Influence of high-fat diet on amine oxidase activity in white adipose tissue of mice prone or resistant to diet-induced obesity

Decreased monoamine oxidase (MAO) activity has been observed in adipose tissue of obese patients. Since substrates of MAO and semicarbazide-sensitive amine oxidase (SSAO) can modify adipocyte metabolism, this work investigates whether changes in amine oxidase activity may occur during white adipose tissue (WAT) development. We evaluated MAO and SSAO activities in WAT of high-fat diet (HFD) and low-fat diet fed mice. To distinguish the effect of HFD on its own from the effect of fat mass enlargement, obesity-prone transgenic line of the FVBn strain lacking beta3-adrenergic receptors (AR) but expressing human beta3-AR and alpha2-AR (mbeta3-/-, hbeta3+/+, halpha2+/-) was compared to its obesity-resistant control (mbeta3-/-, hbeta3+/+). As already reported, the former mice became obese while the latter resisted to HFD. No significant change in SSAO or MAO activity was found in WAT of both strains after HFD when expressing oxidase activity per milligram of protein. However, when considering the overall capacity of the fat depots to oxidize tyramine or benzylamine, there was an increase in MAO and SSAO activity only in the enlarged WAT of HFD-induced obese mice. Therefore, the comparison of these models allowed to demonstrate that the higher amine oxidase capacity hold in enlarged fat stores of obese mice is more likely the consequence of increased fat cell number rather than the result of an increased expression of MAO or SSAO in the adipocyte.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

Influence of prolonged fasting on monoamine oxidase and semicarbazide-sensitive amine oxidase activities in rat white adipose tissue

Monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) activities are very high in white adipose tissue (WAT). SSAO, also known as Vascular Adhesion Protein-1 in vessels, is present at the surface of fat cells and independent approaches have evidenced its impressive increase during adipogenesis. However, the factors that might regulate the expression SSAO and MAO in adipose tissue are still poorly defined. Here, we report the influence of fasting on MAO and SSAO activities in adipose depots. A decrease of MAO activity occurred after three days of starvation in the intra-abdominal adipose tissue (INWAT) of male Wistar rats, regardless of their initial adiposity or fat loss. The reduced fat stores of seven-week old rats, loosing 59% of INWAT mass during fasting, contained only one half of the MAO activity found in fed control. The same reduction of MAO was observed after prolonged fasting in older rats which lose only 26% of their INWAT during the same starvation duration, leading to a fat mass comparable to that of younger fed control rats. It was therefore the endocrine and metabolic changes occurring during fasting that were responsible for the reduced MAO activity and not the amount of INWAT. Surprisingly, SSAO activity remained unchanged during starvation. In subcutaneous WAT, the changes in MAO and SSAO activities exhibited the same tendencies than those found in INWAT. Taken together, these data show that both MAO and SSAO activities increase in INWAT with age-dependent fattening, and indicate that only MAO diminishes during fasting.     

Alteration of Amine Oxidase Activity in the Adipose Tissue of Obese Subjects

Objective: To explore the activity of monoamine oxidases (MAOs) and semicarbazide‐sensitive amine oxidases (SSAOs) in adipose tissue and blood of lean and moderately obese subjects and to study whether there is a link between these hydrogen peroxide‐generating enzymes and blood markers of oxidative stress. Research Methods and Procedures: Nine obese male subjects (BMI 32.6 ± 0.4 kg/m²) and nine controls (BMI 23.4 ± 0.5) of 24‐ to 40‐year‐old subjects were included in the study. MAO and SSAO activities were measured on microbiopsies of abdominal subcutaneous adipose tissue by quantifying ¹⁴C‐tyramine and ¹⁴C‐benzylamine oxidation. Levels of soluble SSAO, lipid peroxidation products, and antioxidant agents were measured in plasma, whereas cytoprotective enzymes were determined in blood lysates. Results: The high MAO activity found in adipose tissue was diminished by one‐half in obese subjects (maximum initial velocity of 1.2 vs. 2.3 nmol tyramine oxidized/mg protein/min). There was no change in SSAO activity, either under its adipose tissue‐bound or plasma‐soluble form. Plasma levels of lipid peroxidation products and antioxidant vitamins remained unmodified, as well as erythrocyte antioxidant enzymes, whereas circulating triglycerides, insulin, and leptin were increased. Discussion: Although they already exhibited several signs of endocrino‐metabolic disorders, the obese men did not exhibit the increase in blood markers of oxidative stress or the decrease in antioxidant defenses reported to occur in very obese or diabetic subjects. The reduced MAO and the unchanged SSAO activities found in obesity suggest that these hydrogen peroxide‐generating enzymes expressed in adipocytes are probably not involved in the onset of the oxidative stress found in severe obesity and/or in its complications.     

