Ctd

ZÉPa

b

c

d

e

f

a

ARR2A

KASMAO

cc[Hioc

ti(

cBT

1d

Pharmacological Research 61 (2010) 355–363

Contents lists available at ScienceDirect

Pharmacological Research

journa l homepage: www.e lsev ier .com/ locate /yphrs

hronic benzylamine administration in the drinking water improves glucoseolerance, reduces body weight gain and circulating cholesterol in high-fatiet-fed mice�

suzsa Iffiú-Soltésza,b, Estelle Wanecqa, Almudena Lombac, Maria P. Portillod, Federica Pellati e,va Szökob, Sandy Boura, John Woodleyf, Fermin I. Milagroc, J. Alfredo Martinezc,hilippe Valeta, Christian Carpénéa,∗

INSERM, U858, Institut de Médecine Moléculaire de Rangueil, IFR 150, Toulouse, FranceDepartment of Pharmacodynamics, Semmelweis University, Budapest, HungaryDepartment of Nutrition and Food Sciences, Physiology and Toxicology, University of Navarra, Pamplona, SpainDepartment of Nutrition and Food Science, Universidad del País Vasco, Vitoria, SpainDepartment of Pharmaceutical Sciences, University of Modena e Reggio Emilia, Modena, ItalyBiopharmtech, Toulouse, France

r t i c l e i n f o

rticle history:eceived 23 September 2009eceived in revised form3 December 2009ccepted 23 December 2009

eywords:dipose tissueemicarbazide-sensitive amine oxidaseonoamine oxidase

ntidiabetic

a b s t r a c t

Benzylamine is found in Moringa oleifera, a plant used to treat diabetes in traditional medicine. In mam-mals, benzylamine is metabolized by semicarbazide-sensitive amine oxidase (SSAO) to benzaldehydeand hydrogen peroxide. This latter product has insulin-mimicking action, and is involved in the effectsof benzylamine on human adipocytes: stimulation of glucose transport and inhibition of lipolysis. Thisstudy examined whether chronic, oral administration of benzylamine could improve glucose toleranceand 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 han-dling 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. HFD-benzylamine mice showed lower body weight gain, fasting blood glucose, total plasma cholesterol and

besity hyperglycaemic response to glucose load when compared to HFD control. In adipocytes, insulin-inducedactivation of glucose transport and inhibition of lipolysis remained unchanged. In aorta, benzylaminetreatment partially restored the nitrite levels that were reduced by HFD. In liver, lipid peroxidationmarkers were reduced. Resistin and uric acid, surrogate plasma markers of metabolic syndrome, weredecreased. In spite of the putaoxidation, and in agreement wbenzylamine could be conside

Abbreviations: ACADM, acyl-coenzyme A dehydrogenase involved in medium-hain fatty acid breakdown; BHLHB2, basic helix-loop-helix domain containing,lass B 2; BZA, benzylamine; CPT1A, carnitine palmitoyl transferase; 2-DG, 2-1,2-3H]-deoxyglucose; FASN, fatty acid synthase; GTT, glucose tolerance test;FD, high-fat diet; INPPL1, inositol polyphosphate phosphatase-like 1; IRS1,

nsulin receptor substrate-1; MAO, monoamine oxidase; ROS/RNS, reactivexygen/nitrogen species; SSAO, semicarbazide-sensitive amine oxidase; VAP-1, vas-ular adhesion protein-1; WAT, white adipose tissue.� A previous account of this work was presented, as a poster, in the 3rd Interna-ional Congress on prediabetes and the metabolic syndrome, held in Nice (France)n 1st–4th April 2009, that subsequently appeared in: Journal of Diabetes (2009) 1suppl. 1):A241.∗ Corresponding author at: Institut National de la Santé et de la Recherche Médi-

ale (INSERM), U858 équipe 3, Institut de Médecine Moléculaire de Rangueil (I2MR),at. L4, CHU Rangueil, F-31432 Toulouse cedex 4, BP 84225 France.el.: +33 5 61 32 56 36; fax: +33 5 61 32 56 23.

E-mail address: christian.carpene@inserm.fr (C. Carpéné).

043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.phrs.2009.12.014

tive deleterious nature of the hydrogen peroxide generated during amineith its in vitro insulin-like actions found on adipocytes, the SSAO-substratered as a potential oral agent to treat metabolic syndrome.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Amine oxidation is considered to be a major metabolic routein the termination of the neurotransmitter function of biogenicamines. Amine oxidation is also involved in the destruction ofdietary amines. The excessive blockade of these reactions is knownas the “cheese effect” leading to fatal hypertensive crises indepressed patients treated with irreversible monoamine oxidase(MAO) inhibitors and consuming amine-containing food. The falseneurotransmitter tyramine has been highly suspected to be detri-mental in these conditions. There is now a growing body of evidence

to indicate that amine oxidases can influence cardiac physiopathol-ogy [1] and have a dual effect in diabetes [2]. Hydrogen peroxide,one of the end-products of the amine oxidase-mediated cataly-sis, is involved in such novel functions. Hydrogen peroxide mimicsinsulin effects in adipocytes and is recognized as an inhibitor of

3 logica

t[(loeaa[i[

saeutctdsimctpfotcmlert[

aaptd

eb[fsilatSrzm

nTigtataf

56 Z. Iffiú-Soltész et al. / Pharmaco

yrosine phosphatases involved in turning-off insulin signalling3]. Substrates of MAO and semicarbazide-sensitive amine oxidasesSSAO) reproduce insulin actions (stimulation of glucose transport,ipolysis inhibition) in an hydrogen peroxide-dependent mannern adipocytes isolated from white adipose tissues (WAT) of differ-nt species [4–6]. SSAO- and MAO-substrates also reproduce short-nd long-term insulin-like actions in preadipocytes [7,8], the SSAOctivity of which strongly increase with adipocyte differentiation9]. Several SSAO substrates have even been reported to exhibitn vivo anti-hyperglycemic effects in rats, mice [10,11] and rabbits6].

