Mouse C2C12 myoblasts (generously provided by Prof. David Yaffe) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 µg/ml streptomycin. When cells reached confluence, the medium was replaced by differentiation medium containing DMEM and 2% horse serum, which was changed every other day. After 4 more days the differentiated C2C12 cells had fused into myotubes. To induce insulin resistance in the differentiated skeletal muscle cells, the medium was replaced by lipid-containing medium. The latter was prepared by addition of FFA (palmitate dissolved in 0.1 M NaOH) to DMEM containing 2% fatty acid-free BSA. Myotubes were incubated for 16 h in the above medium in the presence or absence of 0.75 mM palmitate. To exclude the possibility that any FFA can induce insulin resistance, 0.75 mM oleic acid was also added to myotubes and served as negative control (data not shown).

Following induction of insulin resistance, all culture medium was removed from each well and replaced with 1 ml of fresh culture medium in the absence or presence of 10 µM fluorescent 2-NBDG (Molecular Probes-Invitrogen, CA/Molecular Probes, Eugene, OR), a new fluorescent derivative of glucose with a 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] amino group at the C-2 position ^[33]. For this purpose, the cells were incubated at 37°C with 5% CO₂ for 1 h. The cells were then washed twice with cold phosphate-buffered saline (PBS) and collected for flow cytometric measurement.

To block Ras we transfected differentiated C2C12 cells with 2 7g of green fluorescent protein plasmid (pGFP) or dominant-negative (DN) GFP-Ras (17N), using lipofectamine 2000 reagent according to manufacture's instructions (Invitrogen, Carlsbad, CA, USA). At 48 h post-transfection the cells were subjected to induction of insulin resistance, as described above. The cells were then either harvested and analyzed by Western blotting or tested for glucose uptake using the 2-NBDG method described above.

To examine the impact of Ras inhibition on IκB and NF-κB, we performed Western immunoblotting with specific antibodies. Muscle and fat lysates were obtained from HF-induced mice, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotted as previously described ^[30] with one of the following antibodies: anti-IκB, anti-p-IκB, anti-NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-tubulin (eBioScience, San Diego, CA).

The levels of Ras GTP were determined as described previously ^[30]. Protein bands were visualized with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL) and quantified by densitometry with Image EZQuant-Gel software©.

RNA was extracted from 10⁶ C2C12 muscle cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Reverse transcription (RT)–PCR was performed according to the protocol of the Reverse-iT™ 1^st Strand Synthesis Kit (ABgene, Epsom, UK). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed using the following primers: GAPDH forward [gene: 5′-ACCACAGTCCATGCCATCAC-3′] and GAPDH reverse [gene: 5′-TCCACCACCCTGTTGCTGTA-3′].

PCR was carried out with ReddyMix™ PCR Master Mix (ABgene) on a Programmable Thermal Controller (MJ Research, Waltham, MA) at gene-specific conditions. Primer sequences for Glut4 were: Glut4 forward: [gene: 5′-GATGCCGTCGGGTTTCCAGCA-3′] and Glut4 reverse: [gene: 5′-TGAGGGTGCCTTGTGGGATGG -3′].

The PCR products were subjected to electrophoresis in 2% agarose gel stained with ethidium bromide.

The study was approved by the Institutional Ethics Committee at the Tel Aviv Sourasky Medical Center, Tel Aviv, Israel (Approval ID is L-09-006).

C57Bl/6 mice (n = 8) received hydrodynamic DNA injections, in which 100 µg of pDN-Ras-GFP in 2 ml of PBS was injected intravenously (i.v.) and rapidly (within 5–8 s) under high-pressure into the tail vein. After 48 h the mice were injected i.v with 500 µg of 2-NBDG. Two hours later, muscle, liver and fat tissues were removed and single-cell suspensions from each of those tissues were tested by fluorescence activated cell sorting (FACS) for the presence of 2-NBDG. Mice in a control group (n = 8) were similarly injected and treated, except that they were injected with pGFP instead of pDN-Ras-GFP.

