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Blackwell Science, LtdOxford, UKOBRobesity reviews1467-78812005 The International Association for the Study of Obesity. 64307322Review ArticlePYY3-36 as anti-obesity drug M. M. Boggiano
et al.
obesity reviews
Appetite Regulatory Peptides
PYY3-36 as an anti-obesity drug target
M. M. Boggiano1*, P. C. Chandler1, K. D. Oswald1, R. J. Rodgers2, J. E. Blundell2, Y. Ishii2, A. H. Beattie2,
P. Holch2, D. B. Allison3, M. Schindler4, K. Arndt4, K. Rudolf4, M. Mark4, C. Schoelch4, H. G. Joost5, S. Klaus5,
C. Thöne-Reineke5,6, S. C. Benoit7, R. J. Seeley7, A. G. Beck-Sickinger8, N. Koglin8, K. Raun9, K. Madsen9,
B. S. Wulff9, C. E. Stidsen9, M. Birringer10, O. J. Kreuzer10, X. Y. Deng11, D. C. Whitcomb11, H. Halem12,
J. Taylor12, J. Dong12, R. Datta12, M. Culler12, S. Ortmann13, T. R. Castañeda5,7 and M. Tschöp5,7
1
Department of Psychology, University of
Alabama at Birmingham, Birmingham, AL,
USA; 2Behavioural Neuroscience Laboratory,
Institute of Psychological Sciences, University
of Leeds, UK; 3Section on Statistical Genetics,
Department of Biostatistics & Clinical Nutrition
Research Center, University of Alabama at
Birmingham, Birmingham, AL, USA;
4
Boehringer-Ingelheim Pharma GmbH & Co
KG, Biberach, Germany; 5German Institute
of Human Nutrition, Potsdam-Rehbrücke,
Germany; 6Charité-Universitätsmedizin Berlin,
Institute of Pharmacology and Toxicology,
Center for Cardiovascular Research (CCR),
Berlin, Germany; 7Department of Psychiatry,
University of Cincinnati Genome Research
Institute, Cincinnati, OH, USA; 8Institute of
Biochemistry, University of Leipzig, Leipzig,
Germany; 9Novo Nordisk A/S, Discovery,
Summary
The neuropeptide Y (NPY)/peptide YY (PYY) system has been implicated in
the physiology of obesity for several decades. More recently, Batterham et al. 2002
ignited enormous interest in PYY3-36, an endogenous Y2-receptor agonist, as a
promising anti-obesity compound. Despite this interest, there have been remarkably few subsequent reports reproducing or extending the initial findings, while
at the same time studies finding no anti-obesity effects have surfaced. Out of 41
different rodent studies conducted (in 16 independent labs worldwide), 33 (83%)
were unable to reproduce the reported effects and obtained no change or sometimes increased food intake, despite use of the same experimental conditions (i.e.
adaptation protocols, routes of drug administration and doses, rodent strains,
diets, drug vendors, light cycles, room temperatures). Among studies by authors
in the original study, procedural caveats are reported under which positive effects
may be obtained. Currently, data speak against a sustained decrease in food
intake, body fat, or body weight gain following PYY3-36 administration and
make the previously suggested role of the hypothalamic melanocortin system
unlikely as is the existence of PYY deficiency in human obesity. We review the
studies that are in the public domain which support or challenge PYY3-36 as a
potential anti-obesity target.
Copenhagen, Denmark; 10Peptides &
Elephants GmbH, Potsdam-Rehbrücke,
Keywords: Food intake, obesity, peptide YY, PYY, PYY(3-36), Y-2 receptor.
Germany; 11Department of Medicine, Division
of Gastroenterology, Hepatology and Nutrition,
University of Pittsburgh, Pittsburgh, PA, USA;
12
Biomeasure Incorporated/Ipsen Group,
Milford, MA, USA/Paris, France; 13Institute for
Zoo and Wildlife Research, Berlin, Germany
*Formerly M. M. Hagan.
Received 7 May 2005; revised 31 May 2005;
accepted 7 June 2005
Address for correspondence: MM Boggiano,
415 Campbell Hall, 1300 University Blvd.,
University of Alabama at Birmingham,
Birmingham, AL, 35294-1170, USA. E-mail:
[email protected]
obesity reviews (2005) 6, 307–322
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
307
308
PYY3-36 as anti-obesity drug
M. M. Boggiano et al.
Introduction
Peptide YY (PYY, PYY1-36) is a 36 amino acid hormone,
first isolated from porcine intestine (1) which, together with
pancreatic polypeptide (PP) and neuropeptide Y (NPY),
belongs to the pancreatic polypeptide family. These peptides
are structurally and biologically similar, but are synthesized
and secreted from different sources (2,3). PYY is mainly
present in ileum and expressed by large intestine endocrine
cells (3–5); it is released postprandially in proportion to the
calories ingested (6,7) and acts to inhibit gastric, pancreatic,
and intestinal secretion as well as gastrointestinal motility
(3,8). Moreover, PYY is also present, although in lower
quantity, in the mammalian central nervous system, mainly
in the brain stem (9,10). The effects of centrally administered PYY in rodents are well known and include dramatic
increases in food intake (mainly carbohydrates), body
weight gain, and exploratory activity in rodents (11–15).
These effects are putatively believed to be predominantly
mediated via the Y1 and Y5 receptors (16–18).
The endogenous peptide form, PYY3-36, is a Y2/Y5
receptor agonist and also produces hyperphagia and obesity syndromes in rats and mice when administered centrally (19–23). However, the orexigenic effects of PYY and
NPY, which is equipotent to PYY3-36 in producing hyperphagia, persist in mice with Y1 and Y5 receptor deletions
(21,24,25). The Y2 receptor is a putatively presynaptic
autoreceptor with inhibitory effects on NPY and PYY
release (26,27) and may hence play a role in limiting food
intake. Less is known about the effects of peripherally
administrated PYY or related peptides on ingestive behaviour and energy regulation. The emetic effects of intravenous (i.v.) injection of PYY have been known (28),
but, more recently, intraperitoneal (i.p.) administration of
PYY3-36 was reported to inhibit food intake in rodents
and, after i.v. administration, to decrease appetite in
humans (29–31). However, in comparison with the extensive preclinical testing of other appetite-related hormones
(e.g. leptin, melanocortin receptor ligands, etc.) prior to
human studies, the rapid transition of PYY3-36 from bench
to clinic seems unusual, especially given the scarcity of
published studies replicating the putative anorectic effects
of the peptide in rodents.
Triggered by the exciting findings that peripherally
administered PYY3-36 could indeed lower food intake, not
only in rodents but also in humans (29), others embarked
upon independent investigations. The published anorectic
effect of peripherally administered PYY3-36 was largely
not replicated or extended in other rodent models (23).
Among the few studies reporting anorectic responses, there
are inconsistencies and a lack of robust anti-obesity activity
with PYY3-36 (31–33). The most promising anti-obesity
drug candidates are those that produce their effects reliably
in preclinical models and do so robustly across a range of
obesity reviews
experimental situations (34). Given the number of currently available and regularly discovered new drug targets
for obesity, this discrepancy in preclinical research questions the therapeutic significance of PYY3-36. As negative
results are rarely accepted for publication in visible scientific journals, the hopes of patients and doctors fighting
obesity may be unfounded. In addition, the ability of fellow
scientists to make difficult decisions regarding the identity
of the molecular targets and/or drug candidates in which
to invest may be compromised. The purpose of this review
is to evaluate the anti-obesity potential effect of PYY3-36
by presenting all preclinical evidence available on the efficacy of systemic administration of this peptide to reduce
food intake and body weight in rodents.
