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Adjustments of the enzymatic complement for polyol biosynthesis and accumulation in diapausing cold-acclimated adults of Pyrrhocoris apterus
Journal of Insect Physiology 50 (2004) 303–313 www.elsevier.com/locate/jinsphys
Adjustments of the enzymatic complement for polyol biosynthesis and accumulation in diapausing cold-acclimated adults of Pyrrhocoris apterus V. Kosˇta´l a,, M. Tollarova´ b, J. Sˇula a a
Institute of Entomology, Academy of Sciences of the Czech Republic, Branisˇovska´ 31, 370 05 Cˇeske´ Budeˇjovice, Czech Republic b Faculty of Biological Sciences, University of South Bohemia, Cˇeske´ Budeˇjovice, Czech Republic Received 17 October 2003; received in revised form 8 January 2004; accepted 8 January 2004
Abstract The capacity to accumulate winter polyols (mainly ribitol and sorbitol) during cold-acclimation in Pyrrhocoris apterus is restricted only to the adults that have previously entered diapause. The enzymatic complement involved in polyol biosynthesis was found to differ in a complex manner between diapause and non-diapause adults. Nearly 100% of glycogen phosphorylase (GPase) was present in its active form in non-diapause adults irrespective of their acclimation status. In contrast, less than 40% of GPase was present in its active form in diapause adults prior to cold-acclimation and the inactive form was rapidly activated upon tranv sition from 5 to 0 C, concomitantly with the start of rapid polyol accumulation. The flow of carbon released by activation of glycogen degradation might be routed to the pentose cycle because the activity of glucose-6-P dehydrogenase (G6P-DH) was significantly higher and it increased with cold-acclimation in diapause adults while it was relatively low and it decreased with cold-acclimation in non-diapause adults. Reducing equivalents in the form of NADPH, which were generated in the pentose cycle, might require re-oxidation. Such re-oxidation might be achieved during reduction of sugars to polyols. The activity of NADP(H)-dependent aldose reductase (AR) was about 20-fold higher in diapause than in non-diapause adults. Similarly, the activity of NAD(H)-dependent polyol dehydrogenase (PDH) was higher in diapause adults. In addition, we found a very high activity of an unusual enzyme, NADP(H)-dependent ketose reductase (KR), exclusively in diapause adults. KR might be involved in reduction of fructose to sorbitol. Although its affinity for fructose as a substrate was low (KM ¼ 0:64 M), its activity was about 10-fold higher than that of PDH with fructose. Moreover, the activity of KR significantly increased with cold-acclimation while that of PDH remained unchanged. Different electrophoretic mobilities in PAGE gel suggested that KR and PDH are two different enzymes with specific requirement for NADP(H) or NAD(H), respectively, as co-factors. # 2004 Elsevier Ltd. All rights reserved. Keywords: Diapause; Cold tolerance; Cryoprotectans; Ribitol; Sorbitol; Glycolysis; Pentose cycle; Ketose reductase
1. Introduction The conversion of glycogen into sugar alcohols (polyols) during insect diapause or overwintering was first reported in the late 1950s by several authors (Salt, 1957; Chino, 1957; Wyatt and Meyer, 1959). Slightly later, it became apparent that other diapausing insects prefer to accumulate trehalose (Asahina and Tanno, 1964). Since then, accumulation of different polyols or Corresponding author. Tel.: +420-387-775-229; fax: +420-385300-354. E-mail address: [email protected] (V. Kosˇta´l).
0022-1910/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.01.006
sugars has been confirmed in a large number of diapausing and/or overwintering insects and several different mechanisms of cryoprotection conferred by these compounds were proved or suggested (reviews: Asahina, 1969; So¨mme, 1982, 1999; Zachariassen, 1985; Storey and Storey, 1991). The exact nature of mutual relationships between diapause induction, cold-acclimation, polyol accumulation and cold hardiness is largerly unknown because the topics were often studied separately. The available information was reviewed by Denlinger (1991); Lee (1991); Storey and Storey (1991) and Pullin (1996). Interconversion between glycogen and sugars/polyols is usually temperature-dependent.
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Although in some cases the induction of diapause at relatively high temperature may serve as a sufficient trigger for accumulation of polyols [typically in diapausing embryos of Bombyx mori (Chino, 1957) or in diapausing tropical beetle Stenotarsus rotundus (Pullin and Wolda, 1993)], the process mostly starts only at v temperatures below 15 C for glycerol (Morrissey and Baust, 1976; Storey and Storey, 1983; Renault et al., v 2002) or below 5 C for the other compounds so far studied (Storey and Storey, 1991). Concerted changes in the enzymatic complement of glycolysis and pentose cycle regulate diversion of the carbon flow from the main stream of glycolysis (toward TCA cycle and energy metabolism) to the biosynthesis and accumulation of sugars and polyols (Ziegler and Wyatt, 1975; Hayakawa and Chino, 1981, 1982a,b; Storey and Storey, 1981; Tsumuki et al., 1987). Not only the abundancies of certain enzymes may change seasonally or in response to temperature acclimation but numerous regulatory mechanisms are involved: changing the substrate affinities (KM values) and pH optima with temperature; reversible phosphorylation; changing the concentrations of various modulators and inhibitors (review: Storey and Storey, 1991). Adults of the brachypterous form of the red firebug, Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae), enter a facultative reproductive diapause in response to short-days during the second half of summer in South Bohemia (Czech Republic) (Hodek, 1968, 1983). During relatively warm autumn, the bugs prepare for overwintering, which takes place in the upper litter layer. Overwintering bugs do not tolerate freezing of their v body fluids, supercool to ca. 17 C (with a high indiv vidual variation spanning from 12 to 23 C), and v can survive for approximately two weeks at 15 C (Kosˇta´l and Sˇimek, 2000). When exposed at temperav tures below 5 C, they start to accumulate four specific ‘‘winter polyols’’ (ribitol, sorbitol, arabinitol and mannitol), which probably function as non-colligative cryoprotectants (Kosˇta´l and Sˇlachta, 2001; Kosˇta´l et al., 2001). No capacity for accumulation of winter polyols, however, was detected in the non-diapause, reproducing, adults of P. apterus (Sˇlachta et al., 2002). Thus, the main objective of this study was to contribute to the understanding of physiological mechanisms which allow accumulation of polyols specifically in diapause insects. In this paper, we show that the fat body enzymatic complements differ in several aspects between the diapause and non-diapause adults of P. apterus. The differences were found in: (1) relative proportions of active and inactive forms of glycogen phosphorylase (GPase) and activation of the inactive form by low temperature; (2) activities of glucose-6-P dehydrogenase (G6P-DH), aldose reductase (AR) and polyol dehydrogenase (PDH); (3) presence or absence of
NADP(H)-dependent ketose reductase (KR) activity; (4) direction of changes in the activities of G6P-DH, AR, PDH and KR in response to cold acclimation. The way that such differences could be involved in the accumulation of polyols in diapause insects is discussed.
2. Materials and methods 2.1. Insect rearing and acclimation protocols Adult P. apterus bugs were collected during May 2002 in a field near Chelcˇice, South Bohemia, Czech Republic. Offspring of the field-collected adults were reared under conditions which promote continuous non-diapause development, i.e. long-day photoperiod v (LD) of 18L:6D and a constant temperature of 25 C. The dry seeds of the linden tree (Tilia parviflora Ehrh.) as a food source and water were provided ad libitum. Adults in reproductive diapause were obtained by rearing all larval instars under short-day photoperiod (SD) v of 12L:12D and a constant temperature of 25 C (Hodek, 1968). Only females belonging to the third and fourth laboratory generations were used for analyses. Diapause development can be assessed easily in females (Hodek, 1968, 1983) and no significant differences were found between the two sexes in their ability to accumulate polyols (Kosˇta´l et al., 2001; Sˇlachta et al., 2002) and in the activities of enzymes under study (Kosˇta´l, unpublished results). The young experimental adults were kept under the photoperiods at which they developed and at a conv stant temperature of 20 C for 2 weeks. During this time, non-diapause adults mated and females started to lay eggs or diapause intensified in diapause adults, respectively. After this initial period, at the adult age of 2 weeks, the bugs were subjected to gradual cold-acclimation as depicted in Fig. 1. Alternating temperatures were applied during first two weeks v v v v (thermophase/cryophase: 20 /10 C and 15 /5 C, respectively). Photoperiodic regime (either LD or SD) was maintained during these two weeks. Our earlier results showed that gradual acclimation at alternating temperatures (in comparison to constant temperatures) leads to higher level of cold-hardiness in P. apterus (Kosˇta´l et al., 2001). Later, the insects were kept at a v constant temperature of 5 C/DD (maximum shortv time fluctuations were from 4.6 to 5.8 C) for 3 weeks. v The temperature of 5 C was selected on purpose, because the threshold for rapid polyol accumulation in v P. apterus was shown to be approximately 5 C (Kosˇta´l et al., 2001). Finally, the bugs were transferred to v 0 C/DD for 1 week.
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were measured in 70% ethanol extracts of the whole body samples (one individual was used per each sample). The bug was weighed, submerged in 400 lL of 70% ethanol, cut to pieces, thoroughly homogenized and extracted at room temperature for 5 min with vigorous shaking. The extracts were centrifuged at 20,000 v g/35 C/10 min and the pooled supernatants from two replications of the procedure were used for o-methyloxime trimethylsilyl derivatization and subsequent analysis by gas chromatography coupled to mass spectrometry (Kosˇta´l and Sˇimek, 1995). All chemicals used for analysis of glycogen and polyols were purchased from Sigma-Aldrich Co.
