Pharmacological Research 61 (2010) 355–363
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Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs
Chronic benzylamine administration in the drinking water improves glucose
tolerance, reduces body weight gain and circulating cholesterol in high-fat
diet-fed mice夽
Zsuzsa Iffiú-Soltész a,b , Estelle Wanecq a , Almudena Lomba c , Maria P. Portillo d , Federica Pellati e ,
Éva Szökő b , Sandy Bour a , John Woodley f , Fermin I. Milagro c , J. Alfredo Martinez c ,
Philippe Valet a , Christian Carpéné a,∗
a
INSERM, U858, Institut de Médecine Moléculaire de Rangueil, IFR 150, Toulouse, France
Department of Pharmacodynamics, Semmelweis University, Budapest, Hungary
c
Department of Nutrition and Food Sciences, Physiology and Toxicology, University of Navarra, Pamplona, Spain
d
Department of Nutrition and Food Science, Universidad del País Vasco, Vitoria, Spain
e
Department of Pharmaceutical Sciences, University of Modena e Reggio Emilia, Modena, Italy
f
Biopharmtech, Toulouse, France
b
a r t i c l e
i n f o
Article history:
Received 23 September 2009
Received in revised form
23 December 2009
Accepted 23 December 2009
Keywords:
Adipose tissue
Semicarbazide-sensitive amine oxidase
Monoamine oxidase
Antidiabetic
Obesity
a b s t r a c t
Benzylamine is found in Moringa oleifera, a plant used to treat diabetes in traditional medicine. In mammals, benzylamine is metabolized by semicarbazide-sensitive amine oxidase (SSAO) to benzaldehyde
and hydrogen peroxide. This latter product has insulin-mimicking action, and is involved in the effects
of benzylamine on human adipocytes: stimulation of glucose transport and inhibition of lipolysis. This
study examined whether chronic, oral administration of benzylamine could improve glucose tolerance
and the circulating lipid profile without increasing oxidative stress in overweight and pre-diabetic mice.
The benzylamine diffusion across the intestine was verified using everted gut sacs. Then, glucose handling and metabolic markers were measured in mice rendered insulin-resistant when fed a high-fat diet
(HFD) and receiving or not benzylamine in their drinking water (3600 ␮mol/(kg day)) for 17 weeks. HFDbenzylamine mice showed lower body weight gain, fasting blood glucose, total plasma cholesterol and
hyperglycaemic response to glucose load when compared to HFD control. In adipocytes, insulin-induced
activation of glucose transport and inhibition of lipolysis remained unchanged. In aorta, benzylamine
treatment partially restored the nitrite levels that were reduced by HFD. In liver, lipid peroxidation
markers were reduced. Resistin and uric acid, surrogate plasma markers of metabolic syndrome, were
decreased. In spite of the putative deleterious nature of the hydrogen peroxide generated during amine
oxidation, and in agreement with its in vitro insulin-like actions found on adipocytes, the SSAO-substrate
benzylamine could be considered as a potential oral agent to treat metabolic syndrome.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: ACADM, acyl-coenzyme A dehydrogenase involved in mediumchain fatty acid breakdown; BHLHB2, basic helix-loop-helix domain containing,
class B 2; BZA, benzylamine; CPT1A, carnitine palmitoyl transferase; 2-DG, 2[1,2-3H]-deoxyglucose; FASN, fatty acid synthase; GTT, glucose tolerance test;
HFD, high-fat diet; INPPL1, inositol polyphosphate phosphatase-like 1; IRS1,
insulin receptor substrate-1; MAO, monoamine oxidase; ROS/RNS, reactive
oxygen/nitrogen species; SSAO, semicarbazide-sensitive amine oxidase; VAP-1, vascular adhesion protein-1; WAT, white adipose tissue.
夽 A previous account of this work was presented, as a poster, in the 3rd International Congress on prediabetes and the metabolic syndrome, held in Nice (France)
in 1st–4th April 2009, that subsequently appeared in: Journal of Diabetes (2009) 1
(suppl. 1):A241.
∗ Corresponding author at: Institut National de la Santé et de la Recherche Médicale (INSERM), U858 équipe 3, Institut de Médecine Moléculaire de Rangueil (I2MR),
Bat. L4, CHU Rangueil, F-31432 Toulouse cedex 4, BP 84225 France.
Tel.: +33 5 61 32 56 36; fax: +33 5 61 32 56 23.
E-mail address: [email protected] (C. Carpéné).
1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phrs.2009.12.014
Amine oxidation is considered to be a major metabolic route
in the termination of the neurotransmitter function of biogenic
amines. Amine oxidation is also involved in the destruction of
dietary amines. The excessive blockade of these reactions is known
as the “cheese effect” leading to fatal hypertensive crises in
depressed patients treated with irreversible monoamine oxidase
(MAO) inhibitors and consuming amine-containing food. The false
neurotransmitter tyramine has been highly suspected to be detrimental in these conditions. There is now a growing body of evidence
to indicate that amine oxidases can influence cardiac physiopathology [1] and have a dual effect in diabetes [2]. Hydrogen peroxide,
one of the end-products of the amine oxidase-mediated catalysis, is involved in such novel functions. Hydrogen peroxide mimics
insulin effects in adipocytes and is recognized as an inhibitor of
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tyrosine phosphatases involved in turning-off insulin signalling
[3]. Substrates of MAO and semicarbazide-sensitive amine oxidases
(SSAO) reproduce insulin actions (stimulation of glucose transport,
lipolysis inhibition) in an hydrogen peroxide-dependent manner
on adipocytes isolated from white adipose tissues (WAT) of different species [4–6]. SSAO- and MAO-substrates also reproduce shortand long-term insulin-like actions in preadipocytes [7,8], the SSAO
activity of which strongly increase with adipocyte differentiation
[9]. Several SSAO substrates have even been reported to exhibit
in vivo anti-hyperglycemic effects in rats, mice [10,11] and rabbits
[6].
SSAO is not only present in fat cells but also in blood vessels, in inflamed areas or in lymph nodes where it is known
as vascular adhesion protein-1 (VAP-1), involved in leukocyte
extravasation [12]. SSAO/VAP-1 is also present in the blood as a soluble form, which increases in diabetes [13]. It has been suggested
that its inhibition by SSAO-inhibitors may reduce the vascular
complications of diabetes [14], but it has never been reported
that SSAO-inhibitors exert anti-hyperglycaemic effects [15]. The
dual influence of amine oxidation on diabetes has been demonstrated in experiments consisting of methylamine administration
in mice overexpressing SSAO/VAP-1 at endothelial surfaces [2]:
methylamine improves glucose handling but also worsens diabetic
complications (protein glycation, atherosclerosis and nephropathy). The factors responsible for such deleterious effects are other
products resulting from amine oxidation: the aldehydes, especially
formaldehyde and methylglyoxal originating from the oxidation
of methylamine and aminoacetone. Such aldehydes crosslink proteins and consequently cause endothelial damage [14,16,17]. The
cytotoxicity of these aldehydes can be considered to be even
more harmful than that of hydrogen peroxide, which is peculiarly well tolerated by fat cells [18]. Moreover, oxidation of
exogenous amine substrates can generate aldehydes that are less
reactive than those mentioned above. It has been reported to be
the case for benzaldehyde formed during benzylamine oxidation
[19].
