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Department of Pediatrics, University of Arkansas for Medical Sciences/Arkansas Children’s Hospital Research Institute, Little Rock, AR 72202
3To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT Metabolism of macronutrients in...Mitogenic stimulation of...Activities of biotin-dependent...REFERENCES |
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KEY WORDS: • biotin • carboxylases • humans • lymphocytes • transport
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Metabolism of macronutrients in proliferating lymphocytes |
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TOPABSTRACTMetabolism of macronutrients in...Mitogenic stimulation of...Activities of biotin-dependent...REFERENCES |
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), and the rate of catabolism of glucose (Loos and Roos 1973
, Roos et al. 1972
, Roos and Loos 1973
) increase in response to mitogenic stimulation. These
findings are consistent with the hypothesis that macronutrient demand
varies during the cell cycle.
Clearly, sufficient supplies of these macronutrients (carbohydrates,
amino acids and fatty acids) are required to maintain normal growth and
function of lymphocytes such as mitosis (Lernhardt 1990
,
Loos and Roos 1973
, Waithe et al. 1975
).
Similarly, the micronutrient linoleic acid is required to support
maturation of B lymphocytes to immunoglobulin-secreting cells
(Lernhardt 1990
). In these studies, we sought to
determine whether proliferating lymphocytes exhibit changes in the
transport or utilization of the micronutrient biotin.
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Mitogenic stimulation of lymphocytes causes an increased biotin uptake |
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TOPABSTRACTMetabolism of macronutrients in...Mitogenic stimulation of...Activities of biotin-dependent...REFERENCES |
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). Lipoic
acid, hexanoic acid and bilirubin in the medium did not compete with
biotin for uptake. Biotin transport into nonproliferating lymphocytes
requires metabolic energy and is driven by cotransport of biotin and
sodium.
In the studies presented below, lymphocytes were isolated from
peripheral human blood by gradient centrifugation; proliferation was
stimulated by incubation with mitogens for designated periods of time,
i.e., pokeweed lectin (0.5–2.0 µg/mL), concanavalin A
(5–20 µg/mL) or phytohemagglutinin (5–20
µg/mL) (Zempleni and Mock 1999
).
Proliferation was confirmed by determination of cellular
[3H]thymidine uptake (Zempleni and Mock 1999
); lymphocytes that had been stimulated with mitogens for
3 d accumulated at least 20 times more
[3H]thymidine than unstimulated controls
(P < 0.01). This stimulating index is similar to that
noted by Stites (1987)
.
Then, biotin uptake was quantitated by determination of
[3H] uptake into the lymphocytes during
incubation with [3H]biotin after establishing
that [3H]biotin is not metabolized within the
lymphocytes during the incubation period (<5%) and that
[3H]biotin does not adhere nonspecifically to
lymphocytes (Zempleni and Mock 1998
).
Initially, we sought to determine whether proliferating lymphocytes
exhibit changes in the transport rate of biotin (Zempleni and Mock 1999
). We induced proliferation by incubation with
pokeweed lectin, concanavalin A or phytohemagglutinin for intervals
that varied between 24 and 168 h. Transport rates of
[3H]biotin were determined in both stimulated
lymphocytes and unstimulated controls. When the medium contained 475
pmol/L [3H]biotin (a physiologic
concentration), nonproliferating controls accumulated 406 ± 201
amol [3H]biotin/(106
cells·30 min). In lymphocytes that had been stimulated with mitogens
for 72 h, biotin uptake increased to 278–722% of the control
values for nonproliferating lymphocytes. The percentage of increase of
[3H]biotin uptake was similar among the three
mitogens tested. Activation of biotin transport was maximal at 48–72 h
(Zempleni and Mock 1999
).
The increased biotin uptake into proliferating lymphocytes was not
associated with a global up-regulation of transport activities.
This conclusion is based on the observation that uptake rates of urea
were similar in mitogen-stimulated, proliferating lymphocytes and
unstimulated controls (Zempleni and Mock 1999
).
Next we sought to characterize the mechanisms that mediate the increase
in biotin uptake (Zempleni and Mock 1999
). We
hypothesized that the increase was caused either by an increased number
of one or more types of biotin transporters per cell or by an increase
in the biotin affinity of one or more biotin transporters (or both).
Kinetic studies were performed in lymphocytes induced by pokeweed
lectin for 3 d; controls were not exposed to pokeweed. Biotin
uptake was measured at several distinct biotin concentrations varying
from 238 to 2850 pmol/L. Lineweaver-Burk plots constructed from
these data revealed intersection near the x-axis for the
regression lines of proliferating and nonproliferating lymphocytes;
this observation provides evidence that the increase in biotin uptake
is mediated by an increase in the number of biotin transporters per
cell rather than by an increase in transporter affinity for biotin. The
kinetic parameters derived from the Lineweaver-Burk plot are
consistent with this interpretation. The maximal transport rates
(Vmax) of the biotin transporter in
proliferating lymphocytes and in controls were 9.1 ± 6.6 and 2.3
± 1.6 fmol/(106 cells·30 min),
respectively (P < 0.05). The Michaelis constants
(Km) were 2.4 ± 1.7 and 3.7
± 3.4 nmol/L, respectively, in proliferating lymphocytes and in
controls (P = 0.86, not significant).
