Enhanced insulin sensitivity in mice lacking ganglioside GM3

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 alpha2,3-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.

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^+/+ and GM3S^−/− mice by using GM3S cDNA as a probe. Ethidium bromide-stained gel of RNA before blotting showing 28S and 18S RNA.

Figure 2

Brain gangliosides of GM3S^−/− mice. Gangliosides were isolated from brains of GM3S^+/+ and GM3S^−/− mice. The samples were either untreated (−, lanes 2 and 6) or treated with sialidase (+, lanes 3 and 5). The positions of glycolipid standards are indicated (lane 1, GM1b, GD1α; lane 4, GA1; lane 7, GM1a, GD1a). The bracket (lane 6) indicates the normal major gangliosides in wild-type mice that include GM1a, GD1a, GD1b, and GT1b.

Figure 3

Enhanced insulin receptor phosphorylation in GM3S^−/− mice. The insulin receptor β subunit was analyzed for phosphorylation status in skeletal muscle (A) and adipose tissue extracts (B) by Western blotting. GM3S^+/+, GM3S^+/−, and GM3S^−/− mice, four in each group, were either not injected (−) or injected (+) with insulin. (A and B Upper) Immunoblots developed with antibody to phosphotyrosine (anti-PY). (A and B Lower) Same blot developed with antibody to insulin receptor β subunit (anti-IR-β). (C and D) Quantitation of phosphorylation of insulin receptor β subunit from Western blots of skeletal muscle (C) and adipose tissue (D). Level of phosphorylation (anti-PY) was normalized to amount of total receptor (anti-IR-β). *, P < 0.05 by Student's t test; GM3S^−/− vs. GM3S^+/+ or GM3S^+/−. (E and F) TLC analysis of skeletal muscle (E) and adipose tissue (F) anionic lipids from GM3S^+/+ and GM3S^−/− mice. The anionic lipids were visualized by a phosphoric acid/copper sulfate reagent. Migration positions of free fatty acids, sulfatides, and ganglioside standards (STAND) are shown. The two bands denoted in the adipose tissue profile (F) by the filled circles are unidentified.

Figure 4

Determinations of glucose homeostasis in GM3S^−/− mice. (A) Blood glucose levels of male GM3S^+/+, GM3S^+/−, and GM3S^−/− mice, 6–8 weeks old, during fasting (n = 6, +/+; n = 8, +/−; n = 9, −/−). *, P < 0.05 by Student's t test; GM3S^−/− vs. GM3S^+/+ or GM3S^+/−. (B) Insulin levels of male GM3S^+/+ and GM3S^−/− mice, 6–8 weeks old, when fed and after fasting for 14 h (n = 12 each genotype). (C) Glucose tolerance tests of male GM3S^+/+, GM3S^+/−, and GM3S^−/− mice, 6–8 weeks old (n = 14 each genotype). *, P < 0.05 by Student's t test; GM3S^−/− vs. GM3S^+/+ or GM3S^+/−. (D) Insulin tolerance tests of male mice GM3S^+/+, GM3S^+/−, and GM3S^−/− mice, 6–8 weeks old (n = 6,+/+; n = 8, +/−; n = 8, −/−). *, P < 0.05; GM3S^−/− vs. GM3S^+/+ or GM3S^+/−. (E and F) Sections of pancreas from GM3S^+/+ and GM3S^−/− mice showing islets immunostained for insulin (E) and glucagon (F).

Figure 5

GM3S^−/− mice are protected from high-fat diet-induced insulin resistance. Groups of GM3S^−/− and GM3S^+/+ male mice were placed on a 45% high-fat diet for 10 weeks. The diet was started when the mice were 6–8 weeks old. (A) Glucose tolerance tests on GM3S^+/+ and GM3S^−/− mice after the high-fat diet (n = 8, +/+; n = 10, −/−). *, P < 0.05 by Student's t test; GM3S^−/− vs. GM3S^+/+. (B) Insulin levels of GM3S^+/+ and GM3S^−/− mice after the high-fat diet, when fed and after fasting for 14 h (n = 12 each genotype). *, P < 0.05 by Student's t test; GM3S^−/− vs. GM3S^+/+. (C) Whole body and skeletal muscle glucose uptake of GM3S^+/+ and GM3S^−/− mice after the high-fat diet as determined by euglycemic–hyperinsulinemic clamps (n = 4 each genotype). *, P < 0.05 by Student's t test; GM3S^−/− vs. GM3S^+/+. (E) The percent suppression of endogenous glucose production (EGP) in livers GM3S^+/+ and GM3S^−/− mice after the high-fat diet as determined by euglycemic–hyperinsulinemic clamps (n = 4 each genotype). *, P < 0.05 by Student's t test; GM3S^−/− vs. GM3S^+/+.