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Cachexia (Wasting syndrome)
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Wasting is the process characterized by an involuntary loss of body mass manifested in particular by degeneration of skeletal muscles and adipose tissues. Wasting is not only a physiological condition responding to extremely low energy intake and infection but also part of a complex systemic disorder associated with many diseases, including cancers, chronic obstructive lung disease, congestive heart failure, chronic kidney disease, and other chronic diseases. In particular, 50% of advanced cancer patients are affected by wasting syndrome, which accounts for approximately 20% of cancer death. A number of studies have implicated proinflammatory cytokines, such as tumor necrosis factor α and interleukin 1 and 6, as secreted factors involved in wasting associated with various conditions. Additionally, insulin-like growth factor 1 (IGF-1) signaling is a critical regulator of muscle mass maintenance. Downregulation of IGF-1 signaling in skeletal muscles decreases Akt activity and in turn increases Foxo activity, which induces muscle protein degradation through the ubiquitin-proteasome system and autophagy. Moreover, the transforming growth factor β family members myostatin and activin have been identified as additional secreted factors regulating organ wasting. Stimulation of myostatin/activin signaling in skeletal muscles activates Smad2/3 signaling and inhibits Akt signaling, which increases catabolism of muscle proteins (Kwon, 2015 and references therein).

Cancer is a leading cause of death in industrialized societies, yet the mechanisms by which a tumor claims the life of its host are not always clear. In some cases, the growth of primary or secondary tumors disrupts the function of essential organs, but in other instances, lethality is caused by physiological alterations at a distance from the tumor site. These distant influences, sometimes grouped as “paraneoplastic syndromes,” are major contributors to the morbidity and mortality of cancer patients. A particularly debilitating distant tumor-host interaction is cancer cachexia, which is estimated to occur in >80% of patients with advanced cancers and to account for >20% of cancer deaths. Cancer cachexia is a metabolic disorder that produces progressive tissue wasting, most evident in the loss of adipose and muscle tissue. It is seen frequently with certain types of tumors and only rarely with others. Patient studies are complicated by heterogeneities in patient population, presentation, tumor pathology, comorbidities, and accompanying thereapeutic regimes. Cachectic patients show heightened risk of respiratory failure, increased susceptibility to chemotherapeutic toxicity, and other lethal sequelae. Unlike anorexia, in which caloric intake is reduced, wasting induced by cachexia is not reversed by supplemental nutrition. Available therapies for this clinically critical condition are notably limited in scope and effect (Figueroa-Clarevega, 2015 and references therein).

Bloating syndrome. The “bloating syndrome,” observed in flies transplanted with imaginal discs mutant for the tumor suppressor lethal (2) giant larvae (l(2)gl), is a systemic phenotype relevant to the wasting syndrome. Whereas a wild-type imaginal disc transplanted into the abdomen of an adult fly only grows until it reaches its normal size, a transplanted l(2)gl mutant disc undergoes neoplastic growth and eventually kills the fly. However, before they die, these flies develop the bloating syndrome, whereby the abdomen becomes swollen and translucent and the fat body and ovaries are almost completely degenerated. This degeneration of the fat body and ovaries is reminiscent of the wasting of adipose tissue and skeletal muscles in mammals, because the fat body and ovaries are the organs preserving energy in the forms of lipids and proteins in Drosophila (Kwon, 2015 and references therein).

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Abstract
Organ wasting, related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. This study developed a model for organ wasting in adult Drosophila, whereby overproliferation induced by activation of Yorkie, the Yap1 oncogene ortholog, in intestinal stem cells leads to wasting of the ovary, fat body, and muscle. These organ-wasting phenotypes are associated with a reduction in systemic insulin/IGF signaling due to increased expression of the secreted insulin/IGF antagonist ImpL2 from the overproliferating gut. Strikingly, expression of rate-limiting glycolytic enzymes and central components of the insulin/IGF pathway is upregulated with activation of Yorkie in the gut, which may provide a mechanism for this overproliferating tissue to evade the effect of ImpL2. Altogether, this study provides insights into the mechanisms underlying organ-wasting phenotypes in Drosophila and how overproliferating tissues adapt to global changes in metabolism. (Kwon, 2015).

Highlights

• Localized aberrant cell proliferation induced by activation of Yki in ISCs causes systemic organ wasting.
• Expression of ykiact in midgut causes repression of genes involved in energy metabolism in muscle.
• Overproliferating midgut due to activation of Yki causes hyperglycemia.
• Depletion of ImpL2 from esgts>ykiact midguts rescues systemic reduction of Akt1 phosphorylation and hyperglycemia.
• Depletion of ImpL2 in esgts>ykiact Midguts rescues ovary wasting and muscle degeneration.
• Expression of Insulin/IGF pathway components and glycolytic enzymes is upregulated in the proliferating midgut due to aberrant activation of Yki.

Discussion
This study describes the unexpected observation that the overproliferating midgut due to aberrant Yki activity in ISCs induces the bloating syndrome and systemic organ wasting. Additionally, the overproliferating midgut perturbs organismal metabolism, resulting in an increase of hemolymph trehalose and depletion of glycogen and triglyceride storage. Strikingly, it was shown that the accumulation of hemolymph trehalose and organ-wasting processes are dependent on the antagonist of insulin/IGF signaling, ImpL2, which is specifically upregulated in the proliferating midgut. The study provides strong genetic evidence supporting that systemic organ wasting associated with the aberrant activation of Yki in ISCs cannot be explained solely by the perturbation of general gut function. Based on these findings, the study proposes that ImpL2 is a critical factor involved in systemic organ wasting in Drosophila (Kwon, 2015).