SSAO is not only present in fat cells but also in blood ves-els, in inflamed areas or in lymph nodes where it is knowns vascular adhesion protein-1 (VAP-1), involved in leukocytextravasation [12]. SSAO/VAP-1 is also present in the blood as a sol-ble form, which increases in diabetes [13]. It has been suggestedhat its inhibition by SSAO-inhibitors may reduce the vascularomplications of diabetes [14], but it has never been reportedhat SSAO-inhibitors exert anti-hyperglycaemic effects [15]. Theual influence of amine oxidation on diabetes has been demon-trated in experiments consisting of methylamine administrationn mice overexpressing SSAO/VAP-1 at endothelial surfaces [2]:

ethylamine improves glucose handling but also worsens diabeticomplications (protein glycation, atherosclerosis and nephropa-hy). The factors responsible for such deleterious effects are otherroducts resulting from amine oxidation: the aldehydes, especiallyormaldehyde and methylglyoxal originating from the oxidationf methylamine and aminoacetone. Such aldehydes crosslink pro-eins and consequently cause endothelial damage [14,16,17]. Theytotoxicity of these aldehydes can be considered to be evenore harmful than that of hydrogen peroxide, which is pecu-

iarly well tolerated by fat cells [18]. Moreover, oxidation ofxogenous amine substrates can generate aldehydes that are lesseactive than those mentioned above. It has been reported to behe case for benzaldehyde formed during benzylamine oxidation19].

Our aim was therefore to investigate a potential therapeuticpproach to hyperglycemia and insulin-resistance based on thedministration of an SSAO-substrate that might increase hydrogeneroxide production and mimic insulin action in insulin-sensitiveargets, such as adipocytes, without generating deleterious aldehy-es.

Such a substrate-based approach was reported to functionffectively in rat models of type I and type II diabetes when a com-ination of benzylamine plus vanadate was injected repeatedly10,11,20]. However, two aspects seemed to prevent a straight-orward extrapolation of these findings to clinical use: (1) inpite of its antidiabetic actions, vanadium is suspected of hav-ng toxic side-effects [21], and (2) the effects of benzylamine areess potentiated by vanadium in human adipocytes [5] than in ratdipocytes [10]. Therefore, benzylamine needed to be tested aloneo see whether it was able to acutely improve glucose tolerance.ince we recently obtained encouraging results in normoglycemicats [22] and rabbits [6], we investigated here whether oral ben-ylamine exhibits antidiabetic properties in a type 2 diabetesodel.To this aim, the benzylamine passage across the intesti-

al barrier was first tested in the everted gut sac model.hen, benzylamine was orally administered to mice renderednsulin-resistant by high-fat feeding. Attention was focused onlucose tolerance and insulin sensitivity, circulating lipids, oxida-

ive stress and vascular reactivity. We hypothesized that orallydministered benzylamine was absorbed in sufficient amountso reproduce the insulin-like effects initially observed in vitro ondipocytes, without generating oxidative stress and vascular dys-unction.

l Research 61 (2010) 355–363

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 determi-nation of transport across small intestine as previously described[23]. Briefly, concentrations of [14C]-benzylamine (0.01–1 mM)were applied to the mucosal side of everted gut sacs of approxi-mately 2.5 cm length incubated in tissue culture medium at 37 ◦Cfor the indicated times. Then, the radioactivity found in the serosalfluid was quantified by liquid scintillation counting with or withoutprior extraction with toluene-ethyl acetate, to separate benzy-lamine from its oxidation product, benzaldehyde, as with amineoxidase assays. Viability and protein content of the preparationswere measured as previously reported [23]. Amine oxidase activ-ity was measured in homogenates with 0.1 mM [14C]-benzylaminein 200 mM phosphate buffer, pH 7.5, in the presence of proteaseinhibitors for 30 min as previously described [15]. 15 min pre-incubation with 1 mM semicarbazide or 0.5 mM pargyline was usedto selectively inhibit SSAO or MAO activity, respectively, as previ-ously 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 weeksfor a group of 36 mice housed at 3 animals per cage with free accessto food and water. HFD contained (as percent of kcal): 20% protein,35% carbohydrate and 45% fat. Body weight and water consumptionwere determined weekly, but food intake could not be assessed,due to the sticky nature of the HFD pellets. Three weeks after thenutritional switch, 24 mice sharing the closest body mass weresplit in one group of 12 mice kept without treatment (control),while another group of 12 weight-matched mice received benzy-lamine hydrochloride in the drinking water at 0.44% for a 17-weekperiod (BZA-treated). To achieve this, benzylamine was dissolvedin 400 ml water and used as drinking solution changed every week.For mice weighing 30 g and drinking approx. 3.5 ml/day, the dailydose of ingested benzylamine was 3600 �moles/(kg day). Non-fasting blood glucose was assessed weekly on a drop of bloodobtained between 15:00 and 16:00 h from the tail vein. After 15weeks of treatment, an intraperitoneal glucose tolerance test (GTT)was carried out as follows: mice were fasted for 5 h, then receivedglucose (1 g/kg) by i.p. bolus (t0); blood was collected from thetail vein of conscious mice 15 min before and just prior to theglucose load, and then 15, 30, 45, 60, 90 and 120 min later fordetermination of glucose levels using a Accu-Chek glucose mon-itor (Roche Diagnostic, Meylan, France) as previously reported[24].

2.3. Stability of benzylamine solutions

The benzylamine concentration of the aqueous drinkingsolutions was verified by ion-pair high-performance liquid chro-matography (HPLC) analysis on freshly prepared and 1-week-oldsolutions. HPLC was performed on an Agilent Technologies modu-lar 1100 system (Waldbronn, Germany), using a LiChrospher RP-18column (125 mm × 4 mm i.d. 5 �m), with an isocratic mobile phase

composed of a mixture of water–acetonitrile (65:35, v/v), contain-ing 0.1% H3PO4 and 0.5% SDS at a flow-rate of 1 ml/min and with UVdetection at 205 nm. Quantification was performed by the externalstandard method covering the concentration range 9.8–983 �g/ml(r = 0.999).

logica

2a

wtwo2

2

wtsnpitd(cwflC

2

enptlrMf

2

ouuRautamc(Sda

2

torN

Z. Iffiú-Soltész et al. / Pharmaco

.4. Plasma and tissue sampling, lipolysis and glucose uptake indipocytes

Mice were sacrificed after overnight fasting. Tissues and plasmaere immediately frozen at −80 ◦C. Intra-abdominal white adipose

issues (INWAT: perirenal, retroperitoneal and epididymal fat pads)ere pooled for immediate adipocyte isolation and determination

f lipolysis (glycerol release) and glucose transport activities (by-deoxyglucose uptake assay) as previously described [5].

