Ras Inhibition Induces Insulin Sensitivity and Glucose Uptake

BACKGROUND: Reduced glucose uptake due to insulin resistance is a pivotal mechanism in the pathogenesis of type 2 diabetes. It is also associated with increased inflammation. Ras inhibition downregulates inflammation in various experimental models. The aim of this study was to examine the effect of Ras inhibition on insulin sensitivity and glucose uptake, as well as its influence on type 2 diabetes development. METHODS AND FINDINGS: The effect of Ras inhibition on glucose uptake was examined both in vitro and in vivo. Ras was inhibited in cells transfected with a dominant-negative form of Ras or by 5-fluoro-farnesylthiosalicylic acid (F-FTS), a small-molecule Ras inhibitor. The involvement of IκB and NF-κB in Ras-inhibited glucose uptake was investigated by immunoblotting. High fat (HF)-induced diabetic mice were treated with F-FTS to test the effect of Ras inhibition on induction of hyperglycemia. Each of the Ras-inhibitory modes resulted in increased glucose uptake, whether in insulin-resistant C2C12 myotubes in vitro or in HF-induced diabetic mice in vivo. Ras inhibition also caused increased IκB expression accompanied by decreased expression of NF-κB . In fat-induced diabetic mice treated daily with F-FTS, both the incidence of hyperglycemia and the levels of serum insulin were significantly decreased. CONCLUSIONS: Inhibition of Ras apparently induces a state of heightened insulin sensitization both in vitro and in vivo. Ras inhibition should therefore be considered as an approach worth testing for the treatment of type 2 diabetes

The combined treatment of Copaxone and Salirasib attenuates experimental autoimmune encephalomyelitis (EAE) in mice

EAE is a common animal model for multiple sclerosis (MS). Immunomodulatory treatments such as glatiramer acetate (GA, Copaxone) are beneficial in EAE but are not universally effective in the clinic. The Ras inhibitor farnesylthiosalycylic acid (FTS, Salirasib), efficiently ameliorate EAE as well. Here we demonstrate a synergistic beneficial effect of the combined GA and FTS treatment on EAE; 22.5% of the combined-treatment mice developed disease compared to 87.5%, 77.5% and 82.5% of mice treated with vehicle, GA and FTS, respectively, results supported by MRI, histological, immunological and biochemical data. Such a combined treatment may improve clinical outcome in MS patients

Ras inhibition in HF-induced diabetic mice reduces diabetes incidence and increases the concentration of circulating insulin.

<p><b>A</b>. C57/Bl mice fed on a high-fat diet were treated daily with F-FTS (20 mg/kg body weight; i.p.; <i>n</i> = 30 mice per group) or PBS (<i>n</i> = 30) for 13 weeks. Kaplan-Meier plots of mean incidence of diabetes in each group. <b>B</b>. Blood glucose levels were measured as described in Methods (<i>n</i> = 10 in each group). *** <i>P</i><0.005 compared to control. <b>C</b>. C57Bl/6 mice on a high -fat diet were treated daily with F-FTS (30 mg/kg; <i>n</i> = 10), FTS (60 mg/kg; <i>n</i> = 10) or CMC (<i>n</i> = 10) for 13 weeks. Kaplan-Meier curves record the mean incidence of diabetes in each group. <b>D</b>. Blood glucose levels were measured as described in Methods (<i>n</i> = 10 in each group). *** <i>P</i><0.005 compared to control. <b>E</b>. All treated animals were monitored for weight gain while being fed a high-fat diet. Kaplan-Meier curves record the mean percentage of weight gain in each group. <b>F</b>, <b>G</b>. Serum insulin concentrations were measured by ELISA as described in Methods (<i>n</i> = 10 in each group). ** <i>P</i><0.01 compared to control.</p

Ras inhibition <i>in vivo</i> increases muscle, fat and liver glucose uptake.

