Biotin uptake by human colonic epithelial NCM460 cells: a
carrier-mediated process shared with pantothenic acid
Hamid M.
Said1,2,
Alvaro
Ortiz1,2,
Eric
McCloud1,2,
David
Dyer1,2,
Mary Pat
Moyer3,4, and
Stanley
Rubin5
1 Veterans Affairs Medical
Center, Long Beach 90822;
2 University of California School
of Medicine, Irvine 92717;
5 Veterans Affairs Medical Center,
Sepulveda, California 91343;
3 University of Texas Health
Science Center, San Antonio 78284; and
4 INCELL Corporation, San Antonio,
Texas 78288
 |
ABSTRACT |
Previous studies showed that the normal microflora of the large
intestine synthesizes biotin and that the colon is capable of absorbing
intraluminally introduced free biotin. Nothing, however, is known about
the mechanism of biotin absorption in the large intestine and its
regulation. To address these issues, we used the human-derived,
nontransformed colonic epithelial cell line NCM460. The
initial rate of biotin uptake was found to be
1) temperature and energy dependent,
2)
Na+ dependent (coupling ratio of
1:1), 3) saturable as a function of
concentration [apparent Michaelis constant
(Km) of 19.7 µM], 4) inhibited by
structural analogs with a free carboxyl group at the valeric acid
moiety, and 5) competitively
inhibited by the vitamin pantothenic acid (inhibition
constant of 14.4 µM). Pretreatment with the protein kinase C (PKC)
activators phorbol 12-myristate 13-acetate (PMA) and
1,2-dioctanoyl-sn-glycerol
significantly inhibited biotin uptake. In contrast, pretreatment with
the PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediated
via a marked decrease in maximal uptake velocity and a
slight increase in apparent
Km. Pretreatment
of cells with modulators of the protein kinase A-mediated pathway, on
the other hand, showed no significant effect on biotin uptake. These
results demonstrate, for the first time, the functional existence of a
Na+-dependent, specialized
carrier-mediated system for biotin uptake in colonic epithelial cells.
This system is shared with pantothenic acid and appears to be under the
regulation of an intracellular PKC-mediated pathway.
biotin transport; human colonic epithelial cells; membrane
transport; transport regulation
 |
INTRODUCTION |
BIOTIN IS AN ESSENTIAL micronutrient for normal
cellular functions, growth, and development (3, 9, 36). It acts as a
coenzyme for four carboxylases that catalyze essential steps in
critical cellular metabolic pathways, including fatty acid biosynthesis, gluconeogenesis, and the catabolism of several
branched-chain amino acids and odd-chain fatty acids (3, 9, 36). Biotin deficiency in humans leads to a range of clinical abnormalities, including neurological disorders, growth retardation, and skin abnormalities (3, 9, 36, 39).
Humans and other mammals cannot synthesize biotin and thus must obtain
the vitamin from exogenous sources via intestinal absorption. Biotin is
presented to the intestine from two exogenous sources: from the diet
and as a product of bacterial synthesis by the normal microflora of the
large intestine. Dietary biotin exists in free and protein-bound forms
(14), with the latter requiring conversion to free biotin before
absorption (27, 40). Absorption of free biotin then takes place mainly
in the proximal part of the small intestine via a specialized
Na+-dependent, carrier-mediated
system (5, 21, 23, 26, 28-31). As to the second source of biotin,
previous studies have shown that a substantial portion of the biotin
synthesized by the normal microflora of the large intestine is in the
form of free unbound biotin, i.e., available for absorption (6, 7, 12,
41). Furthermore, in vivo studies in humans, rats, and minipigs have shown that the colon is capable of absorbing significant amounts of
luminally introduced biotin (1, 4, 33). Nothing, however, is known
about the absorption mechanism involved or its cellular regulation.
Addressing this issue is important from a physiological and nutritional
perspective because this latter source of biotin may play a role in the
localized nutrition of colonocytes, in addition to its contribution to
the overall biotin body homeostasis. In this study, we investigated the
mechanism and regulation of biotin transport in the large intestine
using the human-derived, nontransformed, colonic epithelial cell line
NCM460 (18) as an in vitro model system. We chose these cells because
they possess characteristics that are similar to those of normal
colonic epithelial cells, including similar transport processes (18,
20). For example, recent studies in our laboratory have shown that
these cells possess a folate uptake mechanism that is similar to that found in human native colonic apical membrane vesicles (10, 13).
 |
METHODS |
[3H]biotin (specific
activity 58.2 Ci/mmol; radiochemical purity >97%) was obtained from
DuPont NEN (Boston, MA). The culture medium M3:10 was a gift from
INCELL (San Antonio, TX). Other cell culture ingredients were obtained
from Sigma (St. Louis, MO). All other chemicals were of analytical
grade and were obtained from commercial sources.
