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L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

2008; Elsevier BV; Volume: 49; Issue: 8 Linguagem: Inglês

10.1194/jlr.m800046-jlr200

ISSN

1539-7262

Autores

Stephen J. Peterson, George I. Drummond, Dong Hyun Kim, Ming Li, Adam Kruger, Susumu Ikehara, Nader G. Abraham,

Tópico(s)

Regulation of Appetite and Obesity

Resumo

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1β and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1β and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes. We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1β and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1β and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes. Oxidative stress has been implicated in the pathogenesis of insulin resistance and type 2 diabetes and its cardiovascular complications (1Robertson R.P. 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These effects are manifested through inhibition of TNF-α-mediated adhesion of monocytes and vascular cell adhesion molecules to the endothelium, resulting in the modulation of the endothelial inflammatory response. In addition, an increase in adiponectin improved the beneficial effects of antihypertensive agents in hypertensive patients(25Yilmaz M.I. Sonmez A. Caglar K. Celik T. Yenicesu M. Eyileten T. Acikel C. Oguz Y. Yavuz I. Vural A. Effect of antihypertensive agents on plasma adiponectin levels in hypertensive patients with metabolic syndrome.Nephrology (Carlton). 2007; 12: 147-153Crossref PubMed Scopus (103) Google Scholar). PPARγ was shown to regulate the increased expression of both adiponectin (26Iwaki M. Matsuda M. Maeda N. Funahashi T. Matsuzawa Y. Makishima M. Shimomura I. Induction of adiponectin, a fat-derived antidiabetic and antiatherogenic factor, by nuclear receptors.Diabetes. 2003; 52: 1655-1663Crossref PubMed Scopus (632) Google Scholar) and heme oxygenase 1(HO-1) in human vascular cells (27Kronke G. Kadl A. Ikonomu E. Bluml S. Furnkranz A. Sarembock I.J. Bochkov V.N. Exner M. Binder B.R. Leitinger N. Expression of heme oxygenase-1 in human vascular cells is regulated by peroxisome proliferator-activated receptors.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1276-1282Crossref PubMed Scopus (187) Google Scholar). The development of apolipoprotein A-I (apoA-I) mimetic peptides, such as 4F, has previously been reported in detail (28Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.Apolipoprotein A-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (225) Google Scholar). These peptides have equal efficacy whether synthesized from d- or l-amino acids (29Van Lenten B.J. Wagner A.C. Navab M. Anantharamaiah G.M. Hama S. Reddy S.T. Fogelman A.M. Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits.J. Lipid Res. 2007; 48: 2344-2353Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We previously reported that rats made diabetic by an injection of streptozotocin had increased aortic oxidative stress and endothelial sloughing that was ameliorated by D-4F (4Kruger A.L. Peterson S. Turkseven S. Kaminski P.M. Zhang F.F. Quan S. Wolin M.S. Abraham N.G. D-4F induces heme oxygenase-1 and extracellular superoxide dismutase, decreases endothelial cell sloughing, and improves vascular reactivity in rat model of diabetes.Circulation. 2005; 111: 3126-3134Crossref PubMed Scopus (161) Google Scholar, 30Peterson S.J. Husney D. Kruger A.L. Olszanecki R. Ricci F. Rodella L.F. Stacchiotti A. Rezzani R. McClung J.A. Aronow W.S. et al.Long-term treatment with the apolipoprotein A1 mimetic peptide increases antioxidants and vascular repair in type I diabetic rats.J. Pharmacol. Exp. Ther. 2007; 322: 514-520Crossref PubMed Scopus (76) Google Scholar). D-4F treatment was associated with increased levels of aortic HO-1, decreased superoxide, and increased extracellular superoxide dismutase. In the present study, we used a mouse model of obesity and diabetes (ob/ob) that develops spontaneous obesity because of a lack of functional leptin, a protein that regulates appetite. Lack of leptin leads to excessive eating, obesity (31Halaas J.L. Boozer C. Blair-West J. Fidahusein N. Denton D.A. Friedman J.M. