Systemic Administration of Insulin Receptor Antagonist Results in Endothelial and Perivascular Adipose Tissue Dysfunction in Mice

Abstract

Hyperglycemia linked to diabetes results in endothelial dysfunction. In the present work, we comprehensively characterized effects of short-term hyperglycemia induced by administration of an insulin receptor antagonist, the S961 peptide, on endothelium and perivascular adipose tissue (PVAT) in mice. Endothelial function of the thoracic and abdominal aorta in 12-week-old male C57Bl/6Jrj mice treated for two weeks with S961 infusion via osmotic pumps was assessed in vivo using magnetic resonance imaging and ex vivo by detection of nitric oxide (NO) production using electron paramagnetic resonance spectroscopy. Additional methods were used to analyze PVAT, aortic segments and endothelial-specific plasma biomarkers. Systemic disruption of insulin signaling resulted in severe impairment of NO-dependent endothelial function and a loss of vasoprotective function of PVAT affecting the thoracic as well as abdominal parts of the aorta, however a fall in adiponectin expression and decreased uncoupling protein 1-positive area were more pronounced in the thoracic aorta. Results suggest that dysfunctional PVAT contributes to vascular pathology induced by altered insulin signaling in diabetes, in the absence of fat overload and obesity.

Keywords: endothelial function; insulin receptor antagonist; magnetic resonance imaging; perivascular adipose tissue.

Figure 1

Effects of S961 treatment on vascular function of the thoracic and abdominal aorta. (A): Assessment of endothelium-dependent vascular response to acetylcholine (Ach) 30 min after administration in the thoracic aorta (TA, VEH n = 8, S961 n = 7) and abdominal aorta (AA, VEH n = 8, S961 n = 7) (B): Assessment of endothelium-independent vasodilation induced by sodium nitroprusside (SNP) 30 min after iv administration in thoracic aorta (TA, VEH n = 8, S961 n = 8) and abdominal aorta (AA, VEH n = 8, S961 n = 7). (C): Nitric oxide (NO) production in isolated TA (VEH n = 5, S961 n = 7) and AA (VEH n = 8, S961 n = 11), without PVAT. (A,B): significance assessed by Two-way ANOVA (aorta part and treatment being the two factors), normality confirmed by Shapiro-Wilk test, variance by F-test, with post hoc Sidak test. (C): significance assessed by two-sided t-test with Welch’s correction (normality was assessed using the Shapiro–Wilk test). * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 2

Effects of S961 treatment on plasma concentration of nitrite, nitrate and nitrosylhemoglobin content. Plasma concentration of (A): nitrite (VEH n = 8, S961 n = 8), (B): nitrate (VEH n = 7, S961 n = 8), (C): nitrosylhemoglobin (Hb^IINO) content in red blood cells (RBC) (VEH n = 8, S961 n = 9). Statistical significance tested using two-sided Student’s t-test with Welch’s correction, normality confirmed by Shapiro–Wilk test, variance by F-test. Results shown as mean +/− SE. * p < 0.05, ** p < 0.01.

Figure 3

Effects of S961 treatment on selected plasma biomarkers of endothelial dysfunction. Biomarkers of glycocalyx disruption: syndecan-1 (SDC-1), endocan (ESM-1); endothelial permeability: soluble Tie-2 (sTie-2), angiopoietin 1 and 2 (Angpt-1/2), soluble fms-like tyrosine kinase (sFLT-1); endothelial inflammation: soluble form of E-selectin (sE-sel), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1); hemostasis and others: tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) and adiponectin (ADN). VEH n = 6–8, S961 n = 8–11. Statistical significance tested using two-sided Student’s t-test with Welch’s correction, normality confirmed by Shapiro–Wilk test, variance by F-test. Results shown as mean +/− SE. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 4

Effects of S961 treatment on fat depots amount and perivascular adipose tissue (PVAT). (A) images of the mesenteric arteries from vehicle (VEH)- and S961-treated mice, (B) Raman spectroscopy analysis of the intensity of the 1750 nm⁻¹ band, representative of triacylglycerol content in epididymal white adipose (eWAT) tissue (VEH n = 5, S961 n = 6), (C) analysis of PVAT amounts in the thoracic (TA, VEH n = 7, S961 n = 8) and abdominal (AA, VEH n = 7, S961 n = 8) region, expressed as the total area uncoupling protein-1 (UCP1) and perilipin-1 positive staining normalized to vessel lumen, (D) immunohistochemical analysis of the ratio of UCP1 expression to UCP1 + perilipin-1 expression in PVAT of TA (VEH n = 7, S961 n = 11) and AA (VEH n = 8, S961 n = 8), representing the ratio of PVAT showing BAT-like characteristics, (E) decreased adiponectin expression in the TA (VEH n = 7, S961 n = 11) but not in AA (VEH n = 8, S961 n = 8). Statistical significance tested using two-sided Student’s t-test with Welch’s correction, normality confirmed by Shapiro–Wilk test, variance by F-test. not statistically significant (ns), * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001.

Figure 5

Effects of S961 treatment on adipose tissue chemical characteristics by Raman spectroscopy. Analysis (A) and averaged Raman spectra (B,C) of the lipid unsaturation degree (I₁₆₆₀/I₁₄₄₄) in TA and AA PVAT (in C57Bl/6 mice given vehicle (green; n = 5) or S961 (red; n = 5) via osmotic pumps for 2 weeks. Statistical significance tested using two-sided Student’s t-test with Welch’s correction, normality confirmed by Shapiro–Wilk test, variance by F-test: *** p < 0.005.

Figure 6

Effects of S961 treatment on eNOS activity and ROS production in PVAT and adiponectin expression. (A) Reduction eNOS activity in both TA (VEH n = 8, S961 n = 11) and AA (VEH n = 8, S961 n = 9) parts and (B) increased reactive oxygen species activity in PVAT expressed as spin probe oxidation in both TA (VEH n = 7, S961 n = 8) and AA (VEH n = 7, S961 n = 7). Statistical significance tested using two-sided Student’s t-test with Welch’s correction, normality confirmed by Shapiro–Wilk test, variance by F-test: not statistically significant (ns), * p < 0.05, ** p < 0.005, *** p < 0.001.