- Email: [email protected]
Effects of reserpine and octopamine on lindane-induced changes in tissue carbohydrate levels in the American cockroach, Periplaneta americana L
PESTICIDE
BIOCHEMISTRY
AND
19, 151-- 156 (1983)
PHYSIOLOGY
Effects of Reserpine and Octopamine on Lindane-Induced Changes in Tissue Carbohydrate Levels in the American Cockroach, Periplaneta americana L. G.L. of Biology,
Department
ORR AND R.G.H.
University
of Waterloo,
DOWNER Waterloo,
Ontario,
N2L
3GI
Canada
Received July 26, 1982; accepted October 22, 1982 Lindane-induced
effects on carbohydrate reserves were studied in adult cockroaches (Perithat were treated 18 hr prior to experimentation with the amine-depleting drug, reserpine. Reserpine treatment eliminates the lindane-induced reduction in hemolymph trehalose and fat body glycogen levels, but does not affect the depression of muscle glycogen that occurs during lindane poisoning. The results are consistent with the proposal that the effects of lindane on carbohydrate metabolism result from lindane-induced release of aminergic modulators of carbohydrate metabolism. Injection of octopamine into reserpine-treated cockroaches at the time of lindane poisoning restores lindane-induced glycogenolysis within fat body and reduces the extent of glycogenolysis within the thoracic musculature during lindane poisoning, but does not influence hemolymph trehalose levels. Studies on glycogenesis in the musculature and the oxidation of radiolabeled glucose in reserpine-treated insects indicate that a reserpine-sensitive factor may be involved in the regulation of substrate utilization by the muscles.
planetu
americana)
INTRODUCTION
Studies on several insect species including Rhodnius prolixus (I), Schistocerca gregaria (2), Calliphora vicina (3), and Periplaneta americana (4-6) indicate that extraneuronal effects of insecticide poisoning may result from the insecticide’s inducing release of (neuro)hormones. For example, in the American cockroach, Periplaneta americana, topical application of lindane (y-hexachlorocyclohexane) causes reduced glycogen levels in fat bodies and thoracic musculature and a concomitant decrease in the hemolymph concentration of anthrone-positive material (6). These changes may be due to lindaneinduced release of hypertrehalosemic factors from neuroendocrine storage sites. Furthermore, the effects were expressed in head-ligated cockroaches, thus negating the corpus cardiacum as the sole site of release of the active factors. The biogenic amine, octopamine, depletes fat body glycogen and elevates hemolymph trehalose in the cockroach
(7, 8) and
has been
located
in the
ventral cord ganglia of this species (9); therefore, octopamine is a possible effector of the lindane effect on tissue carbohydrate levels. Reserpine causes a reduction of biogenic amine levels in mammalian nervous tissue (10) and appears to have a similar effect in invertebrate systems (11, 12). The treatment of locusts with reserpine depletes neural levels of octopamine (13, 14) and similar observations have been obtained in annelids ( 15). The present study examines the effect of lindane on carbohydrate levels in selected tissues of reserpine-treated cockroaches, with a view to determining the role, if any, of biogenic amines in expressing the symptoms of lindane poisoning. The results demonstrate that prior treatment with reserpine abolishes the lindane-induced depletion of hemolymph and fat body carbohydrate reserves, but does not prevent the reduction in muscle glycogen levels that occurs following lindane treatment; furthermore, reserpine treatment may affect 1.51 004%3575183 $3.00 Copyright All rights
Q 1983 by Academic Ress, Inc. of reproduction in any form reserved.
152
ORR
AND
the ability of muscle to use exogenous sources of carbohydrate. MATERIALS
AND
METHODS
Adult male cockroaches were taken between 1 and 3 months after the final molt from a colony of P. americana maintained under standard conditions in this laboratory. Details concerning colony maintenance, procedures for collecting tissue samples, and for flying insects to exhaustion have been described (16- 18). The methods employed to apply insecticide and to analyze tissues for trehalose (anthrone-positive material) and glycogen have also been described (6). Cockroaches were treated with reserpine 18 hr prior to experimentation by injecting a lO-~1 volume containing 50 pg of the drug in 20% ascorbic acid. The 18-hr period is required to deplete amine stores. Octopamine was administered by injecting 10 ~1, 5 X 10e4 M into the abdominal haemocoel. The evolution of 14C02 following injection of o-[U-14C]glucose (325 mCi/mmol; New England Nuclear, Boston, Mass.) was monitored by radiorespirometry using a slight modification of the procedure of Hoffman and Downer (19), with 14C0, collected every 15 min in 5-ml vol of 25% ethanolamine in methanol. Aliquots from each collecting tube were transferred to scintillation vials containing 12 ml scintillation cocktail [42 ml Liquifluor (New England Nuclear); 100 ml Biosolv (Beckman, Palo Alto, Calif.); 1000 ml toluene (3. T. Baker, Phillipsburg, N. J.)] and radioactivity was monitored on a Searle Analytic Mark III, Model 6880 scintillation counter. Data were analyzed for statistical significance using single-factor ANOVA and Dunnett’s one-tailed or two-tailed a posteriori test (20). Transformations, when appropriate, were computed using Taylor’s power law (20). RESULTS
Adult male cockroaches treated topically with 35 pg lindane in 10 ~1 acetone reach
DOWNER
the prostration stage of poisoning within 3.5 hr of lindane treatment; at this stage, hemolymph trehalose levels and glycogen levels in the fat body and thoracic musculature are markedly depressed. Treatment of the insects with reserpine 18 hr before lindane treatment rendered the animals more sluggish, but did not influence the overall behavioral or temporal sequence of poisoning. However, reserpine eliminated the insecticide-induced reduction of carbohydrate reserves in hemolymph and fat body but did not prevent the lindane effect on glycogen levels in thoracic musculature (Fig. 1). The observation that reserpine treatment abolishes the lindane-induced depletion of carbohydrate reserves in hemolymph and fat body supports the suggestion that the lindane effect on carbohydrate is expressed through the mediation of a reserpinesensitive neuroeffector. The biogenic amine, octopamine, is a possible candidate in this regard and, therefore, insects were injected with octopamine at the time of lindane treatment to determine if the addition
FIG.~I. Effect of reserpine treatment and octopamine injection on lindane-induced changes in carbohydrate levels in tissues of Periplaneta americana. Carbohydrates were estimated3.5 hrfohowing topical treatment with lindane. A, hemolymph trehafose; B, fat body glycogen; C, muscle glycogen. Blank boxes indicate lindane-treated insects, solid black boxes denote insects treated with reserpine prior to lindanepoisoning, and striped boxes describe insects treated with reserpine, lindane, and octopamine. Baseline values for control and reserpine-treated insects were not significantly different and are indicated as zero.