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice.Proc. Natl. Acad. Sci. USA. 1997; 94: 8878-8883Crossref PubMed Scopus (885) Google Scholar), and diabetes, presumably due to an increase of inflammatory cytokines involved in insulin resistance (32M Li Kim D.H. Tsenovoy P. Peterson S.J. Rezzani R. Rodella L.F. Aronow W.S. Ikehara S. Abraham N.G. Treatment of obese diabetic mice with an heme oxygenase inducer reduces visceral and abdominal adiposity increases adiponectin levels and improves insulin sensitivity and glucose tolerance.Diabetes. 2008; (In press)Google Scholar). In this manuscript, we show, in a type 2 diabetic animal model, that treatment with L-4F results in increased HO-1 protein and adiponectin levels, decreased superoxide generation, and improved insulin sensitivity and glucose tolerance. L-4F and all reagents were from sources previously reported (4Kruger A.L. Peterson S. Turkseven S. Kaminski P.M. Zhang F.F. Quan S. Wolin M.S. Abraham N.G. D-4F induces heme oxygenase-1 and extracellular superoxide dismutase, decreases endothelial cell sloughing, and improves vascular reactivity in rat model of diabetes.Circulation. 2005; 111: 3126-3134Crossref PubMed Scopus (161) Google Scholar, 30Peterson S.J. Husney D. Kruger A.L. Olszanecki R. Ricci F. Rodella L.F. Stacchiotti A. Rezzani R. McClung J.A. Aronow W.S. et al.Long-term treatment with the apolipoprotein A1 mimetic peptide increases antioxidants and vascular repair in type I diabetic rats.J. Pharmacol. Exp. Ther. 2007; 322: 514-520Crossref PubMed Scopus (76) Google Scholar) and all other reagents were of the highest available purity. Male obese mice (B6v-Lep ob/J) were purchased from Harlan (Chicago, IL) at the age of 7 weeks, allowed to acclimatize for 1 week, and used at the age of 8 weeks. Age- and sex-matched lean mice (B6.V, lean; Harlan) were used as controls. Mice were fed a normal chow diet and had free access to water and food. Body weight of obese and lean mice at the beginning of the treatment was 34 ± 5 g and 26 ± 3 g, respectively. Beginning at 9 weeks of age, when all obese mice had established diabetes, L-4F [i.e., Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 synthesized from l-amino acids only as previously described (33Navab M. Anantharamaiah G.M. Hama S. Garber D.W. Chaddha M. Hough G. Lallone R. Fogelman A.M. Oral administration of an apo A-I mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol.Circulation. 2002; 105: 290-292Crossref PubMed Scopus (365) Google Scholar) was injected at a dose of 200 μg/100 gdaily in 200 μl vehicle; or vehicle (ABCT: ammonium bicarbonate buffer at pH 7.4 containing 0.01% Tween-20) was administered intraperitoneally for 6 weeks. Blood (50–100 μl) was collected from tailveins of anesthetized obese mice following L-4F or vehicle solution.Glucose monitoring was performed using an automated analyzer (Lifescan, Inc.; Milpitas, CA). Levels were 229±21 and 154 ± 9 mg/dl for obese and lean mice, respectively.There were four groups of animals: A) lean, B) lean+L-4F, C) obese, and D) obese + L-4F. In a separate experiment, vehicle-treated and L-4F-treated animals were housed in metabolic cages. Food intake was monitored daily and was not different in mice receiving vehicle or L-4F. Food intake was monitored less frequently thereafter by measuring remaining chow and body weight. The Animal Care and Use Committee of New York Medical College approved all experiments. At the time of euthanization, subcutaneous and visceral fat in the abdomen (the visible mesenteric fat, fat around the liver, fat around the kidney, and fat around the spleen) were dissected free, pooled for each mouse, weighed, and used to isolate adipocyte mesenchymal stem cells (MSCs). Cells were frozen until needed for protein measurements. Aorta 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 placed in a homogenization buffer (10 mM phosphate buffer, 250 mM sucrose, 1 mM EDTA, 0.1 mM PMSF, and 0.1% tergitol, pH 7.5). Homogenates were centrifuged at 27,000 g for 10 min at 4°C. The supernatant was isolated, and protein levels were assayed (Bradford method). The supernatant was used for measurement of HO-1 and HO-2 (Stressgen Biotechnologies Corp.; Victoria, BC). 