InteractiveFly: GeneBrief

Diabetes- and obesity regulated: Biological Overview | References

BIOLOGICAL OVERVIEW

Mammalian DOR (for 'Diabetes- and Obesity Regulated') was discovered as a gene whose expression is misregulated in muscle of Zucker diabetic rats. Because no DOR loss-of-function mammalian models are available, this study analyzed the in vivo function of DOR by studying flies mutant for Drosophila DOR (dDOR). dDOR is a novel coactivator of ecdysone receptor (EcR) that is needed during metamorphosis. dDOR binds EcR and is required for maximal EcR transcriptional activity. In the absence of dDOR, flies display a number of ecdysone loss-of-function phenotypes such as impaired spiracle eversion, impaired salivary gland degradation, and pupal lethality. Furthermore, dDOR knockout flies are lean. dDOR expression is inhibited by insulin signaling via FOXO. This work uncovers dDOR as a novel EcR coactivator. It also establishes a mutual antagonistic relationship between ecdysone and insulin signaling in the fly fat body. Furthermore, because ecdysone signaling inhibits insulin signaling in the fat body, this also uncovers a feed-forward mechanism whereby ecdysone potentiates its own signaling via dDOR (Francis, 2010).

Thyroid hormone receptor (TR) is an important regulator of development and metabolism in animals. TR is a type II nuclear hormone receptor (NR). It resides in the nucleus and binds DNA regardless of ligand binding, and it heterodimerizes with retinoid X receptor (RXR). In the absence of ligand, TR is complexed with corepressors to inhibit transcription, whereas in the presence of ligand, it binds coactivators and activates transcription. One recently discovered TR coactivator is DOR (Baumgartner, 2007). DOR was first identified as a gene that is downregulated in muscle of diabetic rats. DOR was then shown to have two functions. It acts as a coactivator of thyroid hormone receptor TR_α1, binding TR_α1 and impacting its transcriptional activity. Furthermore, DOR has a second life outside the nucleus, as a regulator of autophagy (Mauvezin, 2010; Nowak, 2009). Together, these data implicate DOR as a regulator of NR function and of metabolism. However, no DOR mutant animals have yet been reported, and the in vivo function of DOR remains to be studied (Francis, 2010).

Drosophila has 18 nuclear receptors, including ecdysone receptor (EcR). EcR shares many commonalities with type II NRs, in that it heterodimerizes with the fly RXR homolog USP, binds DNA constitutively, complexes with either coactivators or corepressors depending on its state of ligand binding, and can form a functional complex with mammalian RXR. The EcR/USP complex senses and responds to the hormone 20-hydroxyecdysone (20E) to regulate developmental timing and metabolism. 20E triggers all developmental transitions, such as the molts from one larval stage to the next, and many events occurring during metamorphosis. These include termination of larval feeding, apoptosis, and elimination of larval salivary glands and larval fat body, as well as many morphological changes in tissues that will give rise to the adult fly. Several EcR corepressors and coactivators have been identified and characterized, including Alien, SMRTER, Bonus, Trithorax-related gene (TRR), Taiman, and rigor mortis. However the coactivator(s) of EcR required for proper pupal development and metamorphosis remain to be described (Francis, 2010).

Interestingly, crosstalk has recently come to light between ecdysone signaling and insulin signaling, which regulates the growth and metabolism of animals. Ecdysone regulates insulin signaling and vice versa. In particular, in the fat body of the fly, ecdysone signaling inhibits PI3K activity and thereby insulin signaling, suggesting an antagonistic relationship between these two hormonal signaling pathways. The molecular mechanisms underlying these regulatory events, however, are not fully understood (Francis, 2010).

In order to study the function of DOR in an in vivo animal model, the Drosophila genome was searched for homologs of human DOR (hDOR). A BLAST search through all predicted Drosophila proteins with the sequence of hDOR yielded CG11347 as the top hit, which was rename Drosophila DOR (dDOR) (Francis, 2010).

