TOXICOLOGY
AND
APPLIED
Further
PHARMACOLOGY
Studies
32,62-12 (1975)
of Methamphetamine-Induced Insulin Release’
E. M. MCMAHON, J. M. FELDMAN~AND S. M. SCHANBERG~ Department of Psychiatry, University of Washington School of Medicine, Seattle, Washington 98195; Veterans Administration Hospital, Durham, North Carolina 27710; and Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 Received June 17,1974; accepted November II,1974
Further
Studies of
Methamphetamine-Induced Insulin Release.
MCMAHON, E. M., FELDMAN, J. M. AND SCHANBERG, S. M. Toxicol. Appl. Pharmacol. 32, 62-72. In the presentstudy we have evaluatedfactors that
modify methamphetamine-induced insulin secretion.While methamphetaminestimulatesinsulinsecretionin intact rats and mice,it stimulatesinsulin secretion in vitro only from mousepancreas.Related drugs such as damphetamine,methylphenidate,and tranylcypromine alsostimulateinsulin secretionin intact rats. Methamphetamine-inducedinsulin secretionis not altered by a-receptorantagonistsor ganglionicblocking agents,but is reduced by B-receptor antagonists.Insulin secretionin responseto methamphetamineis significantly increasedin the hyperthyroid stateand decreased in the hypothyroid state.Chronic methamphetamineadministrationresults in a reduction in the insulin secretedin responseto this agent. We have reported that methamphetamine and amphetamine release insulin from rats and mice in vivo by a mechanism not attributable to hyperglycemia (McMahon et al., 1971). Profound hypoglycemia followed insulin release in mice, although this effect was not observed consistently in normal rats. Similarly, marked species differences in the glycemic response to various drugs have been observed (Feldman and Lebovitz, 1970; Quickel et al., 1971; Mennear et al., 1971), including the effects of methamphetamine on insulin concentrations and carbohydrate tolerance in humans (Sirtori et al. 1971; Citron et al., 1972). The purpose of this report is to characterize further the effect of methamphetamine on insulin and glucose concentrations in the rat. The effect was analyzed in rats with normal or altered thyroid status, after beta-adrenergic receptor blockade, and after a regimen of chronic methamphetamine administration. Insulin responses to methamphetamine were also examined in vitro in the rat, hamster, and rabbit. 1 Portions of this work were supported by U.S. Public Health Service Grants AM-01324, MH13688, MH-11947, and Veterans Administration 2112-01. ‘Dr. Feldman is the recipient of a Clinical Investigatorship (2650-01) from the Veterans Administration. 3 Dr. Schanberg is the recipient of a Career Development Award from the National Institutes of Mental Health, Grant Number K05 MH-06489. 62 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form
reserved.
METHAMPHETAMINE
AND
INSULIN
RELEASE
63
METHODS
Acute Studies-Rats Male Sprague-Dawley rats4 weighing 180 + 10 g were housed in cages 9 by 8 by 6 in, two or three animals per cage, and allowed free access to food and water. Four hours prior to drug administration food was removed and animals were aggregated seven to eight per plastic cage (18 by 10 by 6 in.). Methamphetamine hydrochloride in 0.5 ml of normal saline or saline alone was injected intraperitoneally. At appropriate intervals after injection, rats were killed by decapitation and blood from the carotid arteries was collected in heparinized beakers. Plasma glucose concentrations were measured by a glucose oxidase method (Saifer and Gerstenfeld, 1958). Plasma insulin was measured against mouse insulin standards by a modification of a radioimmunoassay method in which the separation of bound and free insulin was performed by precipitating the bound insulin with a goat anti-serum to guinea pig gamma globulin (Genuth et al., 1965). Methamphetamine had no effect on the measurement of insulin in this system. In some studies, the beta-adrenergic blocking drug propranolol, 0.5 mg/kg,5 was injected intraperitoneally 10 min prior to methamphetamine injection; controls received an equal volume of normal saline at a similar time. To evaluate the effect of ganglionic blockade on methamphetamine-induced insulin release 24 rats were divided into four groups of six animals. Two of the groups received saline pretreatment while the remaining two were pretreated with chlorisondamine chloride.6 Four hours later, one of the saline and one of the chlorisondamine-treated groups received methamphetamine (30 mg/kg, ip). The other two groups were injected again with saline. The animals were killed 15 min later, their blood collected (see above), and the plasma assayed for insulin and glucose. Rats were made hyperthyroid by intraperitoneal administration of Na-l-thyroxine,’ a total of 1.5 mg/kg divided into three equally spaced doses during the 24 hr prior to the experiment. Control animals were injected with an equal volume of saline at corresponding intervals. Surgically thyroidectomized male Sprague-Dawley rats4 were obtained 8-10 days after the operation. These rats weighed 180 f 10 g, 15-20 g less than their age-matched, nonoperated controls. Other rats were made hypothyroid by oral administration of methimazole8 dissolved in drinking water. The concentration was adjusted to achieve an average dose of 10 mg methimazole per rat per day for at least 1 wk. Water consumption did not differ appreciably between animals receiving methimazole and controls receiving water alone. Water was substituted for all methimazole solutions 18 hr prior to these experiments. Plasma calcium determinations were performed by the Duke University Hospital Laboratory of Clinical Chemistry. Chronic Studies-Rats Chronic studies were performed using male Sprague-Dawley rats4 weighing 180 + 10 g, housed three animals per 9 by 8 by 6 in. cage, with free access to food and water. 4 Sprague-Dawley rats obtained from Hormone Assay Corp., Chicago, Illinois. 5 Propranolol (Inderal) obtained from Ayerst Laboratories, Atlanta, Georgia. 6 Chlorisondamine chloride (Ecolid). 7 Na-l-thyroxine obtained from Nutrition Control Products, Division of Pharmex Corp., Hollywood, Florida. 8 Methimazole obtained from Aldrich Chemical Corp., Milwaukee, Wisconsin. 3
64
MCMAHON,
FELDMAN
AND
SCHANBERG
Chronic intraperitoneal administration of methamphetamine was performed as described previously (Schanberg and Cook, 1972) using early morning and late afternoon injections, with the dose increasing 1 mg/kg per injection over a IO-day period. Theinitial dose was 10 mg/kg (20 mg/kg/day) and the final dose on the evening before the experiment was 30 mg/kg (60 mg/kg/day). Control animals received saline injections on a similar schedule; rats were weighed every 3 days for adjustment of drug dosage. The remainder of the experiment was carried out as described for acute studies. Studies of Insulin Release In Vitro Studies of insulin release in vitro were carried out with pancreatic tissue from SwissWebster mice, golden hamsters, New Zealand white albino rabbits, and SpragueDawley rats9 using a slight modification of a previously described incubation system (McMahon et al., 1971; Feldman and Lebovitz, 1970). After an initial 15min preincubation, each piece of pancreas underwent two sequential 30-min incubations, and portions of each incubation medium were assayed for insulin as described above. RESULTS
After methamphetamine injection in the rat, plasma insulin concentrations rose rapidly, increasing 3-500 % over basal values at 15 min (Table 1). This high insulin level decreased rapidly during the next 15 min, but remained eIevated significantly above conTABLE
1
EFFECT OF METHAMPHETAMINE ON PLASMA INSULIN AND GLUCOSE IN THE RAF
Time after injection (min) Experimental group
0
15
Control (saline) Methamphetamine
lOf2
16f3
lOrt2
57 * s*
Control (saline) Methamphetamine
126&4 126+4
124*3 161 + 5*
30
60
Insulin (&J/ml) 14 + 2 15f2 28 + 1” 2112d
90
120
9f7 16f5
17f3 16f 1
Glucose (mg/lOO ml)
118 f 5
118? 4
121f2
129+4
134 + 3c
132+4d
129+5
128+_6
’ Each value representsthe mean + SE of at least eight animals, exceptthe 90-min control group, which contained four animals. Insulin values are expressed as microunits insulin/milliliters plasma; glucose concentrations are expressed as mg glucose/100 ml plasma. The methamphetamine groups received 30 mg/kg ip, in 0.5 ml of normal saline, while controls receive an equal volume of saline. bp < 0.001, different from control group (Students’ t test). ‘p < 0.01, different from control group. “p < 0.05, different from control group. trol values until 60 min after injection. In contrast, plasma glucose concentrations increased only 27 % (Table 1). The effects of various doses of methamphetamine on plasma insulin 15 min after injection are shown in Fig. 1. Methamphetamine caused a dose-related increase in plasma insulin concentration. This effect appeared at dosages as low as 2.5 mg/kg and was maximal after a dosage greater than 10 mg/kg. The effects 9 Sprague-Dawley
rats obtained from Holtzman Company, Madison, Wisconsin.
