Hydrogen peroxide generation by monoamine oxidases in rat white adipocytes: role on cAMP production

In rat, white adipocytes monoamine oxidases (EC 1.4.3.4.) generate hydrogen peroxide (H(2)O(2)). Recent studies suggested that, in addition to its toxic features, H(2)O(2) may behave as a cell second messenger. In the present study, using fluorimetric and chemiluminescence (CL) assays, we showed that tyramine degradation by monoamine oxidases in intact adipocytes resulted in the concentration-dependent generation of H(2)O(2). In addition, we found that, in the presence of low tyramine concentrations, forskolin-dependent cAMP production was significantly increased as compared to that of the control and this increase was prevented by the monoamine oxidase inhibitor pargyline or by the H(2)O(2) trapping system homovanillic acid-peroxidase. Finally, we demonstrated that tyramine degradation by monoamine oxidases increased the ability of isoproterenol to induce cell lipolysis. Taken together, these data suggest that H(2)O(2) produced during substrate degradation by monoamine oxidases may participate in the regulation of adipocyte metabolism.     

Binding of rasagiline-related inhibitors to human monoamine oxidases: a kinetic and crystallographic analysis

Monoamine oxidases A and B (MAO A and B) catalyze the degradation of neurotransmitters and represent drug targets for the treatment of neurodegenerative disorders. Rasagiline is an irreversible, MAO B-selective inhibitor that has been approved as a novel anti-Parkinson's drug. In this study, we investigate the inhibition of recombinant human MAO A and MAO B by several rasagiline analogues. Different substituents added onto the rasagiline scaffold alter the binding affinity depending on the position on the aminoindan ring and on the size of the substituent. Compounds with a hydroxyl group on either the C4 or the C6 atom inhibit both isozymes, whereas a bulkier substituent such as a carbamate is tolerated only at the C4 position. The 1.7 A crystal structure of MAO B in complex with 4-(N-methyl-N-ethyl-carbamoyloxy)-N-methyl-N-propargyl-1(R)-aminoindan shows that the binding mode is similar to that of rasagiline with the carbamate moiety occupying the entrance cavity space. 1(R)-Aminoindan, the major metabolic product of rasagiline, and its analogues reversibly inhibit both MAO A and MAO B. The crystal structure of N-methyl-1(R)-aminoindan bound to MAO B shows that its aminoindan ring adopts a different orientation compared to that of rasagiline.     

microRNA-32 promotes white fat browning through augmenting FGF21 expression in brown adipose tissue

OBJECTIVE To investigate the effect of microRNA-32 on cold-induced thermogenesis and brown adipocyte energy metabolism.METHODS To apply the cold-induced thermogenesis model in mice,8-10 week old male C57Bl6 mice were placed within a 6℃fridge for 7d.Control microRNA inhibitor or miR-32 inhibitor(10mg·kg-1)was administered via intraperitoneal injection 16 hbefore the mice were placed in the fridge.Daily core body temperatures were taken using a rectal temperature probe.Mice were euthanized after 7dand brown adipose tissue(BAT),inguinal and epididymal white adipose tissue(WAT),skeletal muscle and liver tissue analysed for changes in morphology and gene expression.RESULTS miR-32 inhibition in vivoinhibits the emergence of beige cells,which function like BAT cells,within WAT.In silico prediction and gene ontology analysis identified Tob1 as a likely target gene of miR-32.miR-32 inhibition led to increased expression of Tob1 whilst mutation of target sequence abolished this effect.Expression of brown adipose markers such as Ucp1,Pgc1α,Pparαand Prdm16 were significantly reduced in inguinal white adipose tissue(P<0.05).There was also a significant decrease in serumfgf21 levels due to the inhibition of Fgf21 expression in BAT(P<0.05).p38/MAPK signalling in brown adipose tissue was also significantly inhibited within brown adipose tissue leading to decreased fgf21 expression and secretion.CONCLUSION Our study shows that miR-32 plays a crucial role in stimulating beige cell emergence by activating p38/MAPK signalling during cold thermogenesis.miR-32 may prove effective as a treatment for obesity by activating cold-induced thermogenesis leading to increased energy metabolism.     