The human-derived normal colonic epithelial cell line NCM460 was
propagated in the culture medium M3:10 to maintain its colonocyte features (18). The M3:10 medium is M3 base medium supplemented with
10% (vol/vol) fetal bovine serum and antibiotics and contains many
growth factors and nutrients (16, 17, 35). NCM460 cells were used
between passages
32 and
42 in this study. The cells were grown
in 75-cm2 plastic flasks (Costar)
at 37°C in a 5% CO2-95% air
atmosphere, with medium changes every 4 days. NCM460 cells were
subcultured by trypsinization with 0.05% trypsin and 0.9 nM EDTA in
Ca2+- and
Mg2+-free phosphate-buffered
solution and plated onto 12-well plates at a concentration of 5 × 105 cells/well. Uptake of biotin
was studied 2-4 days after confluence. Cell growth was observed by
periodic monitoring with an inverted microscope. Cell viability was
tested by the trypan blue dye exclusion method and found to be >95%.
Uptake experiments were performed at 37°C, unless otherwise stated.
Incubation was performed in Krebs-Ringer buffer containing (in mM) 123 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES (pH 7.4), unless otherwise stated. [3H]biotin was added
to the incubation buffer at the onset of experiments, and uptake was
terminated after 3 min of incubation (unless otherwise specified) by
the addition of 1 ml of ice-cold buffer followed by immediate
aspiration. The monolayers were rinsed twice with ice-cold buffer and
digested with 1 ml of 1 N NaOH, neutralized by HCl, and then counted
for radioactivity in a liquid scintillation counter. Protein contents
of cell digests were estimated on parallel wells by the method of Lowry
et al. (15), using BSA as the standard. Uptake data are means ± SE
of measurements on multiple separate monolayers performed on at least
two different occasions and are expressed in femtomoles or picomoles
per milligram protein per unit time. P
values for experimental vs. simultaneously performed control groups
were calculated using the Student's
t-test. Kinetic parameters of biotin
uptake, i.e., maximal velocity
(Vmax) and the
apparent Michaelis constant
(Km), were
calculated using a computerized nonlinear regression analysis program
of the Michaelis-Menten equation as described previously (38).
 |
RESULTS |
General characteristics of biotin uptake by the human-derived
colonic epithelial cell line NCM460.
Figure 1 shows the uptake of
low (6.4 nM) and high (100 µM) concentrations of biotin by confluent
monolayers of NCM460 cells. At both concentrations, uptake was found to
be linear with time for up to 15 min, and it occurred at rates of 3.53 fmol · mg
protein
1 · min
1
and 9.39 pmol · mg
protein
1 · min
1,
respectively. Thus, in all subsequent studies, 3 min was used as the
standard incubation time (i.e., for initial rate). In another study, we
examined the metabolic form of the radioactivity taken up by the cells
following a 5-min incubation with 21 nM
[3H]biotin.
Cellulose-precoated TLC plates and a solvent system of butanol-acetic
acid-water (4:1:1) were used. The results showed that 95% of the
transported radioactivity was in the form of intact biotin.

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Fig. 1.
Uptake of biotin by NCM460 cells as a function of time. Cells were
incubated at 37°C in Krebs-Ringer buffer (pH 7.4) for different
periods with low (6.4 nM; A) or high
(100 µM; B) concentrations of
biotin. Data are means ± SE of 3-6 separate uptake
determinations performed on 2 separate occasions. When not shown, error
bar is smaller than symbol. For A,
y = 3.53x + 3.82 (r = 0.99). For
B, y = 9.39x + 37.49, (r = 0.99).
|
|
We also examined the effect of temperature on the uptake of biotin (6.4 nM) by NCM460 cells. Uptake was found to significantly decrease when
the incubation temperature was lowered [15.3 ± 0.6 (n = 8), 6.1 ± 0.13 (n = 4;
P < 0.01), and 2.9 ± 0.4 (n = 8;
P < 0.01)
fmol · mg
protein
1 · 3 min
1 at 37, 22, and
4°C, respectively].