Fig. 1. Schematic view of the protocol of cold-acclimation for the adults of Pyrrhocoris apterus (see Materials and methods for detailed description). The non-diapause and diapause insects were acclimated equally. Samples for analyses of glycogen content (Gly), polyol concentrations (Poly) and enzymatic activities (Enz) were taken at different time-phases of cold-acclimation as indicated by arrows. The v temperature threshold of 5 C, below which rapid accumulation of polyols starts, is indicated by a dashed arrow.
2.2. Glycogen and polyols The content of glycogen in the abdominal fat body was measured colorimetrically as glucose released by the phenolic method (Dubois et al., 1956). Fat bodies were dissected under Ringer’s solution. Approximately 80–90% of the abdominal fat body tissue was taken from each female and analyzed as one sample. The tissue was homogenized using Dounce homogenizer, extracted twice in 400 lL of 70% ethanol and centrifuged. After decanting the ethanol, the sample was dissolved in 200 lL of water and boiled for 30 min in 1.1 mL of 30% KOH solution. After cooling and spinning, 500 lL of supernatant was taken for further analysis. Glycogen was precipitaed by adding of 1.5 mL 99.8% ethanol, 250 lL of 10% Na2SO4, keeping the sample on ice for 1 h and spinning. The pellet was re-extracted twice with 70% ethanol and finally dissolved in 1 mL of water. Acidic hydrolysis was achieved by adding 500 lL of water, 200 lL of 5% phenol solution and 1 mL of concentrated H2SO4 to v the 100 lL sample aliquot and keeping at 95 C for 20 min. After 30 min at room temperature, the absorbance of 490 nm light was read and compared to the calibration curve obtained by analysis of (oyster) glycogen standard. Blank samples (no tissue) were run in parallel and subtracted. The concentrations of the four ‘winter’ polyols (arabinitol, ribitol, mannitol and sorbitol), that typically accumulate in diapausing cold-acclimated P. apterus adults (Kosˇta´l and Sˇimek, 2000; Kosˇta´l et al., 2001),
2.3. Enzyme activities Slightly modified methods of Storey and Bailey (1978); Storey and Storey (1981) and Joanisse and Storey (1994) were used. Abdominal fat body was quickly dissected (ca. 3 min) under Ringer solution and submerged in liquid nitrogen immediately. After accumulation of 8–10 fat bodies (pooled to one sample), the nitrogen was evaporated and 600 lL (approximately 8 vol) of homogenization buffer (HB) was added. HB consisted of 100 mM Tris-HCl, pH 8.0, 15 mM mercaptoethanol and 1 mM EDTA. The sample was homogenized for 30 s, while increasing the speed from 10 000–30 000 rpm, using CAT Homogenizer X120 with metal blades (Ingenieurbu¨ro CAT, M. Zipperer GmbH, Germany). After centrifugation at 22 000 v g/20 min/20 C, the supernatant was used as the source of all enzymes. Total protein concentrations in the enzyme preparations were measured by the BCA protein assay (Stoscheck, 1990), and activities were expressed as lmoles of substrate converted to product per min per g of protein. The final activity values were calculated after subtracting blank values. Activities v were measured at 25 C using Pye Unicam SP8-100 spectrophotometer by continuous time scanning at 340 nm. All chemicals and coupling enzymes were purchased from Sigma-Aldrich Co. Glycogen phosphorylase (GPase; E.C. 2.4.1.1) (total aþb). 50 mM potassium phosphate buffer (pH 6.8), 5 mg/mL glycogen (omitted from control), 5 lM glucose-1,6-P2, 0.6 mM NADP+, 2 mM 5’AMP, 15 mM MgCl2, 2 units/mL phosphoglucomutase and 2 units/ mL glucose-6-phosphate dehydrogenase (NADP+ dependent). The active form of the enzyme (a) was measured in the absence of AMP. Phosphofruktokinase-1 (PFK-1; E.C. 2.7.1.11). 20 mM imidazole-HCl buffer (pH 7.2), 10 mM fructose-6phosphate (omitted from control), 2 mM ATP, 0.15 mM NADH, 50 mM KCl, 5 mM MgSO4, 2 units/mL aldolase, 37.5 units/mL triosephosphate isomerase and 4 units/mL glycerol-3-phosphate dehydrogenase.
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Glucose-6-phosphate dehydrogenase (G6P-DH; E.C. 1.1.1.49). 20 mM imidazole-HCl buffer (pH 7.2), 5 mM MgSO4, 1 mM glucose-6-phosphate (omitted from control), 0.6 mM NADP+. Aldose reductase (AR, 1.1.1.21), glucose or ribose activities. 20 mM imidazole-HCl buffer (pH 7.2), 250 mM D-glucose or D-ribose, respectively, (omitted from control), 0.1 mM NADPH. Polyol dehydrogenases (PDH). 20 mM imidazole-HCl buffer (pH 7.2), 250 mM D-fructose (sorbitol dehydrogenase; E.C. 1.1.1.14) or D-ribulose (ribitol dehydrogenase E.C. 1.1.1.56) (the substrates were omitted from controls), 0.15 mM NADH. Ketose reductase (KR; E.C. 1.1.1.?). 20 mM imidazole-HCl buffer (pH 7.2), 250 mM D-fructose (omitted from control), 0.15 mM NADPH.
2.4. Enzyme kinetics and electrophoretic mobility in the native PAGE gel Kinetics and mobilities of selected enzymes were compared in the fat body samples taken from 2-weekold P. apterus adults (without any cold-acclimation) differing in developmental mode, i.e. diapause vs. nondiapause. Michaelis-Menten constants (KM) and maximum velocities of enzymatic reactions (Vmax) were v measured at 25 C in four enzymes with their specific subtrates: G6PDH with glucose-6-phosphate, AR with ribose, PDH with fructose and KR with fructose. KM and Vmax values were determined by non-linear regression analysis of the dependence of activity on substrate concentration using the GraphPad Prism 4 software. Crude enzyme preparations (see above) were loaded onto vertical 4–16% gradient polyacrylamide slab gels (PAGE) prepared according to Williams and Reisfeld (1964) and electrophoresed at 150 V for 80 min. After incubation of the gels in reaction mixtures containing specific substrates and co-factors, the gel areas containing specific enzymes separated during electrophoresis were visualized using the chromogenic reaction of reduced NADH or NADPH with tetrazolium salts, which results in the formation of intensely coloured precipitate, formazan. The methods were as described in Manchenko (2003). Phenazine methosulfate (PMS) was used as an intermediary catalyst. Methyl thiazolyl tetrazolium (MTT) was used to visualize G6P-DH and nitro blue tetrazolium (NBT) was used to visualize PDH and KR. Chemicals were purchased from Sigma-Aldrich Co.
3. Results 3.1. Changes of glycogen content and accumulation of polyols No statistically significant changes were found in the content of fat body glycogen in diapause bugs during cold-acclimation by ANOVA (P ¼ 0:0757) (Fig. 2). This was caused at least partly by a high variation of glycogen content among individual females. Our results indicate that glycogen was not spent during the first part of acclimation, which consisted of a gradual and v thermoperiodic drop of temperature to 5 C. After 3 v weeks at constant 5 C, glycogen content decreased from 512:0 123:1 lg/fat body (mean S:D:, n ¼ 10) to 417:8 164:5 lg (n ¼ 8). Further decrease to 310:9 115:9 (n ¼ 8) was observed after 1 week at conv stant 0 C. In non-diapause adult females, the content of glycogen in abdominal fat body was much lower (ranging between 64.7–92.3 lm/fat body) than in diapause females and its level was maintained relatively constant during cold-acclimation (ANOVA, P ¼ 0:6444) (Fig. 2). Two of the four winter polyols, ribitol and arabinitol, started to accumulate already during the exposure v to constant 5 C. The accumulation was more apparent in ribitol because its levels were approximately 15-fold higher than those of arabinitol (Fig. 2). Significant increases of concentrations were observed at constant v 0 C in all four polyols. Considering 60 mg to represent the average body weight of one female (Kosˇta´l, unpublished data), the total amount of accumulated winter polyols would be approximately 380 lg/female at the end of cold-acclimation. The hydration (proportion of water in the fresh weight) of the whole body in an average diapause cold-acclimated female is 55%
Fig. 2. Changes of glycogen contents (left y axis) and polyol concentrations (right y axis) during the process of cold-acclimation in Pyrrhocoris apterus adults. Glycogen is shown for non-diapause and diapause insects. Four polyols are shown for diapause insects only. Each data point represents mean S:E: of n ¼ 8 10 samples taken from individual females. (FW, fresh weight).
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(Kosˇta´l, unpublished data). It can be deduced from these data that the average sum concentration of winter polyols in the body fluids would reach about 70 mM (380 lg in 33 lL of water). Of course, this is a rough estimation, which supposes that the polyols are evenly distributed in different compartments. Nevertheless, such a value corresponds well to the concentrations that were previously measured directly in haemolymph (up to 100 mM depending on acclimation status; Kosˇta´l et al., 2001). No polyol accumulation was observed in non-diapause females. The concentrations never exceeded 0.01 lg/mg (data not shown).
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they decreased or remained stable in non-diapause bugs. The activity of an unusual enzyme, ketose reductase (KR), was observed exclusively in the diapause specimens. This enzyme required NADPH for conversion of fructose to sorbitol. The activity of KR in reverse direction of the reaction (250 mM sorbitol ! fructose, with 0.15 mM NADP+ as a co-factor) was approximately 20% of that in the direction of fructose reduction (Kosˇta´l, unpublished data). The activity of KR was very high in comparison to the activities of other enzymes, and it further increased in response to cold-acclimation.
