Differential effects of obesity on visceral versus subcutaneous adipose arteries: role of shear-activated Kir2.1 and alterations to the glycocalyx

Abstract

Obesity imposes well-established deficits to endothelial function. We recently showed that obesity-induced endothelial dysfunction was mediated by disruption of the glycocalyx and a loss of Kir channel flow sensitivity. However, obesity-induced endothelial dysfunction is not observed in all vascular beds: visceral adipose arteries (VAAs), but not subcutaneous adipose arteries (SAAs), exhibit endothelial dysfunction. To determine whether differences in SAA versus VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx, mice were fed a normal rodent diet, or a high-fat Western diet to induce obesity. Flow-induced vasodilation (FIV) was measured ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant-negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAAs of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAAs, but had no effect on VAAs, from obese mice and humans. Obesity abolished Kir shear sensitivity in VAA endothelial cells and significantly altered the VAA glycocalyx. In contrast, Kir shear sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were less pronounced. We reveal distinct differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA versus VAA endothelial function with obesity.NEW & NOTEWORTHY We identified a role for endothelial Kir2.1 in the differences observed in VAA versus SAA endothelial function with obesity. The endothelial glycocalyx, a regulator of Kir activation by shear, is unequally perturbed in VAAs as compared with SAAs, which we propose results in a near complete loss of VAA endothelial Kir shear sensitivity and endothelial dysfunction. We propose that these differences underly the preserved endothelial function of SAA in obese mice and humans.

Keywords: Kir2.1; adipose; endothelial dysfunction; glycocalyx; obesity.

Figure 1.

A mouse model of diet-induced obesity promotes endothelial dysfunction in VAAs but not SAAs. FIV was tested in pressurized and preconstricted SAAs and VAAs from the same mice after 4 wk (A and B; n = 4 mice/group), 8 wk (C and D; n = 7 mice/group), or 24 wk (E and F; n = 4 mice/group) on a normal rodent diet (lean) or a high fat, Western diet (obese). FIV was analyzed using a two-way ANOVA to identify significant effects of obesity on endothelial function (P < 0.05). A Bonferroni post hoc test was used to detect significant differences between diet groups at each intraluminal flow administered. *P < 0.01, significantly different vs. obese. FIV, flow-induced vasodilation; SAAs, subcutaneous adipose arteries; VAAs, visceral adipose arteries.

Figure 2.

Endothelial Kir2.1 contributes to FIV in SAAs, but not VAAs, from obese mice. FIV was tested in pressurized and preconstricted SAAs and VAAs incubated with either an Em-AV (Em) or the endothelium-specific DN-Kir2.1-AV (DN) from the same mice following 8 wk on a normal rodent diet (lean) or a high-fat, Western diet (obese). The effect of expressing DN-Kir2.1 in the endothelium of SAA (A and C) and VAA (B and D) on FIV in arteries isolated from lean or obese mice is shown (n = 5 mice/group for all). A two-way ANOVA was used to compare Em vs. DN vs. DN + Ba²⁺ (Ba; 30 μmol/L) in lean vs. obese mice (P < 0.05). A Bonferroni post hoc test was used to detect significant differences between diet groups at each intraluminal flow administered. *P < 0.01, significantly different vs. arteries expressing DN. A two-way repeated measures ANOVA was used to compare DN vs. DN + Ba²⁺ within diet and artery groups. DN, dominant negative; Em-AV, empty adenovirus; FIV, flow-induced vasodilation; SAAs, subcutaneous adipose arteries; VAAs, visceral adipose arteries.

Figure 3.

