Enzymatic regulation of seasonal glycogen cycling in the freeze-tolerant wood frog, Rana sylvatica

Abstract

Liver glycogen is an important energy store in vertebrates, and in the freeze-tolerant wood frog, Rana sylvatica, this carbohydrate also serves as a major source of the cryoprotectant glucose. We investigated how variation in the levels of the catalytic subunit of protein kinase A (PKAc), glycogen phosphorylase (GP), and glycogen synthase (GS) relates to seasonal glycogen cycling in a temperate (Ohioan) and subarctic (Alaskan) populations of this species. In spring, Ohioan frogs had reduced potential for glycogen synthesis, as evidenced by low GS activity and high PKAc protein levels. In addition, glycogen levels in spring were the lowest of four seasonal samples, as energy input was likely directed towards metabolism and somatic growth during this period. Near-maximal glycogen levels were reached by mid-summer, and remained unchanged in fall and winter, suggesting that glycogenesis was curtailed during this period. Ohioan frogs had a high potential for glycogenolysis and glycogenesis in winter, as evidenced by large glycogen reserves, high levels of GP and GS proteins, and high GS activity, which likely allows for rapid mobilization of cryoprotectant during freezing and replenishing of glycogen reserves during thawing. Alaskan frogs also achieved a near-maximal liver glycogen concentration by summer and displayed high glycogenic and glycogenolytic potential in winter, but, unlike Ohioan frogs, started replenishing their energy reserves early in spring. We conclude that variation in levels of both glycogenolytic and glycogenic enzymes likely happens in response to seasonal changes in energetic strategies and demands, with winter survival being a key component to understanding the regulation of glycogen cycling in this species.

The cryoprotectant system of Cope’s gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing

“…Glycogen levels reported in this study were comparable to those found in previous studies, albeit slightly lower, likely due to differences in housing and acclimation procedures (Irwin and Lee 2003 ; Storey and Storey 1985 ). Following several months of cold acclimation and aphagia, glycogen stores did not change, as observed in other freeze-tolerant frogs (Dinsmore and Swanson 2008 ; Costanzo et al 2013 ; do Amaral et al 2016 ). These data suggest that basal metabolism is being supported by other substrates, possibly proteins and lipids, contrasting with previous reports of hepatic glycogen catabolism during cold acclimation and winter months (Koskela and Pasanen 1975 ; Schlaghecke and Blüm 1978 ).…”

“…Cold acclimation was accompanied by a shift to an 8:16 h light–dark regime. Frogs held at 5 °C were not kept in constant darkness (as in other studies, i.e., Costanzo et al 2013 and; do Amaral et al 2016), to replicate acclimation regimes from our laboratory (Zimmerman et al 2007; Goldstein et al 2010), and while photoperiod may affect some responses to cold acclimation (Stevens 1983), its effect on the development of freeze tolerance is unknown. During cold acclimation, each frog was kept in an individual container with access to water in a plastic dish, and was offered food thrice weekly until it no longer fed.…”

“…In addition, levels of glucose, urea, and lactate in plasma and in neutralized tissue homogenates were determined using spectrophotometric assay kits (Pointe Scientific, Canton, MI, USA). Extracts of liver and skeletal muscle were also assayed for glycogen using an enzymatic procedure as previously described (do Amaral et al 2016). Deproteinized liver and skeletal muscle extracts were neutralized with KOH and incubated at 40 °C for 2 h in a 0.2 mol l −1 sodium acetate buffer, pH 4.8, with 1 mg ml −1 amyloglucosidase (Sigma Aldrich).…”

“…The progressive decrease in temperature that precedes winter months influences the accumulation of energetic substrates (Koskela and Pasanen 1975; Scapin and Di Giuseppe 1994; do Amaral et al 2016), and in freeze-tolerant frogs, primes the cryoprotectant system and the associated enzymatic machinery for survival to subzero temperatures (Swanson et al 1996; Costanzo et al 2014; do Amaral et al 2016). …”

“…The ability to survive somatic freezing has a strong seasonal component (Layne and Lee 1989 ; Costanzo et al 2014 ), with winter-conditioned frogs showing enhanced freeze tolerance compared with summer animals (Layne and Lee 1989 ). The progressive decrease in temperature that precedes winter months influences the accumulation of energetic substrates (Koskela and Pasanen 1975 ; Scapin and Di Giuseppe 1994 ; do Amaral et al 2016 ), and in freeze-tolerant frogs, primes the cryoprotectant system and the associated enzymatic machinery for survival to subzero temperatures (Swanson et al 1996 ; Costanzo et al 2014 ; do Amaral et al 2016 ).…”