Laboratoire de Physiopathologie Métabolique et Rénale, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Alexis Carrel, Université Lyon I, 69372 Lyon, France
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ABSTRACT |
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A major shortcoming of renal proximal tubular cells (RPTC) in culture is the gradual modification of their energy metabolism from the oxidative type to the glycolytic type. To test the possible reduction of glycolysis by naturally occurring long-chain fatty acids, RPTC were cultured in a two-chamber system, with albumin-bound palmitate (0.4 mM) added to the basolateral chamber after confluency. Twenty-four hours of contact with palmitate decreased glycolysis by 38% provided that carnitine was present; lactate production was decreased by 38%, and the decrease in glycolysis resulted from a similar decrease of basolateral and apical net uptake of glucose. In contrast to the previously described effect of the nonphysiological oxidative substrate heptanoate, palmitate promoted a long-term decrease in lactate production and sustained excellent cellular growth. After 4 days of contact, decreased glycolysis was maintained even in the absence of carnitine and resulted from a decrease of basolateral uptake only, suggestive of long-term regulation different from the earlier effects. Thus, although cultured RPTC lost their oxidative phenotype, they exhibited a type of regulation (Randle effect) that is found in the oxidative-type but not in the glycolytic-type tissues, therefore unmasking a regulative capacity barely detectable in fresh RPTC. Low PO2 (50 mmHg in the apical chamber) could be a major cause of elevated glycolysis and could hinder the effects of palmitate.
fatty acids; kidney; cell culture; differentiation
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INTRODUCTION |
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A MAJOR SHORTCOMING of renal proximal tubular cell (RPTC) culture is the gradual modification of cellular energy metabolism toward glycolysis (1, 7, 11, 12, 14, 35, 37). In situ or in fresh preparations, RPTC obtain their energy by an oxidative process using substrates other than glucose (lactate, citrate, fatty acids) (15, 23); RPTC are the site of extensive, active uptake and transepithelial transport of glucose (18), but only a small amount of the glucose, if any, that is taken up is metabolized, resulting in CO2 and lactate production (7, 22, 23, 29). In addition, RPTC are gluconeogenic (23). Earlier attempts to improve the metabolic phenotype of cultured tubular proximal cells have shown that glycolysis may be reduced by shaking the medium and thus improving oxygen supply (1, 14, 25, 30); however, conflicting results (35) have shown that it would be useful to measure actual PO2 in this type of experiment. Other factors such as hormone supplementation or nutrient deficiency or excess could be responsible for the modification of energy metabolism in long-term RPTC culture. The widely used insulin supplement was shown to decrease neoglucogenesis, although it was not the cause of increased glycolysis (25); the same study showed that 5 mM lactate increased gluconeogenesis and decreased glycolysis. Glucose itself at high concentrations was reported to induce a loss of differentiation (35), and the excellent oxidative substrate, heptanoate (2 mM), a non-naturally occurring, medium-chain fatty acid, decreased glycolysis in short-term, 24-h cultures but was not found to be able to modify glycolysis in 7-day cultures and decreased growth (1).
In this paper, we report the use of a two-chamber system to test the effect of a long-chain fatty acid on the growth and metabolic phenotype of cultured proximal tubular cells. Indeed, epithelial cells are generally cultured in serum-free, albumin-free medium, which prevents fibroblast growth (9); however, this also prevents the use of insoluble, long-chain fatty acids carried in plasma by albumin. Because fatty acids enter the RPTC at the basolateral side, the luminal side being devoid of albumin and thus of fatty acids, we applied albumin-bound palmitate from the basolateral side of the insert only.
In fact, in mammals, the effects of long-chain fatty acids on glucose utilization vary from inhibition to activation, depending on 1) the organ under consideration, 2) the metabolic nature of the cell (oxidative or glycolytic), and 3) the concentration of fatty acids. In heart (19, 28, 34), liver (13, 31), and lymphocytes (2), fatty acids decrease glycolysis; in addition, in hepatocytes, gluconeogenesis is activated during starvation by free fatty acids that are released from adipocytes (26, 34). In adipocytes, by contrast, glucose uptake and phosphofructokinase are activated by fatty acids (17, 21). In skeletal muscles, fatty acids decrease glucose utilization in oxidative-type muscles only (17, 19); moreover, low concentrations of fatty acids have been shown to inhibit glucose utilization, whereas high concentrations had an activating effect (19). During hyperinsulinemia, muscle glucose uptake is decreased by fatty acids (20). Taken as a whole, these organ specificities meet the demand of glucose exchanges during starvation or during postprandial states. In the kidney proximal tubule, where glycolysis is minimal (23), few data are available; however, it has been shown in rat in vivo that increased plasma concentrations of fatty acids during starvation have no effect on the low renal extraction rate of glucose (15). The aim of this study is to test whether a low physiological concentration of palmitate (0.4 mM) is able to decrease the high rate of glycolysis that is found in cultured RPTC.