Results from this study do not rule out the existence of an additional factor(s) contributing to the bloating syndrome and organ-wasting phenotypes. Indeed, the partial rescue of the bloating syndrome and organ-wasting phenotypes by depletion of ImpL2 in esgts>ykiact midguts suggests the existence of an additional factor(s). Moreover, it was observed that ectopic expression of ImpL2 in ECs is not sufficient to reduce whole-body triglyceride and glycogen levels, although it causes hyperglycemia, reduction of Akt1 phosphorylation, and increase of hemolymph volume. Thus, given the involvement of diverse factors in the wasting process in mammals, it is likely that in addition to ImpL2, another factor(s) contributes to systemic organ wasting in Drosophila (Kwon, 2015).

It was shown that the bloating syndrome caused by esgts>ykiact is associated with ImpL2, as depletion of ImpL2 from esgts>ykiact midguts significantly rescues the bloating phenotype. Given the observation that elevated expression of ImpL2 from esgts>ykiact midgut induces hyperglycemia, it is speculated that the accumulation of trehalose in hemolymph is a factor involved in bloating, because a high concentration of trehalose can cause water influx to adjust hemolymph osmolarity to physiological levels. Interestingly, recent findings have shown that disruption of l(2)gl in discs activates yki, suggesting that the bloating syndrome observed in flies with transplanted l(2)gl mutant discs may be due to aberrant yki activity (Kwon, 2015).

Findings of this study are reminiscent of a previous study showing that in Drosophila, humoral infection with the bacterial pathogen Mycobacterium marinum, which is closely related to Mycobacterium tuberculosis, causes a progressive loss of energy stores in the form of fat and glycogen—a wasting-like phenotype. Infection with M. marinum causes a downregulation of Akt1 phosphorylation. Given this study's observation that ImpL2 produced from esgts>ykiact affects systemic insulin/IGF signaling, it will be of interest to test whether ImpL2 expression is increased upon infection with M. marinum and mediates the effect on the loss of fat and glycogen storage (Kwon, 2015).

yki plays critical roles in tissue growth, repair, and regeneration by inducing cell proliferation, a process requiring additional nutrients to support rapid synthesis of macromolecules including lipids, proteins, and nucleotides. In particular, increased aerobic glycolysis metabolizing glucose into lactate is a characteristic feature of many cancerous and normal proliferating cells. Interestingly, the aberrant activation of yki in ISCs causes a disparity in the gene expression of glycolytic enzymes and the activity of insulin/IGF signaling between the proliferating midgut and other tissues, such as muscle and ovaries. Thus, it is speculated that this disparity favors Yki-induced cell proliferation by increasing the availability of trehalose/glucose to the proliferating midgut, which presumably requires high levels of trehalose/glucose. Additionally, it will be of interest to test whether activation of Yki during tissue growth, repair, and regeneration alters systemic metabolism in a similar manner (Kwon, 2015).

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Wang, X., Li, J., Zhang, W., Wang, F., Wu, Y., Guo, Y., Wang, D., Yu, X., Li, A., Li, F. and Xie, Y. (2023). IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling. Chin Med J (Engl). PubMed ID: 37014770

Abstract

Progressive lipid loss of adipose tissue is a major feature of cancer-associated cachexia. In addition to systemic immune/inflammatory effects in response to tumor progression, tumor-secreted cachectic ligands also play essential roles in tumor-induced lipid loss. However, the mechanisms of tumor-adipose tissue interaction in lipid homeostasis are not fully understood. The yki-gut tumors were induced in fruit flies. Lipid metabolic assays were performed to investigate the lipolysis level of different types of insulin-like growth factor binding protein-3 (IGFBP-3) treated cells. Immunoblotting was used to display phenotypes of tumor cells and adipocytes. Quantitative polymerase chain reaction (qPCR) analysis was carried out to examine the gene expression levels such as Acc1, Acly, and Fasn. This study revealed that tumor-derived IGFBP-3 was an important ligand directly causing lipid loss in matured adipocytes. IGFBP-3, which is highly expressed in cachectic tumor cells, antagonized insulin/IGF-like signaling (IIS) and impaired the balance between lipolysis and lipogenesis in 3T3-L1 adipocytes. Conditioned medium from cachectic tumor cells, such as Capan-1 and C26 cells, contained excessive IGFBP-3 that potently induced lipolysis in adipocyted. Notably, neutralization of IGFBP-3 by neutralizing antibody in the conditioned medium of cachectic tumor cells significantly alleviated the lipolytic effect and restored lipid storage in adipocytes. Furthermore, cachectic tumor cells were resistant to IGFBP-3 inhibition of IIS, ensuring their escape from IGFBP-3-associated growth suppression. Finally, cachectic tumor-derived ImpL2, the IGFBP-3 homolog, also impaired lipid homeostasis of host cells in an established cancer-cachexia model in Drosophila. Most importantly, IGFBP-3 was highly expressed in cancer tissues in pancreatic and colorectal cancer patients, especially higher in the sera of cachectic cancer patients than non-cachexia cancer patients. This study demonstrates that tumor-derived IGFBP-3 plays a critical role in cachexia-associated lipid loss and could be a biomarker for diagnosis of cachexia in cancer patients.

