c9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway
2014; Elsevier BV; Volume: 55; Issue: 5 Linguagem: Inglês
10.1194/jlr.m044032
ISSN1539-7262
AutoresMaria Pina Mollica, Giovanna Trinchese, Gina Cavaliere, Chiara De Filippo, Ennio Cocca, Marcello Gaita, Antonio Della-Gatta, Angela Marano, Giuseppe Mazzarella, Paolo Bergamo,
Tópico(s)Alcohol Consumption and Health Effects
ResumoOxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis. Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis. Nonalcoholic fatty liver disease (NAFLD), which includes steatosis and its progression to nonalcoholic steatohepatitis (NASH), is a liver disorder of clinical significance and one of the most frequent hepatic lesions in Western countries. NAFLD has been described as the hepatic manifestation of metabolic syndrome (1Paredes A.H. Torres D.M. Harrison S.A. Nonalcoholic fatty liver disease.Clin. Liver Dis. 2012; 16: 397-419Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and although its pathogenesis is not fully understood, it is clinically characterized by ectopic lipid accumulation in the liver (steatosis), inflammation, oxidative stress (2Reddy J.K. Rao M.S. Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation.Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 290: G852-G858Crossref PubMed Scopus (646) Google Scholar), and mitochondrial dysfunction (3Mantena S.K. King A.L. Andringa K.K. Eccleston H.B. Bailey S.M. Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases.Free Radic. Biol. Med. 2008; 44: 1259-1272Crossref PubMed Scopus (339) Google Scholar). Mitochondrial function and nuclear factor E2-related factor-2 (Nrf2) have been recognized to play a key role in NAFLD pathogenesis (3Mantena S.K. King A.L. Andringa K.K. Eccleston H.B. Bailey S.M. Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases.Free Radic. Biol. Med. 2008; 44: 1259-1272Crossref PubMed Scopus (339) Google Scholar, 4Bataille A.M. Manautou J.E. Nrf2: a potential target for new therapeutics in liver disease.Clin. Pharmacol. Ther. 2012; 92: 340-348Crossref PubMed Scopus (166) Google Scholar). In particular, defects in mitochondrial performance could contribute to the development of liver disease, and mitochondrial oxidative capacity has been considered a good predictor of liver disease (5Szendroedi J. Roden M. Ectopic lipids and organ function.Curr. Opin. Lipidol. 2009; 20: 50-56Crossref PubMed Scopus (153) Google Scholar). Mitochondria are fundamental organelles involved in providing energetic support through the chemiosmotic process of oxidative phosphorylation. In particular, they generate ATP by oxidizing nutrients (glucose, FAs, and some amino acids), and the energy generated by the electron transport is utilized to phosphorylate ADP to ATP. Electron transport and ATP synthesis are tightly coupled, but some of the energy generated by electron transport is uncoupled from ATP synthesis (6Stucki J.W. The optimal efficiency and the economic degrees of coupling of oxidative phosphorylation.Eur. J. Biochem. 1980; 109: 269-283Crossref PubMed Scopus (306) Google Scholar). Among the factors that affect the degree of mitochondrial coupling, the permeability of the mitochondrial inner membrane to hydrogen ions (leak) plays an important role. The mitochondrial inner membrane exhibits a basal proton leak pathway that has been estimated to contribute 20–25% to the basal metabolic rate in rats (7Rolfe D.F. Brown G.C. Cellular energy utilization and molecular origin of standard metabolic rate in mammals.Physiol. Rev. 1997; 77: 731-758Crossref PubMed Scopus (1370) Google Scholar). In addition, FAs can act as natural uncouplers of oxidative phosphorylation by generating an FA-dependent proton leak pathway (8Skulachev V.P. Fatty acid circuit as a physiological mechanism of uncoupling of oxidative phosphorylation.FEBS