Carbohydrate Metabolism in Drosophila: Reliance on the Disaccharide Trehalose

1. Introduction

Work on Drosophila has been so influential that it has even impinged on human metabolism and health. The reason for this is obvious: There is a strong evolutionary conservation between biological processes of flies and humans. Also, cloning and functional analyses of genes in Drosophila allowing study of many cellular processes have greatly aided in the identification of homologous genes and processes in humans and other organisms, as exemplified by the study of homeotic genes and mutations [1]. A great number of genes required for a myriad different functions (from transcription factors, structural proteins, ion channels and signaling molecules, to those required for behavior and sleep) is common between the two species [2]; in fact, [3] showed that from a pool of 287 genes known to be implicated in humans in cancer and malformations, and neurological, cardiovascular, hematological, immune, endocrine, renal and metabolic diseases, 178 (62%) had their counterparts in flies. These findings underscore the potential impact of Drosophila as a powerful system for gene discovery, but also for study of diseases’ symptoms and complications, and the discovery of therapeutic treatments.

Drosophila melanogaster was originally introduced as a research organism and genetic model at the beginning of the twentieth century by T. H. Morgan and co-workers. From their pioneering studies on heredity and the chromosome theory of inheritance [4], work with this model organism has been the source of biological insights in many areas, including genetics and development [5]. Clearly, Drosophila offers an array of advantages: its cultures in the laboratory are easy and economical, its life cycle is short (approximately ten days at room temperature), its embryonic development lasts only twenty four hours, there is no meiotic recombination in males, it has clearly defined and easily identified structures in the cytoskeleton (subject to mutational change, and, correspondingly, easy to score), has a group of marked balancer chromosomes that impede meiotic recombination in females thus allowing the recovery and maintenance of other mutations of interest, has only four pairs of chromosomes, etc. These characteristics all greatly aid in genetic analyses [6], and since the sequencing of its genome (first released in 2000 [2, 7]), has arguably become the best characterized multicellular eukaryote model system. Critically, there are also large collections of mutant strains, with the one housed at the Bloomington Drosophila Stock Center at Indiana University [8] culturing over 49,000 different mutant stocks. Finally, since the publication of the fly genome, and its refinements with time, emphasis has also been placed on analyses and studies of physiology and signaling pathways involved, rather than just concentrating on ‘gene hunts’ or ‘fishing expeditions’ for candidate genes [9].

Since the discovery and characterization of abnormal phenotypes of insulin pathway mutants in flies [10], one of recent focuses of work in Drosophila have been metabolic studies, especially those of carbohydrates. One of the underlying reasons for this is, no doubt, the recent surge in obesity and diabetes mellitus type 2 among the world’s population in both developing and developed countries [11, 12]. Just as it happens in vertebrates, Drosophila melanogaster regulates its levels of circulating carbohydrates, and stores excesses in the forms of glycogen and lipids, especially of triglycerides, in the fat body [13]. Another fruit of this recent surge in carbohydrate studies has been the elucidation of evolutionarily conserved signaling pathways, like the insulin/IGF/Target of Rapamycin (TOR) pathway [14].

2. The insulin pathway

Glucose homeostasis in mammals is maintained by feedback mechanisms balancing glucose cellular import and replenishment to the blood, so as to maintain a constant 5.5 mM level [15]. This level is the result of glucose cellular entry rates, glucose removal by the liver and new glucose coming from digestion of food glucose sources to the blood. Members of the Glut family of facilitated transporters, of which mammals possess several isoforms and genes, carry out glucose cellular import. It was recognized in the middle nineties that insulin stimulation in adipocytes in culture resulted in activation of a phosphatidyl-inositol-3-kinase (PI3K) isoform, and more glucose cellular import, resulting from Glut4 plasma membrane translocation [16]. Nowadays, Glut4 plasma membrane translocation from an internal vesicle pool is recognized as the rate-limiting step in glucose import by muscle (skeletal and cardiac) cells, and vertebrate adipocytes [17].

