Enzymatic Regulation of Glycogenolysis in a Subarctic Population of the Wood Frog: Implications for Extreme Freeze Tolerance

Abstract

The wood frog, Rana sylvatica, from Interior Alaska survives freezing at –16°C, a temperature 10–13°C below that tolerated by its southern conspecifics. We investigated the hepatic freezing response in this northern phenotype to determine if its profound freeze tolerance is associated with an enhanced glucosic cryoprotectant system. Alaskan frogs had a larger liver glycogen reserve that was mobilized faster during early freezing as compared to conspecifics from a cool-temperate region (southern Ohio, USA). In Alaskan frogs the rapid glucose production in the first hours of freezing was associated with a 7-fold increase in glycogen phosphorylase activity above unfrozen frog levels, and the activity of this enzyme was higher than that of frozen Ohioan frogs. Freezing of Ohioan frogs induced a more modest (4-fold) increase in glycogen phosphorylase activity above unfrozen frog values. Relative to the Ohioan frogs, Alaskan frogs maintained a higher total protein kinase A activity throughout an experimental freezing/thawing time course, and this may have potentiated glycogenolysis during early freezing. We found populational variation in the activity and protein level of protein kinase A which suggested that the Alaskan population had a more efficient form of this enzyme. Alaskan frogs modulated their glycogenolytic response by decreasing the activity of glycogen phosphorylase after cryoprotectant mobilization was well under way, thereby conserving their hepatic glycogen reserve. Ohioan frogs, however, sustained high glycogen phosphorylase activity until early thawing and consumed nearly all their liver glycogen. These unique hepatic responses of Alaskan R. sylvatica likely contribute to this phenotype’s exceptional freeze tolerance, which is necessary for their survival in a subarctic climate.

Hepatocyte responses to in vitro freezing and β‐adrenergic stimulation: Insights into the extreme freeze tolerance of subarctic Rana sylvatica

“…Extreme freeze tolerance in subarctic R. sylvatica derives from myriad adaptations including an enhanced cryoprotectant system (Costanzo et al, ; do Amaral et al, ; Costanzo et al, ; Rosendale et al, ). Our primary objective in the present study was to determine whether this enhanced tolerance is supported by a superior ability of hepatocytes to withstand freezing and rapidly produce glucose, such as occurs during the early hours of freezing.…”

“…At the onset of freezing, glycogenolysis in hepatocytes is heightened, rapidly generating copious amounts of glucose in both Alaskan and Ohioan frogs (do Amaral et al, ). Although the triggering mechanism of freezing‐induced glycogenolysis has not been elucidated, it likely involves β‐adrenergic stimulation of the liver (Storey and Storey, ), which accelerates glycogenolysis through an increased activation of specific enzymes involved in the process (Storey and Storey, ).…”

“…By contrast, frogs indigenous to the Midwestern United States and southern Canada tolerate experimental freezing only to temperatures as low as −4 to −6 °C (Storey and Storey, ; Costanzo and Lee, ). Extreme freeze tolerance in subarctic frogs derives, in part, from an ability to accrue extremely high levels of urea before hibernating and to rapidly mobilize large amounts of glucose during the early stages of freezing (Costanzo et al, ; do Amaral et al, ). Rapid production and distribution of cryoprotectant seems especially important in subarctic frogs, which presumably freeze quickly due to their diminutive size (Martof and Humphries, ) and the severe cold and high cooling rates that characterize their hibernal microhabitat (Larson et al, ).…”

Primeros registros de incremento en los niveles plasmáticos de glucosa en individuos adultos de Pleurodema thaul (Lesson, 1826) (Anura: Leptodactylidae) sometidos a estrés por frío

“…El mecanismo de producción de glucosa extracelular cómo agente crioprotector se activa cuando el anfi bio se enfrenta a condiciones de frío extremo y los individuos son capaces de producir cantidades masivas de glucosa a partir de reservas de glucógeno, en forma súbita frente a la formación de hielo en sus cuerpos (Hill et al 2006, do Amaral et al 2013. Este tipo de crioprotectores han sido reportados como los más recurrentes en anfi bios viviendo en altas latitudes del Hemisferio Norte (Storey & Storey 1996;Hillman et al 2009;) estudios que destacan la glucosa como molécula crioprotectora en al menos seis especies de anfi bios anuros: Rana sylvatica, Lithobates catesbeianus, Pseudacris triseriata, Acris crepitans, Pseudacris crucifer y Rhinella schneideri Layne et al 1996;Irwin et al 1999;Edwards et.al.…”

“…Este tipo de crioprotectores han sido reportados como los más recurrentes en anfi bios viviendo en altas latitudes del Hemisferio Norte (Storey & Storey 1996;Hillman et al 2009;) estudios que destacan la glucosa como molécula crioprotectora en al menos seis especies de anfi bios anuros: Rana sylvatica, Lithobates catesbeianus, Pseudacris triseriata, Acris crepitans, Pseudacris crucifer y Rhinella schneideri Layne et al 1996;Irwin et al 1999;Edwards et.al. 2000;Steiner et al 2000;Jenkins & Swanson 2005;do Amaral et al 2013;Rosendale et al 2014). Recientemente, Sanabria et al (2015) reportaron la respuesta fi siológica de sobreenfriamiento en Rhinella spinulosa en Argentina, indicando como posible mecanismo utilizado por la especie, el incremento en los niveles de glucosa plasmática, lo que no fue confi rmado experimentalmente.…”