special communication

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1 special communication Glucose and glutamine provide similar proportions of energy to mucosal cells of rat small intestine SHARON E. FLEMING, KIRSTEN L. ZAMBELL, AND MARK D. FITCH Department of Nutritional Sciences, University of California, Berkeley, California Fleming, Sharon E., Kirsten L. Zambell, and Mark D. Fitch. Glucose and glutamine provide similar proportions of energy to mucosal cells of rat small intestine. Am. J. Physiol. 27 (Gastrointest. Liver Physiol. 6): G968 G978, The objectives of this study were to establish a reliable method for quantifying glycolytic flux in intestinal epithelial cells, to determine the proportion of energy provided to small intestine epithelial cells by glucose vs. glutamine, and to determine whether there was an energetic advantage to having both substrates present simultaneously. There was substantial retention of H in alanine and lactate when [2- H]glucose was used as tracer for quantifying glycolysis, and the magnitude of the H retention was influenced by the presence of other substrates and metabolites. Detritiation was at least 99% complete, however, when [- H]glucose was used as tracer in this system and the tritium was recovered as H 2 O. Glycolytic flux was six- to sevenfold higher in cells of the proximal than distal small intestine but was not significantly different for young adult (4 mo) vs. aged adult (24 mo) rats. Net ATP production from exogenous substrates was higher when both glucose and glutamine were present simultaneously than when either substrate was present alone, and glucose was calculated to provide 50 60% of the net ATP produced from these two substrates. Most of the energy produced from glucose was produced via the anaerobic metabolic pathways (78% for glucose alone, 95% with glucose and glutamine). Net energy production was calculated to be 10% lower in cells from aged animals than in those from young animals, since CO 2 production from these major substrates was lower in cells from aged animals. detritiation; method GLUCOSE IS UTILIZED BY epithelial cells of the mucosa of the small intestine, and the major products include lactate, carbon dioxide, alanine, and other amino and organic acids (26, 46, 49, 50). Through 1 C nuclear magnetic resonance (NMR) techniques (7), it has been shown that glucose metabolism proceeds primarily via the glycolytic pathway. Other experiments have shown that 5% of the glucose utilization occurs via the pentose phosphate pathway in epithelial cells of both the proximal and distal small intestine (21, 26). It has generally been well recognized that epithelial cells of the proximal small intestine exhibit a high rate of glucose utilization and lactate production, yet glutamine has been increasingly considered to be the major fuel for these cells (2, 45). The major objective of this work was to determine the proportion of energy provided to small intestine epithelial cells by glucose vs. glutamine. To do this, we needed to measure both glycolytic flux and conversion of substrates to metabolites that result in either a net production or utilization of energy. These oxidation and metabolite data could then be used to calculate the relative energetic contributions of glucose and glutamine for cells of the proximal vs. distal small intestine. Also, this approach could be used to assess the influence of senescence on glycolytic flux, since the oxidation of some substrates was reported to be significantly lower in epithelial cells isolated from the proximal small intestine of aged vs. young rats (12). If the metabolic functions of these cells were generally downregulated in senescence, we hypothesized that glycolysis and thus the energy status would be notably lower in cells from aged animals than from young animals. Data that quantify flux of glucose through glycolysis in intestinal cells were not previously available, although glycolytic activity of the rat jejunum was first reported many years ago (9, 41). In those and other studies (, 6, 8, 5, 6, 9, 41), it was established that glycolysis proceeded at a higher rate in the proximal small intestine than in the distal small intestine. This was determined by observing a decreasing distal gradient in oxygen consumption (, 5, 9), decreased glycolytic enzyme activity (6, 8, 6), and a decreased rate of lactate accumulation (, 9). Net lactate production has been used in most cases to estimate glycolysis in intestinal epithelial cells, but this approach is unable to distinguish glucose-derived lactate from lactate produced by other nutrients, such as alanine and glutamine, or from lactate produced after efflux of tricarboxylic acid cycle intermediates. This technique also does not consider the energy contributed by glucose-derived pyruvate when it is channeled into other products, such as alanine. These limitations can be overcome by quantifying the flux of glucose through the glycolytic cycle by using the glucose detritiation technique. This was the approach taken in the experiments that are reported, since this technique allows assessment of the influence of other lactateproducing nutrients on glycolysis. In addition, tritiumlabeled glucose can be used to quantify glycolysis under conditions when there are no appreciable changes in glucose concentration, and also when glycolysis and gluconeogenesis occur simultaneously. Although tritiated glucose had been used previously in studies of intestinal cell metabolism (7, 8), data were not G /97 $5.00 Copyright 1997 the American Physiological Society

