Tribbles activity regulates cell cycle by directly and posttranscriptionally affecting String expression. The Cdc25 protein String achieves mitotic activation by hydrolyzing phospho-tyrosine 15 of the cyclin dependent kinase Cdc2, thus activating Cdc2. string is transcribed in a spatial pattern controlled by the anterior-posterior and dorsoventral patterning systems. Expression of String mRNA in a given mitotic domain precedes mitosis by a few minutes. By analyzing the exception to this rule found in domain 10 on the ventral side at the embryo, the tribbles mode of regulation was uncovered. Although string is expressed in these cells, they do not divide until they are internalized. This delay depends on the activity of the tribbles gene (Seher, 2000) named after the small, round, fictional organisms (from the television series "Star Trek") that proliferate uncontrollably when they contact water. The tribbles effect is restricted to the ventral furrow, even though TRBL mRNA is also present outside of this domain and the trbl mutation can be rescued by uniform exogeneous expression. This suggests that trbl activity is triggered by an input which is present only in the ventral furrow region (Großhans, 2000). Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination (Mata, 2000).

tribbles was identified by three screens: (1) a screen designed to identify loci required for a delayed mitosis in domain 10 (Großhans, 2000); (2) a deficiency screen for mutations that disrupt gastrulation (Seher, 2000), and (3) a screen for genes that when overexpressed in the germline would affect oogenesis (Mata, 2000), In the first screen, embryos deficient for trbl, and for a second uncharacterized mutant frühstart, mitotis takes place prematurely in the ventral domain, such that their pattern of String mRNA expression and the mitotic pattern match each other (Großhans, 2000). In the second screen, the deficiency of tribbles results in the improper formation of the ventral furrow (Seher, 2000). In the third screen (Mata, 2000), an insertion in a vector designed to be overexpressed in the germline produced 32 cell oocytes (cysts) instead of the usual 16 cell oocyte (1 egg and 15 nurse cells).

Tribbles function was examined by assessing the effects of overexpression in imaginal disc cells. The effect of expressing tribbles in wing imaginal discs was assessed using engrailed-GAL4 to drive expression in the posterior compartment. The anterior compartment served as control tissue. The resulting wings had a posterior compartment of close to normal size and pattern, but apparently made up of fewer, larger cells. This interpretation was confirmed by looking directly at the disc, where the cells expressing tribbles, the posterior cells, were found to be fewer and larger than control cells. This type of effect is typical of severe cell cycle delays. Since the effect is confined to one compartment, the wild-type (anterior) cells cannot out-compete the tribbles overexpressing cells. To confirm that tribbles affects the cell cycle and to determine what phase is being affected, cells size analysis, using green fluorescent protein marked cells, was performed on tribbles-expressing cells. Expression of tribbles results in a 4- to 5-fold decrease in the number of cells. DNA content indicates that almost all of these cells are in G2/M. It is concluded that tribbles overexpression slows the G2 phase of wing imaginal disc cells substantially (Mata, 2000).

String/CDC25 phosphatase is required for G2/M progression in imaginal disc cells. Cells homozygous for a hypomorphic string mutation can grow but they divide very slowly and thus become very large, similar to the tribbles overexpression phenotype. Given that string is a limiting regulator in G2 of imaginal disc cells, it was asked whether increased expression of string would affect the tribbles GOF phenotype. Expression of UAS-string under control of engrailed-GAL4 did not give an overt wing phenotype. When UAS-string is coexpressed with UAS-tribbles, the posterior compartment reverts to the normal cell number and density. Thus, overexpression of string suppresses the effect of tribbles overexpression in imaginal disc cells. In the germline of the ovary, overexpression of string also suppresses the extra cystocyte division phenotype caused by tribbles expression. Thus, despite the apparent differences in cell cycle control mechanisms, tribbles opposes or downregulates string/CDC25 in both the wing disc and the germline (Mata, 2000).

