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The heparan sulfate proteoglycan syndecans are transmembrane proteins involved in multiple physiological processes, including cell-matrix adhesion and inflammation. Recent evidence from model systems and humans suggest that syndecans have a role in energy balance and nutrient metabolism regulation. However, much remains to be learned about the mechanisms through which syndecans influence these phenotypes. Previously, we reported that D. melanogaster Syndecan (Sdc) mutants had reduced metabolic activity compared to controls. Here, we knocked down endogenous Sdc expression in the fat body (the functional equivalent of mammalian adipose tissue and liver) to investigate whether the effects on metabolism originate from this tissue. We found that knocking down Sdc in the fat body leads to flies with higher levels of glycogen and fat and that survive longer during starvation, likely due to their extra energy reserves and an increase in gluconeogenesis. However, compared to control flies, they are also more sensitive to environmental stresses (e.g. bacterial infection and cold) and have reduced metabolic activity under normal feeding conditions. Under the same conditions, fat-body Sdc reduction enhances expression of genes involved in glyceroneogenesis and gluconeogenesis and induces a drastic decrease in phosphorylation levels of AKT and extracellular signal regulated kinase 1/2 (ERK1/2). Altogether, these findings strongly suggest that Drosophila fat body Sdc is involved in a mechanism that shifts resources to different physiological functions according to nutritional status.
Excess body fat leads to obesity which has become a serious and increasingly costly worldwide public health problem due to its increased prevalence in adults, children, and adolescents . The health consequences of excess fat are due to a strong correlation between adiposity and the metabolic syndrome, which is a cluster of cardio-metabolic alterations that increases the risk of type 2 diabetes mellitus, cardiovascular disease, and several cancers . In recent years, D. melanogaster has emerged as a powerful model for studying human obesity and metabolic regulation . To this end, earlier quantitative genetic studies performed in our laboratory pinpointed Sdc as a positional candidate gene affecting inter-individual differences in Drosophila fat storage . Syndecans are type I transmembrane proteins that belong to the family of heparan sulfate proteoglycans and together with integrins are the major ECM adhesion receptors . While the number of syndecan proteins varies across species (invertebrates have one, while humans have four encoded by four different genes, SDC1-4) their general structure appears to be conserved . All syndecans are characterized by a core protein with extracellular attachment sites for glycosaminoglycan chains that mediate interactions with a wide array of ligands. Syndecans also contain a highly conserved transmembrane domain and a short cytoplasmic tail that binds to cytoskeleton proteins. This allows them to function independently and in synergy with integrin-mediated signaling to control cell adhesion, behavior, and fate [5, 6].
Our previous work showed that young flies homozygous for a hypomorphic Sdc mutation had lower levels of energy reserves, displayed lower metabolic activity, and reduced survival . Human genetic studies performed in parallel also revealed that genetic polymorphisms in the SDC4 gene were associated with adiposity, fasting plasma glucose, and resting energy expenditure in American children . Studies in mice have also reported a role for members of the syndecan family in adipocyte differentiation and energy balance [7, 8]. However, little is still known about the mechanisms through which syndecans regulate these metabolic phenotypes. Here, we used RNA interference (RNAi) through a UAS-driven transgene (UAS-IR) to down-regulate Sdc expression in the Drosophila fat body. The insect fat body plays a crucial role in nutrient metabolism . Additionally, nutrient-sensing signaling pathways that have been associated with survival are highly active in this tissue . We, therefore, reasoned that by reducing the expression of Sdc specifically in the fat body we could gain insightful information into its role in body composition and metabolism regulation.
We used the GAL4/UAS approach  to knock-down (KD) the expression of Sdc in the fat body using a Larval serum protein 2 (Lsp2)-Gal4 driver [11, 12]. We chose this driver because the Lsp2 gene, although highly expressed in third instar larvae and only moderately in adult flies, is a specific marker of the fat body [11, 12]. The UAS-Sdc-IR (Stock ID: v13322) and w1118 (Stock ID: 5905) lines were obtained from the Vienna Stock Center and the Bloomington Stock Center, respectively. We crossed Lsp2-Gal4 male and UAS-Sdc-IR or w1118 female flies to generate the w1118; Lsp2-Gal4/+; UAS- Sdc-IR/+ (fat body Sdc KD, referred hereafter as lsp2>Sdc-IR) and w1118; Lsp2-Gal4/+; +/+ (control) genotypes. Four to six day old virgin flies of each genotype were assessed for the phenotypic assays described below. The w1118 genotype used in this study is the one in which the transgenic UAS-Sdc-IR stock was originally constructed (http://flystocks.bio.indiana.edu/Browse/VDRCtb.htm), thereby reducing differences in genetic background between lsp2>Sdc-IR and control flies that could affect the phenotypes. Virgin flies were used to minimize the influence of reproduction on energy metabolism. In our previous work we detected female-specific effects of the Sdc mutation on energy storage ; thus, only female flies were used in the present study. All flies were raised at 25°C on a standard cornmeal, molasses, and yeast medium.
