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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:1636-1641, July 2005

Biochemical and Molecular Actions of Nutrients

Impairment of VLDL Secretion by Medium-Chain Fatty Acids in Chicken Primary Hepatocytes Is Affected by the Chain Length1

Kan Sato2, Yositake Cho, Shizuko Tachibana, Tomoyuki Chiba, Wolfgang J. Schneider* and Yukio Akiba

Laboratory of Animal Nutrition, Graduate School of Agricultural Science, Tohoku University, Japan and * Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Austria

2To whom correspondence should be addressed. E-mail: kan{at}bios.tohoku.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
To determine the effect of the chain length of medium-chain fatty acids (MCFAs) on VLDL secretion, the media of chicken hepatocyte cultures were supplemented with hexanoate (6:0), octanoate (8:0), decanoate (10:0), or dodecanoate (12:0). The supplementation of palmitate (16:0) or bovine serum albumin (BSA) alone in media was used as the positive control or the control, respectively. Palmitate significantly increased intracellular triacylglycerol (TG) accumulation and VLDL-TG, -cholesterol, and -apolipoprotein (apo)B secretion. On the other hand, the addition of hexanoate did not affect these variables relative to control cultures supplemented with BSA alone, whereas octanoate, decanoate, and dodecanoate decreased apoB secretion from the chicken hepatocytes. ApoB secretion from hepatocytes cultured with 1.0 mmol/L MCFA, in particular decanoate and dodecanoate, in the presence of 0.2 mmol/L palmitate was significantly lower than that obtained with 0.2 mmol/L palmitate alone. Decanoate at 0.25–1.0 mmol/L dose dependently reduced apoB mRNA expression compared with the control (BSA alone). The levels of 3-hydroxy-3-metylglutaryl-CoA reductase and apoA-I mRNA were significantly lower in cultures supplemented with hexanoate, octanoate, and decanoate than in cultures with dodecanoate and palmitate. These changes did not correspond to the reduction in VLDL-apoB secretion. We suggest that MCFAs with different chain lengths differentially affect apoB secretion and mRNA expression, with decanoate being the most effective at decreasing VLDL-apoB secretion by regulating apoB mRNA expression at the transcriptional level.

KEY WORDS: • apolipoprotein B • chicken hepatocyte primary culture • medium-chain fatty acid • triacylglycerol • VLDL

Apolipoprotein (apo)3 B is an integral protein component of VLDL. When VLDL is metabolized to LDL, apoB is retained. The synthesis of apoB occurs primarily in hepatic and intestinal cells. 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 (1). Thus, the effects of nutritional sources on apoB metabolism, i.e., degradation, assembly, and secretion of VLDL, were studied using cultured hepatocytes.

Medium-chain fatty acids (MCFAs) with 6–12 carbon atoms have several specific biological properties that distinguish them from long-chain fatty acids (LCFAs) (2,3). Basically, MCFAs are transported primarily through the portal vein to the liver after absorption from the intestine because, unlike LCFAs, MCFAs are not incorporated into triacylglycerols (TG) packaged within chylomicrons (4). In peripheral tissues, the oxidation rate of MCFAs is greater than that of LCFA but their esterification rate is low (5). Therefore, the rapid rate of the metabolism of MCFAs increases energy expenditure and decreases the fat deposition in adipose tissue (6). In addition, we reported previously that the addition of octanoate (8:0), an MCFA, to primary cultures of chicken hepatocytes reduced VLDL secretion with respect to both TG and apoB secretion (7). These results suggest that the decrease in fat deposition in birds fed MCFAs relative to birds fed LCFAs is due in part to impairment of VLDL-apoB secretion from liver. Arrol et al. (8) reported that the chain length and degree of saturation of fatty acids affect apoB secretion from HepG2 hepatocytes. However, the effects of MCFAs other than octanoate, for example, hexanoate, decanoate, or dodecanoate, on VLDL-apoB secretion have not been studied to date.

In our present study of chicken primary hepatocytes, we show that the chain length of MCFA (6:0 to 12:0) has specific effects on VLDL secretion compared with the response elicited by exposure of hepatocytes to palmitate (16:0), an LCFA. In addition, possible regulatory mechanisms by which MCFA modulates VLDL secretion from the viewpoint of mRNA expression of apoB are examined.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diets. Male broiler chickens (Ross, provided from Matsumoto Hatchery) consumed ad libitum a commercial grower diet (crude protein 22 g/kg diet, metabolizable energy 13.0 MJ/kg diet) and were housed in wire cages under controlled temperature (23 ± 3°C) conditions. The birds were used for experiments when their body weights were within the range of 1000 to 1200 g (~4 wk of age).

