Myrka et al. Avian Res (2020) 11:44 https://doi.org/10.1186/s40657-020-00231-8 Avian Research RESEARCH Open Access No signature of selection on the C-terminal region of glucose transporter 2 with the evolution of avian nectarivory Alexander M. Myrka1†, Tooba Shah2†, Jason T. Weir2,3 and Kenneth C. Welch Jr.2,3* Abstract Background: Flying birds, especially those that hover, need to meet high energetic demands. Birds that meet this demand through nectarivory face the added challenges of maintaining homeostasis in the face of spikes in blood sugar associated with nectar meals, as well as transporting that sugar to energetically demanding tissues. Nectari- vory has evolved many times in birds and we hypothesized that the challenges of this dietary strategy would exert selective pressure on key aspects of metabolic physiology. Specifcally, we hypothesized we would fnd convergent or parallel amino acid substitutions among diferent nectarivorous lineages in a protein important to sensing, regulating, and transporting glucose, glucose transporter 2 (GLUT2). Methods: Genetic sequences for GLUT2 were obtained from ten pairs of nectarivorous and non-nectarivorous sister taxa. We performed PCR amplifcation of the intracellular C-terminal domain of GLUT2 and adjacent protein domains due to the role of this region in determination of transport rate, substrate specifcity and glucosensing. Results: Our fndings have ruled out the C-terminal regulatory region of GLUT2 as a target for selection by sugar-rich diet among avian nectarivores, though selection among hummingbirds, the oldest avian nectarivores, cannot be discounted. Conclusion: Our results indicate future studies should examine down-stream targets of GLUT2-mediated glucosens- ing and insulin secretion, such as insulin receptors and their targets, as potential sites of selection by nectarivory in birds. Keywords: GLUT2, Nectarivory, Avian, Glucosensing, Glucose, Diet, Insulin signalling Background pathological outcomes from spikes in blood sugar con- To meet the high-energy demands of foraging fight, centration despite their chronic high rates of sugar while relying on relatively dilute sugar solutions (20‒25% intake. Tis metabolic challenge is exacerbated by the sucrose in the case of hummingbirds; Roberts 1996), nec- fact that birds generally maintain higher plasma glucose tarivorous birds must sustain high nectar intake rates. concentrations than other vertebrates of similar body Little is known about how nectarivorous birds are able mass while also storing very little glucose intracellularly to maintain sugar homeostasis and avoid or mitigate as glycogen (Braun and Sweazea 2008). Most work has focused on hummingbirds and comparatively little is known about the metabolic fate of sugars in other necta- *Correspondence: [email protected] rivores, but convergent evolution of dietary ecology sug- † Alexander M. Myrka and Tooba Shah co-frst authors gests that convergent adaptations may be present (Baker 2 Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON M1C 1A4, Canada et al. 1998). Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Myrka et al. Avian Res (2020) 11:44 Page 2 of 11 Physiological and biochemical adaptations for sugar demonstrated that PI3K activity in both cell types is nec- metabolism in birds should not only require the efec- essary for normal insulin-induced activation of both the tive regulation of plasma glucose and fructose levels, but extracellular signal-regulated kinase/mitogen-activated also the capacity for rapid transportation of both sug- protein kinase (ERK/MAPK) pathways and subsequent ars across the intestinal epithelium and to energetically downstream phosphorylation and activation of p70 S6 demanding organs (e.g. fight muscles) (Shen et al. 2012). kinase (S6K1) (Duchêne et al. 2008). Tese pathways An initial avenue of investigation is to look for a signa- upregulate cell growth and protein synthesis in mam- ture of selection on the sequences of proteins related to mals, and likely chickens as well (Duchêne et al. 2008; glucose transport, glucosensing, and sugar homeostasis. Dupont et al. 2009). Together the evidence suggests that Discovery of a signature convergent amino acid sequence despite a lack of GLUT4, insulin regulates cellular energy associated with nectarivorous diets is a frst step in iden- budgets in liver and muscle in chickens. tifying molecular adaptations by which animals subsist It is unknown whether the phenomena impacting on high-sugar diets while avoiding metabolic