Cannabinoid effects on liver glycogen stores

Life Sciences Vol. 12, Part II, pp. 409-418, 1973 Printed in Great Britain

Pergamon Press

CANNABINOID EFFECTS ON LIVER GLYCOGEN STORES Ross A . Sprague, Harris Rosenkrontz and Monique C . Broude Mason Research Institute, Worcester, Massachusetts and National Institute of Mental Health, Rockville, Maryland

(Received 29 November 1972 ; in final form 12 March 1973) SUMMARY Liver glycogen content was determined after administration of D9-tetrohydrocannabinol (p9-THC) to monkeys I . V . for 28 days, to rabbits S . C . for 28 days, and to rots by a single inhalation exposuro . D9-THC doses above 4mg/rcg depressed liver glycogen stores by 60-75% in monkeys with no change in blood glucose . In the rabbit, 15-153mg/kg initiated a 45-72% dose-rotated decline in liver glycogen . Rats subjected to inhalation of 34-IOOmg/kg responded with a dose-rotated 20-26% decroase in liver glycogen within 24 hrs . which intensified to a 33-74% fall 7 days post exposuro . It is suggested that depleted liver glycogen storos arise from both a nonspecific strass and the necessity for oxidizable substrate for detoxification of cannabinoid . THE liver glycogen depot is influenced by a variety of physiological conditions of both a hormonal and non-endocrine naturo .

Stimulation of glycogenesis rosults from dietaryeffects

and gluconeogenesis from the action of glucocorticoids .

On the other hand, glycogenoly-

sis is rogulated by exercise and by eptnephrtne and glucagon through the phosphorylase activation mechanism and by insulin through incroased glucose utilization . Because of the role of adrenocorticotropin (ACTH) and adrenal cortical steroids in stress, pharmacologic challenges may be expected to attenuate carbohydrate metabolism in the Itver .

Drugs that have a primary site of action to the control nervous system may have

the potential of rogulating hypothalmio-roleasing factors that in tum affect levels of pituitary hormones .

Barbiturates, morphine, chlorpromazine and roserpine may inhibit ropro-

duetive function through such a mechanism (I) . Marihuana also exerts its major effect in the central nervous system and several observations such as hypothermia, hypopnea and hypotension suggest some action on the hypothalamus (2,3) .

(n addition, marihuana induced elevation in serum ACTH (4) and adrenal

steroids (5) have been reported .

Because of these findings, the prosent study was conducted

to ascertain the influence of ~9-tetrohydrocannabinol (D9-THC) on liver glycogen levels in monkeys, rots and rabbits .

The cannabinoid was administered by inhalation, intravenously

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~9-THC and Liver Glycogen Stores

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or subcutaneously in acute and subacute investigations . Methods

AI I animals wero housed in steel suspension cages, monkeys individually, and rabbit: and rats throe to a cage . Commercial laboratory chow and wafer wero given _ad Iibitum and monkeys roceived frosh fruit daily . Room temperatures wero maintained at

23 t 2° C . Clinical and physiological measurements wero made weekly but all animals wero observed daily. Proliminary pharmacological and neurochemioal findings have been roporMd elsewhero(6-12) . Synthetic (-)-trons-D9-THC with a purity of 96% was provided by NIMH . Pharmaceutical formulations consisted of solutions of ~9-THC in 100% U. S . P, grade sesame oil and emulsions containing 3-~!% D9-THC in 10% sesame oil-I% Polysorbate 80-isotonic saline for I . V. administration (13). Placebo and marihuana cigarottes containing appraodmately I .2% A9-THC wero also provided by NIMH . Marihuana cigarottes with 996 p9-THC wero proparod by imprognating marihuana cuttings with a chloroform solution of p9-THC, evaporating the solvent and hand-rolling cigarottes.

Monkey 28-Day A9-THC I . V. Troatment: Rhesus monkeys (Maraca mulatto) weighing 3-4 kg wero arranged in groups of 2 males and 2 females each and wero treated I . V, with vehicle, 5, 15 or 45 mg ~9-THC/kg for 28 days. All I. V. injections wero at a rate of 2ml/15 seconds . Except for two un expected deaths due to cumulative toxicity at the 45 ma/rcg dose, no deaths occurred in

the other groups . One animal from each of the other groups was sacrificed at day 21 and the romainder between I-5 days after the last treatment on day 28. Rabbit 2~Day A9-THC S. C. Troatment: Five groups of 3 female New Zealand albino rabbits weighing appraximatelyl .7kg wero injected subcutaneously in a differont rogton of the abdominal surface each day for 28 days. Each group of animals roceived either isotonic saline, sesame oil or D9-THC in sesame oil at a dose of 16, 45 and 153mg/kg. Injection volume was kept constant at 0 .6 ml/kg of body weight. Rat Acute A9-THC Inhalation Study: Groups of 6 male and 6 female Fischer rats of approximately IOOg body weight wero conditioned io the inhalator beforo expasuro to marihuana smoke in a horizontally

modified Walton smoking machine (14, 15) . Animals wero retained in a cassette of cone shaped holders oriented in a circular array which was attached to either side of a 400m1

