Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice
2006; Elsevier BV; Volume: 48; Issue: 2 Linguagem: Inglês
10.1194/jlr.m600393-jlr200
ISSN1539-7262
AutoresAparna Purushotham, Angela A. Wendel, Lifen Liu, Martha A. Belury,
Tópico(s)Fatty Acid Research and Health
ResumoConjugated linoleic acid (CLA) causes insulin resistance and hepatic steatosis in conjunction with depletion of adipokines in some rodent models. Our objective was to determine whether the maintenance of adipokines, mainly leptin and adiponectin, by either removing CLA from diets or using an adiponectin enhancer, rosiglitazone (ROSI), could attenuate CLA-induced insulin resistance. Male C57BL/6 mice were consecutively fed two experimental diets containing 1.5% CLA mixed isomer for 4 weeks followed by a diet without CLA for 4 weeks. CLA significantly depleted adiponectin but not leptin and was accompanied by hepatic steatosis and insulin resistance. These effects were attenuated after switching mice to the diet without CLA along with restoration of adiponectin. To further elucidate the role of adiponectin in CLA-mediated insulin resistance, ROSI was used in a subsequent study in male ob/ob mice fed either control (CON) or CLA diet. ROSI maintained significantly higher adiponectin levels in CON- and CLA-fed mice and prevented the depletion of epididymal adipose tissue and the development of insulin resistance. In conclusion, we show that insulin resistance induced by CLA may be related more to adiponectin depletion than to leptin and that maintaining adiponectin levels alone either by removing CLA or using ROSI can attenuate these effects. Conjugated linoleic acid (CLA) causes insulin resistance and hepatic steatosis in conjunction with depletion of adipokines in some rodent models. Our objective was to determine whether the maintenance of adipokines, mainly leptin and adiponectin, by either removing CLA from diets or using an adiponectin enhancer, rosiglitazone (ROSI), could attenuate CLA-induced insulin resistance. Male C57BL/6 mice were consecutively fed two experimental diets containing 1.5% CLA mixed isomer for 4 weeks followed by a diet without CLA for 4 weeks. CLA significantly depleted adiponectin but not leptin and was accompanied by hepatic steatosis and insulin resistance. These effects were attenuated after switching mice to the diet without CLA along with restoration of adiponectin. To further elucidate the role of adiponectin in CLA-mediated insulin resistance, ROSI was used in a subsequent study in male ob/ob mice fed either control (CON) or CLA diet. ROSI maintained significantly higher adiponectin levels in CON- and CLA-fed mice and prevented the depletion of epididymal adipose tissue and the development of insulin resistance. In conclusion, we show that insulin resistance induced by CLA may be related more to adiponectin depletion than to leptin and that maintaining adiponectin levels alone either by removing CLA or using ROSI can attenuate these effects. Type 2 diabetes is characterized by impaired glucose and lipid metabolism and is associated with obesity (1Kahn B.B. Flier J.S. Obesity and insulin resistance. J. Clin. Invest. 2000; 106: 473-481Crossref PubMed Scopus (2446) Google Scholar). Adipose tissue not only stores excess energy but also has important endocrine functions. Proteins secreted from the adipose tissue, known as adipokines, have important functions in regulating whole body metabolism (2Matsuzawa Y. Funahashi T. Nakamura T. Molecular mechanism of metabolic syndrome X: contribution of adipocytokines adipocyte-derived bioactive substances. Ann. N. Y. Acad. Sci. 1999; 892: 146-154Google Scholar). In particular, the adipokine adiponectin was identified in the adipose tissue and plasma of humans and rodents (3Maeda K. Okubo K. Shimomura I. Funahashi T. Matsuzawa Y. Matsubara K. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1). Biochem. Biophys. Res. Commun. 