Article pubs.acs.org/jpr
Liquid Chromatography−Mass Spectrometry Based Metabolomics Study of Cloned versus Normal Pigs Fed Either Restricted or Ad Libitum High-Energy Diets Kirstine L. Christensen,*,† Mette S. Hedemann,† Henry Jørgensen,† Jan Stagsted,‡ and Knud Erik B. Knudsen† †
Department of Animal Science, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark Department of Food Science, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark
‡
ABSTRACT: Genetically identical cloned pigs should in principle eliminate biological variation and provide more pronounced effects when subjected to, e.g., dietary interventions, but little is known about how phenotype and phenotypic variation is affected by cloning. Therefore, an investigation of the metabolome of cloned pigs compared to normal control pigs was performed to elucidate the variation and possible differences in the metabolic phenotypes during a dietary intervention. A total of 19 control pigs and 17 cloned pigs were given the same high-energy dense diet either ad libitum or in a restricted manner (60% of ad libitum) for ∼6 months, and plasma was subjected to liquid chromatography−mass spectrometry nontargeted metabolomics and biochemical analyses. Low systemic levels of IGF-1 could indicate altered growth conditions and energy metabolism in cloned pigs. In response to ad libitum feeding, clones had a decreased energy intake and lower weight gain compared to controls, and plasma lipid profiles were changed accordingly. Elevated lactate and decreased creatine levels implied an increased anaerobic metabolism in ad libitum fed clones. Less interindividual variation between cloned pigs was however not established, suggesting a strong role for epigenetics and/or the gut microbiota to develop variation. KEYWORDS: cloned pigs, high-energy dense diet, interindividual variation, metabolomics, LC−MS
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insulin sensitivity,8,9 type 2 diabetes,10 atherosclerosis,11 dietary metabolic effects of an oral glucose tolerance test,12 and dietary carbohydrate interventions.13,14 Rodent models have been widely used in obesity and metabolic syndrome research including metabolomics, but metabolic and physiological differences between humans and rodents complicate the translation into effective prevention or intervention therapies.15,16 The pig is an attractive model for nutritional intervention studies since it displays nutritional and digestive aspects comparable with those of humans.15,17 Furthermore, use of genetically identical, cloned pigs could be advantageous, provided they exhibit less interindividual variation compared with normal litter mates. Theoretically, this would allow a more sensitive detection of the effects of dietary interventions and require fewer experimental animals. Clones are expected to be metabolically identical to the controls as no genetic changes have been introduced during the cloning process. However, a recent NMR-based study of biofluids from cloned pigs compared to normal controls revealed differences in the metabolome.18
INTRODUCTION Obesity is a condition in which surplus body fat accumulates to an extent that might affect one’s health.1 It is associated with several comorbidities including the metabolic syndrome that increases the risk of developing type 2 diabetes mellitus and cardiovascular diseases.2,3 The metabolism of obesity and its related dysfunctions together with possible treatment or prevention strategies are being investigated intensively due to the epidemic proportions of the condition. To better understand the underlying biochemical mechanisms, genomics, proteomics, and metabolomics have gained considerable interest. Metabolomics is the study of the metabolome, i.e., the quantification and identification of all metabolites in a biological system at a given time point.4 Liquid chromatography−mass spectrometry (LC−MS) is increasingly applied to metabolomics-driven research, as it is a very useful technique to detect and measure a variety of small biological molecules.5,6 LC−MS is furthermore highly sensitive and provide spectral information that contributes to the identification of metabolites.6 Nontargeted LC−MS metabolomics has been proven in several recent human and animal studies to elucidate biomarkers of dietary and cultural influences related to obesity and the metabolic syndrome. These are, e.g., high-fat diet induced obesity,7 impaired glucose tolerance and reduced © 2012 American Chemical Society
Received: December 22, 2011 Published: May 21, 2012 3573
dx.doi.org/10.1021/pr201253h | J. Proteome Res. 2012, 11, 3573−3580
Journal of Proteome Research
Article
micronutrients. The weight of the pigs was monitored weekly. Blood plasma samples were collected from the jugular vein in heparinised Vacutainers after an overnight fast at 8 and 1/2 months of age; the blood samples were stored on ice and centrifuged within 1.5 h at 1800g for 10 min at 4 °C. The animal experiment was conducted according to protocols approved by the Danish Animal Experiments Inspectorate and complied with the guidelines of the Danish Ministry and Justice concerning animal experimentation and care of animals under study.