In contrast, the relation between insulin signaling and glucose cellular import in insect cells is not that clear. In Drosophila, there is a glucose transport system with similar kinetics to vertebrate Glut transporters [18]. A Drosophila Glut1 homolog cDNA has been cloned and described [19]. Nonetheless, experiments have shown that there is no increase in cellular import of 2-deoxyglucose labeled with ³H in Drosophila Kc cells in culture after activation of the insulin pathway [20], and neither manipulation of activation levels of PI3K or protein kinase B (also called AKT or PKB), both molecules in the insulin pathway, in Drosophila S2 cells augments glucose cellular import [21].

These results could lead one to think that insulin pathway function in vertebrates and insects is not well conserved, and that insect insulin-like-peptides (ILPs) have no role in glucose and / or carbohydrate homeostasis, yet there is ample evidence to the contrary. The Drosophila genome possesses eight insulin-like–peptides (ILPs) [22-25] at last count, and these have been shown to interact with the Drosophila insulin receptor homolog (InR). Moreover, the Drosophila insulin receptor homolog can be functionally exchanged with the vertebrate insulin receptor [26], arguing that functional conservation must be significant.

Furthermore, ablation of the neurosecretory cells (NSC) that secrete DILPs 2, 3 and 5 in larvae gives rise to adults with developmental delays, reduced body size, and high levels of carbohydrates and lipids. Ablation of these same cells in adult flies lead to hypertrehalosemia without growth phenotypes. Knockdown of DILP2 alone by RNAi in NSC results in higher corporal trehalose levels [27, 28]. These effects, due to lower levels of circulating DILPs, causing lipid and carbohydrate accumulation, are analogous to effects observed in diabetic patients or diabetic mice when there is generalized insulin resistance, like in the insulin receptor knockout mice model [13].

Perhaps the clearest demonstration of the insulin pathway’s role in carbohydrate and lipid metabolism comes from studies of Drosophila insulin pathway mutants: From the pioneering studies on Chico, a Drosophila insulin-receptor-substrate (IRS) homolog [10], it has now been demonstrated that practically all viable mutant combinations, that create partial loss-of-function or hypomorphic conditions for the insulin pathway, course with altered lipid and carbohydrate levels [29]. This is also akin to having congenital diabetes mellitus type 2, as these flies are born with a dysfunctional insulin pathway. Besides this metabolic disarray, these mutants have developmental delays, fewer and smaller cells, and concomitantly, smaller sizes, these last being cell independent [10]. In this regard, fly mutant growth phenotypes are similar to IRS1 loss-of-function mice models [30, 31] and other murine diabetic models [32] where growth phenotypes also exist; besides, in flies, the insulin and IGF (insulin growth factor) pathways are mediated by a common route. In addition, fly mutants also have nervous system abnormalities [29].

The insulin and insulin growth factor (IGF) pathways, as stated above, are united in Drosophila (see figure 1). Eight ILPs function as ligands for the sole InR in the fly genome [22, 23]. ILPs 1, 2, 3, 5 and 7 are secreted from NSC in the brain, whereas ILPs 4 and 6 are expressed in the medial intestine and the fat body. Binding of any of these ILPs to the InR causes receptor oligomerization and InR auto-phosphorylation, as the InR is a receptor tyrosine kinase. The InR itself is translated as a single polypeptide, but during its maturation within the cell and before insertion in the plasma membrane, it is proteolytically cleaved, both pieces held together by disulphur bridges. Once phosphorylated, the InRs recruit the IRS-like adaptor proteins, Chico and Lnk [10, 33]. Besides Chico and Lnk, the Drosophila InR has a long cytoplasmic tail that also acts as an IRS, in effect acting as a third IRS [26] (vertebrates have four IRS genes). InR phosphorylates the IRS proteins, like Chico, generating docking sites for other proteins in the pathway.