    Chemicals. Glucagon and bovine serum albumin (BSA; essentially fatty acid–free) were purchased from Sigma Chemical. Basal Medium Eagle and antibiotics were obtained from Gibco BRL. Other reagents and fatty acid sodium salts were obtained from Wako Pure Chemical. Rooster serum was prepared from 6-wk-old male broiler chickens.

    Primary culture of chicken hepatocytes. Liver cells were prepared from chickens that had been food deprived for 12 h and maintained in monolayer culture as described previously (9). Hepatocytes with >90% viability, verified by Trypan blue exclusion test, were used for subsequent plating (5.0 x 105 cells/{phi} 60 mm collagen type I-coated dish) with incubation medium (Basal Medium Eagle supplemented with essential amino acids), containing 75 kU/L penicillin, 75 kU/L streptomycin, 1 mg/L insulin (bovine), 1 mg/L glucagon (human recombinant), and 0.5% rooster serum. After 20 h of incubation (attachment phase), cultures were preincubated for 1 h in serum-free media containing 1 mg/L insulin, 1 mg/L glucagon, and 0.12 mg/L trypsin inhibitor, followed by a 24-h incubation in the presence of media containing 0.25, 0.5, or 1.0 mmol/L of hexanoate (6:0), octanoate (8:0), decanoate (10:0), dodecanoate (12:0), or palmitate (16:0) complexed with essentially fatty acid–free BSA (20 g/L media) as indicated. After the 24-h incubation, the culture media and cells were cooled on ice and collected for the analysis of apoB levels in media, VLDL-TG, VLDL cholesterol, and intracellular-TG and protein, respectively.

To determine the effects of MCFAs on apoB secretion from hepatocytes containing high levels of intracellular TG (lipogenic state) (7), chicken hepatocytes were cultured in the presence of 1.0 mmol/L MCFA together with 0.2 mmol/L palmitate. All analysis was conducted on 4 independent hepatocyte culture preparations.

    Western blot analysis. After incubation with MCFAs, the hepatocyte culture media or cell pellets were separated by 6 or 12% SDS-PAGE in the absence or presence of reducing agents and then transferred to nitrocellulose membrane (Bio-Rad Laboratories). Western blotting experiments utilized a PBS solution containing 7% nonfat dry milk and the strips were incubated with 5 mg/L of chicken apoB–specific monoclonal antibody (CAB4) (10) or rabbit anti- chicken microsomal triglyceride transfer protein (MTP) antisera followed by the corresponding anti-mouse or rabbit IgG conjugated with horseradish peroxidase. After being rinsed 5 times in PBS containing 0.3% Tween 20, the strips were incubated in substrate solution (ECL kit, Amersham Bioscience) for 5–10 min and exposed to Kodak XAR-5 film for 1–10 min. ApoB proteins were detected semiquantitatively with densitometer tracing using Molecular Imager FX (Bio-Rad Laboratories).

    mRNA analysis. Standard molecular biology techniques were used essentially as described by Sambrook et al. (11). Tissues were homogenized in Trizol-Reagent (Gibco BRL) and total RNA isolated by the method of Chomczynski and Sacchi (12). To examine changes in the levels of expression of mRNAs in hepatocytes, total RNA was electrophoresed in a 1.0% agarose gel containing formaldehyde, as described by Lehrach et al. (13), and transferred to Zeta-Probe Membrane (Bio-Rad Laboratories) for hybridization. The probes used for Northern blot analysis included chicken apoB [nucleotides 398-1022, M18421, (14)], fatty acid synthase (FAS) [nucleotides 3076–4054, J03860, (15)], 3-hydroxy-3-metylglutaryl-CoA reductase (HMGR) [nucleotides 1225–2514, AB109635, (16)], apoA-I [nucleotides 52–843, M18746, (17)], and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [nucleotides 400–942, AF047874, (18)]. These cDNA probes were synthesized from chicken liver mRNA and labeled by random priming with [32P]dCTP (6000 Ci/mmol; Takara BcaBEST 228 Labeling Kit). Hybridized RNA blots and quantification of gene expression were carried out as described in our previous report (16). The blots were subsequently hybridized with the GAPDH cDNA probe to correct for differences in the amounts of RNA loaded onto the gel.

