radiolabeled apoB was estimated by subtracting the sum of the apoB secreted into the media and that remaining within the cells after 3 h of chase from the intracellular apoB measured at the end of pulse. There were 0.8, 0.01 and 7.2 103 dpm/mg cell protein in the control, octanoate and palmitate treatments, respectively. In Fig. 3D, the time course of changes in apoB radioactivity in the media and cells following chasing up to 3 h are given as percentages of the total radioactivity measured at the end of the pulse. In control hepatocytes, 80% of the initial radiolabeled apoB was recovered at the end of the 3-h chase
Fig. 3. Effect of octanoate on apoB stability in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. Hepatocytes were pulsed with [35S]-methionine and chased for up to 3 h. Throughout the pulse and chase periods, fatty acids and BSA were present in the media. Media samples and cell lysates were collected at each chase time point, subjected to immunoprecipitation with a specific anti-chicken apoB antibody, and then detected by SDS-PAGE and fluorography (A) or quantitated by scintillation counting (B, C, D). (A) Representative fluorograph at time 0 and 1 h of chase. Treatments are control (C), octanoate (O) and palmitate (P); (B) ApoB accumulation during the pulse (at 0 time); (C) recovery of radiolabeled apoB at 3 h chase time. Distribution of apoB in cells (gray bar), media (hatched bar) in each hepatocyte treatment; (D) results at 0, 0.5, 1, 2, 3 h chase time. Distribution of apoB in cells (open diamond), media (closed squares) as well as the total apoB (closed triangles with dotted lines) in each hepatocyte treatment are expressed as a percentage of labeled apoB at 0 time. Results are expressed as mean T S.D. (n = 4). Different letters indicate significantly difference, P < 0.05.
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(40% in cells and 40% in media), indicating that 20% of newly synthesized apoB was intracellularly degraded during this time. In palmitate-treated hepatocytes, 60% of the initial radiolabeled apoB was recovered at the end of the 3-h chase (30% in cell and 30% in media), indicating that 40% of newly synthesized apoB was intracellularly degraded during this time. On the other hand, in octanoate-treated hepatocytes, almost 100% of newly synthesized apoB was recovered at the end of 3 h chase (60% in cell and 30% in media), indicating that newly synthesized apoB was degraded to a lesser extent than in the other treatments. Octanoate reduced intracellular apoB degradation compared to both the control and palmitate treatments, reducing both the amount and rate of degradation. 3.4. Effect of octanoate on intracellular apoB synthesis in the presence of ALLN The addition of ALLN, a protease inhibitor, to the media tended to increase new synthesis of intracellular apoB protein in all treatments. Octanoate reduced apoB synthesis compared to both control and palmitate treatments in the presence and absence of ALLN (Fig. 4). 3.5. Effect of octanoate on apoB mRNA levels in hepatocytes Addition of octanoate to media significantly decreased the apoB mRNA level compared to the control and palmitate cultures (Fig. 5A). Over the time course apoB mRNA levels in cultures treated with octanoate were reduced to 80, 60 and 20% that of control cultures at 6, 12 and 24 h., respectively (Fig. 5B). On the other hand, no changes were observed in apoB mRNA expression in the palmitate-treated cultures.
Fig. 5. Effect of octanoate on apoB mRNA levels in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for various periods. After a 24-h incubation (A) or after 6-, 12- and 24-h incubations (B), cells were collected, and the total RNA was extracted. ApoB and GAPDH mRNA levels were determined by Northern blotting analysis. Representative illustrations of the Northern blot are documented in the upper panels. Data are expressed as the ratio of apoB/GAPDH. Results are expressed as % of control, mean T S.D. (n = 4). P < 0.05.
3.6. Effect of octanoate on FAS, HMGR reductase and apoA-I mRNA expression in hepatocytes The expression levels of FAS, HMGR reductase and apo AI genes that code for key enzymes of fatty acid synthesis, cholesterol synthesis and protein involved in lipid transport, respectively, did not differ between the control, octanoate and palmitate groups (Fig. 6). 4. Discussion Fig. 4. Effect of octanoate on net apoB synthesis in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. Medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. Hepatocytes were pulsed with [35S]-methionine in the presence or absence of 40 Ag/ml ALLN. Throughout the pulse period, fatty acids and BSA as well as ALLN were present in the medium. After the pulse, cell lysates were collected, subjected to immunoprecipitation with a specific anti-chicken apoB antibody, and then quantitated by scintillation counting. Results are expressed as mean T S.D. (n = 5). Different letters indicate significantly difference, P < 0.05.
VLDL is assembled in hepatocytes from TG, cholesteryl esters, phospholipids and apoB. The assembly is a complex and highly regulated process and a number of factors play key roles [17]. MTP is crucial for VLDL assembly as it catalyzes the transfer of these lipids to nascent apoB molecules [18]. ApoB mRNA expression and apoB protein synthesis in hepatocytes are relatively constitutive and generally does not change under conditions that alter apoB
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Fig. 6. Effect of octanoate on FAS, HMG-CoA reductase and apoA-I mRNA levels in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. After a 24-h incubation, cells were collected and the total RNA was extracted. ApoA-I (A), FAS (B), HMGR (C) and GAPDH mRNA levels were determined by Northern blotting analysis. Representative illustrations of the Northern blot are documented in the upper panels. Data are expressed as the ratio of GAPDH. Results are expressed as % of control, mean T S.D. (n = 4). Different letters indicate significantly difference, P < 0.05.
