Heme Oxygenase-Mediated Increases in Adiponectin Decrease Fat Content and Inflammatory Cytokines Tumor Necrosis Factor- and Interleukin-6 in Zucker Rats and Reduce Adipogenesis in Human Mesenchymal Stem Cells

Adiponectin, an abundant adipocyte-derived plasma protein that modulates vascular function in type 2 diabetes, has been shown to provide cytoprotection to both pancreatic and vascular systems in diabetes. Therefore, we examined whether up-regulation of heme oxygenase (HO)-1 ameliorates the levels of inflammatory cytokines and influences serum adiponectin in Zucker fat (ZF) rats. ZF rats displayed a decrease in both HO activity and HO-1 and HO-2 protein levels and an increase in tumor necrosis factor (TNF)and interleukin (IL)-6 compared with Zucker lean (ZL) rats. Treatment of ZF animals with 2 mg/kg cobalt protoporphyrin IX (CoPP) increased protein levels of HO-1 and HO activity, but HO-2 was unaffected. The increase in HO-1 was associated with a decrease in superoxide levels (p 0.05) and an increase in plasma adiponectin (p 0.005), compared with untreated ZF rats. CoPP treatment decreased visceral and s.c. fat content, and it reduced weight gain (p 0.01). In addition, the inflammatory cytokines TNFand IL-6 were decreased (p 0.04 and p 0.008, respectively). Treatment of human bone marrow-derived adipocytes cultured with CoPP resulted in an increase in HO-1 and a decrease in superoxide levels. Up-regulation of HO-1 caused adipose remodeling, smaller adipocytes, and increased adiponectin secretion in the culture medium of human bone marrow-derived adipocytes. In summary, this study demonstrates that the antiobesity effect of HO-1 induction results in an increase in adiponectin secretion, in vivo and in vitro, a decrease in TNFand IL-6, and a reduction in weight gain. These findings highlight the pivotal role and symbiotic relationship of HO-1 and adiponectin in the modulation of the metabolic syndrome phenotype. Oxidative stress has been implicated in the pathogenesis and cardiovascular complications of insulin resistance in type 2 diabetes (Wellen and Hotamisligil, 2005; Namikoshi et al., 2007). Excessive generation of reactive oxygen species (ROS) is the underlying mechanism of endothelial injury, resulting in an accelerated rate of apoptosis and endothelial cell sloughing (Kruger et al., 2005; Bahia et al., 2006; Kim et al., 2007a). In addition, reduced plasma adiponectin levels have been documented in patients with coronary artery disease and diabetes, presumably as a result of an increases in ROS (Bakkaloglu et al., 2006; Ohashi et al., 2006; Haider et al., 2007). Lin et al. (2005) highlighted the importance of ROS production in adipocytes and the associated insulin resistance and changes in serum levels of adiponectin, suggesting that the increases in ROS are associated with an induced inflammatory response in the adipocyte. Adipose tissue plays an important role in insulin resistance through the production and secretion of a variety of proteins such as tumor necrosis factor (TNF), IL-6, leptin, and adiponectin (Berg and Scherer, 2005). Of these proteins, adiponectin has recently attracted much attention because it has insulin-sensitizing properties that reduce serum triglyceride levels and enhance fatty acid oxidation, insulin activity in the liver, and hepatic glucose uptake (Berg et al., 2001; Kim et al., 2007b). Adiponectin is exclusively secreted from This work was supported by National Institutes of Health Grants DK068134, HL55601, and HL34300. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.135285. ABBREVIATIONS: ROS, reactive oxygen species; TNF, tumor necrosis factor; IL, interleukin; HMW, high molecular weight; PPAR, peroxisome proliferator-activated receptor; HO, heme oxygenase; CoPP, cobalt protoporphyrin IX; SnMP, tin mesoporphyrin; ZL, Zucker lean; ZF, Zucker fat; FBS, fetal bovine serum; MSC, mesenchymal stem cell; O2 ., superoxide; FACS, fluorescence-activated cell sorting; DMEM, Dulbecco’s modified Eagle’s medium; h, human. 0022-3565/08/3253-833–840$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 325, No. 3 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 135285/3340352 JPET 325:833–840, 2008 Printed in U.S.A. 833 at U nirsity of T oedo Liaries on O cber 3, 2009 jpet.asjournals.org D ow nladed fom adipose tissue and its expression is higher in s.c. compared with visceral adipose tissue (Fain et al., 2004). It circulates in the blood, and it is found as both low-molecular-weight oligomers and high-molecular-weight (HMW) multimers (Basu et al., 2007). HMW adiponectin is reported to be more active and to correlate more significantly with glucose and insulin levels compared with both low-molecular-weight and total adiponectin (Lara-Castro et al., 2006). Low plasma levels of HMW adiponectin have been consistently associated with obesity, insulin resistance, type 2 diabetes, and coronary artery disease (Arita et al., 1999). L’Abbate et al. (2007) recently reported that increased adiponectin levels associated with increased expression of HO-1 resulted in enhanced