    Analysis. VLDL in the culture media was collected quantitatively by ultracentrifugation (d < 1.065 g/mL) for 3 h using a KONTRON ultracentrifuge (Kontron Instrument) fitted with a TFT65.13 rotor. Intracellular TG and VLDL-TG were extracted according to the method of Folch et al. (19). The TG concentrations were quantified by the method of Fletcher (20). VLDL cholesterol was measured using the methods of Allain et al. (21). Intracellular protein content was determined by the method of Lowry et al. (22) using BSA as the standard.

    Statistical analysis. The SAS applications software package was used for statistical calculations (SAS Version 6.03, SAS Institute). Data were analyzed by ANOVA using a general linear model procedure followed by Tukey’s test. Intracellular-TG, VLDL-TG, and VLDL cholesterol were analyzed by 2-way ANOVA (5 fatty acids x 2 doses). In addition, to analyze the effect of dose on intracellular-TG, VLDL-TG, and VLDL cholesterol, 1-way ANOVA was conducted using data from the MCFA-treated cultures. Results are expressed as means ± SD of 4 independent hepatocyte culture preparations. Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Intracellular TG accumulation, and secretion of VLDL-TG, VLDL cholesterol, and apoB. Cellular protein concentrations did not differ among the cultures incubated with the different saturated fatty acids (data not shown). Adding palmitate to the incubation media at both 0.5 and 1.0 mmol/L significantly increased intracellular TG accumulation and the secretion of VLDL-TG, VLDL cholesterol (Table 1), and apoB (Fig. 1) compared with other fatty acids. Dodecanoate increased the intracellular TG accumulation but did not increase VLDL-TG and VLDL cholesterol secretion. Secretion of apoB from chicken primary hepatocytes was reduced by dodecanoate treatment. In cultures supplemented with hexanoate, intracellular TG accumulation and VLDL-TG, VLDL cholesterol, and apoB secretions were lower than in those treated with palmitate. These variables, however, did not differ from the control cultures (BSA alone). The effects of octanoate and decanoate were dose dependent. Neither of these fatty acids affected intracellular TG accumulation and VLDL-TG or VLDL cholesterol secretion, compared with control cultures treated with BSA alone. However, apoB secretion from chicken hepatocytes gradually decreased as the concentration of octanoate or decanoate was increased (Fig. 1). Increasing the MCFA concentration from 0.2 to 1.0 mmol/L significantly decreased VLDL-TG secretion (Table 1). The fatty acid x dose interaction was significant for intracellular TG accumulation and for VLDL-TG and VLDL cholesterol secretion (Table 1).


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  		TABLE 1 Effect of MCFA (6:0–12:0) and palmitate (16:0) on intracellular TG accumulation, and secretion of VLDL-TG and VLDL-cholesterol in cultured primary chicken hepatocytes1

 


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  		FIGURE 1 Effect of MCFAs (6:0–12:0) and palmitate (16:0) on apoB secretion in cultured primary chicken hepatocytes. Hepatocytes were incubated for a total of 48 h with 0.25, 0.5, or 1.0 mmol/L of the corresponding fatty acids or BSA alone during the final 24 h of culture. After incubation, 15 µL of media from each treatment was analyzed by Western blotting.

 
    ApoB secretion in the presence of 0.2 mmol/L palmitate. Incubation of chicken hepatocytes with 0.2 mmol/L palmitate increased intracellular TG accumulation by ~200% compared with the palmitate-free treatment (BSA alone). Compared with the 0.2 mmol/L palmitate control, intracellular TG accumulation was higher in hepatocytes incubated with 1.0 mmol/L decanoate or dodecanoate. In contrast, the addition of MCFAs, especially decanoate or dodecanoate, at 1.0 mmol/L significantly reduced apoB secretion relative to the 0.2 mmol/L palmitate control (Fig. 2).



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  		FIGURE 2 Effect of MCFAs (6:0–12:0) on intracellular TG accumulation (A) and apoB secretion (B) in cultured primary chicken hepatocytes in the presence of 0.2 mmol/L palmitate (16:0) and 1.0 mmol/L MCFA during the final 24 h. Values are means ± SD, n = 4 independent hepatocyte cultures. Means without a common letter differ, P < 0.05.

 
    Expression of apoB, FAS, HMG-CoA reductase and apolipoprotein A-I. ApoB mRNA levels did not differ between the control, hexanoate, dodecanoate, and palmitate cultures (Fig. 3A). On the other hand, the addition of 1.0 mmol/L decanoate to the hepatocyte cultures significantly and dose dependently (Fig. 3B) reduced the level of apoB mRNA, and the addition of octanoate also tended (P = 0.06) to reduce apoB mRNA levels.



