Insulin Receptor Substrate 2 Controls Insulin-Mediated Vasoreactivity and Perivascular Adipose Tissue Function in Muscle

Figure 1. Vasoregulatory responses in resistance arteries of IRS2−/− mice without PVAT. In all experiments on tissues of IRS2−/− mice, their IRS2+/+ littermates were the control strain. Acetylcholine, ACh (A); sodium nitroprusside, SNP (B) and ET-1 (C) were similar in IRS2+/+ (°, n = 5) and IRS2−/− resistance arteries (•, n = 6).

Figure 2. IRS2 mediates insulin-stimulated ERK1/2-ET-1 activity in muscle resistance arteries without PVAT. Responses of IRS2+/+ and IRS2−/− RA to insulin (A) and during endothelin receptor blockade using PD142893 (10ulMol/l) (B), n = 5, *p < 0.01 vs. IRS2+/+. (C,D): IRS2 deletion decreases insulin-stimulated phosphorylation of ERK1/2, but not of Akt in muscle RA. ERK1/2 and Akt phosphorylation were compared between insulin stimulation (2 nM) (black bars) and the control situation (white bars) in segments of the gracilis RA from IRS2+/+ (n = 8) and IRS2−/− (n = 9) mice, *p < 0.05; #p < 0.01 vs. control.

Figure 3. IRS2−/− PVAT does not uncover insulin-mediated vasodilation. (A) Arteries obtained from C57/Bl6 mice were incubated with PVAT from IRS2+/+ and IRS2−/− littermates and stimulated with increasing doses of insulin. Incubation with IRS2+/+ PVAT, allowed the artery to dilate in reaction insulin in dose-dependent manner (17.2 ± 4.9% dilation in response to highest (insulin), p < 0.0001, N = 9,) while arteries that were incubated with IRS2−/− PVAT or were deprived of PVAT did not react to insulin (−7.3 ± 4.7%, N = 10 and 0.4 ± 4.1%, N = 9, respectively; p = 0.17). Data are presented as mean ± SEM, tested with two-way ANOVA with Tukey post-hoc correction. (B) At the end of the experiment, endothelial integrity was examined by stimulating the arteries with acetylcholine. All the arteries that were included in the analyses in (A) had at least 60% dilation in response to acetylcholine—cutoff point to rule out endothelialdamage. (C) PVAT did not affect the percentage of arteriolar preconstriction in the organ bath. (D) Adipocyte size in IRS2+/+ PVAT (412.1 ± 58.7 μm) are smaller (p = 0.03) than adipocytes in IRS2−/− PVAT (914.7 ± 63. 2 μm). Data are presented as mean ± SEM, tested with…

Figure 4. IRS2−/−PVAT does not show increased inflammatory gene expression. (A,B) IRS expression in IRS2−/− mice. (C,D) IRS2 deficiency does not alter adiponectin and leptin expression in PVAT. (E,F) IRS2 deficiency does not enhance inflammatory gene expression in PVAT. Data were log-transformed prior to statistical analysis as described in section Methods. APN, Adiponectin.

Figure 5. IRS2−/− PVAT secretes equal amounts of Adiponectin compared to wild-type PVAT. There are no significant differences between IRS2+/+ and IRS2−/− PVAT in (A) adiponectin, (B) IL6, (D) Leptin, (E) PAI-1. (C) Resistin secretion is decreasedin IRS2−/− PVAT. Insulin stimulation did not affect the secretion of the measured adipokines. The amounts of MCP-1 and TNF-α were below the detection limitof the assay.

Figure 6. Normal insulin-stimulated phosphorylation of Akt in arteries incubated with IRS2−/− PVAT. Insulin stimulation for 15 min resulted in strong Akt phosphorylation with no differences between incubation with IRS2−/−or wildtype PVAT.

Figure 7. Dual role of IRS2 in regulation of insulin-induced vasoreactivity in muscle. (A) In health, insulin induces vasodilation by activating its receptor, resulting in phosphorylation and activation of insulin receptor substrates (IRSs) 1 and 2.both IRS isoforms are involved inAkt and endothelial Nitric Oxide Synthase (eNOS) activation, while IRS2 mediates insulin-induced activation of ERK1/2. Perivascular adipose tissue (PVAT) secretes vasodilator adipokines such adiponectin that reduce endothelin (ET-1) secretion (de Boer et al., 2016) and enhance NO production in endothelial cells through AMPK (Eringa et al., 2010). (B) IRS2-deficient PVAT fails to uncover insulin-mediated vasodilation in muscle arteries. Probable mechanisms aredirect mediators that are released from PVAT that interact with eNOS to reduce NO production, such as free fatty acids (FFA) (Previs et al., 2000).