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Lodge, W., Zavortink, M., Golenkina, S., Froldi, F., Dark, C., Cheung, S., Parker, B. L., Blazev, R., Bakopoulos, D., Christie, E. L., Wimmer, V. C., Duckworth, B. C., Richardson, H. E. and Cheng, L. Y. (2021). Tumor-derived MMPs regulate cachexia in a Drosophila cancer model. Dev Cell. PubMed ID: 34473940

Abstract

Cachexia, the wasting syndrome commonly observed in advanced cancer patients, accounts for up to one-third of cancer-related mortalities. A Drosophila larval model of organ wasting was established whereby epithelial overgrowth in eye-antennal discs leads to wasting of the adipose tissue and muscles. The wasting is associated with fat-body remodeling and muscle detachment and is dependent on tumor-secreted matrix metalloproteinase 1 (Mmp1). Mmp1 can both modulate TGF β signaling in the fat body and disrupt basement membrane (BM)/extracellular matrix (ECM) protein localization in both the fat body and the muscle. Inhibition of TGFβ signaling or Mmps in the fat body/muscle using a QF2-QUAS binary expression system rescues muscle wasting in the presence of tumor. Altogether, this study proposes that tumor-derived Mmps are central mediators of organ wasting in cancer cachexia (Lodge, 2021).

Interorgan communication ensures that organs respond in synchrony with alterations in the physiological state of the organism. One disease context where interorgan communication is relevant is cancer cachexia, where tumors cause loss of body fat and skeletal muscle, anorexia, metabolic deregulation, and systemic inflammation. Cachexia is present in up to 80% of advanced-stage cancers, and up to one-third of patients die from complications associated with cachexia. Early detection of the syndrome is further impeded by the lack of effective biomarkers, and it therefore remains a major unmet clinical need. Thus far, cancer cachexia has been studied mainly in rodent models where organ-specific genetic manipulations and large-scale, systematic in vivo functional studies (genetic screens) are challenging. Important parallels exist in how physiology is regulated between the vinegar fly Drosophila melanogaster and humans, whereby evolutionarily conserved enzymes and equivalent organs regulate carbohydrate and lipid metabolism. This makes the fly a valuable model organism for the study of cachexia. The fly fat body stores excess fat as triglycerides, thus functioning as the adipose tissues, while a group of specialized cells, called oenocytes, can function as hepatocytes by mobilizing lipids during periods of starvation. The fly muscles are also critical mediators of systemic energy homeostasis that coordinate energy supply and utilization, and are capable of inducing metabolic changes in distant tissues (Lodge, 2021).

In the last few years, models of cachexia have emerged in both Drosophila and zebrafish, in which several tumor-derived factors have been shown to modulate host physiology. The insulin/insulin growth factor (IGF) antagonist imaginal morphogenesis protein-late 2 (ImpL2) released by neoplastic cancer cells has been shown to suppress insulin signaling by binding to circulating Drosophila insulin-like peptides to cause insulin resistance in peripheral tissues. Gut-derived tumors, induced by the overexpression of the co-transcriptional factor Yorkie (Yki), also produce the PDGF- and VEGF-related factor 1 (Pvf1) ligand to nonautonomously activate host Pvr/MEK signaling and induce tissue wasting. Recently, it was shown that high-sugar-diet (HSD)-enhanced tumorigenesis upregulates Branchless (Bnl), a Drosophila fibroblast growth factor (FGF) ligand, to induce muscle wasting, which in turn releases proline, which fuels tumor growth. However, the signaling regulation that occurs in the tumor to drive tissue remodeling is so far unclear, and whether the remodeled host tissues communicate with each other is poorly understood (Lodge, 2021).

In this study, a larval cachexia model was created, enabling visualization of onset and progression of the disease, including the associated lipid mobilization, muscle detachment, and tissue remodeling within an intact animal. Among several cachectic factors, it was found that tumor-secreted Mmp1 alters the availability of Gbb in the tumor and triggers TGFβ activation in the fat body, resulting in the disruption of basement membrane (BM)/extracellular matrix (ECM) proteins at the adipocyte cell-cell adhesion site. In addition, Mmp1 and Mmp2 also act directly on the fat body and the muscle to trigger their disruption. Interestingly, the disassembly of fat-body cell-cell adhesion in the absence of tumor is sufficient to induce muscle detachment, suggesting that adipocyte breakdown releases signals that culminate in muscle detachment. Together, this study has gained valuable insights into the interplay between tumor, muscle, and adipose tissue during cachexia (Lodge, 2021).

