Previous results showed that the activation of PPARβ/δ reduces vascular
contraction both in aorta and mesenteric arteries (Figure 3.2, Figure 3.3, Figure 3.5, Figure 3.6). Next interest was understanding the molecular mechanism underlying this regulation and the differences, if any, between Naïve vs diabetic tissue as well as aorta vs mesenteric arteries. The regulation of the PI3K/Akt/eNOS pathway and the effects of high glucose concentrations were studied first. With that aim, aorta and mesenteric artery rings from Naïve and STZ-diabetic rats were incubated with different combinations of Insulin, LY29002 (PI3K inhibitor), and GW0742 at normal and high glucose concentrations.
Chapter 3: Non-genomic effects of PPARβ/δ
3.3.4.1 Aorta
Insulin significantly decreases the PE-induced contraction from 1 μM PE on Naïve
aorta rings (Figure 3.10 A, D), while GW0742 does not have a significant effect (Figure 3.10 B, E); however, GW0742 reverses the effect of the Insulin when they are incubated together (Figure 3.10 C, D). Surprisingly, the PI3K inhibitor LY294002 enhances the effect of Insulin and GW0742 blocking the contraction of the artery (Figure 3.10), which is the opposite of the expected response where LY294002 inhibits dilation via PI3K/Akt/eNOS.
Although the acute exposure to high glucose did not have an effect on aorta contraction per se (Figure 3.8) it enhaces the effect of the treatments in Naïve aorta rings (Figure 3.10).
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Figure 3.10 PI3K/Akt/eNOS pathway in Naïve rat aorta. Naïve abdominal aorta rings were
exposed to one of the following treatments: 0.01% DMSO (Vehicle); 1 U/mL Insulin; 1 μM LY29002; 1 U/mL Insulin + 1 μM LY29002, 100 nM GW0742; 100 nM GW0742 + 1 μM LY29002; 1 U/mL Insulin + 100 nM GW0742; 1 U/mL Insulin + 100 nM GW0742 + 1 μM LY29002. Same treatments were performed in normal glucose (NG) concentration of 5 mM and high glucose (HG) concentration of 20 mM. After 30 min incubation a PE dose-response curve was performed. Data are represented as mean ±SEM (n=5-13). Significant difference compared to vehicle controls was analysed by two-way ANOVA followed by Bonferroni’s post hoc test denoted by ***=p<0.001, **=p<0.01, *=p<0.05, ns=non-significance.
Chapter 3: Non-genomic effects of PPARβ/δ
Interestingly, insulin does not have an effect on PE-induced contraction of STZ- diabetic aortic rings (Figure 3.11 A, D), however, it is significantly reduced by GW0742 (Figure 3.11 B-E). Similar to Naïve aorta rings, the co-incubation of insulin and GW0742 reverses any loss of contraction produced by GW0742 (Figure 3.11 C, F). LY294002 reverses the effect of the GW072 to levels similar to control, as predicted (Figure 3.11 B, E), and alike in Naïve tissues, high glucose intensifies the effect of GW0742 (Figure 3.11. B, D, E, F).
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Figure 3.11 PI3K/Akt/eNOS pathway in STZ-diabetic rat aorta. STZ-diabetic abdominal aorta
rings were exposed to one of the following treatments: 0.01% DMSO (Vehicle); 1 U/mL Insulin; 1 μM LY29002; 1 U/mL Insulin + 1 μM LY29002, 100 nM GW0742; 100 nM GW0742 + 1 μM LY29002; 1 U/mL Insulin + 100 nM GW0742; 1 U/mL Insulin + 100 nM GW0742 + 1 μM LY29002. Same treatments were performed in normal glucose (NG) concentration of 5 mM and high glucose (HG) concentration of 20 mM. After 30 min incubation a PE dose-response curve was performed. Data are represented as mean ±SEM (n=4-9). Significant difference compared to vehicle controls was analysed by two-way ANOVA followed by Bonferroni’s post hoc test denoted by ***=p<0.001, **=p<0.01, *=p<0.05, ns=non-significance. Significant difference GW0742 vs GW0742 + LY294002 and Insulin + GW0742 vs Insulin + GW0742 + LY294002 was analysed by Bonferroni’s post hoc test denoted by ff=p<0.01, f=p<0.05.