Lett. 1991; 294: 158-162Crossref PubMed Scopus (393) Google Scholar), which is a function of their unbound amount in the cell. Notably, an inducible proton leak has recently emerged as a major mechanism for the adjustment of the membrane potential to control mitochondrial reactive oxygen species (ROS) emission. By mildly uncoupling, the mitochondria can avoid the oversupply of electrons/reducing equivalents into the respiratory complexes and minimize the likelihood of electron interaction with oxygen (9Mailloux R.J. Harper M.E. Uncoupling proteins and the control of mitochondrial reactive oxygen species production.Free Radic. Biol. Med. 2011; 51: 1106-1115Crossref PubMed Scopus (403) Google Scholar). Oxidative stress results from the imbalance between ROS levels and the ability of antioxidant defenses to fully cope with ROS-mediated oxidative damage (10Finkel T. Holbrook N.J. Oxidants, oxidative stress and the biology of ageing.Nature. 2000; 408: 239-247Crossref PubMed Scopus (7300) Google Scholar); therefore, either excessive ROS production or limited antioxidant defenses contribute to its occurrence in pathophysiological processes. Cells have evolved several mechanisms to neutralize oxidative stress, and, among them, Nrf2 is considered the main mediator of cellular adaptation to redox stress (11Niture S.K.R.K. Jaiswal A.K. Regulation of Nrf2-an update. Regulation of Nrf2—an update.Free Radic. Biol. Med. 2014; 66: 36-44Crossref PubMed Scopus (638) Google Scholar). In normal conditions, the level of Nrf2 protein in the cell is maintained at very low levels by its inhibitor Keap1, which sequesters Nrf2 in the cytosol and facilitates its degradation via the proteasome. Under mild stress conditions, Nrf2 translocates to the nucleus, where it binds to antioxidant responsive elements and activates the transcription of a wide array of enzymes (phase 2 enzymes), the upregulation of which represents an adaptive response leading to improved antioxidant/detoxifying defenses [glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase (NQO1), and γ-glutamylcysteine ligase (GCL)] (12Osburn W.O. Kensler T.W. Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults.Mutat. Res. 2008; 659: 31-39Crossref PubMed Scopus (434) Google Scholar). Nrf2 also modulates genes involved in metabolic regulation, such as fibroblast growth factor 21 (FGF21), a liver-derived prolipolytic hormone, and PPARs, which play an important role in nutrient homeostasis (13Chartoumpekis D.V. Kensler T.W. New player on an old field; the keap1/Nrf2 pathway as a target for treatment of type 2 diabetes and metabolic syndrome.Curr. Diabetes Rev. 2013; 9: 137-145PubMed Google Scholar). Notably, the link between metabolism, ROS homeostasis, and mitochondrial metabolism is further indicated by the role played by PPARγ coactivator-1 (PGC-1). PGC-1α plays an important role in mitochondrial biogenesis and in the regulation of genes responsible for ROS detoxification (14St-Pierre J. Drori S. Uldry M. Silvaggi J.M. Rhee J. Jäger S. Handschin C. Zheng K. Lin J. Yang W. et al.Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators.Cell. 2006; 127: 397-408Abstract Full Text Full Text PDF PubMed Scopus (1774) Google Scholar); whereas PGC-1β is involved in mitochondrial metabolism, and its activation exerted a protective effect from lipid overload (15Bellafante E. Murzilli S. Salvatore L. Latorre D. Villani G. Moschetta A. Hepatic-specific activation of peroxisome proliferator-activated receptor γ coactivator-1β protects against steatohepatitis.Hepatology. 