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Research Communication

Energy Depletion Differently Affects Membrane Transport and Intracellular Metabolism of Riboflavin Taken up by Isolated Rat Enterocytes

Institute of Human Physiology, University of Pavia, 27100 Pavia, Italy, ^a Department of Pharmacology and Physiology, UMDNJ, New Jersey Medical School, Newark, NJ 07103; and ^b Department of General Physiology and Biochemistry, University of Milan, 23100 Milan, Italy

	ABSTRACT

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Isolated rat enterocytes, both normal and those de-energized with rotenone, were used to study the energy dependence of membrane and intracellular intestinal riboflavin transport in vitro. Membrane and intracellular transport were investigated by using short (3 min) and long (20 min) incubation times, respectively. For both types of cells and incubation times, [³H]-riboflavin uptake presented a saturable component prevailing at physiologic intraluminal concentrations. At 3 min incubation, saturable [³H]-riboflavin transport was apparently an energy-independent process with high affinity and low capacity. Values of the saturable component and its apparent constants, K_m and J_max, did not differ in normal and de-energized enterocytes. At 20 min incubation, saturable [³H]-riboflavin transport was a strictly energy-dependent process in which values of the saturable component were significantly greater in normal than in de-energized enterocytes. K_m values did not differ in the two types of cells and were unmodified over 3 min, whereas in normal enterocytes, J_max at 20 min [6.25 ± 0.2 pmol/(mg protein · 20 min)] was significantly greater than at 3 min [2.67 ± 0.33 pmol/(mg protein · 3 min)] and compared with de-energized enterocytes at 20 min [2.54 ± 0.16 pmol/(mg protein · 20 min)]. Both membrane and intracellular events were inhibited by unlabeled riboflavin and analogs, which are good substrates for flavokinase, thus demonstrating the paramount role of this enzyme in riboflavin intestinal transport.

KEY WORDS: • riboflavin • isolated enterocytes • intestinal absorption • energy depletion • rats

	INTRODUCTION

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Different types of absorbing cells have been used to investigate the uptake and the intracellular metabolism of riboflavin (RF).⁵In rat hepatocytes (Aw et al. 1983 ) and renal proximal cells (Bowers-Komro and McCormick 1987 ), guinea pig enterocytes (Hegazy and Schwenk 1983 ), Caco-2 human intestinal epithelial cells (Said and Ma 1994 ) and HK-2 human renal proximal tubule epithelial cells (Kumar et al. 1998 ), RF uptake at physiologic concentrations is temperature and energy dependent, and mainly saturable, whereas the role of Na⁺ is less clear (Aw et al. 1983 , Bowers-Komro and McCormick 1987 , Hegazy and Schwenk 1983 , Kumar et al. 1998 , Said and Ma 1994 ). The intracellular metabolism of RF is characterized by energy-dependent events involving ATP in which RF is transformed into flavin mononucleotide (FMN) by flavokinase and FMN into flavin adenine dinucleotide (FAD) by FAD-synthetase (McCormick and Zhang 1993 ). In isolated rat hepatocytes (Joseph and McCormick 1995 ), isolated guinea pig enterocytes (Hegazy and Schwenk 1983 ), rat renal epithelial cells (Bowers-Komro and McCormick 1987 ), Caco-2 human intestinal epithelial cells (Said and Ma 1994 ) and HK-2 human proximal tubule epithelial cells (Kumar et al. 1998 ), labeled RF has been found, after incubation, to be only partly converted to coenzymes.

In this investigation, isolated enterocytes from the rat small intestine were chosen as a cellular model, allowing parallel study of membrane transport and intracellular metabolism. Short (3-min) and long (20-min) incubation times were used to differentiate membrane from intracellular events (Middleton 1990 ) and normal (NE) as well as rotenone-de-energized (DE) enterocytes (Ricci and Rindi 1992 ) to evaluate the effects of energy depletion. In NE and DE, the time course of [³H]-RF uptake and concentration curves at both incubation times were determined. In addition, the inhibiting power of unlabeled RF and some RF structural analogs on the saturable component of uptake in both NE and DE at short and long incubation times was investigated.