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METHODS |
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Animals. Female New Zealand rabbits, 1.2-1.5 kg (Elevage Scientifique des Dombes, Châtillon-sur-Chalaronne, France), were used for all studies and were killed by an intravenous injection of pentobarbital sodium (5 ml of a 0.03 g/ml solution).
Materials. Culture medium, serum, trypsin-EDTA, and versene were from GIBCO BRL (Inchinnan, UK); hormone supplements, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, carnitine, and lysozyme were from Sigma (St. Louis, MO); bovine fatty acid-free albumin originated from Sigma or from Boehringer (Mannheim, Germany); enzymes were from Boehringer; and palmitate and other chemicals were from Merck (Darmstadt, Germany). Albumin-bound palmitate was prepared as follows: palmitic acid (25 mg/ml) was dissolved in NaOH at 37°C for 30 min and then added (after cooling) to fatty acid-free bovine serum albumin (100 mg/ml); the final solution contained 1 g palmitate/100 g defatted albumin and 0.9 g/l NaCl.
Culture techniques.
Cell preparation and culture have been described in detail in a
previous paper (7); briefly, cells were isolated from rabbit renal
cortex by mechanical dissociation followed by sequential filtration on
polypropylene sieves; cells were cultured in a two-chamber system on
permeable membranes made of pure collagen from rat tail tendon. The
collagen film formed on a 1-mm mesh nylon gauze was mounted on 12-mm
inserts made in our laboratory. The cells were seeded into the inserts
in 0.5 ml of culture medium (Dulbecco's modified Eagle's
medium-Ham's F-12 containing 10 mM HEPES, 25 mM
bicarbonate, 4 mM glutamine, 1 g/l glucose, 5 µg/ml
insulin, 10 ng/ml epidermal growth factor, 108 M
triiodothyronine, 5 × 10
8 M hydrocortisone, 5 µg/ml
transferrin and 3 × 10
8 M sodium selenium), and 1.75 ml of medium was added to the well outside the insert in a 12-well
plate. Plates were maintained at 37°C in an air-CO2
mixture (95:5) saturated with H2O vapor.
Fatty acid exposure protocol. In two preliminary experiments, palmitate (125 mg/l, bound to 7.4 g/l fatty acid-free albumin) was introduced from the first day of culture into serum-free medium in both chambers of the system. The result in both experiments was the inability of cells to attach and spread onto the substratum; cells cultured in a similar system with fatty acid-free albumin only exhibited normal growth. Therefore, the protocol for palmitate provision to the cells was modified as follows.
After 6 days, when confluency was achieved and watertightness was carefully checked (by leaving each filled insert out of the basolateral medium for 1 min), inserts were divided into four groups. Group 1 was cultured with the basal medium inside (i.e., on the apical pole of the cells) and outside the insert (basolateral pole). Group 2 was cultured with 10 g/l fatty acid-free bovine serum albumin added to the medium outside only (basolateral pole). In group 3, albumin-bound palmitate (10 g/l fatty acid-free albumin and 100 mg/l palmitate) was added to the basolateral medium only. Group 4 was cultured like group 3 except that, in addition, 0.05 mM carnitine was introduced into the basolateral medium. On day 7 or on day 10, 100 µl of medium were pipetted into stoppered capillaries for immediate gas determination (the end of the pipette being placed just underneath the insert for the basolateral sampling and just above the monolayer for the apical sampling), and 300 µl of medium were pipetted out into HClO4-containing tubes (2% final content) and stored atBiochemical analysis. PO2 was measured with a blood gas analyser (Radiometer BMS K 30). Cellular aminopeptidase M activity was determined in situ with L-alanine-nitro-4-anilide as substrate with use of the method of S. G. George and A. J. Kenny as previously described (see Ref. 7). Cellular DNA content was measured in the same cells after solubilization of the entire monolayer with the alkaline solution A + B + C of O. H. Lowry, as previously described (see Ref. 7). To eliminate protein contamination of the monolayer by serum albumin, the protein-to-DNA ratio was determined in cells detached from the collagen film by trypsin treatment as follows: after a 10-min rinsing of both sides of the inserts with versene (1:5,000), 2 ml of trypsin (3 g/l)-EDTA (0.2 g/l) were added to both sides of the inserts for 15 min at 37°C, inducing rounding but not detachment of cells. After a rinsing with PBS, most cells could be detached by thorough repeated pipettings. Proteins were determined with the Bradford reagent, and DNA was determined with the method of C. Labarca and K. Paigen, as previously described (see Ref. 7).