TAG, glycogen, glucose, and protein levels were measured using protocols described in . Free glucose concentration was subtracted from the total amount of glucose present in the sample after treatment with the enzyme amyloglucosidase in order to determine glycogen amounts. Trehalose was measured in hemolymph samples using the method described in .
Resting metabolic rate was measured as CO2 production using a flow-through respirometry system (Qubit System Research, Kingston, Ontario, Canada) and the method described in .
Food intake was assessed using the Capillary Feeder (CAFÉ) assay described in  with slight modifications. Groups of five flies were housed in vials containing 1% agar medium and a microcapillary tube filled with liquid food media made up of 5% glucose and 5% autolyzed yeast. Flies were first allowed to acclimate to the capillary system overnight. Food consumption was then quantified for the 12 hour day and night cycles as the change in height of the liquid in the microcapillary tube. Control vials containing no flies were included to estimate the amount of water in the food that evaporated.
Locomotor activity was measured with the Drosophila Activity Monitoring System (Trikinetics, Waltham, MA), which counts the number of times a given fly crosses an infrared beam during a specified time interval .
Survival under starvation conditions was measured by placing 10 flies per genotype on 1.5% agarose medium and recording the number of flies alive at 8-h intervals until all were dead.
Female flies were assayed for their ability to clear infection by Escherichia coli (HB101, a streptomycin resistant strain) using methods described in .
We used a chill-coma recovery assay to test cold tolerance. Groups of 25 females for each genotype were subjected to 0°C for 3 hours. Flies were then returned to room temperature and the time to restore locomotor function was measured.
Flies were dissected into phosphate-buffered saline and abdomens were homogenized in standard lysis buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membrane (Thermo Scientific, Rockford, USA), blocked in 5% BSA, and incubated with 1:1000 rabbit β-Actin (Cell Signaling # 4967); 1:1000 rabbit anti-Phospho-Drosophila Akt (Ser505) (Cell Signaling #4054) and rabbit anti-Akt (Cell Signaling #9272); 1:1000 rabbit anti-Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling #9211) and rabbit anti-p38 MAPK (Cell Signaling #9212); 1:1000 rabbit anti-Phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling #4377) and rabbit anti-p44/42 MAPK (Cell Signaling #4695); and 1:1000 rabbit anti-Phospho-SAPK/JNK(Thr183/Tyr185) (Cell Signaling #4668) and rabbit anti-JNK (Santa Cruz Biotechnology, Inc. #sc-571). The primary antibodies were incubated overnight at 4°C. The secondary antibody was incubated with 1:5000 HRP-conjugated goat anti-rabbit or goat anti-mouse for 1 hour at room temperature and the signal developed using an ECL detection kit (Merk Millipore).
Cox regression as implemented by SAS (V9.1.3) was used to compare survival of lsp2 > Sdc-IR and control flies under starvation conditions. One-way analysis of variance and analysis of covariance (with body weight used as covariate) were used to assess differences between experimental and control groups in locomotor activity and metabolic assays, respectively. Data from the other experimental assays were analyzed by two-tailed Student’s t test.
In the first series of experiments we verified that Sdc is localized to the plasma membrane of fat body cells in adult flies (Supplementary Fig. 1A). In contrast, no visible evidence of Sdc in the fat body of lsp2>Sdc-IR flies was observed by the immunohistochemical stain (Supplementary Fig. 1A). This result was corroborated by qPCR analysis, which showed a 60% reduction in Sdc expression in abdomens of lsp2>Sdc-IR flies compared to controls (Supplementary Fig. 1B). Taken together, these results established that the RNAi approach produced the expected KD of Sdc in the adult fat body.