.5. Circulating endocrine and metabolic parameters

Plasma metabolites (glucose, lactic acid, albumin, bilirubin)ere determined using a Cobas-Mira + multi-analyser, according

o the manufacturer’ instructions (Roche, Neuilly, France). Analy-es of circulating fructosamine (using a colometric assay based onitrotetrazolium blue reduction) and of uric acid (using an uricase-eroxidase method) were adapted to 96-well microplates for use

n the multi-analyser. Plasma lipids were determined as follow:riacylglycerides with the Randox kit for in vitro triacylglycerideiagnostics (Randox Lab. Ltd., Crumlin, UK), plasma free fatty acidsFFA) levels using WAKO NEFA test (Oxoid, Dardilly, France), andholesterol with a BioSystems kit (Barcelona, Spain). Plasma insulinas determined with an Ultrasensitive Insulin Elisa kit obtained

rom Mercodia (Uppsala, Sweden), while immunoreactive resistin,eptin and adiponectin were determined using Linco RIA kits (St.harles, MO, USA).

.6. Tissue markers of reactive nitrogen and oxygen species

Putative changes in the reactive nitrogen species (RNS) werevaluated by the capillary electrophoresis-based determination ofitrate/nitrite content in aorta and muscle samples according to areviously published method [25]. The markers of excessive reac-ive oxygen species (ROS) assessed in liver were: the increase inipid peroxidation products, assayed using a kit for thiobarbituriceactive substances (TBARS) from Cayman Chemical (Ann Harbor,I) and the decrease in aconitase activity [26], assayed using a kit

rom Oxis International (Bioxytech 340, Foster City, CA).

.7. Expression of genes related to energy metabolism in liver

Liver samples were used for RNA isolation and the analysisf gene expression. Total RNA was isolated from frozen samplessing Trizol (Invitrogen, Carlsbad, CA, USA) according to the man-facturer’s instructions. The purified total RNA was treated withNase-free DNase (Ambion, Austin, TX, USA) for 30 min at 37 ◦Cnd used as a template to generate first-strand cDNA synthesissing M-MLV reverse transcriptase (Invitrogen) as described byhe manufacturer. Quantitative real-time PCR was performed usingn ABI PRISM 7000 HT Sequence Detection System using Taq-an probes (Applied Biosystems, Foster City, CA, USA) for mouse

atalase, superoxide dismutase (SOD2), insulin receptor substrateIRS1), BHLHB2 (also known as Sharp-2), INPPL1 (also known aship-2), carnitine palmitoyl transferase (CPT1A), acyl-Coenzyme Aehydrogenase (ACADM), fatty acid synthase (FASN) and 18 s rRNAs internal control.

.8. Statistical analysis.

Results are given as means ± S.E.M. Differences between BZA-reated and control were determined by Student’s t-test for valuesbtained at and after sacrifice or by analysis of variance forepeated observations, regarding data collected during treatment.S denotes a non-significant difference.

l Research 61 (2010) 355–363 357

2.9. Chemicals and reagents

Benzylamine hydrochloride, bovine insulin, fatty acid-freebovine serum albumin, and other reagents were obtained fromSigma–Aldrich (Saint Quentin Fallavier, France, and Milan, Italyfor HPLC grade reagents). Enzymes for the glycerol assay and lib-erase blendzyme 3 were from Roche Diagnostics (Indianapolis,IN, USA). 2-[1,2-3H]-deoxyglucose (2-DG, 29.7 Ci/mmol) was pur-chased from PerkinElmer (Boston, MA, USA), [14C]-benzylaminewas from Amersham Biosciences (Buckinghamshire, UK).

3. Results

3.1. Intestinal transport and oxidation of benzylamine

Initial experiments were carried out to determine whetherbenzylamine could cross the intestinal barrier without beingoxidized by the amine oxidases supposed to protect the bodyagainst dietary amines. Fig. 1A shows that benzylamine was pre-dominantly oxidized by SSAO since 90% of its oxidation by ratintestine was sensitive to 1 mM semicarbazide while it resistedto pargyline inhibition. Moreover, benzylamine oxidation occurredmainly in the muscular layer of small intestine while the mucosawas practically devoid of SSAO-mediated oxidation towards theexogenous amine. It should be noted that the SSAO-dependentactivity was much lower in rat intestine than in rat adipose tis-sue homogenates: 0.13 ± 0.02 nmol oxidized/(mg protein min) vs.1.89 ± 0.25 nmol oxidized/(mg protein min), when 0.1 mM benzy-lamine was used as substrate (n = 5–8, p < 0.001). The trans-mucosalpassage of benzylamine in everted gut sacs was dose- and time-dependent (Fig. 1B) and about 50% of the amine transported to theserosal side was not oxidized, thus justifying the rationale for peros administration.

3.2. Oral benzylamine administration to mice

Benzylamine was dissolved in the drinking water in order toadminister 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 effectsof benzylamine when acutely injected at 70 �mol/kg in overweightmice [27] or orally given at 1900 �mol/(kg day) to rats [22] orat 2000–4000 �mol/(kg day) to mice [27]. The stability of benzy-lamine in the drinking solutions was assessed by HPLC and onlya negligible degradation occurred between freshly prepared solu-tions and after 1 week at room temperature (typically less than 2%decrease in concentration, data not shown). Since the analyticalprocedure used was suitable only for liquid samples, the pres-ence of benzylamine could also be verified in urine, and variedfrom undetectable amounts in controls to 9.6 ± 0.2 �g/ml in micedrinking benzylamine solutions prepared at around 4.8 mg/ml.Therefore, ingested benzylamine was absorbed, metabolized andeven excreted in mice, indicating that in this model, the intesti-nal barrier was not totally insurmountable for this amine oxidasesubstrate.

3.3. Water consumption and body weight gain in mice drinkingbenzylamine

The water consumption was followed weekly and exhibited

small variations (Fig. 2A). However, the overall consumptionwas significantly reduced during the 17-week treatment in micedrinking benzylamine: F(1,102) = 17.1, p < 0.0001. BZA-treated micelimited their daily water consumption by approximately 10% whencompared with control (Fig. 2A).

