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REVIEW
Endocannabinoids, feeding and suckling – from our
perspective
R Mechoulam
1
, EM Berry
2
, Y Avraham
2
, V Di Marzo
3
and E Fride
4
1
Department of Medicinal Chemistry and Natural Products, Pharmacy School, Medical Faculty, Hebrew University,
Jerusalem, Israel;
2
Department of Human Nutrition and Metabolism, Faculty of Medicine, Braun School of Public Health,
Hebrew University, Jerusalem, Israel;
3
Istituto per la Chimica di Molecole di Interesse Biologico, Consglio Nazionale delle
Ricerche, Felice, Naples, Italy and
4
Departments of Behavioral Sciences and Molecular Biology, College of Judea and
Samaria, Ariel, Israel
In this overview we have summarized some aspects of our published work related to the effects of the endocannabinoid system
on appetite and suckling. As noted also by several other groups we have found that anandamide, a major endocannabinoid,
enhances appetite in mice. On partial or full food deprivation over 24 h the levels of 2-arachidonoyl glycerol (2-AG), a second
major cannabinoid, are initially elevated in mouse brain; however, partial food deprivation over a longer period causes reduction
of 2-AG levels. Blocking the endocannabinoid system with a CB1 antagonist on the 1st day after birth leads to inhibition of
suckling; later administration also affects suckling, but does not fully block it.
International Journal of Obesity (2006) 30, S24–S28. doi:10.1038/sj.ijo.0803274
Keywords: appetite; anandamide; 2-arachidonoyl glycerol (2-AG); biphasic effects; starvation
Introduction
Preparations of the Cannabis plant (marijuana, hashish,
ganja, bhang) have been known since times long past,
particularly in India, for their enhancement of appetite.
1
However, almost no research was carried out on this aspect of
Cannabis action until the identification of the psychoactive
constituent, D
9
-tetrahydrocannabinol (THC).
2
Later, interest
in THC as an orexigenic agent rapidly increased when AIDS
patients found that their appetite was enhanced and that
they lost less weight by smoking marijuana. After animal
experiments showed that THC indeed promotes appetite and
clinical trials confirmed its activity, this natural product was
introduced clinically under the generic name Dronabinol.
With the discovery of the cannabinoid CB
1
receptor and
the arachidonic acid derivatives anandamide and 2-arachi-
donoyl glycerol (2-AG) as endogenous cannabinoids, the
field was opened for a thorough investigation of the
molecular basis of the action of the cannabinoid system on
appetite, weight gain and suckling.
In the present overview, we review and summarize some of
the published research carried out in our laboratories in
Jerusalem and Naples in the above areas.
The endocannabinoid system in suckling
Our first foray in this field was the result of a publication in
Nature on the presence of anandamide in chocolate.
3
As
arachidonic acid derivatives are very rare in plants we looked
for anandamide in Israeli-made chocolate and found essen-
tially none. However, we found that oleamide, a sleep
producing endocannabinoid-like compound is present, as it
is in soybean, hazelnuts, oatmeal and millet.
4
Oleamide is an
inhibitor of fatty acid amide hydrolase, the enzyme that
breaks down anandamide. Hence, it seems plausible that
foods may affect endogenous levels of anandamide and its
effects. Surprisingly this line of research has not been
pursued, although we reported that oleamide at high doses
(200 mg/kg) causes anandamide-like effects, associated with
movement, sedation, body temperature and pain. Is it
possible that oleamide and N-acylethanolamines (NAE),
which are present in plants, and which prevent hydrolysis
of anandamide, affect some of the subtle effects of endo-
cannabinoids in man?
The possible effects of food on endocannabinoid levels led
us to take a look at milk – the only food of newborn
mammalians.
