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Hyperglycemia produced by Vespa orientalis venom sac extract
TaxJron. 1973, Vol. 13, p). 221-226. PerQamon Press. Printed in Great Britain.
HYPERGLYCEMIA PRODUCED BY VESPA ORIENTALIS VENOM SAC EXTRACT JACOB ISI-IAY
Department of Physiolo~ry and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel (Accepted for publicatfon 17 December 1974) J~cos Lsxev. Hyperglycemia produced by Vespa orlentalis venom sac extract. Toxlcan 13, 221-226, ]975 . Hornet venom sac extract produces hyperglycemia in cats and in hornet hemolymph. The hyperglycemic factor is probably a protein or a protein bound substance, inasmuch as it is not removed by dialysis and is inactivated by heating at 100°C. HoRx1=r venom sac extract (VSE) has been investigated not only because of its inherent toxicity but also because the venom which the hornet injects into man or animals is a direct excretion of the venom sac. Ideally, the toxic properties of the pure venom should be investigated ; however, in view of the technical difficulty of collecting sufficient amounts of pure venom, an alternative procedure is to study VSE and suppose that its properties parallel those of the venom. There have been numerous studies on the properties of VSE from the Oriental hornet . The various effects of VSE which have been studied include its antigenic properties (IsHAY et al., 1971) ; changes induced in striated muscles (SAlvnsnxx et al., 1971) ; general pharmacological activity (EDERY et al., 1973) ; hemolytic activity (Jos13uA and ISHAY, 1973) ; effect on the cardiovascular system (Knri lxsxY et al., 1974); renal damage (SANDBANK et al., 1973) ; hyaluronidase content (ALLA1.uF et al., 1972); amino acids content (IICAIV and ISHAY, 1973) ; catecholamine content (IsHAY et al., 1974a) ; effects on electrical activity of the cat brain (ISHAY et al., 1974b) and the anti-coagulant activity (JOS1iUA and IsHAY, 1975). All of these studies pertained to VSE and its various effects on mammals. The present paper deals with the hyperglycemic effect of hornet VSE in-vivo in cats and in-vitro in hornet hemolymph. MATERIALS AND METHODS Prepmatiai of VSE Oriental hornets (Vespa orlentalis) are traced to their nests in the field by following their diurnal flight patterns . Once the nests are located, entire colonies are placed under ether anaesthesia at mght, when the adult population is concentrated in the nest, and the workers are then Basily removed and frozen (I3F1AY, 1964). Following frcezing of the worker hornets, their venom sac is unsheathed by pulling on the stringer at the tip of their abdomen. This manoeuvre exposes the sting apparatus and the attached, spindle~ha~ed and whitish sac which is placed in a test tube at -15°C. When enough material has been collected, this is ground and homogenized, dialysed overnight against running water at -4"C and lyophilized. 33efore use, the freeze-dried material is dissolved in a 0" 85 M saline solution and centrifuged to remove all solid particles. Experiments with cats Twelve cats, six of each sea, weighing 2" 32 kg were used . The anaesthesia was initially with ether, followed by Phenobarbital (25 mg per kg, i.v.). Control saline solutions and the test VSE solutions at pH 7 were injected via polyethylene cannulae affixed into the femoral vein . Hlood samples from the cats were collected in heparinized tubes and immediately spun down to remove the erythrocytes and ascertain that TOXICON 1975 Yot. 13
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JACOB ISHAY
there was no hemolysis. The Ln, u of the lyophilized VSE solution was determined by its injection i.v. into male albino mice weighing 20 g and was found to be 50 mg per kg. Cats were given i.v. doses ranging from 2 to 10 mg per kg of lyophilized VSE .
Experiments with hornets
Depending on the caste and stage, hornet larvae at instars 4-5 weigh 600-2400 mg . By puncture through the cuticle, 0"3-0"6 ml of larval hemolymph could be aspirated. The hemolymph was collected in tubes chilled to -4°C and used fresh or was stored at -20°C for later use. In every case a small amount of phenylthiourea was added to the hemolymph in order to prevent blackening . Phenylthiourea prevents the activity of tyrosinase, which is present in the hemolymph and acts upon a variety of phenols such as tyrosine, adrenaline, phenol, catechol, dopa and others to form complex dark pigments, or melanins. In all ]0 incubations were made of hemolymph together with YSE at 37°C using 1"5-33 mg of lyophilized material from fresh or pre-frozen hemolym h per ml . Prior to the incubations the hemolymph was spun down at 2000 g to remove cells and other bris .
