Conceived and designed the experiments: LB MV SM XC AN. Performed the experiments: LB SM MV SS MAR AB. Analyzed the data: MV LB SS MAR GD CL. Contributed reagents/materials/analysis tools: LB SM MV SS MAR AB. Wrote the paper: LB MV AN.
The authors have declared that no competing interests exist.
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.
Type 1 diabetes mellitus (T1D) is a lifelong metabolic disorder of usual acute onset in children, adolescents and young adult people. Over time, micro and macro vascular co-morbidities develop in patients with T1D which are closely related to metabolic control
Exercise plays a crucial role in the prevention and treatment of several chronic diseases, including glucose intolerance states, type 2 diabetes
Metabolomics enables the systematic assessment of the abundant changes of low molecular weight compounds present in biological samples, using high-throughput sample analysis techniques (GC-MS, NMR or HPLC-MS) and computer-assisted multivariate pattern-recognition techniques
Most of the
Ten recreationally active male patients with T1D, recruited by the Department of Endocrinology (Hospital Clinic, Barcelona), and eleven non-diabetic controls matched for sex, age, body mass index (BMI) and similar physical activity, recruited from a research institute (IDIBAPS, Barcelona), were enrolled in the study.
The T1D patients participating in the study had diabetes for a total of 14±8.4 years, undetectable C-peptide levels and good glycemic control, as determined by glycated hemoglobin A1c. Total body composition was measured by densitometry using DXA (Lunar iDXA body composition, GE Healthcare). Patients with chronic complications related to diabetes were excluded. All patients presented microalbuminuria values below 30 mg/L, normal retinal exam by direct and indirect retinoscopy, normal peripheral neurologic evaluation by clinical exploration and biothesiometry (Bio-thesiometer, Bio-Medical Instrument Company, Newbury, OH, U.S.), and normal. resting 12-lead electrocardiogram (ECG) and normal exercise testing by upright cycle-ergometer (25 W/3 min)
All subjects were required to visit the Diabetes and Exercise Research Unit of the Hospital Clínic on two separate occasions. On the first visit, all subjects were fully briefed and familiarized with the experimental procedures. Baseline clinical characteristics such as height, weight, BMI and total and fat body composition where also obtained, and each subject was required to complete an evaluation of current physical activity using the International Physical Activity Questionnaire
On the second visit, conducted at the same time (8:30am) a week later, subjects performed an acute bout of 30 minutes of intense exercise at 80% VO2max (individually calculated during the preliminary session) on a cycle-ergometer. Subjects performed 3 to 6 minutes of warm-up until achieving a fixed cardiac frequency. Prior to exercise, all participants fasted overnight for a 12 hour period, following a balanced meal consisting of approximately 55% carbohydrates, 30% fat, and 15% proteins. Furthermore, T1D patients had received their last short-acting insulin injection before dinner at 8 p.m. and their long-acting insulin injection at 10 p.m. All subjects were asked to avoid strenuous exercise and alcohol consumption the day prior to the acute exercise protocol.
Fasting blood samples were obtained before and after the short-term intensive exercise intervention. Glycemia (glucose-oxidase method, Advia 2400 Siemens Diagnostics, Deerfield, IL, U.S.) and insulinemia (quimioluminiscent method, Siemens Healthcare Diagnostics, Tarrytown, NY, U.S.) were determined in serum samples. For the metabolomic measurements, serum was obtained once blood had been allowed to clot at room temperature for 30 min and after centrifugation at 4°C at 5000 rpm for 10 min. Samples were kept at - 80°C until further metabolomic analysis.
Serum samples were thawed, vortexed and allowed to stand for 10 min prior to NMR analysis. For NMR measurements 430 µl of serum were transferred into 5 mm NMR tubes. A double tube system was used: an internal tube (o.d. 2 mm, supported by a Teflon adapter) containing the reference substance (sodium 3-trimethylsilyl [2, 2, 3, 3-d4] propionate (TSP) 9.9 mmol/l, MnSO4 0.47 mmol/l in 99.9% D2O) was placed coaxially into the NMR sample tube (o.d. 5 mm). This double tube system was kept at 4°C in the sample changer until analysis was performed. Spectra were acquired at a 1H observation frequency of 600.20 MHz at a temperature of 300 K using an Avance III-600 Bruker spectrometer equipped with an inverse TCI 5 mm cryoprobe®. The Carr-Purcell-Meiboom-Gill (cpmg, spin-spin T2 relaxation filter) pulse sequence with a fixed spin-spin relaxation delay of 200 ms was applied to acquire 1H-NMR spectra for all serum samples, in order to minimize the broad signals arising from lipoprotein and albumin in the NMR spectra. For each sample, 128 transients were collected into 32 K data points using a spectral width of 12 kHz with a relaxation delay of 2 s and an acquisition time of 1.36 s. A line-broadening function of 0.3 Hz was applied to all spectra prior to Fourier transform.