EFFECTS
OF
RESERPINE
of octopamine to reserpine-treated cockroaches would restore the hypotrehalosemic and glycogenolytic responses. The results are indicated in Fig. 1 and demonstrate that injection of octopamine does not affect hemolymph trehalose levels in lindane-treated insects that have been injected previously with reserpine; however, octopamine causes pronounced glycogenolysis similar to that observed during lindane-poisoning of cockroaches that were not treated with reset-pine. Figure 1 shows also that octopamine has a slight sparing effect on the lindane-induced depletion of muscle glycogen in normal insects and in those treated previously with reserpine. The results presented in Fig. 1, together with previous studies that relate the period of maximum trehalose depletion from hemolymph with the onset of hyperactivity, suggest that the failure of octopamine to restore lindane-induced hypotrehalosemia to reset-pine-treated insects may be due to the inability of the thoracic musculature in such insects to utilize hemolymph trehalose. This possibility was tested by monitoring the effect of reserpine-treatment on the glycogenic capacity of glycogendepleted muscles. Following flight to exhaustion, glycogen-depleted muscles rapidly take up hemolymph sugar for glycogenesis, thus the amount of glycogen produced under these conditions provides an indirect index of the amount of glucose crossing the muscle membrane. Table 1 demonstrates the glycogen content of thoracic muscles 1 hr after injecting flightexhausted insects with 1.5 mg glucose. The results demonstrate that reset-pine-treated insects incorporate very little glucose into muscle glycogen, whereas normal insects show a significant elevation of muscle glycogen levels within 1 hr. Further evidence in support of the suggestion that reset-pine treatment impairs the uptake of hemolymph carbohydrate by the musculature was obtained by monitoring the evolution of 14C0, following injection of [UJ4C]glucose into the hemocoel of
ON
LINDANE
153
POISONING
TABLE Effect Capacity
of Reserpine of Thoracic
1
Treatment Musculature
on Glycogenic in P. americana
Glycogen concentrationb G.&-e wet wt)
TreatmenP Control Reserpine Reserpine + glucose Control + glucose
6.18 8.06 8.92 12.42
t k r -t
1.82 1.77 (5) 0.63 (8) 1.57 (8)
a Reset-pine or ascorbic acid (control) injected 18 hr prior to insects’ being flown to exhaustion; glucose (1.5 mg) injected 60 minutes prior to dissection of musculature. b Values represent mean ‘- standard error of the mean for the number of determinations shown in parentheses. ANOVA analysis: P = 0.05 (data log transformed); Dunnett’s test (reserpine vs control + glucose): P S 0.05.
reserpine-treated and control cockroaches during the tremor stage of lindane poisoning. It is apparent that the onset of the tremor stage of poisoning results in a pronounced increase in the production of 14C0, in nonreserpine-treated insects, but not in those treated with reset-pine (Table 2). The reduced 14C0, production in reserpinetreated insects may be due to impaired substrate uptake and utilization by the musculature or increased conversion of [14C]glucase into storage reserves by other tissues. Analyses of radioactivity associated with storage reserves in the fat body of reserpine-treated poisoned insects failed to reveal any increased accumulation of label TABLE
2
Effect of Reserpine Treatment on Evolution VO, from Injected [U-W]Glucose during Lindane Poisoning of P. americana
TreatmenP Control + lindane Reset-pine + tindane
of
Percentage increase in WO, evolved* 260 z 118.28 (7)’ 115 2 15.74 (8)
n Reset-pine or ascorbic acid (control) injected 18 hr prior to topical application of lindane. b (dpmmin after onset of tremor stage + dpmimin prior to onset of tremor stage) X 100. r Values represent mean 2 standard error of the mean for the number of determinations shown in parentheses. ANOVA analysis: P = 0.04.