Anatomical distribution of primary amine oxidase activity in four adipose depots and plasma of severely obese women with or without a dysmetabolic profile

Semicarbazide-sensitive amine oxidase (SSAO), identical to primary amine oxidase or vascular adhesion protein-1, is a membrane enzyme that generates hydrogen peroxide. SSAO is highly expressed at the adipocyte surface, and its plasma levels increase with type 2 diabetes. Since visceral adipose tissue (AT) is more tightly associated with obesity complications than subcutaneous (SC) abdominal fat, we compared SSAO activity in plasma and 4 distinct AT locations in 48 severely obese women (body mass index (BMI), averaging 54 ± 11 kg/m²), with or without a dysmetabolic profile. Higher glucose and triacylglycerol levels vs lower high-density lipoprotein (HDL)-cholesterol characterized dysmetabolic women (DYS; n = 25) from non-dysmetabolic (NDYS; n = 23), age- and weight-matched subjects. SC, mesenteric (ME), omental (OM), and round ligament (RL) fat locations were collected during bariatric surgery. SSAO capacity to oxidize up to 1 mM benzylamine was determined in AT and plasma with radiometric and fluorimetric methods. Plasma SSAO was higher in the DYS group. SSAO activity was higher in fat than in plasma, when expressed as radiolabeled benzaldehyde per milligram of protein. In ATs from DYS women, protein content was 10 % higher, and basal hydrogen peroxide release lower than in NDYS subjects, except for RL location. The SSAO affinity towards benzylamine did not exhibit regional variation and was not altered by a dysmetabolic profile (K _m averaging 184 ± 7 μM; n = 183). Although radiometric and fluorimetric methods gave different estimates of oxidase activity, both indicated that AT SSAO activity did not vary according to anatomical location and/or metabolic status in severely obese women.     

Absence of tissue-bound semicarbazide-sensitive amine oxidase activity in carp tissues

We have previously reported that carp (Cyprinus carpio) tissue mitochondria contain a novel form of monoamine oxidase (MAO), which belongs neither to MAO-A nor to MAO-B of the mammalian enzyme. This conclusion results from the findings that the carp MAO was equally sensitive to a selective MAO-A inhibitor clorgyline and to the MAO-B selective inhibitor l-deprenyl, when tyramine, a substrate for both forms, serotonin or beta-phenylethylamine, a substrate for either A or B-form of mammalian MAO, was used. In the present study, we tried to detect another amine oxidase, termed tissue-bound semicarbazide-sensitive amine oxidase (SSAO), activity in carp tissues. As definition of SSAO was used, such as insensitivity to inhibition of the kynuramine oxidizing activity by an MAO inhibitor pargyline and high sensitivity to the SSAO inhibitor semicarbazide. The results indicated that the oxidizing activity was selectively and almost completely inhibited by 0.1 mM pargyline alone or a combination of 0.1 mM pargyline plus 0.1 mM semicarbazide, but not by 0.1 mM semicarbazide alone. We also tried to detect any SSAO activity by changing experimental conditions, such as lower incubation temperature, higher enzyme protein concentration, a lower substrate concentration and different pH's in the reaction, as the enzyme source. However, still no SSAO activity could be detected in the tissues. These results conclusively indicate that carp tissues so far examined do not contain SSAO activity.     

Further studies on semicarbazide-sensitive amine oxidase activities (SSAO) of white adipose tissue.

1. White adipose tissue (WAT) from mice, rabbits, pigs and human subjects was investigated for the characterization of the tissue-bound semicarbazide-sensitive benzylamine oxidase activities (SSAO) present in each species. 2. Enzymes from mice, rabbits and pigs shared similar biochemical characteristics: they exerted histaminase activity, oxidized methylamine and acetylputrescine and were completely blocked by carbonyl reagents and by 3,5-ethoxy-4-aminomethylpyridyne (B24), in a dose-dependent fashion. 3. SSAO activity from human WAT had a lower affinity for benzylamine compared with enzymes in the other species and did not show any histaminase activity. 4. These results show that SSAO from human tissues might have different properties from SSAO of other species.     