cardiac protection from ROS. PPAR -response element has been found to increase expression of adiponectin (Iwaki et al., 2003) and also to regulate the expression of HO-1 in human vascular cells (Krönke et al., 2007). The HO system provides both antioxidant and antiapoptotic properties due to its products bilirubin/biliverdin and CO (Ollinger et al., 2007). HO-1 is induced by oxidant stress, and it plays a crucial role in protection against oxidative insult in diabetes and cardiovascular disease (Abraham and Kappas, 2008). Up-regulation of HO-1 gene expression prevents vascular dysfunction and endothelial cell death through decreases in ROS levels (Abraham et al., 2004). In the present study, we report that HO activity and HO-1 protein expression were decreased in obese rats, and we hypothesized that induction of HO-1 might serve to counteract the negative effects of type 2 diabetes mellitus and the metabolic syndrome. Using cobalt protoporphyrin IX (CoPP), an inducer and tin mesoporphyrin (SnMP, an inhibitor) to manipulate HO activity, we report here, for the first time, that induction of HO-1 was associated with reduced fat content and prevention of weight gain as a result of reduced adipogenesis in both in vitro and in vivo models of type 2 diabetes mellitus. Materials and Methods Animal Protocols. Male Zucker lean (ZL) rats (Charles River Laboratories, Wilmington, MA) and Zucker obese rats (Charles River Laboratories) were maintained on a standard rat diet and tap water ad libitum. In the first protocol, we used Zucker obese but not diabetic rats and in the second protocol, we used 11-week-old Zucker fat (ZF) rats and age-matched ZL controls (six animals/group). In the second protocol, we examined the effects of HO-1 and adiponectin preconditioning on the expression of HO-1, adiponectin, and the vascular phenotype (n 6/group). Chemical structures of most of the compounds were described previously (Martasek et al., 1988). Glucose monitoring was performed using an automated analyzer (Lifescan Inc., Milpitas, CA). Beginning at 11 weeks when all rats had established diabetes, CoPP, SnMP (Frontier Science, Logan, UT), or both were given weekly at a dose of 2 and 5 mg/kg once and three times a week, respectively, by i.p. injection for 6 weeks. Control animals were administered an equal volume of vehicle (0.1 M sodium citrate buffer, pH 7.8) i.p. Six groups of Zucker rats were used: 1) ZL, 2) ZL-CoPP, 3) ZL-CoPP SnMP, 4) ZF, 5) ZF-CoPP, and 6) ZFCoPP SnMP. There was no difference in the food intake among the treatment groups. The Animal Care and Use Committee of New York Medical College approved all experiments. Tissue Preparation for Western Blot of Adipocyte Stem Cells, Heart, Kidney, and Aorta. At the time of sacrifice, s.c. and visceral fat in the abdomen (visible mesenteric fat, fat around the liver, fat around the kidney, and fat around the spleen) was dissected free, pooled for each mouse, weighed, and used to isolate adipocyte mesenchymal stem cells. Cells were frozen until needed for protein measurements. Aorta, heart, and kidney were also harvested, drained of blood, and flash-frozen in liquid nitrogen. Specimens were maintained at 80°C until needed. Frozen aorta and kidney segments were pulverized, and then they were placed in homogenization buffer [10 mM phosphate buffer, 250 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1% Tergitol (Mallinckrodt Baker, Phillipsburg, NJ), pH 7.5] and homogenized using a standard glass homogenizer and pestle. Homogenates were centrifuged at 27,000g for 10 min at 4°C. The supernatant was isolated, and protein levels were assayed by Bradford method (Bradford, 1976). The supernatant was used for measurement of HO-1 and HO-2 (Nventa Biopharmaceuticals, San Diego, CA). Protein levels were visualized by immunoblotting with antibodies against each specific mouse protein. Actin was used to ensure adequate sample loading for all Western blots. Antibodies were prepared in the following dilution: HO-1 and HO-2, 1:1000. In brief, 20 g of lysate supernatant was separated by 12% SDS-polyacrylamide gel electrophoresis, and then it was transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St. Giles, UK) with a semidry transfer apparatus (Bio-Rad, Hercules, CA). The membranes were incubated with 10% milk in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20 buffer at 4°C overnight. After they were washed with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20, the membranes were incubated with anti-HO-1 or anti-HO-2 for 1 h at room temperature, with constant shaking. The filters were washed and subsequently probed with horseradish peroxidase-conjugated donkey anti-rabbit or antimouse IgG (GE Healthcare). Chemiluminescence detection was performed with the Amersham enhanced chemiluminescence detection kit, according to the manufacturer’s instructions. Aortic HO activity was assayed as described previously (Abraham et al., 2003) using a technique in which bilirubin, the end product of heme degradation, was extracted with chloroform, and its concentration