Insulin signaling network. Examined phosphorylation sites in signaling intermediaries are indicated (-P). A, schematic of insulin signaling pathways examined. Blue arrows indicate signaling pathways; green arrow indicates positive feedback signal; gray arrow indicates GLUT4 translocation to the plasma membrane in response to insulin signaling. B, structure of the mechanistic mathematical model of the insulin-signaling network. Model equations are detailed in supplemental text, Section 2–3. Rate constants (at reaction arrows) and measured phosphorylation sites are indicated. The three diabetes parameters introduced in the diabetes state of the model are indicated in red. a, active state; m, plasma membrane-localized state; i, internalized state. AS160, Akt substrate of 160 kDa.

Experimental analysis of insulin signaling normally and in T2D. Freshly isolated mature human adipocytes from non-diabetic (blue) or T2D subjects (red) were incubated with insulin as indicated, and the extent of protein phosphorylation or glucose uptake was determined. a1-a4 show dose-responses and b1-b4 show the corresponding time courses for the indicated insulin signaling intermediaries. a5, rate of glucose uptake with indicated concentration of insulin. b5, rate of glucose uptake with or without insulin (10 nm), as indicated. c1, amount of internalized IR (% of total IR) at the indicated time after addition of 100 nm insulin. Data from (). d1, time course for phosphorylation of IRS1 at tyrosine, with 1.2 nm insulin added at t = 0, and additional insulin to 10 nm added at t = 4 min. Data are from Ref. . c2-c3 and d2-d5, dose-responses and time courses for insulin signaling intermediaries as indicated. a1-a4 and c2-c3, cells incubated with the indicated concentration of insulin for 10 min. b1-b2, cells incubated with 10 nm insulin for the indicated time. Data on normal cells were from Ref. . b3-b4 and d2-d5, cells incubated with 10 nm insulin for the indicated time. Details about patients, and number of patients, donating adipocytes in the different experiments are detailed in supplemental Table S1. Time course experiments show the extent of protein phosphorylation expressed in arbitrary units (a.u.) that in each experiment are the same for non-diabetic and diabetic conditions and thus directly comparable. To compare dose-response/insulin sensitivity/EC₅₀, the extent of protein phosphorylation or rate of glucose uptake is normalized to between 0 and 100% phosphorylation or glucose uptake rate, respectively. Time courses were determined for 60 min, but for clarity only the first 30 min are shown. Blue (non-diabetic) and red (diabetic) dots denote experimental data ± S.E. Lines denote fitting dose-response curves to sigmoidal curves (GraphPad Prism) or for time courses connection of data points.

Comparison of sensitivity to insulin at the different signaling steps, normally and in adipocytes from subjects with T2D. Dose-response effects of the indicated concentrations of insulin on the indicated signaling intermediaries and glucose transport using data from . A, adipocytes from non-diabetic subjects display a shift in insulin sensitivity from IR to IRS1/PKB and from IRS1/PKB to AS160/glucose uptake. B, adipocytes from subjects with T2D display a reduced sensitivity to insulin, most marked for IRS1 and AS160/glucose uptake, but the increased insulin sensitivity from IRS1 to AS160/glucose uptake remains in subjects with T2D. The insulin sensitivity at IR and at PKB is not altered in subjects with T2D. C, a profile likelihood analysis () estimation of mean and variance for the EC₅₀ variables of non-diabetic (blue circles) and diabetic (red circles) subjects shows that EC₅₀ of IRS1-YP and glucose uptake are separated between non-diabetic and diabetic subjects, whereas EC₅₀ of IR-YP and PKB-T308P are clearly overlapping and EC₅₀ of AS160-T642P slightly overlapping. Also, EC₅₀ of IRS1-YP for non-diabetics is lower and separated from EC₅₀ of IR-YP, whereas for diabetics IRS1-YP is higher and also separated. We performed the profile likelihood analysis using a Hill equation to allow for variable slope and maximum values of the parameters of a sigmoid dose-response curve.