The dependence on Na+ of biotin
uptake was also examined by replacing
Na+ in the incubation medium with
an equimolar concentration of Li+,
K+, choline, or mannitol. This
manipulation resulted in a significant (P < 0.01 for each) inhibition in
biotin (6.4 nM) uptake when Na+
was removed, regardless of what was used to replace it (Table 1).
We also examined the effect of buffer pH on the uptake of biotin (6.4 nM) by NCM460 cells. The study was performed both in the presence and
in the absence of Na+ in the
incubation medium (Na+ was
isosmotically replaced by K+).
The results showed that decreasing the incubation buffer pH from 8.5 to
5.5 caused a modest increase in biotin uptake both in the presence and
absence of Na+ (Table
2).
Existence of a carrier-mediated system for biotin uptake by NCM460
cells.
The initial rate of biotin uptake as a function of the substrate
concentration in the incubation medium (0.006-250 µM) was examined. The results showed that biotin uptake includes a saturable component. Uptake by this component was calculated by subtracting diffusion [calculated from the slope of the line between the
point of origin and uptake at a high pharmacological concentration
(1,000 µM) of biotin] from total uptake (Fig.
2). Kinetic parameters of the saturable component were
then calculated as described in METHODS and found to be 19.7 ± 3.1 µM and 38.8 ± 1.9 pmol · mg protein
1 · 3 min
1 for the apparent
Km and
Vmax,
respectively.

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Fig. 2.
Uptake of biotin by NCM460 cells as a function of concentration. Cells
were incubated at 37°C in Krebs-Ringer buffer (pH 7.4) for 3 min
(i.e., initial rate) in presence of different concentrations of biotin
(0.006-250 µM). Uptake shown is that of carrier-mediated
component calculated as described in text. Data are means ± SE of
3-8 separate uptake determinations performed on 2 or 3 separate
occasions. When not shown, error bar is smaller than symbol.
|
|
We also examined the effect of the biotin structural analogs thioctic
acid, desthiobiotin, biocytin, and biotin methyl ester on the uptake of
[3H]biotin (6.4 nM;
Table 3). Thioctic acid and
desthiobiotin caused a marked and concentration-dependent inhibition in
[3H]biotin uptake,
whereas biocytin and biotin methyl ester had no (or minimal) effect.
The effect of unlabeled biotin in the incubation medium on the efflux
of [3H]biotin from
preloaded NCM460 cells was also examined. Preloading with
[3H]biotin was
performed by incubating the cells for 10 min in the presence of 32 nM
[3H]biotin, followed
by washing of the cells and incubation for 10 min with or without 100 µM unlabeled biotin. The cell content of
[3H]biotin was
significantly lower in cells incubated in the presence of unlabeled
biotin in the incubation medium than in cells incubated in the absence
of unlabeled biotin [50.61 ± 3.65 and 63.95 ± 1.3 fmol · mg
protein
1 · 3 min
1, respectively
(n = 3;
P < 0.01) ].
Stoichiometry of
biotin-Na+
carrier-mediated uptake.
The coupling ratio of biotin to
Na+ was investigated using the
"activation method" of Turner and Moran (37) as described by us
before (24). In this method, stimulation in initial rate of biotin (6.4 µM) uptake was determined as a function of increasing the
concentration of the activator, i.e.,
Na+, in the incubation medium
(Fig.
3A).
The K+ ionophore valinomycin (30 µg/ml) was added to the incubation medium as indicated (37). Uptake
data were then applied to the Hill plot [log
Na+ concentration vs. log
(V/Vmax
V), where
V is initial rate of biotin uptake;
Fig. 3B]. The results showed a
linear relationship (r = 0.99) with a
Hill coefficient (i.e., slope) of 1.01, suggesting a
Na+-to-biotin coupling ratio of
1:1.

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Fig. 3.
A: biotin uptake by NCM460 cells as a
function of Na+ concentration in
incubation medium. Cells were incubated for 3 min (i.e., initial rate)
at 37°C in modified Krebs-Ringer buffer (pH 7.4) containing biotin
(6.4 µM) and different Na+
concentrations ([Na+];
NaCl was isosmotically replaced with choline chloride). Valinomycin was
added to incubation medium at 30 µg/ml. Results are means ± SE of
3-6 separate uptake determinations performed on 2 separate
occasions. When not shown, error bar is smaller than symbol.
B: Hill plot of data in
A; log Q = log (V/Vmax V), where
V is initial rate of biotin
uptake in presence of a given Na+ concentration
([Na+]) and
Vmax is maximum
uptake velocity. For Hill plot, y = 1.01x 1.69 (r = 0.99).
|
|
Effect of pantothenic acid and short-chain fatty acids on biotin
uptake by NCM460 cells.
