Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely (P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.     

Metabolism of biotin and analogues of biotin by microorganisms. II. Further studies on the conversion of D-biotin to biotin vitamers by Lactobacillus plantarum

Birnbaum, Jerome (University of Cincinnati, Cinncinati, Ohio), and Herman C. Lichstein. Metabolism of biotin and analogues of biotin by microorganisms. II. Further studies on the conversion of d-biotin to biotin vitamers by Lactobacillus plantarum. J. Bacteriol. 92:913-919. 1966.-Lactobacillus plantarum growing in excess biotin converts a portion to two vitamers (combinable and uncombinable with avidin) not utilizable for growth. These were detected by differential yeast-lactobacillus assay. In the present study, suspensions of 12- and 72-hr cells showed no converting activity. Vitamer formation by nonproliferating 24-hr cells required glucose and exhibited a lag; 17-hr cells showed neither a lag nor a glucose requirement. Iodoacetate and chloramphenicol inhibited vitamer formation by 24-hr cells, but had no effect on 17-hr cells. Addition of hydrolyzed casein or preincubation in biotin decreased the lag and enhanced vitamer formation in 24-hr cells, but had no effect in 17-hr cells. Apparently, 17-hr cells contain the converting enzymes which degenerate as growth proceeds; the lag exhibited by 24-hr cells represents the time necessary to reform the enzymes. Equal amounts of the two vitamers were formed in 17-hr cells; only the avidin-combinable form was produced initially by 24-hr cells, unless hydrolyzed casein was present. Electrophoresis revealed that the avidin-combinable vitamer has the same charge as biotin,whereas the uncombinable form possesses both positive and negative groups. Column chromatography was used to separate the avidin uncombinable material from biotin and the avidin-combinable form. L. plantarum was unable to accumulate the avidin-uncombinable vitamer under conditions permitting good biotin accumulation. It was concluded that L. plantarum sequentially converts biotin to avidin-combinable and -uncombinable vitamers, the latter being impermeable to the cells.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

Brev. lactofermentum rapidly took up biotin from culture medium and stored it in the cells. The saturation level of the stored biotin (3.8 × 10⁴ molecules/cell) exceeded the level required for the maximum growth by ten times, and the minimum level (1.3 × 10³ molecules/cell) was the most adequate to the accumulation of l-glutamic acid. The stored cellular biotin over the minimum level was metabolically available in the subsequent culture lacking in supplemented biotin. The cellular biotin was gradually reduced to the minimum level with the multiplication of the cells, and them the accumulation of l-glutamic acid was observed. This relation between the level of cellular biotin and the accumulation of l-glutamic acid was impaired by the addition of Tween 60 or some saturated fatty acid. In the presence of biotin and Tween 60 the biotin-saturated cells turned into cells capable of accumulating l-glutamic acid keeping the maximum level; and in the same medium the cells having the minimum amount of biotin took up biotin and then were saturated with it, and yet the cells preserved the acid-accumulating property. It was confirmed with the use of bioautographic technique and avidin test that the biotin released from the cells by acid hydrolysis was identical with authentic d-biotin.     

Human peripheral blood mononuclear cells: ; Inhibition of biotin transport by reversible competition with pantothenic acid is quantitatively minor

A transporter present in intestinal cells and in choriocarcinoma cells has been shown to transport both pantothenic acid and biotin at similar transporter affinities. However, the concentration of pantothenic acid in most foods and biological fluids is approximately 200 times the concentration of biotin; theoretically, pantothenic acid might substantially reduce biotin transport via competition. In the present study, we sought to determine whether pantothenic acid reduces biotin transport by the biotin transporter in peripheral blood mononuclear cells (PBMC). PBMC were isolated from human blood by gradient centrifugation. Incubations with [(3)H]biotin and pantothenic acid were conducted at physiologic concentrations. Intracellular [(3)H]biotin was quantified after washing by liquid scintillation counting. Pantothenic acid at 10 to 1,000 nmol/L reduced biotin (475 pmol/L) uptake by less than 12% (P < 0.05). Based on Lineweaver-Burk plots, the competition was reversible. Several structural analogs of pantothenic acid at 1,000 nmol/L reduced biotin transport by only 7 to 15% (P = 0.13). No pattern of molecular structure required for recognition by the transporter was apparent. Extracellular pantothenic acid did not affect biotin efflux from [(3)H]biotin-loaded PBMC (P > 0.05), suggesting that countertransport of extracellular pantothenic acid and intracellular biotin does not increase biotin efflux from PBMC. We conclude that the physiologic effects of pantothenic acid on the transport of biotin in PBMC are likely to be quantitatively minor.     

Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.     

Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum

Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immunohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the Escherichia coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In the other system, biotin ligase was engineered to be retained in the endoplasmic reticulum (ER) and metabolically biotinylates the secretory protein as it passes through the ER. An engineered antibody fragment, a diabody with specificity for carcinoembryonic antigen (CEA) was fused to the biotin acceptor domain (123 amino acid) of Propionibacterium shermanii. Coexpression of the fusion protein with ER retained biotin ligase showed higher biotinylation efficiency than biotinylation by co-secreted ligase. Biotinylation of the anti-CEA diabody tagged with a short (15 amino acid, Biotin Avitag) biotin acceptor peptide was also successful. Utilization of ER retained biotin ligase for biotinylation of protein is an attractive alternative for efficiently producing uniformly biotinylated recombinant proteins for a variety of avidin-biotin technologies.     

Characterization of the biotin transport system in Saccharomyces cerevisiae

The characteristics of the biotin transport mechanism of Saccharomyces cerevisiae were investigated in nonproliferating cells. Microbiological and radioisotope assays were employed to measure biotin uptake. The vitamin existed intracellularly in both free and bound forms. Free biotin was extracted by boiling water. Chromatography of the free extract showed it to consist entirely of d-biotin. Cellular bound biotin was released by treating cells with 6 n H(2)SO(4). The rate of biotin uptake was linear with time for 10 min, reaching a maximum at about 20 min followed by a gradual loss of accumulated free vitamin from the cells. Biotin was not degraded or converted to vitamers during uptake. Transport was temperature- and pH-dependent, optimum conditions for uptake being 30 C and pH 4.0. Glucose markedly stimulated biotin transport. In its presence, large intracellular free-biotin concentration gradients were established. Iodoacetate inhibited the glucose stimulation of biotin uptake. The rate of vitamin transport increased in a linear fashion with increasing cell mass. The transport system was saturated with increasing concentrations of the vitamin. The apparent K(m) for uptake was 3.23 x 10(-7)m. Uptake of radioactive biotin was inhibited by unlabeled biotin and a number of analogues including homobiotin, desthiobiotin, oxybiotin, norbiotin, and biotin sulfone. Proline, hydroxyproline, and 7,8-diaminopelargonic acid did not inhibit uptake. Unlabeled biotin and desthiobiotin exchanged with accumulated intracellular (14)C-biotin, whereas hydroxyproline did not.     

Regulation of Biotin Transport in Saccharomyces cerevisiae

The metabolic control of biotin transport in Saccharomyces cerevisiae was investigated. Nonproliferating cells harvested from cultures grown in excess biotin (25 ng/ml) took up small amounts of biotin, whereas cells grown in biotin-sufficient medium (0.25 ng/ml) accumulated large amounts of the vitamin. Transport was inhibited maximally in cells grown in medium containing 9 ng (or more) of biotin per ml. When avidin was added to biotin-excess cultures, the cells developed the ability to take up large amounts of biotin. Boiled avidin was without effect, as was treatment of cells with avidin in buffer. Avidin did not relieve transport inhibition when added to biotin-excess cultures treated with cycloheximide, suggesting that protein synthesis was required for cells to develop the capacity to take up biotin after removal of extracellular vitamin by avidin. Cycloheximide did not inhibit the activity of the preformed transport system in biotin-sufficient cells. The presence of high intracellular free biotin pools did not inhibit the activity of the transport system. The characteristics of transport in biotin-excess cells (absence of temperature or pH dependence, no stimulation by glucose, absence of iodoacetate inhibition, independence of uptake on cell concentration, and nonsaturation kinetics) indicated that biotin entered these cells by diffusion. The results suggest that the synthesis of the biotin transport system in S. cerevisiae may be repressed during growth in medium containing high concentrations of biotin.     