The aim of this study was to investigate differences in the metabolome of cloned pigs compared to normal control pigs when fed ad libitum or diet restricted with a high-energy dense diet by conventional biochemical parameters as well as LC−MS metabolomics. Our focus was to examine if cloned pigs express less interindividual variation compared with normal litter mates and if they were suitable for nutritional intervention studies in obesity and metabolic syndrome research. Plasma from clones and controls was used for obtaining metabolic fingerprints.
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EXPERIMENTAL SECTION
Analytical Methods
Chemical analyses of the diet were performed with classical methods as described in the paper by Bach Knudsen et al.20 Plasma glucose, nonesterified fatty acids (NEFA), lactate, triglycerides, total cholesterol, low-density lipoproteins (LDL), and high-density lipoproteins (HDL) cholesterol were analyzed using an ADVIA 1650 Chemistry System (Siemens Healthcare Diagnostics, Deerfield, IL, U.S.A.). Plasma glucose was determined using glucose hexokinase II and enzymatic colorimetric determination.21 NEFA were determined using the Wako NEFA C ACS-ACOD assay method (Wako Chemicals GmbH, Neuss, Germany), and lactate was measured according to the method described by Noll.22 Triglycerides were analyzed by determination of glycerol after hydrolysis of mono-, di-, and triglycerides according to Fossati and Prencipe.23 Total cholesterol was determined by the enzymatic hydrolysis of cholesterol esters, oxidation of free cholesterol, and concomitant liberation of hydrogen peroxide.24 LDL and HDL cholesterol were determined after selective isolation and liberation according to the principles of Okada et al.25 and Izawa et al.,26 respectively. Plasma insulin was analyzed by timeresolved fluoroimmunoassay as described by Løvendahl and Purup,27 and insulin-like growth factor 1 (IGF-1) was measured by an EIA kit from R&D Systems, Inc. (Minneapolis, MN, U.S.A.), according to manufacturer’s instructions.
Animals and Sampling
Cloning was performed as previously described by Kragh et al.19 with donor cells from cultured ear fibroblasts of a 65% Danish Landrace × 35% Yorkshire sow. Controls were normal litters (75% Landrace × 25% Yorkshire) obtained by standard artificial insemination. A total of 17 female clones from 5 surrogate sows and 19 female controls from 6 litters were acquired. All pigs were reared in the experimental stables at Aarhus University (AU Foulum, Tjele, Denmark). The pigs were weaned at 28 days of age and fed a standard diet until 3 months of age, where the clones weighed 61.2 kg (±0.3 SEM) and controls 61.2 kg (±0.9 SEM). The next 6 months they all received the same wheat-based high-energy density diet (Table 1). Nine clones and 10 controls were fed ad libitum, whereas the remaining 8 clones and 9 controls were restricted to get 60% of total feed consumed by the ad libitum fed pigs. The diet still provided sufficient amounts of all Table 1. Ingredients and Chemical Composition of the Experimental Diet ingredient, g/kg, as-is white wheat flour wheat bran sugar soy oil casein limestone salt dicalcium phosphate vitamin and mineral premixa chemical composition, % of dry matter dry matter, % ash crude protein crude fat carbohydrates sugars starch nonstarch polysaccharides (NSP) dietary fiber (NSP + lignin) gross energy, MJ/kg DM metabolizable energy,b MJ/kg DM
480 185 100 100 110 7 4 12 2 energy distributionb, % 91.3 3.9 20.7 13.4 57.0 12.1 37.4 7.5 10.1 20.9 18.5
Standards and Chemicals for Metabolomics Analysis
HPLC-grade acetonitrile and methanol were purchased from VWR (West Chester, Pennsylvania, U.S.A.). Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Internal and external standards were obtained from Sigma-Aldrich, Avanti polar lipids (Alabaster, Alabama, U.S.A.), and Merck (Merck, Darmstadt, Germany). Sample Preparation of Blood Plasma for Metabolomics Analysis
Plasma samples of 200 μL were mixed with 20 μL of 100 μg·mL−1 internal standard mix of glycocholic acid (Glycine-1 13 C) and lysophosphatidylcholine (17:0). The samples were added to 600 μL ice-cold methanol, immediately vortexed, and incubated at 4 °C for 20 min for deproteinization. The supernatants were collected after centrifugation at 13 200g for 10 min and evaporated to dryness under a stream of N2. The resulting dry residues were dissolved in 200 μL of water/ acetonitrile/formic acid (95:5:0.1 v/v/v) and filtered through 0.22 μm Corning Spin-X centrifuge tube filters at 10 621g for 10 min. Samples were stored at −20 °C.
