Insect Biochem., Vol. 12, No. 6, pp. 639 642, 1982 0020-1790/82/060639-04503.00/0 Printed in Great Britain. © 1982 Pergamon Press Ltd

PHOSPHOFRUCTOKINASE AS A POSSIBLE KEY ENZYME REGULATING GLYCEROL OR TREHALOSE ACCUMULATION IN DIAPAUSING INSECTS

YOICHI HAYAKAWA and HARUO CHINO Biochemical Laboratory, Institute of Low Temperature Science, Hokkaido University,

Sapporo, Japan

(Received 2 March 1982)

Abstract--The activity of phosphofructokinase in fat body of glycerol-accumulating insects (diapausing pupae of Papilio machaon and diapausing prepupae of Monemaflavescens) was twenty-fold higher than it was in trehalose-accumulating insects (diapausing pupae of Philosamia cynthia and diapausing prepupae of Trichiocamps populi). In addition, the highest activity of glycerol 3-phosphate dehydrogenase was observed in fat body of the Papilio pupae (glycerol-accumulating type) and the lowest activity in fat body of the Trichiocamps prepupae (trehalose-accumulating type), but high activity was also observed in fat body of the Philosamia pupae (trehalose-accumulating type). The above observations strongly suggest that phosphofructokinase acts as a primary key enzyme that regulates the end product, either glycerol or trehalose, of glycogen breakdown during diapause; the relatively high activity of glycerol 3-phosphate dehydrogenase found in the glycerol-accumulating insects may also facilitate the formation of glycerol.

Key Word Index: Diapause, glycogenolysis, glycerol, trehalose, glycerol 3-phosphate dehydrogenase, phosphofructokinase

I N T R O D U C T I O N

IT IS GENERALLY accepted that, in many diapausing insects, glycogen is converted into sugar alcohol such as glycerol and/or sorbitol (CHINO, 1957, 1958; WYATr and MEYER, 1959; ASAHINA, 1969). However, in other insects (for example, prepupae of Trichio- camps populi and pupae of Philosamia cynthia) sugar alcohols do not accumulate appreciably during dia- pause although haemolymph trehalose concentration is considerably elevated concomitant with a decrease of fat body glycogen (AsAHINA and TANNO, 1964; HAYAKAWA and CHINO, 1981). Thus the diapausing insects so far studied may be categorized into two types in terms of glycogen metabolism during dia- pause; one is the sugar alcohol-accumulating type and the other is the trehalose-accumulating type. It has been reported that exposure of some diapausing insects to low temperature accelerates the production of glycerol or trehalose from glycogen (AsAHINA, 1969; ZIEGLER and WYATT, 1975; SHIMADA, 1981; HAYAKAWA and CHXNO, 1981). In addition, we have demonstrated cold activation of fat body glycogen phosphorylase in the trehalose-accumulating pupae, Philosamia cynthia (HAYAKAWA and CHINO, 1982), in a manner similar to that reported for the glycerol- accumulating pupae, Hyalophora cecropia (ZIEGLER and WYATT, 1975).

The present study was designed to assess the bio- chemical basis of the two types of glycogen catab- olism by examining the relative activities of the poten- tial regulatory enzymes, phosphofructokinase and glycerol 3-phosphate dehydrogenase. Evidence is presented to suggest that phosphofructokinase is the primary key enzyme that determines the end product of glycogen breakdown.

MATERIALS AND M ETHODS

Animals

Diapausing pupae of the silkworm, Philosamia cynthia, and full-grown larvae of the sawfly, Trichiocamps populi, were collected from the field. The sawfly larvae entered diapause at a prepupal stage within several days after col- lection. The diapausing pupae of the butterfly, Papilio machaon and diapausing prepupae of the slug caterpillar, Monema flavescens, were kindly provided by Dr Shimada of this Institute and Dr. Tojo of Saga University, respect- ively.

Chemicals Sodium pyruvate and erythritol were purchased from

Wako Chemical Co., Japan. NADH, ATP, fructose-6-phos- phate, dihydroxyacetone phosphate, bovine serum albumin, aldolase, triosephosphate isomerase and glycerol 3-phosphate dehydrogenase were obtained from Sigma Chemical Co., U.S.A. o-(+)-Trehalose and TRI-SIL "Z" were obtained from Nakarai Chemical Co., Japan and Pierce Chemical Co., U.S.A., respectively. All other chemi- cals were of analytical grade.