The activated InR-IRS complex then recruits a PI3K complex (called Dp110 and Dp60, catalytic and regulatory subunits, respectively) to the membrane [14, 34]. Activated PI3K then generates phosphatidyl-inositol 3, 4, 5 trisphosphate (PIP3) from membrane phosphatidyl-inositol 4, 5 biphosphate (PIP2) and ATP. PTEN, a tumor suppressor gene, counteracts this enzymatic activity of PI3K, regulating PI3K output [35]. Plasma membrane accumulation of PIP3 then leads to membrane recruitment and activation by phosphorylation of two kinases, PDK1 and PKB (also called AKT). PKB in particular then phosphorylates and regulates several target proteins involved in metabolic control, including glycogen synthase kinase 3 (GSK-3, or shaggy (sgg), as the gene is called in Drosophila), the transcription factor Foxo, the Tuberous sclerosis complex 2 protein, or Tsc2 (called gigas (gig) in Drosophila) and the salt-inducible kinase 2, or SIK2. PKB’s activity is negatively regulated by a protein phosphatase, of class 2A (PP2A), which de-phosphorylates PKB and inactivates it. PKB can also be phosphorylated and activated by the Target of Rapamycin-complex 2 (TOR-C2) [13].

Activated shaggy then reduces glycogen synthesis by phosphorylation of glycogen synthase, besides participating in several other signaling networks, most notably the Wnt pathway [10, 13]. SIK2 phosphorylates and inactivates the transcriptional coactivator TORC (CRTC2 in mammals), precluding it from acting as a CREB co-activator. Foxo acts as a central catabolic regulatory point in cells; its phosphorylation by PKB enables its cytoplasmic retention, thereby inhibiting its nuclear localization, and transcriptional activity. Part of insulin’s (or ILPs) anabolic effects relies on counteracting Foxo activity, which promotes energy conservation. Foxo also activates InR transcription, as part of a feedback loop.

Gigas’ phosphorylation by PKB stimulates the activity of the Target of Rapamycin Complex 1 (TOR-C1) mediated by the Rheb GTPase. The Target of Rapamycin (TOR) kinase is a central anabolic kinase, regulating many aspects of general metabolism. Besides receiving input from the insulin pathway, TOR also receives input from nutrient sensors like Slimfast, an amino acid plasma membrane transporter. TOR regulates lipid, carbohydrate and autophagy levels. TOR itself forms part of two different complexes, TOR-C1 and TOR-C2. TOR-C1 is anabolic and regulates cellular growth and size, controlling cells’ commitment to growth and energy utilization. Among its phosphorylation targets are the transcription factor SREBP (sterol-regulatory-element-binding-protein), and the ribosomal S6 kinase (S6K). TOR-C2 acts antagonistically to TOR-C1.

SREBP, a target of TOR-C1, regulates expression of genes involved in sterol biosynthesis. S6K, another TOR-C1 target, phosphorylates and regulates several proteins, chief of which is the ribosomal protein S6, besides the initiation and elongation factors elF4B and eEF2K. S6K promotes growth and protein synthesis [13]. In summary, Drosophila and vertebrates have great similarities in the insulin pathway, both at the structural and functional levels.

In vertebrates, glucose homeostasis also depends on another hormone that has opposite effects from those of insulin: Glucagon. Both are secreted from the pancreas. Insulin facilitates glucose translocation from the general circulation to the cell interior via plasma membrane translocation of Glut4 glucose transporters (acting as an anabolic hormone), besides activating growth. In contrast, glucagon activates glycogen hydrolysis (acting as a catabolic hormone), helping to maintain constant glucose levels in the blood. In insects, these basic homeostatic mechanisms are evolutionarily conserved, with a family of eight peptides akin to insulin acting as anabolic hormones in glucose homeostasis, as stated, and the adipokinetic peptides, a subfamily of which constitutes the so-called hypertrehalosemic hormones, playing the role of glucagon.

Despite the close homology throughout the insulin pathway and sugar metabolism, there is one clear and fundamental difference between carbohydrate metabolism of insects and vertebrates: Insects have trehalose (or 1-D-glucopyranosyl-1-D-glucopyranoside; see figure 2). In fact, glucagon’s functions are partially diverted to trehalose synthesis by means of the afore-mentioned hypertrehalosemic hormones. In having trehalose, insects are not alone: Many other species also use trehalose.

