Effect of the insect parasite Hyposoter exiguae (Viereck) on the carbohydrate metabolism of its host, Trichoplusia ni (Hübner)

J. Insect Physiol. Vol. 32, No. 4. pp. 287-293, 1986 Printed in Great Britain. All rights reserved

0022-1910/86 $3.00 + 0.00 Copyright 0 1986 Pergamon Press Ltd

EFFECT OF THE INSECT PARASITE HYPOSUTER EXIGUAE (VIERECK) ON THE CARBOHYDRATE METABOLISM OF ITS HOST, TRICHOPLUSIA NI (HUBNER) S. N. THOMPSON Division

of Biological

Control,

University

of California,

Riverside,

CA 92521 U.S.A.

Abstract-Parasitization of Trichoplusiu ni by Hyposoter exiguue resulted in elevations in the haemolymph trehalose concentration and fat body glycogen level. These increases in tissue carbohydrate reserves were accompanied by an elevation in the maximal velocities of fat body phosphofructokinase and fructose 1,6 bisphosphatase. The activity of the latter, however, was several-fold greater than that of phosphofructokinase and the potential gluconeogenic flux through the fructose phosphate step was, therefore, markedly increased following parasitization. The changes in adenylate levels in parasitized fat body, specifically an increase in AMP and decrease in ATP, were consistent with an elevation in phosphofructokinase activity in viva but not in fructose I,6 bisphosphatase activity. The latter enzyme, however, was shown to be less sensitive to both AMP and fructose 2,6 bisphosphate inhibition in fat body of parasitized individuals. Decreased haemolymph glucogenic amino acid concentrations and an increase in faecal uric acid level accompanied the above effects. It was concluded that parasitization of T. ni by H. exiguae caused a stress-induced hyperglycemia that may result from an alteration in the hormonal regulation over carbohydrate synthesis. Key Word Index: metabolism

Hyposoter

exiguae,

parasitized,

Trichoplusia ni, insect parasite,

carbohydrate

ried out with ease. Following parasitization, insects were placed back in the cups for development. All experimental rearing was carried out at 30°C with a 12 h photoperiod Parasite larvae develop free within the haemocoel of the host and the lst, 2nd and early 3rd instars feed on the host haemolymph (Jowyk and Smilowitz, 1978). Larvae then begin to feed on the fat body and towards the end of their larval development only the gut of the host remains. At the time of emergence the fully developed parasite is comparable in size to the host larvae which dies with only the integument intact.

INTRODUCTION

Qualitative and quantitative changes in tissue methbolite levels occur commonly in animals following parasitic infection (Thompson, 1983a). Such changes reflect alterations in the pattern of metabolism. Although Von Brand (1979) referred to these collectively as “metabolic disturbances” and although some contribute to and reflect pathogenesis, many appear adaptive. Moreover, some physiological and metabolic alterations appear to have provided factors upon which natural selection has acted with the result that the parasite has ultimately derived benefit from the host response. In such cases, host response appears to have assured the survival of the host throughout the course of infection while the parasite is supplied with the factors essential for its successful development. The present report describes the results of studies on the effects of parasitization by the solitary insect endoparasite, Hyposoter exiguae; on the carbohydrate reserves and associated metabolic factors in the host, Trichoplusia ni. The investigations were carried out during the early period of the parasite’s development and prior to any direct host tissue destruction.

Haemolymph

preparation

Larvae were bled by severing the first pair of prolegs and collecting the exuding haemolymph into capillary tubes. Plasma was prepared by centrifuging haemolymph for 2 min in an IEC micro-haematocrit centrifuge. All subsequent analyses were made with plasma rather than whole haemolymph. Non -glycogen

carbohydrate

determination

and

analysis

Non-glycogen carbohydrate was determined by modification of the procedure described by Van Handel (1965a). Samples of haemolymph plasma, 5 ~1, were added to 0.5 ml of 95% ethanol in 1.5 ml polypropylene centrifuge tubes. Tubes were centrifuged at 11,500 rpm for 4 min in a Beckman microfuge B. The supernatant fluid was removed and added to 3 ml of anthrone reagent (500ml cont. H,SO,, 145 ml water, 0.5 g anthrone) in 12ml test tubes. Samples were then heated at 80°C for 45 min in a water bath. After cooling the optical density was

