Endothelial dysfunction due to selective insulin resistance in vascular endothelium: insights from mechanistic modeling

Abstract

Previously, we have used mathematical modeling to gain mechanistic insights into insulin-stimulated glucose uptake. Phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling required for metabolic actions of insulin also regulates endothelium-dependent production of the vasodilator nitric oxide (NO). Vasodilation increases blood flow that augments direct metabolic actions of insulin in skeletal muscle. This is counterbalanced by mitogen-activated protein kinase (MAPK)-dependent insulin signaling in endothelium that promotes secretion of the vasoconstrictor endothelin-1 (ET-1). In the present study, we extended our model of metabolic insulin signaling into a dynamic model of insulin signaling in vascular endothelium that explicitly represents opposing PI3K/NO and MAPK/ET-1 pathways. Novel NO and ET-1 subsystems were developed using published and new experimental data to generate model structures/parameters. The signal-response relationships of our model with respect to insulin-stimulated NO production, ET-1 secretion, and resultant vascular tone, agree with published experimental data, independent of those used for model development. Simulations of pathological stimuli directly impairing only insulin-stimulated PI3K/Akt activity predict altered dynamics of NO and ET-1 consistent with endothelial dysfunction in insulin-resistant states. Indeed, modeling pathway-selective impairment of PI3K/Akt pathways consistent with insulin resistance caused by glucotoxicity, lipotoxicity, or inflammation predict diminished NO production and increased ET-1 secretion characteristic of diabetes and endothelial dysfunction. We conclude that our mathematical model of insulin signaling in vascular endothelium supports the hypothesis that pathway-selective insulin resistance accounts, in part, for relationships between insulin resistance and endothelial dysfunction. This may be relevant for developing novel approaches for the treatment of diabetes and its cardiovascular complications.

Keywords: endothelin-1; endothelium; insulin signaling; mathematical modeling; nitric oxide.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Fig. 1.

Schematic of insulin signaling pathways stimulating nitric oxide (NO) production in endothelial cells. A: postreceptor insulin receptor substrate-phosphatidylinositol 3-kinase (IRS-PI3K)-Akt signaling subsystem. B: endothelial nitric oxide synthase (eNOS)-NO subsystem.

Fig. 2.

Schematic of insulin signaling pathways stimulating endothelin-1 (ET-1) production in endothelial cells. A: postreceptor MAPK signaling subsystem. B: ET-1 subsystem.

Fig. 3.

Complete model of insulin signaling pathways stimulating nitric oxide (NO) and endothelin-1 (ET-1) production in endothelial cells with feedback and cross-talk. Insulin binding to its receptor results in receptor autophosphorylation and the phosphorylation of insulin receptor substrate (IRS-1) by the insulin receptor tyrosine kinase. Phosphorylated IRS-1 activates phosphatidylinositol 3-kinase (PI3K), which increases membrane-bound PI(3,4,5)P3 (PIP3). PIP3 recruits and activates 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which phosphorylates and activates Akt and atypical PKCs. Activated Akt phosphorylates endothelial nitric oxide synthase (eNOS) to increase nitric oxide (NO), a vasodilator. Tyrosine phosphorylation of the insulin receptor (IR) also increases the association of Shc and Grb2 to the IR, leading to the activation of the Ras‐Raf‐MEK‐MAPK1/2 cascade. MAPK increases the production of ET-1, a vasoconstrictor. Positive and negative feedback pathways are indicated by dotted lines. Cross talk between the PI3K and MAPK branches is indicated (-˙˙-˙˙-). Atypical PKC serine phosphorylates IRS-1 to create a negative feedback pathway, and Akt phosphorylates PTP1B to create a positive feedback pathway. Akt directly phosphorylates and attenuates Raf activity to create crosstalk between the PI3K-Akt-NO and MAPK-ET-1 pathways. Under healthy conditions, insulin stimulated vasodilation counteracts the vasoconstrictive effects of insulin-stimulated ET-1.

Fig. 4.

Comparison between model simulations and experimental data from vascular endothelium regarding the dynamics of insulin-stimulated phosphorylation of Akt and eNOS. A: model simulations for intracellular levels of phosphorylated Akt (p-Akt), phosphorylated eNOS (p-eNOS), and nitric oxide (NO) production after a step input of insulin (100 nM, 15 min) plotted as a function of time. B: insulin concentration-response curves generated by model simulations of peak p-Akt were compared with experimental data for p-Akt in response to insulin stimulation as determined by quantification of immunoblotting studies taken from the published literature (43). The line represents the best fit of the simulated data points. C: insulin concentration-response curves generated by model simulations of peak p-eNOS were compared with experimental data for p-eNOS (43). The line represents the best fit of the simulated data points.

Fig. 5.