3. Why trehalose? Physicochemical properties of trehalose

Trehalose was discovered in the mid-nineteen century in rye ergot and then as a component of the cocoons of beetle species of the genus Larinus [36]. Many organisms use trehalose as a go-between between carbohydrate storage as glycogen and the cellular availability of glucose for energy needs (besides the other cellular roles trehalose plays in the cells’ economy as an anti-stress factor): Why is this the case? Vertebrates have foregone altogether the use of trehalose, but still possess unique trehalose-degrading enzymes (trehalases; for example, human trehalase, used to digest trehalose from the diet and garner the glucose moieties within [37]). What advantages do trehalose imparts that has assured its selection in many diverse organisms (bacteria, algae, plants, fungi, invertebrates including insects), yet its loss in the vertebrate lineage? Part of the reason lies in the unique physicochemical properties of trehalose.

Trehalose is a disaccharide formed from two glucose moieties: It is synthesized by the union of two glucopyranose rings at the reducing end of the glycosyl residues. As a consequence, it is a non-reducing sugar. It demonstrates exceptional stability (with a high melting temperature, above 200C, a high glass transition temperature, above 100C, a slow rate of hydrolysis, and very high stability in extreme pHs; more than 99% of trehalose is still present in a pH range from 3.5 to 10 after 24 h at 100C). It is not easily hydrolysed by acid (less so than sucrose, another non-reducing disaccharide found mainly in plant tissues), and its glycosidic bond is not cleaved by -glycosidase, either. It also has low hygroscopicity, normally appearing as a dihydrated form. This form forms hydrogen bonds with the two water molecules, albeit at relatively short distances, making it an unusually strong interaction. In solution, all of the trehalose hydroxide groups make hydrogen bonds with water molecules. These bonds are easier to form than those with sucrose, leading to trehalose solutions with higher viscosity at equivalent concentrations, and less diffusion [36, 38].

The fact that trehalose is so resistant to acid hydrolysis may be key to understanding the use of trehalose in insect blood or hemolymph: Insect hemolymph has high amounts of peptides, free amino acids, and proteins, all of which have amine and amino functional groups. A less resistant sugar might then react with these and be subject to condensation reactions resulting in imines and enamines, altering the structure of proteins and peptides. Also, it is less likely, from the foregoing, to cause ‘browning’, where the sugar’s reactivity ultimately leads to the formation of pigments, and protein degradation, something sucrose is more prone to do. Sucrose circulates in plant sap without these problems mainly because these liquids have much lower levels of proteins, amino acids, and lipids. The slow rate of hydrolysis of trehalose also makes it a very stable compound, and one that is likely to aid in stabilizing other components of mixtures where trehalose is an ingredient [38].

Trehalose is highly soluble in water (up to about 50 g/100 g water at 20C), and even if it is less water-soluble than sucrose (200 g/100g water), solubility is still high enough to allow it to accumulate to the 100 mM range in bodily fluids, like insect hemolymph. This allows trehalose to be readily transported and to accumulate in various extracellular and intracellular milieus. Also, it is a sweet compound, although, again, less so than sucrose. In solution, trehalose does not normally form intra-molecular hydrogen bonds [38].

On the other hand, trehalose also functions as a stabilizer in solutions for other organic molecules, like lipids and proteins. It seems to do so stemming from some of the physicochemical properties discussed: higher hydration capacity, higher viscosity and hydrogen bonding with proteins and other organic molecules, displacing water, than the disaccharide sucrose.

3.4. Trehalose anabolism

The ultimate source of trehalose comes from diet carbohydrates, but their use for trehalose synthesis is not necessarily direct. Under conditions where carbohydrates are abundant in the diet, hexoses are transported in the intestine by means of facilitated diffusion. A Drosophila Glut1 homolog was cloned and sequenced, with a 68% homology to human Glut1 [19]. Glut1 transporters are not specific for glucose, being able to transport other carbohydrates, like mannose, galactose and glucosamine. Insects with protein-rich diets utilize amino acids to synthesize de novo glucose, amino acids being also an indirect source for trehalose synthesis. When not required for energy, glucose from the diet can be stored as glycogen in the fat body. Glycogen is generally a more proximate source of glucose for trehalose synthesis.

