Lactate inhibits citrulline and arginine synthesis from proline in pig enterocytes

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1 Lactate inhibits citrulline and arginine synthesis from proline in pig enterocytes E. LICHAR DILLON, 1 DARRELL A. KNABE, 1 AND GUOYAO WU 1,2 1 Department of Animal Science and Faculty of Nutrition and 2 Departments of Medical Physiology and of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas Dillon, E. Lichar, Darrell A. Knabe, and Guoyao Wu. Lactate inhibits citrulline and arginine synthesis from proline in pig enterocytes. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G1079 G1086, Hypocitrullinemia and hypoargininemia but hyperprolinemia are associated with elevated plasma concentration of lactate in infants. Because the small intestine may be a major organ for initiating proline catabolism via proline oxidase in the body and is the major source of circulating citrulline and arginine in neonates, we hypothesized that lactate is an inhibitor of intestinal synthesis of citrulline and arginine from proline. To test this hypothesis, jejunum was obtained from 14-day-old suckling pigs for preparation of enterocyte mitochondria and metabolic studies. Mitochondria were used for measuring proline oxidase activity in the presence of 0 10 mm L-lactate. For metabolic studies, enterocytes were incubated at 37 C for 30 min in Krebs bicarbonate buffer (ph 7.4) containing 5 mm D-glucose, 2 mm L-glutamine, 2 mm L-[U- 14 C]proline, and 0, 1, 5, or 10 mm L-lactate. Kinetics analysis revealed noncompetitive inhibition of intestinal proline oxidase by lactate (decreased maximal velocity and unaltered Michaelis constant). Lactate had no effect on either activities of other enzymes for arginine synthesis from proline or proline uptake by enterocytes but decreased the synthesis of ornithine, citrulline, and arginine from proline in a concentrationdependent manner. These results demonstrate that lactate decreased intestinal synthesis of citrulline and arginine from proline via an inhibition of proline oxidase and provide a biochemical basis for explaining hyperprolinemia, hypocitrullinemia, and hypoargininemia in infants with hyperlactacidemia. proline oxidase; amino acids; intestine; pigs A SEVERE DEFICIENCY of citrulline and arginine (plasma concentrations below detection limit) has been reported in the infant with elevated plasma concentration of lactate (hyperlactacidemia; up to 14 mm) due to an inherited deficiency of pyruvate dehydrogenase activity (4). Life-threatening hyperammonemia occurs in the patient as a result of arginine deficiency and is effectively prevented by exogenous arginine administration (4), suggesting the presence of intact urea cycle enzymes. A deficiency of arginine has also been reported in adult patients with elevated lactate concentrations (16). It is well documented that hyperprolinemia is associated with hyperlactacidemia in humans (4, 8, 13, 16). Interestingly, Kowaloff et al. (15) observed that The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lactate markedly inhibited the activity of rat liver proline oxidase and suggested that such a regulatory effect of lactate could explain in part the in vivo correlation between hyperprolinemia and elevated plasma concentrations of lactate. However, the mechanism for hypocitrullinemia and hypoargininemia in humans with hyperlactacidemia has not been elucidated. Proline oxidase (a mitochondrial enzyme) oxidizes proline to form pyrroline-5-carboxylate (P5C) and is the first key regulatory enzyme involved in proline degradation in mammals (1). This enzyme has been known to be present in the liver, kidney, and brain, and was traditionally thought to be absent from the small intestine of postnatal animals (14). However, we and others have demonstrated the presence of proline oxidase activity in the pig small intestine (19, 24). Indeed, the activity of proline oxidase (expressed on the basis of tissue weight) was 10- and 6-fold greater in the small intestine than in the liver and kidney of the piglet, respectively (19). Furthermore, we have identified the presence of proline oxidase primarily in enterocytes of the small intestine and demonstrated the synthesis of citrulline and arginine from proline in these cells (24, 25) (Fig. 1). On the basis of tissue distribution of proline oxidase activity in the pig, we suggested that the small intestine is the major organ for initiating proline catabolism in the body (24 26). Both metabolic and enzymological evidence indicates that the small intestine is the major source of circulating citrulline for endogenous synthesis of arginine in neonates and adults (32). In light of the