Uptake of extracellular biotin by Escherichia coli biotin prototrophs.

Uptake of exogenous biotin by two Escherichia coli biotin prototroph strains, K-12 and Crookes, appeared to involve incorporation at a fixed number of binding sites located at the cell membrane. Incorporation was characterized as a binding process specific for biotin, not requiring energy, and stimulated by acidic pH. Constant saturation quantities of exogenous biotin were incorporated by these cells, and the amounts, which were titrated, depended on whether the cells were resting or dividing. Resting cells incorporated exogenous biotin amounting to 2% of their total intracellular biotin content. Fifty percent of the exogenous biotin was incorporated into their free biotin fraction, and 50% was incorporated into their bound biotin fraction. On the other hand, dividing cells incorporated exogenous biotin into all of their intracellular sites, 88% going into the intracellular-bound biotin fraction, and 12% going into the free biotin fraction. Calculations suggested that each cell contained approximately 3,000 binding sites for biotin. It was postulated that biotin incorporation sites might have been components of acetyl coenzyme A carboxylase located at or near the membrane.     

The biotin requirement of human fibroblasts in culture

We have examined the effect of biotin deficiency on the growth, viability, biotin content, and the activities of biotin-dependent and biotin-independent enzymes of human fibroblasts. There was a significant decrease in viability of the biotin-deficient cells even when the medium contained serum lipids. Propionyl CoA carboxylase activity reflected the decreased biotin content of the cells whereas alkaline phosphatase activity was not altered. The inclusion of avidin bound biotin in the growth medium resulted in an increase in biotin content as well as propionyl CoA carboxylase activity over that seen when free biotin was included in the medium. The cells appeared to bind and internalize the avidin-biotin complex by adsorptive pinocytosis. These findings are similar to those demonstrated using HeLa cells.     

Biotin Uptake by Cold-Shocked Cells, Spheroplasts, and Repressed Cells of Saccharomyces cerevisiae: Lack of Feedback Control

Cold-osmotic-shocked cells and spheroplasts of Saccharomyces cerevisiae (ATCC 9896) display a biotin uptake system similar to that observed in intact cells. 2-Mercaptoethanol was found to inhibit biotin transport. Cells repressed for biotin uptake by growth in excess biotin (25 ng/ml) possess an energy-dependent transport system that has a K(m) for biotin of 6.6 x 10(-7) M and a V(max) equal to 39 pmol per mg (dry weight) per min. A similar K(m) (6.4 x 10(-7) M) but a considerably higher V(max) (530 pmol per mg (dry weight) per min) was determined for biotin uptake by cells grown in sufficient biotin (0.25 ng/ml). The V(max) rates of biotin uptake by both repressed and derepressed cells were increased approximately 35-fold in the presence of glucose. These yeast cells appear to regulate their biotin uptake by two mechanisms. An exit system provides for immediate adjustments, whereas turnover of the transport system and repression of new synthesis establishes a slower adaptation to changes in the environment. Feedback inhibition was ruled out as a mechanism of regulation of transport.     

