Fig 1.
TCDD effects on mRNAs for glucose transporters and glycolytic genes in chick embryo (CE) thymus.
CE were treated with TCDD (T) (1nmol/egg) or solvent dioxane (control, C). After 24 hr total RNA was extracted from thymus glands and used to perform RT-qPCRs for known chicken glucose transporters (GLUT) (upper panel) and genes involved in glycolysis (lower panel). CYP1A4 mRNA was measured as an index of AHR activation by TCDD. Bar graphs show means ± SE. Differences between control (C) and TCDD-treated (T) groups was performed using t-test analysis. For this and other figures, *, p≤0.05, **; p, ≤ 0.005; ***, p ≤ 0.001; ****, p ≤ 0.0001; n.s., not significant.
Fig 2.
TCDD suppresses glycolytic hallmarks in thymus.
CE were treated with TCDD or dioxane for 24 hr. a-b, Lymphocytes were extracted from thymus glands and used immediately to measure glucose uptake (a) or lactate release (b) as described in Material and Methods. Graphs for a and b show luminescence detected by a spectrophotometer over 1 hr. Bar graphs show quantification of TCDD effects on glucose uptake and lactate release. Representative experiments are shown (n = 3 for glucose uptake and n = 4 for lactate release; each experiments included 3–4 replicates for each treatment group with each replicate containing pooled thymus lymphocytes from 3–4 CE). c, ATP was measured in lymphocytes extracted from thymus glands from CE treated in ovo with dioxane (control) or TCDD for 24 hr (n = 2; 6–7 replicates for treatment group). d, Total RNA was extracted from thymus glands and RT-qPCR for IL7 was performed as described in Material and Methods. (n = 3; 4–5 replicates for each treatment group). e-f, Thymus glands, homogenized in Laemli buffer, were used for Western blotting with pAKT (S473), total AKT, beta-actin (ACTB) and HIF1A antibodies. Representative Western blots are shown (n = 4; 3 replicates for each treatment group). g, Representative contour plots show populations of CD3- thymocytes analysed for level of expression of CD4 and CD8 markers: lower left quadrant, double negative (CD4-CD8-); lower right quadrant, single positive for CD8 (CD4-CD8+); upper left quadrant, CD4 single positive (CD4+CD8-); upper right quadrant, double positive (CD4+CD8+). n = 4 independent experiments (with each experiment including 5 replicates of pooled lymphocytes from n = 4–5 CE). h, CE were treated with TCDD or dioxane for 72 hr with or without 2-DG (5mg/egg administered 3 times: at the time of the injection of TCDD and 24 hr and 48 hr after TCDD injection). For all panels in this figure, bar graphs represent means ± SE. For panels in a-g, t-test analysis comparing control (C) and TCDD-treated (T) groups was performed. For bar graph in panel h, one-way Anova analysis was used to calculate differences among the means and Tukey's honestly significant difference (HSD) test was used as a post hoc test.
Fig 3.
Correction of TCDD effects on glycolytic endpoints in CE thymus by the NAD+ precursors nicotinamide (NAM) or nicotinamide riboside (NR).
CE were treated with TCDD (T) or solvent (C) for 24 hr. NAM (10mg) or NR (2mg) were administered for the last 4 hr before the chick embryos were sacrificed. Assays for glucose uptake (a) and lactate release (b) by lymphocytes are described in Material and Methods. c. Lymphocytes extracted from thymus glands of CE were used to measure the Extracellular Acidification Rate (ECAR, glycolytic index) by using an Agilent Seahorse XF Stress glycolytic kit. Oxygen Consumption rate (OCR) was measured in the same conditions. Bar graphs, quantification of ECAR and OCR results. d. GAPDH activity was measured using thymus gland lysates as described in Material and Methods (n = 4 or 5 replicates for treatment group). e. ATP measured in lymphocytes extracted from thymus glands of CE treated as above (6 replicates of thymus lymphocytes from a pool of 4–5 CE for each treatment group). For all the panels in this figure, bar graphs represent means ± SE; one-way Anova analysis was used to calculate differences among the means and Tukey's honestly significant difference (HSD) test was used as a post hoc test.
Fig 4.
TCDD effects on mRNAs for glucose transporters and glycolytic genes in CE liver and human primary hepatocytes.
a. CE were treated with TCDD or dioxane and after 24 hr livers were removed and total RNA was extracted and used to perform RT-qPCRs for chicken GLUTs (upper panel) and glycolytic enzymes (lower panel). b. Human primary hepatocytes were plated in a 12-well plate (0.6 million cells/well). After 24 hr they were treated with TCDD (10 nM) for a further 24 hr before being collected for total RNA extraction and analysis by RT-qPCR for human GLUTs (upper panel) and enzymes involved in glycolysis (lower panel). n = 3 replicates for each treatment group. Bar graphs show means ± SE. Differences between control (C) and TCDD-treated (T) groups was performed using t-test analysis.
Fig 5.
TCDD increases glycolytic hallmarks in the liver; NAD+ boosting normalizes glycolysis.
a. CE were treated with TCDD or solvent dioxane and after 24 hr livers were removed and hepatocytes were extracted and assayed for glucose uptake (a). Graph shows luminescence detected by a spectrophotometer over 1 hr period. Bar graph shows quantification of the effect of TCDD on glucose uptake. A representative experiment is shown (n = 4 replicates for each treatment group). b, Upper panels, Livers from CE treated with TCDD (24hr) or solvent (C) were homogenized in Laemli buffer and used for Western blotting with the indicated antibodies. Representative Western blots are shown (n = 3 independent experiments with 3 replicates for each treatment group in each experiment). The bar graph shows relative densitometry units for bands for pAKT over total AKT. Lower panels, Homogenates of livers from CE treated for 24 hr with TCDD or solvent were used for Western blotting with antibodies for HIF1A and beta-actin (30 μg of total protein/lane). c, Total RNA was extracted from livers of CE treated as above and RT-qPCR for IL7 was performed as described in Material and Methods. (n = 4; 3–5 replicates for each treatment group). d, Lactate release from hepatocytes extracted from livers of CE treated with TCDD for 24 hr with and without NR added for the last 4 hr of the TCDD treatment. Graph shows luminescence detected by a spectrophotometer over 1 hr. Bar graph shows quantification of effects of TCDD with or without NR on lactate release. A representative experiment is shown (n = 3–4 replicates for each treatment group; each treatment group contained pooled lymphocytes from 4–5 CE). e, GAPDH activity was measured as described in Material and Methods using lysates of livers from CE treated as described in d (n = 4 or 5 replicates for treatment group). f, CE hepatocytes were cultured and treated for 24 hr with TCDD (1nM). Extracellular Acidification Rate (ECAR) was measured by Seahorse technology as described in Material and Methods. Oxygen Consumption rate (OCR) was measured in the same conditions as ECAR. For all panels, bar graphs represent means ± SE. For a-c, differences between control (C) and TCDD-treated (T) groups was performed using t-test analysis. For d-f, one-way Anova analysis was used to calculate differences among the means and Tukey's honestly significant difference (HSD) test was used as a post hoc test.