METHAMPHETAMINE
AND
INSULIN
65
RELEASE
175r
I 1
I
2253
45 DOSE
II
20 25 30
10
11
40 50
(mg /kg wenght)
FIG. 1. Plasma insulin response to methamphetamine 15 min after injection. Male Sprague-Dawley rats were injected intraperitoneally with various doses of methamphetamine in 0.5 ml normal saline, or an equal volume of saline alone, as described in the text. Each value represents the mean f SE of four observations, and is compared (Student’s t-test) with a control group sacrificed at the same time.
of other structurally related drugs were investigated for their ability to release insulin. As shown in Table 2, all of these agents significantly elevated plasma insulin concentrations as compared to controls. In comparison, the effects of these drugs on plasma glucose was inconsistent. Significant increases occurred only after 10 mg/kg methamphetamine and 30 mg/kg d-amphetamine. TABLE 2 PLASMA INSULIN AND GLUCOSE RESPONSE TO STIMULANT DRUGS 15 MIN AFTER INJECTION'
Plasma glucose Plasma insulin Drug and dose (mg/kg)
W/ml)
Saline Methamphetamine, 10 Methamphetamine, 30 d-Amphetamine, 30 Methylphenidate, 30 Tranylcypromine, 20
12+ 44+ 66 + 62f 26 f 37f
1 12b 15” 1’ 3’ 4”
(‘A of salineinjected control)
loof 132+ 117+ 126+ 106+ 108 +
9b 6 6* 6 9
DEach value represents the mean &- SE of four observations and is compared (Student’s I test) with a control group sacrificed at the same time. *p c 0.05, significantly higher than saline-injected control group. c p c 0.01, significantly higher than saline-injected control group. “p < 0.005, significantly higher than salineinjected control group. c p < 0.001, significantly higher than saline-injected control group.
66
MCMAHON, FELDMAN AND SCHANBERG
The effect of pretreatment with phenoxybenzamine (5 mg/kg, ip) or propranolol(0.5 mg/kg, ip) on plasma insulin or glucose response to methamphetamine in the rat is shown in Table 3. Propranolol (B-blocker) but not phenoxybenzamine (a-blocker) significantly decreased methamphetamine-induced insulin release at 15 min. However, neither antagonist altered the methamphetamine-induced increase in plasma glucose. Control studies showed that basal plasma insulin and glucose concentration were not altered by either propranolol or phenoxybenzamine. The effect of chlorisondamine (a ganglionic blocking agent) on methamphetamine-induced insulin release was tested and it can be seen in Table 4 that chlorisondamine had no apparent effect on this system. This dose of chlorisondamine was demonstrated to block the release of catecholamines from adrenal and nerve endings via ganglionic blockade in this strain of rats (T. Slotkin, personal communication). TABLE
EFFECT OFALIRENERGIC BLOCKING
AGENTS
3
INSULINRELEASES
ON METHAMPHETAMINE-INDUCED
Time after methamphetamine injection (15 min)
Experimental Group Plasma insulin Control (saline) Methamphetamine Propranolol and methamphetamine Phenoxybenzamineand methamphetamine
NJ/ml>
Plasmaglucose (mg/l~ ml)
22+ 4 78 + 12*
123f 9 168 i- 7*
32+
149 f 10d
7’
63 f 12*sd
172 f ll*
c Ratswerepretreatedwith propranolol(0.5 mg/kg,ip), phenoxybenzamine(5mg/kg,ip) or normalsaline10minbeforetheyreceivedmethamphetamine(30mg/kg,ip) or anequalvolumeof saline.Eachvaluerepresents the mean+ SEof eightanimals.Prior to injection,the plasmainsulinwas15.8 ,uU/mlandthe plasmaglucosewas124+ 2 mg/lOOml. * p < 0.01,differentfrom control(Student’sI test). cp < 0.05,differentfrom methamphetamine. dNot significantlydifferentfrom methamphetamine.
Plasma insulin 15 min after methamphetamine (30 mg/kg) in hyperthyroid rats and in rats made hypothyroid by surgical thyroidectomy are shown in Table 5. Hyperthyroid rats showed a greater insulin response to methamphetamine than did euthyroid animals. The insulin response to methamphetamine was reduced markedly in hypothyroid animals. Insulin and glucose responses to methamphetamine in rats made hypothyroid with a regimen of methimazole are shown in Table 6. Rats made hypothyroid by this method weighed an average of 40 g less than their age-matched controls, and showed compensatory thyroid hypertrophy (thyroid wet weight in milligrams : hypothyroid = 33.2 L- 0.2, n = 3; euthyroid 20.0 + 1.O, n = 3, p < 0.001). Methimazole-induced hypothyroidism significantly reduced insulin response to methamphetamine at 15 min postinjection and produced a modest hyperglycemia (Table 6).
METHAMPHETAMINE
AND
TABLE EFFECT
OF CHLORWNDAMINE
INSULIN
67
RELEASE
4
ON METHAMPHETAMINE-INDUCED
Experimental group
Saline and saline Saline and methamphetamine Chlorisondamine and saline Chlorisondamine and methamphetamine
INSULIN
RELEASE”
Plasma insulin W/ml> __..26? 7 97 f 17b 28f 5
Plasma glucose (mg/lOO ml)
100 f 13b.c
165 f 12c
150* 9’ 176t 6 142 k 14’
’ Twenty-four rats were divided into two groups and injected with either saline or chlorisondamine (10 mg/kg, ip). Four hours later, half of each group were given saline (1 ml, ip) and the other half methamphetamine (30 mg/kg, ip). Rats were killed 15 min later and their blood plasma assayed for insulin and glucose as described in Methods. Each value represents the mean + SE of six animals. “p c 0.001, different from saline-saline group (Student’s 1 test). c Not significantly different from saline-methamphetamine group.
