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

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

Co-treatment with FTS and GroA increases the percentage of cells in sub-G1 fraction.

<p>PC-3, DU-145, HCT-116 and DLD-1 were treated for 3 days (DU-145) 4 days (PC-3 and HCT-116) or 5 days (DLD-1) with 50 or 75 µM FTS, in the presence or absence of 10 µM Gro. The cells were then harvested and analyzed for their DNA content by flow cytometry. The percentage of live cells at different cell cycle stages is indicated. (A) Representative results of each cell line. The percentage of live cells at different cell cycle stages is indicated. (B) Statistical analysis of three typical results is presented with p-values: * ,p < 0.1 ; ** p < 0.05 ; *** p < 0.005 each treatment compared to the control ; ^ p < 0.1 ; ^ ^, p < 0.05 ; ^ ^ ^, p < 0.005 combination compared to each treatment alone.</p

GroA enhances the inhibitory effect of FTS on anchorage independent growth.

<p>PC-3, HCT-116 and DLD-1 cells were seeded into soft agar and immediately the cells were treated with FTS (25, 50, 75 µM) in the presence or absence of 10 µM GroA. The extent of colony formation was determined 14 days (PC-3 and DLD-1) or 9 days (HCT-116) later. Colonies were then stained with MTT as described in Materials and Methods. <i>Left </i><i>panels</i>: photomicrographs of typical wells. <i>Right panels</i>: number of colonies (>0.01 mm²) is presented for each treatment as the mean ± SD (n=6; * p<0.1, ** p < 0.05 and *** p < 0.005, each treatment compared to the control; ^ p<0.1, ^ ^ p < 0.05 and ^ ^ ^ p < 0.005 combination compared to each treatment alone).</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

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

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

FTS and GroA treatment affect Ras and Nucleolin localization and reduce their levels.

<p>MDCK and DLD-1 cells were seeded on coverslip coated with poly-L-Lysine and treated with 63 µM FTS (DLD-1) or 75 µM FTS (MDCK) in the presence or absence of 10 µM GroA. After 3 days, cells were fixed and examined by confocal microscopy at 63×magnification using Zeiss 510 META confocal microscope. (A) Representative images of MDCK cells following treatments. (B) Representative images of DLD-1 cells following treatments. (C) DLD-1 cells were seeded at a density of 1.5×10⁵ cells per well in six-well plate in RPMI medium supplemented with 5% FBS. A day later, the cells were treated with 63 μM FTS in the presence or in the absence of 10 μM Gro for 3 days. Total cell lysates were analyzed by Western blotting, using anti-nucleolin and anti- pan Ras Abs. As a control, total cell lysates were immunoblotted with anti-β Actin Abs. Note that GRO and FTS treatments reduced the nucleolin and Ras levels respectively in response to each treatment, however the combined treatment of FTS and GroA reduced the nucleolin and Ras levels simultaneously.</p

QVD rescue from GroA and FTS induced cell death.

<p>(A) DLD-1 cells were treated with 63 µM FTS with or without 10 µM GroA in the presence or absence of 20 µM QVD. The cells were then tested for cell viability using the methylene blue staining assay. (B) DLD-1 cells were treated with 63 µM FTS with or without 10 µM GroA. As positive control, the cells were treated with 200 nM STS (staurosphorine). Total cell lysates were analyzed by Western blotting, using anti-Caspase 3 Abs. As a control, total cell lysates were immunoblotted with anti-Actin Abs.</p