Molecular identification and regulation of hagfish glucose transporters

Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early-vertebrates owing to their basal position in vertebrate phylogeny. Glucose is a ubiquitous cellular energy source that is transported into cells via two classes of carrier proteins: sodium-glucose linked transporters (Sglt; Slc5a) and glucose transporters (Glut; Slc2a). While previous pharmacological evidence has suggested the presence of both sodium-dependent and -independent transport mechanisms in the hagfish, the molecular identities were heretofore unconfirmed. We have identified and phylogenetically characterized both a Slc5a1-like and Slc2a-like gene in the Pacific hagfish (Eptatretus stoutii); the latter sharing common ancestry with other glucose-transporting isoforms of the Slc2a family. To assess the potential post-prandial regulation of these glucose transporters, we examined the abundance and localization of these transporters with qPCR and immunohistochemistry alongside functional studies using radiolabeled 14C-D-glucose. The effects of glucose- or insulin-injection on glucose transport rate and transporter expression were also examined to determine their potential role(s) in the regulation of intestinal glucose carrier proteins. Feeding prompted an increase in glucose uptake across the hindgut at both 0.5 mM (~84%) and 1 mM (~183%) concentrations. Concomitant increases were observed in hindgut Sglt1 protein expression. These effects were not observed following either of glucose- or insulin-injection, indicating these post-prandial factors are not the driving force for transporter regulation over this timeframe. We conclude that hagfish utilize evolutionarily-conserved mechanisms of glucose uptake and so represent a useful model to understand early vertebrate evolution of glucose uptake and regulation.盲鳗（Hagfish）因在脊椎动物系统发育中占据基干类群位置，成为推断早期脊椎动物转运系统演化历程的优质模式物种。葡萄糖是一类广泛存在的细胞能量来源，通过两类载体蛋白介导进入细胞：钠-葡萄糖协同转运蛋白（SGLT；Slc5a家族）与葡萄糖转运蛋白（GLUT；Slc2a家族）。尽管此前的药理学研究证据表明盲鳗体内同时存在钠依赖型与非钠依赖型葡萄糖转运机制，但该过程的分子本质此前尚未得到证实。本研究在太平洋盲鳗（Eptatretus stoutii）中鉴定出Slc5a1样基因与Slc2a样基因，并对二者开展了系统发育特征分析；其中Slc2a样基因与Slc2a家族的其他葡萄糖转运亚型具有共同的演化祖先。为探究上述葡萄糖转运蛋白的餐后调控潜力，本研究通过实时荧光定量PCR（qPCR）与免疫组织化学（immunohistochemistry）技术检测了二者的表达丰度与组织定位，并结合放射性标记¹⁴C-D-葡萄糖开展了功能实验。本研究还检测了葡萄糖注射或胰岛素注射对葡萄糖转运速率及转运蛋白表达的影响，以明确二者在肠道葡萄糖载体蛋白调控中可能发挥的作用。摄食可使后肠在0.5 mM（约84%）与1 mM（约183%）浓度下的葡萄糖摄取量均显著提升；同时后肠的SGLT1蛋白表达量也同步升高。但单独注射葡萄糖或胰岛素均未观察到上述效应，表明在该实验周期内，餐后相关因子并非转运蛋白调控的驱动因素。综上，盲鳗采用了演化保守的葡萄糖摄取机制，因此其是探究早期脊椎动物葡萄糖摄取与调控系统演化的优质模型物种。