TABLE 5 EFFECT
OF ALTERED
THYROID
STATUS
ON METHAMPHETAMINE-INDUCED
INSULIN
RELEASE”
Time after injection (min) Treatment
0
Plasmainsulin @U/ml) Salinealone 15+ 8 Methamphetamine 6+ 2 Hyperthyroid-saline 12!1 8 Hyperthyroid-methamphetamine 12+10 Hyperthyroid-propranolol methamphetamine 12+ 10 Hyperthyroid-saline (2.5 Hypothyroid-methamphetamine ~2.5 Salinealone 124f12 Methamphetamine 149+ 5 Hyperthyroid-saline 1575 6 Hyperthyroid-methamphetamine 157k 6
15 20* 9 55 + 10 16rt 8 107*29
90 9, 7 5+ 2 6rt 2 3f 1
51 ? 14 ~2.5 42 6 8k 6 14f. 4 lo+ 2 125+10 121& 2 162& 8 129?18 16Of. 8 162+ 3 171+ 11 75* 5
a After alteration of thyroid status as described in the text, rats were injected intraperitoneally with methamphetamine (30 mg/kg), decapitated at the indicated times, and blood from the carotid arteries was collected for plasma insulin as described. Where indicated propranolol (0.5 mg/kg, ip) or phenoxybenzamine (5 mg/kg, ip) were administered 10 min prior to the injection of methamphetamine. Each value represents the mean Ifr SE of four determinations.
The hypoglycemic response to methamphetamine which was observed previously in mice (McMahon et al., 1971) was not observed in euthyroid rats injected intraperitoneally with methamphetamine 30 mg/kg (Table 1). Altered thyroid status significantly modified the plasma glucoseresponseto an injection of methamphetamine (30 mg/kg, ip). Hyperthyroid rats showed a depressionof plasma glucoseconcentrations at 90 min similar to that described previously in mice (Table 5).
MCMAHON,FELDMANANDSCHANBERG
68
TABLE 6 PLASMA
INSULIN AND GLUCOSE RESPONSE TO METHAMPHETAMINE METHIMAZOLE-INDUCED HYPOTHYROID RATS'
IN
Injected drug Oral pretreatment
Saline
None
Methamphetamine
Plasmainsulin (&J/ml)
Hz0 alone Hz0 + methimazole Hz0 alone Hz0 + methimazole
17+3
16+ 4
36+ 5 17f 2b 10f 2 Plasmaglucose(mg/lOOml) 170+ 10 204+11 153f 5 146+ 7 150+ 6 180? 3 13f2
DOn the day of the experimentone-thirdof eachpretreatmentgroupwasinjectedintraperitoneallywith methamphetamine in 0.5ml of normalsaline,anequal numberreceivedan equalvolumeof salinealone,anda third groupreceivedno injection.Fifteen minuteslater, animalsweresacrificedandcarotid artery blood wasobtainedfor plasmainsulinandglucose determination.Eachvaluerepresents the meanf SEof sevento ten animals. bp < 0.002,lessthan euthyroid(HZ0 alone)group(Student’st test).
Fifteen minutes after an acute challenge dose of methamphetamine (30 mg/kg), rats chronically injected with methamphetamine released significantly less insulin than rats chronically injected with saline (Table 7). In addition, there was no rise in plasma glucose 15 min after the methamphetamine injection in either group of chronically injected animals. TABLE 7 TACHYPHYLAXISFROMCHRONIC METWHETAMINE ADMINISTRA~ONWITH RWPECTTO MJZTHAM~HETAMINE-INDUCEDINSULINRELEASE“
Challengeinjection
PIetreatment injection
Chronic saline
None (control)
Chronic methamphetamine
27 f 4 16+7
Chronic saline Chronic methamphetamine
100+7 loo&3
Saline
Methamphetamine
Plasmainsulin (&J/ml) 20+2 62+8 14+ 3 34 + 5b Plasmaglucose(% control) 93 + 1 108+4 97 f 3 108* 2
oEighteenhoursafter their lastdose(methamphetamine or saline)animalswereinjectedintraperitoneallywith a challengedoseof methamphetamine (30mg/kg)or with saline(1 ml). Fifteenminutes after the injection,animalsweredecapitatedandbloodfrom the carotidarteriescollectedfor plasma insulinandglucosedeterminations. Valuesrepresent themeanf SEof four to nineanimals. bp c 0.02,lessthanchronicsalinegroup(Student’st test).
69
METHAMPHETAMINEANDINSULINRELEASE
The results of studies in vitro using pancreatic tissue from mice, rats, hamsters, and rabbits are summarized in Table 8. Of the speciesand drug dose studies,only the mouse pancreatic tissuedemonstrated methamphetamine-induced insulin release. TABLE 8 EFFECTOF METHAMPHETAMINEANDTRANYLCYPROMINEONINSULINSECRETIONINVITRO~
Experimental animal Swiss-Webster mice Sprague-Dawley rats (Holtzman)
Glucose cone in incubation media(M) 3.3 x 10-J
3.3 x 10-3
Golden hamster 3.3 x 10-3
1.7 x 10-z Albino rabbits 3.3 x 10-3 1.7 x 10-z
Drug concentration Insulin secretion ,&/lo0 mg/30min Significance (M) None Meth 2.5 x 1O-3 None Meth 1 x 10T4 Meth 1 x 10m3 Tran 1 x 10e3 None Meth 1 x 10e4 Meth 1 x 10e3 Tran 1 x 10V3 None Meth 2.5 x 10e3 None Meth 2.5 x 10-j None Meth 2.5 x 10d3
32f lO(12) 120+ 26 (12) 97 -t- 35 ( 6) 54k 30 ( 6) 44+ 16( 6) 37& 16( 6) 240+ 69 ( 6) 130+ 25 ( 6) 233 If: 62( 6) 182+ 27 ( 6) 443 + 103( 8) 537fllO( 8) 232-t 79 ( 8) 108+ 24 (, 8) 3062+ 788( 6) 1738+ 294 ( 6)
p < 0.02
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
aSegments of pancreas wereremovedand incubatedasdescribed in Methods.Insulinsecretionis reportedasthemean+ SE.Thenumberof pancreas segments studiedisshownin parentheses. Meth = methamphetamine, Tran = tranylcypromine,andN.S. = not significant(Student’st test).
