If the Foxo protein is a bona fide ortholog of mammalian FOXO, insulin should regulate its activity by phosphorylation via a cascade involving Akt (Brunet, 1999; Kops, 1999). Also, phosphorylation of the three specific serine/threonine residues should sequester this transcription factor in the cytoplasm. To test these properties, S2 cells were grown with or without insulin, and endogenous Foxo was detected by Western blot analysis. A single band migrating with molecular mass 113 kD was recognized by both N- and C-terminal Foxo antibodies in the untreated samples. In contrast, the insulin-treated sample revealed the presence of two bands, one migrating with the mobility of 113 kD and a second band with slower mobility. A similar insulin-induced shift was obtained with transfected Foxo-V5 expressed in S2 cells and detected with a V5 antibody. These results suggest that the slower-migrating band may correspond to a phosphorylated form of Foxo (Puig, 2003).
To establish that the slower-migrating form of Foxo induced by insulin treatment is indeed caused by Akt-catalyzed phosphorylation, a mutant form of Foxo was constructed in which all three putative dAkt phosphorylation sites (T44, S190, and S259) were mutated to alanine (FoxoA3). Both wild-type (Foxo-V5) and mutant (FoxoA3-V5) proteins were expressed in S2 cells. After transient expression, the cells were subjected to three different treatments in parallel: insulin; pretreatment with LY294002 (a specific inhibitor of PI3K that counteracts the effects of insulin) followed by insulin treatment, or no treatment control. Extracts derived from cells treated with insulin contained the slower-migrating form of wild-type Foxo when compared with control cells. Pretreatment with the PI3K inhibitor LY294002 reduced the amount of the slower-migrating form of Foxo. In contrast, no slower-migrating species was observed for the triple alanine mutant (FoxoA3) when comparing control, insulin-treated, and LY294002 + insulin-treated samples. To further confirm that the slower-migrating form of Foxo is caused by phosphorylation, cell extracts were incubated with calf intestinal phosphatase (CIP). Western blot analysis showed that the slower-migrating form of Foxo is quantitatively converted to the 113-kD form after CIP treatment. Together, these results indicate that Foxo is phosphorylated by insulin treatment and that this phosphorylation depends on the presence of the dAkt consensus residues T44, S190, and S259 (Puig, 2003).
To test how Foxo subcellular localization is affected by insulin-mediated phosphorylation, S2 cells expressing either wild-type Foxo or mutant FoxoA3 were incubated for 48 h in the absence of serum. Then insulin was added, and localization of transfected Foxo was determined by confocal microscopy after staining with the V5 antibody. When S2 cells are incubated in the absence of serum and insulin, both Drosophila Foxo and dFoxoA3 (constitutively active Drosophila Foxo in which all three putative Akt phosphorylation sites have been mutated to alanine) are found predominantly in the nucleus. After insulin treatment, Foxo is localized in the cytoplasm. In contrast, mutant FoxoA3 remains nuclear even after insulin treatment. This result is consistent with the idea that subcellular localization of Foxo is regulated by insulin (Puig, 2003).
Is Foxo phosphorylation regulated through the PI3K/Akt pathway? A constitutively active form of Drosophila Akt was used in which a myristoylation signal has been fused to the N terminus of Akt. Myr-Akt tagged with V5 epitope was cotransfected in S2 cells grown in the absence of serum and insulin with either Foxo or dFoxoA3, and the phosphorylation state of both proteins was analyzed by Western blot analysis. In the absence of Akt, both Foxo and dFoxoA3 remain unphosphorylated. When Myr-Akt is present in the cells, Foxo but not dFoxoA3 becomes phosphorylated even in the absence of insulin. This result indicates that Myr-dAkt can phosphorylate Foxo in S2 cells. To assess the effect of Foxo phosphorylation by Myr-Akt, use was made of a reporter construct containing four tandem FOXO4-binding sites upstream of the alcohol dehydrogenase distal core promoter driving the luciferase gene (pGL4xFRE). In the absence of Myr-Akt, cells cotransfected with wild-type or mutant Foxo constructs incubated without serum display comparable luciferase activity. In contrast, when Myr-Akt is present, cells cotransfected with wild-type Foxo display luciferase activity that is reduced by more than 65%, whereas activity of the mutant FoxoA3 remains essentially unchanged (Puig, 2003).
These results suggest that insulin induces Foxo phosphorylation through Akt, which leads to cytoplasmic localization and transcriptional inactivation of Foxo. To further confirm that insulin inhibits Foxo activity through Akt, RNAi experiments were performed. S2 cells transfected with either Foxo or FoxoA3 and cotransfected with the luciferase reporter pGL4xFRE were grown in the presence of insulin and treated with dsRNA directed against Drosophila Akt. As a control, dsRNA against lactose repressor (lacI) was used. As expected, Foxo activity is not inhibited by insulin when cells are depleted of Drosophila Akt by dsRNA treatment, but it is inhibited in the lacI control. These results confirm that Akt mediates insulin inhibition of Foxo (Puig, 2003).
The insulin signaling pathway evolved to allow a fast response to changes in nutrient availability while keeping glucose concentration constant in serum. This study shows that, both in Drosophila and mammals, insulin receptor (InR) represses its own synthesis by a feedback mechanism directed by the transcription factor dFOXO/FOXO1. In Drosophila, dFOXO is responsible for activating transcription of dInR, and nutritional conditions can modulate this effect. Starvation up-regulates mRNA of dInR in wild-type but not dFOXO-deficient flies. Importantly, FOXO1 acts in mammalian cells like its Drosophila counterpart, up-regulating the InR mRNA level upon fasting. Mammalian cells up-regulate the InR mRNA in the absence of serum, conditions that induce the dephosphorylation and activation of FOXO1. Interestingly, insulin is able to reverse this effect. Therefore, dFOXO/FOXO1 acts as an insulin sensor to activate insulin signaling, allowing a fast response to the hormone after each meal. These results reveal a key feedback control mechanism for dFOXO/FOXO1 in regulating metabolism and insulin signaling (Puig, 2005).
