System-L amino acid transporters play a key role in pancreatic β-cell signalling and function

Abstract The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into the mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influence β-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets, and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L amino acid transporter 1 (LAT1) is abundantly expressed in the islets, and that knockdown of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the LAT1 is required for regulating β-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes.

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Na(+)-independent transport (uniport) of amino acids and glucose in mammalian cells.

Journal of Experimental Biology ◽

10.1242/jeb.196.1.93 ◽

1994 ◽

Vol 196 (1) ◽

pp. 93-108

Author(s):

D K Kakuda ◽

C L MacLeod

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Mammalian Cells ◽

Glucose Transporter ◽

Sequence Similarity ◽

Amino Acid Sequence Similarity ◽

Amino Acid Transporters ◽

Cationic Amino Acid ◽

Transporter Family ◽

Share Amino Acid Sequence

Recent advances have made possible the isolation of the genes and their cDNAs encoding Na(+)-independent amino acid transporters. Two classes of amino acid 'uniporters' have been isolated. One class contains the mCAT (murine cationic amino acid transporter) gene family that encodes proteins predicted to span the membrane 12-14 times and exhibits structural properties similar to the GLUT (glucose transporter) family and to other well-known transporters. The other class consists of two known genes, rBAT (related to B system amino acid transporters) and 4F2hc, that share amino acid sequence similarity with alpha-amylases and alpha-glucosidases. They are type II glycoproteins predicted to span the membrane only once, yet they mediate the Na(+)-independent transport of cationic and zwitterionic amino acids in Xenopus oocytes. Mutations in the human rBAT gene have been identified by Palacín and his co-workers in several families suffering from a heritable form of cystinuria. This important finding clearly establishes a key role for rBAT in cystine transport. The two classes of amino acid transporters are compared with the well-studied GLUT family of Na(+)-independent glucose transporters.

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The role of amino acid transporters in inherited and acquired diseases

Biochemical Journal ◽

10.1042/bj20101912 ◽

2011 ◽

Vol 436 (2) ◽

pp. 193-211 ◽

Cited By ~ 109

Author(s):

Stefan Bröer ◽

Manuel Palacín

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Mammalian Cells ◽

Neuronal Excitability ◽

Tumour Progression ◽

Building Blocks ◽

Amino Acid Uptake ◽

Whole Body ◽

Amino Acid Transporters ◽

Acid Uptake

Amino acids are essential building blocks of all mammalian cells. In addition to their role in protein synthesis, amino acids play an important role as energy fuels, precursors for a variety of metabolites and as signalling molecules. Disorders associated with the malfunction of amino acid transporters reflect the variety of roles that they fulfil in human physiology. Mutations of brain amino acid transporters affect neuronal excitability. Mutations of renal and intestinal amino acid transporters affect whole-body homoeostasis, resulting in malabsorption and renal problems. Amino acid transporters that are integral parts of metabolic pathways reduce the function of these pathways. Finally, amino acid uptake is essential for cell growth, thereby explaining their role in tumour progression. The present review summarizes the involvement of amino acid transporters in these roles as illustrated by diseases resulting from transporter malfunction.

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Transporters for Cationic Amino Acids in Animal Cells: Discovery, Structure, and Function

Physiological Reviews ◽

10.1152/physrev.1998.78.2.487 ◽

1998 ◽

Vol 78 (2) ◽

pp. 487-545 ◽

Cited By ~ 337

Author(s):