Moreover, mitogenic stimulation of biotin transport was inhibited
completely by the addition of cycloheximide, an inhibitor of protein
synthesis, to the medium (Zempleni and Mock 1999
). This
observation is consistent with the hypothesis that proliferating
lymphocytes increase biotin uptake by increasing synthesis of the
biotin transporter.
Theoretically, the increase in biotin uptake might have been mediated
by increased synthesis of a different biotin transporter than the one
responsible for transport in nonproliferating lymphocytes
(Zempleni and Mock 1998
). Other biotin transporters have
been identified in other tissues. For example, rat placenta transports
biotin by using a relatively unspecific biotin/lipoic acid/pantothenic
acid transporter (Prasad et al. 1998
). Potentially,
proliferating lymphocytes might synthesize this "multivitamin"
transporter at an increased rate rather than the specific biotin
transporter. To investigate this possibility, we examined the effect of
lipoic acid on biotin transport in proliferating and nonproliferating
lymphocytes. Extracellular lipoic acid did not significantly reduce
[3H]biotin uptake into proliferating
lymphocytes or nonproliferating lymphocytes at either physiologic (50
nmol/L) or pharmacologic (5000 nmol/L) concentrations of lipoic acid.
These data provide evidence that the primary transporter of biotin is
the same structurally specific transporter in both proliferating and
nonproliferating lymphocytes.
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Activities of biotin-dependent carboxylases are increased in mitogen-stimulated lymphocytes |
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TOPABSTRACTMetabolism of macronutrients in...Mitogenic stimulation of...Activities of biotin-dependent...REFERENCES |
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, Hymes and Wolf 1999
).
Biotinidase is ubiquitous in mammalian cells and 25% of the cellular
biotinidase activity is located in the nuclear fraction (Pispa 1965
). Biotinylation of histones suggests that biotin might
play some role in DNA transcription and replication. In analogy,
transcription and replication of DNA are affected by acetylation,
methylation phosphorylation or ADP-ribosylation of histones
(Ausio and van Holde 1986
, Boulikas 1988
,
Boulikas et al. 1990
, Hebbes et al. 1988
,
Hohmann 1983
, Kaye and Sheratzky 1969
,
Langan 1970
, Lee et al. 1993
, Paik and Kim 1969
, Roth and Allis 1992
,
Sommerville et al. 1993
). 3) Proliferating
lymphocytes increase biotin uptake to sequester plasma biotin from
invading microorganisms. By analogy, an antimicrobial effect of biotin
withdrawal has been proposed to operate in eggs. Egg white contains the
protein avidin (0.05% of total protein), which binds biotin tightly;
the dissociation constant of the biotin-avidin complex is
10-15 mol/L (Green 1975
). The
tight binding of biotin to avidin prevents diffusion of biotin to the
microbes, and avidin is resistant to a variety of proteases (thus
preventing microbes from releasing biotin from avidin).
We have conducted preliminary studies regarding the effect of
lymphocyte proliferation on activities of biotin-dependent
ß-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) and propionyl-CoA
carboxylase (EC 6.4.1.3) (Zempleni, J. and Mock, D.M., unpublished
observations). In these studies, we stimulated proliferation of
lymphocytes by incubation with either pokeweed lectin (2.0
µg/mL) or concanavalin A (20 µg/mL) for up to
144 h; controls were incubated without mitogens. At timed
intervals, aliquots were collected. Activities of
ß-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase
were assayed using a method based on the incorporation of radiolabeled
bicarbonate into the product of the carboxylase reactions
(Zempleni et al. 1997
).
The activity of ß-methylcrotonyl-CoA carboxylase increased with proliferation, reaching a maximal increase 72 h after addition of either mitogen; at that time, ß-methylcrotonyl-CoA carboxylase activity was ~2.5 times greater than at time 0, i.e., before addition of mitogens to the medium (P < 0.05). At 144 h, ß-methylcrotonyl-CoA carboxylase activity in mitogen-stimulated lymphocytes was not significantly different from time 0 (P > 0.05).
Similarly, the activity of propionyl-CoA carboxylase increased by 10–50% with proliferation; activity reached a maximum at 72 h. The increase did not quite reach statistical significance (P = 0.06).
These pilot studies of carboxylase activities suggest that proliferating lymphocytes increase biotin uptake to provide coenzyme for biotin-dependent carboxylases.
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FOOTNOTES |
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2 Supported by National Institutes of Health grant DK 36823 (to D.M.M.).
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REFERENCES |
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TOPABSTRACTMetabolism of macronutrients in...Mitogenic stimulation of...Activities of biotin-dependent...REFERENCES |
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26.
Zempleni J., Trusty T. A., Mock D. M. Lipoic acid reduces the activities of biotin-dependent carboxylases in rat liver. J. Nutr. 1997;127:1776-1781
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