L-alanine Uptake Research Articles

Differential gene expression for investigation of the effect of germinants and heat activation to induce germination in Bacillus cereus spores

Differential gene expression was used to explore the mechanisms underpinning the differences in the impact of heat activation (70 °C for 30 min) on the germination of Bacillus cereus spores in the presence and absence of a germinant (L-alanine). The number of germinated cells, after heat activation plus L-alanine (3.5 ± 0.02 log CFU/ml) in the spore only initial population was found to be higher than that in only heat activated spores (2.01 ± 0.02 log CFU/ml). The concentration of DPA released by heat activated spores in the presence of L-alanine was 68.3 ± 0.1 and 112.1 ± 0.02 μg/ml after 30 and 60 min, compared to 96.5 and 166.2 ± 0.01 μg/ml after 30 and 90 min, respectively released by spores subjected only to heat activation. Gene (BC0784) encoding for the spore germination protein, gerA operon was up-regulated with a log2-transformed fold change value of 1.2 due to heat activation in the presence of L-alanine. The GerA operon located in the inner membrane is known to be involved in the uptake of L-alanine by B. cereus and has been reported to be involved in L-alanine mediated germination. In addition the up-regulation of genes involved in the uptake of L-alanine is proposed to provide the answer to the synergistic effect of heat and L-alanine in inducing germination in B. cereus spores. In short, heat activation increases the ability of L-alanine to penetrate into the spore's inner membrane, where it can be recognized by the receptors for initiation of the germination pathway. In the current study, the majority of the ribosomal proteins were down-regulated (when spores were heat treated in presence of germinants) this process also appeared to slow down protein synthesis by restricting the protein translation machinery. Differential gene expression revealed the genes responsible for the pathways related to transport and recognition of L-alanine into the spore that could have led to the accelerated germination process along with partial shutting down of protein synthesis pathway and ABC transporters. Knowledge of gene regulation in spores during heat activation will help in the development of approaches to prevent spore germination, which could provide an additional safeguard against bacterial growth and toxin production in improperly cooled heat treated foods.

Determining the functional role of waterborne amino acid uptake in hagfish nutrition: a constitutive pathway when fasting or a supplementary pathway when feeding?

Hagfish are unique among aquatic "vertebrates" in their ability to absorb amino acids directly from the water via skin and gill epithelia, but it is unknown whether this phenomenon extends beyond a few studied substrates; what effect fed state has on absorption; and what functional role this may play in hagfish nutrition. Using in vivo and in vitro transport assays, uptake and tissue distribution of the waterborne amino acids L-alanine, L-lysine, and L-phenylalanine were examined as a function of fed state. All three amino acids were shown to be taken up from the water (lysine and phenylalanine for the first time). Following immersion in radiolabelled solutions for 24h, phenylalanine was the amino acid that accumulated at the highest levels in almost all tissues, with the highest accumulation noted in red blood cells and bile, followed by gill and liver. In general, tissues of fed hagfish displayed a significantly reduced phenylalanine accumulation compared to tissues of hagfish fasted for 3weeks. An in vitro assay showed that phenylalanine was transported across the skin at the highest rate, with the uptake of lysine occurring at the lowest rate. Feeding status had no significant effect on in vitro transport. These data indicate that dissolved organic nutrients are a significant source of nutrition to hagfish, and may be relatively more important during periods of fasting than during periods of feeding when immersed in decaying carcasses.

Selected amino acids uptake by microorganisms in soil of differently managed mountain meadows

This work was aimed at determination of the effect of 13 years abandonment of previously long-term mown mountain meadows on uptake of L-glutamic acid (14CO2H[14CH2]2[NH2]14CO2H) and L-alanine (14CH314CH[NH2]14CO2H) by microbial community of Ap horizon (3–13 cm). The study plots has been located near to the experimental stand “Bílý Kříž“ which is located in the Moravian-Silesian Beskids Mountains (N 49°30’17”, E 18°32’28”), on a slope with an elevation of 825–860 m a. s. l. and southeast orientation, and soil classified as an Oxyaquic Hapludalf. The study was performed to test method for measurement of 14C-labelled amino acids uptake by soil microbial community and to increase knowledge on particular processes of N-transformation in soil of these ecosystems. The results obtained in this work showed that 13 years abandonment of mountain meadow did not significantly (P > 0.05) influence rate of glutamic acid or alanine uptake by soil microbial community. Further research including determination of amino acids use by soil microbial biomass with expression of their partitioning between production of new microbial biomass and energy metabolism is necessary.

