Electrophoretic analysis of liver glycogen phosphorylase activation in the freeze-tolerant wood frog

Crerar, Michael M.; David, Emina S.; Storey, Kenneth B.

doi:10.1016/s0005-2728(88)80010-0

Cited by 9 publications

(13 citation statements)

References 23 publications

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“…Plasma and tissue glucose can rise as high as 200-300mmoll -1 , compared with normal levels of ~5mmoll -1 . This uncharacteristically high breakdown of glycogen and production of glucose requires selective regulation of the enzymes controlling glycogen metabolism (Crerar et al, 1988;Mommsen and Storey, 1992;Russell and Storey, 1995). In particular an 'override' control on normal glucose homeostasis would need to be present to prevent the activation of glycogenesis that is normally triggered when glucose rises above about 10mmoll -1 .…”

Section: Introductionmentioning

confidence: 99%

Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog

Dieni

Bouffard

Storey

2012

Journal of Experimental Biology

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SUMMARYThe terrestrial anuran Rana sylvatica tolerates extended periods of whole-body freezing during the winter. Freezing survival is facilitated by extensive glycogen hydrolysis and distribution of high concentrations of the cryoprotectant glucose into blood and all tissues. As glycogenesis is both an energy-expensive process and counter-productive to maintaining sustained high cryoprotectant levels, we proposed that glycogen synthase kinase-3 (GSK-3) would be activated when wood frogs froze and would phosphorylate its downstream substrates to inactivate glycogen synthesis. Western blot analysis determined that the amount of phosphorylated (inactive) GSK-3 decreased in all five tissues tested in 24h frozen frogs compared with unfrozen controls. Total GSK-3 protein levels did not change, with the exception of heart GSK-3, indicating that post-translational modification was the primary regulatory mechanism for this kinase. Kinetic properties of skeletal muscle GSK-3 from control and frozen frogs displayed differential responses to a temperature change (22 versus 4°C) and high glucose. For example, when assayed at 4°C, the K m for the GSK-3 substrate peptide was ~44% lower for frozen frogs than the corresponding value in control frogs, indicating greater GSK-3 affinity for its substrates in the frozen state. This indicates that at temperatures similar to the environment encountered by frogs, GSK-3 in frozen frogs will phosphorylate its downstream targets more readily than in unfrozen controls. GSK-3 from skeletal muscle of control frogs was also allosterically regulated. AMP and phosphoenolpyruvate activated GSK-3 whereas inhibitors included glucose, glucose 6-phosphate, pyruvate, ATP, glutamate, glutamine, glycerol, NH 4 Cl, NaCl and KCl. The combination of phosphorylation and allosteric control argues for a regulatory role of GSK-3 in inactivating glycogenesis to preserve high glucose cryoprotectant levels throughout each freezing bout.

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Section: Introductionmentioning

confidence: 99%

Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog

Dieni

Bouffard

Storey

2012

Journal of Experimental Biology

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“…Reversible phosphorylation is already known to participate in biochemical regulation during freezing, studies to date having focused mainly on enzymes of carbohydrate metabolism. Enzymes controlled in this manner include glycogen phosphorylase, phosphofructokinase-1 and phosphofructokinase-2 in wood frogs (Crerar et al, 1988;Vazquez Illanes and Storey, 1993), pyruvate kinase in the marine periwinkle,…”

Section: Reversible Protein Phosphorylationmentioning

confidence: 99%

“…As a consequence of the interruption of breathing and heart beat, the frogs experience anoxia and ischemia throughout the freeze followed by oxidative stress when oxygen is reperfused during thawing (Storey and Storey, 2004b;2004c). Wood frogs use glucose as their natural cryoprotectant; levels as high as 200-300 mM accumulate in core organs (control values are ~5 mM) as a result of glucose synthesis from liver glycogen (Storey and Storey, 1985;Storey, 1987a, Crerar et al, 1988.…”

Section: Model Organism: the Freeze-tolerant Wood Frog Rana Sylvaticamentioning

confidence: 99%

“…Winter survival for a large number of terrestrially-hibernating ectothermic organisms relies on freeze tolerance, the ability to endure the conversion of 65-70% of total body water into extracellular ice. To survive, animals strongly suppress their energyconsuming physiological needs and undergo a series of metabolic changes that provide cryoprotection (Crerar et al, 1988Costanzo et al,. 1993King et al, 1993;Swanson et al, 1996;Layne et al, 1989;Storey and Storey, 2004b).…”

Section: Introductionmentioning

confidence: 99%

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Regulation of enzyme function in freeze-tolerance

Dieni¹

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“…It is not surprising, then, to note that RPP of enzymes has been repeatedly demonstrated to play a significant role in the response to environmental stress. For example, in the freeze tolerant wood frog, RPP mediates the activities of enzymes involved in glucose metabolism, including GP, glucose-6-posphate dehydrogenase (G6PDH), and hexokinase (HK), to conserve high levels of cryoprotectants and enable survival of freezing (Crerar et al, 1988;Dieni & Storey, 2008;Dieni & Storey, 2010). Antioxidant defenses such as catalase (CAT) and superoxide dismutase (SOD) are also regulated by RPP to increase free-radical scavenging capacity during freezing (Dawson & Storey, 2016, Dawson et al, 2015, and even protein kinases themselves, which can act on the various proteins requiring phosphorylation during freezing, are regulated in this manner (Dieni & Storey, 2014).…”

Section: Reversible Post-translational Modificationsmentioning

confidence: 99%

Regulation of pyruvate kinase in hypometabolic states

Smolinski¹

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Survival in extreme environments requires drastic changes to animals' physiology and biochemistry, requiring many animals to decrease their metabolic rate. Enzymes are the mediators of metabolic functions and their regulation is critical to surviving these conditions. This thesis explores the properties of a key enzyme of glycolysis, pyruvate kinase (PK), in the freeze tolerant wood frog (Rana sylvatica), the anoxia tolerant snail (Littorina littorea) and the hibernating ground squirrel (Urocitellus richardsonii).The studies showed that temperature, allosteric effectors and other local environmental conditions play a role in regulating pyruvate kinase in response to freezing, anoxia and during hibernation. Furthermore, reversible protein phosphorylation appears to be a common regulatory mechanism of PK by reducing the activity of the enzyme in all three animals. Together, these studies show that PK is a common regulatory target of animals surviving in extreme conditions and that multiple regulatory mechanisms, especially protein phosphorylation, contribute to its regulation. iv

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Electrophoretic analysis of liver glycogen phosphorylase activation in the freeze-tolerant wood frog

Cited by 9 publications

References 23 publications

Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog

Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog

Regulation of enzyme function in freeze-tolerance

Regulation of pyruvate kinase in hypometabolic states

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