Metabolomics Approach for Analyzing the Effects of Exercise in Subjects with Type 1 Diabetes Mellitus

<div><p>The beneficial effects of exercise in patients with type 1 diabetes (T1D) are not fully proven, given that it may occasionally induce acute metabolic disturbances. Indeed, the metabolic disturbances associated with sustained exercise may lead to worsening control unless great care is taken to adjust carbohydrate intake and insulin dosage. In this work, pre- and post-exercise metabolites were analyzed using a ¹H-NMR and GC-MS untargeted metabolomics approach assayed in serum. We studied ten men with T1D and eleven controls matched for age, body mass index, body fat composition, and cardiorespiratory capacity, participated in the study. The participants performed 30 minutes of exercise on a cycle-ergometer at 80% VO₂max. In response to exercise, both groups had increased concentrations of gluconeogenic precursors (alanine and lactate) and tricarboxylic acid cycle intermediates (citrate, malate, fumarate and succinate). The T1D group, however, showed attenuation in the response of these metabolites to exercise. Conversely to T1D, the control group also presented increases in α-ketoglutarate, alpha-ketoisocaproic acid, and lipolysis products (glycerol and oleic and linoleic acids), as well as a reduction in branched chain amino acids (valine and leucine) determinations. The T1D patients presented a blunted metabolic response to acute exercise as compared to controls. This attenuated response may interfere in the healthy performance or fitness of T1D patients, something that further studies should elucidate.</p> </div

CPMG ¹H NMR spectra of a representative PCOS patient's serum.

<p>(<b>A</b>) Comparative spectra at baseline and after 30 months of Pio/Flu/Met polytherapy. CPMG spin echo experiment allows filtering broad signals of lipid and lipoproteins enhancing low-molecular weight metabolites such as amino acids, lactate and intermediate metabolites. The inset displays an expanded δ (0.75–1.5 ppm) spectral region showing two unidentified resonances characteristic of the serum spectra of untreated PCOS patients: a broad singlet arising at 0.85 ppm and three peaks centered at 1.18 ppm. (<b>B</b>) CPMG ¹H-NMR spectra of the same untreated PCOS patient shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029052#pone-0029052-g002" target="_blank">Figure 2A</a> before and after cold acetone precipitation. After acetone precipitation the three signals centered at 1.18 ppm were depleted, confirming the occurrence of oxidized lipoprotein-related structures in the serum of PCOS women.</p

Correlation between IMT and azelaic acid levels in serum.

<p>Positive significant correlation (r = 0.92, p = 3.96×10⁻⁷) between carotid IMT values and azelaic acid levels. Azelaic acid levels were calculated as the ratio of the fragmentation peak of azelaic acid at m/z = 317 (retention time = 16.38 min) and the peak area of the internal standard. Red and green dots represent values of untreated and treated PCOS patients respectively.</p

General significant enrichment in TCA cycle intermediates (TCAIs) in peripheral blood after acute exercise.

<p>Monitored using GC-MS (malate, fumarate, α-ketoglutarate) and NMR (citrate and succinate). Data are mean±sem of net percent variation with exercise. *Indicates a significant variation in metabolic levels with exercise (Wilcoxon rank-summed test for the comparison of a particular metabolite level prior to and after exercise in the independent T1D and control exercisers, q<0.1).</p

Relative changes in lypolysis (A) and BCAA metabolism (B) with acute exercise.

<p>Data are mean±sem of net percent variation. *Indicates a significant variation in metabolic levels with exercise (Wilcoxon rank-summed test for the comparison of a particular metabolite level prior to and after exercise in the independent T1D and control exercisers, q<0.1). #Indicates a significant diabetes×exercise interaction for a particular metabolite (Repeated-measures ANOVA, q<0.1).</p

Summary of the metabolites found to be significantly varied in either analytical platform after 30 months low-dose Pio-Flu-Met polytherapy.

<p>d = doublet, s = singlet, m = multiplet. Percentage of variation was calculated for each patient as the area of the spectral region or selected XCMS feature at baseline minus the area of the same feature or spectral region after the treatment relative to the former. Values are expressed as mean ± SEM. A negative value indicates that levels of the corresponding metabolite resulted significantly decreased with the treatment while positive values indicate a significant increase. p-values correspond to Wilcoxon rank-summed test and FDR correction. Statistical significance was considered for those spectral regions or features having p-corrected values<0.05 and fold changes>2;</p>§<p>Indicates those metabolites whose retention time and mass spectra were checked using pure standard references.</p

Clinical characteristics of T1D patients and control population.

<p>Values are reported as mean values ± SD.</p><p>ns: not significant.</p

Baseline analytical differences of T1D patients and control population.

<p>Values are reported as mean values ±SEM. Selected quantitative ions relative to internal standard areas are used in the case of GC-MS measurements. Selective ¹H-NMR regions relative to ERETIC digital signals are used in the case of NMR measurements. Two-sided p-values are calculated using Mann-Witney test. Statistical significance was set as q<0.1.</p

Lipoprotein ¹H-NMR analysis.

<p>(<b>A</b>) Comparative bipolar-LED diffusion mean spectra of the methyl region (δ 0.85 ppm) for untreated and treated PCOS patients. (<b>B</b>) Bipolar LED pulse sequence ¹H NMR spectra of a treated PCOS serum showing the fitting of the methyl band using the seven Lorentzian functions derived from our previously described methodology. Black line represents the original methyl envelope and green line the reconstructed spectrum after the fitting. (<b>C</b>) The estimated radiuses of lipoprotein particles in serum calculated using the seven Lorentzian functions were compared at baseline and after 30 months of Pio/Flu/Met polytherapy.</p

Linoleic acid oxidation products.

<p>Formation of linoleic acid hydroperoxyderivatives (HPODEs) are further reduced to their corresponding hydroxyderivatives (HODES). Chemical structures of linoleic acid (18∶2); 9- and 13-hydroperoxylinoleic acid (9- and 13-HPODE); and 9 and 13-hydroxylinoleic acid (9- and 13-HODE).</p