The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. The Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of S6 kinase in vitro, and overexpression of S6k in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. These results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability (Zhang, 2000).
The Drosophila genome encodes four members of the PIK-related family: mei41, which encodes an ATR/ATM homolog required for cell cycle arrest in response to DNA damage (Hari, 1995); CG6535, a second ATM-related gene of unknown function; CG4549, whose closest relative encodes the nonsense-mediated mRNA decay protein SMG-1 in C. elegans, and the dTOR. A fifth member of this family, the DNA-dependent protein kinase, is not found in Drosophila or C. elegans (Zhang, 2000).
To begin a mutational analysis of dTOR, the Berkeley Drosophila Genome Project P-element database was searched and two independent homozygous lethal lines bearing P insertions in the dTOR gene were identified (designated here as dTORP1 and dTORP2). Mobilization of these elements restores viability to each chromosome, indicating that the insertions are responsible for the associated lethality. Comparison of the insertion sites of dTORP1 and dTORP2 with the dTOR transcription unit reveal that the P-elements are inserted at 24 and 74 bases downstream of the dTOR transcription start site, respectively, and would likely interfere with normal dTOR expression (Zhang, 2000).
To generate additional dTOR alleles, a series of deletions spanning the dTOR gene was generated by imprecise mobilization of the P-elements. One such mutant, designated dTORDeltaP, was selected for further analysis. Sequence analysis reveals that the dTORDeltaP deletion originates at the dTORP2 insertion site and extends 3514 bp downstream, removing the dTOR translation start site and amino-terminal 902 codons, and thus likely represents a null allele of dTOR. This was confirmed by the absence of detectable dTOR protein in immunoblots of dTORDeltaP larval extracts. A 9.4-kb genomic rescue construct encompassing the dTOR gene and no other predicted transcription units restores full viability and fertility to dTORDeltaP homozygotes (Zhang, 2000).
dTORDeltaP homozygotes hatch at normal rates, but grow more slowly than normal and eventually arrest during larval development, reaching only 24% the mass of wild-type controls. Larvae homozygous for dTORP1 or dTORP2 alleles display a less severe phenotype, eventually growing to approximately 40% and 79% the mass of wild type, respectively, indicating that these alleles likely retain partial dTOR function. In each case, the mutants remain viable and active during an extended larval period of ~30 d, and eventually die without pupating. Larvae heterozygous for dTOR grow at a rate indistinguishable from wild-type controls under normal culture conditions, but are hypersensitive to low concentrations of rapamycin. Thus, dTOR encodes a rapamycin-sensitive protein required for normal growth during larval development (Zhang, 2000).
Overall growth of an organism is generally accompanied by increases in cell number (proliferation), cell size (hypertrophy), or both. To determine how mutations in dTOR inhibit growth, these parameters were examined in a number of tissues. The effect of dTOR on cell size was analyzed in marked clones of dTORDeltaP homozygous cells, which were generated by FLP/FRT-mediated mitotic recombination in dTORDeltaP heterozygous animals. Examination of adult cuticular structures reveal that dTOR homozygous mutant cells are markedly reduced in size. For example, bristles of the wing margin that lack dTOR were both thinner and shorter than adjacent wild-type cell. Area measurements of mutant clones in the wing epithelium show that dTORDeltaP mutant cells are approximately half (56%) the size of controls. Similar effects have been observed in the eye, abdomen, and notum (Zhang, 2000).
To determine whether loss of dTOR affects the size of actively proliferating cells, dTOR mutant clones were examined in the developing imaginal discs, epithelial primordia that proliferate mitotically to give rise to adult structures. Imaginal wing discs containing GFP-marked clones of dTORDeltaP homozygous cells were dissociated into single cells, which were then analyzed by flow cytometry. The mean forward light scatter value (a measure of cell size) of dTOR mutant cells is decreased by 30% compared to wild-type control cells from the same discs. This decrease in cell size is observed in all phases of the cell cycle. Thus, loss of dTOR causes a cell autonomous reduction in the size of both proliferating and postmitotic cells (Zhang, 2000).