Test determinatiazr
The glucose level was determined in triplicate on glucose oxidase (GOD) according to the method of in parallel by the ferricysnide reduction method (total reducing substances, TRS) using a Technicon Autoanalyser (Technicon Bulletin Method, 16B) . Uric acid was determined according to the method of EicxHOxrr et al. (1961) ; sodium and potassium with a Baird Atomic Flame Photometer and chlorides--according to the method of $CHALES and SCHALES (1971) . Quantitative determination of free hemoglobin in plasma was performed as an indicator of intravascular hymolysis (CxossY, 1956). Acid hydrolysis was performed with 0" 1 N HCL in sealed ampoules, heated in a boiling water bath for 3 hr . All data were transformed to logarithms (natural) and then analysis performed using a 3-way ANOVA with L = different dosages (lines), T = times and R = repeat determinations . KINGSLEY Snd G6TCHELL (1960), and
RESULTS
The normal values of glucose, urea, K, Na and Cl in three male cats weighing 3"0, 2"6 and 2"8 kg and determined (a) 20 min and (b) 10 min before i.v. injection of 5 ml of 0"85 per cent saline, and (c) and (d) 10 and 30 min after administration were as follows (mean ~ standard deviation) : glucose (mg ~): (a) 214 ~ l2 (GOD), 230 ~ 15 (TRS), (b) 209 ~ 11 (GOD), 235 ~ 16 (TRS), (c) 206 ~ 11 (GOD), 224 f 17 (TRS) and (d) 203 ~ 12 (GOD) and 226 ~ 18 (TRS). Urea (mg ~) : (a) 41 ~ 2, (b) 41 f 2, (c) 41 ~ 2 and (d) 42 ~ 2. Electrolytes (in m~quiv . per 1) : K: (a) 3 "6 ~ 0"2, (b) 3"7 ~ 0" 2, (c) 3" 8 ~ 0"2, (d) 3"8 ~ 0"2. Na: (a) 145 ~ 7, (b) 145 ~ 8, (c) 145 ~ 7 and (d) 146 ~ 7. C1 : (a) 106 ~ 6, ro) 106 ~ 5 and (c)108 ~ 5. The normal values of the same materials in three female cats weighing 2" 8, 2" 8 and 3"2 kg were as follows : glucose (mg ~): (a) 143 ~ 8 (GOD), 153 ~ 9 (TRS), (b) 144 ~ 8 (GOD), 155 ~ 9 (TRS), (c) 141 ~ 8 (GOD), 150 ~ 9 and (d) 142 ~ 9 (GOD), 151 ~ 9 (T'RS) . Urea (mg ~) : (a) 43 ~ 2, (b) 43 ~ 2, (c) 44 ~ 2 and (d) 44 ~ 2. Electrolytes (in m-equiv. per 1) ; K (a) 4"2 ~ 0"1, (b) 4"3 ~ 0" 1, (c) 4"4 ~ 0" 1 and (d) 4"4 ~ 0"1 . Na: (a) 137 ~ 6, (b) 139 ~ 6, (c) 135 ~ 6 and (d) 131 ~ 6. Cl : (a) 112 ~ 6, (b) 113 ~ 6 and (c) 114 ~ 6. Table 1 summarizes the results obtained following the injection of 10 mg per kg VSE into a male cat weighing 2"8 kg. It should be pointed out that in no instance did the feline blood show hymolysis (plasma hemoglobin values in the arterial blood of the cat was less than 7 mg ~). Boiling of the VSE at pH 7 before its injection eliminated all of the described changes. The results of VSE injection into 5 other cats were similar to those shown in Table 1 . There was no uniformity ofthe glucose level in the test cats (the control values of the glucose level varied as much as 100 per cent from cat to cat). Nevertheless, in all instances there was a sharp rise in the glucose, urea and K levels upon injection of VSE. The other electrolytes, however, did not show marked changes in level . Return of the glucose level to normal occurred in 4-9 hr. TOXICON 1973 Yd. 13
Hyperglycemia by Hornet Venom Sac Extract
22 3
TAHLE 1. EFFECT OP HORNET VENOM SAC EXTRACT 1N]ECTED INTRAVENOUSLY ON GLUCOSE, UREA, K, AND CL LEVELS IN THE HLOOD OF A MALE CAT
1V11IIUteS before (-) and after (-I- ) injection of 10 mg perkg VSE - 30 - 20 - 10 ~- 10 ~- 30 + 60 -F120 -I-180 -F300
Glucose (mg ~) GOD TRS 195 (4) 209 (9) 196 (6) 207 (11) 206 (14) 194 (4) 435 (18) 478 (38) 485 (26) 325 (41) 498 (16) 540 (43) 490 (19) 533 (47) 464 (17) 498 (28) 366 (16) 402 (29)
K
Urea (mg ~) 33 (2) 38 (2) 38 (2) 44 (3) 43 (3) 42 (3) 45 (3) 54 (4) 52 (4)
4~6 (0 "2) 4"7 (0 "2) 4"7 (0 "2) 5" 9 (0 " 3) 4"6 (0" 3) 4" 1 (0~2) 42 (0 "2) 5"0 (0 "3) 9"1 (0~4)
Na (m-equiv . per 1) 140 (7 "5) 140 (7"5) 140 (7 "5) 141 (7 "6) 140 (7"7) 138 (7~0) 136 (7 "2) 143 (7 "2) 143 (7ß)
Na
Cl 106 (4 "0) l06 (4 "0) 106 (4~0) 103 (4~1) 108 (4"2) 113 (4 "2) 109 (4 "3) 112 (4 "4) 114 (4 "5)
All data on glucose determined according to the glucose oxidase method (GOD) or by the total reducing substances method (T1tS) arc significantly higher after injection of VSE than before injection (P < 0"0005). The same significant differences were found for urea . The differences in the level of K after the injection of VSE are not uniform but at 10 and 300 min after injection the values are significantly higher (P < 00005). The Na end CL levels did not significantly change . Numbers in brackets represents 1 standard deviation, based on three determinations per mean . "7 -~"6
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FIa. 1. INCUBATION OF HORNET HEMOLYMPFI, AFTER PROLONGED PREEZWG AT -2O°C, WITH DIALYSED HORNET VENOM 9AC EX7RACI' IN DOSES OF S-lOO mg VSE PER 3 mI HEMOLYMPH.
