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Isopropylnorsynephrine is a stronger lipolytic agent in human adipocytes than synephrine and other amines present in Citrus aurantium

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The weight loss observed in consumers of extracts of Citrus aurantium (bitter orange) has been tentatively attributed to the lipolytic and thermogenic effects of the alkaloids abundant in the unripe fruit. Synephrine, octopamine, tyramine, and other alkaloids have been repeatedly identified and quantified in Citrus members of the Rutaceae family or in their extracts incorporated in dietary supplements for weight management. However, there are only scarce reports on their lipolytic action. This study aimed at comparing the acute lipolytic activity of synephrine, octopamine, tyramine, and N-methyltyramine in rat and human adipocytes. Maximal response to the prototypical β-adrenergic agonist isoprenaline was taken as reference in both species. In rat, octopamine was slightly more active than synephrine while tyramine and N-methyl tyramine did not stimulate-and even inhibited-lipolysis. In human adipocytes, none of these amines stimulated lipolysis when tested up to 10 μg/ml. At higher doses (≥100 μg/ml), tyramine and N-methyl tyramine induced only 20% of the maximal lipolysis and exhibited antilipolytic properties. Synephrine and octopamine were partially stimulatory at high doses. Since synephrine is more abundant than octopamine in C. aurantium, it should be the main responsible for the putative lipolytic action of the extracts claimed to mitigate obesity. Noteworthy, their common isopropyl derivative, isopropylnorsynephrine (also named isopropyloctopamine or betaphrine), was clearly lipolytic: active at 1 μg/ml and reproducing more than 60% of isoprenaline maximal effect in human adipocytes. This compound, not detected in C. aurantium, and which has few reported adverse effects to date, might be useful for in vivo triglyceride breakdown.
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ORIGINAL PAPER
Isopropylnorsynephrine is a stronger lipolytic agent
in human adipocytes than synephrine and other amines
present in Citrus aurantium
Josep Mercader &Estelle Wanecq &Jian Chen &
Christian Carpéné
Received: 10 December 2010 / Accepted: 27 January 2011
#University of Navarra 2011
Abstract The weight loss observed in consumers of
extracts of Citrus aurantium (bitter orange) has been
tentatively attributed to the lipolytic and thermogenic
effects of the alkaloids abundant in the unripe fruit.
Synephrine, octopamine, tyramine, and other alkaloids
have been repeatedly identified and quantified in Citrus
members of the Rutaceae family or in their extracts
incorporated in dietary supplements for weight man-
agement. However, there are only scarce reports on
their lipolytic action. This study aimed at comparing the
acute lipolytic activity of synephrine, octopamine,
tyramine, and N-methyltyramine in rat and human
adipocytes. Maximal response to the prototypical β-
adrenergic agonist isoprenaline was taken as reference
in both species. In rat, octopamine was slightly more
active than synephrine while tyramine and N-methyl
tyramine did not stimulateand even inhibited
lipolysis. In human adipocytes, none of these amines
stimulated lipolysis when tested up to 10 μg/ml. At
higher doses (100 μg/ml), tyramine and N-methyl
tyramine induced only 20% of the maximal lipolysis
and exhibited antilipolytic properties. Synephrine and
octopamine were partially stimulatory at high doses.
Since synephrine is more abundant than octopamine in
C. aurantium, it should be the main responsible for the
putative lipolytic action of the extracts claimed to
mitigate obesity. Noteworthy, their common isopropyl
derivative, isopropylnorsynephrine (also named isopro-
pyloctopamine or betaphrine), was clearly lipolytic:
active at 1 μg/ml and reproducing more than 60% of
isoprenaline maximal effect in human adipocytes. This
compound, not detected in C. aurantium, and which
has few reported adverse effects to date, might be
useful for in vivo triglyceride breakdown.
Keywords Obesity .Isoprenaline .Tyramine .
Octopamine .Rat adipocytes .Dietary supplements
Introduction
Citrus aurantium is a plant belonging to the Rutaceae
family, the fruit of which (bitter orange) has been
used for the preparation of extracts sold worldwide
under the form of phytoproducts claimed to promote
weight loss. The leaves, the peel, and the edible part
J Physiol Biochem
DOI 10.1007/s13105-011-0078-2
J. Mercader :E. Wanecq :C. Carpéné
Institut National de la Santé et de la Recherche Médicale
(INSERM),
U1048 Toulouse, France
J. Mercader :E. Wanecq :C. Carpéné
Université de Toulouse, UPS, Institut de Médecine
Moléculaire de Rangueil,
IFR BMT Toulouse, France
J. Chen
Syntech (SSPF) International Inc,
Montclair, CA 91763, USA
C. Carpéné (*)
INSERM U1048, I2MC, CHU Rangueil,
31432 Toulouse, France
e-mail: christian.carpene@inserm.fr
of the fruits of C. aurantium contain elevated amounts
of phenethylamine alkaloids (i.e. synephrine, octop-
amine, tyramine, N-methyltyramine, and hordenine).
The synephrine content is negatively correlated with
the maturity index of the fruit [10]. Since the content
of such alkaloids is richer in unripe C. aurantium than
in other fruitssuch as mandarins (Citrus reticulata)
or sweet oranges (Citrus sinensis)extracts from the
former are currently included in dietary supplements
aiming at treating obesity.