2 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES G969 available to ensure that these techniques would provide an accurate quantitative analysis of glycolytic flux in these cells. Thus it was necessary to establish the validity of this approach for studies of enterocyte metabolism. Flux of glucose through complex pathways or through specific steps of a single pathway has been assessed by measuring the rate at which H 2 O is produced from specific [ H]glucose isotopes. Detritiation of [2- H]glucose is known to occur during the isomerization of the hexose phosphates, detritiation of [- H]glucose occurs during isomerization of the triose phosphate intermediates and during flux through the pentose phosphate pathway, and detritiation of [5- H]glucose occurs during both the triosephosphate isomerization and the enolase steps (see Refs. 5, 17, and 51 for review). Rates of glycolysis and glucose utilization have been measured by quantifying H 2 O release from [2- H]glucose (5, 16) and [5- H]glucose (, 5). Under conditions in which flux through the pentose phosphate pathway is insignificant, flux through phosphofructokinase-1, the flux-generating step for glycolysis in many tissues, has been measured using [- H]glucose (2, 29, 7, 8). Although this approach has a strong theoretical basis, and the techniques are straightforward, the results may be confounded by incomplete detritiation and isotope discrimination (51). Retention of H in metabolic products other than H 2 O has been reported previously (51), and the presence of nonglucose metabolites has been shown to influence the degree of detritiation (44). To determine the reliability of using H-glucose techniques to quantify flux of glucose through glycolysis in intestinal epithelial cells, it was necessary to determine whether H would be retained in metabolites other than H 2 O, and it was also necessary to determine whether detritiation would be influenced by the presence of nonglucose substrates. From these experiments, we determined that [- H]glucose, but not [2- H]glucose, could be used to reliably estimate flux of glucose through the glycolytic pathway under a range of experimental conditions. Glycolytic flux data and 14 C-metabolite data then could be used to calculate the relative energy contribution of glucose vs. glutamine for intestinal epithelial cells, and differences could be assessed due to intestinal segment, animal age, and substrate availability. MATERIALS AND METHODS Animals. To determine the effect of aging, young (4 mo) and aged (24 25 mo) male Fischer 44 rats were purchased from the National Institute on Aging breeding colony maintained under barrier-reared conditions (Harlan Industries, Indianapolis, IN). They were given NIH 1 stock diet (Western Research Products, Hayward, CA), since they had been fed this diet throughout their lifetimes. For other glycolysis and 14 C-metabolism experiments, Fischer 44 rats (Simonsen Laboratories, Gilroy, CA) weighing g were used. Until the day of experimentation, these animals were allowed free access to commercial diet (Rat Chow no. 5012, Ralston Purina, St. Louis, MO). The animals were anesthetized with pentobarbital sodium (5 mg/100 g rat; Abbott Laboratories, North Chicago, IL) and killed by thoracotomy. Animal handling procedures were approved by the Animal Care and Use Committee, University of California (Berkeley, CA). Chemicals. Radiochemicals obtained from Dupont NEN (Boston, MA) included high-performance liquid chromatography (HPLC)-purified [- H]glucose, HPLC-purified [2- H]glucose, H 2 O, [U- 14 C]aspartate, [U- 14 C]citrulline, [U- 14 C]glucose, [U- 14 C]glutamine, [U- 14 C]glutamate, [U- 14 C]lactate, U- 14 C-labeled -methylglucose, [U- 14 C]ornithine, [U- 14 C]proline, and [1,4-14 C]succinate. [5- H]glucose and [U- 14 C]alanine were obtained from Amersham Life Sciences (Arlington Heights, IL). Ion exchange resins and antibiotics and antimycotics including streptomycin sulfate, penicillin G, kanamycin monosulfate, and amphotericin B were purchased from Sigma Chemical (St. Louis, MO). Other chemicals and reagents were obtained commercially and were of reagent grade. Glucose isotopes were purified using thin-layer chromatography. The labeled isotope was applied to a cm glass plate coated with MN400 microcrystalline cellulose (250 µm, Avicel; Analtech, Newark, NJ) and developed in a solvent system containing n-butanol, glacial acetic acid, and H 2 O (24:4:10, vol/vol/vol), as described previously (49). The presence of radiolabeled compounds was detected using a radioactive plate scanner (Bioscan, Washington, DC). The region of the plate containing glucose was scraped from the plate and the glucose was eluted from the cellulose using Krebs- Henseleit (KH) buffer and stored at 20 C. Radioactivity of the extract was measured, and this information was used to achieve the desired specific activity of the final substratecontaining solutions. In controlled experiments (data not shown), the purification procedure reduced the radioactivity in control flasks (without cells) and reduced variability among replicates but did not significantly alter values for glycolytic flux. Preparation of isolated cells. Animals were killed in the fed state on the morning of experimentation. The entire small intestine was removed, and the appropriate segment was excised (the proximal segment consisted of a 0-cm segment beginning 10 cm distal to the pylorus; the distal segment consisted of a 0-cm segment proximal to the ileocecal junction). Cells were isolated from the mucosa using chemical (EDTA) and gentle mechanical procedures (11, 1, 14, 25). The everted segments were filled with Ca 2 -free KH buffer containing 0.025% bovine serum albumin (BSA). The proximal segment required a 10-min EDTA incubation to completely remove the mucosal cells, whereas the distal segment required a 20-min incubation. The cells were then peeled from the underlying matrix by directing a pressurized stream of ice-cold Ca 2 -containing KH buffer with 0.25% BSA and 5 mmol/l dithiothreitol at the mucosa. Care was taken to maintain the cells at 4 C throughout subsequent preparation steps. Antibiotics were included in the incubation solution (2.5 µg/ml amphotericin B, 100 µg/ml kanamycin monosulfate, 250 U/ml penicillin G, and 250 µg/ml streptomycin sulfate) and in the substrate solutions (0.25 µg/ml amphotericin B, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate). The compositions of these mixtures were based on those used previously for cells isolated from the intestine (25) and for preparing mammalian cell cultures (14). Lactate dehydrogenase (LDH; E.C ) release (Sigma Diagnostics kit DG140-UV) was used to evaluate membrane integrity (1). Our preparations contained primarily intact villi and rafts or clumps of cells. During the 0-min incubation, leakage of LDH into the media averaged 5 6% of the total cell content. Uptake of -methylglucose (22) and 14 CO 2 production were used to assess metabolic integrity (20). The -methyl-glucose uptake ratio averaged 2, indicating that

