Short-chain fatty acids regulate IGF-binding protein secretion by intestinal epithelial cells

Akiyoshi Nishimura1, Mika Fujimoto1,2, Satoshi Oguchi1, Robert D. Fusunyan1, Richard P. MacDermott2, and Ian R. Sanderson1

1 Developmental Gastroenterology Laboratory, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Harvard Clinical Nutrition Research Center, Charlestown 02129; and 2 Gastrointestinal Section, Lahey Hitchcock Clinic, Burlington, Massachusetts 01805

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Gastrointestinal epithelial cells secrete insulin-like growth factor (IGF)-binding proteins (IGFBPs), which modulate the actions of IGFs on cell proliferation and differentiation. Short-chain fatty acids are bacterial metabolites from unabsorbed carbohydrate (including fiber). We hypothesized that they may alter the pattern of IGFBPs secreted by epithelial cells as part of a wider phenomenon by which luminal molecules regulate gastrointestinal epithelial cell signaling. The intestinal epithelial cell line, Caco-2, predominantly secretes IGFBP-3; however, butyrate increased the secretion of IGFBP-2 in a dose-dependent and reversible manner. Butyrate decreased the secretion of IGFBP-3. Butyrate altered only the synthesis and not the cell sorting of IGFBPs because 1) the secretion of IGFBPs remained polarized despite changes in their rates of production, and 2) IGFBP secretion corresponded to mRNA accumulation. The ability of short-chain fatty acids or the fungicide trichostatin A to stimulate IGFBP-2 correlated with their actions on histone acetylation. In conclusion, intestinal epithelial cells respond to short-chain fatty acids by altering secretion of IGFBPs.

Caco-2 cells; acetate; butyrate; propionate; polarity; histone acetylation

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

THE INTESTINAL MILIEU is important in regulating intestinal epithelial cell proliferation and differentiation (24). Variations in the composition of the intestinal lumen may also affect several gastrointestinal disorders (8, 35, 41). However, little is known about how luminal molecules alter the secretion of growth factors by the epithelium.

Short-chain fatty acids are metabolites of intestinal bacteria (15, 16). Carbohydrate intake (including fiber) provides the substrate for their production (15); the species of bacteria in the intestinal microflora determine which short-chain fatty acids are produced (16). Thus short-chain fatty acid production in the intestine is a sensitive indicator of the bacterial ecosystem. Short-chain fatty acids have well-recognized effects on epithelial growth. They act as a primary source of fuel for colonic epithelial cells (38), and they alter their rates of proliferation and differentiation (48). However, their effect on the secretion of growth factors by epithelial cells has not been examined.

Insulin-like growth factors (IGFs) are polypeptides that promote the proliferation and differentiation of cells derived from various organ systems (23, 45). IGF-I exerts a proliferative effect on intestinal epithelial cells (29) and on activated lymphocytes (11) through type 1 IGF receptors (29, 39, 46). IGF-II alters the differentiation of intestinal epithelial cells, acting predominantly at their basolateral surface (50).

The actions of IGFs are modulated by IGF-binding proteins (IGFBPs). Six different types of IGFBPs, IGFBP-1 through IGFBP-6, have been identified and cloned in a variety of organs and cell systems (5-7, 19, 43). The concentrations of IGF-I or -II available for binding to IGF receptors on cell surfaces depend on the affinity by which IGFs bind to IGFBPs. Distinct IGFBPs have differing affinities for IGF-I and -II. For example, IGFBP-3 has a 20-fold greater affinity for IGF-I than does IGFBP-2 (30). IGFBP-2 has a high affinity for IGF-II. IGFBPs also differ from each other by their size and by the chromosomal location of their genes. IGFBP-2, -3, and -4 are encoded on chromosomes 2, 7, and 17, respectively (2, 43).

IGFBPs have been detected in the gastrointestinal tract (1), and they are secreted by derived epithelial cell lines (21, 32). The intestinal epithelial cell line (Caco-2) produces IGFBP-2, -3, and -4 (28). The secretion of IGFBPs is polarized (27, 36), reflecting the localization of IGF actions. Remacle-Bonnet et al. (36) have suggested that the polarity of IGFBP plays a modulatory role in maintaining the functional polarity in differentiated epithelial cells. Because IGFBPs alter cell proliferation and differentiation, we hypothesized that short-chain fatty acids may change the pattern of IGFBP secretion as part of a wider phenomenon by which luminal molecules regulate the expression of signaling molecules by gastrointestinal epithelial cells (31). To examine this hypothesis, we used the intestinal epithelial cell line Caco-2 for the following reasons: 1) the cells grow in chemically defined media, thereby eliminating fetal bovine serum, which contains IGFBPs, many growth factors, and undefined nutrients; 2) they respond to IGFs and express IGF receptors on their cell surfaces (29); and 3) cultured Caco-2 cells spontaneously differentiate into cells with features similar to enterocytes in vivo (34).