Chapter 3: Non-genomic effects of PPARβ/δ
Table 3.11 Emax and EC50 of abdominal aorta from Naïve and STZ-diabetic rats incubated with
1 U/mL Insulin; 1 μM LY29002; 1 U/mL Insulin + 1 μM LY29002, 100 nM GW0742; 100 nM GW0742 + 1 μM LY29002; 1 U/mL Insulin + 100 nM GW0742; 1 U/mL Insulin + 100 nM GW0742 + 1 μM LY29002 in conditions of NG (5 mM) or HG (20 mM) and contracted with PE. Emax is represented as mean ± SEM (n=4-13). Significant difference compared to Vehicle controls was analysed by two-way ANOVA and followed by Bonferroni’s post hoc test denoted by * = p<0.05, **=P<0.01, ***= p<0.001.
Abdominal aorta
NG HG
Treatment Emax (mN) EC50 (nM) Emax (mN) EC50 (nM)
Naïve Vehicle 14.26 ± 1.0 102.2 15.44 ± 1.0 110.1 Insulin 11.05 ± 0.3* 163.7 10.51 ± 1.0** 214.5 Insulin + LY294002 7.85 ± 0.5*** 1023.0 10.27 ± 1.9** 945.1 GW0742 12.55 ± 1.3 109.9 12.62 ± 1.1 100.8 GW0742 + LY294002 10.93 ± 0.7 300.9 12.66 ± 1.0 208.3 Insulin + GW0742 14.05 ± 0.7 200.0 11.51 ± 1.4* 146.3 Insulin + GW0742 + LY294002 10.50 ± 0.9* 686.7 11.50 ± 2.2* 825.6 STZ Vehicle 16.16 ± 1.2 94.4 19.15 ± 1.5 212.1 Insulin 14.16 ± 1.7 134.5 16.33 ± 2.9 306.0 Insulin + LY294002 15.89 ± 3.3 144.2 20.07 ± 3.6 937.8 GW0742 10.15 ± 1.5*** 83.0 12.02 ± 1.4*** 250.1 GW0742 + LY294002 13.84 ± 1.4 50.0 16.89 ± 1.8 115.0 Insulin + GW0742 14.66 ± 1.3 235.9 14.02 ± 1.2* 247.8 Insulin + GW0742 + LY294002 16.12 ± 3.1 74.4 21.54 ± 3.4 486.0
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Insulin and GW0742 significantly reduce PE-induced contraction on Naïve mesenteric artery, which is partially blocked in the presence of LY294002 (Figure 3.12 A, B, D, E). Surprisingly, the incubation of insulin and GW0742 together inhibit each other’s effect resulting in contractions similar to that of the Vehicle (Figure
3.12 C, F). Strangely, the presence of LY294002 together with insulin and GW0742 leads to a loss of contraction compared to Vehicle (Figure 3.12 C, F).
Insulin but not GW0742 significantly reduces PE-induced contraction on STZ- diabetic mesenteric artery, which again, is partially blocked by the presence of LY294002 at low glucose (Figure 3.13 A, B, D, E). Similar to Naïve mesenteric arteries, the presence of insulin and GW0742 together inhibit any effect of the insulin and leads to a contraction similar to Vehicle (Figure 3.13 C, F).
High glucose does not affect the contraction of the mesenteric arteries from Naïve or STZ-diabetic tissues compared to normal glucose incubations, as shown in Figure 3.12 and Figure 3.13.
