John D. McGivan

The effects of ammonium chloride and glucagon on the metabolism of glutamine in isolated liver cells from starved rats

1. Glucagon stimulated gluconeogenesis from glutamine in isolated liver cells to a far greater extent than that from any other amino-acid precursor. 2. Low concentrations of ammonium chloride (less than 1 mM) stimulated glucose production from glutamine. Glucagon further stimulated this glucose production, even in the presence of saturating concentrations of ammonium chloride. 3. In agreement with previous reports, glutamine hydrolysis by isolated mitochondria was found to be stimulated by ammonium chloride. It was found that ammonium chloride activated mitochondrial glutamine hydrolysis at the same concentrations at whict it stimulated glucose production from glutamine in liver cells. The effective activation of glutamine hydrolysis by ammonimum chloride in intact mitochondria was partially inhibited by rotenone and was abolished by uncoupling agents. 4. The addition of glucagon to hepatocytes metabolising glutamine led to a decrease in the intracellular concentration of glutamine and an increase in the intracellular concentration of glutamate. 5. It is likely that glucagon stimulates gluconeogenesis from glutamine by mechanisms which are additional to those that may operate in the stimulation of gluconeogenesis from other amino-acid precursors. It is suggested that both ammonium chloride and glucagon exert their effects on glutamine metabolism by increasing the effective activity of mitochondrial glutaminase (EC 3.5.1.2.).

Evidence for a single common Na+-dependent transport system for alanine, glutamine, leucine and phenylalanine in brush-border membrane vesicles from bovine kidney

The characteristics of the Na+-dependent transport of alanine, glutamine, leucine and phenylalanine were studied in bovine renal brush-border membrane vesicles. Inhibition of the transport of any one of these amino acids by any other was mutually competitive. The Ki value for the inhibition of alanine transport by leucine was similar to the Km for leucine transport; similar interrelationships existed for the other amino acids. Each amino acid was shown to exchange with each of the other amino acids across the membrane. From these and other results it is concluded that the Na+-dependent transport of these four amino acids is catalysed by a single common transport system.

The bovine renal epithelial cell line NBL-1 expresses a broad specificity Na+-dependent neutral amino acid transport system (System B°) similar to that in bovine renal brush border membrane vesicles

(1) In the bovine renal epithelial cell line NBL-1, transport of alanine and glutamine is highly Na(+)-dependent while the transport of leucine and phenylalanine is also stimulated by Na+ although to a much lesser extent. (2) Na(+)-dependent alanine transport is insensitive to inhibition by methyl AIB, lysine and glutamate but is inhibited by a range of other neutral amino acids. (3) Inhibition of Na(+)-dependent alanine transport by glutamine, phenylalanine and leucine is competitive indicating that these amino acids are transported on a common carrier. (4) Amino acid transport in these cells appears to be localised preferentially on the basolateral membrane. (5) The results are consistent with the presence in confluent cells of a broad-specificity Na(+)-dependent neutral amino acid transport system (System Bo) similar to that in bovine BBMV. An Na(+)-independent system with high activity similar to System L is also present. (6) It is argued that previous results in the literature are consistent with the occurrence of this broad-specificity system in other renal cell lines.

Relationships between skatole and androstenone accumulation, and cytochrome P4502E1 expression in Meishan × Large White pigs

The effects of age, castration and diet on accumulation of skatole and androstenone in the backfat of 50% Meishan male pigs, was studied in relation to boar taint. Skatole and androstenone deposition in backfat of entire males was increased with age (114, 144 and 174 days). Castration significantly decreased skatole and androstenone levels in all age groups. The level of cytochrome P4502E1, the initial enzyme involved in hepatic skatole metabolism, was increased by castration at all ages. These results suggest that the decrease in androstenone levels following castration affects the regulation of cytochrome P4502E1 expression, and as a consequence, results in a decrease in skatole levels. The use of sugar beet feed in the diet decreased the level of skatole and increased that of cytochrome P4502E1 expression, but did not affect androstenone levels. It is suggested that skatole accumulation is reduced both by castration and by diet via induction of cytochrome P4502E1. However, the mechanism of induction of cytochrome P4502E1 by diet is different from that involved in its induction by castration.

Glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas.

This paper describes some properties of glutamine oxidation and glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas. The slow-growing adenoma-derived cell line AA/C1, and the rapidly proliferating carcinoma cell line HT29, both required glutamine for growth. The rate of (14)CO(2) production from [U-(14)C]glutamine was faster in AA/C1 cells than in HT29 cells. Conversely HT29 cells showed faster rates of glucose oxidation and lactate production. The activity of glutaminase was 3 times higher in AA/C1 cell extracts than in extracts of HT29 cells. Glutaminase activity in the two cell lines had similar K(m) values for glutamine, but the activity in AA/C1 cells had a higher K(0.5) for activation by phosphate. Glutaminase activity in extracts of both cells was inhibited by glutamate. Western blotting showed the presence, in both cell lines, of isoform(s) of glutaminase with an molecular mass of 63 kDa, intermediate between that of kidney glutaminase and liver glutaminase. PCR-based analysis showed that an mRNA species identical to the kidney-type isoform glutaminase C was present in both cell types as was an additional mRNA species identical to the liver-type glutaminase isoform from human breast tumour cells. Northern blotting using isoform-specific cDNA probes demonstrated that mRNA for both glutaminase isoforms was expressed at significant levels in both cell types. Similar results to those in AA/C1 cells and HT29 cells were obtained in two further adenoma and carcinoma cell lines respectively. These results contrast with those reported previously in hepatocyte/hepatoma model systems with respect to fuel selection, glutaminase activity and isoform expression. They also constitute the first demonstration of simultaneous expression of two glutaminase isoforms in a single cell type.

Evidence for the existence of an ornithine/citrulline antiporter in rat liver mitochondria

In the urea cycle in rat liver, the enzymes carbamoyl phosphate synthase and ornithine transcarbamylase are situated in the mitochondria while the other enzymes of the cycle are cytoplasmic. Thus during the operation of the urea cycle, it is necessary for ornithine to enter the mitochondria and for citrulline to leave. These two amino acids are presumably transported by specific carrier systems, but the systems involved have not been adequately characterised. Citrulline penetrates the mitochondrial membrane relatively slowly at low concentrations. Accordingly, it was originally suggested that citrulline may cross the mitochondrial membrane rapidly only via an ornithine/citrulline antiport system [l]. Gamble and Lehninger ]a] presented evidence which they interpreted to show the existence of an electrogenic ornithine uniporter and a separate citrulline uniporter. However, Bryla and Harris [33 later showed that the transport of ornithine was not energy-dependent and was not linked in an obligatory manner to citrulline transport. McGivan et al. ]4] showed that in the absence of citr&ine, omithine enters the mitochondrial matrix via an ornithine/H’ antiport system. It is possible that two ornithine transport systems exist: an ornithine/H’ antiporter [4] and an ornithinej citrulline exchange system, In this paper, a method for loading mitochondria with citrulline is described, and evidence for an ornith~e~citru~~ne antiporter is presented.

Stimulation of alanine transport and metabolism by dibutyryl cyclic AMP in the hepatocytes from fed rats Assessment of transport as a potential rate-limiting step for alanine metabolism

(1) Cyclic AMP stimulated alanine transport in isolated hepatocytes by approx. 30%, in the range 0.2-5 mM alanine. (2) Alanine utilisation was also stimulated by cyclic AMP. The rates of transport and metabolism were comparable, both in the presence and absence of cyclic AMP. (3) At concentrations of alanine above 1 mM, addition of ouabain, or the reduction of the Na+ concentration, could partially inhibit transport without affecting the rate of metabolism. (4) At these alanine concentrations, stimulation of metabolism by cyclic AMP was associated with a decrease in the intracellular to extracellular alanine concentration ratio. (5) At alanine concentrations below 0.5 mM, or at higher concentrations when transport was inhibited by reducing the Na+ concentration, cyclic AMP caused an increase in the alanine concentration ratio. (6) It is concluded that at concentrations of alanine above 1 mM, alanine transport is not rate-limiting for alanine metabolism in hepatocytes from fed rats, and cyclic AMP stimulates alanine metabolism primarily by an effect on an intracellular reaction. At physiological concentrations of alanine, however, alanine transport appears to be rate-limiting in agreement with a previous report.