R. DEVÉS ◽

C. A. R. BOYD

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Structure And Function ◽

Xenopus Laevis Oocytes ◽

Amino Acid Transporters ◽

Cdna Clones ◽

Animal Cells ◽

Cationic Amino Acid ◽

Cationic Amino Acids ◽

And Function

Devés, R., and C. A. R. Boyd. Transporters for Cationic Amino Acids in Animal Cells: Discovery, Structure, and Function. Physiol. Rev. 78: 487–545, 1998. — The structure and function of the four cationic amino acid transporters identified in animal cells are discussed. The systems differ in specificity, cation dependence, and physiological role. One of them, system y+, is selective for cationic amino acids, whereas the others (B0,+, b0,+, and y+L) also accept neutral amino acids. In recent years, cDNA clones related to these activities have been isolated. Thus two families of proteins have been identified: 1) CAT or cationic amino acid transporters and 2) BAT or broad-scope transport proteins. In the CAT family, three genes encode for four different isoforms [CAT-1, CAT-2A, CAT-2(B) and CAT-3]; these are ∼70-kDa proteins with multiple transmembrane segments ( 12 – 14 ), and despite their structural similarity, they differ in tissue distribution, kinetics, and regulatory properties. System y+is the expression of the activity of CAT transporters. The BAT family includes two isoforms (rBAT and 4F2hc); these are 59- to 78-kDa proteins with one to four membrane-spanning segments, and it has been proposed that these proteins act as transport regulators. The expression of rBAT and 4F2hc induces system b0,+and system y+L activity in Xenopus laevis oocytes, respectively. The roles of these transporters in nutrition, endocrinology, nitric oxide biology, and immunology, as well as in the genetic diseases cystinuria and lysinuric protein intolerance, are reviewed. Experimental strategies, which can be used in the kinetic characterization of coexpressed transporters, are also discussed.

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Increased amino acid supply potentiates glucose-stimulated insulin secretion but does not increase β-cell mass in fetal sheep

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00377.2012 ◽

2013 ◽

Vol 304 (4) ◽

pp. E352-E362 ◽

Cited By ~ 15

Author(s):

Monika M. Gadhia ◽

Anne M. Maliszewski ◽

Meghan C. O'Meara ◽

Stephanie R. Thorn ◽

Jinny R. Lavezzi ◽

...

Keyword(s):

Amino Acids ◽

Insulin Secretion ◽

Amino Acid ◽

Cell Mass ◽

Late Gestation ◽

Insulin Content ◽

Acid Mixture ◽

Β Cell ◽

Fetal Sheep ◽

Glucose Stimulated Insulin Secretion

Amino acids and glucose acutely stimulate fetal insulin secretion. In isolated adult pancreatic islets, amino acids potentiate glucose-stimulated insulin secretion (GSIS), but whether amino acids have this same effect in the fetus is unknown. Therefore, we tested the effects of increased fetal amino acid supply on GSIS and morphology of the pancreas. We hypothesized that increasing fetal amino acid supply would potentiate GSIS. Singleton fetal sheep received a direct intravenous infusion of an amino acid mixture (AA) or saline (CON) for 10–14 days during late gestation to target a 25–50% increase in fetal branched-chain amino acids (BCAA). Early-phase GSIS increased 150% in the AA group ( P < 0.01), and this difference was sustained for the duration of the hyperglycemic clamp (105 min) ( P < 0.05). Glucose-potentiated arginine-stimulated insulin secretion (ASIS), pancreatic insulin content, and pancreatic glucagon content were similar between groups. β-Cell mass and area were unchanged between groups. Baseline and arginine-stimulated glucagon concentrations were increased in the AA group ( P < 0.05). Pancreatic α-cell mass and area were unchanged. Fetal and pancreatic weights were similar. We conclude that a sustained increase of amino acid supply to the normally growing late-gestation fetus potentiated fetal GSIS but did not affect the morphology or insulin content of the pancreas. We speculate that increased β-cell responsiveness (insulin secretion) following increased amino acid supply may be due to increased generation of secondary messengers in the β-cell. This may be enhanced by the paracrine action of glucagon on the β-cell.

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SLC6A14 and SLC38A5 Drive the Glutaminolysis and Serine–Glycine–One-Carbon Pathways in Cancer

Pharmaceuticals ◽

10.3390/ph14030216 ◽

2021 ◽

Vol 14 (3) ◽

pp. 216

Author(s):

