Further studies on amino acid transport in murine P388 leukemia cells in vitro. Presence of system y+

The transport of glycine and L-lysine into murine P388 leukemia cells has been examined. Glycine transport appears to be shared by both systems A and ASC in P388 cells. Glycine transport is Na+-dependent and is effectively blocked by alpha-(methylamino)isobutyric acid, threonine and alanine but only a marginal reduction in transport is seen with 100-fold excess cold 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. System gly is not expressed in P388 cells. Lysine is largely transported by a Na+-independent, pH-insensitive system with a Km of 0.079 mM. Lysine transport is relatively unaffected by the addition of 100-fold excess cold alpha-(methylamino)isobutyric acid, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and the anionic amino acids, L-glutamate and L-aspartate. A partial inhibition of lysine transport was observed with L-threonine and L-leucine while L-arginine and L-histidine radically decreased lysine transport. Lysine appears to be transported by a system similar to the system y+ seen in cultured human fibroblasts, Ehrlich ascites cells, and hepatoma cell lines.     

Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line

The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80–86% and ME1 enzyme activity by 78–86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells.     

Interference of a nonmetabolized analogue of L-leucine with lipid metabolism in tumoral pancreatic islet cells

The nonmetabolized analogue of L-leucine, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), was recently found to inhibit O2 uptake and insulin release from tumoral islet cells of the RINm5F line. BCH inhibited lipogenesis, stimulated lipolysis, and severely decreased the oxidation of endogenous [U-14C]palmitate in prelabelled RINm5F cells. D-Glucose exerted metabolic effects which were sometimes opposite to those caused by BCH and, within limits, protected the islet cells against the inhibitor action of BCH. Since BCH augments NH4+ production and facilitated the catabolism of 14C-labelled amino acids in the prelabelled cells, it is proposed that the unexpected inhibition of O2 uptake by BCH is mainly attributable to a decrease in the oxidation of endogenous fatty acids.     

Alpha-aminocyclic and bicyclic alkane carboxylic acids: differential effects on selected amino acids of rat brain cortex

Abstract— The intraperitoneal administration of 1-aminocyclopentane carboxylic acid, 1-aminocyclohex-ane carboxylic acid, l-aminocycloheptane carboxylic acid, 1-aminocyclooctane carboxylic acid, exo-2-aminobicyclo(2,2. l)heptane-2-carboxylic acid. endo-2-aminobicyclo(2,2.1)heptane-2-carboxylic acid. 2-aminobicyclo(2.2.2)octane-2-carboxylic acid and 2-aminobicyclo(3,2.l)octane-2-carboxylic acid to 18-day-old male rats selectively perturbed the levels of neutral amino acids in the cerebral cortex. While the effect of the above compounds was rather diversified and usually resulted in a reduction of amino acid levels. marked elevations of the levels of valine and isoleucine were also noted. 1-Aminocycloheptane and cyclooctane carboxylic acids were particularly noteworthy, in that they elicited a marked reduction of the levels of cortical phenylalanine.     

Cytotoxicity as an indicator for transport mechanism. Evidence that melphalan is transported by two leucine-preferring carrier systems in the L1210 murine leukemia cell

Melphalan, l-phenylalanine mustard, is transported by the L1210 cell through carriers of the leucine (L) type. Its initial rate of transport is inhibited by both l-leucine, a naturally occurring L system amino acid and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a synthetic amino acid which is transported by the L system in the Ehrlich ascites tumor cell. Both amino acids inhibited melphalan transport comparably in sodium-free medium. However, BCH, in medium containing sodium, was unable to reduce a component of melphalan transport which was readily inhibited by leucine but not by α-aminoisobutyric acid. Inhibition analysis indicated that leucine competes with BCH for transport but that a portion of leucine transport is not readily inhibited by BCH. These results suggest that in the L1210 cell melphalan is transported equally by a BCH-sensitive, sodium-independent L system and a BCH-insensitive, sodium-dependent L system.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Activation of the KATP channel-independent signaling pathway by the nonhydrolyzable analog of leucine,BCH

