E-cigarette constituents propylene glycol and vegetable glycerin decrease glucose uptake and its metabolism in airway epithelial cells in vitro

Abstract

Electronic nicotine delivery systems, or e-cigarettes, utilize a liquid solution that normally contains propylene glycol (PG) and vegetable glycerin (VG) to generate vapor and act as a carrier for nicotine and flavorings. Evidence indicated these "carriers" reduced growth and survival of epithelial cells including those of the airway. We hypothesized that 3% PG or PG mixed with VG (3% PG/VG, 55:45) inhibited glucose uptake in human airway epithelial cells as a first step to reducing airway cell survival. Exposure of H441 or human bronchiolar epithelial cells (HBECs) to PG and PG/VG (30-60 min) inhibited glucose uptake and mitochondrial ATP synthesis. PG/VG inhibited glycolysis. PG/VG and mannitol reduced cell volume and height of air-liquid interface cultures. Mannitol, but not PG/VG, increased phosphorylation of p38 MAPK. PG/VG reduced transepithelial electrical resistance, which was associated with increased transepithelial solute permeability. PG/VG decreased fluorescence recovery after photobleaching of green fluorescent protein-linked glucose transporters GLUT1 and GLUT10, indicating that glucose transport function was compromised. Puffing PG/VG vapor onto the apical surface of primary HBECs for 10 min to mimic the effect of e-cigarette smoking also reduced glucose transport. In conclusion, short-term exposure to PG/VG, key components of e-cigarettes, decreased glucose transport and metabolism in airway cells. We propose that this was a result of PG/VG reduced cell volume and membrane fluidity, with further consequences on epithelial barrier function. Taking these results together, we suggest these factors contribute to reduced defensive properties of the epithelium. We propose that repeated/chronic exposure to these agents are likely to contribute to airway damage in e-cigarette users.

Keywords: airway; electronic cigarettes; glucose; glycerin; propylene glycol.

Fig. 1.

Propylene glycol (PG) and PG mixed with vegetable glycerin (PG/VG) inhibit glucose uptake in proliferating airway cells. Exposure to PG (33–263 mM) for 30 min decreased glucose uptake, shown as %control in a concentration-dependent manner in H441 (A) and BMI-1-transduced (B) human bronchiolar epithelial cells (HBECs). Inhibition of glucose uptake after 30-min exposure to 3% PG (263 mM) or 3% PG/VG (45:55) with and without GLUT inhibitors phloretin (PT) and cytochalasin B (CB), or PT or CB alone in H441 (C) or BMI-1-transduced (D) HBECs. E: exposure of H441 cells to 3% PG/VG (45:55, closed circles) decreased glucose uptake in a time-dependent manner. The effect of PT (open squares) on glucose transport at 24 h is shown for reference. Data are shown as box and whisker plots. Horizontal line, median; box, 25–75th percentiles; whiskers, min and max; +, mean. Significantly different from control: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.

Propylene glycol (PG) and PG mixed with vegetable glycerin (PG/VG) modify airway cell glucose metabolism. The effect of 3% PG or 3% PG/VG, 45:55 or isosmotic mannitol on human bronchiolar epithelial cell (HBEC) metabolism. A. Oxygen consumption rate (OCR; A) and extracellular acidification rate (ECAR; B) after sequential addition of glucose, oligomycin, and 2-deoxy-d-glucose (2-DG). C: mitochondrial ATP production measured as OCR. Glycolysis (D), glycolytic capacity (E), and glycolytic reserve (F) all measured as ECAR. G: ECAR/OCR after glucose injection to initiate metabolism. H: ECAR/OCR after oligomycin injection to inhibit oxidative phosphorylation. Data are shown as box and whisker plots. Horizontal line, median; box, 25–75th percentiles; whiskers, min and max; +, mean. Significantly different from control: ***P < 0.001 and ****P < 0.0001.

Fig. 3.

