Abstract
ABSTRACTGlucose is an essential energy source for both vertebrates and invertebrates. In mammals, glucose uptake is mediated primarily by glucose transporters (GLUTs), members of the major facilitator superfamily (MFS) of passive transporters. Among the GLUTs, GLUT4 is the main glucose transporter in muscles and adipocytes. In skeletal muscle cells, GLUT4 interacts with the lipid raft protein flotillin to transport glucose upon stimulation by insulin. Although several studies have examined GLUT4 function in mammals, few have been performed in crustaceans, which also use glucose as their main energy source. Crustacean hyperglycemic hormone (CHH) is a multifunctional neurohormone found only in arthropods, and one of its roles is to regulate glucose homeostasis. However, the molecular mechanism that underlies CHH regulation and whether GLUT4 is involved in its regulation in crustaceans remain unclear. In the present study, we identified a full-length GLUT4 cDNA sequence (defined herein as EsGLUT4) from the Chinese mitten crab Eriocheir sinensis and analyzed its tissue distribution and cellular localization. By the ForteBio Octet system, two large hydrophilic regions within EsGLUT4 were found to interact with the CHH binding protein (CHHBP), an E. sinensis flotillin-like protein. Interestingly, live-cell imaging indicated that EsGLUT4 and CHHBP responded simultaneously upon stimulation by CHH, resulting in glucose release. In contrast to insulin-dependent GLUT4, however, EsGLUT4 and CHHBP were present within cytoplasmic vesicles, both translocating to the plasma membrane upon CHH stimulation. In conclusion, our results provide new evidence for the involvement of EsGLUT4 and CHHBP in the regulation of glucose homeostasis in crustacean carbohydrate metabolism.
Highlights
BLASTp analysis revealed that the E. sinensis GLUT4 (EsGLUT4) protein shared 58% sequence identity with the Glucose transporter 4 (GLUT4) protein of Phaedon cochleariae, indicating that EsGLUT4 was highly homologous with GLUT4 in arthropoda
We describe a novel assay to study EsGLUT4 trafficking upon stimulation with Crustacean hyperglycemic hormone (CHH), which regulates blood glucose levels and glucose metabolism throughout the crustacean life cycle
Based on the tissue expression analysis in Eriocheir Sinensis (Fig. S3), EsGLUT4 were mainly expressed in muscle but much less in hemolymph
Summary
The uptake of glucose into cells is mediated mainly by sodium-coupled transporters (SGLTs) and glucose transporters (GLUTs) (Sala-Rabanal et al, 2016; Scheepers et al, 2004). GLUT4 plays an important role in regulating glucose transport in muscles and adipocytes, and it functions as a typical facilitative glucose transporter in the uptake of glucose into fat and muscle cells in response to insulin (Abel et al, 2001; Bryant et al, 2002; James et al, 1988, 1989). When an insulin receptor on the cell surface is activated by insulin, downstream signals induce a rapid increase in glucose uptake by inducing GLUT4 translocation from the storage vesicles to the plasma membrane (Dugani and Klip, 2005; Luiken et al, 2015; Malide et al, 2000; Slot et al, 1991). The insulin-dependent transient translocation of GLUT4 plays an important role in the physiological metabolism in mammals
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