secretion. Therefore, VLDL assembly is regulated largely at co- and post-translational levels [19]. ApoB degradation can also occur post-translocationally and even after an apoBcontaining particle is formed at multiple sites in the secretary pathway by a variety of non-proteosomal means [20 –24]. Taking into consideration the data that these previous studies provide on the mechanism of regulation of VLDL secretion, in the present study, we have investigated, in a stepwise manner, whether octanoate reduces VLDL secretion, and how octanoate regulates VLDL secretion in chicken primary hepatocytes. We previously reported that the addition of octanoate to primary cultures of chicken hepatocytes reduced VLDL secretion with respect to both TG and apoB secretion. The effects of octanoate were substantiated even in hepatocytes with high levels of intracellular TG, which was induced by the addition of palmitate. Therefore, we concluded that octanoate decreases secretion of apoB-containing lipoproteins regardless of the intracellular lipogenic state. Our present results confirm those findings, showing that octanoate lowered VLDL-TG, -cholesterol and apoB secretion from primary cultured chicken hepatocytes compared to both control and palmitate cultures (Fig. 1). In this paper, we further suggest that modification of intracellular apoB metabolism is involved in the inhibition of VLDL secretion by octanoate. MTP plays a role in lipoprotein assembly by catalyzing the transfer of lipids to nascent apoB molecules, which is one of the key factors regulating apoB secretion. Recently Marcil et al. reported that butyrate (C4:0) reduced apoB48 synthesis by decreasing MTP protein expression in Caco-2 cells [25]. However, the effect of fatty acids on MTP activity and expression is not clear in hepatocytes, probably because of their long half-life of 4.4 days [26]. Our present results show that octanoate does not change protein level of MTP in chicken hepatocytes, despite octanoate reducing apoB secretion (Fig. 2). It is likely that VLDL assembly itself is not responsible for the impairment of apoB secretion by octanoate.
To date, most reports on the effects of fatty acids on apoB secretion from hepatocytes have viewed these effects as being dependent on apoB degradation and VLDL assembly, while apoB mRNA levels and intracellular apoB synthesis have been reported to be unchanged by fatty acids [27 – 30]. Thus, one might expect that octanoate increases intracellular apoB degradation and consequently decreases apoB secretion. Our results reveal that the addition of 2.0 mM octanoate to the media reduces intracellular apoB degradation compared to fatty acid-free and palmitate-treated control cultures, while it reduces the level of newly synthesized apoB. In addition, in the presence of the proteosomal inhibitor, ALLN, octanoate decreased the level of newly synthesized apoB in hepatocytes. This may be sufficient evidence that decreases in VLDL secretion induced by octanoate treatment are due to decreased intracellular apoB synthesis but not to regulation of apoB degradation. In addition, the present study has shown that octanoate addition not only decreases intracellular apoB synthesis but also significantly reduces apoB mRNA expression, compared to cultures supplemented with BSA alone or with palmitate (Fig. 5A). These results suggest that octanoate can significantly reduce apoB mRNA expression, in turn decreasing apoB synthesis and secretion. Thus, this is the first study to report that octanoate is a unique inhibitor of apoB secretion acting through its effects on apoB mRNA transcription and synthesis. Decreases in apoB mRNA expression induced by octanoate might be due to decreases in apoB gene transcription or increases in apoB mRNA decay. Pullinger et al. reported that the half-life of apoB mRNA is 16 h in HepG2 cells [27]. Here, we carried out a time-course analysis of apoB mRNA levels over 24 h and showed that apoB mRNA levels were reduced by approximately 50% 12 h after the addition of octanoate to the media (Fig. 5B). This might suggest that octanoate downregulates apoB gene transcription. The human apoB-promoter region has binding sites for HNF-4a, C/EBP and other nuclear receptors [31]. Transforming growth factor h (TGF-h) alters apoB secretion from HepG2 cells by changing steady-state
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mRNA levels and protein synthesis through the transcriptional signals, SMADs [32]. It is likely, therefore, that octanoate, itself or in complex with those transcriptional factors, binds to the apoB promoter region and consequently regulates apoB gene expression. Unfortunately, in the present study, direct evidence of whether the decrease in apoB levels induced by octanoate is accounted for by the reduction in transcription from the apoB gene was not obtained. Promotor analysis using apoB gene-transfected cells may help to further elucidate the effects of octanoate on VLDL secretion. In this study, high concentrations of fatty acid, up to 2 mM, were used in the chicken hepatocyte cultures. There are some reports that postprandial serum concentrations of octanoate in pigs fed medium-chain triglyceride increased to 0.6– 0.8 mM [33]. Moreover, we have shown, in the present study that the index of toxicity and mRNA expression of apo A-I, FAS and HMGR are similar in control and palmitate- and octanoatetreated cultures. In addition, some studies in which octanoate was added to a concentration of 4 mM in media [34,35] supports our finding that specific inhibition of apoB mRNA expression by octanoate appears to be without cytotoxicity in chicken hepatocytes. In summary, the present study demonstrates that octanoate lowers VLDL-TG, -cholesterol and -apoB secretion from primary cultured chicken hepatocytes. Moreover, 2.0 mM octanoate, compared not only to palmitate-treated but also to control (BSA alone) cultures, significantly decreased both apoB synthesis and apoB mRNA levels in chicken hepatocytes. This is the first report to show that octanoate impairs VLDL secretion by inducing a reduction in intracellular apoB synthesis via reduction of mRNA levels. It is, therefore, likely that MCT, rich in octanoate, may be used as a novel functional nutrient to control fat deposition and hyperlipidemia. These findings may provide clues not only for the development of new nutritional means for the regulation of VLDL secretion, but also for the identification of factors regulating lipoprotein metabolism. References [1] P.W. Wilson, J.C. Christiansen, K.M. Anderson, W.B. Kannel, Impact of national guidelines for cholesterol risk factor screening. The Framingham Offspring Study, JAMA 262 (1989) 41 – 44. [2] P.O. Kwiterovich, Clinical relevance of the biochemical, metabolic, and genetic factors that influence low-density lipoprotein heterogeneity, Am. J. Cardiol. 90 (2002) 30i – 47i. [3] J.L. Dixon, H.N. Ginsberg, Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells, J. Lipid Res. 34 (1993) 167 – 179. [4] S. Pal, E. Allister, A. Thomson, J.C. Mamo, Cholesterol esters regulate apoB48 secretion in CaCo2 cells, Atherosclerosis 161 (2002) 55 – 63. [5] A.A. Qureshi, D.M. Peterson, The combined effects of novel tocotrienols and lovastatin on lipid metabolism in chickens, Atherosclerosis 156 (2001) 39 – 47. [6] A.C. Bach, Y. Ingenbleek, A. Frey, The usefulness of dietary mediumchain triglycerides in body weight control: fact or fancy? J. Lipid Res. 37 (1996) 708 – 726. [7] A. Geliebter, N. Torbay, E.F. Bracco, S.A. Hashim, T.B. Van Itallie, Overfeeding with medium-chain triglyceride diet results in diminished deposition of fat, Am. J. Clin. Nutr. 37 (1983) 1 – 4.