3.2. Enzyme activities 3.3. Enzyme kinetics and electrophoretic mobilities Results of all assays are summarized in Tables 1 and 2. Activities of GPase (total) and PFK-1 were similar in non-diapause and diapause specimens. As much as 96.2% of total GPase activity was found to be present in active form (a) in non-diapause specimens. In contrast, diapause bugs had only 39.3% of GPase in the a form prior to the start of cold-acclimation and 48.8% v after the acclimation to 5 C. However, after the transv fer to 0 C, the proportion of a form markedly increased to 85.2% within one day. Profound differences were detected between the nondiapause and diapause specimens in the activities of G6P-DH, AR, and PDH. First, the activities were significantly higher in diapause than in non-diapause bugs. Second, the activities tended to increase in response to cold-acclimation in diapause bugs while
The calculated KM values of G6P-DH did not differ statistically and the mobility of the enzyme in a native PAGE gel was also the same in diapause and non-diapause specimens (Table 3, Fig. 3). The Vmax of the glucose-6-P oxidation was more than two-fold higher in the diapause than in the non-diapause bugs (Table 3). Similarly, the KM values of AR and PDH were not different in diapause vs. non-diapause adults, but the Vmax values were more than 20-fold higher in diapause specimens. The KR showed relatively low affinity for fructose as a substrate. The calculated KM value of the fructose reduction by KR was 0.64 M. No activity of KR could be detected in the samples taken from non-diapause bugs (the concentration of fructose in reaction mixture
Table 1 Activities of selected enzymes involved in polyol biosynthesis in Pyrrhocoris apterus: glycogen phosphorylase (GPase), phosphofructokinase-1 (PFK-1), glucose-6-P dehydrogenase (G6P-DH) Enzyme
GPase (a + b form)
GPase (% a form)
PFK-1
G6P-DH
Adult age [weeks]
2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P
Activity [lmol.min/g fat body protein]a non-diapause
diapause
3:8 0:3 3:4 0:1 4:1 0:2 ns 0.1039 94:2 4:1 96:2 1:1 92:8 7:2 ns 0.7930 6:2 0:6 a 4:5 0:4 a 1:3 0:5 b 0.0052 16:2 1:6 11:1 6:6 8:0 0:8 ns 0.2582
4:1 0:1 b 4:8 0:1 a 4:6 0:2 ab 0.0333 39:3 3:1 b 48:8 0:6 b 85:2 4:5 a 0.0014 3:1 0:5 3:1 0:5 2:6 0:1 ns 0.473 21:0 1:3 c 32:5 0:5 b 39:4 0:3 a 0.0004
t-test, P
ns 0.3118 0.0051 ns 0.1296
0.0044 0.0003 ns 0.3331
0.0303 ns 0.0906 ns 0.0691
ns 0.0812 0.0447 0.0004
a Each value represents mean S:D: of two independent activity assays, fat bodies dissected of 8 females were pooled for each assay. ANOVAs were performed for samples taken at different time-phases of cold-acclimation as depicted in Fig. 1 (2w, 7w, 7w + 1d). Means followed by different letters were statistically different (post hoc Tukey’s multiple comparison test). Differences between the activities found in non-diapause and diapause insects were treated by unpaired t-test. (ns, not significant; , P < 0:05; , P < 0:01; , P < 0:001).
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Table 2 Activities of selected enzymes involved in polyol biosynthesis in Pyrrhocoris apterus: aldose reductase (AR), polyol dehydrogenase (PDH), ketose reductase (KR) Enzyme/substrate
AR/Glucose
AR/Ribose
PDH/Fructose
PDH/Ribulose
KR/Fructose
Adult age [weeks]
2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P
Activity [lmol.min/g fat body protein]a non-diapause
diapause
0 0 0:6 0:6 X 3:4 0:1 2:6 0:4 3:2 1:1 ns 0.5430 6:6 0:9 a 5:7 0:5 ab 3:1 0:7 b 0.0340 3:9 0:7 a 3:3 0:2 ab 1:8 0:4 b 0.0461 0 0 0 X
11:8 1:9 12:5 0:5 17:7 1:7 ns 0.0522 56:3 6:2 63:0 8:0 81:4 12:1 ns 0.1397 71:4 16:4 61:5 3:6 74:6 23:3 ns 0.7365 16:9 0:4 28:5 9:2 29:9 6:0 ns 0.2272 149:9 16:6 b 212:9 51:8 ab 316:3 15:5 a 0.0325
t-test, P
X X
0.0055
0.0068 0.0087 0.0119
0.0307 0.0021 0.0493
0.0019 ns 0.0607 0.0221
X X X
a Each value represents mean S:D: of two independent activity assays, fat bodies dissected of 8 females were pooled for each assay. ANOVAs were performed for samples taken at different time-phases of cold-acclimation as depicted in Fig. 1 (2w, 7w, 7w + 1d). Means followed by different letters were statistically different (post hoc Tukey’s multiple comparison test). Differences between the activities found in non-diapause and diapause insects were treated by unpaired t-test. (ns, not significant; , P < 0:05; , P < 0:01; , P < 0:001).
varied between 100 lM–0.5 M). The Vmax value of fructose reduction by KR was unusually high, which indicates that the enzyme was abundantly present in the fat body tissue of diapause bugs (Table 3). Mobilities of the PDH and KR in native PAGE gel differed clearly as shown in Fig. 4.
4. Discussion The capacity to accumulate winter polyols during cold-acclimation is restricted to diapause individuals of P. apterus. As has been shown previously (Sˇlachta et al., 2002), and verified in this study, the reproducing (i.e. non-diapause) individuals can not accumulate polyols.
4.1. Glycogen as a source of carbon for polyol biosynthesis Glycogen deposits in the insect fat body are considered to serve as the principal source of carbon for polyol biosynthesis at low temperatures (Storey and Storey, 1991). We found five-fold higher deposits of glycogen in the abdominal fat bodies of diapause in comparison to non-diapause females of P. apterus. During cold-acclimation, approximately 200 lg (2/5) of fat body glycogen was depleted in an average female (in fact, this amount was probably higher because we sampled only 80–90% of the abdominal fat body). At the same time, an average female accumulated 380 lg of winter polyols in total (mainly ribitol and sorbitol).
Table 3 Kinetics of selected enzymes involved in polyol biosynthesis in Pyrrhocoris apterus Enzyme/(substrate)
G6P-DH AR/Ribose PDH/Fructose KR/Fructose
KM [lM or M]a
Vmax [lmol.min/g fat body protein]
non-diap.
diap.
t-test, P
non-diap.
diap.
t-test, P
20:8 7:9 (4) 0:13 0:05 (3) 0:20 0:06 (4) X
17:4 4:9 (6) 0:11 0:06 (6) 0:15 0:04 (6) 0:64 0:17 (4)
ns 0.4201 ns 0.6368 ns 0.1487 X
12:2 1:8 (4) 3:9 0:4 (3) 4:3 1:9 (4) X
28:0 2:1 (6) 96:8 40:4 (6) 88:8 53:5 (6) 974:3 626 8 (4)
<0.0001 0.0063 0.0148 X
a lM: G6P-DH; M: AR, PDH, KR (the abbreviations of enzyme names are the same as in Tables 1 and 2). Each value represents mean S:D: of (n) substrate affinity/maximum velocity assays performed with independent samples, fat bodies dissected of 8 females were pooled for each sample. Differences between the means found in non-diapause and diapause insects were treated by unpaired t-test. (ns, not significant; , P < 0:05; , P < 0:01; , P < 0:001).
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Fig. 3. Comparison of electrophoretic mobilities of glucose-6-P dehydrogenase (G6P-DH) in non-diapause and diapause adults of Pyrrhocoris apterus. Two independent assays are depicted in (a) and (b). Crude enzyme preparations extracted from fat body were loaded on native PAGE gels (50 lg of fat body protein per each lane), electrophoresed and the areas with G6P-DH activity were visualized after Manchenko (2003) using the chromogenic reaction of reduced NADPH (produced during oxidation of glucose-6-P) with tetrazolium salts.
The discrepancy between the amounts of glycogen depleted and polyols accumulated can be explained at least partly by our fat body tissue sampling procedure, which was not quantitative. It is also possible that a certain proportion of polyols was synthesized in other
Fig. 4. Comparison of electrophoretic mobilities of polyol dehydrogenase (PDH) and ketose reductase (KR) in non-diapause and diapause adults of Pyrrhocoris apterus. Crude enzyme preparations extracted from fat body were loaded on one native PAGE gel (sample X, 100 lg of fat body protein; Y, 50 lg). After electrophoresing, the gel was cut to two pieces and each piece was incubated independently: (a), (b). The areas with PDH or KR activities were visualized after Manchenko (2003) using the chromogenic reaction of reduced NADH or NADPH (produced during oxidation of sorbitol), respectively, with tetrazolium salts.
