Identification of Iodotyrosines as Novel Substrates for the Thyroid Hormone Transporter MCT8.

Background: Monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone transporter identified to date, deficiency of which has been associated with severe intellectual and motor disability and abnormal serum thyroid function tests. However, it is presently unknown if MCT8, similar to other thyroid hormone transporters, also accepts additional substrates, and if disruption of their transport may contribute to the observed phenotype. Methods: In this study, we aimed to identify such substrates by applying liquid chromatography-mass spectrometry-based metabolome analysis in lysates of control and MCT8-overexpressing Xenopus oocytes. A subset of identified candidate substrates were validated by direct transport studies in transiently transfected COS-1 cells and human fibroblasts, which endogenously express MCT8. Moreover, transport characteristics were determined, including transport saturation and cis-inhibition potency of thyroid hormone transport. Results: Metabolome analysis identified 21 m/z ratios, corresponding to 87 candidate metabolites, with a 2.0-times differential abundance in MCT8-injected oocytes compared with controls. These metabolites included 3,5-diiodotyrosine (DIT) and several amino acids, including glutamate and glutamine. In accordance, MCT8-expressing COS-1 cells had 2.2-times lower intracellular accumulation of [125I]-DIT compared with control cells. This effect was largely blocked in the presence of 3,3',5-triiodothyronine (T3) (IC50: 2.5 ± 1.5 µM) or thyroxine (T4) (IC50: 5.8 ± 1.3 µM). Conversely, increasing concentrations of DIT enhanced the accumulation of T3 and T4. The MCT8-specific inhibitor silychristin increased the intracellular accumulation of DIT in human fibroblasts. COS-1 cells expressing MCT8 also exhibited a 50% reduction in intracellular accumulation of [125I]-3-monoiodotyrosine (MIT). In contrast, COS-1 cells expressing MCT8 did not alter the intracellular accumulation of [3H]-glutamate or [3H]-glutamine. However, studies in human fibroblasts showed a 1.5-1.9 times higher glutamate uptake in control fibroblasts compared with fibroblasts derived from patients with MCT8 deficiency, which was not affected in the presence of silychristin. Conclusions: Taken together, our results suggest that the iodotyrosines DIT and MIT can be exported by MCT8. MIT and DIT interfere with MCT8-mediated transport of thyroid hormone in vitro and vice versa. Future studies should elucidate if MCT8, being highly expressed in thyroidal follicular cells, also transports iodotyrosines in vivo.

Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis.

BACKGROUND: The expression of amino acid transporters is known to vary during acute pancreatitis (AP) except for LAT1 (slc7a5), the expression of which remains stable. LAT1 supports cell growth by importing leucine and thereby stimulates mammalian target of rapamycin (mTOR) activity, a phenomenon often observed in cancer cells. The mechanisms by which LAT1 influences physiological and pathophysiological processes and affects disease progression in the pancreas are not yet known. AIM: To evaluate the role of LAT1 in the development of and recovery from AP. METHODS: AP was induced with caerulein (cae) injections in female and male mice expressing LAT1 or after its knockout (LAT1 Cre/LoxP). The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight, assessing microscopical tissue architecture, mRNA and protein expression, protein synthesis, and enzyme activity levels, as well as by testing the recruitment of immune cells by FACS and ELISA. RESULTS: The initial injury, evaluated by measurements of plasma amylase, lipase, and trypsin activity, as well as the gene expression of dedifferentiation markers, did not differ between the groups. However, early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice. Especially in females, we observed less mTOR reactivation and dysfunctional autophagy. The later regeneration phase was clearly delayed in female LAT1 knockout mice, which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein (rbpjl) and basic helix-loop-helix family member A15 (mist1). Amylase mRNA and protein levels remained lower, and, strikingly, female LAT1 knockout mice presented signs of fibrosis lasting until day seven. In contrast, pancreas morphology had returned to normal in wild-type littermates. CONCLUSION: LAT1 supports the regeneration of acinar cells after AP. Female mice lacking LAT1 exhibited more pronounced alterations than male mice, indicating a sexual dimorphism of amino acid metabolism.

