Semicarbazide-sensitive Amine Oxidase Substrates Research Articles

Increased monoamine oxidase activity and imidazoline binding sites in insulin-resistant adipocytes from obese Zucker rats.

BACKGROUNDDespite overt insulin resistance, adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIMTo investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide, since it is produced by monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in adipocytes.METHODS 3H-2-deoxyglucose uptake (2-DG) was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine. 14C-tyramine oxidation and binding of imidazolinic radioligands [3H-Idazoxan, 3H-(2-benzofuranyl)-2-imidazoline] were studied in adipocytes, the liver, and muscle. The influence of in vivo administration of tyramine + vanadium on glucose handling was assessed in lean and obese rats.RESULTS2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats, when compared to their lean littermates. Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited, while MAO was increased and SSAO decreased. These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat, when compared to the lean. In vitro, tyramine precluded the binding to I2 sites, while in vivo, its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese rats. CONCLUSIONThe adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number. However, probably as a consequence of SSAO down-regulation, the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes. The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.

High doses of catecholamines activate glucose transport in human adipocytes independently from adrenoceptor stimulation or vanadium addition.

BACKGROUNDWhen combined with vanadium salts, catecholamines strongly activate glucose uptake in rat and mouse adipocytes.AIMTo test whether catecholamines activate glucose transport in human adipocytes.METHODSThe uptake of 2-deoxyglucose (2-DG) was measured in adipocytes isolated from pieces of abdominal subcutaneous tissue removed from women undergoing reconstructive surgery. Pharmacological approaches with amine oxidase inhibitors, adrenoreceptor agonists and antioxidants were performed to unravel the mechanisms of action of noradrenaline or adrenaline (also named epinephrine).RESULTSIn human adipocytes, 45-min incubation with 100 µmol/L adrenaline or noradrenaline activated 2-DG uptake up to more than one-third of the maximal response to insulin. This stimulation was not reproduced with millimolar doses of dopamine or serotonin and was not enhanced by addition of vanadate to the incubation medium. Among various natural amines and adrenergic agonists tested, no other molecule was more efficient than adrenaline and noradrenaline in stimulating 2-DG uptake. The effect of the catecholamines was not impaired by pargyline and semicarbazide, contrarily to that of benzylamine or methylamine, which are recognized substrates of semicarbazide-sensitive amine oxidase. Hydrogen peroxide at 1 mmol/L activated hexose uptake but not pyrocatechol or benzoquinone, and only the former was potentiated by vanadate. Catalase and the phosphoinositide 3-kinase inhibitor wortmannin inhibited adrenaline-induced activation of 2-DG uptake. CONCLUSIONHigh doses of catecholamines exert insulin-like actions on glucose transport in human adipocytes. At submillimolar doses, vanadium did not enhance this catecholamine activation of glucose transport. Consequently, this dismantles our previous suggestion to combine the metal ion with catecholamines to improve the benefit/risk ratio of vanadium-based antidiabetic approaches.

Oral Supplementation with Benzylamine Delays the Onset of Diabetes in Obese and Diabetic db-/- Mice.

Substrates of semicarbazide-sensitive amine oxidase (SSAO) exert insulin-like actions in adipocytes. One of them, benzylamine (Bza) exhibits antihyperglycemic properties in several rodent models of diabetes. To further study the antidiabetic potential of this naturally occurring amine, a model of severe type 2 diabetes, the obese db-/- mouse, was subjected to oral Bza administration. To this end, db-/- mice and their lean littermates were treated at 4 weeks of age by adding 0.5% Bza in drinking water for seven weeks. Body mass, fat content, blood glucose and urinary glucose output were followed while adipocyte insulin responsiveness and gene expression were checked at the end of supplementation, together with aorta nitrites. Bza supplementation delayed the appearance of hyperglycemia, abolished polydypsia and glycosuria in obese/diabetic mice without any detectable effect in lean control, except for a reduction in food intake observed in both genotypes. The improvement of glucose homeostasis was observed in db-/- mice at the expense of increased fat deposition, especially in the subcutaneous white adipose tissue (SCWAT), without sign of worsened inflammation or insulin responsiveness and with lowered circulating triglycerides and uric acid, while NO bioavailability was increased in aorta. The higher capacity of SSAO in oxidizing Bza in SCWAT, found in the obese mice, was unaltered by Bza supplementation and likely involved in the activation of glucose utilization by adipocytes. We propose that Bza oxidation in tissues, which produces hydrogen peroxide mainly in SCWAT, facilitates insulin-independent glucose utilization. Bza could be considered as a potential agent for dietary supplementation aiming at preventing diabetic complications.

