Leucine competes with kynurenine for blood-to-brain transport and prevents lipopolysaccharide-induced depression-like behavior in mice

Abstract

Inflammation activates indoleamine 2,3-dioxygenase (IDO) which metabolizes tryptophan into kynurenine. Circulating kynurenine is transported into the brain by the large amino transporter LAT1 at the level of the blood-brain barrier. We hypothesized that administration of leucine that has a high affinity for LAT1 should prevent the entry of kynurenine into the brain and attenuate the formation of neurotoxic kynurenine metabolites. To test whether leucine could prevent inflammation-induced depression-like behavior, mice were treated with lipopolysaccharide (LPS, 0.83 mg/kg IP) or saline and treated with L-leucine (50 mg/kg, IP) or vehicle administered before and 6 h after LPS. Depression-like behavior was measured by increased duration of immobility in the forced swim test and decreased sucrose preference. Leucine decreased brain kynurenine levels, blocked LPS-induced depression-like behavior and had antidepressant-like effects in control mice. Leucine had no effect of its own on sickness behavior and neuroinflammation. To confirm that leucine acts by interfering with the transport of kynurenine into the brain, mice were injected with L-leucine (300 mg/kg, IP) immediately before kynurenine (33 mg/kg IP) and brain kynurenine and depression-like behavior were measured 3 h later. Leucine did prevent the entry of exogenous kynurenine into the brain and abrogated depression-like behavior measured by increased duration of immobility in the forced swim test. Additional experiments using an in vitro model of the blood-brain barrier confirmed that kynurenine competes with leucine at the level of the amino acid transporter LAT1 for brain uptake. These experiments also revealed that efflux was the dominant direction of kynurenine transport and was largely independent of LAT1 and leucine, which explains why leucine could block brain uptake of kynurenine without affecting brain clearance. These findings demonstrate that leucine has antidepressant properties vis-à-vis inflammation-induced depression and one mechanism for this is by blocking the ability of kynurenine to enter the brain.

Figure 1. Leucine reduces brain kynurenine:tryptophan ratios and blocks depression-like behavior

A,B Mean sucrose preference (%) and time spent immobile in the forced swim test (s) (± SEM) of mice treated with leucine or vehicle immediately before and 6 h after LPS or PBS (n ≥ 14 per group). C,D Mean kynurenine:tryptophan ratios in plasma and brains (± SEM) of mice treated with 50 mg/kg leucine or vehicle immediately before and 6 h after LPS or phosphate-buffered saline (PBS) (n = 5 per group). **p ≤ 0.01, ***p ≤ 0.0001 for LPS vs PBS main effects. ^#p ≤ 0.05, ^##p ≤ 0.01, ^###p ≤ 0.0001 for leucine vs vehicle main effects.

Figure 2. Leucine blocks the entry of kynurenine into the brain and the depression-like behaviour induced by exogenous administration of kynurenine

A Mean brain kynurenine concentrations (ng/ml) (± SEM) of mice treated with leucine or vehicle immediately before kynurenine or vehicle (n = 6 per group). B Mean time spent immobile in the forced swim test (s) (± SEM) of mice treated with leucine or vehicle immediately before kynurenine or vehicle (n ≥ 9). ^##p ≤ 0.01 for leucine vs vehicle main effects. ^xp ≤ 0.05 for significant differences in Figure B.

Figure 3. Leucine competes with kynurenine brain influx but not efflux via LAT1 in vitro

Tritiated (³H) materials were placed in the luminal chamber and influx into the abluminal chamber measured. A Mean permeability (μl/min/cm²) (± SEM) of tritiated leucine across brain microvascular endothelial cells (BMECs) from the luminal-to-abluminal chamber of a transwell. Unlabelled (cold) leucine (10 mg/ml), BCH (1 mM) and kynurenine (1 mM) inhibited leucine permeability. B Mean permeability (μl/min/cm²) (± SEM) of tritiated (³H) kynurenine across BMECs from the luminal-to-abluminal chamber of a transwell, which indicated the predominance of an efflux system that was unmasked by self-inhibition (C) and by unlabelled (cold) tryptophan (1 mM) but not unlabelled (cold) Leucine or BCH. D Efflux of kynurenine is mainly independent of LAT1. Tritiated (3H) kynurenine was placed in the abluminal chamber and efflux into the luminal chamber measured. Efflux was easily measured for tritiated (3H) kynurenine with modest inhibition by leucine or BCH. Enhanced efflux by unlabeled (cold) Kynurenine is consistent with an influx arm, but one more easily inhibited even by abluminal kynurenine than the efflux arm. E Diagrammatic summary of the findings of Figure 2, showing that leucine is able to inhibit transport of leucine, kynurenine and tryptophan into the brain via LAT1 but does not interfere with transport of tryptophan or excess kynurenine from leaving the brain. 3H = tritiated; UL = unlabelled. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001

Figure 4. Diagrammatic representation of our major finding on leucine blockade of depression-like behavior in mice with or without inflammation

A Overview of pathway of inflammation-induced depression and the anti-depressant effects of leucine. B Overview of kynurenine blood-to-brain transport across BMECs. Inflammation increases blood kynurenine resulting in more kynurenine binding to LAT1 and being transported into the brain, which is blocked by the LAT1 specific antagonist BCH. Leucine competitively inhibits kynurenine binding to LAT1 and thus reduces blood-to-brain transport of kynurenine. An efflux transporter predominates kynurenine brain-to-blood transport, which is not LAT1 and is not bound by leucine. This efflux transporter extrudes kynurenine produced locally by inflammation.