Inhibition of intestinal amino acid transport by blood sugar lowering biguanides

Summary The effect of blood-glucose lowering biguanides (phenethyl- and butylbiguanide) on active intestinal transport of different amino acids has been tested in hamster small intestinein vitro. —.Biguanides inhibited active transport of all amino acids tested. The inhibitory effect of biguanides increased with incubation time, was more pronounced after preincubation of intestinal tissue and was found to be non-competitive. The minimal inhibitory concentration of phenethylbiguanide on amino acid transport was 5×10^–4 M. —¹⁴C-butylbiguanide was found to be transported into hamster small intestine by a concentration-independent, energy-independent uptake mechanism and was accumulated in intestinal tissue against a concentration gradient. — In accord with earlier results on the inhibitory effect of biguanides on active intestinal hexose transport it is concluded that biguanides do not act as specific inhibitors for glucose transport, but rather affect active, energy-requiring intestinal transport mechanisms in general (hexose-, amino acid-, calcium- and myo-inositol transport), most likely due to their known inhibitory effect on mitochondrial respiration, thus depriving mucosal cells of ATP required to translocate substrates against a concentration gradient.Part of this work has been presented at the International Diabetes Congress, Buenos Aires, Argentina, August 1970 and at the Meeting of German and Italian Pharmacology Society, Heidelberg, September 1970.     

Characterization of several amino acid transports and glutamine metabolism in MOLT4 human T4 leukemia cells

The transport system responsible for glutamine, alanine and glutamate in MOLT4 human T4 leukemia cell line were characterized. Kinetic studies of sodium-dependent glutamine and alanine transport exhibited a single saturable high-affinity carrier with a Michaelis constant of 152 +/- 26 microm and 203 +/- 36 microm and a maximal transport velocity of 960 +/- 165 and 1096 +/- 208 nmol/10(9)cells/min, respectively. Glutamate uptake was less than one-tenth of glutamine and alanine, and linearly increased with glutamate concentration which was mediated by diffusion. 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), known as anion channel blockers, inhibited the sodium-dependent glutamine and alanine transport by 40% at 10 microm. Cellular contents of these amino acids in MOLT4 cells revealed glutamate to be the highest among them despite low glutamate influx. A glutamine metabolism study using whole cells indicated this high conversion rate from glutamine to glutamate, but no conversion to another amino acid. Based on these results, the high glutamate concentration in MOLT4 was speculated to be synthesized from transported glutamine by active glutaminase.     

Radioprotection by glutathione ester: transport of glutathione ester into human lymphoid cells and fibroblasts.

Glutathione is not effectively transported into human lymphoid cells, normal human skin fibroblasts, and fibroblasts from patients with genetic deficiencies of gamma-glutamylcysteine synthetase or glutathione synthetase. On the other hand, the monoethyl ester of glutathione, in which the carboxyl group of the glycine residue is esterified, is readily transported into these cells and is hydrolyzed intracellularly. This leads to greatly increased cellular levels of glutathione, which often exceed those found normally. Glutathione ester was found to protect human lymphoid cells of the CEM line against the lethal effects of irradiation. Under the conditions employed, complete protection was found when the ester was added prior to irradiation. Addition of the ester after irradiation was partially effective, suggesting that GSH may also function in repair processes.     

Metabolic fuel and amino acid transport into the brain in experimental diabetes mellitus.

