A tumor-secreted protein utilizes glucagon release to cause host wasting

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Song doi: https://doi.org/10.1101/2024.10.24.619567 Guangming Ding 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China 2 TaiKang Center for Life and Medical Sciences, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yingge Li 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China 2 TaiKang Center for Life and Medical Sciences, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chen Cheng 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China 2 TaiKang Center for Life and Medical Sciences, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kai Tan 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yifei Deng 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Huiwen Pang 3 School of Biomedical Sciences, The University of Queensland , Brisbane, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhongyuan Wang 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China 2 TaiKang Center for Life and Medical Sciences, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Peixuan Dang 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China 2 TaiKang Center for Life and Medical Sciences, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xing Wu 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elisabeth Rushworth 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yufeng Yuan 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China 2 TaiKang Center for Life and Medical Sciences, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: yuanyf1971{at}whu.edu.cn yangzhiyong{at}whu.edu.cn songw{at}whu.edu.cn Zhiyong Yang 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: yuanyf1971{at}whu.edu.cn yangzhiyong{at}whu.edu.cn songw{at}whu.edu.cn Wei Song 1 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University , Wuhan, Hubei, China 2 TaiKang Center for Life and Medical Sciences, Wuhan University , Wuhan, Hubei, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wei Song For correspondence: yuanyf1971{at}whu.edu.cn yangzhiyong{at}whu.edu.cn songw{at}whu.edu.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Tumor-host interaction plays a critical role in malignant tumor-induced organ wasting across multiple species. Despite known regulation of regional wasting of individual peripheral organs by tumors, whether and how tumors utilize critical host catabolic hormone(s) to simultaneously induce systemic host wasting, however, is largely unknown. Using the conserved yki 3SA -tumor model in Drosophila , we discovered that tumors increase the production of adipokinetic hormone (Akh), a glucagon-like catabolic hormone, to cause systemic host wasting, including muscle dysfunction, lipid loss, hyperglycemia, and ovary atrophy. We next integrated RNAi screening and Gal4-LexA dual expression system to identify that yki 3SA -gut tumors secrete Pvf1 to remotely activate its receptor Pvr in Akh-producing cells (APCs), ultimately promoting Akh production. The underlying molecular mechanisms involved the Pvf1-Pvr axis that triggers Mmp2-dependent ECM remodeling of APCs and enhances innervation from the excitatory cholinergic neurons. Interestingly, we also confirmed the similar mechanisms governing tumor-induced glucagon release and organ wasting in mammals. Blockade of either glucagon or PDGFR (homolog of Pvr) action efficiently ameliorated organ wasting in the presence of malignant tumors. Therefore, our results demonstrate that tumors remotely promote neural-associated Akh/glucagon production via Pvf1-Pvr axis to cause systemic host wasting. Introduction Cancer cachexia, also known as tumor-induced host wasting, is a newly-recognized metabolic disorder that typically involves weight decline, loss of muscle and fat tissues, and hyperglycemia 1 . Unlike malnutrition, cancer cachexia can hardly be reversed by nutritional supplementation 2 . Many groups and ours have used rodents, fruit flies, as well as zebrafish, to model tumor-induced host wasting and implicated that, in addition to systemic inflammatory responses 3 , tumor-secreted factors directly target muscle or adipose tissues and impair energy homeostasis there, leading to muscle wasting or lipid loss 4 – 8 . Despite tumor impacts on local metabolism of individual host organs, however, it is not well understood whether and how malignant tumors hijack the essential metabolic hormone(s) from the host to extensively disrupt metabolic homeostasis in multiple organs and lead to systemic host wasting. We have previously established a cancer-cachexia fly model bearing yki 3SA tumors in the gut and further demonstrated that yki 3SA -gut tumors secrete cachectic ligands, such as Imaginal morphogenesis protein-Late 2 (ImpL2), PDGF- and VEGF- related factor 1 (Pvf1), and Unpaired 3 (Upd3), to lead to energy wasting including lipid loss, muscle dysfunction, ovary atrophy, as well as hyperglycemia, probably via impairment of metabolism and homeostasis of muscle and fat tissues 9 – 11 . Importantly, tumor-derived ImpL2/IGFBP2, Upd3/interleukin, and other ligands that result in lipid loss and muscle dysfunction have been consistently found in other tumor-bearing flies and mammals 12 – 16 . Drosophila Akh is a well-established homolog of human glucagon that plays conserved roles in mobilization of systemic energy storages 17 , 18 . Similar to mammalian glucagon that is produced by pancreatic α-cells to activates glucagon receptor (GcgR) in liver and brain and cause energy depletion 19 , 20 , Drosophila Akh is produced by neuroendocrine cells in corpora cardiaca (CC) and activates its receptor AkhR in the fat body and certain neurons in the brain to control homeostasis of systemic lipid, carbohydrate and amino acid metabolism. The molecular mechanisms include at least the AkhR-downstream cAMP and Ca 2+ pathways, which increase glycogenolysis and gluconeogenesis, lipolysis, and amino acid breakdown 21 – 25 . Therefore, proper regulation of Akh release is essential for maintenance of organismal energy balance. Previous studies have indicated APCs sense secreted proteins from distal organs (Upd2, NPF, AstA, and AstC) to regulate Akh release and mobilize energy storages under nutrient deprivation 26 – 29 . Notably, Akh release is also precisely controlled by upstream inhibitory neurons through release of neurotransmitters or neuropeptides (sNPF and Capa) 30 – 32 . In this study, we characterized that Akh release is required for tumor-induced wasting in Drosophila and interestingly uncovered that catabolic ligand Pvf1, which is secreted by yki 3SA tumors, directly activates its receptor Pvr in APCs to promotes Akh secretion. We also demonstrated the molecular mechanisms whereby Pvf1-Pvr axis triggers Mmp2-dependent extracellular matrix (ECM) remodeling of APCs and enhances the innervation to upstream excitatory cholinergic neurons. We further validate the conserved regulation of glucagon release in tumor-induced organ wasting in mice. Results Akh is essential for tumor-induced organ wasting As Akh is associated with metabolic dysregulation such as lipid loss and hyperglycemia, we wondered whether Akh is involved in systemic organ wasting in yki 3SA -tumor-bearing flies. To address this hypothesis, we first examined Akh production in the APCs of yki 3SA -tumor-bearing flies ( esg TS >yki 3SA ). We found that the mass of APCs, intracellular and circulating Akh levels, as well as Akh mRNA level, were all increased in yki 3SA -tumor-bearing flies ( Fig. 1A-E ). We also observed that the expression of tobi , an established Akh target gene as indicated by qPCR 33 , was increased in the yki 3SA -tumor-bearing flies as compared to control flies ( Fig. 1E ). These data reveal that Akh production is enhanced in yki 3SA -tumor-bearing flies. Download figure Open in new tab Figure 1. Akh is essential for tumor-induced host wasting in Drosophila . Representative images of abdomen bloating ( A, up), Akh production in APCs ( A, middle, red, anti-Akh), and gut tumors ( A, bottom, GFP), quantification of APC masses (area) ( B, left, n=3) and intracellular Akh amounts ( B, right, n=3), circulating Akh levels in the hemolymph ( C, left, dot-blot; right, quantification, n=4), quantification of tumor mass (GFP intensity/gut area) ( D , n=3), whole body Akh, AkhR, and tobi mRNA levels ( E, n=4-6, 5 flies/replicate), bloating rates ( F, n=3, 20 flies/replicate), metabolic dysregulation such as triglyceride (TAG) and trehalose (TRE) storages ( G , n=4, 5 flies/replicate), circulating trehalose (TRE) levels in the hemolymph ( H, n=4, 40 flies/replicate) and food intakes ( I, n=4, 20 flies/replicate) of adult yki 3SA -tumor-bearing flies with or without Akh Δ mutation ( Akh SAP /Akh A ) at day 8. ( J ) Heatmap indicating both yki 3SA -tumor- and Akh-dependent differentially expressed genes in the whole-body. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( B, C, E, left) and one-way ANOVA with Bonferroni’s multiple-comparisons test ( D, E, right, F-I ). * p yki 3SA ; Akh Δ ”, to examine whether Akh is essential for yki 3SA -tumor-induced organ wasting. As expected, Akh deficiency significantly reduced Akh production and suppressed tobi gene expression ( Fig. 1E ). We strikingly observed that Akh deficiency robustly improved abdomen bloating, TAG loss, and hyperglycemia (elevation of trehalose, the major circulating carbohydrate composed by two α-glucose in insects) in the yki 3SA -tumor-bearing flies without affecting the gut-tumor growth or food intake ( Fig. 1A , 1D-I and S1C ). Even though Akh does not directly target muscle or ovary tissues due to absent AkhR expression (single-nucleus RNA-seq data, FlyCellAtlas 35 ), we still found that Akh deficiency significantly restored muscle function and ovary homeostasis that were impaired by yki 3SA tumors ( Fig. S1A-B ). We next examined the gene expression in the whole body of yki 3SA -tumor-bearing flies with or without Akh -null mutation using RNA-seq and observed that 194 and 201 genes were up- and down-regulated, respectively, by yki 3SA tumors in a manner dependent on Akh ( Fig. 1J and S1E-F and Table. S1 ). These differentially regulated genes were found to be enriched in biological processes, pathways, and organelles associated with lipid and carbohydrate metabolism, protein homeostasis, as well as immune responses ( Fig. S1F ). Aligned with amelioration of systemic host wasting by Akh deficiency, these genes are specifically expressed in multiple tissues ( Table. S1 ). Notably, the ones expressed in fat body ( Gnmt, CG34136, CG31778, Lsp2 ) are implicated in regulation of energy homeostasis and amino acid metabolism, suggesting they are directly targets of Akh signaling ( Table. S1 ). Taken together, our results demonstrate that yki 3SA tumors remotely promote Akh production to trigger organ wasting. We also investigated whether AkhR deficiency also improved tumor-induced wasting. To do this, we integrated binary expression system 36 , 37 to generate yki 3SA - gut tumors and knock down AkhR expression in the fat body or pan-neurons, two major tissues expressing AkhR (FlyCellAtlas) 35 , using LexA and GAL4 , respectively. We observed that AkhR removal in either tissue at least partially rescued wasting phenotypes including energy loss, abdomen bloating, muscle dysfunction, and ovary atrophy ( Fig. S2 ). These results indicate that AkhR in both the fat body and neurons simultaneously contributes to tumor-induced host wasting. To determine whether Akh gain-of-function in non-tumor control flies sufficiently induce organ wasting, we overexpressed Akh or TrpA1, a heat-activated cation channel that manually promotes peptide hormone release in neurons and endocrinal cells 29 , in APCs. We found that APC overexpression of TrpA1, but not Akh as reported 18 , dramatically increased Akh signaling, leading to lipid loss and trehalose elevation ( Fig. S3A-C and S3F-H ). However, it failed to cause abdomen bloating, muscle dysfunction or ovary atrophy ( Fig. S3A-J ). Similar outcomes were observed when Akh was ectopically overexpressed Akh in fat body or neurons to manually activate local AkhR signaling in adult flies in an autocrine or paracrine manner ( Fig. S3K-P ) 18 . These results indicate that excessive Akh release alone is insufficient to cause organ wasting, despite its roles in carbo-lipid metabolic mobilization. The receptor Pvr promotes Akh release in APCs We hypothesized that yki 3SA tumors secrete cachectic protein(s) to target specific receptors in APCs and regulate Akh release ( Fig. 2A ). To identify these regulatory receptors, we performed an in vivo RNAi screening against transmembrane proteins in the larval APCs by crossing 311 RNAi lines to Akh-GAL4 and measured glycemic changes (circulating trehalose) ( Table. S2 ), a quick and reliable readout of larval Akh response 21 . As expected, RNAi against either Akh or AstA-R2 , which was found to promote Akh production 28 , significantly suppressed glycemic level as compared to control RNAi ( w-i) ( Fig. 2B ). We eventually found 22 and 38 RNAi lines in APCs that down- and up-regulated glycemic levels by >10%, respectively ( Fig. S4A ). These hits included receptors for brain-gut hormones, neurotransmitters, olfactory and gustatory molecules, as well as ion and mechanical channels, suggesting a comprehensive network in APCs that modulates Akh release and glycemic homeostasis ( Fig. S4A and Table. S2 ). Interestingly, RNAi against PDGF- and VEGF-receptor related ( Pvr ), an RTK specifically activated by yki 3SA tumor-derived Pvf1 38 , was a top hit in APCs that dramatically decreased glycemic level (circulating trehalose) ( Fig. 2B ). To validate effects of Pvr in APCs, we examined the expression pattern of Pvr using an endogenous GAL4 line and confirmed Pvr-GAL4 -driven GFP expression in larval APCs ( Fig. S4B ). In addition to Pvr RNAi, overexpression of a dominant negative form of Pvr ( Pvr DN ) in APCs also suppressed larval glycemic level ( Fig. S4C ). These results indicate that Pvr in the larval APCs promotes Akh release. Download figure Open in new tab Figure 2. Pvr signaling regulates Akh release. ( A-B ) Experimental strategy ( A ) and glycemic changes ( B ) of the in vivo RNAi screening against transmembrane proteins in the larval APCs. ( C ) Immunostaining indicating Pvr expression (green, Pvr>GFP; red, anti-Akh) in adult APCs. ( D-F ) Circulating Akh levels in the hemolymph ( D, left, dot-blot; right, quantification, n=3), metabolic changes such as TAG and trehalose (TRE) storages ( E , n=3, 5 flies/replicate), as well as survival rates under starvation ( F , n=4, 20 flies/replicate), of adult flies with Pvr AC overexpression in APCs at day 4. ( G-N ) Bloating phenotype (up) and gut-tumors (bottom, green) ( G, L ), quantification of tumor mass (GFP intensity/gut area) ( H, M, n=3), whole body Akh and tobi expression ( I , N, n=3, 5 flies/replicate), wasting effects such as bloating rates ( J, n=3, 20 flies/replicate), TAG and TRE storages ( K , O , n=3, 5 flies/replicate), and starvation resistance ( P , n=4, 20 flies/replicate) of yki 3SA - tumor-bearing flies with genetic manipulation in APCs at day 6 ( G-K , LexA+GAL4 ) or with tumor Pvf1 knockdown or systemic Akh deficiency ( Akh Δ , Akh SAP /Akh A ) at day 8 ( L-P , GAL4 ). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( D, E ), one-way ANOVA with Bonferroni’s multiple-comparisons test ( H-K, L-O ), or log-rank test ( F, P ). * p < 0.05. We next studied the Pvr functions in adult flies. The Pvr-GAL4 line also confirmed the Pvr expression in adult APCs ( Fig. 2C ). We also found that overexpression of a constitutively active Pvr ( Pvr AC ) in adult APCs increased Akh production, including circulating Akh level in the hemolymph and systemic mRNA levels of both Akh and tobi , enhanced Ca 2+ signaling in APCs, and perturbed carbo-lipid metabolism, including whole-body TAG and trehalose level and survival under starvation ( Fig. 2D-F , S1D, S4C, S4D and S4F ). In contrast, flies with Pvr DN overexpression or Pvr RNAi in APCs exhibited opposite phenotypes ( Fig. S4E-H ). To avoid developmental effects, we further used tub-Gal80 TS to manipulate Pvr in adult APCs only. Consistently, APC Pvr activation for 4 days sufficiently increased Akh production and energy catabolism, while adult Pvr inactivation showed relatively mild effects ( Fig. S4I-L ). Finally, Akh knockdown in the context of Pvr AC overexpression in the APCs dramatically alleviates energy catabolism and restored Akh response ( Fig. S7A-C ), indicating an Akh-dependent metabolic role of Pvr. Taken together, these results demonstrate that Pvr signaling in APCs promotes Akh production. Tumor-derived Pvf1 activate Pvr in APCs to enhance Akh release To verify the physiological effects of Pvr-associated Akh release in the context of yki 3SA -gut tumors, we expressed Pvr DN using Akh - GAL4 to inactivate Pvr specifically in APCs of yki 3SA -tumor-bearing flies. Strikingly, as compared to marginal effects of APC Pvr inactivation in non-tumor adult flies, we observed that Pvr inactivation in APCs of yki 3SA -tumor-bearing flies resulted in a significant decrease in Akh response ( Akh and tobi expression) and robustly improved host wasting, including bloating, lipid loss, hyperglycemia, muscle dysfunction, and ovary atrophy without affecting tumor growth at day 6 ( Fig. 2G-K and S5A-B ). These results were further confirmed by knocking down Pvr expression in APCs of yki 3SA -tumor-bearing flies ( Fig. S5C-G ). Given the distribution Pvr in muscle and adipose tissue 11 , we wondered whether adipose or muscle Pvr signaling also contributes to host wasting. We expressed Pvr DN in muscle and fat body using Mhc- and R4-GAL4 , respectively, in the yki 3SA - tumor-bearing flies. However, Pvr inactivation in neither fat body nor muscle improved yki 3SA -tumor-induced host wasting ( Fig. S6 ), except for a slight rescue in abdomen bloating and hyperglycemia by fat body Pvr inactivation ( Fig. S6C and S6E ). Thus, our data demonstrate that yki 3SA tumors cause host wasting predominantly through Pvr function in APCs. To examine whether tumor-derived Pvf1 functions through Akh production, we performed the genetic interaction between Pvf1 and Akh in yki 3SA -tumor-bearing flies. We found that either Akh -null mutation or tumor-specific Pvf1 knockdown potently diminished wasting effects, including bloating, lipid loss, hyperglycemia, as well as starvation sensitivity, in yki 3SA -tumor-bearing without affecting tumor growth at day 8 ( Fig. 2L-P ). Tumor-specific Pvf1 knockdown also significantly decreased both Akh and tobi mRNA levels, albeit to a less extent than Akh -null mutation ( Fig. 2N ). Importantly, Akh mutation plus tumor- Pvf1 knockdown failed to further significantly alleviate wasting effects as compared to single one(s) ( Fig. 2L-P ). These results demonstrate that tumor-derived Pvf1 functions, at least partially, through Akh to cause host wasting. Pvf1/Pvr axis promotes Akh release via ERK/Mmp2 signaling Previous studies showed that Pvr activates Ras/Raf/MEK/ERK and multiple downstream targets to regulate various events of tissue homeostasis, such as cell proliferation, differentiation, as well as migration 38 . To investigate the molecular mechanisms of Pvr regulation of Akh release, we overexpressed Pvr AC to activate Pvr signaling in APCs and simultaneously knocked down potential downstream targets. We interestingly observed that, in the context of APC Pvr AC overexpression, ERK knockdown significantly increased fly survival rates under starvation, suppressed both Akh and tobi gene expression, increased TAG levels, and decreased trehalose levels ( Fig. 3A-B and S7D ). On the other hand, overexpression of an active Raf ( Raf F179 ) to activate MEK/ERK signaling in the APCs of wild-type flies phenocopied the effects of Pvr activation to promote Akh release, enhance systemic Akh response, and impaired carbo-lipid metabolic homeostasis ( Fig. S7E- G ). We further performed RNAi screening of ERK downstream targets and, strikingly, found that knockdown of Matrix metalloproteinase 2 ( Mmp2 ), but not Mmp1 39 , 40 , potently rescued Pvr AC -associated starvation sensitivity ( Fig. 3A ). Biochemical and metabolic analysis consistently revealed that Mmp2 deficiency suppressed Akh release, decreased tobi expression, as well as abolished lipid loss and hyperglycemia ( Fig. 3B-C , S7D and S7H-I ). Overexpression of Timp, a single homolog of the tissue inhibitors of metalloproteinases (TIMPs) blocking Mmp1/2 in fly 41 , in the context of Pvr AC overexpression in APCs also suppressed Akh release and alleviated subsequent signaling and metabolic outputs ( Fig. 3A-C and S7D ). Note that, neither Mmp2 knockdown or Timp overexpression in APCs of control flies affected systemic Akh signaling or carbo-lipid metabolism ( Fig. S7J-L ), demonstrating their roles in regulating Akh release are contingent upon Pvr activation. APC ERK knockdown in control flies decreased Akh signaling and carbo-lipid mobilization ( Fig. S7J-L ), suggesting that ERK might function through Mmp2 and other targets. Download figure Open in new tab Figure 3. Pvr enhances Akh release via ERK/Mmp2 signaling. ( A-C ) Survival rates under starvation ( A , n=4, 20 flies/replicate), metabolic changes such as TAG and trehalose (TRE) storages ( B , n=4, 5 flies/replicate), and hemolymph Akh levels ( C, left, dot-blot; right, quantification, n=3) of adult flies bearing indicated RNAi in the context of Pvr AC overexpression in APCs at day 4. ( D-G ) Bloating phenotype (up) and gut-tumors (bottom, green) ( D ), quantification of tumor mass (GFP intensity/gut area) ( E, n=3), bloating rates ( F, n=4, 20 flies/replicate), and TAG and TRE storages ( G , n=4, 5 flies/replicate) of yki 3SA -tumor-bearing flies with genetic manipulation in APCs at day 6 ( LexA+GAL4 ). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( C ), one-way ANOVA with Bonferroni’s multiple-comparisons test ( B, E-G ), or log-rank test ( A ). * p < 0.05. We next validated the wasting effects of ERK/Mmp2 axis in APCs in the context of yki 3SA -gut tumors. Using GAL4/LexA binary system, we knocked down expression of ERK or Mmp2 in APCs of yki 3SA -tumor-bearing flies and consistently found that host wasting, including bloating, lipid loss, and hyperglycemia, were robustly improved without affecting yki 3SA tumors in the gut at day 6 ( Fig. 3D-G ). Taken together, these results demonstrate that Pvr-ERK-Mmp2 axis enhances Akh release to cause energy wasting in yki 3SA -tumor flies. Pvf1-Pvr axis enhances Mmp2-dependent ECM remodeling and neuronal innervation of APCs We next investigate the molecular mechanisms by which Mmp2 regulates Akh production. Mmp-mediated homeostasis of ECM is essential for multiple neuronal activities, including neuronal plasticity, synaptic formation and neural innervation 42 . APCs have been reported to be targeted by upstream regulatory neurons to control Akh release 30 , 31 . We therefore wonder whether Pvr regulates Akh release via Mmp2-induced ECM remodeling and neural contact. To address it, we first accessed the ECM homeostasis in the APCs using GFP-tagged collagen IV (Viking, Vkg-GFP) and to label ECM 43 . Interestingly, we observed very strong Vkg-GFP signals around somas of >20 APCs in wild-type adult flies ( Fig. 4A ). Pvr AC overexpression in APCs potently decreased, whereas Mmp2 RNAi in the context of Pvr AC restored, Vkg-GFP signals around APCs at day 4 ( Fig. 4A and 4C ), indicating Pvr-Mmp2 regulation of ECM remodeling. Consistent with Pvr activation by tumor-derived Pvf1, similar patterns of ECM in APCs were observed in flies bearing yki 3SA tumors with or without Pvf1 knockdown at day 8 ( Fig. 4B and 4D ). Similar results were observed using another ECM indicator, integrin βPS (integrin) ( Fig. S8A-B ) 43 . Download figure Open in new tab Figure 4. Pvf1/Pvr axis enhances neural contacts of APCs via ECM remodeling. ( A-D ) ECM homeostasis indicated by Vkg-GFP around the somas of APCs ( A , B, green, GFP; red, anti-Akh) and quantification of APC masses (area), intracellular Akh amounts, as well as extracellular Vkg-GFP amounts ( C, D, n=3) of adult flies with Pvr AC overexpression plus Mmp2 RNAi in APCs at day 4 or flies bearing yki 3SA -tumor plus Pvf1 R NAi at day 8. ( E ) The model showing that ECM (Vkg-GFP) degradation promotes synapse contact including bouton (Syt-GFP) and dendrite (Denmark) formation. ( F-J ) Dendrites labeled by Denmark ( F, G , red; green, anti-Akh) and quantification of dendrite numbers ( H, I, n=4) in APCs of adult flies with Pvr AC overexpression plus Mmp2 RNAi in APCs at day 4 or yki 3SA- tumor-bearing flies ( LexA+GAL4 ) at day 6. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( I ) or one-way ANOVA with Bonferroni’s multiple-comparisons test ( C, D, H ). * p < 0.05. As ECM remodeling promotes synapse formation ( Fig. 4E ), we next examined the synaptic contact using Dendritic Marker (Denmark) to visualize dendrites in the APCs 44 . APC Pvr AC overexpression consistently increased dendrite numbers as indicated by Denmark inside APCs, APC masses, and Akh production in an Mmp2-dependent manner at day 4 ( Fig. 4F and 4H ). yki 3SA -tumor-bearing flies also exhibited an increase in dendrite numbers of APCs as indicated by Denmark puncta ( Fig. 4G and 4I ). These results indicate that Pvf1-Pvr-Mmp2 axis modulates ECM and increases neural innervation of APCs in yki 3SA -tumor-bearing flies. Pvf1-Pvr axis promotes cholinergic innervation of APCs to increase Akh release We next exploit the potential excitatory innervating neuron(s) upstream of APCs by screening different neurotransmitter-producing neurons, including cholinergic ( Cha-GAL4 ), dopaminergic ( TH-GAL4 ), 5-HT ( TRH-Gal4 ), octopaminergic ( Tdc2-GAL4 ), GABAergic ( Gad1-GAL4 ), and glutamatergic ( VGlut-GAL4 ) neurons 45 . We expressed a presynaptic marker, GFP-tagged Synaptotagmin (Syt-GFP), to visualize boutons in these neurons and found strong Syt-GFP signals driven by, at least, Cha-GAL4 in