Para-synephrine has been reported to be the main
alkaloid of C. aurantium fruits and extracts [25],
while the other alkaloids are present in much lower
concentrations [18,20,23]. The contents found in
dried extracts are approximately 3% synephrine, 0.3%
octopamine, 0.06% tyramine, while the other alka-
loids are at the limit of detection. Synephrine is an
alkaloid that is similar in structure to ephedrine,
which has been banned from dietary supplements
inducing weight loss in view of its deleterious actions
on the cardiovascular system. Synephrine is therefore a
major component of ephedra-freeproducts [25]and
has been the subject of numerous analytical determi-
nations in sweet oranges (16 μg/ml of juice),
mandarins (80160 μg/ml of juice), bitter oranges
(56 μg/ml of juice, 1,1203,000 μg/ginpeel),or
especially in multi-component formulations containing
C. aurantium extracts (up to 92,000 μg/g in dried
material) sold as weight-loss and athletic-performance-
enhancement products [2,10,1823]. Evidently, the
amount of (+/) synephrine, the major alkaloid found
in such dietary supplements with supposed slimming
effects, varies largely depending on different factors
such as the grove, the extraction process, or the fact
that the product respects what is on manufacturers'
label or has been adulterated before reaching the
consumer. Such aspects were reviewed elsewhere [12,
20,22,25] and will not be treated in the present study.
The aim of our comparison of the lipolytic
activities of C. aurantium alkaloids was to detect
which of them could be responsible for the fat mass
reduction weakly documented in obese humans [12,
25]. A recent study has reported that the peel or
segment wall extract from Satsuma mandarin orange
is lipolytic in rat adipocytes via β-adrenergic activa-
tion [26]. Therefore, rat adipocytes were firstly used
in our approach, but our attention was focused to
delineate the lipolytic responses in human adipocytes,
since there are interspecies differences regarding
lipolysis control [8]. In fact, we have already reported
that octopamine stimulates lipolysis in rodent white
fat cells, activates oxygen consumption and thermo-
genesis in rat brown fat cells, but is almost inactive
in human adipocytes [9]. Therefore, it appeared
essential to test the putative lipolytic agents in human
adipocytes too.
Thus, different protoalkaloids known to be present
in C. aurantium [23], namely synephrine, octopamine
N-methyltyramine, and tyramine (Fig. 1), were tested
on human fat cell preparations highly responsive to
NH 2
HO
OH
HCl
NHCH3
HOHCl
NH2
HO HCl
NH
HO
OH
HCl
NH
HO
OH
HCl
HO CH3
CH3
NCH3
HO
OH
HCl
Isopropylnorsynephrine hydrochloride
Octopamine hydrochloride
Synephrine hydrochloride
N-methyltryamine hydrochloride
Tyramine hydrochloride
Isoprenaline hydrochloride
Alkaloids present in Citrus aurantium:
Amines not detected in Citrus aurantium:
Fig. 1 Chemical structures
of several protoalkaloids
found in Citrus aurantium
and related amines
J. Mercader et al.
the β-adrenergic receptor (AR) agonist isoprenaline,
in the conditions in which we already investigated the
influence of various pharmacological agents on
adipocyte functions [7,29]. Since inhibition of
lipolysis is also an α
2
-adrenergic response highly
developed in human fat cells [24], we compared the
actions of the above-mentioned protoalkaloids to
bromoxidine and methoxy-idazoxan (α
2
-agonist and
antagonist of reference, respectively).
As there was a clear difference in the respective
potency of isoprenaline and synephrine or octop-
amine, regarding lipolysis stimulation in rat adipo-
cytes, an additional agent that can be considered as a
structural intermediate between these amines was
tested in human adipocytes, isopropylnorsynephrine,
also named isopropyloctopamine or betaphrine
(Fig. 1). This poorly documented synthetic amine
[1] exhibited potent and effective stimulation of fat
mobilization in human fat cells. Lastly, we tested the
capacity of isopropylnorsynephrine and of the bio-
genic amines to be oxidized by the monoamine
oxidase activity (MAO) present in adipose tissue.
Materials and methods
Chemicals
() Isoprenaline hydrochloride, (+/) octopamine
hydrochloride, tyramine hydrochloride, isobutylme-
thylxanthine (IBMX), collagenase (C-6885), and
other reagents were obtained from Sigma-Aldrich
(Saint Quentin Fallavier, F).
3
H-2-deoxyglucose was
from Perkin Elmer (Boston, MA, USA), while
14
C-
tyramine was from Sigma-Aldrich. N-methyltyramine
hydrochloride, (+/) synephrine hydrochloride and
(+/) isopropylnorsynephrine hydrochloride were
synthesized and purified at Syntech SSPF Int'l Inc.
(Montclair, CA, USA). 2-Methoxy-idazoxan (RX
821002) and bromoxidine (UK 14304) were generous
gifts from Dr. H. Paris (INSERM U858, Toulouse).
Lipolytic activity of rat adipocyte preparations
The chosen index of lipolytic activity was the glycerol
released by freshly isolated adipocytes with reference
to the maximal response to () isoprenaline, a widely
recognized lipolytic agent. Adipocytes were isolated
from visceral fat pads of Wistar rats, and the cell
suspension was distributed in incubation vials as
previously described [11]. Briefly, pieces of adipose
tissue were subjected to collagenase digestion at 37°C
in the presence of 3.5% of serum bovine albumin in
the following buffer: KrebsRinger salt solution
containing 15 mM sodium bicarbonate, 10 mM
HEPES, and 5.5 mM glucose (KRBHA). The mean
body weight of the six Wistar rats used was 420 g,
and the mean amount of fat cells distributed into the
incubation vials was 17.3±1.7 mg/400 μl. Tested
amines were dissolved at 100,000 μg/ml in 10%
DMSO (v/v), and subsequent 1/10 dilutions were
done in distilled water. Of such working dilutions,
4μl were added to 400 μl of fat cell suspension in
KRBHA to reach the indicated final concentrations.
