Glycine transporter GLYT1 is essential for glycine-mediated protection of human intestinal epithelial cells against oxidative damage

Abstract

Glycine protects mammalian intestine against oxidative damage caused by ischaemia-reperfusion (IR) injury and prevents or reverses experimentally-induced colitis. However the mechanism of protection remains largely unknown. The objectives of the current study were to demonstrate directly glycine-mediated protection of human intestinal epithelial cells and to determine the requirement for glycine uptake by the specific transporter GLYT1. Exogenous glycine protected human intestinal Caco-2 and HCT-8 cells against the oxidative agent tert-butylhydroperoxide and reduced the intracellular concentration of reactive oxygen species, when applied prior to but not concomitant with the oxidative challenge. Glycine given prior to oxidative challenge preserved intracellular glutathione concentration but had no effect on the rate of glycine uptake. Protection was dependent on GLYT1 activity, being blocked by a specific GLYT1 inhibitor, supporting a requirement for intracellular glycine accumulation. Maintained intracellular glutathione content is indicated as a mechanism through which the protective effect may in part be mediated. However expression of the genes encoding GLYT1 and the glutathione synthesising enzymes glutamate-cysteine ligase, both catalytic and modifier subunits, and glutathione synthetase was not altered by glycine or tert-butylhydroperoxide, suggesting transcriptional regulation is not involved. This work has demonstrated a novel role of GLYT1 in intestine and shown that intestinal epithelial cells respond directly to oxidative challenge without reliance on extra-epithelial tissues or functions such as neurone, blood-flow or immune responses for antioxidant defence. The protective actions of glycine and maintenance of epithelial antioxidant defences suggest it may be beneficial in treatment of inflammatory bowel disease.

Figure 1. Effect of pre- or concurrent treatment with glycine on cell viability in response to t-BOOH treatment

A, glycine pre-treatment: HCT-8 cells were incubated in serum-free medium or serum-free medium containing either 1 mmol l⁻¹ or 5 mmol l⁻¹ glycine for 24 h after which they were transferred to serum-free medium containing 150 μmol l⁻¹ t-BOOH for 24 h. B, concurrent glycine treatment: confluent monolayers of HCT-8 cells were incubated in serum-free medium containing 150 μmol l⁻¹ t-BOOH or serum-free medium containing 150 μmol l⁻¹ t-BOOH and either 1 mmol l⁻¹ or 5 mmol l⁻¹ glycine for 24 h. Cell viability was measured using the MTT assay. Results are shown as a percentage of viability in control cells (open bars), which were maintained in normal medium throughout with changes at appropriate time points, and are means ±s.e.m. of three independent experiments performed on 12 replicates. In each chart, means without a common letter are statistically different (P < 0.05).

Figure 2. Effect of the GLYT1 inhibitor ALX-5407 on glycine-induced cell protection

A, HCT-8 cells were incubated in serum-free medium containing 5 mmol l⁻¹ glycine or 5 mmol l⁻¹ glycine plus 30 nmol l⁻¹ ALX-5407 for 24 h after which they were transferred to serum-free medium containing 150 μmol l⁻¹ t-BOOH for 24 h. Data are compared with those obtained from cells maintained in serum-free medium for 48 h (open bar) and from cells exposed to 150 μmol l⁻¹ t-BOOH following incubation in serum-free medium for 24 h. B, cells were incubated in serum-free medium with or without 30 nmol l⁻¹ ALX-5407 for 24 h after which they were transferred to serum-free medium containing 150 μmol l⁻¹ t-BOOH for 24 h. Control cells (open bar) were maintained in serum-free medium throughout. Cell viability was assessed using the MTT assay. Results are expressed as percent of control value and are means ±s.e.m. from 3 independent experiments performed in triplicate. Means without a common letter are statistically different (P < 0.05).

Figure 3. CLSM images of fixed and permeabilised HCT-8 cells showing GLYT1 staining (green) throughout the cell membrane

Shown are a horizontal section (A) taken below the apical plasma membrane and a vertical section (B) of a polarised monolayer of cells. In the control reaction (C) the GLYT1 specific antibody was omitted. The monlayers were also stained with ethidium homodimer (red, shown in A and C). Bar = 10 μm (A and C).