To see whether overexpression of tribbles affects String protein levels, discs were double-stained with anti-String and anti-Tribbles antibodies. Cells that overexpress tribbles do not show normal levels of endogenous String protein. String expression in third instar larval discs is nonuniform due to asynchronous cycling of the cells. By counting expressing and nonexpressing cells in several discs, it was verified that String-expressing cells do not express Tribbles. The lack of cells coexpressing String and Tribbles could not be explained by cells being in the wrong phase of the cell cycle to accumulate String, since overexpression of tribbles delays cells in G2, the phase of the cell cycle in which String protein normally accumulates. Expression of tribbles also affects the level of String protein when both were overexpressed by the GAL4/UAS system. The overall level of overexpressed String protein is clearly reduced by Tribbles, although not to background levels. The simplest interpretation of these results is that Tribbles directly and posttranscriptionally affects String expression (Mata, 2000).

To investigate the interaction between tribbles and string under more controlled conditions, tissue culture experiments were carried out. The CDC25 homologs String and Twine, and the mitotic Cyclins A and B, were epitope tagged at the N terminus and cloned into the metallothionine expression vector. When cotransfected into Schneider cells, expression of Tribbles severely reduced the level of HA-String. This reduction in protein level was observed without any change in HA-String mRNA levels. Tribbles has a similar effect on HA-Twine protein levels, although less severe, but has no effect on the mitotic Cyclins A and B. Given that the promoter, the 5'UTR and 3'UTRs as well as the initiator methionine and N-terminal epitope tag were identical in the HA constructs, it was reasoned that Tribbles might be acting by increasing String (and Twine) protein turnover. To test this hypothesis, the proteosome inhibitor lactacystin was used. Addition of lactacystin reverses the effect of Tribbles on HA-String protein. This indicates that Tribbles induces degradation of String protein via a proteosome-dependent pathway. To confirm this, a pulse-chase experiment was performed. As expected, metabolically labeled HA-String disappears more rapidly in the presence of Tribbles. Mitotic cyclins are also degraded via the proteosome, stimulated by anaphase-promoting complex (APC). Because Cyclin levels are unaffected by Tribbles, it has been concluded that Tribbles regulates CDC25 protein turnover in a specific manner, not just by generally increasing proteosome activity (Mata, 2000).

The involvment of tribbles in regulating mitosis during gastrulation is documented by Großhans, 2000 and Seher, 2000 (for a description of the Seher study, see Effects of Mutation). During gastrulation, new cells, formed during the first 13 cell cycles, change their shape, and morphogenetic movements rearrange their positions relative to one another. One of the most prominent of these morphogenetic movements is the formation of the ventral furrow, which brings the mesoderm anlage into the interior of the embryo. After the first 13 divisions, cell division ceases, only to resume during gastrulation. During this time mitosis occurs in an asynchronous manner, independently, in at least 25 domains. This asynchrony allows morphogenesis and cell division to occur simultaneously in different regions of the embryo and sequentially in specific primordia. For example, the ventral-most cells first form the ventral furrow and only after this invagination is completed, do they enter mitosis (Großhans, 2000).

Entry into mitosis is positively controlled by String, the homolog of Cdc25, which is both necessary and sufficient for mitosis during gastrulation. The expression pattern of String mRNA closely matches the mitotic pattern. During the cleavage stage String mRNA is uniformly distributed, but then degraded at the pause in mitosis and the transition to cellularization. String mRNA subsequently reappears at the beginning of gastrulation in a pattern preceding the mitotic domains. In all domains except one, mitosis starts a few minutes after String mRNA is expressed. Mitotic domain 10, which comprises most of the mesoderm anlage, behaves differently: the gap between String mRNA expression and entry into mitosis is much longer (Großhans, 2000).

The delay in their mitosis suggests that ventral cells contain a factor lengthening the gap between appearance of String mRNA and entry into mitosis. This delay involves a subtle titration of string activity, since it can be shortened by addition of two more copies of the string chromosomal region, raising the copy number of string to four. Under these conditions the ventral cells divide at about the same time as the cells of domains 1 to 3, which matches the String mRNA pattern more closely than it does in wild-type embryos. Only the mitosis in domain 10 is shifted in these experiments, the order mitosis in the other mitotic domains is not changed (Großhans, 2000).