foregoing, we hypothesized that lactate inhibits enterocyte proline oxidase, thereby suppressing proline catabolism and synthesis of citrulline and arginine from proline in the small intestine. Such a regulatory effect of lactate may offer a biochemical basis for explaining hypocitrullinemia and hypoargininemia as well as hyperprolinemia in the infant with elevated plasma concentrations of lactate (4). This hypothesis was tested with use of the suckling pig, an excellent animal model for studying infant nutrition and metabolism (24, 29). Our results demonstrated that lactate markedly inhibited intestinal proline oxidase and decreased the synthesis of citrulline and arginine from proline in enterocytes. MATERIALS AND METHODS Chemicals. L-Amino acids, L-lactic acid, D-glucose, o-aminobenzaldehyde, ferrocytochrome c (from horse heart), BSA (fraction V, essentially fatty acid free), HEPES, EDTA, dithiothreitol (DTT), phenylmethylsulfonyl fluoride, aprotinin, chymostatin, pepstatin A, inulin, and sucrose were ob /99 $5.00 Copyright 1999 the American Physiological Society G1079

2 G1080 LACTATE AND PROLINE METABOLISM Fig. 1. Synthesis of ornithine, citrulline, and arginine from proline in pig enterocytes. Enzymes that catalyze the indicated reactions are 1) proline oxidase, 2) ornithine aminotransferase, 3) ornithine carbamoyl transferase, 4) argininosuccinate synthase, 5) argininosuccinate lyase, and 6) carbamoyl phosphate synthase I. Reaction 6 requires N-acetylglutamate (synthesized from glutamate and acetyl- CoA) as an essential activator for carbamoyl phosphate synthase I. In pig enterocytes, glutamine metabolism provides glutamate, ammonia, aspartate, and ATP for conversion of proline into citrulline and arginine (30). CP, carbamoyl phosphate. tained from Sigma Chemical (St. Louis, MO). L-[U- 14 C]proline, L-[U- 14 C]glutamine, and [ 3 H]inulin were purchased from American Radiolabeled Chemicals (St. Louis, MO). HPLCgrade methanol and water were obtained from Fisher Scientific (Houston, TX). Dowex AG 50W-X8 (H form) was obtained from Bio-Rad (Richmond, CA) and converted to Na form by eluting a 10-ml resin bed with 30 ml of 1 M NaOH, followed by elution with 30 ml water (until effluent ph 9.0). Animals and preparation of enterocytes. Pigs were offsprings of Yorkshire X Landrace sows and Duroc X Hampshire boars and were housed in Texas A&M University Veterinary Research Park. Suckling pigs were allowed to be nursed freely by their sows and killed at 14 days of age for isolation of the intestine and liver as previously described (24). Briefly, pigs received intramuscular injections of atropine (0.05 mg/kg body wt) and then ketamine and acetylpromazine (4.76 and 0.24 mg/kg body wt, respectively). After this preanesthetic procedure, 5% halothane was administered via a face mask to achieve a surgical plane of anesthesia. The abdomen was opened, and the jejunum was removed. The lumen of jejunum (50 cm) was washed three times with saline and then filled with oxygenated (95% O 2-5% CO 2 )Ca 2 -free Krebs-Henseleit-bicarbonate (KHB) buffer supplemented with 5 mm EDTA and 5 mm glucose. The jejunum was placed in a flask containing Ca 2 -free KHB buffer and incubated in a shaking water bath (37 C, 70 oscillations/min) for 20 min. At the end of the 20-min incubation period, jejunum was patted gently with fingertips for 1 min, and enterocyte suspension was drained into a polypropylene tube. Our cell isolation technique removed enterocytes from along midvillus and villus tip of the jejunum, as determined by examining the morphology of intestinal segments before and after cell isolation (29). Enterocytes were washed three times with oxygenated KHB buffer (EDTA-free) containing 2.5 mm CaCl 2 and 20 mm HEPES (ph 7.4), by centrifugation at 600 g for 2 min and then suspended in this KHB buffer. Determination of proline oxidase activity. Enterocytes ( 50 mg protein) or liver ( 0.5 g) were washed three times in 10 ml buffer (in mm, 250 sucrose, 1 EDTA, 50 potassium phosphate buffer, ph 7.2) by centrifugation at 600 g and 4 C for 2 min. Enterocytes or liver was homogenized in 4-ml homogenization buffer (in mm, 250 sucrose, 1 EDTA, 2.5 DTT, 50 potassium phosphate buffer, ph 7.2). Protease inhibitors (5 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml chymostatin, 5 µg/ml pepstatin A) were added to the homogenization buffer to prevent enzyme degradation. The homogenates were centrifuged at 600 g, 4 C, for 10 min. The supernatant was centrifuged at 12,000 g, 4 C, for 10 min. The resultant mitochondrial pellets were suspended in 1.5 ml of 50 mm potassium phosphate buffer (ph 7.5), stored at 80 C, and used for enzyme assay within 3 days. Proline