The most concentrated assays contained 1,000 μg
amine/ml and 0.1% DMSO at final concentration. All
compounds were incubated with the fat cells during
90 min, and glycerol release was determined on
150 μl of medium by spectrophotometric measure-
ment at 340 nm, as previously described [5].
Subjects and preparation of human adipose cells
for lipolysis measurements
Samples of human subcutaneous adipose tissue were
obtained from ten overweight women undergoing
abdominal lipectomy at the plastic surgery department
of Rangueil hospital (Toulouse, F). Their body mass
index (BMI) was 24.6±0.9 kg/m
2
. The surgical
interventions were conducted under general anesthesia.
The removed pieces of subcutaneous whitish adipose
tissue (WAT) were transferred in less than 30 min to the
laboratory. The ex vivo experiments were performed
under the agreement of the ethics committee in accor-
dance with the principles and guidelines established by
the French National Institute of Medical Research
(INSERM). Once isolated, the human adipocytes were
immediatelywashedinKRBHAandusedat17.6±
1.0 mg lipid/400 μl (approx. 500,000 cells/vial) for
lipolysis measurements, undergoing the same incubation
conditions as described above for rat, while remaining
small pieces of WAT where stored at 80°C before the
assays of MAO activity on tissue homogenates.
Glucose transport assays in isolated adipocytes
3
H-2-deoxyglucose (2-DG) uptake was determined
for 10 min with freshly isolated human fat cells after
Lipolytic effect of synephrine and isopropylnorsynephrine
45 min preincubation with the indicated agents in
400 μl KRBHA (with 2 mM pyruvate replacing
glucose as fuel supply) as previously described [15].
Separation of extracellular and internalized hexose
was performed by centrifugation through dinonyl-
phthalate layer, which allowed to separate buoyant
intact fat cells [11]. Lipid content was determined as
previously reported [4]. For these complementary
determinations, the adipocyte preparations were
obtained from six women undergoing lipectomy and
having a mean BMI of 24.6 ± 1.3 and a mean age of
39 years.
MAO activity on WAT homogenates
Amine oxidation was determined in subcutaneous
WAT homogenates, by incubating 0.5 mM
14
C-
tyramine during 30 min at 37°C together with approx.
100 μg proteins in the absence or the presence of
reference MAO inhibitors as described [27]. For these
determinations, mean BMI of the five female donors
was 25.2, and mean age was 41 years.
Statistical analysis
Results are given as means ± S.E.M. Statistical
significance was assessed by use of Student's ttest
(NS, non-significantly different from respective
control).
Results
Assessment of the lipolytic effect of the main amines
found in C. aurantium extracts
Since most of the reports related to the alkaloid
content of Citrus fruits are giving concentrations as
microgram per gram or microgram per milliliter,
similar units were used in our functional studies. This
allowed to directly compare with the amine richness
in the fruits, juices, or extracts and did not alter the
comparison of the pharmacological properties between
the different amines tested since, with the used salts,
1μg/ml corresponds to 4.9 μM synephrine, 5.3 μM
octopamine, 5.3 μMN-methyltyramine, 5.8 μM tyra-
mine, or 4.3 μM isopropylnorsynephrine. The DMSO
vehicle used for the dissolution of these amines was
without effect on lipolytic activity of rat adipocytes,
even at the highest tested concentration (0.1%), basal
0.27± 0.05, vehicle 0.28±0.05 μmol glycerol/100 mg
lipid/90 min (n=6, NS). As expected, the maximal
lipolytic activation by isoprenaline reached a plateau.
The maximal stimulation elicited by 10 μMisopren-
aline (equivalent to 2.45 μg/ml) increased the basal
activity by sixfold and was taken as 100% reference
(Fig. 2). Tyramine and N-methyltyramine were not
lipolytic. Synephrine was clearly stimulating the
lipolysis in a dose-dependent manner, reaching around
80% of the maximal response to isoprenaline. The
most lipolytic biogenic amine was octopamine, which
reached (or even was greater than) the stimulation
observed with isoprenaline. However, the two latter
amines were only active at elevated doses and
exhibited a very low potency when compared to
isoprenaline: there was a difference of about four
orders of magnitude in their EC
50
.
