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Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
1To whom correspondence should be addressed, (519) 824-4120 Ext. 3742, (519) 763-5902 Fax, kmeckling.ns@aps.uoguelph.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-linolenic 18:3(n-3), 
-linolenic 18:3(n-6)
and arachidonic 20:4(n-6) acids all significantly enhanced cellular
migration in the IEC-6 model of wound healing. Eicosapentanoate,
linoleate, -linolenate, -linolenate and arachidonate are all
capable of improving reconstitution of epithelial integrity following
mucosal injury. Inhibition of eicosanoid synthesis reduced the
enhancement of restitution by n-6 fatty acids back to control
levels.
KEY WORDS: • restitution • fatty acid • rats • IEC-6 cells • wound healing
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The intestinal mucosa is susceptible to a number of
challenges which can compromise the integrity of this very important
tissue in both its barrier and nutrient absorptive functions. Such
challenges include inflammatory bowel disease, ulcers, infection and
radiation and chemotherapy used in cancer treatment. Rapid recovery of
barrier and absorptive function is essential for the treatment and/or
recovery from such insults. The process by which the integrity of the
intestinal epithelium is reestablished following injury has been termed
epithelial restitution (Morris and Wallace 1981
). This
repair process was demonstrated to involve cellular migration and
proliferation. The initial process of migration occurs very rapidly and
is therefore believed to occur independent of cell division
(Silen and Ito 1985
). Cells surrounding the damaged area
migrate to reestablish mucosal integrity. Proliferation of these cells
will then complete the repair process. Proliferation begins 12–16 h
following a mucosal challenge and is complete in 1–2 d (Yeomans et al. 1973
). Our understanding of how restitution and
proliferation of the intestinal epithelia, following injury, are
regulated in response to physical and chemical challenges remains
incomplete.
Endogenous and exogenous bioactive molecules, including those acquired
from an everyday typical diet, can themselves be potent mediators
and/or regulators of many cellular processes. Different types of
dietary fat and more specifically, fatty acids, are such bioactive
molecules. It was demonstrated that the modification of cellular
membrane phospholipids can be achieved, in vitro and in vivo, by
lipid-supplemented media or alteration of dietary fat
(Blackmore and Meckling-Gill 1995
, Philbrick et al. 1987
, Spector et al. 1979
, Vossen et al. 1993
). Therefore, it is reasonable to hypothesize that
alteration of membrane properties due to altered phospholipid
composition is capable of mediating the aforementioned processes
involved in epithelial restitution. Indeed, much literature exists
demonstrating the ability of supplemental fatty acids to modify
cellular proliferation (Rose et al. 1994
, Spector et al. 1979
), growth factor activity (Jiang et al. 1995
, Kaminski et al. 1993
), differentiation
(Awad et al. 1991
, Das 1991
), cell
signaling (Bandyopadhyay et al. 1995
, Hannigan and Williams 1991
) and eicosanoid production (Von Schacky et al. 1985
, Weber 1990
).
We utilized the IEC-6 cell line originally derived from the jejunum of
the rat small intestine and representative of normal crypt cells based
on morphological and immunological criteria (Quaroni et al. 1979
). McCormack et al. (1992)
, as well as other
researchers (Ciacci et al. 1993
, Dignass and Podolsky 1993
, Dignass et al. 1994
), utilized
the IEC-6 cell line to study the process of migration during early
mucosal restitution in an in vitro environment free of nonepithelial
constituents.
Here we describe the effects of various fatty acids on proliferation in subconfluent cultures and on early mucosal restitution using wounded, confluent IEC-6 rat cell cultures.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IEC-6 (ATCC, Rockville, MD) cultures were maintained in Dulbecco's Modified Eagle's Medium (DMEM)2 (Gibco, Burlington, Ontario, Canada), supplemented with 10% of fetal bovine serum (FBS) (Gibco) and 270 U/L of insulin (Gibco) at 37°C in a humidified, atmosphere of 10% of CO2. Media was changed three times weekly and stocks were passaged before confluence. Passages 15–25 were used for all experiments.