2013; 57: 1343-1356Crossref PubMed Scopus (45) Google Scholar). Conjugated linoleic acid (CLA) is the collective name used to indicate a class of positional and geometric conjugated dienoic isomers of linoleic acid. Among the possible isomers, two [namely, cis9,trans11-octadecadienoic acid (9Mailloux R.J. Harper M.E. Uncoupling proteins and the control of mitochondrial reactive oxygen species production.Free Radic. Biol. Med. 2011; 51: 1106-1115Crossref PubMed Scopus (403) Google Scholar, 11Niture S.K.R.K. Jaiswal A.K. Regulation of Nrf2-an update. Regulation of Nrf2—an update.Free Radic. Biol. Med. 2014; 66: 36-44Crossref PubMed Scopus (638) Google Scholar) and trans10,cis12-octadecadienoic acid (10Finkel T. Holbrook N.J. Oxidants, oxidative stress and the biology of ageing.Nature. 2000; 408: 239-247Crossref PubMed Scopus (7300) Google Scholar, 12Osburn W.O. Kensler T.W. Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults.Mutat. Res. 2008; 659: 31-39Crossref PubMed Scopus (434) Google Scholar), which represent ∼90% and 10% of the CLA found in ruminant meats and dairy products, respectively] exhibited characteristic biological activities (16Churruca I. Fernández-Quintela A Portillo M.P. Conjugated linoleic acid isomers: differences in metabolism and biological effects.Biofactors. 2009; 35: 105-111Crossref PubMed Scopus (124) Google Scholar). Consequently, although the isomeric CLA mixture (1:1) has been shown to trigger off the Nrf2 pathway (17Bergamo P. Maurano F. Rossi M. Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress.Free Radic. Biol. Med. 2007; 43: 71-79Crossref PubMed Scopus (58) Google Scholar, 18Bergamo P. Gogliettino M. Palmieri G. Cocca E. Maurano F. Stefanile R. Balestrieri M. Mazzarella G. David C. Rossi M. Conjugated linoleic acid protects against gliadin-induced depletion of intestinal defenses.Mol. Nutr. Food Res. 2011; 55: S248-S256Crossref PubMed Scopus (26) Google Scholar) and to modulate hepatic mitochondrial function (19Pereira A.F. Sá L.L. Reis F.H. Cardoso F.C. Alberici R.M. Prado I.M. Eberlin M.N. Uyemura S.A. Curti C. Alberici L.C. Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins.J. Bioenerg. Biomembr. 2012; 44: 587-596Crossref PubMed Scopus (9) Google Scholar), the efficacy of specific isomers on these mechanisms remains unclear. The modulation of mitochondrial functions and the activation of the Nrf2 pathway have been suggested for the treatment of NAFLD (4Bataille A.M. Manautou J.E. Nrf2: a potential target for new therapeutics in liver disease.Clin. Pharmacol. Ther. 2012; 92: 340-348Crossref PubMed Scopus (166) Google Scholar, 20Rolo A.P. Teodoro J.S. Palmeira C.M. Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis.Free Radic. Biol. Med. 2012; 52: 59-69Crossref PubMed Scopus (654) Google Scholar); therefore, drugs or natural molecules improving the mitochondrial function and Nrf2-activated defenses may prove useful in the treatment/prevention of NAFLD. We assumed that the CLA isomer showing an increased ability to improve mitochondrial function and the Nrf2 pathway would also ameliorate proinflammatory and pro-oxidant signs of nutritionally induced steatohepatitis. To test this hypothesis, Wistar rats were fed a normal diet [control diet (CD)] or a lard-based high-fat diet (L) for 4 weeks, and within each dietary treatment, two subgroups were administered cis9,trans11-CLA (9,11-CLA) or trans10,cis12-CLA (10,12-CLA) (30 mg/day). At the end of this period, proinflammatory [monocyte chemoattractant protein-1 (MCP-1), interleukin-1α (IL-1α), and TNF-α] and liver stress markers [alanine amino transferase (ALT) and γ-glutamyl transpeptidase (GGT)] were measured in the sera; mitochondrial functioning (protein mass, respiratory capacity, β-oxidation, proton leak, aconitase activity, and H2O2 yield), histological parameters, and oxidative stress markers [carbonylated proteins (PCs), and thiobarbituric acid reactive substances (TBARSs)] were analyzed; and the expression of cytoprotective genes (GCL, NQO1, and GST), metabolic genes (FGF21, PPARα, and PPARγ), and genes involved in sensing cellular stress, mitochondrial