5
We first examined the levels of anandamide,
2-AG, oleamide and several NAE’s in human as well as bovine
and goat milk. While the levels of anandamide were
negligible, all milks were found to contain 2-AG (1–9 m/g of
extracted lipids) and considerably higher levels of oleamide,
Correspondence: Professor R Mechoulam, Medical Faculty, Hebrew University,
Ein Kerem campus, Jerusalem 91120, Israel.
E-mail: mechou@cc.huji.ac.il
International Journal of Obesity (2006) 30, S24–S28
&
2006 Nature Publishing Group All rights reserved 0307-0565/06
$
30.00
www.nature.com/ijo
2-palmitoyl glycerol and 2-linoleoyl glycerol. The presence
of 2-AG in milk then led us to examine whether the
cannabinoid CB
1
receptor is involved in suckling. The
cannabinoid CB
1
receptor antagonist, SR141716A (20 mg/
kg), completely inhibited the physical growth of mouse pups
and caused death within 1 week, by depriving them of the
essential benefits of nursing. This devastating effect of
SR141716A was seen after daily injections between days 2
and 8 of life. At least as dramatic an effect was also seen after
a single injection of SR141716A, but only when administered
within the first 24 h after birth (day 1). Administration on
day 2 only, resulted in 50% mortality. Thus, the first 24 h of
life seem to be most critical for the putative endocannabi-
noid-induced growth-promoting effect, which is compatible
with the peak levels of 2-AG. This is also consistent with our
observation that 2-AG is present in mammalian milk from
the 1st day after birth (‘early’ milk or colostrum).
5
Apparently, pup mortality was due to impairment of
suckling. Thus, from day 1 of treatment, no weight was
gained. This effect was also evident from the absence of milk
in the stomachs of the SR141716A-treated pups on each day
of life.
A dose–response relationship was noted. Thus, when half
the dose (10 mg/kg) of SR 141716A was administered on day
1 in the single day regimen, effects of about 50% magnitude
on mortality and milk bands were observed, 5 mg/kg had
almost no effect.
Several experiments were performed in order to investigate
whether the effects of SR141716A were mediated by
cannabinoid CB
1
receptors.
5
When THC was co-adminis-
tered with SR141716A, the detrimental effects on weight
gain and feeding were almost completely reversed. Co-
administration of cannabidiol, a cannabinoid which does
not bind cannabinoid CB
1
receptors, had no influence on
SR141716A-induced effects. 2-Arachidonoyl glycerol by itself
had no effect on the SR141716A-induced growth stunting.
This lack of effect of exogenous 2-AG alone is probably due
to the ease of its degradation by enzymatic hydrolysis of its
ester bond and to its rapid uptake. However, endogenous 2-
AG may be partly protected from hydrolysis and its uptake
may be slowed down by monoacyl glycerols. As mentioned
above, in all milk samples analyzed by us 2-AG is accom-
panied by several 2-acyl glycerols. 2-Palmitoyl glycerol and
2-linoleoyl glycerol, which do not bind to cannabinoid CB
1
receptors cause no cannabis-type effects as evaluated in
binding assays and in several in vivo assays in mice. In all
these assays, however, 2-AG activity was significantly
enhanced by 2-palmitoyl glycerol and 2-linoleoyl glycerol
(an effect named by us ‘entourage effect’). If 2-AG is
consumed by pups with the milk, the ‘entourage’ com-
pounds that are present in milk may enhance its suckling
activity. Therefore, we evaluated the activity of 2-AG in the
presence of the ‘entourage’ compounds. We found that co-
administration of 2-AG (1 mg/kg), 2-palmitoyl glycerol
(10 mg/kg) and 2-linoleoyl glycerol (20 mg/kg) with the
antagonist (20 mg/kg), on day 1, resulted in a significant
delay in mortality rates and an approximately overall one-
third decrease in mortality compared to the treatment effect
of SR141716A alone. A partial improvement in weight gain
was also observed. Thus, the ‘entourage’ effect may enhance
the putative growth-promoting effects of 2-AG. However,
dose ranges may have to be investigated further in order to
find the maximal ‘entourage’ effect, which appears to be a
rather unique mechanism of enhancing biological activity.