3 G970 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES -methyl glucose was concentrated inside the cell by 2-fold and showing that the cell membranes were intact and the cells capable of pumping substrate into the cells against a concentration gradient. Data are reported on a dry weight basis (100 C, 2 h). Incorporation of H from glucose into H 2 O and other metabolites. Cell suspensions (2 4 mg cells, dry wt for ion exchange chromatography analyses; 5 7 mg cells for thinlayer chromatography analyses) were incubated for 0 min at 7 C in KH buffer containing 0.25% BSA, substrates, and trace quantities of [2- H]glucose, [- H]glucose, or [5- H]glucose. The specific activity of tracers ranged from 0.1 to dpm/µmol glucose for ion exchange chromatography and from dpm/µmol glucose for thin-layer chromatography. Each incubation included one of the following substrates: glucose ( mmol/l); glucose (5 mmol/l) and glutamine (5 mmol/l); or glucose (5 mmol/l) and alanine (5 mmol/l). In experiments to determine metabolite production using ion exchange chromatography, cell incubation reactions were stopped by the addition of 5% ZnSO 4 (47) followed by the addition of an equimolar amount of 0. N Ba(OH) 2, according to the method of Bontemp et al. (4), and the mixture was vigorously agitated. In these experiments, an aliquot of the supernatant (attained by centrifugation at 500 g, 2 min), was passed through a column (0.7 ml) of Dowex 1 (borate form) and H 2 O was rinsed from the resin with H 2 O. This technique was used previously to separate H 2 O quantitatively from H-glucose (15). Radioactivity in aliquots of the eluate was determined using liquid scintillation counting (Packard 1600TR, Packard Instruments, Meriden, CT). Dowex was converted to the borate form in 100-g batches by successive washes with 1 N NaOH (2 times), H 2 O (4 times), 0.9 M boric acid (2 times), 0.05 M Na 2 B 4 O 7 (2 times), and H 2 O (4 times). Columns containing 0.7 ml of resin were rinsed with H 2 O before sample loading. Incorporation of glucose and glutamine carbon into metabolites. Epithelial cells from the proximal small intestine of fed Fischer 44 rats were incubated with substrates and trace quantities of [U- 14 C]glucose or [U- 14 C]glutamine. Each incubation included 4 6 mg cells (dry wt), tracer ( dpm to quantify CO 2 production or dpm to quantify other metabolites), and one of the following substrates (in mmol/l): 5 glucose, 5 glutamine, 5 glucose and 5 glutamine, 5 glucose and 0. alanine, or 5 glucose and 5 alanine. After a 0-min incubation, metabolism was stopped by the addition of HClO 4 (10%). CO 2 production was determined using previously described procedures (11, 21). To determine the incorporation of substrate into metabolites other than CO 2, the acidified incubates were centrifuged at 4,00 g for 2 min. Supernatants were neutralized with KOH, lyophilized, ethanol extracted, and chromatographed on cm cellulose thin-layer chromatography plates using two-dimensional chromatography [solvent 1 composed of phenol, H 2 O, and NaCN (:1:0.00, wt/vol/wt; Ref. 48; dried overnight); solvent 2 composed of n-butanol, glacial acetic acid, and H 2 O (24:8:6, vol/vol/vol)]. The R f values for 22 amino acids, lactate, and succinate were previously determined. Samples were spiked with a mixture containing seven unlabeled amino acids (Ala, Gln, Glu, Asp, Pro, Cit, and Orn) to facilitate visualization by spraying the developed plates with 0.05% fluorescamine (48). Radioactivity was visualized using a radioactive plate scanner. Regions of the plate corresponding to known metabolites were scraped and placed in scintillation vials, and compounds were eluted with 400 µl of 0.1 M NaOH, 1 ml H 2 O, and aqueous compatible scintillation cocktail (a70b; RPI, Mt. Prospect, IL). Aspartate and glutamate values were confirmed by processing samples through Dowex 50 and Dowex 1 (formate) ion exchange columns. Effluent containing aspartate and glutamate was lyophilized, redissolved, and analyzed by one-dimensional thin-layer chromatography (solvent 1), then visualized, scraped, and counted as described previously. The specific activity of substrate [dpm/µmol carbon of substrate dpm/(µmol substrate number of carbons/ molecule of substrate)] and the amount of radioactivity in each metabolite were used to calculate the incorporation of substrate carbon into metabolites. Glucose utilization. Net glucose disappearance was determined by measuring glucose concentrations in the media before and after the 0-min incubation. Glucose concentrations were determined enzymatically using assay kits obtained from Sigma Chemical. Statistical analyses. Two-way analysis of variance was used to analyze data describing the effects of substrate and age (or segment) on glycolytic flux. When a factor was found to be statistically significant (P 0.05), follow-up tests were executed using Tukey s Studentized range test at a procedurewise error rate of All analyses were performed using the SPSSX statistical package (40). RESULTS Reliability of detritiation techniques for quantifying glycolytic flux. Differences in the rate of detritiation due to the position of the H on the glucose molecule were investigated. Initially, cells were incubated with 5 mmol/l glucose, and trace quantities of either [2- H]glucose, [- H]glucose, or [5- H]glucose and H 2 O were measured in the eluate after ion exchange chromatography. Production of H 2 O from [2- H]glucose ( µmol g 1 min 1 ; means SE, n ) was significantly lower than from [- H]- and [5- H]glucose isotopes ( and µmol g 1 min 1, respectively). In a subsequent experiment, the rate of detritiation of these three isotopes was measured as a function of glucose concentration, and differences among the isotopes were observed across a wide range of glucose concentrations (Fig. 1). Glucose utilization by the isolated intestinal cells was calculated by measuring the concentration of glu- Fig. 1. Influence of glucose concentration on rate of appearance of H. Epithelial cells, isolated from proximal small intestine of rats, were incubated 0 min in presence of glucose at concentrations ranging from 0.1 to 5.0 mmol/l. Cells were incubated in presence of 1 of the following radioisotopes: [2- H]glucose, [- H]glucose, or [5- H]glucose. Supernatants were applied to Dowex 1 borate columns, and radioactivity was quantified in eluates.

