Luca Mariotta

Kidney amino acid transport

Near complete reabsorption of filtered amino acids is a main specialized transport function of the kidney proximal tubule. This evolutionary conserved task is carried out by a subset of luminal and basolateral transporters that together form the transcellular amino acid transport machinery similar to that of small intestine. A number of other amino acid transporters expressed in the basolateral membrane of proximal kidney tubule cells subserve either specialized metabolic functions, such as the production of ammonium, or are part of the cellular housekeeping equipment. A new finding is that the luminal Na+-dependent neutral amino acid transporters of the SLC6 family require an associated protein for their surface expression as shown for the Hartnup transporter B0AT1 (SLC6A19) and suggested for the l-proline transporter SIT1 (IMINOB, SLC6A20) and for B0AT3 (XT2, SLC6A18). This accessory subunit called collectrin (TMEM27) is homologous to the transmembrane anchor region of the renin–angiotensin system enzyme ACE2 that we have shown to function in small intestine as associated subunit of the luminal SLC6 transporters B0AT1 and SIT1. Some mutations of B0AT1 differentially interact with these accessory subunits, providing an explanation for differential intestinal phenotypes among Hartnup patients. The basolateral efflux of numerous amino acids from kidney tubular cells is mediated by heteromeric amino acid transporters that function as obligatory exchangers. Thus, other transporters within the same membrane need to mediate the net efflux of exchange substrates, controlling thereby the net basolateral amino transport and thus the intracellular amino acid concentration.

Tissue-Specific Alterations in Thyroid Hormone Homeostasis in Combined Mct10 and Mct8 Deficiency

The monocarboxylate transporter Mct10 (Slc16a10; T-type amino acid transporter) facilitates the cellular transport of thyroid hormone (TH) and shows an overlapping expression with the well-established TH transporter Mct8. Because Mct8 deficiency is associated with distinct tissue-specific alterations in TH transport and metabolism, we speculated that Mct10 inactivation may compromise the tissue-specific TH homeostasis as well. However, analysis of Mct10 knockout (ko) mice revealed normal serum TH levels and tissue TH content in contrast to Mct8 ko mice that are characterized by high serum T3, low serum T4, decreased brain TH content, and increased tissue TH concentrations in the liver, kidneys, and thyroid gland. Surprisingly, mice deficient in both TH transporters (Mct10/Mct8 double knockout [dko] mice) showed normal serum T4 levels in the presence of elevated serum T3, indicating that the additional inactivation of Mct10 partially rescues the phenotype of Mct8 ko mice. As a consequence of the normal serum T4, brain T4 content and hypothalamic TRH expression were found to be normalized in the Mct10/Mct8 dko mice. In contrast, the hyperthyroid situation in liver, kidneys, and thyroid gland of Mct8 ko mice was even more severe in Mct10/Mct8 dko animals, suggesting that in these organs, both transporters contribute to the TH efflux. In summary, our data indicate that Mct10 indeed participates in tissue-specific TH transport and also contributes to the generation of the unusual serum TH profile characteristic for Mct8 deficiency.

T‐type amino acid transporter TAT1 (Slc16a10) is essential for extracellular aromatic amino acid homeostasis control

•  The amino acid (AA) transporter TAT1 (Slc16A10) mediates facilitated diffusion of aromatic AAs (AAAs) across membranes. •  TAT1 null mice lack liver control of AAAs and display altered epithelial AA transport. •  The data support the hypothesis that equilibrative transport of essential AAs by TAT1 is crucial for body AA homeostasis control.

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) b0,+AT-rBAT (SLC7A9-SLC3A1) as the luminal intestinal l-DOPA transporter. The major luminal cotransporter (symporter) B0AT1 (SLC6A19) was not involved in levodopa transport. L-Leucine and L-arginine competed with levodopa across the luminal enterocyte membrane as expected for b0,+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.

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

Lat4 (Slc43a2) transports branched‐chain amino acids, phenylalanine and methionine, and is expressed in kidney tubule and small intestine epithelial cells. Using a new knockout model as a negative control, it is shown that Lat4 is expressed at the basolateral side of small intestine enterocytes and kidney epithelial cells of the proximal tubule, thick ascending limb and distal convoluted tubule. In the Xenopus oocyte expression system, Lat4 is shown to function as a uniporter with symmetric intracellular and extracellular apparent affinities for phenylalanine. Mice lacking Lat4 display a slight intrauterine growth restriction, postnatal malnutrition and early death, presumably as a result of defective amino acid (re)absorption. These results demonstrate the crucial role that the uniporter Lat4 plays for amino acid transport across cellular barriers and mouse development.