The dDOR locus is predicted to encode six different transcripts, giving rise to three different polypeptides. The -RA, -RB, -RD, and -RE isoforms encode a 387 amino acid protein hereafter referred to as DOR_long, whereas the -RC isoform encodes a shorter protein, of 273 amino acids, referred to as DOR_short. The -RF isoform encodes an even shorter protein similar to DOR_short but lacking 44 amino acids at the N terminus. While performing RT-PCR with oligonucleotides specific for the long isoform, the presence of two differently sized PCR products was detected. Sequencing revealed that one of the products corresponded to the predicted 'long' isoform. The second product corresponded to an unannotated isoform consisting of the 'long' isoform plus a 90 bp extension of the third exon, resulting from use of an alternate splice donor. As a result, 30 amino acids are inserted in the middle of the dDOR_long protein. Contained in these 30 amino acids is the sequence FENLL, which is similar to the LXXLL nuclear-receptor-interacting motif found in nuclear receptor coactivators. This FENLL sequence aligns to the transactivation domain motif of human DOR (LEDLL) when the two proteins are aligned to each other. This isoform is referred to as dDOR_FENLL. The domain of dDOR_FENLL surrounding the FENLL sequence has 75% identity and 85% homology to human DOR. Three isoforms of dDOR were studied in this work (Francis, 2010).

In order to measure the relative abundance of the three isoforms in vivo, quantitative RT-PCR was performed with isoform-specific primers on RNA extracted from animals of various stages of development. The most abundant isoform is the long one, followed by the FENLL isoform (roughly half the level of the long isoform), whereas the short isoform is expressed at comparatively low levels. This relative expression of the three isoforms is also observed in fat body of wandering third-instar larvae whereas in fat body of early pupae the FENLL isoform strongly predominates. Indeed, the FENLL isoform is highly enriched in fat body of early pupae when compared to the rest of the body (Francis, 2010).

Because expression of human DOR is misregulated in rats, via an unknown mechanism, upon development of diabetes, it was asked whether expression of Drosophila DOR is also regulated by nutritional conditions. Given that the FENLL isoform of dDOR is responsible for the metabolic defects of dDOR mutants, attention was focused on the FENLL isoform. Third-instar larvae were either fasted or fed for 18 hr and then assayed dDOR_FENLL mRNA levels in fat body by quantitative RT-PCR. When control larvae were fasted, dDOR_FENLL expression in fat body increased > 2-fold. One important signaling pathway that is inhibited upon fasting is insulin. It was therefore asked whether dDOR_FENLL expression is inhibited by insulin, because this would explain its upregulation upon fasting. Explanted fat bodies were tested in the presence or absence of 5 μg/ml insulin and dDOR_FENLL expression levels were assayed by quantitative RT-PCR. In the presence of insulin, dDOR_FENLL expression decreased by 73%. dDOR_FENLL expression levels also decreased by 59% in S2 cells treated with 1 μM insulin for 2 hr (Francis, 2010).

One transcription factor mediating much of the transcriptional output of the insulin pathway is FOXO. FOXO activity is suppressed by insulin signaling. Whether regulation of dDOR_FENLL expression is mediated by FOXO was tested by studying animals containing the FOXO^21/25 null allele combination. FOXO^21/25 mutants were starved and it was found that the fasting-induced upregulation of dDOR_FENLL expression in fat body was strongly impaired, indicating that this transcriptional regulation is FOXO dependent. The transcriptional regulation of dDOR_FENLL is analogous to that of a canonical FOXO target gene, 4E-BP. 4E-BP expression is suppressed by insulin in vivo in fat bodies and increases in vivo in fat body upon fasting of wild-type animals but does not increase upon fasting of FOXO mutant animals. It was therefore asked whether dDOR is also a direct transcriptional target of FOXO. In Drosophila, FOXO targets sites are preferentially located within 1 kb of the target promoter. The dDOR promoter region was screened and a perfect consensus FOXO binding site (GTAAACAA) was found 230 nt upstream of the transcription start site of the –RA and –RB transcripts. To test whether FOXO binds this site in vivo, chromatin immunoprecipitation (ChIP) of endogenous FOXO from third-instar larvae was performed. Two negative controls were performed: a mock ChIP using preimmune serum from wild-type animals, and a ChIP using anti-FOXO antibody from FOXO^21/25 null mutant animals. Quantitative PCR (qPCR) on the immunoprecipitated material revealed that the promoter region of 4E-BP, an established direct target of FOXO, was strongly enriched in the FOXO ChIP from wild-type animals, but not in the negative controls. Strikingly, the promoter region of dDOR-RA/B was also strongly enriched in the FOXO ChIP but not in the negative controls, indicating that FOXO binds the dDOR promoter in vivo. As a negative control, the genomic region of mir-278 was not enriched in the FOXO ChIP. Together, these data indicate that expression of dDOR_FENLL is inhibited by insulin signaling as a direct target of FOXO, and identify a molecular mechanism by which insulin signaling inhibits ecdysone signaling in the fat body. Because dDOR is involved in linking nutrient signaling to EcR signaling, whether dDOR mutants have impaired fitness upon nutrient deprivation was tested. Upon removal of food (but not water), dDOR knockout animals died more rapidly than controls.