154
ORR
AND
DOWNER
dently of this (these) factor(s). The lindane-induced depletion of muscle glycogen may be attributed to the reserve being used to fuel the increased muscle activity caused by the excitatory action of lindane; DISCUSSION under these conditions, a glycogenolytic flux is likely to result. Lindane-poisoned cockroaches demonstrate a marked reduction of carbohydrate Injection of octopamine into reserpinereserves in the hemolymph, fat body, and treated cockroaches restores the glycogethoracic musculature with the greatest de- nolytic response of fat body to lindane and reduces the extent of muscle glycopletion in hemolymph reserves occurring genolysis due to lindane, but has no efduring the hyperactive stage of poisoning fect on hemolymph trehalose levels. The (6). The pattern of carbohydrate depletion observations support the proposal that ocis consistent with the proposal that fat body glycogen is mobilized as trehalose which topamine is implicated in the expression of enters the hemolymph and is subsequently the lindane effect on carbohydrate. The taken up and oxidized by the thoracic mus- amine has been shown to interact with an culature to provide energy for the increased octopamine-sensitive adenylate cyclase remuscle activity that is associated with lin- ceptor in cockroach fat body and this indane poisoning. Octopamine causes a teraction has been correlated with inglycogenolytic response in cockroach fat creased glycogenolysis (22, 23); therefore, body (7, 8) and enhances the utilization of octopamine is a probable effector of the lindane-induced glycogenolysis in fat body. metabolic substrates in locust musculature (21); therefore, octopamine was suggested The mobilized glycogen is normally reas a possible effector of the lindane effect leased into the hemolymph in the form of on carbohydrate (6). Because reset-pine has trehalose and this sugar may be used as a been shown to deplete octopamine and substrate for the increased metabolic activother biogenic amines in locust brain (13) ity that is associated with lindane poisoning and corpus cardiacum (14) and in the leech (24, 25). However, octopamine does not renervous system (15), the present study on store the marked depletion of hemolymph lindane poisoning in reserpine-treated in- trehalose levels to reserpine-treated poisects is of value in elucidating the possible soned insects that is observed in linrole of biogenic amines in effecting the lin- dane-poisoned insects that have not been dane effect. treated with reserpine, thereby suggesting The results reported in Fig. 1 clearly that another reset-pine-sensitive factor may demonstrate that prior treatment with re- promote the uptake and utilization of treset-pine abolishes the lindane-induced de- halose by tissues and particularly by the pression of hemolymph trehalose and fat thoracic musculature. body glycogen levels, thereby implicating Evidence that a reset-pine-sensitive factor the involvement of a biogenic amine in the enhances the uptake of carbohydrate into expression of these effects. By contrast, re- flight muscle of the cockroach is provided serpine treatment had no effect on the by the observation that reserpine-treated lindane-induced reduction of muscle glyco- insects which have been flown to exhausgen. The results suggest that a reserpinetion demonstrate decreased glycogenic casensitive factor(s) is responsible for medi- pacity in the musculature compared to that ating the mobilization of fat body glyco- of control flight-exhausted insects (Table gen and the utilization of hemolymph 1). The musculature of flight-exhausted intrehalose during lindane poisoning, but that sects is in a state of glycogenic flux and muscle glycogenolysis proceeds indepentakes up hemolymph sugar to be used for in this tissue with the onset of tremors or, indeed, to demonstrate any differences with non-reserpine-treated poisoned cockroaches.
EFFECTS
OF RESERPINE
replenishment of normal resting levels of glycogen. Therefore, the rate of glycogenesis under these conditions reflects the facility with which sugars cross the muscle membrane. During periods of high metabolic demand, the transport of hemolymph carbohydrate into the muscle cells is facilitated by hydrolysis of the disaccharide, trehalose, into the more mobile monosaccharide, glucose (26); thus, the substrate provided in the present study was glucose rather than trehalose. The results presented in Table I clearly indicate that reserpine treatment reduces the amount of glycogenesis that occurs in the musculature, and therefore suggests that a reserpine-sensitive factor serves to enhance the uptake of hemolymph sugar by this tissue. A similar conclusion may be drawn from the study of 14C0, evolution from [UJ4C]glucose during the course of lindane poisoning in control and reserpine-treated cockroaches. In control insects, the onset of the tremor stage of poisoning is accompanied by a marked increase in evolution of 14COp, presumably due primarily to increased metabolism of radiolabeled substrate by the musculature, although metabolism by other tissues undoubtedly con&ributes some 14C0,. Reserpine-treated insects do not show as great an increase in 14C0, production and this may be due to depressed uptake and utilization of the radiolabeled glucose or to an increase in the rate at which other tissues convert 14Cglucose into storage reserves, thus limiting the availability of radiolabeled substrates for muscle metabolism. The fat body is the major storage organ for glycogen and lipid in the cockroach (27) and studies were conducted to determine if the amount of label incorporated into fat body glycogen and lipid reserves during the course of lindane poisoning was affected by reserpine treatment. As indicated under Results, no appreciable differences were observed, therefore, it is probable that the decreased “CO, production in reserpine-treated insects is due to an effect on the musculature.
ON LINDANE
POISONING
155
No attempt has been made to identify the factor(s) responsible for the effects described in Tables 1 and 2, although biogenic amines must be considered the most probable effector( Candy (21) reported that octopamine increases the rate of oxidation of metabolic substrates by thoracic musculature in the locust. Furthermore, the sparing effect of octopamine injection on lindane-induced glycogenolysis in the musculature of reserpine-treated cockroaches could be explained by proposing that octopamine facilitates the uptake of trehalose and thereby decreases the demand for muscle glycogen. This proposal can also accommodate the observation that octopamine fails to restore lindane-induced hypotrehalosemia in reserpine-treated cockroaches because extramusculature metabolic processes are likely to be depressed in such insects, thus reducing the demand of other tissues for trehalose. Another possibility that warrants further investigation is that octopamine may influence muscle metabolism indirectly by modulating the release of a different effector from neuroendocrine storage organs. In conclusion, the present study supports the proposal that the lindane-induced effect on carbohydrate metabolism is mediated by interference with the release of biogenic amines from neuroendocrine storage sites. The amine(s) involved in the expression of the effect has (have) not been identified, although octopamine is probably implicated. ACKNOWLEDGMENTS The authors thank Dr. S. M. Smith for valuable counsel concerning statistical analyses. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. REFERENCES 1. J. E. Casida and S. H. P. Maddrell, Diuretic hormone release on poisoning Rhodnius with insecticide chemicals. Pesfic. Biochem. Physid. 1, 71 (1971). 2. M. Samaranayaka, Insecticide-induced release of
ORR AND DOWNER
156 hyperglycaemic Schistocerca
and adipokinetic gregaria,
Gen.
