Glycerol contained in vaping liquids affects the liver and aspects of energy homeostasis in a sex-dependent manner

Abstract

Vaping is increasingly popular among the young and adult population. Vaping liquids contained in electronic cigarettes (e-cigarettes) are mainly composed of propylene glycol and glycerol, to which nicotine and flavors are added. Among several biological processes, glycerol is a metabolic substrate used for lipid synthesis in fed state as well as glucose synthesis in fasting state. We aimed to investigate the effects of glycerol e-cigarette aerosol exposure on the aspects of glycerol and glucose homeostasis. Adult and young male and female mice were exposed to e-cigarette aerosols with glycerol as vaping liquid using an established whole-body exposure system. Mice were exposed acutely (single 2-h exposure) or chronically (2 h/day, 5 days/week for 9 weeks). Circulating glycerol and glucose levels were assessed and glycerol as well as glucose tolerance tests were performed. The liver was also investigated to assess changes in the histology, lipid content, inflammation, and stress markers. Lung functions were also assessed as well as hepatic mRNA expression of genes controlling the circadian rhythm. Acute exposure to glycerol aerosols generated by an e-cigarette increased circulating glycerol levels in female mice. Increased hepatic triglyceride and phosphatidylcholine concentrations were observed in female mice with no increase in circulating alanine aminotransferase or evidence of inflammation, fibrosis, or endoplasmic reticulum stress. Chronic exposure to glycerol e-cigarette aerosols mildly impacted glucose tolerance test in young female and male mice. Fasting glycerol, glucose, and insulin remained unchanged. Increased pulmonary resistance was observed in young male mice. Taken together, this study shows that the glycerol contained in vaping liquids can affect the liver as well as the aspects of glucose and glycerol homeostasis. Additional work is required to translate these observations to humans and determine the biological and potential pathological impacts of these findings.

Keywords: glycerol; liver; vaping.

FIGURE 1

Impact of glycerol e‐cigarette aerosol inhalation and glycerol gavage on blood glycerol and glucose concentrations. Six‐week‐old female and male mice (n = 3–4) were administered glycerol in different forms. Mice were exposed for 2 h to glycerol e‐cigarette aerosols (blue circles) or room air (white open circles). Exposure period is represented by the shaded region. Blood glycerol (a, c) and blood glucose (b, d) concentrations were measured. Mice received a glycerol gavage containing 2 g/kg (red), 0.7 g/kg (purple), 0.2 g/kg (dark blue), 0.07 g/kg (light blue), or water (white open circles). Blood glycerol (e, g) and blood glucose (f, h) concentrations were measured. Data are presented as mean ± SEM. Two‐way ANOVA with Šídák's multiple comparison post test was performed comparing experimental groups to control group: *p < 0.05; **p < 0.01; ***p < 0.001. Two‐sided Student's t‐tests were performed for two‐group comparisons: ^† p < 0.05; ^† p < 0.01

FIGURE 2

Glycerol e‐cigarette aerosol exposure does not change body weight. Twelve‐week‐old (adult) and 6‐week‐old (young) female and male mice (n = 9–10) were exposed to room air (white open circles) or glycerol e‐cigarette aerosols (blue circles) for 2 h a day, 5 days a week for 9 weeks. Mice were weighted every morning at the same time to ensure reproducibility (a, c, e, g). Upon euthanasia, ovarian (Ova)/epididymal (Epi), inguinal (Ingui), and retroperitoneal (Retro) adipose tissue were weighted for room air (black and gray boxes) and glycerol e‐cigarette aerosol‐exposed mice (blue boxes) (b, d, f, h). Arrows represent fasting period mice underwent for glycerol and glucose tolerance tests. Gray shaded regions represent weekends, where no exposure took place. Data are presented as mean ± SEM. Two‐way ANOVA with Šídák's multiple comparison post test was performed for body weight curves. Two‐sided Student's t‐tests were performed for two‐group comparisons. *p < 0.05

FIGURE 3

Glycerol e‐cigarette aerosol exposure increases hepatic triglyceride and phosphatidylcholine content in female mice. Twelve‐week‐old (adult) and 6‐week‐old (young) female and male mice (n = 9–10) were exposed to room air (white open circles) or glycerol e‐cigarette aerosols (blue circles) for 2 h a day, 5 days a week for 9 weeks. Upon euthanasia, liver weight was measured (a, e, i, m). Hepatic triglycerides (b, f, j, n) and phosphatidylcholine (c, g, k, o) levels were measured. Hematoxylin and eosin (H&E) staining of formalin‐fixed paraffin‐embedded liver sections was made (d, h, l, p). Data are presented as mean ± SEM. Two‐sided Student's t‐tests were performed for two‐group comparisons. *p < 0.05; **p < 0.01

FIGURE 4

Impact of glycerol e‐cigarette aerosol exposure on liver inflammation, endoplasmic reticulum stress, and remodeling. Twelve‐week‐old (adult) and 6‐week‐old (young) female and male mice (n = 9–10) were exposed to room air (white open circles) or glycerol e‐cigarette aerosol (blue circles) for 2 h a day, 5 days a week for 9 weeks. Blood alanine aminotransferase activity (ALT) was measured (a, e, i, m). Hepatic expression level for inflammatory markers ccl2 and tnf (b, f, j, n), endoplasmic reticulum stress markers atf6 and ddit3 (c, g, k, o), and remodeling marker ctgf was measured by qPCR analysis (b, e, h, k). Data are presented as mean ± SEM. Two‐sided Student's t‐tests were performed for two‐group comparisons. *p < 0.05

FIGURE 5

Impact of glycerol e‐cigarette aerosol exposure on fasting glycerol, glucose, and insulin concentrations. Twelve‐week‐old (adult) and 6‐week‐old (young) female and male mice (n = 9–10) were exposed to room air (white open circles) or glycerol e‐cigarette aerosol (blue circles) for 2 h a day, 5 days a week for 9 weeks. Upon euthanasia, fasting blood glycerol (a, d, g, j), glucose (b, e, h, k), and insulin (c, f, i, l) were assessed. Data are presented as mean ± SEM. Two‐sided Student's t‐tests were performed for two‐group comparisons. *p < 0.05

FIGURE 6

Impact of glycerol e‐cigarette aerosol exposure on glycerol and glucose tolerance. Twelve‐week‐old (adult) and 6‐week‐old (young) female and male mice (n = 9–10) were exposed to room air (white open circles) or glycerol e‐cigarette aerosol (blue circles) for 2 h a day, 5 days a week for 9 weeks. After 6 weeks of exposure, mice were injected with 2 g/kg of glycerol in a saline solution and blood glycerol (a, d, g, j) and blood glucose (b, e, h, k) were assessed. After 7 weeks of exposure, mice were injected with 1 g/kg of d‐glucose in a saline solution and blood glucose (c, f, i, l) was assessed. AUC = area under the curve. Data are presented as mean ± SEM. Two‐way ANOVA with Šídák's multiple comparison post test was performed for tolerance curves: *p < 0.05; **p < 0.01. Two‐sided Student's t‐tests were performed for two‐group comparisons at each timepoint and AUC data: ^† p < 0.05; ^†† p < 0.01