Prolonged treatment with the β3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 2) modulation of glucose uptake and monoamine oxidase activity

Beta3-adrenergic agonists are well-recognited to promote lipid mobilisation and adipose tissue remodeling in rodents, leading to multilocular fat cells enriched in mitochondria. However, effects of beta3-adrenergic agonists on glucose transport are still controversial. In this work, we studied in white adipose tissue (WAT) the influence of sustained beta3-adrenergic stimulation on the glucose transport and on the mitochondrial monoamine oxidase (MAO) activity. As one-week administration of CL 316243 (CL, 1 mg/kg/d) induces beta-adrenergic desensitization in rat but not in guinea pig adipocytes, attention was paid to compare these models. When expressing glucose uptake as nmoles of 2-deoxyglucose/100 mg cell lipids, maximally stimulated uptake was increased in adipocytes of WAT from treated rats but not from treated guinea pigs. However, basal hexose uptake was also increased in CL-treated rats and, as a consequence, the dose-dependent curves for insulin stimulation were similar in control and CL-treated rats when expressed as fold increase over basal. Insulin-induced lipogenesis was unchanged in rat or guinea pig adipocytes after CL-treatment. The glucose carriers GLUT4 and corresponding mRNA were increased in subcutaneous WAT or in brown adipose tissue (BAT) but not in visceral WAT or muscles of CL-treated rats. There was an increase of MAO activity in WAT and BAT, but not in liver, of CL-treated rats while no change was detected in guinea pigs. These findings show that only rat adipocytes, which are beta3-adrenergic-responsive, respond to chronic beta3-AR agonist by an increase of GLUT4 content and MAO activity, despite a desensitization of all beta-adrenoceptor subtypes.     

On the characteristics of mitochondrial monoamine oxidase in pancreas and adipose tissues from genetically obese mice.

The substrate specificity of mitochondrial monoamine oxidase (MAO) in pancreatic and adipose tissues of obese mice and their lean counterparts was determined. The pancreatic MAO of obese mice had a greater specific activity than that of the lean mice. The white adipose tissue MAO was found to be more active than the brown adipose MAO in both groups of mice. While there was no appreciable difference in the MAO activities of brown adipose tissues between obese and lean mice, the enzyme from the white adipose tissue of obese mice had a higher specific activity than that of the lean mice. The higher MAO activity in white adipose tissue was observed when tyramine or serotonin was employed as substrate but not with benzylamine. Examination of mitochondrial MAO from epididymal adipocytes revealed marked differences in the properties of the enzyme between whole adipose tissue and isolated adipocytes. The inhibition characteristics of MAO from these tissues were studied with the specific inhibitors clorgyline and deprenyl.     

Monoamine oxidase and semicarbazide-sensitive amine oxidase in spontaneously hypertensive and in normotensive control rats

The aim of the present work was to compare monoamine oxidase (MAO) and semicarbazide sensitive amine oxidase (SSAO) activity in several tissues from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Contribution of MAO-A, -B and SSAO to the metabolism of each substrate in each tissue was defined from experiments where the decrease of oxidative deamination of each substrate at a given concentration was measured as a function of increasing concentrations of a selective MAO-A, -B or SSAO inhibitor. In the heart, aorta and, to a lesser extent, the femoral arteries MAO-A activity was higher in SHR than in WKY. Similarly in the liver the enzyme activity was higher in SHR than in WKY but was due to the -B form of MAO. In all the other tissues studied (duodenum, brain, lungs, adrenals and kidneys) no difference in MAO-A, MAO-B or SSAO activity was found between SHR and WKY, except for the kidneys and brain, if the differences in the weights of these organs in SHR are taken into account.     

Differences in plasminogen activator inhibitor 1 in subcutaneous versus omental adipose tissue in non-obese and obese subjects.

Human adipose tissue can produce plasminogen activator inhibitor-1 (PAI-1). It has been suggested that high levels of PAI-1 are of importance in enhanced cardiovascular disease observed among obese subjects, especially abdominally obese individuals. In the present study, we investigated the level of mRNA and production of PAI-1 in adipose tissue from two adipose tissue depots (omental vs. subcutaneous). Adipose tissue from both depots was obtained from obese (mean BMI, 46.9 kg/m 2) and non-obese (mean BMI, 23.9 kg/m 2) women. PAI-1 mRNA was measured both in fresh adipose tissue obtained immediately after surgery and after the adipose tissue (fragments) had been incubated for up to 72 h. In immediately frozen adipose tissue, PAI-1 mRNA expression was similar in omental and subcutaneous adipose tissue. No differences between obese and non-obese women were found. However, when adipose tissue fragments were cultured, PAI-1 mRNA and PAI-1 production were significantly higher in omental than in subcutaneous adipose tissue (p < 0.05). In the culture system, the production of PAI-1 in obese subjects was higher than in non-obese subjects in both subcutaneous (p < 0.05) and in omental adipose tissue (p = 0.19). In order to test whether these regional differences observed after incubation of the adipose tissue were due to differences in local accumulation of cytokines that may stimulate PAI-1 by a paracrine or autocrine manner, we investigated the expression of transforming growth factor beta1 (TGF-beta1) mRNA and tumor necrosis factor alpha (TNF-alpha) mRNA and protein. No differences between the two fat depots were found. In conclusion, no differences in PAI-1 expression between omental and subcutaneous adipose tissue were observed in biopsies frozen immediately after removal, but after incubation of adipose tissue (which somehow stimulates PAI-1 production), higher levels of PAI-1 were found in omental adipose tissue than in subcutaneous adipose tissue. Finally, PAI-1 production in adipose tissue from obese women was higher in non-obese women after incubation for 72 h.     