Identification of a Disulfide Bridge Important for Transport Function of SNAT4 Neutral Amino Acid Transporter

SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.

Metabolic Effects of Insulin and IGFs on Gilthead Sea Bream (Sparus aurata) Muscle Cells

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and l-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, l-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K–Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and l-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K–Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture.

Digestion under Duress: Nutrient Acquisition and Metabolism during Hypoxia in the Pacific Hagfish

Hagfish feed by immersing themselves in the body cavities of decaying animals. This ensures a rich nutrient source for absorption via the gills, skin, and gut, but it may also subject hagfish to reduced levels of dissolved oxygen and elevated levels of the products of biological degradation. This study investigated the impacts of hypoxia and ammonia on the assimilation and metabolism of selected nutrients (glycine, l-alanine, and glucose) in Pacific hagfish (Eptatretus stoutii). Throughout exposure to hypoxia, plasma glucose levels increased. This was not accompanied by an increase in gut glucose transport, which suggests mobilization of glucose from body glycogen stores. Hypoxia preexposure enhanced glycine absorption across the gut and the gill, although l-alanine uptake was unchanged in these tissues. A 24-h period of exposure to hypoxia in hagfish concurrently exposed to waterborne radio-labeled glycine led to a large (5.7-fold) increase in brain glycine accumulation. Preexposure to high levels of waterborne ammonia (10 mM) for 24 h had no impact on gut or skin glycine uptake. These results indicate that hagfish are adapted to maintain nutrient assimilation despite environmental stressors and that tissue-specific absorption of key nutrients such as glycine can even be enhanced in order to sustain critical functions during hypoxia.

Characterisation of l-alanine and glycine absorption across the gut of an ancient vertebrate

This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of L-alanine and glycine conformed to Michaelis-Menten kinetics. An uptake affinity (K(m); substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J (max)) of 83 nmol cm(-2) h(-1) were described for L-alanine. The K(m) and J(max) for glycine were 2.2 mM and 11.9 nmol cm(-2) h(-1), respectively. Evidence suggested that the pathways of L-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.

LAT1 overexpression and function compensates downregulation of ASCT2 in an in vitro model of renal proximal tubule cell ageing

Amino acid transporters provide cells neutral amino acids indispensable for growth and proliferation-dependent protein synthesis. This study evaluates whether prolonged serial cell passaging during 6 months (over 50 passages) may induce changes in amino acid transporters properties in Opossum kidney (OK) proximal tubular cells. High passage OK cells exhibit polyploidy, but no difference in the proliferation potential was observed when compared to low passage OK cells. Increased time in culture was accompanied by an increased total, membrane and cytosol protein content. The Na(+)-insensitive [(14)C]-L-leucine uptake was promoted almost exclusively thought LAT1 (~ 90 vs 80%, high versus low passage OK cells). The increased LAT1 protein abundance in high passage OK cells correlated positively with enhanced ability to take up [(14)C]-L-leucine, despite a 4.3-fold decrease in affinity for the substrate. The Na(+)-sensitive [(14)C]-L-alanine transport was decreased by 2.5-fold in high passage OK cells. However, no differences in ASCT2 expression were observed between high and low passage OK cells. It is concluded that OK cells show functional differences in both L-leucine and L-alanine uptake as a function of passage time in culture. The increased expression and activity of LAT1 in high passage OK cells may correspond to a mechanism enabling the cell to develop the hypertrophy response to prolonged cell passaging, when the function of ASCT2 is markedly depressed.

Role of H 2O 2 on the kinetics of low-affinity high-capacity Na +-dependent alanine transport in SHR proximal tubular epithelial cells

The presence of high and low sodium affinity states for the Na +-dependent [ 14C]- l-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538–R547). This study evaluated the role of H 2O 2 on the Na +-dependent [ 14C]- l-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na + dependence of [ 14C]- l-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na + removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H 2O 2 levels in the extracellular medium significantly reduced Na +- K m and V max values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na +-dependent [ 14C]- l-alanine uptake. After removal of apocynin from the culture medium, H 2O 2 levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na +- K m and V max of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H 2O 2 in excess is required for the presence of a low-affinity high-capacity component for the Na +-dependent [ 14C]- l-alanine uptake in SHR PTE cells only. It is suggested that Na + binding in renal ASCT2 may be regulated by ROS in SHR PTE cells.