Next, we examined the effect of reducing Sdc in the fat body on energy stores. Contrary to observations in the Sdc mutant , fat-body specific KD of Sdc increased triacylglycerol (TAG) and glycogen contents by 27% and 37%, respectively (Fig. 1A). No difference in the levels of trehalose, the main circulating sugar, was observed (Fig. 1B). Because changes in energy balance can affect fat storage in flies , we then asked whether the increase in TAG content in lsp2>Sdc-IR flies might be due to an energy imbalance. There was no significant difference in locomotor activity between lsp2>Sdc-IR and control flies (Fig. 1C), but lsp2>Sdc-IR flies displayed a 16% reduction in metabolic rate (Fig. 1D), which could explain their higher TAG content. Interestingly, lsp2>Sdc-IR flies also ingested ~ 15% less food over a 24-hour window (Fig. 1E). To confirm these findings, we investigated two additional GAL4-driver lines - the Yolk-Gal4 and the drug (RU486) inducible S106-GS-Gal4 - that allow the expression of Gal4 specifically in adult fat body. In both cases, as seen in lsp2>Sdc-IR flies, KD of Sdc expression led to increased metabolite storage (Supplementary Fig. 2A and 2B) and decreased resting metabolic rate (Supplementary Fig. 2C). Similarly, KD of Sdc by S106-GS-Gal4 in the presence of RU486 significantly reduced 24-hour food consumption (Supplementary Fig. 2D). Flies carrying Yolk-Gal4 and UAS-Sdc-IR did not display any difference in the amount of food consumed compared to controls (Supplementary Fig. 2D). However, given that Sdc is found in most tissues  and the Yolk-Gal4 line does not drive the expression of Gal4 exclusively in the fat body , it is possible that this latter finding could be a compensatory effect generated from another tissue.
Next, we sought to determine whether the changes in metabolism observed in lsp2>Sdc-IR flies would alter their ability to respond to stress challenges, such as nutrient starvation, bacterial infection, and cold exposure. We observed that starved lsp2>Sdc-IR flies lived longer than controls (Fig. 2A), but they had significantly reduced immune function, with 34% higher bacterial loads than controls 24 hours after infection (Fig. 2B). Additionally, it took lsp2>Sdc-IR flies longer (on average four minutes) to recover from cold-induced paralysis (Fig. 2C) than control flies.
To verify whether starvation resistance resulted from utilization of the higher energy reserves, we measured glycogen levels in flies fasted for 24 and 30 hours. To maintain glucose homeostasis during starvation, wild-type flies use their stored glycogen after a 24 hour fast . Consistent with this previous work, glycogen levels dropped significantly in 24-hour fasted lsp2 > Sdc-IR and control flies (Fig. 2D). However, while glycogen levels of control flies were almost depleted after a 30-hour fast, those of lsp2>Sdc-IR flies were 58% higher in comparison with the 24 hour fast (Fig. 2D). This suggests that the reduction of Sdc in the fat body enhances the gluconeogenic fasting response and promotes glucose storage to glycogen. Concurrently, 30-hour fasted lsp2 >Sdc-IR flies had significantly increased (54%) glucose levels than controls (Fig. 2E). The rate of gluconeogenesis is controlled by the enzyme phosphoenol-pyruvate carboxykinase (PEPCK), which is transcriptionally regulated by a number of physiological signals and plays a key role also in the glyceroneogenesis pathway . D. melanogaster contains two genes encoding PEPCK, CG10924 and Pepck , thus we evaluated the expression of these two genes after a 30 hour fast. We found that the expression of CG10924 and Pepck were significantly increased and decreased, respectively, in lsp2 > Sdc-IR flies compared to controls (Fig. 2F). Since Pepck has a more prominent role in Drosophila glycerol and triglyceride homeostasis than CG10924 , these results corroborate the idea that lsp2 >Sdc-IR flies display enhanced gluconeogenesis under fasting conditions. Additionally, they suggest that KD of Sdc in the fat body might cause an attenuation of glyceroneogenesis to limit free-fatty acids re-esterification, which is a highly ATP-consuming process .
