The previous characterization of an abundant population of non-adrenergic imidazoline-I 2 binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle.Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells.

The hallmarks of insulin action are the stimulation and suppression of anabolic and catabolic responses, respectively. These responses are orchestrated by the insulin pathway and are initiated by the binding of insulin to the insulin receptor, which leads to activation of the receptor's intrinsic tyrosine kinase. Severe defects in the insulin pathway, such as in types A and B and advanced type 1 and 2 diabetes lead to severe insulin resistance, resulting in a partial or complete absence of response to exogenous insulin and other known classes of antidiabetes therapies. We have characterized a novel class of arylalkylamine vanadium salts that exert potent insulin-mimetic effects downstream of the insulin receptor in adipocytes. These compounds trigger insulin signaling, which is characterized by rapid activation of insulin receptor substrate-1, Akt, and glycogen synthase kinase-3 independent of insulin receptor phosphorylation. Administration of these compounds to animal models of diabetes lowered glycemia and normalized the plasma lipid profile. Arylalkylamine vanadium compounds also showed antidiabetic effects in severely diabetic rats with undetectable circulating insulin. These results demonstrate the feasibility of insulin-like regulation in the complete absence of insulin and downstream of the insulin receptor. This represents a novel therapeutic approach for diabetic patients with severe insulin resistance. Diabetes 56: 486 -493, 2007

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

Semicarbazide-sensitive amine oxidases (SSAO) are copper-containing enzymes that oxidatively deaminate primary amines to produce hydrogen peroxide, ammonium, and specific aldehydes. Vascular adhesion protein-1 (VAP-1) is a cell surface and soluble molecule that possesses SSAO activity. VAP-1 protein, SSAO activity, and SSAO reaction products are elevated in the serum of patients with diabetes, congestive heart failure, and specific inflammatory liver diseases. By expressing human VAP-1/SSAO on mouse endothelial cells and subsequently in the serum, and by chronically treating the transgenic mice for 15 months with a high-fat diet and a physiological substrate for SSAO, methylamine, the in vivo roles of SSAO were assessed. The VAP-1 transgene increased the mouse body mass index and subcutaneous abdominal fat pad weights in a manner independent of food consumption. The transgene together with increased SSAO substrate availability enhanced glucose uptake in an SSAO-dependent manner. The increased SSAO activity also led to diabetes-like complications, including advanced glycation end product formation, elevated blood pressure, altered atherosclerosis progression, and nephropathy. These findings suggest that, although manipulation of VAP-1/SSAO has potential to serve as a therapeutic treatment in insulin-resistant conditions, care must be taken to fully understand its impact on obesity and vascular damage.

Aims/hypothesis. Vascular adhesion protein-1 (VAP-1), which is identical to semicarbazide-sensitive amine oxidase (SSAO), is a dual-function membrane protein with adhesion properties and amine oxidase activity. A soluble form of VAP-1 is found in serum, where concentrations are enhanced in diabetes and obesity. In vitro, soluble VAP-1 enhances lymphocyte adhesion to endothelial cells, thus possibly participating in the enhanced lymphocyte adhesion capacity that is implicated in the cardiovascular complications associated with diabetes or obesity. In both, the tissue origin of the soluble VAP-1/SSAO is unknown. We examined whether adipose tissue, which has abundant expression of VAP-1/SSAO, is a source of soluble VAP-1. Methods. We detected VAP-1/SSAO in plasma of diabetic animals, with or without VAP-1 immunoprecipitation, and in culture medium from 3T3-L1 adipocytes and human adipose tissue explants. VAP-1 protein glycosylation was measured.Results. Diabetic and obese animals have increased plasma SSAO activity associated with VAP-1 protein.We also found that 3T3-L1 adipocytes and human adipose tissue explants release a soluble form of VAP-1/SSAO, which derives from the membrane. The release of soluble VAP-1 was enhanced by exposure of murine and human adipocytes to TNF-α and blocked by batimastat, a metalloprotease inhibitor. Partial ablation of adipose tissue reduced plasma SSAO activity in normal and diabetic rats.