hormones of Comp.
Endo-
crinol. 24, 424 (1974). 3. T. C. Normann, Release of neurohormones in the blowfly Calliphora vicina with respect to insecticidal action, in “Insect Neurobiology and Pesticide Action,” SC1 Monograph, p. 305 (1980). 4. J. Granett and N. C. Leeling, Trehalose and glycogen depletion during DDT poisoning of American cockroaches, Periplaneta americana, Ann. Entomol. Sot. Amer. 64, 784 (1971). 5. J. Granett and N. C. Leeling, A hyperglycemic agent in the serum of DDT-prostrate American cockroaches, Periplaneta americana, Ann. Entomol. Sot. Amer. 65, 299 (1972). 6. G. L. Orr and R. G. H. Downer, Effect of lindane (y-hexachlorocyclohexane) on carbohydrate and lipid reserves in the American cockroach, Periplaneta americana L., Pestic. Biochem. Physiol. 17, 89 (1982). 7. R. G. H. Downer, Induction of hypertrehalosemia by excitation in Periplaneta americana, J. Insect Physiol. 25, 59 (1979). 8. R. G. H. Downer, Trehalose production in isolated fat body of the American cockroach, Periplaneta americana, Physiol. C 62,31 (1979).
Comp.
leeches. I. Distribution of octopamine in Macand Erpobdella octoculata, Physiol. C 67, 135 (1980). 16. R. G. H. Downer, Interspecificity of lipid-regulating factors from insect corpus cardiacum, Canad. J. Zool. 50, 63 (1972). 17. J. R. Matthews and R. G. H. Downer, Origin of trehalose in stress-induced hyperglycaemia in the American cockroach, Canad. J. Zool. 52, 1005 (1974). 18. R. G. H. Downer and J. R. Matthews, Glycogen depletion of thoracic musculature during flight in the American cockroach, Periplaneta robdella decora Comp. Biochem.
americana,
19.
20. 21. 22.
Biochem.
9. P. D. Evans, Octopamine distribution in the insect nervous system, J. Neurochem. 30, 1009 (1978). 10. A. Carlsson, N. A. Hillarp, and B. Waldeck, Analysis of the dependent storage mechanism in the amine granules of the adrenal medulla, Acta Physiol. Stand. 59 (Suppl. 215), 5 (1963). 11. N. Frontali, Histochemical localization of catecholamines in the brain of normal and drugtreated cockroaches. J. Insect Physiol. 14, 881 (1968). 12. H. A. Robertson and A. V. Jurio, Octopamine and some related noncatecholic amines in invertebrate nervous systems, Int. Rev. Neurobiol. 19, 173 (1976). 13. H. A. Robertson, Octopamine, dopamine and noradrenaline content of the brain of the locust, Schistocerca gregaria, Experientia 32, 552 (1976). 14. I. Orchard and B. G. Loughton, Is octopamine a transmitter mediating hormone release in insects? J. Neurobiol. 12, 143 (1981). 15. R. A. Webb and I. Orchard, Octopamine in
23.
24.
25.
26.
27.
Comp.
Biochem.
Physiol.
B 55,501
(1976). A. G. D. Hoffman and R. G. H. Downer, Evolution of 14C02from 1-**C-acetate in the American cockroach, Periplaneta americana, Comp. Biochem. Physiol. B 48, 199 (1974). J. H. Zar, “Biostatistical Analyses,” Prentice-Hall, Englewood Cliffs, N.J., 1974. D. J. Candy, The regulation of locust flight muscle metabolism by octopamine and other compounds, Insect Biochem. 8, 177 (1978). J. W. D. Gole and R. G. H. Downer, Elevation of adenosine 3’,5’-monophosphate by octopamine in fat body of the American cockroach, Periplaneta americana L., Comp. Biochem. Physiol. C 64, 223 (1979). R. G. H. Downer, Short-term hypertrehalosemia induced by octopamine in the American cockroach, Periplaneta americana L., in “Insect Neurobiology and Pesticide Action”, SC1 Monograph, p. 321 (1980). D. L. Shankland, Action of dieldrin and related compounds on synaptic transmission. in “Neurotoxicology of Insecticides and Pheromones” (T. Narahashi, Ed.), p. 139, Plenum, New York, 1979. R. Pipa and F. Delcomyn, Nervous system, in “The American Cockroach” (W. J. Bell and K. G. Adiyodi, Eds.), p. 175, Chapman and Hall, London, 1981. R. G. H. Downer and J. R. Matthews, Production and utilisation of glucose in the American cockroach, Periplaneta americana, J. Insect Physiol. 23, 1429 (1977). R. G. H. Downer, Fat body and metabolism. in “The American Cockroach” (W. J. Bell and K. G. Adiyodi, eds.) p. 151, Chapman and Hall, London.