Our aim was therefore to investigate a potential therapeutic
approach to hyperglycemia and insulin-resistance based on the
administration of an SSAO-substrate that might increase hydrogen
peroxide production and mimic insulin action in insulin-sensitive
targets, such as adipocytes, without generating deleterious aldehydes.
Such a substrate-based approach was reported to function
effectively in rat models of type I and type II diabetes when a combination of benzylamine plus vanadate was injected repeatedly
[10,11,20]. However, two aspects seemed to prevent a straightforward extrapolation of these findings to clinical use: (1) in
spite of its antidiabetic actions, vanadium is suspected of having toxic side-effects [21], and (2) the effects of benzylamine are
less potentiated by vanadium in human adipocytes [5] than in rat
adipocytes [10]. Therefore, benzylamine needed to be tested alone
to see whether it was able to acutely improve glucose tolerance.
Since we recently obtained encouraging results in normoglycemic
rats [22] and rabbits [6], we investigated here whether oral benzylamine exhibits antidiabetic properties in a type 2 diabetes
model.
To this aim, the benzylamine passage across the intestinal barrier was first tested in the everted gut sac model.
Then, benzylamine was orally administered to mice rendered
insulin-resistant by high-fat feeding. Attention was focused on
glucose tolerance and insulin sensitivity, circulating lipids, oxidative stress and vascular reactivity. We hypothesized that orally
administered benzylamine was absorbed in sufficient amounts
to reproduce the insulin-like effects initially observed in vitro on
adipocytes, without generating oxidative stress and vascular dysfunction.
2. Materials and methods
2.1. Intestinal benzylamine transport and oxidation
Ten Wistar rats (200–300 g, from Charles River, l’Arbresle,
France) were used for preparation of everted gut sacs and determination of transport across small intestine as previously described
[23]. Briefly, concentrations of [14 C]-benzylamine (0.01–1 mM)
were applied to the mucosal side of everted gut sacs of approximately 2.5 cm length incubated in tissue culture medium at 37 ◦ C
for the indicated times. Then, the radioactivity found in the serosal
fluid was quantified by liquid scintillation counting with or without
prior extraction with toluene-ethyl acetate, to separate benzylamine from its oxidation product, benzaldehyde, as with amine
oxidase assays. Viability and protein content of the preparations
were measured as previously reported [23]. Amine oxidase activity was measured in homogenates with 0.1 mM [14 C]-benzylamine
in 200 mM phosphate buffer, pH 7.5, in the presence of protease
inhibitors for 30 min as previously described [15]. 15 min preincubation with 1 mM semicarbazide or 0.5 mM pargyline was used
to selectively inhibit SSAO or MAO activity, respectively, as previously shown [4].
2.2. Mice, diets and experimental design
C57Bl6 male mice were purchased from Charles River
(l’Arbresle, France). A high-fat diet (HFD) from UAR (UAR, Lyon,
France) replaced the standard rodent chow at the age of 8 weeks
for a group of 36 mice housed at 3 animals per cage with free access
to food and water. HFD contained (as percent of kcal): 20% protein,
35% carbohydrate and 45% fat. Body weight and water consumption
were determined weekly, but food intake could not be assessed,
due to the sticky nature of the HFD pellets. Three weeks after the
nutritional switch, 24 mice sharing the closest body mass were
split in one group of 12 mice kept without treatment (control),
while another group of 12 weight-matched mice received benzylamine hydrochloride in the drinking water at 0.44% for a 17-week
period (BZA-treated). To achieve this, benzylamine was dissolved
in 400 ml water and used as drinking solution changed every week.
For mice weighing 30 g and drinking approx. 3.5 ml/day, the daily
dose of ingested benzylamine was 3600 ␮moles/(kg day). Nonfasting blood glucose was assessed weekly on a drop of blood
obtained between 15:00 and 16:00 h from the tail vein. After 15
weeks of treatment, an intraperitoneal glucose tolerance test (GTT)
was carried out as follows: mice were fasted for 5 h, then received
glucose (1 g/kg) by i.p. bolus (t0 ); blood was collected from the
tail vein of conscious mice 15 min before and just prior to the
glucose load, and then 15, 30, 45, 60, 90 and 120 min later for
determination of glucose levels using a Accu-Chek glucose monitor (Roche Diagnostic, Meylan, France) as previously reported
[24].
2.3. Stability of benzylamine solutions
The benzylamine concentration of the aqueous drinking
solutions was verified by ion-pair high-performance liquid chromatography (HPLC) analysis on freshly prepared and 1-week-old
solutions. HPLC was performed on an Agilent Technologies modular 1100 system (Waldbronn, Germany), using a LiChrospher RP-18
column (125 mm × 4 mm i.d. 5 ␮m), with an isocratic mobile phase
composed of a mixture of water–acetonitrile (65:35, v/v), containing 0.1% H3 PO4 and 0.5% SDS at a flow-rate of 1 ml/min and with UV
detection at 205 nm. Quantification was performed by the external
standard method covering the concentration range 9.8–983 ␮g/ml
(r = 0.999).
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2.4. Plasma and tissue sampling, lipolysis and glucose uptake in
adipocytes
Mice were sacrificed after overnight fasting. Tissues and plasma
were immediately frozen at −80 ◦ C. Intra-abdominal white adipose
tissues (INWAT: perirenal, retroperitoneal and epididymal fat pads)
were pooled for immediate adipocyte isolation and determination
of lipolysis (glycerol release) and glucose transport activities (by
2-deoxyglucose uptake assay) as previously described [5].
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2.9. Chemicals and reagents
Benzylamine hydrochloride, bovine insulin, fatty acid-free
bovine serum albumin, and other reagents were obtained from
Sigma–Aldrich (Saint Quentin Fallavier, France, and Milan, Italy
for HPLC grade reagents). Enzymes for the glycerol assay and liberase blendzyme 3 were from Roche Diagnostics (Indianapolis,
IN, USA). 2-[1,2-3 H]-deoxyglucose (2-DG, 29.7 Ci/mmol) was purchased from PerkinElmer (Boston, MA, USA), [14 C]-benzylamine
was from Amersham Biosciences (Buckinghamshire, UK).
