Adipose Tissue Proadipogenic Redox Changes in Obesity

Abstract

The role of inflammation and oxidative stress in the development of obesity and associated metabolic disorders is under debate. We investigated the redox metabolism in a non-diabetic obesity model, i.e. 11-week-old obese Zucker rats. Antioxidant enzyme activities, lipophilic antioxidant (α-tocopherol, coenzymes Q) and hydrophilic antioxidant (glutathione, vitamin C) contents and their redox state (% oxidized form), were studied in inguinal white fat and compared with blood and liver. The adipose tissues of obese animals showed a specific higher content of hydrophilic molecules in a lower redox state than those of lean animals, which were associated with lower lipophilic molecule content and lipid peroxidation. Conversely and as expected, glutathione content decreased and its redox state increased in adipose tissues of rats subjected to lipopolysaccharide-induced systemic oxidative stress. In these in vivo models, oxidative stress and obesity thus had opposite effects on adipose tissue redox state. Moreover, the increase in glutathione content and the decrease of its redox state by antioxidant treatment promoted in vitro the accumulation of triglycerides in preadipocytes. Taken together and contrary to the emergent view, our results suggest that obesity is associated with an intracellular reduced redox state that promotes on its own the development of a deleterious proadipogenic process. The role of inflammation and oxidative stress in the development of obesity and associated metabolic disorders is under debate. We investigated the redox metabolism in a non-diabetic obesity model, i.e. 11-week-old obese Zucker rats. Antioxidant enzyme activities, lipophilic antioxidant (α-tocopherol, coenzymes Q) and hydrophilic antioxidant (glutathione, vitamin C) contents and their redox state (% oxidized form), were studied in inguinal white fat and compared with blood and liver. The adipose tissues of obese animals showed a specific higher content of hydrophilic molecules in a lower redox state than those of lean animals, which were associated with lower lipophilic molecule content and lipid peroxidation. Conversely and as expected, glutathione content decreased and its redox state increased in adipose tissues of rats subjected to lipopolysaccharide-induced systemic oxidative stress. In these in vivo models, oxidative stress and obesity thus had opposite effects on adipose tissue redox state. Moreover, the increase in glutathione content and the decrease of its redox state by antioxidant treatment promoted in vitro the accumulation of triglycerides in preadipocytes. Taken together and contrary to the emergent view, our results suggest that obesity is associated with an intracellular reduced redox state that promotes on its own the development of a deleterious proadipogenic process. Redox metabolism corresponds to a complex interacting network involving the generation of reactive oxygen species and enzymatic and non-enzymatic cellular antioxidant defenses. Any small and transient disturbance of this balance induces redox signaling, which can act on several transduction pathways or enzyme and transcription factor activities. In contrast, when antioxidant defenses are chronically overwhelmed, the result is an oxidative stress in which free radicals may exert their deleterious effects (1Dröge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7276) Google Scholar). Oxidative stress has been implicated in the pathogenesis of several metabolic diseases as well as in the co-morbidity of diabetes mellitus and atherosclerosis. In such studies, redox metabolism was evaluated by the pattern of various parameters such as manganese-dependent superoxide dismutase, glutathione peroxidase or catalase activities, and glutathione or α-tocopherol content in blood and liver (2Touyz R.M. Schiffrin E.L. Histochem. Cell Biol. 2004; 122: 339-352Crossref PubMed Scopus (504) Google Scholar, 3Faure P. Ramon O. Favier A. Halimi S. Eur. J. Clin. Investig. 