Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

Abstract

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22mumol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7-0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37 degrees C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.

Fuel utilization in colonocytes of the rat

“…The proportion ofglucose converted into lactate was 93 % in the presence of glutamine, 92% in the presence of acetoacetate and 96% in the presence ofn-butyrate (Table 1). Thus it is likely that glucose oxidation in colonocytes accounts for a very small percentage ofglucose utilization and does not replace the oxidation of endogenous fuels, which is similar to the findings with a variety of intestinal and colonic preparations (Windmueller & Spaeth, 1978;Hanson & Parsons, 1978;Watford et al, 1979;Porteous, 1980). The addition of glucose plus glutamine to incubated coloncytes caused an increase in the yield of both aspartate and alanine, but a decrease in glutamate production.…”

“…Thus hexokinase activity may not be indicative of the maximum capacity of glycolysis in colonocytes. This rate ofglucose utilization is considerably higher than that reported for thymoctyes (Culvenor & Weidemann, 1976;Yassmeen et al, 1977;Hume et al, 1978;Brand et al, 1984), lymphocytes (Ardawi & Newsholme, 1983a, 1984a), but about 50%O lower than that reported for enterocytes (Watford et al, 1979;Porteous, 1980). About 83 % of the glucose removed was accounted for as lactate and small amounts of pyruvate.…”

“…Themajor end-products of glutamine metabolism in colonocytes are glutamate, aspartate, alanine and ammonia (Table 1). This is in contrast with the cells of the small intestine, which produce alanine rather than aspartate (Hanson & Parsons, 1977;Watford et al, 1979Watford et al, , 1984Porteous, 1980), and lymphocytes or thymocytes, in which aspartate production predominates (Ardawi & Newsholme, 1983a, 1984aBrand et al, 1984). Of the glutamine utilized by colonocytes, ammonia production accounted for 38% of the glutamine nitrogen, which, since the glutaminase reaction produces ammonia, suggests that glutamate is metabolized via transaminase reactions rather than by glutamate dehydrogenase.…”

“…All spectrophotometric measurements were performed in a Gilford recording spectrophotometer (model 240) at 25°C, except for glucose-6-phosphatase and glutaminase, which were determined at 30 and 37°C respectively. For all enzymes studied, preliminary experiments established that extraction and assay procedures were such as to provide maximum enzyme activities (see Crabtree et al, 1979 (Roediger & Truelove, 1979;Roediger, 1982), but higher than that reported for human colonocytes (Roediger & Truelove, 1979), rat lymphocytes (Ardawi & Newsholme, 1983a) or rat thymocytes (Hume et al, 1978), and similar to that observed in rat enterocytes (Watford et al, 1979;Porteous, 1980). Glutamine, n-butyrate or ketone bodies were the only.substrates that increased the rate of 02 consumption (Table 1).…”

The vascularly and luminally perfused small intestine in vitro: dissection technique and model system

“…Third, for some investigations the perfused intestine seems an inappropriate experimental system, e.g. where it is necessary to isolate epithelial cells or subpopulations of these cells (Towler et al, 1978;Porteous, 1979;Porteous et al, 1979;Porteous, 1980;Morrison & Porteous, 1980;Porteous et al, 1980). Fourth, establishing all the necessary techniques for preparing the intestine for perfusion takes time and particular attention to detail.…”