<p><b>A</b>. HF-induced C57/Bl mice were hydrodynamically injected (i.v.) with DN-GFP-Ras or with pGFP, as described in Methods. Mice were injected with the fluorescent glucose analog 2-[<i>N</i>-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] (2-NBDG) and glucose uptake in muscle, fat and liver tissues was assayed (<i>n</i> = 8). Representative histograms of glucose uptake are presented for each tissue. <b>B.</b> Statistical analysis of the results is presented as means ± S.D. * <i>P</i><0.05, **<i>P</i><0.01. <b>C.</b> Representative gels and densitometry of Ras-GTP are shown (<i>n</i> = 4). * <i>P</i><0.05 compared to control.</p

F-FTS induces glucose uptake <i>in vitro</i> and influences expression of Glut4 mRNA and of IKB/NF-κB protein.

<p><b>A.</b> Insulin-resistant C2C12 myotubes were incubated with or without F-FTS (50 µM), and were then assayed for their ability to absorb fluorescent glucose. Representative histograms of glucose uptake are presented (<i>n</i> = 4) <b>B.</b> Statistical analysis of the results is presented as means ± S.D. * <i>P</i><0.05. <b>C.</b> F-FTS-treated C2C12 cells were tested for Glut4 mRNA and GAPDH mRNA by RT−PCR. Representative gels are shown (<i>n</i> = 4). <b>D.</b> Densitometry of Glut4 is shown. * <i>P</i><0.05 compared to control. <b>E.</b> IKB, NF-κB, p-IKB and tubulin were assayed by western blotting as described in Methods. Representative blots are presented (<i>n</i> = 4) <b>F.</b> Densitometry of IκB, p-IKB and NF-κB expression. * <i>P</i><0.05 compared to control.</p

F-FTS treatment <i>in vivo</i> upregulates glucose uptake by muscle and liver tissues, accompanied by altered IκB/NF-κB expression.

<p><b>A</b>. HF-induced C57/Bl mice treated orally with F-FTS (n = 5) or PBS (control) (n = 5) were injected i.v with 2-NBDG, and glucose uptake in their muscle and liver tissues was tested (<i>n</i> = 5). Representative histograms of glucose uptake are presented for each tissue. <b>B.</b> Statistical analysis of the results is presented as means ± S.D. * <i>P</i><0.05. C. IκB, NF-κB and tubulin in the tissues were assayed by western blotting, as described in Methods. Representative blots are presented (<i>n</i> = 5). D. Densitometry of IκB and NF-κB expression. * <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.005 compared to control.</p

Proposed mechanism explaining the effect of Ras on insulin sensitivity.

<p>Free fatty acids (FFAs) lead to activation of IKK, the inhibitor of IκB kinase. IKK affects insulin sensitivity and glucose uptake via two distinct pathways. First, IKK phosphorylates insulin receptor substrate 1 (IRS-1), resulting in inactivation of insulin signaling through attenuated transcription of glucose transporter 4 (Glut4). Ras inhibition by F-FTS demonstrates enhanced Glut4 transcription, hence also heightened glucose uptake. Second, IKK phosphorylates the inhibitor of κB (IκB), causing it to become detached from nuclear factor κB (NF-κB). NF-κB enters the nucleus and induces transcription of proinflammatory cytokines such as IL-6 and TNF-α. These cytokines leads to deterioration of insulin resistance. Ras inhibition by DN-Ras or by F-FTS augments IκB expression, thereby attenuating the proinflammatory response and enhancing insulin sensitivity and glucose uptake.</p

Inhibition of Ras <i>in vitro</i> by DN-Ras increases glucose uptake and alters IKB/ NF-κB expression.

<p><b>A.</b> Insulin-resistant C2C12 myotubes were transfected with DN-Ras-GFP or GFP plasmid (pGFP) and fluorescent glucose uptake was measured by flow cytometry. Representative histograms of glucose uptake are presented (<i>n</i> = 4) <b>B.</b> Statistical analysis of the results is presented as means ± S.D. * <i>P</i><0.05. <b>C.</b> IκB, NF-κB and tubulin expression in the DN-Ras transfected or GFP-transfected myotubes were assayed by western blotting, as described in Material and Methods. Representative blots are presented (<i>n</i> = 4). <b>D.</b> Densitometry of IκB and NF-κB expression. * <i>P</i><0.05 compared to control.</p