oxidase activity was determined as previously described (24). Briefly, the enzyme assay mixture (1.0 ml), which consisted of 15 mm proline, 20 µm ferrocytochrome c, mitochondrial pellet ( 0.5 mg protein), and 50 mm potassium phosphate buffer (ph 7.5), was incubated at 37 C for 0, 15, or 30 min. Reaction was terminated by addition of 0.5 ml of 10% TCA followed by addition of 0.1 ml of 100 mm o-aminobenzaldehyde. The mixture was allowed to stand at room temperature for 30 min before centrifugation at 600 g for 5 min. The absorbance of the supernatant was measured at 440 nm. Blanks (0-min incubation) were subtracted from sample values before calculating the formation of P5C from proline on the basis of the molar extinction coefficient of P5C ( M 1 cm 1 ). As previously noted (24), blank values of the proline oxidase assay for 0-min incubations were similar to those for incubated blanks without added proline substrate, and sonification of mitochondrial suspension (5 or 10 pulses, output 4, Branson Sonifier- 450, 4 C) after a 3-day storage at 80 C did not further increase proline oxidase activity. Proline oxidase activity was similar between fresh sonicated and 80 C frozen (or 80 C frozen sonicated) mitochondrial suspensions. To determine the Michaelis constant (K m ) and maximal velocity (V max )of proline oxidase, the enzyme assay mixture contained 0.5, 1, 2, 5, 7.5, 10, 15, or 20 mm proline. To determine the effect of lactate on proline oxidase activity, the enzyme assay mixture contained 0, 1, 5, or 10 mm lactate. Lactate stock solution was adjusted to ph 7.5 with 10 mm NaOH before addition to the assay mixture. To determine the effect of pyruvate, ornithine, citrulline, or arginine on proline oxidase activity, these metabolites were added individually to the enzyme assay mixture at 1or5mM. Proline catabolism in enterocytes. Incubations were performed at 37 C for 0 or 30 min in triplicate in 25-ml polypropylene flasks placed in a shaking water bath. Incubation medium (2 ml KHB buffer) contained 1% BSA, 5 mm

3 LACTATE AND PROLINE METABOLISM G1081 D-glucose, 2 mm L-glutamine, 2 mm L-[U- 14 C]proline, or 0, 5, or 10 mm L-lactate. These concentrations of lactate were chosen for enterocyte incubations to mimic plasma lactate concentrations (up to 14 mm) in the infant with hypocitrullinemia and hypoargininemia (4). Lactate stock solution was adjusted to ph 7.4 with 10 mm NaOH before addition to the incubation medium. Glutamine was added to the incubation medium for provision of ammonia, glutamate, aspartate, and ATP, which are all required for conversion of [ 14 C]proline into [ 14 C]ornithine, [ 14 C]citrulline, and [ 14 C]arginine (24). For measurement of P5C in cells plus medium at the end of a 30-min incubation period, 0.5 ml of 10% TCA was added to the incubation medium to terminate the reaction, followed by addition of 0.1 ml of 100 mm o-aminobenzaldehyde. The absorbance of the supernatant at 440 nm was measured, and after subtraction from the blank value (0 min incubation) it was used to calculate net P5C accumulation by enterocytes. To determine amino acids and 14 C-labeled amino acids in cells plus medium at the end of a 30-min incubation period, 0.2 ml of 1.5 M HClO 4 was added to the incubation medium to terminate the reaction, followed by addition of 0.1 ml of 2 M K 2 CO 3. Neutralized extracts were used for amino acid analysis by HPLC and for quantification of [ 14 C]ornithine, [ 14 C]citrulline, and [ 14 C]arginine, as previously described (24). [ 14 C]P5C was separated by anion-exchange chromatography, and its radioactivity was measured using a Packard liquid scintillation counter as previously described (29). Net synthesis of unlabeled ornithine and citrulline (amino acids not found in proteins) from proline and glutamine was calculated on the basis of concentration differences in medium plus cell extracts between 0- and 30-min incubations in the presence of substrates (29). Because arginine can be formed from net proteolysis and catabolized by incubated enterocytes, net synthesis of unlabeled arginine from proline and glutamine was calculated on the basis of concentration differences in medium plus cell extracts between the presence and absence of substrates after 30-min incubation (29). Uptake of glutamine and proline by enterocytes. Uptake of glutamine was measured as described by Bradford and McGivan (3). Briefly, 1 ml of KHB medium (ph 7.4), which contained enterocytes (2 mg protein), 5 mm glucose, 2 mm glutamine plus [U- 14 C]glutamine (0.05 µci/ml), and 1 mm amino-oxyacetate (an inhibitor of glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase) was incubated at 37 C for 2 min. Cell suspension was