Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets.

BACKGROUND/AIMS: Our previous reports suggested that dietary supplementation with lysine influenced intestinal absorption and metabolism of amino acids. In this study, we further investigated the effect of lysine restriction (30%) on feed intake and we also tested the hypothesis that gut microbiome contributed to the potential mechanism of lysine restriction-mediated feeding behavior. Here, we profiled gut microbial communities by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from gut samples as well as growth performance, serum hormones, and intestinal lysine transport in a piglet model. RESULTS: Piglets preferred to the lysine restricted diet when giving three diets and the feed intake was markedly higher in the lysine-restricted group than that in the control group. Altered hormones (leptin, CCK, and ghrelin) might contribute to the feeding behavior caused by lysine restriction. Meanwhile, lysine transporting ability (SLC7A1 and SLC7A2 expression, intestinal electrophysiological changes, and amino acid pool in mesenteric vein) was decreased in response to lysine restriction. Through deep sequencing of bacterial rRNA markers, we observed that bacterial diversity was enhanced in the lysine-restricted group (Shannon H, PD, and Chao1). At the phylum level, lysine restriction enhanced gut Actinobacteria, Saccharibacteria, and Synergistetes abundances. At the family level, Moraxellaceae, Halomonadaceae, Shewanellaceae, Corynebacteriaceae, Bacillaceae, Comamonadaceae, Microbacteriaceae, Caulobacteraceae, and Synergistaceae abundances were increased in response to lysine restriction. Predictive functional profiling of microbial communities by PICRUSt also confirmed that dietary lysine restriction affected gut microbiome, which might further mediate amino acid metabolism, membrane transport, and endocrine system. CONCLUSION: Our results indicated that lysine restriction inhibited intestinal lysine transport and promoted feed intake, which might be associated with gut microbiome.

Deletion of cationic amino acid transporter 2 exacerbates dextran sulfate sodium colitis and leads to an IL-17-predominant T cell response.

L-Arginine (L-Arg) is a semiessential amino acid that has altered availability in human ulcerative colitis (UC), a form of inflammatory bowel disease, and is beneficial in murine colitis induced by dextran sulfate sodium (DSS), a model with similarity to UC. We assessed the role of cationic amino acid transporter 2 (CAT2), the inducible transporter of L-Arg, in DSS colitis. Expression of CAT2 was upregulated in tissues from colitic mice and localized predominantly to colonic macrophages. CAT2-deficient (CAT2-/-) mice exposed to DSS exhibited worsening of survival, body weight loss, colon weight, and histological injury. These effects were associated with increased serum L-Arg and decreased tissue L-Arg uptake and inducible nitric oxide synthase protein expression. Clinical benefits of L-Arg supplementation in wild-type mice were lost in CAT2-/- mice. There was increased infiltration of macrophages, dendritic cells, granulocytes, and T cells in colitic CAT2-/- compared with wild-type mice. Cytokine profiling revealed increases in proinflammatory granulocyte colony-stimulating factor, macrophage inflammatory protein-1α, IL-15, and regulated and normal T cell-expressed and -secreted and a shift from an IFN-γ- to an IL-17-predominant T cell response, as well as an increase in IL-13, in tissues from colitic CAT2-/- mice. However, there were no increases in other T helper cell type 2 cytokines, nor was there a global increase in macrophage-derived proinflammatory cytokines. The increase in IL-17 derived from both CD4 and γδ T cells and was associated with colonic IL-6 expression. Thus CAT2 plays an important role in controlling inflammation and IL-17 activation in an injury model of colitis, and impaired L-Arg availability may contribute to UC pathogenesis.

Pre-treatment of acupuncture stimulation of ST36 (Zusanli) significantly attenuates CAT-2, CAT-2B and GTPCH transcription in septic rat lungs.