Propylene glycol (PG) and PG mixed with vegetable glycerin (PG/VG) did not inhibit airway cell proliferation or elevate cytotoxic markers. Effect of 30-min exposure to 3% PG or 3% PG/VG, 45:55, or isoosmotic mannitol on human bronchiolar epithelial cells (HBECs). A: CyQuant Cell Proliferation Assay of DNA content measured as fluorescence. B: lactate dehydrogenase (LDH) release measured as absorbance at 450–680 nm. Data are shown as box and whisker plots. Horizontal line, median; box, 25–75th percentiles; whiskers, min and max; +, mean. Significantly different from control: *P < 0.05 and ****P < 0.0001.

Fig. 4.

Propylene glycol (PG) and PG mixed with vegetable glycerin (PG/VG) decreases cell surface area but does not elevate p38 mitogen-activated protein kinse (MAPK). A: fluorescence microscopy of calcein-AM-loaded HEK-293 cells before (control) and after exposure to 3% PG/VG or isoosmotic mannitol. Calcein-fluorescent cells are seen as bright white in images. B: surface area (%), of cells exposed to 3% PG/VG or mannitol (as shown in A) over a time course of 120 s. C: collated data of percent change in surface area (ΔSurface Area, %) as shown in B. Data are shown as box and whisker plots. Horizontal line, median; box, 25–75th percentiles; whiskers, min and max; +, mean. D: Western blots of phosphorylated p38 MAPK (p-p38, top) and nonphosphorylated p38 MAPK after exposure to mannitol or 3% PG/VG for 0–60 min. E: quantification of p-p38/total p38MAPK from n = 3 experiments. Data are shown as means ± SD.

Fig. 5.

Propylene glycol mixed with vegetable glycerin (PG/VG) reduced airway epithelial height, decreased transepithelial resistance, and increased epithelial permeability and airway surface liquid (ASL) height A: representative fluorescence microscopy images of calcein-AM-loaded human bronchiolar epithelial cells (HBECs) (green; for cell height measurement) or rhodamine-dextran-labeled airway surface liquid (ASL) overlying HBECs (red; for ASL height measurement) before (control) and after mucosal exposure to 3% PG/VG or mannitol. Effect of control (closed circles), 3% PG/VG (open squares) or isoosmotic mannitol (closed triangles) on cell height (B) and ASL height (C). Transepithelial electrical resistance (TEER; D) and transepithelial permeability (E) measured across H441 cells grown at air-liquid interface. Dotted lines in B and C represent untreated control levels. RFU, relative fluorescence units. Data are shown as means ± SD. Significantly different from control: *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 6.

Propylene glycol mixed with vegetable glycerin (PG/VG) decreases fluorescence recovery after photobleaching (FRAP) of glucose transporters. A: fluorescence of GLUT1-green fluorescent protein (GFP) expressed in HEK-293 cells showing areas measured for background, membrane fluorescence and region of interest (ROI) at baseline, after fluorescent photobleaching (Bleach), and recovery. GLUT10-GFP (B) and GLUT2-mCherry (C) expressed in HEK-293 cells. Graphs showing FRAP in control cells (black line) or after treatment with 3% PG/VG (red line) or mannitol (blue line) over a time course of 90 s for GLUT1-GFP (D), GLUT2-mCherry (E), and GLUT10-GFP (F). RFU, relative fluorescence units. Data are shown as means ± SE, with error bars shown in one direction for clarity. Significantly different from control: as shown or ***P < 0.01 and ****P < 0.0001.

Fig. 7.

Propylene glycol mixed with vegetable glycerin (PG/VG) vaped onto the apical surface of human bronchiolar epithelial cells (HBECs) decreases glucose transport. Effect of 3% PG/VG puffed onto mucosal surface (VAPE) of primary HBECs on cell height: effect of 3% PG/VG (VAPE) (A), or air (AIR) puffed onto mucosal surface or air plus the addition of phloretin (AIR PT) on glucose uptake across the basolateral (serosal) membrane (B) and glucose uptake across the apical (mucosal) membrane (C). Data are shown as box and whisker plots. Horizontal line, median; box, 25–75th percentiles; whiskers, min and max; +, mean. Significantly different from control as shown or *P < 0.05, **P < 0.01, and ***P < 0.001.