Douglas G. Burrin

Replication of human noroviruses in stem cell–derived human enteroids

The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell–derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

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.

Level of nutrition and visceral organ size and metabolic activity in sheep

Thirty-two crossbred wether lambs (initial live-weight 31 kg) were fed on a diet (metabolizable energy (ME) 12.8 MJ/kg) ad lib. (ADLIB) or restricted to maintain body-weight (MAINT) for a 21 d period. On days 0, 7, 14 and 21, four lambs per treatment were slaughtered, visceral organs weighed and tissues sampled. During the 21 d period, ME intake in ADLIB lambs increased quadratically with an average rate of live-weight gain of 425 g/d. In MAINT lambs, live weight (30 kg) was maintained, and daily ME intake (kJ/kg empty body-weight (EBW)0.75) declined (P less than 0.01) quadratically with time. Weights of liver, stomach and small intestines as a percentage of EBW were increased in ADLIB lambs and decreased by 10-33% in MAINT lambs (treatment x day, P less than 0.01). In vitro liver oxygen consumption was not affected by level of feed intake. Estimates of whole-liver O2 consumption (mmol O2/d per kg EBW) increased in ADLIB lambs and were relatively constant in MAINT lambs. These findings suggest that level of feed intake changes the relative proportion of visceral organs to body mass. In addition, the effect of level of feed intake on changes in the relative contribution of visceral organs to whole-body metabolic rate appears to be primarily a result of differences in organ size rather than tissue-specific metabolic activity.

GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets

BACKGROUND & AIMS Our aim was to determine whether the intestinotrophic effects of GLP-2 are mediated by acute up-regulation of intestinal substrate utilization in TPN-fed piglets. METHODS Twenty-four 12-day-old pigs, fitted with a portal flow probe and carotid, jugular and portal catheters, were fed by TPN for 7 days. On day 8, a group of pigs (n = 8) was infused intravenously with saline (control) for 4 hours and then with GLP-2 (500 pmol x kg(-1) x hour(-1), GLP-2) for 4 hours. (2)H-glucose and (13)C-phenylalanine were infused to estimate their kinetics and protein turnover. Another group (n = 8) received consecutive intravenous infusions of saline, GLP-2, and GLP-2 plus N(G)-Nitro-L-arginine methyl ester (L-NAME, 50 micromol x kg(-1) x hour(-1)) for 4 hours each. RESULTS GLP-2 acutely increased portal-drained visceral (PDV) blood flow rate (+25%) and intestinal blood volume (+51%) in TPN-fed piglets. GLP-2 also increased intestinal constitutive nitric oxide synthase (NOS) activity and endothelial NOS protein abundance. GLP-2 acutely increased PDV glucose uptake (+90%) and net lactate production (+79%). Co-infusion of GLP-2 plus L-NAME did not increase either PDV blood flow rate or glucose uptake. GLP-2 increased PDV indispensable amino acid uptake by 220% and protein synthesis by 125%, but did not decrease protein breakdown or phenylalanine oxidation. CONCLUSIONS We conclude that in TPN-fed neonatal pigs, GLP-2 acutely stimulates intestinal blood flow and glucose utilization, and this response is nitric oxide-dependent. These findings suggest that GLP-2 may play an important physiological role in the regulation of intestinal blood flow and that nitric oxide is involved in GLP-2 receptor function.

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.