Fatty acid supplementation.
Fatty acids were introduced into the medium by first incubating the
99% pure fatty acid (Nu-Chek-Prep, Elysian, MN) in FBS for 1 h at
37°C. This incubation results in fatty acid conjugation to bovine
serum albumin present in serum. FBS provides sufficient amounts of
bovine serum albumin to bind fatty acids up to at least 1 mmol/L
(Turcotte and Delcastro 1991
). Fatty acids used in this
work were as follows: docosahexaenoic acid DHA,22:6(n-3),
eicosapentaenoic acid EPA,20:5(n-3), -linolenic acid ALA,18:3(n-3),
linoleic acid LA,18:2(n-6), -linolenic acid GLA,18:3(n-6),
arachidonic acid AA,20:4(n-6), elaidic acid
EA,18:1(n-9trans) and linoelaidic acid
LEA,18:2(n-6)trans.
Effects of fatty acids on growth and survival.
These experiments were carried out in Falcon 96-well tissue culture
plates (Becton Dickinson, Oxnard, CA). Culture medium was supplemented
with the fatty acid of interest and then serial diluted across the
columns of the plate. IEC-6 cells were added at a final density of 3000
cells/well in a total volume of 200 µL. Growth was assessed at
72 h by the use of a Sulforhodamine B (SRB) Protein Dye Binding
Assay (Skehan et al. 1990
). Preliminary experiments
indicated that cultures under these conditions were still in active
growth phase at 72 h post plating. Briefly, cells were fixed with
3 mol/L of trichloroacetic acid (TCA), washed and then stained with 1.7
mmol/L of SRB dye in 0.16 mol/L of acetic acid. The dye was then
solubilized using 10 mmol/L of unbuffered Tris and absorbance read at
570 nm using a Kinetic Microplate Reader (Molecular Devices, Menlo
Park, CA) Absorbance (as a percentage control) was expressed as a
function of fatty acid concentration to establish whether specific
fatty acids stimulated or inhibited growth. Where possible the
concentration of fatty acid that inhibited growth by 50% compared to
unsupplemented cultures (IC50) was estimated from data
derived from growth experiments using computer-generated best
curves software (TableCurve; Jandel Scientific, Corte Madera, CA).
Membrane phospholipid composition.
IEC-6 cells were plated in 100-mm culture dishes (Corning Glass Works,
Corning, NY) at a density of 1.0 x 106 cells/dish.
Fatty acids were supplemented at 30 µmol/L and cultures grown for
96 h with one medium change. Total lipids were extracted by the
method of Bligh and Dyer (1959)
in the presence of
antioxidant butylated hydroxytoluene (2.3 mmol/L). Lipids were
separated by thin-layer chromatography using a solvent of
n-heptane/isopropyl ether/acetic acid (60:40:3 v/v/v) on
Silica Gel G Redi Plates (Fisher Scientific, Nepean, Ontario, Canada).
Lipids were methylated along with 2 µL of a 17:0 lipid standard as an
internal control. Samples were run on a Hewlett-Packard
(Mississauga, ON, Canada) 5890A gas chromatograph and peaks identified
by comparison to a previously run nerve standard.
Wounding assay.
IEC-6 cells were plated in 100-mm culture dishes at a density of 1.0 x 106 cells/dish. Twenty-four hours later medium was aspirated and replaced with media supplemented with the various fatty acids, at 30 µmol/L. Once the cultures had reached confluence, they were wounded with a single-edged razor blade. Wounds were typically 10–15 mm long and the blade was drawn ~10 mm across the plate. Two wounds were produced on each culture dish. Wound lines were immediately assessed by an inverted phase contrast microscope and marked with a permanent marker to assure a well-defined wound line. Cultures were then washed with phosphate buffered saline, and fresh medium supplemented with fatty acid was added. The migration of the cells across the wound line was assessed 24 h later. Cultures were fixed with ice-cold methanol/acetone fixative (4:1), stained with Wright/Giemsa (Sigma Chemical, St. Louis, MO) and photographed using a Nikon, DIAPHOT-TMD inverted microscope (Tokyo, Japan) at a power of 150x. Two photographs of each wound were taken, each at different regions along the wound line. Photographs were divided along the wound line into three 5-cm wide regions. Total number of cells migrating across the wound line was determined in these areas. As well, cell migration was determined in an area extending 2 cm from the wound line and again along 5 cm of photographed wound line. From this area we determined the density of cells migrating across the wound line (number of cells/cm2).