One control and six different doses of VSE were used . Five times of observations and 3 repeats of each value were performed . The effects of the different dosages are significantly different (P < 00005) . There is also asignificant time effect (P < 0"0005). The lines (dosages) can be divided into three groups by a Newman-Keuls multiple range test 1~ (0, S, 10 and 20 mg VSE per ml hemolymph), 4-5 (20 and 30 mg VSE per ml hema lymph), and 5-7 (30, 50 and 100 mg VSEper ml hemolymph) . The mean value of each line increases parallel to the increase in dose o~ venom sac extract (P < 0"003 for this to occur randomly). TIDXICON 1975 Vol. 13
224
JACOB ISHAY
Return to normal of the urea level occurred only after 48 hr. The return of K to its normal level was variable, requiring 6-24 hr. Larval hemolymph that had been stored for two years at -20°C was incubated with lyophilized venom (Fig. 1). Following incubation with VSE, there is an immediate increase of the glucose level, which is dependent on the venom dose . There was, of course, spontaneous, but less marked hyperglycemia with hemolymph alone, a finding noted previously (FISCxL and ISHnY, 1971 ; F~sct« et al., 1974). The same results were obtained by incubation of fresh hemolymph with VSE with the difference that the starting glucose level is lower in fresh hemolymph than in material previously frozen . All samples contained reducing substances (TRS) beside glucose. The ratio of TG :TRS (true glucose as determined by glucose oxidase) was in some cases 95 per cent but usually was between 50 and 75 per cent. Samples of unincubated VSE (19 mg per ml saline), when acid hydrolysed gave a total glucose reading of 10 mg ~. Similar samples of VSE, when incubated at 37°C with saline, gave a constant reading of 10-12 mg ~ glucose. Acid hydrolysis of hemolymph alone, yielded values of 1600 mg ~ glucose. Incubation of hemolymph with fat-body from the vicinity of the venom sacs (10 mg per ml hemolymph) yielded glucose readings close to those of hemolymph alone. This finding rules out the possibility that the hyperglycemic activity of the venom sacs results from their contamination with fat-body tissue possessing such activity. DISCUSSION
Hyperglycemic activity of venoms injected into mammals has been reported earlier. MOHAMEU et al. (1972a) found that dogs treated with a lethal dose of cobra (Naja haje) venom showed a type of diabetes. Similar diabetogenic effects have been produced by Naja nigricollis venom (Moxn~n et a1.,1972b) . Hyperglycemia in rats were produced by scorpion venom but these effects may be mediated by the serotonin present in the venom (MOHAMED et al., 1972c) . The findings in the present study indicate that hornet venom sac extract possesses not only toxic factors, as already shown in previous investigations, but also definite hyperglycemic effects. The hyperglycemia is marked both when VSE is injected i .v. into cats as well as when it is incubated with hornet hemolymph. The rise in glucose level commences shortly after the injection or the incubation and lasts for about 2 hr . In the VSE injected cats, the glucose level drops, after several hr, almost back to normal . Since, the VSE has undergone dialysis, the hyperglycemic effect cannot be attributed to the biogenic amines which are abundant in the venom (HABERMAIVN, 1968 ; OwEx, 1971 ; EnmY et al., 1972 ; ISxAY et al., 1974b). Acid hydrolysis of the venom solution alone, yields low glucose values, so that the glucose cannot derive from precursors in the venom proper . Acid hydrolysis of hornet hemolymph does yield glucose levels higher than those obtained spontaneously in hemolymph alone, but the levels are lower by about 700 mg ~ than that in hemolymph incubated with a high dose of VSE. Saccharolytic enzymes have been detected in hornet venom sacs (Fisexi. et al., 1974) but to the extent that the hemolymph contains poly- and disaccharides which are degraded to glucose by the saccharolytic enzymes, these sugars should also be degraded by acid hydrolysis and should effect the glucose level upon acid hydrolysis. It would seem, therefore, that part of the rise in glucose level in the injected cats, may be attributed to glycogenolysis and the remainder to gluconeogenesis. The latter must be rapid, judging by the rapid rise in blood urea level which accompanies the rise in glucose level. The same considerations should apply also to the recorded rise in the K level of the injected cats . As is known in diabetic TOXICON 1973 Vol. I3
Hyperglycemia by Hornet Venom Sac Extract
225
persons with high blood glucose level, there is also a rise in the level of extracellular K as a consequence of cellular buffering of the metabolic acids ($AY131tS and TRAVLS, 1965 ; VVrr .r .rA~rs,
1968).