2.5. Circulating endocrine and metabolic parameters
3. Results
Plasma metabolites (glucose, lactic acid, albumin, bilirubin)
were determined using a Cobas-Mira + multi-analyser, according
to the manufacturer’ instructions (Roche, Neuilly, France). Analyses of circulating fructosamine (using a colometric assay based on
nitrotetrazolium blue reduction) and of uric acid (using an uricaseperoxidase method) were adapted to 96-well microplates for use
in the multi-analyser. Plasma lipids were determined as follow:
triacylglycerides with the Randox kit for in vitro triacylglyceride
diagnostics (Randox Lab. Ltd., Crumlin, UK), plasma free fatty acids
(FFA) levels using WAKO NEFA test (Oxoid, Dardilly, France), and
cholesterol with a BioSystems kit (Barcelona, Spain). Plasma insulin
was determined with an Ultrasensitive Insulin Elisa kit obtained
from Mercodia (Uppsala, Sweden), while immunoreactive resistin,
leptin and adiponectin were determined using Linco RIA kits (St.
Charles, MO, USA).
2.6. Tissue markers of reactive nitrogen and oxygen species
Putative changes in the reactive nitrogen species (RNS) were
evaluated by the capillary electrophoresis-based determination of
nitrate/nitrite content in aorta and muscle samples according to a
previously published method [25]. The markers of excessive reactive oxygen species (ROS) assessed in liver were: the increase in
lipid peroxidation products, assayed using a kit for thiobarbituric
reactive substances (TBARS) from Cayman Chemical (Ann Harbor,
MI) and the decrease in aconitase activity [26], assayed using a kit
from Oxis International (Bioxytech 340, Foster City, CA).
2.7. Expression of genes related to energy metabolism in liver
Liver samples were used for RNA isolation and the analysis
of gene expression. Total RNA was isolated from frozen samples
using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The purified total RNA was treated with
RNase-free DNase (Ambion, Austin, TX, USA) for 30 min at 37 ◦ C
and used as a template to generate first-strand cDNA synthesis
using M-MLV reverse transcriptase (Invitrogen) as described by
the manufacturer. Quantitative real-time PCR was performed using
an ABI PRISM 7000 HT Sequence Detection System using Taqman probes (Applied Biosystems, Foster City, CA, USA) for mouse
catalase, superoxide dismutase (SOD2), insulin receptor substrate
(IRS1), BHLHB2 (also known as Sharp-2), INPPL1 (also known as
Ship-2), carnitine palmitoyl transferase (CPT1A), acyl-Coenzyme A
dehydrogenase (ACADM), fatty acid synthase (FASN) and 18 s rRNA
as internal control.
3.1. Intestinal transport and oxidation of benzylamine
Initial experiments were carried out to determine whether
benzylamine could cross the intestinal barrier without being
oxidized by the amine oxidases supposed to protect the body
against dietary amines. Fig. 1A shows that benzylamine was predominantly oxidized by SSAO since 90% of its oxidation by rat
intestine was sensitive to 1 mM semicarbazide while it resisted
to pargyline inhibition. Moreover, benzylamine oxidation occurred
mainly in the muscular layer of small intestine while the mucosa
was practically devoid of SSAO-mediated oxidation towards the
exogenous amine. It should be noted that the SSAO-dependent
activity was much lower in rat intestine than in rat adipose tissue homogenates: 0.13 ± 0.02 nmol oxidized/(mg protein min) vs.
1.89 ± 0.25 nmol oxidized/(mg protein min), when 0.1 mM benzylamine was used as substrate (n = 5–8, p < 0.001). The trans-mucosal
passage of benzylamine in everted gut sacs was dose- and timedependent (Fig. 1B) and about 50% of the amine transported to the
serosal side was not oxidized, thus justifying the rationale for per
os administration.
3.2. Oral benzylamine administration to mice
Benzylamine was dissolved in the drinking water in order to
administer approximately 3600 ␮moles/kg of body weight per day
(517 mg of benzylamine /(kg day)) to HFD-fed mice (BZA-treated).
The dose was chosen in relation to the anti-hyperglycemic effects
of benzylamine when acutely injected at 70 ␮mol/kg in overweight
mice [27] or orally given at 1900 ␮mol/(kg day) to rats [22] or
at 2000–4000 ␮mol/(kg day) to mice [27]. The stability of benzylamine in the drinking solutions was assessed by HPLC and only
a negligible degradation occurred between freshly prepared solutions and after 1 week at room temperature (typically less than 2%
decrease in concentration, data not shown). Since the analytical
procedure used was suitable only for liquid samples, the presence of benzylamine could also be verified in urine, and varied
from undetectable amounts in controls to 9.6 ± 0.2 ␮g/ml in mice
drinking benzylamine solutions prepared at around 4.8 mg/ml.
Therefore, ingested benzylamine was absorbed, metabolized and
even excreted in mice, indicating that in this model, the intestinal barrier was not totally insurmountable for this amine oxidase
substrate.
3.3. Water consumption and body weight gain in mice drinking
benzylamine
2.8. Statistical analysis.
Results are given as means ± S.E.M. Differences between BZAtreated and control were determined by Student’s t-test for values
obtained at and after sacrifice or by analysis of variance for
repeated observations, regarding data collected during treatment.
NS denotes a non-significant difference.
The water consumption was followed weekly and exhibited
small variations (Fig. 2A). However, the overall consumption
was significantly reduced during the 17-week treatment in mice
drinking benzylamine: F(1,102) = 17.1, p < 0.0001. BZA-treated mice
limited their daily water consumption by approximately 10% when
compared with control (Fig. 2A).
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Fig. 1. MAO and SSAO activities and transport of benzylamine across the rat small intestine. (A) Freshly prepared homogenates of rat small intestine (whole) and of its epithelial
(epithel) or muscular (muscul) layers separated by scraping were incubated with 0.1 mM [14 C]-benzylamine for the determination of amine oxidation in the absence of any
inhibitor (total oxidation, black columns), with semicarbazide (which blocked SSAO component and left MAO intact, grey columns), or with pargyline (selectively blocking
MAO and leaving SSAO intact, open columns). Mean ± S.E.M. of 4–8 preparations. (B) Time- and concentration-dependence of benzylamine transport across small intestine.
For the time-dependent study, everted gut sacs were incubated with 1 mM [14 C]-benzylamine, while for the concentration-dependence, isotopic solutions of the labelled
amine were incubated with sacs for 45 min at 37 ◦ C. The amount transported into the sacs was expressed as total benzylamine (squares) and as oxidized (into benzaldehyde,
triangles) or non-metabolized fractions (circles). Each point is mean ± S.E.M. of 3–7 determinations.
Although both groups had the same initial body mass,
BZA-treated mice weighed significantly less than the controls
after 1 week of treatment and until the end of experiment:
F(1,374) = 125.3, p < 0.00001 (Fig. 2B). Consequently, the overall body weight gain was clearly limited by the BZA-treatment.
Under non-fasting conditions, benzylamine drinking induced a
modest but significant reduction in blood glucose: F(1,374) = 24.8,
p < 0.00001 (Fig. 2C). Such modest glycaemia reduction is also visualized by the average of weekly measurements in right panel of
Fig. 2C: unfasted blood glucose was 8.18 ± 0.13 mM in control vs.
7.71 ± 0.16 mM in BZA-treated.