2004; 34: 475-481Crossref PubMed Scopus (67) Google Scholar, 4Ford E.S. Mokdad A.H. Giles W.H. Brown D.W. Diabetes. 2003; 52: 2346-2352Crossref PubMed Scopus (350) Google Scholar, 5Yang S. Zhu H. Li Y. Lin H. Gabrielson K. Trush M.A. Diehl A.M. Arch. Biochem. Biophys. 2000; 378: 259-268Crossref PubMed Scopus (321) Google Scholar). A similar pattern was observed in obesity and seemed to indicate that such pathology was related to pro-oxidative context. Indeed, obesity prevalence is correlated with decreased concentrations of plasma antioxidants (6Reitman A. Friedrich I. Ben-Amotz A. Levy Y. Isr. Med. Assoc. J. 2002; 4: 590-593PubMed Google Scholar). An increase in the markers of systemic oxidative stress has therefore been associated with obesity and metabolic syndrome (7Furukawa S. Fujita T. Shimabukuro M. Iwaki M. Yamada Y. Nakajima Y. Nakayama O. Makishima M. Matsuda M. Shimomura I. J. Clin. Investig. 2004; 114: 1752-1761Crossref PubMed Scopus (3869) Google Scholar, 8Urukawa H. Katsuki A. Sumida Y. Gabazza E.C. Murashima S. Morioka K. Maruyama N. Kitagawa N. Tanaka T. Hori Y. Nakatani K. Yano Y. Adachi Y. J. Clin. Endocrinol. Metab. 2003; 88: 4673-4676Crossref PubMed Scopus (343) Google Scholar). Furthermore, it has been suggested that oxidative stress in accumulated fat, assessed by H202 generation and malondialdehyde (MDA), 2The abbreviations used are: MDA, malondialdehyde; IW, inguinal white adipose tissue; EW, epididymal white adipose tissue; Gpx, glutathione peroxidase; SOD, superoxide dismutase; VitC, vitamin C; CoQ, coenzyme Q; αToc, α-tocopherol; LPS, lipopolysaccharide; SVF, stroma vascular fraction; BHA, butylhydroxyanisole. 2The abbreviations used are: MDA, malondialdehyde; IW, inguinal white adipose tissue; EW, epididymal white adipose tissue; Gpx, glutathione peroxidase; SOD, superoxide dismutase; VitC, vitamin C; CoQ, coenzyme Q; αToc, α-tocopherol; LPS, lipopolysaccharide; SVF, stroma vascular fraction; BHA, butylhydroxyanisole. is an early instigator of metabolic syndrome (7Furukawa S. Fujita T. Shimabukuro M. Iwaki M. Yamada Y. Nakajima Y. Nakayama O. Makishima M. Matsuda M. Shimomura I. J. Clin. Investig. 2004; 114: 1752-1761Crossref PubMed Scopus (3869) Google Scholar). Such a conclusion is congruent with the emergent view characterizing obesity as a chronic inflammatory situation (9Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5062) Google Scholar). It is noteworthy that most of these studies were conducted not in situations of strict obesity but with confused metabolic pathologies defined as metabolic syndrome. Taken together, all these reports argue in favor of an oxidative redox state in morbid obesity, although the occurrence of an oxidative stress in adipose tissue during the onset of obesity has never been investigated. Other studies also evidenced the importance of redox metabolism in obesity but in an opposite way. Indeed, we recently demonstrated that in vitro preadipocyte proliferation and differentiation can be controlled by redox metabolism (10Carrière A. Fernandez Y. Rigoulet M. Pénicaud L. Casteilla L. FEBS Lett. 2003; 550: 163-167Crossref PubMed Scopus (95) Google Scholar, 11Carrière A. Carmona M.C. Fernandez Y. Rigoulet M. Wenger R.H. Pénicaud L. Casteilla L. J. Biol. Chem. 2004; 279: 40462-40469Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), while overexpression of cellular glutathione peroxidase has been reported to promote obesity in mice (12McClung J.P. Roneker C.A. Mu W. Lisk D.J. Langlais P. Liu F. Lei X.