Metabolic Correlates to Glycerol Biosynthesis in a Freeze-Avoiding Insect, Epiblema Scudderiana
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Journal of J Comp Physiol B (1989) 15:461 472 Comparative ands..~.,o,~"~ Environ- Physiology B --'PhySiologY @ Springer-Vertag 19139 Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect, Epiblema scudderiana Thomas A. Churchill and Kenneth B. Storey Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada Accepted May 31, 1989 Summary. The course of glycerol biosynthesis, ini- in autumn is one that obligately routes carbo- tiated by exposure to -4 ~ was monitored in hydrate flux through the hexose monophosphate larvae of the goldenrod gall moth, Epiblema scud- shunt. The consequence of this is that fermentative deriana, and accompanying changes in the levels ATP production during anoxia is linked to the ac- of intermediates of glycolysis, adenylates, glyco- cumulation of large amounts of glycerol as tl~e only gen, glucose, fructose-2,6-bisphosphate, and fer- means of maintaining redox balance. mentative end products were characterized. Pro- Key words: Cryoprotectant synthesis - Epiblema duction of cryoprotectant was initiated within 6 h scudderiana - Cold hardiness - Regulation of gly- after a switch from + 16 ~ to -4 ~ with half- colysis - Fructose-2,6-biphosphate maximal levels reached in 30 h and maximal con- tent, 450-500 gmol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at -4 ~ indi- Introduction cated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and gly- The last instar larvae of the gall moth, Epiblema cerol-3-phosphatase. A rapid increase in fructose- scudderiana (Clemens) (Lepidoptera, Olethreuti- 2,6-bisphosphate, an activator of phosphofructoki- dae), overwinter inside spindle-shaped galls on the nase and inhibitor of fructose-l,6-bisphosphatase, stems of goldenrod. The larvae utilize the freeze- appeared to have a role in maintaining flux in the avoidance strategy of cold hardiness. Autumn cold direction of glycerol biosynthesis. Analysis of me- hardening includes a depression of larval super- tabolite changes as glycerol production slowed sug- cooling point to - 38 ~ a decrease in body water gested that the inhibitory restriction of the regula- content, and an accumulation of glycerol to tory enzymes was slightly out of phase. Inhibition amounts that average 18.7% of fresh weight (over at the glycerol-3-phosphatase locus apparently oc- 2000 gmol.g wet weight -1) in mid-winter (Rick- curred first and resulted in a build-up of glycolytic ards et al. 1987). Larvae may also be shielded from intermediates and an overflow accumulation of inoculative freezing by environmental ice by a thin glucose. Glucose inhibition of phosphorylase, cocoon lining the gall cavity. stimulating the conversion of the active a to the Aspects of the biosynthesis of cryoprotectant inactive b forms, appears to be the mechanism that polyols have been investigated by numerous au- shuts off phosphorylase function, counteracting thors. There is general agreement that glycogen the effects of low temperature that are the basis is the substrate used, that glycogen mobilization of the initial enzyme activation. Equivalent experi- is frequently triggered by cold activation of glyco- ments carried out under a nitrogen gas atmosphere gen phosphorylase, that the hexose monophos- suggested that the metabolic make-up of the larvae phate shunt provides the reducing equivalents needed for synthesis, and that changes in el~tzyme Abbreviations. G6P glucose-6-phosphate; F6P fructose-6-phos- activities or regulatory properties can channe,1 car- phate; F1,6P2 fructose-l,6-bisphosphate; F2,6P2 fructose-2,6- bon flux into the appropriate polyol product (for bisphosphate; G3P glycerol-3-phosphate; DHAP dihydroxya- cetonephosphate; GAP glyceraldehyde-3-phosphate; PEP review see Storey and Storey 1988). Questions re- phosphoenolpyruvate; PFK phosphofructokinase; FBPase main, however, about the control of the process fructose-1,6-bisphosphatase; PK pyruvate kinase and the energetics of polyol synthesis. 462 T.A. Churchill and K.B. Storey: Regulation of glycerol synthesis The present experiments were designed to take Results a close look at the initiation of glycerol synthesis Aerobic time course in response to cold exposure. The pattern of chan- ges in glycolytic intermediates and end products Upon exposure to -4 ~ E. scudderiana larvae as well as adenylates was analyzed to provide in- rapidly initiated glycerol synthesis; after a lag time formation on the pathway of glycerol synthesis, of 2 h, levels rose over 4 days to 450 gmol/g wet the regulatory enzymes involved, carbon balance, weight (control=46-t-8 gmol.g -1) (Fig. 1). The and the energetics of the process. The stress of rate of synthesis was highest over the interval be- anoxia was also employed to gain further insights tween 2 and 12 h of cold exposure, at 9.6 gmol into the patterns of