Background
Thiamine is a water-soluble, B-complex vitamin that
cannot be synthesized by mammals, and thus thiamine can be
obtained only from dietary intake. This can lead to severe
consequences in humans when thiamine is limiting; thiamine
deficiency may result in beriberi and the Wernike-Korsakoff
syndrome [ 1 2 ] . Being positively charged and present in
relatively low plasma concentrations, thiamine movement
across cellular membranes requires transporters. Upon being
taken up by a cell, thiamine is rapidly diphosphorylated by
thiamine diphosphokinase to give thiamine diphosphate
(ThDP) [ 3 ] . Thus, thiamine represents only a few percent
of the total cellular thiamine/thiamine phosphate
derivatives. ThDP serves as a cofactor for several enzymes
that are found both in the cytosol (transketolase) and
mitochondria (α-ketoglutarate dehydrogenase complex being
the most studied example). The intracellular concentration
of ThDP has been estimated at 30 μM, with only about 7
percent being free cytosolic and the remainder being
enzyme-bound with much of this within mitochondria [ 4 5 ]
. Previous findings indicate a complex, cell-type dependent
regulation of compartmentalization and intracellular pools
of thiamine and its phosphorylated derivatives in response
to fluctuating extracellular thiamine levels [ 4 5 6 ] .
Hence, thiamine transport, including that by mitochondria,
is of interest.
Thiamine entry into mammalian cells occurs by a
saturable, high affinity transporter that is deficient in
humans with thiamine-responsive megaloblastic anemia (TRMA)
[ 7 8 9 10 11 12 ] . Thiamine uptake by mitochondria has
been demonstrated [ 13 ] , yet thiamine diphosphokinase is
cytosolic and mitochondria cannot convert thiamine to ThDP
[ 14 15 16 ] . Barile and coworkers demonstrated saturable
uptake of ThDP by rat liver mitochondria characterized by a
K
m of around 20 μM. Although the
estimated concentration, in mice and human cells, of
intracellular ThDP is about 30 μM, much of this is found in
a low turnover pool representing enzyme-bound ThDP [ 4 17 ]
. The estimated concentration of free cytosolic ThDP
available for intracellular transport is about 10% of the
total concentration and thus 2 to 3 μM [ 4 17 18 ] . Hence
the physiological significance of the mitochondrial ThDP
uptake just decribed is uncertain. Within mitochondria,
ThDP can be converted to thiamine monophosphate [ 19 ] .
Thiamine or ThDP entry into mitochondria from TRMA cells
has not been studied. Interestingly, ThDP-utilizing enzymes
in mitochondria are much less affected (as revealed by loss
of activity) upon progressive depletion of thiamine
available to TRMA cells than are ThDP-utilizing enzymes of
the cytosol [ 6 ] . For these reasons, we have examined the
uptake of thiamine and especially ThDP by mitochondria from
several human cell types, including cells from TRMA
patients.
Results
Uptake of thiamine by cells and mitochondria
Although our interests primarily were in mitochondrial
uptake of thiamine and its derivatives, we first examined
cellular uptake of thiamine by the lymphoblast cell lines
and found thiamine uptake properties typical of other
mammalian cells. Figure 1aindicates thiamine uptake by
normal lymphoblasts and lymphoblasts derived from a TRMA
patient. The high affinity transport of thiamine by
normal lymphoblasts is abolished in the presence of a 100
fold excess of unlabeled thiamine. Under such conditions,
some uptake continues from a low affinity (K
m in the mM range) transport mechanism
[ 3 ] and/or from diffusion [ 20 ] that characterizes
thiamine uptake in all mammalian cells examined to date.
Using an expanded range of thiamine concentrations from
that shown in fig. 1in multiple experiments resulted in a
K
m of 1.0 ± 0.9 μM for the high
affinity transport by normal lymphoblasts. As expected,
lymphoblasts derived from the TRMA patient showed no high
affinity thiamine transport as revealed by thiamine
uptake being the same in the absence and presence of
excess unlabelled thiamine.
Mitochondria isolated from normal lymphoblasts also
were found to take up thiamine (fig. 1b) in a manner
similar to that of cellular uptake, with both a high and
low affinity component. Using an expanded range of
thiamine concentrations in multiple experiments resulted
in a K
m of 2.1 ± 0.4 μM for high affinity
thiamine uptake by normal lymphoblast mitochondria.
Interestingly, mitochondria from TRMA lymphoblasts did
not possess a "high affinity" thiamine transport capacity
as did mitochondria form normal lymphoblasts. No
difference in uptake of thiamine by TRMA mitochondria was
found in the presence and absence of excess unlabelled
thiamine (fig. 1b).