Effect of systemic administration of PYY3-36 on
food intake and body weight
Attempts to replicate original study yielding
anti-obesity effects of PYY3-36
In order to extend the originally reported anorectic actions
PYY3-36 (29), the present authors and others (23) independently attempted to reproduce the identical original
experimental conditions used by Batterham et al. (Table 1).
This included use of the exact compound (human PYY336), from the same company (Bachem), and with the same
catalogue number (H8585) (23). Habituated animals identical in strain, age and gender were administered identical
doses of the peptide via the same delivery routes in the same
volumes, and under identical light/dark phase, fed/fasted
conditions and number of injections (acute and chronic for
the same number of days). Food intake and body weights
were also measured at the same time points. Despite diligent
adherence to the original methods, the initial published
results could not be replicated. Most importantly, aside
from one study (35) and a second by the authors of the
original report (29), no other lab or experiment observed
a decrease in body weight or body fat in rodents (Table 1).
Extensions of the original protocol to test for antiobesity effects of PYY3-36
Whilst replication of published results is important before
embarking on drug discovery projects, it was not in the
interest of the present authors to challenge the original
findings with PYY3-36, but rather to extend them to other
obesity-relevant experimental conditions and to further
elucidate the peptide’s actions and mediating molecular
mechanisms (36). Since then, a few other study groups have
also performed such studies (31,33,35,37,38). As itemized
in part in Table 1, PYY3-36 was administered to several
rodent strains including Wistar, Long Evans, Lister-hooded,
and Sprague-Dawleys rats, as well as C57BL/6 J mice,
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
Chronic
Chronic
3h
Acute
Acuted
Acute
Acute
Acute
Acute
Acute
Acute
Acute
Chronic
Chronic
Acute
Acute
Acute
Acute
Acute
Chronic (7 d)
i.p.
i.v.
i.v.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
i.p.
i.p.
s.c.
s.c.
s.c.
(2 d)
(7 d)
(7 d)
(2 d)
Chronic (56 d)
Acute
Acuted
Acute
Acute
Chronic (5 d)
Acute
Acute
Acute
Acute
Acute
Acute
pump
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
fa/fa rat
Lister Hooded rat
Long Evans rat
Long Evans rat
Long Evans rat
Long Evans rat
Sprague-Dawley rat
Sprague-Dawley rat
Sprague-Dawley rat
Sprague-Dawley rat
Sprague-Dawley rat
Sprague-Dawley
(diet-induced obese)
Sprague-Dawley rat
Sprague-Dawley rat
Sprague-Dawley rat
Wistar rat
Wistar rat
Wistar rat
Wistar rat
Wistar rat
Wistar rat
Wistar rat
Wistar rat (fasted)
Wistar rat (fasted)
Wistar rat
Wistar rat
Agouti pre and
postobese mice
C57Bl/6 J mice
C57BL/6–129Svj mice
C57BL/6 mouse
C57BL/6 mouse
C57BL/6 mouse
Injection
protocol
Route of
administration
Species (male)
Total # of animals
(control)
20 (14)
4 ¥ 10 (10)
32 (8)
32 (8)
10 (5)
16 (8)
7 (7)
24 (8)
32 (8)
5 ¥ 8 (8)
21 (7)
24 (8)
16 (8)
16 (8)
16 ¥ 6 (16)
24 (8)
10 (5)
16 (8)
24 (12)
24 (8)
32 (8)
24 (6)
29 (13)
75 (19)
32 (8)
8–12
5–6 (5–6)
4–5 (10)
5 (5)j
3 ¥ 6 (6)
4 ¥ 6 (12)
17 (9)
PYY3-36 dose
100 mg (25 nmol)/kg/d
3, 30, 100 mg (0.7, 7.4, 25 nmol)/kg
30, 100, 300 mg (7.4, 25, 74 nmol)/kg
30, 100, 300 mg (7.4, 25, 74 nmol)/kg
30 mg (7.4 nmol)/kg
5 mg (1.2 nmol)/100 g
24, 60, 150 mg (5.9, 14.8, 37 nmol)/kg
3, 10 mg (0.7, 2.5 nmol)/100 g
3 mg (0.7 nmol)/100 g
1.2, 4, 12, 40 mg (0.3, 1, 3, 10 nmol)/kg
300 mg (74 nmol)/kg
3, 10 mg (0.7, 2.5 nmol)/100 g
5 mg (1.2 nmol)/100 g b.i.d.
5 mg (1.2 nmol)/100 g b.i.d.
20–200 ng (5–50 pmol)/kg/min
0.3, 3, 10 mg (0.07, 0.7, 2,5 nmol)/100 g
0.3 mg (0.07 nmol)/100 g
100 mg (25 nmol)/kg
100 mg (25 nmol)/kg
100 mg (25 nmol)/kg
3, 30, 100 mg (0.7, 7.4, 25 nmol)/kg b.i.d.
100 mg (25 nmol)/kg
5 mg (1.2 nmol)/kg
5 mg (1.2 nmol)/kg
30, 100, 300 mg (7.4, 25, 74 nmol)/kg
5 mg (1.2 nmol)/100 g b.i.d.
80 mg (20 nmol)/kg
0.3, 3, 10 mg (0.07, 0.7, 2.5 nmol)/100 g
0.3, 3, 10 mg (0.07, 0.7, 2.5 nmol)/100 g
4.2, 12.5 mg (1, 3 nmol)/kg
4.2 mg (1 nmol)/kg
1 mg (247 nmol)/kg
Table 1 Studies investigating the effects of systemic administration of PYY3-36 in rodents
Increase (~35%)
No effect
NP
NP
ND
ND
ND
ND
ND
ND
ND
ND
ND
Decreased
NP
NP
NP
ND
ND
No effect
Decreasedi
Decreased
No effect
No effect
Transient decreasek
–
–
–
–
–
–
–
–
–
Tschöp et al.
Raun et al.
Raun et al.
Raun et al.
Schindler et al.
Schindler et al.
Whitcomb et al.
Whitcomb et al.
Raun et al.
– Halem et al.
– Heiman et al.
PYY3-36 as anti-obesity drug
(31)
(29)
(23) – Moran et al.
(23) – Moran et al.
(23) – Heiman et al.
(23)
(23)
(63)
(29)
(23)
(23)
(23)
(23)
(23)
(23)
(23)
(23)
(23)
(29)
(38)
– Boggiano (Hagan) et al.
– Boggiano (Hagan) et al.
– Boggiano (Hagan) et al.
– Moran et al.
Ishii et al.
Heiman et al.
Benoit et al.
Benoit et al.
Heiman et al.
(35)
(23)
(23)
(23)
(23)
(23)
(33)
(23)
(23)
(23)
(37)
(23)
Decreasedb
No effect
No effect
No effect
No effect
No effect
NP
No effect
No effect
ND
NP
No effect
–
–
–
–
–
Referencea
Body weight
Increase (~7%)
No effect
Decreased
Decreased
No effect
No effect
No effect
No effect
No effect
No effect
Increased
Increased
No effect
Decreased
Decreasedh
No effect
No effectc
No effect
No effect
No effect
ND
Decreasede
No effect
No effect
Transient decreasef
Decreasedg
No effect
Food intake
obesity reviews
M. M. Boggiano et al.
309
Acute
Acute
Acute
Acute
Chronic (10 d)
Chronic (8 d)
Chronic (7 d)
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
s.c.
pump
ob/ob mice (female)
32 (8)
100, 300, 1000 mg (25, 74, 247 nmol)/kg/d
No effect
Decreasedq
(35)
(23)
(23)
(23)
(23)
(32)
(32)
(35)
(35)
–
–
–
–
Tschöp et al.
Tschöp et al.
Tschöp et al.
Heiman et al.