Vanadium-dependent activation of glucose transport in adipocytes by catecholamines is not mediated via adrenoceptor stimulation or monoamine oxidase activity.

BACKGROUNDBenzylamine and methylamine activate glucose uptake in adipocytes. For tyramine, this effect has even been extended to cardiomyocytes.AIMTo investigate the effects of catecholamines and other amines on glucose uptake.METHODSA screening compared 25 biogenic amines on 2-deoxyglucose (2-DG) uptake activation in rat adipocytes. Pharmacological approaches and transgenic mouse models were then used to decipher the mode of action of several hits.RESULTSIn rat adipocytes, insulin stimulation of 2-DG uptake was reproduced with catecholamines. 100 µmol/L or 1 mmol/L adrenaline, noradrenaline, dopamine and deoxyepinephrine, maximally activated hexose transport only when sodium orthovanadate was added at 100 µmol/L. Such activation was similar to that already reported for benzylamine, methylamine and tyramine, well-recognized substrates of semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidase (MAO). Several, but not all, tested agonists of β-adrenoreceptors (β-ARs) also activated glucose transport while α-AR agonists were inactive. Lack of blockade by α- and β-AR antagonists indicated that catecholamine-induced 2-DG uptake was not mediated by AR stimulation. Adipocytes from mice lacking β1-, β2- and β3-ARs (triple KO) also responded to millimolar doses of adrenaline or noradrenaline by activating hexose transport in the presence of 100 µmol/L vanadate. The MAO blocker pargyline, and SSAO inhibitors did not block the effects of adrenaline or noradrenaline plus vanadate, which were blunted by antioxidants.CONCLUSIONCatecholamines exert unexpected insulin-like actions in adipocytes when combined with vanadium. For limiting insulin resistance by activating glucose consumption at least in fat stores, we propose that catecholamine derivatives combined with vanadium can generate novel complexes that may have low toxicity and promising anti-diabetic properties.

Isolation of Lysyl Oxidase from Human Umbilical Cord

Five isoforms of human lysyl oxidases (LOX, LOXL1‐4) catalyze oxidative deamination of various primary amines and ε‐amino groups of lysine residues in proteins. LOX expression is up‐regulated by hypoxia in normal and cancer tissues. Usually, LOX is isolated from bovine aorta. However, authentic human enzyme should be preferred for pharmacological studies. We have optimized the aorta LOX protocol for human umbilical cord. The procedure includes homogenization, differential washes, urea extraction, absorption on hydroxyapatite and sulfoethylcellulose, followed by chromatography on DEAE‐cellulose and gel filtration. Simultaneously, semicarbazide sensitive amine oxidase (SSAO) can be also isolated using triton extraction followed by concanavalin A and ion exchange chromatography. Amine oxidase activities were measured as hydrogen peroxide release coupled to oxidation of 10‐acetyl‐3,7‐dihydrophenoxazine by horseradish peroxidase with methylamine as a SSAO substrate and free lysine or polyallylamine for LOX. In contrast with placenta, the cord lacks monoamine oxidase and diamine oxidase. In comparison with aorta, umbilical cord is poor in SSAO, whereas the yield of active lysyl oxidase is several folds higher. Immunoblotting using polyclonal antibodies against the five lysyl oxidase isoforms indicate that the isolated preparation contains predominantly LOX. In conclusion, the human umbilical cord is an excellent source of lysyl oxidase. Supported by RFBR grant 13‐04‐01413.