We used the Oldendorf brain uptake index method to study the blood-brain barrier transport of several metabolic substrates in diabetes. Glucose transport into the brain was decreased by 1/3 in rats with moderate diabetes induced by prior injection of streptozotocin (65 mg/kg of body weight). The transports of mannose and the poorly metabolized hexoses 2-deoxyglucose and 3-O-methylglucose were similarly reduced. Likewise, brain glucose transport was decreased in rats with alloxan-induced diabetes. These alterations in brain hexose influx appeared to be related to chronic (1-2 days) hyperglycemia rather than to insulin-lack per se. Thus, starvation of the diabetic rats for 48 hr restored both the plasma glucose concentration and brain hexose transport to normal. Conversely, the substitution of 10% sucrose for their drinking water both increased plasma glucose and decreased hexose transport in insulin-treated diabetic rats. The 45% decrease in maximal glucose transport rate observed and the uniformity of diminished hexose transport probably imply a decrease in the number of available high-affinity transport carriers at the blood-brain barrier. This defect was specific for hexoses in that the transports of neutral and basic amino acids and of beta-hydroxybutyrate were not similarly affected. These results suggest that chronic hyperglycemia decreases the number of hexose carrier molecules available at the blood-brain barrier. Such an adaptation could operate to decrease the net flux of glucose into the brain during sustained hyperglycemia. It also may explain the abnormal sensitivity to abrupt blood glucose lowering in patients with diabetes mellitus.     

Inhibition of system A amino acid transport and hepatocyte proliferation following partial hepatectomy in the rat.

System A, the sodium-dependent neutral amino acid transport activity, has a 3-fold increase in its initial uptake velocity into hepatocytes following partial hepatectomy (PH) in the rat. The purpose of this study was to examine the effect of inhibition of System A-mediated amino acid transport on hepatocyte proliferation and liver regeneration. We describe the in vivo competitive inhibition of System A activity following PH by the nonmetabolizable, System A-specific substrate, alpha-(methylamino)isobutyric acid (MeAIB). Administration of MeAIB 60 minutes before PH decreased the incorporation of [(3)H]thymidine into DNA by 45% +/- 5% and 76% +/- 17% at 24 and 36 hours, respectively. The readministration of MeAIB every 12 hours further decreased DNA synthesis by 92% +/- 18% and 82% +/- 11% at 24 and 36 hours. The recovery of liver mass of rats receiving MeAIB was decreased by 46.4% +/- 5.1% at 24 hours after PH. In vitro, 5 mmol/L MeAIB inhibited proliferation of primary hepatocytes by 56% +/- 4% and 61% +/- 12% 48 hours after incubation with 10% fetal calf serum or epidermal growth factor (5 ng/mL), respectively. Thus, MeAIB inhibition of System A transport activity decreased both in vivo and in vitro inducement of hepatocyte proliferation. Treatment with MeAIB did not significantly change the incorporation of [(3)H]leucine into total liver protein, but changes in serum amino acids and hepatocyte cell volume were observed, suggesting System A transport activity during hepatocyte proliferation functions primarily to provide amino acids to fuel liver-specific biochemical pathways and to increase cell volume.     

Inhibition by colchicine of changes in amino acid transport and initiation of DNA synthesis in regenerating rat liver.

The uptake of amino acids (measured with alpha-aminoisobutyric acid) by hepatocytes stimulated to proliferate by partial hepatectomy followed a biphasic pattern, with an initial peak 8-10 hr after surgery followed by a release of amino acids between 10 and 16 hr and a further increase between 16 and 20 hr after the operation. The second period of increased uptake coincided with the increase in DNA synthesis by the hepatocytes. Colchicine, and other microtubule disrupters administered at the time of partial hepatectomy, abolished the first period of increased amino acid uptake and prevented the cells from initiating DNA synthesis. Pulse-labeling experiments with alpha-aminoisobutyric acid revealed that colchicine did not inhibit amino acid transport per se, but prevented the increased capacity for amino acid transport induced by partial hepatectomy. An injection of colchicine 14 hr after hepatectomy prevented the release of amino acids by the liver and also substantially reduced the initiation of DNA synthesis. The data suggest that the microtubules may play a role in proliferative activation and also in the initiation of DNA synthesis of hepatocytes in vivo and that changes in amino acid transport are linked to these microtubule-requiring processes.     