Fluorescence-activated cell sorter (FACS) analysis has also revealed that the cell cycle phasing of dTOR cells differs significantly from that of controls, with relatively more cells in G1, and fewer in S and G2 phases. This is consistent with the ability of rapamycin to induce G1 arrest in yeast and in mammalian cell culture. To measure proliferation rates of dTOR mutant cells, the number of cells in dTORDeltaP clones were compared with that of their wild-type sister clones (twin spots). Clones of dTORDeltaP mutant cells are similar in size to their twin spots at 48 h after induction, but by 72-96 h they contain significantly fewer cells, indicating that loss of dTOR leads to a reduced rate of cell proliferation. In addition, lone twin spots lacking a corresponding mutant sister clone have occasionally been observed at 96 h after induction, indicating that at some frequency dTORDeltaP homozygous cells are eliminated from the disc epithelium. Because dTORDeltaP cells remain viable for weeks in the context of a homozygous mutant animal, the loss of dTORDeltaP mutant clones is likely the result of cell competition with adjacent wild-type cells, which is known to occur for cells with a growth disadvantage (Zhang, 2000).
Growth properties of cells in the salivary glands of homozygous dTORDeltaP larvae were also examined. The salivary gland is comprised of two cell types: polytene gland cells that undergo multiple rounds of endoreduplication to generate giant nuclei with a ploidy of up to 2048 C, and imaginal ring cells that remain diploid and cycle mitotically. Loss of dTOR affects both cell types. The endoreplicative cells in dTORDeltaP salivary glands undergo only four to five rounds of replication before entering quiescence, reaching a ploidy of 16-32C and a size ~10% that of wild type. The imaginal rings in dTORDeltaP larvae contain approximately fivefold fewer cells than wild type. Together, these results indicate that dTOR is required to promote cell cycle progression in both mitotic and endoreplicative cells, and acts primarily at the G1/S transition (Zhang, 2000).
The cell autonomous reduction in the size of dTOR mutant cells is reminiscent of mutations in components of the PI3K/S6K signaling pathway. Mutations in Pten, the fly homolog of the PTEN tumor suppressor, cause activation of this pathway, leading to increased cell growth. To determine whether dTOR is required for PI3K-dependent signaling, the growth properties of cells lacking both Pten and dTOR were examined. Clonal loss of Pten causes enlargement of imaginal and adult cells, and increases the percentage of cells in the S and G2 phases of the cell cycle. In contrast, cells carrying null alleles of both Pten and dTOR are indistinguishable from cells lacking dTOR alone, with a similar reduction in cell size and accumulation in G1. Loss of dTOR also prevents the increased proliferation caused by mutations in Pten. Cells mutant for weaker alleles of Pten and dTOR (MGH1 and P2, respectively) are intermediate in size, indicating that dTORP2 cells retain partial signaling function. It is concluded that dTOR is epistatic to Pten, and therefore, that dTOR functions at a step downstream of or in parallel to PI3K signaling (Zhang, 2000).
In further tests of dTOR's role in PI3K/S6k signaling, it was found that rapamycin inhibits the serum-dependent phosphorylation of Drosophila p70S6k (S6k) expressed in S2 cells. Dephosphorylation of S6k by rapamycin is prevented by cotransfection of a dTOR point mutant containing a Ser1956 to Thr substitution (dTORRR, which confers rapamycin resistance to mammalian and yeast TOR proteins. Expression of dTORRR carrying an additional point mutation in a residue crucial for kinase activity (dTORRRKD fails to protect S6k from rapamycin-induced dephosphorylation, indicating that the kinase function of dTOR is required to maintain S6k phosphorylation (Zhang, 2000).
To determine whether these biochemical interactions between dTOR and S6k are relevant to their functions in vivo, tests were performed for genetic interactions between them. Remarkably, constitutive overexpression of Drosophila S6k or human p70S6K1 is able to rescue dTORP2/P2 and dTORP1/P2 flies to viability. The greatest degree of rescue is provided by a mutant version of p70S6K1, in which four mitogen-induced phosphorylation sites are mutated to aspartate and glutamate residues (mutant D4). Expression of this construct allowed 74% of expected dTORP1/P2 progeny to survive to adulthood, whereas no dTORP1/P2 animals survived in the absence of S6k overexpression. dTOR flies rescued by S6k overexpression are slightly smaller than wild-type controls, but are fertile and develop at a similar rate as wild type. Although overexpression of S6k does not rescue dTORDeltaP null mutants to adulthood, it does enable them to progress to the pupal stage. Overexpression of S6k in wild-type larvae also confers significant resistance to rapamycin. Again, constitutively active p70S6K1 provides the greatest degree of rapamycin resistance. Together, these results indicate that a major function of dTOR is to maintain levels of active S6k sufficient for normal growth (Zhang, 2000).
Like dTOR mutants, wild-type larvae deprived of amino acids enter an extended larval period with little or no growth. Amino acid deprivation also causes a series of distinctive cellular phenotypes including a reduction in nucleolar area, changes in morphology of the larval fat body, and a cell type-specific cell cycle arrest. Since TOR proteins have been proposed to be regulated in response to amino acid levels, it was of interest to examine whether loss of dTOR mimics these cellular effects (Zhang, 2000).
