Loss of function mutation of the Slc38a3 glutamine transporter reveals its critical role for amino acid metabolism in the liver, brain, and kidney.

Glutamine, the most abundant amino acid in mammals, is critical for cell and organ functions. Its metabolism depends on the ability of cells to take up or release glutamine by transporters located in the plasma membrane. Several solute carrier (SLC) families transport glutamine, but the SLC38 family has been thought to be mostly responsible for glutamine transport. We demonstrate that despite the large number of glutamine transporters, the loss of Snat3/Slc38a3 glutamine transporter has a major impact on the function of organs expressing it. Snat3 mutant mice were generated by N-ethyl-N-nitrosurea (ENU) mutagenesis and showed stunted growth, altered amino acid levels, hypoglycemia, and died around 20 days after birth. Hepatic concentrations of glutamine, glutamate, leucine, phenylalanine, and tryptophan were highly reduced paralleled by downregulation of the mTOR pathway possibly linking reduced amino acid availability to impaired growth and glucose homeostasis. Snat3-deficient mice had altered urea levels paralleled by dysregulation of the urea cycle, gluconeogenesis, and glutamine synthesis. Mice were ataxic with higher glutamine but reduced glutamate and gamma-aminobutyric acid (GABA) levels in brain consistent with a major role of Snat3 in the glutamine-glutamate cycle. Renal ammonium excretion was lower, and the expression of enzymes and amino acid transporters involved in ammoniagenesis were altered. Thus, SNAT3 is a glutamine transporter required for amino acid homeostasis and determines critical functions in various organs. Despite the large number of glutamine transporters, loss of Snat3 cannot be compensated, suggesting that this transporter is a major route of glutamine transport in the liver, brain, and kidney.

Dysregulation of the glutamine transporter Slc38a3 (SNAT3) and ammoniagenic enzymes in obese, glucose-intolerant mice.

BACKGROUND/AIMS: Uric acid nephrolithiasis is prevalent among patients with type 2 diabetes and metabolic syndrome; it is correlated with an acidic urine and lower urinary ammonium excretion and is likely associated with insulin resistance. Insulin stimulates ammoniagenesis in renal cell lines via increased phosphate-dependent glutaminase (PDG) activity and glutamine metabolism. Ammonium excretion into the proximal tubule is mediated at least in part by the Na(+)/H(+)-exchanger NHE3 and in the collecting duct involving the Rhesus protein RhCG. Here we tested, whether obesity and insulin resistance in a diet-induced mouse model could contribute to deranged ammonium excretion. METHODS: Obesity was induced by diet in mice and the impact on key molecules of proximal tubular ammoniagenesis and urinary acid excretion tested. RESULTS: Diet-induced obesity was confirmed by pathological intraperitoneal glucose tolerance tests (IPGTT). Three groups of mice were compared: control mice; obese, glucose-intolerant with abnormal IPGTT (O-GI); or moderate weight with normal IPGTT (Non-Responders, NR). Basal urinary ammonium excretion did not differ among groups. However, acid loading increased urinary ammonium excretion in all groups, but to a lesser extent in the O-GI group. SNAT3 mRNA expression was enhanced in both obese groups. PDG expression was elevated only in acid-loaded O-GI mice, whereas PEPCK was enhanced in both O-GI and NR groups given NH4CI. NHE activity in the brush border membrane of the proximal tubule was strongly reduced in the O-GI group whereas RhCG expression was similar. CONCLUSION: In sum, obesity and glucose intolerance impairs renal ammonium excretion in response to NH4CI feeding most likely through reduced NHE activity. The stimulation of SNAT3 and ammoniagenic enzyme expression may be compensatory but futile.

Potassium restriction, high protein intake, and metabolic acidosis increase expression of the glutamine transporter SNAT3 (Slc38a3) in mouse kidney.