Thus dDOR functions as a novel coactivator of the ecdysone receptor that plays an important role during metamorphosis. Clearly not all EcR functions are impaired in DOR mutants. For instance, very little lethality is seen during larval stages of development, indicating that larval molts are occurring properly. It is possible that different EcR coactivators are important for different aspects of EcR signaling, for instance with rigor mortis plays an important role in the regulation of larval molts (Gates, 2004). Alternatively, because induction of EcR target genes is reduced but not completely eliminated in dDOR knockout animals, this could reflect the differential sensitivity of various biological processes to the degree of EcR activation. Future work may shed more light on this issue. Interesting in this context is that it was possible to rescue the lethality of DOR knockouts by feeding 20E. This suggests that either DOR knockouts also have low ecdysone titers due to impaired expression of E75A, which is involved in an ecdysone feed-forward production pathway, or because the elevated ecdysone titers achieved by supplying exogenous 20E allow other coactivators to compensate for DOR loss of function (Francis, 2010).

This work identifies a new link between ecdysone signaling and insulin signaling. It was previously known that ecdysone signaling inhibits insulin signaling in the fat body. This study shows, conversely, that insulin signaling also inhibits ecdysone signaling. When insulin signaling is high, FOXO activation is low and dDOR expression is low. Conversely, when insulin signaling drops, this allows FOXO to become active, resulting in elevated levels of dDOR expression and maximal activation of EcR target genes. In sum, this study found that there is a mutual antagonistic relationship between insulin signaling and ecdysone signaling in the fat body, possibly creating a system with two equilibrium states -- high ecdysone/low insulin and low ecdysone/high insulin. This makes biological sense because insulin plays an anabolic role in the fat body, whereas ecdysone plays a catabolic role, encouraging lipid mobilization and autophagy. By identifying dDOR as a direct FOXO target, this study has shed light on the molecular mechanism by which part of this antagonistic relationship is achieved (Francis, 2010).

A second consequence of the regulation of dDOR by FOXO is the creation of a feed-forward regulatory mechanism. When ecdysone signaling is activated, it inhibits insulin signaling and activates FOXO, causing increased expression of dDOR. This results in potentiation of the ecdysone signal. This type of mechanism may be important for the dramatic activation of the ecdysone pathway at the end of larval development. Indeed, ecdysone signaling has several autoregulatory positive feedback loops, including EcR-dependent transcription of the EcR gene and downregulation of a microRNA, miR-14, which inhibits EcR expression (Francis, 2010).

DOR was first identified as a gene whose expression is aberrant in Zucker diabetic rats (Baumgartner, 2007). Until DOR knockout mice are analyzed, it is possible that this aberrant regulation is either a cause or a consequence of the diabetes. Because dDOR knockout flies have reduced triglyceride and elevated glycogen stores, it is tempting to speculate that aberrant DOR expression in mammals might actually cause metabolic defects and not simply be a consequence of them. Although DOR expression was downregulated in muscle of diabetic rats, this study found a 2-fold increase in hDOR expression in adipose tissue of type 2 diabetic patients. This indicates that regulation of DOR expression -- and hence the effect on metabolism -- in conditions of metabolic disease in mammals is likely to be tissue specific and complex. The reduction in triglycerides in dDOR knockout flies is also interesting in light of the antagonistic relationship between ecdysone signaling and insulin signaling in the fly. Previous work has shown that flies with systemically reduced insulin signaling have elevated triglyceride levels. Therefore, the leanness of dDOR knockouts would be consistent with increased systemic insulin signaling in dDOR knockout animals (Francis, 2010).

