Glucose Transporter Expression and Regulation Following a Fast in the Ruby-throated Hummingbird, Archilochus colubris

Abstract

Hummingbirds, subsisting almost exclusively on nectar sugar, face extreme challenges to blood sugar regulation. The capacity for transmembrane sugar transport is mediated by the activity of facilitative glucose transporters (GLUTs) and their localisation to the plasma membrane (PM). In this study, we determined the relative protein abundance of GLUT1, GLUT2, GLUT3, and GLUT5 via immunoblot using custom antibodies in whole-tissue and PM fractions of flight-muscle, heart, and liver of ruby-throated hummingbirds (Archilochus colubris). GLUTs examined were detected in nearly all tissues tested. Hepatic GLUT1 was minimally present in whole-tissue and absent in PM fractions. GLUT5 was expressed in flight-muscles at levels comparable to that of their liver, consistent with hummingbird flight-muscles’ hypothesised uniquely high fructose-uptake and oxidation capacity. To assess GLUT regulation, we fed ruby-throated hummingbirds 1M sucrose ad libitum for 24 hours followed by either 1 hour of fasting or continued feeding until sampling. We measured relative GLUT abundance and concentrations of circulating sugars. Blood fructose concentration in fasted hummingbirds declined (∼5 mM to ∼0.18 mM), while fructose-transporting GLUT2 and GLUT5 abundance did not change in PM fractions. Blood glucose concentrations remained elevated in fed and fasted hummingbirds (∼30 mM), while glucose-transporting GLUT1 and GLUT3 in flight muscle and liver PM fractions, respectively, declined in fasted birds. Our results suggest that glucose uptake capacity is dynamically reduced in response to fasting, allowing for maintenance of elevated blood glucose levels, while fructose uptake capacity remains constitutively elevated promoting depletion of blood total fructose within the first hour of a fast.

No signature of selection on the C-terminal region of glucose transporter 2 with the evolution of avian nectarivory

“…No change in plasma membrane recruitment of GLUT2 was seen in hummingbird tissues between fed and fasted states. Whole tissue GLUT2 was, however, greater in the flight muscle of fed hummingbirds than in fasted ones, which may indicate regulation of a recruitable GLUT2 population or, alternatively, regulation of GLUT2 protein turnover (Ali et al 2020). A lack of change in observed plasma membrane GLUT2 suggested that the primary role of this transporter is not in direct modulation of sugar uptake rate, but likely in glucosensing and regulation as described in other systems (Thorens 1996;Thorens and Mueckler 2010;Ali et al 2020).…”

“…As such, there has been ample time for natural selection due to nectarivory to act on these birds and it is not improbable for the two substitutions found only in hummingbirds to be functionally relevant. It is surprising not to find evidence of convergent selection on the regulatory (Katagiri et al 1992) C-terminal region of GLUT2 in nectarivores, given the large variation in blood sugar levels that examined specialist nectarivores experience (17-42 mM glucose in hummingbirds; Beuchat and Chong 1998;11.52-16.51 mM in amethyst sunbirds; Witteveen et al 2014), as well as GLUT2′s posited role in avian insulin signaling and glucosensing (Zhang et al 2013;Ali et al 2020). In interpreting the implications of no signature of selection we first reconsider the state of knowledge of avian GLUT2 in glucose metabolism.…”

“…Whole tissue GLUT2 was, however, greater in the flight muscle of fed hummingbirds than in fasted ones, which may indicate regulation of a recruitable GLUT2 population or, alternatively, regulation of GLUT2 protein turnover (Ali et al 2020). A lack of change in observed plasma membrane GLUT2 suggested that the primary role of this transporter is not in direct modulation of sugar uptake rate, but likely in glucosensing and regulation as described in other systems (Thorens 1996;Thorens and Mueckler 2010;Ali et al 2020). As avian nectarivores, including hummingbirds, share the metabolic challenges of feeding on sugar-rich nectar (Baker et al 1998), we predicted that GLUT2 would have a similar and convergent role in glucosensing and sugar homeostasis in other avian nectarivores.…”

“…Recently it was demonstrated that GLUT2 protein is constitutively expressed in both liver and primary flight muscle of the Ruby-throated Hummingbird (Archilochus colubris); further, dietary status altered GLUT2 protein expression in whole tissue homogenates of flight muscle but not liver (Ali et al 2020). No change in plasma membrane recruitment of GLUT2 was seen in hummingbird tissues between fed and fasted states.…”

“…Given evidence of selection on GLUT4 in relation to dietary ecology and nectarivory in bats (Shen et al 2012), but that birds lack GLUT4 and appear to utilize GLUT2 in insulin responsiveness and glucose homeostasis (Dupont et al 2004;Ali et al 2020), we hypothesized that we would find a signature of nectarivorous dietary ecology in the protein sequence of avian GLUT2.…”