The above data strongly suggest that the antisuckling and
growth-inhibiting effects of SR141716A, are mediated by
blockage of the cannabinoid CB
1
receptors.
The dose of SR141716A needed for a complete inhibition
of food ingestion and survival is relatively high (20 mg/kg).
However, this observation is compatible with the low
responsiveness to cannabinoid ligands during the 1st week
of life in developing pups. Moreover, although lower doses
are often found sufficient to block some THC-induced
effects, many effects of the antagonist require higher doses.
Zimmer et al.
6
have found that cannabinoid CB
1
receptor
knockout mice survive the initial stages of life, which
obviously involve suckling. However, increased mortality
was noted in such mice. Presumably other mechanisms
compensate for the lack of cannabinoid CB
1
receptor-based
suckling. Petrov et al.
7
have reported that endogenous
opioids are involved in early suckling and it is possible that
this, or other systems assume a more prominent role in CB
1
knockout mice. One such system may involve the receptor
gene for lysophosphatidic acid, since Contos et al.
8
have
described a defective suckling response in neonatal mice
with a targeted deletion of this gene. This response may be
related to the observation that the lysophosphatidic acid
receptors have a sequence homology of nearly 30% with the
cannabinoid receptor.
9
There is also a chemical relationship
between the two families of bioactive compounds. The
chemical structures of 2-AG and lysophosphatidic acid (with
2-arachidonoyl as the acyl moiety) only differ by the absence
of a phosphate group in 2-AG.
In a later study,
10
we generalized the above findings to a
different mouse strain (C57BL/6) which is the background
strain for the cannabinoid CB
1
receptor-deficient mice
produced by Zimmer et al. We found that (untreated)
cannabinoid CB
1
receptor-deficient newborns have almost
no milk in their stomachs on day 1 after birth. This
observation supports a critical role for the endocannabi-
noid-CB
1
receptor system in the initiation of milk ingestion
by newborn mice. On the other hand, milk intake from day 2
of life by CB
1
/
mice did not resemble that of SR141716A-
treated normal pups. Thus, on day 2 of life, 75% of the CB
1
/
mice displayed milkbands and by day 3, 100% had ingested
milk, similarly to controls. Despite the ‘catch-up’ in milk
ingestion, however, body weights were significantly lower
throughout life. Rather, the delayed onset in suckling
observed in the knockout pups resembled the transient
effect of cannabinoid CB
1
receptor blockade in normal 5-
day-old pups. Thus, when 5-day-old C57BL/6 pups were
injected with SR141716A, milk ingestion was fully inhibited
Endocannabinoids, feeding and suckling
R Mechoulam et al
S25
International Journal of Obesity
the next day, but overall weight gain and survival were not
affected.
We do not know which compensatory mechanism
enables the CB
1
/
pups to start ingesting milk on the 2nd
day of life in the absence of cannabinoid CB
1
receptors. As
the opioid system plays a regulatory role in milk suckling
(see above) and in view of the rich interactions between the
cannabinoid and opioid systems, it is possible that the
opioid system takes over some of the cannabinoid functions
in CB
1
/
mice.
Thus far, two cannabinoid receptors have been identified
and cloned, CB
1
and CB
2
. The cannabinoid CB
1
receptor is
present in neural and in non-neural tissue, whereas the
cannabinoid CB
2
receptor is detected mainly in immune
cells. Our observations
10
are consistent with the existence of
a third cannabinoid receptor, present in cannabinoid CB
1
receptor-deficient mice, but which, similar to the cannabi-
noid CB
1
receptor, is blocked by SR141716A. Thus, it is
possible that SR141716A affects the CB
1
/
pups by blocking
this putative ‘CB
3
’ receptor, while an injection of SR141716A
in normal mice blocks both cannabinoid CB
1
and ‘CB
3
’
receptors. According to this scenario, the ‘CB
3
’ receptor is
only partially responsible for newborn milk suckling since
the cannabinoid CB
1
knockout mice were only partially
affected by the antagonist. Alternatively, ‘CB
3
’ receptors are
only partially blocked by SR14716A, thus explaining the
partial effect of SR141716A in the knockout pups.