VSE appears to contain a hyperglycemic factor which, similar to glucagon, produces a sharp rise in glucose level both in the blood of vertebrates as well as in the hemolymph of the hornet themselves. In order to obtain hyperglycemia, the cats had to be injected with 2~1 mg per kg VSE, which is a sizable dose from the standpoint of hormonal activity. It should be remembered, however, first, that we are not dealing here with a uniform, chemically pure substance, and secondly, that this hyperglycemic substance was derived from an animal which taxonomically is far removed from mammals and one cannot expect it to have mammalian specificity. We know that in incubations of hemolymph with VSE, the hyperglycemia is maintained for a long time, but we have not yet performed similar tests on live insects. There are, however, reports in the medical literature on the serious consequences of hornet sting. Thus SCHALL11i (1964) described one clinical case of a woman in Europe stung by a hornet who was unconscious upon hospitalization. About 7 hr after the sting, her blood glucose level was 124 mg per cent, which is marginally within the normal range, and so also the levels of the various electrolytes . Similarly, JoxAS and SHLJGAR (1964) report on two children stung by a number of Oriental hornets, one of whom died while the other recovered after several days of severe malaise. Both children underwent tests for blood glucose level a few hr after the sting, but in neither was the glucose level elevated. The children did, however, show damage to the liver and kidneys. Possibly, the small number of stings inflicted in all the described cases was not sufficient to cause hyperglycemia (as we are dealing with anaphylactic shock rather than a systemic toxemia) or simply that any hyperglycemia that did occur disappeared by the time the tests were made. In our experiments with cats, the glucose level returned to normal about 4 hr after the injection of VSE. REFERENCES Au er rrF, D., BER, A. and Iseev, J. (1972) Hyaluronidase activity of extracts of venom sacs of a number of Vespinae-Hymenoptera . Comp. Biochem. Physiol. 43, 119. Cxosev, W. H. (1956) A modiftcation of the benzidine method for the measurement of hemoglobin in plasma and urine. Blood 11, 380. EDErtv, H., ISHAY, J., Less, Y. and Grrrare, S. (1972) Pharmacological activity of Vespa orienealis venom. Toxicon 10,13. Ercrrrroxrr, F., ZEIIdeNOWSRY, S., LEtv, E., RUT6NHER0, A. and Fexres, B. (1961) Improvement of the uric acid determination by the carbonate for serum and urine. I. clin. Path. 14, 450. Frscru., J. and Lsrrev, J. (1972) The glucose levels and carbohydrate sutolysis in Vespa oriennaals. Insectes Soc. 18, 203. Frscrrr., J., ISHAY, J. and RrrrExsEac, A. (1974) Poly- and disaccharidases in Vespa orientalis (Vespinae, Hymenoptera). Comp. Bloclrem . Physiol. 48(B), 299. Hws$a~eivx, E. (1968) Biochemie, Pharmacologie anti Toxicologie tier Inhaltstoffe von Hytnenopterengiften. Ergebn . Physiol. 60, 220. Ixerr, R. and Is~r, J. (1973) Free amino acids in haemolymph and venom of the Oriental hornet (Vespa oriennaals). Comp. Biochem. Physiol. 44(A), 949. Isr;wv, J. (1964) Observations sur la biologie de la guêpe orientale Vespa oriennaals F. Insectes Soc. 11, 193. Isrrw, J., Grr-rEx, S. and Frscm., J. (1971) The antigenic proteins in venom of the Oriental hornet (Vespa orienais. Acta pharm. toi. 31, 71 . IS~rAY, J., AvxexeM, Z., Gath~r.n, Y, and Grues, S. (1974a) Catecholamines in social wasps. Comp. Bloelum. Physiol. 4ß(A), 369. Isrrev, J., Less, Y., Bew~r~ct3en, D., GrrrEa, S. and Serrosexx, U. (1974b) The effects of hornet venom sac extract on the electrical activity of the cat brain. Toxicon 12, 159. Joies, W. and StrvaeR, M. (1964) Severe hepatic and renal damage following wasp sting. Dapim Rejuilm (Folia Med.) 12, 3. ToxrcoN w~s vot. tt
226
JACOB 1SHAY
JosIIVA, H. and IsrtAV, J. (1973) Haemolytic activity of Vespa orientalis venom. Act~ pharm. tox. 33, 42 . JosIIVA, H. and ISFIAY, J. (197 The anticoagulant properties of an extract of the Oriental hornet venom sac. Toxicon 13, 11 . KArLINSxv, E., ISFIAY, J. and Grrreit, S. (1974) Oriental hornet venom: effects on cardiovascular dynamic. Toxicon 12, 69 . KINGSLEY, G. R. and GErcHeLL, G. (1960) Glucose oxidase (GOD) method for determination of glucose in blood and other biological fluids . Clin. Chem . 6, 466. MOFiAAfED, A. H., BESKHAROUN, M. A. Snd EL-DAMARAWY, N. A. (1972a) Effects of cobra (Naja haje) venom on blood glucose, blood phosphate and plasma insulin-like activity in dogs . Toxicon 10, 385. Mo,~n, A. H., MoHAn>E'D, F. A. and EL-DAaIwxnwv, N. A. (1972b) Diabetogenic actions of Naja tligricollis venom I. Effects on glucose tolerance plasma insulin-like activity and blood potassium. Toxicon 10,151 . MoxAM~, A. H., HAM-AYOHE, M., BFSKxAxoux, M. A. and EL-DAMAieawv, N. A. (1972c) Glycemic responses to scorpion venom. Toxicon 10, 139. OweN, M. D. (1971) Insect venoms : identification of dopamine and noradrenaline in wasp and bee sting. Experlentta 27, 544. SANDRANK, U., ISHAY, J. and GrrreR, S. (1971) Mitochondrial changes in the guinea-pig muscle after envenomation with Vespa oritntalis venom. Experientia 27, 303. SANDRANK, U., ISHAY, J. and GrI-reie, S. (1973) Kidney changes in mice due to the Oriental hornet (Vespa orientalls) venom. Histologic and electron-microscopic study. Act~ pharmac. tox. 32, 442. SAYERS, G. and TRAVIS, R. H. (1965) Insulin and oral hypoglycemic drugs. In : The Pharmacological Basis of Therapeutics, pp . 1579-1607, (L . S. GOODMAN and A. GII,MAN, Eds.) . New York : Macmillan. ScxALLaR, H. (1964) Anaphylaktischer Schock and Schwerer Hirnachaden als Komplikation eines Hymenopterenstiches . Schweizer Arch. Neurot. Nturochirurg . Psychiat. 94, 92 . SAALE4, O. and $FIALES, S. (1941) Determination of chlorides by mercury nitrate titration method . J. blot. Chem . 140, 879. WILLIAMS, R. M. (1968) Textbook of Endocrinology. London : Saunders .
