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Generating Ceramide from Sphingomyelin by Alkaline Sphingomyelinase in the Gut Enhances Sphingomyelin-Induced Inhibition of Cholesterol Uptake in Caco-2 Cells

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Abstract

Background

Sphingomyelin (SM) is present in dietary products and cell plasma membranes. We previously showed that dietary SM inhibited cholesterol absorption in rats. In the intestinal tract, SM is mainly hydrolyzed by alkaline sphingomyelinase (alk-SMase) to ceramide.

Aims

We investigated the influence of SM and its hydrolytic products ceramide and sphingosine on cholesterol uptake in intestinal Caco-2 cells.

Methods

Micelles containing bile salt, monoolein, and 14C-cholesterol were prepared with or without SM, ceramide, or sphingosine. The micelles were incubated with Caco-2 cells, and uptake of radioactive cholesterol was quantified.

Results

We found that confluent monolayer Caco-2 cells expressed NPC1L1, and the uptake of cholesterol in the cells was inhibited by ezetimibe, a specific inhibitor of NPC1L1. Incorporation of SM in the cholesterol micelles inhibited cholesterol uptake dose-dependently; 38% inhibition occurred at an equal mole ratio of SM and cholesterol. The inhibition was further enhanced to 45% by pretreating the cholesterol/SM micelles with recombinant alk-SMase, which hydrolyzed SM in the micelles by 85%, indicating ceramide has stronger inhibitory effects on cholesterol uptake. To confirm this, we further replaced SM in the micelles with ceramide and sphingosine, and found that at equal mole ratio to cholesterol, ceramide exhibited stronger inhibitory effect (50% vs 38%) on cholesterol uptake than SM, whereas sphingosine only had a weak effect at high concentrations.

Conclusion

Both SM and ceramide inhibit cholesterol uptake, the effect of ceramide being stronger than that of SM. Alk-SMase enhances SM-induced inhibition of cholesterol uptake by generating ceramide in the intestinal lumen.

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Acknowledgments

Yajun Cheng is thanked for technical assistance. The work was supported by grants from the Albert Påhlsson Foundation, Swedish Nutrition Foundation, and Research Foundation of Lund University and Lund University Hospital. DF is an exchange researcher from the School of Public Health, Sun Yat-sen University, Guangzhou, China.

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Authors and Affiliations

  1. Gastroenterology and Nutrition Laboratory, Biomedical Center B11, Institution of Clinical Sciences, University of Lund, 221 84, Lund, Sweden

    Dan Feng, Lena Ohlsson, Åke Nilsson & Rui-Dong Duan

  2. School of Public Health, Sun Yat-sen University, Guangzhou, China

    Wenhua Ling

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  1. Dan Feng
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  2. Lena Ohlsson
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  3. Wenhua Ling
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Correspondence to Rui-Dong Duan.

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Feng, D., Ohlsson, L., Ling, W. et al. Generating Ceramide from Sphingomyelin by Alkaline Sphingomyelinase in the Gut Enhances Sphingomyelin-Induced Inhibition of Cholesterol Uptake in Caco-2 Cells. Dig Dis Sci 55, 3377–3383 (2010). https://doi.org/10.1007/s10620-010-1202-9

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