BACKGROUND: The isozymes of type-2 cationic amino acid transporter (including CAT-2 and CAT-2B) and guanosine triphosphate cyclohydrolase I (GTPCH) constitute part of the down-stream regulatory pathways that regulate nitric oxide (NO) production mediated by inducible NO synthase (iNOS). We sought to evaluate the effects of acupuncture stimulation of ST36 (Zusanli) on the expression of CAT-2, CAT-2B, and GTPCH in lipopolysaccharide (LPS)-stimulated rat lungs. METHODS: Sixty rats were randomized into 6 groups (n = 10 in each group): 1) LPS, 2) Normal saline (N/S), 3) LPS + ST36, 4) ST36, 5) LPS + Sham, and 6) Sham groups. Manual acupuncture stimulation of ST36 (designated as "ST36") or a "nonacupoint" (designated as "Sham") was performed in lightly immobilized rats for 30 minutes. Then, LPS injection was performed to induce the expressions of iNOS, CAT-2, CAT-2B, and GTPCH in rat lungs. Rats were sacrificed 6 hours after LPS injection and the expressions of these enzymes were assayed. RESULTS: Reverse transcription and polymerase chain reaction (RT-PCR) data revealed that the expressions of iNOS, CAT-2, CAT-2B, and GTPCH in N/S-stimulated rat lungs were low. Exposure to LPS significantly induced the expressions of iNOS, CAT-2, CAT-2B, and GTPCH. In addition, the pre-treatment of ST36 acupuncture significantly attenuated the LPS-induced expressions of iNOS, CAT-2, CAT-2B, and GTPCH in stimulated rat lungs. CONCLUSIONS: Pre-treatment of acupuncture stimulation of ST36 had significantly inhibitory effects on LPS-induced iNOS, CAT-2, CAT-2B, and GTPCH expressions in septic rat lungs.

Rate of transport of l-arginine is independent of the expression of inducible nitric oxide synthase in HEK 293 cells.

Expression of inducible nitric oxide synthase (iNOS) is generally accompanied by a parallel upregulation in l-arginine transport which is dependent, at least in part, on the synthesis of new carrier proteins. It is not clear however whether the induction of iNOS and its subsequent utilisation of l-arginine for NO synthesis contribute to the enhancement in l-arginine transport rates observed following induction of cells with pro-inflammatory mediators. To address this issue, we have transfected an iNOS construct in a pEGFP-N1 vector into HEK-293 cells and investigated the effects this has on l-arginine transport. The expression of iNOS through transfection resulted in the production of significant quantities of NO as detected by the standard Griess assay. Under these conditions, the transport of l-arginine was found to be unaltered, with rate of uptake being comparable in both transfected and non-transfected cells. Characterisation of the transporter(s) involved with uptake of l-arginine revealed features characteristic of the classical cationic amino acid transport system y(+). Further analysis of the expression profile of the cationic amino acid transporter (CAT) involved revealed the presence of transcripts for CAT-1 and CAT-2B. These data demonstrate that iNOS activity does not drive or enhance l-arginine transport despite the fact that HEK-293 cells transport l-arginine via the CATs, including CAT-2B which is thought to be critical for supply of substrate to iNOS.

Garlic attenuates nitric oxide production in rat cardiac myocytes through inhibition of inducible nitric oxide synthase and the arginine transporter CAT-2 (cationic amino acid transporter-2).

It is now accepted that allicin, the main biologically active compound in garlic, exhibits antioxidant activity. The present study was designed to test the hypothesis that the antioxidant activity of garlic can be partially attributed to the inhibition of nitric oxide (NO) production by cytokine-induced NO synthase (iNOS). Cardiac myocytes cultured from neonatal Wistar rats were stimulated by lipopolysaccharide (LPS) and incubated for 24 h with various concentrations of allicin. This resulted in marked inhibition of nitrite production. Interestingly, a low concentration of allicin (10 microM) was significantly more potent in abrogating the effect of LPS on nitrite production than a higher concentration (40 microM). Allicin decreased steady-state iNOS mRNA levels, and this effect was maximal when a lower concentration was used (10 microM compared with 40 microM). In order to explore additional effects of allicin on NO generation that might counteract the effect on iNOS, we assessed the effects of higher allicin concentrations on arginine transport. Allicin inhibited the uptake of 1 mM extracellular arginine in a concentration-dependent manner. The expression of the two arginine transporters that are expressed in cardiac myocytes [CAT-1 (cationic amino acid transporter-1) and CAT-2] was studied using reverse transcription-PCR. A concentration of 200 microM allicin abolished the expression of CAT-2 mRNA, 100 microM significantly attenuated it, whereas 50 microM had no effect. Allicin had no effect on steady-state CAT-1 mRNA levels. Our results suggest that allicin inhibits iNOS activity through two different mechanisms: at lower concentrations it decreases iNOS mRNA levels, whereas at higher concentrations it inhibits arginine transport through down-regulation of CAT-2 mRNA.