BIOCHEMISTRY
AND
19, 151-- 156 (1983)
PHYSIOLOGY
Effects of Reserpine and Octopamine on Lindane-Induced Changes in Tissue Carbohydrate Levels in the American Cockroach, Periplaneta americana L. G.L. of Biology,
Department
ORR AND R.G.H.
University
of Waterloo,
DOWNER Waterloo,
Ontario,
N2L
3GI
Canada
Received July 26, 1982; accepted October 22, 1982 Lindane-induced
effects on carbohydrate reserves were studied in adult cockroaches (Perithat were treated 18 hr prior to experimentation with the amine-depleting drug, reserpine. Reserpine treatment eliminates the lindane-induced reduction in hemolymph trehalose and fat body glycogen levels, but does not affect the depression of muscle glycogen that occurs during lindane poisoning. The results are consistent with the proposal that the effects of lindane on carbohydrate metabolism result from lindane-induced release of aminergic modulators of carbohydrate metabolism. Injection of octopamine into reserpine-treated cockroaches at the time of lindane poisoning restores lindane-induced glycogenolysis within fat body and reduces the extent of glycogenolysis within the thoracic musculature during lindane poisoning, but does not influence hemolymph trehalose levels. Studies on glycogenesis in the musculature and the oxidation of radiolabeled glucose in reserpine-treated insects indicate that a reserpine-sensitive factor may be involved in the regulation of substrate utilization by the muscles.
planetu
americana)
INTRODUCTION
Studies on several insect species including Rhodnius prolixus (I), Schistocerca gregaria (2), Calliphora vicina (3), and Periplaneta americana (4-6) indicate that extraneuronal effects of insecticide poisoning may result from the insecticide’s inducing release of (neuro)hormones. For example, in the American cockroach, Periplaneta americana, topical application of lindane (y-hexachlorocyclohexane) causes reduced glycogen levels in fat bodies and thoracic musculature and a concomitant decrease in the hemolymph concentration of anthrone-positive material (6). These changes may be due to lindaneinduced release of hypertrehalosemic factors from neuroendocrine storage sites. Furthermore, the effects were expressed in head-ligated cockroaches, thus negating the corpus cardiacum as the sole site of release of the active factors. The biogenic amine, octopamine, depletes fat body glycogen and elevates hemolymph trehalose in the cockroach
(7, 8) and
has been
located
in the
ventral cord ganglia of this species (9); therefore, octopamine is a possible effector of the lindane effect on tissue carbohydrate levels. Reserpine causes a reduction of biogenic amine levels in mammalian nervous tissue (10) and appears to have a similar effect in invertebrate systems (11, 12). The treatment of locusts with reserpine depletes neural levels of octopamine (13, 14) and similar observations have been obtained in annelids ( 15). The present study examines the effect of lindane on carbohydrate levels in selected tissues of reserpine-treated cockroaches, with a view to determining the role, if any, of biogenic amines in expressing the symptoms of lindane poisoning. The results demonstrate that prior treatment with reserpine abolishes the lindane-induced depletion of hemolymph and fat body carbohydrate reserves, but does not prevent the reduction in muscle glycogen levels that occurs following lindane treatment; furthermore, reserpine treatment may affect 1.51 004%3575183 $3.00 Copyright All rights
Q 1983 by Academic Ress, Inc. of reproduction in any form reserved.
152
ORR
AND
the ability of muscle to use exogenous sources of carbohydrate. MATERIALS
AND
METHODS
Adult male cockroaches were taken between 1 and 3 months after the final molt from a colony of P. americana maintained under standard conditions in this laboratory. Details concerning colony maintenance, procedures for collecting tissue samples, and for flying insects to exhaustion have been described (16- 18). The methods employed to apply insecticide and to analyze tissues for trehalose (anthrone-positive material) and glycogen have also been described (6). Cockroaches were treated with reserpine 18 hr prior to experimentation by injecting a lO-~1 volume containing 50 pg of the drug in 20% ascorbic acid. The 18-hr period is required to deplete amine stores. Octopamine was administered by injecting 10 ~1, 5 X 10e4 M into the abdominal haemocoel. The evolution of 14C02 following injection of o-[U-14C]glucose (325 mCi/mmol; New England Nuclear, Boston, Mass.) was monitored by radiorespirometry using a slight modification of the procedure of Hoffman and Downer (19), with 14C0, collected every 15 min in 5-ml vol of 25% ethanolamine in methanol. Aliquots from each collecting tube were transferred to scintillation vials containing 12 ml scintillation cocktail [42 ml Liquifluor (New England Nuclear); 100 ml Biosolv (Beckman, Palo Alto, Calif.); 1000 ml toluene (3. T. Baker, Phillipsburg, N. J.)] and radioactivity was monitored on a Searle Analytic Mark III, Model 6880 scintillation counter. Data were analyzed for statistical significance using single-factor ANOVA and Dunnett’s one-tailed or two-tailed a posteriori test (20). Transformations, when appropriate, were computed using Taylor’s power law (20). RESULTS
Adult male cockroaches treated topically with 35 pg lindane in 10 ~1 acetone reach
DOWNER
the prostration stage of poisoning within 3.5 hr of lindane treatment; at this stage, hemolymph trehalose levels and glycogen levels in the fat body and thoracic musculature are markedly depressed. Treatment of the insects with reserpine 18 hr before lindane treatment rendered the animals more sluggish, but did not influence the overall behavioral or temporal sequence of poisoning. However, reserpine eliminated the insecticide-induced reduction of carbohydrate reserves in hemolymph and fat body but did not prevent the lindane effect on glycogen levels in thoracic musculature (Fig. 1). The observation that reserpine treatment abolishes the lindane-induced depletion of carbohydrate reserves in hemolymph and fat body supports the suggestion that the lindane effect on carbohydrate is expressed through the mediation of a reserpinesensitive neuroeffector. The biogenic amine, octopamine, is a possible candidate in this regard and, therefore, insects were injected with octopamine at the time of lindane treatment to determine if the addition
FIG.~I. Effect of reserpine treatment and octopamine injection on lindane-induced changes in carbohydrate levels in tissues of Periplaneta americana. Carbohydrates were estimated3.5 hrfohowing topical treatment with lindane. A, hemolymph trehafose; B, fat body glycogen; C, muscle glycogen. Blank boxes indicate lindane-treated insects, solid black boxes denote insects treated with reserpine prior to lindanepoisoning, and striped boxes describe insects treated with reserpine, lindane, and octopamine. Baseline values for control and reserpine-treated insects were not significantly different and are indicated as zero.