Qualitative model analysis of the initial phase of insulin signaling. A, experimental observations of steady-state phosphorylation of IR (10 nm insulin for the indicated time) and insulin sensitivity for phosphorylation of IRS1 at tyrosine (indicated concentration of insulin for 10 min), normally (blue) and in T2D (red). B, model simulations of time course for phosphorylation of IR, or dose-response curves for phosphorylation of IRS1 at tyrosine in response to increasing concentrations of insulin, with reduced levels of IR or IRS1, or increased feedback to IR. The effects of the indicated perturbations were examined through core prediction analysis () using a model for the initial phase of insulin signaling (). C, model simulations of dose-response curves for phosphorylation of IRS1 at tyrosine in response to increasing concentrations of insulin, with decreased positive or increased negative feedback to IRS1. The effects of the indicated perturbations were examined through qualitative analysis of the minimal model in the figure (supplemental text, Section 1).

Detailed dynamic model of insulin signaling normally and in T2D. Comparison of model simulation (lines) and experimental data (dots) for insulin signaling, normally (blue) and in T2D (red). Model simulations were performed to mimic the experimental conditions, and the same simulations were performed for the normal and the T2D state. a1-a4 and c2-c3, dose-response was simulated using increasing values of the insulin input (0–100 nm as indicated) and the response after 10 min was plotted. The simulated values were normalized between 0 and 100%. a5, dose-response of glucose uptake was simulated using increasing values of the insulin input (0–100 nm as indicated) during 15 min followed by 30 min with a glucose (0.05 mm) input, without removing the insulin input. The simulated values were normalized between 0 and 100%. b5, absolute glucose uptake was simulated using 0 and 100 nm insulin as input for 15 min followed by 30 min with a glucose (0.05 mm) input, without removing the insulin input. We used a normalization constant to fit the normal glucose uptake to data, and the same constant for the T2D glucose uptake. b1-b4, c4-c5, and d2-d5, time course was simulated using 10 nm insulin during 30 min. We used a normalization constant to fit the normal time courses to data, and the same constant for the T2D time course. c1, time course was simulated using 10 nm insulin during 30 min. d1, double-step stimulation was simulated using 1.2 nm insulin as input for 4 min followed by 10 nm insulin for 4 min. We used a normalization constant to fit the normal time course to data, and the same constant for the T2D time course. Experimental details are described as in the legend to .

Model analysis of the individual contribution of the three diabetes parameters to insulin resistance. The simulated effect of reducing the concentration of IR (A) or GLUT4 (B), or reducing the feedback from mTORC1 to IRS1 (C) in the non-diabetic state of the detailed dynamic model (red dotted lines, red open bars) is compared with simulation of normal state (blue lines, blue filled bars) and simulation of diabetic state (red lines, red filled bars). A comprehensive analysis is shown in supplemental Fig. S2.