The nucleolus is the major cellular site of ribosomal assembly, and its size has been shown to correlate with protein synthetic capacity and proliferation rate. To measure nucleolar size in wild-type and dTOR mutant cells, wing imaginal discs containing clones of dTORDeltaP homozygous cells were labeled with an antibody against the nucleolar protein fibrillarin, and examined by confocal sectioning. The nucleolar area in clones of dTOR mutant cells in the wing imaginal disc is approximately half that of surrounding wild-type cells, consistent with a role for dTOR in ribosome biogenesis (Zhang, 2000).
During metamorphosis or starvation, stores of protein, lipid, and glycogen are mobilized from adipose cells of the larval fat body and are used by other tissues as an energy source in place of dietary nutrients. These metabolic effects are visible as changes in appearance of fat body cells. The major visible change in fat body cells in larvae deprived of amino acids is an aggregation of lipid vesicles, and this effect is indistinguishable from that caused by loss of dTOR (Zhang, 2000).
Within 48-72 h of amino acid withdrawal, endoreplicative cells become quiescent, whereas mitotic neuroblasts of the central nervous system continue to cycle for at least 8 d in the absence of amino acids. This pattern of cell cycle responses is distinct from that caused by complete inhibition of protein synthesis, which causes all larval cells to arrest DNA synthesis. To determine whether loss of dTOR causes a cell cycle response similar to that elicited by starvation, cell cycle behavior of these cell types was examined in dTORDeltaP larvae at multiple stages of development. At 3-4 d after egg deposition (AED), both endoreplicative and mitotic tissues were found to cycle normally in dTORDeltaP homozygotes, as measured by incorporation of the nucleotide analog BrdU. In contrast, by 5-6 d AED all endoreplicative tissues including the gut, fat body, and salivary glands fail to incorporate BrdU, whereas neuroblasts continued to cycle. A similar pattern is observed at 10 d AED. Presumably the appearance of this cell cycle arrest at 4-5 d AED results from the perdurance of maternal stores of dTOR mRNA or protein until this time. The cell cycle arrest of dTOR endoreplicative cells can be bypassed by overexpression of the G1/S regulators cyclin E or dE2F/dDP, as has been demonstrated previously for endoreplicative cells arrested by amino acid withdrawal. Thus, amino acid insufficency and loss of dTOR each cause similar growth arrests, changes in cell morphology, and cell type-specific patterns of G1 arrest (Zhang, 2000).
In budding yeast, TOR proteins govern S-phase entry in response to nutrient levels by regulating translation of the G1/S regulator Cln3 (Barbet, 1996 and Polymenis, 1997). Drosophila cyclin E has been proposed to play a role analogous to Cln3, and its abundance increases in response to growth stimuli such as overexpression of activated Ras. Because cyclin E overexpression is able to bypass the cell cycle arrest in dTOR mutants, whether loss of dTOR affects cyclin E expression was examined. Immunoblot analysis of whole larval extracts has revealed that the level of cyclin E protein is reduced ~30-fold in dTORDeltaP mutants, as compared to wild-type larvae of similar stage (Zhang, 2000).
Clonal induction of cells lacking cyclin E in the wing imaginal disc results in a G1 arrest within one to two cell divisions. In contrast, cells mutant for dTOR continue to cycle slowly for several days, and give rise to clones containing multiple cells, indicating that dTOR mutant cells retain at least partial cyclin E activity. Accordingly, cyclin E protein is reduced but not eliminated in dTOR mutant clones in the wing disc. Although 72-h dTORDeltaP clones containing little or no detectable cyclin E immunoreactivity are often observed, many dTOR clones were found containing cells with apparently normal cyclin E levels. It is concluded that the observed reduction in cyclin E protein levels in dTORDeltaP larval extracts is due largely to reduced cyclin E levels in endoreplicating cells, which comprise the majority of larval mass, resulting in a G1 arrest in this cell type. In contrast, dTOR may be required in imaginal cells to maintain normal rates of cyclin E accumulation, rather than absolute levels, consistent with the reduced rate of cell division and extended G1 phase observed in dTOR mutant clones (Zhang, 2000).
dTOR mutants differ from the known PI3K pathway mutants in several important respects. (1) The growth defects caused by loss of dTOR are more severe than those arising from mutations in components of the PI3K signaling pathway. (2) Null mutations in the PI3K subunits Dp110 or p60 allow growth to the third instar larval stage, and chico (IRS-1) and S6k mutants survive to adulthood. At least in the case of chico and S6k, many cells are able to cycle normally throughout development. In contrast, animals lacking dTOR reach only the size of second instar larvae, at which point they undergo cell cycle arrest. (3) Whereas overexpression of Dp110, Akt, or S6k leads to increased growth rate and cell size, dTOR overexpression inhibits growth and reduces cell size. Although variations in genetic background may partially account for some of these phenotypic differences, these results are inconsistent with dTOR acting as an integral component of a linear PI3K/Akt/S6k pathway, and instead argue that dTOR may converge upon this pathway in response to a distinct set of cues (Zhang, 2000 and references therein).
