FoxO1 interacts with transcription factor EB and differentially regulates mitochondrial uncoupling proteins via autophagy in adipocytes

Abstract

Mitochondrial uncoupling proteins (UCPs) are inducible and play an important role in metabolic and redox homeostasis. Recent studies have suggested that FoxO1 controls mitochondrial biogenesis and morphology, but it remains largely unknown how FoxO1 may regulate mitochondrial UCPs. Here we show that FoxO1 interacted with transcription factor EB (Tfeb), a key regulator of autophagosome and lysosome, and mediated the expression of UCP1, UCP2 and UCP3 differentially via autophagy in adipocytes. UCP1 was down-regulated but UCP2 and UCP3 were upregulated during adipocyte differentiation, which was associated with increased Tfeb and autophagy activity. However, inhibition of FoxO1 suppressed Tfeb and autophagy, attenuating UCP2 and UCP3 but increasing UCP1 expression. Pharmacological blockade of autophagy recapitulated the effects of FoxO1 inhibition on UCPs. Chromatin immunoprecipitation assay demonstrated that FoxO1 interacted with Tfeb by directly binding to its promoter, and silencing FoxO1 led to drastic decrease in Tfeb transcript and protein levels. These data provide the first line of evidence that FoxO1 interacts with Tfeb to regulate autophagy and UCP expression in adipocytes. Dysregulation of FoxO1→autophagy→UCP pathway may account for metabolic changes in obesity.

Figure 1

Expression of UCPs during 3T3-L1 adipocyte differentiation. (a and b) Measurement of lipid accumulation during adipocyte differentiation. The cells were cultured and differentiated as described in Materials and methods section, and lipid accumulation was measured by oil red O staining (a) and absorbance at 510 nm (b). (c and d) qPCR analysis of UCP1 (c), UCP2 (d) and UCP3 (e) during adipocyte differentiation. Results were presented as mean±s.d.; n=3–4; *P<0.05; **P<0.01; ***P<0.001.

Figure 2

Effects of FoxO1 inhibition on UCPs and autophagy. (a) Inhibition of FoxO1 upregulated UCP1. (b) Inhibition of FoxO1 down-regulated UCP2. (c) Inhibition of FoxO1 down-regulated UCP3. (d) Inhibition of FoxO1 prevented lipid accumulation in adipocytes. (e and f) Inhibition of FoxO1 attenuated autophagy (p62 degradation). The cells were cultured and treated (days 0–12) as described in Materials and Methods section. DI, differentiation induction; AS, AS1842856 (0.1 μM). Results were presented as mean±s.d.; n=3–4; ***P<0.001.

Figure 3

Autophagy was required for coordinated expression of UCPs in adipocytes. (a–c) Western blot (a) and densitometric (b and c) analysis of Tfeb and p62 suggested that autophagy was upregulated during adipocyte differentiation. (d) Effects of autophagy inhibitors bafilomycin A1 and leupeptin on UCP1 expression. (e) Effects of bafilomycin A1 and leupeptin on UCP2 expression. (f) Effects of bafilomycin A1 and leupeptin on UCP3 expression. (g) Effects of bafilomycin A1 and leupeptin on lipid accumulation. The cells were cultured and treated as described in Materials and Methods section, and the treatment with autophagy inhibitors was conducted on days 0–12. DI, differentiation induction; BL, bafilomycin A1 (4 nM) and leupeptin (0.4 μg/ml). Results were presented as mean±s.d.; n=3–4; *P<0.05; **P<0.01; ***P<0.001.

Figure 4

Nuclear localization and activity of FoxO1 increased during adipogenesis. (a) Western blot analysis of total FoxO1 and phosphorylated FoxO1 (pFoxO1-Thr24) during 3T3-L1 adipocyte differentiation. (b) Measurement of un-phosphorylated FoxO1 (un-p-FoxO1) by densitometric analysis of western blot images. (c) Measurements of FoxO1 activity in the nuclear fractions isolated from adipocytes on days 0, 6 and 12 during differentiation. Results were presented as mean±s.d.; n=3-4; **P<0.01; ***P<0.001.

Figure 5

FoxO1 regulated Tfeb expression. (a) Tfeb transcript was analyzed on days 0, 6 and 12 during adipocyte differentiation. (b) FoxO1 inhibitor AS1842856 (0.1 μM) potently suppressed FoxO1 activity in the nuclear fractions isolated from adipocytes. (c and d) Inhibition of FoxO1 prevented Tfeb upregulation during adipocyte differentiation, both at transcript (c) and protein (d) levels. (e) Tfeb gene contains three FoxO1-binding (i.e., insulin response element, IRE) sites in its promoter region. (f) Chromatin immune-precipitation (ChIP) assay of FoxO1-Tfeb interaction using a FoxO1 specific antibody. DI, differentiation induction; AS, AS1842856. The cells were cultured and treated (days 0–12), and ChIP assay conducted as described in Materials and Methods section. Results were presented as mean±s.d.; n=3–4; *P<0.05; **P<0.01; ***P<0.001.