the APCs ( Fig. 5A and S8C ). Meanwhile, we found that APCs express multiple receptors for acetylcholine from published single-nucleus RNA-seq dataset ( Fig. S8D ) 46 . This cholinergic-APC innervation was further confirmed by the trans-tango system ( Fig. 5B ). We next thermally activated cholinergic neurons by overexpressing TrpA1 at 29 °C and found enhanced systemic Akh response as indicated by tobi expression, as well as lipid loss and hyperglycemia, in the adult flies ( Fig. 5C ). These data indicate that, at least, cholinergic neurons functionally projection onto APCs. Download figure Open in new tab Figure 5. Cholinergic innervation of APCs is essential for yki 3SA -tumor-induced Akh release and host wasting. ( A ) Cholinergic boutons indicated by Cha>Syt-GFP (green) in the somas of adult APCs (red, anti-Akh) at day 4. ( B ) Trans-tango system indicating overlaps between cholinergic innervating neurons (GFP) and APCs (red, anti-Akh). ( C ) Whole body Akh and tobi expression (left, n=3, 5 flies/replicate) and metabolic changes such as TAG and trehalose (TRE) storages (right, n=4, 5 flies/replicate) of adult flies with activation of cholinergic neurons induced at 29°C for 12 hours. ( D ) Upstream cholinergic boutons indicated by Syt-GFP (left, green) in APCs (left, red, anti-Akh) and quantification of bouton numbers (right, n=3) of yki 3SA -tumor-bearing flies ( LexA+GAL4 ) at day 6. ( E-H ) Bloating phenotype ( E, up), gut tumors ( E, middle, green) and quantification ( F, n=3), and APCs ( E, bottom, red, anti-Akh), quantification of APC masses (area) and intracellular Akh amounts ( G , n=3), whole body Akh and tobi expression ( H , n=3, 5 flies/replicate), abdomen bloating rates ( I, left, n=4) and TAG and TRE storages ( I, right, n=4, 5 flies/replicate) of yki 3SA -tumor-bearing flies with synapse disruption in cholinergic neurons at day 6 ( LexA+GAL4 ). ( J ) The schematic model illustrating the release of Akh and its regulation by tumor-secreted Pvf1 and ECM-associated neural innervation in yki 3SA -tumor-bearing flies, leading to organ wasting. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( C, D ), one-way ANOVA with Bonferroni’s multiple-comparisons test ( F- I ). * p < 0.05. We further observed a significant increase in the number of Syt-GFP-labeled cholinergic boutons in the APCs of flies bearing yki 3SA tumors at day 8 ( Fig. 5D ). Finally, to evaluate the functional impacts of cholinergic projections on APCs, we thermally disrupted neurotransmission of cholinergic neurons by expressing a temperature-sensitive mutant of shibire ( shi TS ), the fly homolog of dynamin essential for synaptic vesicle recycling in nerve terminals 47 , at 29 °C. Interestingly, shi TS expression in cholinergic neurons of yki 3SA -tumor-bearing flies significantly suppressed Akh production and alleviated host wasting like bloating, lipid loss, and hyperglycemia at day 6 ( Fig. 5E-I ). Taken together, our data demonstrate that cholinergic innervation of APCs is increased to cause energy wasting in yki 3SA -tumor-bearing flies. Glucagon is required for tumor-induced wasting in mammals Since fly Akh and mammalian glucagon are conserved catabolic hormones, we wonder whether neural-associated glucagon release also participates in tumor-induced host wasting in mammals. To do this, we first assessed the glucagon changes in tumor-bearing mammals. We measured the circulating glucagon levels in the patients bearing pancreatic cancer that is with > 85% chance to develop carbo-lipid wasting and weight decline 48 . Glucagonoma a rare tumor that produces high levels of glucagon, was excluded in advance in this study. Interestingly, as compared to pancreatic benign diseases (n=15), patients bearing pancreatic cancer without (n=18) or with weight decline (>1.5% per month) (n=21) within three months prior to surgery potently exhibited higher serum glucagon concentration ( Fig. 6A and Table. S3 ). Considering the high prevalence of weight loss and body-composition change among pancreatic cancer patients in the end, it is possible that hyperglucagonemia might occur prior to the onset of weight loss or organ wasting. We also observed increased mass of α-cells in normal pancreatic tissues of cancer patients ( Fig. 6B ). Download figure Open in new tab Figure 6. Excessive glucagon release is essential for tumor-induced wasting in mammals. ( A ) Circulating glucagon levels in patients with benign pancreatic disease (n=15) and pancreatic-cancer patients with (n=21) or without (n=18) weight decline. ( B ) Representative islet morphologies (green, Gcg) indicated by confocal images in non-tumor tissue of patients. ( C-D ) Serum glucagon levels ( C ) and islet morphologies indicated by confocal images ( D , green, anti-Gcg; red, anti-insulin) of Apc Min/+ mice after 18 weeks (n=8). ( E-H ) Whole body weight changes ( E ), fed blood glucose ( F ), tissue weights ( G ), liver glycogen contents ( H , PAS staining) and tissue morphologies ( H , Gas, myotube diameters; eWAT, adipocyte sizes) of Apc Min/+ mice from week 18 with or without daily IP injection of 10 mg/Kg/day GRA Ex-25 for two weeks (n=10). ( I-M ) Serum glucagon levels ( I ) islet morphologies (green, anti-glucagon; red, anti-insulin) ( J ), tumor weights ( K ), body weight changes ( L ), and tissue morphologies ( M , Gas, myotube diameters; eWAT, adipocyte sizes) of indicated LLC-tumor-bearing mice ( C57BL/6 , n=5; C57BL/6 +LLC, n=6; Gcg -/- + LLC, n=10) or LLC-tumor-bearing mice with daily IP injection of inhibitors from day 14 for 7 days (PBS, n=6; LLC, n=7; LLC+GRA, n=8, 10 mg/Kg/day). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( C, E-G, I, K ), one-way ANOVA with Bonferroni’s multiple-comparisons test ( A, L ). * p < 0.05. We next examined the glucagon levels in tumor-bearing mice. Apc Min/+ mice are an established colon-cancer model that exhibits weight decline, fat loss, and muscle atrophy at 16-18 weeks 49 , 50 . In line with human observations, we found elevated serum glucagon levels and enlarged α-cells in Apc Min/+ mice when they started showing wasting symptoms ( Fig. 6C-E ). We analyzed the carbo-lipid metabolism in cachectic Apc Min/+ mice after 18 weeks and observed upregulation of glucagon-target genes in the liver, glucose intolerance, higher blood glucose levels, as well as hepatic glycogen depletion ( Fig. 6F-H and S9A-B ), as compared to age-matched wild-type C57BL/6 mice. Previous results have indicated that excessive glucagon response potently also results in weight decline, lipid loss, and muscle atrophy in mice 51 – 55 , we next investigate whether glucagon is essential for tumor-induced systemic wasting. We generated Gcg -null mice with removal of exon 3-6 that contains glucagon coding region and crossed them to Apc Min/+ lines to obtain Apc Min/+ ; Gcg -/- mice. However, Apc Min/+ ; Gcg -/- mice appeared very unhealthy and started dying within week 15 with unknown mechanism (data not shown). We thus intraperitoneally (IP) injected small-molecule inhibitors, GRA Ex25 (GRA), of GCGR in Apc Min/+ mice after 18 weeks to suppress glucagon response instead. Interestingly, injection of GRA for 7 days significantly alleviated weight loss, glucose intolerance, and hyperglycemia, of Apc Min/+ mice ( Fig. 6E-F and S9A-B ). We also observed improvements in the loss of gastrocnemius (Gas) and tibialis anterior (TA) muscle and inguinal (iWAT) and epididymal (e-WAT) white adipose tissues, as well as decreased myofiber cross-sectional area and adipocyte size, of Apc Min/+ mice ( Fig. 6G-H and S10A ). We used another GCGR inhibitor, LGD-6972 (LGD), to avoid off-target effects and consistently observed the improvement of tumor-induced wasting in Apc Min/+ mice without affecting tumor growth ( Fig. S9C-H and S10B ). We also studied mice bearing Lewis Lung Carcinoma (LLC) tumors, an established lung-cancer-cachexia mouse model. Similar to Apc Min/+ mice, we observed hyperglucagonemia, enlarged α-cell mass, weight decline, and loss of muscle and fat tissues in C57BL/6 mice bearing LLC tumors at 21 days after LLC injection ( Fig. 6I-M , S9I-K and S10C ). Injection of LLC cells in Gcg -/- mice, as compared to C57BL/6 mice, did not significantly affect tumor growth but, strikingly, alleviated host wasting including weight decline, hyperglycemia, and loss of fat and muscle ( Fig. 6J-M , S9I-K and S10C ). In addition, we IP injected GRA into LLC-tumor bearing mice to blunt glucagon response for 7 days and observed no effects on tumor growth but significant improvements in weight decline, hyperglycemia, loss of muscle and fat, and hepatic glycogen depletion ( Fig. 6J-M , S9I-K and S10C ). Taken together, our results demonstrate that