In this study, we examined the effect of short-chain fatty acids on IGFBP secretion and compared them with glutamine, another epithelial cell nutrient. We demonstrated that in the absence of short-chain fatty acids, Caco-2 cells predominantly secrete IGFBP-3. However, butyrate and, to a lesser extent, other short-chain fatty acids increased the synthesis of IGFBP-2 while simultaneously decreasing that of IGFBP-3. Glutamine did not affect the profile of IGFBP secretion. The changes in their expression were not related to energy delivery but correlated with their ability to induce histone acetylation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). DMEM, glutamine, nonessential amino acid, penicillin and streptomycin, HEPES buffer solution, iron-saturated human transferrin, and trypsin-EDTA were obtained from GIBCO BRL (Gaithersburg, MD). Fetal bovine serum was supplied by Hyclone Laboratories (Logan, UT). Selenous acid (Na2SeO3) was obtained from Collaborative Biomedical (Bedford, MA). Sodium butyrate, propionate, acetate, isobutyrate, and hexanoate were obtained from Sigma (St. Louis, MO). Twenty-four-well plates, 100-mm polystyrene Petri dishes, and 60-mm dishes were obtained from Falcon (Becton-Dickinson, Franklin Lakes, NJ). Tris · HCl, glycerol, bromphenol blue, and MOPS were obtained from Sigma. SDS, acrylamide, N,N'-methylene-bis-acrylamide, and Trans-Blot nitrocellulose membranes were obtained from Bio-Rad (Hercules, CA). 125I-labeled IGF-I (25 mCi/l), [32P]dCTP (3,000 Ci/mmol), and Gene Screen Plus membranes were obtained from Du Pont-NEN (Boston, MA). Kodak X-OMAT AR film was obtained from Kodak (Rochester, NY). RNAzol kits were obtained from Biotecx Laboratories (Houston, TX). Seakem ME agarose and formaldehyde were obtained from American Bioanalytical (Natick, MA). IGFBP-2- and IGFBP-3-specific oligonucleotide primers were synthesized from published sequences (GenBank M35410 and S56205, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was obtained from the American Type Culture Collection as an insert in a pBR322 plasmid. The PrimeIt II labeling system was obtained from Stratagene (Anaheim, CA). Twelve-millimeter cell culture semipermeable membrane well inserts with a pore size of 0.45 mm were obtained from Millipore (Bedford, MA). Bicinchoninic acid (BCA) protein assay reagents were obtained from Pierce (Rockford, IL). Trichostatin A was purchased from Waco Chemicals (Richmond, VA).

Culture of Caco-2 Cells

Caco-2 cells constitute a cell line derived from a human colon carcinoma that differentiates spontaneously and possesses many characteristics of enterocytes (34). Caco-2 cells were plated onto 2-well plates at an initial density of 5 × 104 cells/cm2. Cells were grown in DMEM containing 10% fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acid, 100,000 U/l penicillin and 100 mg/l streptomycin, and 10 mM HEPES buffer solution under a humidified atmosphere at 37°C and 5% CO2. Cells were used between the 20th and 40th passages.

Experiments were performed on cells at two distinct stages of differentiation, achieved by growing cells for defined time periods after plating (28). Well-differentiated cells were studied on day 15 after plating, whereas less-differentiated cells were used on day 3. On day 2 (less-differentiated cells) or on day 6 (well-differentiated cells), the media were changed from medium containing serum, as described above, to medium without serum (DTS medium). In DTS medium, the 10% FCS was replaced with 5 mg/l iron-saturated human transferrin and 5 mg/l selenous acid (Na2SeO3) (28). Other additives were as described for DMEM with serum. DTS medium supports the normal differentiation and proliferation of Caco-2 cells (28).

To validate the terms less-differentiated and well-differentiated used in this study, sucrase activity was measured spectrophotometrically at day 3 and day 15 after plating on 60-mm dishes (17). Brush-border membrane fractions were prepared by ultracentrifugation, and their protein content was measured by BCA assay according to manufacturer's recommendations. Less-differentiated cells exhibited low sucrase activities (5.01 ± 3.03 mIU/mg brush border protein) at the time at which short-chain fatty acids were added (day 3). Well-differentiated cells (day 15) showed high sucrase activities (64.6 ± 14.3 mIU/mg protein). Cells given butyrate proliferated less than controls, but this did not reach statistical significance over 24 h. All data were expressed per cell.