Long-Term Activation of Semicarbazide-Sensitive Amine Oxidase Lowers Circulating Levels of Uric Acid in Diabetic Conditions

Uric acid is involved in nitrogenous waste in animals, together with ammonia and urea. Uric acid has also antioxidant properties and is a surrogate marker of metabolic syndrome. We observed that the elevated plasma uric acid of high-fat fed mice was normalized by benzylamine treatment. Indeed, benzylamine is the reference substrate of semicarbazide-sensitive amine oxidase (SSAO), an enzyme highly expressed in fat depots and vessels, which generates ammonia when catalysing oxidative deamination. Ammonia interferes with uric acid metabolism/solubility. Our aim was therefore to investigate whether the lowering action of benzylamine on uric acid was related to an improvement of diabetic complications, or was connected with SSAO-dependent ammonia production. First, we observed that benzylamine administration lowered plasma uric acid in diabetic db/db mice while it did not modify uric acid levels in normoglycemic and lean mice. In parallel, benzylamine improved the glycemic control in diabetic but not in normoglycemic mice, while plasma urea remained unaltered. Then, uric acid plasma levels were measured in mice invalidated for AOC3 gene, encoding for SSAO. These mice were unable to oxidize benzylamine but were not diabetic and exhibited unaltered plasma uric levels. Therefore, activated or abolished ammonia production by SSAO was without influence on uric acid in the context of normoglycemia. Our observations confirm that plasma uric acid increases with diabetes and can be normalized when glucose tolerance is improved. They also show that uric acid, a multifunctional metabolite at the crossroads of nitrogen waste and of antioxidant defences, can be influenced by SSAO, in a manner apparently related to changes in glucose homeostasis.

Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1: a patent survey

Introduction: Vascular adhesion protein-1 (VAP-1)/semicarbazide-sensitive amine oxidase (SSAO) is an adhesion protein involved in leukocyte trafficking and inflammatory processes, with a special amine oxidase activity. Inhibitors have been mainly developed for treating chronic inflammatory disorders. The utility of inhibitors as antiangiogenic agents in ophthalmological and oncological diseases is currently under evaluation. SSAO substrates may mimic several insulin effects, although their utility for the treatment of diabetes is still far from being fully understood.Areas covered: This paper reviews the patent literature of SSAO/VAP-1 inhibitors and substrates, for the period of 1990 – 2010. The current stage of SSAO/VAP-1-interacting agents published in patents is described, along with their chemical structures and pharmacological uses.Expert opinion: SSAO/VAP-1 is a promising anti-inflammatory target. Another important field for therapeutic application of these inhibitors may be ophthalmology, due to their antiangiogenic effects. SSAO substrates might also be of therapeutic value in the treatment of diabetes; however, more extensive research has to be undertaken to validate this approach.

Properties of a semicarbazide-sensitive amine oxidase in human umbilical artery.

The metabolism of some aromatic amines by amine oxidase activities in human umbilical artery homogenates has been studied. The inhibitory effects of clorgyline showed that 5-hydroxytryptamine (5-HT) and tryptamine, 1 mM, were predominantly substrates for monoamine oxidase (MAO) type A, whereas MAO-A and B were both involved in the metabolism of beta-phenylethylamine (PEA), 100 microM, and tyramine, 1 mM. About 20-30% of tyramine and PEA metabolism was resistant to 1 mM clorgyline, but sensitive to inhibition by semicarbazide, 1 mM, indicating the presence of a semicarbazide-sensitive amine oxidase (SSAO). Benzylamine, 1 mM, appeared to be metabolized exclusively by SSAO with a Km (161 microM) at pH 7.8 similar to that found for SSAO in other human tissues. Tyramine and PEA were relatively poor substrates for SSAO, with very high apparent Km values of 17.6 and 13.3 mM, respectively, when determined in the presence of clorgyline, 10(-3) M, added to inhibit any metabolism of those amines by MAO activities. However, kinetic studies with benzylamine indicated that clorgyline, 10(-3) M, also appears to inhibit SSAO competitively such that the true Km values for tyramine and PEA may be about 60% of those apparent values given above. No evidence for the metabolism of 5-HT or tryptamine by SSAO was obtained. The aliphatic amine methylamine was recently shown to be a specific substrate for SSAO in umbilical artery homogenates. We have used benzylamine and methylamine as SSAO substrates in histochemical studies to localize SSAO in tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative deamination of aliphatic amines by rat aorta semicarbazide-sensitive amine oxidase

