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1 The Martin Boyer Laboratories, The University of Chicago, Chicago, Illinois 60637; and 2 Section of Gastroenterology, The University of Texas Medical School, Houston, Texas 77225
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Short-chain fatty acids (SCFA), produced by colonic bacterial flora fermentation of dietary carbohydrates, promote colonic Na absorption through mechanisms not well understood. We hypothesized that SCFA promote increased expression of apical membrane Na/H exchange (NHE), serving as luminal physiological cues for regulating colonic Na absorptive capacity. Studies were performed in human colonic C2/bbe (C2) monolayers and in vivo. In C2 cells exposed to butyrate, acetate, proprionate, or the poorly metabolized SCFA isobutyrate, apical membrane NHE3 activity and protein expression increased in a time- and concentration-dependent manner, whereas no changes were observed for NHE2. In contrast, no significant changes in brush-border hydrolase or villin expression were noted. Analogous to the in vitro findings, rats fed the soluble fiber pectin exhibited a time-dependent increase in colonic NHE3, but not NHE2, protein, mRNA, and brush-border activity. These changes were region-specific, as no changes were observed in the ileum. We conclude that luminal SCFA are important physiological cues for regulating colonic Na absorptive function, allowing the colon to adapt to chronic changes in dietary carbohydrate and Na loads.
short-chain fatty acids; butyrate; sodium transport; nutrition; gene induction; intestinal adaptation; dietary fiber; colonocyte; Na/H exchanger 3; Na/H exchanger 2; intestinal flora; bacteria; intestinal mucosa
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SHORT-CHAIN FATTY ACIDS (SCFA) are two- to five-carbon weak acids (pK 4.8) produced by bacterial hydrolysis of carbohydrates (and proteins) that are not digested or absorbed by the small intestine (28). They comprise the major anions of the colonic lumen, with concentrations approaching 100 mM (28). Individual SCFA have distinct physiological effects. Butyrate has been implicated as both a differentiation and trophic factor for intestinal cells (2, 6, 16, 20, 21, 27). In intestinal cell lines, for instance, butyrate decreases colonocyte proliferation and enhances maturation. Although these effects may take time, SCFA, when added to either the mucosal or serosal aspect of the epithelial tissue, can stimulate rapid increases in Na absorption, albeit through mechanisms yet undefined (7, 30).
In this study, we chose to investigate the time- and concentration-dependent effects of SCFA on electroneutral Na absorption, because this pathway represents a major route of Na transport in the proximal colon where SCFA are predominantly formed (13). The colon has a remarkable capacity to adapt to significant variations in luminal water and electrolyte loads, a property that prevents the development of diarrhea in most physiological circumstances (14). Although many systemic signals appear to be involved in regulating colonic Na absorption (8, 9, 31, 34), little attention has been paid to luminal signals or cues that may have an equally important modulatory role, particularly in adaptation to dietary chronic regulation of colonic salt and water transport. We therefore hypothesized that luminal SCFA have a role in modulating the activity and protein expression of apical membrane Na/H exchangers (NHEs), i.e., the predominant transport pathways for Na absorption in the proximal colon (29). There are two NHEs in the apical or brush-border membrane of intestinal cells, NHE2 and NHE3 (4, 17). In the present studies, we demonstrate that SCFA do indeed induce a time- and concentration-dependent increase in NHE3, but not NHE2, activity and protein expression in cultured human colonic epithelial C2/bbe (C2) monolayers. In addition, dietary modification in rats to increase colonic carbohydrate load and hence SCFA production results in increased and specific expression and activity of NHE3 of only the colonic mucosa, independent of changes in colonic epithelial differentiation.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell culture. Confluent C2 monolayers grown on collagen-coated permeable Transwell supports (22) were treated with SCFA 14 days after plating, a time when differentiation is near maximal. SCFA (0-20 mM) were added to complete C2 medium (high-glucose DMEM with 10% FBS supplemented with 10 µg/ml human transferrin, 2 mM glutamine, 50 U/ml streptomycin, and 50 µg/ml penicillin) for varying times (6-48 h). Monolayers were either used for unidirectional 22Na influx measurement of apical NHE activity (22) or for Western blotting to detect NHE3 and NHE2 protein abundance using polyclonal antisera developed and characterized by our laboratory (4, 22).
