Regulation of Semicarbazide-Sensitive Amine Oxidase Expression by Tumor Necrosis Factor- in Adipocytes: Functional Consequences on Glucose Transport

Membrane-associated semicarbazide-sensitive amine oxidase (SSAO) is mainly present in the media of aorta and in adipose tissue. Recent works have reported that SSAO activation can stimulate glucose transport of fat cells and promote adipose conversion. In this study, the murine 3T3-L1 preadipose cell line was used to investigate SSAO regulation by tumor necrosis factor(TNF), a cytokine that is synthesized in fat cells and known to be involved in obesity-linked insulin resistance. SSAO mRNA and protein levels, and enzyme activity were decreased by TNFin a doseand time-dependent manner, without any change of SSAO affinity for substrates or inhibitors. SSAO inhibition caused by TNFwas spontaneously reversed along the time after TNFremoval. The decrease in SSAO expression also occurred in white adipose tissue of C57BL/6 mice treated with mTNF. Overall, we demonstrated that reduction in SSAO expression induced by the cytokine had marked repercussions on amine-stimulated glucose transport, in a doseand time-dependent manner. This effect was more pronounced than the inhibiting effect of TNFon insulin-stimulated glucose transport. Moreover, the peroxisome proliferator-activated receptor agonists thiazolidinediones did not reverse either TNFeffect on amine-sensitive glucose transport or the inhibition of SSAO activity, whereas they antagonized TNFeffects on insulin-sensitive glucose transport. These results demonstrate that TNFcan strongly down-regulate SSAO expression and activity, and through this mechanism can dramatically reduce amine-stimulated glucose transport. This suggests a potential role of this regulatory process in the pathogenesis of glucose homeostasis dysregulations observed during diseases accompanied by TNFoverproduction, such as cachexia or obesity. Copper-containing amine oxidases form a specific family of enzymes that deaminate some aromatic or aliphatic amines to generate ammonia, hydrogen peroxide, and the corresponding aldehydes. An original member of this group is a membrane-bound amine oxidase, highly inhibited by semicarbazide, and often referred to as the “tissue-bound” semicarbazide-sensitive amine oxidase (SSAO) (Lyles, 1996; Jalkanen and Salmi, 2001). This enzyme readily oxidizes exogenous (e.g., benzylamine) or endogenous (e.g., methylamine and aminoacetone) primary amines. SSAO, which is identical to vascular adhesion protein-1, has been cloned in different species (Zhang and McIntire, 1996; Morris et al., 1997; Bono et al., 1998; Smith et al., 1998; Moldes et al., 1999). SSAO activity and transcripts have been detected in a variety of tissues and cell types (Lyles, 1996), but they are prominently expressed in vascular smooth muscle cells (Lyles, 1996), endothelial cells of lymph venules (Jalkanen and Salmi, 2001), and in white and brown adipocytes (Barrand and Callingham, 1982; Raimondi et al., 1991). The physiological and pathophysiological roles of SSAO are still unclear and depend on the cell type on which the enzyme is expressed (Lyles and Pino, 1998; Jalkanen and Salmi, 2001; El Hadri et al., 2002). In fat cells, it has been recently documented that SSAO expression is strongly induced during preadipocyte differentiation (Moldes et al., 1999; Fontana et al., 2001) and that SSAO chronic activation promotes terminal adipocyte maturation (Fontana et al., 2001; Mercier et al., 2001). Furthermore, SSAO is not exclusively present at the plasma membrane of adipocytes, but is also detectable in vesicles containing the insulin-sensitive glucose transporter GLUT4 (Morris et al., 1997; Enrique-Tarancon et al., 1998). The acute effect of insulin on glucose transport can be mimicked by SSAO substrates (Enrique-Tarancon et al., 1998, This work was supported by grants from the Centre National de la Recherche Scientifique and of the Université Paris VI. N.M. is the recipient of a grant of the Ministère de l’Enseignement Supérieur et de la Recherche. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.044420. ABBREVIATIONS: SSAO, semicarbazide-sensitive amine oxidase; IRS, insulin receptor substrate; TNF, tumor necrosis factor; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate buffer saline; G3PDH, glycerol-3-phosphate dehydrogenase; TBS, Tris-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; PCR, polymerase chain reaction; DOG, deoxyglucose. 