Model simulations of intracellular levels of phosphorylated Shc (p-Shc), GTP-bound Ras (Ras-GTP), phosphorylated MAPK (p-MAPK), and ET-1. A: model simulations for intracellular levels of p-Shc, Ras-GTP, and p-MAPK after a step input of insulin (100 nM, 15 min) are plotted as a function of time. B: insulin concentration-response curves generated by model simulations of peak p-Shc, Ras-GTP, and p-MAPK. The lines represent the best fit of the simulated data points. C: model simulations of the time course for ET-1 synthesis/secretion after a step input of insulin (100 nM, 15 min). D: insulin concentration-response generated by model simulations of peak ET-1 synthesis/secretion. Experimental data for ET-1 secreted into conditioned media from bovine aortic endothelial cells (n = 3) in primary culture were obtained, as described in methods.

Fig. 6.

Comparisons between model simulations of insulin-induced changes in vascular tone and experimental data in blood vessels. A: model simulations of vascular tone as a function of time after a step input of insulin (100 nM, 15 min). B: insulin concentration-response generated by model simulations of peak change in vascular tone. Experimental data representing changes in vessel diameter in response to insulin were taken from published studies (12).

Fig. 7.

Model simulations of insulin-concentration response curves under conditions of different phosphatase activities for phosphatase and tensin homolog (PTEN), protein tyrosine phosphatase 1B (PTP1B), and Src homology 2 (SH2)-domain-containing inositol phosphatase 2 (SHIP2A) with the output of peak NO (A), peak ET-1 (B), and peak vascular tone (C).

Fig. 8.

Effects of modulating phosphatase and tensin homolog (PTEN) activity to alter insulin-stimulated vascular tone, and production of nitric oxide (NO) and endothelin (ET-1). A: model simulations of insulin-stimulated changes in vascular tone after a step input of insulin (100 nM, 15 min) with baseline PTEN levels (solid line), a twofold increase and a twofold decrease in PTEN levels (dashed line; [PTEN] = 2 and 0.5, respectively). B and C: bovine aortic endothelial cells (BAEC) in primary culture were transiently cotransfected with an expression vector for red fluorescent protein (RFP) and pCIS2 (empty vector), PTEN-WT, or PTEN-C124S, as described in methods. Cells were then serum-starved overnight, loaded with 4,5-diaminofluorescein diacetate (DAF2-DA), and then stimulated with lysophosphatidic acid (LPA, 5 μM) or insulin (100 nM, 5 min). Production of NO in the cotransfected cells (determined by expression of RFP) was detected using an epifluorescent microscope and quantified using a digital camera, as described in methods. Data are expressed as means ± SE of 3 or 4 independent experiments and are expressed as the percentage of peak NO production in the control group (cells cotransfected with RFP/pCIS2 empty vector and treated with insulin). Overexpression of PTEN-WT attenuated the insulin response while expression of PTEN-C124S augmented this (P < 0.05). Bars labeled with different letters (a, b, c) are significantly different from each other, P < 0.05 (by ANOVA and Bonferroni’s post hoc test).

Fig. 9.

Model simulations of the effect of wortmannin (PI3K inhibitor) treatment on insulin actions. Akt activation rate was restricted to 1/100th of its normal model value (k11) to mimic the behavior of wortmannin. A: effect on peak NO production of insulin+wortmannin compared with insulin alone. B: effect on peak ET-1 production of insulin+wortmannin compared with insulin alone. C: experimental results showing ET-1 concentration in conditioned media after treating endothelial cells with insulin in the absence or presence of wortmannin (n = 4), as described in methods.

Fig. 10.

Effects of glucotoxicity, lipotoxicity, and proinflammatory cytokines on insulin-stimulated phosphorylated endothelial nitric oxide synthase (p-eNOS), p-MAPK, nitric oxide (NO), and endothelin-1 (ET-1) production, and vascular tone: comparisons between experimental data and model predictions. A: high glucose-induced (20 mM, 72 h) impairment of insulin-stimulated p-Akt levels determined from published experiments (20) was used to constrain p-Akt levels in our mathematical model. Then, peak levels of p-eNOS, p-MAPK, and NO production in response to insulin (50 nM, 15 min) were generated from model simulations. These model predictions were plotted next to the published experimental results for insulin-stimulated p-eNOS, p-MAPK, and eNOS activity (NO formation) in endothelial cells exposed to high glucose (20 mM, 72 h) (20). Data are expressed as percent change relative to basal conditions. B: palmitic acid-induced impairment in insulin-stimulated Akt activity derived from published experimental results (90) was used to constrain p-Akt levels in our mathematical model. Then, model-generated levels of insulin-stimulated p-eNOS were compared with corresponding insulin-stimulated p-eNOS levels in endothelial cells exposed to varying concentrations of palmitic acid (0–0.8 mM) from the published literature (3, 90). The solid line represents the linear-least squares regression for the data points shown. C: model-predictions of insulin-stimulated NO and ET-1 levels in cells exposed to varying concentrations of palmitic acid. The solid and dashed lines represent the linear-least squares regression for the data points for NO and ET-1, respectively. D: TNF-α-induced impairment in insulin-stimulated Akt activity in vascular endothelium derived from published experimental results (19) was used to constrain p-Akt levels in our mathematical model. Model-generated changes in insulin-stimulated vascular tone (□) are plotted on the same graph as experimentally determined changes in insulin-stimulated vascular diameter in arteries treated with TNF-α (■) (19). Data are expressed as a percentage of peak response of vascular tone.