Protocol of Caco-2 Cell Incubation

To study the effect of short-chain fatty acids on IGFBP secretion, both less-differentiated (day 3) and well-differentiated (day 15) cells were incubated with butyrate for 24 h at the following concentrations: 0, 0.5, 1, 1.5, 2, 2.5, and 5 mM. (Incubation with butyrate for only 24 h did not result in a detectable increase in sucrase activity compared with control cells without butyrate in either less- or well-differentiated cells.)

To examine whether the effects of butyrate on IGFBP secretion were reversible, conditioned media were collected from Caco-2 cells at additional time points after the removal of butyrate on day 4 (day 3, day 4, day 5, and day 6). After butyrate incubation, each culture well was washed three times by DTS medium to remove the butyrate, and new DTS medium was added to the culture.

Experiments described above examining the effect of butyrate were performed with 2.5 mM glutamine in the medium. Further experiments were undertaken that varied the concentration of glutamine (0, 2, and 4.5 mM) in the absence of butyrate to examine effects of varying energy (or fixed nitrogen) being delivered to the cells. The concentration of 4.5 mM glutamine was chosen because it provides the same energy as 2.5 mM butyrate with 2 mM glutamine. No changes in the pH of the media were observed with the addition of butyrate or glutamine.

Further experiments were performed using other short-chain fatty acids [acetate (C2), propionate (C3), valerate (C5), and hexanoate (C6), each at a concentration of 2.5 mM] to examine whether the profiles of IGFBPs were specific for different short-chain fatty acids. To examine the effect of histone hyperacetylation on IGFBP secretion, cells were incubated with trichostatin A at 0.1, 1, and 10 µM.

Measurement of IGFBP Production by Western Ligand Blotting

IGFBPs in the incubation media collected from Caco-2 cells were analyzed by Western ligand blotting (29). Media were collected and centrifuged at 950 g for 10 min to eliminate cellular material. Samples were kept at -20°C until the IGFBPs were measured. Each point was examined in triplicate wells. One volume of 4× concentrated sample buffer (250 mM Tris · HCl, 40% glycerol, 4% SDS, 0.002% bromphenol blue, pH 6.8) without detergent was added to three volumes of the culture supernatants, and 120 ml of each sample were applied to 10% SDS-polyacrylamide gel. After transfer of proteins to Trans-Blot nitrocellulose membranes, membranes were probed with 125I-IGF-I. They were then exposed to Kodak X-OMAT AR film with an intensifying screen at -70°C. Results were quantified by scanning autoradiograms with IPLab Gel densitometer (Signal Analytics, Vienna, VA) as previously described (28). Because IGFBP-2 and IGFBP-3 secretion increases with differentiation, the density of each band was standardized according to that of a standard sample. The laboratory standard was a single sample of conditioned medium from Caco-2 cells incubated in DTS medium without butyrate. This sample was used on every ligand blot. Cell numbers were measured after trypsinization by counting in a hemacytometer. The IGFBP secretion per 105 cells was calculated for each binding protein.

Examination of IGFBP mRNA in Caco-2 Cells

Separate experiments were performed to examine IGFBP mRNA accumulation by Caco-2 cells. Caco-2 cells were cultured in the presence or absence of butyrate for 24 h in 100-mm polystyrene Petri dishes at an initial density of 5 × 104 cells/cm2. Caco-2 cells were homogenized, and the RNA was extracted, precipitated, washed, and collected as described in the procedures for RNA isolation in the RNAzol kit. IGFBP-2 and -3 mRNA was examined by RNA transfer blot analysis (Northern blots). Thirty micrograms of purified total RNA were electrophoresed in a 2% agarose gel containing 1.8% formaldehyde and 20% MOPS for 18-20 h at 30 V and transferred to Gene Screen Plus membranes by capillary action.

Hybridization experiments were performed using IGFBP-2 cDNA, IGFBP-3 cDNA, and GAPDH cDNA.