Citrulline, Biomarker of Enterocyte Functional Mass and Dietary Supplement. Metabolism, Transport, and Current Evidence for Clinical Use.

L-Citrulline is a non-essential but still important amino acid that is released from enterocytes. Because plasma levels are reduced in case of impaired intestinal function, it has become a biomarker to monitor intestinal integrity. Moreover, oxidative stress induces protein citrullination, and antibodies against anti-citrullinated proteins are useful to monitor rheumatoid diseases. Citrullinated histones, however, may even predict a worse outcome in cancer patients. Supplementation of citrulline is better tolerated compared to arginine and might be useful to slightly improve muscle strength or protein balance. The following article shall provide an overview of L-citrulline properties and functions, as well as the current evidence for its use as a biomarker or as a therapeutic supplement.

ACE2 and gut amino acid transport.

ACE2 is a type I membrane protein with extracellular carboxypeptidase activity displaying a broad tissue distribution with highest expression levels at the brush border membrane (BBM) of small intestine enterocytes and a lower expression in stomach and colon. In small intestinal mucosa, ACE2 mRNA expression appears to increase with age and to display higher levels in patients taking ACE-inhibitors (ACE-I). There, ACE2 protein heterodimerizes with the neutral amino acid transporter Broad neutral Amino acid Transporter 1 (B0AT1) (SLC6A19) or the imino acid transporter Sodium-dependent Imino Transporter 1 (SIT1) (SLC6A20), associations that are required for the surface expression of these transport proteins. These heterodimers can form quaternary structures able to function as binding sites for SARS-CoV-2 spike glycoproteins. The heterodimerization of the carboxypeptidase ACE2 with B0AT1 is suggested to favor the direct supply of substrate amino acids to the transporter, but whether this association impacts the ability of ACE2 to mediate viral infection is not known. B0AT1 mutations cause Hartnup disorder, a condition characterized by neutral aminoaciduria and, in some cases, pellagra-like symptoms, such as photosensitive rash, diarrhea, and cerebellar ataxia. Correspondingly, the lack of ACE2 and the concurrent absence of B0AT1 expression in small intestine causes a decrease in l-tryptophan absorption, niacin deficiency, decreased intestinal antimicrobial peptide production, and increased susceptibility to inflammatory bowel disease (IBD) in mice. Thus, the abundant expression of ACE2 in small intestine and its association with amino acid transporters appears to play a crucial role for the digestion of peptides and the absorption of amino acids and, thereby, for the maintenance of structural and functional gut integrity.

Phosphorylation of mouse intestinal basolateral amino acid uniporter LAT4 is controlled by food-entrained diurnal rhythm and dietary proteins.

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.

Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance.

BACKGROUND: Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2-20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. METHODS: Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. RESULTS: The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2-20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). CONCLUSION: These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.

Mucosal Monosaccharide Transporter Expression in Newborns With Jejunoileal Atresia and Along the Adult Intestine.

OBJECTIVES: In newborn rodents, intestinal maturation involves delayed fructose transporter GLUT5 expression until weaning. In jejunoileal atresia (JIA), distal intestinal segments lack exposure to amniotic fluid-containing carbohydrates. We assessed in human newborns, the impact of intestinal maturation and obstruction on mucosal monosaccharide transporter expression. METHODS: Samples were obtained from 10 newborns operated for small intestinal atresia and from 17 adults undergoing gastroduodenoscopy and/or ileocolonoscopy. mRNA expression of the transporters SGLT1, GLUT1, GLUT2, GLUT5, and GLUT7 was measured in neonate samples proximal and distal of the atresia as well as in adult duodenum, ileum, and colon. Protein expression and localization was assessed using immunofluorescence. RESULTS: Although mRNA expression of monosaccharide transporters did not significantly differ between newborn and adult samples, luminal fructose transporter GLUT5 protein was absent in 0- to 4-day-old neonates, but expressed in adults. The mRNA expression of the 5 tested monosaccharide transporters was unchanged distal from the JIA relative to proximal. Similarly, luminal sodium-dependent glucose transporter SGLT1 and basolateral GLUT2 were expressed proximal and distal to JIA as visualized by immunofluorescence staining. With the exception of glucose transporter GLUT1 that showed highest expression levels in colon, all investigated hexose transporters showed strongest expression in duodenum, lower levels in ileum and lowest in colon. CONCLUSIONS: Human newborns lack small intestinal fructose transporter GLUT5 protein expression and small intestinal atresia does not affect the expression of hexose transporters.