Intriguingly, dDOR shares a number of features with its mammalian homolog. Like hDOR, dDOR functions as a nuclear hormone coactivator. Whereas hDOR binds TR_α1, dDOR binds EcR. TR_α1 and EcR are similar in that they both form heterodimeric complexes with RXR/USP. In fact, EcR can form a functional complex with the human USP homolog RXR in mammalian cells. Furthermore, EcR and TR_α1 both play catabolic roles in some contexts. For instance, ecdysone signaling induces autophagy and lipid mobilization in the fat body and programmed cell death in salivary glands during metamorphosis. Likewise, thyroid hormones increase basal metabolic rates, induce fat mobilization, and enhance fatty acid oxidation. A second similarity between dDOR and hDOR is that both are transcriptionally regulated by nutritional inputs. DOR expression is misregulated in diabetic rats, whereas dDOR expression changes depending on whether the animals are feeding or fasting. Because this study found that regulation of dDOR expression is insulin and FOXO dependent, this raises the possibility that the transcriptional effect on DOR in diabetic rats may also be insulin dependent. A third similarity is that both hDOR and dDOR have two separable functions -- as a nuclear hormone receptor coactivator, and as a regulator of autophagy (this work; Mauvezin, 2010; Nowak, 2009). This makes particular biological sense within the context of the fat body, where ecdysone signaling induces autophagy during metamorphosis. Therefore, the dual functions of dDOR work in parallel, both by potentiating ecdysone signaling and by interacting with the autophagy proteins Atg8a/b (Francis, 2010).

In sum, this work discovers dDOR as a novel EcR coactivator required during fly metamorphosis. Furthermore, it identifies dDOR as a novel component of a gene regulatory network integrating ecdysone and insulin signaling to regulate fly development and metabolism (Francis, 2010).

The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells

The regulation of autophagy in metazoans is only partly understood, and there is a need to identify the proteins that control this process. The diabetes- and obesity-regulated gene (DOR), a recently reported nuclear cofactor of thyroid hormone receptors, is expressed abundantly in metabolically active tissues such as muscle. This study shows that DOR shuttles between the nucleus and the cytoplasm, depending on cellular stress conditions, and re-localizes to autophagosomes on autophagy activation. DOR interacts physically with autophagic proteins Golgi-associated ATPase enhancer of 16 kDa (GATE16) and microtubule-associated protein 1A/1B-light chain 3. Gain-of-function and loss-of-function studies indicate that DOR stimulates autophagosome formation and accelerates the degradation of stable proteins. CG11347, the DOR Drosophila homologue, has been predicted to interact with the Drosophila Atg8 homologues, which suggests functional conservation in autophagy. Flies lacking CG11347 show reduced autophagy in the fat body during pupal development. All together, these data indicate that DOR regulates autophagosome formation and protein degradation in mammalian and Drosophila cells (Mauvezin, 2010).

The Drosophila genome contains two homologues of Atg8 (Atg8a and Atg8b). dDOR is predicted to interact with Atg8a and Atg8b by high-throughput two-hybrid screening, thereby suggesting that the interaction between DOR and Atg8 is conserved from humans to flies (Mauvezin, 2010).

To test whether dDOR controls autophagy during development, three fly strains from the Vienna Drosophila RNAi Center (VDRC) were used: two strains harbouring inducible RNAi expression constructs against the third exon of dDOR (T1 and T4) and one against the fourth exon (T2). Similar results were obtained with all three strains. By crossing the T4 RNAi line to the ubiquitous tubulin-GAL4 driver, in order to express the RNAi construct in the entire animal, and extracting RNA from the resulting larvae, it was confirmed that the RNAi constructs reduced dDOR expression levels by 95% compared with that of control flies. Larvae in which dDOR was knocked down pupated normally and without delay, but died later as pupae (Mauvezin, 2010).

