Peter J. Reeds

Intestinal Glutamate Metabolism

Although it is well known that the intestinal tract has a high metabolic rate, the substrates that are used to generate the necessary energy remain poorly established, especially in fed animals. Under fed conditions, the quantification of substrate used by the gut is complicated by the fact that potential oxidative precursors are supplied from both the diet and the arterial circulation. To circumvent this problem, and to approach the question of the compounds used to generate ATP in the gut, we combined measurements of portal nutrient balance with enteral and intravenous infusions of [U-(13)C]substrates. We studied rapidly growing piglets that were consuming diets based on whole-milk proteins. The results revealed that 95% of the dietary glutamate presented to the mucosa was metabolized in first pass and that of this, 50% was metabolized to CO(2). Dietary glucose was oxidized to a very limited extent, and arterial glutamine supplied no >15% of the CO(2) production by the portal-drained viscera. Glutamate was the single largest contributor to intestinal energy generation. The results also suggested that dietary glutamate appeared to be a specific precursor for the biosynthesis of glutathione, arginine and proline by the small intestinal mucosa. These studies imply that dietary glutamate has an important functional role in the gut. Furthermore, these functions are apparently different from those of arterial glutamine, the substrate that has received the most attention.

Nutrient-independent and nutrient-dependent factors stimulate protein synthesis in colostrum-fed newborn pigs.

ABSTRACT: We hypothesized that nonnutrient components, including growth factors, present in colostrum contribute to the stimulation of protein synthesis in colostrum-fed neonatal pigs. We studied neonatal pigs fed mature milk, colostrum, or a formula containing a macronutrient composition comparable to that of colostrum for 24 h. We measured the circulating concentrations of insulin, insulin-like growth factor I, glucose, and amino acids at intervals throughout the 24-h period, after which we measured in vivo protein synthesis using a flooding dose of [3H]phenylalanine. The rates of protein synthesis in several tissues measured after 24 h of feeding were greater than those we reported previously after 6 h of feeding. The acute (within 6 h) stimulation of protein synthesis in visceral and skeletal muscle tissues of neonatal pigs fed milk, colostrum, or formula was primarily influenced by nutrient intake and associated with rapid secretion of insulin. Indirect evidence suggests that intestinal absorption of ingested colostral insulin was minimal. However, the sustained increase in tissue protein synthesis between 6 and 24 h coincided with an increase in circulating insulin-like growth factor I. We found a novel, specific stimulation of skeletal muscle and jejunal protein synthesis in colostrum-fed pigs that can be attributed to some nonnutrient component of colostrum.

Substrate oxidation by the portal drained viscera of fed piglets

Fully fed piglets (28 days old, 7-8 kg) bearing portal, arterial, and gastric catheters and a portal flow probe were infused with enteral [U-13C]glutamate ( n = 4), enteral [U-13C]glucose ( n = 4), intravenous [U-13C]glucose ( n = 4), or intravenous [U-13C]glutamine ( n = 3). A total of 94% of the enteral [U-13C]glutamate but only 6% of the enteral [U-13C]glucose was utilized in first pass by the portal-drained viscera (PDV). The PDV extracted 6.5% of the arterial flux of [U-13C]glucose and 20.4% of the arterial flux of [U-13C]glutamine. The production of13CO2(percentage of dose) by the PDV from enteral glucose (3%), arterial glucose (27%), enteral glutamate (52%), and arterial glutamine (70%) varied widely. The substrates contributed 15% (enteral glucose), 19% (arterial glutamine), 29% (arterial glucose), and 36% (enteral glutamate) of the total production of CO2 by the PDV. Enteral glucose accounted for 18% of the portal alanine and 31% of the portal lactate carbon outflow. We conclude that, in vivo, three-fourths of the energy needs of the PDV are satisfied by the oxidation of glucose, glutamate, and glutamine, and that dietary glutamate is the most important single contributor to mucosal oxidative energy generation.Fully fed piglets (28 days old, 7-8 kg) bearing portal, arterial, and gastric catheters and a portal flow probe were infused with enteral [U-(13)C]glutamate (n = 4), enteral [U-(13)C]glucose (n = 4), intravenous [U-(13)C]glucose (n = 4), or intravenous [U-(13)C]glutamine (n = 3). A total of 94% of the enteral [U-(13)C]glutamate but only 6% of the enteral [U- (13)C]glucose was utilized in first pass by the portal-drained viscera (PDV). The PDV extracted 6.5% of the arterial flux of [U-(13)C]glucose and 20.4% of the arterial flux of [U-(13)C]glutamine. The production of (13)CO(2) (percentage of dose) by the PDV from enteral glucose (3%), arterial glucose (27%), enteral glutamate (52%), and arterial glutamine (70%) varied widely. The substrates contributed 15% (enteral glucose), 19% (arterial glutamine), 29% (arterial glucose), and 36% (enteral glutamate) of the total production of CO(2) by the PDV. Enteral glucose accounted for 18% of the portal alanine and 31% of the portal lactate carbon outflow. We conclude that, in vivo, three-fourths of the energy needs of the PDV are satisfied by the oxidation of glucose, glutamate, and glutamine, and that dietary glutamate is the most important single contributor to mucosal oxidative energy generation.