Referencea
i.p., intraperitoneal administration; s.c., subcutaneous administration; i.v., intravenous administration; b.i.d., twice a day; ND, not determined; NP, not provided in publication; MW of PYY3-36 is 4050.0 D.
a
For studies with multiple independently run studies, the name preceding the reference number is the lab PI(s).
b
In non-diabetic fa/fa rats; diabetic fa/fa rats had increase in body weight with PYY3-36.
c
No effect on total 1-h intake but a significant increase, ~23–50% in duration of feeding in the first 10 min of the test.
d
Wistar rats were also infused with 10 mg (2.5 nmol)/5 mL PYY3-36 (i.c.v) and increased food intake ~200%; 3, 4 and 8 mg (2 nmol)/3 mL i.c.v. increased food intake ~300%. Long Evans rats also increased
intake with 4.2 mg (1 nmol) PYY3-36 i.c.v. by ~60%. These studies confirm the bioactive viability of the PYY3-36 sample used in peripheral injections.
e
Only the 60 mg (14.8 nmol) dose decreased intake of liquid Ensure and only for 3 h with the suppression appearing to be inversely related to dose. Lesions of the area postrema in a separate group of rats
(n = 7) were found to increase the magnitude and duration of PYY3-36-induced anorectic effects.
f
Transient decrease was ~70% only at 30 min with 4.2 mg (1 nmol)/kg. Other dose and time points were without effect.
g
Rats were maintained on standard chow, or a high-fat diet (HFD), or were 50% chow-restricted. The PYY3-36-induced decreased in 24-h intake was slight, ~10% for chow- and HFD-fed; no effect in 50%
chow-restricted rats.
h
Mice were 24 h fasted; intake of all mice was decreased at 2 h and in only post-obese mice at 24 h.
i
Mice were 16 h fasted; a dose of 0.3 mg (0.07 nmol) actually increased food intake; decreased intake with 3 and 10 mg (0.7 and 2.5 nmol) was reported as statistically significant but represented an
exceedingly small amount of 4-h food intake reduction (within a range of less than one gram).
j
Control rats were Y2-receptor null mice whose intake was not affected by PYY3-36.
k
~30% decrease only in the first 3 d.
l
Food intake was measured on a ‘per cage’, not per mouse basis (4–5 mice per cage).
m
Food intake was reduced only with the highest dose (1 mg or 247 nmol/kg/d); body weight was reduced with a minimum of 300 mg (74 nmol)/kg/d.
n
Suppression of food intake occurred at 6 and 24 h only in 24-h fasted, but not freely fed mice.
o
Effects were obtained only with doses 3 10 mg (2.5 nmol)/kg, highest reduction ~45% with 100 mg (25 nmol)/kg dose, total decreases in intake constituted 0.8–2 g decrease/1 h.
p
~2% decrease on first day only.
q
0 to -4% change from baseline weight with PYY3-36 vs. 6% increase in baseline weight with saline.
(6)
(7)
(4)
(9)
No effect
Increase (~5%)
No effect
Transient
decreasep
18
14
10
17
No effect
Increase (~20%)
No effect
Transient decreasek
No effect
No effect
NP
No effectn
Decreasedn
Decreaseo
8 (8)
8 (8)
192 (90)l
Decreasedm
Decreasedm
98 (42)l
30,100,300,1000 mg
(7.4, 25, 74, 247 nmol)/kg/d
10 mg (2.5 nmol)/100 g
10 mg (2.5 nmol)/100 g
0.1, 1, 3, 10, 30, 100, 300, 1000 mg (0.025,
0.25, 0.7, 2.5, 7.4, 25, 74, 247 nmol)/kg
50, 100 mg (12.3, 25 nmol)/kg b.i.d.
100 mg (25 nmol)/kg b.i.d.
100 mg (25 nmol)/kg
1 mg (247 nmol)/kg
Body weight
Food intake
Total # of animals
(control)
PYY3-36 dose
PYY3-36 as anti-obesity drug
Chronic (28 d)
Chronic (28 d)
pump
C57BL/6 mice on
high-fat diet
I29/J mice
I29/J mice
NIH/Swiss mice
(female)
NMRI mouse
NMRI mouse
NZO mouse
ob/ob mouse
Injection
protocol
Route of
administration
Species (male)
Table 1 Studies investigating the effects of systemic administration of PYY3-36 in rodents (Continued)
310
M. M. Boggiano et al.
obesity reviews
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
obesity reviews
NMRI mice, New Zealand obese mice (23), and non-fasted
129/J mice (32). Various methods of drug delivery were
tested including i.p., i.v., subcutaneous (s.c), once or twice
a day, and continuously via osmotic pump. These treatments were administered prior to both the dark and light
phase, and food intake was measured at 30 min and 1, 2,
3, 4, 6, 8, and 24 h in acute experiments and following
several days in chronic experiments. In 24 of 37 studies,
no difference in food intake was found. Interestingly, in as
many as four of these studies, a statistically significant
increase in food intake was observed. Only 10 separate
studies found a decrease in food intake, but, of these, nine
reported either the need for very high dosing (35), that
animals be fasted (32,38), a paradoxical inverse doseresponse (33), or a weaker (31,37) and more transient (23)
suppression of intake despite chronic dosing, than originally reported (29). Similarly disappointing was the overwhelming lack of suppression in body weight obtained
when several mouse (C57BL/6J, NMRI, New Zealand
obese, ob/ob) and rat (Wistar, Lister-hooded, SpragueDawley, Long Evans) strains were tested. In these studies,
animals were administered 3, 5, 50 and 100 mg (0.7, 1.2,
12.3 and 25 nmol)/kg, 5 mg (1.2 nmol)/100 g, 1 mg
(247 nmol) kg/d, 5 mg (1.2 nmol)/100 g/d; i.p., i.v., s.c.,
once or twice per day, or continuous infusion prior to both
the dark and light phase. Body weight was measured over
2, 5, 7, 8 or 10 d. In 11 of our 14 studies, no change in
body weight was observed in comparison with vehicletreated controls. In one of the experiments, a transient
decrease of body weight was observed on the first day of
treatment, but not at any other time point after continuous
administration with minipump (Table 1). As occurred with
food intake, there was a significant increase of body weight
in two of the studies. Body composition was monitored
daily in two of these mouse experiments using a novel
quantitative magnetic resonance analyser. No change in
lean mass was found in either study, while fat mass actually
increased in one study and did not change in the other.
Another concern with the few experiments reporting
weight loss is the frequent use of very young animals, as
young as 28 d (mice) and 52 d (rats) (29,35). The reported
weight losses, compared with saline-treated control
rodents, may therefore represent a decrease in lean mass.
In the original Batterham et al. 2002 study (29), rapidly
growing rats gained 60 g in 7 d (about 25% of their body
weight), a gain most likely of muscle and bone mass, not
fat mass. Pharmacological blockade of such weight gain
likely represents changes in lean rather than in fat mass.
In the one subsequent study that found decreased body
weight, high doses of PYY3-36 (300–1000 mg or 74–
247 nmol/kg/d) were needed, and these were chronically
infused via minipump for extended durations (28–56 d)
(35). Interestingly, in this same study PYY3-36 accelerated
body weight gain of obese and diabetic fa/fa rats.
PYY3-36 as anti-obesity drug
M. M. Boggiano et al.
311
Some of the present authors were the first to test and
report the effects of PYY3-36 in obese models, observing
no anorectic or weight loss effects in diet-induced obese
Sprague Dawley rats, New Zealand obese mice or ob/ob
mice (23). In an effort to extend the reported effects of
PYY3-36 on food intake and energy balance, feeding
behaviour, spontaneous locomotor activity, energy expenditure (via indirect calorimetry) and other related behaviours, such as rearing, grooming, resting, sniffing and taste
aversion, were also measured (23,39). Contrary to expectations for catabolic actions, there was a significant (though
short-lasting) increase in feeding behaviour, along with
indices of decreased motor activity (Table 2), overall energy
expenditure, and caloric content of the faeces following
treatment with PYY3-36 compared with saline.