was determined spectrophotometrically (dual UV-visible beam spectrophotometer Lambda 25; PerkinElmer Life and Analytical Sciences, Waltham, MA) using the difference in absorbance at a wavelength from 460 to 530 nm, with an extinction coefficient of 40 mM 1 cm . Human Bone Marrow-Derived Adipocyte Mesenchymal Stem Cells. Frozen bone marrow mononuclear cells were purchased from Allcells (Emeryville, CA). After thawing the cells, mononuclear cells were resuspended in an -minimal essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Invitrogen) and 1% antibiotic/antimycotic solution (Invitrogen). The cells were plated at a density of 1 to 5 10 cells per 100-cm dish. The cultures were maintained at 37°C in a 5% CO2 incubator, and the medium was changed after 48 h and every 3 to 4 days thereafter. When the MSCs were confluent, the cells were recovered by the addition of 0.25% trypsin/EDTA (Invitrogen). MSCs (passages 2–3) were plated in a 60-cm dish at a density of 1 to 2 10 and cultured in -minimal essential medium with 10% FBS for 7 days. The medium was replaced with adipogenic medium, and the cells were cultured for an additional 21 days. The adipogenic media consisted of complete culture medium supplemented with DMEM-high glucose, 10% (v/v) FBS, 10 g/ml insulin, 0.5 mM dexamethasone (Sigma-Aldrich, St. Louis, MO), 0.5 mM isobutyl methylxanthine (Sigma-Aldrich), and 0.1 mM indomethacin (Sigma-Aldrich). Oil Red O Staining. For Oil Red O staining, 0.5% Oil Red O solution (Sigma-Aldrich) was used. In brief, adipocytes were fixed in 1% formaldehyde, washed in Oil Red O for 20 min, rinsed with 85% propylene glycol (Sigma-Aldrich) for 3 min, washed in distilled water, and then mounted with aqueous mounting medium (Bavendiek et al., 2005). Measurement of Mesenchymal O2 . Levels and Inflammatory Cytokines and Adiponectin. Using previously described methods, control and fat mesenchymal stem cells, 0.3 mg of protein, approximately 3 10 cells, were placed in plastic scintillation minivials 834 Kim et al. at U nirsity of T oedo Liaries on O cber 3, 2009 jpet.asjournals.org D ow nladed fom containing 5 M lucigenin for the detection of O2 . and other additions in a final volume of 1 ml of air-equilibrated Krebs’ solution buffered with 10 mM HEPES-NaOH, pH 7.4. Lucigenin chemiluminescence was measured in a liquid scintillation counter (LS6000IC; Beckman Coulter, Fullerton, CA) at 37°C, and data are reported as counts per minute per milligram of protein after background subtraction. Adiponectin (HMW), TNF, and IL-6 were determined in rat serum using an enzyme-linked immunosorbent assay. Multiple assay kits were used for quantification of the proteins in rat serum, and assays were conducted according to the manufacturer’s protocol (Pierce, Woburn, MA). Plates were analyzed using a Luminex 100IS analyzer (Luminex Inc., Austin, TX). The data were evaluated as the median fluorescence intensity using appropriate curve-fitting software. A five-parameter logistic method with weighing was used. All measurements were performed in triplicate. Cytokine assays were done according to instructions provided by BD Gentest (Woburn, MA). Detection of MSC Cell Markers by FACS Analysis. Human MSCs are defined by an array of positive and negative markers. MSCs are normally plastic-adherent under standard culture conditions, expressing CD105, CD73, and CD90. MSCs must lack expression of CD45, CD34, CD14, or CD11b, CD79 or CD19, and human leukocyte antigen DR-1. In addition, MSCs must be able to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro (Keating, 2006). Human MSC phenotype was confirmed by flow cytometry (Elite ESP 2358; Beckman Coulter) using several markers known to be found on MSCs. The negative markers used were anti-CD34 and anti-CD45 (BD Biosciences Pharmingen, San Diego, CA), also known to be expressed as hematopoietic stem cell marker and common lymphocyte antigen. CD90, CD105, and CD166 were used as positive markers for MSCs. The data were analyzed using WinMDI 2.8 software (http://facs.scripps.edu/software.html). Culture Conditions of Adipocytes and Effect of HO-1 Inducers and Inhibitors. Adipogenic differentiation of hMSCs was induced by incubation in an adipogenesis induction medium (25 mM DMEM-high glucose supplemented with 10 g/ml insulin, 1 M dexamethasone, 0.2 mM indomethacin, 10% FBS, and 1% antibioticantimycotic solution). Medium was changed every 3 to 4 days (Novikoff et al., 1980; Tondreau et al., 2005). In addition, treatment with 5 M SnMP and glucose was administered every 2 days. CoPP (2 M) treatment and media changes were applied every 4 days. The conditioned media was harvested after 6 days of culture, and the levels of adiponectin were determined. Statistical Analyses. Statistical significance between experimental groups was determined by the Fisher method of analysis of multiple comparisons (p 0.05). For comparison between treatment groups, the null hypothesis was tested by a single-factor analysis of variance for multiple groups or unpaired t test for two groups. Data are presented as mean S.E.