To examine more stringently whether the factor counteracting string is specific for ventral cells, exogeneous String mRNA was expressed at the same level in all cells of the embryo, using a UAS-String transgene driven by a maternally provided Gal4 in embryos otherwise homozygous for a string deletion. Four copies of maternally provided Gal4 produce high levels of string activity, indicated by the uniform entry of all cells into mitosis immediately at the beginning of gastrulation. In these embryos ventral furrow formation is inhibited. Using females with three or two Gal4 insertions, expression of string was gradually lowered. This shifts the onset of mitosis to a time when the first mitoses normally occur in wild-type embryos. Under these conditions differences in the behavior of the cells become apparent. In spite of the uniform string expression, the ventral cells undergoing cell shape changes to form the ventral furrow enter mitosis later than the other cells. This special behavior of the ventral cells is not observed in string heterozygous embryos that have endogeneous as well as exogeneous String mRNA. It is concluded from these experiments that ventral cells contain a dosage-sensitive factor, the ventral inhibitor, that counteracts string activity and that the delay of mitosis in domain 10 of wild-type embryos is due to this factor (Großhans, 2000).

In order to identify components that constitute the ventral inhibitor, a genome-wide screen was carried out for loci that are required for a delayed mitosis in domain 10. By screening 99% of the genome, two novel loci, frühstart and tribbles, were identified. In embryos deficient for either of these genes, cells in the ventral domain are the first to enter mitosis, such that their pattern of String mRNA expression and the mitotic pattern match one another. The order of the other mitotic domains is not altered, suggesting that frs and trbl act specifically in the ventral cells. The double mutant frs trbl shows the same phenotype as the single mutants, suggesting that frs and trbl are nonredundant genes in a common process (Großhans, 2000).

As a consequence of the early mitosis, the mesodermal precursors remain on the surface and do not form a proper ventral furrow. This defect is similar to that observed in embryos in which all cells have been forced into mitosis by increased string dosage or string overexpression. Although other zygotically active genes are known to affect formation of the ventral furrow, frs and trbl are unique in that their defects solely depend on the premature mitosis. In double mutant frs string and trbl string embryos, no mitosis takes place during gastrulation, and the ventral furrow forms as in wild-type. The premature mitosis in frs or trbl embryos is not caused by overexpression of String mRNA in the ventral region, since String mRNA is present in comparable amounts in mutant embryos and with a similar pattern as in their heterozygous siblings or wild-type embryos. Since String is the rate-limiting factor for entry into mitosis during gastrulation, this observation suggests that frs and trbl counteract string via a posttranscriptional mechanism (Großhans, 2000).

In embryos mutant for either snail or twist, no ventral furrow forms and cells are shifted to more lateral fates. String mRNA is not present in domain 10 and mitotic patterns in the ventral region of these mutant embryos are difficult to evaluate. String mRNA is restored to wild-type levels in the prospective domain 10 of snail mutants carrying three copies of wild-type twist. In such mutants, the ventral cells are the first ones to divide, indicating that snail is required for the function of the ventral inhibitor. One possibility would be that the persistence of trbl expression in the ventral region requires mesodermal determination and thus wild-type snail activity. However, snail mutants show a normal pattern of trbl expression and maintain trbl expression in the ventral domain. Similarly, in twist homozygous mutants and in embryos homozygous for deficiencies for frs the expression of trbl is not changed. Because snail embryos do not show a ventral mitotic inhibition, even though their trbl expression is normal, it is concluded that some aspect of mesodermal determination mediated by snail is required for Trbl activation (Großhans, 2000).

To directly investigate the activity of trbl on mitosis, the rapid cell cycle of cleavage stage embryos was used as an assay. trbl is not yet expressed during this stage: it appears only at the beginning of cellularization when the rapid nuclear divisions stop. Large amounts of synthetic Trbl mRNA were injected into the posterior half of the embryos: the subsequent nuclear divisions were followed, and the embryos were fixed before reaching gastrulation. In addition, the progression of the cell cycle after injection was recorded in embryos that express a GFP-Histone fusion protein that labels interphase and mitotic chromosomes. The nuclei at the injection site do not participate in the last mitosis (13th division) resulting in larger nuclei and lower density at the injection site. As a negative control for this assay, mRNA of the unrelated serine-threonine protein kinase Pelle was injected. This did not inhibit mitosis. Since presence of trbl alone is not sufficient to inhibit mitosis and since Trbl requires an additional trigger in the ventral cells, it is possible that this requirement is overridden by providing Trbl mRNA in excess in the microinjection assay (Großhans, 2000).