It is well known that the expression and activity of the InR can be regulated by a wide variety of factors and that changes in the numbers of receptor molecules plays a pivotal role in several physiologic and pathologic states. The lowered sensitivity of cells to insulin and the hyperinsulinemia observed in obesity and type II diabetes mellitus is often accompanied by a reduced number of insulin receptors. Insulin is thought to down-regulate its own receptor by a variety of mechanisms that can influence its synthesis as well as degradation. Interestingly, it has been shown that the number of InR molecules correlates with nutritional conditions both in tissue culture cells and in animals. Thus, levels of InR in growing HepG2 cells are relatively low, and they increase substantially if cells are starved. In addition, states of chronic hyperinsulinemia produce a reduction in the number of InR present in the plasma membrane. InR mRNA levels also change in animals depending on fasting-feeding conditions. For example, rats fed a high-fat diet display a decreased number of InR molecules in liver plasma membranes, and InR mRNA levels in rat skeletal muscle or liver increase after fasting, returning to normal levels after insulin treatment or refeeding. Interestingly, tissues other than muscle or liver might have similar regulation. For example, mRNA and protein levels of rat intestinal InR increase up to 230% in fasting conditions, and these effects are fully reversed by refeeding. Similar observations have been made in other organisms. These effects indicate a nutritional influence on the abundance of the InR. Importantly, insulin levels in serum change in parallel to nutrient availability, both in flies and mammals. Thus, when nutrients are high—that is, after a meal—insulin levels increase, while they decrease upon fasting. In Drosophila it has been shown that the InR/PI3K pathway coordinates cellular metabolism with nutritional conditions. Inhibiting this pathway phenocopies the cellular and organismal effects of starvation, while activating it bypasses the nutritional requirements for cell growth. The InR/PI3K pathway regulates the activity of FOXO1 in mammals, C. elegans, and Drosophila, so nutrient activation of the PI3K pathway results in inactivation of FOXO1 by phosphorylation. However, despite this accumulated base of information, the molecular mechanism linking FOXO1 and InR expression had not been revealed (Puig, 2005).
This study shows that mammalian FOXO1 and its Drosophila counterpart dFOXO directly regulate insulin-signaling response to nutritional conditions through a feedback mechanism that involves activation of transcription from the InR promoter. Incubating C2C12 cells with a balanced salt solution or with serum-free medium up-regulates insulin receptor mRNA. Under these conditions, FOXO1 becomes dephosphorylated and actively binds to the InR promoter. When insulin is added to the medium, InR mRNA is down-regulated, even in the absence of serum, vitamins, amino acids, and glucose. Concomitantly, phosphorylation of FOXO1 increases and binding to InR promoter decreases. These results indicate that FOXO1 regulates InR transcription through a direct feedback mechanism that senses insulin levels in serum, which is, in turn, a reflection of nutrient load. It is important to note that, at this point, it cannot be ruled out that the increased InR protein levels caused by FOXO1 could be due to other mechanisms in addition to increased transcription from the InR promoter (i.e., affecting mRNA stability, or protein translation) (Puig, 2005).
In Drosophila a similar mechanism occurs. Incubation of S2 cells with complete medium keeps dFOXO phosphorylated and inactive, while incubation in HBSS dephosphorylates dFOXO. dInR mRNA is up-regulated only when dFOXO is dephosphorylated and active. In addition, wild-type flies starved for 4 d up-regulate dInR, and this effect requires an intact dfoxo gene. These studies indicate that in Drosophila, the PI3K/Akt pathway also senses insulin levels and regulates binding of dFOXO to the dInR promoter accordingly. These results underscore the importance of the InR/PI3K/Akt pathway in sensing nutrients and insulin, a function that has been conserved during evolution. They also highlight the role of FOXO1 as a sensor for insulin levels, promoting accumulation of InR in the absence of insulin, thereby allowing a fast response to the hormone after each meal. Under conditions in which insulin levels are chronically elevated, for example, in obese animals or patients, down-regulation of InR transcription would occur and insulin sensitivity would be impaired. These results establish the FOXO1 transcription factor as a key player in a feedback control mechanism that regulates metabolism and insulin signaling (Puig, 2005).