0022-3565/03/3043-1197–1208$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 304, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 44420/1047707 JPET 304:1197–1208, 2003 Printed in U.S.A. 1197 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from 2000; Marti et al., 1998; Fontana et al., 2001; Morin et al., 2001) through the release of hydrogen peroxide (EnriqueTarancon et al., 1998; Marti et al., 1998). SSAO substrates can also promote IRS-1 and -3 tyrosine phosphorylation, and stimulate phosphatidylinositol 3-kinase activity and GLUT4 translocation to the plasma membrane (Enrique-Tarancon et al., 2000). A recent study (Marti et al., 2001) has underlined the potential therapeutic interest of SSAO in the control of glycemia: an acute or chronic administration of the synthetic SSAO substrate benzylamine in combination with low doses of vanadate enhances glucose tolerance and reduces hyperglycemia in streptozotocin-induced diabetic rats. Interestingly, several studies have reported that SSAO activity is increased in serum from diabetic (Boomsma et al., 1999; Meszaros et al., 1999) or obese (Meszaros et al., 1999) patients. Taken together, these observations suggest that adipocyte SSAO could play a significant role in the control of glucose homeostasis. TNFis a cytokine involved in the clinical and metabolic disturbances observed in obesity-linked insulin resistance: TNFis overexpressed in adipose tissue of obese rodents or humans, and administration of TNFto animals induces insulin resistance, whereas neutralization of TNFimproves insulin sensitivity (Hotamisligil, 2000; Moller, 2000). Otherwise, results from knockout mice deficient in TNFor its receptors indicate that the cytokine can regulate in vivo insulin sensitivity and is involved at least partly in the onset of obesity-associated insulin resistance (Hotamisligil, 2000; Moller, 2000). However, the exact contribution of TNFto the pathophysiology of insulin resistance observed in human obesity remains controversial (Moller, 2000). Multiple cellular and molecular mechanisms could account for the metabolic effects of TNFon adipose tissue. Thus, the cytokine potently suppresses the expression of genes encoding proteins involved in fatty acid uptake and lipogenesis. TNFalso inhibits preadipocyte differentiation (Torti et al., 1985) and provokes apoptosis (Prins et al., 1997). TNFstimulates lipolysis and increases free fatty acids through different mechanisms. It is also well known that in adipocytes, TNFstrongly inhibits insulin-stimulated glucose transport (Stephens and Pekala, 1991; Szalkowski et al., 1995), through different alterations in insulin signaling pathway (Hotamisligil, 2000; Moller, 2000). Considering the TNF-induced alterations in adipocyte hexose transport, and as regards to the promoting effect of SSAO activation on fat cell glucose uptake, the aim of our study was to investigate the impact of TNFon the expression and function of this membrane-associated amine oxidase. Using the murine preadipose 3T3-L1 cell line, we demonstrate that TNFdecreases SSAO expression without altering adipocyte differentiation level. This effect is also observed in white adipose tissue of TNF-injected mice. Overall, this TNF-induced down-regulation of SSAO expression is involved in a dramatic reduction in amine-stimulated glucose transport. Materials and Methods Cell Culture. Stocks of murine 3T3-L1 preadipocytes were maintained as described previously (Moldes et al., 1999). For experiments, cells were seeded at a density of 10/cm in plastic culture dishes (Falcon, Cowley, UK) and were grown in DMEM supplemented with 10% fetal calf serum (Biomedia, Boussens, France), 100 units/ml penicillin, and 50 g/ml streptomycin (Invitrogen, Carlsbad, CA) in a 10% CO2-humidified atmosphere. Adipocyte differentiation was initiated by administration at confluence of methylisobutylxanthine (100 M), dexamethasone (0.25 M), and insulin (1 g/ml) for 48 h, then cells were refed by DMEM containing 10% fetal calf serum and 1 g/ml insulin. Using this protocol, more than 95% of the cells acquired an adipocyte