In yeast, TOR1 and TOR2 regulate cell growth directly in response to levels of nutrients such as amino acids, rather than in response to intercellular signals. Similarly, human mTOR may also be regulated by nutrient levels. These considerations prompted a comparison of the phenotypes of dTOR mutant animals with the physiological changes caused by nutrient deprivation. By the three criteria examined -- nucleolar size, fat body vesicle formation, and endoreplicative cell cycle arrest -- loss of dTOR precisely mirrors the effects of starvation. An efficient explanation of these results is that dTOR is required for normal responses to changes in nutrient levels. This would be consistent with a model in which full activation of growth targets such as S6k requires two distinct inputs: growth factor-mediated intercellular signals through PI3K, and nutrient-sensing signals through TOR. In this view, TOR proteins may act as part of a checkpoint to attenuate growth factor signaling when local conditions are unfavorable for cell growth (Zhang, 2000).
How TOR proteins might be regulated by nutrient levels is unclear. In the case of amino acids, recent evidence suggests that the primary signal may be uncharged tRNA, which increases in abundance when amino acid levels are low. Interestingly, other members of the PIK-related kinase family such as ATM and DNA-PK also act as checkpoint proteins that are regulated by specific nucleic acid structures. In addition, a recently described member of this family, SMG-1, is involved in the degradation of aberrant mRNAs containing inappropriate nonsense codons. Thus, regulation by nucleic acid may be a common feature of this kinase family (Zhang, 2000 and references therein).
Activation of p70S6K is a common response to virtually all mitogenic stimuli. Phosphorylation of the target of p70S6K, ribosomal protein S6, leads to the selective increase in translation of a subset of transcripts that contain an oligopyrimidine tract at their 5' termini (5' TOP). This class of messages encodes ribosomal proteins and translation elongation factors, and thus p70S6K activation leads to increased ribosomal biogenesis. The demonstration in this study that overexpression of S6k can rescue dTOR mutants to viability indicates that S6k is a critical effector of dTOR, and that one essential function of dTOR is to regulate the activity of S6k (Zhang, 2000).
Despite the central role of S6k in mediating the functions of dTOR, several lines of evidence indicate that dTOR has additional S6k-independent roles. (1) dTOR mutant phenotypes are more severe than those of S6k; lack of dTOR results in growth arrest at the second instar larval stage, whereas S6k mutant animals survive to adulthood, albeit with a delayed development and decreased body size. (2) Although S6k overexpression suppresses the lethality of dTOR mutants, this is only the case for hypomorphic dTOR allelic combinations; dTOR null animals expressing S6k advance only to the pupal stage. (3) dTOR flies rescued by S6k do not grow to the full size of wild-type controls. Thus, some dTOR functions are not fully rescued by S6k. As noted above, in mammalian cells mTOR also stimulates translation through phosphorylation and inactivation of 4E-BP1, an inhibitory binding factor of the translation initiation factor eIF4E. Drosophila eIF4E mutants display a severe growth arrest phenotype, and thus aspects of dTOR function that are not rescued by S6k may reflect diminished eIF4E activity. However, neither overexpression of eIF4E nor mutations in 4E-BP1 detectably alleviate dTOR mutant phenotypes (Zhang, 2000).
In addition to effects on translation, studies in budding yeast have found that inactivation of TOR modulates the level of a number of growth-related or nutrient-regulated transcripts, including those encoding ribosomal proteins (Barbet, 1996; Zaragoza, 1998; Beck, 1999; Cardenas, 1999; Hardwick, 1999; Powers, 1999). A transcriptional role for TOR in higher eukaryotes has been reported as well. Moreover, recent studies have found that mTOR can interact with a number of additional signaling factors including STAT3, protein kinase C, c-Abl, and 14-3-3 (Parekh, 1999; Kumar, 2000a and b; Mori, 2000; Yokogami, 2000). Rapamycin has also recently been shown to disrupt microtubule assembly and function in yeast, independent of its effects on translation (Choi, 2000). Thus, the inability of S6k overexpression to fully supplant dTOR may be due to a requirement for dTOR in multiple cellular functions (Zhang, 2000).