	MATERIALS AND METHODS

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Chemicals.

[³H]-RF (specific activity, 37 GBq/mmol) was purchased from Moravek Biochemicals (Brea, CA). It was periodically analyzed by HPLC and found to be ~95% pure. [¹⁴C]-Carboxyl-dextran (specific activity, 30 MBq/mmol) was from DuPont NEN Research Products, (Boston, MA). Unlabeled RF was obtained from Prodotti Roche (Milan, Italy). Three groups of RF structural analogs were utilized as follows: group 1, modified at the ribityl side chain [5'-deoxy-riboflavin (5'-DORF), 2',3',4',5'-di-O-isopropylidene-riboflavin (DARF) and riboflavin-5'-monosulfate (FMS)]; group 2, modified in C3 of the isoalloxazine moiety [3-methyl riboflavin (3-MRF)]; and group 3, modified in C8 of the isoalloxazine moiety [8-ethoxy-8-demethyl-riboflavin (8-EORF), 8-dimethyl-amino-8-demethyl-riboflavin (roseoflavin; 8-ROF) and 8-chloro-8-demethyl-riboflavin (8-ClRF)]. Analogs were a generous gift, custom-synthesized by Kasai et al. (1988 and 1990). All other chemicals were of analytical grade and supplied by Sigma Chemical (St. Louis, MO) and B.D.H. (Poole, UK).

Animals.

Wistar albino rats of both sexes (300–400 g body weight) of our colony were used throughout. They were fed a standard pellet diet containing 12 mg RF/kg diet (Randoin and Causeret 1947 ), supplied by Laboratorio Dottori Piccioni (Gessate, Italy). All animals were deprived of food for 12 h before the start of the experiments, with free access to water. Animal care was in accordance with NIH guidelines (NRC 1985 ).

Solutions.

Medium A contained the following (mmol/L): 96 NaCl, 1.5 KCl, 27 Na citrate, 0.2 phenyl-methyl-sulfonyl-fluoride, 5.6 K₂HPO₄/ KH₂PO₄, pH 7.3; Medium B contained (mmol/L): 140 NaCl, 1.5 EDTA, 0.5 dithiotreitol, 0.2 phenyl-methyl-sulfonyl-fluoride, 16 K₂HPO₄/ KH₂PO₄, pH 7.3; and Medium C contained (mmol/L): 137 NaCl, 5.2 KCl, 0.6 CaCl₂, 0.8 MgSO₄, 10 D-glucose, 5 glutamine, 0.2 phenyl-methyl-sulfonyl-fluoride, 3 K₂HPO₄/ KH₂PO₄, pH 7.3.

Enterocyte preparation.

Enterocytes were isolated from the small intestine according to Rindi and Laforenza (1997) with minor modifications. For each experiment, one rat was killed by cervical dislocation. The excised entire small intestine was rinsed with oxygenated saline, then filled with and incubated in Medium A at 37°C for 10 min, under oxygenation in a thermostatic shaker (90 oscillations/min). The intraluminal content was discarded and the intestine was filled with and incubated in Medium B, as previously described; this treatment was repeated twice. The intestine was then gently fingered for 2 min and the intraluminal fluid, containing enterocytes, was filtered through 250- and 100-µm mesh nylon filters successively. The filtrate was collected in 50 mL of Medium C; the isolated enterocytes were washed three times with Medium C and centrifuged each time at 50 x g for 2 min. Cellular protein content was measured according to Lowry et al. (1951) with bovine serum albumin as a standard.

Preincubation.

Enterocytes were preincubated differently as follows: 100 µL of final enterocyte suspension (~200 mg protein) was placed in plastic tubes and preincubated at 37°C in a thermostatic shaker (90 oscillations/min) for 10 min using Medium C for NE. Medium C containing 25 µmol/L rotenone was used for DE.

Incubation.