morphology at day 8 after confluence. After washing, mature adipocytes were kept for 16 h in a defined medium consisting of DMEM/Ham’s F-12 medium (2:1, v/v) and 0.1% bovine serum albumin. Thereafter, cells were maintained either in the absence or in the presence of mTNF(Sigma-Aldrich, St. Louis, MO) at concentrations and for periods of time mentioned under Results. Animals. Four-week-old male C57BL/6 mice received a single intraperitoneal injection of 1 g of recombinant mTNF. Forty-eight hours after TNFadministration, animals were killed and epididymal fat pads were quickly excised and immediately frozen in liquid nitrogen until preparation of tissue extracts. Experiments were undertaken according to the Guidelines for the Care and the Use of Experimental Animals. Cell and Tissue Extracts, Biochemical Determinations, and Enzyme Assays. 3T3-L1 cells were washed twice in PBS, harvested, and homogenized in 25 mM Tris, pH 7.5, 1 mM EDTA (20 strokes in a Dounce homogenizer, pestle B). A fraction of the homogenate was stored at 80°C. The remaining fraction was centrifuged at 10,000g for 10 min at 4°C, and the supernatant was kept at 80°C until use. The 10,000g pellet was resuspended in the homogenization buffer and stored at 80°C. Aliquots of homogenates, supernatants, and resuspended pellets were used to determine protein content by the method of Lowry, using bovine serum albumin as a standard. Triglyceride concentration was determined with the Infinity triglycerides kit (Sigma Diagnostics, St. Louis, MO). For preparation of tissue extracts, the epididymal fat depot was homogenized in a buffer consisting of KH2PO4 1 mM, pH 7.8, 250 mM sucrose. After a centrifugation at 600g for 5 min at 4°C, the pellet and the fat cake were discarded, and the supernatant was kept at 80°C until SSAO activity measurement. SSAO activity was tested by measurement of hydrogen peroxide production by the fluorometric method of Matsumoto et al. (1982). Briefly, 25 g of cell homogenates was preincubated for 30 min at 37°C, in a final volume of 100 l containing 40 mM sodium phosphate, pH 7.4, 1 mM homovanillic acid, 1 mM sodium azide, and 1 mM pargyline to inhibit monoamine oxidase A and B. When indicated, an SSAO inhibitor was preincubated with cell extract for 30 min at 37°C before the addition of the substrate. Incubation was initiated by the addition of the SSAO substrate (benzylamine, methylamine, tyramine, or -phenylethylamine) and was carried out in duplicate for 1 h at 37°C. Reaction was stopped with 1 mM semicarbazide, and 1.2 ml of 0.1 N NaOH was added. Fluorescence intensity was measured with excitation at 323 nm and emission at 426 nm. As blank tests, assays were incubated in parallel without substrate addition. Preliminary experiments were performed to ensure that SSAO activity was tested at the initial rate of reaction. Apparent kinetic constants for SSAO substrates were determined within the following concentration ranges: 3 to 300 M for benzylamine, 0.1 to 10 mM for methylamine, 0.1 to 10 mM for tyramine, and 30 M to 3 mM for -phenylethylamine. To determine IC50 and Ki values for amine oxidase inhibitors, SSAO activity was determined in the presence of 30 M benzylamine as a substrate. The concentration ranges of inhibitors used were as follows: 1 M to 1 mM semicarbazide, 1 to 100 M aminoguanidine, 0.1 to 100 nM phenelzine, 1 to 100 nM hydrazine, 3 to 100 nM hydralazine, and 30 nM to 3 M benserazide. Kinetic parameters were determined using the nonlinear regression analysis curve fitting procedure of the ENZFITTER program (Biosoft-Elsevier, Cambridge, UK). Monoamine oxidase activity was tested under conditions identical to those for SSAO, except that pargyline was omitted and replaced by 1198 Mercier et al. at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from 1 mM semicarbazide, and tyramine or serotonin was used as substrate. Polyamine oxidase activity was also tested with the same procedure, using N-acetyl spermidine as a substrate. G3PDH activity was assayed by recording the initial rate of oxidation of NADH at 340 nm at 25°C (Mercier et al., 2001). The standard mixture contained 50 mM triethanolamine-HCl buffer, pH 7.5, 1 mM EDTA, 0.13 mM -NADH, 1 mM dihydroxyacetone phosphate, 1 mM 2-mercaptoethanol, and variable amounts of 10,000g