Rat aorta semicarbazide-sensitive amine oxidase (SSAO) exhibits very high affinity in the deamination of an homologous series of aliphatic amines of 1 to 18 straight chain carbon atoms. The Km value decreases substantially as the chain length of these amines increases. The Vmax values are higher for the short chain amines. Diamines are poor substrates for SSAO or are not acted upon by the enzyme. The substrate preference for SSAO differs from that for monoamine oxidase.

Further studies on the ex-vivo effects of procarbazine and monomethylhydrazine on rat semicarbazide-sensitive amine oxidase and monoamine oxidase activities.

Following administration of the anticancer agent, procarbazine, or one of its metabolites, monomethylhydrazine, to rats, activities of monoamine oxidases A and B (MAO A and MAO B) and of semicarbazide-sensitive amine oxidase (SSAO) were measured ex-vivo. Both compounds were found to be potent inhibitors of SSAO in tissue homogenates, exhibiting ID50 values in most tissues of approximately 8 mg kg-1 (procarbazine) and 0.08 mg kg-1 (monomethylhydrazine). Concurrent dose-dependent inhibition of MAO activities did not occur. However, in liver, potentiation of MAO B activity, to 140% of that in controls, was apparent following monomethyl-hydrazine and this effect was independent of the drug dose. Both compounds produced a dose-dependent potentiation of MAO A in brown adipose tissue, the elevation being more pronounced following monomethylhydrazine, with activity rising to 350% of that in control homogenates. In a parallel in-vitro study, monomethylhydrazine was without effect on MAO A in brown adipose tissue homogenates. By perfusing the SSAO substrate, benzylamine, through the isolated mesenteric arterial bed of the rat, it was found that pretreatment of animals with procarbazine or monomethylhydrazine reduced metabolism of this amine by a similar degree as had been determined ex-vivo in blood vessel homogenates. The results presented suggest that these compounds would be suitable for use as selective inhibitors in pharmacological examinations of SSAO function in isolated tissues and organs.

Benzylamine and methylamine, substrates of semicarbazide-sensitive amine oxidase, attenuate inflammatory response induced by lipopolysaccharide

Current evidence indicates that semicarbazide-sensitive amine oxidase (SSAO) substrates possess insulin-mimic effect, which was thought to play an anti-inflammatory role. The purpose of the present study was to determine whether SSAO substrates benzylamine (BZA) and methylamine (MA) attenuate inflammatory response induced by lipopolysaccharide (LPS). BALB/c mice peritoneal macrophages (PMs) that express SSAO and RAW264.7 mouse macrophages that do not express SSAO were used in vitro studies. Experimental mice were given BZA or MA through intraperitoneal injection before LPS challenge. The results showed that BZA or MA treatment significantly reduced LPS-induced pro-inflammatory mediators (nitric oxide, TNF-α) production, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and glucose consumption in murine PMs, but not in RAW264.7 cell line. The metabolites of BZA or MA catalyzed by SSAO, hydrogen peroxide, formaldehyde, and benzaldehyde could also significantly decrease LPS-induced nitric oxide and TNF-α production, iNOS and COX-2 expression, and glucose consumption in vitro. In addition, BZA or MA administration could significantly decrease plasma pro-inflammatory mediators and the expression of iNOS and COX-2 in liver and lung, and could also attenuate LPS-induced transient hyperglycemia and chronic hypoglycemia. These findings indicated that substrates of SSAO might be involved in the anti-inflammatory effects. The metabolites of BZA and MA catalyzed by SSAO might be responsible for the anti-inflammatory effects. Moreover, BZA or MA administration could be useful for normalization of glucose disposal during endotoxemia.