[8] E. Christensen, T.A. Hagve, M. Gronn, B.O. Christophersen, Betaoxidation of medium chain (C8 – C14) fatty acids studied in isolated liver cells, Biochim. Biophys. Acta 1004 (1989) 187 – 195. [9] S. Tachibana, K. Sato, T. Takahashi, Y. Akiba, Octanoate inhibits very low-density lipoprotein secretion in primary cultures of chicken hepatocytes, Comp. Biochem. Physiol., A Mol. Integr. Physiol. 132 (2002) 621 – 627. [10] T. Chiba, K. Sato, S. Tachibana, K. Takahashi, A. Nishida, Y. Akiba, Preparation of monoclonal antibody against chicken apolipoprotein B and development of enzyme liked immunosolvent assay (ELISA) method with the antibody aiming at the optimization of lipid metabolism in chickens, J. Poult. Sci. 40 (2003) 212 – 220. [11] T. Takahashi, K. Sato, Y. Akiba, A possible procedure to prepare mono fatty acid-enriched plasma membrane in chicken hepatocytes, Anim. Sci. J. 70 (1999) 118 – 123. [12] J.L. Folch, G.H. Sloan-Stanly, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497 – 509. [13] M.J. Fletcher, A colorimetric method for estimating serum triglycerides, Clin. Chim. Acta 22 (1968) 393 – 397. [14] C.C. Allain, L.S. Poon, C.S. Chan, W. Richmond, P.C. Fu, Enzymatic determination of total serum cholesterol, Clin. Chem. 20 (1974) 470 – 475. [15] O.H.R. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275. [16] K. Sato, A. Ohuchi, H.S. Seol, M. Toyomizu, Y. Akiba, Changes in mRNA expression of 3-hydroxy-3-methylglutary coenzyme A reductase and cholesterol 7 alpha-hydroxylase in chickens, Biochim. Biophys. Acta 1630 (2003) 96 – 102. [17] R.A. Davis, Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver, Biochim. Biophys. Acta 1440 (1999) 1 – 31. [18] E.A. Fisher, H.N. Ginsberg, Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins, J. Biol. Chem. 277 (2002) 17377 – 17380. [19] E.A. Fisher, M. Zhou, D.M. Mitchell, X. Wu, S. Omura, H. Wang, A.L. Goldberg, H.N. Ginsberg, The degradation of apolipoprotein B100 is mediated by the ubiquitin – proteasome pathway and involves heat shock protein 70, J. Biol. Chem. 272 (1997) 20427 – 20434. [20] Z. Yao, K. Tran, R.S. McLeod, Intracellular degradation of newly synthesized apolipoprotein B, J. Lipid Res. 38 (1997) 1937 – 1953. [21] J. Twisk, D.L. Gillian-Daniel, A. Tebon, L. Wang, P.H. Barrett, A.D. Attie, The role of the LDL receptor in apolipoprotein B secretion, J. Clin. Invest. 105 (2000) 521 – 532. [22] J.L. Dixon, S. Furukawa, H.N. Ginsberg, Oleate stimulates secretion of apolipoprotein B-containing lipoproteins from Hep G2 cells by inhibiting early intracellular degradation of apolipoprotein B, J. Biol. Chem. 266 (1991) 5080 – 5086. [23] R.A. Borchardt, R.A. Davis, Intrahepatic assembly of very low density lipoproteins. Rate of transport out of the endoplasmic reticulum determines rate of secretion, J. Biol. Chem. 262 (1987) 16394 – 16402. [24] M.J. Bamberger, M.D. Lane, Assembly of very low density lipoprotein in the hepatocyte. Differential transport of apoproteins through the secretory pathway, J. Biol. Chem. 263 (1988) 11868 – 11878. [25] V. Marcil, E. Delvin, C. Garofalo, E. Levy, Butyrate impairs lipid transport by inhibiting microsomal triglyceride transfer protein in Caco-2 cells, J. Nutr. 133 (2003) 2180 – 2183. [26] M.C. Lin, D. Gordon, J.R. Wetterau, Microsomal triglyceride transfer protein (MTP) regulation in HepG2 cells: insulin negatively regulates MTP gene expression, J. Lipid Res. 36 (1995) 1073 – 1081. [27] C.R. Pullinger, J.D. North, B.B. Teng, V.A. Rifici, A.E. Ronhild de Brito, J. Scott, The apolipoprotein B gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mRNA half-life, J. Lipid Res. 30 (1989) 1065 – 1077. [28] E.A. Fisher, M. Pan, X. Chen, X. Wu, H. Wang, H. Jamil, J.D. Sparks, K.J. Williams, The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways, J. Biol. Chem. 276 (2001) 27855 – 27863.