Kidneys produce ammonium to buffer and excrete acids through metabolism of glutamine. Expression of the glutamine transporter Slc38a3 (SNAT3) increases in kidney during metabolic acidosis (MA), suggesting a role during ammoniagenesis. Potassium depletion and high dietary protein intake are known to elevate renal ammonium excretion. In this study, we examined SNAT3, phosphate-dependent glutaminase (PDG), and phosphoenolpyruvate carboxykinase (PEPCK) regulation during a control (0.36%) or low-K(+) (0.02%) diet for 7 or 14 days or a control (20%) or high-protein (50%) diet for 7 days. MA was induced in control and low-K(+) groups by addition of NH(4)Cl. Urinary ammonium excretion increased during MA, after 14-day K(+) restriction alone, and during high protein intake. SNAT3, PDG, and PEPCK mRNA abundance were elevated during MA and after 14-day K(+) restriction but not during high protein intake. SNAT3 protein abundance was enhanced during MA (both control and low K(+)), after 14-day low-K(+) treatment alone, and during high protein intake. Seven-day dietary K(+) depletion alone had no effect. Immunohistochemistry showed SNAT3 staining in earlier parts of the proximal tubule during 14-day K(+) restriction with and without NH(4)Cl treatment and during high protein intake. In summary, SNAT3, PDG, and PEPCK mRNA expression were congruent with urinary ammonium excretion during MA. Chronic dietary K(+) restriction, high protein intake, and MA enhance ammoniagenesis, an effect that may involve enhanced SNAT3 mRNA and protein expression. Our data suggest that SNAT3 plays an important role as the glutamine uptake mechanism in ammoniagenesis under these conditions.

SLC26A7 can function as a chloride-loading mechanism in parietal cells.

To date three potential candidates for parietal cell basolateral Cl(-) entry have been described: the highly 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive Cl(-)/HCO(3)(-) exchanger AE2, the HCO(3)(-) and lowly DIDS-sensitive SLC26A7 protein, and the Na(+)-2Cl(-)K(+) cotransporter (NKCC1). In this study we investigate the contribution of these pathways to secretagogue stimulated acid secretion. Individually hand-dissected rat gastric glands were microfluorimetrically monitored for Cl(-) influx and pH(i) changes. Transporter activity was determined by varying ion content and through the use of pharmacological inhibitors. Expression of SLC26A7 in rat parietal cells was shown by immunohistochemistry and Western blot. SLC26A7 was inhibited by 5-Nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB) (100 microM) in the Xenopus laevis oocyte expression system. Cl(-) influx in parietal cells was enhanced by histamine, depended partially on endogenous HCO(3)(-) synthesis and completely on extracellular Na(+). Removal and subsequent readdition of Cl(-) revealed a low and a high DIDS-sensitive HCO(3)(-) extrusion system contributing to Cl(-) uptake. At acidic pH(i), however, H(+) extrusion via the H(+),K(+)-ATPase depending on Cl(-) uptake was abolished only in the presence of 100 microM (NPPB) and at high (250 microM) DIDS concentration. There was no effect of the NKCC inhibitor bumetanide on stimulated H(+) extrusion. These results would be compatible with SLC26A7 as a Cl(-) uptake system under histamine stimulation.

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.

The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion.

Genes in the KCNE family encode single transmembrane domain ancillary subunits that co-assemble with voltage-gated potassium (Kv) channel alpha subunits to alter their function. KCNE2 (also known as MiRP1) is expressed in the heart, is associated with human cardiac arrhythmia, and modulates cardiac Kv alpha subunits hERG and KCNQ1 in vitro. KCNE2 and KCNQ1 are also expressed in parietal cells, leading to speculation they form a native channel complex there. Here, we disrupted the murine kcne2 gene and found that kcne2 (-/-) mice have a severe gastric phenotype with profoundly reduced parietal cell proton secretion, abnormal parietal cell morphology, achlorhydria, hypergastrinemia, and striking gastric glandular hyperplasia arising from an increase in the number of non-acid secretory cells. KCNQ1 exhibited abnormal distribution in gastric glands from kcne2 (-/-) mice, with increased expression in non-acid secretory cells. Parietal cells from kcne2 (+/-) mice exhibited normal architecture but reduced proton secretion, and kcne2 (+/-) mice were hypochlorhydric, indicating a gene-dose effect and a primary defect in gastric acid secretion. These data demonstrate that KCNE2 is essential for gastric acid secretion, the first genetic evidence that a member of the KCNE gene family is required for normal gastrointestinal function.