IGFBP-2 cDNA. cDNA for IGFPB-2 was made from the PCR products of reverse transcription of human epithelial cell mRNA, using IGFPB-2-specific oligonucleotides as primers. After reverse transcription, using 50 U/ml of reverse transcriptase, cDNA was amplified in four separate reaction tubes with each combination of the following primers (primers are named in the following order: name of the gene and the number of the 1st base pair as reported by GenBank, with "as" at the end if the primer is anti-sense and therefore on the complementary strand from the reported sequences): IGFPB-2 501 (upstream), GGAGCAGGTT GCAGACAATG; IGFPB-2 611 (upstream), CCCTCAAGTC GGGTATGAAG; IGFPB-2 1101as (downstream), ACTCTCCGTT TTCTGCCGGT; and IGFPB-2 1231as (downstream), AAGGAGCAGG TGTGGCATCT. The identity of the four PCR products was confirmed by determining that their sizes on a Southern gel were in accordance with predicted lengths. Only the largest (750 bp) of the four products was used for hybridization experiments.

IGFBP-3 cDNA. cDNA for IGFPB-3 was made by using similar methods as for IGFPB-2 cDNA. cDNA was amplified in four separate reaction tubes with each combination of the following primers: IGFPB-3 11 (upstream), GCCTCCACAT TCAGAGGCAT; IGFPB-3 204 (upstream), AGGAAGGAGG AATGGCTTGC; IGFPB-3 501as (downstream), CCTGACTTTG CCAGACCTTC; and IGFPB-3 561as (downstream), GAGAGCTCTA TGCAGCGTGT. Confirmation of the four PCR products was performed as for IGFPB-2. Again, only the largest (570 bp) of the four products was used for hybridization studies.

GAPDH cDNA. GAPDH cDNA was excised from the pBR322 vector with Pst I. cDNA was labeled with [32P]dCTP (3,000 Ci/mmol) by DNA polymerase (Klenow fragment) after random hexanucleotide priming using the PrimeIt II labeling system. Blots were hybridized and washed according to manufacturer's recommendations (Du Pont-NEN). Stringent washes were performed in 0.5× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and 0.5% SDS at 65°C. Washed blots were autoradiographed between intensifying screens at -70°C. To remove IGFBP-2 cDNA before hybridizing with IGFBP-3 cDNA, blots were incubated for 20 min with 1× SSC at 100°C. The degree of hybridization on autoradiograms was quantified using densitometry as previously described (28).

Polarity of IGFBP Secretion by Caco-2 Cells

Caco-2 cells were grown on 12-mm cell culture semipermeable membrane well inserts with a pore size of 0.45 mm at an initial density of 5 × 104 cells/cm2 (27). The change to serum-free (DTS) medium was made on day 13 in culture cell inserts. Confluence of Caco-2 cell monolayers in the culture inserts was confirmed by measuring the transepithelial electrical resistance with the use of Millicell-ERS (Millipore) on day 13, when cells were confluent and their resistance was >250 Omega  · cm2. On day 14, 2.5 mM butyrate was added to cell culture inserts to either the lower, basolateral or to the upper, apical compartments or simultaneously to both compartments for 24 h. Culture supernatants were then collected from both sides of the epithelium, and IGFBP secretion was examined as in Measurement of IGFBP Production by Western Ligand Blotting. The addition of butyrate did not affect the transmembrane resistance.

Histone Extraction and Separation

Histones were extracted from Caco-2 cells according to Cousens and Alberts (13). Cells were plated at 5 × 104 cells/cm2 in 75-cm2 flasks for 7 days. After 24 h of incubation with either butyrate, trichostatin A (49), or medium alone, cells were removed and the nuclear protein was harvested by centrifugation in MLB buffer (60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 15 mM PIPES, pH 6.5, 0.1% NP 40, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM tetrathionate). The nuclear pellet was suspended in H2SO4 to a final concentration of 0.2 M for 2 h. The suspension was then centrifuged for 15 min, and the supernatant (containing the histones) was removed. The dissolved histones were precipitated with alcohol at -20°C. The precipitant was suspended in water and quantified, and 150 µg of each sample were suspended in running buffer before being loaded onto a Triton X-100-acetic acid-urea gel for histone separation. The gel was prepared (3) by layering an upper gel [1 M acetic acid, 6.3 M urea, 4.4% (wt/vol) acrylamide] onto a separating gel [1 M acetic acid, 8 M urea, 0.5% (vol/vol) Triton X-100, 45 mM NH3, 16% (wt/vol) acrylamide]. Gels were stained with Coomassie blue and destained, and the position of the histone 4 was identified with a histone 4 marker (Boehringer-Mannheim, Indianapolis, IN). We quantified histone acetylation by scanning gel and measuring the density of each histone 4 band. The acetylation of each histone is the product of the band density and the number of histone sites. A ratio of acetylated to unacetylated histone 4 was then calculated.