EFFECTS
OF
RESERPINE
of octopamine to reserpine-treated cockroaches would restore the hypotrehalosemic and glycogenolytic responses. The results are indicated in Fig. 1 and demonstrate that injection of octopamine does not affect hemolymph trehalose levels in lindane-treated insects that have been injected previously with reserpine; however, octopamine causes pronounced glycogenolysis similar to that observed during lindane-poisoning of cockroaches that were not treated with reset-pine. Figure 1 shows also that octopamine has a slight sparing effect on the lindane-induced depletion of muscle glycogen in normal insects and in those treated previously with reserpine. The results presented in Fig. 1, together with previous studies that relate the period of maximum trehalose depletion from hemolymph with the onset of hyperactivity, suggest that the failure of octopamine to restore lindane-induced hypotrehalosemia to reset-pine-treated insects may be due to the inability of the thoracic musculature in such insects to utilize hemolymph trehalose. This possibility was tested by monitoring the effect of reserpine-treatment on the glycogenic capacity of glycogendepleted muscles. Following flight to exhaustion, glycogen-depleted muscles rapidly take up hemolymph sugar for glycogenesis, thus the amount of glycogen produced under these conditions provides an indirect index of the amount of glucose crossing the muscle membrane. Table 1 demonstrates the glycogen content of thoracic muscles 1 hr after injecting flightexhausted insects with 1.5 mg glucose. The results demonstrate that reset-pine-treated insects incorporate very little glucose into muscle glycogen, whereas normal insects show a significant elevation of muscle glycogen levels within 1 hr. Further evidence in support of the suggestion that reset-pine treatment impairs the uptake of hemolymph carbohydrate by the musculature was obtained by monitoring the evolution of 14C0, following injection of [UJ4C]glucose into the hemocoel of
ON
LINDANE
153
POISONING
TABLE Effect Capacity
of Reserpine of Thoracic
1
Treatment Musculature
on Glycogenic in P. americana
Glycogen concentrationb G.&-e wet wt)
TreatmenP Control Reserpine Reserpine + glucose Control + glucose
6.18 8.06 8.92 12.42
t k r -t
1.82 1.77 (5) 0.63 (8) 1.57 (8)
a Reset-pine or ascorbic acid (control) injected 18 hr prior to insects’ being flown to exhaustion; glucose (1.5 mg) injected 60 minutes prior to dissection of musculature. b Values represent mean ‘- standard error of the mean for the number of determinations shown in parentheses. ANOVA analysis: P = 0.05 (data log transformed); Dunnett’s test (reserpine vs control + glucose): P S 0.05.
reserpine-treated and control cockroaches during the tremor stage of lindane poisoning. It is apparent that the onset of the tremor stage of poisoning results in a pronounced increase in the production of 14C0, in nonreserpine-treated insects, but not in those treated with reset-pine (Table 2). The reduced 14C0, production in reserpinetreated insects may be due to impaired substrate uptake and utilization by the musculature or increased conversion of [14C]glucase into storage reserves by other tissues. Analyses of radioactivity associated with storage reserves in the fat body of reserpine-treated poisoned insects failed to reveal any increased accumulation of label TABLE
2
Effect of Reserpine Treatment on Evolution VO, from Injected [U-W]Glucose during Lindane Poisoning of P. americana
TreatmenP Control + lindane Reset-pine + tindane
of
Percentage increase in WO, evolved* 260 z 118.28 (7)’ 115 2 15.74 (8)
n Reset-pine or ascorbic acid (control) injected 18 hr prior to topical application of lindane. b (dpmmin after onset of tremor stage + dpmimin prior to onset of tremor stage) X 100. r Values represent mean 2 standard error of the mean for the number of determinations shown in parentheses. ANOVA analysis: P = 0.04.