Trehalose is synthesized in a condensation reaction of two glucose molecules, generally derived from glycogen (in effect, one of the constituent glucose substrates is in the form of UDP-glucose, derived from glucose-1P, whereas the other glucose substrate is glucose-6P). Since glucose is at the crux of trehalose anabolism, glucose regulation in the fat body impinges directly on trehalose regulation. Both vertebrates and invertebrates, in general, have very similar glucose regulatory mechanisms: insulin signaling and glucagon / hypertrehalosemic (HTH) signaling peptides, as stated above. Hypertrehalosemic hormones in particular are a family of adipokinetic (AKH) neuropeptides whose function is to derive glucose into trehalose synthesis in the fat body.

3.4.1. Insect fat body

Hemolymph circulating trehalose is synthesized in insects’ fat body, a very important and extended organ analogous to both vertebrate liver and adipose tissue in insects [44]. Fat body is distributed throughout the insect body. It is also the principal organ for hemolymph circulating proteins and metabolites syntheses. It performs key roles in energy metabolism, as it both stores lipids and carbohydrates (these last in the form of glycogen), and responds to energy demands liberating both glucose and trehalose to the hemolymph. A Drosophila melanogaster fat body transcriptome analysis has shown that over 2200 genes are expressed in fat body cells, 290 of which attain high levels of expression. Among these are genes required for the insulin pathway, and lipid regulators. Many of the genes identified remain uncharacterized, and so, more work is required to define in greater detail the fat body transcriptome [45].

Several cell types constitute the fat body, the main one being the adipocyte (see figure 3). The adipocyte is a very dynamic cell type, with an active metabolism, regulating organismal carbohydrates and lipids, and also being an important endocrine cell [44].

The fat body is the main insect organ storing energy reserves. Most insect reserves are in the form of lipids, and these, mainly as triacylglycerols (TAGs). TAGs are stored in lipid droplets within adipocytes (figure 3). Lipid droplets have an outer coat of a monolayer of phospholipids and cholesterol molecules enclosing a core of TAGs and cholesterol molecules. In this outer coat some proteins are embedded, especially PATs (perilipin and adipocyte differentiation-related proteins). In insects two PAT proteins have been identified: Lsd1 and Lsd2 [46]. Together, they control synthesis and lipolisis of lipid stores.

Trehalose synthesis in response to energy needs normally courses through glycogen breakdown by glycogen phosphorylases in the fat body (see figure 4). Since the glucose thus obtained can also vie for glycolysis, this last path has to be inhibited. This is achieved in the fat body of cockroaches, where it has been studied, by reduction of fructose-2, 6-bisphosphate, an allosteric regulator of phosphofructokinase, the key regulatory enzyme in glycolysis [15, 47]. The adipokinetic hormones in turn, stimulate this decrease.

3.4.2. Adipokinetic hormones (Hypertrehalosemic hormones)

AKH were originally shown to induce lipid catabolism. In insects, as stated, one subgroup of these hormones produces an increase in circulating trehalose in the hemolymph [48]. About fifty HTH have been reported to date, and it is known that they participate in different functions. They are produced by neurosecretory cells of the corpora cardiaca, part of the ring gland of insects together with the prothoracic gland and the corpus allatum. The ring gland is located surrounding the anterior proventriculus in the thorax [49].

HTHs are synthesized from longer precursor peptides that are proteolitically processed to 8-10 amino acid long peptides. When the HTH neurosecretory cells are ablated, there is a significant drop in circulating trehalose levels in Drosophila larvae; over-expression of these HTHs leads to higher trehalose levels [48]. These hormones are secreted in response to various stimuli that would necessitate high circulating levels of trehalose, like lack of food, dehydration or thermal stress, or during flight. Some insects, like locusts, initiate flight utilizing trehalose, but switch to lipids later, whereas others, like some cockroaches, depend solely on trehalose supply. Still others use proline as an energy source, like some beetle species [44].

HTHs have membrane receptors. The Drosophila and homologous Bombyx mori HTH receptors have been cloned; they are G-protein-coupled-receptors (GPCRs) and are sequence-wise related to the gonadotrophin releasing hormone receptors of mammals [50]. Mutations in the Drosophila receptor show that it indeed mediates the HTH response: Mutants had glycogen and lipid accumulation in the fat body, irrespective of nutritional status, whereas rescue experiments returned glycogen and lipids to wild type levels [51].