19 27 53 11 35 7
a
Provided are the quantities of vitamins and minerals per kilogram of complete diet: 4400 IU of vitamin A, 1000 IU of vitamin D3, 60 mg of alpha-tocopherol, 2.2 mg of menadione, 2.2 mg of thiamin, 4 mg of riboflavin, 3.3 mg of pyridoxine, 11 mg of D-pantothenic acid, 22 mg of niacin, 0.055 mg of biotin, 0.022 mg of vitamin B12, 50 mg of Fe as FeSO4·7H2O, 80 mg of Zn as ZnO, 27.7 mg of Mn as MnO, 20 mg of Cu as CuSO4·5H2O, 0.2 mg of I as KI, and 0.3 mg of Se as Na2SeO3. b Calculated.
Reversed-Phase High Performance Liquid Chromatography Mass Spectrometry
For the LC−MS analysis, an Ultimate 3000 (Dionex, Sunnyvale, CA, U.S.A.) HPLC system was coupled to a MicrOTOF-Q II mass spectrometer (Bruker Daltonik GmbH, Germany) operating in ESI+ and ESI− ion modes. The scan 3574
dx.doi.org/10.1021/pr201253h | J. Proteome Res. 2012, 11, 3573−3580
Journal of Proteome Research
Article
Table 2. Biochemical Characteristics of Clone and Control Pigs at 8 and 1/2 Months of Age; Fasting Values (Mean ± SEM)a ad libitum e
feed intake (kg DM/d) energy intakef (MJ/d) daily weight gaing (kg) glucose (mM) cholesterol (mM) HDL (mM) LDL (mM) LDL/HDL triglyceride (mM) NEFAh (μ ekv./L) lactate (mM) insulin (ng/mL) IGF-1I (ng/mL)
P valuesb
diet restricted c
clone (n = 9)
control (n = 10)
clone (n = 8)
control (n = 9)
type
2.48 ± 0.13b 45.8 ± 2.2b 0.89 ± 0.04b 5.08 ± 0.28a 2.29 ± 0.05b 0.78 ± 0.06b 1.24 ± 0.04b 1.70 ± 0.62a 0.43 ± 0.03 169.7 ± 47.1b 6.23 ± 0.79a 0.14 ± 0.07 113.4 ± 13.2b
2.97 ± 0.16a 54.8 ± 2.7a 1.11 ± 0.03a 4.39 ± 0.16b 3.18 ± 0.14a 1.07 ± 0.05a 1.74 ± 0.09a 1.66 ± 0.36a 0.40 ± 0.05 162.8 ± 32.7b 2.52 ± 0.44b 0.17 ± 0.03 200.6 ± 19.9a
1.37c 25.3c 0.45 ± 0.03c 4.61 ± 0.10b 2.01 ± 0.05c 0.81 ± 0.02b 0.83 ± 0.04c 1.03 ± 0.12b 0.26 ± 0.02 304.2 ± 33.2a 2.08 ± 0.48b 0.03 ± 0.01 55.2 ± 9.2c
1.37c 25.3c 0.37 ± 0.01d 4.50 ± 0.09b 2.06 ± 0.07bc 0.90 ± 0.05b 0.82 ± 0.04c 0.94 ± 0.23b 0.27 ± 0.03 300.1 ± 22.0a 1.42 ± 0.31b 0.07 ± 0.02 123.8 ± 14.3b
