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DEP domain containing mTOR-interacting protein (DEPTOR) inhibits the mechanistic target of rapamycin (mTOR) but its in vivo functions are unknown. Previous work indicates that Deptor is part of the Fob3a quantitative trait locus (QTL) linked to obesity/leanness in mice with Deptor expression being elevated in white adipose tissue (WAT) of obese animals. This relation is unexpected considering the positive role of mTOR in adipogenesis. Here, we dissected the Fob3a QTL and show that Deptor is the highest priority candidate promoting WAT expansion in this model. Consistently, transgenic mice overexpressing DEPTOR accumulate more WAT. Furtheremore, in humans, DEPTOR expression in WAT correlates with the degree of obesity. We show that DEPTOR is induced by glucocorticoids during adipogenesis and that its overexpression promotes, while its suppression blocks, adipogenesis. DEPTOR activates the pro-adipogenic Akt/PKB-PPAR-γ axis by dampening mTORC1-mediated feedback inhibition of insulin signaling. These results establish DEPTOR as a new regulator of adipogenesis.
Obesity, which is defined by excessive white adipose tissue (WAT) accumulation, 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). Although changes in lifestyle are responsible for the increase in incidence of obesity over the last decades, heritability studies provide evidence for a substantial genetic contribution to obesity risk (Bouchard et al., 1990; Maes et al., 1997). Because strategies aiming to reduce obesity have shown limited long-term success, an improvement in our understanding of the molecular mechanisms regulating WAT formation is important for the development of new tools to treat obesity and its related diseases.
The mTOR signaling pathway senses growth factors and nutrients to regulate many biological processes involved in the promotion of cell growth (Laplante and Sabatini, 2012). mTOR interacts with several proteins to form two distinct multiprotein complexes named mTOR complex 1 (mTORC1) and complex 2 (mTORC2). When active, mTORC1 promotes protein synthesis by phosphorylating the eukaryotic initiation factor 4E (eIF4E)-binding protein1 (4E-BP1) and the ribosomal S6 kinase 1 (S6K1), whereas mTORC2 regulates cell survival through the phosporylation of several AGC kinases. It is becoming increasingly clear that, in addition to protein synthesis, mTORC1 also regulates lipid metabolism and adipogenesis (Laplante and Sabatini, 2009). Overactivation of mTORC1 increases adipogenesis in vitro (Zhang et al., 2009) whereas the complete inhibition of mTORC1 by rapamycin or loss of RAPTOR, a key mTORC1 component, blocks this process (Cho et al., 2004; Gagnon et al., 2001; Kim and Chen, 2004; Polak et al., 2008; Yu et al., 2008; Zhang et al., 2009). For these reasons, targeting mTORC1 signaling represents a potential approach for reducing adiposity and improving obesity-related diseases.
Recently, we identified the DEP-domain containing mTOR-interacting protein (DEPTOR, also known as DEPDC6) as a novel protein that represses mTOR signaling (Peterson et al., 2009). DEPTOR interacts with mTORC1 and mTORC2 and its protein stability is reduced when these complexes are active. We observed that DEPTOR is low in most cancer cells but is surprisingly highly expressed in multiple myeloma (Peterson et al., 2009). In these cells, high DEPTOR expression reduces mTORC1 activity and reduces S6K1-mediated feedback inhibition of phosphoinositide-3 kinase (PI3K), which contributes to the activation of protein kinase B (Akt/PKB) and cell survival.
The role of DEPTOR in the regulation of physiological processes in vivo is unknown. Before the identification of DEPTOR as an mTOR-interacting protein, a section on chromosome 15 that includes the Deptor gene was identified as part of the Fob3a quantitative trait locus (QTL) that is linked to obesity/leanness in mice (Stylianou et al., 2005). It was reported that Deptor expression is elevated in the adipose tissues of the mouse line genetically prone to obesity. Because the attention was at that time focused on other candidate genes, the potential role of Deptor in regulating fat accumulation was not examined.