Proliferation during wounding assay.
Determination of proliferation during the wounding assay was performed essentially as described for the migration assay and assessed by immunohistochemistry utilizing bromodeoxyuridine (BrdU) (Boehringer Mannheim). Briefly, at 23 h, cells were incubated with the thymidine analog bromodeoxyuridine (10 µmol/L) for 1 h. BrdU assay was then performed according to manufacturers instructions. Plates were then assessed using phase contrast microscopy for positively stained cells which appear blue/black. A counterstain of Eosin was utilized to provide a favorable contrast and to facilitate the counting of non-BrdU stained cells. Cultures were then photographed in the same manner as migration experiments and the percentage of positively stained cells determined in the same total migration area discussed previously.
Eicosanoid inhibition during wounding assay.
Eicosanoid synthesis was inhibited by the introduction of indomethacin, 1 µmol/L, (Sigma Chemical) into the culture media 4 h prior to wounding. IEC-6 cells were grown in six-well plates and maintained in culture conditions as described for wounding assay in materials and methods. Migration was assessed at 24 h by image analysis using a Nikon DIAPHOT-TMD inverted microscope (Tokyo, Japan) and Northern Exposure software (Empix Imaging Inc., Missassauga, ON, Canada). Migration was expressed as a migration rate: the area covered by migrating cells per µm wound in 24 h (µm/24 h).
Statistics.
Data from proliferation experiments were analyzed by General Linear Models procedure followed by Dunnett post-test using SAS for Windows, release 6.12 (SAS Institute Inc., Cary, NC). Lipid incorporation, migration and BrdU experiments were analyzed for significant differences using General Linear Models procedure followed by Student Newman-Keuls post-test using SAS for Windows, release 6.12 (SAS Institute Inc.). All differences were considered significant at the P < 0.05 level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, panels A and B). The trans fatty acid
LEA significantly inhibited cell growth above 250 µmol/L (Fig. 1
,
panel C). EA was well-tolerated by IEC-6 cultures and had no
significant effect even at concentrations as high as 500 µmol/L.
Inhibition of cell growth by fatty acids was assessed by determination
of IC50 values. IC50 values were resolved using
proliferation data, and outcomes are shown in Table 1.
Information from these curves was used to determine the optimal
(nontoxic but physiologically achievable in whole animal) concentration
of fatty acid that could be used in migration experiments. In order to
maintain equivalent conditions among lipid treatments and avoid growth
inhibition, we chose a fatty acid concentration of 30 µmol/L to be
utilized in phospholipid incorporation and migration experiments.
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Levels (mol/100 mol) of DHA, EPA, LA and AA in unsupplemented control
cultures were 1.9, 0.6, 3.1 and 7.7, respectively. In cultures
supplemented with DHA, EPA, LA and AA there was a 1–5-fold increase in
the supplemental fatty acid into cellular phospholipids. ALA, GLA and
LEA were below the detection limit in unsupplemented control cultures
and rose to 0.4, 0.2 and 10.7 mol/100 mol for supplemented cultures,
respectively. EA eluted at the same retention time as other 18:1
isomers. A larger peak of 18:1 in the supplemented compared to the
unsupplemented culture was assumed to represent incorporation of EA
rather than additional oleic acid. As such EA was estimated to be at
least 8–9 mol/100 mol higher in EA-supplemented cultures.
