Metabolic reprogramming of macrophages: glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype

Abstract

Glucose is a critical component in the proinflammatory response of macrophages (MΦs). However, the contribution of glucose transporters (GLUTs) and the mechanisms regulating subsequent glucose metabolism in the inflammatory response are not well understood. Because MΦs contribute to obesity-induced inflammation, it is important to understand how substrate metabolism may alter inflammatory function. We report that GLUT1 (SLC2A1) is the primary rate-limiting glucose transporter on proinflammatory-polarized MΦs. Furthermore, in high fat diet-fed rodents, MΦs in crown-like structures and inflammatory loci in adipose and liver, respectively, stain positively for GLUT1. We hypothesized that metabolic reprogramming via increased glucose availability could modulate the MΦ inflammatory response. To increase glucose uptake, we stably overexpressed the GLUT1 transporter in RAW264.7 MΦs (GLUT1-OE MΦs). Cellular bioenergetics analysis, metabolomics, and radiotracer studies demonstrated that GLUT1 overexpression resulted in elevated glucose uptake and metabolism, increased pentose phosphate pathway intermediates, with a complimentary reduction in cellular oxygen consumption rates. Gene expression and proteome profiling analysis revealed that GLUT1-OE MΦs demonstrated a hyperinflammatory state characterized by elevated secretion of inflammatory mediators and that this effect could be blunted by pharmacologic inhibition of glycolysis. Finally, reactive oxygen species production and evidence of oxidative stress were significantly enhanced in GLUT1-OE MΦs; antioxidant treatment blunted the expression of inflammatory mediators such as PAI-1 (plasminogen activator inhibitor 1), suggesting that glucose-mediated oxidative stress was driving the proinflammatory response. Our results indicate that increased utilization of glucose induced a ROS-driven proinflammatory phenotype in MΦs, which may play an integral role in the promotion of obesity-associated insulin resistance.

Keywords: Crown-like Structure; Glucose Transport; Glycolysis; Inflammation; Macrophages; Metabolomics; Mitochondrial Metabolism; Obesity; Pentose Phosphate Pathway.

FIGURE 1.

GLUT1 is the primary transporter on proinflammatory MΦ and is detected in obesity-associated crown-like structures. qPCR expression analysis of GLUT1–4 in BMDM (A) and RAW MΦs (B). A, bone marrow was isolated from wild type C57BL/6 mice. BMDMs were left as unstimulated naïve cells (M0) or polarized toward M1 or M2 phenotype. Fold expression from M0 GLUT1 was set to 1. B, RAW MΦs were plated and left unstimulated or stimulated with 100 ng/ml LPS for 24 h. Fold expression from untreated GLUT1 was set to 1. C, GLUT1+ CLS (crown-like structure) (*) and inflammatory loci (→) are indicated in low fat diet-fed lean and high fat diet-fed obese adipose (top images) and livers (bottom images). D, adipose tissue macrophages derived from mice were stained for macrophages (F4/80+), GLUT1 (myc), IL-6 (*, p = 018), and TNFα (*, p = 0.0062). n = 3. Data show the mean ± S.E. of the mean fluorescence intensity (MFI).

FIGURE 2.

Stable overexpression of GLUT1 in RAW264.7 MΦs. A, immunolocalization of GLUT1 to the cell surface (red) in GLUT1-OE compared with GLUT1-EV cells using an anti-FLAG antibody (nuclei are stained blue with DAPI). B, qPCR of GLUT1 and GLUT3 in RAW, GLUT1-EV, and GLUT1-OE. Fold expression is normalized to GLUT1-EV, set to 1. GLUT2 and GLUT4 were not detected. n = 5 separate experiments ± S.E. C, representative Western immunoblot of GLUT1 and loading control actin in RAW, GLUT1- EV, and GLUT1-OE cell lysates ± LPS. D, quantification of immunoblots from three separate experiments ± S.E. For each immunoblot, all lanes are normalized to actin. Unstimulated GLUT1-EV was set to 1.

FIGURE 3.

