Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice
2007; Elsevier BV; Volume: 49; Issue: 1 Linguagem: Inglês
10.1194/jlr.m700195-jlr200
ISSN1539-7262
AutoresAngela A. Wendel, Aparna Purushotham, Lifen Liu, Martha A. Belury,
Tópico(s)Adipose Tissue and Metabolism
ResumoConjugated linoleic acid (CLA) induces insulin resistance preceded by rapid depletion of the adipokines leptin and adiponectin, increased inflammation, and hepatic steatosis in mice. To determine the role of leptin in CLA-mediated insulin resistance and hepatic steatosis, recombinant leptin was coadministered with dietary CLA in ob/ob mice to control leptin levels and to, in effect, negate the leptin depletion effect of CLA. In a 2 × 2 factorial design, 6 week old male ob/ob mice were fed either a control diet or a diet supplemented with CLA and received daily intraperitoneal injections of either leptin or vehicle for 4 weeks. In the absence of leptin, CLA significantly depleted adiponectin and induced insulin resistance, but it did not increase hepatic triglyceride concentrations or adipose inflammation, marked by interleukin-6 and tumor necrosis factor-α mRNA expression. Insulin resistance, however, was accompanied by increased macrophage infiltration (F4/80 mRNA) in adipose tissue. In the presence of leptin, CLA depleted adiponectin but did not induce insulin resistance or macrophage infiltration. Despite this, CLA induced hepatic steatosis. In summary, CLA worsened insulin resistance without evidence of inflammation or hepatic steatosis in mice after 4 weeks. In the presence of leptin, CLA failed to worsen insulin resistance but induced hepatic steatosis in ob/ob mice. Conjugated linoleic acid (CLA) induces insulin resistance preceded by rapid depletion of the adipokines leptin and adiponectin, increased inflammation, and hepatic steatosis in mice. To determine the role of leptin in CLA-mediated insulin resistance and hepatic steatosis, recombinant leptin was coadministered with dietary CLA in ob/ob mice to control leptin levels and to, in effect, negate the leptin depletion effect of CLA. In a 2 × 2 factorial design, 6 week old male ob/ob mice were fed either a control diet or a diet supplemented with CLA and received daily intraperitoneal injections of either leptin or vehicle for 4 weeks. In the absence of leptin, CLA significantly depleted adiponectin and induced insulin resistance, but it did not increase hepatic triglyceride concentrations or adipose inflammation, marked by interleukin-6 and tumor necrosis factor-α mRNA expression. Insulin resistance, however, was accompanied by increased macrophage infiltration (F4/80 mRNA) in adipose tissue. In the presence of leptin, CLA depleted adiponectin but did not induce insulin resistance or macrophage infiltration. Despite this, CLA induced hepatic steatosis. In summary, CLA worsened insulin resistance without evidence of inflammation or hepatic steatosis in mice after 4 weeks. In the presence of leptin, CLA failed to worsen insulin resistance but induced hepatic steatosis in ob/ob mice. Obesity contributes to the etiologies of a variety of comorbid conditions, such as cardiovascular disease, hypertension, and type 2 diabetes. In addition to storing lipid for energy, adipose secretes a variety of adipokines, many of which affect metabolism and inflammation in adipose and nonadipose tissues. Modulation of the endocrine functions of adipose tissue can contribute to a chronic state of inflammation, which leads to the pathogenesis of associated disorders, specifically insulin resistance (1Tilg H. Moschen A.R. Adipocytokines: mediators linking adipose tissue, inflammation and immunity.Nat. Rev. Immunol. 2006; 6: 772-783Crossref PubMed Scopus (2297) Google Scholar). Conjugated linoleic acid (CLA) is a group of dietary fatty acids that modulate adiposity and adipokine levels (2Tsuboyama-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: 1534-1542Crossref PubMed Scopus (485) Google Scholar, 3Wargent E. Sennitt M.V. Stocker C. Mayes A.E. Brown L. O'Dowd J. Wang S. Einerhand A.W. Mohede I. Arch J.R. et al.Prolonged treatment of genetically obese mice with conjugated linoleic acid improves glucose tolerance and lowers plasma insulin concentration: possible involvement of PPAR activation.Lipids Health Dis. 2005; 4: 3Crossref PubMed Scopus (55) Google Scholar, 4Poirier 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: 1059-1065Crossref PubMed Scopus (81) Google Scholar, 5Purushotham A. Wendel A.A. Liu L.F. Belury M.A. Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice.J. Lipid Res. 2007; 48: 444-452Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). CLA exists as positional isomers and stereoisomers of octadecadienoic acid (18:2) and is found naturally in foods derived from ruminants, such as beef, lamb, and dairy products. Commercially, mixed isomer CLA is marketed as a weight-loss supplement (e.g., Tonalin™). Different isomers of CLA have varied biological functions, such as reducing carcinogenesis, decreasing adipose mass, and modulating immune function and type 2 diabetes (6Belury M.A. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action.Annu. Rev. Nutr. 2002; 22: 505-531Crossref PubMed Scopus (710) Google Scholar). Although CLA, specifically the trans-10,cis-12 isomer (7Park 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: 235-241Crossref PubMed Scopus (686) Google Scholar), significantly decreases body weight primarily through a reduction of adipose tissue in a variety of species (8Houseknecht K.L. Vanden Heuvel J.P. Moya-Camarena S.Y. Portocarrero C.P. Peck L.W. Nickel K.P. Belury M.A. Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat.Biochem. Biophys. Res. Commun. 1998; 244: 678-682Crossref PubMed Scopus (567) Google Scholar, 9Ostrowska E. Muralitharan M. Cross R.F. Bauman D.E. Dunshea F.R. Dietary conjugated linoleic acids increase lean tissue and decrease fat deposition in growing pigs.J. Nutr. 1999; 129: 2037-2042Crossref PubMed Scopus (292) Google Scholar, 10Park Y. Albright K.J. Liu W. Storkson J.M. Cook M.E. Pariza M.W. Effect of conjugated linoleic acid on body composition in mice.Lipids. 1997; 32: 853-858Crossref PubMed Scopus (977) Google Scholar), CLA also induces hyperinsulinemia and insulin resistance, primarily in mice (2Tsuboyama-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: 1534-1542Crossref PubMed Scopus (485) Google Scholar, 11Roche 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: 2037-2044Crossref PubMed Scopus (157) Google Scholar, 12Clement 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: 1400-1409Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 13West D.B. Blohm F.Y. Truett A.A. Delany J.P. Conjugated linoleic acid persistently increases total energy expenditure in AKR/J mice without increasing uncoupling protein gene expression.J. Nutr. 2000; 130: 2471-2477Crossref PubMed Scopus (161) Google Scholar, 14Delany 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: R1172-R1179PubMed Google Scholar). In mice, insulin resistance induced by CLA develops in parallel with lipodystrophy (i.e., decreased adipose mass, significant and rapid depletion of the adipokines leptin and adiponectin, and increased hepatic steatosis) (2Tsuboyama-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: 1534-1542Crossref PubMed Scopus (485) Google Scholar, 4Poirier 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: 1059-1065Crossref PubMed Scopus (81) Google Scholar). However, the mechanism by which CLA causes lipodystrophy in mice and the reason this effect is species-specific are not completely understood. Results from several studies emphasize the importance of leptin and adiponectin in the development of CLA-induced insulin resistance: leptin levels and adipose mass were partially preserved when CLA was fed as part of a high-fat diet in C57BL/6J mice. The preservation of leptin may have contributed to improvements in plasma insulin and liver weight also observed in these mice (15Tsuboyama-Kasaoka N. Miyazaki H. Kasaoka S. Ezaki O. Increasing the amount of fat in a conjugated linoleic acid-supplemented diet reduces lipodystrophy in mice.J. Nutr. 2003; 133: 1793-1799Crossref PubMed Scopus (67) Google Scholar). In lean mice supplemented with CLA, adipokines decreased rapidly and before a significant reduction of adipose mass. Furthermore, hyperinsulinemia and increased hepatic lipid concentration accompanied the time-dependent depletion of adiponectin and leptin (4Poirier 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: 1059-1065Crossref PubMed Scopus (81) Google Scholar). These findings indicate that the initial reduction of adipokines by CLA may be independent of reduced adipose mass. In a subsequent study, the reduction of leptin and adiponectin induced by CLA coincided with a proinflammatory state marked by an increased expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and macrophage infiltration in adipose tissue, likely contributing to increased insulinemia (16Poirier H. Shapiro J.S. Kim R.J. Lazar M.A. Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of white adipose tissue.Diabetes. 2006; 55: 1634-1641Crossref PubMed Scopus (162) Google Scholar). These results suggest that the deleterious effects of CLA on insulin sensitivity and insulinemia may be dependent on the alteration of adipokines and the induction of inflammation in adipose tissue. Recent studies show that CLA worsens hyperinsulinemia and insulin resistance in ob/ob mice, which have nonfunctional leptin as a result of a gene mutation (3Wargent E. Sennitt M.V. Stocker C. Mayes A.E. Brown L. O'Dowd J. Wang S. Einerhand A.W. Mohede I. Arch J.R. et al.Prolonged treatment of genetically obese mice with conjugated linoleic acid improves glucose tolerance and lowers plasma insulin concentration: possible involvement of PPAR activation.Lipids Health Dis. 2005; 4: 3Crossref PubMed Scopus (55) Google Scholar, 11Roche 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: 2037-2044Crossref PubMed Scopus (157) Google Scholar). Consequently, insulin resistance induced by CLA cannot be attributed solely to the depletion of leptin. However, because both CLA-fed and ob/ob mice are leptin-deficient, insulin resistance and the other effects of CLA may not necessarily be completely independent of leptin depletion. In this study, we controlled the level of leptin in the ob/ob mouse model with chronic administration of recombinant leptin. By controlling leptin levels and, in effect, negating the leptin depletion effect of feeding CLA, we aimed to determine whether the effects of CLA on insulin resistance, hepatic steatosis, and inflammation occur in a leptin-dependent manner. Six week old male B6.V-Lepob/OlaHsd (ob/ob) mice were obtained through Harlan (Indianapolis, IN) and housed four per cage at 22 ± 0.5°C on a 12 h light/dark cycle. Mice were maintained on isocaloric, modified AIN-93G powdered diets (Bio-Serv, Frenchtown, NJ) containing 6.5% fat by weight. Diets contained either 6.5% soybean oil (CON) or 5% soybean oil and 1.5% CLA mixed triglycerides (CLA). CLA mixed triglycerides (Tonalin™ TG 80; Cognis Corp., Cincinnati, OH) were ∼80% CLA composed of 39.2% cis-9,trans-11- and 38.5% trans-10,cis-12-CLA isomers. In a 2 × 2 factorial design, mice were randomized by body weight and fed either the CON or CLA diet and received intraperitoneal injections of either 1 mg/kg body weight recombinant mouse leptin (R&D Systems, Minneapolis, MN) (CON+ or CLA+) or a similar volume of the vehicle (PBS) (CON− or CLA−) for 4 weeks (n = 8 mice per group). Mice were injected every day, 2 h before the onset of the dark cycle. The leptin dose was based on the lowest dose that induced a reduction in body weight gain and fat and rescued serum insulin levels in ob/ob mice (17Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice.Science. 1995; 269: 540-543Crossref PubMed Scopus (3838) Google Scholar). Four weeks was chosen as the end point because this duration allows time for the development of insulin resistance and hepatic steatosis induced by CLA supplementation in mice (11Roche 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: 2037-2044Crossref PubMed Scopus (157) Google Scholar, 12Clement 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: 1400-1409Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 18Ohashi A. Matsushita Y. Kimura K. Miyashita K. Saito M. Conjugated linoleic acid deteriorates insulin resistance in obese/diabetic mice in association with decreased production of adiponectin and leptin.J. Nutr. Sci. Vitaminol. (Tokyo). 2004; 50: 416-421Crossref PubMed Scopus (44) Google Scholar) and, independently, the correction of metabolic abnormalities of ob/ob mice by leptin (17Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice.Science. 1995; 269: 540-543Crossref PubMed Scopus (3838) Google Scholar, 19Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Weight-reducing effects of the plasma protein encoded by the obese gene.Science. 