Biotin uptake into human peripheral blood mononuclear cells increases early in the cell cycle, increasing carboxylase activities

Abstract

Cells respond to proliferation with increased accumulation of biotin, suggesting that proliferation enhances biotin demand. Here we determined whether peripheral blood mononuclear cells (PBMC) increase biotin uptake at specific phases of the cell cycle, and whether biotin is utilized to increase biotinylation of carboxylases. Biotin uptake was quantified in human PBMC that were arrested chemically at specific phases of the cell cycle, i.e., biotin uptake increased in the G1 phase of the cycle [658 +/- 574 amol biotin/(10(6) cells x 30 min)] and remained increased during phases S, G2, and M compared with quiescent controls [200 +/- 62 amol biotin/(10(6) cells x 30 min)]. The abundance of the sodium-dependent multivitamin transporter (SMVT, which transports biotin) was similar at all phases of the cell cycle, suggesting that transporters other than SMVT or splicing variants of SMVT may account for the increased biotin uptake observed in proliferating cells. Activities of biotin-dependent 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase were up to two times greater in proliferating PBMC compared with controls. The abundance of mRNA encoding 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase paralleled carboxylase activities, suggesting that PBMC respond to proliferation with increased expression of genes encoding carboxylases. Similarly, expression of the gene encoding holocarboxylase synthetase (which catalyzes binding of biotin to carboxylases) increased in response to proliferation, suggesting that cellular capacity to biotinylate carboxylases was increased. In summary, these findings suggest that PBMC respond to proliferation with increased biotin uptake early in the cell cycle, and that biotin is utilized to increase activities of two of the four biotin-requiring carboxylases.

FIGURE 1

Uptake of biotin into proliferating human peripheral blood mononuclear cells (PBMC) increased early in the cell cycle. PBMC were isolated from healthy adults and cultured with 20 mg/L concanavalin A for 30 h to induce proliferation; controls were cultured without concanavalin A. Next, proliferating PBMC were arrested chemically at various phases of the cell cycle (see text) and rates of biotin transport were determined. Data are expressed as means ± sd, n = 5. *Different from quiescent controls, P < 0.01.

FIGURE 2

Expression of the sodium-dependent multivitamin transporter (SMVT) at various phases of the cell cycle in human peripheral blood mononuclear cells (PBMC). Cells were isolated from healthy adults and cultured with 20 mg/L concanavalin A for 30 h to induce proliferation; controls were cultured without concanavalin A. Next, proliferating PBMC were arrested chemically at various phases of the cell cycle (see text). Panel A: The abundance of mRNA encoding SMVT was determined by polymerase chain reaction and gel densitometry. Data are expressed as means ± sd, n = 5. *,**Different from quiescent controls,*P < 0.05, **P < 0.01. Inserts show representative examples of mRNA encoding SMVT and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Panel B: The abundance of SMVT protein was determined in PBMC at various phases of the cell cycle by using an antibody to human SMVT.

FIGURE 3

Activity of 3-methylcrotonyl-CoA carboxylase (MCC) in cultured human peripheral blood mononuclear cells (PBMC). Cells were isolated from healthy adults and cultured in mitogen-containing medium (2.0 mg/L pokeweed lectin; 20 mg/L concanavalin A) for up to 3 d (d 0 = before addition of mitogen). At timed intervals, aliquots were collected to measure MCC activity. Data are expressed as means ± sd, n = 7. *Different from unstimulated cells on d 0, P < 0.01.

FIGURE 4

Activity of propionyl-CoA carboxylase (PCC) in cultured human peripheral blood mononuclear cells (PBMC). Cells were isolated from healthy adults and cultured in mitogen-containing medium (2.0 mg/L pokeweed lectin; 20 mg/L concanavalin A) for up to 3 d (d 0 = before addition of mitogen). At timed intervals, aliquots were collected to measure PCC activity. Data are expressed as means ± sd, n = 7. *Different from unstimulated cells on d 0, P < 0.05.

FIGURE 5

Uptake of [³H]thymidine into cultured human peripheral blood mononuclear cells (PBMC). Cells were isolated from healthy adults and cultured in mitogen-containing medium (2.0 mg/L pokeweed lectin; 20 mg/L concanavalin A) for up to 3 d (d 0 = before addition of concanavalin A). At timed intervals, aliquots were collected to measure [³H]thymidine uptake. Data are expressed as means ± sd, n = 7. Different from unstimulated cells on d 0, *P < 0.05; **P < 0.01.

FIGURE 6

The abundance of mRNA encoding 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and holocarboxylase synthetase in cultured human peripheral blood mononuclear cells (PBMC). Cells were isolated from healthy adults and cultured in mitogen-containing medium (2.0 mg/L pokeweed lectin) for 3 d to induce proliferation; controls were cultured without pokeweed lectin. The abundance of mRNA was determined by polymerase chain reaction; each data point is a single measure. Upper panel: 3-methylcrotonyl-CoA carboxylase; middle panel: propionyl-CoA carboxylase; lower panel: holocarboxylase synthetase. P, pokeweed lectin; C, control.