DISCUSSION The metabolic and behavioral effects of the amphetamines and related compounds have been the subject of several recent publications, and the reader is referred to these for comprehensive review (Ellinwood and Cohen, 1972; Costa and Garattini, 1970). The present studies demonstrate that the magnitude of methamphetamine-induced insulin secretion can be modified by alterations in the animal’s endocrine status, by adrenergic receptor antagonistsand by previous exposure to methamphetamine. Hyperthyroidism enhancesthe lethal effect of amphetamine in aggregatedmice (Moore, 1965) and in Sprague-Dawley rats (J. D. Cook and S. M. Schanberg, unpublished observations:) while the hypothyroid state has been reported to protect rats from amphetamineinduced hyperthermia and lethality (Mantegazza et al., 1968). The short regimen of thyroxine used in the present study doubled the total amount of methamphetaminestimulated insulin release, while thyroidectomy abolished the insulin response (Table 5). These results agree with amphetamine lethality studies, and with a recent report of reversible impairment of insulin secretion in hypothyroid human subjects (Shah et al.,
70
MCMAHON,
FELDMAN
AND
SCHANBERG
1971). Since surgical thyroidectomy was accomplished by removal of the parathyroid glands, serum calcium concentrations were significantly lower(p < 0.001) in the operated group (6.4 f 0.3 mg/lOO ml) as compared to sham operated controls (10.2 + 3.0 mg/ 100 ml). The lack of significant insulin response to methamphetamine in surgically induced hypothyroid rats might have been due to hypocalcemia resulting in altered membrane and receptor excitability, (Camitta and Stoner, 1970; Bressler and VargasCordon, 1970 ; Jaanus et al., 1967). Therefore studies were carried out with methimazoleinduced hypothyroidism. The partial “pharmacological thyroidectomy” achieved with this drug also significantly reduced the insulin response to methamphetamine (Table 6). Considerable evidence has accumulated that insulin release in laboratory animals and in man may be mediated through beta-adrenergic receptors (Turtle and Kipnis, 1967; Porte, 1969; Cerasi et al., 1969; Ashmore, 1970). Most of these studies have used low doses of propranolol which are thought to block beta-adrenergic receptor sites selectively. Results of the present study (Table 3) using a low beta-blocking dose of propranolol suggest that methamphetamine produces insulin release in rats in vivo by a beta-adrenergic mechanism. The studies with phenoxybenzamine suggest that alphaadrenergic receptors are net involved in methamphetamine-stimulated insulin release. Rats injected chronically with gradually increasing doses of methamphetamine have an increased LD50 as compared to controls treated chronically with saline when challenged with acute doses of methamphetamine (J.D. Cook and S. M. Schanberg, unpublished observations). The present studies provide evidence for a similar phenomenon involving methamphetamine-induced insulin release (Table 7). Insulin release after a challenge dose of methamphetamine 30 mg/kg was reduced to 50 % of control levels by the chronic administration of amphetamine. Amphetamine lethality, a process that is potentiated by hyperthyroidism (Moore, 1965) is accompanied by depletion of liver glycogen stores and a depression of the plasma glucose concentrations (Moore et al., 1965). Although significant hypoglycemia was not observed in our euthyroid rats after methamphetamine-induced insulin release (Table l), hyperthyroid animals had augmented methamphetamine-insulin release and showed a relative hypoglycemia 90 min after the methamphetamine injection (Table 5). In contrast, hypothyroid rats had no detectable insulin response at 15 min and demonstrated a mild hyperglycemia (Table 6). These findings support the concept that hypoglycemia secondary to insulin release may be a contributory factor to methamphetamine lethality in hyperthyroid rats. The observed differences in the effect of methamphetamine on insulin secretion in vivo and in vitro in the same species as well as among species cannot be explained by the the present studies. However, it has been shown that species differences in autonomic innervation of the pancreas partially may be responsible for altered insulin reaction to different drugs (Feldman and Lebovitz, 1970; Quickel et al., 1971). It is also possible that methamphetamine-induced insulin release in vivo may be due to the combined effects of indirect as well as direct stimulation, with the former mediated via the central nervous system (McMahon et al., 1971; Kaneto et al., 1965; Frohman et al., 1966; Kaneto et al., 1967; Porte, 1971). However the failure of chlorisondamine, a ganglionic blocking agent, to prevent methamphetamine-induced insulin secretion in the rat suggests that the central nervous system alone does not mediate this effect of methamphetamine (Table 4).
METHAMPHETAMINE
AND
INSULIN
RELEASE
71
Clearly, species differences make it difficult to predict the acute and chronic glucose and insulin responses to methamphetamine in man. However, in this regard, it is of interest that chronic oral amphetamine therapy results in hyperinsulinemia (Sirtori et al., 1971) while abnormal glucosetolerance tests and aberrant insulin values have been reported to occur in humans following the nonmedical use of methamphetamine (Citron et al., 1972). In summary, it appearsthat methamphetamine stimulates insulin secretion in part through a direct effect on the pancreasand this effect can be modulated by hormonal and pharmacological manipulations.
ACKNOWLEDGMENTS We wish to expressour appreciation to Mrs. Agnes Crist, Mrs. June Garrison, and Miss Barbara A. Chapmanfor their excellenttechnical assistance.
REFERENCES J. (1970).Insulin and adrenergicreceptors.Fed. Proc. Fed. Amer. Sot. Exp. Biol. 29, 1386-1387. BRESSLER, R. AND VARGAS-CORDON, M. (1970).The effect of /?-adrenergicreceptor blocking agentson drug inducedinsulin secretion.Advan. Metabol. Dis. Suppl. I, 87-94. CAMITTA, F. D. AND STONER, R. E. (1970). Abnormal glucoseand insulin responseto oral glucoseduring hypocalcemia.Diabetes 19, 380(Abstr.). CERASI, E., EFFENDIC, S. ANDLUFT,R. (1969).Role of adrenergicreceptorsin glucose-induced insulin secretionin man. Lancet 2, 301-302. CITRON, B. P., HALPERN, M. AND MILLER, L. (1972). Abnormal carbohydrate metabolism associatedwith drug abuse.Clin. Res. 20, 236 (Abstr.). COSTA, E. AND GARATTINI, S. (1970). Amphetamines and Related Compounds. Raven Press (New York). ELLINWOOD, E. AND COHEN, S. (1972).Current Concepts OfAmphetamine Abuse. U.S. Government Printing Office (Washington,D.C.). FELDMAN, J. M. AND LEBOVITZ, H. E. (1970).Serotonin inhibition of in vitro insulin release from goldenhamsterpancreas.Endocrinology 86,66-70. FROHMAN, L. A., EZDINILI, E. Z. AND JAVID, R. (1966).Effect of vagal stimulation on insulin secretion.Diabetes 15, 522(Abstr.). GENUTH, S., FROHMAN, L. C. AND LEBOVITZ, H. E. (1965).A radioimmunologicalassaymethod for insulin usinginsulin125Iand gel filtration. J. Clin. Endocrinol. Metab. 25, 1043-1049. JAANUS, S. D., MIELE, E. AND RUBIN, R. P. (1967).The analysisof the inhibitory effect of local anesthetics and propranolol on adrenomedullary secretion evoked by calcium or acetylcholine.Br. J. Pharm. 31, 319-330. KANETO, A., KOSAKA, K. AND NAKAO, K. (1967).Effects of stimulationof the vagusnerve on insulin secretion.Endocrinology 80, 530-536. KANETO,A., MIKI, E., KOSAKA,K., OIUNAKA,S.ANDNAKAO,K. (1965).Effectsof stimulation of the cingulategyrus on insulin secretion.Endocrinology 77, 617-624. MANTEGAZZA, P., NAIMZADA,K. M. ANDRIVA, M. (1968).Activity of amphetaminein hypothyroid rats. Eur. J. Pharmacol. 5, 10-16. MCMAHON, E. M., ANDERSEN, D. K., FELDMAN, J. M. AND SCHANBERG, S.M. (1971).Methamphetamine-inducedinsulin release.Science 174,66-68. MENNEAR, J. H., SPRA~O, G. R. ANDMIYA, T. S. (1971).The comparative glycemiceffectsof catecholaminesin rats and mice. Toxicol. Appl. Pharmacol. 18,835~840. MOORE, K. E., SAWDY, L. G. AND SHAUL, S. R. (1965). Effects of D-amphetamine on blood glucoseand tissueglycogenlevelsof isolatedand aggregatedmice.Biochem. Pharmacol. 14, 197-204. ASHMORE,
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AND
SCHANBERG
K. E. (1965). Amphetamine toxicity in hyperthyroid mice. Biochem. Pharmacol. 14, 1831-1837. PORTE, D., JR. (1969). Sympathetic regulation of insulin secretion. Its relation to diabetes mellitus. Arch. Intern. Med. 123,252-260. PORTE, D. (1971). Neural regulation of insulin secretion. Diabetes 20, 340 (Abstr.). QUICKEL, K. E., FELDMAN, J. M. AND LEBOVITZ, H. E. (1971). Inhibition of insulin secretion by serotonin and dopamine: Species variation. Endocrinology 89, 1295-1302. SAIFER, S. AND GERSTENFELD, B. J. (1958). The photometric microdetermination of blood glucose with glucose oxidase. J. Lab. Clin. Med. 51,448460. SCHANBERG, S. M. AND COOK, J. D. (1972). Effects of acute and chronic methamphetamine on brain norepinephrine metabolism. In Current Concepts of Amphetamine Abuse (E. Ellinwood and S. Cohen, Eds.), U.S. Government Printing Office, Washington, D.C. SHAH, J. H., CERCHIO, G. M. AND POPOVICH, R. A. (1971). Early phase insulin release in hypothyroidism. Diabetes 20, 376 (Abstr.). SIRTORI, C., HURWITZ, H. AND AZARNOFF, D. L. (1971). Hyperinsulinemia secondary to chronic administration of mazindol and d-amphetamine. Amer. J. Med. Sci. 261, 341. TURTLE, J. R. AND KIPNIS, D. M. (1967). An adrenergic receptor mechanism for the control of cyclic 3’5’ adenosine monophosphate synthesis in tissues. Biochem. Biophys. Res. Comm. 28, 797-802. MOORE,
AND
APPLIED
Further
PHARMACOLOGY
Studies
32,62-12 (1975)
of Methamphetamine-Induced Insulin Release’
E. M. MCMAHON, J. M. FELDMAN~AND S. M. SCHANBERG~ Department of Psychiatry, University of Washington School of Medicine, Seattle, Washington 98195; Veterans Administration Hospital, Durham, North Carolina 27710; and Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 Received June 17,1974; accepted November II,1974
Further
Studies of
Methamphetamine-Induced Insulin Release.