1995; 269: 543-546Crossref PubMed Scopus (4184) Google Scholar). Body weights were measured every other day. At 4 weeks, after an overnight (12 h) fast, mice were anesthetized with isoflurane and blood was collected via cardiac puncture. After clotting, blood was centrifuged at 1,500 g for 20 min and sera were used for analyses. Tissues were quickly harvested, weighed, snap-frozen with liquid nitrogen, and stored at −80°C until analyses. All procedures were in accordance with institution guidelines and approved by the Institutional Animal Care and Use Committee of Ohio State University. Glucose levels were measured after an overnight (12 h) fast immediately before (baseline) and at 2 and 4 weeks of experimental treatments via tail vein blood using a One Touch Basic glucometer (Lifescan, Milpitas, CA). An insulin tolerance test (ITT) was conducted 3 days before necropsy. After an overnight (12 h) fast, mice were injected intraperitoneally with 1.5 U/kg body weight insulin (Humulin® R; Eli Lilly and Co., Indianapolis, IN). Tail vein blood was used to measure glucose immediately before injection (time 0) and at 15, 30, 45, 60, 90, and 120 min after the injection. Area under the curve was calculated as the net area contained between individual baselines (set by the glucose value at time 0) and curves using the trapezoidal rule (20Wolever T.M. Jenkins D.J. Jenkins A.L. Josse R.G. The glycemic index: methodology and clinical implications.Am. J. Clin. Nutr. 1991; 54: 846-854Crossref PubMed Scopus (933) Google Scholar). Homeostasis model assessment (HOMA) values were calculated according to Matthews et al. (21Matthews D.R. Hosker J.P. Rudenski A.S. Naylor B.A. Treacher D.F. Turner R.C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.Diabetologia. 1985; 28: 412-419Crossref PubMed Scopus (24799) Google Scholar) as insulin (mU/l)/22.5e−ln glucose (mM). Fasted serum levels of insulin, IL-6, and TNF-α were measured by the LINCOplex Mouse Serum Adipokine Immunoassay kit (Linco Research, Inc., St. Charles, MO). Adiponectin and resistin serum concentrations were determined by ELISA (Linco Research, Inc.) according to the manufacturer's directions. Lipids were extracted from a section of liver with 2:1 (v/v) chloroform and methanol. Final extracts were solubilized in 3:1:1 (v/v/v) tert-butanol, methanol, and Triton X-100 (22Danno 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 (68) Google Scholar). Tissue lipid extracts were analyzed for triglycerides by colorimetric enzymatic hydrolysis (Triglyceride, Free-Glycerol reagents; Sigma, St. Louis, MO). Data are expressed as equivalent triolein concentrations. RNA was extracted from epididymal adipose tissue using the RNeasy® Lipid Tissue Mini kit (Qiagen, Valencia, CA) and from liver using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturers' protocols. RNA was reverse-transcribed with the High-Capacity cDNA Archive kit (ABI, Foster City, CA) according to the directions. cDNA was amplified by real-time PCR in a total reaction volume of 25 μl with TaqMan Gene Expression Assays (ABI) using predesigned and validated primers (FAM probes) from ABI under universal cycling conditions defined by ABI. Target gene expression was normalized to the endogenous control 18S rRNA (VIC probe) amplified in the same reaction and expressed as 2-ΔΔct relative to the CON− group (23Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (116613) Google Scholar). Data are expressed as least square means ± SEM. The main and interaction effects of diet (CON or CLA) and treatment (leptin or vehicle) were analyzed by two-way ANOVA using a complete model in the GLM procedure of the Statistical Analysis System (SAS version 9.1; SAS Institute, Inc., Cary, NC). Body weights over time, fasting glucose levels, and ITT curves were analyzed as repeated measures. Differences of P < 0.05 were considered significant. Before beginning the experimental diets and treatments, all groups had similar average body weights (32.4–32.7 g). After 4 weeks on the experimental diets and treatments, leptin and CLA, both in the absence and presence of leptin, significantly reduced body weights compared with the CON diet alone (Table 1). The reduction in body weight by CLA was significantly more than that by leptin alone; however, there was no significant additive or synergistic effect of CLA and leptin on body weight. Although leptin alone decreased weight gain, CLA, regardless of leptin, induced significant weight loss from initial body weights. Body weights over time are shown in Fig. 1 . Significant differences in weight between mice fed CLA, regardless of leptin treatment, and vehicle-treated mice fed CON were first observed on day 9. On day 15, weights of CLA-fed mice were first significantly