This finding suggests that cellular and mitochondrial
uptake of thiamine may be mediated by the same
transporter since TRMA is defined by mutation within the
thiamine transporter located on the plasma membrane [ 7 8
9 10 11 12 ] . Using antiserum specific for the human
thiamine transporter that is mutated in TRMA individuals,
western analysis consistently resulted in a faint but
detectable band within the isolated mitochondrial
suspension (fig. 1c), even after extensive and multiple
washing.
Uptake of ThDP by mitochondria
Although mitochondria from lymphoblasts (above) and
other mammalian cells [ 13 ] were found to take up
thiamine, the physiological significance of the uptake is
unknown given that thiamine diphosphokinase is cytosolic
and mitochondria cannot convert thiamine to ThDP [ 14 16
21 ] . We thus were interested in uptake of ThDP by
mitochondria, a possibility demonstrated previously with
rat liver [ 14 ] . Mitochondria from normal human
lymphoblasts were able to take up ThDP in a saturable,
biphasic manner (fig. 2) with a first saturation in the
submicromolar range (described below) and a second at
much higher concentrations of ThDP. Time course
experiments indicated ThDP uptake was linear for at least
20 minutes (data not shown), and uptake was linear with
the amount of mitochondrial protein added (inset of fig.
2).
Although the biphasic nature of the uptake is readily
seen in various plots of the data, the existence of the
high affinity component perhaps is best illustrated in
fig. 3in which the uptake at submicromolar concentrations
of ThDP is illustrated (open squares). Uptake of ThDP was
repeated in the presence of 30 μM non-radioactive ThDP, a
concentration that is 100 to about 40 fold excess over
the concentration of radioactive ThDP that was used.
Given the K
m values for the high and low affinity
components (see below), this excess should abolish most
of the high affinity uptake but have little to no effect
on the low affinity uptake. The uptake due solely to the
low affinity component was calculated using the kinetic
parameters determined for this component and was plotted
as open triangles. As seen in fig. 3, uptake in the
presence of 30 μM non-radioactive ThDP (open circles) was
essentially identical to that calculated for the low
affinity component and abolition of the high affinity
uptake indeed was observed.
Using the data from 4 independent experiments resulted
in the determination of a K
m of 0.38 μM for the high affinity
(table 1) and 115 μM for the low affinity uptake
components. The high affinity K
m value compares favorably with the
estimated 2 to 3 μM intracellular concentration of free
ThDP.
Mitochondria isolated from TRMA lymphoblasts took up
ThDP in an essentially identical saturable, biphasic
fashion as uptake by normal mitochondria (fig. 4). The
inset compares the Lineweaver-Burk plot of the high
affinity uptake component for mitochondria of both cell
types. A high affinity K
m of 0.60 (table 1) was calculated for
mitochondrial uptake of ThDP for TRMA lymphoblasts, a
value that is essentially the same as that for
mitochondria isolated from normal lymphoblasts. The
results indicate that although mitochondria from TRMA
lymphoblasts cannot take up thiamine with high affinity,
they can efficiently import ThDP, the active form of
thiamine.
Previous work has indicated that different cell types
may differentially regulate intracellular pools of
thiamine and/or its phosphorylated derivatives [ 6 ] .
Thus, we examined ThDP uptake by several other cell
types. As shown in table 1, high affinity uptake of ThDP
by mitochondria from fibroblasts and neuroblastoma cells
was essentially the same as that for normal and TRMA
lymphoblasts as revealed by the similar K
m values. The apparent affinity for
ThDP characteristic of the high affinity uptake component
was essentially identical for all cell types examined,
however variation was seen in transport capacity, as
revealed by substantially different values for V
max (table 1). Mitochondria from all
of the cell types examined also possessed the low
affinity uptake characterized by K
m 's similar to that of normal
lymphoblasts but with a greater range of values being
found (20 to 115 μM).
The final entry in table 1is for ThDP uptake by
mitochondria isolated from
glyB cells.
GlyB cells are a Chinese hamster
ovary cell line that is deficient in the transport of
folate into mitochondria. For reasons discussed below,
ThDP uptake was examined in these cells. As seen in table
1, high affinity uptake was found with kinetic constants
similar to those of the human cell types. The low
affinity uptake component was also similar to that by
human mitochondria (data not shown).
Discussion + Conclusion
As has been reported for mitochondria of rat liver [ 13
] , mitochondria from human lymphoblasts were found herein
to take up thiamine in a saturable manner characterized by
a K
m of 2.1 μM. Upon entry into a cell,
thiamine is rapidly diphosphorylated to ThDP, resulting in
a low intracellular thiamine concentration [ 4 17 ] . The K
m determined here is about an order of
magnitude greater than the estimated intracellular thiamine
concentration [ 4 ] , raising questions about the
efficiency of such uptake.
