Obesity increases mortality and the risk of developing multiple disorders including insulin resistance, type 2 diabetes, cardiovascular diseases, and cancers (Flegal et al., 2007
; Flegal et al., 2003
; Pischon et al., 2008
; Wang et al., 2005
). Here, we provide evidences that DEPTOR plays a role in regulating fat accumulation in vivo
and in vitro
. Observations made in two independent mouse models, in humans, and in cultured cells, all indicate that DEPTOR expression positively associates with adiposity. These results establish DEPTOR as a new physiological regulator of adipogenesis.
The Fat and Lean mouse lines were previously selected based on their fat content from a genetically highly variable base population for more than 60 generations (Sharp et al., 1984
), resulting in lines that differ in body fat content by more than five fold. The low resolution mapping initially detected the obesity QTL Fob3
in a region on chromosome 15 (Horvat et al., 2000
; Stylianou et al., 2005
). Follow-up crosses of congenic line containing a large segment of chromosome 15 from the Lean line on otherwise Fat line background provided a medium resolution genetic map and revealed that Deptor
is located within the QTL (Stylianou et al., 2005
). We provide a detailed fine genetic mapping with congenic strains carrying overlapping shorter Fob3a
donor genomic segments that further narrowed down Fob3a
candidate interval to below 15Mbp. Expression and bioinformatics analyses confirmed that Deptor
is a high priority candidate gene for fat accumulation in this model.
Using a doxycycline-inducible mouse model, we observed that overexpression of Deptor promotes adiposity in vivo, thus confirming the analyses made in the congenic mouse lines. Interestingly, unlike the Fat congenic mouse lines that gain significantly more weight than the lines carrying Lean-line Deptor allele on a regular diet, iDeptor mice need a higher lipid flux to reproduce this phenotype. The exact reason for this phenomenon is unknown but may be related to differences in DEPTOR expression profile (duration and expression levels) or differences in the genetic background of these models. Nevertheless, these results indicate that DEPTOR overexpression promotes WAT expansion in mice.
In iDeptor mice, increased DEPTOR expression promotes the transcription of many genes regulated by PPAR-γ that favors lipid synthesis and uptake/esterification in WAT. Consistent with these results, we observed that DEPTOR modulates PPAR-γ activity and the expression of many lipogenic genes in vitro. The fact that DEPTOR cell-autonomously promotes adipogenesis in cultured cells strongly suggests that DEPTOR may play a direct role in promoting WAT expansion. In vitro, we noticed that DEPTOR expression triggers fat cell development by promoting preadipocyte commitment as well as lipid synthesis in committed cells. The increase in preadipocyte differentiation coupled to the increase in lipid synthesis in already committed cells may explain why we did not observe a major change in average adipocyte size in WAT of iDeptor mice.
The fact that DEPTOR expression is induced during adipocyte differentiation raises the possibility that elevated DEPTOR levels observed in WAT of the Fat congenic mouse lines and in obese humans could be a consequence rather than a cause of the increased in adipogenesis. Although we cannot rule out the contribution of adipogenesis per se to the increase in DEPTOR levels in WAT of obese, a few facts indicate that adipogenesis may not be the only factor contributing to promote DEPTOR expression in obese. In the congenic mouse lines, elevated DEPTOR expression was observed not only in WAT, but also in the liver of the Fat congenic lines, indicating that high DEPTOR expression in WAT is unlikely to be a consequence of increased adipogenesis. Supporting this observation, preliminary analysis of the Deptor promoter revealed the presence of many polymorphisms between the Fat and the Lean lines that could contribute to the variation in Deptor expression levels between these lines (data not shown). Finally, in WAT of obese humans, we observed that the expression of the classic adipogenic marker aP2 was increased to a much lower extent than DEPTOR, suggesting that the process of adipogenesis is unlikely to be the only factor driving DEPTOR expression.
What promotes DEPTOR expression in WAT of humans is an interesting question. To our knowledge, the Deptor
locus has not been linked to human obesity in any genome wide association studies published so far. Although we do not exclude the possibility that polymorphisms in Deptor
locus or in other elements regulating DEPTOR expression/stability may be found in obese humans, the striking elevation in DEPTOR levels observed here among a population of unrelated individuals suggests that a common mechanism taking place in WAT of obese could promote DEPTOR expression. Interestingly, we show that DEPTOR is highly induced by glucocorticoids, steroid hormones that are secreted by the adrenal cortex in response to stress (Morton, 2010
). Excess of glucocorticoids, as seen in Cushing’s syndrome or in humans chronically treated with exogenous glucocorticoids, causes many adverse effects including obesity (Stanbury and Graham, 1998
). Many reports indicate that the local conversion/activation of glucocorticoids is increased in WAT of obese humans (Morton, 2010
). In this context, it is tempting to speculate that the increase in the local conversion of glucocorticoids in WAT of obese humans may contribute to increase DEPTOR expression, which in turn could facilitate WAT expansion by promoting its ability to store lipids. The increase in fat accumulation observed in response to DEPTOR overexpression in the iDeptor mouse and in iDeptor MEFs in vitro
supports this possibility.