Estrogen regulation of adipose tissue functions: involvement of estrogen receptor isoforms

Adipose tissue has recently been described as one of the major endocrine gland that plays a role in energy homeostasis, lipid metabolism, immune response, and reproduction. An excess of white adipose tissue, caused by a complex interaction between genetic, hormonal, behavioral, and environmental factors, results in obesity: a heterogeneous disorder that predisposes humans to a variety of diseases. Among several hormones, estrogens promote, maintain, and control the typical distribution of body fat and adipose tissue metabolism through still unknown mechanisms. These steroids are known to regulate fat mass, adipose deposition and differentiation, and adipocyte metabolism. Moreover, estrogen deficiency results in increases in adipose tissue, preferentially in visceral fat, which would link obesity to the susceptibility of related disorders. In this review the role of estrogens in adipose tissue differentiation and in the protection against the onset of obesity will be discussed with particular attention being drawn to the underlying molecular mechanisms mediated by estrogen receptor isoforms ERalpha and ERbeta.     

Characterization and quantitation of monoamine oxidases A and B in mitochondria from human placenta

Monoamine oxidases (MAOs) A and B, flavin-containing enzymes found in the outer mitochondrial membrane, oxidize many important biogenic and xenobiotic amines. The two enzymes are expressed in many tissues, with some tissues containing primarily one form and others containing both. Although MAO in placental mitochondria is widely reported to be type A, some investigators have reported low levels of MAO B activity as well. Because placenta is considered the preferred source for purification of type A MAO, we have reinvestigated placental MAO by immunoblotting with monoclonal antibodies and active site labeling with the MAO-specific ligand [3H]pargyline. We have confirmed that placental mitochondrial preparations contain MAO A and low but significant MAO B catalytic activity, as judged by accepted pharmacological criteria (deprenyl-sensitive beta-phenylethylamine and benzylamine oxidation). Immunoblotting revealed polypeptides of sizes expected for both MAO A and B subunits in preparations of placental mitochondria, as well as in preparations of MAO A purified extensively from placenta by partitioning between dextran and polyethylene glycol polymers and chromatography on DEAE-Sepharose CL-6B. Both MAO A and B active sites could be quantitated in placenta by labeling mitochondrial preparations with the MAO-specific affinity ligand [3H] pargyline, followed by immunoprecipitation with MAO A- and MAO B-specific monoclonal antibodies. These results indicate that MAO B activity and protein is consistently present in mitochondrial preparations of human placenta.     

Interactions of monoamine oxidases with the antiepileptic drug zonisamide: specificity of inhibition and structure of the human monoamine oxidase B complex

The binding of zonisamide to purified, recombinant monoamine oxidases (MAOs) has been investigated. It is a competitive inhibitor of human MAO B (K(i) = 3.1 ± 0.3 μM), of rat MAO B (K(i) = 2.9 ± 0.5 μM), and of zebrafish MAO (K(i) = 30.8 ± 5.3 μM). No inhibition is observed with purified human or rat MAO A. The 1.8 ? structure of the MAO B complex demonstrates that it binds within the substrate cavity.     

Effect of ethanol on adrenaline-stimulated glucose uptake in rat white adipose tissue

The effect of ethanol on adrenaline-stimulated glucose uptake by rat white adipose tissue has been examined in vitro. Ethanol (3%) inhibited the stimulatory effect of adrenaline on glucose uptake whereas it failed to inhibit the effect of adrenaline on free fatty acid production. Addition of calcium (12·5mM) to the incubation medium restored adrenaline's effect on glucose uptake. Addition of propranolol also restored the effect of adrenaline inhibited by ethanol. Ethanol did not inhibit insulin-stimulated glucose uptake. These results suggest that ethanol modifies the coupling of the adrenoceptor to the glucose transport system in adipose tissue that is stimulated by adrenaline.     