Leucine and glutamine were used to elicit biphasic insulin release in rat pancreatic islets. Leucine did not mimic the full biphasic response of glucose. Glutamine was without effect. However, the combination of the two did mimic the biphasic response. When the ATP-sensitive K+ (KATP) channel-independent pathway was studied in the presence of diazoxide and KCl, leucine and its nonmetabolizable analog 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) both stimulated insulin secretion to a greater extent than glucose. Glutamine and dimethyl glutamate had no effect. Because the only known action of BCH is stimulation of glutamate dehydrogenase, this is sufficient to develop the full effect of the KATP channel-independent pathway. Glucose, leucine, and BCH had no effect on intracellular citrate levels. Leucine and BCH both decreased glutamate levels, whereas glucose was without effect. Glucose and leucine decreased palmitate oxidation and increased esterification. Strikingly, BCH had no effect on palmitate oxidation or esterification. Thus BCH activates the KATP channel-independent pathway of glucose signaling without raising citrate levels, without decreasing fatty acid oxidation, and without mimicking the effects of glucose and leucine on esterification. The results indicate that increased flux through the TCA cycle is sufficient to activate the KATP channel-independent pathway.     

Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal

Glutamine and glutamate transport activities were measuredin isolated luminal and abluminal plasma membrane vesiclesderived from bovine brain endothelial cells. Facilitativesystems for glutamine and glutamate were almost exclusivelylocated in luminal-enriched membranes. The facilitativeglutamine carrier was neither sensitive to2-aminobicyclo(2,2,1)heptane-2-carboxylic acid inhibition nor did itparticipate in accelerated amino acid exchange; it therefore appearedto be distinct from the neutral amino acid transport system L1. TwoNa-dependent glutamine transporters were found in abluminal-enrichedmembranes: systems A and N. System N accounted for ~80% ofNa-dependent glutamine transport at 100 µM. Abluminal-enriched membranes showed Na-dependent glutamate transport activity. The presence of 1) Na-dependent carrierscapable of pumping glutamine and glutamate from brain into endothelialcells, 2) glutaminase withinendothelial cells to hydrolyze glutamine to glutamate and ammonia, and3) facilitative carriers forglutamine and glutamate at the luminal membrane may provide a mechanismfor removing nitrogen and nitrogen-rich amino acids from brain.

     

Characterization of rabbit aldose reductase-like protein with 3β-hydroxysteroid dehydrogenase activity

To evaluate the role of sphingosine kinase 1 (SphK1) in insulin secretion, we used stable transfection to knock down the expression of the Sphk1 gene in the rat insulinoma INS-1 832/13 cell line. Cell lines with lowered Sphk1 mRNA expression and SphK1 enzyme activity (SK11 and SK14) exhibited lowered glucose- and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine-stimulated insulin release and low insulin content associated with decreases in the mRNA of the insulin 1 gene. Overexpression of the rat or human Sphk1 cDNA restored insulin secretion and total insulin content in the SK11 cell line, but not in the SK14 cell line. The Sphk1 cDNA-transfected SK14 cell line expressed significantly less SphK1 activity than the Sphk1 cDNA-transfected SK11 cells suggesting that the shRNA targeting SK14 was more effective in silencing the exogenous rat Sphk1 mRNA. The results indicate that SphK1 activity is important for insulin synthesis and secretion.     

Blood-Brain Barrier Transport of Basic Amino Acids Is Selectively Inhibited at Low pH

The transport of amino acids across the blood-brain barrier was measured with the single-pass carotid injection method. The pH of the injected bolus varied between 4.5 and 8.5. Arginine and lysine uptakes were inhibited 24% at pH 5.5 and 59% at pH 4.5. The uptakes of 2-aminobicyclo (2,2,1) heptane-2-carboxylic acid and phenylalanine were unaffected at this pH. There were also no changes observed in choline, glucose, or butanol transport. The Ki of arginine transport inhibition by H+ was 2.4 +/- 0.5 microM; i.e., pH 5.6 +/- 0.1. No change with pH occurred in the Km of arginine transport, while a significant decrease (p less than 0.01) was observed in the Vmax (10.2 +/- 2.3 nmol min-1 g-1 and 5.6 +/- 2.3 nmol min-1 g-1 at pH 7.5 and pH 5.5, respectively). This noncompetitive inhibition was found to be transient as arginine uptake at pH 7.5; it was measured by carotid injection 30 sec following a previous bolus which was buffered to pH 4.5, and was not significantly different from the control. This selective inhibition of the blood-brain barrier basic amino acid carrier demonstrates the advantage of the carotid injection approach in exposing the capillary exchange site to extreme alterations in chemical composition which could not be tolerated systemically.     

Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase

Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus beta-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14C into lipid from [U-14C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.     

Characterization of L-threonine and L-glutamine transport in murine P388 leukemia cells in vitro. Presence of an N-like amino acid transport system

The transport of L-threonine and L-glutamine into murine P388 leukemia cells has been characterized. Threonine appears to be a specific substrate for a Na+-dependent amino acid transport system similar to system ASC of the HTC hepatoma cell. Threonine transport is uninhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and alpha-(methylamino)isobutyric acid, shows a pattern of transport similar to that seen in HTC hepatoma cells over the pH range of 5.5-7.5, and is inhibited by L-serine and L-cysteine. Approximately two-thirds of glutamine transport into P388 cells also appears to enter P388 cells via this ASC-analogous system. However, based upon (a) inhibition studies with threonine (where the K1 of threonine inhibition of glutamine transport was 7-fold the Km of threonine transport), (b) inhibition analysis of glutamine transport with various amino acids and amino acid analogues, and (c) different patterns of transport between threonine and glutamine over the pH range of 5.5-7.5, approximately one-third of glutamine transport can be attributed to a second Na+-dependent amino acid transport system. This system appears to be similar to the system N of rat hepatocytes. Glutamine and threonine do not appear to enter P388 cells via systems A or L to any significant degree. P388 cells do not appear to exhibit 'adaptive regulation' of amino acid transport. Differences in 'adaptive regulation' could therefore not be utilized for comparing threonine and glutamine transport.     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.     

SYNTHETIC AMINO ACIDS AND THE NATURE OF l-DOPA TRANSPORT AT THE BLOOD-BRAIN BARRIER

—The blood-brain barrier transport of amino acids has been measured using the carotid injection technique in the rat. The synthetic amino acids, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and α-(methylamino)isobutyric acid (MeAIB), were model substrates in the Ehrlich cell for the leucine (L) and alanine (A) neutral amino acid transport mechanisms, respectively. The uptake (±)b-[carboxyl-¹⁴C]BCH at the same rate for the five brain regions tested suggested a similarity between regions for the L transport mechanism. At injectant concentrations of 0·1 mm (similar to naturally occurring aromatic neutral amino acids), BCH was mainly taken up by a saturable mediated transport mechanism (K₁, 0·16 mm and V_max, 0·03/μmol/g per min). At higher concentrations, uptake by a nonsaturable or diffusional mechanism could be demonstrated. When BCH was added as a second amino acid to l -[3-¹⁴C]DOPA, the saturable component of l -DOPA transport was significantly inhibited. MeAIB had no measurable effect on the rate of l -DOPA transport. These results suggested that the mediated transport mechanism for l -DOPA at the cerebral capillaries is similar to the l -neutral amino acid transport system.     

Studies with leucine, beta-hydroxybutyrate and ATP citrate lyase-deficient beta cells support the acetoacetate pathway of insulin secretion

We hypothesized that contrasting leucine with its non-metabolizable analog 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) might provide new information about metabolic pathways involved in insulin secretion. Both compounds stimulate insulin secretion by allosterically activating glutamate dehydrogenase, which enhances glutamate metabolism. However, we found that leucine was a stronger secretagogue in rat pancreatic islets and INS-1 cells. This suggested that leucine's metabolism contributed to its insulinotropism. Indeed, we found that leucine increased acetoacetate and was metabolized to CO(2) in pancreatic islets and increased short chain acyl-CoAs (SC-CoAs) in INS-1 cells. We then used the leucine-BCH difference to study the hypothesis that acyl groups derived from secretagogue carbon can be transferred as acetoacetate, in addition to citrate, from mitochondria to the cytosol where they can be converted to SC-CoAs. Since BCH cannot form sufficient acetoacetate from glutamate, transport of any glutamate-derived acyl groups to the cytosol in BCH-stimulated cells must proceed mainly via citrate. In ATP citrate lyase-deficient INS-1 cells, which are unable to convert citrate into cytosolic acetyl-CoA, insulin release by BCH was decreased and adding beta-hydroxybutyrate or alpha-ketoisocaproate, which increases mitochondrial acetoacetate, normalized BCH-induced insulin release. This strengthens the concept that acetoacetate-transferred acyl carbon can be converted to cytosolic SC-CoAs to stimulate insulin secretion.     