|
. DHA supplementation increased the level
of EPA in cellular phospholipid compared to control. EPA
supplementation increased levels of the elongation product 22:5(n-3)
when compared to control. Supplementation of ALA increased levels of
elongation and desaturation products, 18:4(n-3), 20:3(n-3), 20:4(n-3)
and EPA as compared to control. In (n-6)-supplemented cultures (LA, GLA
and AA), an increase in (n-6) PUFA content and a corresponding higher
(n-6)/(n-3) ratio was seen. For all three (n-6) fatty acids,
considerable evidence for elongation and desaturation of supplemented
fatty acids was observed. In trans fatty
acid-supplemented cultures, EA supplementation resulted in a
corresponding increased monounsaturated fatty acid (MUFA) content as
well as a slight decrease in saturated fatty acid (SAT) content.
Addition of LEA to the culture media resulted in a considerable
decrease in the MUFA content and an increase in the (n-6) pool,
consequently raising the (n-6)/(n-3) ratio modestly. Supplementation
with (n-3) and (n-6) fatty acids in general resulted in an increase in
SAT content and a decrease in MUFA content.
The effects of fatty acids on epithelial restitution in wounded IEC-6
monolayers are illustrated in Figure 2.
Twenty-four hour, post wounding, provided enough time for cellular
migration such that adequate numbers of cells crossing the wound line
could be assessed. Total number of cells migrating across the wound
line is shown in Figure 2A
. Wounding experiments supplemented with the
(n-3) fatty acid DHA and the trans fatty acids EA and LEA
did not demonstrate any significant effect on the total migration of
IEC-6 cells across the wound line. However, (n-3) fatty acids EPA and
LA as well as the (n-6) fatty acids GLA, ALA and AA did convey a
significant stimulation of total migration of cells across the wound
line as compared to control cultures. Further examination of
photographs was performed to determine the density of cells migrating
across the wound line (Fig. 2B
, defined area described in the Materials
and Methods section). Results from this work demonstrated a pattern of
cell migration stimulation similar to that seen in total cell migration
results. There were no significant effects on the density cells
migrating across the wound line when cultures were supplemented with
the (n-3) fatty acid DHA or trans fatty acids EA and LEA. On
the other hand, supplementation of (n-3) fatty acids EPA and ALA and
the (n-6) fatty acids LA, GLA and AA increased the density of cells
migrating across the wound. Photographs of migrating cultures
(Fig. 3
) demonstrate that the fatty acids EPA, ALA, LA, GLA and AA stimulated
cells adjacent to the wound line to advance farther than in control
cultures.
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). When the percentage of positively stained BrdU cells was assessed in
the total migration area across the wound line, there was no
significant difference between fatty acid-treated cultures and
control (Table 3
).
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Indomethacin treatment had no effect on the migration of (n-3) fatty
acid-, EPA and ALA, treated cultures. Inhibition of eicosanoid
synthesis in (n-6) fatty acid-, LA and AA, treated cultures resulted in
a migration profile similar to that in unsupplemented control cultures.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Blackmore and Meckling-Gill 1995
, Hannigan and Williams 1991
, Martin and Meckling-Gill 1996
).
Differences in eicosanoid synthesis, due to differing precursor pools,
could also result in alterations in cell signaling and metabolism. The
small intestine is the major site of fat absorption and as such is
exposed to potentially high concentrations of fatty acids. The present
study was designed to assess the effects of (n-3), (n-6) and
trans series fatty acids on the process of proliferation and
epithelial restitution utilizing the rat intestinal epithelial cell
line IEC-6.
This lab utilized the SRB assay to assess proliferative and cytotoxic
effects of several drugs and compounds. The SRB assay demonstrates
excellent linearity with cell number, at densities ranging from 1% to
more than 200% confluence, in a number of cell types (Skehan et al. 1990
). Our IC50 values appear consistent with
the work of others in that high levels of PUFA are directly toxic to
cultured cells (Begin et al. 1985
, Begin et al. 1986
). Neoplastic intestinal cell lines are far less
tolerant of such concentrations of fatty acid (Mengeaud et al. 1992
). Selective fatty acid toxicity toward neoplastic cell
lines was suggested to be due to deficiency of 
-6-desaturase
(Dunbar and Bailey 1975
).