Dysfunctional LAT2 Amino Acid Transporter Is Associated With Cataract in Mouse and Humans.

Cataract, the loss of ocular lens transparency, accounts for ∼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 (Slc7a8) and uniporter TAT1 (Slc16a10) are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening SLC7A8 in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects.

Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids.

Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivoMethods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.

Abnormal creatine transport of mutations in monocarboxylate transporter 12 (MCT12) found in patients with age-related cataract can be partially rescued by exogenous chaperone CD147.

Membrane transporters influence biological functions in the ocular lens. Here, we investigate the monocarboxylate transporter 12 (MCT12), also called creatine transporter 2 (CRT2), which is found in the ocular lens and is involved in cataract. As the age-related form affects about half of the population world-wide, understanding relevant pathomechanisms is a prerequisite for exploring non-invasive treatments. We screened the coding exons of the gene SLC16A12 in 877 patients from five cohorts, including Caucasian and Asian ethnicities. A previously identified risk factor, SNP rs3740030, displayed different frequencies in the Asian cohorts but risk could not be established. In 15 patients 13 very rare heterozygous nucleotide substitutions were identified, of which eight led to non-synonymous and four to synonymous amino acid exchanges and one mapped to the canonical splice site in intron 3. Their impact on creatine transport was tested in Xenopus laevis oocytes and human HEK293T cells. Four variants (p.Ser158Pro, p.Gly205Val, p.Pro395Gln and p.Ser453Arg) displayed severe reduction in both model systems, indicating conserved function. Two of these, p.Gly205Val, and p.Ser453Arg, did not localize to the oocyte membrane, suggesting possible impacts on protein interactions for transporter processing. In support, exogenously supplied excess of MCT12's chaperone CD147 in HEK293T cells led to a partial recovery of the defective uptake activity from p.Gly205Val and also from mutant p.Pro395Gln, which did localize to the membrane. Our findings provide first insight in the molecular requirements of creatine transporter, with particular emphasis on rescuing effects by its chaperone CD147, which can provide useful pharmacological information for substrate delivery.

Expression and regulation of the neutral amino acid transporter B0AT1 in rat small intestine.

Absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter B0AT1 (SLC6A19). Its intestinal expression depends on co-expression of the membrane-anchored peptidase angiotensin converting enzyme 2 (ACE2) and is additionally enhanced by aminopeptidase N (CD13). We investigated in this study the expression of B0AT1 and its auxiliary peptidases as well as its transport function along the rat small intestine. Additionally, we tested its possible short- and long-term regulation by dietary proteins and amino acids. We showed by immunofluorescence that B0AT1, ACE2 and CD13 co-localize on the luminal membrane of small intestinal villi and by Western blotting that their protein expression increases in distal direction. Furthermore, we observed an elevated transport activity of the neutral amino acid L-isoleucine during the nocturnal active phase compared to the inactive one. Gastric emptying was delayed by intragastric application of an amino acid cocktail but we observed no acute dietary regulation of B0AT1 protein expression and L-isoleucine transport. Investigation of the chronic dietary regulation of B0AT1, ACE2 and CD13 by different diets revealed an increased B0AT1 protein expression under amino acid-supplemented diet in the proximal section but not in the distal one and for ACE2 protein expression a reverse localization of the effect. Dietary regulation for CD13 protein expression was not as distinct as for the two other proteins. Ring uptake experiments showed a tendency for increased L-isoleucine uptake under amino acid-supplemented diet and in vivo L-isoleucine absorption was more efficient under high protein and amino acid-supplemented diet. Additionally, plasma levels of branched-chain amino acids were elevated under high protein and amino acid diet. Taken together, our experiments did not reveal an acute amino acid-induced regulation of B0AT1 but revealed a chronic dietary adaptation mainly restricted to the proximal segment of the small intestine.

Sirtuin 1 stimulates the proliferation and the expression of glycolysis genes in pancreatic neoplastic lesions.

