NKCC transport mediates the insulinotropic effects of taurine and other small neutral amino acids.

AIMS: Despite its high concentration in pancreatic islets of Langerhans and broad range of antihyperglycemic effects, the route facilitating the import of dietary taurine into pancreatic ß-cell and mechanisms underlying its insulinotropic activity are unclear. We therefore studied the impact of taurine on beta-cell function, alongside that of other small neutral amino acids, L-alanine and L-proline. MAIN METHODS: Pharmacological profiling of insulin secretion was conducted using clonal BRIN BD11 ß-cells, the impact of taurine on the metabolic fate of glucose carbons was assessed using NMR and the findings were verified by real-time imaging of Ca2+ dynamics in the cytosol of primary mouse and human islet beta-cells. KEY FINDINGS: In our hands, taurine, alanine and proline induced secretory responses that were dependent on the plasma membrane depolarisation, import of Ca2+, homeostasis of K+ and Na+ as well as on cell glycolytic and oxidative metabolism. Taurine shifted the balance between the oxidation and anaplerosis towards the latter, in BRIN BD11 beta-cells. Furthermore, the amino acid signalling was significantly attenuated by inhibition of Na+-K+-Cl- symporter (NKCC). SIGNIFICANCE: These data suggest that taurine, like L-alanine and L-proline, acutely induces glucose-dependent insulin-secretory responses by modulating electrogenic Na+ transport, with potential role of intracellular K+ and Cl- in the signal transduction. The acute action delineated would be consistent with antidiabetic potential of dietary taurine supplementation.

Large neutral amino acid supplementation as an alternative to the phenylalanine-restricted diet in adults with phenylketonuria: evidence from adult Pah-enu2 mice.

Phenylketonuria treatment mainly consists of a phenylalanine-restricted diet but still results in suboptimal neuropsychological outcome, which is at least partly based on cerebral monoamine deficiencies, while, after childhood, treatment compliance decreases. Supplementation of large neutral amino acids (LNAAs) was previously demonstrated in young phenylketonuria mice to target all three biochemical disturbances underlying brain dysfunction in phenylketonuria. However, both its potential in adult phenylketonuria and the comparison with the phenylalanine-restricted diet remain to be established. To this purpose, several LNAA supplements were compared with a severe phenylalanine-restricted diet with respect to brain monoamine and amino acid concentrations in adult C57Bl/6 Pah-enu2 mice. Adult phenylketonuria mice received a phenylalanine-restricted diet, unrestricted diet supplemented with several combinations of LNAAs or AIN-93M control diet for 6 weeks. In addition, adult wild-type mice on AIN-93M diet served as controls. The severe phenylalanine-restricted diet in adult phenylketonuria mice significantly reduced plasma and brain phenylalanine and restored brain monoamine concentrations, while brain concentrations of most nonphenylalanine LNAAs remained subnormal. Supplementation of eight LNAAs was similarly effective as the severe phenylalanine-restricted diet to restore brain monoamines, while brain and plasma phenylalanine concentrations remained markedly elevated. These results provide biochemical support for the effectiveness of the severe phenylalanine-restricted diet and showed the possibilities of LNAA supplementation being equally effective to restore brain monoamines in adult phenylketonuria mice. Therefore, LNAA supplementation is a promising alternative treatment to phenylalanine restriction in adult phenylketonuria patients to further optimize neuropsychological functioning.

Presumptive brain influx of large neutral amino acids and the effect of phenylalanine supplementation in patients with Tyrosinemia type 1.

