Additional file 1 of A Descriptive Study of Hot Aches: a Previously Unreported Winter Climbing Phenomenon

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Increasing doses of Allopurinol reduce HIF-1α levels in normoxic HFFs and HUVEC cells.

<p>A. Characterisation of HFF and HUVEC response to hypoxia. Cells were exposed to hypoxia (1% oxygen) for the indicated periods of time. At the end of incubation, protein levels were determined in whole cell extracts by immunoblot analysis using the depicted antibodies. B. Cells were treated with Allopurinol at 10, 100 and 1000 μg/ml for 17 hours. Then the cells were lysed for assessment of the indicated protein levels. Cells were treated with Etoposide (Etop) for 24 hours under normoxia. H-cells exposed to 1% O2 for 16 hours. HIF-1α levels were quantified using ImageJ software and graph depicts mean and standard deviation of a minimum of three independent experiments. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.</p

Allopurinol alters angiogenic traits of HUVEC in an <i>in vitro</i> endothelial tube model.

<p>Cells were treated with Allopurinol at 0, 10, 100, 1000 μg/ml, and analysed at 24 hours. A, control group without Allopurinol. B, 10 μg/ml Allopurinol. C, 100 μg/ml Allopurinol. D, 1000 μg/ml Allopurinol. Graph depicts mean and standard deviation of tube length measures in a minimum of three independent experiments performed in triplicate. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.</p

Allopurinol reduces HIF-1α levels independent of PHD function.

<p>A. Cells were pre-treated with Allopurinol at 10 and 1000 μg/ml for 60 minutes and then were incubated in 1% oxygen or treated with 200 μM DFX for 16 hours. Whole cell lysates were analysed by immunoblot using the indicated antibodies. B. Cells were pre-treated with Allopurinol at 1000 μg/ml for 60 minutes and then were incubated with 20μM MG132 for 3 hours. Whole cell lysates were analysed by immunoblot using the indicated antibodies. HIF-1α levels were quantified using ImageJ software and graph depicts mean and standard deviation of a minimum of three independent experiments. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.</p

Increasing doses of Allopurinol reduce HIF-1α levels in hypoxic HFFs, without changing HIF mRNA levels but have reduced effect in HUVEC cells.

<p>A. Cells were pre-treated with Allopurinol at 10, 100 and 1000 μg/ml for 5, 30 and 60 minutes then were incubated in 1% oxygen for 16 hours. The cells were lysed for assessment of the indicated proteins. HIF-1α levels were quantified using ImageJ software and graph depicts mean and standard deviation of a minimum of three independent experiments. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001. B. HIF-1α and HIF-2α mRNA levels were analysed by quantitative PCR in HFF and HUVEC cells. Graph depicts the mean and standard deviation of a minimum of three independent experiments performed in duplicate. Anova t-test was performed and p values calculated as follows: *p<0.05; **p<0.01; ***p<0.001.</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

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