The results show that in conditions in which insulin levels are low, mammalian FOXO1 activates InR. Interestingly, it was observed that FOXO1 also activates the insulin receptor substrate-2 (IRS-2) promoter under fasting conditions, and, since it occurs with InR, insulin is sufficient to reverse this effect. FOXO1 binds IRS-2 promoter in vitro and in vivo and activates IRS-2 transcription when muscle or liver cells are fasted. In addition, FOXO1 activates IRS-2 promoter in luciferase assays, and this activation depends on the presence of a consensus FRE present in the IRS-2 promoter, because mutating this FRE abolishes FOXO1-dependent activation. Thus, FOXO1 regulation of IRS-2 is parallel to InR regulation. It has also been reported that SREBPs compete with FOXO transcription factors for binding to the IRS-2 promoter in liver; while SREBPs inhibit IRS-2 production, FOXO1 was found to activate IRS-2 transcription. It was also found that fasting promotes binding of FOXO1 to the FRE of the IRS-2 promoter. Therefore, these findings strongly support the conclusions that FOXO1 regulates insulin signaling through a feedback mechanism that impinges on the insulin receptor and at least one of its substrates, IRS-2. After a meal, high levels of insulin peptide hormone activate its cognate receptor, which leads to repression of InR and IRS-2 transcription, resulting in subsequent dampening of the pathway by reducing the number of receptors on the cell surface and by limiting its ability to signal downstream through IRS-2. Conversely, fasting causes reduced levels of InR signaling, which in turn activates FOXO1, leading to increased transcription of InR and IRS-2. Once this transcription mechanism is activated, feedback regulation and phosphorylation of FOXO1 via the insulin signaling cascade automodulates InR expression. Insulin sensitivity could, therefore, be significantly affected by FOXO1 regulation. Regulation of insulin sensitivity by a feedback loop through FOXO1 would allow the cells to keep an exquisite metabolic balance between feeding and fasting states, permitting a faster response of the tissues to insulin changes. This feedback mechanism could well be disrupted in pathological states with abnormally increased insulin levels as is found in the case of insulin-resistant diabetes (Puig, 2005).
Reducing insulin/IGF signaling allows for organismal survival during periods of inhospitable conditions by regulating the diapause state, whereby the organism stockpiles lipids, reduces fertility, increases stress resistance, and has an increased lifespan. The Target of Rapamycin (TOR) responds to changes in growth factors, amino acids, oxygen tension, and energy status; however, it is unclear how TOR contributes to physiological homeostasis and disease conditions. This study shows that reducing the function of Drosophila TOR results in decreased lipid stores and glucose levels. Importantly, this reduction of TOR activity blocks the insulin resistance and metabolic syndrome phenotypes associated with increased activity of the insulin responsive transcription factor, FOXO. Reduction in TOR function also protects against age-dependent decline in heart function and increases longevity. Thus, the regulation of TOR activity may be an ancient 'systems biological' means of regulating metabolism and senescence, that has important evolutionary, physiological, and clinical implications (Luong, 2006).
The major cause of metabolic syndrome (defined as a cluster of metabolic abnormalities such as elevated glucose and lipid levels, related to a state of insulin resistance) and diabetes in humans is reduction of insulin signaling, but the underlying pathways and mechanisms are not completely understood. Likewise, caloric excess can lead to nutrient toxicity and desensitization of insulin signaling. Thus, dysregulation of energy homeostasis can lead to metabolic disturbances and predisposition to a variety of endocrine diseases including diabetes, cardiovascular disease, and cancer (Luong, 2006).
One major system that regulates energy homeostasis in higher metazoa is the insulin/IGF pathway. The functionally conserved components of the insulin/IGF pathway like insulin, the insulin receptor (InR), insulin receptor substrate (IRS), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB, a.k.a. Akt) and the forkhead transcription factor FOXO have been shown to be involved in glucose and lipid homeostasis. Loss of insulin signaling in the periphery and in pancreatic β cells can lead to hyperglycemia and diabetes. For example, disruption of the InsR gene in the pancreatic β cells reduces islet size and insulin secretion. IRS1 knockout mice are hyperglycemic, but their pancreatic β cells hypertrophy to compensate for increased peripheral insulin resistance. In contrast, IRS2 knockout mice are diabetic because their pancreatic β cells are absent due to increased cell death. Additionally, systemic loss of insulin signaling in metazoans leads to elevated lipids as seen in the Daf-2 mutant worms, Chico/IRS mutant flies, and IRS2 ablated mice (Luong, 2006 and references therein).
Many of these insulin/IGF-mediated metabolic effects depend on the winged helix transcription factor, FOXO. FOXO was first identified in the worm, C. elegans as Daf-16, a mutation that can suppress the increased lipid levels and longevity caused by loss of Daf-2, the worm InR ortholog. There is a single evolutionarily conserved Drosophila FOXO ortholog and three mammalian FOXO genes (FOXO1, FOXO3a, and FOXO4). FOXO1 controls glucose homeostasis in both peripheral tissues and pancreatic β cells. For example, expression of a constitutively activated FOXO1 (resistant to insulin/IGF-mediated inactivation) in liver and pancreatic β cells causes hepatic insulin resistance and loss of pancreatic β cells via increased apoptosis, whereas reduction of FOXO1 function can reverse the loss of pancreatic β cells and hyperglycemia seen in the IRS2 ablated mice. Thus, FOXO is a critical mediator of insulin signaling in both insulin sending and receiving tissues (Luong, 2006).
The Tuberous Sclerosis Complex (TSC1-2)/Target of Rapamycin (TOR) pathway responds to changes in insulin/IGF levels, amino acid levels, energy charge, lipid status, mitochondrial metabolites, and oxygen tension by adjusting cell growth. In addition to its well-defined role in controlling cell growth, the TSC1-2/TOR pathway may also potentially be a critical regulator of glucose and lipid homeostasis as TSC1-2/TOR signaling functionally interacts with the insulin/IGF pathway. A role for TOR signaling in glucose and lipid homeostasis in mammalian systems is demonstrated by the S6K1 knockout mice. These mice are hyperglycemic caused by diminished insulin secretion due to reduced pancreatic β cell mass. This result is in keeping with studies that show that rapamycin treatment leads to decreased levels of translation, growth, and survival of pancreatic. However, the mS6K1 mutant mice have low lipid levels because of adipocytes that have increased fatty acid β-oxidation. Additionally, the mS6K1 mutant mice show enhanced glucose uptake upon exogenous insulin addition due to insulin hypersensitivity in peripheral tissues via loss of a negative feedback loop on IRS. Thus, TOR signaling via S6K can modulate insulin sensitivity by altering Ser307 and Ser636/639 phosphorylation and IRS protein levels (Luong, 2006).