S. Tachibana et al. / Biochimica et Biophysica Acta 1737 (2005) 36 – 43 [29] E. Kummrow, M.M. Hussain, M. Pan, J.B. Marsh, E.A. Fisher, Myristic acid increases dense lipoprotein secretion by inhibiting apoB degradation and triglyceride recruitment, J. Lipid Res. 43 (2002) 2155 – 2163. [30] A. Kaptein, E.C. Wit, H.M. Princen, Butyrate stimulates the secretion of apolipoprotein B-100-containing lipoproteins from HepG2 cells by inhibiting the intracellular degradation, Biochim. Biophys. Acta 1213 (1994) 349 – 356. [31] V.I. Zannis, H.Y. Kan, A. Kritis, E.E. Zanni, D. Kardassis, Transcriptional regulatory mechanisms of the human apolipoprotein genes in vitro and in vivo, Curr. Opin. Lipidol. 12 (2001) 181 – 207. [32] K. Singh, O.A. Batuman, H.O. Akman, M.H. Kedees, V. Vakil, M.M. Hussain, Differential, tissue-specific, transcriptional regulation of apoli-
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poprotein B secretion by transforming growth factor beta, J. Biol. Chem. 277 (2002) 39515 – 39524. [33] T.M. Wieland, X. Lin, J. Odle, Utilization of medium-chain triglycerides by neonatal pigs: effects of emulsification and dose delivered, J. Anim. Sci. 71 (1993) 1863 – 1868. [34] J. Han, S.R. Farmer, J.L. Kirkland, B.E. Corkey, R. Yoon, T. Pirtskhalava, Y. Ido, W. Guo, Octanoate attenuates adipogenesis in 3T3-L1 preadipocytes, J. Nutr. 132 (2002) 904 – 910. [35] E. Guillot, P. Legrand, P. Lemarchal, Octanoate metabolism in primary culture of chicken hepatocytes: evidence for high-capacity octanoate esterification, Comp. Biochem. Physiol., B Biochem. Mol. Biol. 118 (1997) 249 – 256.
Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes Shizuko Tachibana a, Kan Sato a,*, Yoshitake Cho a, Tomoyuki Chiba a, Wolfgang J. Schneider b, Yukio Akiba a b
a Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria
Received 11 May 2005; received in revised form 28 August 2005; accepted 6 September 2005 Available online 26 September 2005
Abstract Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucylnorleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids. D 2005 Elsevier B.V. All rights reserved. Keywords: Apolipoprotein B; Chicken hepatocyte primary culture; Octanoate; Very low density lipoprotein
1. Introduction The involvement of increased plasma lipid levels in the development of atherosclerosis has been demonstrated in many studies [1]. Hypertriglyceridaemia often accompanies an increase in small and dense low-density lipoprotein (LDL) and is now generally accepted as a risk factor for coronary heart disease [2]. From a biochemical point of view, regulation
Abbreviations: ALLN, N-acetyl-leucyl-leucyl-norleucinal; apoA, apolipoprotein A; apoB, apolipoprotein B; ER, endoplasmic reticulum; FAS, fatty acid synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; HNF-4, hepatic nuclear factor 4; MTP, microsomal triglyceride transfer protein; TG, triacylglycerol; VLDL, very low-density lipoprotein * Corresponding author. Tel.: +81 22 717 8689; fax: +81 22 717 8691. E-mail address: [email protected] (K. Sato). 1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2005.09.001
of the secretion of apolipoprotein B (apoB), the major protein of atherogenic lipoproteins, from hepatic and intestinal cells has attracted much attention to date [3,4]. Most studies have focused on apoB metabolism in the liver, given the greater contribution to the plasma apoB pool made by that organ and the availability of convenient primary and transformed hepatic cell models [5]. Medium-chain fatty acids with 6 to 12 carbon atoms have been shown to possess several specific biological properties that distinguish them from long chain fatty acids [6]. It has been shown that medium-chain triacylglycerols are potential agents for the prevention of obesity [7]. There is considerable evidence to show that the anti-obesity effect of dietary medium-chain fatty acids is caused by the fast rate of oxidation of these fatty acids, an effect that is not observed with dietary long-chain fatty acids [8]. In addition, we previously reported
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that addition of octanoate (C8:0) to chicken hepatocyte cultures in vitro decreased the secretion of triacylglycerol (TG) and apoB from cells [9] and suggested that the decrease in fat deposition in chickens fed on medium chain fatty acids may be ascribed to impairment of very low density lipoprotein (VLDL)-apoB secretion from the liver. It is, therefore, likely that medium-chain fatty acids, at least octanoate, may be a new nutrient involved in the control of fat deposition and hyperlipidemia. However, the mechanisms by which octanoate inhibits VLDL secretion from hepatocytes are still to be elucidated. The present study was undertaken to clarify the mechanisms underlying the effects of octanoate on VLDL secretion in primary cultures of chicken hepatocytes. We hypothesized that octanoate inhibits VLDL secretion by regulating intracellular apoB metabolism and then investigated the effect of octanoate on three aspects of apoB metabolism: (1) VLDL secretion (TG, cholesterol and apoB secretion); (2) VLDL assembly (apoB synthesis, apoB degradation and microsomal triglyceride transfer protein (MTP) level); and (3) apoB gene expression (apoB mRNA expression). 2. Materials and methods 2.1. Materials Fatty acid sodium salts, bovine serum albumin (BSA, essentially fatty acidfree), glucagon and aprotinin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Basal Medium Eagle, Minimum Medium Eagle without methionine and cysteine and antibiotics were obtained from Gibco BRL (Rockville, MD, USA). Rooster serum was prepared from 6-week-old male broiler chickens. l-[35S]methionine, [a-32P]dCTP, enhance solution and protein A were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL, USA). A monoclonal antibody to chicken apoB was developed in our laboratories [10]. Rabbit anti-chicken MTP antiserum was developed by immunization with synthetic peptide. Other reagents were obtained from Wako Pure Chemical Co. Ltd. (Osaka, Japan).
