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 77843-1817
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ABSTRACT |
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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-14C]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 concentration-dependent 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
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INTRODUCTION |
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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 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).
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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.
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MATERIALS AND METHODS |
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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 obtained from Sigma Chemical (St. Louis, MO). L-[U-14C]proline, L-[U-14C]glutamine, and [3H]inulin were purchased from American Radiolabeled Chemicals (St. Louis, MO). HPLC-grade 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% O2-5% CO2) Ca2+-free Krebs-Henseleit-bicarbonate (KHB) buffer supplemented with 5 mM EDTA and 5 mM glucose. The jejunum was placed in a flask containing Ca2+-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 CaCl2 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
(2.7 × 103
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
(Km) and
maximal velocity
(Vmax) 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 1 or 5 mM.
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 D-glucose, 2 mM L-glutamine, 2 mM L-[U-14C]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 [14C]proline into [14C]ornithine, [14C]citrulline, and [14C]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 14C-labeled amino acids in cells plus medium at the end of a 30-min incubation period, 0.2 ml of 1.5 M HClO4 was added to the incubation medium to terminate the reaction, followed by addition of 0.1 ml of 2 M K2CO3. Neutralized extracts were used for amino acid analysis by HPLC and for quantification of [14C]ornithine, [14C]citrulline, and [14C]arginine, as previously described (24). [14C]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-14C]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 [3H]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 HClO4 (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 14C and 3H using a dual-channel program in a Packard liquid scintillation counter (27). A small amount of 3H radioactivity in the acid layer was used to correct for contamination by the incubation medium, and glutamine uptake was calculated on the basis of 14C radioactivity in the acid layer and the specific activity of [14C]glutamine in the incubation medium. Uptake of proline by enterocytes was measured as described for glutamine uptake, except that 2 mM [U-14C]glutamine and 1 mM amino-oxyacetate were replaced with 2 mM [U-14C]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 glutamine-derived 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 [14C]glutamine-derived [14C]glutamate or [14C]proline-derived [14C]P5C that was released to the incubation medium represented 3.4 and 2.8% of total intracellular 14C radioactivity, respectively, indicating that intracellular accumulation of 14C was a valid indicator of uptake of [14C]glutamine or [14C]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 3.75 -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
MgCl2, 5 mM
N-acetylglutamate, 20 mM
NH4Cl, 5 mM ornithine, 100 mM
NaHCO3, 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 MgSO4, 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.
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RESULTS |
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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 Km and
Vmax values of
proline oxidase in pig enterocytes were determined to be 3.39 ± 0.25 mM and 49.4 ± 9.4 nmol · min1 · mg
protein
1, respectively, in
the absence of lactate. Similarly, apparent Km and
Vmax values of
pig liver proline oxidase were determined to be 2.58 ± 0.21 mM and
2.16 ± 0.14 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
(Vmax value) but
did not affect its affinity for proline (unaltered
Km value) in pig
enterocytes (Fig. 2) and liver (Fig. 3). In contrast, pyruvate or
products of intestinal proline metabolism (ornithine, citrulline, and
arginine) had no effect (P > 0.05)
on enterocyte proline oxidase activity (Table 2).
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Synthesis of ornithine, citrulline, and arginine from proline.
Radiochemical analysis of
[14C]proline products
showed that large amounts of
[14C]ornithine and
[14C]citrulline and to
a lesser extent
[14C]arginine were
formed from 2 mM
[U-14C]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
[14C]ornithine,
[14C]citrulline,
[14C]arginine, and
total [14C]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
concentration-dependent manner (Table 4). Proline utilization, measured
on the basis of proline disappearance from the incubation medium, was
16.0 ± 1.4, 15.3 ± 1.6, 12.7 ± 1.1, and 10.4 ± 0.86 nmol · 30 min1 · 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.
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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.
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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.
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DISCUSSION |
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Endogenous synthesis of arginine has recently attracted considerable interest (32) since the discovery in 1988 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 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 Vmax and unaltered Km) (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 Km value) without altering Vmax (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).