There are additional levels where TSC1-2/TOR signaling may positively and negatively regulate insulin signaling. There are data that suggest that the IRS Ser302 site is required for signaling to TOR and S6K. Thus, ser/thr phosphorylation of the IRS proteins may mediate both positive and negative signals for energy homeostasis. Furthermore, Akt/PKB activity may also be directly regulated by the nutrient-sensitive TOR pathway. Although the insulin/IGF pathway can signal to the TSC1-2/TOR pathway, recent evidence suggests that TOR may directly control Akt/PKB function because Akt/PKB activation depends on TORC2 complex-specific TOR Ser473 phosphorylation of Akt/PKB. Thus, these studies suggest that dysregulation of TSC1-2/TOR signaling may contribute to the pathological progression of metabolic syndrome and diabetes, yet the direct role and function of TOR is unclear in this context (Luong, 2006).
Another functionally conserved energy homeostatic pathway is the AMP-activated protein kinase (AMPK) pathway. This pathway responds to altered energy states caused by cellular stresses like mitochondrial dysfunction, anti-diabetic drugs, and exercise. Activation of the energy sensing AMPK pathway by activated AMPK as well as metformin or AICAR treatment results in decreased lipogenesis and gluconeogenesis via both central. Activated AMPK can phosphorylate TSC2, which inhibits TOR signaling, while loss of AMPK activity causes an increase in TOR signaling. However, the requirement of TOR function for the AMPK energy response is not known. These effects may also be mediated by targets including glycogen synthase, hormone-sensitive lipase, acetyl-CoA carboxylase-2, HMG-CoA reductase, p300, and p53; the different roles of these proteins in the AMPK-mediated low energy response are not well known. Furthermore, activation of AMPK leads to IRS Ser-789 phosphorylation and enhancement of insulin signaling, which suggests that the AMPK response can act separately from the TOR pathway to enhance insulin signaling. Clearly, there is a great need to understand the regulation of TSC1-2/TOR signaling as it relates to the maintenance of energy homeostasis because TOR function is implicated in both insulin/IGF and AMPK signaling (Luong, 2006).
Although TOR occupies a central node that governs catabolic or anabolic responses to different nutritional and energy states, the resultant metabolic effects of altering TOR function in a metazoan are incompletely and poorly understood. This study examines in detail the function of Drosophila TOR in terms of energy homeostasis and senescent responses. Reduction of TOR function is show to result in decreased glucose and lipid levels with concomitant increase of DILP2 from the insulin producing cells. A reduction of TOR function can block activated FOXO-mediated insulin resistance and metabolic syndrome phenotypes. Taken together, these data indicate that TOR function is required for the maintenance of energy homeostasis and organismal senescence. The additional ramifications of this study are that reduction of TOR function may have clinical utility for treating metabolic syndrome and insulin resistance (Luong, 2006).
In contrast to the elevated lipid levels caused by reduction of systemic insulin signaling, the dTOR7/P mutant (containing a P-element insert into TOR) does not show increased lipid levels. Instead, the dTOR7/P mutant shows decreased lipid levels of the fat body that depend on the function of a lipase involved in lipid metabolism. Elevated ketone bodies were observed in the hypoglycemic dTOR7/P mutant, which is indicative of the increased utilization of lipids. Studies in mammalian cardiac tissue have shown that ketone bodies provide their high energy electrons directly to complex I, the NADH dehydrogenase multienzyme complex, of the mitochondrial electron transport chain. Thus, the altered lipid levels show that TOR has a critical role in determining the fate of fats (Luong, 2006).
It has also been shown that 4EBP is involved in lipid metabolism because the increased lipid levels caused by rapamycin treatment are blocked by a 4EBP mutant. Furthermore, loss of the melted mutant has lower lipid levels, due to lowered triglyceride production. This effect is due to increased 4EBP protein levels via FOXO activation in the fatbody. However, there is no change in glucose levels. In this respect, the melted mutant resembles the FIRKO mouse because it shows decreased triglyceride levels without a change in glucose levels. The dTOR7/P mutant has a different lipid phenotype than the one caused by rapamycin treatment, which suggests that rapamycin alters TOR function in a different manner than the dTOR7/P mutant. Additionally, a novel hypomorphic dTOR FAT domain allele in combination with the dTORP allele also shows low glucose and lipid levels, which suggests that partial reduction of TOR activity represents a unique phenotypic class of TOR metabolic effects versus an allele specific phenotype. Although rapamycin affects TORC1 directly, TORC2 may be altered indirectly via TOR depletion and blocking of TORC2 assembly. Thus, it is not currently clear if the effects of rapamycin are due to inhibiting TORC1 and/or TORC2. It is known that rapamycin can impair pancreatic β cell function because it causes decreased growth and survival. Thus, rapamycin treatment can lead to elevated glucose and lipid levels, possibly as a result of systemic insulin loss (Luong, 2006).
Evidence shows that DILP2 levels are increased in the IPCs of the dTOR7/P mutant and the dTOR7/P mutant has lowered glucose levels. Thus, reduction of TOR function can lead to increased DILP2 levels and a reduction of glucose levels. Recent studies with the Drosophila miRNA-278 mutant also showed elevated DILP levels, yet displayed fatbody-mediated insulin resistance as shown by elevated d4EBP and glucose levels. It is believed that the dTOR7/P mutant represents an insulin-sensitized state because the dTOR7/P mutant shows decreased levels of the insulin resistance marker 4EBP, the dTOR7/P mutant shows decreased glucose levels, and, as discussed below, the dTOR7/P mutant blocks activated FOXO-mediated insulin resistance phenotypes. Thus, the dTOR mutant phenotype resembles a whole-animal 'insulin-sensitized' state that can function below the level of constitutive FOXO activity (Luong, 2006).