We produced a model for type 2 diabetes by feeding a high-fat diet (TD.06415, Harlan Laboratories, Madison, WI, USA) to male C57Bl/6 mice, starting when they were 6 weeks old as detailed earlier ^[34]. These high fat-induced diabetic mice were treated orally either with 30 mg/kg of F-FTS (n = 5) or, as a control, with carboxymethyl cellulose (CMC) vehicle (n = 5). Thirteen weeks later, the mice were injected i.v. with 500 µg of 2-NBDG. Two hours after injection, muscle and liver tissues were removed and analyzed for glucose uptake as described above.

To study the effect of F-FTS on type 2 diabetes in high fat diabetic mice, we started to treat them, at the same time as the high-fat diet was initiated, with five different daily treatments, as follows: F-FTS (20 mg/kg body weight), injected intraperitoneally (i.p.; n = 30); F-FTS (30 mg/kg body weight), per os (p.o.; n = 10); FTS (60 mg/kg body weight), p.o. (n = 10); CMC (control), p.o. (n = 10); or PBS (control), i.p. (n = 30). Mice were considered diabetic when two consecutive blood tests, collected from the mice's orbital sinus, yielded glucose concentrations greater than 200 mg/dl glucose. This value was obtained by all mice after 13 weeks on the diet. Prior to euthanasia of mice, fasting serum insulin levels were determined by ELISA according to the manufacture's instructions.

To test the hypothesis that Ras inhibition results in increased uptake of glucose in muscle cells, we induced C2C12 cell differentiation into myotubes (see Methods) and transfected the differentiated muscle cells with DN-GFP-labeled Ras or a GFP-labeled control plasmid. We then induced insulin resistance in all cells by addition of palmitate (see Methods) and assayed their ability to absorb fluorescent glucose. In line with previous reports ^[35], palmitate reduced glucose uptake compared to BSA-treated control cells (not shown). The results clearly demonstrate a significant increase of 160%±10% in glucose uptake in DN-Ras transfected cells treated with palmitate as compared to palmitate-treated GFP-transfected cells (Fig. 1A and B). These results suggest that active Ras inhibition may upregulate glucose absorption.

The nuclear transcription factor NF-κB and its inhibitor IκB have been shown to participate in the induction of insulin resistance by palmitate. To study the effect of Ras inhibition on NF-κB and IκB in the insulin-resistant C2C12 cells, we assayed IκB and NF-κB in these cells following DN-Ras transfection. Compared to the GFP-transfected controls, the DN-Ras transfected cells exhibited a significant increase in their IκB expression (186%±14% increase) as opposed to a decrease in NF-κB expression (45%±10% decrease) relative to GFP-transfected cells (Fig. 1C and D).

To verify the effect of Ras inhibition on glucose uptake, we used F-FTS, a small molecule that interferes with anchorage of Ras to the membrane and hence inhibits Ras function ^[29], ^[31].

First, we examined whether F-FTS mimics the observed effect of DN-Ras on glucose uptake in C2C12 myotubes. Our results showed that F-FTS, like DN-Ras, induced a significant increase in glucose uptake compared to control (151%±7%, Fig. 2A and B). In line with this effect of F-FTS on glucose uptake was the finding that expression of the mRNA glucose transporter 4 (glut-4) was induced by F-FTS. Glut-4 expression in C2C12 myotubes that were rendered insulin-resistant by palmitate and then treated with F-FTS was significantly higher (146%±8%) than in C2C12 myotubes treated with palmitate alone (Fig. 2C and D).

In addition, the level of the proinflammatory transcription factor NF-κB in the insulin-resistant C2C12 cells was significantly lower (by 56%±10%) in the presence of F-FTS than in its absence (Fig. 2E and F). On the other hand, the expression of its inhibitor, IκB, was significantly higher both in its total level and in its phosphorylated form (p- IκB) by 160%±14% and 369%±35%, respectively in the presence of F-FTS compared to control (Fig. 2E and F). These findings are consistent with the results obtained by treatment with DN-Ras.

