Inhibition of rat fat cell lipolysis by monoamine oxidase and semicarbazide-sensitive amine oxidase substrates

Abstract

It has been demonstrated that amine oxidase substrates stimulate glucose transport in cardiomyocytes and adipocytes, promote adipogenesis in pre-adipose cell lines and lower blood glucose in diabetic rats. These insulin-like effects are dependent on amine oxidation by semicarbazide-sensitive amine oxidase or by monoamine oxidase. The present study aimed to investigate whether amine oxidase substrates also exhibit another insulin-like property, the inhibition of lipolysis. We therefore tested the influence of tyramine and benzylamine on lipolytic activity in rat adipocytes. These amines did not modify basal lipolysis but dose-dependently counteracted the stimulation induced by lipolytic agents. The response to 10 nM isoprenaline was totally inhibited by tyramine 1 mM. The blockade produced by inhibition of amine oxidase activity or by 1 mM glutathione suggested that the generation of oxidative species, which occurs during amine oxidation, was involved in tyramine antilipolytic effect. Among the products resulting from amine oxidation, only hydrogen peroxide was antilipolytic in a manner that was potentiated by vanadate, as for tyramine or benzylamine. Antilipolytic responses to tyramine and to insulin were sensitive to wortmannin. These data suggest that inhibition of lipolysis is a novel insulin-like effect of amine oxidase substrates which is mediated by hydrogen peroxide generated during amine oxidation.

Introduction

The presence of amine oxidase activity in adipose cells has been substantiated in different mammalian species Barrand and Callingham, 1982, Raimondi et al., 1991, Tong et al., 1979 including humans Morin et al., 2001, Pizzinat et al., 1999, but its biological role remains to be clarified. Two families of amine oxidases are involved in amine oxidation: the monoamine oxidases A and B (E.C. 1.4.3.4) (Bach et al., 1988), which are flavoproteins present in the outer mitochondrial membrane, and the copper-containing amine oxidases, among which the semicarbazide-sensitive amine oxidases (E.C. 1.4.3.6) are ectoenzymes located at the cell surface (Salminen et al., 1998). Despite their different subcellular location, monoamine oxidase and semicarbazide-sensitive amine oxidase exhibit only discrete differences regarding their substrate specificity. For instance, tyramine, which is present in food and beverages (Kopin, 1993), is a common substrate for monoamine oxidase A, monoamine oxidase B and semicarbazide-sensitive amine oxidase in rats but is only a monoamine oxidase substrate in humans, while benzylamine and methylamine can be considered as relatively selective substrates for semicarbazide-sensitive amine oxidase in both species (Lyles, 1996). The pharmacological distinction between mono- and semicarbazide-sensitive amine oxidases has been essentially established according to inhibitor selectivity: semicarbazide-sensitive amine oxidase is inhibited by carbonyl reagents like semicarbazide but is resistant to most of the selective monoamine oxidase inhibitors (pargyline, clorgyline, deprenyl).

In addition to the classical scavenger function attributed to the amine oxidases of different anatomical locations, the monoamine oxidase and semicarbazide-sensitive amine oxidase present on adipose cells have been recently suspected to participate in the regulation of carbohydrate metabolism on the basis of a growing number of observations. First, a stimulation of glucose transport by benzylamine or tyramine has been reported in rat fat cells Marti et al., 1998, Enrique-Tarancon et al., 1998. Then, semicarbazide-sensitive amine oxidase activity has been proposed to mediate the stimulation of glucose uptake by benzylamine in human adipocytes (Morin et al., 2001), while monoamine oxidase is involved in the serotonin-induced activation of glucose uptake into rat cardiomyocytes (Fischer et al., 1995). Similarly, the insulin-like differentiating effects of benzylamine, tyramine (Fontana et al., 2001) or methylamine (Mercier et al., 2001) on the pre-adipose cells lines 3T3 F442A and 3T3 L1 have been demonstrated to be mediated via semicarbazide-sensitive amine oxidase-dependent oxidation. Finally, tyramine (Morin et al., 2002) and benzylamine (Marti et al., 2001) were found to enhance glucose disposal in vivo. One common mechanism in all these biological responses was the involvement of hydrogen peroxide generated during oxidative deamination, since all these responses were blocked by amine oxidase inhibitors or by catalase and/or glutathione as well as by other antioxidant systems. In rat adipocytes, the presence of vanadate at 0.1 mM allowed both monoamine oxidase and semicarbazide-sensitive amine oxidase substrates to activate the translocation of glucose transporters to the cell surface and to stimulate glucose uptake up to one half the maximal effect of insulin Marti et al., 1998, Enrique-Tarancon et al., 1998. The potentiation of amine effects by vanadate was likely due to an interaction between the hydrogen peroxide resulting from amine oxidase activity and vanadate, and to the subsequent generation of peroxovanadate (Enrique-Tarancon et al., 2000), a powerful tyrosine phosphatase inhibitor (Huyer et al., 1997) and a potent insulin-mimicking agent (Lönnroth et al., 1993).

Since insulin not only stimulates glucose transport but also inhibits lipolysis, we asked ourselves whether tyramine or benzylamine is also able to inhibit lipolysis in a manner dependent on oxidation by monoamine oxidase or semicarbazide-sensitive amine oxidase. Because hydrogen peroxide inhibits lipolysis in rodent fat cells (Little and De Haën, 1980) and because all the metabolic effects of amine oxidase substrates described so far were somewhat insulin-like, an antilipolytic effect was expected. Although this was confirmed by our observation of an antilipolytic effect of benzylamine in human fat cells (Morin et al., 2001), the opposite was reported in the literature, where tyramine has been shown to increase the lipolytic response to isoprenaline in rat adipocytes (Raimondi et al., 2000b). This discrepancy led us to conduct a pharmacological analysis of the effects of tyramine and benzylamine on the lipolytic activity of rat adipocytes. To this end, we tested these monoamine oxidase and semicarbazide-sensitive amine oxidase substrates alone, and against different lipolytic agents. Since the oxidation of a given amine by either monoamine oxidase or semicarbazide-sensitive amine oxidase generates not only hydrogen peroxide but also the corresponding aldehyde and ammonia, we also tested these oxidation products.

The results indicate that 0.1–1 mM tyramine or benzylamine, which are known to activate glucose uptake (Enrique-Tarancon et al., 2000) and to produce hydrogen peroxide (Raimondi et al., 2000b) in rat fat cells, caused an inhibition of lipolysis which was reversed by amine oxidase blockers or by glutathione. Among the end-products of deaminative oxidation, only hydrogen peroxide was able to induce an antilipolytic response that was enhanced by vanadate.