MATERIALS AND METHODS Culture

Stock colonies of the host, Trichoplusia ni, were reared on the artificial diet developed by Shorey and Hale (1965). Host insects were parasitized late during the 3rd stadium. This stage provided suitable rates of successful parasitization and host larvae were large enough that experimental procedures could be car287

288

S. N.

THOMPSON

determined at 620 nm in a Beckman model 25 spectrophotometer. Samples were compared with glucose standards for quantification. Qualitative analysis of sugars in deproteinized plasma was carried out by unidimensional thin-layer chromatography on 250pm layers of Kieselguhr G buffered in sodium phosphate at pH 5 (Waldi, 1965). Visualization was made by spraying developed thin layers with 50% (v/v) sulphuric acid and charring at 115°C for 5 min. The presence of reducing sugars was verified by spraying with aniline-diphenylaminephosphoric acid reagent. Identifications were made by co-chromatography with standards. Amino acid analysis

Physiological fluid analysis was carried out on haemolymph plasma deproteinized in 2.5% sulphosalicylic acid. Samples were applied to a Beckman 6300 amino acid analyzer employing a tri-buffer lithium system. Detection was made with ninhydrin and sample peaks were identified and quantified by comparison with integrated standards. Faecal uric acid analysis

Faecal samples approx 1.0 mg were ground in 10 ml of 0.5% lithium carbonate solution and centrifuged at low speed for 2 min. Nitrogenous excretory products in the supernatant were analyzed by high pressure liquid chromatography as described by Cohen (1983). Detection of purines was made at 254nm. Peaks were compared with standards and were quantified by integration. Fat body preparation

Dissections of fat body were carried out after making a longitudinal mid-ventral incision along the entire body length and carefully pinning back the cuticle. The exposed fat body was washed with physiological saline and small sections removed with forceps onto 25 mm Nucleopore polycarbonate membrane filters. The filters were carefully rinsed and the excess water removed under vacuum. The weight of fat body samples was determined gravimetrically. Glycogen analysis

Fat body glycogen was estimated by modification of the procedure described by Van Handel (1965b). Individual filters with fat body were placed in 12 ml glass centrifuge tubes and 0.4ml of 30% potassium hydroxide added. The tubes were then heated in a water bath at 100°C for 15 min to digest the fat body and after cooling 0.1 ml of saturated sodium sulphate was added to each tube. Glycogen was precipitated by addition of 1 ml of 95% ethanol. After low speed centrifugation the supernatant fluid was discarded and the glycogen-containing pellet redissolved in 0.5 ml of water and 3 ml of anthrone reagent added. The samples were then heated in a water bath at 85°C for 20min and after cooling the optical density was determined at 620 nm as described above. Adenylate analysis

Fat body was excised from insects 3 days after parasitization and was immediately frozen on blocks of dry ice. Frozen tissue was pulverized in a tissue grinder containing 0.5 ml of frozen 3.5% perchloric

acid. Following homogenization, extracts were centrifuged for 5 min at 8730 g and neutralized with 10 N potassium hydroxide. After setting in ice water for a few minutes, the salt precipitate was removed by centrifugation. Analyses were carried by high-pressure liquid chromatography as described by Yamada and Sherman (1980). Adenylate nucleotides were identified by retention time, absorbance ratio and by use of internal standards. Tissue adenylate nucleotide concentrations were based on the level per milligram of total fat body protein. Following perchloric acid precipitation as described above, the resultant pellet was redissolved in 1 N potassium hydroxide, and, after neutralization with hydrochloric acid, the protein content determined by the dye-binding method of Bradford (1976). Enzyme assays