prewarmed to 37 C before addition to KHB medium (prewarmed to 37 C). At the end of a 2-min incubation period, 50 µl of [ 3 H]inulin (0.5 µci/ml) plus 100 µg/ml unlabeled inulin (an extracellular marker) were added to the incubation medium, and 0.25 ml of the mixture was immediately transferred in duplicate to a 1.6-ml microcentrifuge tube, which contained 0.7 ml of an oil mixture of bromododecane and dodecane (20:1, vol/vol) overlaid on 0.2 ml of 1.5 M HClO 4 (27). Cells were rapidly separated from the medium through the oil layer into the acid layer by centrifugation (12,000 g, 1 min). The upper layer (incubation medium) was removed and washed three times with KHB buffer. After the oil layer was removed, the acid layer was assayed for 14 C and 3 H using a dual-channel program in a Packard liquid scintillation counter (27). A small amount of 3 H radioactivity in the acid layer was used to correct for contamination by the incubation medium, and glutamine uptake was calculated on the basis of 14 C radioactivity in the acid layer and the specific activity of [ 14 C]glutamine in the incubation medium. Uptake of proline by enterocytes was measured as described for glutamine uptake, except that 2 mm [U- 14 C]glutamine and 1 mm aminooxyacetate were replaced with 2 mm [U- 14 C]proline (0.05 µci/ml) and 0.1 mm gabaculine [an inhibitor of ornithine aminotransferase (OAT) (9)], respectively. Amino-oxyacetate and gabaculine were used to inhibit catabolism of glutaminederived glutamate and proline-derived P5C, respectively, so as to facilitate the measurement of glutamine and proline transport by enterocytes. Preliminary studies showed that the amount of [ 14 C]glutamine-derived [ 14 C]glutamate or [ 14 C]proline-derived [ 14 C]P5C that was released to the incubation medium represented 3.4 and 2.8% of total intracellular 14 C radioactivity, respectively, indicating that intracellular accumulation of 14 C was a valid indicator of uptake of [ 14 C]glutamine or [ 14 C]proline by pig enterocytes. Our preliminary studies also demonstrated that uptake of glutamine and proline by pig enterocytes was linear for up to 3 min at 37 C. Determination of activities of enzymes converting P5C into arginine. Mitochondria and the cytosol were prepared from jejunal enterocytes for determining activities of the enzymes that convert P5C into arginine as previously described (6). These enzymes include OAT, ornithine carbamoyl transferase (OCT), carbamoyl phosphate synthase I (CPS I), argininosuccinate synthase (ASS), and argininosuccinate lyase (ASL) (24). Mitochondrial extracts were used for assays of OAT, OCT and CPS I, whereas the cytosol was used for assays of ASS and ASL. Enzyme assays were performed at 37 C at two protein levels for 0, 10, and 15 min. Briefly, the OAT assay mixture (2 ml) consisted of (in mm) 75 potassium phosphate buffer (ph 7.5), 20 ornithine, 0.45 pyridoxal phosphate, 0 or ketoglutarate, and 5 o-aminobenzaldehyde, and mitochondrial pellet (0.02 mg protein). The assay medium for OCT (2.0 ml) contained 0.1 M potassium phosphate buffer (ph 7.5), 15 mm ornithine, 40 mm carbamoyl phosphate, and mitochondrial extracts (0.04 mg protein). The assay mixture for CPS I (0.5 ml) consisted of 0.15 M potassium phosphate buffer (ph 7.5), 25 mm ATP, 25 mm MgCl 2,5mMN-acetylglutamate, 20 mm NH 4 Cl, 5 mm ornithine, 100 mm NaHCO 3, 10 units of added OCT (from Streptococcus faecalis, Sigma Chemical), and mitochondrial extracts (0.5 mg protein). The ASS assay mixture (0.2 ml) consisted of (in mm) 75 potassium phosphate buffer (ph 7.5), 10 citrulline, 5 aspartate, 5 ATP, and 5 MgSO 4, and cytosolic extracts (0.4 mg protein). The ASL assay mixture (40 µl) contained (in mm) 129 sodium phosphate buffer (ph 7.0), 10 argininosuccinate, and 65 EDTA, and cytosolic extracts (0.1 mg protein). Protein determination. Protein in enterocytes and mitochondrial extracts was determined by a modified Lowry procedure using BSA as a standard (30). Statistical analysis. Results are expressed as means SE. Data were analyzed by one-way ANOVA and Student- Newman-Keuls multiple comparison test or by paired t-test (21). Probability values 0.05 were used to indicate statistical significance. Table 1. Inhibition of proline oxidase activity by L-lactate in pig enterocytes and liver L-Lactate Concentration, mm 5mM Proline Pig Enterocytes 20 mm Proline 5mM Proline Pig Liver 20 mm Proline a a * a a * b b * b b * c c * c c * d d * d d * Values are means SE in nmol min 1 mg protein 1 ; n 6. Means with different letters in column are significantly different (P 0.05) as analyzed by 1-way ANOVA and Student-Newman-Keuls (SNK) test. *P 0.01 vs. 5 mm proline group as analyzed by paired t-test.