biogenesis, and metabolism (PGC-1α and PGC-1β) were evaluated via biochemical or RT-PCR analysis. Chemicals were of reagent of higher grade from Sigma-Aldrich (St. Louis, MO), unless otherwise specified. Pure 9,11-CLA (#1245) and 10,12-CLA isomer (#1249) were purchased from Matreya LLC (Pleasant Gap, PA). A primary antibody list is shown in supplementary Table I. Peroxidase-conjugated secondary antibodies against rabbit or mouse IgGs were purchased from Dako or Abcam, respectively. Wistar rats (average weight 380 g) (Charles River, Calco, Como, Italy) were divided into two groups (n = 24 each) and individually caged in a temperature-controlled room (24°C), under a 12 h light/12 h dark cycle with free access to water. The first group was fed with a standard rodent diet (CD) (15.88 kJ gross energy/g: 60.4 carbohydrates, 29 protein, and 10.6% fat; Mucedola, Milan, Italy). The second one received L diet (20 kJ/g) in which 40% of metabolizable energy was obtained from lard; the remaining calories were starch (31%) and protein (29%). Each group was further divided in three subgroups (n = 8 each): the first received only CD or L diet, and the two groups were administered daily with 30 mg 9,11-CLA (C9, L9) or 10,12-CLA (C10, L10) isomer corresponding to ∼80 mg/kg body weight or to 0.78 g/day, when expressed in "human equivalent dose" (21Reagan-Shaw S. Nihal M. Ahmad N. Dose translation from animal to human studies revisited.FASEB J. 2008; 22: 659-661Crossref PubMed Scopus (4288) Google Scholar). This dose was chosen because it is comparable with the reported daily intake of 9,11-CLA in humans (22Gebauer S.K. Chardigny J.M. Jakobsen M.U. Lamarche B. Lock A.L. Proctor S.D. Baer D.J. Effects of ruminant trans fatty acids on cardiovascular disease and cancer: a comprehensive review of epidemiological, clinical, and mechanistic studies.Adv. Nutr. 2011; 2: 332-354Crossref PubMed Scopus (195) Google Scholar) and comprises the contribution of trans9-C18:1, which is converted in the 9,11-isomer by liver desaturases (23Turpeinen A.M. Mutanen M. Aro A. Salminen I. Basu S. Palmquist D.L. Griinari J.M. Bioconversion of vaccenic acid to conjugated linoleic acid in humans.Am. J. Clin. Nutr. 2002; 76: 504-510Crossref PubMed Scopus (392) Google Scholar). Throughout the experimental period, body weights and food intakes were monitored daily to allow calculation of body-weight gain and gross energy intake. Spilled food was collected and used for food intake calculation. Gross energy density for rat chow or high-fat diet (HFD; 15.8 or 20.0 kJ/g, respectively) was determined by a bomb calorimeter (Parr adiabatic calorimeter; Parr Instruments Co., Moline, IL). At the end of the experimental period, the rats were anesthetized by an intraperitoneal injection of chloral hydrate (40 mg/100 g body weight) and then euthanized by decapitation. Blood was taken via inferior cava vein. Immediately after blood collection, liver was excised and either immediately processed for the isolation of mitochondria or cut in aliquots and frozen in liquid nitrogen and stored at −80°C for further processing or embedded in OCT compound for histological analysis. Visceral fat pad mass was removed and weighed. All experiments were conducted in compliance with national guidelines for the care and use of research animals (D.L. 116/92, implementation of EEC directive 609/86). In particular, treatment, housing, and euthanizing of animals met the guidelines set by the Italian Health Ministry (Permission n. 176/2005A), and all procedures were approved by the Federico II University Ethical Committee for Animal Research. Total cholesterol and triglyceride concentration and ALT and GGT activities in serum were measured by using standard procedures. IL-1α, interleukin-10 (IL-10), TNF-α, and MCP-1 content in sera was measured by using commercially available