The obesogenic effects of polyunsaturated fatty acids are dependent ...

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hydrolysate (SPH), would be protected from developing high-fat diet-induced pathological characteristics of the metabolic syndrome. We demonstrate that bile acid metabolism can be modulated by the protein source in high-fat fed rats, and that such a modulation may protect against development of the metabolic syndrome. EXPERIMENTAL PROCEDURES Indirect calorimetric measurements – A separate set of rats (n=6/ group) was fed experimental diets for 17 days. Heat production was calculated from gas exchange measurements, as previously described (32). Gas exchange was determined twice, each time for 22 hours. From the average gas exchange measurements, heat production was calculated by the respiratory quotient method, and reported per 24 h. Animals - Male Wistar Hannover GALAS (HanTac:WH) were obtained from Taconic Europe (Ejby, Denmark) and divided into experimental groups (n=6). The rats were kept at a 12-h light/ dark cycle in a temperaturecontrolled room at 22 °C. After acclimatization, the animals were fed experimental diets with either SPH or casein as the sole protein source (Supplemental Table 1). The composition of the protein sources has been described elsewhere (31). Feed intake and body weight were recorded throughout the experiments and faeces were collected the last 5 days. The rats were killed by cardiac puncture under anaesthesia (0.23 mg/ kg BW Fentanyl (Janssen) and 0.45 mg/ kg BW Dormitor Vet (Orion Pharma)). Heparinplasma and EDTA-plasma containing aprotinin were prepared from blood. Tissues were dissected out and weighted. A portion of the liver was used for sub-cellular fractionation and measurement of mitochondrial carnitine palmitoyl transferase-1 capacity, and portions of interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) were homogenized and used for determination of palmitoyl-Coenzyme A oxidation capacity. The rest of the tissues were freeze-clamped and frozen at -80 o C. All animal experiments were approved by the National State Board of Biological Experiments with Living Animals (Norway and Denmark). Whole body composition – After 40 days of feeding, rats were anaesthetized (0.4 mg Dormitor Vet (Medetomidin Hydrochlorid)/ kg BW rat (Orion Pharma, Espoo, Finland), and whole body composition was determined by a dual X-ray absorptiometry scanner equipped with a small animal option (Discovery QDR Series, Hologic, Bedford, MA, USA). 3 Plasma metabolomics - A separate set of rats (n=5/ group) was fed experimental diets for 25 days. After termination in the fed state, 200 µl heparin plasma of each sample was mixed with a solution of 400 µl 0.9% saline and 20% D 2 O. Measurements were performed at 310 K on a on a Bruker Avance III 600 spectrometer, operating at a 1 H frequency of 600.13 MHz, and equipped with a 5-mm 1 H TXI probe (Bruker BioSpin, Rheinstetten, Germany). 1 H NMR spectra were acquired using the Carr- Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence with water suppression. All spectra were referenced to the lactate doublet signal at 1.33 ppm. The spectra were segmented into 0.013 ppm bins and each of the bins was integrated. The reduced spectra excluding the residual water signal were normalized to the whole spectrum. Principal component analysis (PCA) and partial least squares regression discriminate analysis (PLS- DA) was performed using the Unscrambler software version 9.8 (Camo, Oslo, Norway) to elucidate biochemical differences between predefined classes. Martens' uncertainty test was applied to find significant variables on the full cross-validated data (33,34). Bile acid measurements - For total bile acid measurements in feces, bile acids were extracted according to Suckling et al (35). Amounts of total bile acids in fecal extracts and in non-fasted EDTA-plasma were determined enzymatically by the 3-αhydroxysteroid dehydrogenase reaction (Dialab, Vienna, Austria). Bile acids in liver samples were extracted and analyzed using gas-chromatography-mass spectrometry and electrospray-mass spectrometry as previously described in detail (36). Real time RT-qPCR - Total RNA was purified tissues using Trizol, and cDNA was Downloaded from www.jbc.org by guest, on July 7, 2011
synthesized from individual rats. Gene expression was determined in individual samples by real-time qPCR using ABI PRISM 7700 Sequence Detection System (Applied Biosystems) carried out in 96-well plates and in duplicate as earlier described (37). Primers for real-time PCR (Supplemental Table 2) were designed using Primer Express 2.0 (Applied Biosystems). Liver microarray analysis- The following procedures were all performed according to Affymetrix standard procedures. Briefly, equal amounts of RNA isolated from livers were pooled (n=6) and 5 µg of total RNA was used as starting material for the target preparation. First and second strand cDNA synthesis were performed using the SuperScript II System (Invitrogen) according to the manufacturers’ instructions except using an oligo-dT primer containing a T7 RNA polymerase promoter site. Labeled aRNA was prepared using the BioArray High Yield RNA Transcript Labeling Kit (Enzo) using Biotin labeled CTP and UTP (Enzo) in the reaction together with unlabeled NTP´s. Unincorporated nucleotides were removed using RNeasy columns (Qiagen). Fifteen µg of cRNA were fragmented, loading onto the Affymetrix Rat Genome RAE 230 2.0 probe array cartridge and hybridized for 16h. The arrays were washed and stained in the Affymetrix Fluidics Station and scanned using a confocal laserscanning microscope (GeneChip® Scanner 3000 System with Workstation and AutoLoader). The raw images files from the quantitative scanning were analyzed by the Affymetrix Gene Expression Analysis Software (MAS 5.0) resulting in