2.2. Animals and diets Male broiler chickens (Ross, provided from Matsumoto Hatchery, Zao, Japan) were fed ad libitum on a commercial grower’s diet (crude protein 22 g/kg diet, metabolisable energy 13.0 MJ/kg diet) and housed in wire cages under controlled temperature (25 T 3 -C) conditions. The birds were used for experiments when their body weights were within the range of 1000 to 1200 g at about 4 weeks of age.
2.3. Primary culture of chicken hepatocytes Chicken liver cells were prepared and maintained in a monolayer culture as described previously [11]. In brief, male broiler chickens were starved for 3 h and hepatocytes then isolated by perfusion of the liver with 0.05% collagenase. The hepatocytes were plated (5.0 105 cells//60 mm collagen type I-coated dish) with incubation medium for 24 h at 37 -C in 5% CO2 in order to achieve monolayer culture. After a 24-h incubation, the monolayer cultures of hepatocytes were used in experiments.
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Intracellular TG and VLDL-TG were extracted according to the method of Folch and Sloan-Stanly [12]. The TG concentrations were quantified by the method of Fletcher [13]. VLDL-cholesterol was measured using the methods of Allain et al. [14]. Intracellular protein content was determined by the method of Lowry et al. [15] using BSA as the standard.
2.5. Western blot analysis The hepatocyte culture media or cell lysates were separated by 6% SDSpolyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad Laboratories, Hercules, CA, USA). Western blotting experiments utilized phosphate-buffered saline solution (PBS) containing 7% non-fat dry milk to block the membrane. The membranes were then incubated with 5 Ag/ml of specific anti-chicken apoB monoclonal antibody or chicken MTP antisera followed by corresponding antimouse or -rabbit IgG conjugated to horseradish peroxidase. After rinsing five times in PBS containing 0.3% Tween 20, the membranes were incubated in enhanced chemiluminescence solution (Super Signal, Pierce Biotechnology, Inc., Rockford, IL, USA) for 5 to 10 min, and exposed to Kodak XAR-5 film for 1 to 10 min. ApoB proteins were detected semi-quantitatively with densitometer tracing using a Molecular Imager FX (Bio-Rad Laboratories).
2.6. Pulse-chase analysis of apoB metabolism Primary chicken hepatocytes were pre-incubated in methionine-free minimum essential medium supplemented with essential amino acids at 37 -C for 1 h and pulsed with 100 ACi/ml of [35S] methionine/cysteine (redivue Promix l-[35S] in vitro cell labeling mix, Amersham Pharmacia Biotech, Arlington Heights, IL, USA) for 60 min. After the pulse, the cells were washed twice and chased in hepatocyte attachment media supplemented with 10 mM methionine. At various chase times (0.5, 1, 2, 3 h), the cells and media were harvested and the cells were lysed in solubilization buffer (5 mM EDTA) supplemented with a cocktail of protease inhibitors (Complete Protease inhibitor, Roche Diagnostics, Rotkreuz, Switzerland) and homogenized. The homogenate was added to carbonate extraction buffer (100 mM Na2CO3 pH 11.5) and incubated for 30 min on ice. The lysates were centrifuged at 15,000 rpm for 10 min at 4 -C and the supernatants mixed with Tris buffer (500 mM Tris-HCl, pH 6.8) and then subjected to immunoprecipitation. All buffers were supplemented with a cocktail of protease inhibitors. In some experiments, the proteasomal inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) was added to the pulse and chase medium at a concentration of 40 Ag/ml. The pulse and chase times were as described above.
2.7. Immunoprecipitation, SDS-PAGE, fluorography and measurement of labeled apoB The supernatants of hepatocyte lysates and culture media were diluted with IP buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 1% Triton-X-100) supplemented with a protease inhibitor cocktail (Complete Protease inhibitor, Roche Diagnostics). Chicken anti-apoB monoclonal antibody was added to cell lysates and incubated for 4 h at 4 -C. Protein A (Protein A CL-4B, Amersham Pharmacia Biotech, Arlington Heights, IL) was then added to the lysates and the incubation continued for over 15 h at 4 -C with the tube rotating. Protein A – antibody – apoB complexes were collected by centrifugation at 15,000 rpm for 20 min. The immunoprecipitates were washed three times with IP buffer and twice with PBS. To detect the radiolabeled apoB, the pellet was resuspended in sample buffer (62.5 mM Tris-HCl, pH 7.4, 2% SDS, 5% mercaptoethanol) and boiled for 5 min. After centrifugation, supernatants were analyzed by 6% SDS-PAGE. The gels were fixed, and then fluorographed by incubating in enhancer (Amplify, Amersham Pharmacia Biotech). The gels were dried and exposed to X-ray film at 80 -C and the radioactivity of apoB measured using a liquid scintillation counter.
2.4. VLDL isolation and measurements of lipid and protein content 2.8. RNA isolation and Northern blot analysis VLDL in the culture media was quantitatively collected by ultracentrifugation (d < 1.065 g/ml) for 3 h with KONTRON ultracentrifuges (Kontron Instrument K.K., Zurich, Switzerland) fitted with a TFT65.13 rotor.