Surprisingly, mitochondria derived from TRMA
lymphoblasts lacked the high affinity uptake of thiamine.
TRMA is caused by mutations which destroy the high affinity
thiamine transporter of the plasma membrane [ 7 8 9 10 11
12 ] . The similar K
m values found for cellular and
mitochondrial uptake of thiamine for normal lymphoblasts
and the lack of such uptake by TRMA mitochondria and TRMA
cells suggests that high affinity thiamine import into
mitochondria may be carried out by the same transporter or
a variant form, perhaps generated by differential splicing,
of that serving on the plasma membrane. Although western
analysis using anti-human transporter (that is mutated in
TRMA individuals) antiserum supports this interpretation,
further experiments need to be carried out to substantiate
the suggestion of a shared thiamine transporter between
these two membrane systems. Even if true, the physiological
significance of thiamine uptake by mitochondria is unknown
since mitochondria cannot form ThDP from thiamine [ 14 15
16 ] .
We find that mitochondria from a variety of human cell
types efficiently take up ThDP. Uptake is biphasic with a
high and a low affinity component. The K
m values characteristics of the high
affinity uptake component (all around 0.4 μM) are
comparable to the estimated intracellular concentration of
free (non-enzyme bound) cytosolic ThDP of around 3 μM [ 4 ]
. This suggests that the high affinity uptake system is the
physiologically relevant mechanism responsible for ThDP
entry into mitochondria. Earlier work with rat liver
mitochondria identified a ThDP uptake system with an
estimated K
m of around 20 μM [ 14 ] . This is of
the same order of magnitude that we find for the low
affinity component in the human cells examined herein. The
previous work used a less sensitive procedure of examining
ThDP uptake and did not examine uptake below 10 μM. This
would explain the lack of identification in the previous
work of the high affinity uptake component.
There is a high degree of amino acid similarity among
folate transporters and the thiamine transporter of the
plasma membrane [ 7 8 9 10 ] . Recently, it was found that
in murine cells there can be a substantial efflux of ThDP
mediated by the reduced folate carrier protein [ 22 ] .
GlyB cells are a Chinese hamster
ovary cell line derivative that are deficient in the
transport of folates into mitochondria, and the responsible
mitochondrial transporter has recently been identified and
its gene cloned [ 23 ] . We wondered if the mitochondrial
folate transporter was responsible for uptake of ThDP into
mitochondria, making analogies to the ability of the plasma
membrane folate transporter being able to transport ThDP.
However, this is not the case as
glyB cells that lack the
mitochondrial folate transporter were found to take up ThDP
with high affinity kinetics similar to that of mitochondria
of human cells.
Mitochondria from three human cell types - lymphoblasts,
fibroblasts, and neruoblastoma cells - all possessed a high
affinity ThDP uptake component characterized by equivalent
apparent affinity for ThDP as revealed by essentially
identical K
m values. Previous studies indicate the
existence of a complex, cell-type dependent regulation of
compartmentalization and intracellular pools of thiamine
and/or its phosphorylated derivatives in response to
fluctuating extracellular thiamine levels [ 4 6 17 ] . The
cell lines used here were also used in the studies leading
to this conclusion. Clearly, differences in mitochondrial
transporter affinities do not contribute to the cell-type
dependent regulation of ThDP compartmentalization. However,
we did find significant differences in ThDP uptake capacity
with respect to cell type as revealed by variation in the V
max values. Neuroblastoma mitochondria
possessed the largest uptake capacity, having a V
max 4 to 30 fold higher than that of the
other cell types examined. Interestingly, of the three cell
types neuroblastoma cells also are the most resistant to
changes in mitochondrial ThDP-utilizing enzyme activity
upon progressive depletion of the thiamine made available
to the cells [ 6 ] . This suggests that cell-dependent
variation in ThDP uptake capacity by mitochondria may
contribute to the cell-dependent regulation of ThDP
compartmentalization. As such regulation was most clearly
revealed upon progressively depleting thiamine from cells [
6 ] , it will be of interest to examine possible changes in
mitochondrial transport capacity in response to thiamine
depletion.
Studies on the sensitivity of ThDP-utilizing enzymes to
progressive depletion of thiamine that is available to the
cell indicate that such enzymes in mitochondria are
significantly less sensitive than cytosolic enzymes in TRMA
cells [ 6 ] . This could be interpreted as efficient import
of thiamine/ThDP into mitochondria in TRMA cells even
though thiamine inefficiently enters these cells due to the
lack of the high affinity thiamine transporter. Although we
found a lack of mitochondrial uptake of thiamine in TRMA
cells, our finding of an intact, high affinity ThDP
transport mechanism for TRMA mitochondria is consistent
with and offers an explanation for such an
interpretation.