ELISA kits (Thermo Scientific, Rockford, IL; RBMS627R and RBMS629R, Biovendor R and D, Brno, Czech Republic). After the animal was euthanized, small pieces of liver tissue were quickly embedded in OCT compound for cryosectioning with a cryostat. The sections were fixed in acetone and then stained with Sudan Black (24Humasson G.H. Staining lipids and carbohydrates.in: Park R.B. Freeman W.H. In Animal Tissue Techniques. San Francisco, CA1972: 45-47Google Scholar) or hematoxylin-eosin (HE). Immunohistochemical staining to detect macrophages on rat liver cryosections was performed utilizing mouse anti Rat CD68 IgGs and peroxidase-antiperoxidase (Dako) staining. The density of cells expressing CD68 was quantified by counting the number of positive cells from five randomly selected fields with a microscope with a calibrated ocular. Results were expressed as the number of CD68 positive cells per square millimeter. Liver aliquots from the differently treated rats were also used to prepare cytosolic and nuclear extracts (17Bergamo P. Maurano F. Rossi M. Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress.Free Radic. Biol. Med. 2007; 43: 71-79Crossref PubMed Scopus (58) Google Scholar) to be used for Western blotting assays (supplementary Material). Total lipid content, redox status markers, and the activity of GST and NQO1 were measured as described in the supplementary Material. Mitochondria isolation and oxygen consumption (polarographically measured using a Clark-type electrode) were carried out as previously reported (25Lionetti L. Crescenzo R. Mollica M.P. Tasso R. Barletta A. Liverini G. Iossa S. Modulation of hepatic mitochondrial energy efficiency with age.Cell. Mol. Life Sci. 2004; 61: 1366-1371Crossref PubMed Scopus (15) Google Scholar). Oxygen consumption was measured in the presence of substrates and ADP (state 3) and in the presence of substrates alone (state 4), and their ratio [respiratory control ratio (RCR)] was calculated. The rate of mitochondrial FA oxidation was assessed in the presence of palmitoyl-l-carnitine (40 μM). Carnitine-palmitoyl-transferase (CPT) system (CPT1 plus CPT2) and aconitase activity were measured spectrophotometrically (at 412 nm) (26Alexson S.E.H. Nedergaard J. A novel type of short- and medium chain acyl-CoA hydrolases in brown adipose tissue mitochondria.J . Biol. Chem. 1988; 263: 13564-13571Abstract Full Text PDF PubMed Google Scholar, 27Hausladen A. Fridovich I. Measuring nitric oxide and superoxide: rate constants for aconitase reactivity.Methods Enzymol. 1996; 269: 37-41Crossref PubMed Google Scholar). The rate of mitochondrial H2O2 release was assayed by following the linear increase in fluorescence (ex 312 nm and em 420 nm) due to the oxidation of homovanillic acid in the presence of horseradish peroxidase (28Barja G. Mitochondrial free radical production and aging in mammals and birds.Ann. N. Y. Acad. Sci. 1998; 854: 224-238Crossref PubMed Scopus (196) Google Scholar). Mitochondrial protein mass was assessed by evaluating the expression of cytochrome C in isolated mitochondria by Western blotting and by measuring the activity of a mitochondrial marker enzyme, citrate synthase (CS), in liver homogenate and isolated mitochondria (29Srere P.A. Citrate synthase.Methods Enzymol. 