Diabetes and semicarbazide-sensitive amine oxidase (SSAO) activity: a review

The enzyme of semicarbazide-sensitive amine oxidase (SSAO) activity has been reported to be elevated in blood from diabetic patients. SSAO are widely distributed in plasma membranes of various tissues and blood plasma. SSAO-mediated production of toxic aldehydes has been proposed to be related to pathophysiological conditions. Cytotoxic metabolites by SSAO may cause endothelial injury and subsequently induce atherosclerosis. The precise physiological functions of SSAO could play an important role in the control of energy balance in adipose tissue. It is possible that the increased SSAO activity in diabetes may be a result of up-regulation due to increase of SSAO substrates, such as methylamine or aminoacetone. SSAO could play an important role in the regulation of adipocyte homeostasis. Inhibition of SSAO could be of therapeutic value for treatment of diabetic patient.     

高脂饲料诱导的肥胖大鼠网膜素水平的实验研究

人群调查发现肥胖人群网膜素水平较正常人群低,而正常及肥胖大鼠血清网膜素水平及其基因表达情况尚不清楚.将SD大鼠随机分为正常组(n=10)和高脂组(n=30),分别喂养普通饲料和高脂饲料.6 w后从高脂组选取体重增长最快的20只,再从中随机抽取10只继续喂养高脂饲料,12 w后两组各剩9只,采用全自动生化仪ADVIA2400测定血糖及血脂、ELISA检测血清胰岛素及网膜素水平、RT-PCR检测网膜脂肪组织网膜素mRNA表达水平.结果显示高脂组大鼠体重、体重增加值、肥胖指数、低密度脂蛋白、胰岛素、血清网膜素水平及网膜脂肪组织网膜素mRNA表达水平均高于正常组(P<0.05).首次发现肥胖大鼠血清网膜素水平及网膜脂肪组织中网膜素mRNA表达水平较正常大鼠显著增高,与人群调查结果不一致.     

Expression and activity of steroid aldoketoreductases 1C in omental adipose tissue are positive correlates of adiposity in women

We examined expression and activity of steroid aldoketoreductase (AKR) 1C enzymes in adipose tissue in women. AKR1C1 (20alpha-hydroxysteroid dehydrogenase; 20alpha-HSD), AKR1C2 (3alpha-HSD-3), and AKR1C3 (17beta-HSD-5) are involved mainly in conversion of progesterone to 20alpha-hydroxyprogesterone and inactivation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol. Abdominal subcutaneous and omental adipose tissue biopsies were obtained during abdominal hysterectomies in seven women with low visceral adipose tissue (VAT) area and seven age- and total body fat mass-matched women with visceral obesity. Women with elevated VAT areas were characterized by significantly higher omental adipose tissue 20alpha-HSD and 3alpha-HSD-3 mRNA abundance compared with women with low VAT accumulations (1.4- and 1.6-fold differences, respectively; P < 0.05). Omental and subcutaneous adipose tissue 3alpha-HSD activities were significantly higher in women with high vs. low VAT areas (P < 0.05 for both comparisons). Total and visceral adiposities were positively associated with omental 20alpha-HSD mRNA level (r = 0.75, P < 0.003 for fat mass; r = 0.57, P < 0.04 for VAT area) and omental 3alpha-HSD-3 mRNA level (r = 0.68, P < 0.01 for fat mass; r = 0.74, P < 0.003 for VAT area). Enzyme activities in both depots were also positively correlated with adiposity measures. Omental adipose tissue enzyme expression and activity were positively associated with omental adipocyte size and LPL activity. In conclusion, mRNA abundance and activity of AKR1C enzymes in abdominal adipose tissue compartments are positive correlates of adiposity in women. Increased progesterone and/or dihydrotestosterone reduction in abdominal adipose tissue may impact locally on fat cell metabolism.     