358 Z. Iffiú-Soltész et al. / Pharmacological Research 61 (2010) 355–363

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 [14C]-benzylamine for the determination of amine oxidation in the absence of anyinhibitor (total oxidation, black columns), with semicarbazide (which blocked SSAO component and left MAO intact, grey columns), or with pargyline (selectively blockingM ) TimF enzyla e sacst deter

BaFaUmpaF7

3

jcBlumblc1itIta

AO and leaving SSAO intact, open columns). Mean ± S.E.M. of 4–8 preparations. (Bor the time-dependent study, everted gut sacs were incubated with 1 mM [14C]-bmine were incubated with sacs for 45 min at 37 ◦C. The amount transported into thriangles) or non-metabolized fractions (circles). Each point is mean ± S.E.M. of 3–7

Although both groups had the same initial body mass,ZA-treated mice weighed significantly less than the controlsfter 1 week of treatment and until the end of experiment:(1,374) = 125.3, p < 0.00001 (Fig. 2B). Consequently, the over-ll body weight gain was clearly limited by the BZA-treatment.nder non-fasting conditions, benzylamine drinking induced aodest but significant reduction in blood glucose: F(1,374) = 24.8,< 0.00001 (Fig. 2C). Such modest glycaemia reduction is also visu-lized by the average of weekly measurements in right panel ofig. 2C: unfasted blood glucose was 8.18 ± 0.13 mM in control vs..71 ± 0.16 mM in BZA-treated.

.4. Glucose handling by BZA-treated mice

Two weeks before the end of the treatment, mice were sub-ected to GTT after being fasted for 5 h by receiving an i.p. glucosehallenge at 1 g/kg. The overall hyperglycemia was reduced inZA-treated mice as shown by: (i) the more rapid return to basal

evels at almost all the time points tested, (ii) the lower areander the curve (n = 12, p < 0.05) (Fig. 3). Alongside the improve-ent in glucose tolerance, benzylamine treatment also lowered

aseline blood glucose, since the BZA-treated mice exhibitedower values than controls 15 min before and just before the glu-ose load: 8.9 ± 0.7 mM and 10.2 ± 0.7 mM vs. 11.8 ± 0.3 mM and2.9 ± 0.7 mM, respectively (n = 12, p < 0.01) (Fig. 3). Such a decrease

n blood glucose also occurred after overnight fasting, a condi-ion only experienced once in the protocol, just before sacrifice.n fact, glucose measured in drops of blood withdrawn from theail of mice just before sacrifice was 7.29 ± 0.59 mM in controlsnd only 5.61 ± 0.58 mM in the BZA-treated group, correspond-

e- and concentration-dependence of benzylamine transport across small intestine.amine, while for the concentration-dependence, isotopic solutions of the labelledwas expressed as total benzylamine (squares) and as oxidized (into benzaldehyde,

minations.

ing to a reduction at the limit of statistical significance (n = 12,p = 0.055). Further analysis of plasma samples obtained from car-diac puncture after sacrifice definitively showed that glucose levelswere lower in treated mice: they were equivalent to 76 ± 6% ofcontrol values (n = 12, p < 0.02). Immunoreactive insulin levels alsoshowed a tendency to diminish, but, due to high intra-group vari-ability, 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 accompa-nied 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 fatdepots relative to final body weight, was significantly lower thanin the controls (Table 1). This was due to a reduction in both intra-abdominal and subcutaneous white adipose tissues. The liver wasalso smaller in the BZA-treated animals than in control mice butno significant reduction could be detected when hepatic mass wasexpressed as a percentage of body weight (Table 1).

Under fed conditions, no difference in the circulating levels ofFFA was detected between control and BZA-treated groups (respec-tive values found after 15 weeks of treatment were 0.70 ± 0.07and 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 unexpect-edly observed in the plasma from the BZA-treated mice. Such aclear-cut anti-hypercholesterolemic effect of benzylamine, lead-ing to a reduction of 40% in the levels found in the hyperlipidemic

Z. Iffiú-Soltész et al. / Pharmacological Research 61 (2010) 355–363 359

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. Twogroups of age- and weight-matched mice were constituted (4 cages of 3 animals per group) 3 weeks before oral administration of benzylamine (arrow). Benzylamine wasgiven 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 anddrink. (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:a eightp g blow l: overd e righ

meH

3m

i

FtA

verage daily water consumption over the 17-week treatment. (B) Weekly body wanel). Each point/column is the mean ± S.E.M. of 12 determinations. (C) Non-fastineekly in treated mice (HFD-benzylamine) and controls (HFD control). Right paneeterminations performed weekly in each group during the treatment period. For th

ice was not accompanied by a significant decrease of the ben-ficial HDL-cholesterol: 171 ± 21 and 121 ± 18 mg/dl in HFD andFD + BZA, respectively (n = 5, NS).

.6. Influence of chronic benzylamine intake on circulatingarkers of metabolic disturbances

Among the diagnostic parameters given in Table 2, circulat-ng bilirubin remained unchanged, ruling out major impairment

ig. 3. Influence of benzylamine oral treatment on glucose tolerance. After 15 weeks of orime 0, arrow). Left panel: glucose was determined in blood collected from the tail of consrea under the curve of the hyperglycemic excursion (AUC). Mean ± S.E.M. of 12 mice per

changes (left panel) and cumulative body weight gain during the treatment (rightod glucose; left panel: each point is mean ± S.E.M. of 12 determinations performedall value of non-fasting blood glucose; each column is the mean ± S.E.M. of the 17t panels, statistically different from HFD control at: *p < 0.05, **p < 0.01, ***p < 0.001.

concerning liver or red blood cell functions. Similarly, lacticacid levels were not altered by the benzylamine oral treat-ment. Plasma levels of uric acid were increased in HFD-micewhen 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. Fruc-tosamine levels were not modified in the BZA-treated group,suggesting that benzylamine treatment did not increase plasmaprotein glycation. Similarly, albuminemia was not altered. Among

al amine administration, mice were fasted for 5 h prior to glucose load (1 g/kg, i.p. atcious animals at the indicated times before and after the glucose load. Right panel:group. Statistically different from control at: *p < 0.05, **p < 0.01, ***p < 0.001.

360 Z. Iffiú-Soltész et al. / Pharmacological Research 61 (2010) 355–363

Table 1Body mass and proportion of adipose or hepatic tissue in HFD-fed mice after chronicoral benzylamine administration.