Determination of apical membrane NHE activity. Apical NHE activities were measured as previously described (22). Briefly, cells were grown for 14 days on Transwell culture dishes, a time when differentiation is essentially maximal. Cells were then fed with medium with or without SCFA and then either analyzed for NHE proteins by Western blot or used for 22Na flux measurements. All flux data were obtained under basal conditions, i.e., without acid loading through preincubation with ammonium chloride to artificially increase NHE activity. To initiate fluxes, Transwells were washed one time quickly at room temperature with isotonic choline chloride (150 mM choline chloride and 15 mM HEPES, pH 7.4) and immediately used for fluxes. The apical and basolateral media were identical during flux measurement and consisted of (in mmol/l): 20 NaCl, 120 choline chloride, 5 KCl, 1 MgCl2, 2 CaCl2, and 15 HEPES, pH 7.4, with 1 µCi/ml 22Na. Fluxes were allowed to proceed for 10 min, a period during which 22Na influx across the apical membrane is still linear (22). Fluxes were terminated by four rapid washes in ice-cold HEPES-buffered saline. Transwell membranes were cut from the support and solubilized in scintillation fluid for counting. For all experiments, NHE2 and NHE3 activities were defined by the component of the amiloride-inhibitable 22Na influx sensitive or insensitive to HOE-642 inhibition, respectively (22).
In vivo induction of colonic NHE3. To modulate the production of colonic SCFA, rats (250-275 g) were fed specially designed diets containing no fiber (no cellulose) or 5% (wt/wt) pectin over a 2-day period. The diets were obtained from Harlan/Teklad [nos. 97201 and 97202; composition in g/kg: 100 casein, 3 DL-methionine, 335.686 corn starch, 130 maltodextrin, 160 sucrose, 70 soybean oil, 10 vitamin mix TD94707, 2.5 choline bitartrate, 0.014 tert-butylhydroquinone (antioxidant), 13.37 mineral mix, calcium phosphate-deficient TD-79055, 11.43 calcium phosphate, dibasic, and 4.0 calcium carbonate with or without 60 g/kg pectin]. There was no difference in daily chow intake between the two groups of rats (in g chow eaten/2 days, 24.8 ± 2.1 pectin-free chow vs. 25.7 ± 2.9 pectin chow, n = 4). Additionally, over the 2-day period of these two rat diets, there were similar weight gains in both sets of rats (6.2 ± 0.4 and 6.4 ± 0.6 g over 2 days in pectin-free and pectin chows, respectively). The addition of 5% pectin has previously been shown to result in production of SCFA in the colon. To minimize coprophagia, animals were housed in wire-bottomed cages. Mucosal scrapings were then harvested from the ileum (a 15-cm segment >10 cm proximal to the ileocecal valve) and colon (the first 15 cm of colon after the cecum, therefore most of the colon, excluding the rectum). Brush-border membrane vesicles were then prepared for transport studies and analysis of NHE protein, as described previously (9). Briefly, ileal and colonic mucosal scrapings were weighed and added to 30 ml hypotonic lysis buffer (10 mM Tris, pH 7.4, and 3 mM EDTA with protease inhibitors as described previously) and homogenized for 30 s at a speed of 15,000 rpm in an UltraTurrax homogenizer. Samples were taken for enzyme enrichment studies, and the samples were spun at 2,000 g for 5 min at 4°C to remove nuclei and unbroken cells. The supernatants were removed and spun at 10,000 g for 10 min at 4°C to remove mitochondria. The supernatants were removed, and 15 mM CaCl2 was added. Samples were gently stirred in the cold for 15 min and then spun at 8,000 g for 8 min to remove the endoplasmic reticulum, Golgi, and basolateral membranes. The supernatants were spun at 45,000 g for 45 min at 4°C to obtain brush-border membranes. The membranes were resuspended in a small volume of intravesicular transport buffer (10 mM MES, pH 6.1, 3 mM EDTA, and 80 mM mannitol) and resuspended using a Teflon pestle homogenizer. A sample was removed for protein determination and enzyme enrichment studies. Vesicles (5 µl) were added to 45 µl of extravesicular transport buffer [10 mM HEPES, pH 7.4, 1 mM Na with 22Na (1 µCi/ml giving a specific activity of 2,200 dpm/nmol Na), and 80 mM mannitol]. All brush border uptakes were of 10 s duration. Uptake of Na into the vesicles was stopped by addition of 2 ml ice-cold 90 mM mannitol and immediate placement on a 0.45-µm cellulose filter (HAWP; Millipore). The filter was washed one time with 4 ml ice-cold 90 mM mannitol, and the filter was removed and solubilized in liquid scintillation fluid. 22Na was determined by liquid scintillation spectroscopy. For determination of mRNA abundance, intestinal scrapings were homogenized in Trizol and prepared for Northern blots. RNA was extracted one time with acid phenol-chloroform and quantified by absorbance at 260 nm. Twenty micrograms were size-separated on a denaturing formaldehyde agarose gel, transferred to a positively charged nylon membrane by capillary action, and linked to the membrane by ultraviolet light. Blots were prehybridized and hybridized in XOTCH solution (7% wt/vol SDS, 1% wt/vol albumin, 200 mM NaH2PO4, 10 mM EDTA with 15% vol/vol deionized formamide) as previously described (4) using the cDNA probes for rat NHE2 and NHE3. Glyceraldehyde phosphate dehydrogenase was used as a constitutive control to ensure equal loading of lanes. Probes were labeled with 32P by random prime labeling. Blots were hybridized at 55°C overnight and then washed to a stringency of 0.5× saline-sodium citrate-0.5% SDS at 55°C.