Hyperglycemia, hyperlipidemia, and insulin resistance are hallmarks of obesity-induced type 2 diabetes, which is often caused by a high-fat diet (HFD). However, the molecular mechanisms underlying HFD-induced insulin resistance have not been elucidated in detail. This study established a Drosophila model to investigate the molecular mechanisms of HFD-induced diabetes. HFD model flies recapitulate mammalian diabetic phenotypes including elevated triglyceride and circulating glucose levels, as well as insulin resistance. Expression of glass bottom boat (gbb), a Drosophila homolog of mammalian transforming growth factor-β (TGF-β), is elevated under HFD conditions. Furthermore, overexpression of gbb in the fat body produced obese and insulin-resistant phenotypes similar to those of HFD-fed flies, whereas inhibition of Gbb signaling significantly ameliorated HFD-induced metabolic phenotypes. tribbles, a negative regulator of AKT, is a target gene of Gbb signaling in the fat body. Overexpression of tribbles in flies in the fat body phenocopied the metabolic defects associated with HFD conditions or Gbb overexpression, whereas tribbles knockdown rescued these metabolic phenotypes. These results indicate that HFD-induced TGF-β/Gbb signaling provokes insulin resistance by increasing tribbles expression (Hong, 2016).

Abnormally high fat mass is a major risk factor for the development of diabetes. Previous studies emphasize that excess adiposity results in abnormal production of cytokines, growth factors, and hormones, which in turn causes secondary diseases like insulin resistance. This study has demonstrated that HFD-induced obesity triggered TGF-β signaling, which downregulates insulin signaling in the fat body. This study also demonstrated the role of tribbles, a novel target of TGF-β/Gbb signaling, in the development of insulin resistance (Hong, 2016).

Drosophila models were used in several recent studies of diet-induced obesity, insulin resistance, hyperglycemia, and hyperinsulinemia. In Drosophila larvae, a high-sugar diet induces type 2 diabetic phenotypes including hyperglycemia, high TG, and insulin resistance. Likewise, in adult flies, HFD feeding also induces high TG and altered glucose metabolism, and in mammals it causes cardiac dysfunctions like diabetic cardiomyopathy. This study has established a Drosophila model of obesity-induced insulin resistance, which has remarkable parallels with the mammalian system, and used it to observe and investigate the development of insulin resistance under chronic over-nutrition conditions. In addition, to study the Drosophila insulin-resistance phenotype in detail, this study has developed an ex vivo culture system (Hong, 2016).

When adult flies were fed a HFD, their short- and long-term metabolic responses were different: for example, expression and secretion of Dilp2 was increased by short-term HFD but decreased by long-term HFD. Insulin signaling, which was assayed by monitoring pAKT activation and expression of the dFOXO target genes d4E-BP and dInR, was activated in short-term but not long-term HFD, whereas TG and trehalose/glucose levels in hemolymph were increased by long-term HFD. Because these pathological phenotypes in flies were very similar to the phenotypes associated with insulin-resistant diabetes in mammals, it is concluded that HFD adult flies can be used as a model of type 2 diabetes (Hong, 2016).

In addition to increasing TG levels, HFD feeding in flies increased the expression of gbb. In mice, inhibition of TGF-β signaling by knockout of Smad3 protects against diet-induced obesity and diabetes. Inhibition of TGF-β signaling may improve adipose function and reverse the effects of obesity on insulin resistance. The TGF-β/Smad3 signaling also plays a key role in adipogenesis. However, it remains unclear how TGF-β signaling is related to the onset of diet-induced obesity and diabetes. This study examined the effects of Drosophila TGF-β family ligands on obesity. Of the genes that were tested, only gbb was upregulated by HFD. Gab regulates lipid metabolism and controls energy homeostasis by responding to nutrient levels (Ballard, 2010); consequently, gbb mutants have extremely low levels of fat in the fat body, resembling a nutrient-deprived phenotype (Ballard, 2010). On the contrary, gbb overexpression increased the TG level, mimicking the effects of nutrient-rich conditions. These data suggest that TGF-β/Gbb signaling is involved in HFD-induced obesity. Indeed, overexpression of gbb in the fat body phenocopied the TG and trehalose/glucose levels in flies fed a HFD. However, Dilp2 expression was increased by gbb overexpression in the fat body, consistent with the effects of short-term but not long-term HFD (Hong, 2016).