The effect of different concentrations of the anion pantothenate,
(another water-soluble vitamin that is synthesized by the normal
microflora of the large intestine) on the uptake of the anion biotin
was investigated in this experiment. Pantothenic acid produced a
concentration-dependent inhibition of biotin uptake that was found, by
the Dixon method, to be competitive with an inhibition constant
(Ki) of 14.4 µM (Fig. 4).

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Fig. 4.
Dixon plot of effect of pantothenic acid on biotin uptake by NCM460
cells. Cells were incubated at 37°C in Krebs-Ringer buffer (pH 7.4)
containing 0.5 ( ) and 5 ( ) µM
[3H]biotin and
different concentrations of pantothenic acid.
[3H]biotin uptake was
measured over a 3-min incubation (i.e., initial rate), and data were
applied to Dixon plot (i.e., concentration of inhibitor vs.
1/V, where
V is initial rate of
[3H]biotin uptake).
Data are means of 3-6 separate uptake determinations performed on
2 separate occasions.
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|
We also examined the effect of pantothenic acid (20 µM) on the
kinetic parameters of biotin uptake by NCM460 cells. Pantothenic acid
caused a marked increase in the apparent
Km of biotin
uptake with no (or minimal) change in the
Vmax of the
uptake process (apparent
Km of 16.64 ± 1.5 and 59.86 ± 1.9 µM;
Vmax of 35.23 ± 0.93 and 38.9 ± 0.52 pmol · mg
protein
1 · 3 min
1 for control and
presence of pantothenic acid, respectively; Fig. 5).

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Fig. 5.
Effect of pantothenic acid on biotin uptake by NCM460 cells as a
function of biotin concentration. Cells were incubated at 37°C in
Krebs-Ringer buffer (pH 7.4) containing different concentrations of
biotin (1-200 µM) and with ( ) or without ( ) 20 µM
pantothenic acid. Data are means ± SE of 3-6 separate uptake
determinations performed on 2 separate occasions. When not shown, error
bar is smaller than symbol.
|
|
We also tested the effect of the anionic short-chain fatty acids
acetate and butyrate (both at 1 mM) on biotin (6.4 nM) uptake. Neither
of these compounds affected biotin uptake [16.61 ± 0.45, 16.11 ± 0.43, and 17.31 ± 0.63 fmol · mg
protein
1 · 3 min
1 for control and
presence of acetate and of butyrate, respectively (n = 6)].
Effect of metabolic inhibitors on biotin uptake by NCM460 cells.
The effect of preincubating NCM460 cells for 30 min with the metabolic
inhibitors iodoacetate (10 mM), dinitrophenol (DNP; 0.5 mM), and
ouabain (10 mM) on subsequent uptake of
[3H]biotin was
investigated in this experiment. These compounds significantly
(P < 0.01 for each) inhibited biotin
uptake [14.96 ± 0.42 (n = 7), 10.91 ± 0.22 (n = 7), 6.76 ± 0.7 (n = 7), and 8.14 ± 0.12 (n = 7)
fmol · mg
protein
1 · 3 min
1 for control and
presence of DNP, iodoacetate, and ouabain, respectively].
Intracellular regulation of biotin uptake.
We tested in these experiments whether protein kinase C (PKC)- and
protein kinase A (PKA)-mediated pathways are involved in the regulation
of biotin uptake by NCM460 cells. One-hour pretreatment of cells with
the PKC activator phorbol 12-myristate 13-acetate (PMA) led to a
concentration-dependent inhibition in biotin uptake (Table
4), whereas its negative control, i.e.,
4
-PMA, had no effect. Similarly, pretreatment of cells with
1,2-dioctanoyl-sn-glycerol, another
activator of PKC, also led to inhibition in biotin uptake (Table 4). In
contrast, pretreatment of cells with staurosporine and chelerythrine,
inhibitors of PKC, led to a slight but significant stimulation in
biotin uptake (Table 4). When NCM460 cells were pretreated with PMA (1 µM) in the presence of staurosporine (1 µM), the PMA-induced
inhibitory effect on biotin uptake was significantly (P < 0.01) reduced [16.49 ± 0.07, 12 ± 0.25, and 15.7 ± 0.48 fmol · mg
protein
1 · 3 min
1 for control and after
pretreatment with PMA and PMA plus staurosporine, respectively
(n = 3)].