3.4. Glucose handling by BZA-treated mice
Two weeks before the end of the treatment, mice were subjected to GTT after being fasted for 5 h by receiving an i.p. glucose
challenge at 1 g/kg. The overall hyperglycemia was reduced in
BZA-treated mice as shown by: (i) the more rapid return to basal
levels at almost all the time points tested, (ii) the lower area
under the curve (n = 12, p < 0.05) (Fig. 3). Alongside the improvement in glucose tolerance, benzylamine treatment also lowered
baseline blood glucose, since the BZA-treated mice exhibited
lower values than controls 15 min before and just before the glucose load: 8.9 ± 0.7 mM and 10.2 ± 0.7 mM vs. 11.8 ± 0.3 mM and
12.9 ± 0.7 mM, respectively (n = 12, p < 0.01) (Fig. 3). Such a decrease
in blood glucose also occurred after overnight fasting, a condition only experienced once in the protocol, just before sacrifice.
In fact, glucose measured in drops of blood withdrawn from the
tail of mice just before sacrifice was 7.29 ± 0.59 mM in controls
and only 5.61 ± 0.58 mM in the BZA-treated group, correspond-
ing to a reduction at the limit of statistical significance (n = 12,
p = 0.055). Further analysis of plasma samples obtained from cardiac puncture after sacrifice definitively showed that glucose levels
were lower in treated mice: they were equivalent to 76 ± 6% of
control values (n = 12, p < 0.02). Immunoreactive insulin levels also
showed a tendency to diminish, but, due to high intra-group variability, there was no statistically significant effect of the treatment:
23.4 ± 5.9 ␮IU ml−1 vs. 15.2 ± 4.1 ␮IU ml−1 , NS).
3.5. Stored and circulating lipids in BZA-treated mice
The reduced body weight of BZA-treated mice was accompanied by a reduced lipid accumulation since the adiposomatic index,
which is estimated as the percentage of dissected intra-abdominal
(perirenal, retroperitoneal plus epididymal) and subcutaneous fat
depots relative to final body weight, was significantly lower than
in the controls (Table 1). This was due to a reduction in both intraabdominal and subcutaneous white adipose tissues. The liver was
also smaller in the BZA-treated animals than in control mice but
no significant reduction could be detected when hepatic mass was
expressed as a percentage of body weight (Table 1).
Under fed conditions, no difference in the circulating levels of
FFA was detected between control and BZA-treated groups (respective values found after 15 weeks of treatment were 0.70 ± 0.07
and 0.67 ± 0.07 mM, NS). Under fasting condition, neither FFA
nor triglycerides were reduced in the plasma of treated mice
(Table 2). However, a decrease in total cholesterol was unexpectedly observed in the plasma from the BZA-treated mice. Such a
clear-cut anti-hypercholesterolemic effect of benzylamine, leading to a reduction of 40% in the levels found in the hyperlipidemic
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359
Fig. 2. Water consumption, body weight and non-fasting blood glucose of mice fed a high-fat diet (HFD) and treated or not with orally administered benzylamine. Two
groups of age- and weight-matched mice were constituted (4 cages of 3 animals per group) 3 weeks before oral administration of benzylamine (arrow). Benzylamine was
given as a drinking solution in order to achieve an intake of 3600 ␮mol/(kg day) for 17 weeks. Control (CON) and benzylamine-treated mice (BZA) had free access to HFD and
drink. (A) Left panel: water consumption in control (black circles) and BZA-treated mice (open triangles). Each point is the mean ± S.E.M. of 4 determinations. Right panel:
average daily water consumption over the 17-week treatment. (B) Weekly body weight changes (left panel) and cumulative body weight gain during the treatment (right
panel). Each point/column is the mean ± S.E.M. of 12 determinations. (C) Non-fasting blood glucose; left panel: each point is mean ± S.E.M. of 12 determinations performed
weekly in treated mice (HFD-benzylamine) and controls (HFD control). Right panel: overall value of non-fasting blood glucose; each column is the mean ± S.E.M. of the 17
determinations performed weekly in each group during the treatment period. For the right panels, statistically different from HFD control at: *p < 0.05, **p < 0.01, ***p < 0.001.
mice was not accompanied by a significant decrease of the beneficial HDL-cholesterol: 171 ± 21 and 121 ± 18 mg/dl in HFD and
HFD + BZA, respectively (n = 5, NS).
3.6. Influence of chronic benzylamine intake on circulating
markers of metabolic disturbances
Among the diagnostic parameters given in Table 2, circulating bilirubin remained unchanged, ruling out major impairment
concerning liver or red blood cell functions. Similarly, lactic
acid levels were not altered by the benzylamine oral treatment. Plasma levels of uric acid were increased in HFD-mice
when compared with age-matched controls fed a standard chow
(365.9 ± 32.4 ␮mol/l vs. 250.2 ± 22.7 ␮mol/l, n = 10–12, p < 0.01)
and BZA-treatment significantly lowered this parameter. Fructosamine levels were not modified in the BZA-treated group,
suggesting that benzylamine treatment did not increase plasma
protein glycation. Similarly, albuminemia was not altered. Among
Fig. 3. Influence of benzylamine oral treatment on glucose tolerance. After 15 weeks of oral amine administration, mice were fasted for 5 h prior to glucose load (1 g/kg, i.p. at
time 0, arrow). Left panel: glucose was determined in blood collected from the tail of conscious animals at the indicated times before and after the glucose load. Right panel:
Area under the curve of the hyperglycemic excursion (AUC). Mean ± S.E.M. of 12 mice per group. Statistically different from control at: *p < 0.05, **p < 0.01, ***p < 0.001.
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Table 1
Body mass and proportion of adipose or hepatic tissue in HFD-fed mice after chronic
oral benzylamine administration.
Experimental group
HFD
Body mass (g)
INWAT mass (g)
SCWAT mass (g)
Adiposomatic index (% of body mass)
Liver weight (% of body mass)
36.0
2.59
1.72
11.6
3.6
HFD + BZA
±
±
±
±
±
1.8
0.27
0.22
0.9
0.1
28.9
1.23
0.82
6.6
3.4
±
±
±
±
±
1.3**
0.26**
0.17**
1.1**
0.1
Mice fed a high-fat diet alone (HFD) or in combination with benzylamine in drinking
water (HFD + BZA) were sacrificed after 17 weeks of treatment. The adiposomatic
index is the sum of dissected intra-abdominal plus subcutaneous white adipose
tissues (INWAT + SCWAT) given as percentage of body mass. Mean ± S.E.M. of 12
mice per group.
**
Significantly different from HFD at: p < 0.01.
the three adipokines measured, only resistin showed a significant decrease while adiponectin and leptin plasma levels remained
unaltered in BZA-treated mice (Table 2).