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 dilutions: HO-1 and HO-2, 1:1,000 (Upstate Cell Signaling Solutions; Chicago, IL). Briefly, 20 μg of lysate supernatant was separated by 12% SDS/PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences; Uppsala, Sweden) with a semi-dry transfer apparatus (Bio-Rad; Hercules, CA). The membranes were incubated with 10% milk in 10 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, and 0.05% Tween-20 (TBST) buffer at 4°C overnight. After they were washed with TBST, the membranes were incubated with either 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 anti-mouse IgG (Amersham). Chemiluminescence detection was performed with the Amersham ECL detection kit according to the manufacturer's instructions. Bone marrow and aortic HO activity were assayed as previously described (34Abraham N.G. Kushida T. McClung J. Weiss M. Quan S. Lafaro R. Darzynkiewicz Z. Wolin M. Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endothelial cells.Circ. Res. 2003; 93: 507-514Crossref PubMed Scopus (143) Google Scholar) using a technique in which bilirubin, the end product of heme degradation, was extracted with chloroform and its concentration determined spectrophotometrically (Dual UV/VIS Beam Spectrophotometer Lambda 25; Perkin-Elmer, Norwalk, CT) using the difference in absorbance between 464 nm and 530 nm and an extraction coefficient of 40 mM−1 cm−1. To isolate mouse adipocyte MSCs, adipose tissues were washed with PBS and digested at 37°C for 30 min with 0.075% type II collagenase (35Lee R.H. Kim B. Choi I. Kim H. Choi H.S. Suh K. Bae Y.C. Jung J.S. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.Cell. Physiol. Biochem. 2004; 14: 311-324Crossref PubMed Scopus (807) Google Scholar). Enzyme activity was inhibited with α-MEM containing 10% FBS and 1% antimitotic/antimycotic solution (Invitrogen; Carlsbad, CA), and centrifuged at 1,200 × g for 10 min. The pellet was then resuspended in MEM media as described above. Bone marrow smears made from the fibia were stained with 0.5% Oil Red O in isopropanol (w/v) for 10 min, and lipid droplets were then evaluated using a light microscope digitalized with a charge-coupled device camera and an image analysis system (Imaging and Computers; Milan, Italy). The mean number of lipid droplets was calculated from six different fields. Frozen bone marrow mononuclear cells were purchased from Allcells (Emeryville, CA). After thawing, mononuclear cells were resuspended in α-MEM (Invitrogen) supplemented with 10% heat-inactivated FBS (Invitrogen), and 1% antibiotic/antimycotic solution (Invitrogen). The cells were plated at a density of 1–5 × 106 cells per 100 cm2 dish. The cultures were maintained at 37°C in a 5% CO2 incubator. The medium was changed after 48 h and every 3–4 days thereafter. When the MSCs were confluent, the cells were recovered by the addition of 0.25% trypsin/EDTA (Life Technologies; Frederick, MD). Passage 2–3 MSCs were plated in a 60 cm2 dish at a density of 1–2 × 104 and cultured in α-MEM 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 μg/ml insulin, 0.5 mM dexamethasone (Sigma-Aldrich; St. Louis, MO), 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and 0.1 mM indomethacin (Sigma-Aldrich). Subcutaneous and visceral fats were pooled for each mouse and used to isolate adipocyte stem cells. Cells were frozen until needed for protein measurements and O2− levels. Employing previously described methods, MSCs from lean, obese vehicle-treated, and L-4F-treated mice were placed in plastic scintillation minivials containing 5 μm lucigenin for the detection of O2− 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 Instruments, San Diego, CA) at ∼37°C; data are reported as counts/min/mg protein after background subtraction. Human MSCs are defined by an array of positive and negative markers. Human MSCs are normally plastic-adherent under standard culture conditions and express CD105, CD73, and CD90. To be classified as an MSC, the cell must also lack expression of CD45, CD34, CD14 or CD11b, CD79 or CD19, and HLA-DR. In addition, MSCs must be able to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro (36Keating A Mesenchymal stromal cells.Curr. Opin. Hematol. 2006; 13: 419-425Crossref PubMed Scopus (249) Google Scholar). Human MSC phenotype was confirmed by flow cytometry (Elite ESP 2358; Beckman-Coulter, Miami, FL) using several markers known to be found on MSCs. The negative markers used were anti-CD34 and anti-CD45 (BD-Pharmingen; Palo Alto, CA), which are known to be expressed on hematopoietic stem cells and common lymphocytes. CD90, CD105, and CD166 were used as positive markers for human MSCs. The data were analyzed using WinMDI 2.8 software. Adipogenic differentiation of human MSCs was induced by incubation in an adipogenesis induction medium [DMEM-high glucose (37Novikoff A.B. Novikoff P.M. Rosen O.M. Rubin C.S. Organelle relationships in cultured 3T3-L1 preadipocytes.J. Cell Biol. 1980; 87: 180-196Crossref PubMed Scopus (260) Google Scholar, 38Tondreau T. Meuleman N. Delforge A. Dejeneffe M. Leroy R. Massy M. Mortier C. Bron D. Lagneaux L. Mesenchymal stem cells derived from CD133-positive cells in mobilized peripheral blood and cord blood: proliferation, Oct4 expression, and plasticity.Stem Cells. 