iNOS gene expression in cardiac tissue of experimental animals.

<p>Cardiac tissue homogenates were subjected to RT-PCR. Results were normalized to ribosomal gene Rn18S as housekeeping gene. Abbreviations are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. Mean ± SEM of 4–6 mice for each group. *p<0.05, at least, <i>vs</i> WT.</p

Body weight and basal blood glucose levels during the 16 weeks of controlled diet in all groups of animals.

<p>WT: wild type controls HFD: high fat diet.</p><p>*p<0.05, at least, vs WT;</p>§<p>p<0.05, at least, vs eNOS^+/−.</p><p>Just before starting diet regimens (at 4 weeks of age), body weights of animals’ groups were: WT, 22.1±0.9 g; eNOS^+/−, 21.7±1.2 g; eNOS^−/−, 22.0±1.3 g; blood glucose levels were: WT, 142±4 mg/dL; eNOS^+/−, 138±9 mg/dL; eNOS^−/−, 140±8 mg/dL.</p

Coronary resistance (CR) in Langendorff configuration in basal conditions (a); b) Percent change in CR during 3 minutes of acetylcholine (Ach) infusion with respect to the corresponding basal value; c) percent change in CR during 3 minutes of sodium nitroprusside (SNP) infusion.

<p>Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. Mean ± SEM of at least 6 mice for each group. *p<0.05, at least, <i>vs</i> WT; §p<0.05, at least <i>vs</i> eNOS^+/−.</p

Percent increase (16 versus 4 weeks) of body weight (a) and blood glucose levels (b) during the 16 weeks of monitoring in all groups of animals, indicated as follows: WT, healthy controls fed with standard diet; eNOS^+/−, partial knockout eNOS mice fed with standard diet; eNOS^−/−, total knockout eNOS mice fed with standard diet; WT HFD, healthy controls fed with high-fat diet (HFD); eNOS^+/− HFD, partial knockout eNOS mice fed with HFD.

<p>Mean ± SEM of 15–18 mice for each group, except eNOS^−/− mice (n = 10). *p<0.05, at least, <i>vs</i> WT; §p<0.05, at least, <i>vs</i> eNOS^+/−.</p

eNOS expression in cardiac tissue of experimental animals.

<p>Cardiac tissue homogenates were subjected to Western blotting for eNOS, peNOS and α-tubulin protein determination. Representative immunoblots (inferior left panel) are shown for all groups of mice. Abbreviations are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. Mean ± SEM of 4–6 mice for each group. *p<0.05, at least, <i>vs</i> WT; §p<0.05, at least, <i>vs</i> eNOS^+/−.</p

Blood glucose and plasma insulin levels during glucose tolerance test.

<p>Abbreviations are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. a) Blood glucose levels were determined during a glucose tolerance test (glucose 1.5 g/kg i.p.) performed after 15 weeks of HFD diet. b) values of blood glucose at baseline and 15 minutes after the glucose load. c) the area under the curve (AUC) for glycaemic values. d) Plasma insulin levels were determined during a glucose tolerance test (glucose 1.5 g/kg i.p.) performed after 15 weeks of HFD diet. e) Plasma insulin levels at baseline and 15 minutes after the glucose load (insulin peak). f) % increase of 15-min post-loading plasma insulin level with respect to the corresponding basal value. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. In the panels a) and d) empty circles are WT mice, empty triangles are eNOS^+/− mice, empty squares are eNOS^−/− mice, filled circles are WT HFD mice, and filled triangles are eNOS^+/− HFD. Mean ± SEM of 15–18 mice for each group, except for eNOS^−/− mice (n = 4). *p<0.05, at least, vs WT; §p<0.05, at least, vs eNOS^+/−.</p

Expression of ERK1-2 and Akt in cardiac tissue of experimental animals.

<p>Cardiac tissue homogenates were subjected to Western blotting for Akt, pAkt, ERK1-2, and pERK1-2 protein determination. Representative immunoblots (inferior left panel) are shown for all groups of mice. Abbreviations are defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. Histograms showing the double pERK/p-Akt ratio have been reported. Mean ± SEM of 4–6 mice for each group. *p<0.05, at least, <i>vs</i> WT; §p<0.05, at least <i>vs</i> eNOS^+/−.</p

Blood glucose levels during insulin tolerance test.

<p>Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. a) Blood glucose levels were determined during an insulin tolerance test (insulin 0.75 U/kg i.p.) performed after 16 weeks of diet. b) percent decrements of blood glucose values with respect to basal at 15 minutes after the insulin administration. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104156#pone-0104156-g001" target="_blank">Fig. 1</a>. Empty circles are WT mice, empty triangles are eNOS^+/− mice, empty squares are eNOS^−/− mice, filled circles are WT HFD mice, and filled triangles are eNOS^+/− HFD Mean ± SEM of 15–18 mice for each group, except for eNOS^−/− mice (n = 4). *p<0.05, at least, <i>vs</i> WT; §p<0.05, at least, <i>vs</i> eNOS^+/−.</p

Diagrammatic representation of proposed mechanisms of interaction between eNOS deficiency, metabolic stress and insulin-dependent signaling pathway in coronary endothelial cells.

<p>Diagrammatic representation of proposed mechanisms of interaction between eNOS deficiency, metabolic stress and insulin-dependent signaling pathway in coronary endothelial cells.</p