Oral Administration of Semicarbazide Limits Weight Gain together with Inhibition of Fat Deposition and of Primary Amine Oxidase Activity in Adipose Tissue

An enzyme hitherto named semicarbazide-sensitive amine oxidase (SSAO), involved in the oxidation of primary amines, is abundantly expressed in adipocytes. Although SSAO physiological functions remain unclear, several molecules inhibiting its activity have been described to limit fat accumulation in preadipocyte cultures or to reduce body weight gain in obese rodents. Here, we studied whether oral administration of semicarbazide, a prototypical SSAO inhibitor, limits fat deposition in mice. Prolonged treatment with semicarbazide at 0.125% in drinking water limited food and water consumption, hampered weight gain, and deeply impaired fat deposition. The adiposomatic index was reduced by 31%, while body mass was reduced by 15%. Such treatment completely inhibited SSAO, but did not alter MAO activity in white adipose tissue. Consequently, the insulin-like action of the SSAO substrate benzylamine on glucose transport was abolished in adipocytes from semicarbazide-drinking mice, while their insulin sensitivity was not altered. Although semicarbazide is currently considered as a food contaminant with deleterious effects, the SSAO inhibition it induces appears as a novel concept to modulate adipose tissue development, which is promising for antiobesity drug discovery.

Antioxidant and Amine Oxidase Inhibitory Activities of Hydroxyurea

Hydroxyurea (HU, NH(2)CONHOH), or hydroxycarbamide, is a hydroxamic acid derivative used as a drug for anti-neoplasm and sickle-cell disease. In this study, HU was found to have antioxidant activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals and dose-dependent inhibitory activities against monoamine oxidase (MAO)-A, MAO-B, and semicarbazide-sensitive amine oxidase (SSAO) as compared to controls of clorgyline, deprenyl, and semicarbazide respectively. HU showed mixed-type, competitive-type, and competitive-type inhibition, respectively, with respect to substrates of MAO-A, MAO-B, and SSAO with apparent inhibition constants (Ki) of 19.46, 5.38, and 1.84 microM.

Modulation of Endothelium-Dependent Relaxation of Equine Digital Arteries By Insulin and Hypoxia

Modulation of Endothelium-Dependent Relaxation of Equine Digital Arteries By Insulin and Hypoxia

Arylalkylamine vanadium salts as new anti-diabetic compounds

Vanadium compounds show insulin-like effects in vivo and in vitro. Several clinical studies have shown the efficacy of vanadium compounds in type 2 diabetic subjects. However, a major concern is safety, which calls for the development of more potent vanadium compounds. For that reason different laboratories develop strategies to decrease the therapeutic dose of vanadate. One of these strategies use substrates of semicarbazide-sensitive amine oxidase (SSAO)/vascular adhesion protein-1 (VAP-1), a bifunctional protein with amine oxidase activity and adhesive properties implicated in lymphocyte homing at inflammation sites. Substrates of SSAO combined with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 glucose transporter recruitment to the plasma membrane in 3T3-L1 adipocytes and in rat adipocytes. This combination also shows anti-diabetic effects in various animal models of type 1 and type 2 diabetes. Benzylamine/vanadate administration generates peroxovanadium locally in pancreatic islets, which stimulates insulin secretion, and also produces peroxovanadium in adipose tissue, thereby activating glucose metabolism in adipocytes and in neighboring muscle. This opens up the possibility of using the SSAO/VAP-1 activity as a local generator of protein tyrosine phosphatase inhibitors in anti-diabetic therapy. More recently a novel class of arylalkylaminevanadium salts have shown potent insulin-mimetic effects downstream of the insulin receptor. Administration of these compounds lowers glycemia and normalizes the plasma lipid profile in type 1 and type 2 models of diabetes. The combination of different approaches to decrease vanadium doses, among them chelating agents and SSAO substrates, should permit to develop safe and efficient vanadium based agents safe for diabetes treatment.