Overexpression of activated FOXO in peripheral and IPC tissues results in elevated glucose and lipid levels. Although TOR signaling can alter insulin signaling upstream of FOXO, reducing TOR function is able to reverse these effects. Thus, the results show that reduction of TOR activity can block the activated FOXO-mediated insulin resistance and metabolic syndrome phenotypes. These results suggest that strategies to dampen, reduce, or block TOR signaling may be able to overcome insulin resistance (i.e., hyperglycemia and hypertriglyceridemia) below the level of increased FOXO activity in mammalian systems (Luong, 2006).
Although FOXO has >100 potential targets that might contribute to the metabolic phenotype, this study identified Fatty acid synthase (FAS) and DILP2 as candidate mediators of the TOR effect on the FOXO metabolic phenotypes. The effect on FAS is interesting because it is upregulated by FOXO overexpression and in an IRS/chico mutant and may be an important determinant of the lipid levels. It has also been shown that activation of daf-16/FOXO can decrease the mRNA levels of a worm insulin gene, ins-7 . This result is consistent with results showing that DILP2 mRNA levels are decreased and reducing TOR activity can reverse this FOXO-mediated reduction of DILP2. These results might have parallels with a role for FOXO and TOR in the regulation of insulin levels in mammals (Luong, 2006).
A selective and unexpected regulation of TOR effectors is also seen: loss of 4EBP protein and a mild effect on S6K Ser389 phosphorylation. It has been recently shown that the 4EBP gene is a target of FOXO in Drosophila and thus may represent one of the TOR targets responsible for contributing to the FOXO-mediated metabolic phenotypes. It has also been shown that daf-15/Raptor is a target of daf-16/FOXO in C. elegans and may also contribute to the TOR metabolic and senescent phenotypes. Raptor may also account for the selective difference in the regulation of 4EBP and S6K function by TOR because Raptor binds to both S6K and 4EBP and loss of 4EBP may allow for more S6K binding to Raptor for TOR-mediated phosphorylation. Thus, these results suggest that reduction of TOR function may have selective effects on translation (Luong, 2006).
Reduction of TOR function does not provide resistance against acute stresses or cause sterility. This result is in contrast to the yeast TOR1 mutant, which shows elevated stress resistance, and the d4EBP mutant, which shows stress and starvation sensitivity. Nevertheless, the dTOR7/P mutant has an increased lifespan. This result is in keeping with the yeast, worm and fly studies that show that loss of TOR signaling can increase lifespan, as a major mediator of caloric restriction. Thus, alterations of TOR signaling contribute to the regulation of lifespan (Luong, 2006).
It is also seen that reduction of TOR activity prevents age-dependent functional decline of heart performance. It is not currently clear how TOR is regulating these organ and organismal responses, but the altered lipid metabolism may underlie these changes. For example, changes in lipid metabolism can both autonomously and non-autonomously affect heart function. Thus, reduction of TOR function may reallocate energy stores preferentially for the control of ‘long-term’ responses such as lifespan and organ maintenance. Importantly, there are many potential links between changes in energy homeostasis with alterations in aging and organ senescence. Channelling diverse stimuli like amino acids, growth factors, oxygen tension, and energy charge into the TOR pathway may be an economic method to mobilize fuel stores like lipids to counteract these fluctuations (Luong, 2006).
The conservation of basic mechanisms between Drosophila and mammals is well established. It has been shown that disruption of insulin signaling in non-mammalian systems like Drosophila results in altered glucose and lipid levels. Reducing TOR function can reverse activated FOXO-mediated insulin resistance phenotypes induced in both insulin producing and insulin receiving tissues, and thus this study provides the first direct evidence that reducing TOR function may have a clinical benefit to counter insulin resistance, metabolic syndrome, and/or diabetes. Furthermore, altering TOR signaling may underlie the benefits of various diet and nutritional regimens. These results demonstrate the utility of using the powerful genetics of this system to unravel the complex pathways involved in maintaining glucose and lipid homeostasis. In unraveling the complex genetic network of TOR and InR signaling, although far from completion, the Drosophila model has been indispensable in finding critical components and uncovering functionally important genetic interactions between these two pathways. Thus, the basic mechanisms controlling glucose and lipid homeostasis, including mechanisms by which the TSC1-2/TOR pathway influences insulin signaling as well as the influence of TSC1-2/TOR signaling on peripheral tissue and IPC physiology, are also functionally conserved (Luong, 2006).
This study has described a new use for reducing TOR activity to block insulin resistance, metabolic syndrome, and diabetic-like phenotypes downstream of activated FOXO, underlining the utility of the Drosophila model to identify and analyze components and compounds that block insulin resistance and metabolic syndrome phenotypes as well as pathological aspects of aging and organ senescence (Luong, 2006).
Foxo regulates cell cycle arrest possibly by transcriptionally activating genes implicated in cell division or in cell growth. As an initial attempt to identify target genes of Foxo, DNA microarrays were used to assess gene expression profiles in S2 cells stably transfected with mutant Foxo and grown in the presence of insulin. Cells expressing wild-type Foxo or untransfected S2 cells subjected to the same treatment were assayed as controls (Puig, 2003).