2005; 23: 1105-1112Crossref PubMed Scopus (383) Google Scholar), supplemented with 10 μg/ml of insulin, 1 μmol/l of dexamethasone, 0.2 mmol/l of indomethacin, 10% FBS, and 1% antibiotic–antimycotic solution]. The medium was changed every 3–4 days (37Novikoff A.B. Novikoff P.M. Rosen O.M. Rubin C.S. Organelle relationships in cultured 3T3-L1 preadipocytes.J. Cell Biol. 1980; 87: 180-196Crossref PubMed Scopus (260) Google Scholar, 38Tondreau T. Meuleman N. Delforge A. Dejeneffe M. Leroy R. Massy M. Mortier C. Bron D. Lagneaux L. Mesenchymal stem cells derived from CD133-positive cells in mobilized peripheral blood and cord blood: proliferation, Oct4 expression, and plasticity.Stem Cells. 2005; 23: 1105-1112Crossref PubMed Scopus (383) Google Scholar), in the presence of vehicle alone or vehicle containing L-4F. At 50% confluence, L-4F and vehicle solutions were added and adipogenesis was measured using Oil Red O as described (38Tondreau T. Meuleman N. Delforge A. Dejeneffe M. Leroy R. Massy M. Mortier C. Bron D. Lagneaux L. Mesenchymal stem cells derived from CD133-positive cells in mobilized peripheral blood and cord blood: proliferation, Oct4 expression, and plasticity.Stem Cells. 2005; 23: 1105-1112Crossref PubMed Scopus (383) Google Scholar). Briefly, cells were fixed in ice-cold 10% formalin in PBS for 10 min, rinsed with distilled water, and stained with Oil Red O solution for 20 min. Cells were placed in absolute propylene glycol for 5 min, rinsed in 85% propylene glycol, followed by distilled water, and air dried. Oil Red O stain was extracted with isopropanol, and optical absorbance was measured at 490–520 nm. For Oil Red O staining, 0.5% Oil Red O solution (Sigma-Aldrich) was used. Briefly, 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 mounted with aqueous mounting medium (39Bavendiek U. Zirlik A. LaClair S. MacFarlane L. Libby P. Schonbeck U. Atherogenesis in mice does not require CD40 ligand from bone marrow-derived cells.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1244-1249Crossref PubMed Scopus (53) Google Scholar). Adiponectin (HMW), IL-6, and IL-1β were determined in mouse serum using an ELISA assay (Pierce Biotechnology, Inc.; Woburn, MA), and insulin was measured using an ELISA kit (Millipore; Billerica, MA). After a 12 h fast, mice were injected intraperitoneally with glucose (2.0 g/kg body weight). Blood samples were taken at various time points (0–120 min), and blood glucose levels and serum insulin levels were measured. For determination of insulin tolerance, mice were injected intraperitoneally with insulin (2.0 U/kg). Blood samples were taken at various time points (0–90 min), and blood glucose levels were measured. 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 ANOVA for multiple groups or unpaired t-test for two groups. Data are presented as mean ± SEM. Prior to treatment, the ratio of HO-1 to actin in the aorta of obese mice was significantly less (P < 0.05) when compared with lean mice, using Western blot analysis. The ratio of HO-2 to actin was slightly lower in obese mice compared with lean (0.95 ± 0.05 and 1.10 ± 0.06) (Fig. 1A), but the difference was not significant. In a separate experiment, daily injection of L-4F for 6 weeks resulted in a significant increase in HO-1 expression in both lean and obese mice (Fig. 1B). Densitometry scanning showed that the ratio of HO-1 to actin was significantly increased by L-4F (lean vs. 4F-treated lean mice, P < 0.05; vehicle-treated obese mice vs. L-4F-treated obese mice, P < 0.008; results not shown). HO-2 levels were unaffected by L-4F treatment in both lean and obese animals. There was no difference in HO-1 and HO-2 levels in vehicle-treated and untreated animals. HO activity was measured in aortas isolated from L-4F- and vehicle-treated animals (Fig. 1C). There was a significant (P < 0.05) decrease in HO activity in vehicle-treated

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