Glutamine and the Bowel

Since the pioneering work of Windmueller and Spaeth, the importance of glutamine to the support of intestinal mucosal metabolic function has become generally accepted. Nevertheless, the mechanisms underlying this role still remain obscure. This paper explores a number of questions: 1) Is glutamine essential for intestinal function? 2) To what extent does this relate to its intermediary metabolism? 3) What is the importance of glutamine as a biosynthetic precursor? 4) Is glutamine supplementation of the nutrient mixture presented to patients of any metabolic or clinical benefit? As a result of this exploratory exercise, the following general conclusions were reached: 1) Much suggestive biochemical and physiologic evidence exists that implies that glutamine, especially systemic glutamine, supports the function of the intestinal mucosal system. 2) Despite the extensive metabolism of this amino acid by the intestinal tissues, most evidence suggests that if glutamine does play a physiologic role in the bowel, it is not compellingly related to its intermediary metabolism. 3) There is, on the other hand, evidence that the mucosal cells not only utilize extracellular glutamine but synthesize the amino acid. Given that inhibition of glutamine synthesis inhibits both proliferation and differentiation of mucosal cell cultures, this suggests some more subtle regulatory role. This notion is supported by the demonstration that glutamine will activate a number of genes associated with cell cycle progression in the mucosa. 4) Despite the accumulated evidence, the mechanisms underlying glutamines function and the question whether glutamine supplementation uniformly benefits mucosal health remain equivocal at best.

Response of skeletal muscle protein synthesis to insulin in suckling pigs decreases with development

The elevated rate of muscle protein deposition in the neonate is largely due to an enhanced stimulation of skeletal muscle protein synthesis by feeding. To examine the role of insulin in this response, hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs. Pigs were infused with 0, 30, 100, or 1,000 ng ⋅ kg-0.66 ⋅ min-1of insulin to mimic the plasma insulin levels observed under fasted, fed, refed, and supraphysiological conditions, respectively. Whole body amino acid disposal was determined from the rate of infusion of an amino acid mixture necessary to maintain plasma essential amino acid concentrations near their basal fasting levels. A flooding dose ofl-[4-3H]phenylalanine was used to measure skeletal muscle protein synthesis. Whole body amino acid disposal increased progressively as the insulin infusion rate increased, and this response was greater in 7- than in 26-day-old pigs. Skeletal muscle protein synthesis was stimulated by insulin, and this response was maximal at a low insulin infusion rate (30 ng ⋅ kg-0.66 ⋅ min-1). The stimulation of muscle protein synthesis by insulin was also greater in 7- than in 26- day-old pigs. These data suggest that muscle protein synthesis is more sensitive to insulin than whole body amino acid disposal. The results further suggest that insulin is a central regulatory factor in the elevated rate of muscle protein deposition and the increased response of skeletal muscle protein synthesis to feeding in the neonate.The elevated rate of muscle protein deposition in the neonate is largely due to an enhanced stimulation of skeletal muscle protein synthesis by feeding. To examine the role of insulin in this response, hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs. Pigs were infused with 0, 30, 100, or 1,000 ng . kg-0.66 . min-1 of insulin to mimic the plasma insulin levels observed under fasted, fed, refed, and supraphysiological conditions, respectively. Whole body amino acid disposal was determined from the rate of infusion of an amino acid mixture necessary to maintain plasma essential amino acid concentrations near their basal fasting levels. A flooding dose of L-[4-3H]phenylalanine was used to measure skeletal muscle protein synthesis. Whole body amino acid disposal increased progressively as the insulin infusion rate increased, and this response was greater in 7- than in 26-day-old pigs. Skeletal muscle protein synthesis was stimulated by insulin, and this response was maximal at a low insulin infusion rate (30 ng . kg-0.66 . min-1). The stimulation of muscle protein synthesis by insulin was also greater in 7- than in 26- day-old pigs. These data suggest that muscle protein synthesis is more sensitive to insulin than whole body amino acid disposal. The results further suggest that insulin is a central regulatory factor in the elevated rate of muscle protein deposition and the increased response of skeletal muscle protein synthesis to feeding in the neonate.

Parenteral nutrition selectively decreases protein synthesis in the small intestine