P4-299: Neuron-binding autoantibodies in human sera enhance Abeta42 accumulation in adult mouse brain neurons

of Alzheimer’s disease (AD), and it is characterized by the deposition of beta-amyloid (A ) in cerebral cortical and meningeal blood vessels (Kawai M. et al., 1993), inducing degeneration of vascular cells. Semicarbazide-sensitive amine oxidase (SSAO) [E.C. 1.4.3.6] is present in vascular cells and in plasma. It metabolizes primary amines (Lyles GA., 1996) generating hydrogen peroxide (H2O2), ammonia (NH3) and the corresponding aldehyde, that contribute to the oxidative stress, advanced glycation end-product generation (GubisneHaberle D. et al., 2004), and beta amyloid aggregation (Chen K. et al., 2006). Furthermore in endothelial cells, SSAO is induced under inflammatory conditions (Smith D.J. et. al., 1998). We have reported that SSAO is overexpressed in cerebrovascular tissue of patients with CAA-AD, and that it colocalizes with beta-amyloid deposits (Ferrer I. et al., 2002). This over-expression correlates with high SSAO activity in plasma of severe AD patients (del Mar Hernandez M. et al., 2005). We have also described that plasma SSAO is able to induce apoptosis in vascular cells (Hernandez M. et.al., 2006). The aim of this work is to demonstrate whether A is able to induce SSAO overexpression in HUVEC cells as vascular cell type. Methods: Because of the SSAO/ VAP-1 expression fenotype is lost in cultured cells, HUVEC (human umbilical vein endothelial cells) cells, were stably transfected with vector pcDNA 3.1 containing hVAP-1/SSAO. Cells were treated with A 1-40 Dutch type (mutation E22Q) and/or Methylamine as SSAO substrate. Cell viability, SSAO activity and its expression were determined using specific antibodies against SSAO. Results: Herein we report that A 1-40 E22Q induces the SSAO activity and expression in HUVEC cells. This increasing activity promotes oxidative stress that enhances the toxicity generated by A alone. This toxicity is reverted by specific SSAO inhibitors, confirming SSAO as the responsible of such effect. Conclusions: These results allow us to postulate that deposits of A in cerebrovascular tissue, induce SSAO expression that may contribute to the vascular damage associated to CAA-AD.

Reduction of fat deposition by combined inhibition of monoamine oxidases and semicarbazide-sensitive amine oxidases in obese Zucker rats

Semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidases (MAO) are highly expressed in adipocytes and generate hydrogen peroxide when activated. Consequently, high concentrations of MAO- or SSAO-substrates acutely stimulate glucose transport and inhibit lipolysis in isolated adipocytes in a hydrogen peroxide-dependent manner. Chronic treatments with MAO and SSAO substrates also increase in vitro adipogenesis and in vivo glucose utilization and fat deposition in diabetic rodents. To further investigate the interplay between amine oxidases, energy balance and fat deposition, prolonged MAO and/or SSAO blockade was performed in obese rats. Pargyline (P, MAO inhibitor), semicarbazide (S, SSAO inhibitor), alone or in combination (P + S), were daily i.p. administered for 3–5 weeks to obese Zucker rats at doses ranging from 20 to 300 μmol/kg. P + S treatments abolished MAO and SSAO activities in any tested tissue. P and S led to a 12–17% reduction of food intake when given in combination but were inactive when given separately. Despite a similar body weight gain reduction in P + S-treated and pair-fed rats, the mitigation of fat deposition was greater in rats receiving both inhibitors. Adipocytes from P + S-treated rats responded as control to insulin but exhibited impaired responses to tyramine, benzylamine or methylamine plus vanadate when considering glucose transport activation or lipolysis inhibition. Although our results did not directly demonstrate that amines are able to spontaneously produce in vivo the insulin-like effects described in vitro, we propose that P + S-induced reduction of fat deposition results from decreased food intake and from impaired MAO- and SSAO-dependent lipogenic and antilipolytic actions of endogenous or alimentary amines.