different from leptin-treated mice fed CON. A significant difference in weight between vehicle-treated and leptin-treated mice fed CON was first detected on day 17. All weights among the aforementioned comparisons remained significantly different through the end of the study. CLA-fed groups, whether in the absence or presence of leptin, never differed significantly from each other over the duration of the study. Differences in body weight were reflected by differences in epididymal adipose mass (Table 1). Leptin and CLA, in the absence and presence of leptin, significantly reduced epididymal adipose mass compared with the CON diet alone, and the reduction of epididymal adipose mass by CLA was significantly greater than that of leptin alone. Leptin treatment, regardless of diet, significantly decreased liver weight, whereas CLA did not have a significant effect.TABLE 1Effects of CLA and leptin on body and tissue weightsDiet ± LeptinFactors (P)VariableCON−CLA−CON+CLA+DietTrtIntFinal body weight (g)42.20 ± 0.97c28.98 ± 0.97a34.79 ± 0.97b28.41 ± 0.97a<0.001<0.0010.001Change in body weight (g)9.74 ± 0.56c−3.54 ± 0.56a2.39 ± 0.56b−4.30 ± 0.56a<0.001<0.001<0.001Epididymal adipose weight (g)2.96 ± 0.14c1.71 ± 0.14a2.16 ± 0.14b1.36 ± 0.14a<0.001<0.0010.004Liver weight (g)2.76 ± 0.17b3.18 ± 0.17b1.29 ± 0.17a1.56 ± 0.17a0.057<0.0010.694CLA, conjugated linoleic acid (and CLA diet); CON, control diet. Values represent least square means ± SEM, with significant differences (P < 0.05) within rows denoted by different letters. Factors include the main effects of diet and leptin treatment (Trt) and the interaction (Int) between diet and leptin treatment. Open table in a new tab CLA, conjugated linoleic acid (and CLA diet); CON, control diet. Values represent least square means ± SEM, with significant differences (P < 0.05) within rows denoted by different letters. Factors include the main effects of diet and leptin treatment (Trt) and the interaction (Int) between diet and leptin treatment. After 2 weeks of diets and treatments, CLA significantly increased fasting glucose levels in the absence of leptin (Table 2). Increased fasting glucose levels were maintained, but not increased, by CLA after 4 weeks of diets and treatments. In the presence of leptin, CLA did not significantly increase fasting glucose levels at 2 or 4 weeks. Leptin also prevented an increase in fasting glucose in mice fed the CON diet by 4 weeks. In the absence of leptin, CLA significantly increased fasting insulin levels compared with CON, whereas leptin significantly reduced fasting insulin levels in both diet groups (Table 2).TABLE 2Effects of CLA and leptin on fasted serum analytesDiet ± LeptinFactors (P)VariableCON−CLA−CON+CLA+DietTrtIntFasting glucose (mg/dl) Baseline115.12 ± 10.38101.25 ± 10.38117.00 ± 10.38107.38 ± 10.38 2 weeksaThere were no significant differences (P < 0.05) between 2 weeks and 4 weeks within any diet ± leptin group.130.25 ± 10.38a,b161.59 ± 12.10b106.25 ± 10.38a102.50 ± 10.38a 4 weeksaThere were no significant differences (P < 0.05) between 2 weeks and 4 weeks within any diet ± leptin group.122.87 ± 10.38b153.63 ± 10.38c89.50 ± 10.38a93.38 ± 10.38a,bInsulin (pmol/l)441.24 ± 49.45b771.05 ± 58.51c94.99 ± 46.25a209.00 ± 46.25a<0.001<0.0010.042Adiponectin (μg/ml)9.37 ± 0.57b3.91 ± 0.57a10.61 ± 0.57b5.23 ± 0.57a<0.0010.5020.405Resistin (ng/ml)22.13 ± 2.20b14.67 ± 2.20a9.17 ± 2.20a13.91 ± 2.20a0.5470.0090.017Interleukin-6 (pg/ml)25.89 ± 5.01b1.22 ± 5.01a5.87 ± 4.68a4.41 ± 4.68a0.0120.0940.024Tumor necrosis factor-α (pg/ml)2.01 ± 0.990.20 ± 0.991.18 ± 0.922.85 ± 0.920.9400.3470.079Values represent least square means ± SEM, with significant differences (P < 0.05) within rows denoted by different letters. Factors include the main effects of diet and leptin treatment (Trt) and the interaction (Int) between diet and leptin treatment. Fasting glucose levels were analyzed by repeated-measures ANOVA; only differences within diet ± leptin group and within time are shown.a There were no significant differences (P < 0.05) between 2 weeks and 4 weeks within any diet ± leptin group. Open table in a new tab Values represent least square means ± SEM, with significant differences (P < 0.05) within rows denoted by different letters. Factors include the main effects of diet and leptin treatment (Trt) and the interaction (Int) between diet and leptin treatment. Fasting glucose levels were analyzed by repeated-measures