Discrepancies within studies reporting anti-obesity
effects of PYY3-36
There are a number of discrepancies among the few studies
suggesting that PYY3-36 decreases food intake. Specifically, the reported duration of PYY3-36 anorexia was
much less persistent than originally reported (31,32,38).
There was also a smaller magnitude of these effects, which
were only observed after pre-fasting, as well as the finding
of no effect at all in freely fed animals (32). An anti-obesity
drug should be robust across fed and fasted states and
should remain potent for extended periods of time. In one
study (31), food intake suppression did not last beyond
12 h, and the suppression was in the range of only 0.2 g in
groups of four to six mice. Measuring such small differences would require very precise instrumentation, but the
mice were simply presented with standard laboratory chow
pellets in Petri dishes that where manually weighed. Several
studies trying to replicate the reported anorectic effects of
PYY3-36 used state-of-the-art computerized online monitoring equipment to measure food intake. One investigation (Table 2) that included video recording and treatmentblind ethological analyses revealed that PYY3-36 dosedependently reduced the latency and increased the frequency and duration of eating in tests with palatable mash
(39). Of those that did find a hypophagic effect with PYY336, a surprising inverse dose effect trend was noted (33),
while others reported previously effective doses to be ineffective in reducing food intake and body weight (31). A
study by Nordheim and colleagues investigated cardiovascular effects in rats after PYY3-36 treatment and observed
‘slight’ reductions in food intake (37), but ones that were
not comparable with those reported by Batterham et al.
However, in mice, any feeding-suppression by PYY3-36
was more likely to occur when they were hungry (16- and
24-h fasted) prior to injection (Table 1) (32). This would
suggest that the peptide has significant limitations in its
actions, interacting only with physiological signals specifi-
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
312
PYY3-36 as anti-obesity drug
obesity reviews
M. M. Boggiano et al.
Table 2 Effects of PYY3-36 on feeding latency, locomotor, and other behaviours of rodents
Behavioural measure
Saline
3 mg/kg
30 mg/kg
100 mg/kg
Statistics
Eat latency seconds
Eat duration seconds
Eat frequency
38.70 ± 8.15
744.45 ± 62.20
51.20 ± 8.24
18.48 ± 4.72
758.40 ± 57.71
53.00 ± 9.02
15.90 ± 2.19
738.59 ± 55.14
45.40 ± 6.88
12.46 ± 1.86*
804.54 ± 47.99
51.10 ± 7.35
F(3,21) = 6.45, P = 0.025
F(3,27) = 1.27, NS
F(3,27) = 1.60, NS
0.11 ± 0.35
0.10 ± 0.10
0.00 ± 0.00
0.00 ± 0.00
0.73 ± 1.53
0.20 ± 0.13
0.43 ± 1.14
0.20 ± 0.13
F(3,27) = 1.20, NS
F(3,27) = 1.00, NS
Locomotion duration seconds
Locomotion frequency
912.64 ± 65.85
223.60 ± 14.41
906.33 ± 84.74
205.50 ± 19.08
892.17 ± 91.62
200.70 ± 11.73
799.59 ± 96.05
203.80 ± 17.01
F(3,27) = 1.45, NS
F(3,27) = 1.25, NS
Rear duration seconds
Rear frequency
653.92 ± 51.54
156.00 ± 10.37
554.16 ± 66.46
138.20 ± 12.60
470.91 ± 52.88**
117.70 ± 10.63**
424.85 ± 73.22**
109.30 ± 13.04**
F(3.27) = 12.39, P < 0.001
F(3.27) = 8.34, P < 0.001
Groom duration seconds
Groom frequency
234.46 ± 20.11
23.60 ± 2.82
314.37 ± 52.30
23.30 ± 2.72
274.81 ± 36.19
23.00 ± 2.26
265.09 ± 32.92
23.50 ± 2.20
F(3,27) = 0.85, NS
F(3,27) = 0.01, NS
Rest duration seconds
Rest frequency
216.94 ± 84.87
11.30 ± 3.84
340.01 ± 142.31
10.60 ± 3.80
241.39 ± 94.15
13.50 ± 4.02
209.77 ± 102.85
10.60 ± 3.18
F(3,27) = 0.81, NS
F(3,27) = 0.47, NS
Sniff duration seconds
Sniff frequency
771.43 ± 88.76
127.40 ± 12.51
696.78 ± 62.52
110.30 ± 13.56
861.41 ± 89.40
126.90 ± 13.45
104.15 ± 137.85*
140.20 ± 16.20
F(3,27) = 6.12, P < 0.003
F(3,27) = 3.32, P < 0.035
Drink duration seconds
Drink frequency
Doses of 3, 30, 100 mg are equivalent to 0.7, 7.4, and 25 nmol respectively; *P < 0.05, **P < 0.03 vs. saline; measures were during a 1-h-test with
palatable mash in adult male Lister-hooded rats initially habituated to the test arena and test food for 1 h daily for 5 d prior to the experiment, and with
additional basal sessions run weekly throughout the study. Human PYY3-36 (Bachem) was used and given in a complete repeated measures design
(n = 10). Videotapes were scored blind by two highly trained observers (intra-rater and inter-rater reliability 3 0.8 [Ishii et al. (23,39)].
NS, not significant.
cally activated in states of negative energy and with receptors more available in mouse vs. rat physiology. If this is
true, this poses a problem for PYY3-36 as an anti-obesity
agent, because efficacy would be contingent on obese
patients’ ability to limit their intake and on human receptor
distribution mimicking that of mice. Nonetheless, although
Batterham et al. 2002 obtained anorectic effects in freely
feeding rats, it is unfortunate that many have been unable
to reproduce these effects in fed and fasted mice and rats
using the same doses of PYY3-36.
Factors potentially explaining the discrepant
findings with PYY3-36 on food intake and body
weight
Species-specificity and bioactivity of PYY3-36
Those unable to replicate the anorectic effects of PYY3-36
considered the importance of species-specificity in compounds used in in vivo pharmacology. Although Batterham
et al. 2002 did not specify the species form of PYY3-36
used in their rodent studies, they cited Bachem as the commercial source and, at that time, Bachem supplied only
human PYY3-36. For this reason, human PYY3-36 was
used in subsequent attempts to reproduce the first reported
anorectic effects of the peptide. Since then, the original
authors have confirmed their use of human PYY3-36 (personal emails and communication at the Endocrine Society
Meeting, San Francisco, June 2002). Regardless of the
exact compound used, it has routinely failed to suppress
food intake. Some investigators also tested rat PYY3-36
[synthesized by the UCLA Peptide Synthesis Laboratory, a
generous gift from Dr J. Reeves (23)] and, as with human
PYY3-36, no decrease in food intake was observed.