1996; 221: 286-289Google Scholar, 4Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. A novel serum protein similar to C1q, produced exclusively in adipocytes. J. Biol. Chem. 1995; 270: 26746-26749Google Scholar, 5Hu E. Liang P. Spiegelman B.M. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J. Biol. Chem. 1996; 271: 10697-10703Google Scholar, 6Nakano Y. Tobe T Choi-Miura N.H. Mazda T. Tomita M. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. (Tokyo). 1996; 120: 803-812Google Scholar) and is inversely associated with obesity and type 2 diabetes (7Hotta K. Funahashi T. Arita Y. Takahashi M. Matsuda M. Okamoto Y. Iwahashi H. Kuriyama H. Ouchi N. Maeda K. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler. Thromb. Vasc. Biol. 2000; 20 (et al.): 1595-1599Google Scholar, 8Berg A.H. Combs T.P. Du X. Brownlee M. Scherer P.E. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat. Med. 2001; 7: 947-953Google Scholar). Administration of adiponectin attenuates insulin resistance by decreasing tissue triglyceride (TG) levels as a result of increased fatty acid oxidation (9Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat. Med. 2002; 8 (et al.): 1288-1295Google Scholar, 10Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat. Med. 2001; 7 (et al.): 941-946Google Scholar). In addition, adiponectin decreases hyperglycemia by suppressing hepatic glucose production along with increasing glucose uptake by the skeletal muscle (8Berg A.H. Combs T.P. Du X. Brownlee M. Scherer P.E. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat. Med. 2001; 7: 947-953Google Scholar, 9Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat. Med. 2002; 8 (et al.): 1288-1295Google Scholar). Conjugated linoleic acid (CLA) consists of positional and geometric isomers of octadecadienoate that are naturally found in foods such as beef, lamb, milk, and other dairy products (11Pariza M.W. Ha Y.L. Conjugated dienoic derivatives of linoleic acid: a new class of anticarcinogens. Med. Oncol. Tumor Pharmacother. 1990; 7: 169-171Google Scholar). It is well established that cis9,trans11 (c9t11)-CLA and trans 10,cis 12 (t10c12-CLA) have unique effects on lipid metabolism (12Brown J.M. Boysen M.S. Jensen S.S. Morrison R.F. Storkson J. Lea-Currie R. Pariza M. Mandrup S. McIntosh M.K. Isomer-specific regulation of metabolism and PPARgamma signaling by CLA in human preadipocytes. J. Lipid Res. 2003; 44: 1287-1300Google Scholar), and it is the t10c12-CLA isomer that is mainly associated with decreases in body fat in experimental rodent models (13Hargrave K.M. Li C. Meyer B.J. Kachman S.D. Hartzell D.L. Della-Fera M.A. Miner J.L. Baile C.A. Adipose depletion and apoptosis induced by trans-10,cis-12 conjugated linoleic acid in mice. Obes. Res. 2002; 10: 1284-1290Google Scholar, 14Park Y. Storkson J.M. Albright K.J. Liu W. Pariza M.W. Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids. 1999; 34: 235-241Google Scholar, 15Ryder J.W. Portocarrero C.P. Song X.M. Cui L. Yu M. Combatsiaris T. Galuska D. Bauman D.E. Barbano D.M. Charron M.J. Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression. Diabetes. 2001; 50 (et al.): 1149-1157Google Scholar, 16Roche H.M. Noone E. Sewter C. McBennett S. Savage D. Gibney M.J. O'Rahilly S. Vidal-Puig A.J. Isomer-dependent metabolic effects of conjugated linoleic acid: insights from molecular markers sterol regulatory element-binding protein-1c and LXRalpha. Diabetes. 2002; 51: 2037-2044Google Scholar) as well as in some but not all human studies (17Blankson H. Stakkestad J.A. Fagertun H. Thom E. Wadstein J. Gudmundsen O. Conjugated linoleic acid reduces body fat mass in overweight and obese humans. J. Nutr. 2000; 130: 2943-2948Google Scholar, 18Belury M.A. Mahon A. Banni S. The conjugated linoleic acid (CLA) isomer, t10c12-CLA, is inversely associated with changes in body weight and serum leptin in subjects with type 2 diabetes mellitus. J. Nutr. 2003; 133 (Suppl.): 257-260Google Scholar, 19Terpstra A.H. Effect of conjugated linoleic acid on body composition and plasma lipids in humans: an overview of the literature. Am. J. Clin. Nutr. 2004; 79: 352-361Google Scholar), independently of energy intake (20DeLany J.P. Blohm F. Truett A.A. Scimeca J.A. West D.B. Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake. Am. J. Physiol. 1999; 276: 1172-R–R1179Google Scholar). Feeding CLA to mice is associated with lipodystrophy and worsening of insulin sensitivity (16Roche H.M. Noone E. Sewter C. McBennett S. Savage D. Gibney M.J. O'Rahilly S. Vidal-Puig A.J. Isomer-dependent metabolic effects of conjugated linoleic acid: insights from molecular markers sterol regulatory element-binding protein-1c and LXRalpha. Diabetes. 2002; 51: 2037-2044Google Scholar, 21Tsuboyama-Kasaoka N. Takahashi M. Tanemura K. Kim H.J. Tange T. Okuyama H. Kasai M. Ikemoto S. Ezaki O. Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. Diabetes. 2000; 49: 1534-1542Google Scholar, 22Clement L. Poirier H. Niot I. Bocher V. Guerre-Millo M. Krief S. Staels B. Besnard P. Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse. J. Lipid Res. 2002; 43: 1400-1409Google Scholar, 23Poirier H. Rouault C. Clement L. Niot I. Monnot M.C. Guerre-Millo M. Besnard P. Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse. Diabetologia. 2005; 48: 1059-1065Google Scholar, 24Poirier H. Niot I. Clement L. Guerre-Millo M. Besnard P. Development of conjugated linoleic acid (CLA)-mediated lipoatrophic syndrome in the mouse. Biochimie. 2005; 87: 73-79Google Scholar). These effects have been attributed to a rapid and significant reduction of adipose tissue and a sharp decline in insulin-sensitizing adipokines such as adiponectin and leptin (23Poirier H. Rouault C. Clement L. Niot I. Monnot M.C. Guerre-Millo M. Besnard P. Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse. Diabetologia. 2005; 48: 1059-1065Google Scholar). Although feeding dietary CLA has been shown previously to deplete adipokines and cause hyperinsulinemia (23Poirier H. Rouault C. Clement L. Niot I. Monnot M.C. Guerre-Millo M. Besnard P. Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse. Diabetologia. 2005; 48: 1059-1065Google Scholar), it is unknown whether removing CLA from the diet can restore the level of adipokines and attenuate insulin resistance, showing that there is, in fact, a causal link between adipokine depletion by feeding dietary CLA and the development of hepatic steatosis and insulin resistance in mice. Furthermore, dietary CLA has been shown to induce insulin resistance in ob/ob mice, which lack functional leptin (16Roche H.M. Noone E. Sewter C. McBennett S. Savage D. Gibney M.J. O'Rahilly S. Vidal-Puig A.J. Isomer-dependent metabolic effects of conjugated linoleic acid: insights from molecular markers sterol regulatory element-binding protein-1c and LXRalpha. Diabetes. 2002; 51: 2037-2044Google Scholar), raising the possibility that adipokines other than leptin may be more important in CLA-mediated insulin resistance in mice. Thus, we further investigated the role of adiponectin in insulin resistance mediated by CLA in leptin-deficient ob/ob mice. To this end, we used the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (ROSI) in conjunction with CLA and hypothesized that maintaining serum adiponectin in ob/ob mice, which lack functional leptin, attenuates the effects of CLA on insulin resistance and hyperglycemia. Diet components were purchased from Research Diets (New Brunswick, NJ) and Bio-Serv (Frenchtown, NJ) for studies 1 and 2, respectively. CLA-mixed TGs (39.2% c9t11-CLA and 38.5% t10c12-CLA) were obtained from Cognis (Cincinnati, OH). ROSI was obtained form Cayman Chemical (Ann Arbor, MI). Eleven week old male C57BL/6 mice and 6 week old male ob/ob mice were purchased from Harlan (Indianapolis, IN) and Charles River Laboratories, Inc. (Wilmington, MA), respectively. Mice were housed four per cage at 22 ± 0.5°C on a 12 h day/night cycle. Mice received standard chow for 1 week while adjusting to their new environment. All