malignant tumors cause organ wasting via elevation of glucagon production. PDGFR/VEGFR blockade alleviates cholinergic-α-cell contacts, glucagon production and wasting in tumor-bearing mice Similar to Akh secretion modulated by cholinergic neurons, mammalian glucagon release is controlled by acetylcholine probably derived from parasympathetic nerves 56 . Published single-cell RNA-seq data reveal that α-cells express acetylcholine receptors as well as VEGFRs and PDGFRs (VEGFR/PDGFR), the homolog of Drosophila Pvr 57 , 58 ( Fig. S10E ). Because mouse Apc Min/+ colon, LLC lung, and human pancreatic tumors were found to produce large amounts of VEGF/PDGF 59 – 66 , we wonder whether tumors promote neural-associated glucagon secretion via VEGFR/PDGFR signaling in a manner similar to fly Pvr signaling. To address this hypothesis, we treated cultured glucagon-producing αTC1 cells with synthetic PDGF-BB to activate PDGFR/VEGFR signaling and observed an increase in expression of multiple Mmp genes and a decrease in Timp genes ( Fig. S10F ). In line with this, we next examined islet morphologies in Apc Min/+ mice and found less ECM contents in α-cells that were indicated by versican staining ( Fig. 7A ). Meanwhile, we strikingly observed increased overlapping between α-cells and intrapancreatic nerves (PGP9.5 + ), especially nerves (AchT + ), in Apc Min/+ mice, indicating cholinergic innervation of α-cells ( Fig. 7B and S10G ). The cholinergic innervation was further associated with the regions of α-cell expansion ( Fig. 7B ). Download figure Open in new tab Figure 7. PDGFR/VEGFR blockade alleviates hyperglucagonemia and cholinergic-α-cell contacts in tumor-bearing mice. ( A-I ) ECM levels of α- cells ( A , green, anti-Gcg; red, anti-Versican) and islet morphologies ( B , green, Gcg; left, red, anti-PGP9.5; right, red, anti-ChAT) indicated by confocal images, serum glucagon levels ( C ), body weights ( D ), GTT ( E ), fed blood glucose ( F ), tissue weights ( G ), forelimb grip strength ( H ), and tissue morphologies ( I , Gas, myotube diameters; eWAT, adipocyte sizes) of indicated mice that were performed daily IP injection of PDGFR/VEGFR inhibitors from week 16 for 2 weeks. C57BL/6 mice (control), Apc Min +veh, Apc Min +Ax (30 mg/Kg/day), and Apc Min +Reg (20 mg/Kg/day) (n=5). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by one-way ANOVA with Bonferroni’s multiple-comparisons test. * p < 0.05. To investigate the impacts of PDGFR/VEGFR signaling on cholinergic innervations and glucagon secretion, we IP injected VEGFR/PDGFR inhibitors, axitinib (Ax) or regorafenib (Reg), into Apc Min/+ mice to block VEGFR/PDGFR signaling at week 18. We found that short-term administration of either inhibitor robustly restored ECM contents of α-cells and diminished cholinergic innervation and α-cell expansion to alleviate hyperglucagonemia ( Fig. 7A-B ). In support of glucagon catabolic effects, we subsequently found that either Ax or Reg administration hardly affected tumor growth but significantly improved the weight loss, glucose intolerance, loss of fat and muscle tissues, and forelimb weakness in Apc Min/+ mice ( Fig. 7C-I , S10D and S10H ). Collectively, these results demonstrate that PDGFR/VEGFR blockade decreases cholinergic innervation on α-cells, glucagon release, and host wasting in Apc Min/+ mice. Discussion Tumor-induced host organ wasting is a general phenomenon in both vertebrates and invertebrates. We have previously revealed that yki 3SA -gut tumors cause host wasting partially through secretion of ImpL2 and Upd3 to impair local metabolism of muscle or fat tissue using Drosophila as a conserved cancer model 9 , 10 . In this study, we further uncovered that yki 3SA tumors extensively result in systemic energy wasting via secretion of another ligand Pvf1 to hijack neuronal-associated release of Akh, a critical metabolic hormone from host. The molecular mechanisms include that Pvf1-Pvr axis triggers ECM remodeling of APCs and enhances their innervation of excitatory cholinergic neurons ( Fig. 5I ). We also identified the similar neural regulation of glucagon production regarding organ wasting in tumor-bearing mice. It is interesting to find that Akh deficiency robustly improved muscle dysfunction and ovary atrophy, in addition to established lipid loss and hyperglycemia, in tumor-bearing flies, even though AkhR is not detectable in either muscle or ovary cells (FlyCellAtlas 35 ). We speculate an indirect regulation of muscle and ovary homeostasis by Akh. Note that, AkhR removal in both fat body and neurons partially alleviated host wasting. It could be possible that AkhR signaling in the fat body causes systemic amino acid consumption 25 , leading to and subsequent ovary and muscle degeneration. AkhR + neurons might promote muscle or ovary wasting through directly or indirectly projections in response to excessive Akh as well. However, Akh gain-of-function in control non-tumor flies failed to affect organ wasting, such as muscle dysfunction and ovary atrophy, suggesting that Akh might collaborate with other tumor-associated factors like inflammatory responses to regulate organ wasting in yki3SA tumor-bearing flies. We have previously characterized the catabolic roles of tumor-derived Pvf1 and speculated that fat body and muscle as the major responding tissues based on Pvr gain-of-function in non-tumor flies 11 . However, the real functional target organ(s) of Pvf1 validated by Pvr loss-of-function in the yki 3SA -tumor-bearing flies are still unclear. In this study, we demonstrated APCs, but not adipose or muscle, as the predominant tissues that respond to tumor-derived Pvf1 to cause wasting. This is because Pvr inactivation in either muscle or fat body in flies bearing Pvf1-inducing tumors rarely shows wasting improvement. The discrepancies of Pvr effects between APCs and fat body/muscle would be caused by differential in vivo Pvf1 delivery, abundance of Pvr expression, and/or intratissue signaling regulation in yki 3SA -tumor-bearing flies. In line with our speculations, recent evidence showed that Pvf1-Pvr signaling in Malpighian tubules contributes to yki 3SA -tumor-induced organ wasting. It would be interesting to investigate the impacts of Pvr signaling in additional tissues like oenocytes 67 and neurons of yki 3SA -tumor-bearing flies using LexA/GAL4 binary expression system in future studies. Pvf1-Pvr activation in APCs increases both mRNA level and release of Akh. We investigated Pvr downstream regulators and found that ERK knockdown restores both of them, while Mmp2 knockdown in APCs only alleviates Akh release without affecting Akh transcription, in the context of Pvf1-Pvr activation. These results suggest that Pvf1-Pvr axis in APCs promotes Akh release via not only ERK- associated Akh synthesis, but also ERK-Mmp2-induced Akh secretion. Consistent with this notion, our results further revealed that tumor-derived Pvf1 activates Pvr in APCs to increase Mmp2-dependent ECM degradation and APC innervation to upstream neurons, promoting Akh release. Previous studies have reported that upstream inhibitory neurons project to APCs to suppress Akh release in response to nutrient availability 30 – 32 , however, the existence of upstream excitatory neurons of APCs to promote Akh release in Drosophila was largely unknown. By screening multiple neurotransmitters in this study, we identified cholinergic neurons as the major excitatory neurons innervating APCs. Overexpression of TrpA1 and Shi TS to activate cholinergic neurons and impair the projection to APCs, respectively, further confirmed their pivotal roles in promoting Akh release and enhancing systemic energy wasting in the context of yki 3SA tumors. Because multiple acetylcholine receptors were found to be expressed in APCs, cholinergic neurons most likely release acetylcholine to directly impact on APC function. In addition, as compared to cholinergic neurons, we also observed relatively weaker contacts between APCs and neurons labelled by TRH- , Gad1- , and Tdc2-GAL4 . A recent study also indicated that Tdc2-GAL4- labelled octopaminergic neurons project on APCs 68 . The potential of contacts between APCs and these neurons might impact Akh release as well in normal or yki 3SA -tumor-bearing flies. On the other hand, the single-nucleus RNA-seq that indicates moderate Pvr expression in, at least, cholinergic and serotonergic neurons ( FlyCellAtlas.org 35 ) raises the possibility that Pvf1 might also modulate the activity of these upstream innervating neurons to coordinate Akh production. One of our important findings here includes that glucagon is essential for muscle and fat wasting in tumor-bearing mice. The plausible mechanisms