Analysis of differentiation by butyrate. To determine if butyrate had any differentiating effects either in vitro or in vivo, the differentiation markers alkaline phosphatase and sucrase (when appropriate) were measured. For the C2 cells, cells were harvested from Transwells after 14 days postplating and then with or without 5 mM butyrate for 2 days. Cells were scraped from the filter in PBS and lysed in 10 vol hypotonic buffer (10 mM Tris and 3 mM EDTA with protease inhibitors as described previously). Nuclei, unbroken cells, and mitochondria were removed by centrifugation (10,000 g for 5 min), and the microsomal membrane fraction was obtained by centrifugation (100,000 g for 20 min). The amount of protein in this membrane fraction was measured using the bicinchoninic acid procedure, and alkaline phosphatase and sucrase activities were measured immediately. Alkaline phosphatase was measured colorimetrically using para- nitrophenol phosphate and measuring absorbance at 490 nm as described by Cox and Griffin (12). Sucrase activities were measured as described in the microassay procedure of Messer and Dahlquist (23) by measuring the glucose generated by sucrase activity. Glucose oxidase and ortho-dianisidine were used to generate a colored end product, and the absorbance was measured at 450 nm. To determine the effect of pectin on differentiation in the rat, alkaline phosphatase activities were measured in brush borders of both the ileum and colon.
As another marker of differentiation, the C2 cells and brush-border membranes from rat ileum and colon were analyzed for villin by Western blotting. The protocol used was essentially the same as used for NHE Western blots, except that a monoclonal anti-villin antibody (Transduction Laboratories, Lexington, KY) was used.Transmural 22Na fluxes. To determine if pectin increased the absorption of Na in the colon, colonic mucosa from the identical region used for vesicle studies (the transverse colon) were used. Rats were fed the pectin-enriched or control diets for 2 days, the colonic segments were removed, and the external muscle layer was removed by dissection (taking <2 min). Sheets of stripped mucosa were mounted in Ussing chambers with a 1-cm2 aperture. The solution used for the flux contained (in mmol/l): 25 NaHCO3, 1 MgCl2, 1.25 CaCl2, 5 KCl, 1 NaH2PO4, 34 NaCl, and 80 N-methyl-D-glucamine chloride. The solution was gassed with 5% CO2-95% O2, thereby yielding a pH of 7.4 at 37°C. After being mounted in Ussing chambers, tissues were allowed to equilibrate for 10 min, and 1 µCi/ml 22Na was added to either mucosal or serosal solution. After 10 min, flux periods were initiated. All flux periods were of 20-min duration. The first flux period was a control; therefore, there were no additions. The amiloride analog HOE-694 was then added to the mucosal side of each chamber to a final concentration of 100 µM. The HOE-694 concentration was increased in the transmural flux studies since the amiloride analogs are competitive inhibitors of NHEs. After 10 min, fluxes were measured over 20 min. Dimethylamiloride was then added to 500 µM concentration to inhibit all NHEs. After 10 min, a third flux period of 20 min was measured. Samples were taken from the "hot" side (to which the 22Na had been added) at the beginning and end of each experiment to confirm that the specific activity had not changed and that no leaks existed for the tracer. Fluxes were calculated from samples taken from the "cold" sides at the beginning and end of each of the three flux periods. To ensure that the tissues were still viable at the end of the experiment, 10 µM PGE2 was added to the serosal side, and any tissue that did not demonstrate a significant increase in short-circuit current was excluded as being nonviable.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Butyrate causes a time- and dose-dependent increase in apical
membrane NHE3 activity and expression.
Shown in Fig. 1A are the
unidirectional lumen-to-cell membrane 22Na influxes that
reflect apical membrane NHE activity. As described in MATERIALS
AND METHODS, NHE2 activity is defined as the component of the
amiloride-inhibitable 22Na influx that is sensitive to
HOE-642. In contrast, NHE3 is defined as the HOE-642-insensitive
component of the amiloride-inhibitable 22Na influx.