GLUT1 up-regulated glycolytic rates and capacity and blunted respiratory capacity. ECAR and OCR were measured using the Seahorse Bioanalyzer. Glycolytic rate (A; *, p = 0.0003) and glycolytic capacity (B; *, p = 0.01) were measured. Glycolytic reserve was equal in GLUT1-EV and GLUT1-OE (not shown). Oxygen consumption was measured using the Seahorse before glucose addition to the media (C, − glucose, *, p = 0.002) and after glucose injection into the assay (D, + glucose, *, p = 0.0005). n = 3 experiments with n = 6 replicates were conducted, and data were normalized to protein in each well.

FIGURE 4.

GLUT1 gain of function up-regulates glucose metabolism in GLUT1-OE MΦs compared with GLUT1-EV. A, [2-³H]DG uptake was measured in GLUT1-EV and GLUT1-OE cells. *, p < 0.0001 GLUT1-OE versus GLUT1-EV. B, [¹⁴C] ubiquitously labeled glucose oxidation was measured. *, p = 0.001 GLUT1-OE versus GLUT1-EV. C, [¹⁴C] ubiquitously labeled glucose incorporation into glycogen was measured as above. *, p = 0.03 GLUT1-OE versus GLUT1-EV. D, lactate was measured in conditioned media of GLUT-EV and GLUT1-OE MΦs cultured for 24 h. *, p = 0.005 GLUT1-OE versus GLUT1-EV.

FIGURE 5.

GLUT1-OE drives pentose phosphate pathway. A, metabolomic analysis of cell lysates reveals increases in glycolysis, the pentose phosphate pathway, and purine/pyrimidine metabolism. B, diagram of pentose phosphate pathway (PRPP, phosphoribosyl pyrophosphate). Red and green-shaded cells indicate p ≤ 0.05; red indicates that the mean values are significantly higher, and green values significantly lower for GLUT1-OE versus GLUT1-EV. Ribulose (C) and ribose (*, p = 4.55 × 10⁻⁶) (D), sedoheptulose-7-phosphate (E), ribose-5-phosphate (F), coenzyme A (*, p = 0.0002) (G), and pantothenate (*, p = 0.00007) (H) were modulated by GLUT1 expression.

FIGURE 6.

GLUT1 gain of function drives increased expression of inflammatory mediators. A, microarray analysis of untreated GLUT1-EV and GLUT1-OE MΦs was conducted. Supervised two-class significance analysis of microarray analysis identified 1,547 genes differentially regulated by GLUT1 at a false discovery rate of 3.2% (supplemental Table 2). 775 were down-regulated and 772 were up-regulated in a GLUT1-dependent manner. Cluster analysis revealed two clusters of genes regulated by GLUT1. B, media from GLUT1-EV and GLUT1-OE MΦs were examined for secretion of inflammatory mediators using a proteome profiler array and normalized to protein lysates. Significantly (p < 0.05) regulated proteins are shown with the exception of eotaxin and IL-10.

FIGURE 7.

GLUT1 overexpression inflammatory mediator production is dependent upon glycolysis and ROS production. A, qPCR of PAI-1 was measured in GLUT1-EV and GLUT1-OE MΦs left unstimulated or treated with 100 ng/ml LPS for 24 h. Graphs are of combined data from three independent experiments ± S.E. *, p < 0.05 versus unstimulated; ^, p < 0.05 versus GLUT1-EV). B, qPCR of PAI-1 was measured in GLUT1-EV and GLUT1-OE MΦs left untreated or co-treated with 1 mm 2-DG to inhibit glycolysis or 5 mm NAC to quench ROS for 24 h. Graphs are of combined data from three independent experiments ± S.E. GLUT1-EV is set to 1. *, p = 0.0001. C, ROS production was measured in unstimulated RAW, GLUT1-EV, and GLUT1-OE cells. MFI, mean fluorescence intensity. Total ROS (C) and superoxide (D) in RAW, GLUT1-EV, and GLUT1-OE were detected. Graphs shown are representative of three separate experiments done in triplicate. Means ± S.E. are shown. *, p < 0.0005 versus RAW and GLUT1-EV.