Understanding the Mechanism of Action of the Novel SSAO Substrate (C7NH10)6(V10O28)·2H2O, a Prodrug of Peroxovanadate Insulin Mimetics

A new vanadium salt, hexakis(benzylammonium) decavanadate (V) dihydrate (C(7)NH(10))(6)(V(10)O(28)).2H(2)O (1), has been synthesized as well as characterized chemically and biologically. An in vitro enzyme assay revealed that compound 1 is oxidized to the same extent as a combination of benzylamine and vanadate by the enzyme semicarbazide-sensitive amine oxidase (SSAO), and therefore can be considered an SSAO substrate. It also stimulates glucose uptake in isolated rat adipocytes in a dose-dependent manner. We describe here the results of (51)V-NMR experiments that, combined with the in vitro results, corroborate that compound 1 could act as a prodrug of di-peroxovanadate ([V(OH)(2)(OO)(2)(OH)(2)](2-)) insulin mimetics.

Prolonged treatment with aminoguanidine strongly inhibits adipocyte semicarbazide-sensitive amine oxidase and slightly reduces fat deposition in obese Zucker rats

Beneficial effects of aminoguanidine (AG) on diabetic vascular complications result from prevention of protein glycation, inhibition of inductible NO synthase, and inhibition of vascular semicarbazide-sensitive amine oxidase (SSAO). However, influence of AG on adipose tissue deposition has been poorly investigated in obesity. Considering that SSAO is highly expressed in fat cells, and that a SSAO blocker has been recently reported to reduce body weight gain in obese mice, this work aimed to investigate the influence of AG on adipose tissue functions. First, AG was shown to directly inhibit SSAO activity in cultured adipocytes. Although AG did not directly alter lipolytic activity in human adipocytes, it inhibited benzylamine-induced antilipolysis via SSAO (but not NO synthase) inhibition. When AG was i.p. administered to obese Zucker rats (270 μmol kg −1 day −1 for 3 weeks), treated rats lost their capacity to oxidize benzylamine in a SSAO-dependent manner in adipose tissues and in cerebral vessels. Monoamine oxidase activity was unmodified in liver, skeletal muscles or adipose tissues and tended to increase in brain vessels. AG-treatment did not change body weight gain or hyperinsulinemic state of obese rats but slightly reduced subcutaneous fat deposition. AG did not modify insulin responsiveness in adipocytes but impaired the effects of SSAO substrates, such as glucose transport activation and lipolysis inhibition by methylamine or benzylamine plus vanadate. These results show that complete impairment of SSAO activity produced by AG-treatment in obese rats was likely responsible for a weak limitation of fat deposition. Previously proposed for prophylaxis in diabetes, AG may be useful for treating obesity via its SSAO blocking properties.

Semicarbazide-sensitive amine oxidase substrates fail to induce insulin-like effects in fat cells from AOC3 knockout mice

Substrates of semicarbazide-sensitive amine oxidases (SSAO) stimulate glucose transport in adipocytes. To definitively demonstrate the involvement of SSAO in this insulin-like effect, glucose transport has been studied in fat cells from mice with a targeted deletion of AOC3, a gene encoding a SSAO called vascular adhesion protein-1. SSAO activity was present in white adipose tissues of wild type (WT) but was absent in AOC3KO mice. The SSAO-substrates benzylamine and methylamine were unable to stimulate hexose transport in adipocytes isolated from AOC3KO mice while they were active in WT adipocytes, especially in combination with vanadate. Impairment of amine-dependent glucose uptake was also observed with tyramine while there was no change in insulin responsiveness. These observations prove that the effects of exogenous or biogenic amines on glucose transport are not receptor-mediated but are oxidation-dependent. They also confirm that the major SSAO form expressed in mouse adipocytes is encoded by the AOC3 gene.