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tissues (muscles, gut), which contain rich glycogen deposits. Approximately 90 lg of glycogen, which was present in the fat body of an average non-diapause female, would theoretically suffice for production of a slightly less amount of sorbitol and ribitol (as some carbon atoms are lost as CO2 during passage through the pentose cycle, which produces reducing power). Total activities of GPase, a glycogen degrading enzyme, were similar in non-diapause and diapause insects. Also the activities of PFK-1, a rate limiting enzyme of the upper part of glycolysis, decreased to a similar level after the cold-acclimation in both groups of insects. Despite these similarities, ribitol and sorbitol remained at trace levels and little or no glycogen was lost during cold-acclimation. Our results do not allow us to decide with certainty if the breakdown of glycogen was stopped/slowed down, or if the carbon flow from glycogen was directed primarily to the TCA cycle in non-diapause insects. In the former case, the small amount of glycogen would not be the main cause of the incapability to accumulate polyols. In the latter case, however, the relatively small glycogen deposits found in non-diapause adults could compromise the availability of carbon atoms for biosynthesis of polyols. 4.2. Diapause-related changes in enzymatic complement This study showed that the enzymatic complement involved in glycogen degradation, glycolysis, pentose cycle and final reduction of sugars to polyols in diapause P. apterus adults differs from the one which is found in non-diapause adults in several important aspects. Results are summarized in Fig. 5. Close to 100% of GPase was present in its active (a) form in non-diapause adults while it was less than 40% in diapause adults. The inactive form (b) of phosphorylase was rapidly (within 1 day) activated upon transition of v diapause adults from 5 to 0 C, concomitantly with the abrupt start of accumulation of all four winter polyols. Such results suggest that activation of GPase by subv threshold temperatures (< 5 C) could be the initial step in polyol accumulation in diapause P. apterus. Cold activation of fat body GPase has been well documented in many insects (Ziegler et al., 1979; Storey and Storey, 1981; Hayakawa, 1985; Chen and Denlinger, 1990; Li et al., 2002). However, other concerted changes in the enzymatic complement are required in order to exploit the carbon flow released from glycogen for polyol biosynthesis. For instance, low temperature inactivation of PFK-1 was shown to result in a severe restriction of carbon flux through the PFK-1 locus, which potentiates production of sorbitol in overwintering insects (Hayakawa and Chino, 1982b; Storey, 1982). Another change, which is typically observed in polyol-accumulating insects and nematodes, is the relative activation
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Fig. 5. Summary of the results of enzymatic activity assays performed in this study. The relative activities of enzymes (shown in bold italics, abbreviations as in the Tables 1 and 2) extracted from the fat body of cold-acclimated Pyrrhocoris apterus are depicted as arrows in the schematic metabolic map. The width of each arrow is proportional to the enzyme’s activity according to the scale shown in the lower left corner. Arrows with solid border-lines or dashed border-lines represent diapause or non-diapause, respectively, insects.
of the pentose cycle at low temperatures (Tsumuki et al., 1987; Storey et al., 1991; Holden and Storey, 1994; Jagdale and Gordon, 1997). In accordance with that, we found that activity of G6P-DH, the first and rate-limiting enzyme of the pentose cycle (Levy, 1979), almost doubled during the cold-acclimation of diapause adults while it halved in the non-diapause adults. As a result, the activity of G6P-DH was 5-fold higher at the end of the acclimation procedure in diapause adults than in non-diapause ones (Fig. 5). Oxidation of glucose-6-P catalyzed by G6P-DH is one of the two NADPH-generating steps of the pentose cycle. Reducing equivalents in the form of NADPH are critically needed for production of polyols. In fact, when a high proportion of carbon flow is routed through the pentose cycle (as a result of GPase and G6P-DH activations and PFK-1 inhibition), the NADPH generated must be reoxidized in order to maintain redox balance, and the reduction of sugars to polyols effectively fullfils that task (Storey and Storey, 1990). We found that the conventional enzymes with capacity to reduce sugars to polyols (AR, PDH) were abundantly present (highly active) in diapause adults while their activities were low in the non-diapause adults of P. apterus (Fig. 5). The apparent affinities of the P. apterus’s AR and PDH for
their preferred substrates were relatively low (KM: ARRibose, 0.11 M; PDHFructose, 0.15 M), which is characteristic for similar enzymes from different sources (Yaginuma and Yamashita, 1979; Jefferry and Jo¨rnvall, 1988; Ng et al., 1992). Neither of the two substrates (ribose and fructose) was detected to occur at concentrations close to KM values in P. apterus; the concentrations never exceeded 0.01 M (Kosˇta´l and Sˇimek, unpublished data). Perhaps high abundancies of the enzymes found in diapause insects help to counteract the low substrate concentrations and low enzyme affinities and thus allow the synthesis of polyols from v their respective sugar precursors. At 25 C and our experimental conditions, the AR with ribose showed approximately 2–3-fold higher activity than the PDH with ribulose (Fig. 5). Such data suggest that synthesis of ribitol could be accomplished preferably via the reduction of ribose by NADPH-dependent AR in P. apterus. But, additional experiments are needed to verify functionality of that pathway in vivo. Sorbitol is typically synthesized by reduction of glucose by an NADPH-dependent AR in most animals (Jefferry and Jo¨rnvall, 1988) including insects (Yaginuma and Yamashita, 1979; Storey and Storey, 1981, 1983). In P. apterus, activity of AR with glucose as a substrate was
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relatively low at our experimental conditions; the PDH activity with fructose was 4–5-fold higher (Fig. 5). However, NAD(H)-dependent PDH is generally considered to function mainly in sorbitol catabolism (Jefferry and Jo¨rnvall, 1988; Storey and Storey, 1981, 1983). Our data suggest that the preferred route for sorbitol synthesis in P. apterus could use the activity of an unusual enzyme, NADP(H)-dependent KR. 4.3. NADP(H)-dependent ketose reductase Sorbitol biosynthesis via reduction of fructose by the NADP(H)-dependent KR activity in diapause P. apterus adults seems to be supported by the following facts: (1) the activity of ‘‘conventional’’ AR, which reduces glucose to sorbitol, was approximately 18-fold lower than that of KR. (2) KR was highly active (abundant?) in diapause adults but its activity could not be detected in non-diapause adults. (3) KR requires NADPH as a co-factor and relative production of NADPH is likely to increase in diapause adults in response to cold-acclimation as a result of increasing activity of G6P-DH. (4) The generation of NADH, a co-factor needed for reducing activity of PDH, could be relatively limited at low temperatures when the carbon flow through the lower part of glycolysis is slowed down by inhibition of the PFK-1 locus. (5) With the decreasing temperature of acclimation, the activity of KR increased in diapause adults. The affinity of KR for fructose as a substrate, however, was very low (KM ¼ 0:64 M). Concentrations of fructose were typically below 0.01 M in P. apterus independent of its physiological state (Kosˇta´l and Sˇimek, unpublished data). As in the case of AR, we suggest that the high abundance of the enzyme might help to overcome such a limited availability of substrate. Compartmentalization, which causes elevation of substrate concentrations in the restricted locality of enzyme presence, might be another way that can help to explain how an enzyme with low affinity for its substrate might be functional even if overall concentration of substrate in the tissue is low. NADP+-dependent enzymatic conversion of sorbitol has been reported previously in Drosophila melanogaster (Bischoff, 1976). More recently, NADP(H)-dependent KR, which catalyzes reduction of fructose to sorbitol, was purified and characterized in the silverleaf whitefly, Bemisia argentifolii (Wolfe et al., 1998; Salvucci et al., 1998), its cDNA sequence was obtained (Wolfe et al., 1999) and the crystal structure of the protein was resolved (Banfield et al., 2001). These results revealed that the whitefly’s KR is closely similar to NAD(H)-dependent polyol dehydrogenases, members of MDR superfamily (Wolfe et al., 1999) and clearly distinct from NADPH-dependent aldose reductases, members of AKR superfamily (Bohren et al., 1989). Relatively minor changes in the amino-acid
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sequence of the catalytic domain allowed the whitefly’s KR to use NADP(H) as a co-factor (Wolfe et al., 1999). Interestingly, the whitefly’s KR also showed extremly low affinity for fructose. The apparent KM v was 0.62 M at 24 C (Salvucci et al., 1998), which is almost equal to the value obtained in this study for P. apterus. Similarly, as in our study, the activity of KR in whitefly was 16-fold higher than the maximal activity of PDH with the same substrate, fructose. Whiteflies use the KR to convert this substrate, which is found abundantly in their diet, to sorbitol, which functions as thermoprotectant under heat-stress conditions (Wolfe et al., 1998). The potential relationships of the P. apterus’s KR with either MDR or AKR protein superfamilies remain uncertain. We showed that the electrophoretic mobility of P. apterus’s KR on the PAGE gel differs substantially from that of PDH. It suggests that KR and PDH are two different enzymes with relatively distinct structure and not just one enzyme with low selectivity for NAD(H)/NADP(H) as potential co-factors.
5. Conclusions P. apterus adults are able to accumulate winter polyols, chiefly ribitol and sorbitol, in response to cold-acclimation only if they have previously entered diapause (Sˇlachta et al., 2002). Here we showed that activities of several enzymes involved in polyol biosynthesis significantly differ between diapause and nondiapause insects. In diapause insects, the process of polyol accumulation is probably triggered by activation of GPase upon transition to sub-threshold temperature v of 5 C. Increasing activity of G6P-DH with decreasing temperature of acclimation suggests that the activity of the pentose cycle and production of reducing power in the form of NADPH is relatively elevated at low temperatures in diapause insects. Such a reducing power can be used to convert sugars to ribitol or sorbitol. Enzymes that can catalyze these reductions had either significantly higher activity in diapause than in nondiapause insects [NADP(H)-dependent AR] or the activity occurred specifically in diapause insects [NADP(H)-dependent KR].
Acknowledgements The concentrations of polyols were analyzed in cooperation with Petr Sˇimek and Helena Zahradnı´cˇkova´ (Institute of Entomology AS CR). This study was supported by the Grant Agency of the Czech Republic (grant no. 206/03/0099) and by the Academy of Sciences of the Czech Republic (project no. K 600 5114).