include at least glucagon-associated hepatic amino acid catabolism remotely causes systemic amino acid loss and organ wasting 54 , 69 . Taken together previous studies that demonstrate glucagon’s roles in systemic wasting regulation in type 2 diabetic and sleep-deprived mice 29 , 54 , we propose that glucagon also functions as a general cachectic hormone in chronic conditions, beyond its established role in short-term hyperglycemic regulation. While anorexia resulted from malignant cancer invariably increases glucagon release that potentially contribute to energy wasting 70 , elucidating the pathogenic mechanisms that stimulate glucagon production by critical tumor-associated cachectic factors remains a crucial research endeavor. Ex vivo studies have revealed that multiple mammalian neurotransmitters including acetylcholine control glucagon secretion 56 , 71 . However, there are very limited in vivo evidence. Thus, it is striking to observe cholinergic-α-cell contact in the context of malignant tumors. We further revealed the VEGFR/PDGFR signaling promotes cholinergic-α-cell interaction through ECM remodeling of α-cells in tumor-bearing Apc Min/+ mice, leading to excessive glucagon release and host wasting. Even though we observed PDGF-BB-induced MMPs expression in cultured α-TC1 cells, incorporating both α-cells and cholinergic neurons into organoids would help provide a more definitive confirmation of neural-associated glucagon release in α-cells in the future. Note that, beside glucagon secretion, α-cell proliferation is also correlated to cholinergic contact with unknown mechanisms. Given large amounts of VEGF/PDGFs produced in both rodent and human malignant tumors 59 – 62 , we therefore conclude a novel mechanism of tumor-host interaction whereby tumors remotely enhance cholinergic-α-cell innervation via VEGFR/PDGFR signaling to promote glucagon release and systemic energy loss. Author Contributions G.D., Y.L. and C.C. designed and performed experiments, including metabolic assays, qPCR, genetic manipulation, and neuronal analysis. G.D., Y.L., C.C. and K.T. performed mouse work. Y.D., H.P., Z.W., P.D., E. R., and X.W. helped perform mouse work and fly genetics. Y.Y., Z. Y. and W.S. discussed results. C.C. and W.S. wrote the manuscript. Declaration of interests The authors declare no competing interests. Supplemental Figure Legends Supplemental Figure 1. Akh is essential for tumor-induced host wasting in Drosophila . ( A-B ) Representative images of muscle degeneration ( A, up) indicated by swollen mitochondria (M) and gaps (G) between mitochondria and myofibril (F), ovary atrophy ( A, down), and climbing rates ( B, n=17) of adult yki 3SA -tumor-bearing flies with or without Akh Δ mutation ( Akh SAP /Akh A ) at day 8. ( C-D ) Metabolic changes including TAG and trehalose (TRE) storages in 8-day old indicated control adult flies ( C, n=5; D, n=6). ( E ) PCA analysis of whole-body gene expression of yki 3SA -tumor-bearing flies with or without Akh Δ mutation ( Akh SAP /Akh A ) at day 8. ( F ) Gene Ontology Enrichment analysis of 395 differentially expressed genes that are both yki 3SA -tumor- and Akh-dependent indicating that the following terms of biological process, cellular compartment, and KEGG pathways are significantly enriched. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( C ) or one-way ANOVA with Bonferroni’s multiple-comparisons test ( B, D ). * p < 0.05. Supplemental Figure 2. AkhR in either fat body or brain contributes to tumor-induced host wasting. Representative images of abdomen bloating ( A, F, up), gut tumors and mass quantification ( A, F, middle, GFP; B, G, n=3), ovary atrophy ( A, F, bottom), bloating rates ( C, H, n=3), climbing rates ( D, I, n=20), and global storages of TAG and trehalose (TRE) ( E, J, n=6) of adult yki 3SA tumor-bearing flies (LexA+ GAL4 ) with AkhR RNAi in either fat body ( R4-GAL4> ) ( A-E ) or pan-neuron ( elav-GAL4> ) ( F-J ) at day 4. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by one-way ANOVA with Bonferroni’s multiple-comparisons test. * p < 0.05. Supplemental Figure 3. Excessive Akh release causes lipid and carbohydrate mobilization but not organ wasting. Representative images of abdomen ( A, F, K, N ), whole-body gene expression ( B, G, n=4, 5 flies/replicate), whole-body TAG and TRE levels ( C, H, n=4, 5 flies/replicate), climbing rates ( D, I, L, O, n=20), as well as ovary images and size quantification ( E, J, M, P, n>10), of adult flies with TrpA1 overexpression in the APCs ( A-E ), Akh overexpression in the APCs ( Akh-GAL4>, F-J ), fat body ( R4-GAL4, K-M ), or pan-neurons ( elav-GAL4, N-P ) at 29 degree at day 8 ( A-J ) or 25 degree at day 4 ( K-P ). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test. * p 10% in APCs. ( B ) Immunostaining indicating Pvr expression in the larval APCs (green, Pvr>GFP; red, anti-Akh). ( C ) Glycemic (circulating trehalose) levels of indicated flies (left, larvae at day 5, n=8, 10 flies/replicate; right, adult flies at day 4, n=3, 30 flies/replicate). ( D ) Representative images of CalexA.GFP indicating intracellular Ca 2+ flux in adult flies with Pvr AC overexpression in the APCs (left) and GFP quantification (right, n=6). ( E-H ) Circulating Akh levels ( E, left, dot-blot; right, quantification, n=3), systemic mRNA levels of Akh and tobi ( F , n=3, 5 flies/replicates), TAG and trehalose (TRE) storages ( G , n=4, 5 flies/replicates), and survival under starvation ( H, n=4, 20 flies/replicate) of indicated flies at day 4. ( I-L ) Circulating Akh levels ( I, dot-blot), Akh production in APCs ( J , left) and quantification of APC masses and intracellular Akh amounts ( J, right, n=3), TAG and trehalose (TRE) ( K , n=4, 5 flies/replicates), and survival under starvation ( L, n=4, 20 flies/replicate) of indicated flies with Pvr manipulation only in adult APCs using tub-Gal80 TS at day 5 after transgene induction. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( D-G, J-K ) or log-rank test ( H, L ). * p < 0.05. Supplemental Figure 5. Pvr blockade in APCs alleviates tumor-induced wasting. Representative images of abdomen bloating ( C, up), gut tumors and mass quantification ( C, middle, GFP; D, n=3), ovary atrophy ( A, up; C, middle) and muscle degeneration ( A, C, bottom) indicated by swollen mitochondria (M) and gaps (G) between mitochondria and myofibril (F), climbing rates ( B, n=17; F, n=17), bloating rates ( E, n=4), and metabolic changes including TAG and trehalose (TRE) storages ( G, n=4) of yki 3SA -tumor-bearing flies ( LexA+GAL4 ) with APC Pvr inactivation (Pvr DN , A-B ) or Pvr RNAi ( C-G ) at day 6. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by one-way ANOVA with Bonferroni’s multiple-comparisons test. * p < 0.05. Supplemental Figure 6. Pvr blockade in muscle or fat body hardly alleviates tumor-induced wasting. Representative images of abdomen bloating ( A, F, up), gut tumors and mass quantification ( A, F, middle, GFP; B, G , n=3), muscle degeneration ( A, F, bottom) indicated by swollen mitochondria (M) and gaps (G) between mitochondria and myofibril (F), bloating rates ( C, n=4; H, n=3), climbing rates ( D, n=17; I, n=20), and metabolic changes including TAG and trehalose (TRE) storages ( E, n=4; J, n=6) of yki 3SA -tumor-bearing flies ( LexA+GAL4 ) with Pvr inactivation (Pvr DN ) in the fat body ( R4>, A-E ) or muscle (Mhc>, F-J ) at day 6. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by one-way ANOVA with Bonferroni’s multiple-comparisons test. * p < 0.05. Supplemental Figure 7. Pvr regulation of Akh production. Survival under starvation ( A, E, H, L, n=4, 20 flies/replicate), whole body Akh or tobi expression ( B, D, J, n=3 or 5, 5 flies/replicate), metabolic changes such as TAG or TRE storages ( C, F, I, K, n=3 or 5, 5 flies/replicate) and circulating Akh levels in the hemolymph ( G ) of adult flies with indicated genotypes at day 4 ( A-I ) or 8 ( J-L ). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( B-D, F, I, J, left, K, left), one-way ANOVA with Bonferroni’s multiple-comparisons test ( J, righ , K, right), or log-rank test ( A, E, H, L ). * p < 0.05. Supplemental Figure 8. ECM homeostasis and neural innervation of APCs. ( A-B ) Representative images of ECM homeostasis indicated by Integrin βPS ( A, B, left, red, anti-integrin) around the somas of APCs ( A , left, green, GFP; B, left, green, anti-Akh) and quantification of extracellular integrin amounts ( A, B, n=5-9) of adult flies with Pvr AC overexpression in APCs at day 4 or flies bearing yki 3SA -tumor plus Pvf1 R NAi at day 7. ( C ) Representative images of boutons that are indicated by Syt-GFP (green) driven by indicated GAL4 lines in the somas of adult APCs (red, Akh) at day 4. ( D ) Published snRNA-seq data indicating that multiple neurotransmitter receptors are expressed in adult CCs or APCs. Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( A ) or one-way ANOVA with Bonferroni’s multiple-comparisons test ( B ). * p < 0.05. Supplemental Figure 9. Blockade of glucagon response alleviates wasting in tumor-bearing mice. ( A-B ) Hepatic gene expression ( A ) and glucose tolerance test (GTT) ( B ) of Apc Min/+ mice from week 18 with or without daily IP injection of 10 mg/Kg/day GRA Ex-25 (GRA) for two weeks (n=10). ( C-H ) Gut tumors ( C ), body weights ( D ), fed blood glucose ( E ), tissue weights ( F ), forelimb grip strengths ( G ), and tissue morphologies ( H , Gas, myotube diameters; eWAT, adipocyte sizes) of Apc Min/+ mice from week 18 with or without IP injection of 6 mg/Kg/day LGD-6972 (LGD) for 10 days (two injections in every three days) for two weeks (n=5). ( I-K ) Fed blood glucose levels ( I ), tissue weights ( J ), and liver glycogen contents ( K , PAS staining) of indicated LLC-tumor-bearing mice ( C57BL/6 , n=5; C57BL/6 +LLC, n=6; Gcg -/- + LLC, n=10) or LLC-tumor-bearing mice with daily IP injection of GRA from day 14 for 7 days (PBS, n=6; LLC, n=7; LLC+GRA, n=8, 10 mg/Kg/day). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( A-B ) or one-way ANOVA with Bonferroni’s multiple-comparisons test ( D-G, I-J ). * p < 0.05. Supplemental Figure 10. PDGFR/VEGFR express in α-cells. ( A-D ) Quantification of gastrocnemius myofiber cross-sectional area, epididymal white adipocyte size, as well as hepatic glycogen content (PAS intensity) in the indicated mice (n=5). ( E ) Gene expression in both mouse α- and β-cells as indicated by RPKM in a published dataset (GSE54973). Genes with RPKM > 1 are considered as functional expressed. ( F ) Gene expression in α- TC1 cells that were treated with 10 ng/mL PDGF-BB for 3 hours (n=3). ( G ) Surface rendering of confocal images merging 8 sections of neve-α-cell contact the islet from the top side (green, Gcg; red, PGP9.5). ( H ) Tumor morphologies indicated by Methylene-blue staining in the colon (arrows indicate tumors) of that were performed daily IP injection of PDGFR/VEGFR inhibitors from week 16 for 2 weeks. C57BL/6 mice (control), Apc Min +Veh, Apc Min +Ax (30 mg/Kg/day), and Apc Min +Reg (20 mg/Kg/day) (n=5). Data are presented as mean ± SEM. Each dot represents one biological replicate. Statistical analysis was conducted by two-tailed unpaired t-test ( F ) or one-way ANOVA with Bonferroni’s multiple-comparisons test ( A-D ). * p < 0.05. Supplemental Table Legends Supplemental Table 1. The FPKM values of gene expression in the whole body of yki 3SA -tumor-bearing flies with or without Akh mutation. Supplemental Table 2. The glycemic changes of larvae with APCs bearing different RNAi against transmembrane proteins. Supplemental Table 3. Clinical characteristics of patients with pancreatic cancer and benign diseases. Experimental Methods Fly strains Files were raised on fly food (5 g agar, 25 g dry yeast, 75 g corn flour, 90 g sucrose, 1.5 g Methylparaben, 4 mL propionic acid per liter) in incubator 12 h light /12 h dark cycle at 25°C. Transgenic and mutant flies were used as below: View this table: View inline View popup To induce gut tumors, we followed the experimental procedures described previously 1 . Briefly, different UAS and LexAop insertions were crossed to esg-GAL4, tub-GAL80 TS , UAS- GFP and esg-LexA, tub-GAL80 TS , LexAop-GFP at 18°C, respectively, to inactivate GAL4/LexA. 4-day-old virgin adult progenies were placed at 29°C to induce the transgenes (day 0 for tumor induction). Flies were transferred onto fresh food every 2 days. For the in vivo RNAi screening against transmembrane proteins in larval APCs, different RNAi lines from DRSC/TRiP center of Harvard Medical School were crossed to Akh-GAL4 at 25 °C. The off-spring larvae were allowed to grow at 25 °C for 5 days to reach late 3 rd -instar for glycemic measurements as previously reported 5 . To access the regulation of Akh release in adult flies, different UAS lines were crossed to Akh-GAL4 at 25 °C. The virgin progenies were collected and maintained at 25 °C for 4 days for metabolic and biochemical measurements. Negative controls, w 1118 and UAS-w-RNAi, exhibited similar phenotypes and only w 1118 is shown in the figures. The 3 rd instar larvae and female adult flies were used for metabolic and wasting examination in this study. Lipid and carbohydrate measurements in flies We measured fly TAG and carbohydrates as described previously 1 . Briefly, 5 female flies from each group were homogenized with 0.5 mL PBS containing 0.2% Triton X-100 using Multi-sample tissuelyser-24 (Shanghai Jingxin Technology) and heated at 70°C for 5 min. The supernatant was collected after centrifugation at 12,000 X g for 10 min at 4°C. 10 μL of supernatant was used for protein quantification using Bradford Reagent (Sigma, B6916- 500ML). Whole body trehalose levels were measured from 10 μL of supernatant treated with 0.2 μL trehalase (Megazyme, E-TREH) at 37°C for 30 min using glucose assay reagent (Megazyme, K-GLUC) following the manufacturer’s protocol. We subtracted the amount of free glucose from the measurement and then normalized the subtracted values to protein levels in the supernatant. To measure whole body triglyceride levels, we processed 10 μL of supernatant using a Serum Triglyceride Determination kit (Sigma, TR0100), subtracted the amount of free glycerol in the supernatant from the measurement, and then normalized to protein levels in the supernatant. Dot-blot analysis of hemolymph Akh Hemolymph of 60–80 female adult flies was collected and 1:100 diluted with PBS. 10 μL of diluted hemolymph was dropped on nitrocellulose membrane (GE Healthcare) and air dried at room temperature for 5 min. The membrane was then boiled in PBS for 3 min and subsequently fixed with 4% PFA in PBS for 20 min. The membrane was blocked with 3% BSA in PBS for 30 min at room temperature and then incubated with rabbit anti-Akh antibody (1:1000, Abclonal, A22867) in 3% BSA in PBS at 4 °C for overnight followed by incubation with HRP-conjugated secondary antibodies in 3% BSA in PBS for 1 h at room temperature. The intensities of the black dots were considered as the amounts of Akh in the hemolymph. Ponceau Red staining prior to blocking was used as the loading control. Climbing, food intake and starvation assays Female adult flies, which were placed in an empty vial and tapped down to the bottom for climbing, were allowed to climb for 2 s. Flies were filmed to quantify climbing height and speed using ImageJ. A minimum of 15 flies and 3 independent trials were performed for each condition. 5 female adult flies, which were cultured on normal food containing 1% (w/v) bright-blue dye for 24h, were homogenized in 1 mL PBS and centrifuged at 12,000 X g for 10 min at 4°C to remove the debris. Protein concentration in the 10 μL lysate was measured using Bradford Reagent (Sigma, B6916-500ML). The food intake was measured by quantifying concentration of bright-blue dye in 200 μL lysate at 595 nm with normalization to protein concentration. 80 female adult flies of genotype were cultured on starvation food (1% agar in H 2 O). Dead flies were counted every 12 h. Mice strains and cell lines All mouse work was approved by the Animal Care and Ethical Committee at Wuhan University. C57BL/6, Apc Min/+ (T001457), and Gcg -/- (T014382) mice were obtained from Gempharmatech, China and housed individually at 22-24°C with a 12 h light/dark cycle with water and food ad libitum. Sample size, determined empirically via performing preliminary experiments, was chosen to be at least five to ensure that adequate statistical power was achieved. Male mice were grouped with similar average body weight one week prior for inhibitor administration. Tumor-bearing mice received daily intraperitoneal injections of Axitinib (T1452, Targetmol) (30 mg/Kg/day), Regorafenib (T1792, Targetmol) (20 mg/Kg/day), GRA-Ex-25 (T3422, Targetmol) (10 mg/Kg/day) or vehicle from 16-18 weeks. LGD-6972 (T25711, Targetmol) (6 mg/Kg/day, two injections every three days) or vehicle was intraperitoneally injected into mice from 18-20 weeks. As for LLC-tumor-bearing mice, PBS or 5 million LLC cells per mouse were injected subcutaneously over the flank (Day 0). After LLC- cell progression for 14 days (Day 14), mice received daily intraperitoneal injections of vehicle or 10 mg/kg GRA-Ex-25 (T3422, Targetmol) for 7 days (Day 21). All mice were sacrificed in the late light cycle (3 p.m.