1969; 13: 3-5Crossref Scopus (2031) Google Scholar). CS activity, measured in the homogenate and expressed per gram wet liver, reflects the product of mitochondrial protein mass and specific activity of the CS enzyme. To determine CS-specific activity, measurements were made in isolated mitochondria, and the results were expressed per milligram of mitochondrial proteins. Finally, mitochondrial protein mass, expressed as milligram per gram of wet liver, was calculated as the ratio between CS activity in the homogenate and isolated mitochondria. Total RNA was isolated from liver of rats fed with the different diets with or without CLA supplement by using the NucleoSpin RNA II kit (Macherey-Nagel), with an on-column DNase I step. RNA concentrations were given using a Qubit Fluorometer (Invitrogen). RNAs were then reverse transcribed using the SuperScript VILO MasterMix (Invitrogen). A total of 100 ng of cDNA and its dilution series to calculate the efficacy of primers were amplified by quantitative RT-PCR on an iCycler iQTM (Bio-Rad) using 300 nM gene-specific primers, Maxima SYBR Green/Fluorescein qPCR Master Mix (26Alexson S.E.H. Nedergaard J. A novel type of short- and medium chain acyl-CoA hydrolases in brown adipose tissue mitochondria.J . Biol. Chem. 1988; 263: 13564-13571Abstract Full Text PDF PubMed Google Scholar) (Thermo Scientific), and the following PCR conditions: 1 cycle at 95°C for 10 min, and 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The expression level of β-actin gene was used as an internal control for normalization. Raw cycle threshold values (Ct values) obtained for target genes were subtracted from the Ct value obtained for the reference gene. The final graphical data were derived from the R = (Etarget)ΔCt_target (control - sample)/(Eref)ΔCt_ref (control - sample) formula (30Pfaffl M.W. A new mathematical model for relative quantification in realtime RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25420) Google Scholar). Universal Probe Library Assay Design Center (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000) was used for designing primers (supplementary Table II). All data, obtained from triplicate analyses, were presented as mean ± standard error. Differences among groups were compared by ANOVA followed by the Newman-Keuls test to correct for multiple comparisons. Differences were considered statistically significant at P < 0.05. All analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA). The effects produced by the supplementation of individual CLA isomers on lipid metabolism (total cholesterol and triglycerides), liver damage marker enzymes (ALT and GGT), proinflammatory cytokines (IL-1α and TNF-α), chemokine (MCP-1), and IL-10 were measured in the serum of rats maintained on CD. No differences in total cholesterol, triglycerides, IL-10, MCP-1 levels, or GGT activity were found in the C9 and C10 groups; however, in the C9 animals, a significant decrease of IL-1α and TNF-α levels (27% and 35%, respectively) was found compared with the C10 and the CD rats. In contrast, the ALT level in the C10 rats was significantly higher (47%, P < 0.01) compared with the CD animals (Table 1). These results demonstrate that, due to the low doses used, the administration of individual isomers to CD-fed rats produces differential effects on blood serum parameters and that 9,11-CLA intake is accompanied by a mild but significant decrease in proinflammatory cytokine levels.TABLE 1CLA isomer supplementations have different impacts on serum biochemical markers of CD-fed ratsSerum ParametersCDC9C10Triglycerides (mg/dl)100 ± 4106 ± 5110 ± 6Total cholesterol (mg/dl)66 ± 362 ± 265 ± 2TNF-α (pg/ml)110 ± 10a71 ± 5b109 ± 12aMCP-1 (ng/ml)3.3 ± 0.23.7 ± 0.23.8 ± 0.3IL-1α (pg/ml)56 ± 3a41 ± 1b51 ± 3aIL-10 (pg/ml)56 ± 160 ± 457 ± 3ALT (U/l)42 ± 2a47 ± 2a62 ± 2bGGT (U/l)0.54 ± 0.070.48 ± 0.040.56 ± 0.08The results are expressed as the means ± SD from triplicate analyses from n = 7 animals/group. Differing superscript letters indicate statistically significant differences (P < 0.05). Open table in a new tab The results are expressed as the means ± SD from triplicate analyses from n = 7 animals/group. Differing superscript letters indicate statistically significant differences (P < 0.05). The increased mitochondrial state 3 and state 4 respiration rates measured in C10 rats using succinate or palmitoyl-carnitine (FA