Glucose, lactate, and fatty acids were determined enzymatically with spectrophotometric methods, and changes in the concentration of NADH or NADPH (7, 33) were recorded after neutralization of samples with a mixture of KOH (20%, wt /vol) H3PO4 (0.15 M).Calculations. Metabolite consumption or production was calculated as the difference between the total content (luminal + basolateral) of control medium from inserts incubated without cells and the total content of medium from inserts incubated with cells.
Statistical analyses were performed with the use of analysis of variance followed by protected tests of Fisher. ![]() |
RESULTS |
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Effect of palmitate on renal tubular cell growth. Twenty-four hour contact of cultured cells with palmitate induced no change in DNA content (Table 1); a slight, nonsignificant increase in protein-DNA was seen when carnitine was provided in addition to palmitate. The cellular DNA content increased from day 7 to day 10 in the basal medium, showing that, under these conditions, growth was not arrested at day 7 despite monolayer confluency and watertightness; however, this late growth was suppressed by palmitate in the presence of carnitine. The protein-to-DNA ratio was increased by palmitate at day 10, i.e., after 4 days of supplementation, irrespective of the presence of carnitine.
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Effect of palmitate on glycolysis.
Our cultured cells under basal conditions exhibited a very high
glycolysis rate with >100 µmol glucose
consumed · day1 · mg
protein
1. The lactate production-to-glucose consumption
ratio was increased to ~1.4 [normal ratio in freshly isolated RPTC,
0.5 (7)]; therefore, glucose was not totally converted into lactate.
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Transepithelial distribution of glucose and lactate. In our system, there was a transepithelial glucose gradient of 3.43 mM/0.95 mM (basolateral content /apical content, P < 0.001) across the cellular monolayer 24 h after medium replacement at day 6 in basal medium. This probably resulted from differences in glucose uptake from each chamber, although transepithelial transport cannot be excluded. Table 3 shows that the decrease in glycolysis induced by 24 h of palmitate plus carnitine provision resulted from a similar decrease in glucose net uptake from both sides of the monolayer, because basolateral uptake decreased by 41%, whereas apical uptake decreased by 28%. On the other hand, after 4 days of provision of palmitate, the 20% decrease of glycolysis induced by palmitate only resulted from a decrease of net basolateral uptake.
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Oxygenation status of the system. There was a PO2 gradient between the apical and basolateral sides of the inserts (apical < basolateral, error P < 0.001; Fig. 1) as soon as cells were present, including at day 3, long before confluency. In fact, at day 7, cell respiration induced a sharp decrease of apical PO2 to far below the normal plasma value, whereas basolateral PO2 remained within the normal range in basal conditions.
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Effect of palmitate on aminopeptidase and transepithelial resistance. RPTC in culture gradually lose brush borders and brush-border enzyme activity (1, 7, 37). Because this could result from the absence of lipidic nutrients in the culture medium, aminopeptidase, a brush-border enzyme, was determined after palmitate provision to our cells; however, as shown in Table 4, no improvement was produced by palmitate, either in the presence or absence of carnitine. Similarly, no change in electrical resistance of the monolayer was induced by the presence of palmitate.
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DISCUSSION |
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Evidence of early glycolysis regulation by fatty acids in cultured proximal tubular renal cells. The fact that in cultured RPTC glycolysis may be decreased by 24-h exposure to palmitate is interesting for several reasons. First, it shows for the first time that cultured RPTC have the capacity to regulate glycolysis by means of the naturally occuring long-chain fatty acids (LCFA); this capacity is a typical feature of hepatocytes, heart, and oxidative-type muscle (13, 19, 20, 27, 28, 34). In these organs, three possible sites of inhibition have been identified; the main site is phosphofructokinase, but short- and long-term inhibition of pyruvate dehydrogenase has also been described (13, 16, 27, 34). Both effects were suppressed by inhibition of fatty acid mitochondrial transport or oxidation (13, 16, 27, 31); in addition, in muscle that is insulin responsive, insulin-sensitive glucose transport was shown to be inhibited by fatty acids (17). In our study with cultured RPTC, the fact that lactate production is lowered by palmitate to the same extent as glycolysis, together with the fact that glucose uptake from both sides of the cell is lowered by palmitate, is consistent with the view that an intracellular step, such as phosphofructokinase inhibition, mediates the palmitate effect on glycolysis and that the oxidative pathway is not affected by palmitate. However, in view of the relatively long duration (24 h) of the palmitate exposure, allowing slight changes in the protein/DNA content, it is not excluded that genomic regulation of other sites might add its effects to pure enzymic-stage regulation.