INTRODUCTION: Hereditary Tyrosinemia type 1 (HT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Current treatment consists of 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and a tyrosine and phenylalanine restricted diet. Recently, neuropsychological deficits have been seen in HT1 patients. These deficits are possibly associated with low blood phenylalanine concentrations and/or high blood tyrosine concentrations. Therefore, the aim of the present study was threefold. Firstly, we aimed to calculate how the plasma amino acid profile in HT1 patients may influence the presumptive brain influx of all large neutral amino acids (LNAA). Secondly, we aimed to investigate the effect of phenylalanine supplementation on presumptive brain phenylalanine and tyrosine influx. Thirdly, we aimed to theoretically determine minimal target plasma phenylalanine concentrations in HT1 patient to ensure adequate presumptive brain phenylalanine influx. METHODS: Data of plasma LNAA concentrations were obtained. In total, 239 samples of 9 HT1 children, treated with NTBC, diet, and partly with phenylalanine supplementation were collected together with 596 samples of independent control children. Presumptive brain influx of all LNAA was calculated, using Michaelis-Menten parameters (Km) and Vmax-values obtained from earlier articles. RESULTS: In HT1 patients, plasma concentrations and presumptive brain influx of tyrosine were higher. However, plasma and especially brain influx of phenylalanine were lower in HT1 patients. Phenylalanine supplementation did not only tend to increase plasma phenylalanine concentrations, but also presumptive brain phenylalanine influx, despite increased plasma tyrosine concentrations. However, to ensure sufficient brain phenylalanine influx in HT1 patients, minimal plasma phenylalanine concentrations may need to be higher than considered thus far. CONCLUSION: This study clearly suggests a role for disturbed brain LNAA biochemistry, which is not well reflected by plasma LNAA concentrations. This could play a role in the pathophysiology of the neuropsychological impairments in HT1 patients and may have therapeutic implications.

Large neutral amino acids: dietary effects on brain neurochemistry and function.

The ingestion of large neutral amino acids (LNAA), notably tryptophan, tyrosine and the branched-chain amino acids (BCAA), modifies tryptophan and tyrosine uptake into brain and their conversion to serotonin and catecholamines, respectively. The particular effect reflects the competitive nature of the transporter for LNAA at the blood-brain barrier. For example, raising blood tryptophan or tyrosine levels raises their uptake into brain, while raising blood BCAA levels lowers tryptophan and tyrosine uptake; serotonin and catecholamine synthesis in brain parallel the tryptophan and tyrosine changes. By changing blood LNAA levels, the ingestion of particular proteins causes surprisingly large variations in brain tryptophan uptake and serotonin synthesis, with minimal effects on tyrosine uptake and catecholamine synthesis. Such variations elicit predictable effects on mood, cognition and hormone secretion (prolactin, cortisol). The ingestion of mixtures of LNAA, particularly BCAA, lowers brain tryptophan uptake and serotonin synthesis. Though argued to improve physical performance by reducing serotonin function, such effects are generally considered modest at best. However, BCAA ingestion also lowers tyrosine uptake, and dopamine synthesis in brain. Increasing dopamine function in brain improves performance, suggesting that BCAA may fail to increase performance because dopamine is reduced. Conceivably, BCAA administered with tyrosine could prevent the decline in dopamine, while still eliciting a drop in serotonin. Such an LNAA mixture might thus prove an effective enhancer of physical performance. The thoughtful development and application of dietary proteins and LNAA mixtures may thus produce treatments with predictable and useful functional effects.

Surplus dietary tryptophan inhibits stress hormone kinetics and induces insulin resistance in pigs.