Defective intestinal amino acid absorption in Ace2 null mice.

Mutations in the main intestinal and kidney luminal neutral amino acid transporter B(0)AT1 (Slc6a19) lead to Hartnup disorder, a condition that is characterized by neutral aminoaciduria and in some cases pellagra-like symptoms. These latter symptoms caused by low-niacin are thought to result from defective intestinal absorption of its precursor L-tryptophan. Since Ace2 is necessary for intestinal B(0)AT1 expression, we tested the impact of intestinal B(0)AT1 absence in ace2 null mice. Their weight gain following weaning was decreased, and Na(+)-dependent uptake of B(0)AT1 substrates measured in everted intestinal rings was defective. Additionally, high-affinity Na(+)-dependent transport of L-proline, presumably via SIT1 (Slc6a20), was absent, whereas glucose uptake via SGLT1 (Slc5a1) was not affected. Measurements of small intestine luminal amino acid content following gavage showed that more L-tryptophan than other B(0)AT1 substrates reach the ileum in wild-type mice, which is in line with its known lower apparent affinity. In ace2 null mice, the absorption defect was confirmed by a severalfold increase of L-tryptophan and of other neutral amino acids reaching the ileum lumen. Furthermore, plasma and muscle levels of glycine and L-tryptophan were significantly decreased in ace2 null mice, with other neutral amino acids displaying a similar trend. A low-protein/low-niacin diet challenge led to differential changes in plasma amino acid levels in both wild-type and ace2 null mice, but only in ace2 null mice to a stop in weight gain. Despite the combination of low-niacin with a low-protein diet, plasma niacin concentrations remained normal in ace2 null mice and no pellagra symptoms, such as photosensitive skin rash or ataxia, were observed. In summary, mice lacking Ace2-dependent intestinal amino acid transport display no total niacin deficiency nor clear pellagra symptoms, even under a low-protein and low-niacin diet, despite gross amino acid homeostasis alterations.

Exocrine pancreas glutamate secretion help to sustain enterocyte nutritional needs under protein restriction

Glutamine (Gln) is the most concentrated amino acid in blood and considered conditionally essential. Its requirement is increased during physiological stress, such as malnutrition or illness, despite its production by muscle and other organs. In the malnourished state, Gln has been suggested to have a trophic effect on the exocrine pancreas and small intestine. However, the Gln transport capacity, the functional relationship of these two organs, and the potential role of the Gln-glutamate (Glu) cycle are unknown. We observed that pancreatic acinar cells express lower levels of Glu than Gln transporters. Consistent with this expression pattern, the rate of Glu influx into acinar cells was approximately sixfold lower than that of Gln. During protein restriction, acinar cell glutaminase expression was increased and Gln accumulation was maintained. Moreover, Glu secretion by acinar cells into pancreatic juice and thus into the lumen of the small intestine was maintained. In the intestinal lumen, Glu absorption was preserved and Glu dehydrogenase expression was augmented, potentially providing the substrates for increasing energy production via the TCA cycle. Our findings suggest that one mechanism by which Gln exerts a positive effect on exocrine pancreas and small intestine involves the Gln metabolism in acinar cells and the secretion of Glu into the small intestine lumen. The exocrine pancreas acinar cells not only avidly accumulate Gln but metabolize Gln to generate energy and to synthesize Glu for secretion in the pancreatic juice. Secreted Glu is suggested to play an important role during malnourishment in sustaining small intestinal homeostasis. NEW & NOTEWORTHY Glutamine (Gln) has been suggested to have a trophic effect on exocrine pancreas and small intestine in malnourished states, but the mechanism is unknown. In this study, we suggest that this trophic effect derives from an interorgan relationship between exocrine pancreas and small intestine for Gln-glutamate (Glu) utilization involving the uptake and metabolism of Gln in acinar cells and secretion of Glu into the lumen of the small intestine.