The glutamate analog 2-amino-4-phosphonobutyrate antagonizes synaptic transmission from cones to horizontal cells in the goldfish retina

In the retina, the glutamate analog 2-amino-4-phosphonobutyrate (APB) distinguishes a class of glutamate receptors that is thought to be found only on depolarizing bipolar cells (DBCs). We now report that APB is a potent antagonist of cone-driven horizontal cells in the goldfish retina. APB hyperpolarized the membrane to the same potential as cobalt Ringer's and blocked the light responses. APB acted specifically on the cone pathway, as it had no effect on rod-driven horizontal cells. The lowest effective APB concentration for antagonistic action on the horizontal cells (approximately 2 microM) was similar to the concentration for agonist action on DBCs. APB was not able to block the actions of exogenous glutamate or kainate on horizontal cells. We propose that the action of APB on the cone-horizontal cell synapse is mediated at a site that is distinct from the glutamate and kainate binding site. Therefore, APB is most probably acting at a different locus on the synaptic glutamatergic receptors of the horizontal cells or at presynaptic receptors located on the cones themselves.     

Cloning a plant amino acid transporter by functional complementation of a yeast amino acid transport mutant.

Amino acids are transported across the plasma membrane of plant cells by proton-amino acid symports. We report here the successful cloning of a neutral amino acid carrier by functional complementation. A histidine transport deletion mutant of Saccharomyces cerevisiae was transformed with an Arabidopsis thaliana cDNA library constructed in a yeast expression vector. Forty transformants, out of 10(5), allowed growth on a histidine-limiting medium. The acquired ability to grow on low histidine was shown to be strictly dependent on the protein encoded by the expression plasmid. Histidine and alanine transport activity were 10- to 20-fold greater in the transformants. The transport kinetics, inhibitor sensitivity, and substrate specificity match those of neutral system II, a neutral amino acid carrier we previously described in plasma membrane vesicles isolated from leaf tissue. The cDNA insert is 1.7 kb with an open reading frame that codes for a protein containing 486 amino acids with a calculated molecular mass of 52.9 kDa and three sites of potential N-linked glycosylation. Hydropathy analysis of the deduced amino acid sequence suggests this is an integral membrane protein with 10-12 membrane-spanning alpha-helices. Overall, the sequence of this amino acid carrier is not closely related to any other protein sequences in the GenBank data base. Interestingly, however, there are small regions of sequence that exhibit significant levels of similarity with at least seven other integral membrane proteins.     

Inhibition of 5-Oxoprolinase by 2-Imidazolidone-4-Carboxylic Acid

L-2-Imidazolidone-4-carboxylic acid is an effective competitive inhibitor of the reaction catalyzed by 5-oxoprolinase, in which 5-oxo-L-proline (L-pyroglutamic acid, L-2-pyrrolidone-5-carboxylic acid, L-5-oxopyrrolidine-2-carboxylic acid) is converted to L-glutamate, with concomitant cleavage of ATP to ADP and orthophosphate. L-2-Imidazolidone-4-carboxylate decreased the rate of metabolism of 5-oxo-L-[(14)C]proline to (14)CO(2) by rat-kidney slices but had no effect on the metabolism of [(14)C]glutamate. Mice injected with L-2-imidazolidone-4-carboxylate exhibited greatly reduced ability to metabolize 5-oxo-L-proline, but metabolized glutamate at an essentially normal rate. The findings provide an approach to an animal model for the human condition 5-oxoprolinuria, in which there is apparently a deficiency of renal 5-oxoprolinase activity. The evidence indicates that 5-oxoproline is a normal metabolite.     

Relationship of insulin binding to amino acid transport by cultured 14-day embryonic chick heart cells.