Characterization and astrocytic modulation of system L transporters in brain microvasculature endothelial cells

Brain trafficking of amino acids is mainly mediated by amino acids transport machineries of the blood-brain barrier (BBB), where astrocytes play a key maintenance role. However, little is known about astrocytes impacts on such transport systems, in particular system L that consists of large and small neutral amino acids (NAAs) transporters, that is, LAT1/4F2hc and LAT2/4F2hc, respectively. In the current investigation, functionality and expression of system L were studied in the immortalized mouse brain microvascular endothelial b.End3 cells cocultured with astrocytes or treated with astrocyte-conditioned media (ACM). LAT2/4F2hc mediated luminal uptake of L-phenylalanine and L-leucine resulted in significantly decreased affinity of system L in b.End3 cells treated with ACM, while LAT2/4F2hc mediated luminal uptake of L-alanine remained unchanged. Gene expression analysis revealed marked upregulation of LAT1 and 4F2hc, but downregulation of LAT2 in b.End3 cells cultured with ACM. The basal to apical transport of L-phenylalanine and L-alanine appeared to be significantly greater than that of the apical to basal direction in b.End3 cells indicating an efflux functionality of system L. No marked influence was observed for transport of L-phenylalanine in b.End3 cells cocultured with astrocytes, while a slight decrease was seen for L-alanine in the basal to apical direction. Based on our findings, we propose that system L functions as influx and/or efflux transport machinery displaying a greater propensity for the outward transport of large and small NAAs. Astrocytes appeared to modulate the transcriptic expression and uptake functionalities of system L, but not the transport activities.

Functional activity of a large neutral amino acid transporter (LAT) in rabbit retina: A study involving the in vivo retinal uptake and vitreal pharmacokinetics of l-phenyl alanine

Purpose The purpose of this study is to elucidate the functional activity of large neutral amino acid transporter (LAT) in rabbit retina and to delineate its role in the retinal uptake and intravitreal pharmacokinetics of l-phenylalanine ( l-Phe). Methods In vivo retinal uptake of l-Phe and l-alanine ( l-Ala) was determined in the presence and absence of specific transport inhibitors following intravitreal administration. l and d isomers of amino acids were employed as inhibitors to determine the stereo-selectivity of LAT. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out for LAT isoforms (LAT1 and LAT2). Vitreal disposition of l-Phe following administration in rabbit vitreous was studied in the presence of an other competing LAT substrate d-methionine using microdialysis. Results Retinal uptake of l-Phe was significantly inhibited in presence of a specific LAT inhibitor, 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH), l isomers of large neutral amino acids and LAT1 specific but not by LAT2 specific and charged amino acids. No significant inhibition of l-Ala retinal uptake was observed with LAT substrates. LAT isoforms (LAT1 and LAT2) were identified by RT-PCR in rabbit retina. The mean residence time (MRT) and area under curve (AUC) values of l-Phe following intravitreal adminstration were significantly increased in the presence of d-methionine, a LAT substrate. Conclusions This study demonstrates the functional activity and molecular expression of large neutral amino acid transporter in the rabbit retina. Furthermore, based on these studies it can be concluded that LAT is involved in the retinal uptake and intravitreal elimination of l-Phe.

Underexpression of the Na+-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

Mouse Oocytes Regulate Metabolic Cooperativity Between Granulosa Cells and Oocytes: Amino Acid Transport1