Gas-liquid chromatography (GLC) for determining haemo- lymph trehalose and sugar alcohol

Standard erythritol solution (0.1 ml of 0.01% solution) was added to 0.1 ml freshly collected haemocyte free hae- molymph and the mixture was evaporated at 60°C using a nitrogen evaporator. The residue was dried overnight under reduced pressure over P2Os; and 0.4ml TRI-SIL "Z" was added. The solution was heated at 65°C for 2 hr to prepare the trimethylsilylated derivatives. The resulting de- rivatives were then applied to GLC (Shimadzu, model GC-4CMPF) using a glass column, 3m x 3mm i.d. packed with 1.5% (w/w) OV-1 on Chromosorb W. The column was run from 130 to 270 at 5°C/rain.

639

640 YOICHI HAYAKAWA and HARUO CHIN()

Preparation and assay ~?/" #lyeerol 3-phosphate dehydro- ~,]enase

Dissected fat bodies were rinsed gently in saline {0.16 M NaCI, 0.02 M KCI), blotted with a filter paper and weighed. The fresh tissue was homogenized in a chilled motor-driven glass~ Teflon homogenizer in buffer (20 mM Tris-HC1, 0.25 M sucrose, pH 7.5). After centrifugation at 8000 g at 4 'C for 10 min, the infranatant between the pellet and fat-rich top layer was withdrawn and filtered through Whatman No. 2 paper. The filtrate was used as enzyme source. The enzyme was measured by a method based on CHINO (1960). The reaction mixture comprised the follow- ing in a final volume of 1.0 ml: 20mM Tris HCI buffer (pH 7.5), 6mM MgSO,,, 0.04mM NADH. 18mM dihydroxy acetone phosphate. The reaction mixture was prepared freshly for each determination and was pre-incubated at 25 C for 10min. The reaction was started by adding the enzyme preparation and the oxidation of NADH was measured by recording absorbance at 340 nm in Shimadzu UV-240 spectrophotometer. One unit of glycerol 3-phos- phate dehydrogenase activity was defined as lhe amount of enzyme which converts l/l-mole of dihydroxyacetone phosphate per rain in the above conditions.

Preparation and assay q[ phosph~fi'uctokinase In most assays, the enzyme preparation was obtained by

the procedure described for glycerol 3-phosphate dehydro- genase although a different homogenizing buffer (100 mM triethanolamine acetate buffer, pH 7.61 was employed. In some experiments, the enzyme was partially purified by a method slightly modified from UNDERWOOD and NEWS- HOLME (1965); saturated ammonium sulphate was added to the filtrate to produce a 40'}~. saturation. The precipitate collected by centrifugation at 10,000 ,q for 10 rain was redis- solved in homogenizing buffer.

The activity of phosphoffuctokinase was assayed with a coupled enzyme system slightly modified from the method of ATZPODIEN and BODE (1970). It has been reported that phosphofructokinase of locust fat body or flight muscle is considerably inhibited by ATP that itself is one of the sub- strates of this enzyme (WALKER and BAILEY, 1969). In a preliminary experiment, therefore, we tested the effect of ATP on phosphofructokinase of fat bodies of the Philosa- mia and Papilio pupae and the maximum activity was ob- served at about 3.3 mM ATP. Thus, the following con- dition was used throughout the determination of the enzyme activity. The reaction mixture contained the fol- lowing components in a final volume of 1.2ml:167mM triethanolamine acetate buffer {pH 7.6), 8.6 mM MgSO4, 0.33 mM NADH, 3.3 mM ATP, 0.56 units glycerol 3-phos- phate dehydrogenase, 0.56 units aldolase, 0.56 units triose- phosphate isomerase. The reaction mixture was freshly p~e- pared before use and was equilibrated to 25'C for 10 rl~n prior to assay. The reaction was started by adding NADH and the oxidation of NADH was measured by recording absorbance at 340 nm in a Shimadzu UV-240 spectropho- tometer. One unit of activity catalyzed the formation of 1 it-mole fructose 1.6-diphosphate per min under the con- ditions described.

Protein determination The protein content of enzyme solution (lO #l) was deter-

mined by the Bio-Rad assay with bovine serum albumin as standard.

RESULTS

Preliminary experiments using two species (P. cyn- thia and P. machaon pupae) confirmed that conver- sion of fat body glycogen into trehalose or glycerol is considerably accelerated by exposure of these insects to low temperature (2°C) and that the concomitant

activation of glycogen phosphorylase in fat body always occurs, irrespective of the end product of gly- cogen breakdown (data not shown, see also HAYAK- AWA and CHINO, 1981, 1982}.

