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Medical Research Service, VA Medical Center, Long Beach, CA, 90822 and Departments of Medicine and Physiology/Biophysics, University of California School of Medicine, Irvine, CA 92697
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMECHANISM AND REGULATION OF...MECHANISM OF TRANSPORT OF...REFERENCES |
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KEY WORDS: • biotin • intestinal absorption • colonic transport • transport mechanism • transport regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMECHANISM AND REGULATION OF...MECHANISM OF TRANSPORT OF...REFERENCES |
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); therefore, in this paper, we will focus our
presentation on new findings regarding the mechanisms and regulation of
biotin transport in mammalian small and large intestine. The intestine is exposed to biotin from two sources, the diet and the bacterially synthesized biotin in the large intestine. We will discuss current findings on transport of biotin from these two sources in the small and large intestine, respectively.
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MECHANISM AND REGULATION OF BIOTIN TRANSPORT IN THE SMALL INTESTINE: ABSORPTION OF DIETARY BIOTIN |
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TOPABSTRACTINTRODUCTIONMECHANISM AND REGULATION OF...MECHANISM OF TRANSPORT OF...REFERENCES |
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). Protein-bound biotin is digested by
gastrointestinal proteases and peptidases to biocytin and
biotin-containing short peptides (Wolf et al. 1984
). These biotin
derivatives are then converted to free biotin by the action of the
enzyme biotinidase (Wolf et al. 1984
). Studies in our laboratory have
shown that, at least in rats, this conversion step to free biotin is
essential for efficient absorption and optimal bioavailability of
dietary protein-bound biotin (Said et al. 1993
). The mechanism of
transport of free biotin in the small intestine has been studied by us
and others with the use of intact intestinal tissue preparations (Brown et al. 1986
, Said et al. 1987
). Transport was found to occur via a
specialized, carrier-mediated process which is Na+
dependent in nature (Brown et al. 1986
, Said et al. 1987
). These
studies, however, did not tell us about the mechanism of biotin
transport across the individual membrane domain of the polarized
intestinal absorptive epithelial cells, i.e., the brush border and
basolateral membranes. These two membrane domains have different
functions, structures and permeabilities. Thus, for detailed
understanding of the intestinal absorption process, knowledge about the
mechanism of the individual membrane transport event is essential. To
address these issues, purified brush border membrane vesicles
(BBMV)3and basolateral membrane vesicles (BLMV) were used. Studies with
purified intestinal BBMV have shown biotin transport at this membrane
domain to occur via a specialized, Na+ gradient-driven,
carrier-mediated system (Said et al. 1987
, Said and Redha 1988
, Said et
al. 1988
, Said and Derweesh 1991
). Furthermore, the transport process
was found to be electroneutral in nature, suggesting a coupling ratio
of biotin to Na+ of 1:1 (Said et al. 1987
, Said and Redha 1988
, Said and Derweesh 1991
). Exit of biotin from the intestinal
absorptive epithelial cells across the basolateral membrane was
examined by using purified intestinal BLMV and found to be via a
Na+-independent, carrier-mediated system (Said et al. 1988
,
Said 1991).
The involvement of a carrier-mediated system in the intestinal
absorption process of biotin has been found in different species
including humans, rats and rabbits (Said et al. 1987
, Said and Redha 1988
, Said and Derweesh 1991
). Therefore, the previous notion that
species variation may exist in the mechanism of intestinal transport of
biotin (Spencer and Brody 1964
) is no longer supported.
Regional differences in the ability of the small intestine to absorb
biotin have been observed and were found to vary, depending on the
developmental stage of the intestine. In the intestine of adult humans
and rats, biotin uptake was found to be higher in the duodenum that in
the jejunum, which was in turn higher than uptake in the ileum (Said and Redha 1987
, Said et al. 1988
). This difference was found to be due
to differences in the Vmax of the biotin uptake
process and not the apparent Km (Said et al.
1988
), suggesting a higher carrier density/activity in the proximal
compared with the distal small intestine.
The transport process of biotin in the small intestine was found to be
adaptively regulated by the extracellular substrate levels (Said et al. 1989
). Biotin deficiency was found to lead to a specific and
significant up-regulation in the substrate intestinal uptake compared
with controls. On the other hand, biotin supplementation at
pharmacologic amounts leads to a specific and significant
down-regulation in the biotin uptake process. These changes were
mediated through changes in the Vmax but not the
apparent Km of the biotin uptake process,
suggesting that the effect was mediated through changes in the
number/activity and not the apparent affinity of the biotin uptake
system.
