Ganglioside GM1 contributes to extracellular/intracellular regulation of insulin resistance, impairment of insulin signaling and down-stream eNOS activation, in human aortic endothelial cells after short- or long-term exposure to TNFα

Figures

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  Figure 1. Ganglioside GM1 levels increase in TNFα-treated HAECs (A) Levels of GM1 in TNFα-treated HAECs analyzed by flow cytometry. Representative results are shown. GM1 expression in control cells and negative controls is depicted by black dot lines and thin gray lines, respectively. (B) Mean fluorescence intensities (MFIs) of GM1 expression relative to control HAECs. (C) Representative photos of HAECs 3 days after exposure to TNFα (0.1 ng/ml–10 ng/ml). Inset image is at high magnification. (D) Cell surface levels of gangliosides in short-term (3 days) 1 ng/ml TNFα-treated HAECs analyzed by flow cytometry using antibodies against each ganglioside. Representative results are shown. Negative controls are depicted by thin gray lines. (E) MFIs relative to control HAECs. Results are presented as means ± SD from three independent experiments. (F) Pathways of gangliosides and glycosyltransferases contributing to each synthetic pathway. Gangliosides highlighted in red fonts were examined in this study. (G) Real-time PCR analysis of the glycosyltransferases shown in (F) and NEU3 using cDNA derived from control and short-term (3 days) 1 ng/ml TNFα-treated HAECs. The results are shown after…

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  Figure 2. Increased GM1 levels in 1 ng/ml TNFα-treated HAECs reduce insulin signaling (A) At day 0, cells were reseeded with 1 ng/ml TNFα and with or without AMP-dNM in HAEC medium and incubated for 3 days. At day 3, cells were harvested and analyzed (B–F). Control cells were grown in HAEC medium. (B) Real-time PCR analysis of IRS2 and eNOS performed using cDNA derived from control and TNFα-treated HAECs. Results shown were normalized against values obtained for control HAECs (value = 1). (C) Cell surface levels of GM1 in TNFα-treated HAECs with or without AMP-dNM treatment analyzed by flow cytometry. MFIs of three independent experiments relative to control HAECs are shown. (D) Western blot analysis of insulin signaling performed in TNFα-treated HAECs with or without AMP-dNM treatment. (E) Histograms show mean densitometric readings ± SD of phosphorylated or non-phosphorylated proteins in insulin-stimulated cells normalized to loading control (β-ACTIN). All values were obtained from three independent experiments. (F) Immunocytochemical staining performed in TNFα-treated HAECs with or without AMP-dNM treatment. Representative images are shown (GM1, green; IRα, red; DAPI, blue; GM1 and IRα…

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  Figure 3. Reduction of eNOS levels in long-term 1 ng/ml TNFα-treated HAECs is inhibited by AMP-dNM treatment (A) At day 0, cells were reseeded with 1 ng/ml TNFα and with or without AMP-dNM in HAEC medium and incubated for 3 days. At day 3, cells were reseeded with 1 ng/ml TNFα and with or without AMP-dNM in HAEC medium and subsequently incubated for 4 days. At day 7, cells were harvested and analyzed (B–F). Control cells were grown in HAEC medium. (B) Real-time PCR analysis of IRS2 and eNOS performed using cDNA derived from control and TNFα-treated HAECs with or without AMP-dNM treatment. Results shown were normalized against values obtained for control HAECs (value = 1). (C) Cell surface levels of GM1 in TNFα-treated HAECs with or without AMP-dNM treatment analyzed by flow cytometry. MFIs relative to control HAECs of three independent experiments are shown. (D) Western blot analysis of insulin signaling performed in TNFα-treated HAECs with or without AMP-dNM treatment. (E) Histograms show mean densitometric readings ± SD of phosphorylated or non-phosphorylated proteins in insulin-stimulated cells normalized to loading controls (β-ACTIN). All values were obtained from three independent…