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 [14C]ornithine, [14C]citrulline, [14C]arginine, and total [14C]P5C from [U-14C]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.
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ACKNOWLEDGEMENTS |
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We thank Wene Yan, Edward Gregg, and Sean Flynn for technical assistance and Frances Mutscher for secretarial support.
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FOOTNOTES |
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This research was supported in part by grants from the United States Department of Agriculture (97-35206-5096) and the American Heart Association (9740124N). G. Wu is an established investigator of the American Heart Association.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Wu, 212 Kleberg Bldg., Dept. of Animal Science, Texas A&M Univ., College Station, TX 77843-2471 (E-mail: g-wu{at}tamu-edu).
Received 17 October 1998; accepted in final form 22 December 1998.
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REFERENCES |
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---|
1.
Adams, E.,
and
L. Frank.
Metabolism of proline and the hydroxyprolines.
Annu. Rev. Biochem.
49:
1005-1061,
1980[Medline].
2.
Alican, I.,
and
P. Kubes.
A critical role for nitric oxide in intestinal barrier function and dysfunction.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G225-G237,
1996
3.
Bradford, N. M.,
and
J. D. McGivan.
The transport of alanine and glutamine into isolated rat intestinal epithelial cells.
Biochim. Biophys. Acta
689:
55-62,
1982[Medline].
4.
Byrd, D. J.,
H.-P. Krohn,
L. Winkler,
C. Steinborn,
M. Hadam,
J. Brodehl,
and
D. H. Hunneman.
Neonatal pyruvate dehydrogenase deficiency with lipoate responsive lactic acidaemia and hyperammonaemia.
Eur. J. Pediatr.
148:
543-547,
1989[Medline].
5.
Cerra, F. B.,
J. Caprioli,
J. H. Siegel,
R. R. McMenamy,
and
J. R. Border.
Proline metabolism in sepsis, cirrhosis and general surgery.
Ann. Surg.
190:
577-586,
1979[Medline].
6.
Davis, P. K.,
and
G. Wu.
Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
119B:
527-537,
1998[Medline].
7.
Davis, T. A.,
H. V. Nguyen,
R. Garcia-Bravo,
M. L. Fiorotto,
E. M. Jackson,
D. S. Lewis,
D. R. Lee,
and
P. J. Reeds.
Amino acid composition of human milk is not unique.
J. Nutr.
124:
1126-1132,
1994[Medline].
8.
DeVivo, D. C.,
M. W. Haymond,
M. P. Leckie,
Y. L. Bussmann,
D. B. McDougal, Jr.,
and
A. S. Pagliara.
The clinical and biochemical implications of pyruvate carboxylase deficiency.
J. Clin. Endocrinol. Metab.
45:
1281-1296,
1977[Abstract].
9.
Flynn, N. E.,
and
G. Wu.
An important role for endogenous synthesis of arginine in maintaining arginine homeostasis in neonatal pigs.
Am. J. Physiol.
271 (Regulatory Integrative Comp. Physiol. 40):
R1149-R1155,
1996
10.
Gianotti, L.,
J. W. Alexander,
T. Pyles,
and
R. Fukushima.
Arginine-supplemented diets improve survival in gut-derived sepsis and peritonitis by modulating bacterial clearance.
Ann. Surg.
217:
644-654,
1993[Medline].
11.
Halestrap, A. P.,
and
R. M. Denton.
Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by -cyano-4-hydroxycinnamate.
Biochem. J.
138:
313-316,
1974[Medline].
12.
Halestrap, A. P.,
and
R. C. Poole.
The transport of pyruvate and lactate across mitochondrial and plasma membranes.
In: Anion Transport Protein of the Red Blood Cell Membrane, edited by N. Hamasaki,
and M. L. Jennings. Amsterdam: Elsevier, 1989, p. 73-86.
13.
Haworth, J. C.,
T. L. Perry,
J. P. Blass,
S. Hansen,
and
N. Urquhart.
Lactic acidosis in three sibs due to defects in both pyruvate dehydrogenase and -ketoglutarate dehydrogenase complexes.
