Ann Dermatol.  2012 Feb;24(1):39-44. 10.5021/ad.2012.24.1.39.

Inhibitory Effect of Vitamin U (S-Methylmethionine Sulfonium Chloride) on Differentiation in 3T3-L1 Pre-adipocyte Cell Lines

• Lee NY
• Park KY
• Min HJ
• Song KY
• Lim YY
• Park J
• Kim BJ
• Kim MN

• KMID: 2156854
• DOI: http://doi.org/10.5021/ad.2012.24.1.39

Abstract

BACKGROUND
S-methylmethionine sulfonium chloride was originally called vitamin U because of its inhibition of ulceration in the digestive system. Vitamin U is ubiquitously expressed in the tissues of flowering plants, and while there have been reports on its hypolipidemic effect, its precise function remains unknown.
OBJECTIVE
This study was designed to evaluate the anti-obesity effect of vitamin U in 3T3-L1 pre-adipocyte cell lines.
METHODS
We cultured the pre-adipocyte cell line 3T3L1 to overconfluency and then added fat differentiation-inducing media (dexamethasone, IBMX [isobutylmethylxanthine], insulin, indomethacin) and different concentrations (10, 50, 70, 90, 100 mM) of vitamin U. Then, we evaluated changes in the levels of triglycerides (TGs), glycerol-3-phosphate dehydrogenase (G3PDH), AMP-activated protein kinase (AMPK), adipocyte-specific markers (peroxisome proliferator-activated receptor gamma [PPAR-gamma], CCAAT/enhancer-binding protein alpha [C/EBP-alpha], adipocyte differentiation and determination factor 1 [ADD-1], adipsin, fatty acid synthase, lipoprotein lipase) and apoptosis-related signals (Bcl-2, Bax).
RESULTS
There was a gradual decrease in the level of TGs, C/EBP-alpha, PPAR-gamma, adipsin, ADD-1 and GPDH activity with increasing concentrations of vitamin U. In contrast, we observed a significant increase in AMPK activity with increasing levels of vitamin U. The decrease in bcl-2 and increase in Bax observed with increasing concentrations of vitamin U in the media were not statistically significant.
CONCLUSION
This study suggests that vitamin U inhibits adipocyte differentiation via down-regulation of adipogenic factors and up-regulation of AMPK activity.

Keyword

Adipocyte differentiation; S-methylmethionine sulfonium chloride; Vitamin U

MeSH Terms

1-Methyl-3-isobutylxanthine
Adipocytes
AMP-Activated Protein Kinases
Cell Line
Complement Factor D
Digestive System
Down-Regulation
Fatty Acid Synthetase Complex
Flowers
Glycerolphosphate Dehydrogenase
Insulin
Lipoproteins
Triglycerides
Ulcer
Up-Regulation
Vitamin U
Vitamins
1-Methyl-3-isobutylxanthine
AMP-Activated Protein Kinases
Complement Factor D
Fatty Acid Synthetase Complex
Glycerolphosphate Dehydrogenase
Insulin
Lipoproteins
Triglycerides
Vitamin U
Vitamins

Figure

• 

  Fig. 1 Cell viability of vitamin U at different concentrations in 3T3-L1 cell lines. The results show a slight decrease in viability with higher concentrations of vitamin U. However, no statistically significant differences were observed.

• 

  Fig. 2 Effect of vitamin U on the inhibition of adipocyte differentiation in Oil Red O staining, triglyceride (TG) assay and glycerol-3-phosphate dehydrogenase (G3PDH) activity assay. Oil droplets were abundant in differentiated adipocytes, and the number of oil droplets decreased with vitamin U treatment in a concentration-dependent manner (A, B). Consistent with the appearance of formed oil droplets, vitamin U significantly reduced cellular TG levels in a concentration-dependent manner (C). We found that vitamin U resulted in a significant inhibition of intracellular G3PDH (D). *Significant at p<0.05. ND: non-differentiated, NT: non-treated, Vit U: vitamin U, OD: optical density, G3PDH: glycerol-3-phosphate dehydrogenase.