The efflux of biotin from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PMBCs) are readily available for sampling and are a useful model for studying biotin metabolism in human cells. To better understand biotin handling by PMBCs, we investigated the mechanism(s) and kinetics of biotin efflux from PMBCs. Human PMBCs were incubated with [(3)H]biotin at 475 pmol/L to load the cells. The [(3)H]biotin-loaded cells were then harvested and incubated in [(3)H]biotin-free media for up to 20 hours. At various intervals, aliquots of the PMBC suspensions were collected and analyzed for intracellular [(3)H]biotin. [(3)H]Biotin efflux from cells at 37 degrees C was fast and triphasic; the half-lives for the three elimination phases were 0.2 +/- 0.02 hours, 1.2 +/- 0.1 hours, and 21.9 +/- 13.6 hours. Such a triphasic [(3)H]biotin efflux could reflect (1) rapid efflux of free biotin, (2) slower release of biotin bound to intracellular molecules, and (3) even slower release from carboxylases in cellular organelles. Incubation at 4 degrees C rather than 37 degrees C increased the [(3)H]biotin retained at 20 hours from 27% to 85%. This observation is consistent with transporter-mediated efflux. When cellular glucose utilization was reduced by 2-deoxy-d-glucose and sodium fluoride, [(3)H]biotin efflux was similar to controls, suggesting that biotin efflux does not directly require metabolic energy. When [(3)H]biotin-loaded cells were incubated in external medium containing unlabeled biotin analogs, [(3)H]biotin efflux was accelerated approximately two times compared with incubation in a biotin-free medium. This observation suggests that biotin efflux is mediated by the same transporter that mediates biotin uptake from the extracellular medium (i.e., classic countertransport).     

Electrogenic nature of rat sodium-dependent multivitamin transport

We report on the electrogenic nature of the transport process mediated by the rat sodium-dependent multivitamin transporter. In Cos-7 cells, the relationship of Na(+) concentration versus biotin and pantothenate uptake rate was sigmoidal with a Na(+):substrate stoichiometry of 2:1. In Cos-7 cells expressing rat SMVT biotin transport was significantly higher when the membrane was hyperpolarized and considerably reduced when the membrane was depolarized. Similarly, biotin uptake in X. laevis oocytes expressing rat SMVT was inhibited with depolarized oocyte membrane by altering the K(+) permeability across the membrane. It is concluded that the transport of biotin and pantothenate mediated by rat SMVT is electrogenic with a Na(+):substrate coupling ratio of 2:1 and that the transport process is associated with the transfer of one net positive charge across the membrane per transport cycle.     

Biotin uptake, utilization, and efflux in normal and biotin-deficient rat hepatocytes.

Biotin uptake, utilization, and efflux were studied in normal and biotin-deficient cultured rat hepatocytes. Biotin-deficient cells accumulate about 16-fold more biotin than do normal cells when incubated with a physiological concentration of biotin for 24 h. This difference is due to the greater amount of protein-bound biotin relative to free biotin in biotin-deficient hepatocytes, and is attributable to the presence of more apocarboxylases in deficient cells. The rate of biotin uptake and the rate of activation of the carboxylases, acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase, are proportional to the concentration of exogenous biotin. Increases in carboxylase activities are proportional to the concentration of biotin only at exogenous biotin concentrations of less than 410 nM. Concentrations of 410 nM or more biotin increase carboxylase activities to normal or near normal. Biocytin inhibits biotin uptake at very high concentrations, whereas desthiobiotin and lipoic acid have no effect. Biocytin in the medium results in carboxylase activation either intracellularly or extracellularly by conversion to biotin by biotinidase. Investigation of the efflux of biotin from normal and biotin-deficient cells preincubated with the vitamin showed greater retention of biotin by biotin-deficient cells than by normal cells over 24 h. Retention of free biotin is similar in biotin-deficient and normal cells. The greater amount of biotin retained by biotin-deficient cells is accounted for by the greater amount of bound biotin in these cells. These results suggest that the free and bound biotin pools are independently regulated. The ready loss of free biotin from these cells has implications for the treatment of inherited, biotin-responsive carboxylase deficiencies.     

