Enhanced insulin sensitivity in mice lacking ganglioside GM3

Tadashi Yamashita

Akira Hashiramoto

Martin Haluzik

Hiroki Mizukami

Jose Daniotti+4 authors

^{Genetics of Development and Disease and}^{Diabetes Branches, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;}^{Molecular Glycobiology, The Glycoscience Institute, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan;}^{Centro de Investigaciones en Quimica Biologica de Cordoba, Departamento de Quimica Biologica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000 Cordoba, Argentina;}^{Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany; and}^{Deutsches Krebsforschungszentrum Heidelberg, Abteilung für Zelluläre und Molekulare Pathologie, INF 280, 69120 Heidelberg, Germany}

^{To whom correspondence should be addressed at: Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive, MSC 1821, Building 10, Room 9N-314, Bethesda, MD 20892. E-mail:}vog.hin@aiorp.

Edited by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved January 22, 2003

Received 2002 Sep 30

Abstract

Gangliosides are sialic acid-containing glycosphingolipids that are present on all mammalian plasma membranes where they participate in recognition and signaling activities. We have established mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide α2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. Our results show that GM3 ganglioside is a negative regulator of insulin signaling, making it a potential therapeutic target in type 2 diabetes.

Abstract

Gangliosides are sialic acid-containing glycosphingolipids that are ubiquitously distributed on vertebrate plasma membranes. They are synthesized in the Golgi apparatus by the sequential transfer of carbohydrate residues onto a ceramide lipid anchor. A combinatorial biosynthetic pathway results in a large diversity of oligosaccharide structures on gangliosides (1). GM3 ganglioside is a key structure; it contains the simplest ganglioside oligosaccharide in this pathway (Fig. (Fig.11A), a trisaccharide composed of glucose, galactose, and sialic acid. It is the most widely distributed ganglioside among tissues, and it serves as a precursor for most of the more complex ganglioside species.

An external file that holds a picture, illustration, etc.
Object name is pq0635898001.jpg

Figure 1

Genetic disruption of GM3 ganglioside synthesis. (A) Pathway of ganglioside synthesis showing the block in GM3S^{mice. Cer, ceramide. (}B) Strategy to disrupt GM3S locus. (Top) Targeting vector. Asterisk indicates that the HindIII site was disrupted. (Middle) Locus organization. (Bottom) Targeted locus. The approved gene name for GM3S is Siat9. (C) Southern blot of tail DNA showing HindIII cleaved DNA fragments corresponding to the wild-type (6.0 kb) and targeted (10.5 kb) GM3S alleles. (D) Northern blot of brain RNA from GM3S^andGM3S^{mice by using}GM3S cDNA as a probe. Ethidium bromide-stained gel of RNA before blotting showing 28S and 18S RNA.

Gangliosides are involved in cell-signaling functions as ligands and as modulators of receptor activity (2–5). In the case of the insulin receptor, sialylparagloboside and the structurally related GM3 ganglioside have been found to inhibit the intrinsic tyrosine kinase activity of soluble receptors (6, 7). Furthermore, GM3 ganglioside depressed insulin-mediated signaling in cultured cells (6, 8). These results and the finding of enhanced expression of GM3 synthase in models of insulin resistance have led to the suggestion that GM3 ganglioside overexpression may play a role in the pathogenesis of type 2 diabetes through a negative modulation on insulin receptor signaling (6). To determine the physiological role of GM3 ganglioside and whether insulin receptor signaling is influenced by GM3 ganglioside in vivo, we established mice with a disrupted gene (GM3S; also known as Siat9) encoding GM3 synthase. We have found that mice without the capacity to synthesize GM3 ganglioside had enhanced phosphorylation of the skeletal muscle insulin receptor after ligand binding. Correspondingly, they showed heightened responses in glucose and insulin tolerance tests. Furthermore, these mutant mice were protected from high-fat diet-induced insulin resistance. The results indicate that GM3 ganglioside has an important role in regulating insulin sensitivity.

Acknowledgments

We thank Chuxia Deng for expertise in establishment of chimeric mice, Dr. M. Kiso (Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan) for generosity in supplying us with synthetic ganglioside GM1b and GD1α standards, and Katie Prothero for help in preparing the figures. We are grateful to the Fonds der Chemischen Industrie for financial support.

Acknowledgments

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Footnotes

References

1. Kolter T, Proia R L, Sandhoff K. J Biol Chem. 2002;277:25859–25862.[PubMed]
2. Hakomori S I. Proc Natl Acad Sci USA. 2002;99:225–232.
3. Hakomori S. Glycoconj J. 2000;17:627–647.[PubMed]
4. Allende M L, Proia R L. Curr Opin Struct Biol. 2002;12:587–592.[PubMed]
5. Miljan E A, Bremer E G. Sci STKE. 2002;2002:RE15.[PubMed]
6. Tagami S, Inokuchi Ji J, Kabayama K, Yoshimura H, Kitamura F, Uemura S, Ogawa C, Ishii A, Saito M, Ohtsuka Y, et al J Biol Chem. 2002;277:3085–3092.[PubMed][Google Scholar]
7. Nojiri H, Stroud M, Hakomori S. J Biol Chem. 1991;266:4531–4537.[PubMed]
8. Tsuruoka T, Tsuji T, Nojiri H, Holmes E H, Hakomori S. J Biol Chem. 1993;268:2211–2216.[PubMed]
9. Liu Y, Wada R, Kawai H, Sango K, Deng C, Tai T, McDonald M P, Araujo K, Crawley J N, Bierfreund U, et al J Clin Invest. 1999;103:497–505.[Google Scholar]
10. Sandhoff R, Hepbildikler S T, Jennemann R, Geyer R, Gieselmann V, Proia R L, Wiegandt H, Grone H J. J Biol Chem. 2002;277:20386–20398.[PubMed]
11. Steppan C M, Bailey S T, Bhat S, Brown E J, Banerjee R R, Wright C M, Patel H R, Ahima R S, Lazar M A. Nature. 2001;409:307–312.[PubMed]
12. Haluzik M, Dietz K R, Kim J K, Marcus-Samuels B, Shulman G I, Gavrilova O, Reitman M L. Diabetes. 2002;51:2113–2118.[PubMed]
13. Youn J H, Kim J K, Buchanan T A. Diabetes. 1994;43:564–571.[PubMed]
14. Kim J K, Michael M D, Previs S F, Peroni O D, Mauvais-Jarvis F, Neschen S, Kahn B B, Kahn C R, Shulman G I. J Clin Invest. 2000;105:1791–1797.
15. Saltiel A R, Kahn C R. Nature. 2001;414:799–806.[PubMed]
16. Vainio S, Heino S, Mansson J E, Fredman P, Kuismanen E, Vaarala O, Ikonen E. EMBO Rep. 2002;3:95–100.
17. Simons K, Toomre D. Nat Rev Mol Cell Biol. 2000;1:31–39.[PubMed]
18. Anderson R G. Annu Rev Biochem. 1998;67:199–225.[PubMed]
19. Moller D E. Trends Endocrinol Metab. 2000;11:212–217.[PubMed]
20. Saltiel A R. Cell. 2001;104:517–529.[PubMed]