3.7. Unaltered insulin responsiveness and insulin-mimicking
properties of benzylamine in adipocytes isolated from
BZA-drinking mice
The dose-dependent stimulation of lipolysis by isoprenaline was
similar in adipocytes from HFD control and BZA-treated mice (not
shown). In control group, the submaximal lipolysis stimulation
by 10 nM isoprenaline was partly inhibited with 100 nM insulin
(44 ± 6% inhibition) and with the combination of 0.1 mM benzylamine and vanadate (38 ± 7% inhibition). These antilipolytic effects
were equivalent in treated group: 40 ± 8 and 46 ± 4% inhibition,
respectively (n = 12, NS).
Insulin dose-dependent stimulation of glucose transport was
unaltered (not shown) and reached maximum at 100 nM: 5.9 ± 0.7
and 5.2 ± 0.4-fold increase over basal in control and BZA-treated,
respectively (n = 12, NS). Synergism between benzylamine and
vanadate reproduced such 2-DG uptake activation in both HFD
control and BZA-treated mice: 82 ± 7 and 90 ± 10% of maximal
insulin stimulation (n = 12, NS). Therefore, no alteration in the
adipocyte responses to insulin or benzylamine plus vanadate could
be detected after benzylamine administration.
3.8. Influence of benzylamine intake on liver gene expression
Chronic benzylamine treatment did not change the expression of three genes related to insulin signalling (IRS1, BHLHB2,
INPPL1) in liver (not shown). The expression of a key gene related
to lipogenic activity (FASN, encoding for fatty acid synthase) was
also unchanged in the BZA-treated mice while the number of the
Table 2
Effect of chronic benzylamine ingestion on plasma metabolites and cytokines.
Experimental group
HFD
FFA (mM)
Triglycerides (mg/dl)
Total cholesterol (mg/dl)
Total bilirubin (␮mol/l)
Lactic acid (mmol/l)
Fructosamine (␮mol/l)
Uric acid (␮mol/l)
Albumin (␮mol/l)
Leptin (ng/ml)
Adiponectin (␮g/ml)
Resistin (ng/ml)
0.61
69.2
218.2
6.2
8.4
200.4
365.9
448.5
9.78
12.92
5.93
HFD + BZA
±
±
±
±
±
±
±
±
±
±
±
0.06
5.9
10.2
2.4
0.8
11.2
32.4
20.8
2.46
1.16
0.51
0.64
58.0
131.0
4.5
8.5
193.5
234.1
411.8
9.31
12.64
4.45
±
±
±
±
±
±
±
±
±
±
±
0.07
3.2
15.7***
0.9
0.5
6.9
13.6***
14.0
2.35
1.03
0.29*
Mice fed a high-fat diet (HFD) and drinking benzylamine (HFD + BZA) were sacrificed
after overnight fasting. Mean ± S.E.M. of 12 mice.
*
Significantly different from HFD at: p < 0.05.
***
Significantly different from HFD at: p < 0.001.
Table 3
Effect of chronic benzylamine ingestion on the concentration of stable end-products
of NO metabolism.
Experimental group
HFD
Nitrite (nmol/g aorta)
Nitrate (nmol/g aorta)
Nitrite (nmol/g muscle)
Nitrate (nmol/g muscle)
53.5
99.2
19.7
27.5
HFD + BZA
±
±
±
±
2.7
9.2
3.5
3.7
66.2
95.8
17.9
27.3
±
±
±
±
4.4*
6.4
1.7
4.3
Mean ± S.E.M. of 8–9 aortas and 6–7 hind leg muscles per group.
*
Statistical difference between mice fed a high-fat diet (HFD) and drinking benzylamine (HFD + BZA) at: p < 0.05.
transcripts of carnitine palmitoyl transferase (CPT1A), involved
in long-chain fatty acid transport into the mitochondria and ␤oxidation, tended to increase twofold (range 2.05–2.79 relative to
control set at 1, n = 10). Taken together, these results seemed to
indicate that insulin sensitivity remained unchanged in the liver of
BZA-treated mice. On the contrary, the balance between synthesis and degradation of fatty acids appeared to be in favour of an
enhanced catabolism and could rely with the decreasing tendencies of hepatic triglyceride content (from 58.9 ± 8.9 in control to
42.9 ± 5.5 mg/g in BZA-treated, n = 10, p < 0.10). Nevertheless, the
absence of change in ACADM expression (acyl-Coenzyme A dehydrogenase involved in medium-chain fatty acid breakdown) and
in the hepatic phospholipids (17.4 ± 0.8 mg/g vs. 19.0 ± 1.2 mg/g,
n = 12, NS) or cholesterol content (3.2 ± 0.3 mg/g vs. 4.2 ± 0.5 mg/g,
NS) revealed that not all the aspects of hepatic lipid handling were
modified by benzylamine treatment.
3.9. Influence of benzylamine intake on RNS and ROS
Chronic benzylamine treatment did not modify the nitrate
content in aortic tissue (Table 3). This finding argued against a
putative exaggerated oxidative stress induced by the hydrogen
peroxide production accompanying sustained benzylamine ingestion and oxidation, that could contribute to an elevated oxidative
conversion of NO to RNS. On the contrary, nitrite concentration was increased in the aortas of BZA-treated mice, indicating
that benzylamine administration slightly improved the reduced
NO bioavailability found in the vasculature of HFD-mice: aorta
nitrite levels were 125 ± 18.1 nmol/g in mice fed a normal chow;
only 53.5 ± 2.7 nmol/g in HFD-fed mice, and 66.2 ± 4.4 nmol/g in
HFD + BZA mice. The similar nitrite and nitrate concentrations
found in the muscle of HFD and BZA-treated mice pointed out that
neither oxidative stress or NO bioavailability was altered in this
tissue by the treatment (Table 3).
In liver homogenates, the concentration of lipid peroxidation
markers was decreased from 5.6 ± 0.4 to 3.9 ± 0.1 ␮M TBARS in
control and BZA-treated respectively (n = 12, p < 0.001). The lack
of significant decrease in hepatic aconitase activity (110 ± 6 vs.
95 ± 5 arbitrary units, n = 12, NS), also indicated that chronic benzylamine was not promoting an easily detectable oxidative stress.
Accordingly, no change of expression was found in the liver for
the mitochondrial superoxide dismutase that protects cells against
superoxide toxicity, and for catalase that catalyzes the decomposition of hydrogen peroxide (not shown).
4. Discussion
The present study shows that chronic benzylamine administration improves glucose tolerance in mice fed a diabetogenic,
high-fat diet, at least when considering their lower non-fasting
and fasting glycaemia and their reduced hyperglycaemic responses.
Moreover, mice drinking benzylamine reduced their weight gain,
plasma cholesterol, resistin and uric acid levels without exhibiting
systemic or tissular signs of excessive oxidative stress.
Z. Iffiú-Soltész et al. / Pharmacological Research 61 (2010) 355–363
Benzylamine, also called moringine (CAS 100-46-9/CAS 328799-8) is an aromatic monoamine present in the plant Moringa
oleifera, known as the “miracle tree” and used in folk medicine as an
antidiabetic agent, a property confirmed in a pharmacological survey of medicinal plants [28]. However, it has never been directly
proved that benzylamine is an orally active antidiabetic molecule.