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8852-8857Crossref PubMed Scopus (399) Google Scholar). The aim of this work was to investigate changes in adipose tissue redox metabolism in an obese model without the confounding factors of diabetes and other metabolic dysregulations, i.e. the Zucker rat (13Tofovic S.P. Jackson E.K. Methods Mol. Med. 2003; 86: 29-46PubMed Google Scholar). The putative changes in redox defenses of the adipose tissues between obese and lean Zucker rats were compared with those occurring in blood and liver and also compared with those of rats subjected to an experimental known oxidative stress (14Ben-Shaul V. Lomnitski L. Nyska A. Zurovsky Y. Bergman M. Grossman S. Toxicol. Lett. 2001; 123: 1-10Crossref PubMed Scopus (100) Google Scholar). Additionally, the importance of the redox state in the development of fat stores was assessed by studying the effect of antioxidants on lipid synthesis in cultured preadipocytes. Animals—Animals were acclimated for 7 days to 22 ± 1 °C in individual cages with a 12-h light-dark cycle and given commercial food and water ad libitum. There were 11-week-old obese and lean Zucker rats (Charles River Laboratories, L'Arbresle, France), 11-week-old Wistar rats (Janvier, Le Genest-St-Isle, France) intraperitoneally injected with 5 mg of LPS/kg (LPS dissolved in water) for 12 h, and C57 black 6N mice (Harlan, Gannat, France) for primary cultures of inguinal white adipose tissue (IW). Animals were euthanized after anesthesia with 100 mg of ketamine and 10 mg of xylazine/kg body weight. All experiments were conducted in accordance with guidelines for animal care in the Institut Federatif de Recherche Louis Bugnard, Rangueil Hospital, Toulouse. Tissues, Adipocytes, and Stroma Vascular Fraction (SVF) Isolation—Plasmas from abdominal aorta blood of anesthetized Zucker rats were collected into heparinated or EDTA tubes. Liver and epididymal and inguinal adipose tissues (EW and IW) that were considered as intra-abdominal and subcutaneous fat, respectively, were removed, washed in phosphate-buffered saline medium, blotted, weighed, and immediately frozen in liquid nitrogen. Prior to biochemical measurements, frozen aliquots were pulverized. Mature adipocytes and SVF were isolated from finely minced tissues as previously described (15Prunet-Marcassus B. Moulin K. Carmona M.C. Villarroya F. Pénicaud L. Casteilla L. FEBS Lett. 1999; 464: 184-188Crossref PubMed Scopus (17) Google Scholar). Circulating Glucose and Insulin—Plasma glucose and insulin were measured by glucose-dye-oxidoreductase with reactive strips (Accu-Chek Active, Roche Diagnostics) and radioimmunoassay (RIA kit; Diasorin), respectively. Adipocyte Glucose Uptake—Adipocyte suspensions were incubated with or without insulin for 45 min at 37 °C in a final volume of 400 μl. Then, 2-deoxy-d-[3H]glucose (0.4 μCi) was added at a final concentration of 0.1 mm for 10 min. Assays were stopped with 100 μl of 100 μm cytochalasin B, and then aliquots were counted (16Morin N. Lizcano J.M. Fontana E. Marti L. Smih F. Rouet P. Prévot D. Zorzano A. Unzeta M. Carpéné C. J. Pharmacol. Exp. Ther. 2001; 297: 563-572PubMed Google Scholar). Cell Culture and Adipogenesis Assessment—3T3-F442A preadipocytes were routinely cultivated in a 21% O2, 74% N2, and 5% CO2 humidified atmosphere. At confluence, the medium was changed and antioxidant was added. Cell cultures were maintained for 7 days after confluence (the medium and antioxidant were changed every 2 days). For primary culture of mice, IW SVF cells were purified and cultured as previously described (17Planat-Benard V. Silvestre J.S. Cousin B. André M. Nibbelink M. Tamarat R. Clergue M. Manneville C. Saillan-Barreau C. Duriez M. Tedgui A. Levy B. Pénicaud L. Casteilla L. Circulation. 2004; 109: 656-663Crossref PubMed Scopus (1184) Google Scholar). At confluence, preadipocytes were induced to differentiate in the presence of 850 nm insulin, 2 nm triiodothyronine, 33 nm dexamethasone, and 1 μg/ml of transferrin. They were treated with antioxidant as described for 3T3-F442A preadipocytes. Cellular triglyceride content was determined using a commercially available test combination (Triglyceride enzymatique PAP150; bioMérieux). Triglycerides were also stained with Oil Red O (18Ramirez-Zacarias J.L. Castro-Munozledo F. Kuri-Harcuch W. Histochemistry. 