All probes were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) from chicken hepatocyte RNA. All PCR primers were
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designed based on published sequences from Genbank as follows; apoB (M18421), apolipoprotein A-I (apoA-I, M18746), fatty acid synthase (FAS, J03860), 3-hydroxy-3-metylglutaryl-CoA reductase (HMGR, AB109635) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AF047874). Total RNA was isolated from the culture dishes using Trizol-Reagent (Invitrogen, San Diego, CA, USA). For Northern blot analysis, 20 Ag of total RNA were resolved using glyoxal solution and electrophoresed on a 1.0% agarose gel, and transferred to membrane (Zeta-Probe Blotting Membranes, Bio-Rad Laboratories, Hercules, CA). Hybridized RNA blots and quantification of gene expression were carried out as described by our previous report [16]. The blots were subsequently hybridized with a GAPDH cDNA probe to correct for differences in the amounts of RNA loaded onto the gel.
2.9. Statistical analysis A computer-generated SAS applications package was used for statistical calculations (Statistical Analysis System Version 6.03, SAS Institute Inc., Cary, NC). Group data for multiple comparisons were analyzed by ANOVA using a general linear model procedure followed by Duncan’s multiple range tests.
3. Results 3.1. Effect of octanoate on intracellular TG accumulation and VLDL-TG, VLDL-cholesterol and apoB secretion No changes in cellular protein concentrations, cell number, and results of the trypan blue exclusion test were observed in cultures incubated with up to 2.0 mM of fatty acids for 5 days incubation (data not shown). Intracellular TG accumulation in hepatocytes was not affected by the addition of fatty acids (Fig.
Fig. 2. Effect of octanoate on microsomal triglyceride transfer protein expression in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. MTP expression was determined by Western blotting analysis. Representative illustration of the Western blot is documented in the upper panel. Results are expressed as mean T S.D. (n = 3).
1A). In contrast, VLDL-TG secretion from hepatocytes incubated with 2.0 mM octanoate was significantly lower than both control and palmitate-treated cultures (Fig. 1B). In addition, both VLDL-cholesterol and apoB secretion from hepatocytes incubated with 2.0 mM octanoate were significantly reduced compared to both control and palmitate-treated cells (Fig. 1C and D). These results confirm that octanoate
Fig. 1. Effect of octanoate on intracellular TG accumulation and VLDL-TG, VLDL-cholesterol and apoB secretion. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing various concentrations of octanoate (0, 0.2, 1.0, 2.0 mM) or 0.2 mM palmitate complexed with 1.88% BSA and the cells incubated for 24 h. Intracellular TG accumulation (A), VLDL-TG secretion (B), VLDL-cholesterol secretion (C) were measured as described in Section 2. ApoB secretion (D) was determined by Western blotting analysis. Representative illustration of the Western blot is documented in the upper panel (D). Results are expressed as mean T S.D. (n = 4). Different letters indicate significantly difference, P < 0.05.
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reduces VLDL secretion in the aspects of TG, cholesterol and apoB from chicken hepatocytes. 3.2. Effect of octanoate on MTP in hepatocytes Fig. 2 shows that MTP levels in the hepatocytes were not changed by any of the treatments. No differences in cellular protein concentrations were observed in the cultures supplemented with saturated fatty acids. 3.3. Effect of octanoate on apoB synthesis, degradation and secretion in hepatocytes The level of radiolabeled apoB following a 1 h pulse of [35S] methionine/cysteine was increased by palmitate addition and reduced by octanoate addition, relative to the control. Octanoate treatment significantly reduced intracellular apoB accumulation, compared to both control and palmitate
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treatments, 1 h into the chase period. (Fig. 3A, B). In culture media, the apoB concentrations in cultures supplemented with octanoate were also reduced. Three hours into the chase period, intracellular apoB and apoB accumulation in the media (apoB secretion) in the octanoate-treated group were reduced compared to the control and palmitate groups (Fig. 3C). The degradation of radiolabeled apoB was estimated by subtracting the sum of the apoB secreted into the media and that remaining within the cells after 3 h of chase from the intracellular apoB measured at the end of pulse. There were 0.8, 0.01 and 7.2 103 dpm/mg cell protein in the control, octanoate and palmitate treatments, respectively. In Fig. 3D, the time course of changes in apoB radioactivity in the media and cells following chasing up to 3 h are given as percentages of the total radioactivity measured at the end of the pulse. In control hepatocytes, 80% of the initial radiolabeled apoB was recovered at the end of the 3-h chase
Fig. 3. Effect of octanoate on apoB stability in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. Hepatocytes were pulsed with [35S]-methionine and chased for up to 3 h. Throughout the pulse and chase periods, fatty acids and BSA were present in the media. Media samples and cell lysates were collected at each chase time point, subjected to immunoprecipitation with a specific anti-chicken apoB antibody, and then detected by SDS-PAGE and fluorography (A) or quantitated by scintillation counting (B, C, D). (A) Representative fluorograph at time 0 and 1 h of chase. Treatments are control (C), octanoate (O) and palmitate (P); (B) ApoB accumulation during the pulse (at 0 time); (C) recovery of radiolabeled apoB at 3 h chase time. Distribution of apoB in cells (gray bar), media (hatched bar) in each hepatocyte treatment; (D) results at 0, 0.5, 1, 2, 3 h chase time. Distribution of apoB in cells (open diamond), media (closed squares) as well as the total apoB (closed triangles with dotted lines) in each hepatocyte treatment are expressed as a percentage of labeled apoB at 0 time. Results are expressed as mean T S.D. (n = 4). Different letters indicate significantly difference, P < 0.05.
