Targeting FoxO1 with AS1842856 suppresses adipogenesis

Abstract

Hyperplasia (i.e., increased adipogenesis) contributes to excess adiposity, the hallmark of obesity that can trigger metabolic complications. As FoxO1 has been implicated in adipogenic regulation, we investigated the kinetics of FoxO1 activation during adipocyte differentiation, and tested the effects of FoxO1 antagonist (AS1842856) on adipogenesis. We found for the first time that the kinetics of FoxO1 activation follows a series of sigmoid curves, and reveals the phases relevant to clonal expansion, cell cycle arrest, and the regulation of PPARγ, adiponectin, and mitochondrial proteins (complexes I and III). In addition, multiple activation-inactivation transitions exist in the stage of terminal differentiation. Importantly, persistent inhibition of FoxO1 with AS1842856 almost completely suppressed adipocyte differentiation, while selective inhibition in specific stages had differential effects on adipogenesis. Our data present a new view of FoxO1 in adipogenic regulation, and suggest AS1842856 can be an anti-obesity agent that warrants further investigation.

Keywords: AS1842856; AS1842856, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid; BMI, basal media I; BMII, basal media II; C1, mitochondrial complex I; C3, mitochondrial complex III; DMI, differentiation media I; DMII, differentiation media II; FoxO1; FoxO1, forkhead box O1; G6P, glucose 6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; PPARγ, peroxisome proliferator-activated receptor gamma; T2DM, type 2 diabetes mellitus; adipogenesis; mitochondria; obesity.

Figure 1.

Persistent inhibition of FoxO1 by AS1842856 largely suppressed adipogenesis. (A–B) Microscopy images of preadipocytes that were maintained in basal medium (A), and that underwent differentiation induced with the protocol as described in Materials and Methods (B). (C) Microscopy images of cells that were treated with AS1842856 (1.0 μM, days 0–12) and underwent differentiation induction as in (B). All the images were captured on day 12, with the microscope set at 100X. (D) Measurement of extracted Oil red O retained in cells by the absorbance (O.D.) at 510 nm (n = 6). (E) Representative western blot showing that AS1842856 inhibited FoxO1 through binding the de-phosphorylated sites, and to a lesser extent suppressed FoxO1 protein expression. β-actin was probed as the loading control. DI, differentiation induction; AS, AS1842856. (F and G) Densitometric analysis of protein gel blot images with NIH ImageJ software; n = 3−5. * P < 0 .05, and ***, P < 0 .0001.

Figure 2.

AS1842856 suppressed PPARγ and mitochondrial protein expression. (A) Western blots showing the effect of AS1842856 on PPARγ, adiponectin, mitochondrial proteins C1 and C3. β-actin was probed as the loading control. DI, differentiation induction; AS, AS1842856. (B–D) Densitometric analysis of western blot images with NIH ImageJ software; n = 3−5. * P < 0 .05; **, P < 0 .01; and ***, P < 0 .0001.

Figure 3.

The kinetics of FoxO1 activation followed a series of sigmoid curves during adipogenesis. (A) Western blots showing FoxO1 expression (i.e., tFoxO1), activation (i.e., dephosphorylation) and inactivation (i.e., phosphorylation) during adipogenesis. β-actin was probed as the loading control. (B) Densitometric analysis of protein gel blot images with NIH ImageJ software; n = 3−5. AP, activation peak; IP, inactivation peak. * P < 0 .05; and ***, P < 0 .0001.

Figure 4.

Kinetics of FoxO1-regulated protein expression during adipogenesis. (A) Western blots showing the expression of PPARγ, adiponectin, mitochondrial proteins C1 and C3. β-actin was probed as the loading control. (B) Densitometric analysis of western blot images for PPARγ and adiponectin with NIH ImageJ software. (C) Densitometric analysis of protein gel blot images for C1 and C3 with NIH ImageJ software. n = 3−5. * P < 0 .05; NS, not significant.

Figure 5

(See previous page). Effects of phase-specific FoxO1 inhibition on adipogenesis. (A and B) Images of cells that were maintained in basal medium (A), and that underwent differentiation induced with the protocol as described in Materials and Methods (B). (C) Images of cells that were treated with AS1842856 during stage BMII (days 7–12) and underwent differentiation induction as in (B). (D) Images of cells that were treated with AS1842856 during stage DMI (days 3–4) and underwent differentiation induction as in (B). (E) Images of cells that were treated with AS1842856 during stage BMI (days 1–2) and underwent differentiation induction as in (B). (F) Images of cells that were treated with AS1842856 during stage DMII (days 5–6) and underwent differentiation induction as in (B). The final concentration of AS1842856 was 0.1 μM. All the images were captured on day 12, and the microscope was set at 100X. (G) Measurement of extracted Oil red O retained in cells by the absorbance (O.D.) at 510 nm (n = 6). Treatments of ‘A’–‘F’ refer to the individual treatments as described in panels A-F. * P < 0 .05; **, P < 0 .01; and ***, P < 0 .0001.