Metabolic fate and function of dietary glutamate in the gut

Glutamate is a main constituent of dietary protein and is also consumed in many prepared foods as an additive in the form of monosodium glutamate. Evidence from human and animal studies indicates that glutamate is a major oxidative fuel for the gut and that dietary glutamate is extensively metabolized in first pass by the intestine. Glutamate also is an important precursor for bioactive molecules, including glutathione, and functions as a key neurotransmitter. The dominant role of glutamate as an oxidative fuel may have therapeutic potential for improving function of the infant gut, which exhibits a high rate of epithelial cell turnover. Our recent studies in infant pigs show that when glutamate is fed at higher (4-fold) than normal dietary quantities, most glutamate molecules are either oxidized or metabolized by the mucosa into other nonessential amino acids. Glutamate is not considered to be a dietary essential, but recent studies suggest that the level of glutamate in the diet can affect the oxidation of some essential amino acids, namely leucine. Given that substantial oxidation of leucine occurs in the gut, ongoing studies are investigating whether dietary glutamate affects the oxidation of leucine in the intestinal epithelial cells. Our studies also suggest that at high dietary intakes, free glutamate may be absorbed by the stomach as well as the small intestine, thus implicating the gastric mucosa in the metabolism of dietary glutamate. Glutamate is a key excitatory amino acid, and metabolism and neural sensing of dietary glutamate in the developing gastric mucosa, which is poorly developed in premature infants, may play a functional role in gastric emptying. These and other recent reports raise the question as to the metabolic role of glutamate in gastric function. The physiologic significance of glutamate as an oxidative fuel and its potential role in gastric function during infancy are discussed.

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.

Effect of level of nutrition on splanchnic blood flow and oxygen consumption in sheep

The objective of the present study was to measure changes in splanchnic blood flow and oxygen consumption in sheep fed on a high-concentrate diet ad lib. (ADLIB) or an amount sufficient to maintain body-weight (MAINT) for 21 d. Eleven ram lambs were surgically implanted with chronic indwelling catheters in the portal, hepatic and mesenteric veins and mesenteric artery to measure blood flow and net O2 flux through the liver and portal-drained viscera (PDV). During the 21 d period, PDV (P less than 0.05) and liver (P less than 0.01) blood flow increased in ADLIB and decreased in MAINT lambs (treatment x day, linear). After 21 d, O2 consumptions in PDV and liver of MAINT lambs were 37 and 63% lower than in ADLIB lambs. In the control period, total splanchnic tissues represented an average of 52% of whole body O2 consumption. After 21 d, the relative contributions of PDV and liver to whole-body O2 consumption were 28 and 41% in ADLIB and 19 and 22% in MAINT lambs respectively. Allometric regression variables indicate that liver O2 consumption responds more rapidly to changes in metabolizable energy intake than portal O2 consumption. These results indicate that blood flow and O2 consumption in both PDV and liver are related to level of nutrition. Furthermore, splanchnic tissues represent a significant component of whole-body O2 consumption that is subject to manipulation by level of nutrition.

Enteral feeding induces diet-dependent mucosal dysfunction, bacterial proliferation, and necrotizing enterocolitis in preterm pigs on parenteral nutrition

Preterm neonates have an immature gut and metabolism and may benefit from total parenteral nutrition (TPN) before enteral food is introduced. Conversely, delayed enteral feeding may inhibit gut maturation and sensitize to necrotizing enterocolitis (NEC). Intestinal mass and NEC lesions were first recorded in preterm pigs fed enterally (porcine colostrum, bovine colostrum, or formula for 20-40 h), with or without a preceding 2- to 3-day TPN period (n = 435). Mucosal mass increased during TPN and further after enteral feeding to reach an intestinal mass similar to that in enterally fed pigs without TPN (+60-80% relative to birth). NEC developed only after enteral feeding but more often after a preceding TPN period for both sows colostrum (26 vs. 5%) and formula (62 vs. 39%, both P < 0.001, n = 43-170). Further studies in 3-day-old TPN pigs fed enterally showed that formula feeding decreased villus height and nutrient digestive capacity and increased luminal lactic acid and NEC lesions, compared with colostrum (bovine or porcine, P < 0.05). Mucosal microbial diversity increased with enteral feeding, and Clostridium perfringens density was related to NEC severity. Formula feeding decreased plasma arginine, citrulline, ornithine, and tissue antioxidants, whereas tissue nitric oxide synthetase and gut permeability increased, relative to colostrum (all P < 0.05). In conclusion, enteral feeding is associated with gut dysfunction, microbial imbalance, and NEC in preterm pigs, especially in pigs fed formula after TPN. Conversely, colostrum milk diets improve gut maturation and NEC resistance in preterm pigs subjected to a few days of TPN after birth.

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.