Clearly, data from Table 2
indicate that -6-desaturase activity is
intact in this nontransformed cell line and is reflected in IEC-6
tolerance to relatively high concentrations of fatty acid. Tolerance of
such high concentrations of fatty acids by normal intestinal epithelial
cells may also be indicative of the type of lipid environment these
cells would be subject to in the lumen of the small intestine.
Concentrations of fatty acids in the blood plasma following
supplementation could easily be 10–100 µmol/L and much higher in the
intestinal lumen. The effects of this entire range of concentrations
were examined in the current experiment.
The IEC-6 cell line was effectively utilized as a model of wound repair
to study the process of intestinal epithelial cell restitution. This
model demonstrated many similarities to in vivo wound repair including
independence from cell proliferation and dependence on microfilaments
and polyamine synthesis (McCormack et al. 1992
).
Therefore we chose this model to assess the effects of a variety of
fatty acids on the process of epithelial restitution. Other researchers
used shorter migration times (6 h) (McCormack et al. 1992
). However, such short migration times were assessed in
conjunction with the use extracellular matrix-coated culture dishes
which in itself enhances migration. Twenty-four hour post migration
provided enough time for an adequate number of cells to migrate into
the wounded area without the confounding use of extracellular matrix.
Supplementation of the trans fatty acids, LEA and EA, had no
effect on the process of epithelial restitution as measured in wounded,
confluent IEC-6 monolayers. However, supplementation of (n-3) fatty
acids EPA and ALA, as well as the cis (n-6) fatty acids LA,
GLA and AA, did convey a significant enhancement of the total number of
cells migrating across the wound line during the restitution process.
As expected, where there were more cells migrating across the wound
line, a larger area was covered by the moving cells. Indeed,
photographs indicate that cells of wounded cultures supplemented with
EPA, LA, ALA, GLA and AA migrate a greater distance from the wound
line. Furthermore, quantitation of cells migrating under these
conditions indicates that more cells migrate into equivalent areas
(i.e., the density of cells). Given that the IEC-6 model retains many
of the features of a normal intestinal mucosa, this suggests that
supplementation with these fatty acids could produce a more rapid and
complete sealing of the wounded area and may aid in protecting the
mucosa from deleterious lumenal contents in an intact intestine. This
data also suggest that the trans conformers are inactive as
modulators of the restitution process. The observed isometric's
specificity is not surprising given that there is evidence that
trans fatty acids are less efficiently converted to
eicosanoid products and poor regulators of signaling molecules such as
protein kinase C (Murakami and Routtenberg 1985
,
Srivastava and Awasthi 1982
).
The process of epithelial restitution was demonstrated to occur
independent of cell division (McCormack et al. 1992
,
Silen and Ito 1985
). Data from the BrdU assay supported
the idea that fatty acids promote early repair by a mechanism
independent of cell proliferation.
Eicosanoids, especially prostaglandins (PG), are produced by intestinal
epithelial cells and are capable of influencing wound repair in the
gastrointestinal tract (Dubois et al. 1994
,
Eberhart and Dubois 1995
, Mizuno et al. 1997
, Reuter et al. 1996
, Wang et al. 1989
). The use of drugs which act as inhibitors of PG formation
results in ulcers and impaired wound healing in the gastrointestinal
tract (Mizuno et al. 1997
, Reuter et al. 1996
, Soll et al. 1991
, Wang et al. 1989
). In our work we have clearly demonstrated incorporation
of these PG precursors into cellular phospholipid. It is possible that
such fatty acid modification is capable of altering PG synthesis and
influencing the process of epithelial restitution during wound healing.