oxidation) as substrates (Fig. 1A, B) were further increased in C9 animals compared with controls. The CPT activity measured in the C9 and C10 rats (6.4 ± 0.5 or 6.2 ± 0.3 nmol/min/mg) was not different from that measured in the CD rats (6.0 ± 0.3 nmol/min/mg). All mitochondrial preparations, regardless of rat treatment, were able to produce RCRs of ≥6, indicating that the mitochondria were not damaged during the isolation procedure. After 4 weeks of treatment, the CS-specific activity (per milligram protein in the isolated mitochondria) was invariable, whereas significantly higher CS activity (per gram of tissue homogenate) was found. Therefore, the significantly higher mitochondrial protein (calculated as CS activity/CS-specific activity) (Fig. 1C) and cytochrome C found in CLA-treated rats (C9 > C10) (Fig. 1C, insert) indicate that the improved oxidative capacity appears to be supported (at least in part) by an increased mitochondrial mass. In addition, the significant increase of PGC-1α and PGC-1β in C9 rats, as compared with CD or C10 animals (Fig. 1D, E), clearly indicates the 9,11-CLA ability to upregulate mitochondrial biogenesis and metabolism. Mitochondrial proton leak was assessed via the titration of the steady respiratory rate (state) as a function of mitochondrial membrane potential in liver mitochondria. This titration curve is an indirect measurement of proton leak because a steady oxygen consumption rate (i.e., proton efflux rate) in nonphosphorylating mitochondria is equal to the proton influx rate due to proton leak. To determine the individual effect of CLA isomers on mitochondrial leakage, both the basal and FFA-induced proton leakage were measured in differently treated animals. When the basal proton leakage was evaluated in the CD rat groups, the mitochondria from the CLA-treated animals exhibited increased proton leakage (C9 > C10); the mitochondria from the C9 rats exhibited the highest oxygen consumption to maintain the same membrane potential compared with the controls and C10 animals (Fig. 1F). In FFA-induced conditions, the mitochondria from the CLA-supplemented animals exhibited comparable kinetic curves (Fig. 1G). No changes were observed in the expression of uncoupling protein 2 (UCP2) (data not shown). Next, the H2O2 yield in the mitochondria was measured. Aconitase is very sensitive to superoxide exposure; thus, its activity represents a valuable index of ROS-dependent injury. Interestingly, the H2O2 yield was significantly reduced in the mitochondria from the C9 and C10 groups (C9 > C10; P < 0.05) (Fig. 1H), and, similarly, a higher aconitase activity was found in the C9 and C10 rats compared with CD (C9 > C10) (Fig. 1I), thus supporting the link between mitochondrial leakage and redox status (8Skulachev V.P. Fatty acid circuit as a physiological mechanism of uncoupling of oxidative phosphorylation.FEBS Lett. 1991; 294: 158-162Crossref PubMed Scopus (393) Google Scholar). Significantly lower liver lipid contents were measured in the C9 and C10 rats maintained on CD (3.3 ± 0.1% and 3.1 ± 0.2%, respectively) compared with controls (4.0 ± 0.1%). The oxidative stress marker (PC and TBARS) levels measured in C9 and C10 animals were similar to the controls (data not shown). To investigate the ability of individual isomers to activate the Nrf2 pathway, the mRNA expression of antioxidant/detoxifying enzymes (GCL, GST, and NQO1) in the liver was compared between the groups maintained on CD. Interestingly, their levels were significantly elevated in C9 rats, but no difference in mRNA expression was found in the C10 animals compared with CD (Fig. 2A). No change in the GST or NQO1 enzyme activity was found in differently treated rats (data not shown); however, the activation of Nrf2 pathway in C9 rats was e
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