Focused was placed on three negative regulators of insulin signaling, PTP1b, PTEN, and tribbles 3 (TRB3), which are involved in insulin resistance in obese mammals. tribbles was upregulated in gbb-overexpressing cells and flies. In mammals, Tribbles encodes an evolutionarily conserved kinase that plays multiple roles in development, tissue homeostasis, and metabolism. A mammalian Tribbles homolog, Tribbles homolog 3 (TRB3), is highly expressed in liver tissue under fasting and diabetic conditions, and inhibits insulin signaling by direct binding to Akt and blocking phosphorylation-dependent Akt activation. Indeed, the expression level of TRB3 is elevated in patients with type 2 diabetes and animal models of this disease. In the systemic sclerosis model, TGF-β signaling can induce mammalian TRB3 and activates TGF-β signaling-mediated fibrosi. Recent work showed that Drosophila tribbles, like mammalian TRB3, inhibits insulin-mediated growth by blocking Akt activation. In this study, tribbles expression was increased in HFD conditions in both mice and flies, as well as in TGF-β-treated human liver cells. tribbles knockdown rescued the diabetic phenotypes caused by HFD, consistent with previous findings in mammals. In addition, tribbles knockdown rescued the diabetic phenotypes caused by gbb overexpression. These data strongly suggest that the evolutionarily conserved tribbles gene is a novel downstream target of Gbb signaling, and that tribbles knockdown rescues diabetic phenotypes in flies. Therefore, future studies should seek to elucidate TGF-β-Trb3 signaling and its functions in mammalian adipocytes; the resultant findings could suggest new strategies for preventing type 2 diabetes (Hong, 2016).

In summary, This study established a Drosophila insulin-resistance model and demonstrated that Gbb signaling in the fat body plays a critical role in obesity-mediated insulin resistance by regulating tribbles expression. These results provide insights regarding the function of Gbb/TGF-β signaling in metabolic disease, and suggest that this pathway represents a promising therapeutic target for treatment of obesity and diabetes (Hong, 2016).

The deviations from the consensus of protein kinases make it unlikely that Trbl is a functional protein kinase. To test this possibility further, an allele of Trbl (K266R) was generated, mutated at a crucial lysine of the catalytic center thought to govern the specificity of kinases for their targets. This lysine was changed to an arginine, a change which should maintain the configuration of the active site but alter its specificity. A lysine is present in all serine-threonine protein kinases, whereas an arginine is characteristic of conventional tyrosine kinases. When injected into embryos, this allele also induces a premature pause in the cell cycle (20 out of 41 embryos scored), confirming that Trbl does not function as a serine-threonine protein kinase (Großhans, 2000).

Evolutionary Homologs

TRB1 negatively regulates gluconeogenesis by suppressing the transcriptional activity of FOXO1

Tribbles related homolog 1 is the mammalian ortholog of Tribbles, which controls cell division and migration during development in Drosophila. TRB1 is a pseudokinase and functions as a scaffold protein. Recent findings suggest that TRB1 plays important roles in hepatic lipid metabolism and participates in insulin resistance. However, the underlying mechanisms have not yet been elucidated. This study demonstrates that TRB1 suppresses FOXO1 (see Drosophila Foxo) transcriptional activity to downregulate the expression of G6Pase and PEPCK, which encode gluconeogenic rate-limiting enzymes. TRB1 knockdown enhances FOXO1 binding to the gluconeogenic gene promoters. It also increases FOXO1 acetylation and recruits CBP to the binding sequence of FOXO1. These results suggest that TRB1 suppresses the expression of G6Pase and PEPCK by attenuating FOXO1 transcriptional activity and negatively regulates gluconeogenesis (Tsuzuki, 2018).

date revised: 5 August 2023

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