An amino acid transporter involved in gastric acid secretion.

Gastric acid secretion is regulated by a variety of stimuli, in particular histamine and acetyl choline. In addition, dietary factors such as the acute intake of a protein-rich diet and the subsequent increase in serum amino acids can stimulate gastric acid secretion only through partially characterized pathways. Recently, we described in mouse stomach parietal cells the expression of the system L heteromeric amino acid transporter comprised of the LAT2-4F2hc dimer. Here we address the potential role of the system L amino acid transporter in gastric acid secretion by parietal cells in freshly isolated rat gastric glands. RT-PCR, western blotting and immunohistochemistry confirmed the expression of 4F2-LAT2 amino acid transporters in rat parietal cells. In addition, mRNA was detected for the B(0)AT1, ASCT2, and ATB(0+) amino acid transporters. Intracellular pH measurements in parietal cells showed histamine-induced and omeprazole-sensitive H+-extrusion which was enhanced by about 50% in the presence of glutamine or cysteine (1 mM), two substrates of system L amino acid transporters. BCH, a non-metabolizable substrate and a competitive inhibitor of system L amino acid transport, abolished the stimulation of acid secretion by glutamine or cysteine suggesting that this stimulation required the uptake of amino acids by system L. In the absence of histamine glutamine also stimulated H+-extrusion, whereas glutamate did not. Also, phenylalanine was effective in stimulating H+/K+-ATPase activity. Glutamine did not increase intracellular Ca2+ levels indicating that it did not act via the recently described amino acid modulated Ca2+-sensing receptor. These data suggest a novel role for heterodimeric amino acid transporters and may elucidate a pathway by which protein-rich diets stimulate gastric acid secretion.

L-type amino acids stimulate gastric acid secretion by activation of the calcium-sensing receptor in parietal cells.

Parietal cells are the primary acid secretory cells of the stomach. We have previously shown that activation of the calcium-sensing receptor (CaSR) by divalent (Ca(2+)) or trivalent (Gd(3+)) ions stimulates acid production in the absence of secretagogues by increasing H(+),K(+)-ATPase activity. When overexpressed in HEK-293 cells, the CaSR can be allosterically activated by L-amino acids in the presence of physiological concentrations of extracellular Ca(2+) (Ca(o)(2+); 1.5-2.5 mM). To determine whether the endogenously expressed parietal cell CaSR is allosterically activated by L-amino acids, we examined the effect of the amino acids L-phenylalanine (L-Phe), L-tryptophan, and L-leucine on acid secretion. In ex vivo whole stomach preparations, exposure to L-Phe resulted in gastric luminal pH significantly lower than controls. Studies using D-Phe (inactive isomer) failed to elicit a response on gastric pH. H(+)-K(+)-ATPase activity was monitored by measuring the intracellular pH (pH(i)) of individual parietal cells in isolated rat gastric glands and calculating the rate of H(+) extrusion. We demonstrated that increasing Ca(o)(2+) in the absence of secretagogues caused a dose-dependent increase in H(+) extrusion. These effects were amplified by the addition of amino acids at various Ca(o)(2+) concentrations. Blocking the histamine-2 receptor with cimetidine or inhibiting system L-amino acid transport with 2-amino-2-norbornane-carboxylic acid did not affect the rate of H(+) extrusion in the presence of L-Phe. These data support the conclusion that amino acids, in conjunction with a physiological Ca(o)(2+) concentration, can induce acid secretion independent of hormonal stimulation via allosteric activation of the stomach CaSR.