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  Figure 4. Induction of GM1 expression is reversible in 1 ng/ml TNFα-treated HAECs (A) At day 0, cells were reseeded with 1 ng/ml TNFα in HAEC medium and incubated for 3 days. At day 3, cells were reseeded without TNFα. Control cells were grown in HAEC medium. (B) Cell surface levels of GM1 in short-term (3 days) 1 ng/ml TNFα-treated HAECs 4 days after removal of TNFα analyzed by flow cytometry. MFIs relative to control HAECs of three independent experiments are shown. (C) Real-time PCR analysis of B4GALNT1 performed using cDNA derived from control and short-term (3 days) 1 ng/ml TNFα-treated HAECs 4 days after removal of TNFα. Results shown were normalized against the values obtained for control HAECs (value = 1). Results are presented as means ± SD from three independent experiments. (D) Western blot analysis of insulin signaling performed for short-term (3 days) 1 ng/ml TNFα-treated HAECs 4 days after removal of TNFα. (E) At day 0, cells were reseeded with 1 ng/ml TNFα in HAEC medium and incubated for 3 days. At day 3, cells were reseeded with 1 ng/ml TNFα in HAEC medium and further incubated for 4 days. At day 7, cells were reseeded without TNFα. Control cells were grown in HAEC medium.…

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  Figure 5. eNOS activation is attenuated independently of GM1 in 10 ng/ml TNFα-treated HAECs (A) At day 0, cells were reseeded with 10 ng/ml TNFα and with or without AMP-dNM in HAEC medium and incubated for 3 days. At day 3, cells were harvested and analyzed (B–F). Control cells were grown in HAEC medium. (B) Real-time PCR analysis of IRS2 and eNOS performed using cDNA derived from control and TNFα-treated HAECs with or without AMP-dNM treatment. The results shown were normalized against the values obtained for control HAECs (value = 1). (C) Cell surface levels of GM1 in TNFα-treated HAECs with and without AMP-dNM treatment analyzed by flow cytometry. MFIs relative to control HAECs of three independent experiments are shown. (D) Western blot analysis of insulin signaling performed for TNFα-treated HAECs with or without AMP-dNM treatment. (E) Histograms show mean densitometric readings ± SD of phosphorylated or non-phosphorylated proteins in insulin-stimulated cells normalized to loading controls (β-ACTIN). All values were obtained from three independent experiments. (F) Immunocytochemical staining performed in TNFα-treated HAECs with or without AMP-dNM treatment. Representative images are…

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  Figure 6. Induction of GM1 expression is stable in 10 ng/ml TNFα-treated HAECs (A) At day 0, cells were reseeded with 10 ng/ml TNFα in HAEC medium and incubated for 3 days. At day 3, cells were reseeded without TNFα and with or without AMP-dNM in HAEC medium and further incubated for 4 days. At day 7, cells were harvested and analyzed (B-G). Control cells were grown in HAEC medium. (B) Real-time PCR analysis of IRS2 and eNOS performed using cDNA derived from control, 3-day TNFα-treated HAECs and 3-day TNFα-treated HAECs 4 days after removal of TNFα. Results shown were normalized against values obtained for control HAECs (value = 1). (C) FACS analysis of cell surface GM1 performed in control and TNFα-treated HAECs with or without AMP-dNM treatment for 4 days after removal of TNFα. MFIs relative to control HAECs are shown. (D) Real-time PCR analysis of B4GALNT1 performed using cDNA derived from control and TNFα-treated HAECs 4 days after removal of TNFα. Results shown were normalized against values obtained for control HAECs (value = 1). (E) Western blot analysis of insulin signaling performed for TNFα-treated HAECs with or without AMP-dNM treatment 4 days after removal of TNFα. (F)…

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  Figure 7. Aging and senescence affect eNOS levels due to increased GM1 levels in short-term 1 ng/ml TNFα-treated HAECs (A) At day 0, aged/senescent HAECs were reseeded with 1 ng/ml TNFα in HAEC medium with or without AMP-dNM and incubated for 3 days. At day 3, cells were harvested and analyzed (B–E). (F) At day 0, non-aged HAECs were reseeded with or without 25 nM GM1 in HAEC medium. At day 1, cells were treated with 1 ng/ml TNFα with or without 25 nM GM1 in HAEC medium and incubated for 3 days. At day 4, cells were harvested and analyzed (G and H). Control cells were grown in HAEC medium. (B, D and G) Real-time PCR analysis of eNOS performed using cDNA derived from short-term (3 days) 1 ng/ml TNFα-treated senescent HAECs with or without AMP-dNM treatment (B), short-term (3 days) 1 ng/ml TNFα-treated aged HAECs with or without AMP-dNM treatment (D), and short-term (3 days) 1 ng/ml TNFα-treated GM1-overexpressing HAECs (G). Results shown were normalized against values obtained for control HAECs (value = 1). (C, E and H) Western blot analysis of eNOS performed for short-term (3 days) 1 ng/ml TNFα-treated senescent HAECs with or without AMP-dNM treatment (C), short-term (3 days) 1 ng/ml…