The possibility that Deptor could favor adiposity is counterintuitive considering the well-established positive role of mTORC1 in regulating 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). However, because DEPTOR impairs mTORC1 action to a much lower degree than rapamycin or RAPTOR loss, its impact on the regulation of adipogenesis and WAT accumulation may be different. Here, we used several congenic lines for dissecting the Fob3a QTL and confirmed that Deptor is a high priority candidate regulating WAT accumulation in the polygenic mouse model of obesity/leanness. To demonstrate the positive role of Deptor in regulating adiposity, we generated a doxycycline-inducible transgenic mouse model for Deptor overexpression and found that Deptor promotes WAT expansion. Consistent with these observations, we noted that DEPTOR expression is elevated in WAT of obese humans and positively correlates with the degree of obesity. DEPTOR is induced during adipogenesis through a glucocorticoid receptor-dependent mechanism and its expression facilitates adipocyte differentiation in a cell-autonomous fashion. We observe that DEPTOR positively regulates adipogenesis by promoting the activity of the pro-adipogenic factors Akt/PKB and peroxisome proliferator-activated receptor-γ (PPAR-γ). These results establish DEPTOR as a new physiological regulator of adipogenesis.
A polygenic mouse model of obesity was previously developed by divergent selective breeding and resulted in strains differing substantially in fat content (Horvat et al., 2000; Sharp et al., 1984). The mice were selected for high fat (Fat line) or low fat (Lean Line) 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 Fob3 QTL is one of several chromosomal regions controlling obesity in the Fat and Lean mouse lines. The Fob3 region has been dissected into distinct obesity QTLs called Fob3a, Fob3b1, and Fob3b2 (Prevorsek et al., 2010; Stylianou et al., 2005). Genetic mapping and microarray studies showed that that Deptor, together with many other genes, is part of the Fob3a QTL and could be involved in regulating adiposity (Stylianou et al., 2004; Stylianou et al., 2005).
To examine the contribution of the Deptor-containing Fob3a segment in the regulation of fat accumulation in the Fat mouse strains, the Fob3a interval was genetically dissected by analyzing the phenotypes of several congenic lines (P, V, W, and Z) with overlapping Fob3a regions of various lengths (Figure 1A). Congenic lines P and Z carrying the Fat-line derived Fob3a-Deptor region had significantly increased weights of WAT compared to lines V and W that carry the Fob3a-Deptor region from the Lean line (Figure 1B, left panel). These results were confirmed in independent F2 congenic intercrosses (Figure 1B middle and right panels) and were not dependent on changes in food intake (Figure S1A). A cross-section analysis of overlapping congenic segments fine mapped the Fob3a region down to a ~15 Mbp region containing the Deptor gene (Figure 1C).
To further narrow down the genetic interval likely to contain causal Fob3a QTL polymorphism(s), we performed Fob3a-interval-specific haplotype analysis that identified genomic blocks which are not identical by descent (non-IBD). Two non-IBD regions within the Fob3a segment were identified (Figure 1C). Of the 29 genes included in this region, 22 genes were considered as potential candidate (see methods). Strikingly, Deptor was highly expressed in WAT of the Fat line (8.9 fold, p<0.001) and in the congenic line Z (8.3 fold, p<0.001) carrying the Fob3a-Deptor segment derived from the Fat line compared to the line V carrying the Fob3a-Deptor segment derived from the Lean line (Figure 1D). Deptor was similarly regulated in the liver (Figure 1E). Microarray analyses also confirmed the differential regulation of Deptor in WAT of an independent F2 congenic intercross (Table S1). No other candidate gene showed such a consistent differential regulation between the lean and obese lines (Figure S1B to S1D and Table S1). In order to narrow down the list of potential candidate genes, we looked at the function (GO profile), the tissue distribution (BioGPS), and the available transgenic/knockout data for all the candidate genes included in the Fob3a region (see methods). Together with the expression results, these analyses revealed that Deptor is the highest priority candidate gene regulating fat accumulation in this model (Figure 1F and Table S2).