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Santabarbara-Ruiz, P. and Leopold, P. (2021).An Oatp transporter-mediated steroid sink promotes tumor-induced cachexia in Drosophila. Dev Cell 56(19): 2741-2751.e2747. PubMed ID: 34610327

Abstract

Cancer cachexia is associated with many types of tumors and is characterized by a combination of anorexia, loss of body weight, catabolic alterations, and systemic inflammation. A tumor model was developed in Drosophila larvae that causes cachexia-like syndrome, and it was found that cachectic larvae show reduced levels of the circulating steroid ecdysone (Ec). Artificially importing Ec in the tumor through the use of the EcI/Oatp74D importer aggravated cachexia, whereas feeding animals with Ec rescued cachectic defects. This suggests that a steroid sink induced by the tumor promotes catabolic alterations in healthy tissues. This study found that Oatp33Eb, a member of the Oatp transporter family, is specifically induced in tumors promoting cachexia. The overexpression of Oatp33Eb in noncachectic tumors induced cachexia, whereas its inhibition in cachectic tumors restored circulating Ec and reversed cachectic alterations. Oatp transporters are induced in several types of hormone-dependent tumors, and this result suggests that a similar sink effect could modify hormonal balance in cachectic cancer patients (Santabarbara-Ruiz, 2021).

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Hodgson, J. A., Parvy, J. P., Yu, Y., Vidal, M. and Cordero, J. B. (2021). Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia. Int J Mol Sci 22(15). PubMed ID: 34361081

Abstract

Cancer cachexia is a common deleterious paraneoplastic syndrome that represents an area of unmet clinical need, partly due to its poorly understood aetiology and complex multifactorial nature. This study interrogated multiple genetically defined larval Drosophila models of tumourigenesis against key features of human cancer cachexia. The results indicate that cachectic tissue wasting is dependent on the genetic characteristics of the tumour and demonstrate that host malnutrition or tumour burden are not sufficient to drive wasting. This study shows that JAK/STAT and TNF-α/Egr signalling are elevated in cachectic muscle and promote tissue wasting. Furthermore, a dual driver system is introduced that allows independent genetic manipulation of tumour and host skeletal muscle. Overall, this study presents a novel Drosophila larval paradigm to study tumour/host tissue crosstalk in vivo, which may contribute to future research in cancer cachexia and impact the design of therapeutic approaches for this pathology (Hodgson, 2021).

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Karekar, P., Jensen, H. N., Russart, K. L. G., Ponnalagu, D., Seeley, S., Sanghvi, S., Smith, S. A., Pyter, L. M., Singh, H. and Gururaja Rao, S. (2021). Tumor-Induced Cardiac Dysfunction: A Potential Role of ROS. Antioxidants (Basel) 10(8). PubMed ID: 34439547

Abstract
Cancer and heart diseases are the two leading causes of mortality and morbidity worldwide. Many cancer patients undergo heart-related complications resulting in high incidences of mortality. It is generally hypothesized that cardiac dysfunction in cancer patients occurs due to cardiotoxicity induced by therapeutic agents, used to treat cancers and/or cancer-induced cachexia. However, it is not known if localized tumors or unregulated cell growth systemically affect heart function before treatment, and/or prior to the onset of cachexia, hence, making the heart vulnerable to structural or functional abnormalities in later stages of the disease. This study incorporated complementary mouse and Drosophila models to establish if tumor induction indeed causes cardiac defects even before intervention with chemotherapy or onset of cachexia. Focus was placed on one of the key pathways involved in irregular cell growth, the Hippo-Yorkie (Yki) pathway. The transcriptional co-activator of the Yki signaling pathway was overexpressed to induce cellular overgrowth; Yki overexpression in the eye tissue of Drosophila results in compromised cardiac function. These cardiac phenotypes were rescued using antioxidant treatment, with which it is concluded that the Yki induced tumorigenesis causes a systemic increase in ROS affecting cardiac function. These results show that systemic cardiac dysfunction occurs due to abnormal cellular overgrowth or cancer elsewhere in the body; identification of specific cardiac defects associated with oncogenic pathways can facilitate the possible early diagnosis of cardiac dysfunction.

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Figueroa-Clarevega, A. and Bilder, D. (2015). Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting. Dev Cell 33: 47-55. PubMed ID: 25850672

Abstract
Tumors kill patients not only through well-characterized perturbations to their local environment but also through poorly understood pathophysiological interactions with distant tissues. This study uses a Drosophila tumor model to investigate the elusive mechanisms underlying such long-range interactions. Transplantation of tumors into adults induces robust wasting of adipose, muscle, and gonadal tissues that are distant from the tumor, phenotypes that resemble the cancer cachexia seen in human patients. Notably, malignant, but not benign, tumors induce peripheral wasting. The insulin growth factor binding protein (IGFBP) homolog ImpL2, an antagonist of insulin signaling, was identified as a secreted factor mediating wasting. ImpL2 is sufficient to drive tissue loss, and insulin activity is reduced in peripheral tissues of tumor-bearing hosts. Importantly, knocking down ImpL2, specifically in the tumor, ameliorates wasting phenotypes. The study proposes that the tumor-secreted IGFBP creates insulin resistance in distant tissues, thus driving a systemic wasting response (Figueroa-Clarevega, 2015).

Highlights

• Tumor induces cachexia-like wasting in host tissues.
• Tumor-induced wasting is not due to starvation.
• ImpL2 is secreted by malignant tumors and is sufficient to induce wasting.
• Tumor reduces insulin signaling pathway sctivity in host tissues.
• ImpL2 is necessary for robust tumor-induced cachexia-like wasting.