Glycerol 3-phosphate dehydrogenase

Low activity of glycerol 3-phosphate dehydro- genase in trehalose-accumulating insects is likely to impair the formation of glycerol 3-phosphate from dihydroxyacetone phosphate and therefore, reduce the accumulat ion of glycerol.

Preliminary experiments indicated thak unlike gly- cogen phosphorylase, the activities of glycerol 3-phos- phate dehydrogenase and phosphofructokinase are not affected appreciably by the temperature to which the diapausing insects are exposed. Therefore, the enzyme was prepared from diapausing pupae or pre- pupae that had been kept at room temperature (18.-24 C).

The experimental data given in Table l demon- strate that glycerol 3-phosphate dehydrogenase is present in the fat body of all species tested, a l though the activity of the enzyme varies according to species. Highest activity is demonst ra ted in the P. machaon pupae (glycerol-accumulating type) and very low ac- tivity in the T. populi prepupae (trehalose-accumulat- ing type), but high activity was also observed in the fat body of the P. cynthia pupae (trehalose-accumulat- ing type). Thus, this enzyme does not appear to have an impor tan t role as a key enzyme in regulating the end product of glycogen breakdown during diapause.

With dihydroxyacetone phosphate as substrate, no difference in the K~ value was observed with prep- arat ion from P. machaon pupae and P. cynthia pupae.

PhosphoJ?uctokinase

Phosphofructokinase as well as glycogen phos- phorylase is generally considered to function as a key enzyme of glycolysis in many organisms and there- fore, may serve also to regulate the end product of glycogen breakdown during diapause. The specific ac- tivities of fat body phosphofructokinase of four dia- pausing insects are shown in Table 2. A consistent difference in the specific activity is evident between the two categories of insects, with trehalose-accumu- lating species demonst ra t ing only one twentieth of the activity observed in glycerol-accumulating insects. This difference was retained after the enzyme was par- tially purified. The data in Table 2 also demonstra te practically no difference in the K,,~ value between the two types with fructose 6-phosphate as substrate, thus indicating that the differences in specific activity are not due to differences in Km.

The relationship between phosphofructokinase ac- tivity and trehalose accumulat ion was examined in Trichiocamps populi, which were collected at the full grown larval stage and entered diapause within a few days after collection. As shown in Fig. 1, the accumu- lation of trehalose in haemolymph of this insect occurs without prior exposure of the animals to cold; the haemolymph trehalose level at the larval stage was rather low (less than 3 5 mg/ml) and it increased steadily, even at room temperature (18-24~'C), to reach a maximum level (60 70 mg/ml} at about eight to ten days after collection. The elevation of trehalose concentrat ion occurs concomitant ly with decreased

Phosphofructokinase and diapause

Table 1. Specific activity and K m value with dihydroxyacetone phosphate of fat body glycerol 3-phosphate dehydrogenase

641

Specific activity K,. Types Insects (U/rag protein + S.D3 (raM)

Glycerol-accumulating Papilio machaon 4.12 + 0.43 0.09, 0.12 type Monema flavescens 0.891 _+ 0.239 - -

Trehalose-accumulating Philosamia cynthia 0.419 + 0.184 0.10, 0.13 type Trichiocamps populi 0.091 +_ 0,004 - -

Insects were placed at room temperature (18-24°C) and were used ten days or later after the beginning of diapause or pupation. Values of specific activities are means _+ S.D. of five determinations. The direct results from two determinations are given for Km values.

Table 2, Specific activity and Km value with fructose 6-phosphate of fat body phosphofructokinase

Specific activity (U/g protein Km

Types Insects Preparation + S.D.) (raM)

f Crude 127 _ 32(n = 5) 0.56 _+ 0.17 Papilio machaon

Glycerol accumulating t PPT 206 (n -- 2) 0.60 type

Monemaflavescens Crude 148 _ 21 (n = 6)

f Crude 6.3 + 2.1 (n = 6) 0.55 _+ 0.21

Trehalose accumulating Philosamia cynthia [ PPT 14 + 5.8 (n = 6) 0.62 +_ 0.49

type Trichiocamps populi Crude 7.9 _+ 1.6(n = 7) 0.66 + 0.31

Each animal was placed under the nearly same condition as that for glycerol 3-phosphate dehydro- genase. Values of specific activities are means +_ S,D. of experiments (experimental number in paren- theses), K,, values are means -+_ S,D. of three experiments. PPT, precipitate by ammonium sulphate. Other explanations as in Table 1,

activity of phosphofructokinase in the fat body and the low activity of this enzyme persists throughout diapause,

DISCUSSION

In a previous paper (HAVAKAWA and C~IINo, 1981), we proposed that diapausing insects may be classified according to the pattern of carbohydrate metabolism that they exhibit during diapause; thus, those that accumulate sugar alcohol constitute one type and those that accumulate trehalose form the other type. It has also been demonstrated in many diapausing insects that glycogen phosphorylase acts as a key enzyme and its activation is essential for the initiation of glycogen breakdown, irrespective of the nature of the end product (YAMASHITA etal . 1975; ZIEGLER and WYATT, 1975; I"IAYAKAWA and CmNO, 1982).