This is the first study that shows the somewhat beneficial effects
of chronic benzylamine ingestion on glucose handling, circulating
cholesterol and nitrogen monoxide bioavailability. Such observations are in perfect agreement with a preliminary study reporting
a reduced hyperglycaemic response during GTT in non-obese nondiabetic rats receiving benzylamine per os [22]. Our findings also
extend previous observations made in mice rendered obese after 3
months of very-high-fat diet (72% energy from fat), showing that
benzylamine was equally efficient alone or when associated with
vanadium in reducing the hyperglycemic response to a glucose
load [27]. Noteworthy, repeated benzylamine administration has
already been reported to decrease the elevated blood glucose of
type 1 and type 2 diabetic rats, but only when injected/perfused
together with vanadate [10,11]. Since the therapeutic applications
of the association of benzylamine with vanadium could be limited
by the toxicity of the transition metal (in spite of its antidiabetic properties), we have focused our interest on the influence
of benzylamine alone on glucose handling. In vivo, an acute antihyperglycemic action was observed with benzylamine doses as low
as 7 ␮mol/kg after injection in mice fed a very-high-fat diet or after
intravenous infusion in conscious rabbits [6,27]. In these models,
the acute anti-hyperglycemic effect of benzylamine was abolished
after SSAO/VAP-1 blockade by semicarbazide treatment [6].
The benzylamine diffusion across everted gut sacs and the benzylamine detection in the urine of BZA-drinking mice show that
the intestinal barrier is not insurmountable for this amine oxidase
substrate, supporting the idea that oral administration could result
in tissue benzylamine levels sufficient to reproduce some of the
insulin-like actions observed in vitro.
In fact, orally given benzylamine produced the same effects on
glucose homeostasis as when injected at lower doses. Benzylamine
ingestion also led to somewhat unexpected effects such as reduction in water consumption. This can be considered as a reduced
fluid intake only if spillage and dressing can be ruled out. Similar
reduction had already been observed in non-obese, non-diabetic
rats supplemented with 0.29% benzylamine drinking solution for 7
weeks [22] or in normoglycaemic mice drinking 0.5% benzylamine
(Carpéné et al., unpublished data). It is difficult to assess whether
benzylamine exerted an aversive influence on drinking behaviour
or a pharmacologic effect on fluid homeostasis. The bad smell of
benzylamine solutions, smelling like putrefied fish at higher concentrations than those given to rodents favours the hypothesis of a
distasteful drinking having an aversive effect.
The body weight gain reduction observed in BZA-drinking mice
was also unexpected since the acute effects of benzylamine found
in adipocytes (lipolysis inhibition, glucose uptake and lipogenesis
activation) were supposed to lead to fattening and increased weight
gain once benzylamine was chronically ingested. The reduced
body weight gain is however agreeing with the already reported
inhibitory effects of benzylamine on appetite, mediated by potassium channel blockade in central nervous system [29]. As far as we
know, the hypophagic effects of benzylamine have been described
only in mid-term experiments in behavioural studies [29,30] and
no clear reduction of food intake or body weight after prolonged
treatment has been reported to date, after benzylamine drinking at
0.3% in rats [22], or at 0.5% in mice (Carpéné et al., unpublished data)
fed a standard diet. Unfortunately, in the present study, it was difficult to assess whether a reduction in calorie intake was responsible
for the decreased body weight gain during BZA-treatment because
the given high-fat chow was particularly sticky and could not be
361
accurately weighed. If such a central hypophagic action of chronically ingested benzylamine persisted in the treated HFD-fed mice,
it could be largely responsible for the weight gain normalization
and for its concomitant beneficial effects, including the reduced
non-fasting glycaemia and improved glucose tolerance.
Another unexpected effect of oral benzylamine administration
was the reduction in circulating cholesterol levels. Although the
decrease was mainly observed in the non-HDL fraction, there was
no rationale to support such a beneficial effect. It can just be noted
that, in a very recent study, various hydrazine derivatives used
as carbonyl scavengers, but known to inhibit SSAO and/or MAO
activity, such as hydralazine, phenelzine and iproniazid, have been
shown to exert anti-atherogenic effects, but without lowering circulating cholesterol [31]. Whether there is a putative link between
amine oxidase activity and cholesterol metabolism deserves to be
clarified.
A decrease in FFA was expected, owing to the antilipolytic effects
of benzylamine previously found in human and rat adipocytes
[5,32] and confirmed here for murine fat cells. In fact, the limitation of fat deposition observed in treated mice is in apparent
contradiction with the benzylamine antilipolytic effect detected
on adipocytes in the presence of vanadate but fits with its central hypophagic action [29,30]. Also unpredicted was the decrease
in plasma resistin found in BZA-drinking mice, while a decrease in
leptin was expected, due to the reduced adiposity of the treated
animals. Nevertheless, the unaltered levels of this latter adipokine,
together with unchanged adiponectin, reduced resistin and the
slightly reduced insulin levels are consistent with an improvement
of blood glucose control in BZA-treated mice.
Considering the deleterious vascular effects of methylamine administration reported in mice overexpressing vascular
SSAO/VAP-1 [2], oxidative stress or diabetic-like vascular injury
could have been predicted to occur in HFD-fed mice upon BZAtreatment. However, no evident signs of excessive glycation or
vascular damage consequent to excessive activation of soluble
or vascular SSAO/VAP-1 and aldehyde-induced cross-linking
[17], were detected after benzylamine administration. Firstly,
fructosamine, which reflects glycated plasma protein levels, was
not altered by BZA-treatment. Secondly, elevated plasma uric
acid, that accompanies the onset of metabolic syndrome [33], was
decreased in BZA-treated mice. Instead, the lowered levels of this
independent marker of cardiovascular disease risk [34] could be
regarded as protective for cardiac and renal function. Thirdly, benzylamine administration lowered the levels of lipid peroxidation
markers, and did not modify the aconitase activity [26] or the
expression of the antioxidant enzymes catalase and SOD2 in liver,
suggesting that the treatment did not promote oxidative stress.
Finally, when considering the ratio nitrate/nitrite as an indicator
of the conversion of NO to RNS by excessive ROS, there was no
indication of such oxidative stress in the aorta or skeletal muscle
of benzylamine drinking mice. Moreover, nitrite concentration,
which was significantly elevated, suggested an increased NO
production. Since insulin has been reported to increase nitrite
concentration in the aorta by enhancing eNOS expression [35],
the influence of BZA-treatment may be considered beneficial and
related to the insulin-mimicking properties of the amine. Lowered
plasma uric acid and increased nitrite concentration in the aorta
may therefore indicate an improved endothelial function in BZAtreated animals in place of the feared damage. Alternatively, it can
be an indirect effect secondary to the reductions of overweight,
hyperglycemia and hypercholesterolemia.