4 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES G971 cose in the media both before and after incubation. Glucose utilization averaged 1.5 µmol g 1 min 1 when cells were incubated in glucose alone. This agrees well with glycolytic flux data when measured using [- H]- or [5- H]glucose. However, these data suggested that glycolytic flux was being underestimated when the [2- H]glucose isotope was used. The efficiency with which the Dowex 1-borate ion exchange resin separated glucose from its metabolites was evaluated by quantifying the recovery of radiolabeled standards from the columns. Glucose was found to be entirely retained by the resin, and H 2 O was found to be entirely excluded (Table 1). Lactate and alanine standards were not clearly discriminated, however, and these two major metabolites of glucose were found in the eluate at 5% and 50%, respectively, using conditions identical to those for eluting H 2 O. This raised the question as to whether, in cell incubations, there was incomplete detritiation of some isotopes of glucose resulting in retention of label as lactate or alanine and inaccurate measures of glycolytic flux. To ascertain this, radioactivity was determined in lactate and alanine. In parallel experiments using [U- 14 C]glucose as tracer, we found that 76 78% of the metabolized glucose carbon could be recovered in these products (Fig. 2). When [2- H]glucose was used as tracer and cell supernatants were analyzed by quantitative two-dimensional thin-layer chromatography, H was detected in both alanine and lactate (Table 2). The retention of tritium in alanine ranged from 0.2 to 0.5 µmol g 1 min 1 under the three substrate conditions evaluated. The retention of tritium in lactate differed by substrate, however, and ranged from 0.95 µmol g 1 min 1 in the presence of 5 mmol/l each of glucose and alanine, to 4.8 µmol g 1 min 1 in the presence of glucose alone. When [- H]glucose was used as tracer, retention of H in either alanine or lactate was negligible under the range of conditions evaluated. The rates at which the total of all H-labeled metabolites of glucose were produced was calculated using the following data: amount of H eluted from the Dowex 1 column, the efficiency with which the major metabolites of glucose were excluded from or retained by the Dowex resin, and the amounts of H-labeled alanine and lactate produced. Using [2- H]glucose as tracer, we found that the rate of H-elution from the Dowex column was significantly higher in the presence of glutamine or alanine vs. glucose alone (Table ). When the retention of H-labeled alanine and lactate by the Table 1. Discrimination of glucose and its metabolites by Dowex 1 (borate form) ion exchange resin In eluate %Distribution Retained on resin Glucose H 2 O Lactate 5 95 Alanine Glucose, lactate, and alanine were determined using 14 C-labeled substrate. H 2 O was determined using H 2 O. Fig. 2. Incorporation of glucose carbon into metabolites. Epithelial cells, isolated from proximal small intestine of rats, were incubated 0 min in presence of 5 mmol/l glucose, 5 mmol/l each of glucose and glutamine, 5 mmol/l glucose and 0. mmol/l alanine, and 5 mmol/l each of glucose and alanine. Trace quantities of [U- 14 C]glucose were present also. Specific activity of glucose in incubation medium was used to calculate incorporation of glucose carbon into metabolites as indicated ( others refers to fraction of radioactive glucose that was detected on regions of the plate not ascribed to substrate or to 1 of the identified metabolites). Results are reported as µmol glucose carbon atoms incorporated g dry wt isolated cells 1 min 1. Dowex column was considered, however, the major metabolites of glucose (sum of H 2 O, [ H]lactate, and [ H]alanine) were produced at similar rates for [2- H]glucose under the conditions evaluated. Because [ H]lactate and [ H]alanine were minor metabolites of [- H]glucose, values for H eluted from the Dowex column and for total glucose metabolized were similar when [- H]glucose was used as tracer (Table ). Also, calculated values for total H-metabolite production were similar when either [2- H]glucose or [- H]glucose were used as tracers, and these values were unaffected by the presence of the nonglucose substrates glutamine Table 2. Appearance of H in alanine and lactate after incubation of isolated jejunal epithelial cells with [2- H]- or [- H]glucose in presence of glucose with or without other substrates Isotope/Substrate H-Containing Metabolites Alanine Lactate [2- H]glucose Glucose Glucose glutamine Glucose alanine [- H]glucose Glucose Glucose glutamine Glucose alanine Values (in µmol glucose g dry wt cells 1 min 1 ) are means; coefficients of variation averaged 10%. Concentrations of substrates were 5 mmol/l. To establish the validity of two-dimensional thin-layer chromatography (TLC) analytical procedures, lactate production was quantified in a separate experiment using [U- 14 C]glucose as label. Supernatants were analyzed using both two-dimensional TLC and high-performance liquid chromatography (followed by fraction collection of effluent and analysis using scintillation counting). Values determined by these 2 techniques agreed to within % (data not shown).

5 G972 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES Table. Total H-labeled metabolite production from glucose after incubation of isolated jejunal epithelial cells with [2- H]- or [- H]glucose in presence of glucose with or without other substrates Tracer/Substrates Eluted from Dowex Metabolites Retained on Dowex 1 Alanine Lactate Total Glucose Metabolized (%) [2- H]glucose Glucose (69%) Glucose glutamine * (84%) Glucose alanine * (94%) [- H]glucose Glucose (99%) Glucose glutamine (99%) Glucose alanine (99%) Values are in µmol g dry wt cells 1 min 1. Concentrations of substrates were 5 mmol/l. Measurement of alanine and lactate was calculated by multiplying rate of production (Table 2) by fractional retention on Dowex 1 resin (Table 1). Total glucose metabolized was calculated as the sum of data in the previous columns and represents conversion of [ H]glucose to H 2 O, alanine, and lactate. Retention of H in other metabolites was not detected. Values in parentheses represent %total accounted for in Dowex eluate (column 1/column 4 100). Values for Dowex eluate are means SE; n 4. *P 0.05, significantly different from values for glucose controls using paired t-test. Table 4. Glycolytic flux rate using [- H]glucose for mucosal cells of the proximal and distal small intestine from young and aged rats and alanine. Based on these results, future glycolytic flux experiments were conducted using [- H]glucose, since the tritium did not appear to be carried into major products such as lactate and alanine, as was observed when [2- H]glucose was used. Influence of animal age and intestinal segment on glycolytic flux. Glycolytic flux in cells of the proximal small intestine was not significantly different for young (4 mo) vs. aged (24 mo) animals, when determined with glucose at 5 mmol/l (Table 4). Also, adding glutamine to the media at 5 mmol/l had no significant effect on flux of glucose through the glycolysis pathway for animals at either of these two ages. When cells of the distal small intestine were incubated in glucose at 5 mmol/l, glycolytic flux was only 15% of the rate in cells of the proximal small intestine (Table 4). A similar degree of difference between segments was observed when both glucose and glutamine were present. Metabolites of glucose and glutamine using 14 C-labeled substrates. The formation of products from the metabolism of glucose and glutamine was measured to consider both energy-producing and energy-consuming processes when calculating the energetic contribution to intestinal epithelial cells of these two major substrates. Major metabolites of glucose included lactate, alanine, and CO 2 (Fig. 2). The relative proportions and absolute amounts of these metabolites were influenced by the composition of the media. In the presence of glucose alone, lactate and CO 2 predominated. Including glutamine in the media reduced the formation of lactate and CO 2 from glucose and increased alanine formation. Including alanine in the media had similar effects to including glutamine, but the magnitude of the effects was influenced by alanine concentration; increasing alanine concentration decreased glucose conversion to lactate and CO 2 and increased conversion of glucose to alanine. The presence of alanine did not influence production of citrulline and CO 2 from glucose. For all four substrate treatments, quantifiable but insignificant amounts of glucose carbon (a total of 1 µmol carbon atoms g 1 min 1 ) were detected in compounds including ornithine, proline, glutamate, aspartate, and succinate. Major metabolites of glutamine included glutamate and CO 2, which were produced in similar quantities regardless of whether or not glucose was added to the media (Fig. ). Lactate and alanine were produced to lesser extents, and the addition of glucose appeared to increase incorporation of glutamine carbon into lactate but decrease incorporation into alanine. Small amounts of glutamine carbon were also incorporated into aspartate, citrulline, proline, ornithine, and succinate. Overall, the presence of exogenous glucose did not influence Glucose Glucose Glutamine Young Aged Young Aged Proximal segment Distal segment Values (in µmol glucose g 1 min 1 ) are means SE for Fischer 44 rats; n. Young, 4-mo-old rats; aged, 24-mo-old rats. Within proximal segment data set, overall mean value for young animals (pooled mean 15.8) was not significantly different from that for aged animals (pooled mean 15.4). Overall mean value with glucose as substrate (pooled mean 15.7) was not significantly different from that with glucose glutamine as substrates (pooled mean 15.5). For young animals, overall mean value for the proximal segment (pooled mean 15.6) was significantly higher than that for the distal segment (pooled mean 2.7). Fig.. Incorporation of glutamine carbon into metabolites. Epithelial cells, isolated from proximal small intestine of rats, were incubated 0 min in presence of 5 mmol/l glutamine or 5 mmol/l each of glucose and glutamine. Trace quantities of [U- 14 C]glutamine were present also. Specific activity of glutamine in incubation medium was used to calculate incorporation of glutamine carbon into metabolites as indicated ( others refers to fraction of radioactive glutamine that was detected on regions of the plate not ascribed to substrate or to 1 of the identified metabolites). Results are reported as µmol glutamine carbon atoms incorporated g dry wt of isolated cells 1 min 1.

6 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES G97 the incorporation of glutamine carbon into these metabolites. Energetic contribution of glucose vs. glutamine. The relative energy contributed by the metabolism of glucose vs. glutamine was calculated using glycolytic flux and substrate oxidation data (Table 4, Figs. 2 and ). Several assumptions were used in these calculations. In intestinal cells, as in other cell types, the conversion of glucose to pyruvate would produce 8 mol ATP/mol glucose, assuming that reducing equivalents were transferred into the mitochondria via the malate/aspartate shuttle. The conversion of 1 mol pyruvate to mol CO 2 was assumed to produce 15 mol ATP/mol pyruvate. The production of lactate from pyruvate was assumed to require mol ATP/mol lactate since 1 mol NADH would be utilized. Also, 1 mol glutamine was assumed to be oxidized to 2 mol CO 2, with an energy yield of 9 ATP/2 CO 2. This low rate of oxidation was based on values for A T reported previously for intestinal cells incubated under similar conditions (1). Values for A T are calculated from either the acetate or succinate CO 2 ratios (e.g., 14 CO 2 from [1-14 C]acetate/ 14 CO 2 from [2-14 C]acetate) and predict the probability that a molecule entering the tricarboxylic acid cycle will be oxidized to CO 2 (18). Values for A T averaged 0.4 for cells of the proximal small intestine when incubated in 5 mmol/l glutamine or in 5 mmol/l of both glutamine and glucose (1). This finding was recently verified in our laboratory by calculating the ratio of 14 CO 2 from [U- 14 C]glutamine/[1-14 C]glutamine (data not shown). Similar observations were reported previously by Watford (45). We assumed that the remaining three carbons of the glutamine molecule would be detected in other metabolites, allowing independent consideration of their energetic value. The energy cost of producing lactate was considered. However, due to the low flux rate of glutamine into other metabolites that was observed (Fig. ), the energy cost of producing compounds such as citrulline was ignored in these calculations. The formulae used to perform these calculations are provided in the Table 5 legend. Table 5. Contribution of glucose and glutamine to the energy supply of small intestine mucosal cells from young rats Glucose Proximal Small Intestine Glucose glutamine Glutamine Glucose Distal Small Intestine Glucose glutamine Glutamine Glycolytic flux, µmol glucose g 1 min CO 2 from [U- 14 C]glucose, µmol CO 2 produced g 1 min CO 2 from [U- 14 C]glutamine, µmol CO 2 produced g 1 min ATP production, µmol g 1 min 1 From glucose Glucose to pyruvate Pyruvate to CO Pyruvate to lactate* ND ND ATP production, µmol g 1 min 1 From glutamine Glutamine to CO Glutamine to lactate ND ND Net ATP production, µmol g 1 min 1 From glucose If glycerol phosphate shuttle ( 94.7) ( 70.5) From glutamine Total If glycerol phosphate shuttle ( 94.7) ( 129.8) Unless otherwise noted, it is assumed that H is transported into the mitochondria via the malate-aspartate shuttle. Data for glycolytic flux are taken from Table 4. Data for CO 2 from [U- 14 C]glucose and from [U- 14 C]glutamine for proximal small intestine were determined in separate experiments using previously described techniques (20, 21). Data for distal small intestine were previously reported (20, 21). Positive signs denote net energy production. ATP