SDS gel electrophoresis was performed using a 4% by 20% gradient gel 8. Quantification of Western blots was performed using Image J Processing and

Transcription

1 Supplemental Material: Western blot: SDS gel electrophoresis was performed using a 4% by 20% gradient gel 8. Quantification of Western blots was performed using Image J Processing and Analysis (NIH). Quantitative RT-PCR Analyses RNA was extracted using RNAeasy kit (Qiagen). For gene expression analysis, real-time RT-PCR reactions were conducted using TaqMan Gene Expression Analysis as described previously 8 (Applied Biosystems) and normalized to GAPDH levels. Nitric oxide measurement NO content was measured in epididymal adipose tissue using Electron Spin Resonance Spectroscopy technique as described previously 8. Mice: Male db/db and age-matched lean controls, db/+m (2 3 mo of age; Harlan) were housed in a temperature-controlled room with a 12:12-h light-dark cycle and maintained with access to food (rodent chow) and water ad libitum. Insulin sensitivity testing: Insulin sensitivity test was performed after an overnight fast. Mice were injected intraperitoneally with human insulin (0.75 unit/kg body weight), after which blood was collected from a tail vein and blood glucose levels were monitored 0, 15, 30, 60, 90 and 120 minutes after injection using glucometer (FreeStyle, 1

2 TheraSense). Plasma Insulin and Blood glucose measurement: Blood samples were placed on ice until separation of plasma by centrifugation. Insulin levels were determined using an ELISA kit (Crystal Chem Inc), and glucose levels were determined by glucometer. Supplementary results: Effect of Sildenafil on enos null mice: To test the hypothesis that enos-derived NO (and cgmp) is required for the protective effect of sildenafil in the adipose tissue, 8 week old enos-/- mice on a low fat diet were administered either vehicle or sildenafil (30 mg/kg/day) for 4 weeks. At the end of 4 weeks, we assessed the metabolic parameters (supplementary figure IVA), and found that there was no effect on body weight and fasting plasma glucose. Further, in the adipose tissue in enos-/- mice that had received sildenafil or vehicle, we found that there was no difference in the sildenafil treated animals relative to controls at the level of NF-!B activation as assessed by phosphorylation of p65 subunit or levels of mac-2 protein. Further, there was no increase in phospho-vasp protein levels. The proinflammatory/macrophage mrna expression (TNF-", MCP-1, IL-6, CCR-2, CD11c and F480) was found to be unchanged between the sildenafil and vehicle treated animals (supplementary figure IVB &C). Taken together, these observations suggest that enos-derived NO-cGMP signaling is crucial for the 2

3 anti-inflammatory effects of sildenafil exerted by phosphorylation of VASP. Effect of sildenafil on db/db mice: We asked if sildenafil is able to protect a model of genetic obesity and insulin resistance such as db/db mice. We treated 12 week old db/db obese mice and db/+m, their lean controls, on chow, with 30 mg/kg/day sildenafil for 4 weeks. At the end of the study period, we assessed the metabolic parameters such as weight and fasting glucose, and found no differences between the vehicle and sildenafil treated mice (supplementary figure VA). We also performed insulin tolerance test and found that there was no effect of sildenafil (data not shown). We next examined the effect on adipose tissue inflammation. Relative to db/+m lean mice, the db/db mice showed extensive inflammation as assessed by mrna expression of inflammatory genes: TNF-", IL-6, MCP-1 and inflammatory myeloid markers such as CD11b, CD11c, CCR2, CD68 and F480, and protein levels of p-p65 subunit of NF-!B and Mac-2. While there was no appreciable effect of the sildenafil on the db/+m mice, there was a significant decrease in mrna expression levels of inflammatory and immune cell markers (Supplementary figure VB), and also phosphorylation of p65 subunit of NF-!B and mac-2 protein levels in db/db mice with sildenafil treatment relative to vehicle treated db/db mice (Supplementary figure VC). Further, we find that the levels of phospho-vasp at ser-239 were significantly elevated in the sildenafil treated db/db mice compared to vehicle treated db/db mice (Supplementary figure VC), suggesting that the anti-inflammatory effects of sildenafil were mediated by 3

4 activation of the NO-cGMP-VASP pathway. Supplementary Figure legends: Supplementary figure I: Densitometric quantitation of enos or VASP from LF and HF diet fed mice: A. enos levels were compared to #-actin, B. VASP levels were compared to #-actin (n=3-4). Supplementary Figure II: Glucose homeostasis in high-fat fed vehicle versus sildenafil treated mice. A: Insulin sensitivity test: B. % drop of the initial glucose. Vehicle treated (black circles) and sildenafil treated (black squares) animals on 14 weeks of high fat diet are shown. IP denotes intraperitoneal injection. *P<0.05 vehicle vs. sildenafil treated high-fat fed mice. (n=4-5 per group). Supplementary figure III. Effect of sildenafil on phosphorylation of VASP at Ser-239 on mice fed LF and HF for 14 weeks: Cell lysates from vehicle or sildenafil treated LF and HF-fed mice were analysed for phosphorylation of VASP by immunoblotting. Densitometric quantitation showing fold difference in phosphorylated VASP relative to GAPDH in EWAT of vehicle and sildenafil treated LF and HF mice. *p<0.05 vehicle LF vs. HF. P<0.05 vehicle vs. sildenafil treated HF-fed mice. Supplementary figure IV: Effect of sildenafil on enos null mice: A. Metabolic 4