During insect metamorphosis, larval tissues such as the salivary gland, fat body and midgut undergo precisely timed periods of autophagy. As dDOR expression by quantitative PCR is much higher in the fat body than in the salivary gland, the fat body was chosen as a model system to study autophagy, and Lysotracker was used as a reporter. At the end of larval development, third-instar larvae cease eating, leave the food and start wandering to find a dry place for pupation. During this wandering stage, autophagy is induced to high levels in the larval fat body and it has been proposed that it participates in the removal of this tissue by programmed cell death events that occur during metamorphosis. In agreement with previous observations, this study detected numerous Lysotracker-positive puncta in the fat body of control larvae at the wandering stage, just before the onset of pupation. By comparison, few puncta were observed in control larvae in early third instar, when they were still feeding, and there was no activation of autophagy. Compared with control animals, wandering third-instar dDOR knockdown animals showed a 40% decrease in Lysotracker-positive dots in the fat body. Moreover, electron microscopy studies showed an abundance of autophagosomes and autolysosomes in the fat bodies of wandering third-instar control animals, whereas a strong reduction was detected in the dDOR knockdown group. Together, these data indicate that dDOR is required in Drosophila for developmentally regulated autophagy at the onset of metamorphosis (Mauvezin, 2010).

It is proposed that DOR is a new regulator of autophagy in mammalian and Drosophila cells. The observations that support this idea are as follows: (1) mammalian DOR interacts with LC3 and GATE16, two homologous proteins that participate as lipidated forms in the formation of autophagosomes in mammalian cells; (2) DOR does not colocalize with LAMP1 protein or with Lysotracker, thereby indicating that it is a marker of early autophagosomes; (3) DOR does not localize with autophagosomes when HeLa cells are treated with chloroquine, thereby indicating that its role is restricted to induced autophagy; (4) DOR gain-of-function enhances the abundance of autophagosomes and accelerates protein degradation in HeLa cells; (5) DOR loss-of-function reduces autophagosome formation and protein degradation in muscle cells; and (6) DOR loss-of-function represses autophagy of the fat body during Drosophila pupation (Mauvezin, 2010).

These data also indicate that DOR protein localization is sensitive to cellular stress. In particular, activation of autophagy by either amino-acid starvation or rapamycin treatment -- the inhibition of the mammalian target of rapamycin (mTOR) -- drives DOR into autophagosomes in a reversible manner. On this basis, it is proposed that the shuttling of DOR between the nucleus and cytosol is mTOR activity-dependent. Interestingly, the effect of amino-acid starvation on the efflux of DOR from the nucleus was abolished in the presence of 3-methyladenine, an inhibitor of class III phosphatidylinositol 3-kinase (Vps34), which indicates the existence of a functional crosstalk between mTOR and Vps34 in the regulation of the subcellular localization of DOR. In mammals, Vps34 participates in a complex that is essential for an early nucleation step in autophagosome formation. The interaction between Vps34 and mTOR is complex and poorly understood, and although Vps34 is required for insulin-stimulated and amino-acid-stimulated activation of mTOR, it also stimulates autophagy (Mauvezin, 2010 and references therein).

The DOR protein has a short half-life and undergoes degradation through the proteasome. A functional interaction between autophagy and proteasomal activities has recently been reported. On the basis of this observation, it is proposed that DOR is one of the proteins that control autophagy and is regulated by proteasomal activity (Mauvezin, 2010).

These results show that DOR is a bifunctional protein that operates both in the nucleus and in the cytosol. In the former, DOR acts as a nuclear co-factor, and binds to and co-activates the thyroid hormone receptor TRα1. In the latter, DOR participates in autophagosome formation and, consequently, causes enhanced autophagy and accelerated protein degradation. It is therefore proposed that DOR is a component of the regulatory machinery of autophagy in metazoans (Mauvezin, 2010).

Search PubMed for articles about Drosophila Dor

Baumgartner, B. G., et al. (2007). Identification of a novel modulator of thyroid hormone receptor-mediated action, PLoS ONE 2: e1183. PubMed ID: 18030323

Francis, V. A., Zorzano, A. and Teleman, A. A. (2010). dDOR is an EcR coactivator that forms a feed-forward loop connecting insulin and ecdysone signaling. Curr. Biol. 20(20): 1799-808. PubMed ID: 20888228

Gates, J., Lam, G., Ortiz, J. A., Losson, R. and Thummel, C. S. (2004). rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development. Development 131: 25-36. PubMed ID: 14645129

Mauvezin, C., et al. (2010). The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells. EMBO Rep. 11: 37-44. PubMed ID: 2001080

Nowak, J., et al. (2009). The TP53INP2 protein is required for autophagy in mammalian cells. Mol. Biol. Cell 20: 870-881. PubMed ID: 19056683

date revised: 10 July 2021

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