O9- THC and Liver Glycogen Stores

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cylindrical smoke chamber . Every minute a 60m1 puff volume of 2-second duration was automatically introduced into the smoke chamber from 3 simultaneously smoked marihuana cigorottes. Ths smoke romained in the chamber for 30 seconds and was then actively purged with frosh air beforo the commencement of the next puff. The dose of marihuana was rogulated by varying the number of puffs administered . Rats wero exposed to09-THC doses of 18, 34, 68 or IOOmg/kg or placebo smoke. Half the animals wero sacrificed 24 hours later and the other half after 7 days post troatment .

Biochemical Determinations : Blood samples from monkeys wero taken from the medial cubical vein beforo and during troatment; rabbits wero exsanguinated by cardiac puncturo beforo necropsy. Blood

glucose was determined by the hexakinase reaction (16). At necropsy the liver weights wero roaorded and an approximate 6g samples wero digssted.in IOmI of hot 30% potassium hydrnoride . Aliquot of a water-~diluMd digestis wero reacted with anthrare In sulfuric acid fa the estimation of glycogen (17) .

Result The m=int ara shown in the following Tables (I for the monkey, 2 for the rabbit and 3 for the rat) . TABLE I The Effect of Inhavenous A9-Tetrahydrocannabinol on Monkey Liver Wst Weight, Carbohydrate Content and Blood Glucose D9-THC

No. monkeys (ma/k~da ) sex Vehicle x 28°

Liver mean t S . D. gl wet weight m

g)

2M + 2F

87.3 f 9.5

5 x 28 15x 28

2M + 2F 2M+2F

84 .4 t 10 . 0 100.4f 4.5c

5. Of 2 .8 b 4.3t2.1b

45x 28

IMd

119.Ot21 .5c

3.4

13.6f 6.9

~ Blood glucose mean t S. D . protest Mst (mg/IOOmI) (mg/IOOmI)

I

65 t 5

69 t 5

76 f 6 77f 9

84 f 4 79f 4

66f 5

65f 2

a. Vehicle contained 1096 sesame oil - I% Polysorbate 80 in isotonic saline b . P< 0.01 c. Livers slightly edematous d. CMe male and one female died unexpectedly and a second female was sacrificed promaturoly

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The data in Table I reveal a 60-75% depression (P< 0.01) to the concentration of liver glycogen at all doses of e9-THC with no apparent change in blood gluwae ievela . The highest doses of e9-THC induced a 29-36% elevatïon in wet weight of munkdy livers . Similar results were obtained in another study where 4, 16 or 64 but not I mg,/ky was given for 10 days . Subcutaneous administration of e9-THC in the rabbit for 28 days evoked signiti~wnt parallel dose-related decreases of 25-55% (P< 0.01) in liver wet weight and dec:raases of 45-72% (P< 0.01) in glycogen conront (Table 2) . Thero was also a dose-rolared signifi cant decline of 15-6396 in final body weight to initial body weight ratio . Blood glucose remained unchanged. TABLE 2 The Effect of

e9-Tetrahydrocannabinol

Cüven 28 Days Subcutans~ously

on Rabbit Body Weight, Liver Glycogen and Blood Cslacose°

e9-THc (m9~9)

Ratio of final body weight to initial body weight

Liver mean f S. D.

Saline

I .84 t 0. 12

98 .7 f 19 .5

158 t 24

Sesame-oi I

I .64 t 0. 06

43 .2 f 7 .2

89 .3 f I .5

175 f 14

15 .9

I .53 f 0. 03

66 . 5 f 9. 5 6

4 I . 8 f I .5

45 .0

1 .39f 0.03c

153 .4

0 .94t 0.066

wet weight (g)

glycogen (mg~9)

Blood glucose mean f S. D. (mg/100 m I)

23 .2 t 4. 5 b

18% t 18

62 .7f 3.06

14 .9t 2. 16

172f 21

40 .4f I .Sb

I I .9f I .3b

128t 21

a . All values are a mean of three female animals per group b . P< 0 .01 c . P< 0 .05 Fischer rats subjected to acute inhalation of smoke from marihuana cigorettes also responded with a depression in liver glycogen (Table 3) . Since the fivers of molt and fe male animals gave similar results, the values for both sexes were combined . The day following a single inhalation, liver glycogen levels fell from control values by 20-2b% at doses above 34mg/kg (P< 0.05) . Corresponding detanninations of liver waiyht and ylyc;ogen following a 7-day recovery period revealed a dose-related decrease in gly~ugen . The decline in the concentration of liver glycogen was 33%, 45% and 74% fur e9-7NC ocses of 34, 68 and IOOmg/kg, respectively (P< 0 .01 to< 0.05) . Such aecroments wore in

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addition to those induced by placebo smoke itself . TABLE 3 Liver Glycogen Levels in Fischer Rats Exposed to a Single Inhalation of A9-Tetrahydrocannabinol a

~9 -THC ~r^9~9 x d°Ys)

Liver mean f S . D. wet weight da y I posttreat.