Tyler Sniegowski ◽

Ksenija Korac ◽

Yangzom D. Bhutia ◽

Vadivel Ganapathy

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cancer Cells ◽

Critical Role ◽

Amino Acid Transporters ◽

Extracellular Milieu ◽

Methyl Transfer ◽

Functional Features ◽

Metabolic Reactions ◽

Carbon Pathways

The glutaminolysis and serine–glycine–one-carbon pathways represent metabolic reactions that are reprogramed and upregulated in cancer; these pathways are involved in supporting the growth and proliferation of cancer cells. Glutaminolysis participates in the production of lactate, an oncometabolite, and also in anabolic reactions leading to the synthesis of fatty acids and cholesterol. The serine–glycine–one-carbon pathway is involved in the synthesis of purines and pyrimidines and the control of the epigenetic signature (DNA methylation, histone methylation) in cancer cells. Methionine is obligatory for most of the methyl-transfer reactions in the form of S-adenosylmethionine; here, too, the serine–glycine–one-carbon pathway is necessary for the resynthesis of methionine following the methyl-transfer reaction. Glutamine, serine, glycine, and methionine are obligatory to fuel these metabolic pathways. The first three amino acids can be synthesized endogenously to some extent, but the need for these amino acids in cancer cells is so high that they also have to be acquired from extracellular sources. Methionine is an essential amino acid, thus making it necessary for cancer cells to acquire this amino acid solely from the extracellular milieu. Cancer cells upregulate specific amino acid transporters to meet this increased demand for these four amino acids. SLC6A14 and SLC38A5 are the two transporters that are upregulated in a variety of cancers to mediate the influx of glutamine, serine, glycine, and methionine into cancer cells. SLC6A14 is a Na+/Cl− -coupled transporter for multiple amino acids, including these four amino acids. In contrast, SLC38A5 is a Na+-coupled transporter with rather restricted specificity towards glutamine, serine, glycine, and methionine. Both transporters exhibit unique functional features that are ideal for the rapid proliferation of cancer cells. As such, these two amino acid transporters play a critical role in promoting the survival and growth of cancer cells and hence represent novel, hitherto largely unexplored, targets for cancer therapy.

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Detailed characterization of the UMAMITs provides insight into their evolution, functional properties as amino acid transporters and role in the plant

10.1101/2021.02.18.431885 ◽

2021 ◽

Author(s):

Chengsong Zhao ◽

Réjane Pratelli ◽

Shi Yu ◽

Brett Shelley ◽

Eva Collakova ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Functional Properties ◽

Critical Role ◽

Amino Acid Transporters ◽

Cell Compartments ◽

Transporter Family ◽

Insight Into

AbstractAmino acid transporters play a critical role in distributing amino acids within the cell compartments and between the plant organs. Despite this importance, relatively few amino acid transporter genes have been characterized and their role elucidated with certainty. Two main families of proteins encode amino acid transporters in plants: the Amino Acid-Polyamine-Organocation superfamily, containing mostly importers, and the Usually Multiple Acids Move In and out Transporter family, apparently encoding exporters, totaling about 100 genes in Arabidopsis alone. Knowledge on UMAMITs is scarce, focused on six Arabidopsis genes and a handful of genes from other species. To get insight into the role of the members of this family and provide data to be used for future characterization, we studied the evolution of the UMAMITs in plants, and determined the functional properties, the structure, and the localization of the 44 Arabidopsis UMAMITs. Our analysis showed that the AtUMAMIT are essentially localized at the tonoplast or the plasma membrane, and that most of them are able to export amino acids from the cytosol, confirming a role in intra- and inter-cellular amino acid transport. As an example, this set of data was used to hypothesize the role of a few AtUMAMITs in the plant and the cell.

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Amino acid transporters in the regulation of insulin secretion and signalling

Biochemical Society Transactions ◽

10.1042/bst20180250 ◽

2019 ◽

Vol 47 (2) ◽

pp. 571-590 ◽

Cited By ~ 6

Author(s):

Kiran Javed ◽

Stephen J. Fairweather

Keyword(s):

