Intriguingly, inhibition of autophagy significantly increased UCP1 transcript (by 2.2-fold, em P /em 0.001) but reduced the expression of UCP2 (by 38%, em P /em 0.01) and UCP3 (by 89%, em P /em 0.001) in adipocytes (Figures 3dCf), concomitant with suppression of adipogenesis (Figure 3g). These data recapitulated the effects of inhibiting FoxO1 on UCPs during adipocyte differentiation (Figure 2), thereby underlining the importance of FoxO1-autophagy axis in the coordinated expression of UCP1, UCP2 and UCP3. Open in a separate window Figure 3 Autophagy was required for coordinated expression of UCPs in adipocytes. (aCc) Western blot (a) and densitometric (b and c) analysis of Tfeb and p62 suggested that autophagy was upregulated during adipocyte differentiation. demonstrated that FoxO1 interacted with Tfeb by directly binding to its promoter, PS 48 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 FoxO1autophagyUCP pathway may account for metabolic changes in obesity. Introduction Obesity is one of the most pressing health concerns in the US.1C3 The growing epidemic of obesity has been attributed largely to modern lifestyle characteristic of energy overconsumption and physical inactivity.3,4 As such, the strategies limiting energy intake or increasing energy expenditure have been proposed for obesity prevention.3C5 Mitochondria play a central role in cellular energy homeostasis.3,6C8 In particular, induction of mitochondrial uncoupling protein (UCP) in mice promotes energy dissipation and protects against obesity, while genetic UCP deficiency causes obesity.5,9,10 In line with these findings, UCP polymorphisms have been increasingly reported in obese humans,11,12 and adipose UCP gene expression is significantly lower in morbidly obese people than in lean individuals. 13 These studies suggest that dysregulation of UCPs contributes to development of obesity, and understanding the mechanism of regulation of UCPs in adipocytes may lead to new options for obesity prevention and treatment. UCPs are a family of mitochondrial transporters (or mitochondrial anion carriers) located in the inner membrane.14,15 In adipocytes or adipose tissue, three isoforms of UCP have been identified, UCP1, UCP2 and UCP3, although their expression levels vary.14C18 PS 48 UCP1 is primarily expressed in brown adipose tissue, and it uncouples mitochondrial respiration from ATP production/oxidative phosphorylation, dissipating energy in the form of heat.14,15 Under certain conditions (e.g., cold exposure), UCP1 expression in white adipocytes can be significantly induced, leading to a browning phenotype.17 UCP2 and UCP3 share amino acid identity with UCP1 (59 and 57%, respectively), and their function in mitochondrial uncoupling is still under investigation.14,15,18 Although some studies suggested that UCP2 and UCP3 were proton channels like UCP1, others regarded them as ion channels that limit the production of reactive oxygen species, and export toxic fatty acid anions and peroxides from mitochondrial matrix.14,15,18,19 FoxO1 is a transcription factor that regulates mitochondrial function and adipocyte differentiation.2,20C23 Activation of FoxO1 in liver alters mitochondrial biogenesis, morphology and function in the insulin resistant mice, while genetic ablation of FoxO1 significantly normalizes mitochondria and metabolism.21,24 In adipocytes, silencing FoxO1 with specific antagonist or siRNA potently inhibits cell differentiation and lipid accumulation, accompanied with changes in expression of mitochondrial respiration chain proteins.2,22,23 Recently we found that FoxO1 controlled lipid droplet growth and adipose autophagy, the cellular process that has been implicated in adipocyte differentiation.25C29 Moreover, genetic and pharmacological inhibition of autophagy leads to browning of white adipose tissue, characteristic of increased UCP1 expression.26C29 However, it is unknown how mechanistically FoxO1 regulates autophagy and other UCPs (i.e., UCP2 and UCP3). In the present work, we show that FoxO1-mediated autophagy upregulates UCP2 and UCP3 in adipocytes but downregulates UCP1. Mechanistically, FoxO1 interacted with transcription Factor EB (Tfeb), a key regulator of autophagosome and lysosome, 30 by directly binding to the promoter and regulating its expression. Results Expression patterns of UCPs during adipocyte differentiation Following an established protocol, we Rabbit polyclonal to AdiponectinR1 cultured 3T3-L1 preadipocytes and induced cell differentiation.2,31 Maturation of adipocytes was paralleled with significant lipid accumulation as measured by oil red O staining and spectrophotometric reading at 510?nm (Figures 1a and b).2,32,33 Interestingly, the expression of UCP1, UCP2 and UCP3 showed distinctive kinetics during adipocyte differentiation (Figures 1cCe). In contrast.Results were presented as means.d.; em n /em =3C4; ***P 0.001. Suppression of autophagy recapitulated the effects of FoxO1 inhibition on UCPs To examine the role of autophagy in UCP regulation, we measured kinetics of autophagy during adipogenesis (Figure 3, Supplementary Figure 1). 