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© 2003 The American Society for Nutritional Sciences J. Nutr. 133:2180-2183, July 2003

Biochemical and Molecular Actions of Nutrients

Butyrate Impairs Lipid Transport by Inhibiting Microsomal Triglyceride Transfer Protein in Caco-2 Cells

Valérie Marcil, Edgard Delvin*, Carole Garofalo and Emile Levy2

Departments of Nutrition and * Biochemistry, Centre de Recherche, Hôpital Sainte-Justine, Université de Montréal, Montréal, QC, Canada, H3T 1C5

2To whom correspondence should be addressed. E-mail: levye{at}justine.umontreal.ca.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Recently, the idea was advanced that short-chain fatty acids (SCFA) may potentially regulate intestinal fat absorption. The aim of this investigation was to examine the effects of butyrate on the intracellular events governing the assembly of triglyceride-lipoproteins in enterocytes. To this end, differentiated human Caco-2 cells were exposed to 10 or 20 mmol/L butyrate for 20 h. The incubation of Caco-2 cells with butyrate decreased cholesteryl ester (P < 0.005) export in the basolateral medium, probably due to reduced activity of DL-3-hydroxy-3-methyl-glutaryl-CoA reductase (P < 0.02), the rate-limiting enzyme in cholesterol synthesis. Furthermore, a drop was noted in the protein expression of microsomal triglyceride transfer protein (P < 0.03), concomitant with the inhibition of de novo apolipoprotein B-48 synthesis (P < 0.02) and triglyceride-rich lipoprotein output (P < 0.03). Our results support the hypothesis that SCFA can influence lipoprotein concentrations by limiting lipid release from the small intestine into the circulation.

KEY WORDS: • HMG-CoA reductase • apolipoprotein B-48 • microsomal triglyceride transfer protein • fat absorption

Butyrate constitutes the major energy fuel for the colon. Of all short-chain fatty acids (SCFA), it has the greatest effect on colonocyte biology, including cell maturation, cycle arrest, differentiation and apoptosis (1–3). Not only does butyrate affect the physiology of the colonic epithelium, but it also exhibits beneficial effects on the intestine in various pathologic states such as colonic neoplasia, ulcerative colitis, diversion colitis and colonic injury (4). Moreover, butyrate may improve some small intestine functions, and its use with other SCFA has been recommended to supplement the parenteral nutrition solutions of individuals with short bowel syndrome or intestinal malabsorption syndromes (5,6).

The beneficial effects of complex carbohydrate and high fiber diets on carbohydrate and lipid metabolism have been suggested to be mediated by SCFA metabolism in the liver. SCFA may indirectly alter carbohydrate and lipid metabolism (7,8). However, our recent findings indicate that butyrate may directly influence lipid metabolism in Caco-2 cells (9), through a putative regulation of intestinal fat absorption and circulating lipoprotein concentrations. In this report, we investigated the role of regulatory key proteins in the decreased triglyceride (TG)-rich lipoprotein production mediated by butyrate.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
Cell culture.

Caco-2 cells were grown at 37°C with 5% CO2 in minimum essential medium (MEM; GIBCO-BRL, Grand Island, NY), containing penicillin/streptomycin (100 kU/L), MEM nonessential amino acids (0.1 mmol/L) and supplemented with 10% decomplemented fetal bovine serum (FBS; Flow, McLean, VA). Caco-2 cells (passages 30- 40) were maintained in 17.5-cm2 flasks (Corning, NY). Cultures were split (1:3 to 1:6) when they reached 70–90% confluence, using trypsin-EDTA (50 g/L-0.5 mmol/L; GIBCO-BRL). For individual experiments, cells were plated at a density of 1 x 106 cells/well on 24.5-mm polycarbonate Transwell filter inserts with 0.4-µm pores (Costar, Cambridge, MA), in MEM (as above), supplemented with 5% FBS. The inserts were placed into six-well culture plates, permitting separate access to the upper and lower compartments of the monolayers. Cells were cultured for 20 d, a period at which the Caco-2 cells are highly differentiated and suitable for lipid synthesis (10,11). Butyrate (20 mmol/L) was added to the upper chamber in serum-free MEM. Lactic dehydrogenase was measured as described previously (9) and proteins were determined by BIORad kit (Montreal, Quebec, Canada).

Measurement of cholesterol synthesis and secretion.

Cholesterol biogenesis was evaluated, employing [14C]-acetate as a precursor (1.9943 TBq) for a 20-h incubation period, as described previously (9,12). Free cholesterol (FC) and cholesteryl ester (CE) were separated by TLC.

Lipid carrier.

Blood was drawn 2 h after the oral intake of a fat meal by human volunteers, and postprandial plasma was prepared to serve as a carrier for the lipoproteins synthesized by Caco-2 cells. The TG-enriched plasma was incubated at 56°C for 1 h to inactivate enzymatic activity in the presence of antiproteases (phenylmethylsulfonyl fluoride, pepstatin, EDTA, aminocaproic acid, chloramphenicol, leupeptin, glutathione, benzamidine, dithiothreitol, sodium azide and Trasylol, all at a final concentration of 1 mmol/L).