–6 p.m.) and tissues were weighed and frozen immediately in liquid nitrogen for future tests. αTC1 cells were cultured in DMEM (PM150211, ProCell, China) supplemented with 1g/L glucose, 10% FBS and antibiotics. After incubation with 10 ng/mL PDGF-BB (AP002631HU, Cusabio, China) for 3 hours, αTC1 cells were washed and lysed using Trizol (15596018, Thermo Fisher) for RNA extraction. Data of patients with pancreatic cancer The study was performed in accordance with the Helsinki Declaration and approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (KELUN2021042). All participants gave their written informed consent. Patient-, surgery- and oncology-related data were obtained from medical records. Weight loss > 1.5% per month within three months before surgery was considered as weight decline. Peripheral blood was drawn from patients with pancreatic cancer and benign diseases who were diagnosed by pathological examination at Department of Hepatobiliary and Pancreatic Surgery of Zhongnan Hospital of Wuhan University. The plasma was separated and stored at −80 °C until analysis. Immunostaining and electronic microscopy Adult midguts, larval and adult brains were dissected in PBS and fixed for 15 min in PBS containing 4% paraformaldehyde. After fixation, the samples were washed with PBST (0.2% Triton-X100 in PBS) and blocked with 1% BSA in PBST. After incubation with primary antibodies anti-Akh (1:10000, this study) or integrin βPS (1:100, DSHB, CF.6G11) overnight at 4°C, the tissues were washed and then incubated with Alexa fluorescence secondary antibody (1:1000, A32742, Thermo Fisher) and DAPI (1:1000, D1306, ThermoFisher) for 1 h at room temperature then washed. Images of fly appearances were performed on a Nikon SMZ18 or Nikon Eclipse Ts2 and confocal images were obtained using a Zeiss LSM880. For quantification of Akh staining and CaLexA-GFP intensities, stacks were Z-projected and the signals of and Akh or CaLexA-GFP in the whole CC were quantified using Integrated Density in ImageJ. The background was subtracted to give the net signal. APC masses were quantified using ImageJ to analyze the APC areas. Thoraces from adult flies were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde, 2% paraformaldehyde overnight and were performed EM analysis following standard protocols by Servicebio at Wuhan, China. Mouse and human pancreatic samples were fixed in PBS containing 4% paraformaldehyde and cyrosections were performed following standard protocols by Servicebio at Wuhan, China. Primary antibodies including anti-glucagon (1:500, Servicebio, GB12097), anti-PGP9.5 (1:200, Servicebio, GB11159-1), anti-Versican (6 mg/mL, Sigma, AB1033), biotinylated HABP (1:500, Sigma, 385911); Alexa fluorescence secondary antibodies (1:300, Servicebio, GB21303) and (1:400, Servicebio, GB25301) together with DAPI (1:1000, Servicebio, G1012-100ML) and HRP-labeled Streptavidin (1:2000, Beyotime, A0303) were used. Confocal images were obtained using a Zeiss LSM880. Confocal images of 8 sections of a pancreas/islet were merged to generate the 3D-view image. Adipose, liver, and gut tissues were fixed in PBS containing 4% paraformaldehyde and were performed H&E, PAS, or Methylene-blue staining following standard protocols by Servicebio at Wuhan, China. RNA Extraction and qPCR 5 female adult flies of each genotype and αTC1 cells were lysed with Trizol (Thermo Fisher, 15596018) for RNA extraction and cDNA was transcribed using HiScript II Q RT Supermix (Vazyme, R222-01). qPCR was then performed using ChamQ SYBR qPCR Master Mix (Vazyme, Q311-03) on a CFX96 Real-Time System/C1000 Thermal Cycler (Bio-Rad). Expression levels of target genes in fly and mouse were normalized to RpL32 and β-actin, respectively. The following primers were used in this study. View this table: View inline View popup RNA-seq analysis of gene expression in adult flies 5 female adult flies were dissected for total RNA extraction. After assessing RNA quality with Agilent Bioanalyzer (RIN > 7), mRNAs were enriched by poly-A pull-down. Sequencing libraries were prepared with Illumina Truseq RNA preparation kits and were sequenced using Illumina HiSeq 2000 by Benagen (Wuhan, China). We multiplexed samples in each lane, which yields target number of single-end 100-bp reads for each sample, as a fraction of 180 million reads for the whole lane. After trimming, sequence reads were mapped to the Drosophila genome (FlyBase genome annotation version r6.48) using Tophat. With the uniquely mapped reads, gene expression was quantified using Cufflinks (FPKM values) and HTseq (read counts per gene). Differentially expressed genes were analyzed based on both adjusted p value using DSeq2 as well as fold change cut-off. Prior to fold change calculation, we set to a value of “1” for any FPKM value between 0 and 1 to reduce the possibility that we get large ratio values for genes with negligible levels of detected transcript in both the experimental sample and the wildtype control (e.g. FPKM 0.1 vs. 0.0001), as those ratios are unlikely to have biological relevance. A cut-off of 2-fold change consistently observed among replicates and the adjusted p value of 0.1 or lower from DSeq2 analysis were used. Heatmap was generated using MEV_4_7 based on FPKM change. To analyze the data under biological context, we assembled pathway annotation, including biological processes, KEGG pathways and cellular compartment, from DAVID bioinformatics resources. We calculated enrichment p value of gene sets among the up or down regulated genes using hyper-geometric distribution and selected gene sets significantly enrichment with p value less than 0.05. Measurements of serum glucagon Serum from blood samples was collected after centrifugation at 3,000 rpm for 15 min at room temperature. 50μL serum was 1:4 diluted with 150 μL dilution buffer to measure glucagon concentration using commercial ELISA kits (Mouse, DGCG0, R&D Systems; human, D711361, Sangon Biotech). All procedures were performed by following the manufacturer’s protocol. Glucose Tolerance Test (GTT) GTTs were performed in 16-hour-fasted male animals as previously reported 6 . Each animal received an injection of 1 g/Kg glucose in sterile saline. Blood glucose levels were measured at different time points using a glucometer. Mouse forelimb grip strength was measured using Grip Strength Meter (HANDPI, HP-10). Statistical Analysis Data are presented as the mean ± SEM. Unpaired Student’s t test and one-way ANOVA followed by post-hoc test were performed to assess differences. The P value of < 0.05 was considered statistically significant. Acknowledgements We thank the DRSC/TRiP at Harvard Medical School, Bloomington Drosophila Stock Center, NIG in Japan, and Vienna Drosophila Resource Center for providing fly stocks; Sheng Li (South China Normal University) for Vkg-GFP and UAS-Timp flies and integrin βPS antibodies; Ronald Kühnlein (Max-Planck Institute) for Akh SAP and Akh A flies; Norbert Perrimon (Harvard Medical School) for Akh-GAL4 flies; Wei Zhang (Tsinghua University) for Cha-GAL4; Yufeng Pan (Southeast University, China) for Th-GAL4, Tdc2-GAL4, Trh-GAL4 and Oct-TyrR-GAL4 lines; Yong Liu and Baoliang Song (Wuhan University, China) and Liangyou Rui (Michigan University) for insightful comments. Work in the Song lab was supported by the Chinese National Natural Science Foundation (92357303, 91957118, 32350013, 32425029), Chinese Ministry of Science and Technology (National Key R&D Program, 2021YFC2700700) and the Fundamental Research Funds for the Central Universities (2042022dx0003). 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Share A tumor-secreted protein utilizes glucagon release to cause host wasting Guangming Ding , Yingge Li , Chen Cheng , Kai Tan , Yifei Deng , Huiwen Pang , Zhongyuan Wang , Peixuan Dang , Xing Wu , Elisabeth Rushworth , Yufeng Yuan , Zhiyong Yang , Wei Song bioRxiv 2024.10.24.619567; doi: https://doi.org/10.1101/2024.10.24.619567 Share This Article: Copy Citation Tools A tumor-secreted protein utilizes glucagon release to cause host wasting Guangming Ding , Yingge Li , Chen Cheng , Kai Tan , Yifei Deng , Huiwen Pang , Zhongyuan Wang , Peixuan Dang , Xing Wu , Elisabeth Rushworth , Yufeng Yuan , Zhiyong Yang , Wei Song bioRxiv 2024.10.24.619567; doi: https://doi.org/10.1101/2024.10.24.619567 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Physiology Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17728) Bioengineering (13916) Bioinformatics (42037) Biophysics (21489) Cancer Biology (18637) Cell Biology (25553) Clinical Trials (138) Developmental Biology (13401) Ecology (19941) Epidemiology (2067) Evolutionary Biology (24367) Genetics (15622) Genomics (22547) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88747) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9835) Zoology (2272)