1992; 97: 493-497Crossref PubMed Scopus (801) Google Scholar) and visualized with phase contrast microscopy. Real-time PCR—Total RNA from cells was extracted by the Extract-all solution (Eurobio). Complementary DNA was synthesized with SuperScript™ reverse transcriptase and poly(dT) primers (Invitrogen). The real-time PCR measurement of individual cDNAs was performed using SYBR green dye to measure duplex DNA formation with the Gene Amp 5700 (Applied Biosystem) and normalized to the amount of 36B4 RNA. Increases of peroxisome proliferator-activated receptor γ2 and lipoprotein lipase and decrease of β actin mRNA expression were linked with adipocyte differentiation (19Rosen E.D. Walkey C.J. Puiserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307PubMed Google Scholar, 20Spiegelman B.M. Farmer S.R. Cell. 1982; 29: 53-60Abstract Full Text PDF PubMed Scopus (200) Google Scholar). Primers used were the following: 36B4 sense 5′-AGTCGGAGGAATCAGATGAGGAT-3′, antisense 5′-GGCTGACTTGGTTGCTTTGG-3′; PPARγ2 sense 5′-AGTGTGAATTACAGCAAATCTCTGTTTT-3′, antisense 5′-GCACCATGCTCTGGGTCAA-3′; LPL sense 5′-GTGGCCGAGAGCGAGAAC-3′, antisense 5′-CCACCTCCGTGTAAATCAAGAAG-3′; and β actin sense 5′-CCGTGAAAAGATGACCCAGATC-3′, antisense 5′-GCCTGGATGGCTACGTACATG-3′. Antioxidant Enzymes—Powdered frozen tissues were in mm mm for for IW or The were at for 10 and at for 45 min these were performed at Antioxidant enzyme were assessed in and in or catalase were performed in and A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were by of S. G. Eur. J. Biochem. PubMed Scopus Google Scholar). was determined by of at nm of Methods for Scholar). peroxidase was measured using as H. L. Chem. Biochem. Google Scholar). and frozen SVF, and plasma were used to measure α-tocopherol and coenzyme and in and were by liquid with on the A. A. Fernandez Y. A.M. S. B. M. J.P. Casteilla L. FEBS Lett. 2004; PubMed Scopus Google Scholar). and vitamin were also from tissue and glutathione was evaluated in and in of reduced oxidized VitC, and its oxidized were by liquid with S. M. I. J. Biochem. 1999; PubMed Scopus Google Scholar) and J. W.H. J. PubMed Scopus Google Scholar), respectively. For glutathione and VitC, we the redox state as was from the formation of K. Methods PubMed Scopus Google Scholar). ± were and between were determined by at Antioxidant in and Liver of Zucker enzymatic were lower in IW than in liver and in IW lower content and higher redox state of the oxidized than liver For lipophilic molecules content was lower in IW, content was higher than in liver. content was lower in IW than in liver We then investigated the of antioxidants between the adipocyte and stroma vascular expected, the lipophilic and αToc, were in adipocytes the hydrophilic glutathione and VitC, were also in adipocytes and Adipocyte of Zucker the of animal model was to investigate the between obesity and we the insulin of adipose tissue of 11-week-old obese Zucker rats. rats had ± that not from lean rats ± and higher ± ± for lean that these animals were obese but not The glucose mg of was lower in obese ± than in lean rats ± Furthermore, insulin of glucose was at 100 nm in lean and obese isolated this was lower in obese ± than in lean ± the increase was to ± and ± in obese and not that isolated adipocytes from obese rats were to insulin but of lower insulin in Liver and Redox of Zucker the of obese Zucker rats were and showed not was observed for all redox parameters a decrease of the content glutathione and plasma contents were lower in obese than in lean rats. Moreover, glutathione redox state was higher in obese non-enzymatic and lipid in liver and blood of obese Zucker rats In enzymatic and glutathione