For both NE and DE, incubation at 37°C was started by adding 100 µL of Medium C, containing either 0.25 µmol/L or different final concentrations of [³H]-RF. Incubation was stopped by adding 1 mL ice-cold Medium C; subsequently, cell suspensions were centrifuged according to Kimmich and Randles (1982) . The amount of [³H]-RF taken up by enterocytes was measured radiometrically by using a Packard Tri-Carb 2000 CA Analyzer (Packard Instruments, Downers Grove, IL) and expressed as pmol /mg protein after correction for adherent water. This was evaluated in each experiment by using [¹⁴C]-carboxyl-dextran and separate flasks (Wilson and Treanor 1975 ). The saturable component of RF uptake was determined at each concentration by subtracting nonsaturable uptake (calculated from the slope of the linear portion of the cumulative uptake at high RF concentrations) from the cumulative uptake. Apparent kinetic constants were calculated from the experimental data by a least-squares regression program (GraphPad Prism 2.01 for Windows 3.1, GraphPad Software, San Diego, CA).

Inhibition power of unlabeled RF and structural analogs on the saturable component of [³H]-RF uptake was studied by adding 100 µL cell suspension to 100 µL of Medium C containing the following: 0.25 µmol/L [³H]-RF (control) or 0.25 µmol/L [³H]-RF plus 2.5 µmol/L unlabeled RF or RF structural analogs. Incubation times were 3 or 20 min.

Statistical methods.

All values given are the means of triplicate determinations for each different preparation of isolated enterocytes ± SEM. The significance of differences between means was determined by ANOVA followed by Newman-Keuls Q test using a computerized program (Glantz 1988 ). Significant differences between the means of two samples were accepted at P < 0.05 (Snedecor and Cochran 1967 ).

	RESULTS

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NE viability, metabolic state and transport efficiency, determined by assessing trypan blue exclusion, basal O₂ consumption and ATP content, and valine uptake (Ricci and Rindi 1992 ), showed that they were suitable for transport studies. The addition of 25 µmol/L rotenone (DE) drastically reduced both ATP content and functional properties (see Ricci and Rindi 1992 ). The amount of [³H]-RF in adherent water was ~2% of total radioactivity of the cells. The average value of cell protein content was 2.5 ± 0.15 g/L cellular suspension (mean ± SEM, n = 6).

Effects of energy depletion.

Time course. In NE, uptake was linear for the first 12 min of incubation (r = 0.997, P < 0.03), whereas in DE, uptake was linear up to 6 min (r = 0.987, P < 0.02) and increased slowly thereafter (Fig. 1 ).Moreover, both the line fits of the initial uptake data showed a significant, positive y-intercept, possibly due to a binding process. However, in this study, we have made no correction of the RF uptake values for this binding component because kinetic results obtained with corrected data did not differ significantly. After 6 min of incubation, uptake rates were significantly higher in NE than in DE.

View larger version (17K): [in this window] [in a new window]   Figure 1. Time course of [3H]-riboflavin uptake in isolated rat enterocytes. Values are means ± SEM, n= 5; when not shown, SEM was within the symbol area. [3H]-Riboflavin final concentration in the incubation medium was 0.25 µmol/L. *Significantly different (P < 0.05) from de-energized enterocytes.

After 3 min incubation, the values of cumulative uptake, their two components and the apparent kinetic constants (Table 1 )did not differ in NE and DE. At 0.25 µmol/L [³H]-RF concentration, the saturable component was 75% of the cumulative uptake (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 2. Kinetics of [3H]-riboflavin uptake at the long incubation time (20 min) in isolated rat enterocytes. Incubations were conducted with different initial [3H]-riboflavin concentrations (0.125–3 µmol/L). Values are means, n = 5; SEMwere within 10% of the mean values.

At 3- and 20-min incubation times, unlabeled RF significantly inhibited the saturable component rate of 0.25 µmol/L [³H]-RF uptake in both NE and DE; DARF, FMS and 3-MRF were ineffective (data not shown), whereas 5'-DORF significantly lowered the saturable component of uptake at both incubation times only in NE. 8-EORF, 8-ROF and 8-ClRF behaved like unlabeled RF; in particular, 8-EORF and 8-ROF significantly lowered the saturable component of uptake in NE and DE at both incubation times. Significant reductions were also observed with 8-ClRF except in DE at 3 min incubation (Fig. 3 ).