Measurements were performed without acid-loading cells to assess the
innate functional activities of these brush-border NHEs. Under
pretreatment conditions (no SCFA), NHE2 activity can be readily
measured, but little endogenous NHE3 is expressed. Treatment of C2
cells with 10 mM butyrate stimulates a time-dependent and specific
induction of NHE3 activity (Fig. 1) in which increased activity can be
observed as early as 6 h and is near maximal by 12 h. In
contrast, no significant changes in NHE2 activity were observed.
Similar findings were noted when measurements were performed under
acid-loaded conditions, i.e., in which maximal NHE activity is induced
(data not shown). Butyrate induced NHE3 activity whether it was added
to the apical or basolateral medium. After 12 h, basolateral
butyrate (10 mM) induced NHE3 to 6.7 ± 0.7 nmol per Transwell for
10 min (n = 4), which was not different from the induction observed after apical addition. All subsequent experiments were done by adding SCFA to the apical medium, since physiologically the SCFA are generated in the colonic lumen.
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Other SCFAs induce NHE3.
To determine whether other SCFA are capable of inducing C2 NHE3, cell
monolayers were treated with 10 mM acetate or proprionate for 48 h. As shown in Fig. 4, both agents induce
significant increases in NHE3 activity and protein expression, whereas
no changes in NHE2 were observed (Fig. 4). To determine whether
metabolism of these fatty acids might play a role in the induction of
NHE3, cells were treated with the poorly metabolized fatty acid
isobutyrate at 10 mM for 48 h. As shown in Fig. 4, isobutyrate
also induces NHE3 activity and protein, suggesting that metabolism is
not responsible for the observed effects. It should be noted that
butyrate is considered to be the preferred metabolic fuel for
colonocytes (18).
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In vivo generation of fatty acids by altering dietary fiber induces
colonic NHE3.
To determine whether the generation of SCFA in vivo alters NHE3
activity, rats were fed specially prepared diets containing no dietary
fiber or 6% pectin. Pectin is resistant to digestion but is readily
metabolized to SCFA by colonic bacteria. After the rats were on these
diets for 2 days, NHE2 and NHE3 brush-border membrane activity, mRNA
abundance, and protein expression were determined. A large increase in
NHE3, but not NHE2, activity was observed in colonocytes of pectin-fed
rats (Fig. 5). The increase in NHE3
activity was 246 ± 33% (n = 5) compared with
control. There was a small increase in colonic NHE2 (17.7 ± 14.3%, n = 4). This response was region specific, as
no changes in NHE3 or NHE2 activity were observed in the small
intestine (a maximal change of 12% was noted in all cases in ileal
transport activity of all animals). Parallel increases in colonic
brush-border NHE3 protein of 182 ± 29% (n = 5)
were observed (Fig. 5), whereas no changes in colonic NHE2 protein were
observed (4.8 ± 11.2%, n = 4). As with activity,
no changes in NHE3 or NHE2 protein expression were observed in the
ileum (<8% changes were noted in all tissues examined). To determine
whether the increased NHE3 protein level was due to increased mRNA
levels, Northern blots were generated from RNA extracted from colonic
and ileal mucosa. NHE3 mRNA in the colon of pectin-fed rats increased
281 ± 40% (n = 3) over control diet rats,
whereas no change in NHE2 mRNA was observed (Fig.
6; increase of 12.7 ± 16.9% for
NHE2 mRNA in pectin-fed rat colon, n = 4). NHE3
and NHE2 mRNA abundance in ileal samples was not different between
pectin-fed and control animals (a maximum of 12% increase was noted
for either NHE2 or NHE3 mRNA in all ileal samples analyzed).
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Induction of Na transport by pectin diet.