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V. Kosˇta´l et al. / Journal of Insect Physiology 50 (2004) 303–313 Storey, K.B., 1982. Phosphofructokinase from the overwintering gall fly larva, Eurosta solidaginis: control of cryoprotectant polyol synthesis. Insect Biochemistry 12, 501–505. Storey, K.B., Bailey, E., 1978. The intracellular distribution of enzymes of carbohydrate degradation in the fat body of the adult male cockroach. Insect Biochemistry 8, 73–79. Storey, K.B., Keefe, D., Kourtz, L., Storey, J.M., 1991. Glucose-6phosphate dehydrogenase in cold hardy insects: kinetic properties, freezing stabilization, and control of hexose monophosphate shunt activity. Insect Biochemistry 21, 157–164. Storey, K.B., Storey, J.M., 1981. Biochemical strategies of overwintering in the gall fly larva, Eurosta solidaginis: effect of low temperature acclimation on the activities of enzymes of intermediary metabolism. Journal of Comparative Physiology B 144, 191–199. Storey, K.B., Storey, J.M., 1991. Biochemistry of cryoprotectants. In: LeeJr., R.E., Denlinger, D.L. (Eds.), Insects at Low Temperature. Chapman and Hall, New York and London, pp. 64–93. Stoscheck, C.M., 1990. Quantitation of proteins. In: Deutscher, M.P. (Ed.), Methods in Enzymology, Vol. 182. Academic Press Limited, London, pp. 50–68. Tsumuki, H., Rojas, R.R., Storey, K.B., Baust, J.G., 1987. The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch). Insect Biochemistry 17, 347–352.
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Adjustments of the enzymatic complement for polyol biosynthesis and accumulation in diapausing cold-acclimated adults of Pyrrhocoris apterus V. Kosˇta´l a,, M. Tollarova´ b, J. Sˇula a a
Institute of Entomology, Academy of Sciences of the Czech Republic, Branisˇovska´ 31, 370 05 Cˇeske´ Budeˇjovice, Czech Republic b Faculty of Biological Sciences, University of South Bohemia, Cˇeske´ Budeˇjovice, Czech Republic Received 17 October 2003; received in revised form 8 January 2004; accepted 8 January 2004
Abstract The capacity to accumulate winter polyols (mainly ribitol and sorbitol) during cold-acclimation in Pyrrhocoris apterus is restricted only to the adults that have previously entered diapause. The enzymatic complement involved in polyol biosynthesis was found to differ in a complex manner between diapause and non-diapause adults. Nearly 100% of glycogen phosphorylase (GPase) was present in its active form in non-diapause adults irrespective of their acclimation status. In contrast, less than 40% of GPase was present in its active form in diapause adults prior to cold-acclimation and the inactive form was rapidly activated upon tranv sition from 5 to 0 C, concomitantly with the start of rapid polyol accumulation. The flow of carbon released by activation of glycogen degradation might be routed to the pentose cycle because the activity of glucose-6-P dehydrogenase (G6P-DH) was significantly higher and it increased with cold-acclimation in diapause adults while it was relatively low and it decreased with cold-acclimation in non-diapause adults. Reducing equivalents in the form of NADPH, which were generated in the pentose cycle, might require re-oxidation. Such re-oxidation might be achieved during reduction of sugars to polyols. The activity of NADP(H)-dependent aldose reductase (AR) was about 20-fold higher in diapause than in non-diapause adults. Similarly, the activity of NAD(H)-dependent polyol dehydrogenase (PDH) was higher in diapause adults. In addition, we found a very high activity of an unusual enzyme, NADP(H)-dependent ketose reductase (KR), exclusively in diapause adults. KR might be involved in reduction of fructose to sorbitol. Although its affinity for fructose as a substrate was low (KM ¼ 0:64 M), its activity was about 10-fold higher than that of PDH with fructose. Moreover, the activity of KR significantly increased with cold-acclimation while that of PDH remained unchanged. Different electrophoretic mobilities in PAGE gel suggested that KR and PDH are two different enzymes with specific requirement for NADP(H) or NAD(H), respectively, as co-factors. # 2004 Elsevier Ltd. All rights reserved. Keywords: Diapause; Cold tolerance; Cryoprotectans; Ribitol; Sorbitol; Glycolysis; Pentose cycle; Ketose reductase
1. Introduction The conversion of glycogen into sugar alcohols (polyols) during insect diapause or overwintering was first reported in the late 1950s by several authors (Salt, 1957; Chino, 1957; Wyatt and Meyer, 1959). Slightly later, it became apparent that other diapausing insects prefer to accumulate trehalose (Asahina and Tanno, 1964). Since then, accumulation of different polyols or Corresponding author. Tel.: +420-387-775-229; fax: +420-385300-354. E-mail address: [email protected] (V. Kosˇta´l).
0022-1910/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.01.006
sugars has been confirmed in a large number of diapausing and/or overwintering insects and several different mechanisms of cryoprotection conferred by these compounds were proved or suggested (reviews: Asahina, 1969; So¨mme, 1982, 1999; Zachariassen, 1985; Storey and Storey, 1991). The exact nature of mutual relationships between diapause induction, cold-acclimation, polyol accumulation and cold hardiness is largerly unknown because the topics were often studied separately. The available information was reviewed by Denlinger (1991); Lee (1991); Storey and Storey (1991) and Pullin (1996). Interconversion between glycogen and sugars/polyols is usually temperature-dependent.
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Although in some cases the induction of diapause at relatively high temperature may serve as a sufficient trigger for accumulation of polyols [typically in diapausing embryos of Bombyx mori (Chino, 1957) or in diapausing tropical beetle Stenotarsus rotundus (Pullin and Wolda, 1993)], the process mostly starts only at v temperatures below 15 C for glycerol (Morrissey and Baust, 1976; Storey and Storey, 1983; Renault et al., v 2002) or below 5 C for the other compounds so far studied (Storey and Storey, 1991). Concerted changes in the enzymatic complement of glycolysis and pentose cycle regulate diversion of the carbon flow from the main stream of glycolysis (toward TCA cycle and energy metabolism) to the biosynthesis and accumulation of sugars and polyols (Ziegler and Wyatt, 1975; Hayakawa and Chino, 1981, 1982a,b; Storey and Storey, 1981; Tsumuki et al., 1987). Not only the abundancies of certain enzymes may change seasonally or in response to temperature acclimation but numerous regulatory mechanisms are involved: changing the substrate affinities (KM values) and pH optima with temperature; reversible phosphorylation; changing the concentrations of various modulators and inhibitors (review: Storey and Storey, 1991). Adults of the brachypterous form of the red firebug, Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae), enter a facultative reproductive diapause in response to short-days during the second half of summer in South Bohemia (Czech Republic) (Hodek, 1968, 1983). During relatively warm autumn, the bugs prepare for overwintering, which takes place in the upper litter layer. Overwintering bugs do not tolerate freezing of their v body fluids, supercool to ca. 17 C (with a high indiv vidual variation spanning from 12 to 23 C), and v can survive for approximately two weeks at 15 C (Kosˇta´l and Sˇimek, 2000). When exposed at temperav tures below 5 C, they start to accumulate four specific ‘‘winter polyols’’ (ribitol, sorbitol, arabinitol and mannitol), which probably function as non-colligative cryoprotectants (Kosˇta´l and Sˇlachta, 2001; Kosˇta´l et al., 2001). No capacity for accumulation of winter polyols, however, was detected in the non-diapause, reproducing, adults of P. apterus (Sˇlachta et al., 2002). Thus, the main objective of this study was to contribute to the understanding of physiological mechanisms which allow accumulation of polyols specifically in diapause insects. In this paper, we show that the fat body enzymatic complements differ in several aspects between the diapause and non-diapause adults of P. apterus. The differences were found in: (1) relative proportions of active and inactive forms of glycogen phosphorylase (GPase) and activation of the inactive form by low temperature; (2) activities of glucose-6-P dehydrogenase (G6P-DH), aldose reductase (AR) and polyol dehydrogenase (PDH); (3) presence or absence of
NADP(H)-dependent ketose reductase (KR) activity; (4) direction of changes in the activities of G6P-DH, AR, PDH and KR in response to cold acclimation. The way that such differences could be involved in the accumulation of polyols in diapause insects is discussed.
2. Materials and methods 2.1. Insect rearing and acclimation protocols Adult P. apterus bugs were collected during May 2002 in a field near Chelcˇice, South Bohemia, Czech Republic. Offspring of the field-collected adults were reared under conditions which promote continuous non-diapause development, i.e. long-day photoperiod v (LD) of 18L:6D and a constant temperature of 25 C. The dry seeds of the linden tree (Tilia parviflora Ehrh.) as a food source and water were provided ad libitum. Adults in reproductive diapause were obtained by rearing all larval instars under short-day photoperiod (SD) v of 12L:12D and a constant temperature of 25 C (Hodek, 1968). Only females belonging to the third and fourth laboratory generations were used for analyses. Diapause development can be assessed easily in females (Hodek, 1968, 1983) and no significant differences were found between the two sexes in their ability to accumulate polyols (Kosˇta´l et al., 2001; Sˇlachta et al., 2002) and in the activities of enzymes under study (Kosˇta´l, unpublished results). The young experimental adults were kept under the photoperiods at which they developed and at a conv stant temperature of 20 C for 2 weeks. During this time, non-diapause adults mated and females started to lay eggs or diapause intensified in diapause adults, respectively. After this initial period, at the adult age of 2 weeks, the bugs were subjected to gradual cold-acclimation as depicted in Fig. 1. Alternating temperatures were applied during first two weeks v v v v (thermophase/cryophase: 20 /10 C and 15 /5 C, respectively). Photoperiodic regime (either LD or SD) was maintained during these two weeks. Our earlier results showed that gradual acclimation at alternating temperatures (in comparison to constant temperatures) leads to higher level of cold-hardiness in P. apterus (Kosˇta´l et al., 2001). Later, the insects were kept at a v constant temperature of 5 C/DD (maximum shortv time fluctuations were from 4.6 to 5.8 C) for 3 weeks. v The temperature of 5 C was selected on purpose, because the threshold for rapid polyol accumulation in v P. apterus was shown to be approximately 5 C (Kosˇta´l et al., 2001). Finally, the bugs were transferred to v 0 C/DD for 1 week.