Pediatrics
58:
564-572,
1976[Abstract].
14.
Herzfeld, A.,
V. A. Mezl,
and
W. E. Knox.
Enzymes metabolizing 1-pyrroline-5-carboxylate in rat tissues.
Biochem. J.
166:
95-103,
1977[Medline].
15.
Kowaloff, E. M.,
J. M. Phang,
A. S. Granger,
and
S. J. Downing.
Regulation of proline oxidase activity by lactate.
Proc. Natl. Acad. Sci. USA
74:
5368-5371,
1977[Abstract].
16.
Marliss, E. B.,
T. T. Aoki,
C. J. Toews,
P. Felig,
J. J. Connon,
J. Kyner,
W. E. Huckabee,
and
G. F. Cahill, Jr.
Amino acid metabolism in lactic acidosis.
Am. J. Med.
52:
474-481,
1972[Medline].
17.
Moncada, S.,
and
A. Higgs.
The L-arginine-nitric oxide pathway.
N. Engl. J. Med.
329:
2002-2012,
1993
18.
Raul, F.,
M. Galluser,
R. Schleiffer,
F. Gosse,
M. Hasselmann,
and
N. Seiler.
Beneficial effects of L-arginine on intestinal epithelial restitution after ischemic damage in rats.
Digestion
56:
400-405,
1995[Medline].
19.
Samuels, S. E.,
K. S. Acton,
and
R. O. Ball.
Pyrroline-5-carboxylate reductase and proline oxidase activity in the neonatal pig.
J. Nutr.
119:
1999-2004,
1989[Medline].
20.
Segel, I. H.
Simple inhibition systems.
In: Enzyme Kinetics. New York: Wiley-Interscience, 1975, p. 100-160.
21.
Steel, R. G. D.,
and
J. H. Torrie.
Principles and Procedures of Statistics. New York: McGraw-Hill, 1980.
22.
Tamion, F.,
V. Richard,
S. Lyoumi,
M. Daveau,
G. Bonmarchand,
J. Leroy,
C. Thuillez,
and
J. P. Lebreton.
Gut ischemia and mesenteric synthesis of inflammatory cytokines after hemorrhagic or endotoxic shock.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G314-G321,
1997
23.
Tiao, G.,
S. Hobler,
J. J. Wang,
T. A. Meyer,
F. A. Luchette,
J. E. Fischer,
and
P.-O. Hasselgren.
Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.
J. Clin. Invest.
99:
163-168,
1997
24.
Wu, G.
Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1382-G1390,
1997
25.
Wu, G.
Intestinal mucosal amino acid catabolism.
J. Nutr.
128:
1249-1252,
1998
26.
Wu, G.,
P. K. Davis,
N. E. Flynn,
D. A. Knabe,
and
J. T. Davidson.
Endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in postweaning growing pigs.
J. Nutr.
127:
2342-2349,
1997
27.
Wu, G.,
and
N. E. Flynn.
Regulation of glutamine and glucose by cell volume in lymphocytes and macrophages.
Biochim. Biophys. Acta
1243:
343-350,
1995[Medline].
28.
Wu, G.,
and
D. A. Knabe.
Free and protein-bound amino acids in sow's colostrum and milk.
J. Nutr.
124:
415-424,
1994[Medline].
29.
Wu, G.,
and
D. A. Knabe.
Arginine synthesis in enterocytes of neonatal pigs.
Am. J. Physiol.
269 (Regulatory Integrative Comp. Physiol. 38):
R621-R629,
1995
30.
Wu, G.,
D. A. Knabe,
and
N. E. Flynn.
Synthesis of citrulline from glutamine in pig enterocytes.
Biochem. J.
299:
115-121,
1994[Medline].
31.
Wu, G.,
D. A. Knabe,
N. E. Flynn,
W. Yan,
and
S. P. Flynn.
Arginine degradation in developing porcine enterocytes.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G913-G919,
1996
32.
Wu, G.,
and
S. M. Morris, Jr.
Arginine metabolism: nitric oxide and beyond.
Biochem. J.
336:
1-17,
1998[Medline].