The present study was designed to determine the biochemical basis for the observed dichotomy of end product resulting from phosphorylase-initiated glyco- lysis. This study has examined the relative specific activities of two potential regulatory enzymes, gly- cerol 3-phosphate dehydrogenase and phosphofructo- kinase, in two trehalose-accumulating and two glycer- ol-accumulating species.

The data on glycerol 3-phosphate dehydrogenase appear to suggest that diapausing insects which have

3O 2e 10

• "- 20 .~_ w

1

o f °

I I .

(A)

o

O

O ~ c , o , , o o 6 o

0 5 lo 15 Days after collection

Fig. 1, Changes in haemolymph trehalose content (A) and fat body phosphofructokinase activity (B) of Trichiocamps populi at various times following collection in the field, Full grown larvae collected from the field were kept at room temperature until use, and entered prepupal diapause within a few days after collection, Each point represents the value of one determination in which five to ten animals

were used,

642 YO1CHI HAYAKAWA and HARt ~ ('l~llNo

G Iyc,o gen ~ Iqmwkw,~u

G- I -P ~ UDPG -, . . . . . Trehalose it J G-6-P ' ~t

F-6-P A're \ 1 i~ ~ m o ~ ] ~ Trehalose jep1~jy acc~vm~l~ting t y p e

F-I 6-DP

, , A,_ = Glyceraldehyde 3-P . . . . . . Dihydroxyacelone-P

-":Jl OIAO,.I I e~wo! :1-Nnpato do~ydeqa~

V l Glycerol 3 4

Glycerol GI ycerol accumulat ing type

Fig. 2. Glycogenolysis during diapause. The reaction is blocked at step of phosphofructokinase (star) in the treha- lose-accumulating insects while it proceeds beyond this

step in the glycerol-accumulating insects.

high glycerol 3-phosphate dehydrogenase activity in the fat body accumulate glycerol whereas insects which have low activity of this enzyme accumulate trehalose. However, the results are not consistent, and high activity is detected also in the trehalose-accumu- lating insect, P. cynthia (Table 1). Thus, it seems that glycerol 3-phosphate dehydrogenase is not the enzyme which is primarily responsible for regulating the end product of glycogen breakdown during dia- pause. By contrast, the observed twenty-fold differ- ence in the activity of phosphofructokinase in fat body occurs consistently between the two types of insects with very high activity observed in glycerol- accumulating insects but extremely low levels in tre- halose-accumulating insects. The low activity of phos- phofructokinase in the latter may cause an interrup- tion of glycolysis at this step and may result in the accumulation of fructose 6-phosphate. This inter- mediate would then be available to form trehalose via glucose 6-phosphate and glucose 1-phosphate (see Fig. 2). When phosphofructokinase activity is suffi- ciently high as observed for glycerol-accumulating insects, glycogenolysis proceeds beyond this step without any interruption and finally produces glycerol with the aid of a relatively high activity of glycerol 3-phosphate dehydrogenase. The above interpretation is supported by the observation that in trehalose-accumulating insects such as T. populi pre- pupae, the trehalose accumulation in haemolymph occurs concomitantly with the rapid fall of phospho- fructokinase activity in the fat body (Fig. 1).

Another possibility is that trehalose synthase may be absent in the fat body of glycerol-accumulating insects. However, this seems very unlikely because a small amount of trehalose (about I mg/ml) is always observed in haemolymph of the diapausing pupae of

P. machaotl (see also HAYAKAWA and ('HIN(), 1982). In conclusion, fat body phosphofructokinase acts as a primary key enzyme to regulate the end product of glycogen metabolism during diapause, either glycerol or trehalose, whereas glycerol 3-phosphate dehydro- genase is of minor importance in this regulation but high activily of this enzyme is required for gb'cerol formation.

Ackm~wled~jonents The aulhors wish to thank Dr. R. G. H. DOWNER, University of Waterloo, for reading the manuscript. This study was partly supported by a research grant from Ministry of Education of Japan (434037).

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