Isolation of lipoproteins.

The determination of secreted lipoproteins was performed as described previously (13,14). Briefly, radiolabeled [14C]-oleic acid (specific activity, 1.961 TBq; Amersham, Oakville, Canada) was added to unlabeled oleic acid and then solubilized in fatty acid–free bovine serum albumin (BSA) [BSA/oleic acid, 1:5 (mol/mol)]. The final oleic acid concentration was 0.8 mmol/L (16.65 kBq/well). Cells were first washed with PBS (GIBCO), and the [14C]-oleic acid–containing medium was added to the upper compartment. Caco-2 cells were incubated with the lipid substrate as described above, in the presence or absence of butyrate. The medium supplemented with antiproteases (as described above) was first mixed with a plasma lipid carrier (4:1, v/v) to efficiently isolate de novo synthesized lipoproteins. The lipoproteins were then isolated by ultracentrifugation using a TL-100 ultracentrifuge (Beckman Instruments, Montreal, Quebec, Canada), as described previously (13–15).

De novo apolipoprotein synthesis.

The effect of butyrate on newly secreted apolipoprotein (apo) B-48 was assessed as described previously (16,17). To first induce apolipoprotein synthesis, cells were incubated apically with unlabeled oleic acid bound to albumin in serum-free medium, 20 h before [35S]-methionine incubation. The concentration of the unlabeled lipid was equivalent to the labeled substrate described above. During this time, butyrate was again added to the apical chamber. After a 20-h incubation, cells as well as the outer chambers were rinsed twice with PBS. The apical compartment was replaced with 1.5 mL of methionine-free medium containing the unlabeled substrate and [35S]-methionine (3.7 GBq/L) (Amersham, 1.85 GBq/mmol). After incubation for 3 h at 37°C with 5% CO2, the medium from the basolateral compartment was collected and supplemented with the antiprotease cocktail and unlabeled methionine to act as a carrier (final concentration, 0.1 mmol/L). Immunoprecipitation and SDS were performed as described previously (16,17). Apo B-48 slices were sectioned from the gel and counted after an overnight incubation with 1 mL of Beckman tissue solubilizer and 10 mL of liquid scintillation fluid (Ready Organic, Beckman). Results were expressed as % TCA/mg protein to assess the specific effect of butyrate on apolipoprotein synthesis and secretion.

Microsomal triglyceride transfer protein (MTP) expression.

To assess MTP mass, Caco-2 cells were homogenized and adequately prepared for Western blotting as described previously (18,19). MTP was quantitated using an HP Scanjet scanner equipped with a transparency adapter and software.

HMG-CoA reductase activity assay.

Enzymatic activity was assayed as described previously (10,12). The reaction mixture contained 100 mmol/L potassium phosphate (pH 7.4), 150 µg cellular protein, 20 mmol/L glucose-6-phosphate, 12.5 mmol/L dithiothreitol, 2.5 mol/L NADP and 1.2 U glucose-6-phosphate dehydrogenase. The reaction was initiated by the addition of [14C]-3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) (200 Bq/nmol). After incubation for 30 min at 37°C, the [14C]-mevalonate formed was converted into lactone, isolated by TLC, and counted using an internal standard to correct for incomplete recovery.

ACAT activity assay.

The standard acyl-CoA:cholesterol acyltransferase (ACAT) determination was based on our previous assay (10,12). We added 5 nmol [14C]-oleoyl CoA (specific activity, ~167 Bq/nmol) to the mixture containing 150 µg cellular protein to initiate the reaction in a buffer solution (pH 7.5) consisting of cholesterol, 0.04 mol/L KH2PO4, 50 mmol/L NaF, 0.25 mol/L sucrose and 1 mmol/L EDTA. After incubation for 10 min at 37°C, the reaction was stopped by adding chloroform/methanol (2:1, v/v) followed by [3H]-cholesteryl oleate as an internal standard to estimate recovery.

Statistical analysis.

All values were expressed as mean ± SEM. Data were analyzed by two-tailed Student’s t test. Differences were considered significant at P < 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
The present work employed Caco-2 cells to study the modulation of lipid transport by butyrate. In the presence of this SCFA, no cytotoxic effect was noted. After the incubation of Caco-2 with physiologic concentrations of butyrate, cell integrity, differentiation and viability were not altered as assessed by sucrase and lactic dehydrogenase activities, cell monolayer transepithelial resistance, and cell protein and DNA contents (Table 1). In addition, cell viability by trypan blue exclusion was also assessed and was uniformly >90% in the absence or presence of butyrate at the same concentrations.


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  		TABLE 1 Effect of butyrate on Caco-2 cell integrity1

 
The incubation of fully differentiated Caco-2 cells with butyrate decreased the secretion of CE in the basolateral medium, confirming our recent data (9) (Fig. 1). Because HMG-CoA reductase and ACAT control cholesterol synthesis and esterification, respectively, we determined the activity of these two key regulatory enzymes. A marked inhibitory effect of butyrate was recorded on HMG-CoA reductase without any change in ACAT activity (Fig. 2).