Two-hundered and seventy-seven genes were found to be up-regulated in Foxoa3-expressing cells when compared with Foxo-expressing cells or untransfected S2 cells. Interestingly, two genes that were consistently and specifically up-regulated in these conditions were the Drosophlia InR gene (13.5-fold) and the Drosophila 4EBP/Thor gene (25-fold). Both genes have been implicated in the regulation of cell growth by insulin. To confirm that InR and 4EBP are bona fide transcriptional targets of Foxo, the same experiment described above was performed but in the presence of cycloheximide to inhibit translation. As expected, both InR and 4EBP continue to be transcriptionally activated (2.5- and 3.1-fold, respectively) by FOXOA3 but not Foxo in the insulin-repressed state. This result suggests that Foxo, when released from control by the insulin/dAkt cascade, is involved in transcription from the InR and 4EBP promoters (Puig, 2003).
To confirm these microarray results and to independently quantitate the increase in mRNA transcription, RNase protection assays were performed with mRNAs extracted from cells stably transfected with either Foxo or FoxoA3. Indeed, FoxoA3 stimulates transcription of Drosophila 4EBP and InR by 16.3- and 11-fold, respectively. A time-course experiment confirmed that Drosophila InR mRNA increases rapidly upon FoxoA3 expression: 3 h after CuSO4 addition, there is already an 8-fold increase, reaching 20-fold after 9 h of CuSO4 induction. Similar results were obtained for Drosophila 4EBP. These experiments suggest that Foxo expression specifically activates both Drosophila InR and 4EBP transcription, thus unmasking an important feedback control mechanism in this pathway involving Foxo and InR (Puig, 2003).
Having obtained evidence that exogenously transfected Foxo responds to insulin and regulates both the downstream target gene 4EBP and the feedback control target InR, it was of interest to know if endogenous Foxo would also activate transcription of these genes. The PI3K inhibitor LY294002 was used to activate endogenous Foxo or insulin to deactivate it. S2 cells grown in the absence of serum for 48 h were treated either with LY294002 or insulin. Total RNA was extracted and RNase protection was performed to detect Drosophila InR and 4EBP mRNAs. Both mRNA levels are significantly increased after LY294002 treatment (5.3-fold for dInR and 4-fold for d4EBP) when compared with insulin treatment. This result provides further evidence indicating that the PI3K–Akt pathway regulates InR and 4EBP transcription via Foxo (Puig, 2003).
It was of interest to determine whether Foxo directly binds to the promoters of Drosophila 4EBP and InR. To identify the DNA region recognized by Foxo in these two promoters, a 1708-bp fragment of the 4EBP promoter and a 1562-bp fragment of the InR promoter were inserted into a luciferase reporter vector. When transfected into S2 cells, these fragments responded to Foxo activation (3-fold for 4EBP, >200-fold for InR. A series of deletions lacking upstream sequences still responded to Foxo activation, albeit more weakly, suggesting that Foxo can bind the DNA in a region close to the start of transcription (485 bp for the d4EBP promoter and 194 bp for the dInR promoter). In contrast, Foxo completely fails to activate a reporter construct in which upstream activating sequences (UAS) for the transcription factor GAL4 are fused to the luciferase gene, confirming that transcription activation is specific for both 4EBP and InR promoters (Puig, 2003).
Interestingly, 125 bp upstream of the transcription start site of the d4EBP promoter there are three tandem copies of a putative FOXO4 recognition element (FRE). These elements are reminiscent of the ones present in the human glucose-6-phosphatase promoter, previously shown to bind FOXO4 (Yang, 2002). This was reassuring because Foxo and FOXO4 share 85% identity in the core of the forkhead DNA-binding domain. Similarly, several putative FRE sequences appear in the InR promoter in the region comprising nucleotides -1434 to -70 (Puig, 2003).
To determine whether Foxo binds these putative FREs, band shift experiments were performed with a 113-bp DNA probe encompassing the 4EBP FRE motifs and with 12 separate DNA probes (ranging from 100 to 150 bp) spanning a region of 1.4 kb from the InR promoter. Purified recombinant Foxo expressed in Escherichia coli efficiently binds the 113-bp FRE-containing fragment from the 4EBP promoter compared with control DNA fragments. Furthermore, Foxo binding to the 4EBP promoter fragment can be efficiently competed with an unlabeled 113-bp 4EBP promoter fragment but not with nonspecific DNA. Similarly, purified recombinant Foxo binds efficiently to 5 out of 12 of the DNA fragments located within the InR promoter. As expected, each of the five DNA fragments bound by Foxo contains putative FREs. Thus, Foxo can specifically bind to both promoters in vitro. To determine whether Foxo also binds these same DNA regions in vivo, chromatin immunoprecipitation (ChIP) experiments were performed with S2 cells expressing either Foxo or dFoxoA3. Cells were incubated with serum, and Foxo expression was induced with the addition of CuSO4. After 6 h, cells were cross-linked with formaldehyde, and extracts were prepared and immunoprecipitated. After reversal of cross-links, DNA was recovered, and PCR was performed with primers encompassing regions containing putative FREs in both promoters. The results indicate that Foxo can directly bind to both the 4EBP and InR promoters in vivo. These results establish that Foxo can specifically bind the 4EBP and InR promoters both in vitro and in vivo (Puig, 2003).
To demonstrate that Foxo can directly activate transcription of these promoters in vitro, the constructs were used that contain 485 bp of the 4EBP promoter region and 514 bp of the InR promoter region, respectively. Addition of purified recombinant Foxo to in vitro reactions activates transcription of these promoters by at least 3-fold (4EBP) and 5.5-fold (InR), which is comparable to the activation observed in vivo. Under in vitro transcription conditions, activation of the 4EBP promoter by Foxo becomes rapidly saturated with increasing amounts of Foxo. As expected, Foxo also activates (up to sixfold) a synthetic promoter bearing four FOXO4-binding sites placed upstream of the alcohol dehydrogenase distal promoter. Together these results show that transcription of 4EBP and InR can be directly activated by Foxo in vitro (Puig, 2003).