Further analysis was performed by mixing increas-
ing concentrations of the biogenic amines together
with a submaximal dose of isoprenaline (10 nM,
inducing a fivefold stimulation). Octopamine was
clearly additive to isoprenaline, regarding lipolysis
activation. This was not observed with synephrine
(Fig. 3). Again, tyramine and N-methyltyramine
behaved differently from the former amines, since
they dose-dependently inhibited the submaximal
10001001010.10.01
-20
0
20
40
60
80
100
a
g
ent, µ
g
/ml
*** ***
***
bas 0.001
isoprenaline
synephrine
N-methyltyramine
% of maximal isoprenaline-induced lipolysis
***
***
*** ***
tyramine
octopamine
***
***
*
rat adipocytes
Fig. 2 Comparison of the effects of the biogenic amines
detected in Citrus aurantium on lipolysis in rat adipocytes. Rat
adipocytes were incubated for 90 min with the indicated doses
of the hydrochloride salts of isoprenaline (closed circles),
octopamine (closed squares), synephrine (open squares),
tyramine (open triangles)orN-methyl tyramine (closed
triangles). Isoprenaline was used as the lipolytic agent of
reference: its maximal effect (reaching 1.50± 0.15 μmol glyc-
erol/100 mg cell lipids/90 min) was set at 100%. Mean ± SEM
of six observations. Different from basal (open circle)at*p<
0.05; ***p<0.001
J. Mercader et al.
effect of isoprenaline. They can be qualified as
antilipolytic agents, which stimulate receptors nega-
tively coupled to the adenylyl cyclase or act as partial
agonistsor even antagonistsat β-ARs. Irrespec-
tive of their mechanism of action, these amines can
hardly be suspected to promote the lipolytic action of
C. aurantium and its related dietary supplements.
Taken together, the observations obtained with rat
adipocytes validated that octopamine and synephrine
could be the components responsible of the claimed
activation of lipid mobilization by C. aurantium
extracts, if the case of their ingested dose is sufficient
enough to reach 1 μg/ml in the interstitial fluid of fat
depots. They also confirmed that tyramine exerts
antilipolytic effects, as previously reported [28].
Then, we compared in human adipocytes the
effects of the C. aurantium alkaloids, but we also
investigated the effects of isopropylnorsynephrine, on
the basis of its resemblance to isoprenaline, synephrine,
and octopamine, as shown in Fig. 1.
Lipolytic effect of tyramine, synephrine, and related
derivatives in human subcutaneous adipose cells
DMSO vehicle was without noticeable effect while
isoprenaline provoked a dose-dependent stimulation
of lipolysis in human adipocytes, which resembled to
that obtained in rat, with a maximum of sevenfold
increase over basal observed at 10 μM (Fig. 4). All
the tested amines increased lipolysis but with clearly
lower intrinsic activity compared with isoprenaline.
According to the relative order of potency or to the
ranking of maximal activity, the classification was
0
50
100
150
n=6
***
***
***
***
%of10nMisoprenalinelipolytice
ffect
10 100 100010 100 100010 100 100010 100µg/ml:
control octopamine synephrinemet
hyltyramine tyramine
-
300
***
***
Fig. 3 Effect of octopamine, synephrine, tyramine, and N-
methyltyramine on lipolysis in rat adipocytes in the presence of
10 nM isoprenaline. Lipolytic activity was submaximally
stimulated by 10 nM isoprenaline without or with the indicated
amines. Results as percentage of lipolysis obtained with 10 nM
isoprenaline alone (control), set at 100% with basal set at 0%.
Mean ± SEM of six observations. Different from control at
***p<0.001
isoprenaline
N-methyltyramine
synephrine
tyramine
isopropylnorsynephrine
0
20
40
60
80
100
%ofmaxim
al isoprenaline-induced lipolysis
10001001010.10.01
**
*
***
***
***
*** ***
**
*
**
**
*
**
*
a
g
ent, µ
g
/mL
0.001
octopamine
bas
***
human adipocytes
Fig. 4 Lipolytic responses of human adipose cells to the amines
detected in Citrus aurantium and derivatives. Freshly isolated
adipocytes were incubated for 90 min with increasing concen-
trations of isoprenaline (black circles) or the indicated amines.
Results are expressed as percentage of 10 μM (2.4 μg/ml)
isoprenaline-induced lipolysis. Mean ± SEM of five determina-
tions. Different from basal (open circle)at*p<0.05; **p<0.01;
***p<0.001
Lipolytic effect of synephrine and isopropylnorsynephrine
(with maximal percent of isoprenaline-dependent
activation given between parentheses): isoprenaline
(100%)>>isopropylnorsynephrine (59%)octop-
amine (52%)>synephrine (33%) > tyramine (25%) =
N-methyltyramine (20%). Tyramine and N-methyltyr-
amine were poorly lipolytic at 100 μg/ml only, with
an effect that decreased at the highest dose tested
(1,000 μg/ml, approx. 5 mM). The effects of
synephrine and octopamine were more impressive
but only detected at concentrations above 10 μg/ml.
The most lipolytic amine was isopropylnorsynephrine,
characterized by a plateau of maximal effect between 1
and 1,000 μg/ml, equivalent in amplitude to one half of
isoprenaline maximal stimulation. Notably, isopropyl-
norsynephrine elicited lipolytic responses with a high
inter-individual variability but with an approximately
100-fold higher potency than synephrine.
Synephrine, isopropylnorsynephrine, and the lipolysis
control in human adipocytes
The amines were then compared under a condition of
mild-stimulated lipolysis, in the presence of 10 μM
adrenaline. None of the amines was able to amplify
the response to adrenaline, but all of them tended to
inhibit it, contrarily to the α
2
-AR antagonist methoxy-
idazoxan, which clearly potentiated it (Table 1). Since
only the latter was able to block the α
2
-adrenegic
antilipolysis and to reveal the β-adrenergic compo-
nent of adrenaline, it can be concluded that the tested
alkaloids were unable to block α
2
-ARs. However, the
amines could bind to the α
2
-ARs in an agonistic
manner. This eventuality was tested by comparing
their putative antilipolytic action to that of bromox-
idine, a well-known α
2
-AR agonist. While the latter
totally inhibited the IBMX-induced lipolysis in a
manner that was reversed by the α
2
-AR antagonist
methoxy-idazoxan (Table 2), tyramine and its methyl-
ated derivative exhibited partialbut significant
antilipolytic action. Such antilipolysis was not added
to that of bromoxidine (Table 2), leading to suspect
either a partial agonism at α
2
-AR or a completely
different antilipolytic action. Again, isopropylnorsy-
nephrine was the most lipolytic of the tested amines,
exhibiting a tendency to enhance the IBMX lipolytic
effect.