The characteristics of insulin receptors were studied in cultured embryonic chick heart cells which demonstrated insulin-responsive amino acid transport. Binding of [125I]iodoinsulin was time dependent, reversible, saturable, species specific, and proportional to cell number. Optimum binding occurred at pH 7.8 in the presence of 0.1% bovine serum albumin. Curvilinear Scatchard plots were found for chicken and bovine insulin binding at 15 and 30 C. Equilibrium association constants (Kas) and maximum capacities were calculated based on a two-receptor model. When studied at 15 C, chicken insulin was bound with Kas of 5.0 and 0.026 nM-1 for the high and low affinity receptors, respectively. Bovine insulin bound with Kas of 2.1 and 0.03 nM-1. The binding capacities of 600 and 9000 molecules/cell for the high and low affinity receptors, respectively, were the same for both species of ligand. At 30 C, the Ka of the high affinity chicken insulin receptor interaction decreased to 1.6 nM-1, whereas the low affinity Ka was not changed. In competitive binding assays, chicken insulin was 4 and 250 times more potent than bovine and guinea pig insulin, respectively. Human GH and mouse epidermal growth factor did not compete with chicken insulin. Although mouse epidermal growth factor did not compete with insulin, it did stimulate 2-aminoisobutyric acid accumulation. The maximal stimulation by this hormone was less than and additive to that produced by the maximal stimulation of insulin. These data indicate that insulin and epidermal growth factor acted through different receptors to stimulate amino acid transport. When insulin-stimulated functional response was compared to calculated receptor occupancy in this two-receptor model, stimulation of amino acid transport paralleled occupancy of the low affinity receptor such that at half-maximal transport stimulation, approximately 50% of these receptors were occupied. Half-maximal stimulation of 2-aminoisobutyric acid transport occurred at 18- and 20-nM concentrations of bovine and chicken insulins, respectively. Guinea pig insulin failed to stimulate 2-aminoisobutyric acid uptake even at 350 nM. Both the affinity of binding in the low affinity class and the insulin concentration for half-maximal stimulation of 2-aminoisobutyric acid transport were independent of temperature in the temperature ranges studied. We conclude that 14-day embryonic chick heart cells possess at least two classes of receptors which bind insulin. Occupancy of the lower affinity class of insulin receptors correlates quantitatively with insulin stimulation of 2-aminoisobutyric acid transport.     

3'':5''-cyclic AMP: independent induction of amino acid transport by epinephrine in primary cultures of adult rat liver cells.

Liver cells were obtained from adult rats by a collagenase perfusion technique and cultured as monolayers in serum-free media. Epinephrine and isoproterenol both induced large increases in intracellular adenosine 3':5'-monophosphate (cAMP) within 1-2 min whereas epinephrine (but not isoproterenol) induced 2- to 3-fold increases in the rate of alpha-aminoisobutyric acid transport within 2-4 hr after a 1 hr lag. Propranolol abolished the increase in cAMP elicited by epinephrine and isoproterenol, but did not block the induction of alpha-aminoisobutyric acid transport by epinephrine. In contrast, dihydroergotamine abolished and phentolamine diminished the induction of alpha-aminoisobutyric acid transport by epinephrine but did not decrease the stimulation of cAMP levels by epinephrine. Epinephrine dose response curves for cAMP and alpha-aminoisobutyric acid transport were similar. Once exposed to epinephrine, cells became refractory to further stimulation of cAMP levels by epinephrine.     

Sodium-dependent amino acid transport by cultured hamster cells: membrane vesicles retain transport changes due to glucose starvation and cycloheximide.

Enhanced alpha-aminoisobutyric acid transport by hamster cells cultured in the absence of D-glucose has been demonstrated in isolated membrane vesicles. The observed enhancement was seen in the presence but not in the absence of Na+. Kinetic analysis of transport using both the intact cells and the membrane vesicles showed that the overall enhancement was associated with an increase in Vmax. Decreases in transport activity by intact cells resulting from extended exposure of the cells to inhibitors of protein synthesis, such as cycloheximide, were also evident in membrane vesicles. The use of metabolically inactive membrane vesicles demonstrated that amino acid uptake by intact cells is a transport property of the plasma membrane. In addition, this study shows that membrane vesicle preparations can be exploited for the purpose of studying the regulation of amino acid transport. Taken together, the data suggest that carrier turnover is involved in the regulation of amino acid transport in animal cells.     