Fat body tissue from animals parasitized for three days was placed in ice-cold 0.2 M triethanolamine buffer, pH 7.5, containing 0.5 mM EDTA and 1.OmM magnesium chloride, and homogenized in a micro-tissue grinder. The homogenate was centrifuged at 1830g for 20 min at 4°C. The supernatant was removed and centrifuged at 37,000 g for 40 min at 4°C. Maximal activities of phosphofructokinase (PFKase) and fructose 1,6 bisphosphatase (FBPase) were determined in the second supernatant fraction immediately after preparation. Protein concentration was determined as above. PFKase was assayed at 30°C by the enzymecoupled NADH oxidation method described by Soling et al. (1970). The rate of change in optical density at 340 nm was monitored continuously with a Beckman 25 spectrophotometer. A 1 ml standard assay 0.02 mmol triethanolamine, mixture contained: 0.5pmol EDTA, l.Opmol MgCl,, 0.62pmol ATP, 2.5 pmol AMP, 0.2 pmol NADH, 0.15 units of aldolase, 5.5 units of triose phosphate isomerase and 1.9 units of glycerol phosphate dehydrogenase. The reaction was initiated by the addition of 3.1 pmol of fructose 6 phosphate (F6P). The effects of substrate, ATP and AMP levels on enzyme activity were examined. FBPase activity was assayed at 30°C by the enzyme-coupled NADP reduction method as outlined by Rosen et al. (1965). The reaction was monitored spectrophotometrically as described above. A 1 ml standard assay mixture contained: 0.02 mmol triethanolamine, 0.5 pmol EDTA, 1.0 pmol MgCl,, 0.5 pmol NADP, 1.7 units glucose 6 phosphate dehydrogenase and 3.5 units phosphoglucose isomerase. The reaction was initiated by addition of 0.2pmol fructose 1,6 bisphosphate (F1,6BP). The effects of substrate, AMP and fructose 2,6 bisphosphate (F2,6BP) were determined. Enzyme velocities were expressed as nmol product formed/min/mg fat body protein. Maximum net gluconeogenic flux was estimated by subtracting the maximal velocity of PFKase from that of FBPase. A maximum gross gluconeogenic flux based on fresh body weight was also calculated after considering the fat body protein level per unit weight. The fluxes reported are “potential” ones only, reflecting the maximal possible gluconeogenic flux

Effects of parasitism on host metabolism

289

I

0

POST

OVIPOSITION

2

4

6

6

(DAYS)

Fig. 1. The effects of parasitization on the tissue carbohydrate levels of Trichoplusiuni parasitized late during the third stadium by the insect parasite, Hyposoter exiguae. (A) Haemolymph non-glycogen carbohydrate concentration. (B) Fat body glycogen level. e-parasitized, O-control.

in potential net gluconeogenic flux. Moreover, the elevated enzyme activities in parasitized individuals were accompanied by an increase in the level of fat body protein per mg fresh body weight. On this basis, the difference in gross gluconeogenic flux between parasitized and control larvae was greater. In the presence of AMP, the activity of phosphofructokinase increased with F6P concentration in a hyperbolic fashion (Fig. 2A). In the absence of AMP, however, the substrate-velocity relationship was sigmoidal in nature and the maximal velocity was significantly reduced. Moreover, the curve was displaced to the right with increased ATP concentration. PFKase activity was maximal at low ATP levels. At higher ATP concentrations inhibition occurred (Fig. 2B). The inhibition by ATP was reduced by increasing F6P concentration and was relieved in the presence of AMP. The activity of FBPase increased rapidly with F1,6BP concentration but substrate became inhibitory above 0.05 mM (Fig. 3A). AMP and F2,6BP were inhibitory even at low concentrations (Fig. 3B, 4). Moreover, the inhibitory effects of either AMP or F2,6BP were enhanced in the presence of the other (Fig. 4). The inhibition brought about by AMP or F2,6BP, however, was in all cases less in the enzyme preparations from parasitized larvae.

through the fructose-phosphate step if the enzymes were operating at their maximal velocities. It is not possible to estimate the actual maximal gluconeogenic flux without knowledge of the activities of the other rate-controlling enzymes of the pathways and identification of the rate-limiting step in vivo. RESULTS

Tissue carbohydrate levels Parasitization increased the haemolymph nonglycogen carbohydrate level in T. ni throughout the entire developmental period (Fig. 1A) (Thompson,

1982a). Chromatographic analysis demonstrated that trehalose comprised the bulk of the non-glycogen carbohydrate (Thompson, 1982a; Thompson and Binder, 1984). The level of fat body glycogen was also elevated in parasitized hosts (Fig. 1B). In this case, however, the concentration decreased sharply after 4 days after infection. This decrease occurred when the parasite began to feed directly on host tissues. Fat body enzyme activities and kinetics

The maximal velocities of both PFKase and FBPase were markedly elevated in T. ni fat body 3 days after parasitization [Table l] (Thompson and Binder, 1984). The changes resulted in a several-fold increase

A

x

‘OOr i-’

[FRUCTOSE

6 PHOSPHATE]

1

mM

[ ADENOSINE

5’ TRIPHOSPHATE]

mM

in the fat body of Trichoplusiani. (A) Effect of substrate concentration on maximal velocity; e-with 1.86mM ATP, O-with 0.62 mM ATP, x -with 0.62 mM ATP and 2.5 mM AMP. (B) Effect of ATP concentration on maximal enzyme velocity; @-with 2.0 mM fructose 6 phosphate, O-with 5 mM fructose 6 phosphate, x -with 5 mM fructose 6 phosphate and 2.5 mM AMP. Fat bodies from 10 individuals were pooled for each assay. (After Thompson and Binder, 1984).