5 parameters were assessed. Weight of enos null mice treated with vehicle or sildenafil. (n=3-6 each). Fasting blood glucose levels of enos null mice treated with vehicle and sildenafil. (n=5-6 each). B. mrna expression analysis of the enos null mice administered either vehicle or sildenafil. Genes such as TNF-", MCP-1, IL-6, CCR-2, CD11c and F480 were assessed. (n=5-6 each). C. Protein analysis of the enos deficient mice treated with vehicle or sildenafil. i. phosphorylated-p65 subunit of NF-!B. ii. Mac-2, iii. phosphorylated-vasp (Ser239). Densitometric quantitation of proteins relative to GAPDH are shown (n=5-6 each). Supplementary figure V: Effect of administration of sildenafil or vehicle on db/+m and db/db mice: A. Metabolic parameters were assessed: Weight (n=6, db/db mice, n=3, db/+m) fasting blood glucose (n=6, db/db mice, n=3, db/+m). * p<0.05, significant difference between db/+m and db/db vehicle treated animals. B. mrna expression analysis of the db/+m and db/db animals, such as TNF-", MCP-1, IL-6, CCR-2, CD68, F480, CD11c and CD11b. p<0.05 *, significant difference between db/+m and db/db vehicle treated animals, p<0.05, significant difference between db/db vehicle treated animals and db/db sildenafil treated mice. (n=6 each db/db mice, n=3 each db/+m). C. Protein analysis of the db/db mice treated with vehicle or sildenafil. i. phosphorylated-p65 subunit of NF-!B, ii. Mac-2, iii. phosphorylated-vasp (Ser239). Densitometric quantitation of proteins relative to GAPDH are shown (n=4 each). p<0.05 *, significant difference between db/db mice treated with sildenafil and db/db animals treated with 5

6 vehicle. 6

7 A. B. p= Relative VASP levels LF p=0.75 HF Supplementary figure I: Densitometric quantitation of enos or VASP from LF and HF diet fed mice: A. enos levels were compared to!-actin, B. VASP levels were compared to!-actin. p values are shown.

8 A. B.!"!" "!"!"!" Supplementary Figure II: Glucose homeostasis in high-fat fed vehicle versus sildenafil treated mice. A: Insulin sensitivity test: B. % drop of the initial glucose. Vehicle treated (black circles) and sildenafil treated (black squares) animals on 14 wk of high fat diet are shown. IP denotes intraperitoneal injection. *P<0.05 vehicle vs. sildenafil treated high-fat fed mice. (n=4-5 per group).!"##$%&%'()*+,-."*%,//,

9 #123!4 567,#*8(%9',!" 0, Supplementary figure III. Effect of sildenafil on phosphorylation of VASP at Ser-239 on mice fed LF and HF for 14 wk: Cell lysates from vehicle or sildenafil treated LF and HF-fed mice were analysed for phosphorylation of VASP by immunoblotting. Densitometric quantitation showing fold difference in phosphorylated VASP relative to GAPDH in EWAT of vehicle and sildenafil treated LF and HF mice. *p<0.05 vehicle LF vs. HF. P<0.05 vehicle vs. sildenafil treated HF-fed mice(n=3).!"##$%&%'()*+,-."*%,///,

10 A. B. C. Supplementary figure IV: Effect of sildenafil on enos null mice: A. Metabolic parameters were assessed. Weight of enos null mice treated with vehicle or sildenafil. (n=3-6 each). Fasting blood glucose levels of enos null mice treated with vehicle and sildenafil. (n=5-6 each). B. mrna expression analysis of the enos null mice administered either vehicle or sildenafil. Genes such as TNF-!, MCP-1, IL-6, CCR-2, CD11c and F480 were assessed. (n=5-6 each). C. Protein analysis of the enos deficient mice treated with vehicle or sildenafil. i. phosphorylated-p65 subunit of NF- "B. ii. Mac-2, iii. phosphorylated-vasp (Ser239). Densitometric quantitation of proteins relative to GAPDH are shown (n=5-6 each). Supplementary IV

11 !"#.,., $"# &############# '# &############'# %"#.,.,., Supplementary figure V: Effect of administration of sildenafil or vehicle for 4 weeks on db/+m and db/db mice: A. Metabolic parameters were assessed: Weight (n=6, db/db mice, n=3, db/+m) fasting blood glucose (n=6, db/db mice, n=3, db/+m). * p<0.05, significant difference between db/+m and db/db vehicle treated animals. B. mrna expression analysis of the db/+m and db/db animals, such as TNF-!, MCP-1, IL-6, CCR-2, CD68, F480, CD11c and CD11b. p<0.05 *, significant difference between db/+m and db/db vehicle treated animals, p<0.05, significant difference between db/db vehicle treated animals and db/db sildenafil treated mice. (n=6 each db/db mice, n=3 each db/+m). C. Protein analysis of the db/db mice treated with vehicle or sildenafil. i. phosphorylated-p65 subunit of NF- "B, ii. Mac-2, iii. phosphorylated-vasp (Ser239). Densitometric quantitation of proteins relative to GAPDH are shown (n=4 each). p<0.05 *, significant difference between db/db mice treated with sildenafil and db/db animals treated with vehicle.!"##$%&%'()*+,-,