Discussion
Cachexia remains a major obstacle to cancer treatment, in part because the molecular mechanisms that drive it remain uncertain. This study describes a fly model that mimics certain aspects of human cachexia and utilizes this model to identify a specific cachectic mediator. The tumor-induced wasting described herein in flies resembles cancer cachexia in its independence from food consumption, its target tissues, its progressive nature, and its induction by certain but not all types of tumors. The fly model does not parallel all features associated with the human condition; for instance, only slight upregulation of putative fly orthologs of mammalian regulators implicated in muscle catabolism are detected. Human cancer cachexia is clearly a heterogeneous and multifactorial condition, and this complexity has impeded progress in its understanding. This study uses a reductionist system to identify a single tumor-derived factor that can drive the robust deterioration of peripheral tissues (Figueroa-Clarevega, 2015).

Insulin signaling is a central regulator of tissue mass in both flies and humans. Data demonstrate that ImpL2, a secreted insulin antagonist produced by malignant tumors, is a major mediator that is both necessary and sufficient for wasting. It is known that ImpL2 is also a systemic wasting factor in a different fly tumor model. Reduced insulin signaling is further responsible for wasting induced by mycobacterial infection of flies; whether ImpL2 is the relevant mediator in this case is not known. ImpL2 is the single fly homolog of mammalian IGFBPs and can bind to systemic insulin-like ligands to antagonize insulin signaling. By this mechanism, the tumor effectively induces insulin resistance in peripheral tissues (Figueroa-Clarevega, 2015).

Insulin resistance is a frequent feature of both cachectic patients and rodent cachexia models; indeed, some evidence suggests that exogenous insulin can ameliorate tissue loss in these contexts. The seven mammalian IGFBPs are variously upregulated or downregulated in different tumors, but they have been evaluated in cancer, primarily with respect to their affects on tumor growth. Data from this study motivate assessments of whether highly cachectogenic human tumors, such as pancreatic and gastric cancers, display elevated expression of IGFBPs and how therapies designed to correct insulin resistance might be used to treat such tumors (Figueroa-Clarevega, 2015).

ImpL2 joins the list of effectors induced by neoplastic transformation in fly tumors, including mitogens and pro-invasive factors. Recent work shows that the Upd3 mitogen is upregulated by dual activity of JNK and Hippo signaling. The ImpL2 regulatory region, like that of Upd3, contains evolutionarily conserved binding sites for AP-1 and Sd transcription factors, suggesting that it may also be synergistically regulated by these pathways that monitor epithelial integrity. Despite the reduced insulin signaling in neoplastic tumors themselves (e.g., 4EBP levels are elevated ∼21-fold, and they are hypersensitive to PI3K reduction), the tumors nevertheless robustly proliferate. How ImpL2-upregulating tumors escape insulin resistance remains an unanswered question, although metabolic changes suggested by transcriptome alterations may be a possible mechanism (Figueroa-Clarevega, 2015).

While tumor-specific inhibition of ImpL2 causes a significant amelioration of the wasting phenotype, rescue is not complete, suggesting that other aspects of tumor-host interaction remain to be uncovered. It is known that a fly homolog of IL-6, a molecule implicated in several rodent cachexia models, is not sufficient to induce wasting, while partial ablation of host innate immune cells does not qualitatively alter wasting phenotypes; however, contributing roles for these factors have not been ruled out. Future work will analyze other tumor-produced factors, including metabolites generated by anabolic and catabolic alterations in the tumor, to evaluate their involvement as well. The manipulability of the simple model developed here, including the ability to rapidly assess fully defined combinations of host and tumor genotypes, opens the door to candidate as well as forward genetic approaches to identify additional factors mediating tumor-host interactions (Figueroa-Clarevega, 2015).

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Zuberova, M., Fenckova, M., Simek, P., Janeckova, L. and Dolezal, T. (2010). Increased extracellular adenosine in Drosophila that are deficient in adenosine deaminase activates a release of energy stores leading to wasting and death. Dis Model Mech 3: 773-784. PubMed ID: 20940317

Abstract
Extracellular adenosine is an important signaling molecule in neuromodulation, immunomodulation and hypoxia. Adenosine dysregulation can cause various pathologies, exemplified by a deficiency in adenosine deaminase in severe combined immunodeficiency. This study established a Drosophila model to study the effects of increased adenosine in vivo by mutating the main Drosophila adenosine deaminase-related growth factor (ADGF-A). Using a genetic screen, it was shown that the increased extracellular adenosine in the adgf-a mutant is associated with hyperglycemia and impairment in energy storage. The adenosine works in this regard through the adenosine receptor as an anti-insulin hormone in parallel to adipokinetic hormone, a glucagon counterpart in flies. If not regulated properly, this action can lead to a loss of energy reserves (wasting) and death of the organism. Because adenosine signaling is associated with the immune response and the response to stress in general, these results mark extracellular adenosine as a good candidate signal involved in the wasting syndrome that accompanies various human pathologies (Zuberova, 2010).

Highlights

• Mutation in Phosphorylase Kinase suppresses the adgf-a mutant phenotype.
• The adgf-a mutant is extremely sensitive to diet restriction.
• Sensitivity to dietary restriction is specific for adgf-a.
• Sensitivity to dietary restriction is suppressed by blocking the signaling through the adenosine receptor.
• Extracellular adenosine stimulates energy release from the fat body in parallel to adipokinetic hormone (AKH).

