Plasma Acylcarnitines and Amino Acid Levels As an Early Complex Biomarker of Propensity to High-Fat Diet-Induced Obesity in Mice

<div><p>Obesity is associated with insulin resistance and impaired glucose tolerance, which represent characteristic features of the metabolic syndrome. Development of obesity is also linked to changes in fatty acid and amino acid metabolism observed in animal models of obesity as well as in humans. The aim of this study was to explore whether plasma metabolome, namely the levels of various acylcarnitines and amino acids, could serve as a biomarker of propensity to obesity and impaired glucose metabolism. Taking advantage of a high phenotypic variation in diet-induced obesity in C57BL/6J mice, 12-week-old male and female mice (<i>n</i> = 155) were fed a high-fat diet (lipids ~32 wt%) for a period of 10 weeks, while body weight gain (<b>BWG</b>) and changes in insulin sensitivity (<b>ΔHOMA-IR</b>) were assessed. Plasma samples were collected before (week 4) and after (week 22) high-fat feeding. Both univariate and multivariate statistical analyses were then used to examine the relationships between plasma metabolome and selected phenotypes including BWG and ΔHOMA-IR. Partial least squares-discrimination analysis was able to distinguish between animals selected either for their low or high BWG (or ΔHOMA-IR) in male but not female mice. Among the metabolites that differentiated male mice with low and high BWG, and which also belonged to the major discriminating metabolites when analyzed in plasma collected before and after high-fat feeding, were amino acids Tyr and Orn, as well as acylcarnitines C16-DC and C18:1-OH. In general, the separation of groups selected for their low or high ΔHOMA-IR was less evident and the outcomes of a corresponding multivariate analysis were much weaker than in case of BWG. Thus, our results document that plasma acylcarnitines and amino acids could serve as a gender-specific complex biomarker of propensity to obesity, however with a limited predictive value in case of the associated impairment of insulin sensitivity.</p></div

Body weight of mice before and during the development of obesity.

<p>Body weight of mice was monitored at the 2-week intervals starting at 2 weeks of age and continuing until the end of experiment (week 24). All mice were fed a standard laboratory chow (STD) until the age of 12 weeks, when most of the mice received a high-fat diet (HFD); the remaining mice continued on the STD diet until the end of study. Both male (<b>A</b>) and female (<b>B</b>) mice were followed during the experiment. The data are means ± SD (STD, empty circles, <i>n</i> = 14 (male) or 11 (female); HFD, black circles, <i>n</i> = 82 (male) or 73 (female)).</p

Multivariate analysis of plasma metabolome.

<p>Plasma levels of AC species and amino acids in the fasting state were analyzed by the PLS-DA analysis in groups of mice selected for the highest and lowest BWG. Score plots resulting from the PLS-DA analysis of plasma metabolite levels measured at week 4 (<b>A</b>, <b>C</b>) and week 22 (<b>B</b>, <b>D</b>) of the study were generated for both male (<b>A</b>, <b>B</b>) and female (<b>C</b>, <b>D</b>) mice. Plasma metabolites that were identified as those most discriminating between the groups of „high gainers”(red marks) vs. „low gainers”(green marks) in both genders are listed in the tables under the respective score plots; the metabolites are ranked according to their variable influence on projection (VIP) scores, and only those with VIP scores greater than 1 are shown. Three outliers based on PCA analysis and two animals due to missing values were excluded from further analysis. </p

An overview of the experimental setup.

<p>Mice were weaned at the age of 4 weeks and fed a standard laboratory chow (STD) until 12 weeks of age, when they began to be fed a high-fat diet (HFD) until the end of study at the age of 24 weeks. Intraperitoneal glucose tolerance test (IPGTT) was performed before (week 12) and towards the end of high-fat feeding (week 22).</p

Body weight and metabolic phenotypes of mice before and after high-fat feeding.

<p>Body weight and metabolic phenotypes of mice before and after high-fat feeding.</p

The distribution of selected obesity-associated phenotypes within the groups of HFD mice.

<p>The following parameters including body weight at week 24 (<b>A</b>) as well as body weight gained between the week 12 and 24 of the experiment (<b>B</b>), glucose tolerance assessed as the area under the glucose curve (AUC) at week 22 (<b>C</b>) as well as its change (ΔAUC) between the week 12 and 22 (<b>D</b>), and insulin resistance assessed as HOMA-IR at week 22 (<b>E</b>) as well as its change (ΔHOMA-IR) between week 12 and 22 (<b>F</b>) were assessed and their intra-group distributions were plotted. Both male (black bars; <i>n</i> = 82) and female (white bars; <i>n</i> = 73) mice were analyzed.</p

Correlation analysis of plasma AC species and amino acids with the degree of obesity and insulin resistance.

<p>Plasma levels of individual metabolites assessed in both male and female mice (M and F, respectively) at week 4 and 22 were correlated with the parameters of body weight gain during a 12-week period of high-fat feeding (i.e. week 12–24; <b>A</b>) and with the change in insulin sensitivity during a 10-week-period (i.e. week 12–22; <b>B</b>). Data are presented as heatmaps, where different colors correspond to coefficient values of the respective correlations (Spearman's rank correlation coefficients).</p

Correlation analysis of various metabolic parameters assessed before and after high-fat feeding.

<p>Before the induction of obesity by a high-fat diet (week 12–24), mice were fed a standard laboratory chow from weaning (week 4) until week 12. Spearman's rank correlation coefficients were generated to characterize the relationships between different variables associated with the weight gain and glucose homeostasis in both male (<b>A</b>) and female (<b>B</b>) mice. The data are presented as heatmaps, where different colors correspond to coefficient values of the respective correlations.</p

Plasma levels of selected metabolites showing a significant difference between the groups of mice with the lowest and highest BWG.

<p>Plasma levels of C16-DC (<b>A</b>), C18:1-OH (<b>B</b>), C18:1 (<b>C</b>), and C3 (<b>D</b>) were assessed at week 4 and week 22 (i.e. after 10 weeks of high-fat feeding) in groups of male and female mice showing the lowest (green marks) and highest (red marks) BWG. ^a<i>p</i><0.05 vs. „low gainers”at week 22; ^b<i>p</i><0.05 vs. „high gainers”at week 22; ^c<i>p</i><0.05 vs. „low gainers”at week 4.</p

Oral glucose tolerance test after 22 weeks of HF feeding.

<p>HFpu and HFs mice were fasted for 5 hours at the start of the light phase, after which mice received glucose via oral gavage. Blood glucose levels were measured before glucose administration and during the 2 hours thereafter (A), and were expressed as iAUC (B).</p