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were measured in 70% ethanol extracts of the whole body samples (one individual was used per each sample). The bug was weighed, submerged in 400 lL of 70% ethanol, cut to pieces, thoroughly homogenized and extracted at room temperature for 5 min with vigorous shaking. The extracts were centrifuged at 20,000 v g/35 C/10 min and the pooled supernatants from two replications of the procedure were used for o-methyloxime trimethylsilyl derivatization and subsequent analysis by gas chromatography coupled to mass spectrometry (Kosˇta´l and Sˇimek, 1995). All chemicals used for analysis of glycogen and polyols were purchased from Sigma-Aldrich Co.
Fig. 1. Schematic view of the protocol of cold-acclimation for the adults of Pyrrhocoris apterus (see Materials and methods for detailed description). The non-diapause and diapause insects were acclimated equally. Samples for analyses of glycogen content (Gly), polyol concentrations (Poly) and enzymatic activities (Enz) were taken at different time-phases of cold-acclimation as indicated by arrows. The v temperature threshold of 5 C, below which rapid accumulation of polyols starts, is indicated by a dashed arrow.
2.2. Glycogen and polyols The content of glycogen in the abdominal fat body was measured colorimetrically as glucose released by the phenolic method (Dubois et al., 1956). Fat bodies were dissected under Ringer’s solution. Approximately 80–90% of the abdominal fat body tissue was taken from each female and analyzed as one sample. The tissue was homogenized using Dounce homogenizer, extracted twice in 400 lL of 70% ethanol and centrifuged. After decanting the ethanol, the sample was dissolved in 200 lL of water and boiled for 30 min in 1.1 mL of 30% KOH solution. After cooling and spinning, 500 lL of supernatant was taken for further analysis. Glycogen was precipitaed by adding of 1.5 mL 99.8% ethanol, 250 lL of 10% Na2SO4, keeping the sample on ice for 1 h and spinning. The pellet was re-extracted twice with 70% ethanol and finally dissolved in 1 mL of water. Acidic hydrolysis was achieved by adding 500 lL of water, 200 lL of 5% phenol solution and 1 mL of concentrated H2SO4 to v the 100 lL sample aliquot and keeping at 95 C for 20 min. After 30 min at room temperature, the absorbance of 490 nm light was read and compared to the calibration curve obtained by analysis of (oyster) glycogen standard. Blank samples (no tissue) were run in parallel and subtracted. The concentrations of the four ‘winter’ polyols (arabinitol, ribitol, mannitol and sorbitol), that typically accumulate in diapausing cold-acclimated P. apterus adults (Kosˇta´l and Sˇimek, 2000; Kosˇta´l et al., 2001),
2.3. Enzyme activities Slightly modified methods of Storey and Bailey (1978); Storey and Storey (1981) and Joanisse and Storey (1994) were used. Abdominal fat body was quickly dissected (ca. 3 min) under Ringer solution and submerged in liquid nitrogen immediately. After accumulation of 8–10 fat bodies (pooled to one sample), the nitrogen was evaporated and 600 lL (approximately 8 vol) of homogenization buffer (HB) was added. HB consisted of 100 mM Tris-HCl, pH 8.0, 15 mM mercaptoethanol and 1 mM EDTA. The sample was homogenized for 30 s, while increasing the speed from 10 000–30 000 rpm, using CAT Homogenizer X120 with metal blades (Ingenieurbu¨ro CAT, M. Zipperer GmbH, Germany). After centrifugation at 22 000 v g/20 min/20 C, the supernatant was used as the source of all enzymes. Total protein concentrations in the enzyme preparations were measured by the BCA protein assay (Stoscheck, 1990), and activities were expressed as lmoles of substrate converted to product per min per g of protein. The final activity values were calculated after subtracting blank values. Activities v were measured at 25 C using Pye Unicam SP8-100 spectrophotometer by continuous time scanning at 340 nm. All chemicals and coupling enzymes were purchased from Sigma-Aldrich Co. Glycogen phosphorylase (GPase; E.C. 2.4.1.1) (total aþb). 50 mM potassium phosphate buffer (pH 6.8), 5 mg/mL glycogen (omitted from control), 5 lM glucose-1,6-P2, 0.6 mM NADP+, 2 mM 5’AMP, 15 mM MgCl2, 2 units/mL phosphoglucomutase and 2 units/ mL glucose-6-phosphate dehydrogenase (NADP+ dependent). The active form of the enzyme (a) was measured in the absence of AMP. Phosphofruktokinase-1 (PFK-1; E.C. 2.7.1.11). 20 mM imidazole-HCl buffer (pH 7.2), 10 mM fructose-6phosphate (omitted from control), 2 mM ATP, 0.15 mM NADH, 50 mM KCl, 5 mM MgSO4, 2 units/mL aldolase, 37.5 units/mL triosephosphate isomerase and 4 units/mL glycerol-3-phosphate dehydrogenase.
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Glucose-6-phosphate dehydrogenase (G6P-DH; E.C. 1.1.1.49). 20 mM imidazole-HCl buffer (pH 7.2), 5 mM MgSO4, 1 mM glucose-6-phosphate (omitted from control), 0.6 mM NADP+. Aldose reductase (AR, 1.1.1.21), glucose or ribose activities. 20 mM imidazole-HCl buffer (pH 7.2), 250 mM D-glucose or D-ribose, respectively, (omitted from control), 0.1 mM NADPH. Polyol dehydrogenases (PDH). 20 mM imidazole-HCl buffer (pH 7.2), 250 mM D-fructose (sorbitol dehydrogenase; E.C. 1.1.1.14) or D-ribulose (ribitol dehydrogenase E.C. 1.1.1.56) (the substrates were omitted from controls), 0.15 mM NADH. Ketose reductase (KR; E.C. 1.1.1.?). 20 mM imidazole-HCl buffer (pH 7.2), 250 mM D-fructose (omitted from control), 0.15 mM NADPH.
2.4. Enzyme kinetics and electrophoretic mobility in the native PAGE gel Kinetics and mobilities of selected enzymes were compared in the fat body samples taken from 2-weekold P. apterus adults (without any cold-acclimation) differing in developmental mode, i.e. diapause vs. nondiapause. Michaelis-Menten constants (KM) and maximum velocities of enzymatic reactions (Vmax) were v measured at 25 C in four enzymes with their specific subtrates: G6PDH with glucose-6-phosphate, AR with ribose, PDH with fructose and KR with fructose. KM and Vmax values were determined by non-linear regression analysis of the dependence of activity on substrate concentration using the GraphPad Prism 4 software. Crude enzyme preparations (see above) were loaded onto vertical 4–16% gradient polyacrylamide slab gels (PAGE) prepared according to Williams and Reisfeld (1964) and electrophoresed at 150 V for 80 min. After incubation of the gels in reaction mixtures containing specific substrates and co-factors, the gel areas containing specific enzymes separated during electrophoresis were visualized using the chromogenic reaction of reduced NADH or NADPH with tetrazolium salts, which results in the formation of intensely coloured precipitate, formazan. The methods were as described in Manchenko (2003). Phenazine methosulfate (PMS) was used as an intermediary catalyst. Methyl thiazolyl tetrazolium (MTT) was used to visualize G6P-DH and nitro blue tetrazolium (NBT) was used to visualize PDH and KR. Chemicals were purchased from Sigma-Aldrich Co.
3. Results 3.1. Changes of glycogen content and accumulation of polyols No statistically significant changes were found in the content of fat body glycogen in diapause bugs during cold-acclimation by ANOVA (P ¼ 0:0757) (Fig. 2). This was caused at least partly by a high variation of glycogen content among individual females. Our results indicate that glycogen was not spent during the first part of acclimation, which consisted of a gradual and v thermoperiodic drop of temperature to 5 C. After 3 v weeks at constant 5 C, glycogen content decreased from 512:0 123:1 lg/fat body (mean S:D:, n ¼ 10) to 417:8 164:5 lg (n ¼ 8). Further decrease to 310:9 115:9 (n ¼ 8) was observed after 1 week at conv stant 0 C. In non-diapause adult females, the content of glycogen in abdominal fat body was much lower (ranging between 64.7–92.3 lm/fat body) than in diapause females and its level was maintained relatively constant during cold-acclimation (ANOVA, P ¼ 0:6444) (Fig. 2). Two of the four winter polyols, ribitol and arabinitol, started to accumulate already during the exposure v to constant 5 C. The accumulation was more apparent in ribitol because its levels were approximately 15-fold higher than those of arabinitol (Fig. 2). Significant increases of concentrations were observed at constant v 0 C in all four polyols. Considering 60 mg to represent the average body weight of one female (Kosˇta´l, unpublished data), the total amount of accumulated winter polyols would be approximately 380 lg/female at the end of cold-acclimation. The hydration (proportion of water in the fresh weight) of the whole body in an average diapause cold-acclimated female is 55%
Fig. 2. Changes of glycogen contents (left y axis) and polyol concentrations (right y axis) during the process of cold-acclimation in Pyrrhocoris apterus adults. Glycogen is shown for non-diapause and diapause insects. Four polyols are shown for diapause insects only. Each data point represents mean S:E: of n ¼ 8 10 samples taken from individual females. (FW, fresh weight).
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(Kosˇta´l, unpublished data). It can be deduced from these data that the average sum concentration of winter polyols in the body fluids would reach about 70 mM (380 lg in 33 lL of water). Of course, this is a rough estimation, which supposes that the polyols are evenly distributed in different compartments. Nevertheless, such a value corresponds well to the concentrations that were previously measured directly in haemolymph (up to 100 mM depending on acclimation status; Kosˇta´l et al., 2001). No polyol accumulation was observed in non-diapause females. The concentrations never exceeded 0.01 lg/mg (data not shown).
