13 research outputs found
Indirect calorimetry.
<p>At 6 weeks after the initiation of the experiment, oxygen consumption (<i>V</i>O<sub>2</sub>) and carbon dioxide production were recorded every 2 min using indirect calorimetry. The measurements were performed following the diet-switch protocol in individual mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1B</a>). During the first part of the measurements (between 6.00 p.m. and 8.00 a.m.), animals had <i>ad libitum</i> access to water and various cHF-based diets. After that period, the animals were fasted for 10 hours. At the beginning of the dark cycle at 6.00 p.m., all subgroups were switched to Chow diet, and the measurements continued for 20 more hours (‘Re-feeding Chow ‘). The measurements were performed under the 12-hour light-dark cycle (lights on from 6∶00 a.m.) at ambient temperature of 22°C. Data are means±SE (<i>n</i> = 5; mice randomly chosen from each subgroup, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t001" target="_blank">Table 1</a>) expressed for the following three time-periods (i) from 0.00 p.m. to 8.00 a.m., feeding various cHF-based diets; (ii) from 9.00 a.m. to 5 p.m., fasting; and (iii) from 0.00 p.m. to 8.00 a.m., re-feeding Chow. ΔRER, the difference in RER between mice re-fed Chow diet and fasted mice.</p>a<p>Significantly different from cHF diet;</p>b<p>significantly different from cHF+ROSI diet (ANOVA).</p
Content of PDK4 protein in gastrocnemius muscle.
<p>Mice were killed either without any additional manipulations, that is, while offered the ‘original’ cHF-based diet in fed state (crossed bars), or following the diet-switch protocol when re-fed Chow diet (full bars). <b>A</b>. Representative Western blot analysis. <b>B</b>. Quantification of PDK4 protein in skeletal muscle. Values are means±S.E. (<i>n</i> = 5–8).<sup> a</sup>Significantly different from mice offered the cHF+F+ROSI diet (ANOVA).</p
Growth characteristics and plasma parameters.
<p>Three-month-old mice were placed on various diets and killed 8 weeks thereafter. Plasma parameters were followed as described in Methods, either in mice with free access to various cHF-based diets, or when mice were re-fed Chow (using the diet-switch protocol; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1C</a>). BHB, β-hydroxybutyrate in the animals re-fed Chow.</p>a<p>Significantly different from cHF;</p>b<p>significantly different from cHF+F;</p>c<p>significantly different from cHF+ROSI;</p>d<p>significantly different from cHF+F+ROSI (ANOVA).</p>f<p>Significantly different from Chow (<i>t</i>-test).</p
Synopsis of the results of metabolomic and gene expression analyses in the muscle.
<p>Metabolomic and gene expression analyses suggested complementary effects of the single interventions, with rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism, especially when combined with <i>n</i>-3 LC-PUFA (1), and <i>n</i>-3 LC-PUFA supporting specifically complete oxidation of fatty acids in mitochondria (7). These beneficial metabolic effects were associated with inhibition of low grade tissue inflammation (5) and the activation of the switch between glycolytic and oxidative muscle fibers (8), especially in the combined intervention. Moreover, rosiglitazone inhibited gene expression of fructose-1,6-bisphosphatase 2 - a key enzyme of gluconeogenesis (2), while the concentrations of most of lysophosphatidylcholines were reduced in response to <i>n</i>-3 LC-PUFA (4). Glucogenic amino acids, namely glycine and serine, were affected by the combined intervention (6). As we published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone.0043764-Kuda1" target="_blank">[14]</a>, the combined intervention also exerted synergistic stimulatory effect on muscle glycogen synthesis (3). Altered metabolites (bold font) and altered transcripts (bold font, rectangle) are marked. BCAA, branched-chain amino acids; C, acylcarnitine; CPT1b, carnitine palmitoyltransferase 1b; CYP1a1, member of the cytochrome P450 family genes; FBP2, fructose-1,6-bisphosphatase 2; GLUT4, glucose transporter 4; lysoPCs, lysophosphatidylcholines; PD, pyruvate dehydrogenase; PDK4 pyruvate dehydrogenase kinase 4; PFK, phosphofructokinase; MYH6 and MYH7, myosin heavy polypeptide; TCA cycle, tricarboxylic acid cycle; TNNC1, troponin C1.</p
Indirect calorimetry.