There are scant data on the signaling pathways activated by HTHs. In cockroaches, HTH signaling increases inositol trisphosphate levels, leading to intracellular high Ca⁺² concentrations and higher activity of protein kinase C (PKC). This produces an increase in the activity of glycogen phosphorylase, and trehalose synthesis. Inhibitors of PKC reduce trehalose production [52]. On the other hand, in locusts, AKH stimulation leads to cAMP production [53].

3.4.3. Final reactions of trehalose synthesis

Trehalose phosphate synthase catalyses condensation of glucose 6-P and UDP-glucose to generate trehalose 6-P in the fat body. Trehalose 6-P is then de-phosphorylated by the protein phosphatase coded by the trehalose phosphate phosphatase (tpp) gene to generate trehalose [54]. Both trehalose phosphate synthase and glycogen synthase use UDP-glucose as substrate, so besides competition for glucose between trehalose synthesis and glycolysis, the opposing trehalose phosphate synthase and glycogen synthase enzymes must be regulated too in order to shunt glucose to one or the other metabolic outcome [55]. Km values for trehalose phosphate synthase vary in different species where it has been studied (the moth Hyalophora cecropia, the cockroach Periplaneta americana, the blowfly Phormia regina, the sleeping chironomid Polypedilum vanderplanki, and the fruit fly Drosophila melanogaster). trehalose phosphate synthase 1 (tps1) overexpression in flies results in elevated trehalose levels, whereas mutations in this gene are lethal in the first larval instar. Trehalose phosphate synthases have also been studied in other organisms, like Escherichia coli, the yeast Saccharomyces cerevisiae, and plants like Selaginella lepidophylla, Myrothamus spp. and Arabipdosis thaliana. In A. thaliana, whose genome has been sequenced, there are eleven tps and ten tpp genes, suggesting that trehalose metabolism in plants is also very important [55, 56].

The aforementioned synthesis pathway is the one most commonly found; but there are, nevertheless, four other biosynthetic routes, three of them only present in prokaryotes. Some prokaryotes can use up to three different synthesis routes. The genes encoding trehalose synthase (treS), maltooligosyl-trehalose synthase (treY), maltooligosyl-trehalohydrolase (treZ) and trehalose glycosyltransfering synthase (treT) are prokaryotic, whereas trehalose phosphorylase (treP) is present in both [56] (see figure 5).

4. Conclusion

Why has trehalose become an evolutionary ‘winner’ molecule? And this, despite the energetic costs associated with its reasonably convoluted metabolism, as a central go-between between the diet and other storage carbohydrates. The reasons seem to stem from the fact that trehalose is not only a high energy storage molecule, with low reactivity and high stability, but also, and precisely because of this stability and lack of reactivity, because it can stabilize and preserve other biomolecules, like proteins and lipids subjected to stressful environments, besides environments; besides, it can be transported it can be transported and accumulated to high concentrations without toxic effects.

Its prevalence in many different organisms of varied evolutionary origins and relatedness strongly suggest that trehalose has been retained in most lineages, rather than introduced, and that it is only in the vertebrate lineage where it is conspicuously missing. Why this is so in vertebrates, is difficult to explain.

Nowadays, trehalose has been subject to a revival in interest, since many of the same characteristics of non reactivity, stability and no toxicity that have propelled it to such a prominent role in many species in the natural world are now being newly appreciated by the pharmaceutical and cosmetic industries. Trehalose is being featured more and more in the excipients of drugs and pills, in creams, and as a sugar substitute in processed food formulas, as it is an energetic, non-toxic, stable meal ingredient, and has a sugary taste. Especially in Japan, where a procedure for industrial synthesis of trehalose was invented and is now being commercially applied, the allure of trehalose is proving to be irresistible, with promises in the near future to likewise take on the world as a whole [38].

The corresponding author’s laboratory is funded by PAPIIT # IN203110 and CONACYT # 81864. A. R. D. is funded by CONACYT scholarship # 369737.