To test the hypothesis that upregulation of Deptor plays a causal role in obesity, we generated a doxycycline-inducible DEPTOR transgenic mouse model (Figure 2A). For ease, we refer to this model as iDeptor mice. This transgenic approach was selected because it allowed a modest overexpression of DEPTOR in many tissues including WAT (Figure 2B, S2A), thus grossly mimicking the differences observed between the Lean and Fat congenic mouse lines. iDeptor mice treated with doxycycline did not show changes in body weight, body weight gain, and tissue weight compared to control mice (Figure S2B, S2C). When fed a high fat diet containing doxycycline, iDeptor mice gained more weight than controls (Figure 2C). The increase in body weight in iDeptor mice was not caused by changes in food intake or locomotion but it was associated with an increase in food efficiency (Figure S2D to S2F). The weights of WAT and liver were significantly increased in iDeptor mice (Figure 2D). Histological analyses revealed that, despite the increase in WAT weight, DEPTOR expression did not change average adipocyte size (Figure S2G and S2H), indicating that DEPTOR affected WAT mass at least by increasing the number of fat cells. Consistent with the increase in WAT mass, plasma leptin was increased in iDeptor mice (Figure S2I). Lipid accumulation in the liver, a condition known as hepatosteatosis, was also increased by DEPTOR overexpression (Figure S2J and S2K).
In order to evaluate if DEPTOR modulates the ability of WAT to accumulate lipids, the expression of numerous genes encoding proteins regulating lipogenesis and lipid uptake/retention was measured in WAT of iDeptor mice. DEPTOR overexpression significantly increased the mRNA levels of many of these genes (Figure 2E). Interestingly, most of the genes increased by DEPTOR have been previously identified as targets of the master regulator of adipogenesis, PPAR-γ (Berger and Moller, 2002; Guan et al., 2002; Nishino et al., 2008). These results indicate that DEPTOR promotes WAT expansion at least in part by improving the ability of this tissue to synthesize and accumulate lipids. Importantly, DEPTOR overexpression also induced the transcription of many lipogenic genes in the liver, which likely contributed to promote hepatic triglyceride accumulation and weight gain in iDeptor mice (Figure S2L).
Because DEPTOR overexpression promotes adiposity and the expression of genes facilitating lipid accumulation in mice, we asked if DEPTOR expression might also be associated with obesity in humans. DEPTOR protein levels were measured in WAT of humans with body mass indexes (BMI) ranging from 22 to 80. Strikingly, DEPTOR protein expression was strongly elevated in obese humans (3.47 fold in BMI >30, p=0.0001) (Figure 3A and 3B). Additionally, we noted a significant correlation between DEPTOR protein levels and BMI (r=0.42, p<0.005). Importantly, no such correlation was observed with the expression of other proteins, such as aP2 or Akt/PKB (Figure S3A and S3B). Together with the results obtained in the mouse models, these observations support the possibility that DEPTOR could play an important role in regulating WAT expansion in humans.
In order to better understand the biological function of DEPTOR in adipose tissue/adipocytes, we examined if DEPTOR is regulated during fat cell formation in vivo and in vitro. In WAT collected from murine embryos and pups at different stages of development, DEPTOR levels increased significantly during the WAT formation that occurs as mice begin to suckle and accumulate fat (Figure 4A and Figure S4A). Consistent with these results, DEPTOR levels also increased in mouse embryonic fibroblasts (MEFs) and 3T3-L1 cells treated with an adipogenic cocktail that promotes their differentiation into the mature adipocytes (Figure 4B, 4C and Figure S4B). Thus, in vivo and in two well-established in vitro models, DEPTOR expression increases during adipogenesis.