Fig. 2. Kinetic behaviour of phosphofrucktokinase

290

S. N. THOMPSON 0

I

OO

I

I

,

I

I

I

I

I

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

I

0.1

[FRUCTOSE

I ,6

BISPHOSPHATE] mM

J

1.0 [ADENOSINE

5’ MONOPHOSPHATE~~M

Fig. 3. Kinetic behaviour of fructose 1,6 bisphosphatase in the fat body of Trichoplusia ni. (A) Effect of substrate concentration on maximal enzyme velocity. (B) Effect of AMP concentration on maximal enzyme velocity. Fat bodies from 10 individuals were pooled for each assay. (After Thompson and Binder, 1984). Fat body adenylate and haemolymph free amino acid levels

Considerable changes in AMP and ATP levels occurred in T. ni fat body 3 days after parasitization [Table l] (Thompson and Binder, 1984). In general, the level of AMP was increased and ATP reduced. The haemolymph of T. ni had high levels of free amino acids (Table 2). The three major amino acids were proline, histidine and glutamine. Glutamine represented 25% or more of the total. Parasitization resulted in a decline in total haemolymph glucogenic amino acid level and the level of glutamine decreased sharply. Faecal uric acid levels

Uric acid was the major nitrogenous excretory product of T. ni larvae. Xanthine and hypoxanthine, however, were detected in small amounts in some individuals. The level of excreted uric acid was sharply elevated in parasitized T. ni larvae and the highest level occurred 2 days after parasitization [Fig. 51 (Thompson and Cohen, 1984).

DISCUSSION Changes in host tissue carbohydrate levels have been reported during several parasite-host associations involving cestode, trematode, nematode, as well as insect endoparasites (Von Brand, 1979; Thompson, 1983a). In most cases the levels are decreased and this has often been attributed to high rates of carbohydrate consumption by the developing parasite(s). The present finding of elevated carbohydrate levels during the host association of H. exiguae was, therefore, unusual. Similar elevated host

carbohydrate reserves were, however, previously reported by Dahlman and Vinson (1975, 1976) in Heliothis virescens parasitized by the insect parasites Microplitis croceipes or Campolitis sonorensis. With M. croceipes the pattern of increase in host haemolymph trehalose level during the course of infection was similar to that in the T. ni-H. exiguae relationship. In the case of C. sonorensis, however, an increase parasite

in host trehalose was not observed until the moulted to the second instar, 5 days after

parasitization.

Table 1. Activities of phosphofructokinase (PFKase) and fructose 1,6 bisphosphatase (FBPase), potential gluconeogenic flux and adenylate levels in the fat body of Trichoplusia ni 3 days after parasitization late during the third stadium by the insect parasite Hyposoter exiguae. Maximal enzyme velocity

Potential

gluconeogenetic

flux* Adenylate

nmol product formed/min/mg fat body protein Replicate experiments

FBPase

PFKase

Control (4O)t Parasitized (40) % Change

17.9 98.0 + 547

4.5 20.1 +441

Control (15) Parasitized (15) % Change

16.8 98.5 +586

0.8 10.7 +I338

Control (30) Parasitized (30) % Change

11.4 89.3 f783

3.8 21.8 + 574

*Calculated by subtracting the velocity of PFKase tNumbers of pooled individuals in parentheses. #Determined in separate experiments.

Net (nmol glucose formed/min/mg fat body protein) Replicate 13.5 77.9 + 577

Gross (nmol glucose formed/min/g fresh body weight)

levels5

(nmol/mg fat body protein) AMP

ATP

1 19.5 165.9 +850

C(lO)t3.2 P(10) 6.5 flO0

42.2 34.3 -19

Replicate 2 16.0 87.8 + 549

18.0 225.8 +I254

C(10) 3.2 P(10) 4.9 +50

43.8 36.2 -17

Replicate 7.6 67.5 +888

14.0 251.7 +1798

C(10) 5.9 P(10) 11.9 +101

29.2 30.3 0

3

from that of FBPase.