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they decreased or remained stable in non-diapause bugs. The activity of an unusual enzyme, ketose reductase (KR), was observed exclusively in the diapause specimens. This enzyme required NADPH for conversion of fructose to sorbitol. The activity of KR in reverse direction of the reaction (250 mM sorbitol ! fructose, with 0.15 mM NADP+ as a co-factor) was approximately 20% of that in the direction of fructose reduction (Kosˇta´l, unpublished data). The activity of KR was very high in comparison to the activities of other enzymes, and it further increased in response to cold-acclimation.
3.2. Enzyme activities 3.3. Enzyme kinetics and electrophoretic mobilities Results of all assays are summarized in Tables 1 and 2. Activities of GPase (total) and PFK-1 were similar in non-diapause and diapause specimens. As much as 96.2% of total GPase activity was found to be present in active form (a) in non-diapause specimens. In contrast, diapause bugs had only 39.3% of GPase in the a form prior to the start of cold-acclimation and 48.8% v after the acclimation to 5 C. However, after the transv fer to 0 C, the proportion of a form markedly increased to 85.2% within one day. Profound differences were detected between the nondiapause and diapause specimens in the activities of G6P-DH, AR, and PDH. First, the activities were significantly higher in diapause than in non-diapause bugs. Second, the activities tended to increase in response to cold-acclimation in diapause bugs while
The calculated KM values of G6P-DH did not differ statistically and the mobility of the enzyme in a native PAGE gel was also the same in diapause and non-diapause specimens (Table 3, Fig. 3). The Vmax of the glucose-6-P oxidation was more than two-fold higher in the diapause than in the non-diapause bugs (Table 3). Similarly, the KM values of AR and PDH were not different in diapause vs. non-diapause adults, but the Vmax values were more than 20-fold higher in diapause specimens. The KR showed relatively low affinity for fructose as a substrate. The calculated KM value of the fructose reduction by KR was 0.64 M. No activity of KR could be detected in the samples taken from non-diapause bugs (the concentration of fructose in reaction mixture
Table 1 Activities of selected enzymes involved in polyol biosynthesis in Pyrrhocoris apterus: glycogen phosphorylase (GPase), phosphofructokinase-1 (PFK-1), glucose-6-P dehydrogenase (G6P-DH) Enzyme
GPase (a + b form)
GPase (% a form)
PFK-1
G6P-DH
Adult age [weeks]
2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P
Activity [lmol.min/g fat body protein]a non-diapause
diapause
3:8 0:3 3:4 0:1 4:1 0:2 ns 0.1039 94:2 4:1 96:2 1:1 92:8 7:2 ns 0.7930 6:2 0:6 a 4:5 0:4 a 1:3 0:5 b 0.0052 16:2 1:6 11:1 6:6 8:0 0:8 ns 0.2582
4:1 0:1 b 4:8 0:1 a 4:6 0:2 ab 0.0333 39:3 3:1 b 48:8 0:6 b 85:2 4:5 a 0.0014 3:1 0:5 3:1 0:5 2:6 0:1 ns 0.473 21:0 1:3 c 32:5 0:5 b 39:4 0:3 a 0.0004
t-test, P
ns 0.3118 0.0051 ns 0.1296
0.0044 0.0003 ns 0.3331
0.0303 ns 0.0906 ns 0.0691
ns 0.0812 0.0447 0.0004
a Each value represents mean S:D: of two independent activity assays, fat bodies dissected of 8 females were pooled for each assay. ANOVAs were performed for samples taken at different time-phases of cold-acclimation as depicted in Fig. 1 (2w, 7w, 7w + 1d). Means followed by different letters were statistically different (post hoc Tukey’s multiple comparison test). Differences between the activities found in non-diapause and diapause insects were treated by unpaired t-test. (ns, not significant; , P < 0:05; , P < 0:01; , P < 0:001).
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Table 2 Activities of selected enzymes involved in polyol biosynthesis in Pyrrhocoris apterus: aldose reductase (AR), polyol dehydrogenase (PDH), ketose reductase (KR) Enzyme/substrate
AR/Glucose
AR/Ribose
PDH/Fructose
PDH/Ribulose
KR/Fructose
Adult age [weeks]
2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P 2 7 7 þ 1 day ANOVA, P
Activity [lmol.min/g fat body protein]a non-diapause
diapause
0 0 0:6 0:6 X 3:4 0:1 2:6 0:4 3:2 1:1 ns 0.5430 6:6 0:9 a 5:7 0:5 ab 3:1 0:7 b 0.0340 3:9 0:7 a 3:3 0:2 ab 1:8 0:4 b 0.0461 0 0 0 X
11:8 1:9 12:5 0:5 17:7 1:7 ns 0.0522 56:3 6:2 63:0 8:0 81:4 12:1 ns 0.1397 71:4 16:4 61:5 3:6 74:6 23:3 ns 0.7365 16:9 0:4 28:5 9:2 29:9 6:0 ns 0.2272 149:9 16:6 b 212:9 51:8 ab 316:3 15:5 a 0.0325
t-test, P
X X
0.0055
0.0068 0.0087 0.0119
0.0307 0.0021 0.0493
0.0019 ns 0.0607 0.0221
X X X
a Each value represents mean S:D: of two independent activity assays, fat bodies dissected of 8 females were pooled for each assay. ANOVAs were performed for samples taken at different time-phases of cold-acclimation as depicted in Fig. 1 (2w, 7w, 7w + 1d). Means followed by different letters were statistically different (post hoc Tukey’s multiple comparison test). Differences between the activities found in non-diapause and diapause insects were treated by unpaired t-test. (ns, not significant; , P < 0:05; , P < 0:01; , P < 0:001).
varied between 100 lM–0.5 M). The Vmax value of fructose reduction by KR was unusually high, which indicates that the enzyme was abundantly present in the fat body tissue of diapause bugs (Table 3). Mobilities of the PDH and KR in native PAGE gel differed clearly as shown in Fig. 4.
4. Discussion The capacity to accumulate winter polyols during cold-acclimation is restricted to diapause individuals of P. apterus. As has been shown previously (Sˇlachta et al., 2002), and verified in this study, the reproducing (i.e. non-diapause) individuals can not accumulate polyols.
4.1. Glycogen as a source of carbon for polyol biosynthesis Glycogen deposits in the insect fat body are considered to serve as the principal source of carbon for polyol biosynthesis at low temperatures (Storey and Storey, 1991). We found five-fold higher deposits of glycogen in the abdominal fat bodies of diapause in comparison to non-diapause females of P. apterus. During cold-acclimation, approximately 200 lg (2/5) of fat body glycogen was depleted in an average female (in fact, this amount was probably higher because we sampled only 80–90% of the abdominal fat body). At the same time, an average female accumulated 380 lg of winter polyols in total (mainly ribitol and sorbitol).
Table 3 Kinetics of selected enzymes involved in polyol biosynthesis in Pyrrhocoris apterus Enzyme/(substrate)
G6P-DH AR/Ribose PDH/Fructose KR/Fructose
KM [lM or M]a
Vmax [lmol.min/g fat body protein]
non-diap.
diap.
t-test, P
non-diap.
diap.
t-test, P
20:8 7:9 (4) 0:13 0:05 (3) 0:20 0:06 (4) X
17:4 4:9 (6) 0:11 0:06 (6) 0:15 0:04 (6) 0:64 0:17 (4)
ns 0.4201 ns 0.6368 ns 0.1487 X
12:2 1:8 (4) 3:9 0:4 (3) 4:3 1:9 (4) X
28:0 2:1 (6) 96:8 40:4 (6) 88:8 53:5 (6) 974:3 626 8 (4)
<0.0001 0.0063 0.0148 X
a lM: G6P-DH; M: AR, PDH, KR (the abbreviations of enzyme names are the same as in Tables 1 and 2). Each value represents mean S:D: of (n) substrate affinity/maximum velocity assays performed with independent samples, fat bodies dissected of 8 females were pooled for each sample. Differences between the means found in non-diapause and diapause insects were treated by unpaired t-test. (ns, not significant; , P < 0:05; , P < 0:01; , P < 0:001).
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Fig. 3. Comparison of electrophoretic mobilities of glucose-6-P dehydrogenase (G6P-DH) in non-diapause and diapause adults of Pyrrhocoris apterus. Two independent assays are depicted in (a) and (b). Crude enzyme preparations extracted from fat body were loaded on native PAGE gels (50 lg of fat body protein per each lane), electrophoresed and the areas with G6P-DH activity were visualized after Manchenko (2003) using the chromogenic reaction of reduced NADPH (produced during oxidation of glucose-6-P) with tetrazolium salts.
The discrepancy between the amounts of glycogen depleted and polyols accumulated can be explained at least partly by our fat body tissue sampling procedure, which was not quantitative. It is also possible that a certain proportion of polyols was synthesized in other
Fig. 4. Comparison of electrophoretic mobilities of polyol dehydrogenase (PDH) and ketose reductase (KR) in non-diapause and diapause adults of Pyrrhocoris apterus. Crude enzyme preparations extracted from fat body were loaded on one native PAGE gel (sample X, 100 lg of fat body protein; Y, 50 lg). After electrophoresing, the gel was cut to two pieces and each piece was incubated independently: (a), (b). The areas with PDH or KR activities were visualized after Manchenko (2003) using the chromogenic reaction of reduced NADH or NADPH (produced during oxidation of sorbitol), respectively, with tetrazolium salts.
