GLUT-1 Enhances Glycolysis, Oxidative Stress, and Fibroblast Proliferation in Keloid

Abstract

A keloid is a fibroproliferative skin tumor. Proliferating keloid fibroblasts (KFs) demand active metabolic utilization. The contributing roles of glycolysis and glucose metabolism in keloid fibroproliferation remain unclear. This study aims to determine the regulation of glycolysis and glucose metabolism by glucose transporter-1 (GLUT-1), an essential protein to initiate cellular glucose uptake, in keloids and in KFs. Tissues of keloids and healthy skin were explanted for KFs and normal fibroblasts (NFs), respectively. GLUT-1 expression was measured by immunofluorescence, RT-PCR, and immunoblotting. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured with or without WZB117, a GLUT-1 inhibitor. Reactive oxygen species (ROS) were assayed by MitoSOX immunostaining. The result showed that glycolysis (ECAR) was enhanced in KFs, whereas OCR was not. GLUT-1 expression was selectively increased in KFs. Consistently, GLUT-1 expression was increased in keloid tissue. Treatment with WZB117 abolished the enhanced ECAR, including glycolysis and glycolytic capacity, in KFs. ROS levels were increased in KFs compared to those in NFs. GLUT-1 inhibition suppressed not only the ROS levels but also the cell proliferation in KFs. In summary, the GLUT-1-dependent glycolysis and ROS production mediated fibroblast proliferation in keloids. GLUT1 might be a potential target for metabolic reprogramming to treat keloids.

Keywords: glucose transporter 1 (GLUT-1); glycolysis; keloids; proliferation; reactive oxygen species (ROS).

Figure 1

Enhanced glycolysis in keloid fibroblasts (KF). (A) The continuous ECARs and OCRs were recorded and analyzed using an XF94 extracellular flux analyzer. The scheme of ECARs shows the section of the time trace corresponding to each module. Subsequently, 10 mM glucose, 1 µM oligomycin, and 75 mM 2-DG were injected to the medium. (B) Representative ECAR was measured in KFs and NFs (1 × 10⁴ cells/well). Data are presented as mean ± SEM. (N = 6, * p < 0.05, ** p < 0.01, *** p < 0.001.). The representative data from 3 independent experiments are shown. (C) Continuous OCRs values were recorded and analyzed. The scheme of continuous of OCRs shows the section of the time trace corresponding to each module. Subsequently, 1 µM oligomycin, 1 µM FCCP, and 0.5 µM rotenone/antimycin A were injected to the medium. (D) Representative OCRs were measured in KFs and NFs (1 × 10⁴ cells/well). Data are presented as mean ± SEM (N = 6, * p < 0.05, ** p < 0.01, *** p < 0.001.). Shown is the representative data from 3 independent experiments.

Figure 2

Glycolytic enzymes were upregulated in KFs. RNA was purified and the mRNA level of glycolytic enzymes was determined by RT-PCR. Data are presented as mean ± SEM. (N = 6, * p < 0.05, ** p < 0.01) from representative data of 3 independent experiments. Abbreviations: GLUT-1, glucose transporter 1; GPI, phosphoglucose isomerase; PFK, phosphofructokinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PKM2, pyruvate kinase M2; PDK1, pyruvate dehydrogenase kinase 1; LDH, lactate dehydrogenase.

Figure 3

GLUT-1 expression was increased in both Fs and hyperfibrotic regions in keloid tissues. (A) Immunohistochemically staining for GLUT-1 in normal skin, and keloids (N = 5, normal skin; N = 5, keloids). Scale bar = 500 μm. Right panels are high-power views of tissues. Scale bar = 200 μm. (B) Scatterplots of expression levels; the middle line represents the mean value (* p < 0.05). (C) The immunofluorescence staining for GLUT-1 in NFs and KFs pretreated with or without 10 µM WZB117 for 48 h. Scale bar = 200 μm. The white rectangle in (C) indicates the GLUT-1 morphology for high magnifications at right panels. (D) is the quantification of IF from (C). Five random fields of each section were selected and more than 35 cells of each field were counted. Data are presented as mean ± SEM from 3 or more independent experiments (N = 6, * p < 0.05).

Figure 4

GLUT-1 mediated the enhanced glycolysis in KFs. NFs and KFs were pretreated with or without 10 µM of WZB117 for 48 h and then incubated with base medium at 37 °C in a non-CO₂ incubator. Subsequently, 10 mM glucose, 1 µM oligomycin, and 75 mM 2-DG were injected to the medium. The ECARs were recorded and analyzed by the Seahorse XF-24 software, including (A) changes in the glycolytic function, (B) glycolysis, (C) glycolytic capacity, and (D) glycolytic reserve. Data are presented as mean ± SEM from 3 or more independent experiments. (N = 6, * p < 0.05, ** p < 0.01).

Figure 5

GLUT-1 inhibition increased the mitochondrial OXPHOS in NFs. NFs and KFs were pretreated with or without 10 µM of WZB117 and then incubated at 37 °C in a non-CO₂ incubator. Subsequently, 1 µM oligomycin, 1 µM FCCP, and 0.5 µM rotenone/antimycin A were injected to the medium. The OCRs were recorded and analyzed by the Seahorse XF-24 software, including (A) changes in the mitochondrial respiration, (B) basal respiration, (C) ATP production, (D) proton leak, (E) maximal respiration, and (F) spare respiratory capacity. Data are presented as mean ± SEM from 3 or more independent experiments. (N = 6, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

GLUT-1 regulated the enhanced ROS development in KFs. NFs and KFs were pretreated with or without 10 µM of WZB117 for 48 h. (A) The ROS at baseline. (B) The ROS in NFs and KFs after treatment with WZB117. Measures shown are the representative data from 3 independent experiments. Scale bar = 200 μm. (C) is the quantification of relative level from (B). Five random fields of each section were selected and more than 50 cells of each field were counted. Data are presented as mean ± SEM (N = 6, * p < 0.05, KFs control compared with NFs control; # p< 0.05, ## p < 0.01, KFs WZB117 compared with KFs control).

Figure 6

GLUT-1 regulated the enhanced ROS development in KFs. NFs and KFs were pretreated with or without 10 µM of WZB117 for 48 h. (A) The ROS at baseline. (B) The ROS in NFs and KFs after treatment with WZB117. Measures shown are the representative data from 3 independent experiments. Scale bar = 200 μm. (C) is the quantification of relative level from (B). Five random fields of each section were selected and more than 50 cells of each field were counted. Data are presented as mean ± SEM (N = 6, * p < 0.05, KFs control compared with NFs control; # p< 0.05, ## p < 0.01, KFs WZB117 compared with KFs control).

Figure 7

Blocking GLUT-1 decreased proliferation of KFs and NFs, to a greater extent with KFs. NFs and KFs were pretreated with or without 10 µM of WZB117 for 48 h. (A) The WST-1 assay was used to determine cell proliferation and cell viability at different time-points. (N = 6, * p < 0.05, KFs WZB117 compared with KFs control, NFs WZB117 compared with NFs control). (B) The immunofluorescence staining for Ki67+cells in NFs and KFs showed that WZB117-treated fibroblasts had decreased proliferation rates compared controls. Data shown are representative from 3 independent experiments. Scale bar = 150 μm. (C) is the quantification of Ki67+ fibroblasts from (B). Five random fields of each section were selected and more than 30 cells of each field were counted. Data are presented as mean ± SEM of 3 or more independent experiments (N = 6, * p < 0.05, ** p < 0.01).

Figure 8

The hypothesized regulatory role of GLUT-1 in glycolysis and metabolic reprograming in the pathogenesis of keloids.