The transport of branched-chain amino acids into isolated rat liver cells

Isolated liver cells may constitute a useful system for the study of the penetration of metabolites across the liver cell plasma membrane. Hepatocytes isolated from rat liver have been used to study the transport of taurocholic acid [I] and of hexoses [2] . Recently, the uptake of the non-metabolisable amino acid analogues a-aminobutyrate and cycloleucine has been investigated in detail [3,4] . Although the liver is one of the major sites of amino acid metabolism in mammalian systems, relatively little is known about the quantitative characteristics of the transport of naturally-occurring amino acids across the liver cell plasma membrane. The kinetics of amino acid transport can studied in isolated liver cells using radioactive substrates, but a major problem in this approach is the prevention of the metabolism of the labelled compound after it has been transported into the cell. However, it is known that the activity of the branched-chain amino acid transaminase in rat liver is very low [5] . The transport of the branched chain amino acids can therefore be studied in liver cells over short time intervals when no significant breakdown of these compounds will occur. In this paper, the characteristics of the uptake of L-leucine, L-isoleucine and L-valine into isolated liver cells are described. 2. Experimental

Induction of High Affinity Glutamate Transport Activity by Amino Acid Deprivation in Renal Epithelial Cells Does Not Involve an Increase in the Amount of Transporter Protein

In renal epithelial cells amino acid deprivation induces an increase in L-Asp transport with a doubling of the V and no change in K (4.5 μM) in a cycloheximide-sensitive process. The induction of sodium-dependent L-aspartate transport was inhibited by single amino acids that are metabolized to produce glutamate but not by those that do not produce glutamate. The transaminase inhibitor aminooxyacetate in glutamine-free medium caused a decrease in cell glutamate content and an induction of glutamate transport. In complete medium aminooxyacetate neither decreased cell glutamate nor increased transport activity. These results are consistent with a triggering of induction of transport by low intracellular glutamate concentrations. High affinity glutamate transport in these cells is mediated by the excitatory amino acid carrier 1 (EAAC1) gene product. Western blotting using antibodies to the C-terminal region of EAAC1 showed that there is no increase in the amount of EAAC1 protein on prolonged incubation in amino acid-free medium. Conversely, the induction of high affinity glutamate transport by hyperosmotic shock was accompanied by an increase in EAAC1 protein. It is proposed that low glutamate levels lead to the induction of a putative protein that activates the EAAC1 transporter. A model illustrating such a mechanism is described.

The transport of alanine and glutamine into isolated rat intestinal epithelial cells.

(1) The transport of alanine and glutamine into isolated rat intestinal epithelial cells, prepared as described previously (Watford, M., Lund, P. and Krebs, H.A. (1979) Biochem. J. 178, 589-596), was studied. (2) Cells isolated by this method accumulated alanine 7-fold from an external concentration of 0.5 mM, and by this criterion appear more suitable for transport studies than do previous rat intestinal cell preparations. (3) In these cells, it was shown using several different approaches that the major part of the transport of alanine and glutamine is mediated by a common carrier which is Na+ dependent and is sensitive to inhibition by 2-methylaminoisobutyric acid. (4) These results are in contrast to the situation in isolated hepatocytes were glutamine is transported by a carrier system (System N) distinct from that that which mediates the transport of alanine. (5) It is suggested that a major metabolic function of this transport system in intestinal cells is the exchange of extracellular glutamine for intracellular alanine, which is a major product of glutamine metabolism in the gut.