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(40% in cells and 40% in media), indicating that 20% of newly synthesized apoB was intracellularly degraded during this time. In palmitate-treated hepatocytes, 60% of the initial radiolabeled apoB was recovered at the end of the 3-h chase (30% in cell and 30% in media), indicating that 40% of newly synthesized apoB was intracellularly degraded during this time. On the other hand, in octanoate-treated hepatocytes, almost 100% of newly synthesized apoB was recovered at the end of 3 h chase (60% in cell and 30% in media), indicating that newly synthesized apoB was degraded to a lesser extent than in the other treatments. Octanoate reduced intracellular apoB degradation compared to both the control and palmitate treatments, reducing both the amount and rate of degradation. 3.4. Effect of octanoate on intracellular apoB synthesis in the presence of ALLN The addition of ALLN, a protease inhibitor, to the media tended to increase new synthesis of intracellular apoB protein in all treatments. Octanoate reduced apoB synthesis compared to both control and palmitate treatments in the presence and absence of ALLN (Fig. 4). 3.5. Effect of octanoate on apoB mRNA levels in hepatocytes Addition of octanoate to media significantly decreased the apoB mRNA level compared to the control and palmitate cultures (Fig. 5A). Over the time course apoB mRNA levels in cultures treated with octanoate were reduced to 80, 60 and 20% that of control cultures at 6, 12 and 24 h., respectively (Fig. 5B). On the other hand, no changes were observed in apoB mRNA expression in the palmitate-treated cultures.
Fig. 5. Effect of octanoate on apoB mRNA levels in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for various periods. After a 24-h incubation (A) or after 6-, 12- and 24-h incubations (B), cells were collected, and the total RNA was extracted. ApoB and GAPDH mRNA levels were determined by Northern blotting analysis. Representative illustrations of the Northern blot are documented in the upper panels. Data are expressed as the ratio of apoB/GAPDH. Results are expressed as % of control, mean T S.D. (n = 4). P < 0.05.
3.6. Effect of octanoate on FAS, HMGR reductase and apoA-I mRNA expression in hepatocytes The expression levels of FAS, HMGR reductase and apo AI genes that code for key enzymes of fatty acid synthesis, cholesterol synthesis and protein involved in lipid transport, respectively, did not differ between the control, octanoate and palmitate groups (Fig. 6). 4. Discussion Fig. 4. Effect of octanoate on net apoB synthesis in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. Medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. Hepatocytes were pulsed with [35S]-methionine in the presence or absence of 40 Ag/ml ALLN. Throughout the pulse period, fatty acids and BSA as well as ALLN were present in the medium. After the pulse, cell lysates were collected, subjected to immunoprecipitation with a specific anti-chicken apoB antibody, and then quantitated by scintillation counting. Results are expressed as mean T S.D. (n = 5). Different letters indicate significantly difference, P < 0.05.
VLDL is assembled in hepatocytes from TG, cholesteryl esters, phospholipids and apoB. The assembly is a complex and highly regulated process and a number of factors play key roles [17]. MTP is crucial for VLDL assembly as it catalyzes the transfer of these lipids to nascent apoB molecules [18]. ApoB mRNA expression and apoB protein synthesis in hepatocytes are relatively constitutive and generally does not change under conditions that alter apoB
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Fig. 6. Effect of octanoate on FAS, HMG-CoA reductase and apoA-I mRNA levels in the hepatocytes. Hepatocytes were incubated for 25 h as described in Section 2. The medium was then changed to 4 ml of serum-free medium containing 0 and 2.0 mM of octanoate or 0.2 mM palmitate complexed with 1.88% BSA and incubated for 24 h. After a 24-h incubation, cells were collected and the total RNA was extracted. ApoA-I (A), FAS (B), HMGR (C) and GAPDH mRNA levels were determined by Northern blotting analysis. Representative illustrations of the Northern blot are documented in the upper panels. Data are expressed as the ratio of GAPDH. Results are expressed as % of control, mean T S.D. (n = 4). Different letters indicate significantly difference, P < 0.05.
secretion. Therefore, VLDL assembly is regulated largely at co- and post-translational levels [19]. ApoB degradation can also occur post-translocationally and even after an apoBcontaining particle is formed at multiple sites in the secretary pathway by a variety of non-proteosomal means [20 –24]. Taking into consideration the data that these previous studies provide on the mechanism of regulation of VLDL secretion, in the present study, we have investigated, in a stepwise manner, whether octanoate reduces VLDL secretion, and how octanoate regulates VLDL secretion in chicken primary hepatocytes. We previously reported that the addition of octanoate to primary cultures of chicken hepatocytes reduced VLDL secretion with respect to both TG and apoB secretion. The effects of octanoate were substantiated even in hepatocytes with high levels of intracellular TG, which was induced by the addition of palmitate. Therefore, we concluded that octanoate decreases secretion of apoB-containing lipoproteins regardless of the intracellular lipogenic state. Our present results confirm those findings, showing that octanoate lowered VLDL-TG, -cholesterol and apoB secretion from primary cultured chicken hepatocytes compared to both control and palmitate cultures (Fig. 1). In this paper, we further suggest that modification of intracellular apoB metabolism is involved in the inhibition of VLDL secretion by octanoate. MTP plays a role in lipoprotein assembly by catalyzing the transfer of lipids to nascent apoB molecules, which is one of the key factors regulating apoB secretion. Recently Marcil et al. reported that butyrate (C4:0) reduced apoB48 synthesis by decreasing MTP protein expression in Caco-2 cells [25]. However, the effect of fatty acids on MTP activity and expression is not clear in hepatocytes, probably because of their long half-life of 4.4 days [26]. Our present results show that octanoate does not change protein level of MTP in chicken hepatocytes, despite octanoate reducing apoB secretion (Fig. 2). It is likely that VLDL assembly itself is not responsible for the impairment of apoB secretion by octanoate.