Shear sensitivity of Kir2.1 channels is abolished in VAA, but not SAA, endothelial cells of obese mice. Freshly isolated endothelial cells were isolated from SAA and VAA of lean and obese mice and seeded into the parallel plate flow chamber for electrophysiological recordings under fluid shear. Representative baseline Kir currents (I_Kir) recorded in a static bath from endothelial cells isolated from SAA (A) and VAA (B) of lean and obese mice are shown. C: group data (n = 6–10 cells from 3 to 5 mice/group) reveals significant effects of obesity on the baseline I_Kir densities at −100 mV. A Kruskal–Wallis test (P < 0.05) was followed by Dunn–Sidak post hoc analysis to determine significant differences between groups (*P < 0.0125). Representative recordings from endothelial cells isolated from SAAs (D and F) and VAAs (E and G) of lean and obese mice are shown before (static) and during (shear) application of flow to the chamber. D–G, insets: shear-induced increase in I_Kir (I_Sh-St) from the representative recordings. H: Kruskal–Wallis test for multiple comparisons (P < 0.05) was followed by Dunn–Sidak post hoc analysis to identify significant differences in shear-induced I_Kir between groups. *P < 0.0125, significantly different vs. respective lean control; †P < 0.0125, significantly different vs. SAA within diet group; n = 6–10 cells from 3 to 5 mice/group. Kir current (pA) was normalized to cell capacity (pF). Endothelial cell capacity was not different between artery type or diet groups tested by two-way ANOVA. pA/pF , cell capacitance to current densities; SAAs, subcutaneous adipose arteries; VAAs, visceral adipose arteries; Sh-St, static − shear.

Figure 4.

Obesity-induced biophysical alterations are more pronounced in VAA glycocalyx. Adipose arteries were isolated and open en face so that biophysical properties of the glycocalyx could be assessed using atomic force microscopy. A: significant increases in SAA and VAA endothelial glycocalyx elastic modulus (EM) were detected with obesity following an independent samples Mann–Whitney test. *P < 0.05, compared with arteries isolated from the same adipose tissue of lean mice. A paired Wilcoxon rank-sum test was used to compare the glycocalyx EM of adipose arteries isolated from the same lean or obese mice. †P < 0.05, compared with arteries isolated from different adipose depots within diet group. B: significant decreases in SAAs and glycocalyx thickness with obesity were observed following an independent samples Mann–Whitney test. *P < 0.05 compared with arteries isolated from the same adipose tissue of lean mice. A paired Wilcoxon rank-sum test was used to compare the glycocalyx thickness of adipose arteries isolated from the same lean or obese mice. †P < 0.05, compared with arteries isolated from different adipose depots within diet group. For A and B, 15–20 measurements were averaged from each VAA and SAA from the same mice (n = 6–8 arteries from 4 to 5 mice/group) and are shown as connected dot plots. Bar graphs show average and means ± SE for each artery type from lean and obese mice. ns, not significant; SAAs, subcutaneous adipose arteries; VAAs, visceral adipose arteries.

Figure 5.

Endothelial Kir2.1 contributes to SAA, but not VAA, FIV in obese humans. A: FIV was tested in pressurized and preconstricted SAAs and VAAs incubated with either an Em-AV (Em) or the endothelium-specific DN-Kir2.1-AV (DN) from obese human subjects (n = 7). Subcutaneous and visceral (mesenteric) adipose biopsies were collected at the time of planned bariatric surgery and SAA and VAA were isolated and incubated with respective AVs for 48 h before FIV. The effect of expressing DN-Kir2.1 (DN) vs. Em-AV (Em) in SAAs and VAAs from obese human subjects on FIV is shown. A two-way ANOVA was used to detect significant differences in FIV (P < 0.05). A Bonferroni post hoc test was used to detect significant differences between diet groups at each intraluminal flow administered. *P < 0.01, significantly different vs. arteries expressing SAA-DN; †P < 0.01, significantly different vs. VAA-Em. B: individual data points collected from each obese subject and artery tested show the percent dilation at the maximum flow rate of Δ100. Averages are shown as horizontal bars with means ± SE. DN, dominant negative; Em-AV, empty adenovirus; FIV, flow-induced vasodilation; ns, not significant; SAAs, subcutaneous adipose arteries; VAAs, visceral adipose arteries.