To determine whether the increased activity of NHE3 resulted in
increased Na absoption by the colon, transmural 22Na fluxes
were measured in vitro in Ussing chambers. The Na concentration of the
solution used in the chambers was set to 60 mM so that the contribution
of the paracellular flux would be less than in a normal Ringer
solution, which contains ~140 mM Na. The reported affinities of NHE2
and NHE3 for Na are ~25 and 12 mM. Therefore, 60 mM Na should be
sufficient to measure absorptive fluxes measured through these
exchangers. The pectin diet caused a large increase in the total Na
mucosal-to-serosal fluxes but did not increase the serosal-to-mucosal
flux (Table 1). HOE-694, the amiloride analog that inhibits NHE2 much better than NHE3, caused a similar decrease in the mucosal-to-serosal Na flux, independent of the chow
fed. Addition of dimethylamiloride caused a greater decrease in the
pectin chow-fed rats, demonstrating that there is greater Na absorption
mediated through NHE3.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SCFA are important to normal intestinal physiology, underscoring the essential symbiotic relationship between colonic bacterial flora and human hosts (5, 26). Cell culture studies suggest that a number of functions of the colonic epithelial cell, including cell migration (32) and cytokine secretion (15), are potently modulated by SCFA. This study proposes yet another important role for these agents in regulating colonic Na absorption, allowing this region of the gut to adapt to chronic perturbations in Na and dietary carbohydrate load. Because the apical membrane transporters NHE2 and NHE3 play a major role in mediating colonic Na absorption, their function and expression must be tightly regulated by physiological stimuli that are received systemically and luminally. Systemic regulatory mechanisms and signals are far better understood and include processes that can acutely alter apical membrane NHE activity, such as stimulated increases in second messengers, or chronically by affecting the availability of NHE transporters in the apical membrane. Mineralocorticoids, for instance, have little immediate effect on colonic NHE activity but gradually increase NHE3 activity through region- and isoform-specific stimulation of NHE3 protein expression (8). This study is the first to demonstrate that chronic luminal SCFA are physiologically important signals that play an important role in modulating colonic NHE3 activity and hence overall Na absorption. They substantiate the pivotal role of the colonic bacterial flora in modulating normal digestive and absorptive function. Finally, our findings support the possibility of using dietary manipulation to regulate intestinal absorptive function in health and in disease (26). Indeed, the inclusion of amylase-resistant starch has recently been demonstrated to be of significant beneficial effect in diarrhea (25). The effects of the amylase-resistant starch may well be due to colonic generation of SCFA from this carbohydrate source.
The induction of NHE3 by SCFA is quite specific because associated alterations in NHE2 or in mucosal differentiation were not observed. The effect of SCFA on NHE3 does not appear to require metabolism, as demonstrated by the in vitro effect of the poorly metabolized SCFA isobutyrate. However, SCFA induction of NHE3 may be related to cellular acidification, as decreased intracellular pH has been observed in isolated colonic crypts acutely exposed to SCFA (7, 10, 30). The induction of acidosis in a renal culture cell line (opposum kidney), for instance, chronically increases the expression and function of NHE3 (1). The mechanism by which cell pH may induce NHE3 appeared to involve a c-src-dependent pathway, as the transfection of a specific inhibitor of c-src abolished stimulated increases in NHE3 expression (33). Alternatively, butyrate has been shown to be a potent inhibitor of histone deacetylases (3) and has effects on DNA methylation (11). Proprionate is also a deacetylase inhibitor, albeit far less potent. Finally, it is possible that SCFA directly stimulate NHE3 gene transcription. For instance, a butyrate response element (BRE) has been identified that may be involved in regulating the expression of many genes (19, 24). Within the 2-kB 3'-flanking promoter region of the human NHE3 gene, there are at least three potential butyrate response elements. Whether these transcription elements are involved in SCFA induction of NHE3 remains to be determined.
The region-specific induction of NHE3 is not surprising in light of our current understanding of SCFA bioavailability. Because SCFA are exclusively made by bacterial metabolism of dietary carbohydrates, SCFA production would not be expected in the small bowel. We presume that levels of serum SCFA that may be achieved after a meal are not sufficient to stimulate small intestinal NHE3 expression. Nevertheless, we cannot rule out the possibility that the small bowel enterocytes are inherently different from colonocytes in their NHE3 response to SCFA.
In conclusion, we believe the regulation of colonic NHE3 expression by luminal SCFA has an important and major role in modulating colonic Na absorptive capacity relative to perturbations in luminal SCFA and Na loads. The notion that intestinal transport functions are responsive to luminal cues is novel and clinically relevant, particularly in designing measures to improve mucosal absorptive functions, particularly in diseases in which mucosal function may be impaired or compromised.
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ACKNOWLEDGEMENTS |
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This work was performed in the Martin Boyer Laboratories and was supported by National Institute of Diabetes and Digestive and Kidney Disease Grants DK-38510, DK-47722, and Core Grant DK-42086 and by the Gastrointestinal Research Foundation of Chicago.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. B. Chang, The Univ. of Chicago, MC 6084, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail:echang{at}medicine.bsd.uchicago.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 August 2000; accepted in final form 22 November 2000.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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)
butyrate (But) or ileum and colon of rats on control or pectin-enriched
diets. C2 cell data are presented on top, and rat intestinal
data are on bottom. In both cases data are means ± SE
for 3 separate experiments.
