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. 2017 Nov 27;128(1):309–322. doi: 10.1172/JCI89333

Figure 5. IgG from HFD-fed mice attenuates insulin-induced eNOS activation and transendothelial transport of insulin via FcγRIIB and eNOS antagonism, and IgG from T2DM subjects blunts insulin action in human endothelium via FcγRIIB.

Figure 5

(A) BAECs were preincubated for 30 minutes with IgG isolated from control diet–fed (Con-IgG) or HFD-fed (HFD-IgG, 10 μg/ml) mice, and eNOS activity stimulated by insulin (100 nM) was measured. n = 6. (B and C) BAECs were transfected with or without control RNAi or RNAi targeting FcγRIIB. Downregulation of FcγRIIB was assessed by immunoblotting (B) using anti-FcγRIIB or anti-eNOS antibody to evaluate protein loading. (C) Forty-eight hours after transfection, cells were pretreated with CRP (25 μg/ml), Con-IgG, or HFD-IgG (10 μg/ml) for thirty minutes, and eNOS activity stimulated by insulin was measured. n = 16. (D) HAECs were preincubated with Con-IgG or HFD-IgG (10 μg/ml), and eNOS activity under basal conditions or with insulin treatment was measured. n = 3. (E) Confluent HAEC monolayers on Transwells were preincubated for 30 minutes with CRP, Con-IgG, or HFD-IgG in the absence or presence of the NOS inhibitor L-NAME (2 mM) or the NO donor SNAP (100 nM). FITC-conjugated insulin (50 nM) was added to the upper chamber, and the amount of insulin transcytosed to the lower chamber was evaluated after 2 hours. n = 7. (F) Using the study design and methods described for E, endothelial cell insulin transcytosis was evaluated without versus with CRP, Con-IgG, or HFD-IgG treatment, in the presence of subtype-matched control antibody (C, 10 μg/ml) or the FcγRIIB-blocking antibody AT10 (BL, 10 μg/ml). n = 6. (G) Insulin-induced eNOS activation was evaluated in HAECs with or without treatment with IgG from nondiabetic individuals (Con) or T2DM patients. n = 6. (H) Additional NOS activity assays were performed in the presence of the subtype-matched control antibody (C) or the FcγRIIB-blocking antibody (BL). n = 3. Values represent the mean ± SEM. *P < 0.05, ***P < 0.005, and ****P < 0.001, by 1-way ANOVA with Tukey’s post-hoc test.