To gain insight into how KD of Sdc in the fat body affects nutrient metabolism and the response to environmental stresses under fed conditions, we analyzed the transcriptome profile of abdomens (where the fat body is more conspicuous) of lsp2 > Sdc-IR and control flies by RNA-sequencing (Supplementary Table 2) (GEO accession number: GSE67001). We then selected 22 transcripts among those with fold changes greater than 1.3 between genotypes (at an uncorrected P-value of <0.05) for validation by qPCR using independent mRNA samples. We chose transcripts expressed in the fat body with known biological functions relevant to the traits affected by fat body-specific Sdc reduction. For 11 of the 22 genes, we confirmed significant differences in expression levels between lsp2>Sdc-IR and control flies (Fig. 3). Genes significantly up-regulated in lsp2>Sdc-IR flies include those involved in glycerol and fat metabolism (Pepck, Glycerol kinase-Gyk, Microsomal triacylglycerol transfer protein-Mtp, and Adipokinetic hormone receptor-AkhR), gluconeogenesis and glycogen metabolic process (CG10924 and Glycogen binding subunit 70E-Gbs-70E), response to oxidative stress (Ecdysone-induced protein 28/29kD-Eip71CD and Glutathione S transferase E1-GstE1), positive regulation of circadian sleep/wake and sleep (Angiotensin-converting enzyme related-Acer), and integrin-mediated cell adhesion (Tiggrin-Tig) . qPCR analysis did not detect differential expression of NF-κB pathway controlled antimicrobial genes between lsp2 > Sdc-IR and control flies (Fig. 3). But, the expression of the gene Listericin, which encodes a protein that suppresses the growth of Gram-negative bacteria , was significantly reduced in lsp2 > Sdc-IR flies (Fig. 3) and so may contribute to the reduced bacterial clearance ability of these flies.
The transcriptional up-regulation of genes involved in gluconeogenesis under fed conditions raised the possibility that fat body-specific KD of Sdc might affect PI3K/AKT signaling. Thus, we assessed phosphorylation/activation of AKT (Ser505) in fly abdomens. As predicted, AKT phosphorylation levels were decreased by 43% in lsp2>Sdc-IR flies compared to control flies (Fig. 4). This finding echoes work in mammals showing that syndecan-4 activates the phosphorylation of AKT at Ser473 by regulating the assembly and plasma membrane recruitment of the mammalian Target of Rapamycin Complex 2 in endothelial cells .
The mammalian “stress regulated” mitogen-activated protein kinase (MAPK) cascades, which are activated by a variety of environmental stresses, interact at different levels with the PI3K/AKT pathway to control cell survival and proliferation . Thus, we further investigated whether the phosphorylation status of p42/p44 MAPK, p38 MAPK, and JNK was altered in lsp2>Sdc-IR flies compared to controls. Whereas no change in phospho-p38 MAPK levels were observed between genotypes (Fig. 4), we detected a significant increase (35%) in the phosphorylation of JNK (Fig. 4). Additionally, we found a dramatic decrease (75%) in the phosphorylation of p42/p44 MAPK (Fig. 4), which is consistent with the cross-inhibition between Ras-ERK and PI3-AKT pathways seen in mammals .
Altogether, our results indicate that reduced levels of Sdc in the fat body promotes an energy-saving mechanism for survival during periods of reduced food availability. We propose that this mechanism allows fat-body Sdc KD flies to allocate resources to storage and maintain circulating sugar (trehalose) homeostasis when food is available. This, in turn, triggers metabolic adaptations that lead to reduced metabolic activity, food intake, and ability to respond to other energy-demanding activities, such as immune function and cold tolerance. At the molecular level, this notion is supported by our finding of decreased phosphorylation of AKT and up-regulation of genes involved in glyceroneogenesis (Gyk and Pepck), gluconeogenesis (CG10924), and glycogen synthesis (Gbs-70E) in lsp2>Sdc-IR flies compared to controls under fed conditions. Of note, Gbs-70E encodes a protein similar to mammalian protein targeting to glycogen (PTG) whose overexpression in mouse liver has been recently reported to increase liver glycogen and reduce food intake . Furthermore, we observed that fed lsp2>Sdc-IR flies had higher transcript levels of Eip71CD (encoding the Drosophila homolog of Methionine Sulfoxide Reductase A) than controls. Earlier work identified Eip71CD as a cold-responsive gene in two D. virilis groups , with expression levels of Eip71CD being consistently lower during acclimation to low temperature. Given that overexpression of Eip71CD also confers resistance to oxidative stress , it is possible that its higher expression levels in lsp2>Sdc-IR flies may be the result of an increase in oxidative stress in these flies. The trade-off, however, is an enhanced sensitivity to cold. The JNK signaling has been reported to trigger a stress-activated pathway that promotes Drosophila tolerance to oxidative stress and starvation . Thus, the increase in JNK phosphorylation in lsp2>Sdc-IR flies further supports this view. These findings significantly increase our understanding of the role of syndecans in nutrient metabolism regulation.
We are grateful to Michael Crowley and David K. Crossman at the UAB Heflin Center for Genomic Sciences for performing RNA-sequencing analysis. This work was supported by the National Institutes of Health (R01 DK084219) and UAB Diabetes Research Center Pilot & Feasibility Program (P30 DK079626) grants to MD.
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