Increased Hepatic Insulin Action in Diet-Induced Obese Mice Following Inhibition of Glucosylceramide Synthase

Obesity is characterized by the accumulation of fat in the liver and other tissues, leading to insulin resistance. We have previously shown that a specific inhibitor of glucosylceramide synthase, which inhibits the initial step in the synthesis of glycosphingolipids (GSLs), improved glucose metabolism and decreased hepatic steatosis in both ob/ob and diet-induced obese (DIO) mice. Here we have determined in the DIO mouse model the efficacy of a related small molecule compound, Genz-112638, which is currently being evaluated clinically for the treatment of Gaucher disease, a lysosomal storage disorder.DIO mice were treated with the Genz-112638 for 12 to 16 weeks by daily oral gavage. Genz-112638 lowered HbA1c levels and increased glucose tolerance. Whole body adiposity was not affected in normal mice, but decreased in drug-treated obese mice. Drug treatment also significantly lowered liver triglyceride levels and reduced the development of hepatic steatosis. We performed hyperinsulinemic-euglycemic clamps on the DIO mice treated with Genz-112638 and showed that insulin-mediated suppression of hepatic glucose production increased significantly compared to the placebo treated mice, indicating a marked improvement in hepatic insulin sensitivity.These results indicate that GSL inhibition in obese mice primarily results in an increase in insulin action in the liver, and suggests that GSLs may have an important role in hepatic insulin resistance in conditions of obesity

Reducing Glycosphingolipid Content in Adipose Tissue of Obese Mice Restores Insulin Sensitivity, Adipogenesis and Reduces Inflammation

Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (LepOb) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of LepOb mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) γ, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals

Glycogen content of livers and muscles after treatment with Genz-112638.

<p>C57BL/6 mice that were fed a normal diet (Lean) or a HFD (DIO) were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011239#pone-0011239-g001" target="_blank">Fig. 1</a>. Data shown as mean ± SEM (n = 6–9 mice per group). <i>P</i>>0.05 between all groups.</p

Effect of Genz-112638 on hepatic steatosis.

<p>DIO mice were treated with Genz-112638 or placebo (water) by daily oral gavage at 75 or 125 mg/kg/day for 16 weeks. A) Representative liver sections stained with hematoxylin and eosin (H&E). B) Fatty change score of the livers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011239#s4" target="_blank">Methods</a>). C) Liver triglyceride levels. Data shown as mean ± SEM (n = 6–10 mice per group). **<i>P</i><0.01 Genz-112638 DIO vs. Water DIO.</p

Sphingolipid levels in the livers of DIO mice after treatment with Genz-112638.

<p>C57BL/6 mice were fed a normal diet (Lean) or a HFD (DIO) for 8 weeks. The DIO mice were then treated with Genz-112638 or placebo (water) by twice daily oral gavage (120 mg/kg/day) for 22 weeks, while remaining on the HFD. Sphingolipid levels were determined by high performance liquid chromatography – tandem mass spectrometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011239#s4" target="_blank">Materials and Methods</a>). Data shown as mean ± SEM (n = 6–9 mice per group). *<i>P</i><0.05, **<i>P</i><0.01 Genz-112638 DIO vs. Water DIO.</p

Effect of Genz-112638 on A) food consumption and B) body weight.

<p>C57BL/6 mice were fed a normal diet (Lean) or a HFD (DIO) for 8 weeks. The DIO or Lean mice were then treated with Genz-112638 or placebo (water) by daily oral gavage (125 mg/kg/day) for 12 weeks, while remaining on their respective diets. Data shown as mean ± SEM (n = 7–14 mice per group). *<i>P</i><0.05 Genz-112638 DIO (both doses) vs. Water DIO, weeks 0–9. There was no significant difference in body weights between the Water DIO and drug treated groups.</p