Journal of Comparative Physiology B

, Volume 159, Issue 4, pp 461–472 | Cite as

Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

  • Thomas A. Churchill
  • Kenneth B. Storey
Article

Summary

The course of glycerol biosynthesis, initiated by exposure to −4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to −4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 μmol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at −4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.

Key words

Cryoprotectant synthesis Epiblema scudderiana Cold hardiness Regulation of glycolysis Fructose-2,6-biphosphate 

Abbreviations

G6P

glucose-6-phosphate

F6P

fructose-6-phosphate

F1, 6P

fructose-1,6-bisphosphate

F2,6P2

fructose-2,6-bisphosphate

G3P

grycerol-3-phosphate

DHAP

dinydroxyacetonephosphate

GAP

glyceraldehyde-3-phosphate

PEP

phosphoenolpyruvate

PFK

phosphofructokinase

FBPase

fructose-1,6-bisphosphatase

PK

pyruvate kinase

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Atkinson DE (1977) Cellular energy metabolism and its regulation. Academic Press, New YorkGoogle Scholar
  2. Bergmeyer HU, Gruber W, Gutman I (1974) D-Sorbitol. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic Press, New York, pp 1323–1326Google Scholar
  3. Churchill TA, Storey KB (1989) Seasonal variation in the temperature-stimulated interconversion of glycogen and glycerol pools in a freeze avoiding moth larva. Cryo Lett 10:127–136Google Scholar
  4. Eggstein M, Kuhlmann E (1974) Triglycerides and glycerol: determination after alkaline hydrolysis. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic Press, New York, pp 1825–1831Google Scholar
  5. Gäde G (1984) Anaerobic energy metabolism. In: Hoffmann KH (ed) Environmental physiology and biochemistry of insects. Springer, Berlin Heidelberg New York, pp 119–136Google Scholar
  6. Hayakawa Y (1985) Activation mechanism of insect fat body phosphorylase by cold. Insect Biochem 15:123–128Google Scholar
  7. Hayakawa Y, Chino H (1982) Temperature-dependent activation or inactivation of glycogen phosphorylase and synthase of fat body of the silkwormPhilosamia cynthia: the possible mechanism of the temperature-dependent interconversion between glycogen and trehalose. Insect Biochem 12:361–366Google Scholar
  8. Hers H-G (1976) The control of glycogen metabolism in the liver. Annu Rev Biochem 45:167–189Google Scholar
  9. Hue L, Rider MH (1987) Role of fructose-2,6-bisphosphate in the control of glycolysis in mammalian tissues. Biochem J 245:313–324Google Scholar
  10. Kellher MJ, Rickards J, Storey KB (1987) Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: laboratory investigations of temperature cues in the regulation of cold hardiness. J Insect Physiol 33:581–586Google Scholar
  11. Keppler D, Decker K (1974) Glycogen: determination with amyloglucosidase. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic Press, New York, pp 1127–1131Google Scholar
  12. Lowry OH, Passonneau JV (1972) A flexible system of enzymatic analysis. Academic Press, New YorkGoogle Scholar
  13. Meyer SGE (1978) Effects of heat, cold, anaerobiosis and inhibitors on metabolite concentrations in larvae ofCallitroga macellaria. Insect Biochem 6:471–477Google Scholar
  14. Rickards J, Kelleber MJ, Storey KB (1987) Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: winter profiles of a natural population. J Insect Physiol 33:443–450Google Scholar
  15. Schaftingen E van (1984) d-Fructose-2,6-bisphosphate. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Verlag Chemie, Weinheim, pp 335–341Google Scholar
  16. Sonobe H, Matsumoto A, Fukuzaki Y, Fujiwara S (1979) Carbohydrate metabolism and restricted oxygen supply in the eggs of the silkwormBombyx mori. J Insect Physiol 25:381–388Google Scholar
  17. Storey JM, Storey KB (1983) Regulation of cryoprotectant metabolism in the overwintering gall fly larva,Eurosta solidaginis: temperature control of glycerol and sorbitol levels. J Comp Physiol 149:495–502Google Scholar
  18. Storey KB, Storey JM (1984) Biochemical adaptation for freezing tolerance in the wood frog,Rama sylvatica. J Comp Physiol 155:29–36Google Scholar
  19. Storey KB, Storey JM (1988) Freeze tolerance in animals. Physiol Rev 68:27–84Google Scholar
  20. Storey JM, Storey KB (in press) Energetics and carbon balance of polyol synthesis inEurosta solidaginis: responses of the system to perturbation by anoxia stress. J Comp Physiol BGoogle Scholar
  21. Storey KB, Baust JG, Storey JM (1981) Intermeadiary metabolism during low temperature acclimation in the overwintering gall fly larva,Eurosta solidaginis. J Comp Physiol B 144:183–190Google Scholar
  22. Tsumuki H, Rojas RR, Storey KB, Baust JG (1987) The fate of [14C]glucose during cold-hardening inEurosta solidaginis (Fitch). Insect Biochem 17:347–352Google Scholar
  23. Williamson JR (1970) General features of metabolic control as applied to the erythrocyte. Adv Biol Med 6:117–136Google Scholar
  24. Wood FE, Nordin JH (1980) Activation of the hexose monophosphate shunt during cold-induced glycerol accumulation byProtophormia terranovae. Insect Biochem 10:87–93Google Scholar
  25. Ziegler R, Ashida M, Fallon AM, Wimer LT, Silver Wyatt S, Wyatt GR (1979) Regulation of glycogen phosphorylase in fat body ofCecropia silkmoth pupae. J Comp Physiol 131:321–332Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Thomas A. Churchill
    • 1
  • Kenneth B. Storey
    • 1
  1. 1.Institute of Biochemistry and Department of BiologyCarleton UniversityOttawaCanada

Personalised recommendations