MCMAHON, E. M., FELDMAN, J. M. AND SCHANBERG, S. M. Toxicol. Appl. Pharmacol. 32, 62-72. In the presentstudy we have evaluatedfactors that
modify methamphetamine-induced insulin secretion.While methamphetaminestimulatesinsulinsecretionin intact rats and mice,it stimulatesinsulin secretion in vitro only from mousepancreas.Related drugs such as damphetamine,methylphenidate,and tranylcypromine alsostimulateinsulin secretionin intact rats. Methamphetamine-inducedinsulin secretionis not altered by a-receptorantagonistsor ganglionicblocking agents,but is reduced by B-receptor antagonists.Insulin secretionin responseto methamphetamineis significantly increasedin the hyperthyroid stateand decreased in the hypothyroid state.Chronic methamphetamineadministrationresults in a reduction in the insulin secretedin responseto this agent. We have reported that methamphetamine and amphetamine release insulin from rats and mice in vivo by a mechanism not attributable to hyperglycemia (McMahon et al., 1971). Profound hypoglycemia followed insulin release in mice, although this effect was not observed consistently in normal rats. Similarly, marked species differences in the glycemic response to various drugs have been observed (Feldman and Lebovitz, 1970; Quickel et al., 1971; Mennear et al., 1971), including the effects of methamphetamine on insulin concentrations and carbohydrate tolerance in humans (Sirtori et al. 1971; Citron et al., 1972). The purpose of this report is to characterize further the effect of methamphetamine on insulin and glucose concentrations in the rat. The effect was analyzed in rats with normal or altered thyroid status, after beta-adrenergic receptor blockade, and after a regimen of chronic methamphetamine administration. Insulin responses to methamphetamine were also examined in vitro in the rat, hamster, and rabbit. 1 Portions of this work were supported by U.S. Public Health Service Grants AM-01324, MH13688, MH-11947, and Veterans Administration 2112-01. ‘Dr. Feldman is the recipient of a Clinical Investigatorship (2650-01) from the Veterans Administration. 3 Dr. Schanberg is the recipient of a Career Development Award from the National Institutes of Mental Health, Grant Number K05 MH-06489. 62 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form
reserved.
METHAMPHETAMINE
AND
INSULIN
RELEASE
63
METHODS
Acute Studies-Rats Male Sprague-Dawley rats4 weighing 180 + 10 g were housed in cages 9 by 8 by 6 in, two or three animals per cage, and allowed free access to food and water. Four hours prior to drug administration food was removed and animals were aggregated seven to eight per plastic cage (18 by 10 by 6 in.). Methamphetamine hydrochloride in 0.5 ml of normal saline or saline alone was injected intraperitoneally. At appropriate intervals after injection, rats were killed by decapitation and blood from the carotid arteries was collected in heparinized beakers. Plasma glucose concentrations were measured by a glucose oxidase method (Saifer and Gerstenfeld, 1958). Plasma insulin was measured against mouse insulin standards by a modification of a radioimmunoassay method in which the separation of bound and free insulin was performed by precipitating the bound insulin with a goat anti-serum to guinea pig gamma globulin (Genuth et al., 1965). Methamphetamine had no effect on the measurement of insulin in this system. In some studies, the beta-adrenergic blocking drug propranolol, 0.5 mg/kg,5 was injected intraperitoneally 10 min prior to methamphetamine injection; controls received an equal volume of normal saline at a similar time. To evaluate the effect of ganglionic blockade on methamphetamine-induced insulin release 24 rats were divided into four groups of six animals. Two of the groups received saline pretreatment while the remaining two were pretreated with chlorisondamine chloride.6 Four hours later, one of the saline and one of the chlorisondamine-treated groups received methamphetamine (30 mg/kg, ip). The other two groups were injected again with saline. The animals were killed 15 min later, their blood collected (see above), and the plasma assayed for insulin and glucose. Rats were made hyperthyroid by intraperitoneal administration of Na-l-thyroxine,’ a total of 1.5 mg/kg divided into three equally spaced doses during the 24 hr prior to the experiment. Control animals were injected with an equal volume of saline at corresponding intervals. Surgically thyroidectomized male Sprague-Dawley rats4 were obtained 8-10 days after the operation. These rats weighed 180 f 10 g, 15-20 g less than their age-matched, nonoperated controls. Other rats were made hypothyroid by oral administration of methimazole8 dissolved in drinking water. The concentration was adjusted to achieve an average dose of 10 mg methimazole per rat per day for at least 1 wk. Water consumption did not differ appreciably between animals receiving methimazole and controls receiving water alone. Water was substituted for all methimazole solutions 18 hr prior to these experiments. Plasma calcium determinations were performed by the Duke University Hospital Laboratory of Clinical Chemistry. Chronic Studies-Rats Chronic studies were performed using male Sprague-Dawley rats4 weighing 180 + 10 g, housed three animals per 9 by 8 by 6 in. cage, with free access to food and water. 4 Sprague-Dawley rats obtained from Hormone Assay Corp., Chicago, Illinois. 5 Propranolol (Inderal) obtained from Ayerst Laboratories, Atlanta, Georgia. 6 Chlorisondamine chloride (Ecolid). 7 Na-l-thyroxine obtained from Nutrition Control Products, Division of Pharmex Corp., Hollywood, Florida. 8 Methimazole obtained from Aldrich Chemical Corp., Milwaukee, Wisconsin. 3
64
MCMAHON,
FELDMAN
AND
SCHANBERG