produced from flux of glucose to pyruvate glycolytic flux (µmol g 1 min 1 ) 8 µmol ATP produced/µmol glucose converted to pyruvate. ATP produced from flux of pyruvate to CO 2 glucose oxidized (µmol CO 2 produced from [U- 14 C]glucose g 1 min 1 ) 1 µmol pyruvate oxidized/ µmol CO 2 15 µmol ATP produced/µmol pyruvate oxidized. Assume that conversion of pyruvate to lactate results in net utilization of mol ATP/mol lactate formed. ATP utilized to convert pyruvate to lactate µmol C atoms of glucose in lactate g 1 min 1 1 µmol lactate/ µmol C atoms µmol ATP utilized/µmol lactate produced. Negative signs denote net energy utilization. *Lactate data are taken from Fig. 2. Assume that 1 mol glutamine is oxidized to 2 mol CO 2 (based on CO 2 ratio data from Ref. 7 and data from Ref. 1) with an energy yield of 9 ATP/2 CO 2. Also, assume that some glutamine-derived pyruvate would be converted to lactate with an energy cost of µmol ATP utilized/µmol lactate produced. Due to low flux rates of glutamine carbon into other metabolites, the energy cost of producing compounds such as citrulline has been ignored. ATP produced from flux of glutamine to CO 2 glutamine oxidized (µmol CO 2 produced g 1 min 1 ) 9 µmol ATP produced/2 µmol CO 2 produced from glutamine. ATP utilized for flux of glutamine carbon from pyruvate to lactate µmol C atoms lactate produced g 1 min 1 1 µmol lactate/ µmol lactate C atoms µmol ATP utilized/µmol lactate produced. Lactate data are taken from Fig.. For net ATP production, if we assume that the glycerol phosphate shuttle, rather than the malate-aspartate shuttle, facilitates transfer of reducing equivalents from the cytosol to the mitochondria, then all NADH produced by glycolysis but not consumed in reduction of pyruvate to lactate would be transported into the mitochondria with a net energy yield of 2 ATP/NADH, rather than at ATP/NADH. Values in parentheses reflect net energy production assuming that the glycerol phosphate shuttle, not the malate-aspartate shuttle, functions in intestinal epithelial cells. When assuming reducing equivalent transfer via the glycerol phosphate shuttle, net ATP produced from glucose (µmol ATP produced in conversion of glucose to pyruvate µmol ATP utilized in conversion of pyruvate to lactate) 2/ µmol ATP produced in conversion of pyruvate to CO 2. ND, not determined.

7 G974 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES In cells of the proximal small intestine, total ATP production from exogenous substrates was higher when both substrates were present than with either glucose alone (0% higher) or glutamine alone (161% higher, using values in Table 5). With both substrates present, glucose provided 62% of the net ATP so that the relative energy contribution from glucose:glutamine was 62:8 for cells of the proximal intestine (Table 6). Because lactate production from glucose and glutamine was not determined for cells of the distal small intestine, only a maximum ATP production from these two substrates could be estimated. Using this approach, it appears that net ATP production from glucose and/or glutamine would be at least two times higher for cells of the proximal small intestine than for cells of the distal segment (Table 5). The glycolytic flux data reported here (Table 4) and the glucose and glutamine oxidation data reported previously (1) were used to calculate energy production from glucose by jejunal epithelial cells of aged vs. young animals under conditions in which both substrates were present at 5 mmol/l. As a percentage of ATP from glucose and glutamine, glucose contributed 55% vs. 58% of net ATP from young vs. aged animals, respectively, assuming that lactate production was not influenced by the aging process. Total energy produced from exogenous substrates by cells of aged animals was calculated to average 90% of the value for young animals, and this difference was due to lower oxidative decarboxylation of both glucose and glutamine. Table 6. Contribution of glucose to the energy supply for mucosal cells of the proximal small intestine of young rats Exogenous Substrate Glucose Glucose glutamine Net energy from exogenous substrates µmol ATP g 1 min * 155.5* Net energy produced from glucose metabolism µmol ATP g 1 min * 96.2* %net ATP 100% 62% Net energy produced from anaerobic metabolism of glucose µmol ATP g 1 min %glucose ATP derived from glycolysis 78% 95% Assume that the malate-aspartate shuttle transfers reducing equivalents into the mitochondria. Anaerobic metabolism of glucose refers to cytosolic metabolism of glucose to lactate, via pyruvate, in which the pyruvate molecule does not enter the mitochondrial tricarboxylic acid cycle. Previous studies using 1 C nuclear magnetic resonance techniques showed that this pathway resulted in production of 70% of lactate derived from glucose, regardless of whether glutamine was present or not (7). Net ATP produced from anaerobic metabolism of glucose ATP produced in conversion of glucose to pyruvate (µmol ATP g 1 min 1 ) [0.7 ATP consumed in conversion of pyruvate to lactate (µmol ATP g 1 min 1 )]. *Data are taken from Table 5. DISCUSSION [- H]glucose but not [2- H]glucose can be used to quantify glycolytic flux in intestinal cells. The production of H 2 O from glucose, measured using traditionally accepted procedures, differed among the three [ H]glucose isotopes that were used in these experiments. Using epithelial cells from the proximal small intestine, we found that the rates of detritiation were significantly higher for the [- H]- and [5- H]glucose isotopes than for the [2- H]glucose isotopes (see RE- SULTS and Fig. 1). Neither purifying these isotopes using conventional thin-layer chromatography techniques nor purchasing HPLC-purified isotopes altered this finding, and this difference could not be explained using the technical information available from the suppliers regarding position of the H-label. For several reasons, these results were surprising and not readily explained. First, the rate of detritiation is usually expected to occur at a greater rate (not lesser rate as we observed) for the [2- H]glucose isotope than for the [- H]glucose or [5- H]glucose isotopes. The production of H 2 O has been found to be higher from [2- H]glucose than from [- H]glucose or [5- H]glucose in studies of muscle (28), hepatocytes (10, 0), and oocytes (19). Detritiation of the [2- H]glucose isotope occurs during hexose phosphate isomerization, producing fructose-6-phosphate that could have other fates, in addition to proceeding through the glycolytic cycle and being converted to pyruvate and other metabolites (as discussed earlier). By comparison, detritiation of the [- H]glucose isotope occurs both during the pentose phosphate pathway and during flux through glycolysis, whereas detritiation of the [5- H]glucose isotope occurs predominantly during flux through glycolysis (reviewed in Refs. 17 and 51). Secondly, the apparent stimulatory effects of glutamine and alanine on H 2 O production using the [2- H]glucose isotope were unexpected, since