Companies were also solicited to synthesize PYY3-36
species-independent compounds, and these were tested
separately in multiple mice and rat bioassays for relevant
receptor sub-types, namely Y1, Y2 and Y5. In addition,
some investigators have used pure (tested by HPLC and
GCMS) PYY3-36. Bioactivity was confirmed for all of
these PYY3-36 compounds, in which they either increased
food intake following intracerebroventricular (i.c.v)
administration or caused delayed gastric emptying following peripheral administration, both well established effects
of PYY3-36 (8,11,12,40–42). Delayed gastric emptying is
believed to be mediated via Y2 receptors (43), while Y1
and Y5 receptors have been implicated in the centrally
induced hyperphagic response (16–18). Together, these
measures attest to the functional integrity of the compounds tested (Fig. 1A–D), and indicate that, independent
of the source (e.g. Bachem, Eli Lilly, Polypeptides, Neosystems, Tocris, Peptides & Elephants) and after confirmation
of their central and systemic bioactivity in rodents, PYY336 did not decrease food intake in mice and rats (Figs 2A–
D and 3A–D respectively) or decrease body weight
(Fig. 4). As such, concerns about diverging sub-type specificity and bioactivity seem unlikely to be a potential explanation for the discrepant results. Another aspect to
consider is the pharmacokinetic properties of PYY3-36.
Little is known regarding the relevant plasma levels of
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
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M. M. Boggiano et al.
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Saline
2h
3 nmol PYY (3-36)
Time access to food (h)
Figure 1 (A) Quality and identity check of 38.9 mg (9.6 nmol)/mL PYY3-36 used for in vivo studies. PYY3-36 (Bachem) was dissolved in phosphatebuffered saline, stored at room temperature and analysed 3 d later. Sample was analysed by HPLC and detected by UV at 214 nm. Peptide purity was
estimated to ~94% and the concentration to 38.6 mg (9.5 nmol)/mL. Insert: The identity of the sample was analysed by LC-MS to yield a MH3+ ion with
m/z of 1350.8 corresponding to a MW of 4049.4; calculated MW 4049.6 [Madsen & Kjellander (23)]. (B) Comparison of solutions of PYY3-36 (Bachem
lot no. 522282) prepared fresh () or the remainder from in vivo studies () analysed in human NPY Y2 receptor binding assays using 125I-PYY (Perkin
Elmer) as radioligand. Both solutions gave essentially the same biological activity, 0.8 mg (0.2 nmol) vs. 1.6 mg (0.4 nmol), P < 0.05, n = 2. Peptide was
reconstituted in phosphate buffered saline and analysed immediately or following 3 d at room temperature. Assays were performed using recombinant
human NPY Y2 receptors expressed in and extracted from BHK tk-ts13 cells. 1.5 mg membrane protein was mixed with 0.2 mg (50 pM) 125I-PYY and
varying concentrations of PYY3-36 in buffer (25 mM HEPES, 2.5 mM CaCl2, 1.2 mM MgCl2, 0.1% BSA, pH 7.4). Radioligand binding was determined
using WGA-coated SPA beads and TopCount NTX [Stidsen, Wulff, & Schiødt (23)]. (C) Stimulation of food intake after i.c.v. administration of 1 mg
(0.25 nmol)/kg PYY3-36 (Saxon) or 0.5 mg (0.12 nmol)/kg NPY (Polypeptide Laboratories) in the lateral ventricle of fed, female Wistar rats (230–250 g,
12/12 h light cycle), P < 0.05, n = 10. Compounds had been administered 2 h before the beginning of the dark phase. Rats had been daily trained for
handling for 2 weeks [Schoelch, Arndt, Rudolph, Mark & Schindler (23)]. (D) Gastric emptying slowing by 12 mg (3 nmol) i.p. PYY3-36 (Bachem) in n = 8
male Sprague Dawley rats adapted to a daily intragastric tubing procedure for one week prior to start of testing; *P < 0.05 [Kinzig, Behles, Scott & Moran
(23)].
PYY3-36 achieved after i.p. or i.v. administration. A high
initial Cmax of the peptide, for example, may exert
anorectic effects that would explain why after i.v. application some investigators have found transient anorectic
effects. I.p. injection mimics the more natural (postprandial) condition of more slowly rising plasma PYY3-36 levels. Additional pharmacokinetic data are needed to
evaluate this as a possible explanation for the discrepant
results.
Are non-PYY3-36-responding animals stressed?
The possibility has been suggested that insufficient habituation procedures can preclude the detection of an anorectic response to PYY3-36 (44). The merit of this as a
putative reason for discrepant PYY3-36 seems unlikely on
several levels. First, in the original publication (29), Batterham et al. state that their rats were accustomed to i.p.
injections of 0.5 mL of saline for 2 d prior to the study.
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Cumulative food intake (g)
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3
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Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
11 13 15 17 19 21 23
Time after injection (h)
*P < 0.05 ANOVA SNK
Figure 2 (A) Intraperitoneal (i.p.) administration of 100 mg (25 nmol)/kg PYY3-36 (Peptides & Elephants), twice per day for 10 d in male NMRI mice
increased food consumption (P < 0.05, n = 7). (B) I.p. administration of 100 mg (25 nmol)/kg PYY3-36 (Bachem), twice per day for 8 d in male New
Zealand obese (NZO) mice failed to decrease food consumption; n = 4–6 [Castañeda, Thöne-Reineke, Ortmann, Klaus, Birringer, Kreuzer, Joost &
Tschöp (23)]. (C) I.p. PYY3-36 (Bachem) on cumulative food intake in male C57BL/6 J mice did not decrease food intake; n = 6 [Kinzig, Behles, Scott,
& Moran (23)]. (D) Continuous subcutaneous (s.c) injections of 1 mg (247 nmol)/kg/d PYY3-36 (Lilly) for 7 d in male C57BL/6 mice decreased food
consumption during the first 3 d of treatment; P < 0.05; n = 8–9, but the effect was not sustained thereafter [Craney, Smiley, Flora & Heiman (23)].
Later, Halatchev et al. (31) specifically compared injection-acclimated with non-injection-acclimated mice and
reported anorectic effects of PYY3-36 only in injectionacclimated mice. More recently, researchers at Amylin
Pharmaceuticals testing PYY3-36 in rodents stated that
their animals were acclimated for one week, but did not
describe what this involved, short of acclimation to the
light/dark cycle (35). In direct contrast, studies failing to
find anorectic effects included standard protocols for
thorough habituation to chronic and repeated handling,
mock injections and food intake measures (23). The
animals were extensively adapted to the experimental
environment and showed no signs of stress before
experimentation. More important however, is the issue of
acclimatization to drug delivery in humans. If a drug is so
sensitively contingent on several first experiences with an
inactive agent, it will require a most unusual protocol for
patients.
Secondly, if stress of injection affects appetite, it should
do so equally, regardless of the anorectic agents that are
administered in the same manner. In this context, some
experiments included acute systemic administration of
cholecystokinin (CCK), MT-II (melanocortin 3/4-receptor
agonist), and/or naloxone (opioid receptor antagonist) as
positive controls to PYY3-36 administration. While the
former agents effectively suppressed appetite under the
exact same experimental conditions, PYY3-36 did not.
Thirdly, although any non-specific effects of stress on
appetite and body weight gain should have been evident in
saline-treated controls, these animals did not lose weight
or decrease their food intake (Figs 2–4). Fourthly, it is
curious that, when PYY3-36 has been reported to decrease
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
PYY3-36 as anti-obesity drug
a
2 h chow intake (kcal)
30
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20
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30.00
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Food intake (g)
Food intake (g)
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Figure 3 (A) Lack of effect of single intraperitoneal (i.p.) 3–100 mg (0.7–25 nmol)/kg dose PYY3-36 (Bachem) on 1-h intake of palatable mash of male
Lister-hooded rats; n = 10 [Ishii, Beattie, Holch, Blundell & Rodgers (23,39)]. (B) Lack of effect of i.p. 3 mg (0.7 nmol)/100 g dose of PYY3-36 (Bachem)
compared with i.p. 0.5 mg (123.5 nmol)/kg; opioid antagonist, naloxone HCl (Sigma) in male Sprague-Dawley rats a, maintained on chow (3.3 kcal/g;
3.5%fat), b, resistant to obesity on a high-fat diet (HFD: 4.41 kcal/g, 31.8%fat), and c, obese from a HFD; NAL + P = naloxone + PYY3-36; n = 8 per drug/
group; *P < 0.05, **P < 0.01, ***P < 0.001 [Boggiano (Hagan), Chandler & Oswald (23)]. (C) Lack of effect of i.p. 3–100 mg (0.7–25 nmol) PYY3-36
(Neosystems, Inc.) in fasted male Wistar rats when administered twice a day (AM and PM before start of the night phase); n = 8 [Arndt; Rudolf, Mark &
Schindler (23)]. (D) Lack of effect of i.p. PYY3-36 from three different suppliers (Tocris, Bachem or Neosystems) on food intake of male Wistar rats; n = 6
[Arndt, Rudolf, Mark & Schindler (23)].
food intake, it apparently does so exclusively, or at least
more effectively, in mice that are pre-fasted for at least 24 h
(Table 1 footnotes). Based on the much faster metabolism
of rodents, this would translate into a period of complete
fasting for about one week for humans before PYY3-36
would have an effect on the size of a following meal.