Chronic intraperitoneal administration of methamphetamine was performed as described previously (Schanberg and Cook, 1972) using early morning and late afternoon injections, with the dose increasing 1 mg/kg per injection over a IO-day period. Theinitial dose was 10 mg/kg (20 mg/kg/day) and the final dose on the evening before the experiment was 30 mg/kg (60 mg/kg/day). Control animals received saline injections on a similar schedule; rats were weighed every 3 days for adjustment of drug dosage. The remainder of the experiment was carried out as described for acute studies. Studies of Insulin Release In Vitro Studies of insulin release in vitro were carried out with pancreatic tissue from SwissWebster mice, golden hamsters, New Zealand white albino rabbits, and SpragueDawley rats9 using a slight modification of a previously described incubation system (McMahon et al., 1971; Feldman and Lebovitz, 1970). After an initial 15min preincubation, each piece of pancreas underwent two sequential 30-min incubations, and portions of each incubation medium were assayed for insulin as described above. RESULTS
After methamphetamine injection in the rat, plasma insulin concentrations rose rapidly, increasing 3-500 % over basal values at 15 min (Table 1). This high insulin level decreased rapidly during the next 15 min, but remained eIevated significantly above conTABLE
1
EFFECT OF METHAMPHETAMINE ON PLASMA INSULIN AND GLUCOSE IN THE RAF
Time after injection (min) Experimental group
0
15
Control (saline) Methamphetamine
lOf2
16f3
lOrt2
57 * s*
Control (saline) Methamphetamine
126&4 126+4
124*3 161 + 5*
30
60
Insulin (&J/ml) 14 + 2 15f2 28 + 1” 2112d
90
120
9f7 16f5
17f3 16f 1
Glucose (mg/lOO ml)
118 f 5
118? 4
121f2
129+4
134 + 3c
132+4d
129+5
128+_6
’ Each value representsthe mean + SE of at least eight animals, exceptthe 90-min control group, which contained four animals. Insulin values are expressed as microunits insulin/milliliters plasma; glucose concentrations are expressed as mg glucose/100 ml plasma. The methamphetamine groups received 30 mg/kg ip, in 0.5 ml of normal saline, while controls receive an equal volume of saline. bp < 0.001, different from control group (Students’ t test). ‘p < 0.01, different from control group. “p < 0.05, different from control group. trol values until 60 min after injection. In contrast, plasma glucose concentrations increased only 27 % (Table 1). The effects of various doses of methamphetamine on plasma insulin 15 min after injection are shown in Fig. 1. Methamphetamine caused a dose-related increase in plasma insulin concentration. This effect appeared at dosages as low as 2.5 mg/kg and was maximal after a dosage greater than 10 mg/kg. The effects 9 Sprague-Dawley
rats obtained from Holtzman Company, Madison, Wisconsin.
METHAMPHETAMINE
AND
INSULIN
65
RELEASE
175r
I 1
I
2253
45 DOSE
II
20 25 30
10
11
40 50
(mg /kg wenght)
FIG. 1. Plasma insulin response to methamphetamine 15 min after injection. Male Sprague-Dawley rats were injected intraperitoneally with various doses of methamphetamine in 0.5 ml normal saline, or an equal volume of saline alone, as described in the text. Each value represents the mean f SE of four observations, and is compared (Student’s t-test) with a control group sacrificed at the same time.
of other structurally related drugs were investigated for their ability to release insulin. As shown in Table 2, all of these agents significantly elevated plasma insulin concentrations as compared to controls. In comparison, the effects of these drugs on plasma glucose was inconsistent. Significant increases occurred only after 10 mg/kg methamphetamine and 30 mg/kg d-amphetamine. TABLE 2 PLASMA INSULIN AND GLUCOSE RESPONSE TO STIMULANT DRUGS 15 MIN AFTER INJECTION'
Plasma glucose Plasma insulin Drug and dose (mg/kg)
W/ml)
Saline Methamphetamine, 10 Methamphetamine, 30 d-Amphetamine, 30 Methylphenidate, 30 Tranylcypromine, 20
12+ 44+ 66 + 62f 26 f 37f
1 12b 15” 1’ 3’ 4”
(‘A of salineinjected control)
loof 132+ 117+ 126+ 106+ 108 +
9b 6 6* 6 9
DEach value represents the mean &- SE of four observations and is compared (Student’s I test) with a control group sacrificed at the same time. *p c 0.05, significantly higher than saline-injected control group. c p c 0.01, significantly higher than saline-injected control group. “p < 0.005, significantly higher than salineinjected control group. c p < 0.001, significantly higher than saline-injected control group.
66
MCMAHON, FELDMAN AND SCHANBERG
The effect of pretreatment with phenoxybenzamine (5 mg/kg, ip) or propranolol(0.5 mg/kg, ip) on plasma insulin or glucose response to methamphetamine in the rat is shown in Table 3. Propranolol (B-blocker) but not phenoxybenzamine (a-blocker) significantly decreased methamphetamine-induced insulin release at 15 min. However, neither antagonist altered the methamphetamine-induced increase in plasma glucose. Control studies showed that basal plasma insulin and glucose concentration were not altered by either propranolol or phenoxybenzamine. The effect of chlorisondamine (a ganglionic blocking agent) on methamphetamine-induced insulin release was tested and it can be seen in Table 4 that chlorisondamine had no apparent effect on this system. This dose of chlorisondamine was demonstrated to block the release of catecholamines from adrenal and nerve endings via ganglionic blockade in this strain of rats (T. Slotkin, personal communication). TABLE
EFFECT OFALIRENERGIC BLOCKING
AGENTS
3
INSULINRELEASES
ON METHAMPHETAMINE-INDUCED
Time after methamphetamine injection (15 min)
Experimental Group Plasma insulin Control (saline) Methamphetamine Propranolol and methamphetamine Phenoxybenzamineand methamphetamine
NJ/ml>
Plasmaglucose (mg/l~ ml)
22+ 4 78 + 12*
123f 9 168 i- 7*
32+
149 f 10d
7’
63 f 12*sd
172 f ll*
c Ratswerepretreatedwith propranolol(0.5 mg/kg,ip), phenoxybenzamine(5mg/kg,ip) or normalsaline10minbeforetheyreceivedmethamphetamine(30mg/kg,ip) or anequalvolumeof saline.Eachvaluerepresents the mean+ SEof eightanimals.Prior to injection,the plasmainsulinwas15.8 ,uU/mlandthe plasmaglucosewas124+ 2 mg/lOOml. * p < 0.01,differentfrom control(Student’sI test). cp < 0.05,differentfrom methamphetamine. dNot significantlydifferentfrom methamphetamine.