4 G1082 LACTATE AND PROLINE METABOLISM Fig. 2. Double reciprocal plot of 1/S vs. 1/V for proline oxidase in pig enterocytes. Data represent means SE, n 6. Enterocyte mitochondria were used for measuring proline oxidase activity in the presence of 0, 1, 5, or 10 mm L-lactate and 1, 2, 5, 10, and 20 mm L-proline, as described in text. Lactate deceased (P 0.05) maximal velocity (V max ) and had no effect (P 0.05) on the Michaelis constant (K m ) of proline oxidase, as analyzed by ANOVA and Student-Newman-Keuls (SNK) test. RESULTS Proline oxidase activity. Large amounts of proline oxidase activity were found in enterocyte mitochondria (Table 1). As previously reported (19, 26), proline oxidase activity in the pig liver was much less (P 0.01) than in enterocytes (Table 1). Proline oxidase activity in pig enterocytes and liver was inhibited (P 0.05) by lactate in a concentration-dependent manner (Table 1). From the double reciprocal plot of 1/S vs. 1/V (Fig. 2), apparent K m and V max values of proline oxidase in pig enterocytes were determined to be Fig. 3. Double reciprocal plot of 1/S vs. 1/V for proline oxidase in pig liver. Data represent means SE, n 5. Liver mitochondria were used for measuring proline oxidase activity in presence of 0, 5, or 10 mm L-lactate and 1, 2, 5, 10, and 20 mm L-proline, as described in text. Lactate deceased (P 0.05) V max and had no effect (P 0.05) on K m of proline oxidase, as analyzed by ANOVA and SNK test. mm and nmol min 1 mg protein 1, respectively, in the absence of lactate. Similarly, apparent K m and V max values of pig liver proline oxidase were determined to be mm and nmol min 1 mg protein 1, respectively, in the absence of lactate (Fig. 3). Results of enzyme kinetics indicated that lactate decreased the maximum velocity of enzyme activity (V max value) but did not affect its affinity for proline (unaltered K m value) in pig enterocytes (Fig. 2) and liver (Fig. 3). In contrast, pyruvate or products of intestinal proline metabolism (ornithine, citrulline, and

5 LACTATE AND PROLINE METABOLISM G1083 Table 2. Effect of pyruvate, ornithine, citrulline, and arginine on proline oxidase activity in pig enterocytes Addition of Amino Acid to Assay Mixture 5 mm Proline 20 mm Proline None * 1 mm Pyruvate * 5 mm Pyruvate * 1mML-Ornithine * 5mML-Ornithine * 1mML-Citrulline * 5mML-Citrulline * 1mML-Arginine * 5mML-Arginine * Values are means SE in nmol min 1 mg protein 1 ; n 4. Addition of each amino acid had no significant effect (P 0.05) on proline oxidase activity as analyzed by 1-way ANOVA. *P 0.01 vs. 5 mm proline group as analyzed by paired t-test. arginine) had no effect (P 0.05) on enterocyte proline oxidase activity (Table 2). Synthesis of ornithine, citrulline, and arginine from proline. Radiochemical analysis of [ 14 C]proline products showed that large amounts of [ 14 C]ornithine and [ 14 C]citrulline and to a lesser extent [ 14 C]arginine were formed from 2 mm [U- 14 C]proline in pig enterocytes (Table 3). Consistent with the inhibition of proline oxidase activity, increasing extracellular lactate concentrations from 0 to 5 or 10 mm decreased (P 0.05) the synthesis of [ 14 C]ornithine, [ 14 C]citrulline, [ 14 C]arginine, and total [ 14 C]P5C by enterocytes in a concentration-dependent manner (Table 3). HPLC analysis of amino acids also revealed that lactate markedly decreased (P 0.05) the formation of ornithine, citrulline, and arginine by enterocytes incubated with proline and glutamine (Table 