Indeed, ongoing work in our lab indicated a role for PG in this fatty
acid-supplemented wound-healing model. In data presented here the
cyclooxygenase inhibitor indomethacin attenuated the stimulatory effect
of LA and AA on restitution but had no effect on this process in
cultures supplemented with EPA and ALA. Therefore it seems likely that
enhanced restitution in response to n-6 fatty acids is mediated through
cyclooxygenase products. Furthermore, it appears that n-3 fatty acids
are working through alternative pathways that may include growth factor
modulation.
A number of regulatory cytokines produced by the intestinal mucosa were
studied to determine if they play a role in the process of epithelial
restitution. Several of these stimulate the restitution process and a
central role for transforming growth factor-ß (TGF-ß) was described
in the IEC-6 model by other researchers (Ciacci et al. 1993
, Dignass and Podolsky 1993
, Dignass et al. 1994
). It should be noted that these previous studies
demonstrating a potent effect of growth factors were completed in
low-serum or serum-free conditions. Serum alone significantly
stimulates the restitution process in wounded confluent IEC-6
monolayers (Zushi et al. 1996
) which may explain the
more modest differences in treatments observed in our experiments with
fatty acid supplementation in 10% serum containing media. We
contend that presentation of fatty acids in serum better represents the
complex environment of intestinal mucosal cells in vivo. It has
been demonstrated that a fish oil-supplemented diet, rich in DHA
and EPA, is capable of increasing TGF-ß mRNA and protein in mouse
spleen (Fernandes et al. 1994
). In the same study, corn
oil diets demonstrated increased incorporation of LA and AA into
tissues with no affect on TGF-ß. It is possible that EPA and ALA, in
the present work, are exerting their enhanced cellular migration
effects through TGF-ß. EPA, but not DHA nor AA, stimulated migration
of bovine endothelial cells and is capable of potentiating the
migratory effects of basic fibroblast growth factor and tumor necrosis
factor- (Kanayasu et al. 1991
). Therefore, it is
possible that fatty acid supplemented in IEC-6 culture media are also
capable of potentiating the effects of cytokines through modulation of
receptor function or post receptor pathways. The abovementioned study
corroborates our work, demonstrating enhanced migration of
EPA-enriched cells and the lack of effect of DHA. EPA differs from
DHA in that it is an effective inhibitor of both cyclooxygenase and
lipoxygenase while DHA inhibits only the cyclooxygenase pathway. Also,
EPA can be metabolized into eicosanoid products while DHA is not.
Furthermore, in our IEC-6 cells, EPA was elongated to docosapentaenoic
acid 22:5(n-3) while DHA was not retroconverted to this metabolite.
Docosapentaenoic acid was shown to be a potent stimulator of
endothelial cell migration (Kanayasu-Toyoda et al. 1996
). The balance of these products could be critical to the
activity as modifiers of the restitution process.
In conclusion, we demonstrated that cis n-6 and n-3 fatty acid supplementation is capable of modulating both proliferation and injury repair processes in rat IEC-6 intestinal epithelial cells. This data suggests that fatty acid supplementation could play an important adjuvant role in enhancing mucosal recovery and protection from harmful luminal contents following surgical stress, radiation and chemotherapy. Work is ongoing to identify the mechanism(s) involved in this process.
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FOOTNOTES |
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-linolenic acid; BrdU, bromodeoxyuridine; DHA, docosahexaenoic acid;
DMEM, Dulbecco's modified Eagle's medium; EA, elaidic acid; EPA,
eicosapentaenoic acid; FBS, fetal bovine serum; GLA, -linolenic
acid; IC50, concentration inhibiting 50% cell growth; LA,
linoleic acid; LEA, linoelaidic acid; MUFA, monounsaturated fatty acid;
PG, prostaglandin; PUFA, polyunsaturated fatty acid; SAT, saturated
fatty acid; SRB, sulforhodamine B; TCA, trichloroacetic acid; TGF-ß,
transforming growth factor-ß. 
Manuscript received January 13, 1999. Initial review completed May 3, 1999. Revision accepted June 29, 1999.
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