TOXICON 1973 Vol. 19
HYPERGLYCEMIA PRODUCED BY VESPA ORIENTALIS VENOM SAC EXTRACT JACOB ISI-IAY
Department of Physiolo~ry and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel (Accepted for publicatfon 17 December 1974) J~cos Lsxev. Hyperglycemia produced by Vespa orlentalis venom sac extract. Toxlcan 13, 221-226, ]975 . Hornet venom sac extract produces hyperglycemia in cats and in hornet hemolymph. The hyperglycemic factor is probably a protein or a protein bound substance, inasmuch as it is not removed by dialysis and is inactivated by heating at 100°C. HoRx1=r venom sac extract (VSE) has been investigated not only because of its inherent toxicity but also because the venom which the hornet injects into man or animals is a direct excretion of the venom sac. Ideally, the toxic properties of the pure venom should be investigated ; however, in view of the technical difficulty of collecting sufficient amounts of pure venom, an alternative procedure is to study VSE and suppose that its properties parallel those of the venom. There have been numerous studies on the properties of VSE from the Oriental hornet . The various effects of VSE which have been studied include its antigenic properties (IsHAY et al., 1971) ; changes induced in striated muscles (SAlvnsnxx et al., 1971) ; general pharmacological activity (EDERY et al., 1973) ; hemolytic activity (Jos13uA and ISHAY, 1973) ; effect on the cardiovascular system (Knri lxsxY et al., 1974); renal damage (SANDBANK et al., 1973) ; hyaluronidase content (ALLA1.uF et al., 1972); amino acids content (IICAIV and ISHAY, 1973) ; catecholamine content (IsHAY et al., 1974a) ; effects on electrical activity of the cat brain (ISHAY et al., 1974b) and the anti-coagulant activity (JOS1iUA and IsHAY, 1975). All of these studies pertained to VSE and its various effects on mammals. The present paper deals with the hyperglycemic effect of hornet VSE in-vivo in cats and in-vitro in hornet hemolymph. MATERIALS AND METHODS Prepmatiai of VSE Oriental hornets (Vespa orlentalis) are traced to their nests in the field by following their diurnal flight patterns . Once the nests are located, entire colonies are placed under ether anaesthesia at mght, when the adult population is concentrated in the nest, and the workers are then Basily removed and frozen (I3F1AY, 1964). Following frcezing of the worker hornets, their venom sac is unsheathed by pulling on the stringer at the tip of their abdomen. This manoeuvre exposes the sting apparatus and the attached, spindle~ha~ed and whitish sac which is placed in a test tube at -15°C. When enough material has been collected, this is ground and homogenized, dialysed overnight against running water at -4"C and lyophilized. 33efore use, the freeze-dried material is dissolved in a 0" 85 M saline solution and centrifuged to remove all solid particles. Experiments with cats Twelve cats, six of each sea, weighing 2" 32 kg were used . The anaesthesia was initially with ether, followed by Phenobarbital (25 mg per kg, i.v.). Control saline solutions and the test VSE solutions at pH 7 were injected via polyethylene cannulae affixed into the femoral vein . Hlood samples from the cats were collected in heparinized tubes and immediately spun down to remove the erythrocytes and ascertain that TOXICON 1975 Yot. 13
221
222
JACOB ISHAY
there was no hemolysis. The Ln, u of the lyophilized VSE solution was determined by its injection i.v. into male albino mice weighing 20 g and was found to be 50 mg per kg. Cats were given i.v. doses ranging from 2 to 10 mg per kg of lyophilized VSE .
Experiments with hornets
Depending on the caste and stage, hornet larvae at instars 4-5 weigh 600-2400 mg . By puncture through the cuticle, 0"3-0"6 ml of larval hemolymph could be aspirated. The hemolymph was collected in tubes chilled to -4°C and used fresh or was stored at -20°C for later use. In every case a small amount of phenylthiourea was added to the hemolymph in order to prevent blackening . Phenylthiourea prevents the activity of tyrosinase, which is present in the hemolymph and acts upon a variety of phenols such as tyrosine, adrenaline, phenol, catechol, dopa and others to form complex dark pigments, or melanins. In all ]0 incubations were made of hemolymph together with YSE at 37°C using 1"5-33 mg of lyophilized material from fresh or pre-frozen hemolym h per ml . Prior to the incubations the hemolymph was spun down at 2000 g to remove cells and other bris .