content were measured in and the other parameters were measured in were as of the for lean and ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a in Redox of in and Oxidative IW showed between obese and lean antioxidant metabolism was observed for and SOD, but catalase was higher in obese than in lean rats In IW, glutathione and contents were higher in obese rats and their redox were lower than in lean rats In contrast, obese rats had lower lipophilic molecule content as well as lower than lean rats Taken together, these results that fat accumulation was associated with a of hydrophilic antioxidants in their reduced state and lower lipid peroxidation. We then to glutathione metabolism as a of redox The of glutathione in IW and of obese rats showed that glutathione content is higher in the fat when the lower redox state in obese rats is specific to to in obesity, IW and glutathione contents were decreased in situations of systemic oxidative stress and inflammation induced by treatment Redox and Adipocyte the of the redox state to a reduced we treated cultured 3T3-F442A preadipocytes with an treated with concentrations showed a triglyceride increase results were with and with primary culture of IW SVF cells not by Oil Red O and adipocyte expression the adipocyte differentiation and lipid increase in cells and expected, glutathione content was increased while its redox state was decreased in cells treated with in glutathione in its reduced was with increase in triglyceride content and adipocyte In this work we demonstrated in obesity, adipose tissue is not associated with an oxidized redox state as observed in other tissues during inflammatory In contrast, obesity is associated with a reduced redox state that promotes in vitro accumulation of is considered a chronic inflammatory associated with a decrease in antioxidant defenses and the of markers of oxidative stress in blood and liver (2Touyz R.M. Schiffrin E.L. Histochem. Cell Biol. 2004; 122: 339-352Crossref PubMed Scopus (504) Google Scholar, 3Faure P. Ramon O. Favier A. Halimi S. Eur. J. Clin. Investig. 2004; 34: 475-481Crossref PubMed Scopus (67) Google Scholar, 4Ford E.S. Mokdad A.H. Giles W.H. Brown D.W. Diabetes. 2003; 52: 2346-2352Crossref PubMed Scopus (350) Google Scholar, 5Yang S. Zhu H. Li Y. Lin H. Gabrielson K. Trush M.A. Diehl A.M. Arch. Biochem. Biophys. 2000; 378: 259-268Crossref PubMed Scopus (321) Google Scholar, D. T. B. J. Metab. 2004; PubMed Scopus Google Scholar). view is not with the recently observed of obesity in mice of the most antioxidant glutathione peroxidase (12McClung J.P. Roneker C.A. Mu W. Lisk D.J. Langlais P. Liu F. Lei X.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8852-8857Crossref PubMed Scopus (399) Google Scholar). Moreover, reactive oxygen species been demonstrated to in vitro A. Carmona M.C. Fernandez Y. Rigoulet M. Wenger R.H. Pénicaud L. Casteilla L. J. Biol. Chem. 2004; 279: 40462-40469Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In in antioxidant defenses or markers of lipid was evidenced in the blood of obese when compared with their V. Prévot D. N. C. J. P. Zorzano A. Carpéné C. 2004; PubMed Scopus Google Scholar). A in studying the between obesity and oxidative stress is that the animal used the of congruent metabolic such as insulin The obese Zucker rats the to this it is well known on their can be considered as obese without Indeed, such obese animals but without although lower insulin was in isolated adipocytes of obese these enzymatic activities, hydrophilic antioxidant molecule and lipophilic content were determined in adipose tissue of lean Zucker rats and were to be lower than in liver. for be related to the described for white adipose tissues C. B. S. A. A. B. J. S. Fernandez Y. D. Pénicaud L. Casteilla L. Rigoulet M. Biochem. Pharmacol. PubMed Scopus Google Scholar). The lower of lipid in adipose tissues when