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Additional file 1: of Cognitive impairment in metabolically-obese, normal-weight rats: identification of early biomarkers in peripheral blood mononuclear cells

Nucleotide sequences of primers used for real-time RT-qPCR amplification. (DOCX 17 kb

Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice

<div><p>The gastrointestinal tract constitutes a physiological interface integrating nutrient and microbiota-host metabolism. Conjugated linoleic acids (CLA) have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach proteins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute to body weight regulation. Adult mice were fed either a normal fat (NF, 12% kJ content as fat) or a high-fat (HF, 43% kJ content as fat) diet. In the latter case, half of the animals received daily oral supplementation of CLA. Expression and content of stomach proteins and specific bacterial populations from caecum were analysed. CLA supplementation was associated with an increase in stomach protein expression, and exerted a prebiotic action on both <i>Bacteroidetes/Prevotella</i> and <i>Akkermansia muciniphila</i>. However, CLA supplementation was not able to override the negative effects of HF diet on <i>Bifidobacterium</i> spp., which was decreased in both HF and HF+CLA groups. Our data show that CLA are able to modulate stomach protein expression and exert a prebiotic effect on specific gut bacterial species.</p></div

DNA levels of representative bacterial species in mice caecum are altered by diet.

<p>Bacterial species from caecum content was analysed in all groups. (A) Bacterial DNA levels in caecum content (μg bacterial DNA/g caecum content) was increased significantly by HF diet. DNA abundance of 16S rRNA gene of representative bacterial species (B) Firmicutes, (C) Actinobacteria, (D) Bacteroidetes, (E) Verrucomicrobia and (F) Proteobacteria was analysed in mouse caecum and normalised with the average of the NF group (2^−ΔΔCt). Fold change respect to NF group was calculated (Log₂ FC) and is indicated below each column. Data are mean ± SEM of 6–8 animals/group. Letters indicate differences amongst groups; one-way ANOVA followed by Bonferroni test (p<0.05). When homogeneity of variances was not assumed, data were log transformed.</p

Nucleotide sequences of primers used for qPCR amplification in mouse stomach.

<p>Forward and reverse sequences designed for qPCR amplification in stomach samples of mice.</p><p>Nucleotide sequences of primers used for qPCR amplification in mouse stomach.</p

Sequence of primers used for bacterial profiling in caecum content.

<p>Forward and reverse sequences for qPCR amplification in mouse caecal content.</p><p>Sequence of primers used for bacterial profiling in caecum content.</p

Circadian changes of milk and plasma ghrelin concentration.

<p>A: day 5, B: day 10, C: day 15 of lactation. X axis: time points. The shading part of the graphs indicates the dark period. The result of each time-point represents the mean value ± SEM of 5 dams. ANOVA, LSD test (a≠b) and cosinor analysis were performed as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145376#pone.0145376.g001" target="_blank">fig 1</a> and materials and methods section.</p

Changes in milk and plasma leptin (A), ghrelin (B) and adiponectin (C) concentration throughout lactation (days 5, 10 and 15).

<p>Each bar in the graphs represents the mean value ± SEM of 30 dams. ANOVA analysis was performed, followed by LSD test for <i>post hoc</i> comparisons (a≠b).</p

Nucleotide sequences of primers and amplicon size.

<p>Nucleotide sequences of primers and amplicon size.</p

mRNA expression levels of Leptin in WAT in 25 d and 6 m old animals.

<p>mRNA expression levels of Leptin in inguinal and retroperitoneal white adipose tissue depots (iWAT and rpWAT respectively), in 25 d and 6 m old male and female offspring of controls (C) and 20% caloric restricted dams during gestation (CR). mRNA expression levels in male and female controls are set to 100% and values of CR animals expressed relative to their controls. Each group is made up of animals from at least four different litters. Data are mean ± s.e.m.</p><p>*Different from their respective C group (p<0.05; Student's t -test).</p