SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity.

Cancer cells evade T cell-mediated killing through tumour-immune interactions whose mechanisms are not well understood1,2. Dendritic cells (DCs), especially type-1 conventional DCs (cDC1s), mediate T cell priming and therapeutic efficacy against tumours3. DC functions are orchestrated by pattern recognition receptors3-5, although other signals involved remain incompletely defined. Nutrients are emerging mediators of adaptive immunity6-8, but whether nutrients affect DC function or communication between innate and adaptive immune cells is largely unresolved. Here we establish glutamine as an intercellular metabolic checkpoint that dictates tumour-cDC1 crosstalk and licenses cDC1 function in activating cytotoxic T cells. Intratumoral glutamine supplementation inhibits tumour growth by augmenting cDC1-mediated CD8+ T cell immunity, and overcomes therapeutic resistance to checkpoint blockade and T cell-mediated immunotherapies. Mechanistically, tumour cells and cDC1s compete for glutamine uptake via the transporter SLC38A2 to tune anti-tumour immunity. Nutrient screening and integrative analyses show that glutamine is the dominant amino acid in promoting cDC1 function. Further, glutamine signalling via FLCN impinges on TFEB function. Loss of FLCN in DCs selectively impairs cDC1 function in vivo in a TFEB-dependent manner and phenocopies SLC38A2 deficiency by eliminating the anti-tumour therapeutic effect of glutamine supplementation. Our findings establish glutamine-mediated intercellular metabolic crosstalk between tumour cells and cDC1s that underpins tumour immune evasion, and reveal glutamine acquisition and signalling in cDC1s as limiting events for DC activation and putative targets for cancer treatment.

Maternal Exposure to Oxidized Soybean Oil Impairs Placental Development by Modulating Nutrient Transporters in a Rat Model.

INTRODUCTION: As an exogenous food contaminant, dietary oxidized lipid impairs growth and development, and triggers chronic diseases in humans or animals. This study explores the effects of soybean oil with different oxidative degree on the placental injury of gestational rats. METHODS AND RESULTS: Thirty-two female adult rats are randomly assigned to four groups. The control group is fed the purified diet with fresh soybean oil (FSO), and the treatment groups are fed purified diets with lipid content replaced by oxidized soybean oil (OSO) at 200, 400, and 800 mEqO2 kg-1 from conception until delivery. On day 20 of gestation, OSO decreased placental and embryonic weights as the oxidative degree increased linearly and quadratically. The expression of Bax showed a linear increase, and Bcl-2 decreased as the oxidative degree increased. The expression of Fosl1 and Esx1 is linearly and quadratically decreased in OSO-treated groups than FSO group. OSO decreased the level of IL-10 but increased expression of IL-1ß in placenta and plasma. OSO remarkably upregulates levels of Fatp1 and Glut1 and decreases expression of Snat2 and Glut3. CONCLUSION: OSO aggravates placental injury by modulating nutrient transporters and apoptosis-related genes, impedes placental growth and development, and ultimately leads to the decrease of fetal weight.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 µmol·kg-1·h-1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

System A amino acid transporters regulate glutamine uptake and attenuate antibody-mediated arthritis.

Proliferation of rapidly dividing bone marrow-derived cells is strongly dependent on the availability of free glutamine, whose uptake is mediated through different amino acid transporters. The sodium-coupled neutral amino acid transporter (SNAT) family was previously reported to be associated with the development of collagen-induced arthritis in mice. Here, we tested the hypothesis whether impairment of SNAT proteins influences immune cell function and in turn alters arthritis development. The 2-(methylamino)isobutyric acid (MeAIB), a SNAT-specific substrate, was used to modulate the function of SNAT proteins. We demonstrate that glutamine uptake by murine naive lymphocytes, and consequent cell proliferation, is strongly associated with system A transporters. Physiological impairment of SNAT proteins reduced the antibody-initiated effector phase of arthritis, mainly by affecting the levels of circulating monocytes and neutrophils. MeAIB was also shown to affect the proliferation of immortalized cells, through trans-inhibition of SNAT proteins. Based on our observations, we conclude that SNAT proteins regulate the initial stages of lymphocyte activation by regulating glutamine uptake, and that the effector phase of arthritis can be affected by non-metabolized SNAT substrates. Most probably, metabolically active cells within both the adaptive and the innate immune systems are regulated by SNAT proteins and play a role in modifying arthritis development.

Effects of dietary n-6:n-3 PUFA ratio on fatty acid composition, free amino acid profile and gene expression of transporters in finishing pigs.

Revealing the expression patterns of fatty acid and amino acid transporters as affected by dietary n-6:n-3 PUFA ratio would be useful for further clarifying the importance of the balance between n-6 and n-3 PUFA. A total of ninety-six finishing pigs were fed one of four diets with the ratio of 1:1, 2·5:1, 5:1 and 10:1. Pigs fed the dietary n-6:n-3 PUFA ratio of 5:1 had the highest (P< 0·05) daily weight gain, and those fed the dietary n-6:n-3 PUFA ratio of 1:1 had the largest loin muscle area (P< 0·01). The concentration of n-3 PUFA was raised as the ratio declined (P< 0·05) in the longissimus dorsi and subcutaneous adipose tissue. The contents of tryptophan, tasty amino acids and branched-chain amino acids in the longissimus dorsi were enhanced in pigs fed the dietary n-6:n-3 PUFA ratios of 1:1-5:1. The mRNA expression level of the fatty acid transporter fatty acid transport protein-1 (FATP-1) was declined (P< 0·05) in the longissimus dorsi of pigs fed the dietary n-6:n-3 PUFA ratios of 1:1-5:1, and increased (P< 0·05) in the subcutaneous adipose tissue of pigs fed the dietary n-6:n-3 PUFA ratios of 5:1 and 10:1. The expression profile of FATP-4 was similar to those of FATP-1 in the adipose tissue. The mRNA expression level of the amino acid transceptors LAT1 and SNAT2 was up-regulated (P< 0·05) in the longissimus dorsi of pigs fed the dietary n-6:n-3 PUFA ratios of 1:1 and 2·5:1. In conclusion, maintaining the dietary n-6:n-3 PUFA ratios of 1:1-5:1 would facilitate the absorption and utilisation of fatty acids and free amino acids, and result in improved muscle and adipose composition.

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

Functional properties and cellular distribution of the system A glutamine transporter SNAT1 support specialized roles in central neurons.

Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 approximately 0.3 mm), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax approximately 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ >> H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via alpha-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.