Experimental group HFD HFD + BZA

Body mass (g) 36.0 ± 1.8 28.9 ± 1.3**

INWAT mass (g) 2.59 ± 0.27 1.23 ± 0.26**

SCWAT mass (g) 1.72 ± 0.22 0.82 ± 0.17**

Adiposomatic index (% of body mass) 11.6 ± 0.9 6.6 ± 1.1**

Liver weight (% of body mass) 3.6 ± 0.1 3.4 ± 0.1

Mice fed a high-fat diet alone (HFD) or in combination with benzylamine in drinkingwater (HFD + BZA) were sacrificed after 17 weeks of treatment. The adiposomaticitm

tcu

3pB

ssb(lwr

uarvciab

3

sIta

TE

Ma

Table 3Effect of chronic benzylamine ingestion on the concentration of stable end-productsof NO metabolism.

Experimental group HFD HFD + BZA

Nitrite (nmol/g aorta) 53.5 ± 2.7 66.2 ± 4.4*

Nitrate (nmol/g aorta) 99.2 ± 9.2 95.8 ± 6.4Nitrite (nmol/g muscle) 19.7 ± 3.5 17.9 ± 1.7Nitrate (nmol/g muscle) 27.5 ± 3.7 27.3 ± 4.3

ndex is the sum of dissected intra-abdominal plus subcutaneous white adiposeissues (INWAT + SCWAT) given as percentage of body mass. Mean ± S.E.M. of 12

ice per group.** Significantly different from HFD at: p < 0.01.

he three adipokines measured, only resistin showed a signifi-ant decrease while adiponectin and leptin plasma levels remainednaltered in BZA-treated mice (Table 2).

.7. Unaltered insulin responsiveness and insulin-mimickingroperties of benzylamine in adipocytes isolated fromZA-drinking mice

The dose-dependent stimulation of lipolysis by isoprenaline wasimilar in adipocytes from HFD control and BZA-treated mice (nothown). In control group, the submaximal lipolysis stimulationy 10 nM isoprenaline was partly inhibited with 100 nM insulin44 ± 6% inhibition) and with the combination of 0.1 mM benzy-amine and vanadate (38 ± 7% inhibition). These antilipolytic effects

ere equivalent in treated group: 40 ± 8 and 46 ± 4% inhibition,espectively (n = 12, NS).

Insulin dose-dependent stimulation of glucose transport wasnaltered (not shown) and reached maximum at 100 nM: 5.9 ± 0.7nd 5.2 ± 0.4-fold increase over basal in control and BZA-treated,espectively (n = 12, NS). Synergism between benzylamine andanadate reproduced such 2-DG uptake activation in both HFDontrol and BZA-treated mice: 82 ± 7 and 90 ± 10% of maximalnsulin stimulation (n = 12, NS). Therefore, no alteration in thedipocyte responses to insulin or benzylamine plus vanadate coulde detected after benzylamine administration.

.8. Influence of benzylamine intake on liver gene expression

Chronic benzylamine treatment did not change the expres-ion of three genes related to insulin signalling (IRS1, BHLHB2,NPPL1) in liver (not shown). The expression of a key gene relatedo lipogenic activity (FASN, encoding for fatty acid synthase) waslso unchanged in the BZA-treated mice while the number of the

able 2ffect of chronic benzylamine ingestion on plasma metabolites and cytokines.

Experimental group HFD HFD + BZA

FFA (mM) 0.61 ± 0.06 0.64 ± 0.07Triglycerides (mg/dl) 69.2 ± 5.9 58.0 ± 3.2Total cholesterol (mg/dl) 218.2 ± 10.2 131.0 ± 15.7***

Total bilirubin (�mol/l) 6.2 ± 2.4 4.5 ± 0.9Lactic acid (mmol/l) 8.4 ± 0.8 8.5 ± 0.5Fructosamine (�mol/l) 200.4 ± 11.2 193.5 ± 6.9Uric acid (�mol/l) 365.9 ± 32.4 234.1 ± 13.6***

Albumin (�mol/l) 448.5 ± 20.8 411.8 ± 14.0Leptin (ng/ml) 9.78 ± 2.46 9.31 ± 2.35Adiponectin (�g/ml) 12.92 ± 1.16 12.64 ± 1.03Resistin (ng/ml) 5.93 ± 0.51 4.45 ± 0.29*

ice fed a high-fat diet (HFD) and drinking benzylamine (HFD + BZA) were sacrificedfter 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.

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 ben-

zylamine (HFD + BZA) at: p < 0.05.

transcripts of carnitine palmitoyl transferase (CPT1A), involvedin long-chain fatty acid transport into the mitochondria and �-oxidation, tended to increase twofold (range 2.05–2.79 relative tocontrol set at 1, n = 10). Taken together, these results seemed toindicate that insulin sensitivity remained unchanged in the liver ofBZA-treated mice. On the contrary, the balance between synthe-sis and degradation of fatty acids appeared to be in favour of anenhanced catabolism and could rely with the decreasing tenden-cies of hepatic triglyceride content (from 58.9 ± 8.9 in control to42.9 ± 5.5 mg/g in BZA-treated, n = 10, p < 0.10). Nevertheless, theabsence of change in ACADM expression (acyl-Coenzyme A dehy-drogenase involved in medium-chain fatty acid breakdown) andin 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 weremodified by benzylamine treatment.

3.9. Influence of benzylamine intake on RNS and ROS

Chronic benzylamine treatment did not modify the nitratecontent in aortic tissue (Table 3). This finding argued against aputative exaggerated oxidative stress induced by the hydrogenperoxide production accompanying sustained benzylamine inges-tion and oxidation, that could contribute to an elevated oxidativeconversion of NO to RNS. On the contrary, nitrite concentra-tion was increased in the aortas of BZA-treated mice, indicatingthat benzylamine administration slightly improved the reducedNO bioavailability found in the vasculature of HFD-mice: aortanitrite 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 inHFD + BZA mice. The similar nitrite and nitrate concentrationsfound in the muscle of HFD and BZA-treated mice pointed out thatneither oxidative stress or NO bioavailability was altered in thistissue by the treatment (Table 3).

In liver homogenates, the concentration of lipid peroxidationmarkers was decreased from 5.6 ± 0.4 to 3.9 ± 0.1 �M TBARS incontrol and BZA-treated respectively (n = 12, p < 0.001). The lackof significant decrease in hepatic aconitase activity (110 ± 6 vs.95 ± 5 arbitrary units, n = 12, NS), also indicated that chronic ben-zylamine was not promoting an easily detectable oxidative stress.Accordingly, no change of expression was found in the liver forthe mitochondrial superoxide dismutase that protects cells againstsuperoxide toxicity, and for catalase that catalyzes the decomposi-tion of hydrogen peroxide (not shown).

