Biochem Biophys Res Commun. 2016 Aug 12;477(1):103-8.
Knockdown expression of Syndecan in the fat body impacts nutrient metabolism and the organismal response to environmental stresses in Drosophila melanogaster.
Eveland M1, Brokamp GA1, Lue CH2, Harbison ST3, Leips J2, De Luca M4.
1Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL, USA.
2Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA.
3Laboratory of Systems Genetics, National Heart Lung and Blood Institute, Bethesda, MD, USA.
4Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: email@example.com.
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 Drosophila 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.
KEYWORDS: AKT; ERK1/2; Fat body; Metabolism; Survival; Syndecans
The ability to store energy as fat is required for the life cycle of many organisms . Tightly interrelated physiological pathways have evolved to regulate the balance between energy intake and expenditure at an optimal level, so that fat storage is increased (or decreased) in fat tissue . Mounting evidence suggests that fat tissue serves as a critical link between the mechanisms and pathways contributing to longevity and “healthspan” (reviewed in [2, 3]. This development has been driven by the recognition that mammalian white adipose tissue (WAT) is not just a storage site for energy, but a metabolically active tissue secreting bioactive molecules, called adipokines, that affect local cells, peripheral organs, and the brain. It is the interplay of the bioactive molecules produced by the WAT cells with growth factors, exogenous hormones (e.g. insulin), and several adipocyte transcription factors members that contributes to the remodeling of WAT and therefore to its capability to expand and regress in response to changes in energy balance throughout life . In recent years, it has also become clear that the “healthy” expansion and contraction of WAT requires extracellular matrix (ECM) and cell cytoskeleton modifications . The ECM is an intricate network that provides structural and anchoring support to the cells in order to stabilize cell morphology and tissue architecture. However, it also controls many aspects of the cell’s dynamic behavior (including cell proliferation and growth, cell shape, migration, and differentiation) by binding to specific integrin cell-surface receptors and/or cell-surface syndecans . The latter are members of the heparan sulfate proteoglycan family of glycoproteins and, like integrins, are cell-adhesion molecules present on the cell surface of a wide range of invertebrate and vertebrate tissues . While invertebrates have only one Syndecan (Sdc) gene that is expressed in most tissues, mammalian genomes contain four different genes (SDC1-4), with respective proteins . Previously, we and others reported that mutations in the Sdc gene affected energy metabolism and life span in the fruit fly Drosophila melanogaster [8, 9]. Parallel human genetic studies also showed that genetic variants in the human SDC4 gene were associated with intra-abdominal fat and energy expenditure in American children . Specifically, we found that children homozygous for the minor G-allele of SDC4 rs1981429 had more intra-abdominal fat than those homozygous for the T-allele . Notably, in a follow-up genetic study conducted in a cohort of healthy elderly Italian subjects (age 64 to 107 years) we observed that the rs1981429 G-allele was associated with higher levels of fasting plasma triglycerides (a risk factor for cardiovascular disease) and a lower likelihood of becoming a centenarian (subjects aged 100 or older) . Based on these observations, we hypothesized that syndecans might have a role in a conserved mechanism linking life span to adipose tissue. To test our hypothesis, we used a well-established genetics approach in D. melanogaster, called GAL4/UAS-RNA interference (RNAi), to knockdown the expression of the Sdc gene specifically in the fat body, the functional equivalent of mammalian WAT and liver. In the GAL4/UAS-RNAi system, the transcription of an RNAi hairpin (the responder), which in turn suppresses the expression of a target gene, is controlled by the presence of a UAS (Upstream Activation Sequence) enhancer element. Since the transcription of the responder is activated in the presence of the GAL4 protein, male flies of the responder line ought to mate with female flies expressing GAL4 in a specific tissue (the driver). In the resulting progeny, the responder is then expressed only in the cells that express the driver . In our study, we first crossed transgenic flies carrying the GAL4 component driven by the Lsp2 promoter for expression in the fat body (lsp2-GAL4) over UAS- Sdc Inverted repeat (IR) transgenic flies to produce the fat body-specific Sdc knockdown flies (hereafter indicated as lsp2>Sdc-IR) or w1118 flies to produce the controls (Figure 1). Subsequently, we performed a set of experiments using the resulting progeny. We found that while fat body-specific Sdc knockdown flies showed no difference in trehalose (the circulating sugar in insects) levels compared to controls under ad libitum conditions, they had more fat and glycogen. Additionally, compared to control flies, fat body-specific Sdc knockdown flies had increased survival under starvation conditions, most likely as a consequence of using their extra energy reserves and producing more glucose. However, they were more sensitive to infection and cold stress, ate less, and had reduced metabolic activity than controls when fed ad libitum . Collectively, this suggests that reduced Sdc levels in the fat body promotes an energy-saving mechanism for survival during periods of reduced food availability. This mechanism allows flies with reduced levels of Sdc in the fat body to allocate resources to storage and maintain circulating sugar (trehalose) homeostasis when food is available. But, it triggers metabolic adaptations that lead to decreased metabolic rate and other energy-demanding activities. These metabolic changes could ultimately impair cellular/tissue functions and thereby reduce life span, as seen in the hypomorphic Sdc mutant flies . This idea is further corroborated by recent work in the laboratory of Dr. Jeff Leips (who has been part of our study) showing that the life span of lsp2>Sdc-IR flies was significantly reduced in both sexes compared to controls (Figure 2). The specific mechanism through which reduction of the Sdc gene in the fat body affects nutrient metabolism and life span remains elusive. Nevertheless, one exciting finding of our study is that lsp2>Sdc-IR flies had lower levels of phosphorylated prosurvival/proliferation mediators, Akt and Erk1/2. Additionally, transcript levels of the Listericin gene, a downstream effector of the JAK/STAT signaling pathway , were significantly reduced in lsp2>Sdc-IR flies . Taken together, these results suggest that Sdc might influence cellular energy level through effects on signaling pathways involved in cell survival and proliferation.
The importance of the study: To the best of our knowledge, ours is the first study to identify Sdc as a mediator linking fat tissue metabolism and survival in D. melanogaster. This finding provides additional evidence to the growing body of research in humans and model systems suggesting a critical role of adipose tissue in the systemic regulation of aging and longevity.
Figure 1. Mating schemes used in the study. See text for details.
Figure 2. Survival curves of fat body-specific Sdc knockdown (lsp2< Sdc-IR) and control flies under ad libitum feeding conditions. The adult life span of lsp2>Sdc-IR flies was significantly reduced in both sexes compared to controls (females: log-rank test, chi square statistic (c2) = 18.63, P<0.0001; males: c2 = 59.22, P<0.0001).
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