View larger version (41K): [in this window] [in a new window]   Figure 3. Inhibition of the saturable component of [3H]-riboflavin uptake in isolated rat enterocytes. Unlabeled riboflavin and structural analogs were added to the incubation medium at an initial concentration 10 times higher than that of [3H]-riboflavin (0.25 µmol/L) and incubated for 3 or 20 min. Values are means ± SEM, n = 5. *Significantly different from analogs (P < 0.05). Unlabeled compounds were as follows: A, riboflavin; B, 5'-deoxy-riboflavin; C, 8-ethoxy-8- demethyl-riboflavin; D, 8-dimethyl-amino-8-demethyl-riboflavin; E, 8-chloro-8-demethyl-riboflavin.

	DISCUSSION

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At 3 min incubation time, [³H]-RF uptake can be considered prevalently, but not exclusively energy independent on the following basis: the time course profile (Fig. 1) , the uptake concentration curves, their saturable component (prevailing at low concentrations) (data not shown) and the relevant K_m and J_max values (Table 1) are similar in both NE and DE. In rat intestinal sacs (Middleton 1990 ) and in brush border membrane vesicles (BBMV) (Casirola et al. 1993 ) the saturable components of the initial rate of RF uptake had apparent K_m values ranging from 0.12 and 0.38 µmol/L, similar to those found for NE and DE. However, in isolated enterocytes (Hegazy and Schwenck 1983 ), hepatocytes (Aw et al. 1983 ) and kidney cells (Bowers-Komro and McCormick 1987 ), RF initial uptake was greatly reduced in the presence of metabolic inhibitors, suggesting an energy-dependent process. The disagreement regarding energy dependence at short incubation times can be ascribed to the different experimental conditions used (a different kind of cell and metabolic inhibitor, and the different preincubation time with the metabolic inhibitor).

Over a long incubation time (20 min), [³H]-RF uptake rate was strictly energy dependent because the time course profile, cumulative uptake and saturable component values were consistently significantly higher in NE than in DE (Figs. 1 and 2) . In NE, in particular, the saturable component at 20 min was 63% higher than at 3 min. Because it was only 27% higher than at 3 min in DE, the increase was due mainly to an energy-dependent process involved in the intracellular metabolism of RF (probably enzymatic transformation of RF to FMN and to FAD). K_m values did not differ in NE and DE at 3- or 20-min incubation times (Table 1) , indicating that the affinity of RF for the enterocytic binding sites in membranes and possibly in flavin enzymes was unaffected by either incubation time or energy depletion. In contrast, in NE, the J_max value at 20 min was significantly greater than at 3 min and than that determined in DE. Moreover, in DE, J_max was unmodified by incubation time and was similar to the B_max (maximal binding) value for rat BBMV at equilibrium [1.3 pmol/(mg protein · 20 min); Casirola et al. 1993]. These results support the hypothesis that the rate of [³H]-RF uptake by NE over a long incubation time involves a strictly energy-dependent saturable process of transport, with a significantly higher capacity than in DE.