154
ORR
AND
DOWNER
dently of this (these) factor(s). The lindane-induced depletion of muscle glycogen may be attributed to the reserve being used to fuel the increased muscle activity caused by the excitatory action of lindane; DISCUSSION under these conditions, a glycogenolytic flux is likely to result. Lindane-poisoned cockroaches demonstrate a marked reduction of carbohydrate Injection of octopamine into reserpinereserves in the hemolymph, fat body, and treated cockroaches restores the glycogethoracic musculature with the greatest de- nolytic response of fat body to lindane and reduces the extent of muscle glycopletion in hemolymph reserves occurring genolysis due to lindane, but has no efduring the hyperactive stage of poisoning fect on hemolymph trehalose levels. The (6). The pattern of carbohydrate depletion observations support the proposal that ocis consistent with the proposal that fat body glycogen is mobilized as trehalose which topamine is implicated in the expression of enters the hemolymph and is subsequently the lindane effect on carbohydrate. The taken up and oxidized by the thoracic mus- amine has been shown to interact with an culature to provide energy for the increased octopamine-sensitive adenylate cyclase remuscle activity that is associated with lin- ceptor in cockroach fat body and this indane poisoning. Octopamine causes a teraction has been correlated with inglycogenolytic response in cockroach fat creased glycogenolysis (22, 23); therefore, body (7, 8) and enhances the utilization of octopamine is a probable effector of the lindane-induced glycogenolysis in fat body. metabolic substrates in locust musculature (21); therefore, octopamine was suggested The mobilized glycogen is normally reas a possible effector of the lindane effect leased into the hemolymph in the form of on carbohydrate (6). Because reset-pine has trehalose and this sugar may be used as a been shown to deplete octopamine and substrate for the increased metabolic activother biogenic amines in locust brain (13) ity that is associated with lindane poisoning and corpus cardiacum (14) and in the leech (24, 25). However, octopamine does not renervous system (15), the present study on store the marked depletion of hemolymph lindane poisoning in reserpine-treated in- trehalose levels to reserpine-treated poisects is of value in elucidating the possible soned insects that is observed in linrole of biogenic amines in effecting the lin- dane-poisoned insects that have not been dane effect. treated with reserpine, thereby suggesting The results reported in Fig. 1 clearly that another reset-pine-sensitive factor may demonstrate that prior treatment with re- promote the uptake and utilization of treset-pine abolishes the lindane-induced de- halose by tissues and particularly by the pression of hemolymph trehalose and fat thoracic musculature. body glycogen levels, thereby implicating Evidence that a reset-pine-sensitive factor the involvement of a biogenic amine in the enhances the uptake of carbohydrate into expression of these effects. By contrast, re- flight muscle of the cockroach is provided serpine treatment had no effect on the by the observation that reserpine-treated lindane-induced reduction of muscle glyco- insects which have been flown to exhausgen. The results suggest that a reserpinetion demonstrate decreased glycogenic casensitive factor(s) is responsible for medi- pacity in the musculature compared to that ating the mobilization of fat body glyco- of control flight-exhausted insects (Table gen and the utilization of hemolymph 1). The musculature of flight-exhausted intrehalose during lindane poisoning, but that sects is in a state of glycogenic flux and muscle glycogenolysis proceeds indepentakes up hemolymph sugar to be used for in this tissue with the onset of tremors or, indeed, to demonstrate any differences with non-reserpine-treated poisoned cockroaches.
EFFECTS
OF RESERPINE
replenishment of normal resting levels of glycogen. Therefore, the rate of glycogenesis under these conditions reflects the facility with which sugars cross the muscle membrane. During periods of high metabolic demand, the transport of hemolymph carbohydrate into the muscle cells is facilitated by hydrolysis of the disaccharide, trehalose, into the more mobile monosaccharide, glucose (26); thus, the substrate provided in the present study was glucose rather than trehalose. The results presented in Table I clearly indicate that reserpine treatment reduces the amount of glycogenesis that occurs in the musculature, and therefore suggests that a reserpine-sensitive factor serves to enhance the uptake of hemolymph sugar by this tissue. A similar conclusion may be drawn from the study of 14C0, evolution from [UJ4C]glucose during the course of lindane poisoning in control and reserpine-treated cockroaches. In control insects, the onset of the tremor stage of poisoning is accompanied by a marked increase in evolution of 14COp, presumably due primarily to increased metabolism of radiolabeled substrate by the musculature, although metabolism by other tissues undoubtedly con&ributes some 14C0,. Reserpine-treated insects do not show as great an increase in 14C0, production and this may be due to depressed uptake and utilization of the radiolabeled glucose or to an increase in the rate at which other tissues convert 14Cglucose into storage reserves, thus limiting the availability of radiolabeled substrates for muscle metabolism. The fat body is the major storage organ for glycogen and lipid in the cockroach (27) and studies were conducted to determine if the amount of label incorporated into fat body glycogen and lipid reserves during the course of lindane poisoning was affected by reserpine treatment. As indicated under Results, no appreciable differences were observed, therefore, it is probable that the decreased “CO, production in reserpine-treated insects is due to an effect on the musculature.
ON LINDANE
POISONING
155
No attempt has been made to identify the factor(s) responsible for the effects described in Tables 1 and 2, although biogenic amines must be considered the most probable effector( Candy (21) reported that octopamine increases the rate of oxidation of metabolic substrates by thoracic musculature in the locust. Furthermore, the sparing effect of octopamine injection on lindane-induced glycogenolysis in the musculature of reserpine-treated cockroaches could be explained by proposing that octopamine facilitates the uptake of trehalose and thereby decreases the demand for muscle glycogen. This proposal can also accommodate the observation that octopamine fails to restore lindane-induced hypotrehalosemia in reserpine-treated cockroaches because extramusculature metabolic processes are likely to be depressed in such insects, thus reducing the demand of other tissues for trehalose. Another possibility that warrants further investigation is that octopamine may influence muscle metabolism indirectly by modulating the release of a different effector from neuroendocrine storage organs. In conclusion, the present study supports the proposal that the lindane-induced effect on carbohydrate metabolism is mediated by interference with the release of biogenic amines from neuroendocrine storage sites. The amine(s) involved in the expression of the effect has (have) not been identified, although octopamine is probably implicated. ACKNOWLEDGMENTS The authors thank Dr. S. M. Smith for valuable counsel concerning statistical analyses. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. REFERENCES 1. J. E. Casida and S. H. P. Maddrell, Diuretic hormone release on poisoning Rhodnius with insecticide chemicals. Pesfic. Biochem. Physid. 1, 71 (1971). 2. M. Samaranayaka, Insecticide-induced release of
ORR AND DOWNER
156 hyperglycaemic Schistocerca
and adipokinetic gregaria,
Gen.
hormones of Comp.