Discussion
It has been previously shown that a deregulation of the adenosine levels due to the deficiency of Drosophila ADGF-A leads to larval or pupal death associated with a degeneration of larval fat body. This study used a genetic screen and found that a mutation in the γ-subunit of the phosphorylase kinase (PhK-γ) suppresses the adgf-a mutant phenotype. PhK activates through the phosphorylation of glycogen phosphorylase (glycogenolysis). Thus, the suppressive effect of PhK-γ mutation corresponds to the observation that the fat body, the main organ for glycogen storage, does not degenerate in the rescued larvae. This rescue leads to a speculation that carbohydrate metabolism might be affected in the adgf-a mutant larvae (Zuberova, 2010).

The striking sensitivity of the adgf-a mutant to dietary restriction strongly supports this conclusion. A pure yeast diet, which is rich in proteins but poor in carbohydrates, was used. The use of this diet leads to enhanced larval lethality of the adgf-a mutant, whereas the wild-type larvae develop normally on this food. Supplementing the pure yeast diet with 5% sucrose gives larvae resembling the adgf-a mutant phenotype on the cornmeal-based fly food. An increase of sucrose to 10% then leads to a significantly higher mutant survival rate, with a greatly improved morphology of the mutant larvae and most of them not showing any sign of fat body degeneration. Thus, although lowering carbohydrates enhances the mutant phenotype, increasing the amount of carbohydrate in the diet most probably compensates the loss of energy reserves or allows the saving of more stores than in the restrictive conditions. Saving stores of energy is crucial for the larvae to continue in their development. The survival of the adgf-a mutant larvae is thus critically dependent on the amount of carbohydrate in the diet (Zuberova, 2010).

Finally, an analysis of carbohydrate concentrations in the hemolymph demonstrates that the balance between energy storage and release is indeed shifted in the adgf-a mutant towards release because the mutant larvae present doubled levels of glucose in the hemolymph compared with the wild type. The elevated levels of extracellular adenosine in the adgf-a mutant are thus associated with hemolymph hyperglycemia. Extracellular adenosine is not lowered in the mutant larvae rescued on a 10%-sucrose diet compared with mutants on a 5%-sucrose diet, demonstrating that the rescue by increased sugar is not due to lowering of the extracellular adenosine level. The level of glucose in the hemolymph is also similarly increased, most probably because the elevated adenosine increases the circulating glucose to these levels. Therefore, it is plausible that increasing the amount of carbohydrates in the diet allows the saving of more stores, while still keeping the hemolymph glucose levels high. This in turn is expressed as a thick fat body of the rescued larvae on 10% sucrose. The rescue by diets higher in sucrose further demonstrates that it is not hyperglycemia (at this level) that kills the adgf-a mutants (Zuberova, 2010).

The measurement of carbohydrate stores demonstrate that the accumulation of both glycogen and trehalose during feeding period is much slower in the adgf-a mutant larvae than in heterozygous siblings most probably because the elevated adenosine forces the circulating glucose levels to be kept high. This might have fatal consequences for larvae on a diet poor in carbohydrates that does not allow larvae to save enough stores to proceed normally in development, as seen with the adgf-a mutant on a pure yeast diet. Slowing down the glycogen breakdown (by mutation in PhK-γ) does not help much in this situation; EP779 increases the survival of adgf-a poorly on a pure yeast diet. However, it does help larvae on a 5%-sucrose diet, which seems to provide adgf-a with just the minimum amount of carbohydrate, sufficient to save enough stores, at least for some larvae (about a third of them) to proceed in development. Probably any situations requiring a mobilization of the energy from the glycogen stores shifts the thin balance on 5% sucrose so it does not allow the adgf-a mutants to pupate. In these conditions, slowing down the glycogen breakdown helps the adgf-a mutants, as demonstrated by a significant increase in pupariation rate of the EP779; adgf-a mutants on 5% sucrose (Zuberova, 2010).

The main storage organ in insects is the fat body, a counterpart of mammalian liver and adipose tissue. The deposition of stores as well as the stimulation of their release is intensively studied in many insect models, which show that the basic regulation of carbohydrate metabolism is similar to that in higher organisms. Glycogen synthesis and breakdown is regulated by glycogen synthase and glycogen phosphorylase. The energy-demanding processes in insects stimulate the release of energy from the fat body stores through the action of AKHs. AKH, a counterpart of mammalian glucagon, binds to G-protein-coupled receptors and, in the case of carbohydrate metabolism, activates glycogen phosphorylase via cAMP production and Ca2+ (binding to Gs and Gq subunits). This includes regulation by Ca2+-dependent PhK (Zuberova, 2010).

Extracellular adenosine can affect carbohydrate metabolism in the fat body by two possible mechanisms: signaling through AdoR, and intracellularly by transport via adenosine transporters and a conversion to AMP by adenosine kinase inside the cell. Glycogen phosphorylase is, in addition to phosphorylation by PhK, also activated by allosteric effectors including AMP. AMP can also inhibit glycogen synthase through the action of AMP-activated protein kinase (AMPK). However, it was shown that the blockage of AdoR signaling almost completely rescues the mutant larvae on a pure yeast diet. This is supported by resetting the glucose level in the hemolymph of the adoR adgf-a double mutants back to normal levels. These results clearly demonstrate the role of AdoR signaling in the regulation of carbohydrate metabolism in Drosophila. A similar role for adenosine signaling through adenosine receptors stimulating the glucose release has been described for mammalian liver cells, suggesting that this role of extracellular adenosine and the mechanism of action are evolutionary conserved from flies to mammals (Zuberova, 2010).