Metabolic reprogramming is a feature of neoplasia and tumor growth. Sirtuin 1 (SIRT1) is a lysine deacetylase of multiple targets including metabolic regulators such as p53. SIRT1 regulates metaplasia in the pancreas. Nevertheless, it is unclear if SIRT1 affects the development of neoplastic lesions and whether metabolic gene expression is altered.To assess neoplastic lesion development, mice with a pancreas-specific loss of Sirt1 (Pdx1-Cre;Sirt1-lox) were bred into a KrasG12D mutant background (KC) that predisposes to the development of pancreatic intra-epithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC). Similar grade PanIN lesions developed in KC and KC;Sirt1-lox mice but specifically early mucinous PanINs occupied 40% less area in the KC;Sirt1-lox line, attributed to reduced proliferation. This was accompanied by reduced expression of proteins in the glycolysis pathway, such as GLUT1 and GAPDH.The stimulatory effect of SIRT1 on proliferation and glycolysis gene expression was confirmed in a human PDAC cell line. In resected PDAC samples, higher proliferation and expression of glycolysis genes correlated with poor patient survival. SIRT1 expression per se was not prognostic but low expression of Cell Cycle and Apoptosis Regulator 2 (CCAR2), a reported SIRT1 inhibitor, corresponded to poor patient survival.These findings open perspectives for novel targeted therapies in pancreatic cancer.

Real-time functional characterization of cationic amino acid transporters using a new FRET sensor.

L-arginine is a semi-essential amino acid that serves as precursor for the production of urea, nitric oxide (NO), polyamines, and other biologically important metabolites. Hence, a fast and reliable assessment of its intracellular concentration changes is highly desirable. Here, we report on a genetically encoded Förster resonance energy transfer (FRET)-based arginine nanosensor that employs the arginine repressor/activator ahrC gene from Bacillus subtilis. This new nanosensor was expressed in HEK293T cells, and experiments with cell lysate showed that it binds L-arginine with high specificity and with a K d of ∼177 µM. Live imaging experiments showed that the nanosensor was expressed throughout the cytoplasm and displayed a half maximal FRET increase at an extracellular L-arginine concentration of ∼22 µM. By expressing the nanosensor together with SLC7A1, SLC7A2B, or SLC7A3 cationic amino acid transporters (CAT1-3), it was shown that L-arginine was imported at a similar rate via SLC7A1 and SLC7A2B and slower via SLC7A3. In contrast, upon withdrawal of extracellular L-arginine, intracellular levels decreased as fast in SLC7A3-expressing cells compared with SLC7A1, but the efflux was slower via SLC7A2B. SLC7A4 (CAT4) could not be convincingly shown to transport L-arginine. We also demonstrated the impact of membrane potential on L-arginine transport and showed that physiological concentrations of symmetrical and asymmetrical dimethylarginine do not significantly interfere with L-arginine transport through SLC7A1. Our results demonstrate that the FRET nanosensor can be used to assess L-arginine transport through plasma membrane in real time.

Essential amino acid transporter Lat4 (Slc43a2) is required for mouse development.

Amino acid (AA) uniporter Lat4 (Slc43a2) mediates facilitated diffusion of branched-chain AAs, methionine and phenylalanine, although its physiological role and subcellular localization are not known. We report that Slc43a2 knockout mice were born at expected Mendelian frequency but displayed an ∼10% intrauterine growth retardation and low amniotic fluid AAs, suggesting defective transplacental transport. Postnatal growth was strongly reduced, with premature death occurring within 9 days such that further investigations were made within 3 days of birth. Lat4 immunofluorescence showed a strong basolateral signal in the small intestine, kidney proximal tubule and thick ascending limb epithelial cells of wild-type but not Slc43a2 null littermates and no signal in liver and skeletal muscle. Experiments using Xenopus laevis oocytes demonstrated that Lat4 functioned as a symmetrical low affinity uniporter with a K0.5 of ∼5 mm for both in- and efflux. Plasma AA concentration was decreased in Slc43a2 null pups, in particular that of non-essential AAs alanine, serine, histidine and proline. Together with an increased level of plasma long chain acylcarnitines and a strong alteration of liver gene expression, this indicates malnutrition. Attempts to rescue pups by decreasing the litter size or by nutrients injected i.p. did not succeed. Radioactively labelled leucine but not lysine given per os accumulated in the small intestine of Slc43a2null pups, suggesting the defective transcellular transport of Lat4 substrates. In summary, Lat4 is a symmetrical uniporter for neutral essential AAs localizing at the basolateral side of (re)absorbing epithelia and is necessary for early nutrition and development.

Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors.

Sodium-dependent neutral amino acid transporter B(0)AT1 (SLC6A19) and imino acid (proline) transporter SIT1 (SLC6A20) are expressed at the luminal membrane of small intestine enterocytes and proximal tubule kidney cells where they exert key functions for amino acid (re)absorption as documented by their role in Hartnup disorder and iminoglycinuria, respectively. Expression of B(0)AT1 was shown in rodent intestine to depend on the presence of the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). This enzyme belongs to the renin-angiotensin system and its expression is induced by treatment with ACE-inhibitors (ACEIs) or angiotensin II AT1 receptor blockers (ARBs) in many rodent tissues. We show here in the Xenopus laevis oocyte expression system that human ACE2 also functionally interacts with SIT1. To investigate in human intestine the potential effect of ACEIs or ARBs on ACE2, we analysed intestinal biopsies taken during routine gastroduodenoscopy and ileocolonoscopy from 46 patients of which 9 were under ACEI and 13 ARB treatment. Analysis of transcript expression by real-time PCR and of proteins by immunofluorescence showed a co-localization of SIT1 and B(0)AT1 with ACE2 in the brush-border membrane of human small intestine enterocytes and a distinct axial expression pattern of the tested gene products along the intestine. Patients treated with ACEIs displayed in comparison with untreated controls increased intestinal mRNA levels of ACE2, peptide transporter PEPT1 (SLC15A1) and AA transporters B(0)AT1 and PAT1 (SLC36A1). This study unravels in human intestine the localization and distribution of intestinal transporters involved in amino acid absorption and suggests that ACEIs impact on their expression.

The molecular mechanism of intestinal levodopa absorption and its possible implications for the treatment of Parkinson's disease.

Levodopa (L-DOPA) is the naturally occurring precursor amino acid for dopamine and the main therapeutic agent for neurologic disorders due to dopamine depletion, such as Parkinson's disease. l-DOPA absorption in small intestine has been suggested to be mediated by the large neutral amino acids transport machinery, but the identity of the involved transporters is unknown. Clinically, coadministration of l-DOPA and dietary amino acids is avoided to decrease competition for transport in intestine and at the blood-brain barrier. l-DOPA is routinely coadministered with levodopa metabolism inhibitors (dopa-decarboxylase and cathechol-O-methyl transferase inhibitors) that share structural similarity with levodopa. In this systematic study involving Xenopus laevis oocytes and Madin-Darby canine kidney epithelia expression systems and ex vivo preparations from wild-type and knockout mice, we identified the neutral and dibasic amino acids exchanger (antiporter) b(0,+)AT-rBAT (SLC7A9-SLC3A1) as the luminal intestinal l-DOPA transporter. The major luminal cotransporter (symporter) B(0)AT1 (SLC6A19) was not involved in levodopa transport. L-Leucine and L-arginine competed with levodopa across the luminal enterocyte membrane as expected for b(0,+)AT-rBAT substrates, whereas dopa-decarboxylase and cathechol-O-methyl transferase inhibitors had no effect. The presence of amino acids in the basolateral compartment mimicking the postprandial phase increased transepithelial levodopa transport by stimulating basolateral efflux via the antiporter LAT2-4F2 (SLC7A8-SLC3A2). Additionally, the aromatic amino acid uniporter TAT1 (SLC16A10) was shown to play a major role in l-DOPA efflux from intestinal enterocytes. These results identify the molecular mechanisms mediating small intestinal levodopa absorption and suggest strategies for optimization of delivery and absorption of this important prodrug.

Amino acids regulate transgene expression in MDCK cells.

Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.

Transport of amino acids in the kidney.