Amino Acids ◽

Small Intestine ◽

Insulin Secretion ◽

Amino Acid ◽

Insulin Signalling ◽

Amino Acid Transporter ◽

Fibroblast Growth Factor 21 ◽

Amino Acid Transporters ◽

Acid Transporter

Abstract Amino acids are increasingly recognised as modulators of nutrient disposal, including their role in regulating blood glucose through interactions with insulin signalling. More recently, cellular membrane transporters of amino acids have been shown to form a pivotal part of this regulation as they are primarily responsible for controlling cellular and circulating amino acid concentrations. The availability of amino acids regulated by transporters can amplify insulin secretion and modulate insulin signalling in various tissues. In addition, insulin itself can regulate the expression of numerous amino acid transporters. This review focuses on amino acid transporters linked to the regulation of insulin secretion and signalling with a focus on those of the small intestine, pancreatic β-islet cells and insulin-responsive tissues, liver and skeletal muscle. We summarise the role of the amino acid transporter B0AT1 (SLC6A19) and peptide transporter PEPT1 (SLC15A1) in the modulation of global insulin signalling via the liver-secreted hormone fibroblast growth factor 21 (FGF21). The role of vesicular vGLUT (SLC17) and mitochondrial SLC25 transporters in providing glutamate for the potentiation of insulin secretion is covered. We also survey the roles SNAT (SLC38) family and LAT1 (SLC7A5) amino acid transporters play in the regulation of and by insulin in numerous affective tissues. We hypothesise the small intestine amino acid transporter B0AT1 represents a crucial nexus between insulin, FGF21 and incretin hormone signalling pathways. The aim is to give an integrated overview of the important role amino acid transporters have been found to play in insulin-regulated nutrient signalling.

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Prolonged L-alanine exposure induces changes in metabolism, Ca2+ handling and desensitization of insulin secretion in clonal pancreatic β-cells

Clinical Science ◽

10.1042/cs20080138 ◽

2009 ◽

Vol 116 (4) ◽

pp. 341-351 ◽

Cited By ~ 10

Author(s):

Neville H. McClenaghan ◽

Siobhan M. Scullion ◽

Brian Mion ◽

Chandralal Hewage ◽

J. Paul G. Malthouse ◽

...

Keyword(s):

Amino Acids ◽

Insulin Secretion ◽

Amino Acid ◽

Pyruvate Dehydrogenase ◽

Metabolic Flux ◽

Prolonged Exposure ◽

Area Under The Curve ◽

Control Culture ◽

Β Cells ◽

Β Cell

Acute insulin-releasing actions of amino acids have been studied in detail, but comparatively little is known about the β-cell effects of long-term exposure to amino acids. The present study examined the effects of prolonged exposure of β-cells to the metabolizable amino acid L-alanine. Basal insulin release or cellular insulin content were not significantly altered by alanine culture, but acute alanine-induced insulin secretion was suppressed by 74% (P<0.001). Acute stimulation of insulin secretion with glucose, KCl or KIC (2-oxoisocaproic acid) following alanine culture was not affected. Acute alanine exposure evoked strong cellular depolarization after control culture, whereas AUC (area under the curve) analysis revealed significant (P<0.01) suppression of this action after culture with alanine. Compared with control cells, prior exposure to alanine also markedly decreased (P<0.01) the acute elevation of [Ca2+]i (intracellular [Ca2+]) induced by acute alanine exposure. These diminished stimulatory responses were partially restored after 18 h of culture in the absence of alanine, indicating reversible amino-acid-induced desensitization. 13C NMR spectra revealed that alanine culture increased glutamate labelling at position C4 (by 60%; P<0.01), as a result of an increase in the singlet peak, indicating increased flux through pyruvate dehydrogenase. Consistent with this, protein expression of the pyruvate dehydrogenase kinases PDK2 and PDK4 was significantly reduced. This was accompanied by a decrease in cellular ATP (P<0.05), consistent with diminished insulin-releasing actions of this amino acid. Collectively, these results illustrate the phenomenon of β-cell desensitization by amino acids, indicating that prolonged exposure to alanine can induce reversible alterations to metabolic flux, Ca2+ handling and insulin secretion.

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Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

Methods and Protocols ◽

10.3390/mps4030051 ◽

2021 ◽

Vol 4 (3) ◽

pp. 51

Author(s):

Satish Kantipudi ◽

Daniel Harder ◽

Sara Bonetti ◽

Dimitrios Fotiadis ◽

Jean-Marc Jeckelmann

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Protein Complexes ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Expression Host ◽

Amino Acids And Derivatives ◽

Heterodimeric Complex ◽

Acid Transporter ◽

And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

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