To determine whether the enhanced glucose uptake resulting from treatment with DN-Ras in vitro (Figs. 1 and 2) is also observed in a short-term in vivo model, we examined the effects of Ras inhibition on the uptake of fluorescent glucose by muscle, fat and liver tissues in mice. Hydrodynamic injection of DN-Ras into the tail veins of wild-type C57Bl/6 mice resulted in a significant increase in fluorescent glucose uptake in these tissues (an increase of 214%±10%, 150%±8%, and 157%±16% relative to the GFP-treated controls, respectively) (Fig. 3A, B).

Testing of the above tissues for Ras-GTP expression revealed a significant decrease, concomitantly with the effects on glucose uptake (Fig.3C).

To verify the above in vivo findings in a relevant type 2 diabetes model, we treated 6-week-old C57Bl/6 mice fed on a high fat diet with F-FTS or, as a control, with PBS for 13 weeks, as described above, and then examined the ability of their muscle, fat and liver tissues to absorb intravenously injected fluorescent glucose. Cells obtained from the muscle and liver tissues of F-FTS-treated mice exhibited a significant increase in fluorescent glucose uptake compared to control (178±18% and 153±7%, respectively; Fig. 4A, B). No significant differences in glucose uptake were observed in the fat tissues (data not shown).

Immunoblot assays for IκB and NF-κB expression showed increased IκB expression compared to controls in muscle (by 158±10%) and fat (by 181±13%) tissues obtained from F-FTS treated mice, as opposed to a significant decrease compared to controls in NF-κB expression by 30±10% and by 38±8%, respectively (Figure 4C, D). No differences were found in the liver tissue (data not shown). Taken together, these results showed that Ras inhibition caused an increase in IκB/NF-κB-dependent glucose uptake in vivo.

Having shown that Ras inhibition by F-FTS mimics the effect of DN-Ras on glucose uptake both in vitro and in vivo, we next examined the effect of long-term treatment with F-FTS or FTS on the development of hyperglycemia in an experimental model of type 2 diabetes.

To examine how the effectiveness of the drugs was influenced by the route of administration, we treated the mice either intraperitoneally (i.p.) or per os (p.o.). While being fed from the age of 6 weeks with a high-fat diet to induce diabetes, C57Bl/6 mice were also treated i.p., daily for 13 weeks, with either 20 mg/kg of F-FTS (n = 30) or with PBS (n = 30). F-FTS treatment resulted in a significant decrease in the incidence of diabetes, with 20% of the F-FTS treated mice developing diabetes as compared to 60% in the PBS-treated group (Fig. 5A). Similar results were observed when mice were treated p.o with either 30 mg/kg F-FTS or 60 mg/kg FTS compared to CMC treatment: diabetes developed in 18% of the F-FTS-treated and in 30% of the FTS-treated mice, whereas in the CMC-treated mice 72% developed diabetes (Fig. 5C). Animals were considered diabetic when blood glucose levels exceeded 200 mg/dl (Fig 5B, D).

As expected, the high-fat diet caused a significant increase in body weight in all treated groups ^[36]. No significant differences in weight gain were observed between the i.p. F-FTS-treated and PBS-treated groups or between the p.o FTS-treated, F-FTS-treated and CMC-treated groups (Fig. 5E).

We also assayed circulating insulin in the different groups of mice. After i.p. treatment, the levels of circulating insulin were significantly decreased in the F-FTS-treated group (4.27±1.4 ng/ml relative to 14±4.9 ng/ml in the PBS-treated group). In the orally treated mice, insulin concentrations in the F-FTS-treated and the FTS-treated groups were also significantly reduced (7.9±1.3 ng/ml and 6.8±1.1 ng/ml, respectively, relative to 14.6±2 ng/ml in the CMC-treated group; Fig. F,G).