We next took advantage of the in vitro adipogenesis systems to investigate the mechanisms that regulate DEPTOR expression. Each component of the adipogenic cocktail was removed individually and DEPTOR levels were measured after the induction of adipogenesis. Only the removal of dexamethasone, a synthetic glucocorticoid, blocked the induction of DEPTOR that occurs during differentiation (Figure 4D and S4C). The addition of dexamethasone or corticosterone increased DEPTOR levels even in undifferentiated MEFs (Figure 4E to 4G) or 3T3-L1 cells (Figure S4D). Analyses of publicly available transcriptional profiling data showed that glucocorticoids induce Deptor mRNA levels in mouse primary chondrocytes in vitro and the placenta in vivo, indicating that this mechanism of regulation is conserved in various conditions and cell types (Figure S4E and S4F). We observed that inhibition of transcription by actinomycin D or of the glucocorticoid receptor (GR) by RU-486 completely abrogated the dexamethasone-induced increases in Deptor mRNA and DEPTOR protein (Figure 4H). These results are consistent with glucocorticoids directly controlling Deptor expression through a transcriptional mechanism that depends on the GR. Bioinformatics analyses revealed the presence of a conserved glucocorticoid response element (GRE) in mouse and human Deptor promoter (Figure 4I). In order to demonstrate a physical interaction between the GR and the Deptor promoter, we treated MEFs with dexamethasone and performed chromatin immunoprecipitations (ChIP) using an antibody against the GR. Using this approach, we observed that dexamethasone promotes GR binding to the Deptor promoter (Figure 4J). Importantly, binding of GR to the Deptor promoter was enriched when the PCR was performed close to the potential GRE, which supports the possibility that this specific site may play a role in the control of Deptor expression by glucocorticoids.
An important question arising from this last set of results relates to the temporal regulation of DEPTOR by glucocorticoids during adipogenesis. If glucocorticoid signaling is an important regulator of DEPTOR expression during adipogenesis, why is the increase in DEPTOR levels relatively weak during the first two days of differentiation (Figure 4B)? Because in undifferentiated MEFs dexamethasone induced DEPTOR expression within 5 hours of addition (Figure 4E and 4F), we hypothesized that a component of the adipogenic cocktail might counteract the positive effects of dexamethasone on DEPTOR expression at early time points. Indeed, we observed that IBMX dose-dependently blocked the stimulatory effect of dexamethasone on DEPTOR protein and Deptor mRNA levels (Figure S4G). Consistent with this observation, IBMX removal from the adipogenic cocktail promoted Deptor mRNA expression (Figure S4C). As a non-selective phosphodiesterase inhibitor, IBMX is thought to exert some of its effects by raising intracellular cAMP levels. Treating cells with various doses of the cAMP analog dibutyryl-cAMP or with the adenylate cyclase activator forskolin did not reduce Deptor expression in response to dexamethasone (not shown), indicating that IBMX competes with dexamethasone for the control of Deptor expression by a mechanism that does not depend on variation in cAMP levels.
To better understand the role of DEPTOR in adipogenesis, MEFs isolated from iDeptor mice were treated or not with doxycycline and were differentiated into mature adipocytes. Consistent with the in vivo findings, DEPTOR overexpression significantly increased triglyceride accumulation in MEFs (Figure 5A and Figure S5A). Moreover, we noted a dose-dependent effect of DEPTOR on the promotion of adipogenesis in this model (Figure S5B). Importantly, doxycycline per se had no effect on adipocyte differentiation in wild-type MEFs (Figure S5C). Similar to what was observed in WAT in vivo, DEPTOR overexpression induced the expression of many genes regulated by PPAR-γ (Figure 5B, 5C and Figure S5D to S5F). In order to determine which step in the adipogenic program is affected by DEPTOR overexpression (commitment vs. terminal differentiation), DEPTOR was expressed in iDeptor MEFs during the early (day −2 to day +2) or the late (day +4 to day +12) stage of adipogenesis. Both treatments led to a significant increase in triglyceride accumulation, indicating that DEPTOR expression triggers fat cell development by promoting preadipocyte commitment as well as lipid synthesis in committed cells (Figure 5D and 5E).
We next determined if DEPTOR expression is necessary for adipocyte differentiation in vitro. Using 3T3-L1 cells, we observed that suppression of DEPTOR expression by short-hairpin RNA (shRNA) significantly reduced adipogenesis (Figure 5F and S5G). This was associated with a reduction in PPAR-γ protein levels and mRNA expression of genes controlled by PPAR-γ (Figure 5G to 5I and Figure S5H). Together, these results indicate that DEPTOR controls adipogenesis in a cell-autonomous fashion by modulating the expression of PPAR-γ and its target genes.