ANOVA; only differences within diet ± leptin group and within time are shown. After 4 weeks of diets and treatments, an ITT was conducted to assess the response to insulin. In the absence of leptin, glucose levels in mice fed CLA did not decrease at any time point after the administration of insulin, indicating unresponsiveness to insulin (Fig. 2A). In the presence of leptin, glucose levels in mice fed CLA decreased significantly after insulin injection, as they did in mice fed the CON diet. In Fig. 2B, the net areas contained within the ITT curves were quantified. As implied by the positive area, in the absence of leptin, mice fed CLA were unresponsive to insulin, which was significantly different from the other groups. There were no significant differences among the other groups, and the net areas were negative, indicating insulin responsiveness. A second estimation of insulin resistance by HOMA showed that in the absence of leptin, CLA worsened insulin resistance. Leptin decreased insulin resistance in both diet groups (Fig. 2C). Together, the ITT and HOMA data show that in the absence of leptin, CLA worsens insulin resistance, but it does not do so in the presence of leptin. Serum adiponectin levels were reduced significantly by CLA in both the absence and presence of leptin (Table 2). Leptin had no effect on serum adiponectin levels in either diet group. Serum resistin, however, was significantly decreased by leptin and CLA in the absence and presence of leptin. Similar to serum adiponectin, CLA, in the absence and presence of leptin, significantly reduced epididymal white adipose tissue (WAT) adiponectin mRNA expression compared with the respective CON diet groups (Fig. 3A). Unlike serum levels, however, leptin significantly increased WAT adiponectin mRNA expression in CON-fed mice. Both leptin and CLA significantly decreased WAT resistin mRNA expression; however, the reduction induced by CLA, regardless of leptin treatment, was significantly greater than with leptin alone (Fig. 3B). CLA had no effect on hepatic triglyceride concentrations in the absence of leptin (Fig. 4A). However, in the presence of leptin, CLA increased hepatic TG. Leptin significantly reduced hepatic triglyceride levels in both diet groups; however, leptin only partially reduced hepatic triglycerides in mice fed CLA compared with CON. Hepatic mRNA expression of markers of lipogenesis [sterol-regulatory element binding protein-1 (SREBP-1) and FAS] and lipid transport (FAT/CD36) was not altered by CLA (Fig. 4B) but was reduced by leptin in both diet groups. In the absence of leptin, CLA did not alter markers of fatty acid oxidation [peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1α, fatty acid binding protein 1, and acyl-CoA oxidase 1] compared with vehicle-treated mice fed the CON diet. These markers were increased by leptin in CON-fed mice. In the presence of leptin, CLA decreased these markers to levels of vehicle-treated mice in either diet group. Both CLA and leptin significantly reduced serum IL-6 compared with CON− mice (Table 2). Serum TNF-α did not significantly change with any diet or treatment. Contrary to the trend in serum IL-6, only leptin significantly decreased WAT IL-6 mRNA expression compared with vehicle-treated mice on either diet (Fig. 5A). Likewise, only leptin significantly decreased WAT TNF-α mRNA expression compared with vehicle-treated mice (Fig. 5B). CC chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1, a macrophage recruiter, was decreased significantly by leptin treatment regardless of diet but was not altered by CLA (Fig. 5C). In the absence of leptin, CLA significantly increased F4/80 mRNA expression, a macrophage-specific marker, nearly 2-fold compared with all other groups (Fig. 5D). Adipokines, particularly leptin and adiponectin, produced by adipose tissue are recognized as key mediators in both insulin sensitivity and inflammation and provide an important link of communication among tissues. Rapid depletion of both adiponectin and leptin coincides with increased inflammation and subsequent insulin resistance and hepatic steatosis with CLA supplementation in mice (4Poirier 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: 1059-1065Crossref PubMed Scopus (81) Google Scholar, 16Poirier H. Shapiro J.S. Kim R.J. Lazar M.A. Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of white adi
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