Test determinatiazr
The glucose level was determined in triplicate on glucose oxidase (GOD) according to the method of in parallel by the ferricysnide reduction method (total reducing substances, TRS) using a Technicon Autoanalyser (Technicon Bulletin Method, 16B) . Uric acid was determined according to the method of EicxHOxrr et al. (1961) ; sodium and potassium with a Baird Atomic Flame Photometer and chlorides--according to the method of $CHALES and SCHALES (1971) . Quantitative determination of free hemoglobin in plasma was performed as an indicator of intravascular hymolysis (CxossY, 1956). Acid hydrolysis was performed with 0" 1 N HCL in sealed ampoules, heated in a boiling water bath for 3 hr . All data were transformed to logarithms (natural) and then analysis performed using a 3-way ANOVA with L = different dosages (lines), T = times and R = repeat determinations . KINGSLEY Snd G6TCHELL (1960), and
RESULTS
The normal values of glucose, urea, K, Na and Cl in three male cats weighing 3"0, 2"6 and 2"8 kg and determined (a) 20 min and (b) 10 min before i.v. injection of 5 ml of 0"85 per cent saline, and (c) and (d) 10 and 30 min after administration were as follows (mean ~ standard deviation) : glucose (mg ~): (a) 214 ~ l2 (GOD), 230 ~ 15 (TRS), (b) 209 ~ 11 (GOD), 235 ~ 16 (TRS), (c) 206 ~ 11 (GOD), 224 f 17 (TRS) and (d) 203 ~ 12 (GOD) and 226 ~ 18 (TRS). Urea (mg ~) : (a) 41 ~ 2, (b) 41 f 2, (c) 41 ~ 2 and (d) 42 ~ 2. Electrolytes (in m~quiv . per 1) : K: (a) 3 "6 ~ 0"2, (b) 3"7 ~ 0" 2, (c) 3" 8 ~ 0"2, (d) 3"8 ~ 0"2. Na: (a) 145 ~ 7, (b) 145 ~ 8, (c) 145 ~ 7 and (d) 146 ~ 7. C1 : (a) 106 ~ 6, ro) 106 ~ 5 and (c)108 ~ 5. The normal values of the same materials in three female cats weighing 2" 8, 2" 8 and 3"2 kg were as follows : glucose (mg ~): (a) 143 ~ 8 (GOD), 153 ~ 9 (TRS), (b) 144 ~ 8 (GOD), 155 ~ 9 (TRS), (c) 141 ~ 8 (GOD), 150 ~ 9 and (d) 142 ~ 9 (GOD), 151 ~ 9 (T'RS) . Urea (mg ~) : (a) 43 ~ 2, (b) 43 ~ 2, (c) 44 ~ 2 and (d) 44 ~ 2. Electrolytes (in m-equiv. per 1) ; K (a) 4"2 ~ 0"1, (b) 4"3 ~ 0" 1, (c) 4"4 ~ 0" 1 and (d) 4"4 ~ 0"1 . Na: (a) 137 ~ 6, (b) 139 ~ 6, (c) 135 ~ 6 and (d) 131 ~ 6. Cl : (a) 112 ~ 6, (b) 113 ~ 6 and (c) 114 ~ 6. Table 1 summarizes the results obtained following the injection of 10 mg per kg VSE into a male cat weighing 2"8 kg. It should be pointed out that in no instance did the feline blood show hymolysis (plasma hemoglobin values in the arterial blood of the cat was less than 7 mg ~). Boiling of the VSE at pH 7 before its injection eliminated all of the described changes. The results of VSE injection into 5 other cats were similar to those shown in Table 1 . There was no uniformity ofthe glucose level in the test cats (the control values of the glucose level varied as much as 100 per cent from cat to cat). Nevertheless, in all instances there was a sharp rise in the glucose, urea and K levels upon injection of VSE. The other electrolytes, however, did not show marked changes in level . Return of the glucose level to normal occurred in 4-9 hr. TOXICON 1973 Yd. 13
Hyperglycemia by Hornet Venom Sac Extract
22 3
TAHLE 1. EFFECT OP HORNET VENOM SAC EXTRACT 1N]ECTED INTRAVENOUSLY ON GLUCOSE, UREA, K, AND CL LEVELS IN THE HLOOD OF A MALE CAT
1V11IIUteS before (-) and after (-I- ) injection of 10 mg perkg VSE - 30 - 20 - 10 ~- 10 ~- 30 + 60 -F120 -I-180 -F300
Glucose (mg ~) GOD TRS 195 (4) 209 (9) 196 (6) 207 (11) 206 (14) 194 (4) 435 (18) 478 (38) 485 (26) 325 (41) 498 (16) 540 (43) 490 (19) 533 (47) 464 (17) 498 (28) 366 (16) 402 (29)
K
Urea (mg ~) 33 (2) 38 (2) 38 (2) 44 (3) 43 (3) 42 (3) 45 (3) 54 (4) 52 (4)
4~6 (0 "2) 4"7 (0 "2) 4"7 (0 "2) 5" 9 (0 " 3) 4"6 (0" 3) 4" 1 (0~2) 42 (0 "2) 5"0 (0 "3) 9"1 (0~4)
Na (m-equiv . per 1) 140 (7 "5) 140 (7"5) 140 (7 "5) 141 (7 "6) 140 (7"7) 138 (7~0) 136 (7 "2) 143 (7 "2) 143 (7ß)
Na
Cl 106 (4 "0) l06 (4 "0) 106 (4~0) 103 (4~1) 108 (4"2) 113 (4 "2) 109 (4 "3) 112 (4 "4) 114 (4 "5)
All data on glucose determined according to the glucose oxidase method (GOD) or by the total reducing substances method (T1tS) arc significantly higher after injection of VSE than before injection (P < 0"0005). The same significant differences were found for urea . The differences in the level of K after the injection of VSE are not uniform but at 10 and 300 min after injection the values are significantly higher (P < 00005). The Na end CL levels did not significantly change . Numbers in brackets represents 1 standard deviation, based on three determinations per mean . "7 -~"6
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FIa. 1. INCUBATION OF HORNET HEMOLYMPFI, AFTER PROLONGED PREEZWG AT -2O°C, WITH DIALYSED HORNET VENOM 9AC EX7RACI' IN DOSES OF S-lOO mg VSE PER 3 mI HEMOLYMPH.