4). Net accumulation of P5C was also reduced (P 0.05) by lactate in a concentrationdependent manner (Table 4). Proline utilization, measured on the basis of proline disappearance from the incubation medium, was , , , and nmol 30 min 1 mg protein 1 (means SE, n 6), respectively, in the presence of 0, 1, 5, and 10 mm lactate. Lactate at 5 and 10 mm decreased (P 0.05) proline utilization by 21 and 35%, respectively. It should be noted that, in enterocytes from 14-dayold pigs, some of the P5C derived primarily from proline was converted to ornithine, citrulline, and arginine in pig enterocytes (Fig. 1), and thus the P5C measured at the end of 30-min incubation represented Table 4. Effects of lactate on the net accumulation of pyrroline-5-carboxylate (P5C) and net synthesis of ornithine, citrulline, and arginine in pig enterocytes incubated in presence of 2 mm proline and 2 mm glutamine Medium L-Lactate, mm its net accumulation but not its total synthesis (Table 4). Total synthesis of P5C should include net P5C accumulation and formation of ornithine, citrulline, plus arginine. Pig enterocytes contain both proline oxidase (a mitochondrial enzyme) (24) and P5C reductase (a cytosolic enzyme converting P5C to proline) (31), which constitute a potential intracellular proline-p5c cycle. Thus, to quantify P5C at the end of the 30-min incubation, we prefer not to use the term net synthesis of P5C from proline because it may imply a net result of the potential proline-p5c cycle. On the other hand, because ornithine, citrulline, and arginine were further metabolized in enterocytes (32), the measurements of these three amino acids represented their net synthesis but not their total synthesis. Uptake of glutamine and proline. These data are summarized in Table 5. Uptake of glutamine by enterocytes was greater (P 0.01) than that of proline. Lactate had no effect (P 0.05) on uptake of glutamine or proline by enterocytes. Activities of enzymes converting P5C into arginine. These data are summarized in Table 6. OAT and OCT activities were particularly high in pig enterocytes, compared with CPS I, ASS, and ASL. In contrast to proline oxidase, lactate had no effect (P 0.05) on activities of all these enzymes in enterocytes. DISCUSSION Net Accumulation of P5C Net Synthesis Ornithine Citrulline Arginine a a a a a a a a b b b b c c c c Values are means SE in nmol 30 min 1 mg protein 1 ; n 6. Enterocytes were incubated for 30 min in presence of 2 mm L-glutamine, 2 mm L-proline, and 0, 1, 5, or 10 mm L-lactate. Means with different letters in column are significantly different (P 0.05) as analyzed by 1-way ANOVA and SNK test. Endogenous synthesis of arginine has recently attracted considerable interest (32) since the discovery in Table 3. L-Lactate inhibits net synthesis of [ 14 C]ornithine, [ 14 C]citrulline, [ 14 C]arginine, and total synthesis of [ 14 C]pyrroline-5-carboxylate from [U- 14 C]proline in pig enterocytes Medium L-Lactate, mm Net Accumulation of [ 14 C]P5C Net Synthesis From [ 14 C]proline [ 14 C]Orn [ 14 C]Cit [ 14 C]Arg Total Synthesis of [ 14 C]P5C From [ 14 C]proline a a a a a a a a a a b b b b b c c c c c Values are means SE in nmol 30 min 1 mg protein 1 ; n 6. Enterocytes were incubated for 30 min in presence of 2 mm L-glutamine, 2 mm L-[U- 14 C]proline, and 0, 1, 5 or 10 mm L-lactate. [ 14 C]P5C, [ 14 C]pyrroline-5-carboxylate. Means with different letters in column are significantly different (P 0.05) as analyzed by 1-way ANOVA and SNK test.