The positive role of DEPTOR in regulating adipogenesis, lipogenesis, and fat accumulation is counterintuitive considering that DEPTOR was reported to inhibit mTORC1 (Peterson et al., 2009
), a protein complex known to promote adipogenesis and adipose cell maintenance (Cho et al., 2004
; Gagnon et al., 2001
; Kim and Chen, 2004
; Polak et al., 2008
; Yu et al., 2008
; Zhang et al., 2009
). We showed in the first DEPTOR report that this protein inhibits S6K1 activation, which reduces the negative feedback loop on IRS1/PI3K and promotes Akt/PKB activity (Peterson et al., 2009
). Here, we confirm that DEPTOR promotes Akt/PKB action but, unexpectedly, found that this was not associated with severe inhibition of the classical downstream effectors of mTORC1 (S6K1/S6, 4E-BP1), which have been implicated in the regulation of adipogenesis downstream of mTORC1 (Carnevalli et al., 2010
; Le Bacquer et al., 2007
). Instead, we observed that DEPTOR reduces the phosphorylation of IRS1 on S636/639, a site directly targeted by mTORC1 that promotes IRS1 degradation (Tzatsos, 2009
; Tzatsos and Kandror, 2006
). These results indicates that DEPTOR selectively relieves the negative feedback loop on IRS1, which promotes the action of the pro-adipogenic Akt/PKB, while preserving the function of mTORC1 towards other substrates. From that perspective, it is clear that DEPTOR does not block mTORC1 to nearly the same degree than rapamycin or RAPTOR loss, which have both been show to block adipogenesis. The incomplete inhibition of mTORC1 action probably explains why DEPTOR does not blocks adipogenesis. How DEPTOR selectively regulates IRS1 without impairing S6K1/S6 or 4E-BP1 is unclear. It is possible that chronic modulation of DEPTOR expression could rewire the mTOR signaling pathway by modulating the relation between mTORC1 and the numerous feedback loops (Laplante and Sabatini, 2012
). Such reorganization in the pathway could lead to a new signaling equilibrium where the elevation in Akt/PKB could reactivate the action of mTORC1 towards some substrates.
To dissect the respective contribution of mTORC1 and Akt/PKB in the control of adipogenesis, Zhang et al. used Tuberous sclerosis 2 (Tsc2)
null MEFs (Zhang et al., 2009
). Loss of TSC2 activates mTORC1 and, through mTORC1-dependent feedback mechanism, completely inhibits Akt/PKB. Using this model, Zhang et al. showed that adipogenesis is enhanced when mTORC1 is constitutively activated. These results differ from our findings by suggesting that adipogenesis depends on high mTORC1 activation and that the mTORC1-dependent negative feedback loop on PI3K-Akt/PKB axis is not playing a significant inhibitory role on this process. Importantly, the supraphysiological activation of mTORC1 creates a signaling context that might override regulatory processes that normally take place during adipogenesis. For instance, basal PPAR-γ expression is induced by more than 30 fold in Tsc2
null cells (Zhang et al., 2009
). Overexpression of PPAR-γ is sufficient to induce adipogenesis and can partially correct the adipogenic defect caused by the loss of Akt/PKB (Peng et al., 2003
; Tontonoz et al., 1994
; Yun et al., 2009
). The difference in PPAR-γ expression levels likely account for the different results observed between the present study and the one from Zhang et al.
DEPTOR overexpression increased glucose uptake, lipogenesis, and PPAR-γ activation, which all represent key processes controlled by Akt/PKB contributing to adipogenesis. Interestingly, examples of proteins affecting adipogenesis through the modulation of Akt/PKB and PPAR-γ exist in the literature. The Tribbles homologue 2 (TRB2) and TRB3 are pseudokinases acting as dominant-negative regulators of several kinases, including Akt/PKB (Du et al., 2003
). TRB2/3 expression is reduced during adipogenesis and their overexpression blocks Akt/PKB and PPAR-γ activation and adipogenesis (Bezy et al., 2007
; Naiki et al., 2007
; Takahashi et al., 2008
). The coordinated increase in DEPTOR levels and the decrease in TRB2/3 during adipogenesis indicate that pre-adipocytes trigger various signaling events to insure high activation of pro-adipogenic signals.
In conclusion, we show that DEPTOR cell-autonomously promotes adipogenesis and that elevated expression of DEPTOR associates with obesity in mice and humans. These findings improve our understanding of the molecular mechanisms regulating WAT formation and may ultimately contribute to the development of new tools to treat obesity and its related diseases.