Endo-
crinol. 24, 424 (1974). 3. T. C. Normann, Release of neurohormones in the blowfly Calliphora vicina with respect to insecticidal action, in “Insect Neurobiology and Pesticide Action,” SC1 Monograph, p. 305 (1980). 4. J. Granett and N. C. Leeling, Trehalose and glycogen depletion during DDT poisoning of American cockroaches, Periplaneta americana, Ann. Entomol. Sot. Amer. 64, 784 (1971). 5. J. Granett and N. C. Leeling, A hyperglycemic agent in the serum of DDT-prostrate American cockroaches, Periplaneta americana, Ann. Entomol. Sot. Amer. 65, 299 (1972). 6. G. L. Orr and R. G. H. Downer, Effect of lindane (y-hexachlorocyclohexane) on carbohydrate and lipid reserves in the American cockroach, Periplaneta americana L., Pestic. Biochem. Physiol. 17, 89 (1982). 7. R. G. H. Downer, Induction of hypertrehalosemia by excitation in Periplaneta americana, J. Insect Physiol. 25, 59 (1979). 8. R. G. H. Downer, Trehalose production in isolated fat body of the American cockroach, Periplaneta americana, Physiol. C 62,31 (1979).
Comp.
leeches. I. Distribution of octopamine in Macand Erpobdella octoculata, Physiol. C 67, 135 (1980). 16. R. G. H. Downer, Interspecificity of lipid-regulating factors from insect corpus cardiacum, Canad. J. Zool. 50, 63 (1972). 17. J. R. Matthews and R. G. H. Downer, Origin of trehalose in stress-induced hyperglycaemia in the American cockroach, Canad. J. Zool. 52, 1005 (1974). 18. R. G. H. Downer and J. R. Matthews, Glycogen depletion of thoracic musculature during flight in the American cockroach, Periplaneta robdella decora Comp. Biochem.
americana,
19.
20. 21. 22.
Biochem.
9. P. D. Evans, Octopamine distribution in the insect nervous system, J. Neurochem. 30, 1009 (1978). 10. A. Carlsson, N. A. Hillarp, and B. Waldeck, Analysis of the dependent storage mechanism in the amine granules of the adrenal medulla, Acta Physiol. Stand. 59 (Suppl. 215), 5 (1963). 11. N. Frontali, Histochemical localization of catecholamines in the brain of normal and drugtreated cockroaches. J. Insect Physiol. 14, 881 (1968). 12. H. A. Robertson and A. V. Jurio, Octopamine and some related noncatecholic amines in invertebrate nervous systems, Int. Rev. Neurobiol. 19, 173 (1976). 13. H. A. Robertson, Octopamine, dopamine and noradrenaline content of the brain of the locust, Schistocerca gregaria, Experientia 32, 552 (1976). 14. I. Orchard and B. G. Loughton, Is octopamine a transmitter mediating hormone release in insects? J. Neurobiol. 12, 143 (1981). 15. R. A. Webb and I. Orchard, Octopamine in
23.
24.
25.
26.
27.
Comp.
Biochem.
Physiol.
B 55,501
(1976). A. G. D. Hoffman and R. G. H. Downer, Evolution of 14C02from 1-**C-acetate in the American cockroach, Periplaneta americana, Comp. Biochem. Physiol. B 48, 199 (1974). J. H. Zar, “Biostatistical Analyses,” Prentice-Hall, Englewood Cliffs, N.J., 1974. D. J. Candy, The regulation of locust flight muscle metabolism by octopamine and other compounds, Insect Biochem. 8, 177 (1978). J. W. D. Gole and R. G. H. Downer, Elevation of adenosine 3’,5’-monophosphate by octopamine in fat body of the American cockroach, Periplaneta americana L., Comp. Biochem. Physiol. C 64, 223 (1979). R. G. H. Downer, Short-term hypertrehalosemia induced by octopamine in the American cockroach, Periplaneta americana L., in “Insect Neurobiology and Pesticide Action”, SC1 Monograph, p. 321 (1980). D. L. Shankland, Action of dieldrin and related compounds on synaptic transmission. in “Neurotoxicology of Insecticides and Pheromones” (T. Narahashi, Ed.), p. 139, Plenum, New York, 1979. R. Pipa and F. Delcomyn, Nervous system, in “The American Cockroach” (W. J. Bell and K. G. Adiyodi, Eds.), p. 175, Chapman and Hall, London, 1981. R. G. H. Downer and J. R. Matthews, Production and utilisation of glucose in the American cockroach, Periplaneta americana, J. Insect Physiol. 23, 1429 (1977). R. G. H. Downer, Fat body and metabolism. in “The American Cockroach” (W. J. Bell and K. G. Adiyodi, eds.) p. 151, Chapman and Hall, London.