The effects of synephrine and isopropylnorsynephrine
on glucose transport in human adipocytes
Then, we studied the capacity of synephrine and
isopropylnorsynephrine to regulate glucose uptake in
human fat cells since octopamine has been already
described to partially activate (at 0.1 and 1 mM)
glucose transport in such cells or in adipocytes from
β
3
-AR KO mice [27]. Neither synephrine nor
isopropylnorsynephrine were able to provoke a
noticeable stimulation of glucose transport when
incubated for 45 min with adipocytes, while 100 nM
insulin enhanced the basal uptake by fourfold (not
shown). However, a difference was found between
Table 1 Effect of a combination of 10 μM adrenaline and
biogenic amines or isopropylnorsynephrine on lipolysis
Incubation condition Glycerol release, μmol/
100 mg lipid/90 min
Basal 0.12± 0.01 (10)**
Adrenaline 10 μM 0.40± 0.07 (10)
Adrenaline+ tyramine 0.31± 0.02 (5)
Adrenaline+ N-methyltyramine 0.31± 0.02 (5)
Adrenaline+ synephrine 0.33± 0.02 (5)
Adrenaline+ isopropylnorsynephrine 0.25± 0.06 (5)
Adrenaline+ methoxy-idazoxan
10 μM
0.75± 0.08 (10)**
The amines were added at 100 μg/ml. The number of determi-
nations is given in parentheses
**p<0.01, Different from adrenaline alone
Table 2 Antilipolytic effect of tyramine, bromoxidine, and
related amines
Incubation condition IBMX alone IBMX+ bromoxidine
1μM
Control 100.0±8.2 6.0±2.4***
+Methoxy-idazoxan
10 μM
97.7± 2.2 95.4 ± 7.7
+Tyramine 100 μg/ml 63.7±9.0* 22.7± 5.8***
+N-methyltyramine
100 μg/ml
56.7± 4.5 ** 18.6 ± 3.5 ***
+Synephrine 100 μg/ml 125.8± 21.8 70.3± 5.7 **
+Isopropylnorsynephrine
100 μg/ml
121.8± 10.9 120.6 ± 8.4
IBMX 1 mM stimulated the lipolysis from 0.13 ± 0.02 to 0.85 ±
0.10 μmol glycerol/100 mg lipid/90 min and was set as 100%
control. In parallel conditions, the other agents (amines, α
2
-AR
agonist or antagonist) were added at the indicated final
concentrations. Mean ± SEM of four determinations
*p<0.05; **p< 0.01; ***p< 0.001, Different from control
J. Mercader et al.
synephrine and its derivative, regarding the acute
influence on insulin responsiveness. Only the high
dose (1 mM) of synephrine hampered the submaximal
activation of uptake by 10 nM insulin (reducing it
from 2.38±0.27 to 1.22± 0.10 nmol 2-DG/100 mg
lipid/10 min, n=6, p<0.01). Such inhibitory behavior
was not observed with lower doses of synephrine
(0.010.1 mM) or with isopropylnorsynephrine, since
the uptake in the presence of 10 nM insulin plus 1 mM
of this substituted amine was equivalent to 2.28±
0.24 nmol 2-DG/100 mg lipids/10 min (n=6, NS).
Lack of isopropylnorsynephrine oxidation by MAO
in human WAT
Tyramine and octopamine are MAO substrates in human
adipocytes [27], which highly express MAO-A. We
investigated whether the other amines interacted with
MAO. While N-methyltyramine was clearly undergoing
oxidation by MAO, isopropylnorsynephrine did not
behave as a MAO substrate since, when tested from
100 nM to 10 mM on human WAT homogenates, it did
not compete for
14
C-tyramine oxidation. More precisely,
the oxidation of 0.5 mM
14
C-tyramine was producing
1.5±0.1 nmol of labeled aldehyde per milligram protein
per minute and was inhibited by approx. 90% by
10 mM cold tyramine or N-methyltyramine, while it
was only inhibited by 20±2% by 10 mM isopropylnor-
synephrine. In this view, isopropylnorsynephrine was
resembling to the β-AR agonist isoprenaline, which was
also unable to inhibit
14
C-tyramine oxidation (only 5±
4% inhibition, n=5, NS).
Discussion
Our comparative study showed that in rat adipocytes,
octopamine and synephrine exhibit lipolytic activities
that may constitute the basis for an anti-obesity effect
of Citrus extracts. When considering the numerous
qualitative and quantitative determinations of the
alkaloids present in the diverse multi-component
preparations containing Citrus aurantium or in the
fruits themselves, it appears that octopamine, which is
only present in trace amounts [21], cannot be the major
active component responsible for the lipid-mobilizing
activity ascribed to such preparations [12,25]. In spite
of having a lower intrinsic activity than octopamine,
synephrine can be considered as the predominant active
component, since it is the most abundant alkaloid found
in such preparations [20]. Unfortunately, synephrine
(like octopamine) is clearly less lipolytic in human
adipocytes than in rat, and the reduction in body weight
gain observed without overtly declared alteration in
oxidative stress in rodents after its subchronic admin-
istration [3] is probably not as evident in man. Since it
is known that high doses of C. aurantium extracts
(5,000 mg/kg) or of synephrine (2,000 mg/kg) exert in
mouse toxic effects that are reversible and related to
adrenergic overstimulation [2], the benefit/risk ratio of
increasing the dietary intake of these biogenic amines in
overweight or obese subjects to limit WAT extension
remains to be established.