Figure 4. Expression and localisation of GLYT1 in human large intestine

A, agarose gel elctrophoresis of products from PCR amplification of human intestinal cDNAs using GLYT1-specific primers. A single PCR product of ∼400 bp is visible for each sample. Lanes are: I, ileum; I-C, ileo-caecum; C, caecum; A, ascending colon; T, transverse colon; D, descending colon; R, rectum; L, liver; K, kidney; N, negative control (ultra-pure water replaced cDNA in the reaction). B, CLSM images of frozen sections of human descending colon stained for GLYT1. a, optical section (low power view) showing GLYT1 expression in both apical and basal membranes of cells throughout the colonic crypt. c–d, optical sections (high power views), again showing GLYT1 expression in both apical and basolateral membranes of cells in the crypt base. In the absence of anti-GLYT1 antibody (b) staining by the AlexaFluor 488-conjugated detecting antibody is negligible. a and b, which show the same region of consecutive sections, were collected and displayed using identical parameters. L, crypt lumen. Bar = 50 μm (a and b); 10 μm (c and d).

Figure 5. Inhibition of Na⁺- and Cl⁻-dependent basolateral glycine uptake by a GLYT1-specific inhibitor in HCT-8 cells

Effect of GLYT1 inhibitor, ALX-5407, and GLYT2 inhibitor, ALX-1393, on basolateral, alanine-insensitive, Na⁺- and Cl⁻-dependent glycine uptake in HCT-8 cells. Glycine uptake was measured in Krebs buffer in the presence of 5 μmol l⁻¹ glycine, 5 mmol l⁻¹ alanine and increasing concentrations of either ALX-5407 (□) or ALX-1393 (▪) over 10 min. Results are expressed as means with error bars of ±s.e.m., n= 6.

Figure 6. Effect of glycine and t-BOOH on basolateral glycine uptake

Caco-2 cells were exposed to 5 mmol l⁻¹ glycine, 150 μmol l⁻¹ t-BOOH or glycine followed by t-BOOH, and basolateral glycine uptake measured over 15 min in Krebs buffer containing 5 mmol l⁻¹ alanine. Results are expressed as means ±s.e.m., n= 6. Means without a common letter are statistically different (P < 0.05).

Figure 7. Effect of glycine and GLYT-1 inhibition on ROS concentration

A, Caco-2 cells were incubated in serum-free medium containing 1 or 5 mmol l⁻¹ glycine with or without 30 nmol l⁻¹ ALX-5407 prior to incubation with t-BOOH and determination of ROS concentration. Control cells (maximum ROS concentration, open bar) were exposed only to t-BOOH. Results are expressed as a percentage of maximum and are means ±s.e.m. of three independent experiments performed in triplicate. Bars without a common letter are statistically different (P < 0.05). B, CLSM images of Caco-2 (a and c) and HCT-8 cells (b and d) preloaded with carboxy-H₂DCFDA and exposed to 100 μmol l⁻¹ t-BOOH (A and B) or normal medium (C and D) for 1 h. In the presence of ROS the fluorescein compound is oxidised to carboxy-DCF and emits bright green fluorescence. Images are shown at ×100 (a and c) and ×20 (b and d) magnification. Bar = 10 μm.

Figure 8. Effect of glycine and GLYT-1 inhibition on intracellular GSH concentration during oxidative stress

GSH concentration was measured in HCT-8 cell monolayers incubated in serum-free medium or serum-free medium containing 5 mmol l⁻¹ glycine for 24 h followed by a further 24 h in serum-free medium or serum-free medium containing 150 μmol l⁻¹ t-BOOH (A), Caco-2 cell monolayers incubated with serum-free medium alone or containing 5 mmol l⁻¹ glycine or l-alanine for 4 h prior to exposure to 100 μmol l⁻¹ t-BOOH for 1 h (B), and Caco-2 cells incubated in serum-free medium containing 30 nmol l⁻¹ ALX-5407 with or without 5 mmol l⁻¹ glycine for 4 h and subsequent exposure to 100 μmol l⁻¹ t-BOOH for 1 h (C). Results are shown relative to cells incubated in serum-free medium throughout (open bars) and are means ±s.e.m. of three independent experiments performed in quadruplicate. Means without a common letter are statistically different (P < 0.05).