The measure of the inhibiting power by unlabeled RF and structural analogs of the saturable [³H]-RF uptake in NE and DE provides an insight into the energy dependence of the saturable process. Unlabeled RF and analogs modified at C8 of the isoalloxazine moiety (8-EORF, 8-ROF, 8-ClRF), inhibit the saturable transport of RF in BBMV (Casirola et al. 1994 ) and are good substrates for flavokinase (Kasai et al. 1990 , McCormick 1975 ). Over both incubation times, they significantly reduced the saturable component of [³H]-RF uptake in NE as well as (except 8-ClRF) in DE. This suggests that these compounds inhibited both types of saturable process over both short and long incubation times. The same compounds (except 8-ClRF) also inhibited the saturable component of [³H]-RF uptake in DE at both incubation times. The analogs modified at the ribityl group (DARF, FMS) or at C3 of the isoalloxazine moiety (3-MRF), which do not inhibit RF transport by BBMV (Casirola et al. 1993 ) and are not substrates for flavokinase (Kasai et al. 1990 , McCormick 1975 ), had no effect when taken up by enterocytes. The exception was 5'-DORF, which inhibited the saturable component of [³H]-RF uptake in NE over each incubation time. Two hypotheses can be formulated to explain this behavior. First, 5'-DORF could associate with protein binding sites present inside the enterocyte or in the basolateral, but not in the microvillous membrane. In fact, in intact rat intestinal tissue, 5'-DORF, which is absorbed only via simple diffusion, can interfere with the specific absorption of RF at low concentrations (Kasai et al 1988 ), whereas it does not inhibit RF absorption in rat intestinal BBMV (Casirola et al. 1994 ). The assumption that some form of flavoprotein binding must take place for antagonistic activity to occur, as assumed by Lambooy (1975) , could support this hypothesis. Second, 5'-DORF could also affect the intracellular metabolism of [³H]-RF because a significant inhibition was found only in NE. However 5'-DORF cannot serve as a substrate or even as an effective competitive inhibitor in the subsequent conversion of FMN to FAD as catalyzed by FAD-synthetase. This enzyme, purified from the liver by Oka and McCormick (1987) , has stringent requirements for dianionic charge of the RF 5'-phosphate (Bowers-Komro et al. 1989 ).

In conclusion, these results show that in isolated rat enterocytes, RF uptake is characterized by the presence of a saturable mechanism, which prevails at physiologic intraluminal concentrations (0.125–2 µmol/L) and exhibits high affinity for the RF binding sites that are located in cellular membranes and cytosolic enzymes (flavokinase and FAD-synthetase). In the initial phase (3 min) of uptake, when membrane events prevail, the saturable uptake appears mainly as an energy-independent process with low capacity. In the later phase (20 min), when intracellular metabolic events are predominant, the saturable uptake has an increased capacity (see J_max values). However, this may be significantly reduced by both those structural analogs that are good substrates for flavokinase and by energy depletion, suggesting that it could be due to the flavokinase and/or FAD-synthetase activities. The presence of a saturable mechanism even when cellular metabolism is virtually blocked, as in DE, suggests that the transport across the membrane is an energy-independent process that is due solely to RF binding to the membranes (Casirola et al. 1993 ). Finally, these results show that rat enterocytes can accumulate [³H]-RF because at 20 min incubation, the intracellular content of [³H]-RF in NE and DE was 240 and 70% greater, respectively, than at an external concentration of 0.25 µmol/L (Fig. 1) , assuming an enterocyte water content of 3.7 µL/mg protein (Baur et al 1978 ).

	ACKNOWLEDGMENTS

 
We express our gratitude to Dr. S. Kasai, Osaka City University (Japan), for his invaluable advice.

	FOOTNOTES

 
¹ To whom correspondence and reprint requests should be addressed.

¹ Preliminary data of this research appeared in abstract form [Gastaldi, G., Casirola, D., Ferrari, G., Ricci, V. & Rindi, G. (1993) Riboflavin uptake by isolated enterocytes: effect of energy depletion and structural analogs. Pfluegers Arch. 423: R12 (abs.)].

² Supported by MURST, Rome, 1991.

³ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement'' in accordance with 18 USC section 1734 solely to indicate this fact.

⁴ Abbreviations used: BBMV, brush border membrane vesicles; 8-ClRF, 8-chloro-8-demethyl-riboflavin; DARF, 2',3',4',5'-di-O-isopropylidene-riboflavin; DE, rotenone-de-energized enterocytes; 5'-DORF, 5'-deoxy-riboflavin; 8-EORF, 8-ethoxy-8-demethyl-riboflavin; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; FMS, riboflavin-5'-monosulphate; 3-MRF, 3-methyl-riboflavin; NE, normal enterocytes; RF, riboflavin; 8-ROF, 8-dimethyl-amino-8-demethyl-riboflavin (roseoflavin).

Manuscript received July 21, 1998. Revision accepted October 29, 1998.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gastaldi, G. Articles by Rindi, G. Search for Related Content PubMed PubMed Citation Articles by Gastaldi, G. Articles by Rindi, G.