The reduced growth of dTOR mutants reflects reductions in both cell size and cell number. Because direct inhibition of proliferation increases rather than decreases cell size, it is proposed that the primary function of dTOR is to promote cell growth, and that the decreased proliferation of dTOR mutant cells is a secondary effect in response to their reduced rate of growth. The accumulation of dTOR mutant cells in the G1 phase of the cell cycle suggests that the G1/S transition is particularly sensitive to growth rate. This is consistent with previous observations that stimulation of cell growth by overexpression of dMyc, PI3K, or activated Ras promotes progression through the G1/S but not G2/M transition (Zhang, 2000 and references therein).
A factor likely to be involved in coupling growth and division rates is the G1/S regulator cyclin E. Cyclin E is rate limiting for G1/S progression in imaginal discs, and its levels increase by a post-transcriptional mechanism in response to stimulation of cell growth. The demonstration that dTOR is required for normal accumulation of cyclin E suggests that translational control is likely to play an important role in cyclin E regulation. In budding yeast, translation of the G1 cyclin Cln3 is regulated by a leaky scanning mechanism involving a small upstream ORF (Polymenis, 1997). In this regard, it is interesting to note that the 5'-untranslated region of the Drosophila cyclin E message contains multiple upstream ORFs. Thus, the mechanisms connecting G1/S progression to cell growth may be conserved between yeast and multicellular organisms (Zhang, 2000).
Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. Rheb (Ras homolog enriched in brain), a novel, highly conserved member of the Ras superfamily of G-proteins, promotes cell growth. Overexpression of Rheb in the developing fly causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, Rheb overexpression results in accumulation of cells in S phase and an increase in cell size. Rheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of Rheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that Rheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that Rheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, flies with reduced Rheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator target of rapamycin(TOR), a kinase required for growth factor-dependent phosphorylation of ribosomal S6 kinase (S6K). In cultured cells, the effect of overexpressing Rheb was blocked by the addition of rapamycin. These results imply that Rheb is involved in TOR signaling (Patel, 2003). Additional studies show that Rheb functions downstream of the tumor suppressors Tsc1 (tuberous sclerosis 1)-Tsc2, with Tsc2 functioning as a GAP for Rheb (Saucedo, 2003; Zhang, 2003), and that a major effector of Rheb function in controlling growth is, in fact, ribosomal S6 kinase (Stocker, 2003). It is still not clear, however, how Rheb signals to TOR (Zhang, 2003).
Given the similarities between Rheb and mutants in the InR and TOR signalling pathways, it is conceivable that Rheb represents a novel component of one of these growth control pathways. To test this possibility, a detailed epistasis analysis was performed. Examined first was whether the negative regulators of InR and TOR signalling (PTEN and Tsc1-Tsc2, respectively) could counteract the effects of Rheb overexpression. All overexpression experiments were performed in the eye using the GMR-Gal4 driver line. Expression of either PTEN or Tsc1-Tsc2 alone results in a very similar size reduction of the ommatidia when compared with control ommatidia. However, whereas expression of PTEN has no influence on the increase in ommatidial size caused by Rheb overexpression, co-expression of Tsc1-Tsc2 results in ommatidia of approximately wild-type size, indicating that the activities of Rheb and Tsc1-Tsc2 can counteract each other. Next, the enlarged ommatidia phenotype of GMR-Rheb was assayed in a number of mutant backgrounds. Reducing the activity of Drosophila protein kinase B (PKB) has no effect on ommatidial size. Similar results were obtained with hypomorphic mutations in InR and Dp110, respectively. In contrast, ommatidial size is dominantly reduced by a mutation in TOR (TOR2L1), and a suppression to wild-type size is observed in a S6K mutant background. Thus, the Rheb overexpression phenotype is dependent on TOR and S6K function, but is independent of InR signal strength. Finally, the behaviors of Rheb PTEN and Rheb Tsc1 double mutants were examined. The phenotypic consequences were assayed in mosaic animals using the ey-Flp method. As expected, the Rheb PTEN double-mutant tissue clearly displays a Rheb phenotype. The Rheb Tsc1 mutant tissue also resembles Rheb single mutants, indicating that Rheb is epistatic over (functions downstream of) Tsc1 (Stocker, 2003).
In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).
Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).
In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).
The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).
Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).
Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).
The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).
How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).
In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).
Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).
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).
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