procedures were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Ohio State University. To determine the role of adipokine depletion by CLA in the development of insulin resistance and hepatic steatosis, 12 week old male C57BL/6 mice (n = 10) were consecutively fed two different experimental diets. For the first 4 weeks, mice were fed a 1.5% CLA experimental diet, which contained 5% soybean oil plus 1.5% CLA-TG mix by weight, for a total of 6.5% fat. This dose of CLA provided ∼0.6% (by weight) each of the c9t11-CLA and t10c12-CLA isomers. Supplementation with either 0.5% purified t10c12-CLA isomer or 1% CLA mixture has been shown to effectively reduce adipose tissue in mice and produce liver steatosis (15Ryder J.W. Portocarrero C.P. Song X.M. Cui L. Yu M. Combatsiaris T. Galuska D. Bauman D.E. Barbano D.M. Charron M.J. Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression. Diabetes. 2001; 50 (et al.): 1149-1157Google Scholar, 23Poirier H. Rouault C. Clement L. Niot I. Monnot M.C. Guerre-Millo M. Besnard P. Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse. Diabetologia. 2005; 48: 1059-1065Google Scholar, 24Poirier H. Niot I. Clement L. Guerre-Millo M. Besnard P. Development of conjugated linoleic acid (CLA)-mediated lipoatrophic syndrome in the mouse. Biochimie. 2005; 87: 73-79Google Scholar). At the end of the first 4 week diet period, half of the mice were euthanized, and the remaining mice were switched to a diet without CLA (chow diet containing 4–5% total fat). All mice had free access to food and water. Body weights were measured at the indicated time points. Six week old male ob/ob mice were randomized by body weight and fed experimental diets containing 6.5% total fat for 4 weeks. The diets contained 6.5% soybean oil [control (CON) diet; n = 8] by weight. Additionally, 10 mice were maintained on the CON diet for the first 2 weeks, after which 6 mice were switched to the CLA diet and 4 mice were continued on the CON diet for the last 2 weeks of the study period. During the last 2 weeks, mice received daily intraperitoneal injections of PBS (vehicle control: 10% DMSO and 90% PBS solution) or 10 mg/kg body weight/day ROSI (25Chaput E. Saladin R Silvestre M. Edgar A.D. Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight. Biochem. Biophys. Res. Commun. 2000; 271: 445-450Google Scholar, 26Carmona M.C. Louche K. Nibbelink M. Prunet B. Bross A. Desbazeille M. Dacquet C. Renard P. Casteilla L. Penicaud L. Fenofibrate prevents rosiglitazone-induced body weight gain in ob/ob mice. Int. J. Obes. 2005; 29: 864-871Google Scholar). Body weights were measured weekly. Insulin sensitivity was determined for studies 1 and 2 using an insulin tolerance test at the indicated times. Mice were fasted overnight and received intraperitoneal injections of insulin [Humulin R (Eli Lily, Inc.)] at doses of 0.75 U/kg body weight for C57BL/6 mice and 1.5 U/kg body weight for ob/ob mice. Insulin-stimulated glucose clearance was determined by tail vein bleeding at 0, 15, 30, 45, 60, 90, and 120 min after insulin injection. Insulin sensitivity was determined by calculating the areas under the curves, and individual baselines were used to normalize data. Fasting blood glucose (FBG) levels were measured at baseline, 2 weeks, and 4 weeks for study 2. Mice were fasted overnight for 12 h, and tail vein blood was used to analyze FBG using a One Touch Basic glucose analyzer (Lifescan, Milpitas, CA). To avoid the effects of injected insulin on gene expression, mice were anesthetized by isoflurane in the fasted state 2 days after the insulin tolerance test. Blood was collected by heart puncture, centrifuged at 1,500 g at 4°C to isolate serum, and stored at −80°C for hormone and metabolite analyses. Liver, epididymal adipose, and gastrocnemius muscle tissues were weighed, snap-frozen in liquid nitrogen, and stored at −80°C for further analysis. Time course depletion-repletion of adipokines from study 1 was determined using 4 