The sirtuin inhibitor cambinol reduces intracellular glucosylceramide with ceramide accumulation by inhibiting glucosylceramide synthase

Figures & data

Figure 1. Effect of cambinol on the cellular UGCG activity.

(a) The effect of resveratrol and cambinol on the cellular levels of UGCG or SMS activity. NIH3T3 cells were treated with either resveratrol (200 μM) or resveratrol in combination with 100 μM of cambinol for 8 h. Cell-permeable fluorescent ceramide was then added to cells to measure the cellular levels of UGCG and SMS activity. Resveratrol and cambinol were dissolved in EtOH and DMSO, respectively. The same amount of EtOH and DMSO was added to the control experiment. (b) TLC showing the inhibition of the cellular UGCG activity by cambinol. TLC was applied using a solution of chloroform/methanol/water (65/25/4, v/v/v) for 25 min. The cellular UGCG activity of HEK293 cells was measured after treatment with 100 μM of cambinol for 8 h. The concentration (c) and time (d)-dependent inhibition of cellular UGCG activity by cambinol. After treatment with cambinol, NBD C6-ceramide conjugated to BSA was added to HEK293 cells and incubated for 60 min before analyzing by TLC.

Figure 2. Measurement of sphingolipid levels of cambinol-treated cells.

Quantification of cellular sphingolipid levels of cambinol-treated cells by LC-ESI MS/MS analysis. The bar graphs show the amount of cellular GlcCer (a), ceramide (b) (c) LacCer, and (d) sphingomyelin in HEK293 cells. Lipids were extracted from cells treated with 25, 50, or 100 μM of cambinol for 8 h and then applied to LC-ESI MS/MS.

Figure 3. Inhibitory efficiency and mechanism of cambinol on UGCG activity.

The effects of D-PDMP, AMP-DNM, and cambinol on in vitro UGCG (a) and SMS (b) activity. The corresponding concentrations of D-PDMP, AMP-DNM, and cambinol were added to a reaction mixture composed of the cell lysates of HEK293 (enzyme source), NBD-ceramide, and UDP-glucose. The reaction products were analyzed by TLC. The kinetic parameters for UDP-glucose (c) and NBD-ceramide (d) of UGCG treated with or without cambinol. The fluorescent intensities of NBD-GlcCer synthesized by UGCG were fitted into competitive (dotted line) or noncompetitive (solid line) models in GraphPad Prism. (e) The structures of cambinol and sirtinol. (f) The effects of cambinol and sirtinol on in vitro UGCG activity. In vitro UGCG activity was measured with 62.5 μM of cambinol or sitrinol.

Figure 4. Dependency of histidine 193 in UGCG for the inhibition by cambinol.

(a) Immunoblot analysis showing the expression of FLAG-tagged UGCG WT and H193A. HEK293 cells were transfected with mock-, UGCG WT-FLAG-, or UGCG H193A-FLAG-expressing vectors. The effects of D-PDMP (b), cambinol (c), and AMP-DNM (d) on the UGCG WT and H193A activity. Cell lysates of UGCG WT- or H193A-expressing cells were incubated with 20 μM of D-PDMP, 50 μM of cambinol, or 10 μM of AMP-DNM. The in vitro UGCG activities were measured using NBD ceramide and UDP-glucose.

Figure 5. Involvement of UGCG in ceramide accumulation caused by cambinol.

The effects of cambinol or AMP-DNM on the cellular GlcCer (a) and ceramide (b) levels. HEK293 cells were treated with 100 μM of cambinol or 10 μM of AMP-DNM for 14 h. The effect of cambinol on the amount of GlcCer (c) and ceramide (d) of ugcg (+/+) or (-\-) MEF. MEF cells were incubated with or without 100 μM of cambinol for 8 h. The cellular GlcCer and ceramide levels were measured using LC-ESI MS/MS analysis. The sirtuin inhibitor cambinol inhibits the GlcCer synthase (UGCG) activity in an unconventional manner.