• 

  Fig. 3 Effects of vitamin U on the expressions of PPAR-γ, C/EBP-α, ADD-1, adipsin, FAS and LPL in 3T3-L1 adipocytes. The mRNA levels of C/EBP-α, ADD-1 and adipsin decreased, and both the mRNA and protein levels of PPAR-γ remarkably decreased with vitamin U treatment (A, B). DM: differentiated media, Vit U: vitamin U, PPAR-γ: peroxisome proliferator-activated receptor γ, C/EBP-α: CCAAT/enhancer binding protein α, FAS: fatty acid synthase, ADD-1: adipocyte differentiation and determination factor 1, LPL: lipoprotein lipase.

• 

  Fig. 4 Effect of vitamin U on activation of AMP-activated protein kinase (AMPK) detected using western blotting. Protein levels of phosphorylated AMPK increased with vitamin U treatment in a dose-dependent manner during adipogenic differentiation (A, B). DM: differentiated media, Vit U: vitamin U, AMPK: AMP-activated protein kinase, ND: non-differentiated, NT: non-treated. *Significant at p<0.05.

Reference

1. Spiegelman BM, Flier JS. Obesity and the regulation of energy balance. Cell. 2001. 104:531–543.

2. Spiegelman BM, Hu E, Kim JB, Brun R. PPAR gamma and the control of adipogenesis. Biochimie. 1997. 79:111–112.

3. Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. 1994. 79:1147–1156.

4. Umek RM, Friedman AD, McKnight SL. CCAAT-enhancer binding protein: a component of a differentiation switch. Science. 1991. 251:288–292.

5. Yeh WC, Cao Z, Classon M, McKnight SL. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev. 1995. 9:168–181.

6. Kim JB, Spiegelman BM. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev. 1996. 10:1096–1107.

7. Ranganathan G, Unal R, Pokrovskaya I, Yao-Borengasser A, Phanavanh B, Lecka-Czernik B, et al. The lipogenic enzymes DGAT1, FAS, and LPL in adipose tissue: effects of obesity, insulin resistance, and TZD treatment. J Lipid Res. 2006. 47:2444–2450.

8. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature. 1998. 395:763–770.

9. Rosen BS, Cook KS, Yaglom J, Groves DL, Volanakis JE, Damm D, et al. Adipsin and complement factor D activity: an immune-related defect in obesity. Science. 1989. 244:1483–1487.

10. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science. 1993. 259:87–91.

11. Ahima RS, Flier JS. Adipose tissue as an endocrine organ. Trends Endocrinol Metab. 2000. 11:327–332.

12. Sartipy P, Loskutoff DJ. Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci USA. 2003. 100:7265–7270.

13. Hardie DG, Scott JW, Pan DA, Hudson ER. Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett. 2003. 546:113–120.

14. Fryer LG, Carling D. AMP-activated protein kinase and the metabolic syndrome. Biochem Soc Trans. 2005. 33:362–366.

15. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 2005. 1:15–25.

16. Roche-VITEC . Vitamin U. Pig News Info. 1990. 11:329–330.

17. Gessler NN, Kharchenko LI, Pavlovskaia TE, Bykhovskiĭ VIa. Radiation-protective effect of S-methylmethionine (vitamin U). Prikl Biokhim Mikrobiol. 1996. 32:666–668.

18. Tomiyama K, Nakata H, Sasa H, Arimura S, Nishio E, Watanabe Y. Wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, inhibits adipocytic differentiation of 3T3-L1 cells. Biochem Biophys Res Commun. 1995. 212:263–269.

19. Towler MC, Hardie DG. AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res. 2007. 100:328–341.

20. Dolinsky VW, Dyck JR. Role of AMP-activated protein kinase in healthy and diseased hearts. Am J Physiol Heart Circ Physiol. 2006. 291:H2557–H2569.

21. Hwang JT, Park IJ, Shin JI, Lee YK, Lee SK, Baik HW, et al. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase. Biochem Biophys Res Commun. 2005. 338:694–699.