In our conditions, which can be considered as
highly responsive to isoprenaline (sixfold stimula-
tion), the lipolytic action of octopamine found in rat
adipocytes is in complete agreement with its stimulatory
action already reported in rodents [9,27], including
hibernators [5]. The mechanism of action of octop-
amine is related to the activation of β
3
-ARs, and a very
low lipolytic response to this amine has been observed
in guinea pig and in human adipocytes [9], known to
express much less functional β
3
-ARs than the rat. A
minor discrepancy appeared when comparing the
maximal intrinsic activity of octopamine in human
adipocytes observed in the present study (reaching 48%
of maximal isoprenaline effect) to the faint activity
previously reported (accounting for 12% of maximum)
[9]. In spite of this difference which remains explained,
the overall maximal octopamine-induced lipolysis
remains much lower than that of isoprenaline in human
adipocytes. Therefore, the capacity of repeated i.p.
injections of octopamine to lower the body weight gain
of obese rats [6] remains far for being extrapolated into
a mere prediction of a lipid mobilization provocked by
octopamine supplementation in obese patients.
The inhibition of lipolysis observed with tyramine
in rat adipocytes is completely confirmatory of that
already reported in the same model for millimolar
doses of tyramine and other amine oxidase substrates
[28]. This antilipolytic effect has been shown to be
mediated by the hydrogen peroxide produced during
amine oxidation occurring in fat cells [28]. The
antilipolytic effect of N-methyl tyramine, which
resembles to that of tyramine, does not seem to be
mediated by an activation of α
2
-ARs since the α
2
-
adrenergic antilipolytic component is very weak in rat
adipocytes [27]. A very weak lipolytic effect of
Lipolytic effect of synephrine and isopropylnorsynephrine
tyramine and of its methylated derivative was
evidenced for the first time in human adipocytes.
However, more remarkable was the capacity of these
amines to exert a substantial antilipolysis in human
cells when IBMX is present. A partial agonism at β-
ARs could be excluded since IBMX activates lipolysis
without stimulating adrenergic receptors. Because tyra-
mine and its methylated derivative hardly added their
effects to the antilipolytic α
2
-AR agonist bromoxidine,
a partial agonism to α
2
-ARs cannot be excluded in
human adipocytes. However, this hypothesis appears
unlikely when considering that: (1) the amines were
also antilipolytic in rat adipocytes lacking functional
α
2
-ARs, (2) tyramine is readily oxidized by human
adipocytes and may inhibit triglyceride breakdown in a
hydrogen peroxide-mediated manner, as evidenced in
rat adipocytes [28]. Whether the antilipolytic effect of
tyramine exists under physiological situations deserves
to be studied, since tyramine is widely distributed in
numerous foods and beverages other than Citrus fruits
(e.g., seafood, cheese, wine, beer).
Synephrine has been supposed to act as an
antagonist on presynaptic α-receptors [17]. Our
observations disagree with an α
2
-AR antagonist
component since: (1) synephrine was unable to
improve adrenaline-induced lipolysis in human fat
cells, and (2) synephrine did not totally prevent
bromoxidine-induced antilipolysis on IBMX stimula-
tion. In the same conditions, the α
2
-adrenergic
antagonist methoxy-idazoxan clearly potentiated the
lipolytic effect of adrenaline (by blocking its α
2
-
antilipolytic component, predominant in obese
patients [24]) and totally reversed the α
2
-adrenergic
antilipolytic effect of bromoxidine. A partial agonism
at α
2
-ARs cannot be excluded but did not allow
synephrine to notably inhibit IBMX-induced lipolysis.
To verify whether such partial agonism exists for this
amine, as for the other protoalkaloids, it should be
necessary to test whether the addition of a β-blocker in
the medium reveals a clearer antilipolytic behavior on
IBMX. However, such verifications seem poorly
mandatory in view of recent observations made on
cells expressing the different subtypes of human α-ARs
and showing the poor affinity of synephrine for α
1
-,
α
2A
-, and α
2C
-ARs [17]. More informative can be to
unravel how synephrine impairs insulin activation of
glucose transport. One possible reason could be given
by the fact that β
3
-ARs agonists, including octopamine,
hamper the insulin action in rat fat cells [11,27], but
such event remains unlikely in human fat cells, which
have only a weak β
3
-adrenergic responsiveness.
Another concern that can be raised is that (+/)
synephrine was used in our study while the naturally
occurring amine is mainly R-()-synephrine. However,
it has been recently shown that this natural form of
synephrine undergoes high risks of racemization
during the extraction procedure from the plant material
as the result of an acceleration of this process by high
temperatures and acidic or basic pH values [22].