Chapter 3: Non-genomic effects of PPARβ/δ
Figure 3.12 PI3K/Akt/eNOS pathway in Naïve rat mesenteric arteries. Naïve mesenteric artery
rings were exposed to one of the following treatments: 0.01% DMSO (Vehicle); 1 U/mL Insulin; 1 μM LY29002; 1 U/mL Insulin + 1 μM LY29002, 100 nM GW0742; 100 nM GW0742 + 1 μM LY29002; 1 U/mL Insulin + 100 nM GW0742; 1 U/mL Insulin + 100 nM GW0742 + 1 μM LY29002. Same treatments were performed in normal glucose (NG) concentration of 5 mM and high glucose (HG) concentration of 20 mM. After 30 min incubation a PE dose-response curve was performed. Data are represented as mean ±SEM (n=4-13). Significant difference compared to vehicle controls was analysed by two-way ANOVA followed by Bonferroni’s post hoc test denoted by ***=p<0.001, **=p<0.01, *=p<0.05, ns=non-significance. Significant difference Insulin + GW0742 vs Insulin + GW0742 + LY294002 was analysed by Bonferroni’s post hoc test denoted by fff=p<0.001, ff=p<0.01, f=p<0.05.
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Figure 3.13 PI3K/Akt/eNOS pathway in STZ-diabetic rat mesenteric arteries. STZ-diabetic
mesenteric artery rings were exposed to one of the following treatments: 0.01% DMSO (Vehicle); 1 U/mL Insulin; 1 μM LY29002; 1 U/mL Insulin + 1 μM LY29002, 100 nM GW0742; 100 nM GW0742 + 1μM LY29002; 1 U/mL Insulin + 100 nM GW0742; 1 U/mL Insulin + 100 nM GW0742 + 1 μM LY29002. Same treatments were performed in normal glucose (NG) concentration of 5 mM and high glucose (HG) concentration of 20 mM. After 30 min incubation a PE dose-response curve was performed. Data are represented as mean ±SEM (n=4-9). Significant difference compared to vehicle controls was analysed by two-way ANOVA followed by Bonferroni’s post hoc test denoted by ***=p<0.001, **=p<0.01, *=p<0.05, ns=non-significance.
Chapter 3: Non-genomic effects of PPARβ/δ
Table 3.12Emax and EC50 of mesenteric arteries from Naïve and STZ-diabetic rats incubated
with 1 U/mL Insulin; 1 μM LY29002; 1 U/mL Insulin + 1 μM LY29002, 100 nM GW0742; 100 nM GW0742 + 1 μM LY29002; 1 U/mL Insulin + 100 nM GW0742; 1 U/mL Insulin + 100 nM GW0742 + 1 μM LY29002 in conditions of NG (5 mM) or HG (20 mM) and contracted with PE. Emax is represented as mean ± SEM (n=4-13). Significant difference compared to Vehicle controls was analysed by two-way ANOVA and followed by Bonferroni’s post hoc test denoted by * = p<0.05, **=P<0.01, ***= p<0.001.
Mesenteric arteries
NG HG
Treatment Emax (mN) EC50 (μM) Emax (mN) EC50 (μM)
Naïve Vehicle 14.14 ±1.1 6.1 13.73 ± 1.1 6.6 Insulin 10.23 ± 1.8** 8.2 9.34 ± 1.2*** 8.4 Insulin + LY294002 10.73 ± 1.6 16.6 10.35 ± 1.3* 8.1 GW0742 11.26 ± 1.0* 7.2 11.12 ± 0.8* 7.1 GW0742 + LY294002 11.96 ± 1.2 8.5 12.23 ± 1.8 8.5 Insulin + GW0742 16.84 ± 0.9 7.4 12.83 ± 1.0 8.8 Insulin + GW0742 + LY294002 10.31 ± 1.5** 21.5 10.07 ± 1.5** 19.6 STZ Vehicle 17.18 ± 1.5 1.8 18.17 ± 1.6 1.6 Insulin 9.87 ± 1.1*** 5.7 12.64 ± 1.7* 6.8 Insulin + LY294002 12.51 ± 1.7* 4.3 13.26 ± 1.6* 7.0 GW0742 15.01 ± 1.4 3.2 15.53 ±1.4 1.9 GW0742 + LY294002 15.46 ± 1.4 2.8 15.45 ± 1.3 3.2 Insulin + GW0742 15.97 ± 2.0 3.9 16.86 ± 2.2 3.7 Insulin + GW0742 + LY294002 17.05 ± 2.2 4.8 16.02 ± 1.7 7.2
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