h fasted serum from retro-orbital eye bleeds at the indicated time points. Mice were anesthetized using isoflurane. Additionally, fasted serum insulin, adiponectin, and resistin levels from study 2 were determined using ELISAs (LINCO Research, St. Charles, MO). Fasting serum TGs and NEFAs from study 2 were measured using spectrophotometric assays from Sigma (St. Louis, MO) and Wako Chemicals (Richmond, VA), respectively. Liver and muscle tissues were homogenized and lysed in 10× Tris (w/v) buffer containing 20 mM trizma base, 1% Triton X-100, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM Na4P2O7·10H2O, and protease inhibitors. TGs were extracted with 2:1 (v/v) chloroform-methanol, final extracts were dissolved in 3:1:1 (v/v/v) tert-butanol-methanol-Triton X-100 (27Danno H. Jincho Y. Budiyanto S. Furukawa Y. Kimura S. A simple enzymatic quantitative analysis of triglycerides in tissues. J. Nutr. Sci. Vitaminol. (Tokyo). 1992; 38: 517-521Crossref PubMed Scopus (69) Google Scholar), and TGs were quantitatively measured with an enzymatic colorimetric kit (Sigma). Values are expressed as percentage tissue weight. Sections from liver and muscle tissue were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA), and RNA was isolated using the manufacturer's protocol. RNA from adipose tissue was isolated using the RNeasy lipid extraction kit (Qiagen, Valencia, CA). RNA was diluted in RNase-free water and quantified by spectrophotometry. RNA integrity was assessed by electrophoresis using agarose gel and ethidium bromide staining. The first transcripts were reverse-transcribed using reverse transcriptase (Invitrogen), and cDNA was amplified using real-time PCR with FAM-labeled TaqMan gene expression assays (Applied Biosystems, Foster City, CA). In short, 5 ng of the reverse transcription reaction was amplified in a total reaction volume of 25 μl using predesigned and validated primers for liver fatty acid synthase (FAS), the fatty acid transporter CD36, acetyl-coenzyme A oxidase (AOX), and tumor necrosis factor-α (TNF-α) using universal cycling conditions. Target gene expression was normalized to Vic-labeled 18S, which was used as an endogenous control and amplified in the same reaction as the target gene. All data are presented as means ± SEM. Data were analyzed using MINITAB (version 14). Data from study 2 were analyzed by one-way ANOVA. Posthoc analysis was performed using Tukey's test. Other comparisons were analyzed by Student's t-test as appropriate. Weight gain and serum adipokine concentrations over time were analyzed by repeated-measures ANOVA using SAS (version 9.1; Cary, NC). Differences were considered significant at P < 0.05. Body weights and weight gain were reduced significantly in C57BL/6 mice after 4 weeks of supplementation with dietary CLA (Table 1 , Fig. 1A ). Supplementation with dietary CLA significantly decreased epididymal adipose mass and increased liver weight. When CLA was removed from the diets, body weight and adipose tissue mass significantly increased (Table 1). Concomitant with increased body weight and adipose tissue weight, liver weights decreased significantly after 4 weeks on the diet without CLA (Table 1).TABLE 1Body weights and organ weights, study 1VariableBaseline+CLA−CLABody weight (g)28.08 ± 0.7327.211 ± 0.90aP < 0.05 versus baseline.30.33 ± 1.60bP < 0.05 versus +CLA.Liver weight (% body weight)—7.56 ± 0.834.28 ± 0.08bP < 0.05 versus +CLA.Epididymal adipose (g)—0.233 ± 0.030.38 ± 0.01bP < 0.05 versus +CLA.Insulin-stimulated glucose uptake (area under the curve)−6272.14 ± 793.51−1,864.69 ± 581.99aP < 0.05 versus baseline.