compared with liver be to a lower content L. H. A. O. PubMed Scopus (95) Google Scholar) and to a higher antioxidant defenses adipose tissue seemed to to lipid peroxidation. then showed that most of the hydrophilic and lipophilic of the antioxidant defenses were in the adipose tissue of obese rats and of an increase in glutathione and vitamin content in their reduced changes were specific to adipose tissues the liver antioxidant defenses of obese rats while blood markers the of an oxidative stress. The observed between obese and lean adipose tissue can be to adipocytes of their in the contents of antioxidant The most of this was that the adipose tissue of obese rats showed of lipid than that of lean as well as a higher content of hydrophilic molecules in a reduced state a lower content of lipophilic The reduced state of glutathione and be to their Indeed, the reduced of was by reduced by glutathione in the presence of a reduced by the in adipocytes I. E. A. Biochem. Biophys. PubMed Scopus Google Scholar). antioxidant defenses in the lipid phase of adipose tissue were lower in obese than in lean rats. was lower in adipose tissues of obese than of lean Zucker rats C. M. M. F. M. Biol. PubMed Scopus Google Scholar), these in the results for lipid peroxidation. The lipid in obese adipose tissue was also related to the content of the reduced of glutathione and VitC, the to from changes are contrary to those observed under systemic oxidative stress induced by Indeed, oxidative stress (14Ben-Shaul V. Lomnitski L. Nyska A. Zurovsky Y. Bergman M. Grossman S. Toxicol. Lett. 2001; 123: 1-10Crossref PubMed Scopus (100) Google Scholar) as expected, associated with an oxidized obese Zucker rats were in a reduced state. results are with our in vitro the of antioxidants increased glutathione content in its reduced in promoted triglyceride reports that an increase in reactive oxygen species or a decrease in antioxidant defenses preadipocyte intracellular reactive oxygen species in vitro preadipocyte differentiation by expression of the A. Carmona M.C. Fernandez Y. Rigoulet M. Wenger R.H. Pénicaud L. Casteilla L. J. Biol. Chem. 2004; 279: 40462-40469Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). in vivo treatment of rats with an of glutathione decreased glutathione and in adipocytes and lower body T. T. H. H. M. N. H. M. A. M. N. H. Y. I. Fujita T. 2004; PubMed Scopus Google Scholar). our conclusion is by studies at or overexpression of in of in increased oxidative stress and decreased body L.A. E. B. Biol. Med. 2000; PubMed Scopus Google Scholar). In contrast, overexpression of in an obese (12McClung J.P. Roneker C.A. Mu W. Lisk D.J. Langlais P. Liu F. Lei X.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8852-8857Crossref PubMed Scopus (399) Google Scholar). It is noteworthy that in this the their on liver and and demonstrated that overexpression reduced intracellular reactive oxygen our conclusion to be to the (7Furukawa S. Fujita T. Shimabukuro M. Iwaki M. Yamada Y. Nakajima Y. Nakayama O. Makishima M. Matsuda M. Shimomura I. J. Clin. Investig. 2004; 114: 1752-1761Crossref PubMed Scopus (3869) Google Scholar). be by the obese model We that the obese rats used in our are lower insulin but not mice, their are obese and A. H. K. K. S. E. H. M. T. T. H. K. Clin. Full Text Full Text PDF PubMed Scopus Google Scholar). We also demonstrated that adipose tissue of obese an and redox into our results and other we can that obesity is by a reduced redox state in adipose reduced redox state in proliferation and differentiation by of transcription factors A. Carmona M.C. Fernandez Y. Rigoulet M. Wenger R.H. Pénicaud L. Casteilla L. J. Biol. Chem. 2004; 279: 40462-40469Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). A deleterious cycle thus that promotes and the onset of obesity We J. for in and M. C. Y. D. V. and C. for We also N. for in of the