To date, most reports on the effects of fatty acids on apoB secretion from hepatocytes have viewed these effects as being dependent on apoB degradation and VLDL assembly, while apoB mRNA levels and intracellular apoB synthesis have been reported to be unchanged by fatty acids [27 – 30]. Thus, one might expect that octanoate increases intracellular apoB degradation and consequently decreases apoB secretion. Our results reveal that the addition of 2.0 mM octanoate to the media reduces intracellular apoB degradation compared to fatty acid-free and palmitate-treated control cultures, while it reduces the level of newly synthesized apoB. In addition, in the presence of the proteosomal inhibitor, ALLN, octanoate decreased the level of newly synthesized apoB in hepatocytes. This may be sufficient evidence that decreases in VLDL secretion induced by octanoate treatment are due to decreased intracellular apoB synthesis but not to regulation of apoB degradation. In addition, the present study has shown that octanoate addition not only decreases intracellular apoB synthesis but also significantly reduces apoB mRNA expression, compared to cultures supplemented with BSA alone or with palmitate (Fig. 5A). These results suggest that octanoate can significantly reduce apoB mRNA expression, in turn decreasing apoB synthesis and secretion. Thus, this is the first study to report that octanoate is a unique inhibitor of apoB secretion acting through its effects on apoB mRNA transcription and synthesis. Decreases in apoB mRNA expression induced by octanoate might be due to decreases in apoB gene transcription or increases in apoB mRNA decay. Pullinger et al. reported that the half-life of apoB mRNA is 16 h in HepG2 cells [27]. Here, we carried out a time-course analysis of apoB mRNA levels over 24 h and showed that apoB mRNA levels were reduced by approximately 50% 12 h after the addition of octanoate to the media (Fig. 5B). This might suggest that octanoate downregulates apoB gene transcription. The human apoB-promoter region has binding sites for HNF-4a, C/EBP and other nuclear receptors [31]. Transforming growth factor h (TGF-h) alters apoB secretion from HepG2 cells by changing steady-state
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mRNA levels and protein synthesis through the transcriptional signals, SMADs [32]. It is likely, therefore, that octanoate, itself or in complex with those transcriptional factors, binds to the apoB promoter region and consequently regulates apoB gene expression. Unfortunately, in the present study, direct evidence of whether the decrease in apoB levels induced by octanoate is accounted for by the reduction in transcription from the apoB gene was not obtained. Promotor analysis using apoB gene-transfected cells may help to further elucidate the effects of octanoate on VLDL secretion. In this study, high concentrations of fatty acid, up to 2 mM, were used in the chicken hepatocyte cultures. There are some reports that postprandial serum concentrations of octanoate in pigs fed medium-chain triglyceride increased to 0.6– 0.8 mM [33]. Moreover, we have shown, in the present study that the index of toxicity and mRNA expression of apo A-I, FAS and HMGR are similar in control and palmitate- and octanoatetreated cultures. In addition, some studies in which octanoate was added to a concentration of 4 mM in media [34,35] supports our finding that specific inhibition of apoB mRNA expression by octanoate appears to be without cytotoxicity in chicken hepatocytes. In summary, the present study demonstrates that octanoate lowers VLDL-TG, -cholesterol and -apoB secretion from primary cultured chicken hepatocytes. Moreover, 2.0 mM octanoate, compared not only to palmitate-treated but also to control (BSA alone) cultures, significantly decreased both apoB synthesis and apoB mRNA levels in chicken hepatocytes. This is the first report to show that octanoate impairs VLDL secretion by inducing a reduction in intracellular apoB synthesis via reduction of mRNA levels. It is, therefore, likely that MCT, rich in octanoate, may be used as a novel functional nutrient to control fat deposition and hyperlipidemia. These findings may provide clues not only for the development of new nutritional means for the regulation of VLDL secretion, but also for the identification of factors regulating lipoprotein metabolism. References [1] P.W. Wilson, J.C. Christiansen, K.M. Anderson, W.B. Kannel, Impact of national guidelines for cholesterol risk factor screening. The Framingham Offspring Study, JAMA 262 (1989) 41 – 44. [2] P.O. Kwiterovich, Clinical relevance of the biochemical, metabolic, and genetic factors that influence low-density lipoprotein heterogeneity, Am. J. Cardiol. 90 (2002) 30i – 47i. [3] J.L. Dixon, H.N. Ginsberg, Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells, J. Lipid Res. 34 (1993) 167 – 179. [4] S. Pal, E. Allister, A. Thomson, J.C. Mamo, Cholesterol esters regulate apoB48 secretion in CaCo2 cells, Atherosclerosis 161 (2002) 55 – 63. [5] A.A. Qureshi, D.M. Peterson, The combined effects of novel tocotrienols and lovastatin on lipid metabolism in chickens, Atherosclerosis 156 (2001) 39 – 47. [6] A.C. Bach, Y. Ingenbleek, A. Frey, The usefulness of dietary mediumchain triglycerides in body weight control: fact or fancy? J. Lipid Res. 37 (1996) 708 – 726. [7] A. Geliebter, N. Torbay, E.F. Bracco, S.A. Hashim, T.B. Van Itallie, Overfeeding with medium-chain triglyceride diet results in diminished deposition of fat, Am. J. Clin. Nutr. 37 (1983) 1 – 4.
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