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  		FIGURE 3 Effect of MCFAs (6:0–12:0) and palmitate (16:0) on apoB mRNA levels (A) and the dose-dependent changes in apoB mRNA levels by decanoate (B) in cultured primary chicken hepatocytes. (A) Hepatocytes were incubated with 1.0 mmol/L MCFA or palmitate during the final 24 h of culture. Values for apoB mRNA are means ± SD, n = 4 independent hepatocyte cultures. Means without a common letter differ, P < 0.05. (B) Hepatocytes were incubated with 0, 0.25, 0.5 or 1.0 mmol/L decanoate during the final 24 h of culture. Total RNA (30 µg) prepared from cultured cells was hybridized with a chicken apoB or GAPDH probe and visualized by molecular imager.

 
The expression levels of FAS, HMGR, and apoA-I, genes that code for key enzymes of fatty acid synthesis, cholesterol synthesis, and for protein involved in lipid transport, respectively, did not differ among control, hexanoate, octanoate, and decanoate groups. However, HMGR and ApoA-I levels in the cells treated with dodecanoate and palmitate were significantly increased relative to control cultures (Fig. 4).



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  		FIGURE 4 Effect of MCFAs (6:0–12:0) and palmitate (16:0) on FAS, HMGR, and apoA-I mRNA levels in cultured primary chicken hepatocytes. Values for FAS (A), HMGR (B) and apoA-I (C) mRNAs are means ± SD, n = 4 independent hepatocyte cultures. Means without a common letter differ, P < 0.05.

 
    Intracelluer microsomal triglyceride transfer protein levels. The MTP immunoreactive protein level did not differ in chicken hepatocytes cultured with 1.0 mmol/L fatty acids compared with control (BSA alone) (Fig. 5).



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  		FIGURE 5 Effect of MCFAs (6:0–12:0) and palmitate (16:0) on intracellular MTP levels in cultured primary chicken hepatocytes. Hepatocytes were incubated with 1.0 mmol/L corresponding fatty acids or BSA alone during the final 24 h. After incubation, cells were subjected to Western blot analysis. Values are means ± SD, n = 4 independent hepatocyte cultures. Means without a common letter differ, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Hepatic VLDL secretion is modulated by nutritional states, particularly by the fatty acid profiles of rations (23,24). In cultured Hep G2 cells, Arrol et al. (8) showed that the addition of oleate (18:1) and palmitate (16:0) increased apoB secretion by 140 and 50%, respectively, above the control group level and was accompanied by an increase in intracellular TG. Kummrow et al. (25) reported that docosahexaenoic acid-treated McA-RH7777 cells decreased apoB secretion to 26% of the untreated control, whereas myristate (14:0) resulted in a 50% increase. Thus, fatty acids of varying chain lengths and saturation differentially affect plasma apoB-100 levels via VLDL secretion from hepatocytes. We reported previously that the addition of octanoate to primary cultures of chicken hepatocytes reduced VLDL secretion with respect to TG and apoB secretion (7). Therefore, the present experiments with chicken hepatocytes were undertaken to clarify the effect of chain length of MCFAs on VLDL secretion and the related mechanisms.

A novel finding of the present study is that decanoate dramatically modulates VLDL secretion (Table 1, Fig. 1). Decanoate at 1.0 mmol/L extensively reduced apoB secretion and mRNA expression compared with a control treated with BSA alone, whereas it did not modulate intracellular TG accumulation, MTP immunoreactive protein levels, or mRNA expression of FAS, HMGR, and apoA-I. The apoB-containing pre-VLDL particle is formed during translation and translocation of apoB into the lumen of the rough endoplasmic reticulum (26) and is further modified during a short post-translational period (27) to form a mature VLDL particle that is secreted. VLDL secretion, in particular apoB secretion, has been considered to be modulated by the degree of intracellular apoB degradation (28). Dixon et al. (29) demonstrated that oleic acid stimulates secretion of apoB-containing lipoproteins from HepG2 cells by inhibiting early intracellular degradation of apoB. The (n-3) PUFAs, eicosapentaenoic acid and docosahexaenoic acid, decreased apoB secretion by increasing apoB degradation in hamster hepatocytes (30). Therefore, reduction of apoB secretion from hepatocytes was considered to be dependent on apoB degradation and VLDL assembly, whereas apoB mRNA levels were reported to be unchanged. Recently, however, Xu et al. (31) showed that supplementation with adrenocorticotropic hormone reduced apoB mRNA levels in HepG2 cells, consistent with a direct inhibitory effect on apoB synthesis in hepatocytes. The present study showed that 1.0 mmol/L decanoate, when added to media, significantly decreased apoB mRNA levels in hepatocytes, compared with the addition of palmitate or BSA alone. Octanoate also tended to decrease apoB mRNA expression (Fig. 3). These results suggest that MCFAs, especially decanoate, are able to reduce apoB mRNA expression, which in turn might decrease apoB synthesis and secretion. However, in the present study, it is unclear whether the decrease in apoB mRNA levels induced by decanoate is accounted for by reduced apoB gene transcription or enhanced apoB mRNA degradation. Pulse-chase experiments and promotor analysis using apoB gene transfected cells may help to further elucidate the effects of MCFAs on VLDL secretion.