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tissues (muscles, gut), which contain rich glycogen deposits. Approximately 90 lg of glycogen, which was present in the fat body of an average non-diapause female, would theoretically suffice for production of a slightly less amount of sorbitol and ribitol (as some carbon atoms are lost as CO2 during passage through the pentose cycle, which produces reducing power). Total activities of GPase, a glycogen degrading enzyme, were similar in non-diapause and diapause insects. Also the activities of PFK-1, a rate limiting enzyme of the upper part of glycolysis, decreased to a similar level after the cold-acclimation in both groups of insects. Despite these similarities, ribitol and sorbitol remained at trace levels and little or no glycogen was lost during cold-acclimation. Our results do not allow us to decide with certainty if the breakdown of glycogen was stopped/slowed down, or if the carbon flow from glycogen was directed primarily to the TCA cycle in non-diapause insects. In the former case, the small amount of glycogen would not be the main cause of the incapability to accumulate polyols. In the latter case, however, the relatively small glycogen deposits found in non-diapause adults could compromise the availability of carbon atoms for biosynthesis of polyols. 4.2. Diapause-related changes in enzymatic complement This study showed that the enzymatic complement involved in glycogen degradation, glycolysis, pentose cycle and final reduction of sugars to polyols in diapause P. apterus adults differs from the one which is found in non-diapause adults in several important aspects. Results are summarized in Fig. 5. Close to 100% of GPase was present in its active (a) form in non-diapause adults while it was less than 40% in diapause adults. The inactive form (b) of phosphorylase was rapidly (within 1 day) activated upon transition of v diapause adults from 5 to 0 C, concomitantly with the abrupt start of accumulation of all four winter polyols. Such results suggest that activation of GPase by subv threshold temperatures (< 5 C) could be the initial step in polyol accumulation in diapause P. apterus. Cold activation of fat body GPase has been well documented in many insects (Ziegler et al., 1979; Storey and Storey, 1981; Hayakawa, 1985; Chen and Denlinger, 1990; Li et al., 2002). However, other concerted changes in the enzymatic complement are required in order to exploit the carbon flow released from glycogen for polyol biosynthesis. For instance, low temperature inactivation of PFK-1 was shown to result in a severe restriction of carbon flux through the PFK-1 locus, which potentiates production of sorbitol in overwintering insects (Hayakawa and Chino, 1982b; Storey, 1982). Another change, which is typically observed in polyol-accumulating insects and nematodes, is the relative activation
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Fig. 5. Summary of the results of enzymatic activity assays performed in this study. The relative activities of enzymes (shown in bold italics, abbreviations as in the Tables 1 and 2) extracted from the fat body of cold-acclimated Pyrrhocoris apterus are depicted as arrows in the schematic metabolic map. The width of each arrow is proportional to the enzyme’s activity according to the scale shown in the lower left corner. Arrows with solid border-lines or dashed border-lines represent diapause or non-diapause, respectively, insects.
of the pentose cycle at low temperatures (Tsumuki et al., 1987; Storey et al., 1991; Holden and Storey, 1994; Jagdale and Gordon, 1997). In accordance with that, we found that activity of G6P-DH, the first and rate-limiting enzyme of the pentose cycle (Levy, 1979), almost doubled during the cold-acclimation of diapause adults while it halved in the non-diapause adults. As a result, the activity of G6P-DH was 5-fold higher at the end of the acclimation procedure in diapause adults than in non-diapause ones (Fig. 5). Oxidation of glucose-6-P catalyzed by G6P-DH is one of the two NADPH-generating steps of the pentose cycle. Reducing equivalents in the form of NADPH are critically needed for production of polyols. In fact, when a high proportion of carbon flow is routed through the pentose cycle (as a result of GPase and G6P-DH activations and PFK-1 inhibition), the NADPH generated must be reoxidized in order to maintain redox balance, and the reduction of sugars to polyols effectively fullfils that task (Storey and Storey, 1990). We found that the conventional enzymes with capacity to reduce sugars to polyols (AR, PDH) were abundantly present (highly active) in diapause adults while their activities were low in the non-diapause adults of P. apterus (Fig. 5). The apparent affinities of the P. apterus’s AR and PDH for
their preferred substrates were relatively low (KM: ARRibose, 0.11 M; PDHFructose, 0.15 M), which is characteristic for similar enzymes from different sources (Yaginuma and Yamashita, 1979; Jefferry and Jo¨rnvall, 1988; Ng et al., 1992). Neither of the two substrates (ribose and fructose) was detected to occur at concentrations close to KM values in P. apterus; the concentrations never exceeded 0.01 M (Kosˇta´l and Sˇimek, unpublished data). Perhaps high abundancies of the enzymes found in diapause insects help to counteract the low substrate concentrations and low enzyme affinities and thus allow the synthesis of polyols from v their respective sugar precursors. At 25 C and our experimental conditions, the AR with ribose showed approximately 2–3-fold higher activity than the PDH with ribulose (Fig. 5). Such data suggest that synthesis of ribitol could be accomplished preferably via the reduction of ribose by NADPH-dependent AR in P. apterus. But, additional experiments are needed to verify functionality of that pathway in vivo. Sorbitol is typically synthesized by reduction of glucose by an NADPH-dependent AR in most animals (Jefferry and Jo¨rnvall, 1988) including insects (Yaginuma and Yamashita, 1979; Storey and Storey, 1981, 1983). In P. apterus, activity of AR with glucose as a substrate was
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relatively low at our experimental conditions; the PDH activity with fructose was 4–5-fold higher (Fig. 5). However, NAD(H)-dependent PDH is generally considered to function mainly in sorbitol catabolism (Jefferry and Jo¨rnvall, 1988; Storey and Storey, 1981, 1983). Our data suggest that the preferred route for sorbitol synthesis in P. apterus could use the activity of an unusual enzyme, NADP(H)-dependent KR. 4.3. NADP(H)-dependent ketose reductase Sorbitol biosynthesis via reduction of fructose by the NADP(H)-dependent KR activity in diapause P. apterus adults seems to be supported by the following facts: (1) the activity of ‘‘conventional’’ AR, which reduces glucose to sorbitol, was approximately 18-fold lower than that of KR. (2) KR was highly active (abundant?) in diapause adults but its activity could not be detected in non-diapause adults. (3) KR requires NADPH as a co-factor and relative production of NADPH is likely to increase in diapause adults in response to cold-acclimation as a result of increasing activity of G6P-DH. (4) The generation of NADH, a co-factor needed for reducing activity of PDH, could be relatively limited at low temperatures when the carbon flow through the lower part of glycolysis is slowed down by inhibition of the PFK-1 locus. (5) With the decreasing temperature of acclimation, the activity of KR increased in diapause adults. The affinity of KR for fructose as a substrate, however, was very low (KM ¼ 0:64 M). Concentrations of fructose were typically below 0.01 M in P. apterus independent of its physiological state (Kosˇta´l and Sˇimek, unpublished data). As in the case of AR, we suggest that the high abundance of the enzyme might help to overcome such a limited availability of substrate. Compartmentalization, which causes elevation of substrate concentrations in the restricted locality of enzyme presence, might be another way that can help to explain how an enzyme with low affinity for its substrate might be functional even if overall concentration of substrate in the tissue is low. NADP+-dependent enzymatic conversion of sorbitol has been reported previously in Drosophila melanogaster (Bischoff, 1976). More recently, NADP(H)-dependent KR, which catalyzes reduction of fructose to sorbitol, was purified and characterized in the silverleaf whitefly, Bemisia argentifolii (Wolfe et al., 1998; Salvucci et al., 1998), its cDNA sequence was obtained (Wolfe et al., 1999) and the crystal structure of the protein was resolved (Banfield et al., 2001). These results revealed that the whitefly’s KR is closely similar to NAD(H)-dependent polyol dehydrogenases, members of MDR superfamily (Wolfe et al., 1999) and clearly distinct from NADPH-dependent aldose reductases, members of AKR superfamily (Bohren et al., 1989). Relatively minor changes in the amino-acid
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sequence of the catalytic domain allowed the whitefly’s KR to use NADP(H) as a co-factor (Wolfe et al., 1999). Interestingly, the whitefly’s KR also showed extremly low affinity for fructose. The apparent KM v was 0.62 M at 24 C (Salvucci et al., 1998), which is almost equal to the value obtained in this study for P. apterus. Similarly, as in our study, the activity of KR in whitefly was 16-fold higher than the maximal activity of PDH with the same substrate, fructose. Whiteflies use the KR to convert this substrate, which is found abundantly in their diet, to sorbitol, which functions as thermoprotectant under heat-stress conditions (Wolfe et al., 1998). The potential relationships of the P. apterus’s KR with either MDR or AKR protein superfamilies remain uncertain. We showed that the electrophoretic mobility of P. apterus’s KR on the PAGE gel differs substantially from that of PDH. It suggests that KR and PDH are two different enzymes with relatively distinct structure and not just one enzyme with low selectivity for NAD(H)/NADP(H) as potential co-factors.
5. Conclusions P. apterus adults are able to accumulate winter polyols, chiefly ribitol and sorbitol, in response to cold-acclimation only if they have previously entered diapause (Sˇlachta et al., 2002). Here we showed that activities of several enzymes involved in polyol biosynthesis significantly differ between diapause and nondiapause insects. In diapause insects, the process of polyol accumulation is probably triggered by activation of GPase upon transition to sub-threshold temperature v of 5 C. Increasing activity of G6P-DH with decreasing temperature of acclimation suggests that the activity of the pentose cycle and production of reducing power in the form of NADPH is relatively elevated at low temperatures in diapause insects. Such a reducing power can be used to convert sugars to ribitol or sorbitol. Enzymes that can catalyze these reductions had either significantly higher activity in diapause than in nondiapause insects [NADP(H)-dependent AR] or the activity occurred specifically in diapause insects [NADP(H)-dependent KR].
Acknowledgements The concentrations of polyols were analyzed in cooperation with Petr Sˇimek and Helena Zahradnı´cˇkova´ (Institute of Entomology AS CR). This study was supported by the Grant Agency of the Czech Republic (grant no. 206/03/0099) and by the Academy of Sciences of the Czech Republic (project no. K 600 5114).
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