To better understand the mechanism by which DEPTOR promotes adipogenesis, we examined the effect of DEPTOR expression or depletion on mTOR signaling. Overexpression of DEPTOR in MEFs reduced the phosphorylation of insulin receptor substrate 1 (IRS1) at S636/639, a site directly targeted by mTORC1 that favors IRS1 degradation (Tzatsos, 2009; Tzatsos and Kandror, 2006), and promoted the phosphorylation/activation of Akt/PKB (Figure 6A). We also observed that dexamethasone and corticosterone, which both promote DEPTOR expression, also induced Akt/PKB phosphorylation in MEFs (Figure S6A and S6B). Moreover, the induction of DEPTOR expression during adipogenesis was associated with an increase in Akt/PKB activation (Figure S6C and S6D). Such an elevation in Akt/PKB phosphorylation was also observed in adipose tissue of mice overexpressing DEPTOR (Figure S6E). Conversely, suppression of DEPTOR expression in 3T3-L1 cells increased IRS1 phosphorylation at S636/639, reduced IRS1 levels, and blocked Akt/PKB activation (Figure 6B). DEPTOR depletion also reduced Akt/PKB-mediated Forkhead box O1/3a (FoxO1/3a) phosphorylation and nuclear exclusion (Figure S6F). Importantly, Akt/PKB activation was impaired in DEPTOR-depleted 3T3-L1 cells treated with insulin-like growth factor-1 (IGF-1), a key molecule promoting preadipocyte differentiation (Figure S6G) (Smith et al., 1988). We also noted a significant reduction in Akt/PKB activation over the differentiation process in DEPTOR depleted 3T3-L1 cells (Figure S6H).
It was reported that the complete inhibition of mTORC1 blocks WAT accumulation in vivo and adipogenesis in vitro (Cho et al., 2004; Gagnon et al., 2001; Kim and Chen, 2004; Polak et al., 2008; Yu et al., 2008; Zhang et al., 2009). Because DEPTOR inhibits mTORC1 (Peterson et al., 2009), it might be expected that DEPTOR expression would repress lipid accumulation in adipocytes. Importantly, although DEPTOR had a significant impact on the phosphorylation of IRS1 S636/639, we noted that the activation state of other classic downstream effectors of mTORC1 (S6K1, S6 or 4E-BP1) was not severely affected by the overexpression/knockdown of DEPTOR (Figure 6A and 6B). This indicates that DEPTOR does not impair mTORC1 activity to the same extent than rapamycin or RAPTOR loss, which probably explains why DEPTOR does not similarly affect adipogenesis.
Adipogenesis is a cellular process that requires the activation of Akt/PKB (Kohn et al., 1996; Magun et al., 1996; Menghini et al., 2005; Nakae et al., 2003; Peng et al., 2003). Akt/PKB induces glucose uptake, its metabolism and incorporation into lipids and promotes the activation of PPAR-γ through various mechanisms (Rosen and MacDougald, 2006). Because DEPTOR positively regulates Akt/PKB and the expression of many genes downstream of PPAR-γ, we tested the possibility that DEPTOR could affect adipogenesis by modulating the Akt/PKB-PPAR-γ axis. Consistent with the role of Akt/PKB in regulating glucose uptake and lipogenesis, we observed that modulation in Akt/PKB activation by DEPTOR was associated with significant changes in glucose uptake and incorporation into lipids (Figure 6C and S6I). Importantly, we also observed that expression of a constitutively active Akt/PKB corrected the adipogenic defect observed when DEPTOR was depleted, indicating that DEPTOR affects adipogenesis upstream of Akt/PKB (Figure 6D). Because Akt/PKB is a strong activator of PPAR-γ, we also looked at the impact of DEPTOR on the activity of this transcription factor. DEPTOR depletion in 3T3-L1 cells was associated with a significant reduction in the transactivation capability of PPAR-γ (Figure 6E). Interestingly, the inclusion of the potent PPAR-γ agonist rosiglitazone corrected adipogenesis and PPAR-γ expression in cells with suppressed DEPTOR levels (Figure 6F and Figure S6J). Together, these results indicate that DEPTOR controls adipogenesis by regulating the activation of the pro-adipogenic factors Akt/PKB and PPAR-γ. We propose a model in which the induction of DEPTOR during adipogenesis functions to reduce the feedback loop from mTORC1 to IRS1, which facilitates adipogenesis by upregulating the action of Akt/PKB and PPAR-γ.