The lack of deleterious effects of BZA-treatment on vasculature
is in agreement with the paradigm proposing that benzaldehyde,
the product of SSAO/VAP-1-mediated benzylamine oxidation, is
less cytotoxic than formaldehyde produced by methylamine oxidation [17,19]. Therefore, we suggest that among the different
362
Z. Iffiú-Soltész et al. / Pharmacological Research 61 (2010) 355–363
possible fates for ingested benzylamine, a competition with methylamine for SSAO/VAP-1-mediated oxidation can occur, as observed
in vitro on cultured adipocytes [8]. As a result, less cytotoxic aldehyde could be generated in BZA-drinking mice. In this context,
it should be mentioned that benzylamine can also be oxidatively
deaminated by reactive aldehydes themselves such as methylglyoxal (produced in part by SSAO/VAP-1-dependent aminoacetone
oxidation which is increased in diabetic plasma) and may limit their
toxicity by acting like a scavenger [36]. Obviously, benzylamine can
be oxidized not only in blood and vessels, but in other tissues, especially WAT, that is dramatically enlarged in overweight rodents and
one of the richest in SSAO and MAO activities [24]. It is therefore
conceivable that direct insulin-like actions of orally given benzylamine could have occurred in WAT, owing to its SSAO richness,
but it is hardly compatible with the observed decrease in adiposity,
except if increased energy expenditure or decreased calorie intake
were sustained throughout the treatment, via central benzylamine
effects elsewhere described to be independent on amine oxidation
[29,30].
In liver, the expression of genes related to insulin signalling
(such as IRS1, BHLHB2, INPPL1) was not modified, confirming that
benzylamine did not reverse or amplify insulin-resistance. However, the increased expression of liver CPT1A, the enzyme that
regulates the transport of long-chain acyl-CoA into the mitochondria to produce energy through beta-oxidation, indicated that an
increase in fatty acid oxidation might occur in BZA-treated hepatocytes. Similar increases have been reported in situations of fasting,
diabetes, and fat feeding while it is known that insulin represses
the transcription of CPT1A in liver [37]. Therefore benzylamine did
not exert direct insulin-like effects on liver, an observation that has
to be connected with the lack of SSAO in hepatocytes. Moreover,
increase in CPT1A gene can also be associated with fat loss [38], a
situation provoked by benzylamine ingestion.
5. Conclusions
Recent findings on the anti-inflammatory properties of a new
generation of SSAO/VAP-1-inhibitors [39] did not indicate whether
exogenous substrates of the same enzyme could exhibit interesting properties regarding metabolic control. Although it remains
to verify whether the immune system was influenced by benzylamine treatment, the present findings show that oral benzylamine
administration merits testing in other diabetes/obesity models,
including the db/db mice a valuable model for testing glucose
lowering capacities of antidiabetic drug candidates [40]. Finally,
the reported weight gain-reducing, anti-hyperglycemic and hypocholesterolemic actions of benzylamine warrant future searches for
amine oxidase substrates more active than benzylamine, as it has
been initiated towards human SSAO by Yraola et al. [41]. Such soluble amines may be endowed with more powerful antidiabetic
properties and yet devoid of adverse effects.
Acknowledgments
The authors express gratitude to D. Prévot and A. Desquesnes
for biochemical analyses at ANEXPLO platform, Y. Barreira and the
staff of animal unit of I2MR, Toulouse, for invaluable help. The
expertise of S. Benvenuti regarding amine chromatography is gratefully acknowledged. This work was partly supported by an R&D
grant from the “Communauté de Travail des Pyrénées” (project
MP #05018677), the Hungarian Scientific Research Fund (OTKA
K63415) and by “Programme Balaton” (project Egide #14117SA
and Hungarian National Office of Research and Technology OMFB00491/2007) for Franco-Spanish and Franco-Hungarian exchanges,
respectively.
References
[1] Bianchi P, Kunduzova O, Masini E, Cambon C, Bani D, Raimondi L, et al. Oxidative
stress by monoamine oxidase mediates receptor-independent cardiomyocyte apoptosis by serotonin and postischemic myocardial injury. Circulation
2005;112:3297–305.
[2] Stolen CM, Madanat R, Marti L, Kari S, Yegutkin GG, Sariola H, et al. Semicarbazide sensitive amine oxidase overexpression has dual consequences: insulin
mimicry and diabetes-like complications. FASEB J 2004;18:702–4.
[3] Goldstein BJ, Mahadev K, Wu X, Zhu L, Motoshima H. Role of insulin-induced
reactive oxygen species in the insulin signaling pathway. Antioxid Redox Signal
2005;7:1021–31.
[4] Marti L, Morin N, Enrique-Tarancon G, Prévot D, Lafontan M, Testar X, et al. Tyramine and vanadate synergistically stimulate glucose transport in rat adipocytes
by amine oxidase-dependent generation of hydrogen peroxide. J Pharmacol
Exp Ther 1998;285:342–9.
[5] Morin N, Lizcano JM, Fontana E, Marti L, Smih F, Rouet P, et al. Semicarbazidesensitive amine oxidase substrates stimulate glucose transport and inhibit
lipolysis in human adipocytes. J Pharmacol Exp Ther 2001;297:563–72.
[6] Iglesias-Osma MC, Garcia-Barrado MJ, Visentin V, Pastor-Mansilla MF, Bour S,
Prévot D, et al. Benzylamine exhibits insulin-like effects on glucose disposal,
glucose transport, and fat cell lipolysis in rabbits and diabetic mice. J Pharmacol
Exp Ther 2004;309:1020–8.
[7] Mercier N, Moldes M, El Hadri K, Fève B. Semicarbazide-sensitive amine
oxidase activation promotes adipose conversion of 3T3-L1 cells. Biochem J
2001;358:335–42.
[8] Carpéné C, Daviaud D, Boucher J, Bour S, Visentin V, Grès S, et al. Shortand long-term insulin-like effects of monoamine oxidase and semicarbazidesensitive amine oxidase substrates in cultured adipocytes. Metabolism
2006;55:1397–405.
[9] Bour S, Daviaud D, Gres S, Lefort C, Prévot D, Zorzano A, et al. Adipogenesisrelated increase of semicarbazide-sensitive amine oxidase and monoamine
oxidase in human adipocytes. Biochimie 2007;89:916–25.
[10] Marti L, Abella A, Carpéné C, Palacin M, Testar X, Zorzano A. Combined
treatment with benzylamine and low dosages of vanadate enhances glucose
tolerance and reduces hyperglycemia en streptozotocin-induced diabetic rats.
Diabetes 2001;50:2061–8.
[11] Abella A, Marti L, Camps M, Claret M, Fernandez-Alvarez J, Gomis R, et al.
Semicarbazide-sensitive amine oxidase/Vascular adhesion protein-1 activity
exerts an antidiabetic action in Goto-Kakizaki rats. Diabetes 2003;52:1004–
13.
[12] Salmi M, Yegutkin GG, Lehvonen R, Koskinen K, Salminen T, Jalkanen S. A
cell surface amine oxidase directly controls lymphocyte migration. Immunity
2001;14:265–76.