Drosophila embryonic Kc167 cells respond to insulin stimulation with upregulated activities of PKB and S6K. mRNA profiling experiments were performed using the Affymetrix GeneChip system to measure on a genome-wide scale the transcriptional changes induced by insulin in these cells. On the basis of the currently held model that FOXO transcription factors are transcriptional activators that are negatively regulated by insulin, potential Foxo target genes were expected to be repressed in Kc167 cells upon insulin stimulation. Foxo target gene candidates were selected that are transcriptionally downregulated by a factor of two or more upon insulin stimulation and whose promoter regions contain one or more conserved forkhead-response elements (FHREs) with the consensus sequence (G/A)TAAACAA. Three of these candidate gene products are each involved in one of two biological processes known to be negatively regulated by insulin, namely gluconeogenesis (PEPCK) and lipid catabolism (CPTI and long-chain-fatty-acid-CoA-ligase). The remaining candidates are involved in stress responses (cytochrome P450 enzymes), DNA repair (DNA polymerase iota), transcription and translation control (4E-BP and CDK8), and cell-cycle control (centaurin gamma and CG3799). Several of the insulin-repressed genes have been reported to be transcriptionally induced in Drosophila larvae under conditions of complete starvation (4E-BP and PEPCK) or sugar-only diet (CPTI and long-chain-fatty-acid-CoA-ligase) (Jünger, 2003).
4E-BP was chosen for further investigation, because it has previously been reported to be insulin-regulated at the level of protein phosphorylation, but not at the level of gene expression. The 4E-BP gene encodes a translational repressor and was initially identified as the immune-compromised Thor mutant in a genetic screen for genes involved in the innate immune response to bacterial infection. There are several FHREs in the genomic region around the 4E-BP locus. The 4E-BP protein is negatively regulated by insulin through LY294002- and rapamycin-sensitive phosphorylation, suggesting involvement of the Dp110 and TOR signaling pathways. Phosphorylation of 4E-BP leads to the dissociation of 4E-BP from its binding partner, the translation initiation factor eIF4E, which then participates in the formation of a functional initiation complex. Positive transcriptional regulation of 4E-BP by Foxo, which corresponds to negative transcriptional regulation by insulin, would be a complementary mechanism of regulation (Jünger, 2003).
Whether overexpression of endogenous foxo can induce transcriptional upregulation of the 4E-BP gene was investigated. On the basis of overexpression results, the Dp110DN-Foxo coexpression was used to efficiently activate Foxo. Eye imaginal discs from Dp110DN-expressing third instar larvae display a low level of basal 4E-BP transcription throughout the disc, which is not induced by the driver construct alone. Coexpression of foxo elicits a dramatic upregulation of 4E-BP transcription posterior to the morphogenetic furrow. Consistent with this observation, it was possible to induce expression of the 4E-BP enhancer trap line Thor1 with human FOXO3a-TM . It remains unclear, however, whether regulation of d4E-BP expression by Foxo is of physiological relevance (Jünger, 2003).
Overexpression of 4E-BP partially suppresses the PKB overexpression phenotype, but since ectopic expression experiments have to be interpreted with some caution, whether loss of 4E-BP function suppresses the cell-number reduction in insulin-signaling mutants as does loss of Foxo function was investigated. Double-mutant flies were generated for PKB and 4E-BP and it was observed that the Thor1 mutation slightly but significantly suppressed the reduced cell-number phenotype in a dose-dependent manner. The Thor1 mutation itself had no effect on ommatidial number compared to wild-type flies, so additive effects of d4E-BP and dPKB can be ruled out. These observations strongly argue that under conditions of reduced insulin-signaling activity, the Foxo-dependent reduction in cell number is in part mediated by the transcriptional upregulation of its target 4E-BP. Microarray studies in both mammalian and Drosophila cells imply that FOXO transcription factors exert their physiological functions by modulating expression of large sets of target genes (Jünger, 2003).
The messenger RNA 5' cap-binding protein eIF4E is regulated by its binding protein (4E-BP), a downstream target of phosphatidylinositol-3-OH kinase [PI(3)K] signaling. Drosophila 4E-BP (d4E-BP) activity becomes critical for survival under dietary restriction and oxidative stress, and is linked to life span. The Drosophila forkhead transcription factor (dFOXO) activates d4E-BP transcription. Ectopic expression of d4E-BP in dFOXO-null flies restores oxidative stress resistance to control levels. Thus, d4E-BP is an important downstream effector of a dFOXO phenotype, and regulation of translation by eIF4E is vital during environmental stress (Tettweiler, 2005).
A rapid response is a crucial early line of defense in preventing cellular death in situations of stress. Translational regulation allows an organism to generate quick responses to environmental cues by controlling the expression of protein from existing cellular mRNAs. Translation initiation of most eukaryotic mRNAs requires binding of eIF4F, a protein complex made up of eIF4A, eIF4G, and eIF4E, to the 5' cap structure. eIF4E activity is highly regulated both by Mnk1/Mnk2-dependent phosphorylation and by repressor proteins termed eIF4E-binding proteins (4E-BPs), which compete with eIF4G for the same binding site on eIF4E. 4E-BPs themselves are negatively regulated by phosphorylation, and are downstream effectors of the PI3K/TOR pathway. Under nutritionally favorable conditions, the evolutionarily conserved TOR pathway is active and results in 4E-BP phosphorylation. This prevents 4E-BP binding to eIF4E, thus upregulating translation. Conversely, poor nutrition causes inhibition of the TOR pathway, such that unphosphorylated 4E-BP represses translation through eIF4E binding (Tettweiler, 2005 and references therein).