6 G1084 LACTATE AND PROLINE METABOLISM Table 5. Effect of L-lactate on glutamine and proline uptake by pig enterocytes Lactate Concentration, mm Glutamine Uptake Proline Uptake * * * * Values are means SE in nmol min 1 mg protein 1 ; n 6. Enterocytes were incubated for 2 min in presence of 2 mm L-[U- 14 C]glutamine or [U- 14 C]proline. Incubation medium also contained 0, 1, 5, or 10 mm L-lactate. Intracellular 14 C radioactivity was measured as an indicator of glutamine or proline uptake. L-Lactate had no significant effect (P 0.05) on glutamine or proline uptake by enterocytes as analyzed by 1-way ANOVA. *P 0.01 vs. glutamine uptake as analyzed by paired t-test of arginine as the physiological precursor of nitric oxide (NO), a free radical with enormous physiological, immunological, and pathological importance (17). In addition to glutamine as a substrate for intestinal citrulline synthesis (30), we have recently demonstrated a novel pathway for the synthesis of citrulline and arginine from proline via proline oxidase in enterocytes (24), the major source of circulating citrulline for endogenous arginine synthesis in neonates and adults (32). Because intestinal synthesis of citrulline and arginine from glutamine decreases progressively in neonatal pigs during the suckling period (29, 30), proline is the major substrate for citrulline and arginine synthesis in enterocytes during this period (24 26). The endogenous synthesis of arginine from proline as well as glutamine plays an important role in maintaining arginine homeostasis in neonates (9), because arginine is remarkably deficient in the milk of most mammals, including humans, pigs, and mice (7), and both proline and glutamine are abundant amino acids in the milk (28). As a result, hypocitrullinemia and hypoargininemia occur in animals or humans with intestinal resection or with a defect in intestinal synthesis of citrulline (32). Thus regulation of intestinal synthesis of citrulline and arginine from proline is of great nutritional and physiological significance. Proline oxidase activity is much greater in pig enterocytes than in liver (Table 1) and is greater in the small Table 6. Effect of L-lactate on activities of enzymes converting pyrroline-5-carboxylate into arginine in pig enterocytes Enzyme Lactate, mm OAT OCT CPSI ASS ASL Values are means SE in nmol min 1 mg protein 1 ; n 5. OAT, ornithine aminotransferase; CPSI, carbamoyl-phosphate synthase I; OCT, ornithine carbamoyltransferase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase. L-Lactate had no significant effect (P 0.05) on activities of these enzymes as analyzed by 1-way ANOVA. intestine than in all other porcine tissues examined (26). These results suggest that the small intestine is the major organ for initiating proline catabolism in the body. However, little is known about the regulation of intestinal proline metabolism in animals or humans. The recent report that a severe deficiency of citrulline and arginine occurs in the infant with hyperlactacidemia (4) prompted us to investigate whether lactate inhibited intestinal synthesis of citrulline and arginine from proline. Marliss et al. (16) have also reported an arginine deficiency in adult humans with elevated plasma concentrations of lactate. Interestingly, lactate inhibited proline oxidase activity in the rat liver (15), as in the pig liver (Table 1). Although this result may explain in part hyperprolinemia under conditions associated with high plasma concentrations of lactate (4, 8, 13, 16), the mechanism responsible for hypocitrullinemia and hypoargininemia in the patient with hyperlactacidemia remains unknown. Because there is no net production of citrulline or arginine by the liver due to a very high arginase activity and tight channeling of hepatic urea cycle enzymes (32), inhibition of proline catabolism by lactate in the liver is not likely to contribute to citrulline or arginine deficiency in animals or humans with hyperlactacidemia. Therefore, we determined a possible role of lactate in regulating intestinal proline oxidase and other enzymes that synthesize arginine from proline. An important finding of this study is that lactate at concentrations found in patients with hyperlactacidemia (4, 8, 13, 16) markedly inhibited mitochondrial proline oxidase in pig enterocytes (Table 1, Fig. 2). Among all enzymes that synthesize arginine from proline, proline oxidase was the only enzyme whose activity was inhibited by lactate (Table 6). Neither pyruvate (the immediate product of lactate) nor proline metabolites (ornithine, citrulline, and arginine) affected intestinal proline oxidase activity (Table 2). Because lactate is virtually not metabolized by mitochondria, an inhibition of proline oxidase activity in lysed mitochondria (Table 1) suggests that lactate directly inactivates the enzyme. Kinetics analysis indicated noncompetitive inhibition of intestinal proline oxidase by lactate (decreased V max and unaltered K m ) (Fig. 2), as described by Segel (20). In this class of enzyme inhibition, an inhibitor (e.g., lactate) bears no structural resemblance to the substrate (e.g., proline) but binds to either free enzyme (e.g., proline oxidase) or to enzyme-substrate complex, thus reducing enzyme activity (20). Lactate was also a noncompetitive inhibitor of pig liver proline oxidase (Fig. 3). In contrast, lactate appeared to inhibit rat liver proline oxidase by decreasing the affinity of the enzyme for proline (increased K m value) without altering V max (competitive inhibition) (15). In competitive inhibition, an inhibitor of the enzyme is structurally similar to its substrate (20). Lactate does not resemble proline in structure, and thus it is not clear how lactate could be a competitive inhibitor of rat liver proline oxidase in the previous study (15).