−4,735.00 ± 514.13bP < 0.05 versus +CLA.Liver TG (% liver weight)—12.4089 ± 0.49682.8534 ± 1.1637bP < 0.05 versus +CLA.CLA, conjugated linoleic acid; TG, triglyceride. Body and organ weights of male C57BL/6 mice at baseline (n = 10), after 4 weeks of feeding diet with (+) CLA (n = 5), and after an additional 4 weeks of feeding diet without (−) CLA (n = 5). Values represent means ± SEM. Superscripts represent significant differences between treatments.a P < 0.05 versus baseline.b P < 0.05 versus +CLA. Open table in a new tab Fig. 1.Effect of dietary conjugated linoleic acid (CLA) on weight gain from studies 1 and 2. A: Male C57BL/6 mice were fed a diet containing 1.5% CLA (+CLA; n = 10) for 4 weeks followed by 4 weeks without CLA (−CLA; n = 5). * P < 0.05 versus baseline; § P < 0.05 versus the last time point on the diet containing CLA. B: Male ob/ob mice were fed either control (CON) or CLA-supplemented (CLA) diets and received either PBS or rosiglitazone (ROSI) for 2 weeks by intraperitoneal injection daily. Closed circles, CON-PBS (n = 8); open circles, CON-ROSI (n = 4); closed triangles, CLA-ROSI (n = 6). Data shown are means ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CLA, conjugated linoleic acid; TG, triglyceride. Body and organ weights of male C57BL/6 mice at baseline (n = 10), after 4 weeks of feeding diet with (+) CLA (n = 5), and after an additional 4 weeks of feeding diet without (−) CLA (n = 5). Values represent means ± SEM. Superscripts represent significant differences between treatments. Levels of adiponectin decreased over time in C57BL/6 mice on the diet containing CLA. Significant differences were observed at day 6, and adiponectin levels continued to decrease over time (Fig. 2B ). Switching mice to the diet without CLA significantly increased adiponectin levels; however, levels remained significantly lower (50% of baseline) than in C57BL/6 mice. In contrast, leptin levels were less responsive to dietary CLA and were not significantly different compared with baseline (Fig. 2A).Fig. 2.Effect of CLA on time-course depletion/repletion of adipokines from study 1. Male C57BL/6 mice were fed a diet containing 1.5% CLA (+CLA; n = 10) for 4 weeks followed by 4 weeks without CLA (−CLA; n = 5). A: Serum leptin in male C57BL/6 mice. B: Serum adiponectin in male C57BL/6 mice. Adipokine concentrations were determined at the indicated times. * P < 0.05 versus baseline; § P < 0.05 versus the last time point on the diet containing CLA. Data shown are means ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Insulin sensitivity was significantly worsened in C57BL/6 mice after 4 weeks on the CLA diet (area under the curve) (Table 1, Fig. 3A ). Switching mice to the diet without CLA significantly improved insulin sensitivity. The improvement in insulin sensitivity was significant at 2 weeks after the switch to the diet without CLA.Fig. 3.Effect of dietary CLA on insulin sensitivity. A: Male C57BL/6 mice from study 1. Insulin sensitivity was measured at baseline (n = 10; closed circles), after 4 weeks on dietary CLA (+CLA; n = 10; open circles), and after 2 weeks on the diet without CLA (−CLA; n = 5; closed triangles). B: Male ob/ob mice from study 2. Insulin sensitivity was measured after 2 weeks on either CON or CLA-supplemented diets and injected with PBS or ROSI. Closed circles, CON-PBS (n = 8); open circles, CON-ROSI (n = 4); closed triangles, CLA-ROSI (n = 6). Significance was determined using area under the curve (Tables 1, 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Corresponding to increased liver weights, 4 weeks of feeding dietary CLA significantly increased hepatic TG in C57BL/6 mice. Switching to the diet without CLA for 4 weeks significantly attenuated hepatic steatosis (Table 1). Muscle TG levels were not significantly different between the two diet groups (data not shown). ROSI administration for 2 weeks prevented weight loss in male ob/ob mice fed dietary CLA, and weight gain was comparable to that in CON-PBS mice in both ROSI-treated groups (Fig. 1B). Epididymal adipose mass was also not significantly different in the CLA-ROSI group compared with the CON-ROSI and CON-PBS groups (Table 2 ).TABLE 2Body weights, organ weights, and serum metabolites, study 2VariableCON-PBSCON-ROSICLA-ROSIFinal body weight (g)42.20 ± 1.547.58 ± 2.1246.5 ± 