In recent studies in our laboratory, we have focused on possible
regulation of the biotin uptake process in the small intestine by
specific intracellular protein kinase–mediated pathways. We used the
human-derived cultured intestinal epithelial cells, Caco-2, as an in
vitro model system for the enterocyte. We used Caco-2 cells because
previous studies have demonstrated that these cells serve as a good in
vitro model system for studying the finer details of the biotin
intestinal uptake process, i.e., this cell line possesses a biotin
uptake mechanism similar to that of the native enterocyte (Ma et al. 1994
). We found that pretreatment of these cells for 1 h with the
protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA)
led to a concentration-dependent inhibition in biotin uptake by Caco-2
cells (Table 1
).On the other hand, pretreating the cells with 4
-PMA, the negative
control of PMA, did not affect biotin uptake, demonstrating the
specificity of PMA action on PKC (Table 1)
. Similarly, pretreatment of
cells with the PKC activator sn-1,2-dioctanoylglycerol
caused a significant (P < 0.01) inhibition in biotin
uptake (Table 1)
. In contrast, pretreatment of Caco-2 cells with the
PKC inhibitors staurosporine and chelerythrin led to a small, but
significant stimulation in biotin uptake (Table 1)
. The mechanism
through which PMA pretreatment, i.e., PKC activation, inhibits biotin
uptake is not known, but the inhibition appears to be mediated through
a decrease in the number/activity, but not the affinity of the
functional biotin uptake carriers. This suggestion is on the basis of
the observation that PMA pretreatment leads to a marked decrease in the
Vmax , but not the apparent
Km of the biotin uptake process, i.e.,
Vmax was 255 ± 33.3 and 157.6 ± 15.3
pmol/(mg protein · 3 min); apparent Km was 11.7 ± 3.1 and 10.4 ± 1.5 µmol/L for control and PMA-pretreated
cells, respectively (Fig. 1
).
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),
the heart (Beinlich et al. 1990
) and the placenta (Grassl 1992
). The
physiologic and nutritional implications of this interaction deserve
further investigation.
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MECHANISM OF TRANSPORT OF THE BACTERIALLY SYNTHESIZED BIOTIN IN THE LARGE INTESTINE AND ITS CELLULAR REGULATION |
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TOPABSTRACTINTRODUCTIONMECHANISM AND REGULATION OF...MECHANISM OF TRANSPORT OF...REFERENCES |
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, Wrong et al. 1981
). In vivo studies in
humans, rats and minipigs have shown that the colon is indeed capable
of absorbing considerable amounts of lumenal biotin (Barth et al. 1986
,
Brown and Rosenberg 1987
, Sorrell et al. 1971
). Very little is known,
however, about the transport mechanism involved and its cellular
regulation. Using the human-derived, nontransformed colonic epithelia
cell line NCM460 (Kumar et al. 1997
, Moyer et al. 1996
), we have
recently performed studies to address these issues (Said et al. 1997
).
Our results showed that biotin uptake by these cells is Na+
dependent in nature as indicated by the significant inhibition in
biotin uptake when Na+ in the incubation medium is replaced
with other monovalent cations or with mannitol. Na+
dependence was further confirmed by the finding that ouabain, an
inhibitor of Na+-K+ ATPase, causes a
significant inhibition in biotin (Said et al. 1997
). Initial rate of
biotin uptake as a function of concentration was found to include a
saturable component with an apparent Km of
19.7 ± 3.1 µmol/L and a Vmax of
38.8 ± 1.9 pmol/(mg protein · 3 min), respectively (Said 1991
). Furthermore, uptake of 3H-biotin was found to be
significantly inhibited by structural analogs with a free carboxyl
group at the valeric acid moiety (such as thioctic acid and
desthiobiotin), but not affected by biocytin, an analog with a blocked
carboxyl group at this moiety. Collectively, these findings suggest the
involvement of a specific, Na+-dependent, carrier-mediated
system for biotin uptake by colonic epithelial cells NCM460.
After the characterization of the mechanism of biotin uptake by
the human-derived colonic epithelial cells NCM460, we investigated
possible regulation of the uptake process by specific intracellular
protein kinase–mediated pathways (Said et al. 1997
). Our results again
showed that pretreatment of cells with the PKC activators PMA and with
sn-1,2-dioctanoylglycerol led to a significant inhibition in
biotin uptake. Again the effect appeared to be mediated mainly via a
decrease in the Vmax of the biotin uptake
process (i.e., a decrease in the number/activity of the biotin uptake
carriers) with less changes in its apparent Km(i.e., less change in its affinity). On the other hand, no role
for a PKA-mediated pathway in the regulation of biotin uptake was
observed as indicated by the lack of significant effect on biotin
uptake by pretreatment of NCM460 cells with modulators of this pathway
(Said et al. 1997
).
Similar to the findings with Caco-2 cells, biotin uptake by the
NCM460 cells was also found to be inhibited in a
concentration-dependent manner by pantothenic acid (unpublished
observations). This observation is of potential nutritional
significance because pantothenic acid is also synthesized in
considerable amounts by the normal microflora of the large intestine
(Wrong et al. 1981
). Again, the inhibition in biotin uptake by
pantothenic acid was found from the Dixon plot to be competitive in
nature; the Ki of inhibition was similar to the
apparent Km of the biotin uptake process by
these cells. This suggested that the two vitamins utilize the same
uptake carrier in these colonic cells. This suggestion was further
supported by the finding that biotin also inhibited the uptake of
pantothenic acid by these cells (unpublished observations).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants from the Department of
Veterans Affairs and by the National Institutes of Health (DK-39501 and
DK-47203).
3 Abbreviations used: BBMV, brush border membrane
vesicles; BLMV, basolateral membrane vesicles; IBMX,
3-isobutyl-1-methylxanthine; PKA, protein kinase A; PKC, protein kinase
C; PMA, phorbol 12-myristate 13-acetate.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMECHANISM AND REGULATION OF...MECHANISM OF TRANSPORT OF...REFERENCES |
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