Regulation of amino acid transport in rat and human thyroid cells

To clarify the role of insulin, IGF-I and TSH in thyroid cell regulation, their effects on amino acid transport were studied separately. These effects were noted and compared using both the Wistar rat thyroid cell line and human thyroid cell cultures. Insulin, IGF-I and TSH were able independently to induce the radiolabelled alpha-aminoisobutyric acid transport within the rat thyroid cells: TSH stimulated the amino acid transport in rat thyroid cells in a dose-dependent way from 1 pmol/l to 10 nmol/l. Similarly, insulin increased amino acid transport significantly from 0.17 nmol/l up to 0.17 mumol/l and IGF-I from 0.13 pmol/l up to 0.13 mumol/l. The combined effects of insulin and TSH on amino acid transport were equal to the theoretical sum of the activities, whereas those of IGF-I and TSH were greater than the theoretical one. When human thyroid cell cultures were used, a significant increase of labelled amino acid transport was induced by TSH, i.e. from 0.1 pmol/l to 10 pmol/l; IGF-I stimulated amino acid transport in a range from 0.13 pmol/l to 13 pmol/l, under the same conditions. Conversely, only large doses of insulin, i.e. 1.7 nmol/l, were able weakly to stimulate amino acid transport. When submaximal TSH and IGF-I doses were co-incubated in human thyroid cells, an additive effect on amino acid transport was observed.     

Role of sialic acid in synaptosomal transport of amino acid transmitters.

Active, high-affinity, sodium-dependent uptake of gamma-aminobutyric acid and of the acidic amino acid D-aspartate was inhibited by pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. Inhibition was of a noncompetitive type and was related to the amount of sialic acid released. The maximum accumulation ratios of both amino acids (intracellular [amino acid]/extracellular [amino acid]) remained largely unaltered. Treatment with neuraminidase affected neither the synaptosomal energy levels nor the concentration of internal potassium. It is suggested that the gamma-aminobutyric acid and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of the carrier proteins directly and not through modification of driving forces responsible for amino acid uptake.     

Stimulation of hepatic glutathione formation by administration of L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-prolinase substrate.

5-Oxo-L-prolinase, the enzyme that catalyzes the conversion of 5-oxo-L-proline to L-glutamate coupled to the cleavage of ATP to ADP and Pi, also acts on L-2-oxothiazolidine-4-carboxylate (an analog of 5-oxoproline in which the 4-methylene moiety is replaced by sulfur) and ATP to yield cysteine and ADP. The enzyme, which exhibits an affinity for the analog similar to that for the natural substrate, is inhibited by the analog in vitro and in vivo. L-2-oxothiazolidine-4-carboxylate thus serves as a potent inhibitor of the gamma-glutamyl cycle at the step of 5-oxoprolinase. Administration of L-2-oxothiazolidine-4-carboxylate to mice that had been depleted of hepatic glutathione led to restoration of normal hepatic glutathione levels. Since L-2-oxothiazolidine-4-carboxylate is an excellent substrate of the enzyme, it may serve as an intracellular delivery system for cysteine and thus has potential as a therapeutic agent for conditions in which there is depletion of hepatic glutathione.     

Clinical experience with the testing of serum proteinase activity by cathepsin B-like sensitive amino acid derivatives of 7-amino-4-methylcoumarine

The proteolytic activity of serum against two synthetic polypeptide substrates conjugated with 7-amino-4-methylcoumarine which are known to be highly specific substrates for lysosomal cathepsin B-like cysteine proteinase, was studied in normal individuals and in patients with breast and colorectal cancer. The results showed that both substrates are cleaved in different ways and their hydrolysis is very poorly sensitive to inhibitors of cysteine proteinases. On the other hand, cleavage is intensively inhibited by disodium-EDTA which is known as an activator of cysteine proteinases. This indicates that lysosomal conditions are quite different from serum which is an unstable mixture of various enzymes and their substrates. It was also demonstrated that hydrolysis of the tested substrates is not probably realized by cysteine proteinases but it can be performed by cooperation of other serum proteinases. Moreover, our results confirmed that serum activity of these enzymes can be significantly higher in patients with breast cancer, particularly with metastasis, than in healthy women. A slight insignificant increase was observed also in patients with colorectal cancer.