We also examined the effect of PMA on the kinetic parameters of biotin
uptake by NCM460 cells. This was done by examining the effect of
pretreatment of cells with 1 µM PMA on the uptake of biotin as a
function of concentration. The results (Fig.
6) showed that PMA cause a marked decrease
in the Vmax of
biotin uptake with a slight increase in the apparent
Km of the uptake process (Vmax of
40.76 ± 1.58 and 25.21 ± 0.63 pmol · mg
protein
1 · 3 min
1 and apparent
Km of 18.68 ± 2.36 and 23.85 ± 2.19 µM for control and pretreatment with PMA,
respectively).

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Fig. 6.
Effect of pretreatment of NCM460 cells with phorbol 12-myristate
13-acetate (PMA) on biotin uptake as a function of concentration. Cells
were pretreated for 1 h with 1 µM PMA at 37°C in Krebs-Ringer
buffer (pH 7.4). , PMA; , control. Different concentrations of
[3H]biotin
(0.006-250 µM) were then added to incubation medium, and uptake
was determined after 3 min of incubation. Data are means ± SE of
3-6 separate uptake determinations performed on 2 separate
occasions. When not shown, error bar is smaller than symbol.
|
|
In another experiment, we examined the effect of pretreatment of NCM460
cells with modulators of the PKA-mediated pathway on biotin uptake.
None of these modulators significantly affected biotin uptake (Table
4).
 |
DISCUSSION |
The normal microflora of the large intestine synthesizes biotin (6, 7,
12, 41), and human, rat, and minipig colon are capable of absorbing
significant amounts of luminally introduced biotin (1, 4, 33). The
mechanism of biotin absorption in the large intestine and the
intracellular regulation of that process, however, are not known. In
the present study, we used the human-derived, normal colonic epithelial
cell line NCM460 to address these issues. Uptake of biotin by these
cells was found to be appreciable and linear with time for up to 15 min
of incubation and was temperature dependent. No metabolic alteration in
the transported substrate was observed following 5 min of incubation with [3H]biotin. The
uptake process of biotin by NCM460 cells was dependent on the presence
of Na+ in the incubation medium,
as indicated by the drastic inhibition in the vitamin uptake observed
when Na+ was replaced in the
incubation medium with other monovalent cations or with mannitol. This
suggestion was further supported by the observation of a significant
inhibition of the vitamin uptake when cells were treated with the
Na+-K+-ATPase
inhibitor ouabain. Incubation buffer pH was found to have some effect
on biotin uptake by these cells, as suggested by the modest increase in
the substrate uptake when the incubation buffer pH was decreased from
8.5 to 5.5. This trend of pH effect was observed both in the presence
and in the absence of Na+ in the
incubation medium, suggesting that the effect is not mediated through
an effect of pH on the
Na+-dependent component of the
vitamin uptake process. A similar type of effect of pH on biotin uptake
has been seen previously in brush-border membrane vesicles of human
jejunum and was attributed to the possible effect of pH on the ionic
status of biotin
(pKa of biotin is
4.51) (26).
Biotin uptake by NCM460 cells apparently involves a specialized,
carrier-mediated system, as indicated by the saturation in the vitamin
uptake as a function of increasing the substrate concentration in the
incubation medium. This suggestion was further supported by the marked
cis-inhibition in
[3H]biotin uptake by
certain structural analogs (thioctic acid and desthiobiotin) and by the
stimulation of
[3H]biotin efflux from
preloaded cells by unlabeled biotin in the incubation medium. The
contribution of this carrier-mediated system to the overall absorption
process of biotin in the colon depends on the prevailing vitamin
concentration in the colonic lumen, being higher at low physiological
concentrations and lower at high concentrations. It is worth noting
here that structural analogs with a free carboxyl group at the valeric
acid moiety of the biotin molecule, such as desthiobiotin and thioctic
acid, were potent inhibitors of
[3H]biotin uptake
compared with analogs with a blocked carboxyl group at this moiety,
such as biocytin and biotin methyl ester, which showed no (or minimal)
effect on [3H]biotin
uptake. This demonstrates the importance of this carboxyl moiety of the
biotin molecule in the recognition of and interaction with the
substrate uptake carrier in these cells. Similar structural requirements in the biotin molecule have been reported for biotin transport in other cellular systems (8, 25, 34).