A third cannabinoid receptor, not blocked by SR141716A,
has been proposed in previous in vivo and in vitro studies
using the same cannabinoid CB
1
receptor knockout mice as
in our study.
11
The cannabinoid CB
1
receptor agonist
WIN55,212-2 displayed specific binding to CB
1
/
brain
tissue, whereas SR141716A did not specifically affect
WIN55,212-2-induced stimulation of [
35
]GTPgS binding in
brains of CB
1
/
mice. In our study, we attempted to
encourage CB
1
/
pup growth with WIN55,212-2 or with 2-
AG which, as mentioned above, is found in high quantities
in milk. We also treated CB
1
/
knockout pups with the
potent agonist CP55,940 at two doses (5 and 20 mg/kg).
However, none of these treatments enhanced body weight
gain in the CB
1
/
pups. Therefore, the putative third ‘CB
3
’
receptor suggested by us has different pharmacological
properties compared to the ‘CB
3
’ receptor proposed pre-
viously.
In conclusion of this portion of our studies, we can state
that we have confirmed a critical role for cannabinoid CB
receptors for the initiation of milk suckling within the first
24 h of birth. Timing is essential: administration of the
cannabinoid CB receptor antagonist on a later day results in
a partial effect or no effect at all. We speculate that normally,
without experimental interference, endocannabinoids, in
particular, 2-AG from the pup’s brain which peaks on the 1st
day of life, are required to initiate the suckling response/milk
ingestion. We further postulate that from day 2, when levels
of 2-AG are lower again, endocannabinoids from maternal
milk, maintain the suckling process.
Surprisingly, milk intake and survival were also impaired
upon administration of the CB
1
receptor antagonist in CB
1
receptor-deficient pups although not as dramatically as in
wild type pups. These results support previous evidence for
the existence of additional cannabinoid receptor(s). We
suggest two interpretations, both consistent with our finding
of a partial effect of SR141716A in the cannabinoid CB1
knockout pups: either SR141716A partially blocks the
putative ‘CB
3
’ receptor present on cannabinoid CB
1
knock-
out mice, or SR141716A is a full antagonist of the ‘CB
3
’, but
this receptor only partially controls the initial stages of milk
ingestion in the newborn mouse.
Effects of endocannabinoids on appetite
The research on the effect of cannabinoids on feeding and
appetite has been summarized in several reviews.
12,13
Of
particular relevance to our work is the report by Williams
and Kirkham
14
who found that anandamide (0.5, 1, 5,
10 mg/kg) increased food intake in male rats during a 3-h
feeding period and that this effect was blocked by the CB
1
antagonist, SR 141716A. In a different protocol (female mice
on a 40% diet restriction) we found that doses as low as
0.001 mg/kg enhanced food intake.
15
Anandamide-treated
mice consumed 44% more food during 1 week of 2.5 h
feeding each day. Doses of 0.7 and 4 mg/day did not cause
any significant change. This unexpected behavior has
precedence. We have shown that anandamide produces a
bi-phasic dose–response in both behavior and neuro-bio-
chemistry.
16
Thus, at low doses, it stimulated ambulation
and rearing, as well as gut motility in the open field
situation, decreased the rate of immobility on ring standing,
and analgesia on a hot plate; it also stimulated aggressive
behavior in timid mice and phagocytosis by mouse leuko-
cytes. At high doses, opposite effects of inhibition were
observed.