1.73Liver weight (% body weight)6.50 ± 0.35a6.91 ± 0.49a,b8.35 ± 0.41bEpididymal adipose (g)2.96 ± 0.183.00 ± 0.273.15 ± 0.22Serum insulin (pg/ml)3,099.64 ± 440.20a2,180.24 ± 525.05a,b1,015.95 ± 80.95bResistin (ng/ml)22.14 ± 1.02a14.72 ± 1.35b20.31 ± 1.54a,bAdiponectin (ng/ml)10,751.00 ± 1530.2a55,996.00 ± 1459.0b56,023 ± 1349.6bFasting blood glucose (mmol/l)6.82 ± 0.66a3.93 ± 0.98b4.70 ± 0.80bInsulin-stimulated glucose uptake (area under the curve)−1,735 ± 1088.03a−6,760.00 ± 962.85b−5,576.25 ± 920.97bSerum TG (mg/dl)88.13 ± 11.85a33.89 ± 3.89b44.27 ± 8.84bNEFA (mEq/l)0.90 ± 0.09a0.40 ± 0.12b0.51 ± 0.09bLiver TG (% liver weight)18.1527 ± 1.9909a18.2921 ± 2.3035a10.2850 ± 0.5803bROSI, rosiglitazone. Male ob/ob mice were fed experimental diets containing 6.5% soybean oil (CON diet; n = 8) for 4 weeks. Additionally, 10 mice were maintained on the CON diet for the first 2 weeks, after which 6 mice were switched to the CLA diet containing 5% soybean oil plus 1.5% CLA-mixed TG and 4 mice were continued on the CON diet for the last 2 weeks of the study period and injected with either PBS (CON-PBS) or ROSI (CON-ROSI and CLA-ROSI). Values represent means ± SEM. Superscripts represent significant differences between treatments. Differences between means were calculated using one-way ANOVA, and values were considered significant at P < 0.05. Open table in a new tab ROSI, rosiglitazone. Male ob/ob mice were fed experimental diets containing 6.5% soybean oil (CON diet; n = 8) for 4 weeks. Additionally, 10 mice were maintained on the CON diet for the first 2 weeks, after which 6 mice were switched to the CLA diet containing 5% soybean oil plus 1.5% CLA-mixed TG and 4 mice were continued on the CON diet for the last 2 weeks of the study period and injected with either PBS (CON-PBS) or ROSI (CON-ROSI and CLA-ROSI). Values represent means ± SEM. Superscripts represent significant differences between treatments. Differences between means were calculated using one-way ANOVA, and values were considered significant at P < 0.05. Two weeks of treatment with ROSI not only prevented increases in both glucose and insulin in mice fed dietary CLA (CLA-ROSI vs. CON-ROSI and CON-PBS) but also significantly decreased FBG levels compared with CON-PBS mice (Table 2). ROSI administration significantly increased adiponectin levels in mice fed the CON or CLA diet compared with mice fed the CON-PBS diet (Table 2). Serum resistin levels were significantly higher in CON-PBS mice compared with CON-ROSI mice; however, ROSI administration did not have an effect on resistin levels in mice fed CLA. Furthermore, ROSI administration significantly decreased serum levels of TG and NEFA in both CON- and CLA-fed mice compared with the CON-PBS group (Table 2). Compared with the CON-PBS group, ROSI significantly improved insulin sensitivity in both CON- and CLA-fed ob/ob mice, and CLA-ROSI-fed mice had insulin sensitivity comparable to CON-ROSI mice (area under the curve) (Table 2, Fig. 3B). Although ROSI treatment did not decrease hepatic lipids in CON-fed mice, interestingly, there was a significant reduction in liver TG in the CLA-ROSI group compared with the CON-PBS and CON-ROSI groups after 2 weeks of ROSI treatment (Table 2). There was no effect of diet or treatment on muscle TG in ob/ob mice (data not shown). Because the combination of CLA with ROSI led to significantly lower liver TG, we measured mRNA levels of genes indicative of lipid oxidation and lipid synthesis in the liver. ROSI treatment significantly increased mRNA levels of liver AOX in both CON- and CLA-fed mice compared with CON-PBS mice (Fig. 4A ). There were no significant differences in the mRNA levels of PPARα and carnitine palmitoyl transferase between the groups (data not shown). Although ROSI treatment significantly increased mRNA levels of FAS in the CON-fed mice, interestingly, the combination of CLA with ROSI led to significantly low
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