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Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO, such as benzylamine, in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in 3T3-L1 and rat adipocytes. Here we examined whether acute and chronic administration of benzylamine and vanadate in vivo enhances glucose tolerance and reduces hyperglycemia in diabetic rats. Acute intravenous administration of these drugs enhanced glucose tolerance in nondiabetic rats and in streptozotocin (STZ)-induced diabetic rats. This occurred in the absence of changes in plasma insulin concentrations. However, the administration of benzylamine or vanadate alone did not improve glucose tolerance. The improvement caused by benzylamine plus vanadate was abolished when rats were pretreated with the SSAOinhibitor semicarbazide. Chronic administration of benzylamine and vanadate exerted potent antidiabetic effects in STZ-induced diabetic rats. Although daily administration of vanadate alone (50 and 25 mol ⅐ kg ؊1 ⅐ day ؊1 i.p.) for 2 weeks had little or no effect on glycemia, vanadate plus benzylamine reduced hyperglycemia in diabetic rats, enhanced basal and insulin-stimulated glucose transport, and upregulated GLUT4 expression in isolated adipocytes. In all, our results substantiated that acute and chronic administration of benzylamine with low dosages of vanadate have potent antidiabetic effects in rats.

In this study we have explored whether the bifunctional protein semicarbazide-sensitive amine oxidase (SSAO)/ vascular adhesion protein-1 (VAP-1) represents a novel target for type 2 diabetes. To this end, Goto-Kakizaki (GK) diabetic rats were treated with the SSAO substrate benzylamine and with low ineffective doses of vanadate previously shown to have antidiabetic effects in streptozotocin-induced diabetic rats. The administration of benzylamine in combination with vanadate in type 2 diabetic rats acutely stimulated glucose tolerance, and the chronic treatment normalized hyperglycemia, stimulated glucose transport in adipocytes, and reversed muscle insulin resistance. Acute in vivo administration of benzylamine and vanadate stimulated skeletal muscle glucose transport, an effect that was also observed in incubated muscle preparations coincubated with adipose tissue explants or with human recombinant SSAO. Acute administration of benzylamine/vanadate also ameliorated insulin secretion in diabetic GK rats, and this effect was also observed in incubated pancreatic islets. In keeping with these observations, we also demonstrate that pancreatic islets express SSAO/ VAP-1. As far as mechanisms of action, we have found that benzylamine/vanadate causes enhanced tyrosine phosphorylation of proteins and reduced protein tyrosine phosphatase activity in adipocytes. In addition, incubation of human recombinant SSAO, benzylamine, and vanadate generates peroxovanadium compounds in vitro. Based on these data, we propose that benzylamine/vanadate administration generates peroxovanadium locally in pancreatic islets, which stimulates insulin secretion and also produces peroxovanadium in adipose tissue, activating glucose metabolism in adipocytes and in neighboring muscle. This opens the possibility of using the SSAO/VAP-1 activity as a local SSAO/VAP-1 is expressed in a variety of tissues, and under normal conditions expression is high in adipose cells. In isolated rat adipocytes, SSAO/VAP-1 is mainly at the plasma membrane, and nearly 17 ϫ 10 6 copies of this protein are present at the cell surface in a single adipocyte (5,6). In contrast, SSAO activity is very low or absent in other insulin-responsive tissues such as skeletal muscle or heart (7). In fact, it has been demonstrated that SSAO/ VAP-1 is not expressed in 3T3-L1 fibroblasts and that SSAO/VAP-1 gene expression is induced during adipogenesis (8). This finding is in complete agreement with the previous observation of an increase in the SSAO activity of stroma-vascular preadipocytes from rat adipose tissue during their conversion into adipocytes when cultured in vitro (9). This suggests that SSAO/VAP-1 is a member of the adipogenic gene program and, in addition, that SSAO/ VAP-1 may contribute to the acquisition of some final characteristics of fully differentiated adipose cells. Most of the SSAO/VAP-1 expressed in rat adipocytes is found in plasma membrane (5,6). Thus, subcellular fractionation of membranes from 3T3-L1 adipocytes or isolated rat adipocytes has demonstr...