Furthermore, fasting is known to increase plasma corticosteroid levels in rodents, a well-known stress response (45).
Hence, if PYY3-36 effects are inhibited by stress, any
anorectic effects of the peptide should be more readily
detected in fed, not fasted, animals. The opposite, however,
seems to be the case. Moreover, the possibility that one
must follow a strict injection habituation protocol in order
to observe PYY3-36-induced hypophagia (31) is also challenged by the findings of Nordeim et al. (37). They detected
slight 24 h reductions in intake with PYY3-36 in rats that
had only one week of acclimation to the environment (no
mock saline injections) prior to extensive surgical procedures (abdominal incision, intestinal retraction, arterial
re-sectioning and telemetry transmitter transplantation)
and introduction to unfamiliar dietary regimens (some that
included restriction). Clearly, these animals were not without stress.
In summary, it seems highly unlikely that stress arising
from inadequate habituation procedures can explain the
discrepant results with PYY3-36, given that so many studies have systematically controlled for such effects. It is also
unrealistic to expect all laboratories worldwide to follow
identical protocols when testing any anti-obesity drug.
Rather, drugs with expected effects on rodents’ ingestive
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
PYY3-36 as anti-obesity drug
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Change in body weight (g)
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Day of treatment
Figure 4 (A) Intraperitoneal (i.p.) administration of 100 mg (25 nmol)/kg PYY3-36 (Peptides & Elephants) twice per day for 10 d in male NMRI mice
increased body weight; P < 0.05, n = 7 [Castañeda, Thöne-Reineke, Ortmann, Klaus, Birringer, Kreuzer, Joost & Tschöp (23)]. (B) Intraperitoneal (i.p.)
administration of 100 mg (25 nmol)/kg PYY3-36 (Bachem) twice per day for 8 d in male New Zealand obese mice failed to decrease body weight; n = 4–
6 [Castañeda, Thöne-Reineke, Ortmann, Klaus, Birringer, Kreuzer, Joost & Tschöp (23)]. (C) Lack of effect of i.p. 3–100 mg (0.7–25 nmol)/kg PYY3-36 on
post-treatment bodyweight gain in adult male Lister-hooded rats [Ishii, Beattie, Holch, Blundell & Rodgers (23,39)]. (D) I.p. administration of 5 mg
(1.2 nmol)/100 g/d PYY3-36 (Lilly) for 5 d in male Long Evans rats did not change body weight; n = 8 [Craney, Smiley, Flora & Heiman (23)].
behaviour should decrease food intake robustly across
diverse environments. More importantly, a potential antiobesity drug should work robustly against similarly varying
circumstances in humans. Stress is certainly a relevant
condition in human obesity (46) and can, under certain
circumstances, even be a salient stimulus for overeating
(47,48). Hence, if stress were playing a role in precluding
PYY3-36-induced hypophagia, even in the absence of evidence for such a causal relationship, then long-term treatment of obesity with this peptide poses quite a challenge,
a concern recently echoed by O’Rahilly and colleagues
(44).
Statistical issues: power and significance
Can the discrepancy in responses with PYY3-36 be
explained simply by reference to stochastic factors?
Although a few of the present reviewers’ studies produced
results in the direction predicted by Batterham et al. (29),
their number, relative to the total number of these investigator’s independent experiments conducted (assuming all
tests were done at a 2-tailed 0.05 level), was not significantly greater than expected, when referred to the binomial
distribution (for food intake 2 of 41 yields P = 0.273; for
body weight 1 of 11 yields P = 0.243). In contrast, the
number in the unexpected direction was significantly
greater than expected by chance, given the number of
hypotheses tested (for food intake 4 of 41 yields P = 0.019;
for body weight 2 of 11 yields P = 0.030). Might one
conjecture that failure to reproduce an anorectic effect with
PYY3-36 is the result of insufficient power or type II
errors? In their original publication, Batterham et al. (29)
stated that ‘PYY3-36 that was administered i.p. twice daily
for 7 d reduced cumulative food intake (7-d cumulative
food intake: PYY3-36, 187.6 ± 2.7 g; saline, 206.8 ± 2.3 g;
n = 8 per group, P < 0.0001) and decreased body weight
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
obesity reviews
gain (PYY3-36, 48.2 ± 1.3 g; saline, 58.7 ± 1.9 g; n = 8 per
group, P < 0.002; Fig. 1b)’.
Using standard power analysis software and computations (49), these numbers imply that a replication study
with n = 8 per group would have (i) well over 90% power
to reject the null hypothesis of no anorectic effect on 7-d
food intake; and (ii) approximately 80% power to reject
the null hypothesis of no decrease in body weight gain.
Although not every study conducted by the present reviewers and others (23) was designed exactly the same, this
suggests that power could conservatively be expected to be
at least 0.80 in most cases. For food intake, out of 41
experiments, only two yielded significant results in the predicted direction at the 2-tailed 0.05 alpha level. If the
population effect of PYY3-36 were equivalent to the sample estimates reported by Batterham et al. (29) and the
power were truly on the order of 0.80 per experiment, then
the probability of obtaining no more than two significant
results in such a situation is approximately 2.9 ¥ 10-25 by
the binomial distribution. For body weight, out of 11
experiments only one significant result in the predicted
direction was obtained at the 2-tailed 0.05 alpha level.
Again, if power were on the order of 0.80 per experiment,
then the probability of obtaining no more than one significant result in such a situation is approximately 9.2 ¥ 10-7.
To be extremely conservative, if it is assumed that each
experiment conducted only had 50% power, then the probability of obtaining only one significant result out of 11
trials is still only 0.006 and the probability of obtaining no
more than two significant results out of 41 trials is only
3.9 ¥ 10-10. Thus, by any reasonable assumptions about
power, the results of the reviewers and others (23) can be
seen as significantly discordant with those of Batterham
et al. (29) and cannot be easily explained on the basis of
low power.
Molecular analysis of PYY3-36: possible
explanations for inconsistent responses
A more productive and valuable strategy in speculating on
the reasons for lack of PYY3-36 in so many investigators’
hands is to consider the peptide’s molecular and physiological properties.
The role of DPP-IV in the conversion of PYY1-36 to
PYY3-36
It has recently been reported that mice deficient for dipeptidyl peptidase (DPP-IV), the enzyme that cleaves PYY1-36
into PYY3-36, which should suffer from complete PYY336 deficiency, are not obese, but are protected against dietinduced obesity and hyperinsulinemia. These PYY3-36
deficient mice also show reduced food intake and increased
energy expenditure. Although this finding (50) may reflect
PYY3-36 as anti-obesity drug
M. M. Boggiano et al.
317
not only lack of PYY3-36, but also an increased presence
of active glucagon-like peptide 1 (GLP-1) and glucosedependent insulinotropic polypeptide (GIP), this observation is in strong contrast with an essential role for PYY336 as a physiological satiety factor. However, the results
reported on DPP-IV knockout mice are consistent with
observations showing that chronic administration of
PYY3-36 in NMRI mice increase food intake and body
weight (23) and with why acute treatment with PYY3-36
attenuates activity (Table 2).