dysfunc- insulin responsiveness in chickens are at work in other tion. Selection of a target protein for screening necessi- avian species. With the genetic absence of GLUT4, an tates consideration of how avian blood glucose regulation open question is whether a nectar diet-induced selec- difers from that of mammals and other vertebrates, par- tive pressure acts on another GLUT isoform involved ticularly with regard to glucose transporters (GLUTs). in sugar homeostasis in avian nectarivores, and in turn GLUTs are key players in sugar homeostasis, able to on the insulin sensitivity of avian liver and muscle. We move hydrophilic sugars across the lipid bilayer (Uldry turned our attention to the glucosensing isoform neces- and Torens 2004). In addition to transporting glucose sary for insulin and glucagon secretion in mice, GLUT2 and fructose, GLUTs also play vital roles in glucosens- (Wu et al. 1998; Torens and Mueckler 2010; Long and ing and blood glucose regulation, and facilitate insulin Cheeseman 2015). signalling (Taniguchi et al. 2006; Zhao and Keating 2007; GLUT2 is most highly expressed in the gut and in Torens and Mueckler 2010). key tissues of sugar homeostasis, such as intestines, Te insulin signalling pathway is not thoroughly char- liver, kidneys, and pancreatic beta islet cells of mam- acterized in non-mammal species, but the role of insu- mals, and expression is similar in Chickens (Gallus gal- lin appears broadly conserved in non-avian vertebrates lus) (Kono et al. 2005; Long and Cheeseman 2015; Byers (Polakof et al. 2011). In mammals, the insulin pathway et al. 2018). In rodent models, presence and activity of is as follows: circulating insulin binds to extracellu- GLUT2 are required for normal glucose sensing, which lar insulin receptors (IRs) of target tissues, triggering precedes insulin and glucagon secretion (Torens and phosphorylation of IR substrates, which in turn activate Mueckler 2010), and it is one of the few GLUTs to trans- the phosphatidyl inositol 3′-kinase-protein kinase B port both glucose and fructose, albeit with relatively (PI3K-Akt) pathway. In response, glucose transporter 4 low binding afnities (Wu et al. 1998; Long and Cheese- (GLUT4)-bearing intracellular vesicles are translocated man 2015). Te high mammalian Km values of GLUT2 to the cell membrane (Taniguchi et al. 2006; Dupont (17 mM for glucose and 76 mM for fructose in humans; et al. 2009) resulting in increased glucose uptake capacity Uldry and Torens 2004) mean that transport capacity (Huang and Czech 2007). is not saturated at the range of serum sugar levels typi- While the insulin-mediated GLUT4 response is broadly cally experienced in mammals. Tis ensures that mam- conserved among fsh, reptiles, and mammals (Shepherd malian glucose uptake rate in glucose-sensing tissues is and Kahn 1999; Polakof et al. 2011; OsorIo-Fuentealba always reactive to changes in circulating glucose (To- et al. 2013), GLUT4 is absent from the genomes of all rens and Mueckler 2010). Te mammalian protein also birds examined (Carver et al. 2001; Seki et al. 2003; responds to glucose binding through interprotein inter- Sweazea and Braun 2006; Braun and Sweazea 2008). actions, leading to transcriptional regulation (Leturque Because birds lack GLUT4, insulin does not induce et al. 2009). Broiler chickens injected with insulin dem- uptake of blood glucose into avian muscle, heart or adi- onstrated reduced liver expression of GLUT2 and IR pose tissues (Braun and Sweazea 2008), though birds do transcripts, suggesting that GLUT2 is insulin sensitive in retain sensitivity to glucagon (Hazelwood 1973). avian liver (Dupont et al. 2004). Decreased glucose trans- Despite the insensitivity of avian blood glucose to insu- port capacity in liver in response to insulin signalling may lin and hyperactivity of PI3K in chicken muscle (Dupont serve to make glucose more available to peripheral tis- et al. 2004), evidence in chickens has shown that insulin sues (Zhang et al. 2013). Caution must be taken in inter- still has a regulatory role over cell metabolism in chicken preting the results of Zhang et al. (2013), as much greater hepatoma cells and primary myocytes (Duchêne et al. than physiologically relevant levels of insulin were 2008; Dupont et al. 2009). Inhibition of chicken PI3K administered to achieve a response. Still, the capacity for Myrka et al. Avian Res (2020) 11:44 Page 3 of 11 insulin-stimulated GLUT2 regulation was nonetheless on the C-terminal region and adjacent primary protein demonstrated.