Recently we have shown that surplus dietary tryptophan (TRP) reduced the plasma concentrations of cortisol and noradrenaline in pigs. Stress hormones are known to affect insulin sensitivity and metabolism. We now investigated the long-term effects of surplus dietary TRP on 1) plasma and urinary stress hormone kinetics, 2) insulin sensitivity for glucose and amino acid clearance, and 3) whole body nitrogen balance. Pigs were fed for 3weeks a high (13.2%) vs normal (3.4%) TRP to large neutral amino acids (LNAA) diet, leading to reduced fasting (14 h) plasma cortisol (17.1+/-3.0 vs 28.9+/-4.3 ng/mL, p<0.05) and noradrenaline (138+/-14 vs 225+/-21 pg/mL, p<0.005) concentrations, lower daily urinary noradrenaline (313+/-32 vs 674+/-102 ng/kg day, p<0.001) and adrenaline (124+/-13 vs 297+/-42 ng/kg day, p<0.001) but higher dopamine (5.8+/-0.5 vs 1.5+/-0.2 microg/kg day, p<0.001) excretions, respectively. Insulin sensitivities for both glucose and amino acid clearance, (as measured by the intraportal hyperinsulinaemic (1 mU/kg min) euglycaemic euaminoacidaemic clamp technique), were lower by 22% in pigs on the high vs normal TRP/LNAA diet (14.8+/-1.4 vs 18.9+/-0.9, p<0.05 and 69.7+/-4.3 vs 89.7+/-6.8 mL/kg min, p<0.05, respectively) without affecting urinary nitrogen excretion (35.5+/-1.0 vs 36.6+/-1.0% of dietary nitrogen intake, p=ns). In conclusion, long-term feeding of surplus dietary TRP inhibits both baseline adrenocortical and sympathetic nervous system activity, it induces insulin resistance for both glucose and amino acid clearance but it does not affect whole body protein catabolism. This indicates that the bioactive amino acid TRP contributes to homeostasis in neuroendocrinology and insulin action and that low baseline adrenocortical and sympatho-adrenal axis activity are associated with insulin resistance.

Large neutral amino acids supplementation in phenylketonuric patients.

Phenylketonuria is an inborn error of amino acid metabolism that results in severe mental retardation if not treated early and appropriately. The traditional treatment, consisting of a low-phenylalanine diet, is usually difficult to maintain throughout adolescence and adulthood, resulting in undesirable levels of blood phenylalanine and consequent neurotoxicity. The neurotoxicity of phenylalanine is enhanced by its transport mechanism across the blood-brain barrier, which has the highest affinity for phenylalanine compared with the other large neutral amino acids that share the same carrier. The supplementation of large neutral amino acids in phenylketonuric patients has been showing interesting results. Plasma phenylalanine levels can be reduced, which may guarantee important metabolic and clinical benefits to these patients. Although long-term studies are needed to determine the efficacy and safety of large neutral amino acids supplements, the present state of knowledge seems to recommend their prescription to all phenylketonuric adult patients who are non-compliant with the low-phenylalanine diet.

Brain dysfunction in phenylketonuria: is phenylalanine toxicity the only possible cause?

In phenylketonuria, mental retardation is prevented by a diet that severely restricts natural protein and is supplemented with a phenylalanine-free amino acid mixture. The result is an almost normal outcome, although some neuropsychological disturbances remain. The pathology underlying cognitive dysfunction in phenylketonuria is unknown, although it is clear that the high plasma concentrations of phenylalanine influence the blood-brain barrier transport of large neutral amino acids. The high plasma phenylalanine concentrations increase phenylalanine entry into brain and restrict the entry of other large neutral amino acids. In the literature, emphasis has been on high brain phenylalanine as the pathological substrate that causes mental retardation. Phenylalanine was found to interfere with different cerebral enzyme systems. However, apart from the neurotoxicity of phenylalanine, a deficiency of the other large neutral amino acids in brain may also be an important factor affecting cognitive function in phenylketonuria. Cerebral protein synthesis was found to be disturbed in a mouse model of phenylketonuria and could be caused by shortage of large neutral amino acids instead of high levels of phenylalanine. Therefore, in this review we emphasize the possibility of a different idea about the pathogenesis of mental dysfunction in phenylketonuria patients and the aim of treatment strategies. The aim of treatment in phenylketonuria might be to normalize cerebral concentrations of all large neutral amino acids rather than prevent high cerebral phenylalanine concentrations alone. In-depth studies are necessary to investigate the role of large neutral amino acid deficiencies in brain.

Functional properties and cellular distribution of the system A glutamine transporter SNAT1 support specialized roles in central neurons.

Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 approximately 0.3 mm), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax approximately 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ >> H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via alpha-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.