Biotin uptake by isolated rat intestinal cells

Isolated intestinal mucosa cells of rats were used to investigate the intestinal transport of biotin. This method utilizing a double-label isotope technique showed that uptake could not be saturated, even in a wide range of biotin concentrations (0.01-2 microM). A metabolic inhibitor (antimycin A) did not prevent cell uptake of biotin. The transport mechanism was independent of temperature (Q10 = 1.04). When excess biotin was added to the incubation medium, there was no efflux of the vitamin from intestinal cells. The results also showed that the cells did not concentrate the vitamin, regardless of its concentration in the incubation medium. The mechanism of biotin uptake by rat cells at physiological concentrations is thus a passive diffusion phenomenon.     

Receptor Binding Activities of Biotinylated Derivatives of β-Nerve Growth Factor

beta-nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C-bio-NGF) using biotin hydrazide and the coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution, using N-hydroxysuccinimidyl biotin, to produce derivatives with ratios of one, two, and four biotin moieties per NGF subunit (N-bio-NGF). The various biotinylated NGF derivatives were compared with native NGF for their capacity to compete with 125I-NGF for binding to NGF receptors on rat pheochromocytoma (PC12) cells at 4 degrees C. On the basis of such radioreceptor assays, C-bio-NGF was as effective as native NGF in binding to NGF receptors. C-bio-NGF was also as effective as native NGF in promoting neurite outgrowth from PC12 cells. In contrast, N-bio-NGF containing one biotin per NGF subunit was only 28% as active in binding as native NGF. Increasing the biotin:NGF ratio to 2 to 4 further decreased receptor binding to 13% and 6%, respectively, as compared to native NGF. Once bound to cells, C-bio-NGF had the capacity to mediate the specific binding of 125I-streptavidin to PC12 cells. This binding of streptavidin was prevented by excess native NGF and by antiserum to NGF, but not by RNase A, insulin, cytochrome c, or nonimmune serum. In addition, a variant PC12 line lacking functional NGF receptors was not labeled by 125I-streptavidin after prior incubation with C-bio-NGF.     

Active Transport of Biotin in Escherichia coli K-12

The transport of [(14)C]biotin into cells of a biotin prototroph, Escherichia coli K-12 strain Y10-1, was investigated. The vitamin taken up by the cells in this strain existed primarily in the free form. Addition of glucose enhanced the rate of uptake six- to eightfold and the steady level was reached in 2 to 3 min resulting in accumulation of biotin against a concentration gradient. The uptake showed marked dependence on temperature (Q(10), 2.3; optimum, 37 C) and pH (optimum 6.6) and was inhibited by iodoacetate. Energy of activation for glucose-dependent uptake was calculated to be 16,200 cal per mol. The rate of biotin uptake with increasing biotin concentrations showed saturation kinetics with an apparent K(m) and V(max) values of 1.4 x 10(-7) M and 6.6 pmol per mg of dry cells per min respectively. The cells also accumulated biotin against a concentration gradient in the absence of added glucose, although at a much lower rate. This accumulation was much more susceptible to inhibition by azide and uncouplers of oxidative phosphorylation suggesting that the energy source was supplied through the electron-transport chain. Inhibition studies with a number of biotin analogues indicated the requirement for an intact ureido ring. The biotin uptake was inhibited in cells grown in biotin-containing medium and was shown to be the result of repression of the transport system, suggesting the control of the biotin transport.     

Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.     

The biotin requirement of HeLa cells

We have examined the effect of alterations in the biotin content of the medium on the growth, viability, biotin content, and the activities of biotin-dependent and biotin-independent enzymes of the HeLa cells. The inclusion in the growth medium of avidin, which almost irreversibly binds with biotin (K_d, 10^?15 M), results in an increase in cellular biotin content and biotin enzyme activity over that seen when the cells are grown in a biotin-depleted medium. The addition of avidin-bound biotin to the growth medium led to a forty-fold increase in cellular biotin when compared to the inclusion of an equivalent amount of free biotin in the medium. HeLa cells are able to internalize avidin-bound biotin. Biotin is released from this complex to function as the prosthetic group of biotin enzymes. HeLa cells do have a nutritional requirement for biotin.