Statistics

Each point was examined using triplicate wells. Results were expressed as means ± SE. The statistical significance of short-chain fatty acids or glutamine on IGFBP secretion was assessed by Student's t-test. Dose responses were analyzed by linear regression. Results were considered significant when P < 0.05.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Butyrate Reversibly Alters the Profile of IGFBPs Secreted by Intestinal Epithelial Cells

To examine the effects of butyrate on IGFBP secretion from Caco-2 cells, cells were incubated with and without butyrate for 24 h. Autoradiograms of Western ligand blots showed four distinct bands with relative molecular weights of 44,000, 39,500 (IGFBP-3), 37,000 (IGFBP-2), and 24,500 (IGFBP-4) (Fig. 1). The identity of these binding proteins on ligand blots of conditioned media from Caco-2 cells has previously been confirmed using immunoblotting (28). A concentration of 2.5 mM butyrate increased the secretion of IGFBP-2 (P < 0.0001) but decreased the secretion of IGFBP-3 (P < 0.0005) (Fig. 1). The changes in IGFBP secretion induced by butyrate were greater in less-differentiated cells than in well-differentiated cells (Fig. 2, A and B). The low level of IGFBP-4 secreted by Caco-2 cells was not altered by butyrate.


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Fig. 1.   Autoradiogram of a Western ligand blot of Caco-2 cell-conditioned medium collected from less-differentiated cells (day 3 after plating) 24 h after addition of butyrate (0 or 2.5 mM). Insulin-like growth factor (IGF)-binding proteins (IGFBPs) were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were probed with 125I-labeled IGF-I. (This result was representative of 9 separate experiments.)


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Fig. 2.   Densitometry of IGFBP secretion by Caco-2 cells at different stages of Caco-2 cell differentiation. A: less-differentiated Caco-2 cells (day 3) with 2.5 mM butyrate. Conditioned media were collected over 24 h. The 2.5 mM butyrate significantly enhanced IGFBP-2 secretion (P < 0.0001, over 3 consecutive experiments in triplicate wells), whereas it inhibited IGFBP-3 secretion (P < 0.0005). B: well-differentiated Caco-2 cells (day 15) with 2.5 mM butyrate. Conditioned media were collected over 24 h. The 2.5 mM butyrate significantly enhanced IGFBP-2 secretion (P < 0.0001). Samples were analyzed by Western ligand blotting. Autoradiograms were examined by densitometry, and IGFBP secretion was expressed in units relative to a laboratory standard based on densitometry per 105 cells. Results are expressed as means ± SE of triplicate wells in 1 experiment.

The secretion of IGFBP production was affected by butyrate at concentrations up to 2.5 mM (Fig. 3) in a dose-dependent manner. Higher concentrations of butyrate resulted in little additional effect (data not shown). To examine whether butyrate reversibly altered IGFBP secretion, culture supernatants were sampled for a further two days after butyrate had been removed from the culture media (Fig. 4). Within 48 h after removal of butyrate, IGFBP secretion had returned to the level observed before the addition of butyrate. IGFBP-3 returned to its original level more rapidly than IGFBP-2.


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Fig. 3.   Dose-response curve of IGFBP-2 and -3 secretion after butyrate in Caco-2 cells given butyrate on day 3. Effects of butyrate were dose dependent. Results are means ± SE of triplicate wells and are representative of 3 experiments. Kinetics analysis: maximal transport rate (Vmax) for IGFBP-2 = 37 units/cell, substrate dissociation constant (Ks) = 2 mM; minimal transport rate (Vmin) for IGFBP-3 = 17 units/cell, Ks = 2 mM.


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Fig. 4.   Effect of butyrate on secretion of IGFBPs from Caco-2 cells is reversible. On day 3, conditioned media were collected, and 5 mM butyrate was added to each culture for 24 h. On day 4, conditioned media were collected, each culture well was washed to remove butyrate, and new medium without butyrate was added to culture. Results are means ± SE of triplicate wells and are representative of 3 experiments.

