17β-Estradiol Treatment Improves Acetylcholine-Induced Relaxation of Mesenteric Arteries in Ovariectomized UC Davis Type 2 Diabetes Mellitus Rats in Prediabetic State

Figures

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  FIGURE 1. Metabolic parameters of ovariectomized (OVX) and E2 replaced (OVX+E2) control and prediabetic (PD) rats. (A) Plasma estradiol (E2) concentration (B) Body weight (C) Blood glucose level (D) HbA1c (E) Plasma insulin level (F) Abdominal adipose tissue percentage to total body weight (G) Circulating triglyceride level. Values are represented as mean ± SEM. Each bar represents the values obtained from n = 5–6 animals per group. Capped lines indicate significant differences between two groups (* p < 0.05, *** p < 0.001, **** p = 0.0001), analyzed using one-way ANOVA followed by Tukey’s post hoc test.

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  FIGURE 2. Western blot analysis of (A) IRS1 (B) GLUT-4 (C) AMPK-α (D) p-AMPK-α expression in skeletal muscle of ovariectomized (OVX) and E2 replaced (OVX+E2) control and prediabetic (PD) rats. Protein level was quantified by densitometry and normalized to corresponding GAPDH. Values are represented as mean ± SEM. Each bar represents the values obtained from n = 4–5 animals per group. Representative bands of target and housekeeping protein (GAPDH) were shown from the same membranes. Capped lines indicate significant differences between two groups (* p < 0.05, ** p < 0.01, *** p < 0.001), analyzed using one-way ANOVA followed by Tukey’s post hoc test. IRS1, insulin receptor substrate 1; GLUT-4, glucose transporter-4; AMPK-α, AMP-activated protein kinase-α; p-AMPK-α, phosphorylated-AMP-activated protein kinase-α.

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  FIGURE 3. Concentration-response curve (CRC) to acetylcholine (ACh, 10−8 to 10−5 M) in intact mesenteric arterial rings precontracted with phenylephrine (PE, 2 μM) from ovariectomized (OVX) and E2 replaced (OVX+E2) control and prediabetic (PD) rats. Data are expressed as mean ± SEM. n = 5–6 animals per group. Brackets indicate significant differences between groups (p < 0.05), analyzed using 2-way ANOVA with repeated measures followed by Tukey’s post hoc test.

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  FIGURE 4. Effects of inhibiting cyclooxygenase (COX), nitric oxide synthase (NOS), small conductance and intermediated conductance calcium activated potassium channels (SKca and IKca) on acetylcholine (ACh)-induced vasorelaxation in mesenteric arteries taken from ovariectomized (OVX) and E2 replaced (OVX+E2) control and prediabetic (PD) rats; (A) OVX (B) OVX+E2 (C) PD OVX (D) PD OVX+E2. Concentration response curve (CRC) to ACh (10−8 to 10−5) was generated in the absence (No drug) or in the presence of indomethacin (Indo), Indo+L-NAME (I+L), and Indo+L-NAME+Apamin+TRAM-34 (I+L+A+T) in mesenteric arterial rings. Indo (indomethacin; 10μM), L-NAME (Nω-nitro-L-arginine methyl ester; 200 μM), Apamin (1 μM), TRAM-34; [1-{(2-Chlorophenyl) diphenylmethyl}-1H-pyrazole; 1 μM]. Data are expressed as mean ± SEM. n = 5–6 animals per group. * (P < 0.05) vs. no drug; # (P < 0.05) vs. Indo; $ (P < 0.05) vs. Indo+L-NAME, analyzed using 2-way ANOVA repeated measures followed by Bonferroni’s post hoc test. NO, nitric oxide; EDH, endothelium-dependent hyperpolarization.

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  FIGURE 5. Concentration-response curve (CRC) to sodium nitroprusside (SNP, 10−9 to 10−5 M) in intact mesenteric arterial rings precontracted with phenylephrine (PE, 2 μM) from ovariectomized (OVX) and E2 replaced (OVX+E2) control and prediabetic (PD) rats. Data are expressed as mean α SEM. n = 4–5 per group. Brackets indicate significant differences (p < 0.05) between groups, analyzed using 2-way ANOVA with repeated measures followed by Tukey’s post hoc test.

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  FIGURE 6. Concentration-response curve (CRC) to phenylephrine (PE, 10−8 to 10−5 M) in intact mesenteric arterial rings of ovariectomized (OVX) and E2 replaced (OVX+E2) control and prediabetic (PD) rats. Data are expressed as mean ± SEM. n = 4–5 per group. Brackets indicate significant differences between groups (p < 0.05), analyzed using two-way ANOVA with repeated measures followed by Tukey’s post hoc test.

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  FIGURE 7. Western blot analysis of (A) eNOS and (B) NOX-1 expression in mesenteric arteries of ovariectomized (OVX) and E2 replaced (OVX+E2) control and prediabetic (PD) rats. Protein levels were quantified by densitometry and normalized to corresponding GAPDH. Values are represented as mean ± SEM. Each bar represents the values obtained from n = 3–4 animals per group. Representative bands of target and housekeeping protein (GAPDH) were shown from the same membranes. Capped lines indicate significant differences between two groups (** p < 0.01, **** p < 0.0001), analyzed using one-way ANOVA followed by Tukey’s post hoc test. eNOS, endothelial nitric oxide synthase; NOX-1, NADPH oxidase-1.