these results imply that glutamine and alanine stimulate flux of glucose through glycolysis in epithelial cells of the proximal small intestine. In other studies with these cells, glutamine did not increase net glucose utilization (7, 21), and glutamine also did not influence relative flux of glucose through the pentose phosphate pathway (21). The apparent stimulatory effect of alanine on glycolysis was unexpected since alanine is known to inhibit glycolysis by inhibiting pyruvate kinase in a variety of cells, including intestinal epithelial cells (42). The unexpected results with the [2- H]glucose isotope are shown in these studies to be due to incomplete detritiation of the glucose molecule, resulting in partial retention of the tritium in lactate and alanine (Table 2). Furthermore, the completeness of the detritiation of the [2- H]glucose isotope was influenced by the substrates present in the media (Table 2), suggesting that the presence of glutamine and alanine exerted effects on the transfer of HtoH 2 O during the hexose phosphate isomerization reaction. As much as 1% of the H-label from glucose passing through the glycolytic cycle could be retained on the Dowex 1-borate resin,

8 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES G975 resulting in gross underestimation (Table ). Consequently, the [2- H]glucose isotope, when used in conjunction with traditional ion exchange chromatography techniques, could not be used to quantify flux of glucose through glycolysis in epithelial cells isolated from the proximal small intestine. The extent to which these same phenomena occur in other systems is not known, as data demonstrating the validity of this procedure have not generally been presented, and warnings (17) regarding the limitations of the detritiation techniques for assessing metabolic flux have been generally ignored. Our experiences with this system suggest the following: the [2- H]glucose isotope is incompletely detritiated during glycolysis in some metabolic systems, allowing the label to remain in lactate and alanine; the completeness of detritiation is not consistent across a range of metabolic conditions (e.g., variations in the presence and concentrations of metabolites and substrates); and the traditional ion exchange chromatography techniques do not allow H 2 O to be quantitatively separated from all H-labeled metabolites of glucose (Table 1). When these factors were accounted for, flux of glucose through glycolysis (calculated as the sum of the three major metabolites of [2- H]glucose: H 2 O, [ H]alanine, and [ H]lactate) was unaffected by glutamine or alanine (Table ), which is as we would expect. The problems observed with using [2- H]glucose to measure glycolytic flux were avoided by using [- H]glucose as tracer. Consequently, [- H]glucose can be used in conjunction with Dowex 1 ion exchange chromatography to quantify flux of glucose through glycolysis in intestinal epithelial cells. The close agreement in glycolytic rates that were measured using [- H]glucose and [5- H]glucose (Fig. 1) supports the validity of the use of [- H]glucose for quantifying glycolysis in intestinal epithelial cells. This close agreement is expected in tissues in which only a small fraction of glucose utilization occurs via the pentose phosphate pathway, such as in isolated intestinal epithelial cells (21, 26). Glycolysis is an important energy-producing pathway for intestinal epithelial cells. When both glucose and glutamine were present at 5 mmol/l, the metabolism of glucose by cells of the proximal small intestine was calculated to provide 62% of the net ATP produced from these two substrates (Table 6). Previously, glucose was found, in neonatal pig enterocytes, to contribute 40% of the ATP produced from glucose and glutamine when both were present at 2 mmol/l (4) and 75% of ATP when glucose and glutamine were present at 5 and 2 mmol/l, respectively (52). Glycolysis makes an important contribution to the energy supply of enterocytes, but this fact is not reflected in the CO 2 data alone. To illustrate, the relative CO 2 production from glucose:glutamine has been reported to be 20:80 and 0:70 in the proximal and distal small intestine, respectively (20, 21, 50), yet our calculations suggest that glucose may provide at least 60% of the net ATP under similar conditions. Furthermore, the results of 1 C NMR studies using intestinal epithelial cells provided data that could be used to calculate the proportion of glucose-derived lactate formed directly from the pyruvate produced via glycolysis (7). These data showed that 70% of the lactate was produced directly via glycolysis when mucosal cells of the proximal small intestine were incubated either in glucose alone or in a media containing both glucose and glutamine. These earlier findings were used to calculate the contribution of anaerobic glucose metabolism (defined here as cytosolic metabolism of glucose to lactate via pyruvate) to the overall energy production from glucose metabolism. Analysis of ATP yield showed that anaerobic glucose metabolism was responsible for 78% of the energy produced from glucose when glucose was the sole exogenous substrate and 95% of the energy produced from glucose when both glucose and glutamine were present (Table 6). These calculations emphasize that the glycolytic pathway is much more important than the tricarboxylic acid cycle in producing energy from glucose in these cells. Cells in culture are commonly found to rely more heavily on anaerobic metabolism than intact tissue. Substantial lactate and alanine production from glucose has been reported for the jejunal epithelium when studied in vivo (50), however, and the ratio of glucose carbon in (lactate alanine)/co 2 was similar for the in vivo study (ratio of 5.2; Ref. 50) and for our in vitro study (ratio of 6.6; Fig. 2). This suggests that the high anaerobic metabolism reported here (Table 6) may reflect enterocyte metabolism in vivo. The glycolytic flux data used in the calculations presented in Tables 5 and 6 were determined with glucose and glutamine at equimolar concentrations of 5 mmol/l. The concentration was set at 5 mmol/l, the postprandial plasma glucose concentration, and this concentration also approximates the luminal glutamine concentration after consumption of a high-protein meal (1). To simulate a condition in which luminal nutrients are unavailable to jejunal epithelial cells, the energy yield from glucose could be calculated using data that would mimic plasma glucose and plasma glutamine concentrations at 5.0 and 0.5 mmol/l, respectively (50). Under these conditions, flux of glucose through glycolysis would be unaffected (interpolation of data in Table 4), glucose oxidation would be increased (21), and glutamine oxidation would be decreased (21). Thus, whereas glucose contributed 62% of the ATP when glucose and glutamine were both present at 5 mmol/l, glucose is expected to contribute an even greater proportion of the ATP under fasting conditions. The energy yield from glucose immediately after meal consumption could be estimated also. After food consumption, glucose concentrations in the jejunal lumen have been reported to reach mmol/l (9, 1), and luminal glutamine concentrations may be as high as 5 mmol/l (1). We found that flux through glycolysis was relatively stable at glucose concentrations exceeding 5 mmol/l (Fig. 1), and we reported previously the influence of high glucose concentrations on glucose and glutamine oxidation (20, 21). If the calculations are made using estimated values for glyco-