One control and six different doses of VSE were used . Five times of observations and 3 repeats of each value were performed . The effects of the different dosages are significantly different (P < 00005) . There is also asignificant time effect (P < 0"0005). The lines (dosages) can be divided into three groups by a Newman-Keuls multiple range test 1~ (0, S, 10 and 20 mg VSE per ml hemolymph), 4-5 (20 and 30 mg VSE per ml hema lymph), and 5-7 (30, 50 and 100 mg VSEper ml hemolymph) . The mean value of each line increases parallel to the increase in dose o~ venom sac extract (P < 0"003 for this to occur randomly). TIDXICON 1975 Vol. 13
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JACOB ISHAY
Return to normal of the urea level occurred only after 48 hr. The return of K to its normal level was variable, requiring 6-24 hr. Larval hemolymph that had been stored for two years at -20°C was incubated with lyophilized venom (Fig. 1). Following incubation with VSE, there is an immediate increase of the glucose level, which is dependent on the venom dose . There was, of course, spontaneous, but less marked hyperglycemia with hemolymph alone, a finding noted previously (FISCxL and ISHnY, 1971 ; F~sct« et al., 1974). The same results were obtained by incubation of fresh hemolymph with VSE with the difference that the starting glucose level is lower in fresh hemolymph than in material previously frozen . All samples contained reducing substances (TRS) beside glucose. The ratio of TG :TRS (true glucose as determined by glucose oxidase) was in some cases 95 per cent but usually was between 50 and 75 per cent. Samples of unincubated VSE (19 mg per ml saline), when acid hydrolysed gave a total glucose reading of 10 mg ~. Similar samples of VSE, when incubated at 37°C with saline, gave a constant reading of 10-12 mg ~ glucose. Acid hydrolysis of hemolymph alone, yielded values of 1600 mg ~ glucose. Incubation of hemolymph with fat-body from the vicinity of the venom sacs (10 mg per ml hemolymph) yielded glucose readings close to those of hemolymph alone. This finding rules out the possibility that the hyperglycemic activity of the venom sacs results from their contamination with fat-body tissue possessing such activity. DISCUSSION
Hyperglycemic activity of venoms injected into mammals has been reported earlier. MOHAMEU et al. (1972a) found that dogs treated with a lethal dose of cobra (Naja haje) venom showed a type of diabetes. Similar diabetogenic effects have been produced by Naja nigricollis venom (Moxn~n et a1.,1972b) . Hyperglycemia in rats were produced by scorpion venom but these effects may be mediated by the serotonin present in the venom (MOHAMED et al., 1972c) . The findings in the present study indicate that hornet venom sac extract possesses not only toxic factors, as already shown in previous investigations, but also definite hyperglycemic effects. The hyperglycemia is marked both when VSE is injected i .v. into cats as well as when it is incubated with hornet hemolymph. The rise in glucose level commences shortly after the injection or the incubation and lasts for about 2 hr . In the VSE injected cats, the glucose level drops, after several hr, almost back to normal . Since, the VSE has undergone dialysis, the hyperglycemic effect cannot be attributed to the biogenic amines which are abundant in the venom (HABERMAIVN, 1968 ; OwEx, 1971 ; EnmY et al., 1972 ; ISxAY et al., 1974b). Acid hydrolysis of the venom solution alone, yields low glucose values, so that the glucose cannot derive from precursors in the venom proper . Acid hydrolysis of hornet hemolymph does yield glucose levels higher than those obtained spontaneously in hemolymph alone, but the levels are lower by about 700 mg ~ than that in hemolymph incubated with a high dose of VSE. Saccharolytic enzymes have been detected in hornet venom sacs (Fisexi. et al., 1974) but to the extent that the hemolymph contains poly- and disaccharides which are degraded to glucose by the saccharolytic enzymes, these sugars should also be degraded by acid hydrolysis and should effect the glucose level upon acid hydrolysis. It would seem, therefore, that part of the rise in glucose level in the injected cats, may be attributed to glycogenolysis and the remainder to gluconeogenesis. The latter must be rapid, judging by the rapid rise in blood urea level which accompanies the rise in glucose level. The same considerations should apply also to the recorded rise in the K level of the injected cats . As is known in diabetic TOXICON 1973 Vol. I3
Hyperglycemia by Hornet Venom Sac Extract
225
persons with high blood glucose level, there is also a rise in the level of extracellular K as a consequence of cellular buffering of the metabolic acids ($AY131tS and TRAVLS, 1965 ; VVrr .r .rA~rs,
1968).