~9)

gly~~n

day 7 posttroat.

da y I ttreat .

~m9~9)

da y 7 rear .

Placebo x I

5 .79 t I . 16

5 .99 1 0 .72

56 .5 f 9. I

28 .5 1 6 .3

18x1

5 .071 1 .20

6.25E 0.51

51 .7E 3 .7

29 .0E 6 .1

34 x 1

5 .47 f 0. 85

6. 051 0.64

61 .4 f 14 .6

19 . 21 4. 8

68x1

5 .81E I .II

5 .55f0.90

41 .5E 4.8 6

15 .815 .6 6

IOOxI

5 .80E 1 .50

5 .74E 0.85

45 .1E 11 .0 6

7.31 1 .6 0

a . Each group contained 6 M and 6 F mts: glycogen determined either 24 hours after the last exposure or after a one week recovery period b . P< 0.05 c . P< 0 .01 D lscussion That marihuana and its major pharmacological component ~9-THC have a marked effect on liver glycogen stores was evident under a variety of treafinent regimens . L1~THC subacute treatment by the I . V. route in monkeys, acute inhalation in the rat, and S. C. administration in the rabbit diminished liver glycogen depots . In the instance of the~inhalation route, the effect on liver glycogen persisted and intensified during a 7-day recovery period . Generally, the changes in liver glycogen were dose-rotated and Independent of

changes in blood glucose . Alteration in glycogen levels did not correlate with changes in liver weight since monkeys and rats rosponded with glycogen changes in the absence of

signifcant liver weight changes . Moreover, in the rabbit, a marked decline in liver glycogen stores occrmAd before a significant decroment in body weight .

The doses of pure synthetic D9-THC used in the prosent study were considerably

higher than thate generated by smoking American marihuana with a I .5% D9-THC content

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or European hashish with an average 7 .5% content. In man, a usual inhalation dose of D9-THC is known to be approximately 0.2 mg/Icg from marihuana or I mg/kg from hashish (3). On the basis of surface area (18), equivalent Inhalation doses for the rot would be I .4 and

7mg/kg, rospectively . The inhalation doses needed to alter liver glycogen content in the rat wero respectively 20-and 4-fold greater than the hunan doses. It was not surprising that high doses wero necessary for evoking a glycogen rosponse by inhalation in the rat since this animal, an obligatory nose-broather, has a highly efficient filtration apparatus in its nasal passages . Since the I. V. route closely simulates the inhalation route, marihuana and hashish doses in the monkey, calculated on the basis of surface area and equivalent to that used by man, would be 0 .6 and 3mg/kg, rospectively . Thus, in the present study, I .V . doses affecting liver glycogen wero only 2-6 times groater than those used by man . This was also true for the S . C. doses used to the rabbit study . Responses of liver glycogen levels to drugs affecting the CNS have been inve:tlgated infroquently . Cannabinol given intramuscularly has been roported to deplete liver glycogen in the rabbit (20) . Subacute IntragaaMc doses of ethanol did not alter Ilver glycogen in the rat (21) . Cigarotte tobacco whole smoke has been shown to decrease liver glycogen In mice (14) . In man, marihuana inhalation deteriorated the glucose tolerance test (22) and some claim of hepatotoxiclty has been made (23) . Marihuano-stimulated appetite in man (24) and rat (25) without a change in blood glucose is known . Diminution of liver glycogen depots under acute inhalation circumstances may be a nonspecific stress rosponse like that observed for mice exposed to tobacco smoke (14) . On the other hand, the liver itself may roquiro incroased chemical energy to metabolize the large quantities of marihuana . Unlea a romarkably increased tumaver rate of blood glucase existed that provented detection of blood glucose formation from glycogen, it seems reasonable that Ilver glycogen was providing oxldizable subatr+ate for synthesis of hydr+oocylases and chemical energy for the hydroocylation reactions associated with cannabinoids (26, 2~ . Whether marihuana has a diroct effect on liver phosphorylaae or an indlroct effect through rolease of epinephrine, glucagon or insulin romains udcnawn (28) . Conclusions It may be concluded that high doses of A9-THC given LV ., S. C. or by inhalation for a suitable number of troatments can deplete liver glycogen depots . Reduced glyoogsn levels may be nonspecific stross response and/or stimulated glycogenolysis may provide axidizable substrate needed for detcodtlcation of cannabinoids .

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