The isopropyl derivative of demethylated synephr-
ine or octopamine was the most lipolytic of the agents
tested on human adipocytes. Its potency and intrinsic
activitiy were more attractive than those of the
protoalkaloids found in C. aurantium. However, this
derivative has never been evidenced in bitter oranges
(F. Pellatti, personal communication) and cannot be
considered as a naturally occurring alkaloid. The
lipolytic effect of isopropylnorsynephrine has been
reported by Wenkeová and coworkers in their
pioneering studies on pieces of human adipose tissue
in vitro [30]. As in our present studies made on highly
responsive isolated adipocytes, these authors found
35 years ago that isopropylnorsynephrine produced
about 40% of the maximal lipolytic effect of
isoprenaline on WAT pieces. A direct activation of
β-ARs, but not of α-ARs by this amine has been
evidenced on cardiovascular functional responses [1].
In the human adipocytes, the complete reversion of
the bromoxidine-induced antilipolysis, observed with
isopropylnorsynephrine was probably not due to an
α
2
-AR antagonist property since at the same concen-
tration, the agent did not potentiate the adrenaline
effect. A more likely interpretation is that isopropyl-
norsynephrine has stimulated the β-ARs, leading to
an activated lipolysis that was further enhanced by
IBMX, resulted in a cAMP overproduction that
cannot be lowered by bromoxidine. The impressive
lipolytic capacities of isopropylnorsynephrine (half-
maximal effect of isoprenaline reached with only
1μg/ml) encourage to consider it as a good candidate
to promote lipid mobilization. A drug-based approach
using isopropylnorsynephrine rather than a diet
supplementation may constitute a novel safe basis of
managing body weight. However, facing to these
lipolytic properties, the lack of serious adverse actions
reported so far for this synephrine derivative have to
be scrupulously verified before developing such novel
lipid-mobilizing treatment.
J. Mercader et al.
Meanwhile, the fact that isopropylnorsynephrine
does not undergo amine oxidation, as measured in our
competition experiments of
14
C-tyramine oxidation
completely fits its lack of activation of glucose
transport. It is also consistent with very ancient
findings indicating that the lipolytic response to this
adrenergic drug was not differing when using albu-
mins of different origin (human vs. bovine) in the
incubation medium, while the potency of catechol-
amines (e.g., adrenaline) was different from one
preparation to another [16]. Our current interpretation
of such findings is that, in this period, albumin
preparations were less purified than at the present
time, and the bovine one was more contaminated by
soluble amine oxidase, the activity of which is more
abundant in bovine than in human plasma [14].
Consequently, the catecholamines were more oxidized
in the former preparation and their lipolytic properties
were altered, when compared to incubations with
human albumin. We therefore propose that the
lipolytic action of isopropylnorsynephrine was the
same irrespective of the source of albumine tested
because isopropylnorsynephrine, like isoprenaline,
was not oxidized during the incubations.
To conclude, synephrine, which is quantitatively the
most represented alkaloid in C. aurantium extracts could
be endowed of the putative lipid-mobilizing effects of
such extracts in man. Only a direct in situ analyis of
lipolysis, owing to the microdalysis technique [24]
could definitively demonstrate such property. However,
such investigations must be performed not only with
multi-component dietary supplements but also with
purified alkaloids. A recent clinical study showed that
dietary supplements increased blood pressure and
plasma glucose post-exercise, and modestly improved
exercise tolerance [13], but it was impossible to make
the part due to synephrine since such products contained
synephrine and caffeine. Regardless of this issue, our
observations have confirmed that isoprenaline, and to a
lesser extent, isopropylnorsynephrine, are lipolytic drugs
which are much more active than the dietary amines
synephrine or octopamine, while tyramine and N-
methyl tyramine exhibit antilipolytic actions.
Acknowledgements This work was partly supported by
Communauté de Travail des Pyrénées and the DIOMED project
(INTERREG IVB-SUDOE-FEDER, SOE1/P1/E178). The
authors express gratitude to Virgile Visentin and Danielle
Prévot for their help. They also acknowledge Philippe Valet
(Univ. Toulouse, France) and Federica Pellati (Univ. Modena,
Italy) for their respective knowledge on human adipocyte
biology or Citrus biogenic amines and the staff of plastic
surgery of Rangueil hospital for facilitating access to surgical
wastes. In memoriam to Hervé Paris.
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J. Mercader et al.
... In a subsequent study, the lipolytic activity of p-synephrine, poctopamine, tyramine and N-methyltyramine were compared in rat and human adipocytes based on β-3 adrenergic receptor binding (Mercader, Wanecq, Chen, & Carpene, 2011). In rat fat cells, at a concentration of 10 μg/ml both p-synephrine and p-octopamine exhibited approximately 60% of the lipolytic activity of 1 nM/ml of isoprenaline while tyramine and N-methyltyramine exhibited no effect or were weakly antagonistic. ...
... Various studies have shown that p-synephrine binds to β-3 adrenergic receptors, resulting in an increase in the body's ability to breakdown fats (Carpene' et al., 1999;Carpene' et al., 2014;Mercader et al., 2011). Binding to β-3 adrenergic receptors does not influence heart rate or blood pressure, although it may be speculated that cardiovascular down-regulation due to β-3 adrenergic receptor binding may result in small decreases in diastolic blood pressure, which has been demonstrated (Ratamess et al., 2018;Shara et al., 2016). ...
... Because p-synephrine and p-octopamine bind at least 10 times more readily to α-1, α-2, β-1 and β-2 adrenergic receptors from rodents than humans (Carpene' et al., 1999;Carpene' et al., 2014;Mercader et al., 2011), the small but clinically insignificant cardiovascular effects seen in rodents at high doses (Hansen et al., 2012(Hansen et al., , 2013 Hansen, Juliar, White, & Pellicore, 2011) cannot be extrapolated to humans. As a consequence, based on these receptor binding studies, cardiovascular effects are not predicted or expected to occur in humans. ...