In an early review on the effects of Cannabis, Paton and
Pertwee
17
wrote: ‘Nor does one readily find another
substance so contradictory, capable of taming yet producing
aggressiveness, of both enhancing and depressing sponta-
neous activity, of being anticonvulsant yet generating
epileptiform cortical discharges.’ Another early example of
a cannabinoid biphasic effect is reported by McLaughlin
et al.
18
They showed that THC, when administered to sheep,
initially increased food intake, but later the intake decreased.
Endocannabinoid levels in brain – relevance to
appetite
Several groups have looked into the presence and levels of
endocannabinoids in the brain and have established ex-
cellent methods for analyses of endocannabinoids. Endo-
cannabinoid levels are very sensitive to biochemical and
Endocannabinoids, feeding and suckling
R Mechoulam et al
S26
International Journal of Obesity
pathological changes in the brain. Thus, Di Marzo et al. have
reported
19
the presence of anandamide and 2-AG in two
regions of the basal ganglia, the globus pallidus and
substantia nigra. In the reserpine-treated rat, an animal
model of Parkinson’s disease, suppression of locomotion was
accompanied by a sevenfold increase in the levels of the 2-
AG (up to around 5.0 nmol/g) in the globus pallidus, but not
in the other five brain regions analyzed. Defective leptin
signalling is also associated with elevated hypothalamic, but
not cerebellar, levels of endocannabinoids in obese db/db
and ob/ob mice and Zucker rats. Acute leptin treatment of
normal rats and ob/ob mice reduces anandamide and 2-AG
levels in the hypothalamus. These findings indicate that
endocannabinoids in the hypothalamus may tonically
activate CB
1
receptors to enhance food intake and thus form
part of the neural circuitry regulated by leptin.
In our original publication, in which we described 2-AG as
an endogenous cannabinoid, we recorded that this com-
pound is present in about 5.0 nmol/g wet weight of mouse
spleen tissue.
20
In a later study, we found that in isolated rat
aorta under cholinergic stimulation by carbachol the
production of 2-AG was enhanced about fivefold (from
0.41 to 2.0 nmol/g wet weight).
21
We have also shown that,
after injury to the mouse brain, 2-AG levels were signifi-
cantly elevated (in 4 h the level of 2-AG increased on average
from 9.18 to 105.4 nmol/g and after 24 h it still remained at a
level of 56.0 nmol/g).
22
The first direct evidence of altered brain levels of
endocannabinoids (2-AG particularly) during fasting and
feeding was reported by Kirkham et al.,
23
who measured
anandamide and 2-AG levels in brain regions of rats, during
fasting (24 h), feeding of palatable food, or after satiation.
Endocannabinoid levels were compared to those in rats fed
ad libitum. Fasting increased levels of anandamide and 2-AG
in the limbic forebrain and, to a lesser extent, of 2-AG in the
hypothalamus. By contrast, hypothalamic 2-AG declined as
animals ate. No changes were detected in satiated rats.
Endocannabinoid levels in the cerebellum, a control region
not directly involved in the control of food intake, were
unaffected by any manipulation. 2-Arachidonoyl glycerol
was sensitive to variations during feeding and this observa-
tion supports a role for endocannabinoids in the control of
appetitive motivation. It was also demonstrated that 2-AG
can reliably stimulate eating.
Our aim was to establish whether a 12-day food restriction
regimen in mice affects 2-AG levels in the whole brain, in the
hypothalamus, a brain area associated with feeding, and in
the hippocampus, a brain area associated with cognitive
functions.
24
We assumed that the results of such measure-
ments may be of some relevance to starvation and/or to
anorexia nervosa – a psychiatric condition in which the
patients impose diet restrictions on themselves resulting not
only in reduced food intake but also in multiple endocrine
abnormalities. Our results showed that diet restriction over
12 days lowered the levels of 2-AG both in the hippocampus
and the hypothalamus, although differences were observed.