Amino acids are the building blocks of proteins and key intermediates in the synthesis of biologically important molecules, as well as energy sources, neurotransmitters, regulators of cellular metabolism, etc. The efficient recovery of amino acids from the primary filtrate is a well-conserved key role of the kidney proximal tubule. Additionally, renal metabolism participates in the whole body disposition of amino acids. Therefore, a wide array of axially heterogeneously expressed transporters is localized on both epithelial membranes. For transepithelial transport, luminal uptake, which is carried out mainly by active symporters, is coupled with a mostly passive basolateral efflux. Many transporters require partner proteins for appropriate localization, or to modulate transporter activity, and/or increase substrate supply. Interacting proteins include cell surface antigens (CD98), endoplasmic reticulum proteins (GTRAP3-18 or 41), or enzymes (ACE2 and aminopeptidase N). In the past two decades, the molecular identification of transporters has led to significant advances in our understanding of amino acid transport and aminoacidurias arising from defects in renal transport. Furthermore, the three-dimensional crystal structures of bacterial homologues have been used to yield new insights on the structure and function of mammalian transporters. Additionally, transgenic animal models have contributed to our understanding of the role of amino acid transporters in the kidney and other organs and/or at critical developmental stages. Progress in elucidation of the renal contribution to systemic amino acid homeostasis requires further integration of kinetic, regulatory, and expression data of amino acid transporters into our understanding of physiological regulatory networks controlling metabolism.

Implementation of a geographic information system (GIS) for the planning of beekeeping in the west region of Paraná.

This study aimed to develop a Geographic Information System (GIS), for storage of information and geographic location of apiaries in eight counties in western Paraná; study the local flora; the land used; and the honey productivity in the harvest of 2010 in two of these areas: Marechal Cândido Rondon and Santa Helena. In order to do so we used the software SPRING, delimiting a radius of action of bees of three kilometers around the apiaries. We interviewed and registered 126 beekeepers with 383 apiaries. By using the images we selected areas with greater and lower overlap of hives in Marechal Cândido Rondon (144 and 44 hives, respectively) and Santa Helena (165 and 40 hives, respectively), in a three kilometers radius, selecting 15 colonies in each area, for the study of the parameters cited. In the multivariate analysis of the grouping, five groups were formed, by their similarity of management, indicating the higher average production in the hives of the most populated area of Santa Helena and lower average production in the most populated of Marechal Cândido Rondon. The grouping of hives, the differences in the production of honey and floristic survey indicated that these differences could be associated with management, floristic and climatic differences recorded in the period of production, in the areas studied.

Amino acid transporters expression in acinar cells is changed during acute pancreatitis.

Pancreatic acinar cells accumulate amino acids against a marked concentration gradient to synthesize digestive enzymes. Thus, the function of acinar cells depends on amino acid uptake mediated by active transport. Despite the importance of this process, pancreatic amino acid transporter expression and cellular localization is still unclear. We screened mouse pancreas for the expression of genes encoding amino acid transporters. We showed that the most highly expressed transporters, namely sodium dependent SNAT3 (Slc38a3) and SNAT5 (Slc38a5) and sodium independent neutral amino acids transporters LAT1 (Slc7a5) and LAT2 (Slc7a8), are expressed in the basolateral membrane of acinar cells. SNAT3 and SNAT5, LAT1 and LAT2 are expressed in acinar cells. Additional evidence that these transporters are expressed in mature acinar cells was gained using acinar cell culture and acute pancreatitis models. In the acute phase of pancreatic injury, when acinar cell loss occurs, and in an acinar cell culture model, which mimics changes occurring during pancreatitis, SNAT3 and SNAT5 are strongly down-regulated. LAT1 and LAT2 were down-regulated only in the in vitro model. At protein level, SNAT3 and SNAT5 expression was also reduced during pancreatitis. Expression of other amino acid transporters was also modified in both models of pancreatitis. The subset of transporters with differential expression patterns during acute pancreatitis might be involved in the injury/regeneration phases. Further expression, localization and functional studies will follow to better understand changes occurring during acute pancreatitis. These findings provide insight into pancreatic amino acid transport in healthy pancreas and during acute pancreatitis injury.