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... In rats, synephrine (54) (0.01-1000 lg/mL) was clearly stimulatingthe lipolysis in a dose-dependent manner (p \ 0.001), reaching around 80% of the maximal response to isoprenaline, while N-methyltyramine (55) was not lipolytic. In human adipocytes which were obtained from ten overweight women, although both synephrine (54) and N-methyltyramine (55) increased lipolysis, the lipolysis of synephrine (54) (33%) was still more than that of N-methyltyramine (55) (20%) (Mercader et al. 2011). ...
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Citrus aurantium extract and its primary alkaloidal constituent, synephrine, are widely used in weight management products and as thermogenic agents. Citrus aurantium extract is also known as bitter orange extract, a product that is derived from the unripe (green) fruits. In traditional Chinese medicine, it is known as “Chih-shi” or “Zhi shi.” Synephrine is a phenylethylamine derivative, also known as oxedrine or p -synephrine due to the hydroxy group in the para position on the benzene ring. In recent years, bitter orange extract has been used in weight management products due to its putative stimulant effects on metabolic processes, including increased lipolysis and thermogenesis as well as its mild appetite suppressant effects.
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Citrus aurantium (bitter orange) is a plant belonging to the family Rutaceae, whose fruit extracts have been used recently for the treatment of obesity. The most important biologically active constituents of the C. aurantium fruits are phenethylamine alkaloids (i.e. octopamine, synephrine, tyramine, N-methyltyramine and hordenine). Synephrine is a primary synthesis compound with pharmacological activities such as vasoconstriction, elevation of blood pressure and relaxation of bronchial muscle. Synephrine is present in the peel and the edible part of Citrus fruit. Of the adrenergic amines of natural origin, synephrine has been found to be the main constituent of C. aurantium fruits and extracts; the other alkaloids are either absent or present in only low concentrations. It is known that synephrine and the other amines found in C. aurantium have adverse effects on the cardiovascular system, owing to adrenergic stimulation. In light of the great commercial proliferation of C. aurantium herbal medicines in recent years, this review provides an overview of various extraction, separation and detection techniques employed for the qualitative and quantitative determination of the alkaloids in C. aurantium and related species. The application of chromatographic and electrophoretic methods for the separation and determination of these active components in C. aurantium plant material and derivatives are described. Since synephrine is a chiral compound, enantioselective chromatographic and electrophoretic techniques for the analysis of synephrine enantiomers in natural products are presented. Furthermore, examples of identification of these active compounds in complex matrices by hyphenated methods, such as gas chromatography–mass spectrometry and high-performance liquid chromatography–mass spectrometry, are described. The advantages and limitations of these separation and identification methods are assessed and discussed.
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In this study, the racemization kinetic parameters of R-(-)-synephrine, the active phenethylamine alkaloid of Citrus aurantium L., were determined by means of an off-column HPLC method. Enantioseparation was carried out in different buffer solutions and solvents on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH, 100 mm x 4.0 mm i.d., 5 microm). The mobile phase was 10 mM sodium phosphate buffer (pH 6.0)-2-propanol (95:5, w/w), with 50 microM disodium EDTA, at 0.8 mL/min. The column was thermostatted at 20 degrees C and detection was set at 225 nm. The influence of pH value, ionic strength, temperature and addition of organic modifier on the rate constant, the half-life of racemization and the free energy barrier of racemization of R-(-)-synephrine were determined. Among the different chemical and physical parameters evaluated as affecting the racemization of naturally occurring R-(-)-synephrine, pH, temperature and addition of an organic co-solvent appear to have the strongest effect, while ionic strength does not exert a significant influence on the racemization rate. The results of the present study indicated that synephrine racemization is possible at high temperature at both acidic and basic pH values; therefore, the extraction procedure of R-(-)-synephrine from the plant material should be carried out under specific conditions to preserve the stereochemical integrity and the biological activity of this secondary metabolite.
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Visfatin, a protein identified as a secretion product of visceral fat in humans and mice, is also expressed in different anatomical locations, and is known as pre-B cell-colony enhancing factor (PEBF1). It is also an enzyme displaying nicotinamide phosphoribosyltransferase activity (Nampt). The evidence that levels of visfatin correlate with visceral fat mass has been largely debated and widely extended to other regulations in numerous clinical studies and in diverse animal models. On the opposite, the initial findings regarding the capacity of visfatin/Nampt/PEBF1 to bind and to activate the insulin receptor have been scarcely reproduced, and even were contradicted in recent reports. Since the putative insulin mimicking effects of visfatin/Nampt/PEBF1 have never been tested on mature human adipocytes, at least to our knowledge, we tested different human visfatin batches on human fat cells freshly isolated from subcutaneous abdominal fat and exhibiting high insulin responsiveness. Up to 10 nM, visfatin was devoid of clear activatory action on glucose transport in human fat cells while, in the same conditions, insulin increased by more than threefold the basal 2-deoxyglucose uptake. Moreover, visfatin was unable to mimic the lipolysis inhibition induced by insulin. Visfatin definitively cannot be considered as a direct activator of insulin signalling in human fat cells. Nevertheless itsin vivo effects on insulin release and on glucose handling deserve to further study the role of this multifunctional extracellular enzyme in obese and diabetic states.