Because AdoR is expressed in the Drosophila ring gland where the AKH-producing cells are located, it was speculated that the effect of adenosine could be triggered by AKH release in response to AdoR signaling in the ring gland. However, the ablation of AKH-producing cells does not rescue the adgf-a mutant, which demonstrates that AKH is not involved in the observed effect of adenosine on carbohydrate metabolism. Previous analysis of AdoR expression by in situ hybridization did not reveal its expression in the fat body. However, using a more sensitive method of tissue-specific RT-PCR, it was shown that AdoR is indeed expressed in the fat body. Although this study did not a definite proof, the clear suppressive effect of adoR mutation and AdoR expression in the fat body suggest that the direct signaling of extracellular adenosine to the fat body via AdoR might lead to the increase of glucose in the hemolymph. It has been previously shown that Drosophila AdoR is, similarly to the AKH receptor, associates with Gs and Gq proteins, leading to formation of cAMP and Ca2+ release. This leads to the conclusion that AdoR could serve as an additional receptor along with the AKH receptor for activating glycogenolysis via G-protein/PhK stimulation in the fat body (Zuberova, 2010).

Glycogenolysis is not the only way to increase glucose in the hemolymph, especially during feeding. The mutation in PhK-γ does not decrease the glucose level in the adgf-a mutant whereas the adoR mutation does. The extracellular adenosine thus instructs the organism to keep hemolymph glucose (originating most probably from dietary sucrose) levels high, while lowering glycogen storage. Therefore, there might be an additional AdoR signaling effect, besides that on glycogenolysis, that influences how much of the carbohydrate obtained from food will be used for storage and how much will be kept in the hemolymph for immediate use. Extracellular adenosine can be then seen as a true anti-insulin hormone. This agrees with a concept proffered earlier that adenosine can play an even stronger anti-insulin hormone-like role in the mammalian system under certain conditions than classical anti-insulin hormones such as adrenaline or glucagon. It is important to stress that although this study focuses solely on carbohydrate metabolism, adenosine might have a similar effect on lipid metabolism (Zuberova, 2010).

The observation in this study that accumulated extracellular adenosine can lead to death of the organism through an impairment to the process of saving energy stores might help to better understand certain pathologies. The immune response is energetically costly and a progressive loss of energy stores – also called wasting – is observed during certain chronic infections in both flies and humans. In spite of a potentially high biomedical importance, the network of the molecular signals leading to wasting is largely unexplored. Results of this work suggest that extracellular adenosine could be one of those signals. Adenosine is well known as a stress hormone and it is produced during the immune response; therefore it could play an important hormonal role in energy allocation during stress conditions such as infection. Earlier studies suggest a connection between adenosine signaling and Toll signaling, which is an important regulator of the immune response in flies. A requirement to use virtually sterile conditions to obtain reproducible results in this work suggests a high sensitivity of the adgf-a mutants to infectious stress. Microarray analysis in the fly model for wasting also shows changes in the expression of the adenosine-regulating enzymes. It is important to further investigate this intriguing role for extracellular adenosine, and this study demonstrates that Drosophila can be now utilized as a useful, genetically tractable model to investigate the stress-hormone-like roles of extracellular adenosine in energy allocation in different conditions in vivo (Zuberova, 2010).

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Dionne, M.S., Pham, L.N., Shirasu-Hiza, M. and Schneider, D.S. (2006). Akt and FOXO dysregulation contribute to infection-induced wasting in Drosophila. Curr Biol 16: 1977-1985. PubMed ID: 17055976

Abstract
Studies in Drosophila have taught us a great deal about how animals regulate the immediate innate immune response, but we still know little about how infections cause pathology. This study examines the pathogenesis associated with Mycobacterium marinum infection in the fly. M. marinum is closely related to M. tuberculosis, which causes tuberculosis in people. A microarray analysis shows that metabolism is profoundly affected in M. marinum-infected flies. A genetic screen identifies foxo mutants as slower-dying after infection than wild-type flies. FOXO activity is inhibited by the insulin effector kinase Akt; it was shown that Akt activation is systemically reduced as a result of M. marinum infection. Additionally, flies infected with Mycobacterium marinum undergo a process like wasting: they progressively lose metabolic stores, in the form of fat and glycogen. They also become hyperglycemic. In contrast, foxo mutants exhibit less wasting. In people, many infections—including tuberculosis—can cause wasting, much as is seen in Drosophila. This study is the first examination of the metabolic consequences of infection in a genetically tractable invertebrate and gives insight into the metabolic consequences of mycobacterial infection, implicating impaired insulin signaling as a key mediator of these events. These results suggest that the fly can be used to study more than the immediate innate immune response to infection; it can also be used to understand the physiological consequences of infection and the immune response (Dionne, 2006).

