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, 36 (11), 959-965

Reduction of Syndecan Transcript Levels in the Insulin-Producing Cells Affects Glucose Homeostasis in Adult Drosophila Melanogaster

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Reduction of Syndecan Transcript Levels in the Insulin-Producing Cells Affects Glucose Homeostasis in Adult Drosophila Melanogaster

Jonathan L Warren Jr et al. DNA Cell Biol.

Abstract

Signaling by direct cell-matrix interactions has been shown to impact the transcription, secretion, and storage of insulin in mammalian β cells. However, more research is still needed in this area. Syndecans are transmembrane heparan sulfate proteoglycans that function independently and in synergy with integrin-mediated signaling to mediate cell adhesion to the extracellular matrix. In this study, we used the model organism Drosophila melanogaster to determine whether knockdown of the Syndecan (Sdc) gene expression specifically in the insulin-producing cells (IPCs) might affect insulin-like peptide (ILP) production and secretion. IPCs of adult flies produce three ILPs (ILP2, ILP3, and ILP5), which have significant homology to mammalian insulin. We report that flies with reduced Sdc expression in the IPCs did not show any difference in the expression of ilp genes compared to controls. However, they had significantly reduced levels of the circulating ILP2 protein, higher circulating carbohydrates, and were less glucose tolerant than control flies. Finally, we found that IPCs-specific Sdc knockdown led to reduced levels of head Glucose transporter1 gene expression, extracellular signal-regulated kinase phosphorylation, and reactive oxygen species. Taken together, our findings suggest a cell autonomous role for Sdc in insulin release in D. melanogaster.

Keywords: AKT; ERK; glucose-induced insulin secretion; syndecan.

Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
IPCs-specific Sdc knockdown leads to significantly decreased circulating ILP2 protein levels. (A, B) Despite no decrease in ilps mRNA levels (A), circulating ILP2 levels were significantly lower in IPC-specific Sdc knockdown (ilp2<Sdc-RNAi) flies compared to controls (ilp2<w1118) (B). Gene expression levels were measured by qPCR using mRNA extracted from heads of males (n = 6). All genes were normalized to rp49. Circulating ILP2 levels were measured by ELISA and values represent the means of four independent replicates. **p < 0.01 relative to control. Error bars represent SE. ELISA, enzyme-linked immunosorbent assay; ILP, insulin-like peptide; IPC, insulin-producing cell; qPCR, quantitative polymerase chain reaction; SE, standard error.
<b>FIG. 2.</b>
FIG. 2.
IPCs-specific Sdc knockdown increases circulating trehalose and stored glycogen, and provides survival advantage to starvation independent of changes on energy balance. (A) Values represent the mean of circulating trehalose levels in IPCs-specific Sdc knockdown (ilp2>Sdc-RNAi) and control (ilp2>w1118) flies (n ≥ 7). (B) Levels of triacylglycerol (TAG, left) and glycogen (GLYC, right) in homogenates of decapitated flies (n = 10 independent replicates). Measurements were normalized to total protein levels. (C) Survival curves under water-only starvation conditions. Starvation survival times were significantly longer in ilp2>Sdc-RNAi males compared to controls [log-rank test, chi square statistic (χ2) = 16.3, p < 0.0001]. (D) Values represent the average amount of food ingested by five housed flies from 10 independent replicates. (E) Values represent the least-square means of whole-body CO2 production (VCO2), an index of resting metabolic rate, adjusted for live body weight (n = 10 independent replicates). *p < 0.05 and ****p < 0.0001 relative to control. Error bars represent SE.
<b>FIG. 3.</b>
FIG. 3.
IPCs-specific Sdc knockdown leads to glucose intolerance. (A) Oral glucose tolerance test data (see text for details). Values represent the means of glucose in homogenates of decapitated flies (n = 10). (B) Left panel: quantification of phosphorylated AKT from western blot assays following glucose feeding (“fed,” n = 4). Right panel: representative western blots images in ilp2>w1118 and ilp2>Sdc-RNAi flies. β-actin was used as loading control. *p < 0.05 and **p < 0.01 relative to control. Error bars represent SE. MW, molecular weight.
<b>FIG. 4.</b>
FIG. 4.
IPCs-specific Sdc knockdown reduces levels of head Glut1 gene expression, ERK phosphorylation, and brain reactive oxygen species. (A) Gene expression levels were measured by qPCR using mRNA isolated from heads of ilp2>w1118 and ilp2>Sdc-RNAi flies (n = 6). Transcript levels of each target gene were normalized to rp49. (B) Left panel: quantification of phosphorylated ERK from western blot assays (n = 4). Right panel: representative western blots images in ilp2>w1118 and ilp2>Sdc-RNAi flies. β-Actin was used as loading control. (C) Values represent the means of H2O2 in fly brains (n = 16). In all panels, *p < 0.05 and **p < 0.01 relative to control. Error bars represent SE. ERK, extracellular signal-regulated kinase.

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