In a separate study, we investigated the uptake coupling ratio of
biotin to Na+ using the activation
method of Turner and Moran (37). The ratio was found to be 1:1,
suggesting that one Na+ is
transported with one biotin molecule, and that the event is electroneutral in nature. In another study, the uptake process of
biotin was found to be dependent on cellular energy, as indicated by
the significant inhibition in the vitamin uptake by pretreatment of
cells with the metabolic inhibitors iodoacetate and DNP.
The normal microflora of the large intestine synthesizes significant
quantities of not only biotin but also other substrates, such as the
water-soluble vitamin pantothenic acid and the short-chain fatty acids
acetate and butyrate. These substrates, like biotin, also exist as
anions at the physiological pH of the large intestinal lumen, and thus
might be expected to interact or interfere with uptake of the anionic
biotin. Because of this, and because pantothenic and fatty acids have
been shown to inhibit biotin uptake in other systems (2, 11, 19, 22,
34), we examined their effect on biotin uptake by NCM460 cells.
Pantothenic acid caused a concentration-dependent competitive
inhibition in biotin uptake. The fact that pantothenic acid also caused
a marked increase in the apparent
Km of biotin uptake (from 16.64 µM for control to 59.86 µM in the presence of
pantothenic acid), with no (or minimal) effect on the
Vmax of the
uptake process, further confirms the competitive nature of the
inhibition. It is also worth mentioning here that the
Ki for
pantothenic acid (14.4 µM) is close to the apparent
Km of biotin
uptake by these cells (range between 16.64 and 19.7 µM). These
findings suggest that biotin and pantothenic acid share the same uptake
system in these cells. Similar findings have been reported in the heart
(2), the placenta (11, 19), and the small intestine (22). In contrast,
pantothenic acid did not appear to affect the transport of biotin
across the brain microvessel endothelial cells (32). The physiological
and nutritional implications of interactions between these vitamins
deserves further investigation. As to the effects of the
short-chain fatty acids acetate and butyrate on biotin uptake, no
inhibition was observed with either compound. This is unlike biotin
transport through the blood-brain barrier, which was inhibited by
nonanoic acid, a straight-chain fatty acid (34).
In separate studies, we investigated the potential for intracellular
regulation of the biotin uptake process of NCM460 cells by PKC- and
PKA-mediated pathways. Our findings showed that pretreatment of cells
with PMA (but not with its negative control, 4
-PMA) and with
1,2-dioctanoyl-sn-glycerol, activators
of PKC, caused significant inhibition in biotin uptake. On the other
hand, pretreatment of cells with staurosporine and chelerythrine,
inhibitors of PKC, produced a slight but significant stimulation in
biotin uptake. Furthermore, the inhibition by PMA was significantly
reduced when staurosporine was added to the pretreatment buffer. These
findings point toward the involvement of a PKC-mediated pathway in the regulation of biotin uptake by these cells. The effect of PKC activation by PMA on biotin uptake was found to be mediated through marked inhibition in the
Vmax of the
uptake process and a slight increase in the apparent
Km. This suggests
that PKC activation leads to a marked decrease in the activity
and/or number of the functional biotin uptake carriers and a
slight decrease in their affinity, respectively. In contrast to the
role of PKC, no role for a PKA-mediated pathway in the regulation of
biotin uptake by NCM460 cells was found. This conclusion is based on
the observations that specific modulators of this pathway did not
significantly affect biotin uptake in these cells.
The above-described findings on the mechanism of biotin uptake by the
human-derived colonic epithelial cells NCM460, showing involvement of a
specialized, carrier-meditated and
Na+-dependent process that is also
shared by the vitamin pantothenic acid and appears to be under the
regulation of a PKC-mediated pathway, are similar to those reported
elsewhere for the vitamin uptake in small intestinal epithelial cells
(22, 23, 28, 30, 31). Thus it is reasonable to suggest that absorption of dietary biotin in the small intestine and that of the bacterially synthesized vitamin in the large intestine occur via a similar cellular
mechanism. In summary, our findings show for the first time the
functional existence of a specialized carrier-mediated, Na+-dependent system for biotin
uptake in colonic epithelial cells. This system is shared by the
vitamin pantothenic acid and appears to be under the regulation of an
intracellular PKC-mediated pathway.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from the Department of Veterans
Affairs and by National Institute of Diabetes and Digestive and Kidney
Diseases Grants DK-47203 and DK-02357.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: H. M. Said, VA Medical Center 151, Long
Beach, CA 90822.
Received 24 April 1998; accepted in final form 12 August 1998.
 |
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