4. Discussion

The present study shows that chronic benzylamine adminis-tration improves glucose tolerance in mice fed a diabetogenic,

high-fat diet, at least when considering their lower non-fastingand fasting glycaemia and their reduced hyperglycaemic responses.Moreover, mice drinking benzylamine reduced their weight gain,plasma cholesterol, resistin and uric acid levels without exhibitingsystemic or tissular signs of excessive oxidative stress.

logica

9oavpToctadembvlattobbohaita

ztsii

gitflrrw(bobcd

wiagbiskont0fcft

Z. Iffiú-Soltész et al. / Pharmaco

Benzylamine, also called moringine (CAS 100-46-9/CAS 3287-9-8) is an aromatic monoamine present in the plant Moringaleifera, known as the “miracle tree” and used in folk medicine as anntidiabetic agent, a property confirmed in a pharmacological sur-ey of medicinal plants [28]. However, it has never been directlyroved that benzylamine is an orally active antidiabetic molecule.his is the first study that shows the somewhat beneficial effectsf chronic benzylamine ingestion on glucose handling, circulatingholesterol and nitrogen monoxide bioavailability. Such observa-ions are in perfect agreement with a preliminary study reportingreduced hyperglycaemic response during GTT in non-obese non-iabetic rats receiving benzylamine per os [22]. Our findings alsoxtend previous observations made in mice rendered obese after 3onths of very-high-fat diet (72% energy from fat), showing that

enzylamine was equally efficient alone or when associated withanadium in reducing the hyperglycemic response to a glucoseoad [27]. Noteworthy, repeated benzylamine administration haslready been reported to decrease the elevated blood glucose ofype 1 and type 2 diabetic rats, but only when injected/perfusedogether with vanadate [10,11]. Since the therapeutic applicationsf the association of benzylamine with vanadium could be limitedy the toxicity of the transition metal (in spite of its antidia-etic properties), we have focused our interest on the influencef benzylamine alone on glucose handling. In vivo, an acute anti-yperglycemic action was observed with benzylamine doses as lows 7 �mol/kg after injection in mice fed a very-high-fat diet or afterntravenous infusion in conscious rabbits [6,27]. In these models,he acute anti-hyperglycemic effect of benzylamine was abolishedfter SSAO/VAP-1 blockade by semicarbazide treatment [6].

The benzylamine diffusion across everted gut sacs and the ben-ylamine detection in the urine of BZA-drinking mice show thathe intestinal barrier is not insurmountable for this amine oxidaseubstrate, supporting the idea that oral administration could resultn tissue benzylamine levels sufficient to reproduce some of thensulin-like actions observed in vitro.

In fact, orally given benzylamine produced the same effects onlucose homeostasis as when injected at lower doses. Benzylaminengestion also led to somewhat unexpected effects such as reduc-ion in water consumption. This can be considered as a reduceduid intake only if spillage and dressing can be ruled out. Similareduction had already been observed in non-obese, non-diabeticats supplemented with 0.29% benzylamine drinking solution for 7eeks [22] or in normoglycaemic mice drinking 0.5% benzylamine

Carpéné et al., unpublished data). It is difficult to assess whetherenzylamine exerted an aversive influence on drinking behaviourr a pharmacologic effect on fluid homeostasis. The bad smell ofenzylamine solutions, smelling like putrefied fish at higher con-entrations than those given to rodents favours the hypothesis of aistasteful drinking having an aversive effect.

The body weight gain reduction observed in BZA-drinking miceas also unexpected since the acute effects of benzylamine found

n adipocytes (lipolysis inhibition, glucose uptake and lipogenesisctivation) were supposed to lead to fattening and increased weightain once benzylamine was chronically ingested. The reducedody weight gain is however agreeing with the already reported

nhibitory effects of benzylamine on appetite, mediated by potas-ium channel blockade in central nervous system [29]. As far as wenow, the hypophagic effects of benzylamine have been describednly in mid-term experiments in behavioural studies [29,30] ando clear reduction of food intake or body weight after prolongedreatment has been reported to date, after benzylamine drinking at

.3% in rats [22], or at 0.5% in mice (Carpéné et al., unpublished data)ed a standard diet. Unfortunately, in the present study, it was diffi-ult to assess whether a reduction in calorie intake was responsibleor the decreased body weight gain during BZA-treatment becausehe given high-fat chow was particularly sticky and could not be

l Research 61 (2010) 355–363 361

accurately weighed. If such a central hypophagic action of chroni-cally ingested benzylamine persisted in the treated HFD-fed mice,it could be largely responsible for the weight gain normalizationand for its concomitant beneficial effects, including the reducednon-fasting glycaemia and improved glucose tolerance.

Another unexpected effect of oral benzylamine administrationwas the reduction in circulating cholesterol levels. Although thedecrease was mainly observed in the non-HDL fraction, there wasno rationale to support such a beneficial effect. It can just be notedthat, in a very recent study, various hydrazine derivatives usedas carbonyl scavengers, but known to inhibit SSAO and/or MAOactivity, such as hydralazine, phenelzine and iproniazid, have beenshown to exert anti-atherogenic effects, but without lowering cir-culating cholesterol [31]. Whether there is a putative link betweenamine oxidase activity and cholesterol metabolism deserves to beclarified.

A decrease in FFA was expected, owing to the antilipolytic effectsof benzylamine previously found in human and rat adipocytes[5,32] and confirmed here for murine fat cells. In fact, the limi-tation of fat deposition observed in treated mice is in apparentcontradiction with the benzylamine antilipolytic effect detectedon adipocytes in the presence of vanadate but fits with its cen-tral hypophagic action [29,30]. Also unpredicted was the decreasein plasma resistin found in BZA-drinking mice, while a decrease inleptin was expected, due to the reduced adiposity of the treatedanimals. Nevertheless, the unaltered levels of this latter adipokine,together with unchanged adiponectin, reduced resistin and theslightly reduced insulin levels are consistent with an improvementof blood glucose control in BZA-treated mice.