VSE appears to contain a hyperglycemic factor which, similar to glucagon, produces a sharp rise in glucose level both in the blood of vertebrates as well as in the hemolymph of the hornet themselves. In order to obtain hyperglycemia, the cats had to be injected with 2~1 mg per kg VSE, which is a sizable dose from the standpoint of hormonal activity. It should be remembered, however, first, that we are not dealing here with a uniform, chemically pure substance, and secondly, that this hyperglycemic substance was derived from an animal which taxonomically is far removed from mammals and one cannot expect it to have mammalian specificity. We know that in incubations of hemolymph with VSE, the hyperglycemia is maintained for a long time, but we have not yet performed similar tests on live insects. There are, however, reports in the medical literature on the serious consequences of hornet sting. Thus SCHALL11i (1964) described one clinical case of a woman in Europe stung by a hornet who was unconscious upon hospitalization. About 7 hr after the sting, her blood glucose level was 124 mg per cent, which is marginally within the normal range, and so also the levels of the various electrolytes . Similarly, JoxAS and SHLJGAR (1964) report on two children stung by a number of Oriental hornets, one of whom died while the other recovered after several days of severe malaise. Both children underwent tests for blood glucose level a few hr after the sting, but in neither was the glucose level elevated. The children did, however, show damage to the liver and kidneys. Possibly, the small number of stings inflicted in all the described cases was not sufficient to cause hyperglycemia (as we are dealing with anaphylactic shock rather than a systemic toxemia) or simply that any hyperglycemia that did occur disappeared by the time the tests were made. In our experiments with cats, the glucose level returned to normal about 4 hr after the injection of VSE. REFERENCES Au er rrF, D., BER, A. and Iseev, J. (1972) Hyaluronidase activity of extracts of venom sacs of a number of Vespinae-Hymenoptera . Comp. Biochem. Physiol. 43, 119. Cxosev, W. H. (1956) A modiftcation of the benzidine method for the measurement of hemoglobin in plasma and urine. Blood 11, 380. EDErtv, H., ISHAY, J., Less, Y. and Grrrare, S. (1972) Pharmacological activity of Vespa orienealis venom. Toxicon 10,13. Ercrrrroxrr, F., ZEIIdeNOWSRY, S., LEtv, E., RUT6NHER0, A. and Fexres, B. (1961) Improvement of the uric acid determination by the carbonate for serum and urine. I. clin. Path. 14, 450. Frscru., J. and Lsrrev, J. (1972) The glucose levels and carbohydrate sutolysis in Vespa oriennaals. Insectes Soc. 18, 203. Frscrrr., J., ISHAY, J. and RrrrExsEac, A. (1974) Poly- and disaccharidases in Vespa orientalis (Vespinae, Hymenoptera). Comp. Bloclrem . Physiol. 48(B), 299. Hws$a~eivx, E. (1968) Biochemie, Pharmacologie anti Toxicologie tier Inhaltstoffe von Hytnenopterengiften. Ergebn . Physiol. 60, 220. Ixerr, R. and Is~r, J. (1973) Free amino acids in haemolymph and venom of the Oriental hornet (Vespa oriennaals). Comp. Biochem. Physiol. 44(A), 949. Isr;wv, J. (1964) Observations sur la biologie de la guêpe orientale Vespa oriennaals F. Insectes Soc. 11, 193. Isrrw, J., Grr-rEx, S. and Frscm., J. (1971) The antigenic proteins in venom of the Oriental hornet (Vespa orienais. Acta pharm. toi. 31, 71 . IS~rAY, J., AvxexeM, Z., Gath~r.n, Y, and Grues, S. (1974a) Catecholamines in social wasps. Comp. Bloelum. Physiol. 4ß(A), 369. Isrrev, J., Less, Y., Bew~r~ct3en, D., GrrrEa, S. and Serrosexx, U. (1974b) The effects of hornet venom sac extract on the electrical activity of the cat brain. Toxicon 12, 159. Joies, W. and StrvaeR, M. (1964) Severe hepatic and renal damage following wasp sting. Dapim Rejuilm (Folia Med.) 12, 3. ToxrcoN w~s vot. tt
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JosIIVA, H. and IsrtAV, J. (1973) Haemolytic activity of Vespa orientalis venom. Act~ pharm. tox. 33, 42 . JosIIVA, H. and ISFIAY, J. (197 The anticoagulant properties of an extract of the Oriental hornet venom sac. Toxicon 13, 11 . KArLINSxv, E., ISFIAY, J. and Grrreit, S. (1974) Oriental hornet venom: effects on cardiovascular dynamic. Toxicon 12, 69 . KINGSLEY, G. R. and GErcHeLL, G. (1960) Glucose oxidase (GOD) method for determination of glucose in blood and other biological fluids . Clin. Chem . 6, 466. MOFiAAfED, A. H., BESKHAROUN, M. A. Snd EL-DAMARAWY, N. A. (1972a) Effects of cobra (Naja haje) venom on blood glucose, blood phosphate and plasma insulin-like activity in dogs . Toxicon 10, 385. Mo,~n, A. H., MoHAn>E'D, F. A. and EL-DAaIwxnwv, N. A. (1972b) Diabetogenic actions of Naja tligricollis venom I. Effects on glucose tolerance plasma insulin-like activity and blood potassium. Toxicon 10,151 . MoxAM~, A. H., HAM-AYOHE, M., BFSKxAxoux, M. A. and EL-DAMAieawv, N. A. (1972c) Glycemic responses to scorpion venom. Toxicon 10, 139. OweN, M. D. (1971) Insect venoms : identification of dopamine and noradrenaline in wasp and bee sting. Experlentta 27, 544. SANDRANK, U., ISHAY, J. and GrrreR, S. (1971) Mitochondrial changes in the guinea-pig muscle after envenomation with Vespa oritntalis venom. Experientia 27, 303. SANDRANK, U., ISHAY, J. and GrI-reie, S. (1973) Kidney changes in mice due to the Oriental hornet (Vespa orientalls) venom. Histologic and electron-microscopic study. Act~ pharmac. tox. 32, 442. SAYERS, G. and TRAVIS, R. H. (1965) Insulin and oral hypoglycemic drugs. In : The Pharmacological Basis of Therapeutics, pp . 1579-1607, (L . S. GOODMAN and A. GII,MAN, Eds.) . New York : Macmillan. ScxALLaR, H. (1964) Anaphylaktischer Schock and Schwerer Hirnachaden als Komplikation eines Hymenopterenstiches . Schweizer Arch. Neurot. Nturochirurg . Psychiat. 94, 92 . SAALE4, O. and $FIALES, S. (1941) Determination of chlorides by mercury nitrate titration method . J. blot. Chem . 140, 879. WILLIAMS, R. M. (1968) Textbook of Endocrinology. London : Saunders .
TOXICON 1973 Vol. 19