A method for stabilization of monoamine oxidases in homogenates of rat intestine epithelium

In a homogenate of epithelium isolated from the small intestine of male Wistar rats, the amine oxidase activity with 10(-3)M tyramine was 9200 +/- 200 nmol (g tissue)-1 h-1 of which 91% was due to the A form of monoamine oxidase (MAO) and 9% to the B form. Semicarbazide-sensitive amine oxidase activity was not detected with either 10(-3)M tyramine or 10(-4)M benzylamine as substrate. However, it was detectable in the homogenate of the gut residue where the activity with 10(-4)M benzylamine was 3600 +/- 200 nmol (g tissue)-1 h-1. The MAO activity, in homogenates of epithelium prepared with 0.1 M sodium phosphate pH 7.4, was stable at 4 degrees C for at least 6 h whilst at minus 20 degrees C it decreased by 70% within 24 h. Incorporation of 10% (v/v) glycerol into the homogenization medium stabilized the enzymes. The total activity and proportions due to MAO-A and MAO-B and kinetic constants for tyramine and 5-hydroxytryptamine, did not alter during 5 weeks storage at -20 degrees C. The ability to store tissue homogenates should facilitate studies of intestinal amine oxidases.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Cold-induced changes in gene expression in brown adipose tissue: implications for the activation of thermogenesis

Brown adipose tissue (BAT) is the site of heat production (thermogenesis). This unique function is performed by uncoupling protein 1 (UCP1) specifically expressed in mitochondria of BAT. UCP1 dissipates the driving force of ATP synthesis, and thus causes heat production followed by energy expenditure. The thermogenic function of BAT has the role of maintaining body temperature under cold conditions. When animals are exposed to cold, the expression of UCP1 gene is increased to activate thermogenesis. To date, functional analysis of BAT has been focused on UCP1, because it plays an indispensable role in thermogenesis. However, the gene expression of not only UCP1 but also that of other genes in BAT is expected to be regulated to achieve effective thermogenesis. Our previous investigations showed increased expression of genes that encode several energy metabolic enzymes in the BAT of rats kept in the cold. These changes in gene expression imply that the enhancement of energy metabolism is needed to activate thermogenesis. Furthermore, various reports from studies focused on genes whose expression is changed in response to cold stimulation have provided new insights into the function of BAT. In this review, to understand the thermogenic function of BAT systematically, we have provided an overview of previous findings on changes in the expression of genes thought to be related to the activation of thermogenesis in BAT.     

Effect of ethanol on adrenaline-stimulated glucose uptake in rat white adipose tissue.

The effect of ethanol on adrenaline-stimulated glucose uptake by rat white adipose tissue has been examined in vitro. Ethanol (3%) inhibited the stimulatory effect of adrenaline on glucose uptake whereas it failed to inhibit the effect of adrenaline on free fatty acid production. Addition of calcium (12.5 mM) to the incubation medium restored adrenaline's effect on glucose uptake. Addition of propranolol also restored the effect of adrenaline inhibited by ethanol. Ethanol did not inhibit insulin-stimulated glucose uptake. These results suggest that ethanol modifies the coupling of the adrenoceptor to the glucose transport system in adipose tissue that is stimulated by adrenaline.     

Metabolism of zaleplon by human liver: evidence for involvement of aldehyde oxidase

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in precision-cut human liver slices and liver cytosol preparations. 2. Human liver slices metabolized ZAL to a number of products including 5-oxo-ZAL (M2), N-desethyl-5-oxo-ZAL (M1) and N-desethyl-ZAL (DZAL), the latter metabolite being known to be formed by CYP3A forms. 3. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (± SEM) K_m and V_max of 93 ± 18 mm and 317 ± 241 pmol/min/mg protein, respectively. 4. Using 16 individual human liver cytosol preparations a 33-fold variability in the metabolism of 80µM ZAL to M2 was observed. Correlations were observed between M2 formation and the metabolism of the aldehyde oxidase substrates phenanthridine (r² = 0.774) and phthalazine (r² = 0.460). 5. The metabolism of 80µM ZAL to M2 in liver cytosol preparations was markedly inhibited by the aldehyde oxidase inhibitors chlorpromazine, promethazine, hydralazine and menadione. Additional kinetic analysis suggested that chlorpromazine and promethazine were non-competitive inhibitors of M2 formation with K_i of 2.3 and 1.9 µM, respectively. ZAL metabolism to M2 was also inhibited by cimetidine. 6. Incubations conducted with human liver cytosol and H₂¹⁸O demonstrated that the oxygen atom incorporated into ZAL and DZAL to form M2 and M1, respectively, was derived from water and not from molecular oxygen. 7. In summary, by correlation analysis, chemical inhibition and H₂¹⁸O incorporation studies, ZAL metabolism to M2 in human liver appears to be catalysed by aldehyde oxidase. With human liver slices, ZAL was metabolized to products dependent on both aldehyde oxidase and CYP3A forms.     