ITIH-5 expression in human adipose tissue is increased in obesity

Adipocytes secrete many proteins that regulate metabolic functions. The gene inter-α (globulin) inhibitor H5 (ITIH-5) encodes a secreted protein and is known to be expressed abundantly in the placenta. However, using gene expression profiles data we observed high expression of ITIH-5 in adipose tissue. The aim of this study was to test the hypothesis that ITIH-5 is strongly expressed in human adipocytes and adipose tissue, and is related to obesity and clinical metabolic variables. ITIH-5 adipose tissue mRNA expression was analyzed with DNA microarray and real-time PCR, and its association with clinical variables was examined. ITIH-5 protein expression was analyzed using western blot. ITIH-5 mRNA expression was abundant in human adipose tissue, adipocytes, and placenta, and higher in subcutaneous (sc) compared to omental adipose tissue (P < 0.0001). ITIH-5 mRNA and protein expression in sc adipose tissue were higher in obese compared to lean subjects (P < 0.0001 and P < 0.001, respectively). ITIH-5 mRNA expression was reduced after diet-induced weight loss (P < 0.0001). ITIH-5 mRNA expression was associated with anthropometry and clinical metabolic variables. In conclusion, ITIH-5 is highly expressed in sc adipose tissue, increased in obesity, down regulated after weight loss, and associated with measures of body size and metabolism. Together, this indicates that ITIH-5 merits further investigation as a regulator of human metabolism.     

Adipocyte triglyceride lipase expression in human obesity

We have investigated the gene and protein expression of adipose triglyceride lipase (ATGL) and triglyceride (TG) lipase activity from subcutaneous and visceral adipose tissue of lean and obese subjects. Visceral and subcutaneous adipose tissue was obtained from 16 age-matched lean and obese subjects during abdominal surgery. Tissues were analyzed for mRNA expression of lipolytic enzymes by real-time quantitative PCR. ATGL protein content was assessed by Western blot and TG lipase activity by radiometric assessment. Subcutaneous and visceral adipose tissue of obese subjects had elevated mRNA expression of PNPLA2 (ATGL) and other lipases including PNPLA3, PNPLA4, CES1, and LYPLAL1 (P < 0.05). Surprisingly, ATGL protein expression and TG lipase activity were reduced in subcutaneous adipose tissue of obese subjects. Immunoprecipitation of ATGL reduced total TG lipase activity in adipose lysates by 70% in obese and 83% in lean subjects. No significant differences in the ATGL activator CGI-58 mRNA levels (ABHD5) were associated with obesity. These data demonstrate that ATGL is important for efficient TG lipase activity in humans. They also demonstrate reduced ATGL protein expression and TG lipase activity despite increased mRNA expression of ATGL and other novel lipolytic enzymes in obesity. The lack of correlation between ATGL protein content and in vitro TG lipase activity indicates that small decrements in ATGL protein expression are not responsible for the reduction in TG lipase activity observed here in obesity, and that posttranslational modifications may be important.     

Benzylamine antihyperglycemic effect is abolished by AOC3 gene invalidation in mice but not rescued by semicarbazide-sensitive amine oxidase expression under the control of aP2 promoter

Semicarbazide-sensitive amine oxidase (SSAO) is a transmembrane enzyme that metabolizes primary amines from endogenous or dietary origin. SSAO is highly expressed in adipose, smooth muscle and endothelial cells. In each of these cell types, SSAO is implicated in different biological functions, such as glucose transport activation, extracellular matrix maturation and leucocyte extravasation, respectively. However, the physiological functions of SSAO and their involvement in pathogenesis remain uncompletely characterized. To better understand the role of adipose tissue SSAO, we investigated whether it was necessary and/or sufficient to produce the antihyperglycemic effect of the SSAO-substrate benzylamine, already reported in mice. Therefore, we crossed SSAO-deficient mice invalidated for AOC3 gene and transgenic mice expected to express human SSAO in an adipocyte-specific manner, under the control of aP2 promoter. The aP2?Chuman AOC3 construct (aP2?ChAOC3) was equally expressed in the adipose tissue of mice expressing or not the native murine form and almost absent in other tissues. However, the corresponding SSAO activity found in adipose tissue represented only 20?% that of control mice. As a consequence, the benzylamine antihyperglycemic effect observed during glucose tolerance test in control was abolished in AOC3-KO mice but not rescued in mice expressing aP2?ChAOC3. The capacity of benzylamine or methylamine to activate glucose uptake in adipocytes exhibited parallel variations in the corresponding genotypes. Although the aP2?ChAOC3 construct did not allow a total rescue of SSAO activity in adipose tissue, it could be assessed from our observations that adipocyte SSAO plays a pivotal role in the increased glucose tolerance promoted by pharmacological doses of benzylamine.