Effects of parasitism on host metabolism

291 B

Fig. 4. The effect of fructose 2,6 bisphosphate (A) and AMP (B) on fat body fructose 1,6 bisphosphatase activity in Trichoplusiu ni parasitized late during the third stadium by the insect parasite, Hyposoter exiguae. e-parasitized, n-control; O-parasitized, [7--control with 2.5 PM F2,6BP (A) and 10 PM AMP (B). Calculation of percent activities following AMP (A) and F2,6BP (B) additions were based on the units of enzyme activity with and without 2.5 PM F2,6BP (A) and 10 PM AMP (B) prior to AMP and F2,6BP addition ai 100% activity.

50 rA

LL

3

POST OVIPOSITION

(DAYS)

Fig. 5. Uric acid excretion by Trichoplusia ni parasitized late during the third stadium by the insect parasite, Hyposoter exiguae. (A) Faecal uric acid concentration: (B) total uric acid excreted. O--control, +--parasitized ( + SE, N = lo), 1X = mean of $prox 30 individuals.

The investigations described in the present report were aimed at determining the contribution of de nova synthesis to blood sugar and the results were consistent with the conclusion that parasitization resulted in a stress-induced hyperglycemia due to a net increase in the rate of gluconeogenesis. Gluconeogenesis, the major pathway for the net synthesis of carbohydrate from non-carbohydrate substrate, occurs via a reversal of the glycolytic scheme. Three steps of the latter pathway, however, including the interconversion of F6P and F1,6BP, are non-equilibrium and irreversible. These three ratecontrolling steps are potentially rate limiting and the enzymes catalyzing the opposing reactions, PFKase for glycolysis and FBPase for gluconeogenesis in the above step, are regulatory. Elevation in the maximal activity of both enzymes in parasitized 7’. ni (Table 1) suggested an increased cycling rate may occur following parasitization (Thompson and Binder, 1984). Moreover, because the activity of FBPase exceeded that of PFKase, the results indicate a sharp increase in the potential rate of gluconeogenesis. The major carbon source for gluconeogenesis is amino acid and the decreased concentration of free amino acids in the haemolymph of T. ni following parasitization suggested that this is providing the substrate for de nova glucose synthesis in the fat body of parasitized individuals (Table 2). The elevated faecal uric acid levels indicated a high level of amino acid deamination (Fig. 5).

The kinetic behaviour of both PFKase and FBPase from T. ni fat body was similar to that described for these enzymes isolated from other gluconeogenic tissues (Thompson and Binder, 1984). The effect of parasitization on adenylate levels, specifically, decreased ATP and increased AMP (Table l), was consistent with an elevation in PFKase activity (Fig. 2). Although the increase in AMP level was inconsistent with an elevated FBPase activity in vivo (Fig. 3), the decreased sensitivity of the latter enzyme from parasitized larvae to both AMP and F2,6BP inhibition (Fig. 4) would explain how the activity of FBPase could be elevated in vivo when the AMP level was simultaneously increased. The basis for this decreased sensitivity in parasitized fat body is unexplained. Studies with mammalian systems, however, which have demonstrated regulation of the fructose phosphate step by glucagon, suggest that hormonal action may be involved and that an upset in hormone production or activity may have resulted in the observed changes in carbohydrate metabolism in parasitized T. ni. The roles of AMP and F2,6BP in regulating gluconeogenesis were described by Van Schaftingen and Hers (1981). Both metabolites were shown to inhibit FBPase purified from rat liver. Moreover, it was shown that both act synergistically such that the inhibition brought about by one was enhanced in the presence of the other. Thus, AMP inhibition was decreased in response to a decreased F2,6BP level.