Materials and Methods
Radiochemicals
[ 3H]thiamine (1 Ci/mmol, radiochemical purity greater
than 97%) and [ 3H]thiamine diphosphate (1.4 Ci/mmol,
radiochemical purity greater than 98 %) were purchased
from Moravek Biochemical Inc (Brea, CA).
Cell culture
Normal lymphoblasts, TRMA lymphoblasts and fibroblasts
cell lines were obtained and have been characterized as
described [ 6 ] . The human neuroblastoma cells were an
SY-SY5Y cell line, a thrice-cloned subline of SK-N-SH [
24 ] .
GlyB cells, a Chinese hamster ovary
K1 subline, are deficient in the transport of folate into
mitochondria [ 23 ] and were a kind gift from L. Chasin
(Columbia University). All cell types were growth at 37°C
in the presence of 10 μM thiamine in RPMI 1640 medium
supplemented with 10% heat-inactive fetal calf serum, 2
mM L-glutamine and 1 g/L penicillin/streptomycin, except
glyB cells which were grown in MEM
medium. Cells were grown and used at late log phase or at
80-90% confluency.
Cellular thiamine transport
Cells were harvested, washed four times with 40 ml of
ice-cold transport buffer (145 mM NaCl, 1 mM MgCl
2 , 1 mM CaCl
2 , 10 mM glucose, 10 mM HEPES, pH
7.4), titered, and preincubated for 30 min. at 37°C after
resuspending 3 × 10 7cells in 1 ml of transport buffer.
Various amounts of [ 3H]thiamine (to give submicromolar
and micromolar final concentrations) were added and the
reactions were incubated for 30 min. The specific
activity of the radioactive thiamine was the same for
each concentration used. The cells were collected by
rapid filtration onto glass fiber filters (type A/E,
Gelman Sciences, Ann Arbor, MI) and washed via filtration
with 10 ml of cold transport buffer. After thorough
drying (overnight at 60°C), the amount of labeled
thiamine taken up by the cells was determined by
scintillation counting [ 25 ] . For each concentration,
the uptake in the presence of a 100-fold excess of
unlabelled thiamine was performed to assess the
contribution to uptake from a low affinity (K
m in the mM range) component [ 3 ]
and/or from diffusion [ 20 ] .
Isolation of mitochondria
Mitochondria were isolated from about 3 × 10 8cells
according to published procedures [ 26 27 ] . The final
mitochondrial pellet was suspended in suspension buffer
(140 mM KCl, 0.3 mM EDTA, 5 mM MgCl
2 , 10 mM HEPES, pH 7.4) to give a
protein concentration of 3-4 mg/ml. Protein concentration
was determined using the Bio-Rad DC Protein Assay Kit
(Bio-Rad Laboratories, Hercules, CA). The isolation and
purity of the mitochondrial preparations were monitored
by western analysis using anti-cytochrome C antiserum and
by microscopy. Western analysis also was performed on the
subcellular fractions using antiserum raised against a
human thiamine transporter-specific peptide. The
antiserum detects a protein of 55 KD (predicted size of
the plasma membrane thiamine transporter) that is not
detected in cells from TRMA individuals that possess a
premature stop codon within the transporter gene
(unpublished results).
Uptake of thiamine and ThDP by mitochondria
Uptake of thiamine and ThDP by mitochondria was
determined by a rapid filtration procedure [ 26 28 ] .
Incubations were performed at 37°C by rapidly mixing 30
μl of mitochondrial suspension (ca. 100 micrograms of
protein) with 220 μl of incubation buffer (140 mM KCl,
0.3 mM EDTA, 5 mM MgCl
2 , 10 mM Mes, pH 6.5) containing
labeled thiamine or ThDP at various concentrations. The
uptake was stopped at 15 min. by the addition of 2 ml of
ice-cold stop buffer (100 mM KCl, 100 mM mannitol, 10 mM
potassium phosphate, pH 7.4) and the mitochondria were
collected by rapid filtration on 0.45 μM Millipore
membrane filters. The filters were immediately washed
with 5 ml of stop buffer via filtration, and they were
subsequently dried at 60°C overnight. The amount of
labeled thiamine or ThDP taken up by the mitochondria was
determined by scintillation counting. Background binding
was determined by using a 100 fold excess of unlabelled
thiamine or ThDP in parallel reactions.
Authors' Contributions
QS carried out most of the experiments and participated
in writing the manuscript. CKS conceived of the study,
participated in its design and coordination, performed a
few of the thiamine uptake by cells experiments, and
participated in writing the manuscript.
Abbreviations
thiamine diphosphate (ThDP); thiamine-responsive
megaloblastic anemia (TRMA)