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  		FIGURE 1 Cholesterol synthesis and secretion in Caco-2 cells incubated with 20 mmol/L butyrate for 20 h in the presence of 0.37 MBq [14C]-acetate (200Bq/nmol) in the apical compartment. Values are means ± SEM for 3 independent experiments. *Different from control, P < 0.005. TC, total cholesterol; FC, free cholesterol; CE, cholesteryl ester.

 


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  		FIGURE 2 Activities of key sterol regulatory enzymes in Caco-2 cells incubated with 10 mmol/L (A) or 20 mmol/L (B) butyrate for 20 h at 37°C. Cell homogenates were assayed for DL-3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and acyl-CoA:cholesterol acyltransferase (ACAT). Values are means ± SEM for 6 independent experiments. *Different from control, P < 0.02; **different from control, P < 0.0005.

 
The CE present in the intestine are generated after cholesterol synthesis and a reaction catalyzed by ACAT. Because of their limited solubility in cell membranes, CE accumulate as lipid droplets in the cytoplasm, from which they can be incorporated into TG-rich lipoproteins after hydrolysis and reesterification. The availability of cholesterol and/or CE may also influence apo B synthesis and secretion by the intestine and liver. The inhibition of intestinal and hepatic cholesterol synthesis or cholesterol esterification has been shown in various studies to reduce apo B secretion (20–23). Therefore, we determined apo B production and TG-rich lipoprotein output in a subsequent step. The addition of butyrate reduced the delivery of newly synthesized apo B-48 and the exocystosis of de novo TG-rich lipoprotein in Caco-2 cells (Fig. 3).



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  		FIGURE 3 Apolipoprotein (apo) B-48 synthesis (A) and triglyceride (TG)-rich lipoprotein secretion (B) in Caco-2 cells incubated with butyrate in the apical compartment for 20 h with either [14C]-oleic acid or [35S]-methionine. TG-rich lipoproteins were separated at 100,000 x g for 2.26 h by ultracentrifugation. Values are means ± SEM for 3 independent experiments. *Different from control, P < 0.02; **different from control, P < 0.03.

 
The assembly of TG-rich lipoproteins involves a complex process in which apo B is packaged with lipids. Lipoprotein formation is initiated as apo B is translocated into the lumen of the endoplasmic reticulum, and MTP appears to be essential to this process to form stable nascent apo B–containing particles, which otherwise would be degraded (24–26). The current experiments demonstrated that butyrate decreased the protein expression of MTP (Fig. 4). We therefore suggest that the negative modulation of cholesterol production and MTP synthesis by butyrate reduced the lipidation of apo B-48, thereby resulting in intracellular apo B-48 degradation leading to a decline in chylomicron and VLDL secretion.



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  		FIGURE 4 Microsomal triglyceride transfer protein (MTP) protein expression in Caco-2 cells cultured in the presence of 10 mmol/L butyrate (A) or 20 mmol/L butyrate (B). Representative illustrations of the Western blot are documented in the upper panel. Values are means ± SEM for 6 independent experiments. *Different from control, P < 0.03; **different from control, P < 0.0003.

 
A decrease in fiber intake has been shown to contribute to the development of cardiovascular diseases (27,28). Thus, diet modification involving a substantial increase in fiber consumption has been recommended, given the potential of fiber in lowering circulating lipids (28,29). Several mechanisms have been proposed to explain the hypocholesterolemic effect of dietary fiber, i.e., the alteration of gastric emptying, modification of bile acid absorption and metabolism, production of SCFA from fermentation of fiber in the colon, up-regulation of the hepatic LDL receptor and changes in plasma concentration or tissue sensitivity to insulin or other hormones (30,31). Our results, using Caco-2 cells, indicate that butyrate directly affects the absorptive and metabolic functions of enterocytes. A number of important observations were made in the current investigation that support the effectiveness of butyrate to slow down intestinal fat transport.


   ACKNOWLEDGMENTS
 
The authors thank Schohraya Spahis for secretarial assistance.


   FOOTNOTES
 
1 Supported by the Canadian Institutes of Health Research (CIHR), the Canadian Foundation for Crohn’s and Colitis, the Dairy Farmers of Canada and by a research scholarship award from Fonds de la Recherche Scientifique du Québec (FRSQ). Back

3 Abbreviations used: ACAT, acyl-CoA:cholesterol acyltransferase; apolipoprotein, apo; BSA, bovine serum albumin; CE, cholesteryl ester; FBS, fetal bovine serum; FC, free cholesterol; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-CoA; MEM, minimal essential medium; MTP, microsomal triglyceride transfer protein; TC, total cholesterol; TG, triglyceride. Back

Manuscript received 18 December 2002. Initial review completed 24 January 2003. Revision accepted 18 April 2003.


   LITERATURE CITED
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 MATERIALS AND METHODS
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