Model analysis and experimental validation with inhibition of mTORC1. Model simulations of the effect of rapamycin inhibition of mTORC1 (cyan) before stimulation with insulin (left panels) and experimental validation (right panels). The different lines in the model simulations represent the effect of 50, 75, 83, 88, 90, 92, and 93% inhibition of mTORC1. Simulations of the normal state (blue) are the same as in . A comprehensive model analysis with inhibition of mTORC1 is shown in supplemental Fig. S3. A, left panel, model simulation for phosphorylation of S6 at Ser-235/236 with (cyan) or without (blue) rapamycin inhibition of mTORC1. Right panel, adipocytes from non-diabetic subjects were preincubated with (cyan) (n = 3) or without (blue) (n = 6) 50 nm rapamycin for 30 min and then stimulated with 10 nm insulin for the indicated time, when cells were analyzed for the extent of phosphorylation of S6 at Ser-235/236 by SDS-PAGE and immunoblotting (mean ± S.E.) B, left panel, model simulation for phosphorylation of IR at tyrosine with (cyan) or without (blue) rapamycin inhibition of mTORC1. Right panel, adipocytes from non-diabetic subjects were preincubated with (cyan) or without (blue) 10 nm rapamycin for 15 min and then with 1000 microunits/ml of insulin for 15 min, when phosphorylation of IR at Tyr-1146 was analyzed by SDS-PAGE and immunoblotting. Data are from Ref. . C, left panel, model simulation for phosphorylation of IRS1 at tyrosine with (cyan) or without (blue) rapamycin inhibition of mTORC1. Right panel, adipocytes from non-diabetic subjects (n = 5) were preincubated with (cyan) or without (blue) 50 nm rapamycin for 30 min and then with the indicated concentration of insulin for 10 min, when phosphorylation of IRS1 at tyrosine was analyzed by SDS-PAGE and immunoblotting (mean ± S.E.). Data are from Ref. . D, left panel, model simulation for phosphorylation of IRS1 at Ser-307 with (cyan) or without (blue) rapamycin inhibition of mTORC1. Right panel, adipocytes from non-diabetic subjects (n = 5) were preincubated with (cyan) or without (blue) 50 nm rapamycin for 30 min and then with 100 nm insulin for 10 min, when phosphorylation of IRS1 at Ser-307 was analyzed by SDS-PAGE and immunoblotting (mean ± S.E.). Data are from Ref. . E, left panel, model simulation for phosphorylation of AS160 at Thr-642 with (cyan) or without (blue) rapamycin inhibition of mTORC1. Right panel, adipocytes from non-diabetic subjects were preincubated with (cyan) or without (blue) 10 nm rapamycin for 15 min and then with 1000 microunits/ml of insulin for 15 min, when phosphorylation of AS160 at Thr-642 was analyzed by SDS-PAGE and immunoblotting. Data are from Ref. . F, left panel, model simulation for insulin stimulation of glucose uptake with rapamycin inhibition of mTORC1. Right panel, human subcutaneous adipocytes from non-diabetic subjects (n = 23) were preincubated with 10 nm rapamycin for 15 min and then with 1000 microunits/ml of insulin for 15 min, when [¹⁴C]glucose uptake was determined for 45 min. Data are from Ref. . G, left panel, model simulation for phosphorylation of PKB at Ser-473 with (cyan) or without (blue) rapamycin inhibition of mTORC1. Right panel, adipocytes from non-diabetic subjects were preincubated with (cyan) (n = 3) or without (blue) (n = 8) 50 nm rapamycin for 30 min and then stimulated with 10 nm insulin for 10 min, when cells were analyzed for the extent of phosphorylation of PKB at Ser-473 by SDS-PAGE and immunoblotting (mean ± S.E.).

Effects on glucose uptake at the cellular and whole body levels when treating T2D by increasing the feedback from mTORC1 to IRS1. A and B, model simulations with the detailed mechanistic model of insulin signaling for the diabetic state, with a lowered rate of dephosphorylation of IRS1 at Ser-307 (v2d in B). A, glucose uptake by adipocytes in response to the indicated concentrations of insulin (red dashed lines) shows normalized insulin sensitivity. B, basal and maximal insulin-stimulated uptake of glucose (red open bars) are only slightly affected by the perturbation. Continuous blue/red lines and filled blue/red bars indicate model simulations of normal/diabetic adipocytes. C and D, model simulations, with the comprehensive combined whole body model, of glucose uptake by the adipose tissue in response to a meal. C, model simulations for normal subjects (blue line) compared with diabetic subjects (red line). Also indicated are calculated data from the whole body level models for normal non-diabetic (blue dots) and diabetic subjects (red dots). D, model simulations for diabetic subjects with a 10 times increased feedback from mTORC1 to IRS1 (red dashed line) compared with normal (blue line) and diabetic (red line) subjects.

Attenuated mTORC1 signaling in adipocytes from T2D patients with or without metformin treatment. Adipocytes were obtained from non-diabetic or T2D subjects (as indicated). Patients with T2D had not received any medication for T2D or had received metformin (as indicated) as part of their treatment prior to surgery and tissue biopsy. The adipocytes were incubated with insulin at 10 nm until steady-state phosphorylation (40 min for S6 and 10 min for IRS1), when cells were analyzed by SDS-PAGE and immunoblotting for the extent of phosphorylation of S6 at Ser-235/236 (A) and phosphorylation of IRS1 at Ser-307 (B). Results are presented as mean ± S.E. for triplicate cell determinations of 7 subjects (non-diabetic), 3 subjects (T2D with metformin treatment prior to surgery), and 3 subjects (T2D without metformin treatment).