A Rhodnius prolixus Insulin Receptor and Its Conserved Intracellular Signaling Pathway and Regulation of Metabolism

Abstract

The insulin signaling pathway is a modulator of metabolism in insects and can regulate functions associated with growth and development, as well as lipid and carbohydrate balance. We have previously reported the presence of an insulin-like peptide and an insulin-like growth factor in Rhodnius prolixus, which are involved in the homeostasis of lipids and carbohydrates in post-feeding and non-feeding periods. In the present study, we have characterized the first insulin receptor (IR) to be discovered in R. prolixus, Rhopr-IR, and investigated its intracellular signaling cascade and its role in nutrient control. We identified a candidate protein sequence within R. prolixus putative peptidome and predicted its conserved features using bioinformatics. Tissue-specific expression analyses indicated that the Rhopr-IR transcript is differentially-expressed in all tissues tested, with the highest values observed in the central nervous system (CNS). Treatment of insects with the IR kinase activator BpV(phen), glucose, or porcine insulin resulted in the activation of protein phosphorylation in the fat body, and stimulated the phosphorylation of protein kinase Akt, an evolutionarily conserved key regulator of the intracellular insulin signaling cascade. We also observed activation of Akt and phosphorylation of its downstream targets glycogen synthase kinase 3 β (GSK3β) and the transcription factor FOXO for several days following a blood meal. We used dsRNA to knockdown transcript expression and examined the resulting effects on metabolism and intracellular signaling. Furthermore, knockdown of the Rhopr-IR transcript increased lipid levels in the hemolymph, while reducing lipid content in the fat body. Interestingly, the levels of carbohydrates in the hemolymph and in the fat body did not show any alterations. The activation of Akt and phosphorylation of FOXO were also reduced in knockdown insects, while the phosphorylation pattern of GSK3β did not change. Our results support the identification of the first IR in R. prolixus and suggest that Rhopr-IR signaling is involved in hemolymph nutrient homeostasis and fat body storage both in post-feeding and in non-feeding stages. These metabolic effects are likely regulated by the activation of Akt and downstream cascades similar to mammalian insulin signaling pathways.

Transcriptomic analysis of regulatory pathways involved in female reproductive physiology of Rhodnius prolixus under different nutritional states

“…After blocking, the blots were incubated overnight at 4 °C, with primary antibody (1:1,000 dilution in PBS-T with 3% BSA): anti-phospho-Akt (ser473); anti-phospho-GSK3β (ser9); anti-phospho-FoxO (ser256); anti-phospho-ToR (ser2448); anti-phospho-p70S6K (ser434); anti-phospho-4E-BP1 (Thr37/46) (all rabbit antibodies from Cell Signaling Technology, MA, USA) and anti-tubulin (mouse monoclonal antibody from Life Technologies, ON, CA). The specificity of these antibodies has been previously reported 12 . Primary antibodies were washed-off with PBS-T followed by incubation in secondary antibody (1:5,000, horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibodies, from Cell Signaling Technology) for 1 h at room temperature with constant agitation.…”

“…Gene specific primers (GSP) were combined with GSPs containing the T7 RNA polymerase promoter sequence ( Supplementary Table S2). As an experimental control, a dsRNA molecule based on the Ampicillin Resistance Gene (dsARG) from the pGEM-T Easy Vector system (Promega, WI, USA) was used throughout the study 12 .…”

“…Together, ILP/ToR signaling represents a nutritional sensing mechanism and plays a crucial role in determining the tradeoff between reproductive success and survival in some insect species 6 . Recently, in juvenile stages of R. prolixus, we identified ILP, IGF and InR 10 – 12 . ILP is only produced by a small group of medial neurosecretory cells in the brain.…”

The Role of Peptide Hormones in Insect Lipid Metabolism

“…Interestingly, the levels of carbohydrates in the hemolymph and the fat body were found to be unchanged. The activation of AKT and phosphorylation of foxO were also reduced in knockdown insects (Defferrari et al, 2018). InR expression was found to be up-regulated in response to the reduction of ILPs in 5th instar M. sexta larvae (Walsh and Smith, 2011).…”

“…Larvae over-expressing InR accumulate more fat in the fat body (Britton et al, 2002). Additionally, InR knockdown increases lipid levels in the hemolymph, while reducing lipid content in the fat body in R. prolixus (Defferrari et al, 2018). Interestingly, the levels of carbohydrates in the hemolymph and the fat body were found to be unchanged.…”