Hypothalamic receptor sub-types activated by
PYY3-36
PYY3-36 is a potent agonist of Y2 and Y5 receptors
(22,51,52). Some of the present authors conducted their
own in vitro binding, i.e. using SMS-KAN-, HEC1b-Hy5or SK-N-MC-cells to perform classic ligand-receptor binding studies (UCLA Peptide Synthesis Laboratory). They
observed equally strong binding of PYY3-36 and PYY1-36
at Y2-receptors, moderate binding at Y5-receptors, and
substantially weaker binding of PYY3-36 as compared
with PYY1-36 at Y1-receptors (23). Both Y2 and Y5 receptor types are present in the rodent brain in specific loci with
varying density (3). While Y5 receptors are believed to be
responsible, at least in part, for the orexigenic effects of
NPY and PYY in the CNS, Y2 are putative presynaptic
autoreceptors that may mediate negative autofeedback of
NPY onto NPY expressing neurones (26,27). Batterham
and colleagues put forward the very elegant hypothesis that
PYY3-36, which easily crosses the blood brain barrier (53),
acts predominantly on Y2 receptors in the arcuate nucleus
of the hypothalamus to reduce food intake (29). Y2 ligands
administered centrally may suppress food intake in rodents
(54), although evidence on action of such compounds still
is more than scarce, and mice with complete Y2-receptor
deletion are reported to have increased body weight and
food intake (27). Y5 receptors are more concentrated in
the area postrema (AP) than in the hypothalamus (55), and
lesions of the AP have recently been shown to transiently
enhance possible anorectic potency of PYY3-36 (33). These
biological observations are principally consistent with the
hypothesis that PYY3-36 may be inhibiting food intake via
more selective stimulation of Y2-receptors than PYY1-36.
However, the PYY3-36 compounds used in the studies
where investigators have been unable to observe reductions
in food intake had been successfully tested for binding
capabilities at Y2-R in vitro. It is also unclear why, despite
evidence of a greater presence of Y2 than Y5 receptors
throughout brain and within the hypothalamus (56), i.c.v.
administered PYY (11–15) and i.c.v. PYY3-36 (19–23)
were equally or, in some cases, more potent than NPY in
producing robust hyperphagia (21). Interestingly, there are
also species-specific differences: e.g. mice are less sensitive
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318
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M. M. Boggiano et al.
to changes in metabolic parameters after centrally administered PYY3-36 than after centrally administered NPY
(27). In actuality, the exact role of Y2 receptors in feeding
and energy regulation remains unknown. Deletion of the
Y2-receptor targeted mouse mutagenesis resulted in transiently increased food intake but decreased body weight
(57). The investigators of this study also found that complete germ-line Y2 deletion resulted in decreased body
weight and decreased food intake (57). Although Batterham and colleagues (29) concluded that Y2 receptors mediate PYY3-36-induced hypophagia, based on its lack of
effect in Y2 deficient mice (29), the strong inconsistencies
throughout all published wildtype rodent studies challenge
the specificity of the lack of response in Y2-knock-out mice
and therefore the above mentioned conclusion. It also
seems relevant to note that mice fasted for 48 h do not
exhibit any change in arcuate nucleus Y2 receptor expression (58).
Other molecular mechanisms that could
mediate PYY3-36 induced satiety
Peripheral actions of PYY3-36 may also play a role in the
transient reductions in food intake observed in some laboratories. Decreased gut motility and faecal output is mediated via Y2 receptor activation (3), and it is well known
that PYY slows gastric emptying and increases intestinal
transit time. For example, Y2 agonists and PYY applied in
the dorsal vagal complex suppress thyrotropin-releasing
hormone (TRH) stimulated gastric motility (26). A temporary decrease in food intake may therefore also be the
result of visceral illness, because it has been reported that
PYY induces or is closely correlated with induction and
severity of emesis (28,59). Although not significant, a trend
for conditioned taste aversion with 30 mg (7.4 nmol)/kg
i.p. PYY3-36 has been reported, while early human trials
report that nausea is the most common side effect of treatment with PYY3-36 (60). PYY3-36 infusion in humans
was accompanied by a decrease in plasma ghrelin levels
(30), a hormone signalling hunger and metabolic efficiency
(61). However, how precisely PYY3-36 interacts with ghrelin is unknown at present and a possible role for circulating human ghrelin in food intake regulation is still
controversial.
Little is also known about the exact central mechanisms
by which PYY3-36 might induce satiety. Impressive and
elaborate electrophysiology studies reported by Batterham
and colleagues strongly suggested activation of proopiomelanocortin (POMC) neurones as a central mechanism
for PYY-induced satiety (29). However, more recently, food
intake and body weight in POMC-deficient mice have been
found to be unaffected by peripheral administration of
PYY3-36 (62). Consistent with these findings, PYY3-36
also has no effect on food intake in mice deficient for the
obesity reviews
melanocortin-4 receptor (31), which is known to ultimately
mediate changes in POMC expression after activation by
POMC derived a-MSH. The discrepancies in studies implicating a role for the melanocortin system may be the result
of species-specific differences between rats (in which the
electrophysiology studies were performed) and mice (in
which the pharmacological studies after targeted mutagenesis were performed). However, such differences are not
typical, and there is no other evidence to support such an
explanation. Coll et al. (2004) offer the possibility that the
anorectic cocaine- and amphetamine-regulated transcript
(CART) and the neurotransmitter gamma-aminobutyric
acid (GABA) may be candidates to mediate PYY3-36induced effects. These substances are known to be partially
co-expressed with and released by POMC neurones respectively (44); however, there is no known colocalization
between Y2-receptors, GABA and CART. Thus far, the
most consistent central changes evoked by PYY3-36
administration seem to be reductions in hypothalamic NPY
expression, supporting a putative role of Y2 activity in
effects of PYY3-36 (29,32). Most recently, however, R.
Cone and colleagues concluded, via an elaborate series of
studies, that PYY3-36 causes visceral illness and conditioned taste aversion as well as c-fos expression in the area
postrema and nucleus tractus solitarius, brain regions
known to mediate these responses (63). These findings may
explain why some groups have observed small transient
effects on food intake without body weight effects following systemic PYY3-36. This is an effect that is very different
from the anorectic and catabolic effects of melanocortin
agonist compounds such as MT-II.
Some have questioned whether it is important at all to
understand the molecular mechanisms mediating PYY3-36
action, if it successfully treats obesity (44). At one level, if
a compound is clinically efficacious (and of course safe),
understanding its physiology might be secondary. However,
to date, no peer-reviewed report has described actual
weight loss following administration of PYY3-36 in
humans.
Comparison of PYY3-36 with the status of other
satiety agents as anti-obesity targets
If PYY3-36 is truly a powerful satiety agent with the potential to treat obesity, conditions under which it successfully
works should not have to be defined down to the most
meticulous details. Before it can reasonably be chosen as a
clinical candidate, there should be repeatable and robust
dose-dependent effects across multiple animal models in
multiple independent laboratories. A notable difference
between the bench-to-clinic evolution of PYY3-36 and
other anti-obesity candidates is the paucity of such published preclinical investigations, which typically precedes a
human drug target. For example, 2 years after the first
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
obesity reviews
reports of leptin-induced satiety and ghrelin-induced food
intake, numerous reports (over 500 for leptin and 200 for
ghrelin) from many independent laboratories had supported and extended the original findings in rodents. In
contrast, since the initial report on PYY3-36 (29), only six
supporting articles have been published, and the majority
of these document various experimental contingencies and
caveats (e.g. dependence on unique habituation protocol,
transience of effects, lack of effect in fed animals, absence
of clear dose-dependency, no effect on body weight, dependence on extensive pre-fasting).