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.
Antibodies were obtained from the following sources: antibody to DEPTOR (09-463) from Upstate/Millipore; antibodies to phospho-S473 Akt/PKB(4058), phospho-T308-Akt/PKB (2965), Akt/PKB (4691), ap2 (2120), C/EBP-α (2295), C/EBP-δ (2318), PPAR-γ (2443), mTOR (2983), Rictor (2140), Raptor (2280), phospho-S636/639-IRS1 (2388), IRS1 (2382), 4E-BP1 (9452), phospho-T37/46-4E-BP1 (2855), S6K1 (9202), phospho-T389 S6K1 (9205), S6 (2212), phospho-S240/24-S6 (2215), NDRG1 (5196), phospho-T346-NDRG1 (3317), phospho-PKCαβ-T636/641 (9375), FoxO1 (9462), phospho-T24 FoxO1/3a (9464), PTEN (9559), from Cell Signaling Technology; antibodies to C/EBP-β (sc-150), Actin (sc-1616), PKCα (SC-208), GR (SC-1004), IgGs (sc-2027), and Lamin A/C (sc-6215) from Santa Cruz Biotechnology. Secondary antibodies were all purchased from Santa Cruz Biotechnology. 3T3-L1 cells were purchased from ATCC and cultured as indicated. Primary mouse fibroblasts were established from E13.5 embryos.
For MEF differentiation, at 2 days post confluence (day 0), cells were treated with the adipogenic cocktail containing containing DMEM, FBS (10%), insulin (830 nM), dexamethasone (1 µM), 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), and rosiglitazone (5 µM). Two days later, cells were switched to maintenance medium containing DMEM, 10% FBS, and 830 nM insulin for the remaining duration of differentiation. The maintenance medium was changed every 48 hours. 3T3-L1 cells were differentiated the same way but rosiglitazone was not included in the adipogenic cocktail.
C57BL6/J females mice were mated with C57BL6/J males and plugs were checked to identify potential pregnant mice. Embryos were collected 18.5 days post-impregantion (E18.5) and pups were sacrificed 2, 5, and 12 days after birth. Subcutaneous adipose tissue was collected form the inguinal region using microdissection tools and samples were frozen in liquid nitrogen for later analysis (western blotting and qRT-PCR) or directly used for imaging. Small pieces of tissue were incubated in PBS containing Hoechst (1:1000) (33342, Invitrogen), HCS LipidTOX™ (1:2000) (Invitrogen, H34476), and isolectin GS-IB4M (1:5000) (I21411, Invitrogen) for 20 minutes. Tissues were then washed twice with PBS, mounted, and analyzed by confocal microscopy.
The mean values for biochemical data from each group were compared by Student's t test were p < 0.05 was considered significant.
We thank members of the Sabatini lab for helpful discussions. We also thank Gregor Gorjanc and Peter Juvan for help and consultations on data analyses of the polygenic mouse model. This research was supported by fellowships from the Canadian Institutes of Health Research (CIHR) and from Les Fonds de la Recherche en Santé du Québec (FRSQ) to M.L.; grants from the Slovenian Research Agency (core funding program P4-0220 and project Syntol) to S.H.; a Young Scientist Fellowship and Grant from the São Paulo Research Foundation (FAPESP 2009/15354-7 and 2010/10909-8) to W.T.F.; the Jane Coffin Childs Memorial Fund for Medical Research to K.B.; the Young researcher scholarship (Slovenian Research Agency) to Z.P.; the Human Frontier Science Program to A.E. ; and NIH grants CA103866, CA129105, and AI47389 to D.M.S. D.M.S. is an investigator of the Howard Hughes Medical Institute.
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