We investigated the effects of an elemental diet fed parenterally or enterally on total mucosal protein and lactase phlorizin hydrolase (LPH) synthesis. Catheters were placed in the stomach, jugular vein, and carotid artery of 12 3-day-old pigs. Half of the animals were given an elemental regimen enterally and the other half parenterally. Six days later, animals were infused intravenously with [2H3]leucine for 6 h and killed, and the midjejunum of each animal was collected for analysis. The weight of the midjejunum was 8 +/- 1.5 and 17 +/- 1.6 g in parenterally fed and enterally fed piglets, respectively. LPH activities (mumol.min-1.g protein-1) were significantly higher in parenterally vs. enterally fed piglets. Total small intestinal LPH activities were lower in parenterally vs. enterally fed animals. The abundance of LPH mRNA relative to elongation factor-1 alpha mRNA was not different between groups. The fractional synthesis rate of total mucosal protein and LPH was significantly lower in parenterally fed animals (67 +/- 7 and 66 +/- 7%/day, respectively) than in enterally fed animals (96 +/- 7 and 90 +/- 6%/day, respectively). The absolute synthesis rate (the amount of protein synthesized per gram of mucosa) of total mucosal protein was significantly lower in parenterally fed than in enterally fed piglets. However, the absolute synthesis rate of LPH was unaffected by the route of nutrient administration. These results suggest that the small intestine partially compensates for the effects of parenteral feeding by maintaining the absolute synthesis rate of LPH at the same levels as in enterally fed animals.

Amino acid composition of the milk of some mammalian species changes with stage of lactation

To determine whether the amino acid composition of milk changes during lactation, we compared the amino acid pattern (concentration of each individual amino acid relative to the total amino acid concentration) of colostrum with that of mature milk in six mammalian species. In the human, horse, pig and cow, the pattern of amino acids changed between colostrum and mature milk: glutamate, proline, methionine, isoleucine and lysine increased; cystine, glycine, serine, threonine and alanine decreased. In these four species, the total amino acid concentration also decreased 75% between colostrum and mature milk. In the baboon (Papio cynocephalus anubis and Papio cynocephalus anubis/Papio cynocephalus cynocephalus) and rhesus monkey (Macaca mulatta), however, there was little change in the pattern of amino acids between colostrum and mature milk, and total amino acid concentration decreased only about 25% between colostrum and mature milk. Mature milk rather than colostrum was the most similar among the three primates in both amino acid pattern and total amino acid concentration. We conclude, in those species in which total amino acid concentrations decline substantially between colostrum and mature milk, amino acid patterns also change. The presence of a change in amino acid pattern and total amino acid concentration during lactation appears to be unrelated to phylogenetic order.

Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo

The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.

Phenylalanine utilization by the gut and liver measured with intravenous and intragastric tracers in pigs

To investigate intestinal and hepatic metabolism of phenylalanine, four conscious pigs (7.5 kg), bearing arterial, venous, and hepatic portal catheters, were fasted for 12 h and infused with [ phenyl-2H5]phenylalanine via a peripheral vein and [ carboxyl-13C]phenylalanine via the stomach. During the first 6 h of the infusion, the pigs remained fasted and received only the intravenous tracer. During the second 6 h, they received an intragastric infusion of milk replacer and both tracers. In the fasted state, the portal-drained viscera extracted 10% ( P < 0.025) of the arterial [2H5]phenylalanine flow of the pigs. In the fed state, the splanchnic tissues metabolized 45% of the enteral tracer and intestinal metabolism accounted for 76% of the total splanchnic extraction. The tracer-to-tracee ratio of both tracers in apolipoprotein B-100 (apo B-100) phenylalanine was twofold ( P < 0.001) higher than that of hepatic free phenylalanine. The ratios of the two tracers in portal (13C/2H; 1.66) and apo B-100 (1.76) phenylalanine were similar but higher ( P < 0.05) than that of arterial phenylalanine (1.29). We conclude that intestinal metabolism dominates the splanchnic extraction of enteral phenylalanine and that in the fed state, the hepatic protein synthetic precursor pool derives from portal phenylalanine.

Dietary and systemic phenylalanine utilization for mucosal and hepatic constitutive protein synthesis in pigs

The objective of this study was to quantify the utilization of dietary and systemic phenylalanine for mucosal and hepatic constitutive protein synthesis in piglets. Seven female piglets (7.6 kg) bearing arterial, portal, peripheral venous, and gastric catheters were fed a high-protein diet and infused intragastrically with U-13C-labeled protein and intravenously with [2H( phenyl)5]phenylalanine ([2H5]phenylalanine) for 6 h. The isotopic enrichment of the two phenylalanine tracers was measured in arterial and portal blood, in mucosal and hepatic-free and protein-bound phenylalanine, and in very low-density apolipoprotein B-100, albumin, and fibrinogen. The relative isotopic enrichments of the tracers in mucosal-free (ratio of2H5- to U-13C-labeled = 0.20 ± 0.05) and protein-bound (0.32 ± 0.08) phenylalanine differed significantly ( P < 0.01). Although this suggests preferential use of arterial phenylalanine for mucosal protein synthesis, on a molar basis, 59 ± 6% of the mucosal protein was derived from dietary phenylalanine. There were significant differences ( P < 0.025) between the relative labeling of the two tracers in arterial (ratio of2H5- to U-13C-labeled = 1.25 ± 0.48) and portal (ratio of2H5- to U-13C-labeled = 0.72 ± 0.18) phenylalanine. The mean ratio of the two tracers in all proteins of hepatic origin that were analyzed (0.69 ± 0.18) was similar to that of portal phenylalanine. We conclude that in the fed state portal phenylalanine is preferentially used for constitutive as well as secreted hepatic protein synthesis.