<p>During week 6 of the experiment, in mice fed cHF diet , or mice subjected to various interventions (cHF+F, cHF+ROSI, and cHF+F+ROSI diets; <i>n</i> = 5; mice randomly chosen from each subgroup, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t001" target="_blank">Table 1</a>), indirect calorimetry was performed using the diet-switch protocol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1B</a>). Thus, during the first part of the measurements (between 6.00 p.m. and 8.00 a.m.), animals had <i>ad libitum</i> access to water and various cHF-based diets. After that period, the diets were removed and the animals fasted for 10 hours (between 8.00 a.m. and 6.00 p.m.). At the beginning of the dark phase of the day cycle (at 6.00 p.m.), all the subgroups were offered Chow diet and the measurements continued for 20 more hours. <b>A</b> and <b>B</b>. Oxygen consumption (<b>A</b>) and RER values (<b>B</b>) during re-feeding Chow diet (mean values). <b>C</b>. Plots of PRCF of RER values during the periods of fasting (broken lines; data collected between 9.00 a.m. and 5.00 p.m.) and re-feeding Chow (solid lines; data collected between 0.00 p.m. and 8.00 a.m.). Each curve represents the data pooled from all mice within a given group (<i>n</i> = 5; see above; ∼1,200 RER measurements per curve). For the means over different periods of the measurements and for the statistical analysis of these data, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t002" target="_blank">Table 2</a>.</p
Expression of selected genes in gastrocnemius muscle.
<p>Mice were killed either without any additional manipulations, that is, while offered various ‘original’ cHF-based diets (<b>OrD</b>; crossed bars), or following the diet-switch protocol when re-fed Chow diet (full bars); see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1</a>. <b>A</b>.Genes involved in carbohydrate metabolism: pyruvate dehydrogenase kinase isozyme 4 (<i>Pdk4</i>); fructose-1,6-bisphosphatase isoenzyme 2 (<i>Fbp2</i>); and glucose transporter type 4 (<i>Glut4</i>). <b>B</b>. Genes involved in lipid metabolism: acyl-CoA thioesterase 1 (<i>Acot1</i>); carnitine palmitoyltransferase 1b (<i>Cpt1b</i>); and CD36 antigen (<i>Cd36</i>). <b>C</b>. Slow muscle (oxidative) fiber genes: myosin, heavy polypeptide 6 (<i>Myh6</i>); myosin, heavy polypeptide 7 (<i>Myh7</i>); and troponin C type 1 (<i>Tnnc1</i>). <b>D. </b><i>Pgc1α</i>. <b>E.</b> Cytochrome P450, family 1, subfamily a, polypeptide 1 (<i>Cyp1a1</i>). Data are means±SE (<i>n</i> = 7–8). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone.0043764.s007" target="_blank">Table S6</a>. <sup>a</sup>Significantly different from cHF, OrD; <sup>b</sup>significantly different from cHF+F, OrD; <sup>c</sup>significantly different from cHF+ROSI, OrD; <sup>d</sup>significantly different from cHF+F+ROSI, OrD; <sup>e</sup>significantly different from cHF+F+ROSI, re-fed Chow (ANOVA).</p
Concentrations of selected metabolites in gastrocnemius muscle extracts.