A search for genes expressed more highly in mouse cumulus cells than mural granulosa cells by subtraction hybridization yielded Slc38a3. SLC38A3 is a sodium-coupled neutral amino acid transporter having substrate preference for l-glutamate, l-histidine, and l-alanine. Detectable levels of Slc38a3 mRNA were found by in situ hybridization in granulosa cells of large preantral follicles, but levels were higher in all granulosa cells of small antral follicles; expression became limited to cumulus cells of large antral follicles. Expression of Slc38a3 mRNA in granulosa cells was promoted by fully grown oocytes from antral follicles but not by growing oocytes from preantral follicles. Fully grown oocytes were dependent on cumulus cells for uptake of l-alanine and l-histidine but not l-leucine. Fully grown but not growing oocytes secreted one or more paracrine factors that promoted cumulus cell uptake of all three amino acids but of l-alanine and l-histidine to a much greater extent than l-leucine. Uptake of l-leucine appeared dependent primarily on contact-mediated signals from fully grown oocytes. Fully grown oocytes also promoted elevated levels of Slc38a3 mRNA and l-alanine transport by preantral granulosa cells, but growing oocytes did not. Therefore, fully grown oocytes secrete one or more paracrine factors that promote cumulus cell uptake of amino acids that oocytes themselves transport poorly. These amino acids are likely transferred to oocytes via gap junctions. Thus, oocytes use paracrine signals to promote their own development via metabolic cooperativity with cumulus cells. The ability of oocytes to mediate this cooperativity is developmentally regulated and acquired only in later stages of oocyte development.

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H(+)/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5' and 3' untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na(+)-independent uptake of proline, glycine, l-alanine and alpha-(methylamino)isobutyric acid. Proline uptake was maximal at pH 5.0 (K(m) 2.2+/-0.7 mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na+ but in the presence of an inwardly directed H+ gradient. In the presence of Na+ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.

Identification and Characterization of a Na+-Dependent Neutral Amino Acid Transporter, ASCT1, in Rabbit Corneal Epithelial Cell Culture and Rabbit Cornea

Purpose: The aim of this study was to investigate the presence of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit primary corneal epithelial cell culture and rabbit cornea. Methods: Uptake studies were carried out on rabbit primary corneal epithelial culture (rPCEC) cells using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34°C. Uptake and transport of L-alanine was determined at various concentrations. Inhibition studies were conducted in presence of various L- and D-amino acids, metabolic inhibitors like ouabain and sodium azide, and in the absence of sodium to delineate the functional characteristics of L-alanine uptake and transport. Reverse transcription–polymerase chain reaction (RT-PCR) was performed on total RNA harvested from rabbit cornea and rPCEC cells for identification of ASCT1. Results: Uptake of L-Ala was found to be saturable with a Km of 0.71 mM and a Vmax value of 0.84 μ moles min−1 mg−1 protein. Uptake was independent of pH and energy but depends on sodium. It was inhibited by serine, threonine, cysteine, and glutamine but did not respond to BCH (2-aminobicyclo [2,2,1] heptane-2-carboxylic acid) and MeAIB (α -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (Km 6.52 mM and Vmax 1.09 × 10−2 μ moles min−1 cm−2), energy independent, and subject to similar competitive inhibition. Presence of ASCT1 on rPCEC and on rabbit cornea was identified by RT-PCR. Conclusions: L-Alanine, the chosen model substrate, was actively transported by Na+-dependent, neutral amino acid exchanger ASCT1, which was identified and functionally characterized on rPCEC cells and rabbit cornea.

Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout.

The relative function of IGF-I and insulin on fish muscle metabolism and growth has been investigated by the isolation and culture at different stages (myoblasts at day 1, myocytes at day 4, and myotubes at day 10) of rainbow trout muscle cells. This in vitro model avoids interactions with endogenous peptides, which could interfere with the muscle response. In these cells, the effects of IGF-I and insulin on cell proliferation, 2-deoxyglucose (2-DG), and l-alanine uptake at different development stages, and the use of inhibitors were studied and quantified. Insulin (10-1,000 nM) and IGF-I (10-100 nM) stimulated 2-DG uptake in trout myocytes at day 4 in a similar manner (maximum of 124% for insulin and of 142% for IGF-I), and this stimulation increased when cells differentiated to myotubes (maximum for IGF-I of 193%). When incubating the cells with PD-98059 and especially cytochalasin B, a reduction in 2-DG uptake was observed, suggesting that glucose transport takes place through specific facilitative transporters. IGF-I (1-100 nM) stimulated the l-alanine uptake in myocytes at day 4 (maximum of 239%), reaching higher values of stimulation than insulin (100-1,000 nM) (maximum of 160%). This stimulation decreased when cells developed to myotubes at day 10 (118% for IGF-I and 114% for insulin). IGF-I (0.125-25 nM) had a significant effect on myoblast proliferation, measured by thymidine incorporation (maximum of 170%), and required the presence of 2-5% fetal serum (FBS) to promote thymidine uptake. On the other hand, insulin was totally ineffective in stimulating thymidine uptake. We conclude that IGF-I is more effective than insulin in stimulating glucose and alanine uptake in rainbow trout myosatellite cells and that the degree of stimulation changes when cells differentiate to myotubes. IGF-I stimulates cell proliferation in this model of muscle in vitro and insulin does not. These results indicate the important role of IGF-I on growth and metabolism of fish muscle.