Highlights

• Mycobacterial infection induces widespread alterations in expression of genes involved in energy storage.
• foxo mutants die more slowly than wild-type flies but show no change in bacterial numbers.
• Insulin signaling via Akt is reduced in M. marinum-infected flies.
• The disruption of signaling via Akt is not like that seen in type-2 diabetics.
• M. marinum-infected flies progressively lose glycogen and fat and are hyperglycemic.
• The extent of wasting is reduced in hypomorphic foxo mutants.

Discussion
This study shows that flies infected with M. marinum undergo a process like wasting. This wasting response is in part the consequence of systemic failure of Akt activation and consequent activation of the transcription factor FOXO, resulting in a diabetes-like state. The observed failure of Akt activation is not caused by traditional insulin resistance mechanisms. Data suggest that mycobacterial infection causes a systemic reduction in Akt activation, either by reducing the level of circulating insulin or by increasing the turnover of activated Akt, or both. This results in excessive Gsk-3 and FOXO activity, which causes the progressive loss of energy stores. The study argues that the impaired insulin signaling seen in M. marinum-infected Drosophila is likely to represent a common response to many infections in many hosts, including humans, and moreover is likely a significant cause of disease morbidity (Dionne, 2006).

It was shown that foxo mutants are longer-lived when infected with M. marinum. This makes an intriguing contrast with earlier observations that flies overexpressing foxo are longer-lived than wild-type flies when uninfected. Moreover, foxo mutant flies die more rapidly than wild-type flies under conditions of oxidative stress. These observations suggest that death from old age or oxidative stress is mechanistically different from death from M. marinum infection (Dionne, 2006). 

In humans, insulin is among the most important anabolic signals, and it is also a satiety signal: type-1 diabetics (who progressively lose the insulin-producing cells of the pancreas) exhibit increases in appetite and consequently in food intake, even as they are progressively losing body mass. In fly larvae, insulin-like peptides (ILPs) appear to play a role roughly analogous to the metabolic role seen in humans: Larvae in which the insulin receptor (InR) or PI3 kinase (PI3K) are overexpressed accumulate excessive levels of fat and show reduced feeding. Conversely, larvae that lack ILP-producing cells (IPCs) in the brain become hyperglycemic (Dionne, 2006). 

In adult flies, the metabolic role of ILPs appears to be more complex. The loss of ILP signaling via IPC ablation or loss-of-function mutations in chico (the fly homolog of mammalian IRS proteins) or InR results in increased triglyceride and glycogen storage—although, in the case of IPC ablation, this increase in energy stores is still accompanied by hyperglycemia. Studies using a temperature-sensitive InR allele show that the critical period for the increase in metabolic storage is during pupariation, indicating that the storage effect should be regarded as a developmental defect rather than a physiological one. This study is the first examination of the results of insulin inhibition in wild-type adult flies without developmental perturbation. It suggests that the insulin signaling pathway acts in adult flies to drive glucose uptake and energy storage in a manner analogous to its action in mammals and larval Drosophila (Dionne, 2006). 

Immune responses pose significant costs for the host. One hypothesis suggests that the primary cost of immune responses is energetic: that metabolic energy used by the immune system is being taken from other important systems. This has been easiest to observe in cases where animals are placed under energy constraints and then forced to raise an immune response: In these situations, the induced immune responses have easily observable deleterious effects on other physiological processes. Conversely, this cost is also visible as immunosuppression in animals that are carrying out other energy-intensive activities (Dionne, 2006). 

These observations suggest that there should be mechanisms for direct control of energy allocation to the immune response. Data from this study indicate that Akt and Foxo form an important component in this regulation. The study speculates that the systemic disruption of insulin signaling may be a mechanism by which insects reduce energy allocation to nonimmune tissues; moreover, a similar mechanism might operate in mammals. The clinical literature is rich with examples of metabolic changes resulting from a variety of infections. Tuberculosis and other chronic infections can be associated with slow wasting of fatty and lean tissues and glucose intoleranc; acute bacteremia tends to be associated with rapid wasting and full-scale hyperglycemia. Resting insulin levels are an excellent predictor of survival in septic patients, and aggressive treatment of septic hyperglycemia with exogenous insulin dramatically increases survival. Wasting alone could be accounted for simply by the energetic cost of the immune response; however, infections often cause hyperglycemia as well, suggesting that systemic changes in metabolic regulation may be the underlying cause of infection-induced wasting. That is, wasting may be a pathological consequence of regulated energy reallocation (Dionne, 2006).

Other possibilities should not be overlooked: in particular, the possibility that the metabolic changes observed in the fly might be part of a pathogenic strategy on the part of the bacterium. Organisms living in the circulation can easily double their local glucose concentration simply by degrading circulating insulin. In this reading, the fact that a wide variety of infections cause hyperglycemia in mammals would be a result of the fact that this strategy is such an attractive one for a pathogen that it has been selected many times independently. However, the apparent connection between increased levels of proinflammatory cytokines and cachexia in mammals leads to the connection that infection-induced wasting is primarily a consequence of the host response—though one that is ripe for exploitation by some classes of pathogen (Dionne, 2006).

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Reviews

Piccirillo, R., Demontis, F., Perrimon, N. and Goldberg, A.L. (2014). Mechanisms of muscle growth and atrophy in mammals and Drosophila. Dev Dyn 243: 201-215. PubMed ID: 24038488

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Discussion on role of Runt in Cachexia

Back to Drosophila as a Model for Human Diseases

Date revised: 20 June 2015

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