Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor - PubMed (original) (raw)

Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor

Eva Gonzalez et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Insulin resistance is a component of the metabolic syndrome and Type 2 diabetes. It has been recently shown that in liver insulin resistance is not complete. This so-called selective insulin resistance is characterized by defective insulin inhibition of hepatic glucose output while insulin-induced lipogenesis is maintained. How this occurs and whether uncoupled insulin action develops in other tissues is unknown. Here we show in a model of chronic hyperinsulinemia that adipocytes develop selective insulin resistance in which translocation of the GLUT4 glucose transporter to the cell surface is blunted yet nuclear exclusion of the FoxO1 transcription factor is preserved, rendering uncoupled insulin-controlled carbohydrate and lipid metabolisms. We found that in adipocytes FoxO1 nuclear exclusion has a lower half-maximal insulin dose than GLUT4 translocation, and it is because of this inherent greater sensitivity that control of FoxO1 by physiological insulin concentrations is maintained in adipocytes with compromised insulin signaling. Pharmacological and genetic interventions revealed that insulin regulates GLUT4 and FoxO1 through the PI3-kinase isoform p110α, although FoxO1 showed higher sensitivity to p110α activity than GLUT4. Transient down-regulation and overexpression of Akt isoforms in adipocytes demonstrated that insulin-activated PI3-kinase signals to GLUT4 primarily through Akt2 kinase, whereas Akt1 and Akt2 signal to FoxO1. We propose that the lower threshold of insulin activity for FoxO1's nuclear exclusion is in part due to its regulation by both Akt isoforms. Identification of uncoupled insulin action in adipocytes suggests this condition might be a general phenomenon of insulin target tissues contributing to insulin resistance's pathophysiology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Hyperinsulinemia leads to selective insulin resistance in adipocytes. (A) Immunoblot analyses and densitometry of Akt phosphorylation from adipocytes unstimulated or stimulated with 1 nM insulin for 30 min. Each point is the mean ± SEM, n = 3. CHI, chronic hyperinsulinemia (10 nM insulin for 16 h). (B) Surface-to-total distribution of HA-GLUT4-GFP in unstimulated and 1 nM insulin-stimulated adipocytes. After exposure to 10 nM insulin for 16 h, cells were washed, serum-starved for 8 h, and 1 nM insulin-stimulated GLUT4 translocation assayed. Insulin-stimulated GLUT4 translocation was also measured in cells that after the 16 h incubation with 10 nM insulin were recultured in normal growth medium for 16 h or 40 h to measure reversibility of insulin resistance. Each point is the mean ± SEM, _n_≥3. (C) Percentage of adipocytes with cytosolic FoxO1-GFP. Each bar is the mean ± SEM, n = 5. (D) Micrographs of adipocytes stained for neutral lipids (LipidTox, red) and ATGL expression (green). Nuclei are stained with Hoechst 3322 (blue). (E) Immunoblot and densitometry analyses of ATGL expression. Chronic Hyperinsulinemia (CHI). Each point is the mean ± SEM, n = 5. (F) Neutral lipid accumulation in adipocytes. Neutral lipids were stained with LipidTox neutral lipid stain and the fluorescence intensity per cell was determined by quantitative fluorescence microscopy. Chronic Hyperinsulinemia (CHI). Each bar is the mean ± SE of five experiments. Approximately 200 cells per experiment were quantified. (G) Glycerol release in control adipocytes (CT) and adipocytes exposed to chronic hyperinsulinemia (CHI). Each bar is the mean ± SE of four experiments. *p < 0.05 versus control (_t_-test).

Fig. 2.

Fig. 2.

GLUT4 translocation and FoxO1 nuclear exclusion display distinct sensitivities to insulin. (A) Insulin-dose response for HA-GLUT4-GFP translocation and FoxO1 nuclear exclusion. Each point is the mean ± SEM, _n_≥5. (B) Immunoblot of insulin signaling intermediates in adipocytes stimulated with insulin for 30 min. (C) Densitometry analyses of experiments like those shown in B. Each point is mean ± SEM, n = 3. Values were normalized to that of 10 nM insulin treated cells. (D) ED50s for insulin-induced GLUT4 plasma membrane translocation, FoxO1 nuclear exclusion, and Akt, TBC1D4, and FoxO1 phosphorylation. The values were derived from the data in A and C using a dose-response logistic curve fit. (E) Insulin-dose response for HA-GLUT4-GFP translocation and FoxO1-GFP nuclear exclusion in control adipocytes and adipocytes exposed to chronic hyperinsulinemia (CHI). Each point is the mean ± SEM, _n_≥3.

Fig. 3.

Fig. 3.

GLUT4 and FoxO1 regulation by insulin display distinct sensitivities to p110α activity. (A) Surface-to-total distribution of HA-GLUT4-GFP and percentage of cells with cytosolic FoxO1-GFP in adipocytes treated with p110 isoform inhibitors (30 min) followed by 1 nM insulin stimulation (30 min). Each point is the mean ± SEM, _n_≥3. *p < 0.05 (_t_-test). (B) Immunoblot and densitometry analyses of Akt phosphorylation in adipocytes. Each bar is the mean ± SEM, n = 3. Cells were treated as in A. (C) Immunoblot and densitometry analyses of cells lysates from adipocytes treated with control or PTEN siRNA. (D) Surface-to-total distribution of HA-GLUT4-GFP in adipocytes treated with control or PTEN siRNA. Each point is the mean ± SEM, n = 3. (E) Percentage of control or PTEN KD adipocytes with cytosolic FoxO1. Each point is the mean ± SEM, n = 2–4.

Fig. 4.

Fig. 4.

GLUT4 and FoxO1 display distinct Akt isoform requirements and sensitivities in response to insulin. (A) Insulin-dose response for surface-to-total distribution of HA-GLUT4-GFP in adipocytes treated with control or Akt isoform-specific siRNAs. Each point is the mean ± SEM, _n_≥3. (B) Insulin-dose response for FoxO1-GFP nuclear exclusion in adipocytes treated with control or Akt isoform-specific siRNAs. Each bar is the mean ± SEM, _n_≥3. (C) Effect of the Akt inhibitor Akti1/2 (1 μM) on 1 nM insulin-mediated FoxO1 nuclear exclusion. Each bar is the mean ± SEM, n = 3. (D) Surface-to-total distribution of HA-GLUT4-GFP in adipocytes overexpressing either Flag-Akt1 or Flag-Akt2 by electroporation. Each bar is the mean ± SEM, n = 3. (E) Percentage of adipocytes overexpressing either Akt1 or Akt2 by electroporation displaying cytosolic FoxO1-GFP. Flag-Akt1 and Flag-Akt2 expression was more than 10-fold higher than the endogenous kinases. 1 μM Akt inhibitor Akti1/2 for 30 min as noted. Each bar is the mean ± SEM, n = 3. (F) Immunoblot and densitometry analyses of immunoprecipitated Flag-Akt1 and Flag-Akt2. CHI: chronic hyperinsulinemia. Each point is the mean ± SEM, n = 3. (G) Schematic of insulin signaling to GLUT4 and FoxO1 in control adipocytes and adipocytes exposed to chronic hyperinsulinemia. Dashed lines indicate impaired function.

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