Butyrate Increases IGFBP-2 mRNA and Decreases IGFBP-3 mRNA

The changes in IGFBP expression induced by butyrate and observed by ligand blotting could be explained by alterations either in IGFBP gene expression or in posttranslational processing. For example, changes in the affinity of individual binding proteins to IGF-I, directly induced by butyrate, would alter the binding of ligand to the blot. To exclude this, IGFBP mRNA was examined by RNA transfer (Northern) blots, and the changes were quantified by densitometry (Fig. 5). Butyrate stimulated the accumulation of IGFBP-2 mRNA in Caco-2 cells (P < 0.01) and reduced IGFBP-3 mRNA (P < 0.05). These observations corresponded to the changes seen in IGFBP secretion detected on Western ligand blots. IGFBP-4 mRNA was not detectable by Northern blots, in accordance with the low rate of production of IGFBP-4 protein. Because the secretion of IGFBP-4 was not significantly altered by short-chain fatty acids, more sensitive techniques to detect IGFBP-4 mRNA were not developed.


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Fig. 5.   Changes in IGFBP mRNA of Caco-2 cells treated with 2.5 mM butyrate on day 3. After 24 h, Caco-2 cells were homogenized and RNA was extracted. mRNA was examined by RNA transfer blot analysis (Northern blots). Thirty milligrams of purified total RNA were separated by electrophoresis, and membranes were hybridized with labeled IGFBP-2 cDNA, followed by IGFBP-3 cDNA and later glyceraldehyde-3-phosphate dehydrogenase (GAPDH). cDNA probes were removed from membrane between hybridizations. Autoradiograms were exposed for 18 h (IGFBP-2), 8 h (IGFBP-3), or 2 h (GAPDH). Densitometry was measured on autoradiograms as described in Ref. 28. Results are means ± SE of 4 wells and are representative of 3 experiments. NS, not significant.

Glutamine Does Not Alter the Ratio of IGFBPs Secreted by Caco-2 Cells But Enhances IGFBP-2, -3, and -4 in Equal Proportion

The effect of butyrate on Caco-2 cell secretion could be due either to delivery of increased energy to Caco-2 cells or to some specific effect of the butyrate molecule that distinguishes it from other epithelial cell nutrients. Glutamine, another important epithelial cell nutrient, was therefore given to Caco-2 cells at varying concentrations. Glutamine enhanced the secretion of both IGFBP-2 and IGFBP-3 (Fig. 6) but did not alter the relative ratio of their secretion. Similarly, the concentration of glutamine (0, 2, and 4.5 mM) did not affect the relative accumulation of IGFBP mRNA (data not shown).


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Fig. 6.   Glutamine does not alter profile of IGFBPs secreted by less-differentiated Caco-2 cells. On day 3, medium was changed to DTS medium (DMEM containing 5 mg/ml iron-saturated human transferrin) without glutamine or DTS medium with glutamine (2 or 4.5 mM) in each culture. On day 4, conditioned media were collected 24 h after incubation. Results are means ± SE of triplicate wells. Glutamine significantly increased IGFBP secretion (ANOVA: IGFBP-2, P < 0.005; IGFBP-3, P < 0.005; IGFBP-4, P < 0.05). Glutamine did not significantly alter the ratio between different IGFBPs.

Butyrate Alters the Rate But Not the Polarity of IGFBP Secretion

Caco-2 cells secrete IGFBPs in a polarized fashion. IGFBP-2 is predominantly secreted at the basolateral pole, whereas IGFBP-3 is apically secreted (27). To examine the effect of butyrate on the polarity of IGFBP secretion, cells were grown to confluence on semipermeable membranes. On day 14, butyrate was added to the upper chamber (Fig. 7). Butyrate increased IGFBP-2 secretion (P < 0.01, n = 3), but IGFBP-2 was still secreted in greater amounts on the basolateral side. Similarly, IGFBP-3 per chamber decreased with butyrate (P < 0.05, n = 3), but it was still predominantly secreted at the apical pole. Thus butyrate does not alter the mechanism(s) that directs different IGFBPs to distinct poles of the epithelial cell but only the rate of IGFBP synthesis. In further experiments, the effects of butyrate did not depend on which side of the epithelial cell monolayer it was given (data not shown). Butyrate caused no change in transepithelial resistance; the changes induced by butyrate were, therefore, not due to an alteration in the paracellular permeability of the cells to secreted IGFBPs.


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Fig. 7.   Secretion of IGFBPs remains polarized after addition of butyrate. Cells were grown in 12-mm cell culture inserts at an initial density of 5 × 104 cells/cm2. Change to DTS medium was made on day 13 in culture cell inserts. Confluence of Caco-2 cell monolayers in culture inserts was confirmed by measuring transepithelial electrical resistance on day 13. On day 14, butyrate (2.5 mM) was added to cell culture inserts to either upper, apical or lower, basolateral compartments or simultaneously to both compartments for 24 h. Conditioned media were then collected from both sides of epithelium. Samples were analyzed by Western ligand blotting (with 125I-IGF-I). Experiments were repeated on 3 occasions.