Mouse system-N amino acid transporter, mNAT3, expressed in hepatocytes and regulated by insulin-activated and phosphoinositide 3-kinase-dependent signalling.

Amino acid transporters are essential for normal cell function and physiology. In the present study, we report the identification and functional and regulatory characterization of a mouse system-N amino acid transporter, mNAT3. Expression of mNAT3 in Xenopus oocytes revealed that the strongest transport activities were preferred for L-alanine. In addition, mNAT3 is an Na(+)- and pH-dependent low-affinity transporter and it partially tolerates substitution of Na(+) by Li(+). mNAT3 has been found to be expressed predominantly in the liver, where it is localized to the plasma membrane of hepatocytes, with the strongest expression in those cells adjacent to the central vein, decreasing gradually towards the portal tract. Treatment of mouse hepatocyte-like H2.35 cells with insulin led to a significant increase in the expression of mNAT3, and this stimulation was associated closely with an increase in the uptake of L-alanine. Interestingly, this insulin-induced stimulatory effect on mNAT3 expression was attenuated by the phosphoinositide 3-kinase inhibitor LY294002, but not by the mitogen-activated protein kinase inhibitor PD98059, although both kinases were fully activated by insulin. The results suggest that insulin-mediated regulation of mNAT3 is likely to be mediated through a phosphoinositide 3-kinase-dependent signalling pathway. The unique expression pattern and insulin-mediated regulatory properties of mNAT3 suggest that this transporter may play an important role in liver physiology.

Inhibitory Effects of Basic Drugs on the Sodium-Dependent Transport of L-Alanine via System B0 in the Small Intestine

To elucidate the mechanism of the interaction of basic drugs with Na(+)-dependent L-alanine absorption from the small intestine, we investigated the effect of imipramine on the transport of L-alanine via system B(0), which is thought to be one of the main Na(+)-dependent systems for intestinal absorption of short-chain neutral amino acids, including L-alanine. The uptake of L-alanine by cells that express hATB(0) (human amino acid transporter B(0)) was inhibited in the presence of imipramine. The Eadie-Hofstee plot showed that the inhibition was not competitive with the substrate (L-alanine) but competitive with Na(+). When rat intestinal brush border membrane vesicles were used, several basic drugs had inhibitory effects. Inhibition was also observed in intestinal absorption evaluated by an in situ single-pass perfusion technique. However, the potencies of inhibition were different. The potency of inhibition was dependent on the lipophilicity of the drugs.

Epidermal Growth Factor Regulation of System L Alanine Transport in Undifferentiated and Differentiated Intestinal Caco-2 Cells

Epidermal growth factor (EGF) in intestinal lumen regulates many gut epithelial cell functions. Influenced by growth factors at various differentiation stages, enterocytes execute the major task of absorbing nutrient amino acids. The purpose of this study was to investigate the effects of EGF on Na +-independent L-alanine transport in intestinal epithelial cells. Na +-independent [ 3H]-L-alanine transport was measured in the differentiating Caco-2 cells. In both the undifferentiated and differentiated states, L-alanine uptake occurred via a single saturable Na +-independent system L plus simple passive diffusion. System L activity decreased as the cells progressed from the undifferentiated to the differentiated state. Prolonged incubation with EGF (>30 hours) resulted in a 70% increase in system L activity in both undifferentiated and differentiated cells. EGF stimulated the system L V max without affecting K m. System L activity stimulation was inhibited by chelerythrine chloride, cycloheximide, or actinomycin D. These data suggest that intestinal epithelial cell differentiation is associated with a decrease in system L transport capacity. EGF activates system L transport activity through a signaling mechanism involving protein kinase C, independent of cell differentiation state. Both cell differentiation and EGF regulation of system L activity occur via alteration of functional copies of the system L transporter. ( J Gastrointest Surg 2002;6:410–417.)

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.