Neutral amino acid transport at the human blood-brain barrier

The kinetics of human blood-brain barrier neutral amino acid transport sites are described using isolated human brain capillaries as an in vitro model of the human blood-brain barrier. Kinetic parameters of transport (Km, Vmax, and KD) were determined for eight large neutral amino acids. Km values ranged from 0.30 +/- 0.08 microM for phenylalanine to 8.8 +/- 4.6 microM for valine. The amino acid analogs N-methylaminoisobutyric acid and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid were used as model substrates of the alanine- and leucine-preferring transport systems, respectively. Phenylalanine is transported solely by the L-system (which is sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and leucine is transported equally by the L- and ASC-system (which is sodium-dependent and N-methylaminoisobutyric acid-independent). Dose-dependent inhibition of the high affinity transport system by p-chloromercuribenzenesulfonic acid is demonstrated for phenylalanine, similar to the known sensitivity of blood-brain barrier transport in vivo. The Km values for the human brain capillary in vitro correlate significantly (r = 0.83, p less than 0.01) with the Km values for the rat brain capillary in vivo. The results show that the affinity of human blood-brain barrier neutral amino acid transport is very high, i.e. very low Km compared to plasma amino acid concentrations. This provides a physical basis for the selective vulnerability of the human brain to derangements in amino acid availability caused by a selective hyperaminoacidemia, e.g. hyperphenylalaninemia.     

The stimulus-secretion coupling of amino acid-induced insulin release. XI. Kinetics of deamination and transamination reactions

L-Leucine and its nonmetabolized analogue, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH) activate glutamate dehydrogenase in pancreatic islets, whether the reaction velocity is measured in the direction of glutamate synthesis or glutamate deamination. The rate of glutamate oxidative deamination is increased by ADP and inhibited by 2-ketoglutarate, NH4+ and GTP. The islet homogenate catalyzes the transamination between L-glutamate and either 2-ketoisocaproate or pyruvate, and between 2-ketoglutarate and L-leucine, L-aspartate, L-alanine, L-isoleucine, L-valine, L-norvaline or L-norleucine, but not b (+/-) BCH. The glutamate-aspartate transaminase is preferentially located in mitochondria relative to other transaminases. The parallel effects of L-leucine and BCH on glutamate dehydrogenase and their vastly different abilities to act as transamination partners may account for both analogies and discrepancies in the metabolic and functional responses of the islets to these two branched-chain amino acids.     

Na+-dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na+-dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo [3,2,1]octane-3-carboxylic acid (BCO) by blastocysts was largely Na+ dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na+-dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo [2,2,1]heptane-2-carboxylic acid (Ki approximately 1.0 mM) and L-valine (Ki approximately 0.10 mM) appeared to inhibit Na+-dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, we conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system.     

Reconstitution of the L-leucine-H+ cotransporter of the plasma membrane from Chang liver cells into proteoliposomes

L-Leucine is cotransported with H+ in the plasma membrane of Chang liver cells (Mitsumoto, Y. et al. (1986) J. Biol. Chem. 261, 4549). The leucine transport system was solubilized from the plasma membrane of the cells with ocytl glucoside and reconstituted in proteoliposomes prepared by a rapid dilution of a mixture of the solubilized proteins, octyl glucoside and liposomes. The proteoliposomes exhibited H(+)-gradient and electrical potential-stimulated leucine uptake. The H(+)-gradient-stimulated leucine uptake could be completely inhibited by carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH). The stimulatory effect of H+ gradient on leucine uptake was shown to be mainly due to decrease of the Km, but not to change of the Vmax, of the transport kinetics. These results suggest that the leucine-H+ cotransporter is solubilized and reconstituted into proteoliposomes.     

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of α-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released α-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.