<p>Analysis was performed in mice re-fed Chow diet (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g003" target="_blank">Fig. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t001" target="_blank">Table 1</a>). <b>A.</b> Carnitines: propionyl-L-carnitine and isovalerlycarnitine (C3+C5); malonyl-L-carnitine (C4-OH); and various even-chain monounsaturated acylcarnitines (C10∶1, C14∶1, C16∶1, and C18∶1; individual acylcarnitines are denoted by their side chain; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone.0043764.s002" target="_blank">Table S1</a>). <b>B</b>. Amino acids. <b>C.</b> Lysophosphatidylcholines: stearoyl lysophasphatidylcholine (lysoPC C18∶0); linoleoyl lysophosphatidylcholine (lysoPC C18∶2); and arachidonoyl lysophasphatidylcholine (lysoPC C20∶4). <b>D.</b> Sphingolipids: palmitoyl sphingomyeline (SM C16∶0); stearoyl sphingomyeline (SM C18∶0); and hydroxysphingomyeline [SM(OH) C24∶1]. <b>D.</b> Data are means±SE (<i>n</i> = 7–8). Dietary groups are: cHF (black bars), cHF+F (blue bars); cHF+ROSI (green bars) and cHF+F+ROSI (violet bars). <sup>a</sup>Significantly different from cHF; <sup>b</sup>significantly different from cHF+F; <sup>c</sup>significantly different from cHF+ROSI (ANOVA).</p
Differentially expressed, unique genes classified in biological processes.
<p>Single interventions, unique genes with absolute FC ≥1.2 significantly different from cHF; combined intervention, unique genes with absolute FC ≥1.2 significantly different from cHF+F (<i>t</i>-test). Genes were manually classified in biological processes using scientific literature and bioinformatical resources, following initial MetaCore pathway analysis.</p
Overview of experimental setup.
<p> Starting at 3 months of age, subgroups of mice were fed either Chow or cHF diets, or subjected to various interventions (cHF+F, cHF+ROSI, and cHF+F+ROSI diets), which lasted for 8 weeks (<b>A</b>). During week 6 of the experiment, indirect calorimetry was performed using the ‘diet-switch protocol’ (<b>B</b>). At the end of the experiment, animals were killed either following the diet-switch protocol when re-fed Chow diet (<b>C</b>), or without any additional manipulations, while offered various cHF-based diets (not shown). White and grey background (<b>B, C</b>), light and dark phase of the day, respectively. Dotted arrow lines (<b>B</b>), periods of data collection for calculation of the mean values of <i>V</i>0<sub>2</sub>, RER, and PRCF (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g002" target="_blank">Fig. 2</a>).</p
The effects of various interventions on gastrocnemius muscle metabolome.
<p>At 3 month of age, subgroups of mice were fed cHF diet, or subjected to various interventions using cHF-based diets (cHF+F, cHF+ROSI, and cHF+F+ROSI). Animals were killed while re-fed Chow diet (see the diet-switch protocol and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-g001" target="_blank">Fig. 1</a>). Targeted metabolomics analysis was performed in gastrocnemius muscle extracts. In total, concentrations of 163 metabolites were determined using FIA-MS with the Biocrates AbsoluteIDQ™ technology (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone.0043764.s002" target="_blank">Table S1</a>) and PLS-DA was performed. <b>A</b>. 2D-score scatter plot of the first (X-axis) and the second (Y-axis) PLS-DA component are shown for selected groups of mice (<i>n</i> = 7–8; mice randomly chosen from each subgroup, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone-0043764-t001" target="_blank">Table 1</a>). Mice were fed cHF (black circles), cHF+ROSI (green circles), cHF+F (blue circles), or cHF+F+ROSI (violet circles) diets. <b>B</b>. Corresponding loading scatter plot. Acylcarnitines (green triangle), amino acids (inverted blue triangle), glycerophospholipids (yellow circles), sphingolipids (violet circles) and sum of hexoses (red diamante) are shown. The score (<b>A</b>) and loading (<b>B)</b> plots complement each other. The position of objects (muscle sample) in a given direction in the score plot is determined by variables (metabolites) lying in same direction in the loading plot. For identification of the individual metabolites shown in <b>B</b>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043764#pone.0043764.s001" target="_blank">Fig. S1</a>.</p