Considering the deleterious vascular effects of methy-lamine administration reported in mice overexpressing vascularSSAO/VAP-1 [2], oxidative stress or diabetic-like vascular injurycould have been predicted to occur in HFD-fed mice upon BZA-treatment. However, no evident signs of excessive glycation orvascular damage consequent to excessive activation of solubleor vascular SSAO/VAP-1 and aldehyde-induced cross-linking[17], were detected after benzylamine administration. Firstly,fructosamine, which reflects glycated plasma protein levels, wasnot altered by BZA-treatment. Secondly, elevated plasma uricacid, that accompanies the onset of metabolic syndrome [33], wasdecreased in BZA-treated mice. Instead, the lowered levels of thisindependent marker of cardiovascular disease risk [34] could beregarded as protective for cardiac and renal function. Thirdly, ben-zylamine administration lowered the levels of lipid peroxidationmarkers, and did not modify the aconitase activity [26] or theexpression 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 indicatorof the conversion of NO to RNS by excessive ROS, there was noindication of such oxidative stress in the aorta or skeletal muscleof benzylamine drinking mice. Moreover, nitrite concentration,which was significantly elevated, suggested an increased NOproduction. Since insulin has been reported to increase nitriteconcentration in the aorta by enhancing eNOS expression [35],the influence of BZA-treatment may be considered beneficial andrelated to the insulin-mimicking properties of the amine. Loweredplasma uric acid and increased nitrite concentration in the aortamay therefore indicate an improved endothelial function in BZA-treated animals in place of the feared damage. Alternatively, it canbe an indirect effect secondary to the reductions of overweight,hyperglycemia and hypercholesterolemia.

The lack of deleterious effects of BZA-treatment on vasculatureis in agreement with the paradigm proposing that benzaldehyde,the product of SSAO/VAP-1-mediated benzylamine oxidation, isless cytotoxic than formaldehyde produced by methylamine oxi-dation [17,19]. Therefore, we suggest that among the different

3 logica

plihidootbcoclbewe[

(berdicdtntis

5

geitlailthabup

A

fsefgMKa0r

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

62 Z. Iffiú-Soltész et al. / Pharmaco

ossible fates for ingested benzylamine, a competition with methy-amine for SSAO/VAP-1-mediated oxidation can occur, as observedn vitro on cultured adipocytes [8]. As a result, less cytotoxic alde-yde could be generated in BZA-drinking mice. In this context,

t should be mentioned that benzylamine can also be oxidativelyeaminated by reactive aldehydes themselves such as methylgly-xal (produced in part by SSAO/VAP-1-dependent aminoacetonexidation which is increased in diabetic plasma) and may limit theiroxicity by acting like a scavenger [36]. Obviously, benzylamine cane oxidized not only in blood and vessels, but in other tissues, espe-ially WAT, that is dramatically enlarged in overweight rodents andne of the richest in SSAO and MAO activities [24]. It is thereforeonceivable that direct insulin-like actions of orally given benzy-amine could have occurred in WAT, owing to its SSAO richness,ut it is hardly compatible with the observed decrease in adiposity,xcept if increased energy expenditure or decreased calorie intakeere sustained throughout the treatment, via central benzylamine

ffects elsewhere described to be independent on amine oxidation29,30].

In liver, the expression of genes related to insulin signallingsuch as IRS1, BHLHB2, INPPL1) was not modified, confirming thatenzylamine did not reverse or amplify insulin-resistance. How-ver, the increased expression of liver CPT1A, the enzyme thategulates the transport of long-chain acyl-CoA into the mitochon-ria to produce energy through beta-oxidation, indicated that an

ncrease in fatty acid oxidation might occur in BZA-treated hepato-ytes. Similar increases have been reported in situations of fasting,iabetes, and fat feeding while it is known that insulin represseshe transcription of CPT1A in liver [37]. Therefore benzylamine didot exert direct insulin-like effects on liver, an observation that haso be connected with the lack of SSAO in hepatocytes. Moreover,ncrease in CPT1A gene can also be associated with fat loss [38], aituation provoked by benzylamine ingestion.

. Conclusions

Recent findings on the anti-inflammatory properties of a neweneration of SSAO/VAP-1-inhibitors [39] did not indicate whetherxogenous substrates of the same enzyme could exhibit interest-ng properties regarding metabolic control. Although it remainso verify whether the immune system was influenced by benzy-amine treatment, the present findings show that oral benzylaminedministration merits testing in other diabetes/obesity models,ncluding the db/db mice a valuable model for testing glucoseowering capacities of antidiabetic drug candidates [40]. Finally,he reported weight gain-reducing, anti-hyperglycemic and hypoc-olesterolemic actions of benzylamine warrant future searches formine oxidase substrates more active than benzylamine, as it haseen initiated towards human SSAO by Yraola et al. [41]. Such sol-ble amines may be endowed with more powerful antidiabeticroperties and yet devoid of adverse effects.

cknowledgments

The authors express gratitude to D. Prévot and A. Desquesnesor biochemical analyses at ANEXPLO platform, Y. Barreira and thetaff of animal unit of I2MR, Toulouse, for invaluable help. Thexpertise of S. Benvenuti regarding amine chromatography is grate-ully acknowledged. This work was partly supported by an R&Drant from the “Communauté de Travail des Pyrénées” (project

P #05018677), the Hungarian Scientific Research Fund (OTKA

63415) and by “Programme Balaton” (project Egide #14117SAnd Hungarian National Office of Research and Technology OMFB-0491/2007) for Franco-Spanish and Franco-Hungarian exchanges,espectively.

[

[

l Research 61 (2010) 355–363

References

[1] Bianchi P, Kunduzova O, Masini E, Cambon C, Bani D, Raimondi L, et al. Oxidativestress by monoamine oxidase mediates receptor-independent cardiomy-ocyte apoptosis by serotonin and postischemic myocardial injury. Circulation2005;112:3297–305.

[2] Stolen CM, Madanat R, Marti L, Kari S, Yegutkin GG, Sariola H, et al. Semicar-bazide sensitive amine oxidase overexpression has dual consequences: insulinmimicry and diabetes-like complications. FASEB J 2004;18:702–4.

[3] Goldstein BJ, Mahadev K, Wu X, Zhu L, Motoshima H. Role of insulin-inducedreactive oxygen species in the insulin signaling pathway. Antioxid Redox Signal2005;7:1021–31.

[4] Marti L, Morin N, Enrique-Tarancon G, Prévot D, Lafontan M, Testar X, et al. Tyra-mine and vanadate synergistically stimulate glucose transport in rat adipocytesby amine oxidase-dependent generation of hydrogen peroxide. J PharmacolExp Ther 1998;285:342–9.