Histone Deacetylase Inhibitor Trichostatin A Induces IGFBP-2 Secretion

We wished to determine whether the effect of butyrate on IGFBPs was due to its known inhibition of histone deacetylation or to some other gene-specific mechanism. Caco-2 cells were therefore treated with trichostatin A, a specific inhibitor of histone deacetylase. The effect of this agent on histone acetylation (49) was verified on Triton X-100-acetic acid-urea gels (Fig. 8). Both butyrate and trichostatin A enhanced histone acetylation. In addition, trichostatin A, a fungicide with no chemical homology to butyrate, increased IGFBP-2 secretion (Fig. 9).


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Fig. 8.   Butyrate and trichostatin A induce acetylation of histone 4 (H4). Caco-2 cells were grown in media only or were given butyrate (2.5 mM) or trichostatin A (0.1, 1, and 10 µM) on day 3 after plating. After 24 h, histones were extracted and separated on a Triton X-100-acetic acid-urea-polyacrylamide gel. Cells in media (control) contain unacetylated (H4-0) or monoacetylated (H4-1) histone 4. Bands of higher molecular weight are seen in cells treated with butyrate and trichostatin A. They include additional di- (H4-2), tri- (H4-3), and tetra-acetylated (H4-4) forms. H2b, histone 2b.


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Fig. 9.   IGFBP-2 secretion is enhanced by both butyrate and trichostatin A (TSA). Butyrate and TSA were given on day 3, and cells and supernatants were collected on day 4. Secretion of IGFBP-2 was significantly increased (P < 0.01) by both agents. Each histogram is the mean ± SE of measurements on 3 lanes. OD, optical density.

Short-Chain Fatty Acids Enhance IGFBP-2 Secretion According to Their Ability to Induce Histone Acetylation

Bacteria in the intestinal lumen produce other short-chain fatty acids, particularly acetate and propionate. The relative concentration of these short-chain fatty acids depends on both carbohydrate intake and bacterial flora. Short-chain fatty acids had differing effects on IGFBP-2 secretion (Fig. 10), with butyrate (C4) having the greatest effect (P < 0.001). Valerate (C5; P < 0.01) and propionate (C3; P < 0.001) also increased IGFBP-2 but were less potent than butyrate, and acetate and hexanoate stimulated the least. The pattern of short-chain fatty acid stimulation on histone acetylation was the same as on IGFBP-2 secretion. There was a significant correlation between histone acetylation and IGFBP-2 secretion (r2 = 0.936).


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Fig. 10.   Pattern of IGFBP-2 secretion induced by short-chain fatty acids reflects their effect on histone acetylation. Caco-2 cells on day 3 were incubated with either acetate (C2), propionate (C3), butyrate (C4), valerate (C5), or hexanoate (C6). Supernatants and cells were collected for IGFBPs on day 4, and, at the same time, histones were extracted to assess acetylation. Acetylation of histone 4 was quantified by densitometry and expressed as a ratio of acetyl groups to unacetylated histone 4. Values are means ± SE of triplicate wells in 2 experiments. Secretion of IGFBP-2 correlated with histone acetylation.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The present study shows that short-chain fatty acids regulate the secretion of IGFBPs by intestinal epithelial cells. Because short-chain fatty acids are the products of bacterial metabolism, the epithelial secretion of IGFBPs responds to fermentation products. This is the first demonstration that molecules produced by resident bacteria at concentrations found in the intestinal lumen (15, 16, 38, 40) may regulate the secretion of growth factors by epithelial cells.

IGFBPs inhibit the binding of IGFs to IGF receptors in the intestine (12, 32), thereby altering cell proliferation and differentiation. IGF receptors are present both on epithelial cells (29, 39, 25, 33) and on activated lymphocytes (46). Three IGFBPs have been identified in the conditioned media of Caco-2 cells (28), and each has a distinct affinity for IGF-I and -II (30). For example, IGFBP-3 binds to IGF-I with an affinity manyfold greater than that of IGFBP-2 (30). The specificity of the effect of different binding proteins on the concentration of free IGFs is exaggerated by the presence of proteases, which are IGFBP specific (18). IGFBP-3 is particularly susceptible to the actions of such proteases. Together, these data indicate that altering the profile of IGFBPs has profound effects on the bioavailability of IGF-I and -II.