9 G976 GLYCOLYSIS IN ISOLATED RAT ENTEROCYTES lytic flux, glucose oxidation, and glutamine oxidation, glucose is still shown to contribute 60% of the net ATP for jejunal epithelial cells under these fed conditions, also. Thus it appears that glucose may provide at least 60% of the net ATP from these two major substrates under a range of usual physiological conditions. Substantially more energy was produced from exogenous substrates when both glucose and glutamine were available to the cells than when only glutamine was present (Table 5). Whether the presence of exogenous fuels increases the total energy available (via metabolism of both endogenous and exogenous fuels) is unknown, although previous work by us (1), Ashy and Ardawi (2), Mallet et al. (27), and Watford et al. (46) has shown that glucose and glutamine increase oxygen uptake by small intestine epithelial cells. Providing exogenous substrates has been shown to increase ATP turnover and spare endogenous fuels in newborn pig enterocytes (4). Alanine does not inhibit glycolysis in intestinal epithelial cells. Alanine is known to inhibit pyruvate kinase in a variety of cells, including intestinal epithelial cells (42). Because of the potential for alanine concentrations in and around these cells to be elevated by the presence of glutamine (Fig. ), the potential for alanine to influence energy production was considered. Alanine did not influence flux of glucose through glycolysis (Table ) even when alanine was present at concentrations greatly exceeding the plasma concentration of 0. mmol/l (2, 50). Also, alanine did not influence the glucose oxidation to CO 2 (Fig. 2). Thus it appears that alanine does not inhibit glycolysis in intestinal epithelial cells as it does in other organs. This difference may be of strategic importance to intestinal cells since the intestine serves the body by metabolizing glutamine, and alanine is a metabolite of both glucose and glutamine (Figs. 2 and ). The malate/aspartate shuttle vs. glycerol phosphate shuttle. The relative importance of the malate/aspartate shuttle vs. the glycerol phosphate shuttle has been considered. For most of the calculations (Table 5), the malate/aspartate shuttle was assumed to predominate. The general conclusions are unaltered, however, when it is assumed that reducing equivalents are transported from the cytosol into the mitochondria via the glycerol phosphate shuttle. To reach this conclusion, net ATP from glucose metabolism was recalculated assuming that NADH, produced in the cytosol via glycolysis but not reconsumed in the conversion of pyruvate to lactate, would be oxidized in the mitochondria with a net ATP production of 2 mol ATP/mol NADH, rather than ATP as for the malate/aspartate shuttle. The relative contribution of glucose:glutamine was 54:46 for the glycerol phosphate shuttle vs. 62:8 for the malate/aspartate shuttle (Table 5). This demonstrates that glucose generates considerable energy in intestinal epithelial cells, regardless of which shuttle predominates. We expect that the malate/aspartate shuttle will predominate, however, since the jejunal mucosa has been reported to have similar activities for aspartate aminotransferase (also known as glutamateoxaloacetate transaminase) and alanine aminotransferase (also known as glutamate-pyruvate transaminase), and enzyme activities have been reported in both the cytosolic and mitochondrial fractions (4). Our data show, however, that negligible quantities of aspartate are released as a metabolite of glucose or glutamine by the epithelial cells, whereas alanine is a major metabolite of both substrates (Figs. 2 and ). The most likely role for the aspartate aminotransferase enzyme, therefore, is to facilitate the interconversions needed for the malate/aspartate shuttle. It is possible that the glycerol phosphate shuttle is present in these cells also, since glycerol phosphate dehydrogenase activity, the ratelimiting enzyme for this shuttle, has been reported in the mitochondrial fraction of the rat small intestine (24). Because of the high proportion of glucose-derived pyruvate that is converted to lactate, however, the relative flux through the two major shuttles appears to exert little influence on the calculated relative energy contribution of glucose and glutamine to jejunal epithelial cells. Glycolytic flux is not different for aged vs. young rats. Aging (4 vs. 24 mo) did not significantly influence the rate of glycolytic flux in cells of the proximal small intestine (Table 4), and the influence of glutamine was also not different for cells taken from young adult vs. aged adult animals. We reported previously that, in comparison to cells from young animals, jejunal epithelial cells from aged animals consume oxygen at a lower rate (1) and produce CO 2 from glucose at a lower rate (12, 1). Our calculations show that net ATP production from exogenous glucose and glutamine by jejunal cells of aged animals was 90% of the value calculated for jejunal cells of young animals (data in RESULTS). Nonetheless, the proportion of energy calculated to be available to cells from glucose and glutamine was similar for young vs. aged animals (data in RESULTS). Taken together, these data show that all metabolic processes in intestinal epithelial cells are not influenced similarly by the aging process since CO 2 production, but not glycolysis, is influenced by aging. In these studies, [- H]glucose detritiation was found to reliably quantify flux of glucose carbon through glycolysis by isolated rat small intestine epithelial cells. This technique was used to determine the influence of other pyruvate- or acetyl CoA-producing substrates on the conversion of glucose to pyruvate. Glycolytic flux was six- to sevenfold higher in cells of the proximal small intestine than in cells of the distal small intestine, yet, in both segments, glucose was calculated to provide 50% of the net ATP derived from the two major exogenously provided substrates, glucose and glutamine. In the presence of both glucose and glutamine, anaerobic metabolism of glucose provided 95% of the energy derived from glucose. We thank Dr. John D. Cremin, Jr. for helpful discussions and review of this manuscript. We also thank J. Quan, R. Gill, and B. Young for technical assistance, M. Hudes for statistical consultation, I. Hincenbergs for animal care, and Professor B. O. de Lumen for making the radioactive plate scanner available to us. In addition, we

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