[5] Morin N, Lizcano JM, Fontana E, Marti L, Smih F, Rouet P, et al. Semicarbazide-sensitive amine oxidase substrates stimulate glucose transport and inhibitlipolysis 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 PharmacolExp Ther 2004;309:1020–8.

[7] Mercier N, Moldes M, El Hadri K, Fève B. Semicarbazide-sensitive amineoxidase activation promotes adipose conversion of 3T3-L1 cells. Biochem J2001;358:335–42.

[8] Carpéné C, Daviaud D, Boucher J, Bour S, Visentin V, Grès S, et al. Short-and long-term insulin-like effects of monoamine oxidase and semicarbazide-sensitive amine oxidase substrates in cultured adipocytes. Metabolism2006;55:1397–405.

[9] Bour S, Daviaud D, Gres S, Lefort C, Prévot D, Zorzano A, et al. Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamineoxidase in human adipocytes. Biochimie 2007;89:916–25.

10] Marti L, Abella A, Carpéné C, Palacin M, Testar X, Zorzano A. Combinedtreatment with benzylamine and low dosages of vanadate enhances glucosetolerance 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 activityexerts 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. Acell surface amine oxidase directly controls lymphocyte migration. Immunity2001;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 semicarbazide-sensitive 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 handlingin streptozotocin-induced diabetic rats is improved by tyramine but not by theamine 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 NeuralTransm Suppl 1998;52:201–16.

17] Gubisne-Haberle D, Hill W, Kazachkov M, Richardson JS, Yu PH. Proteincross-linkage induced by formaldehyde derived from semicarbazide-sensitiveamine oxidase-mediated deamination of methylamine. J Pharmacol Exp Ther2004;310:1125–32.

18] Lei T, Xie W, Han J, Corkey BE, Hamilton JA, Guo W. Medium-chain fatty acidsattenuate 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 cellularsemicarbazide-sensitive amine oxidase to amine metabolism and cardiovas-cular 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 ofinsulin. Diabetes 2007;56:486–93.

21] Srivastava AK. Anti-diabetic and toxic effects of vanadium compounds. Mol CellBiochem 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 lipidmetabolism in rats. J Physiol Biochem 2005;61:371–80.

23] Barthe L, Woodley JF, Kenworthy S, Houin G. An improved everted gut sac asa simple and accurate technique to measure paracellular transport across thesmall 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. Tyraminestimulates glucose uptake in insulin-sensitive tissues in vitro and in vivo viaits 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 tran-sient iotachphoretic sample stacking. J Chromatogr A 2004;1051:177–83.

26] Nulton-Persson AC, Szweda LI. Modulation of mitochondrial function by hydro-gen peroxide. J Biol Chem 2001;276:23357–61.

27] Iffiú-Soltész Z, Prévot D, Grès S, Bour S, Szöko É, Knauf C, et al. Influence ofbenzylamine acute and chronic administration of benzylamine on glucose tol-

logica

[

[

[

[

[

[

[

[

[

[

[

[

[and glucose lowering effects of a novel indole derivative in vitro and in vivo.Pharmacol Res 2007;56:335–43.

Z. Iffiú-Soltész et al. / Pharmaco

erance in diabetic and obese mice fed on very high-fat diet. J Physiol Biochem2007;63:305–16.

28] Kar A, Choudhary BK, Bandyopadhyay NG. Comparative evaluation of hypo-glycaemic activity of some Indian medicinal plants in alloxan diabetic rats. JEthnopharmacol 2003;84:105–8.

29] Pirisino R, Ghelardini G, Banchelli G, Galeotti N, Raimondi L. Methylamineand benzylamine induced hypophagia in mice: modulation by semicarbazide-sensitive benzylamine oxidase inhibitor and aODN towards Kv1.1 channels. BrJ Pharmacol 2001;134:880–6.

30] Pirisino R, Galeotti N, Livi S, Raimondi L, Ghelardini G. 4-Methyl benzy-lamine stimulates food consumption and counteracts the hypophagic effectsof amphetamine acting on brain Shaker-like Kv1.1 channels. Br J Pharmacol2006;147:218–24.

31] Galvani S, Coatrieux C, Elbaz M, Grazide M-H, Thiers J-C, Parini A, et al. Carbonylscavenger and antiatherogenic effects of hydrazine derivatives. Free Rad BiolMed 2008;45:1457–67.

32] Visentin V, Prévot D, Marti L, Carpéné C. Inhibition of rat fat cell lipolysis bymonoamine oxidase and semicarbazide-sensitive amine oxidase substrates.

Eur J Pharmacol 2003;466:235–43.

33] Yoo TW, Sung KC, Shin H, Kim BJ, Kim BS, Kang JH, et al. Relationship betweenserum uric acid concentration and insulin resistance and metabolic syndrome.Circ J 2005;69:928–33.

34] Gagliardi AC, Miname MH, Santos RD. Uric acid: a marker of increased cardio-vascular risk. Atherosclerosis 2009;202:11–7.

[

l Research 61 (2010) 355–363 363

35] Kobayashi T, Kamata K. Effect of chronic insulin treatment on NO pro-duction and endothelium-dependent relaxation in aortae from establishedSTZ-induced diabetic rats. Atherosclerosis 2001;155:313–20.

36] Akagawa M, Sasaki T, Suyama K. Oxidative deamination of benzylamine byglycoxidation. Bioorg Med Chem 2003;11:1411–7.

37] Louet JF, Le May C, Pégorier JP, Decaux JF, Girard J. Regulation of liver carnitinepalmitoyltransferase I gene expression by hormones and fatty acids. BiochemSoc Trans 2001;29:310–6.

38] Shin ES, Cho SY, Lee EH, Lee SJ, Chang IS, Lee TR. Positive regulation of hepaticcarnitine palmitoyl transferase 1A (CPT1A) activities by soy isoflavones andl-carnitine. Eur J Nutr 2006;45:159–64.

39] Salter-Cid LM, Wang E, O’Rourke AM, Miller A, Gao H, Huang L, etal. Anti-inflammatory effects of Inhibiting the amine oxidase activity ofsemicarbazide-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

41] Yraola F, Garcia-Vicente S, Fernandez-Recio J, Albericio F, Zorzano A, Marti L, etal. New efficient substrates for semicarbazide-sensitive amine oxidase/VAP-1 enzyme: analysis by SARs and computational docking. J Med Chem2006;49:6197–208.