Human studies with PYY3-36
The first report on PYY plasma levels (total PYY; immunoassays to specifically quantify PYY3-36 are not yet available) suggested that PYY is low in obese individuals, that
a PYY-deficiency may be contributing to their obesity, and,
hence, that PYY replacement therapy might be useful (30).
However, the same investigators recently reported that,
using the same assay, PYY levels in obese individuals were
either normal or slightly increased (64). The same was
recently reported by some of the present authors in a much
larger population of lean, obese and morbidly obese individuals (65).
Based on the first very promising and enthusiastic report
of PYY3-36 as an anti-obesity agent, several research
groups have explored its effects in humans. In a first (double-blind crossover) study, described alongside the original
report of anorectic effects in rodents (29), 12 fasting nonobese young adults, infused with 3.2 ng (0.8 pmol)/kg
PYY3-36 over 90 min, ate 33% less food over 24 h than
when infused with saline. In the second study, 12 fasted
obese and non-obese young adults were i.v. infused with
8 mg (2 nmol)/m2 body surface area PYY3-36 over 90 min.
Compared with saline infusion, obese and non-obese subjects reduced their 24-h intake by 26% and 34% respectively, with suppression occurring within and not beyond
the first 12 h post-infusion (30). These studies were criticized in regard to the reported 100% efficacy in all treated
subjects with only a single dose administration of PYY,
which is unusual in such studies (L. A. Campfield, Health
SCOUT; Health Day News, September 3, 2004). The
reported suppression of food intake also seems incongruent
with the only slight reduction in hunger, suggesting that
other factors such as nausea or alterations in food hedonics
may have been involved. Nausea has been the most frequent side effect reported in human PYY studies to date
(60). Based on the known strong potency of PYY to inhibit
intestinal secretion (3,8), systemic infusion of PYY3-36
seems likely to cause obstipation. Obstipation, however, is
known to stimulate satiety mechanisms mildly and transiently via vagal reflexes. A tendency toward decreased
caloric content in faeces would be consistent with these
PYY3-36 as anti-obesity drug
M. M. Boggiano et al.
319
actions (66). In a separate report, baseline PYY levels in an
obese group were reported to be 40% lower than in a nonobese group (30). This is suggested to explain impaired
satiety in this group, but another plausible explanation is
that lower PYY in this group serves to trigger overeating
(D. Cummings, Health SCOUT; Health Day News, September 3, 2004).
The crucial question is whether chronic peripheral infusion of PYY3-36 will decrease body fat mass in humans.
PYY3-36 would have to override massive compensatory
activity of numerous other orexigenic pathways (34).
Although no scientific peer-reviewed information is available, Nastech Pharmaceutical Co., Inc., has developed a
PYY3-36 nasal spray and has released the only report of
reduced food intake and weight loss in PYY3-36-treated
obese subjects (mean body mass index = 33.3; n = 37; six
per treatment condition). An average decrease of 77 cal vs.
648 cal was reported for subjects given intranasal PYY336 before one meal a day vs. before all three meals a day
for six consecutive days respectively, while an average
weight loss of 1.3 pounds was reported only for those
subjects on the maximum dosing condition over the 6-d
study. However, it was not reported if that weight loss was
the result of decreased fat mass, decreased muscle mass, or
loss of water. While one other study reported decreased
food intake in humans treated with PYY3-36 (29,30), this
is the only press release reporting any body weight loss
(67). Whether weight loss is sustained with repeated treatment remains to be determined. Chelikani et al. found rats
to decrease food intake with continuous i.v. infusion of
PYY3-36 (68), but no change in body weight was reported.
Moran and colleagues recently found that, in primates,
single doses of 4 and 12 mg (1 and 3 nmol)/kg PYY3-36
reduced food intake acutely but this suppression was not
sustained despite multiple administrations over days (69).
Unless the peptide substantially increases energy expenditure, a steady and sustained decrease of food intake will be
critical to lasting weight loss. Still, although the large
majority of studies in rodents did not evidence anorectic
PYY3-36 effects, to date studies in humans and primates
have yielded more promising results. Thus, failed rodent
studies cast doubt on the value of PYY3-36 as a therapeutic
agent, but in no way are they sufficient to rule out such
putative value. Until there are more data in human and
non-human primates, one cannot rule out possible species
differences. Nevertheless, while such interspecies differences are conceivable, given the typical interspecies conservation of effects with respect to substances influencing
ingestive behaviour, we find this somewhat unlikely.
Conclusions
This review aimed to critically evaluate all available information on a possible role of PYY3-36 as an endogenous
© 2005 The International Association for the Study of Obesity. obesity reviews 6, 307–322
320
PYY3-36 as anti-obesity drug
obesity reviews
M. M. Boggiano et al.
satiety agent and as an effective anti-obesity drug. Several
observations have decreased initial enthusiasm about the
potential of PYY3-36 as a magic bullet against the consequences of today’s hypercaloric environment. First, there is
an unusually large number of failed attempts (we estimate
about 90% of all studies) to replicate and extend the first
reported anorectic effects of PYY3-36. Secondly, while the
original studies presented a very elegant hypothesis and an
intriguing collection of data (29), it is unfortunate that
these original findings have not been reproduced by some
42 investigators and that very few supporting publications
have appeared in the literature since then (over 3 years). In
contrast, after the discovery of the ob/ob protein, leptin,
though it was eventually found to vary in anorectic potency
in some models of obesity (70), not a single group was
reportedly unable to reproduce the original observations of
reduced food intake and body weight. Moreover, despite
the fact that leptin is incomparably more difficult to generate and more expensive to obtain, literally hundreds of
studies have since supported the hormone’s catabolic role
in energy balance. Thirdly, data are now emerging to indicate that basal PYY levels are apparently not abnormally
low in obesity and that any small anorectic effects observed
may have been evoked by visceral illness. Importantly, these
data are being provided by independent laboratories,
including laboratories of the authors of the original report
of the anorectic actions of PYY3-36 (29).
Nevertheless, Batterham and colleagues have offered the
scientific community an important set of observations.
Their data suggesting a role for PYY3-36 as an endogenous
anorectic substance brought attention to this peptide and
provided an impetus for vigorous experimental efforts to
reproduce and extend their results. The results of many
subsequent investigations, however, have failed to confirm
the original observations and have called the potency and,
indeed, the effectiveness of PYY3-36 as an anorectic peptide into question. At this juncture, further clarity can best
come from additional experimental and clinical data collected under the most rigorous conditions with sample sizes
sufficient to yield definitive conclusions. However, in
designing such studies, it may be worth considering possible explanations for why results appear so discrepant and,
in that light, we offer the observations collected in this
review article. We hope that the as-yet unexplained discrepancies in results serve to stimulate further research geared
towards resolving the circumstances, if any, under which
this peptide might produce consistent, physiologically
meaningful, and behaviourally selective effects on human
food intake and body fat mass.
Acknowledgements
Independent studies were supported by grants: RO3
DK066007-01
(M.M.
Boggiano),
CNRU
grant
P30DK056336 (D.B. Allison), DK54080 and DK54890 (R.
J. Seeley), and DK45781-01A1 (D. Whitcomb). We thank
Dr Paul Blanton of UAB for his adept editing assistance.
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