7 LACTATE AND PROLINE METABOLISM G1085 To demonstrate physiological or pathophysiological relevance of inhibition of proline oxidase by lactate, metabolic studies were conducted with enterocytes incubated in the presence of 1 mm lactate (physiological plasma concentrations of lactate) and elevated lactate concentrations (5 and 10 mm) found in plasma of humans with hyperlactacidemia, hypoargininemia, and hyperprolinemia (4). Lactate is readily transported across plasma membrane and mitochondrial membrane (11, 12). Thus increasing extracellular lactate concentrations results in an increase in intracellular and intramitochondrial lactate concentrations (11). Because lactate had no effect on uptake of glutamine or proline by enterocytes (Table 5), proline uptake by mitochondria (15), or enzymes converting P5C to arginine (Table 6), inhibition of intestinal proline catabolism by lactate likely occurs at the level of proline oxidase. Consistent with inhibition of proline oxidase, lactate decreased the synthesis of [ 14 C]ornithine, [ 14 C]citrulline, [ 14 C]arginine, and total [ 14 C]P5C from [U- 14 C]proline by pig enterocytes in a concentration-dependent manner (Table 3). HPLC analysis of amino acids also indicated a decrease in the formation of ornithine, citrulline, and arginine by enterocytes incubated in the presence of 5 and 10 mm lactate (Table 4). Physiological plasma concentrations of lactate (1 mm) had no effect on the synthesis of citrulline and arginine from proline in pig enterocytes (Tables 3 and 4). Because there is limited synthesis of P5C, ornithine, citrulline, and arginine from glutamine in enterocytes of 14-day-old pigs (29, 30), the net accumulation of large amounts of these metabolites in cells incubated in the presence of both glutamine and proline was derived mainly from proline. Thus both radiochemical and HPLC analyses demonstrated a concentration-dependent inhibition by lactate of the synthesis of ornithine, citrulline, arginine, and P5C from proline in pig enterocytes. Similar percentage of proline-derived P5C converted into ornithine (28 30%), citrulline (30 32%), and arginine (5 6%) suggests that lactate did not affect conversion of P5C to these amino acids in enterocytes. This is consistent with our results that lactate had no effect on activities of OAT, OCT, CPS I, ASS, or ASL in enterocytes (Table 6). Because the small intestine may be the major organ for initiating proline catabolism in the body on the basis of tissue distribution of proline oxidase activity in the pig and is almost the exclusive source of circulating citrulline for endogenous arginine synthesis (24 26), our finding of inhibition of intestinal proline catabolism and synthesis of citrulline and arginine from proline provides a hitherto unrecognized metabolic basis for explaining hypocitrullinemia, hypoargininemia, and hyperprolinemia in humans with hyperlactacidemia (4, 16). Results of this study may also have implications to understanding impaired intestinal function and pathophysiology in ischemia and sepsis that are associated with elevated plasma lactate concentrations. When the small intestine is subject to endotoxin or ischemia, local production of lactate by enterocytes or infiltrating immunocytes and plasma concentrations of lactate (up to 10 mm) is markedly increased due to enhanced glycolysis (22). In light of our present findings, an increase in lactate concentrations would result in an inhibition of proline oxidase and a decrease in the synthesis of citrulline and arginine from proline by enterocytes. This would lead to a local deficiency of arginine in intestinal mucosa and consequently to decreased NO synthesis. Inasmuch as NO plays an important role in regulating intestinal barrier function (2), decreased intestinal proline metabolism may contribute to impaired intestinal integrity and injury. In this regard, it is noteworthy that provision of exogenous arginine prevents intestinal damage associated with gut ischemia or sepsis (10, 18). In addition, an inhibition of intestinal proline catabolism may help to explain hypoargininemia (23) and hyperprolinemia (5) associated with elevated plasma concentrations of lactate in humans with sepsis. In summary, results of both enzymological and metabolic studies demonstrate that lactate inhibited intestinal synthesis of citrulline and arginine from proline via an inhibition of proline oxidase. This study of intestinal proline catabolism and the previous study of hepatic proline oxidase (15) together help explain the in vivo correlation between hyperprolinemia and elevated plasma concentrations of lactate in animals and humans. Our findings also provide a novel biochemical basis for explaining hypocitrullinemia, hypoargininemia, and hyperprolinemia in infants with hyperlactacidemia. We thank Wene Yan, Edward Gregg, and Sean Flynn for technical assistance and Frances Mutscher for secretarial support. This research was supported in part by grants from the United States Department of Agriculture ( ) and the American Heart Association ( N). G. Wu is an established investigator of the American Heart Association. Address for reprint requests and other correspondence: G. 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