Plasma insulin 15 min after methamphetamine (30 mg/kg) in hyperthyroid rats and in rats made hypothyroid by surgical thyroidectomy are shown in Table 5. Hyperthyroid rats showed a greater insulin response to methamphetamine than did euthyroid animals. The insulin response to methamphetamine was reduced markedly in hypothyroid animals. Insulin and glucose responses to methamphetamine in rats made hypothyroid with a regimen of methimazole are shown in Table 6. Rats made hypothyroid by this method weighed an average of 40 g less than their age-matched controls, and showed compensatory thyroid hypertrophy (thyroid wet weight in milligrams : hypothyroid = 33.2 L- 0.2, n = 3; euthyroid 20.0 + 1.O, n = 3, p < 0.001). Methimazole-induced hypothyroidism significantly reduced insulin response to methamphetamine at 15 min postinjection and produced a modest hyperglycemia (Table 6).
METHAMPHETAMINE
AND
TABLE EFFECT
OF CHLORWNDAMINE
INSULIN
67
RELEASE
4
ON METHAMPHETAMINE-INDUCED
Experimental group
Saline and saline Saline and methamphetamine Chlorisondamine and saline Chlorisondamine and methamphetamine
INSULIN
RELEASE”
Plasma insulin W/ml> __..26? 7 97 f 17b 28f 5
Plasma glucose (mg/lOO ml)
100 f 13b.c
165 f 12c
150* 9’ 176t 6 142 k 14’
’ Twenty-four rats were divided into two groups and injected with either saline or chlorisondamine (10 mg/kg, ip). Four hours later, half of each group were given saline (1 ml, ip) and the other half methamphetamine (30 mg/kg, ip). Rats were killed 15 min later and their blood plasma assayed for insulin and glucose as described in Methods. Each value represents the mean + SE of six animals. “p c 0.001, different from saline-saline group (Student’s 1 test). c Not significantly different from saline-methamphetamine group.
TABLE 5 EFFECT
OF ALTERED
THYROID
STATUS
ON METHAMPHETAMINE-INDUCED
INSULIN
RELEASE”
Time after injection (min) Treatment
0
Plasmainsulin @U/ml) Salinealone 15+ 8 Methamphetamine 6+ 2 Hyperthyroid-saline 12!1 8 Hyperthyroid-methamphetamine 12+10 Hyperthyroid-propranolol methamphetamine 12+ 10 Hyperthyroid-saline (2.5 Hypothyroid-methamphetamine ~2.5 Salinealone 124f12 Methamphetamine 149+ 5 Hyperthyroid-saline 1575 6 Hyperthyroid-methamphetamine 157k 6
15 20* 9 55 + 10 16rt 8 107*29
90 9, 7 5+ 2 6rt 2 3f 1
51 ? 14 ~2.5 42 6 8k 6 14f. 4 lo+ 2 125+10 121& 2 162& 8 129?18 16Of. 8 162+ 3 171+ 11 75* 5
a After alteration of thyroid status as described in the text, rats were injected intraperitoneally with methamphetamine (30 mg/kg), decapitated at the indicated times, and blood from the carotid arteries was collected for plasma insulin as described. Where indicated propranolol (0.5 mg/kg, ip) or phenoxybenzamine (5 mg/kg, ip) were administered 10 min prior to the injection of methamphetamine. Each value represents the mean Ifr SE of four determinations.
The hypoglycemic response to methamphetamine which was observed previously in mice (McMahon et al., 1971) was not observed in euthyroid rats injected intraperitoneally with methamphetamine 30 mg/kg (Table 1). Altered thyroid status significantly modified the plasma glucoseresponseto an injection of methamphetamine (30 mg/kg, ip). Hyperthyroid rats showed a depressionof plasma glucoseconcentrations at 90 min similar to that described previously in mice (Table 5).
MCMAHON,FELDMANANDSCHANBERG
68
TABLE 6 PLASMA
INSULIN AND GLUCOSE RESPONSE TO METHAMPHETAMINE METHIMAZOLE-INDUCED HYPOTHYROID RATS'
IN
Injected drug Oral pretreatment
Saline
None
Methamphetamine
Plasmainsulin (&J/ml)
Hz0 alone Hz0 + methimazole Hz0 alone Hz0 + methimazole
17+3
16+ 4
36+ 5 17f 2b 10f 2 Plasmaglucose(mg/lOOml) 170+ 10 204+11 153f 5 146+ 7 150+ 6 180? 3 13f2
DOn the day of the experimentone-thirdof eachpretreatmentgroupwasinjectedintraperitoneallywith methamphetamine in 0.5ml of normalsaline,anequal numberreceivedan equalvolumeof salinealone,anda third groupreceivedno injection.Fifteen minuteslater, animalsweresacrificedandcarotid artery blood wasobtainedfor plasmainsulinandglucose determination.Eachvaluerepresents the meanf SEof sevento ten animals. bp < 0.002,lessthan euthyroid(HZ0 alone)group(Student’st test).
Fifteen minutes after an acute challenge dose of methamphetamine (30 mg/kg), rats chronically injected with methamphetamine released significantly less insulin than rats chronically injected with saline (Table 7). In addition, there was no rise in plasma glucose 15 min after the methamphetamine injection in either group of chronically injected animals. TABLE 7 TACHYPHYLAXISFROMCHRONIC METWHETAMINE ADMINISTRA~ONWITH RWPECTTO MJZTHAM~HETAMINE-INDUCEDINSULINRELEASE“
Challengeinjection
PIetreatment injection
Chronic saline
None (control)
Chronic methamphetamine
27 f 4 16+7
Chronic saline Chronic methamphetamine
100+7 loo&3
Saline
Methamphetamine
Plasmainsulin (&J/ml) 20+2 62+8 14+ 3 34 + 5b Plasmaglucose(% control) 93 + 1 108+4 97 f 3 108* 2
oEighteenhoursafter their lastdose(methamphetamine or saline)animalswereinjectedintraperitoneallywith a challengedoseof methamphetamine (30mg/kg)or with saline(1 ml). Fifteenminutes after the injection,animalsweredecapitatedandbloodfrom the carotidarteriescollectedfor plasma insulinandglucosedeterminations. Valuesrepresent themeanf SEof four to nineanimals. bp c 0.02,lessthanchronicsalinegroup(Student’st test).
69
METHAMPHETAMINEANDINSULINRELEASE
The results of studies in vitro using pancreatic tissue from mice, rats, hamsters, and rabbits are summarized in Table 8. Of the speciesand drug dose studies,only the mouse pancreatic tissuedemonstrated methamphetamine-induced insulin release. TABLE 8 EFFECTOF METHAMPHETAMINEANDTRANYLCYPROMINEONINSULINSECRETIONINVITRO~
Experimental animal Swiss-Webster mice Sprague-Dawley rats (Holtzman)
Glucose cone in incubation media(M) 3.3 x 10-J
3.3 x 10-3
Golden hamster 3.3 x 10-3
1.7 x 10-z Albino rabbits 3.3 x 10-3 1.7 x 10-z
Drug concentration Insulin secretion ,&/lo0 mg/30min Significance (M) None Meth 2.5 x 1O-3 None Meth 1 x 10T4 Meth 1 x 10m3 Tran 1 x 10e3 None Meth 1 x 10e4 Meth 1 x 10e3 Tran 1 x 10V3 None Meth 2.5 x 10e3 None Meth 2.5 x 10-j None Meth 2.5 x 10d3
32f lO(12) 120+ 26 (12) 97 -t- 35 ( 6) 54k 30 ( 6) 44+ 16( 6) 37& 16( 6) 240+ 69 ( 6) 130+ 25 ( 6) 233 If: 62( 6) 182+ 27 ( 6) 443 + 103( 8) 537fllO( 8) 232-t 79 ( 8) 108+ 24 (, 8) 3062+ 788( 6) 1738+ 294 ( 6)
p < 0.02
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
aSegments of pancreas wereremovedand incubatedasdescribed in Methods.Insulinsecretionis reportedasthemean+ SE.Thenumberof pancreas segments studiedisshownin parentheses. Meth = methamphetamine, Tran = tranylcypromine,andN.S. = not significant(Student’st test).