[13] Obata T. Diabetes and semicarbazide-sensitive amine oxidase (SSAO) activity:
a review. Life Sci 2006;79:417–22.
[14] Ekblom J. Potential therapeutic value of drugs inhibiting semicarbazidesensitive amine oxidase: vascular cytoprotection in diabetes mellitus.
Pharmacol Res 1998;37:87–92.
[15] Visentin V, Bour S, Boucher J, Prévot D, Valet P, Ordener C, et al. Glucose handling
in streptozotocin-induced diabetic rats is improved by tyramine but not by the
amine oxidase inhibitor semicarbazide. Eur J Pharmacol 2005;522:139–46.
[16] Yu PH. Deamination of methylamine and angiopathy; toxicity of formaldehyde,
oxidative stress and relevance to protein glycoxidation in diabetes. J Neural
Transm Suppl 1998;52:201–16.
[17] Gubisne-Haberle D, Hill W, Kazachkov M, Richardson JS, Yu PH. Protein
cross-linkage induced by formaldehyde derived from semicarbazide-sensitive
amine oxidase-mediated deamination of methylamine. J Pharmacol Exp Ther
2004;310:1125–32.
[18] Lei T, Xie W, Han J, Corkey BE, Hamilton JA, Guo W. Medium-chain fatty acids
attenuate agonist-stimulated lipolysis, mimicking the effects of starvation.
Obes Res 2004;12:599–611.
[19] Conklin DJ, Langford SD, Boor PJ. Contribution of serum and cellular
semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity. Toxicol Sci 1998;46:386–92.
[20] Garcia-Vicente S, Yraola F, Marti L, Gonzalez-Munoz E, Garcia-Barrado MJ,
Canto C, et al. Oral insulin-mimetic compounds that act independently of
insulin. Diabetes 2007;56:486–93.
[21] Srivastava AK. Anti-diabetic and toxic effects of vanadium compounds. Mol Cell
Biochem 2000;206:177–82.
[22] Bour S, Visentin V, Prévot D, Daviaud D, Saulnier-Blache J-S, Guigné C, et al.
Effects of oral administration of benzylamine on glucose tolerance and lipid
metabolism in rats. J Physiol Biochem 2005;61:371–80.
[23] Barthe L, Woodley JF, Kenworthy S, Houin G. An improved everted gut sac as
a simple and accurate technique to measure paracellular transport across the
small intestine. Eur J Drug Metab Pharmacokinet 1998;23:313–23.
[24] Morin N, Visentin V, Calise D, Marti L, Zorzano A, Testar X, et al. Tyramine
stimulates glucose uptake in insulin-sensitive tissues in vitro and in vivo via
its oxidation by amine oxidases. J Pharmacol Exp Ther 2002;303:1238–47.
[25] Szökö E, Tabi T, Halasz AS, Palfi M, Magyar K. High sensitivity analysis of nitrite
and nitrate in biological samples by capillary zone electrophoresis with transient iotachphoretic sample stacking. J Chromatogr A 2004;1051:177–83.
[26] Nulton-Persson AC, Szweda LI. Modulation of mitochondrial function by hydrogen peroxide. J Biol Chem 2001;276:23357–61.
[27] Iffiú-Soltész Z, Prévot D, Grès S, Bour S, Szökő É, Knauf C, et al. Influence of
benzylamine acute and chronic administration of benzylamine on glucose tol-
Z. Iffiú-Soltész et al. / Pharmacological Research 61 (2010) 355–363
[28]
[29]
[30]
[31]
[32]
[33]
[34]
erance in diabetic and obese mice fed on very high-fat diet. J Physiol Biochem
2007;63:305–16.
Kar A, Choudhary BK, Bandyopadhyay NG. Comparative evaluation of hypoglycaemic activity of some Indian medicinal plants in alloxan diabetic rats. J
Ethnopharmacol 2003;84:105–8.
Pirisino R, Ghelardini G, Banchelli G, Galeotti N, Raimondi L. Methylamine
and benzylamine induced hypophagia in mice: modulation by semicarbazidesensitive benzylamine oxidase inhibitor and aODN towards Kv1.1 channels. Br
J Pharmacol 2001;134:880–6.
Pirisino R, Galeotti N, Livi S, Raimondi L, Ghelardini G. 4-Methyl benzylamine stimulates food consumption and counteracts the hypophagic effects
of amphetamine acting on brain Shaker-like Kv1.1 channels. Br J Pharmacol
2006;147:218–24.
Galvani S, Coatrieux C, Elbaz M, Grazide M-H, Thiers J-C, Parini A, et al. Carbonyl
scavenger and antiatherogenic effects of hydrazine derivatives. Free Rad Biol
Med 2008;45:1457–67.
Visentin V, Prévot D, Marti L, Carpéné C. Inhibition of rat fat cell lipolysis by
monoamine oxidase and semicarbazide-sensitive amine oxidase substrates.
Eur J Pharmacol 2003;466:235–43.
Yoo TW, Sung KC, Shin H, Kim BJ, Kim BS, Kang JH, et al. Relationship between
serum uric acid concentration and insulin resistance and metabolic syndrome.
Circ J 2005;69:928–33.
Gagliardi AC, Miname MH, Santos RD. Uric acid: a marker of increased cardiovascular risk. Atherosclerosis 2009;202:11–7.
363
[35] Kobayashi T, Kamata K. Effect of chronic insulin treatment on NO production and endothelium-dependent relaxation in aortae from established
STZ-induced diabetic rats. Atherosclerosis 2001;155:313–20.
[36] Akagawa M, Sasaki T, Suyama K. Oxidative deamination of benzylamine by
glycoxidation. Bioorg Med Chem 2003;11:1411–7.
[37] Louet JF, Le May C, Pégorier JP, Decaux JF, Girard J. Regulation of liver carnitine
palmitoyltransferase I gene expression by hormones and fatty acids. Biochem
Soc Trans 2001;29:310–6.
[38] Shin ES, Cho SY, Lee EH, Lee SJ, Chang IS, Lee TR. Positive regulation of hepatic
carnitine palmitoyl transferase 1A (CPT1A) activities by soy isoflavones and
l-carnitine. Eur J Nutr 2006;45:159–64.
[39] Salter-Cid LM, Wang E, O’Rourke AM, Miller A, Gao H, Huang L, et
al. Anti-inflammatory effects of Inhibiting the amine oxidase activity of
semicarbazide-sensitive amine oxidase. J Pharmacol Exp Ther 2005;315:553–
62.
[40] Li YY, Wu HS, Tang L, Feng CR, Yu JH, Li Y, et al. The potential insulin sensitizing
and glucose lowering effects of a novel indole derivative in vitro and in vivo.
Pharmacol Res 2007;56:335–43.
[41] Yraola F, Garcia-Vicente S, Fernandez-Recio J, Albericio F, Zorzano A, Marti L, et
al. New efficient substrates for semicarbazide-sensitive amine oxidase/VAP1 enzyme: analysis by SARs and computational docking. J Med Chem
2006;49:6197–208.