Metabolism of zaleplon by human liver: evidence for involvement of aldehyde oxidase

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in precision-cut human liver slices and liver cytosol preparations. 2. Human liver slices metabolized ZAL to a number of products including 5-oxo-ZAL (M2), N-desethyl-5-oxo-ZAL (M1) and N-desethyl-ZAL (DZAL), the latter metabolite being known to be formed by CYP3A forms. 3. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (+/- SEM) K(m) and V(max) of 93 +/- 18 mm and 317 +/- 241 pmol/min/mg protein, respectively. 4. Using 16 individual human liver cytosol preparations a 33-fold variability in the metabolism of 80 micro M ZAL to M2 was observed. Correlations were observed between M2 formation and the metabolism of the aldehyde oxidase substrates phenanthridine (r(2) = 0.774) and phthalazine (r(2) = 0.460). 5. The metabolism of 80 micro M ZAL to M2 in liver cytosol preparations was markedly inhibited by the aldehyde oxidase inhibitors chlorpromazine, promethazine, hydralazine and menadione. Additional kinetic analysis suggested that chlorpromazine and promethazine were non-competitive inhibitors of M2 formation with K(i) of 2.3 and 1.9 micro M, respectively. ZAL metabolism to M2 was also inhibited by cimetidine. 6. Incubations conducted with human liver cytosol and H(2)(18)O demonstrated that the oxygen atom incorporated into ZAL and DZAL to form M2 and M1, respectively, was derived from water and not from molecular oxygen. 7. In summary, by correlation analysis, chemical inhibition and H(2)(18)O incorporation studies, ZAL metabolism to M2 in human liver appears to be catalysed by aldehyde oxidase. With human liver slices, ZAL was metabolized to products dependent on both aldehyde oxidase and CYP3A forms.     

Neuronal responses to noradrenaline in the cerebral cortex: evidence against the involvement of alpha 2-adrenoceptors.

The technique of microelectrophoresis was used to test the hypothesis that alpha 2-adrenoceptors are involved in mediating the excitatory responses of single neurones to noradrenaline in the somatosensory cerebral cortex of the rat. In the first series of experiments the effects of two alpha 2-adrenoceptor antagonists, yohimbine and idazoxan (RX-781094), were compared on excitatory responses to noradrenaline, phenylephrine and acetylcholine. The response to noradrenaline was not more susceptible to antagonism by these drugs than the response to the alpha 1-adrenoceptor stimulant, phenylephrine. Yohimbine antagonized responses to all three agonists equally, while idazoxan antagonized responses to noradrenaline and phenylephrine equally with relative preservation of responses to acetylcholine. In the second series of experiments the effects of the selective alpha 2-adrenoceptor stimulant, UK-14304, were examined. UK-14304 produced weak and inconsistent excitations on a small number of cells; however, most of the cells did not respond to this drug. When applied continuously using low ejection currents, UK-14304 selectively and reversibly antagonized responses to noradrenaline and phenylephrine without affecting responses to acetylcholine. These results suggest that, in the somatosensory cortex of the rat, neuronal excitation to noradrenaline is unlikely to be mediated either wholly or partly by alpha 2-adrenoceptors. The antagonism of neuronal responses to noradrenaline and phenylephrine by idazoxan probably reflects the alpha 1-adrenoceptor antagonistic properties of the drug which is known to occur at higher concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)