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 FoxO1autophagyUCP pathway may account for metabolic changes in obesity. Introduction Obesity is one of the most pressing health concerns in the US.1C3 The growing epidemic of obesity has been attributed largely to modern lifestyle characteristic of energy overconsumption and physical inactivity.3,4 As such, PS 48 the strategies limiting energy intake or increasing energy expenditure have been proposed for obesity prevention.3C5 Mitochondria play a central role in cellular energy homeostasis.3,6C8 In particular, induction of mitochondrial uncoupling protein (UCP) in mice promotes energy dissipation and protects against obesity, while genetic UCP deficiency causes obesity.5,9,10 In line with these findings, UCP polymorphisms have been increasingly reported in obese humans,11,12 and adipose UCP gene expression is significantly lower in morbidly obese people than in lean individuals.13 These studies suggest that dysregulation of UCPs contributes to development of obesity, and understanding the mechanism of regulation of UCPs in adipocytes may lead to new options for obesity prevention and treatment. UCPs are a family of mitochondrial transporters (or mitochondrial anion carriers) located in the inner membrane.14,15 In adipocytes or adipose tissue, three isoforms of UCP have been identified, UCP1, UCP2 and UCP3, although their expression levels vary.14C18 UCP1 is primarily expressed in brown adipose tissue, and it uncouples mitochondrial respiration from ATP production/oxidative phosphorylation, dissipating energy in the form of heat.14,15 Under certain conditions (e.g., cold exposure), UCP1 expression in white adipocytes can be significantly induced, leading to a browning phenotype.17 UCP2 and UCP3 share amino acid identity with UCP1 (59 and 57%, respectively), and their function in mitochondrial uncoupling is still under investigation.14,15,18 Although some studies suggested that UCP2 and UCP3 were proton channels like UCP1, others regarded them as ion channels that limit the production of reactive oxygen species, and export toxic fatty acid anions and peroxides from mitochondrial matrix.14,15,18,19 FoxO1 is a transcription factor that regulates mitochondrial function and adipocyte differentiation.2,20C23 Activation of FoxO1 in liver alters mitochondrial biogenesis, morphology and function in the insulin resistant mice, while genetic ablation of FoxO1 significantly normalizes mitochondria and metabolism.21,24 In adipocytes, silencing FoxO1 with specific antagonist or siRNA potently inhibits cell differentiation and lipid accumulation, accompanied with changes in expression of mitochondrial respiration chain proteins.2,22,23 Recently we found that FoxO1 controlled lipid droplet growth and adipose autophagy, the cellular process that has been implicated in adipocyte differentiation.25C29 Moreover, genetic and pharmacological inhibition of autophagy leads to browning of white adipose tissue, characteristic of increased UCP1 expression.26C29 However, it is unknown how mechanistically FoxO1 regulates autophagy and other UCPs (i.e., UCP2 and UCP3). In the present work, we show that FoxO1-mediated autophagy upregulates UCP2 and UCP3 in adipocytes but downregulates UCP1. Mechanistically, FoxO1 interacted with transcription Factor EB (Tfeb), a key regulator of autophagosome and lysosome,30 by directly binding to the promoter and regulating its expression. Results Expression patterns of UCPs during adipocyte differentiation Following an established protocol, we cultured 3T3-L1 preadipocytes and induced cell differentiation.2,31 Maturation of adipocytes was paralleled with significant lipid accumulation as measured by oil red O staining and spectrophotometric reading at 510?nm (Figures 1a and b).2,32,33 Interestingly, the expression of UCP1, UCP2 and UCP3 showed distinctive kinetics during adipocyte differentiation (Figures 1cCe). In contrast to UCP1 that underwent downregulation (Figure 1c), UCP2 and UCP3 were upregulated drastically (Figures 1d and e). These data support the notion that upregulation of UCP1 counteracts lipid accumulation in adipocytes,34,35 and that UCP2 and UCP3 are required for lipid metabolism.14,15,19,36 Open in a separate window PS 48 Number 1 Manifestation of UCPs during.