Using a two-hybrid screen, we identified a Foxo1-CoRepressor termed as FCoR that is expressed in adipose tissue, inhibits Foxo1 transcriptional activity, and is phosphorylated by PKA and then translocated into the nucleus. FCoR is activated in a fasting or starved state, or during cold exposure, all conditions in which PKA is activated by glucagon or adrenergic stimuli. Thus, FCoR may be an important metabolic regulator that coordinates the insulin/cAMP response by interacting with Foxo. However, at fasting state, expression level of activated FCoR protein is decreased. We speculate that the active, presumed phosphorylated, FCoR protein is unstable and turned over rapidly.
Experiments showed that FCoR prevents Sirt1 binding to Foxo1, thus enhancing Foxo1 acetylation and inhibiting its transcriptional activity. Several proteins have been reported to affect Foxo1 and Sirt1. In C. elegans
, two 14-3-3 proteins bind to SIR-2.1 and DAF-16 and are required for SIR-2.1-induced transcriptional activation of DAF-16 (Berdichevsky et al, 2006
). Furthermore, four-and-a-half LIM 2 (FHL2) also interacts with FOXO1; FHL2 enhances the interaction between FOXO1 and SIRT1 and therefore enhances the deacetylation of FOXO1 (Yang et al, 2005
). To date, FCoR is the only protein that is known to inhibit the interaction of Sirt1 and Foxo1.
Recently, it has been reported that class IIa HDACs 4/5/7 and class I HDAC (HDAC3) are critical components of the transcriptional response to fasting in liver, shuttling into the nucleus in response to forskolin or glucagon. HDAC4 and 5 induces the acute transcription of gluconeogenic enzymes such as G6Pase via deacetylation and activation of Foxo family transcription factors (Mihaylova et al, 2011
; Wang et al 2011
). In the present study, we used trichostatin A (TSA) for the prevention of deacetylation of Foxo1. Therefore, we cannot exclude the possibility that FCoR works on the regulatory complex with the class IIa and I HDACs. It is established that in hepatocytes, Foxo is actively promoting transcription of its target genes after forskolin, glucagon, or cAMP treatment. However, Fcor
is expressed little in liver of wild-type mice under normal chow diet. If FCoR is expressed in liver, then the predicted acetylation and inhibition of Foxo may run counter to the known activation of Foxo by forskolin/cAMP agonists.
Interestingly, FCoR itself has intrinsic acetyltransferase activity. Acetylation represents a fail-safe mechanism for preventing excessive Foxo1 activity (Banks et al, 2011
). Therefore, FCoR may maintain Foxo1 acetylation by direct acetylation in the cytosol in the fed state and by interruption of the association of Foxo1 with Sirt1 in the nucleus in a fasting or starved state or during cold exposure. This is consistent with the idea that FCoR acts to fine-tune Foxo1 activity (). FCoR can also acetylate other transcription factors and cofactors (Nakae et al,
unpublished observation). Therefore, the sudden decline of body weight in WFCoR
mice may be due to acetylation of other binding partners of FCoR. Further investigation of FCoR-binding proteins is needed to identify such partners.
Figure 9 A model for the roles of FCoR in fine-tuning of Foxo1 activity. (A) When mice are in the fed state, Foxo1 is phosphorylated and FCoR expression is increased. Both proteins remain in the cytosol. FCoR acetylates Foxo1 directly and keeps it in the acetylated (more ...)
The finding that knockdown of endogenous FCoR inhibited the differentiation of 3T3-F442A cells suggests that FCoR is indispensable for adipocyte differentiation. This is in keeping with our previous findings that overexpression of CN Foxo1 (ADA) inhibits the differentiation of 3T3-F442A cells (Nakae et al, 2003
). Jing et al
reported that the KR mutant of Foxo1, which is activated and thus mimics the deacetylated protein (Banks et al, 2011
), inhibits adipocyte differentiation (Jing et al, 2007
). Therefore, knockdown of FCoR may accelerate Foxo1 deacetylation and so inhibit adipogenesis.
The present study demonstrated that while FCoR overexpression decreased the adipocyte size in transgenic mice, FcorKO
mice exhibited increased adipocyte size, consistent with findings for Foxo1 haploinsufficiency or transgenic mice overexpressing a dominant-negative Foxo1 mutant in fat cells (aP2-FLAG-Δ256
) (Nakae et al, 2003
; Kim et al, 2009
). Namely, inhibition of Foxo1 leads to smaller adipocytes. Therefore, inhibition of Foxo1 by overexpression of FCoR reduces adipocyte size. On the other hand, activation of Foxo1 by deletion of FCoR increases adipocyte size. Foxo1 is involved in the early stages of adipose conversion (Nakae et al, 2003
). Therefore, it is speculated that generation of adipocytes in FcorKO
mice is decreased due to activation of Foxo1. Previous reports showed that Foxo1 inhibits PPARγ activity (Dowell et al, 2003
; Armoni et al, 2006
; Fan et al, 2009
). Knockdown of FCoR should enhance Foxo1 activity, leading to inhibition of PPARγ and adipogenesis. However, in the present study, FCoR did not prevent Foxo1-induced inhibition of PPARγ activity. More than that, knockdown of Fcor
gene expression in 3T3-F442A cells and overexpression of FCoR in WAT significantly increased Pparg
expression. Foxo1 represses transcription from either Pparg1
promoter (Armoni et al, 2006
). Therefore, inhibition of Foxo1 by FCoR may work on Pparg
promoter and increase Pparg
expression, leading to enhanced adipogenesis and smaller adipocytes.
We found that FCoR expression in BAT of transgenic mice led to cold intolerance and to decreased expression levels of Ppargc1a
, suggesting that FCoR regulates brown adipocyte function. FCoR expression is induced by cold exposure. Therefore, FCoR in BAT may restrict mitochondrial biogenesis during cold exposure by inhibition of Ppargc1a
expression. However, the findings are inconsistent with our previous report that aP2-FLAG-
Δ256 mice, in which a transactivation-defective Foxo1 (Δ256) is expressed in both WAT and BAT, showed increased oxygen consumption accompanied by increased expression of PGC-1α, uncoupling protein (UCP)-1, UCP-2, and β3-adrenergic receptor (β3-AR) (Nakae et al, 2008a
). If FCoR works by inhibiting Foxo1 activity in BAT, then overexpression of FCoR should enhance the thermogenic function of BAT, leading to increased expression of Ppargc1a
and increased energy expenditure. Therefore, FCoR may inhibit Ppargc1a
expression via other partners but not via inhibition of Foxo1 in BAT. However, in liver, Ppargc1a
is a transcriptional target that is induced by the dephosphorylation mutant of Foxo1 (Daitoku et al, 2003
; Matsumoto et al, 2006
). Further investigation is needed to clarify the molecular target of FCoR in the Ppargc1a
promoter in BAT.
FCoR acts as a ‘repressor' of Foxo1 and Foxo3a but not of Foxo4. The failure to identify homologous genes in C. elegans, along with the repression of FCoR expression in states of food deprivation, indicates that FCoR may act as an anti-thrifty gene. FCoR regulates insulin sensitivity by altering the size of white adipocytes in WAT and induces cold intolerance by inhibiting Ppargc1a expression. FCoR is thus an attractive therapeutic target for treatment of obesity and type 2 diabetes.