DISCUSSION The metabolic and behavioral effects of the amphetamines and related compounds have been the subject of several recent publications, and the reader is referred to these for comprehensive review (Ellinwood and Cohen, 1972; Costa and Garattini, 1970). The present studies demonstrate that the magnitude of methamphetamine-induced insulin secretion can be modified by alterations in the animal’s endocrine status, by adrenergic receptor antagonistsand by previous exposure to methamphetamine. Hyperthyroidism enhancesthe lethal effect of amphetamine in aggregatedmice (Moore, 1965) and in Sprague-Dawley rats (J. D. Cook and S. M. Schanberg, unpublished observations:) while the hypothyroid state has been reported to protect rats from amphetamineinduced hyperthermia and lethality (Mantegazza et al., 1968). The short regimen of thyroxine used in the present study doubled the total amount of methamphetaminestimulated insulin release, while thyroidectomy abolished the insulin response (Table 5). These results agree with amphetamine lethality studies, and with a recent report of reversible impairment of insulin secretion in hypothyroid human subjects (Shah et al.,
70
MCMAHON,
FELDMAN
AND
SCHANBERG
1971). Since surgical thyroidectomy was accomplished by removal of the parathyroid glands, serum calcium concentrations were significantly lower(p < 0.001) in the operated group (6.4 f 0.3 mg/lOO ml) as compared to sham operated controls (10.2 + 3.0 mg/ 100 ml). The lack of significant insulin response to methamphetamine in surgically induced hypothyroid rats might have been due to hypocalcemia resulting in altered membrane and receptor excitability, (Camitta and Stoner, 1970; Bressler and VargasCordon, 1970 ; Jaanus et al., 1967). Therefore studies were carried out with methimazoleinduced hypothyroidism. The partial “pharmacological thyroidectomy” achieved with this drug also significantly reduced the insulin response to methamphetamine (Table 6). Considerable evidence has accumulated that insulin release in laboratory animals and in man may be mediated through beta-adrenergic receptors (Turtle and Kipnis, 1967; Porte, 1969; Cerasi et al., 1969; Ashmore, 1970). Most of these studies have used low doses of propranolol which are thought to block beta-adrenergic receptor sites selectively. Results of the present study (Table 3) using a low beta-blocking dose of propranolol suggest that methamphetamine produces insulin release in rats in vivo by a beta-adrenergic mechanism. The studies with phenoxybenzamine suggest that alphaadrenergic receptors are net involved in methamphetamine-stimulated insulin release. Rats injected chronically with gradually increasing doses of methamphetamine have an increased LD50 as compared to controls treated chronically with saline when challenged with acute doses of methamphetamine (J.D. Cook and S. M. Schanberg, unpublished observations). The present studies provide evidence for a similar phenomenon involving methamphetamine-induced insulin release (Table 7). Insulin release after a challenge dose of methamphetamine 30 mg/kg was reduced to 50 % of control levels by the chronic administration of amphetamine. Amphetamine lethality, a process that is potentiated by hyperthyroidism (Moore, 1965) is accompanied by depletion of liver glycogen stores and a depression of the plasma glucose concentrations (Moore et al., 1965). Although significant hypoglycemia was not observed in our euthyroid rats after methamphetamine-induced insulin release (Table l), hyperthyroid animals had augmented methamphetamine-insulin release and showed a relative hypoglycemia 90 min after the methamphetamine injection (Table 5). In contrast, hypothyroid rats had no detectable insulin response at 15 min and demonstrated a mild hyperglycemia (Table 6). These findings support the concept that hypoglycemia secondary to insulin release may be a contributory factor to methamphetamine lethality in hyperthyroid rats. The observed differences in the effect of methamphetamine on insulin secretion in vivo and in vitro in the same species as well as among species cannot be explained by the the present studies. However, it has been shown that species differences in autonomic innervation of the pancreas partially may be responsible for altered insulin reaction to different drugs (Feldman and Lebovitz, 1970; Quickel et al., 1971). It is also possible that methamphetamine-induced insulin release in vivo may be due to the combined effects of indirect as well as direct stimulation, with the former mediated via the central nervous system (McMahon et al., 1971; Kaneto et al., 1965; Frohman et al., 1966; Kaneto et al., 1967; Porte, 1971). However the failure of chlorisondamine, a ganglionic blocking agent, to prevent methamphetamine-induced insulin secretion in the rat suggests that the central nervous system alone does not mediate this effect of methamphetamine (Table 4).
METHAMPHETAMINE
AND
INSULIN
RELEASE
71
Clearly, species differences make it difficult to predict the acute and chronic glucose and insulin responses to methamphetamine in man. However, in this regard, it is of interest that chronic oral amphetamine therapy results in hyperinsulinemia (Sirtori et al., 1971) while abnormal glucosetolerance tests and aberrant insulin values have been reported to occur in humans following the nonmedical use of methamphetamine (Citron et al., 1972). In summary, it appearsthat methamphetamine stimulates insulin secretion in part through a direct effect on the pancreasand this effect can be modulated by hormonal and pharmacological manipulations.
ACKNOWLEDGMENTS We wish to expressour appreciation to Mrs. Agnes Crist, Mrs. June Garrison, and Miss Barbara A. Chapmanfor their excellenttechnical assistance.
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K. E. (1965). Amphetamine toxicity in hyperthyroid mice. Biochem. Pharmacol. 14, 1831-1837. PORTE, D., JR. (1969). Sympathetic regulation of insulin secretion. Its relation to diabetes mellitus. Arch. Intern. Med. 123,252-260. PORTE, D. (1971). Neural regulation of insulin secretion. Diabetes 20, 340 (Abstr.). QUICKEL, K. E., FELDMAN, J. M. AND LEBOVITZ, H. E. (1971). Inhibition of insulin secretion by serotonin and dopamine: Species variation. Endocrinology 89, 1295-1302. SAIFER, S. AND GERSTENFELD, B. J. (1958). The photometric microdetermination of blood glucose with glucose oxidase. J. Lab. Clin. Med. 51,448460. SCHANBERG, S. M. AND COOK, J. D. (1972). Effects of acute and chronic methamphetamine on brain norepinephrine metabolism. In Current Concepts of Amphetamine Abuse (E. Ellinwood and S. Cohen, Eds.), U.S. Government Printing Office, Washington, D.C. SHAH, J. H., CERCHIO, G. M. AND POPOVICH, R. A. (1971). Early phase insulin release in hypothyroidism. Diabetes 20, 376 (Abstr.). SIRTORI, C., HURWITZ, H. AND AZARNOFF, D. L. (1971). Hyperinsulinemia secondary to chronic administration of mazindol and d-amphetamine. Amer. J. Med. Sci. 261, 341. TURTLE, J. R. AND KIPNIS, D. M. (1967). An adrenergic receptor mechanism for the control of cyclic 3’5’ adenosine monophosphate synthesis in tissues. Biochem. Biophys. Res. Comm. 28, 797-802. MOORE,