Cellular synthesis and accumulation of TG and cholesterol modulate VLDL secretion from hepatocytes (28). Here, we demonstrated that 1.0 mmol/L MCFA reduced apoB secretion from cultures in the presence of 0.2 mmol/L palmitate, compared with cultures supplemented with 0.2 mmol/L palmitate alone, whereas decanoate and dodecanoate increased intracellular TG accumulation (Table 1). It is likely, therefore, that octanoate, decanoate, and dodecanoate reduce the secretion of apoB-containing lipoproteins regardless of the intracellular lipogenic state. In addition, dodecanoate and decanoate increased the expression of HMGR and did not modify FAS mRNA levels but lowered apoB secretion (Figs. 1 and 4). These results suggest that the impairment of VLDL secretion by MCFAs was not associated with changes in the cellular synthesis of fatty acids and cholesterol. The levels of MTP, a regulator of VLDL assembly, were not modified by any of the fatty acid treatments. These results suggest that VLDL assembly itself does not account for the impairment of apoB secretion by MCFAs. Moreover, the mRNA level of apoA-I was not affected by MCFAs, with the exception of dodecanoate, suggesting that MCFAs specifically modulate apoB gene expression. Thus, MCFAs, particularly decanoate, are novel nutrients that decrease VLDL secretion in a manner that differs from the effects of nutrients and hormones reported to date.

In this study, high concentrations of fatty acid, up to 1.0 mmol/L, were used in the chicken hepatocyte cultures. We assayed the index of toxicity resulting from the addition of fatty acids and showed that cellular protein concentration, cell number, and the results of the trypan blue exclusion test were not affected in cultures incubated with up to 1.0 mmol/L of fatty acid for 5 d (data not shown). In addition, intracellular ATP levels of 1.0 mmol/L hexanoate-, octanoate-, decanoate-, dodecanoate-, or palmitate-treated cells were 6.3 ± 0.7, 6.1 ± 1.1, 7.8 ± 1.2, 7.5 ± 0.7, or 6.3 ± 0.9 pmol/dish, respectively, and were not affected by the fatty acid treatment. Moreover, in the present study, we showed that mRNA expression of apoA-I, FAS, and HMGR was equal in the control and decanoate-treated cultures. In addition, there are studies in the literature in which octanoate was added to the culture media at concentrations as high as 4 mmol/L (32), indicating that specific inhibition of apoB mRNA expression by decanoate may be evaluated in chicken hepatocytes without cytotoxicity problems.

In conclusion, MCFAs of varying chain lengths differentially affect apoB-100 secretion and mRNA expression. MCFAs lower VLDL-TG and VLDL cholesterol secretion from primary cultured chicken hepatocytes, relative to cultures treated with palmitate. Octanoate, decanoate, and dodecanoate, compared not only with palmitate but also with the BSA control, all decrease apoB secretion. Secretion of apoB, however, is not affected by the addition of hexanoate. Moreover, apoB mRNA levels in chicken hepatocytes are reduced by decanoate treatment. This is the first report to show that decanoate impairs VLDL secretion by reducing the levels of apoB mRNA. These findings may provide a clue not only to the development of new nutritional means for the regulation of VLDL secretion, but also to ways of identifying factors regulating lipoprotein metabolism.


   FOOTNOTES
 
1 Supported by Grants-in-Aid (#14760175 and 13556043) from The Ministry of Education, Science and Culture of Japan, and from the Austrian Science Fund (FWF) to W.J.S. Back

3 Abbreviations used: apo, apolipoprotein; BSA, bovine serum albumin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMGR, 3-hydroxy-3-metylglutaryl-CoA reductase; LCFA, long-chain fatty acid; MCFA, medium-chain fatty acid; MTP, microsomal triglyceride transfer protein; TG, triacylglycerol. Back

Manuscript received 6 January 2005. Initial review completed 6 March 2005. Revision accepted 12 April 2005.


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