The second point of interest is that RPTC in culture, despite their apparent loss of the oxidative phenotype, exhibit a type of regulation that is found in oxidative-type but not in glycolytic-type tissues (4, 13, 19, 31, 34). In contrast, in hepatoma cells, which exhibit the same glycolytic metabolism as our cells and which also originate from an oxidative tissue, glycolysis does not decrease but increases in the presence of palmitate (6); this indicates that the loss of differentiation of energy metabolism in RPTC in primary culture is not so profound as that in transformed cells and suggests that this loss is reversible. It should also be noticed that this inhibitory effect on glycolysis has never been described in noncultured RPTC, in which glycolysis is either absent, as in the rat (23), or low, as in the rabbit (7, 8, 22); the only study dealing with the effect of palmitate on glycolysis, which used rabbit cortex (which is not pure RPTC), showed no effect on lactate production, although glucose uptake or accumulation was decreased (22). Thus our results show that the glycolysis increase resulting from culture uncovers a regulation capacity typical of oxidative tissue.Effect of palmitate on renal proximal cell differentiation. Our results are in good agreement with those previously obtained by Aleo and Schnellman (1); these authors partially prevented glycolysis increase in cultured RPTC by adding 2 mM heptanoate. However, from our results, the use of the LCFA palmitate provides two advantages over heptanoate. First, palmitate added from confluency sustained excellent growth for at least 4 days although it arrested DNA increase, and, despite the presence of albumin, the carrier of palmitate, the growth of a possible fibroblastic population was not demonstrated, as shown by the RPTC marker aminopeptidase. In contrast, heptanoate added from day 0 was inhibitory to growth (1); this inhibition could be due in part to the timing of the application of the fatty acid, perhaps as a result of a physical mechanism, because, as indicated in METHODS, when we tried to introduce palmitate at day 0, the cells failed to spread and grow. The role of fatty acids during initiation of growth has also been documented by experiments showing that the C4 fatty acid butyrate improves attachment but is inhibitory for initial growth at high concentrations (36). A second advantage of palmitate is that it causes a long-term decrease of glycolysis, whereas heptanoate was reported to decrease glycolysis for only 1 day and to induce a paradoxical rise in lactate production after 7 days (1). These discrepancies probably result from the different metabolic fates of LCFA and medium-chain fatty acids (MCFA); indeed MCFA are mostly oxidized in a carnitine-independent process, whereas LCFA enter into the mitochondria more slowly via carnitine palmitoyl transferase, a key regulating enzyme of lipid metabolism. In parallel, LCFA are rapidly recycled into cellular lipids and lipoproteins (3). Thus differences in long-term regulation by these two fatty acid types are not to be excluded. Besides, the dicarboxylic acids formed after several hours by MCFA only have been shown to decrease mitochondrial ATP production (3); in addition, after several days of MCFA supply, adaptive changes have been found, decreasing oxidation of these fatty acids (3) and possibly causing the paradoxical rise of lactate in heptanoate-supplemented RPTC (1). The carnitine requirement for palmitate's early effect is not surprising, because this carrier is not synthesized by RPTC (5). Unexpectedly, palmitate slightly decreased glycolysis at day 10 in the absence of carnitine, suggesting the existence of a long-term effect of palmitate independent of its oxidation. This long-term decrease of glycolysis, in contrast to that of the early effect, resulted from decreased uptake from the basolateral side only, suggesting a membrane effect restricted to this pole of the cell, which could be a lesser transport rate by the facilitated glucose transporter (GLUT). In fact, this transport in RPTC is restricted to the basolateral membrane, whereas sodium-coupled active glucose transporter is localized in the apical membrane (18); in cultured rabbit RPTC, these two types of transporters have been shown to collaborate for glucose uptake (25). Therefore, long-term effects of palmitate could possibly reflect not just a simple continuation of short-term effects but a completely different mechanism of action.
The palmitate effect, although interesting, was not sufficient to lower glycolysis to the fresh tissue level, which is ~2 µmol · mg ![]() |
ACKNOWLEDGEMENTS |
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We thank Prof. G. Baverel for helpful discussions.
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FOOTNOTES |
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This work was supported by Institut National de la Santé et de la Recherche Médicale (Contrat de Recherche 950201) and by a grant from the Ministère de l'Environnement (95008).
Address for reprint requests: H. Simonnet, Mécanismes Moléculaires du Diabete, Institut National de la Santé et de la Recherche Médicale, U449, Faculté de Médicine Alexis Carrel, 12 rue Guillaume Paradin, 69372 Lyon Cédex 08, France.
Received 30 September 1996; accepted in final form 16 July 1997.
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