cell files containing background corrected probe values. Liver microarray KEGG pathway analysis – Liver microarray data was used to identify metabolic pathways regulated by treatments, using the KEGG resource (38,39), release 43. Genes with at least two-fold change in expression level between the two treatment groups were taken as differentially expressed genes (DEGs). The enrichment of these DEGs among KEGG pathways was measured by twotailed Fisher’s exact test. The pathways with P- value less than 0.05 were considered as statistically significant and were further studied. Histological analyses – Lipids in cryosections of frozen liver samples were stained by the standard Oil Red-O method. Parts of the epididymal white adipose tissue (eWAT) were subjected to standard hematoxylin and eosin staining as previously described (40). Plasma measurements - Lipids, metabolites and enzyme activities in plasma were determined by commercially available enzymatic kits: alanine aminotransferase (ALAT), total cholesterol, HDL-cholesterol, LDL-cholesterol (Dialab, Vienna, Austria), glucose, TAG (MaxMat, Montpellier, France), OH-butyrate (Randox, Crumlin, United Kingdom). Hormones were measured in EDTA plasma containing aprotonin: insulin by an ELISA kit (DRG Diagnostics, Germany) and glucagon by a Radioimmunoassay (RIA) Kit (LINCO Research, ST. Charles, MO, USA). Liver glycogen content – Glycogen in liver samples was converted to glucose by treatment with amyloglucosidase as described elsewhere (41). Glucose content after the treatment was determined using a commercial glucoses quantification kit (Dialab, Vienna, Austria). Liver lipid analyses - Total lipids were extracted from liver samples with chloroform: methanol, 2:1 (v/v). The lipid classes (TAG, cholesterol and sterol esters) were analyzed on an automated Camaq HPTLC system and separated on HPTLC silica gel 60 F plates as previously described (42). Liver mitochondrial preparation and CPT I assay – Liver mitochondria-enriched fractions were prepared as previously described (30). Freshly prepared mitochondrial enriched fractions were used for determination of carnitine-palmitoyl transferase capacity, as previously described (43), with or without addition of the CPTI inhibitor malonyl-CoA (5µM). Liver cytosolic preparation and total glutathione determination – After removal of the mitochondrial enriched fraction, the homogenates were further centrifuged at 100 000g av for 90 minutes at 4 o C. The supernatant collected after this centrifugation Downloaded from www.jbc.org by guest, on July 7, 2011 4
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The obesogenic effects of polyunsat
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Table of Contents Acknowledgement .
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Abstract in Danish Polyumættede n-
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Polyunsaturated fatty acids in the
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was more than 3 times higher in the
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gluconeogenesis and ureagenesis (Fi
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high dietary PUFAs, the modulation
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In a pair of population studies, an
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18. Massiera, F. et al. Arachidonic
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54. Xu, J. et al. Polyunsaturated f
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Annexes 1. Ma T, Liaset B, Hao Q, P
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Fish Oil, Sucrose and Obesity Obesi
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Fish Oil, Sucrose and Obesity Figur
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Fish Oil, Sucrose and Obesity Table
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Fish Oil, Sucrose and Obesity 6. Ma
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Carbohydrate source and insulin sec
Page 37 and 38: 20 21 22 23 24 25 26 27 28 29 30 31
Page 39 and 40: 68 69 70 71 72 73 74 75 76 77 78 79
Page 41 and 42: 114 115 116 117 118 119 120 121 122
Page 43 and 44: 160 161 162 163 164 165 166 167 168
Page 45 and 46: 208 209 210 211 212 213 214 215 216
Page 47 and 48: 256 257 258 259 260 261 262 263 264
Page 49 and 50: 302 303 304 305 306 307 308 309 310
Page 51 and 52: 350 351 FIGURE LEGENDS 352 353 354
Page 53 and 54: 398 399 400 401 402 403 404 subunit
Page 55 and 56: 446 447 448 449 450 451 gamma, coac
Page 57 and 58: 484 485 486 TABLE 1. Macronutrient
Page 59 and 60: Energy (kJ/g) 17.16 25.12 25.12 25.
Page 61 and 62: 561 562 563 564 565 566 567 568 569
Page 63 and 64: 635 636 637 638 639 640 641 642 643
Page 65: 711 712 713 714 715 716 717 718 719
Page 74 and 75: Cyclooxygenases and UCP1 Although i
Page 76 and 77: Cyclooxygenases and UCP1 Figure 2.
Page 78 and 79: Cyclooxygenases and UCP1 Figure 4.
Page 80 and 81: Cyclooxygenases and UCP1 PLoS ONE |
Page 82 and 83: Cyclooxygenases and UCP1 E synthase
Page 84 and 85: Cyclooxygenases and UCP1 29. Granne
Page 86 and 87: JBC Papers in Press. Published on J
Page 90 and 91: was used as the liver cytosolic fra
Page 92 and 93: In order to demonstrate that bile a
Page 94 and 95: Nutritional regulation of endogenou
Page 96 and 97: in increased whole body energy expe
Page 98 and 99: 38. Kanehisa M, and Goto S. (2000)
Page 100 and 101: FOOTNOTES This work was carried out
Page 102 and 103: fed the same diets, plus the SPH-di
Page 104 and 105: Figure 2 A nmol/ min/ mg prot 2,4 1
Page 106 and 107: Figure 4 A 6 Liver Pparα B 21 Live
Page 108 and 109: Figure 7 A Plasma ALAT B Liver Ucp2
Page 110 and 111: Ruzzin et al. investigated the meta
Page 112 and 113: Ruzzin et al. CD14, and rho kinases
Page 114 and 115: Ruzzin et al. salmon oil induced a
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