carbon flux and the energetic g- ~. h-*, achieving half-maximal concentrations requirements of polyol synthesis. of glycerol within about 30 h. Glycogen content of the larvae was quite variable, but by 3648 h Materials and methods it had dropped to 50%-60% of the control level, a net decrease sufficient to account for the carbon Chemicals and animals. All chemicals and biochemicals were needs of glycerol synthesis. Glycerol was the only purchased from Sigma Chemical Co., St. Louis, MO or Boehr- polyol produced; sorbitol did not accumulate (lev- inger Mannheim Co., Montreal, PQ. Galls containing E. scud- deriana larvae were collected in Ottawa, Canada, in mid-Sep- els less than 0.5 gmol-g-t; data not shown) even tember 1987 and held in the laboratory for 2.5 weeks at when glucose content peaked at high values. 16~ 1 ~ prior to experimentation. Larvae were removed from Changes in the activity state of regulatory en- their galls and placed in either covered petri dishes or sealed zyme(s) in a pathway alter the distribution of car- Erlenmeyer flasks for the aerobic versus anoxic experiments, respectively. bon between substrates and products of these loci. For example, enzyme activation typically produces Animal experimentation. Experiments were designed to monitor a decrease in substrate content and an increase in the metabolic events accompanying the activation of glycerol product content as flux through the locus is acti- synthesis by low temperature exposure. Larvae, previously ac- vated; these changes are most pronounced over climated to 16 ~ were moved to a second incubator at -4~ 1 ~ and were sampled at set time intervals of up to the transition time while the pathway flux changes 14 days. At each sampling time, larvae were rapidly removed from one steady state to another. Such changes from the incubator, frozen in liquid nitrogen, and then trans- in substrate and product contents in a pathway ferred to -80 ~ for storage. Experiments were carried out can be used to diagnose enzyme activation or inhi- under either aerobic or anoxic conditions. For the anoxic treat- ments, Erlenmeyer flasks containing larvae were flushed with bition in response to an external trigger/stress and a mixture of 95% N2/5% CO2 for 30rain and then sealed to identify the rate-controlling enzyme in a path- and transferred to the -4 ~ incubator. way (Williamson 1970). When applied to the glyco- lytic pathway during the active synthesis of gly- Sample preparation and metabolite assay. Samples of 6 frozen cerol by E. scudderiana, this metabolic control larvae were pooled, ground to a powder using a mortar and pestle cooled in liquid nitrogen, and then weighed and extracted theory can help to pinpoint the control points and 1:5 w/v in 6% perchloric acid (containing 1 mM EDTA) the sequence of activation/inhibition of these loci (Storey et al. 1981); an aliquot of the well-mixed homogenate over the time course of glycerol biosynthesis. For was removed for glycogen determination (Keppler and Decker aerobic larvae exposed to -4 ~ the course of 1974). Precipitated protein was removed by centrifugation and extracts were neutralized by the addition of 3 N KOH/0.4 M glycerol biosynthesis can be broken into four dis- TRIS/0.3 M KC1 and recentrifuged. Aliquots of neutralized ex- tinct time periods. tract were immediately used for assays of pyruvate, ATP, ADP, and AMP assays; the remaining extract was frozen at -80 ~ 0 to 2 h. Cold exposure resulted in distinct changes for subsequent use. Metabolites were assayed enzymatically us- in the contents of glycolytic intermediates and re- ing a Pye Unicam SP8-100 spectrophotometer. Assays used were: glycerol (Eggstein and Kuhlman 1974), sorbitol (Berg- lated metabolites in E. scudderiana. Within the first meyer et al. 1974), and all other metabolites (Lowry and Pas- 2 h the content of G3P, the immediate precursor SOlmeau 1972). of glycerol, had decreased significantly (P<0.05) F2,6P2 content was assayed in alkaline extracts of individ- by 50% (Fig. 1), suggesting an increased flux ual larvae (1:20 w/v in 50 M NaOH) as described by van through glycerol-3-phosphatase. Over the same Schaftingen (1984). Extraction and assay of glycogen phosphorylase was as time G6P content rose by 4.5-fold. F6P content in Storey and Storey (1984) with the exception that enzyme was assayed, but in all cases levels were below the activity was measured in the well-suspended homogenate. limits of detection (less than 0.05 gmol. g- ~). How- Data are reported as means _+ SEM for n = 4 samples with ever, levels of the product of PFK, F1,6P2, showed 6 larvae pooled per sample (or 1 larva per sample for F2,6P;). a significant rise of 60%, and along with a rise Statistically significant differences, where reported, were per- formed using the Student's t-test with a significance level of in GAP, this suggests an increase in flux through P<0.05. the PFK locus to provide triose phosphates for T.A.