Short-chain fatty acids may alter IGFBP expression by three possible mechanisms. They include 1) an alteration in binding of nuclear proteins to IGFBP gene promoters (14, 44), 2) changes in chromatin structure (9, 26, 37), and 3) alterations in enterocytic differentiation (10, 20). We have shown that the effect of short-chain fatty acids on IGFBP-2 expression correlates with their ability to induce histone acetylation (Fig. 10). To exclude the possibility that histone acetylation and IGFBP-2 secretion were independent mechanisms stimulated from a common pathway, we treated cells with trichostatin A. This fungicide is a specific inhibitor of histone deacetylase that induces histone acetylation (49). Trichostatin A also induced IGFBP-2 secretion (Fig. 9). Trichostatin A has no chemical similarity to short-chain fatty acids. Short-chain fatty acids thus induced IGFBP-2 secretion through histone acetylation.

The changes observed in IGFBPs after treatment with butyrate were not due to the changes in enterocyte differentiation induced by this molecule (20). First, the differentiation of Caco-2 cells is associated with an increase of both IGFBP-3 and IGFBP-2 (28), whereas butyrate enhanced IGFBP-2 and inhibited IGFBP-3. Second, the effects of butyrate were rapidly reversible, but its effects on differentiation of Caco-2 cells continue to increase after its removal (48). Third, propionate altered IGFBP expression in a manner similar to that of butyrate, but propionate does not have a comparable effect to butyrate on enterocyte differentiation (48). The alterations in IGFBP secretion induced by short-chain fatty acids, therefore, are the result of a generalized change in differentiation. Nevertheless, the state of differentiation of the epithelial cells was a factor in the degree to which the cells responded to butyrate (Fig. 2). The increase in IGFBP-2 secretion induced by butyrate in well-differentiated cells (190%) is smaller than in less-differentiated cells (330%). Similarly, the inhibition of IGFBP-3 was observed only in less-differentiated cells. It is possible, therefore, that epithelial cells are more susceptible to short-chain fatty acid regulation at early stages of differentiation.

Changes in IGFBP secretion reflected changes in IGFBP mRNAs (Fig. 5). The changes observed in detection of IGFBPs by ligand blots were therefore not due to posttranscriptional modifications or to cleavage of IGFBPs to low-affinity forms (4, 42). IGFBP secretion was much more readily detectable in conditioned media than was mRNA in Northern blots of cellular RNA. Thirty micrograms of RNA were required per sample to obtain a sufficient signal to subject the results to densitometry and statistical analysis. The scarcity of the IGFBP transcripts was particularly evident when they were compared with GAPDH mRNA, used as a standard in our assay. Even after membranes had been boiled twice to remove two IGFBP cDNA probes, membranes hybridized with GAPDH cDNA required 5- to 10-fold less exposure to obtain autoradiograms with signal comparable to IGFBP cDNAs. Although this cannot be taken as an accurate quantitative comparison, it does point to the difficulty of using IGFBP mRNA to study IGFBP expression in intestinal epithelial cells.

Because the secretion of IGFBPs is polarized (27, 36), the secretion of these proteins is subject to two distinct control mechanisms. Transcription and translation regulate the IGFBP synthesized, but there must also be a separate mechanism that directs the IGFBP either to the apical or to the basolateral pole. Butyrate alters only the first of these two processes, the synthesis of the IGFBPs, because their secretion remains polarized with butyrate (Fig. 7). Thus the sorting of IGFBPs is due to a butyrate-independent process. Glycosylation of IGFBPs may dictate the polarity of IGFBP secretion. Such a mechanism would not be expected to depend on butyrate.

In conclusion, short-chain fatty acids alter the profile of IGFBP secretion in a stereospecific manner. The epithelial cell responds to bacterial products by secreting proteins that may modulate factors that affect their own proliferation and that of other cell types in the intestine.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DK-47753, DK-21474, DK-40561, and HD-12437 and by grants from the Hood Foundation. A. Nishimura was supported by an Eleanor Naylor Dana and Bernard F. Gordon Research Fellowship from the Lahey Hitchcock Clinic.

    FOOTNOTES

Address for reprint requests: I. R. Sanderson, Dept. of Paediatric Gastroenterology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Dominion House, West Smithfield, London EC1A 7BE, UK (E-mail: i.r.sanderson{at}mds.qmw.ac.uk).

Received 29 October 1997; accepted in final form 1 April 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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