292

S. N. THOMPSON

Table 2. Haemolymph concentrations

Major glueegenic amino acids Glutamine Histidine Proline Arginine Glycine Serine Lysine Total glucogenic amino acid

of glucogenic amino acids in Trichoplusiani 3 days after parasitization late during the third stadium bv the insect uarasite Hvuosoterexkuae Haemolymph concentration Replicate 2

Replicate 1

(bM,) Replicate 3

Parasitized %

Change

Parasitized %

Change

Parasitized %

Change

19,980 12,960 11,160 9900 9840 11,340

21,675 15,750 11,325 3750 3600 6660 6900

-44 -21 -13 -66 -64 -33 -39

37,250 15,875 7815 9250 5188 8125 15,000

23,475 14,775 9375 2850 3375 6150 7275

-37 -7 +19 -69 -35 -24 -52

40,188 18,250 8688 10,750 5625 8313 11,438

22, I25 16,125 10,350 3000 2775 5850 6900

-45 -12 +19 -72 -33 -30 -40

151,938

95,296

-37

123,659

84,454

-32

134,809

87,147

-35

Control 39,000

Control

Previously, F2,6BP was shown to greatly decrease in hepatocytesincubatedwithglucagon(VanSchaftingen et al., 1980a). In contrast to its effects on FBPase, F2,6BP is a potent stimulator of phosphofructokinase (PFKase) which catalyzes the opposing reaction of this substrate cycle (Van Schaftingen et al., 1980b). Thus, the overall effect of glucagon in stimulating gluconeogenesis at the fructosephosphate step results from an inhibition of PFKase and a stimulation of FBPase through the action of F2,6BP. Elevation in the rate of gluconeogenesis has been shown in other animals to be responsible for observed hyperglycemia during abnormal physiological conditions. For example, Chan et al. (1975) reported increased gluconeogenesis as well as the simultaneous elevation of glycolytic enzyme activity in genetically diabetic mice and suggested glucagon involvement. A hormonal upset was also suggested by Long et al. (1971) to be responsible for the hyperglycemia commonly observed during bacterial infection of human patients, and Rocha et al. (1973) subsequently reported that glucagon levels were elevated during sepsis. Although the changes in carbohydrate metabolism in T. ni following parasitization by H. exiguae were consistent with pathological host response to stress as described above, they may, nevertheless, have adaptive significance for the parasite-host association. In vitro studies with two species of insect parasites have demonstrated that increased dietary sugar levels of the magnitude occurring in the haemolymph of T. ni following parasitization did provide significant nutritional benefit and increased growth rates (Thompson, 1982b, 1983b). Previous studies on the host associations of several parasitic Hymenoptera have demonstrated that many effects of parasitization are caused by virus-like particles transmitted with the egg at the time of oviposition (Krell and Stoltz, 1980). Because the effects of parasitization by H. exiguae are observable within 24 h and prior to hatching of the parasite, it is likely that the H. exiguae-associated virus rather than the developing parasite itself is ultimately responsible for the metabolic changes observed. Indeed, Dahlman and Vinson (1977) reported that elevated haemolymph trehalose levels were observed in H. virescens following injection of a virus particle preparation from M. croceipes.

Control

Unfortunately, few studies are available on the effects of viral infection on intermediary host cell metabolism. In higher animals, however, viruses have been shown to infect pancreatic cells, resulting in diabetes mellitus (Notkins and Yoon, 1984). Because the physiological mechanism by which parasitization alters the carbohydrate metabolism of T. ni may involve hormonal regulation, it may be that the H. exiguae virus alters the activity of certain cells in the insect endocrine system responsible for the regulation of carbohydrate metabolism. In any case, the above studies suggest that parasitization of T. ni by H. exiguae results in a stress-induced hyperglycemia that may have a similar origin to that described in higher animals and, moreover, that this may result from a hormonal imbalance and an alteration in the mechanism of regulation over carbohydrate synthesis. REFERENCES Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. Biochem. 12,

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J. Physiil. i9, i702-1712. Cohen A. C. (1983) A simole, ranid and highlv sensitive method of separation and quantification of -uric acid, hypoxanthine and xanthine by HPLC. Experimentia 39,

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Effects of parasitism on host metabolism Long C. L., Spencer J. L., Kinney J. M. and Geiger J. W. (1971) Carbohydrate metabolism in man: effect of elective operations and major surgery. J. appl. Physiol. 31, 110-116.

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785-786. Van Handel E. (1965a) Estimation of glycogen in small amounts of tissue. Analyt. Biochem. 11, 256-265. Van Handel E. (1965b) Microseparation of glycogen, sugars and lipids. Analyt. Biochem. 11, 266-271. Van Schaftingen E. and Hers H.-G. (1981) Inhibition of fructose I,6 bisphosphatase by fructose 2,6 bisphosphate. Proc. natn. Acad. Sci. U.S.A. 78, 2861-2863.

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