The amounts of 2-AG in the hypothalamus depended on the
severity of the diet restriction, while in the hippocampus the
amounts of 2-AG fell to a common level, irrespective of the
diet restriction protocol.
24
Our results differ from those reported from Kirkham et al.
presumably due to the non-identical experimental condi-
tions.
23
A major difference between the experiments de-
scribed by Kirkham et al. and ours is the food restriction
protocol. While Kirkham et al. report that the rats used in
their experiment were under severe food restriction (20% of
their normal daily intake) and were killed after 24 h, the mice
used in the experiments described here were on a less severe
food restriction (see above) for a considerably longer period
(12 days). Thus, Kirkham et al. apparently measure the effects
of hunger, while we measure presumably the effect of
semistarvation and our observations may be relevant to the
physiology of starvation and anorexia nervosa. In order to
reconcile our results with those of Kirkham et al. we analyzed
mouse brain after 24 h of full starvation (fasting). Indeed, as
in the British report, we found significant enhancement of 2-
AG levels, contrary to the decrease of 2-AG levels after 12
days of diet restriction described above. It is well established
that endocannabinoids enhance appetite and it is reasonable
to expect that over short periods of hunger 2-AG levels will
be enhanced, as reported by Kirkham et al., and confirmed by
us. In our view, the lowering of 2-AG levels after 12 days is
compatible with the needs of an animal on starvation.
Potentiation of hunger, caused by high levels of endocanna-
binoids, presumably will be detrimental in these conditions.
We assumed that supplementation of food such as soya
containing polyunsaturated fatty acids, in particular linole-
ate (18:2n6) which may be converted into arachidonate,
would elevate the 2-AG levels and could counteract the effect
of diet restriction. We were surprised to find that such
supplementation actually significantly lowered 2-AG levels
in non-diet-restricted mice and in the hippocampus in mice
on 50% diet restriction. We also assumed that 20% soya oil
might cause end product inhibition of 2-AG synthetic
enzymes by excess arachidonate and therefore decreased
the soya oil level to 5%. Although a strong lowering trend of
2-AG levels in total brain was noted, this effect did not reach
statistical significance. Other types of oil also did not cause
any significant change in 2-AG levels. Berger et al.
25
in a
study on piglets also reported that 2-AG levels exhibited a
strong trend towards decrease if the milk was supplemented
with the long chain unsaturated fatty acids.
If the above observations on diet restriction in mice
parallel the human condition, we can expect that diet
restriction self-imposed by humans, as in anorexia, may
cause lowering of 2-AG levels leading to further reduction of
food consumption, thus perpetuating the clinical condition.
The observed lowering of 2-AG levels in mice on
prolonged diet restriction may also represent a general
coping (psychobehavioral) strategy for intermittent starva-
tion when food is scarce – most wild animal species certainly
undergo periods of starvation. As we are all well aware
Endocannabinoids, feeding and suckling
R Mechoulam et al
S27
International Journal of Obesity
starvation was also a common occurrence in human
populations in former times, or even today, in certain parts
of the world.
Possible therapeutic implications
Patients with anorexia nervosa refuse to eat and show
decreased concentrations of tyrosine, norepinephrine meta-
bolites in the cerebrospinal fluid and a variety of endocrine
abnormalities. Some patients have memory and learning
deficits. Two brain areas are prominent in these processes –
the hippocampus, which is involved in aspects of cognitive
function and the hypothalamus, the center for the regula-
tion of systemic energy balance. In our study, anandamide
administration was able to reverse many of the neurochem-
ical and behavioral deficits following semistarvation, even
before weight gain. Such properties could have therapeutic
potential in the treatment of cachexia associated with cancer
and AIDS, the side effects of weight loss and in the
maintenance of a reduced body weight and also in the
extreme case of anorexia nervosa. However, much further
work is required in studying the effects of chronic cannabi-
noid treatment on these functions in animals at different
levels of systemic energy balance.
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