Whether d4E-BP is essential under starvation and oxidative stress conditions was investigated, because dFOXO activates the transcription of d4E-BP and d4E-BP mRNA levels increase upon starvation. Evidence is provided that d4E-BP activity is linked to life span, since overexpression of dFOXO is linked to increased longevity. This work establishes that d4E-BP is the critical effector of the dFOXO-induced stress-sensitive phenotype (Tettweiler, 2005).
The insulin/PI3K signaling pathway controls both tissue growth and metabolism. Melted has been identified as a new modulator of this pathway in Drosophila. Melted interacts with both Tsc1 and Foxo and can recruit these proteins to the cell membrane. Evidence is provided that in the melted mutant, Tor activity is reduced and Foxo is activated. The melted mutant condition mimics the effects of nutrient deprivation in a normal animal, producing an animal with 40% less fat than normal (Teleman, 2005).
As a means to identify possible functions of Melted, the Eukaryotic Linear Motif server) was used to look for functional motifs conserved between fly and human Melted. The only conserved motifs found in the N-terminal region of these proteins were two Forkhead-associated domain ligand domains (LIG_FHA_1). Forkhead transcription factors FoxA2, FoxA3, FoxC2, and FoxO1 are involved in glucose and fat metabolism. Insulin signaling activates Akt, which phosphorylates Foxo and leads to its retention in the cytoplasm. It was therefore asked if Melted affects the subcellular localization of a Foxo-GFP fusion protein. Foxo-GFP is predominantly nuclear in the absence of insulin stimulation in serum-starved S2 cells and increases in the cytoplasm after insulin stimulation. In serum-starved cells cotransfected to express Melted, Foxo-GFP is still primarily nuclear, but much of the nonnuclear protein appears at the membrane colocalized with Melted. Upon insulin stimulation, a robust increase in the level of Foxo-GFP was observed at the cell membrane. The interaction was confirmed by coimmunoprecipitation of Melted with Foxo in insulin-stimulated S2 cells (Teleman, 2005).
The observation that insulin stimulation induces a shift toward membrane localization of Foxo in the presence of Melted in S2 cells raised the possibility that melted regulates Foxo activity in vivo. To address this, expression of the Foxo target 4E-BP was examined in wild-type and melted mutant animals. Under fed conditions, insulin signaling is active and 4E-BP transcript levels are relatively low. In wild-type flies that were starved for 24 hr to reduce insulin levels and thereby activate Foxo, 4E-BP transcript increased ~4-fold. In starved flies lacking Melted, 4E-BP transcript increased over 25-fold. This increase in 4EBP transcription was absent in the starved melted/Foxo double mutant, confirming that it is Foxo dependent. Thus, in the absence of Melted, Foxo activity is higher than normal, suggesting that Melted limits Foxo activity in vivo (Teleman, 2005).
To determine whether the elevated Foxo activity observed in melted mutants contributes to the lean phenotype of these animals, the normalized triglyceride levels of melted mutant and melted foxo double-mutant flies were compared. Reducing Foxo activity suppresses the leanness of the melted mutant to a considerable degree, reaching near normal fat levels. The rescue was highly statistically significant. foxo mutants did not show higher-than-normal fat levels compared to wild-type. These observations suggest that Melted acts by regulating Foxo activity to control expression of genes important in fat metabolism (Teleman, 2005).
The Tor pathway integrates information on cellular nutritional status and stress from the heterodimeric Tsc1/2 complex. melted mutants exhibit reduced Tor activity. By recruiting Foxo to the membrane in an insulin-regulated manner Melted influences expression of Foxo targets. By reducing Tor activity and at the same time increasing Foxo activity, the melted mutant mimics the effects of nutrient deprivation in a normal animal, producing a lean phenotype (Teleman, 2005).
To determine whether Tor activity affects fat accumulation, the effects were tested of increasing Tor activity in wild-type and melted mutant adipose tissue. Use was made of a UAS-Tor transgene that can provide Tor activity in vivo when expressed at appropriate levels. It was confirmed that expression of UAS-Tor under ppl-Gal4 control in adipose tissue leads to increased total body fat, as does increasing PI3K activity. In contrast, a comparable elevation of Tor expression in melted mutant flies has no effect on fat levels. Both this result and the significant rescue caused by removal of Foxo indicate that in the melted mutant, the Foxo branch of the pathway becomes limiting for fat accumulation. In view of this finding, it was next asked whether elevated Tor pathway activity could increase fat levels in the melted mutant if Foxo activity was simultaneously reduced. To do so, use was made of the catalytic subunit of PI3K (Dp110) to inactivate Foxo and simultaneously activate Tor. The fat body driver lsp2-Gal4 or the UAS-Dp110 transgenes have little effect on their own in the melted mutant background, but when combined, the elevated PI3K activity in the fat body increases fat levels of the melted mutant. The effect is stronger than that of removing Foxo only, increasing fat levels to above normal. Taken together, these observations suggest that the Tor branch of the pathway contributes to the control of fat levels under conditions in which Foxo activity levels are low. This is normally the case in feeding animals in which insulin levels are relatively high (Foxo activity is elevated under starvation conditions: as seen by comparing 4E-BP levels in fed versus starved wild-type and foxo mutant flies). Under conditions in which insulin levels are low or in the melted mutant, in which Foxo activity is elevated, the effects of Foxo appear to dominate (Teleman, 2005).
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