A second aliquot of serum (100µL) was used for GC-MS analysis according to Agilent’s specifications
Written informed consent was obtained from all subjects prior to participation. The experimental protocol was approved by the Research and Ethics committees of the Hospital Clínic de Barcelona, in accordance with the Declaration of Helsinki.
The acquired CPMG 1H-NMR spectra were phased, baseline-corrected and referenced to the chemical shift of the α-glucose anomeric proton doublet at 5.23 ppm. Pure standards compound reference in BBioref AMIX (Bruker) was used; HMDB and Chenomx databases were used for metabolite identification. After baseline correction, intensities of each 1H-NMR region identified in the CPMG 1D-NMR spectra were integrated using the AMIX 3.8 software package (Bruker, GmBH). Each region was normalized to the ERETIC (Electronic REference To access
Raw GC/MS files were exported into the platform-independent netCDF (*.cdf) and loaded into XCMS software (version 1.6.1) based on R-program version 2.4.0 (R-Foundation for statistical computing,
Baseline metabolic differences between T1D and control groups were evaluated using the Mann-Witney U run test. Effects of exercise intervention on the independent control and T1D groups were assessed using the Wilcoxon exact rank sum tests. Repeated measures ANOVA were used to determine diabetes×exercise interactions. A statistically significant interaction indicates that control and T1D responded differently to the acute exercise protocol for a given metabolite. To account for multiple testing, q-values were computed for all systematic univariate tests outlined above by applying the FDR (False Discovery Rate) procedure described by Storey et al
Anthropometric and fitness data are summarized in
Control | T1D | p-values | ||
Subjects | 11 | 10 | ns | |
Age (years) | 32.5±8.8 | 35.1±8.4 | ns | |
Evolution of diabetes (years) | – | 14±8.4 | – | |
Height (m) | 1.76±0.06 | 1.76±0.05 | ns | |
Weight (kg) | 75.9±8.6 | 75.6±5.8 | ns | |
BMI (kg/m2) | 24.7±2.6 | 24.3±1.7 | ns | |
Fat percentage (%, by DXA) | 23.9±5.7 | 21.7±6.5 | ns | |
IPAQ (METs min/week) | 2550±995 | 2630±241 | ns | |
VO2max (mL/kg/min) | 34±9.1 | 35±6.5 | ns | |
Units of insulin glargine | – | 31±7.9 | – |
Values are reported as mean values ± SD.
ns: not significant.
At baseline (
Control(n = 11) | T1D(n = 10) | q-values | |
|
|||
Glucose (mg/dl) | 90.27±2.25 | 202.7±24.36 | 5.52×10−4 |
Insulin (UI/L) | 6.96±1.31 | 18.62±4.64 | 0.022 |
C-peptide (ng/ml) | – | undetectable | – |
Glycated hemoglobin (%) | – | 6.9±1 | – |
|
|||
Lysine | 0.039±0.0036 | 0.025±0.0029 | 0.023 |
Glycerol | 0.039±0.0022 | 0.045±0.0039 | 0.064 |
Citrate | 0.006±0.0004 | 0.008±0.0005 | 0.052 |
Malate | 0.001±0.0001 | 0.002±0.0002 | 0.083 |
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.
Relative changes in insulin and glucose (A) and in gluconeogenic precursors (B) in response to 30 minutes of acute exercise (80% VO2max). Percentage of variation was calculated for each individual as the levels of a certain metabolite after exercise minus the levels of the same metabolite prior to exercise relative to the former. Data are shown as mean±sem of net percent variation for T1D and control groups. A positive value of percentage of variation indicates that metabolic levels have increased in mean with exercise, whereas a negative mean denotes the opposite. *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). Insulin and glucose data correspond to biochemical measurements, whereas lactate, alanine, and pyruvate were evaluated in 1H-NMR spectra according to
A significant enrichment in TCA cycle intermediates citrate, malate, fumarate, and succinate was observed after acute exercise in the peripheral blood of both control and T1D groups (
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).
Exercise significantly increased glycerol and oleic and linoleic acid concentrations in the control group exclusively. This effect was attenuated in the T1D group (
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).
Metabolite identification parameters are presented in supporting table (
The principal aim of this study was to analyze the metabolomic profile at rest and after a short period of intense exercise in patients affected by T1D to provide a comprehensive insight into the physiological effects of exercise on this particular population. Based on serum sample analysis, we have compared the metabolic response to short term acute exercise in T1D and healthy subjects using an untargeted metabolomics approach (GC-MS and 1H-NMR). Our findings revealed similar metabolic events in T1D patients and their control matched exercisers, although the T1D patients showed an attenuation of overall metabolic response after intense short-term exercise.
As it is commonly known, in order to increase energy supply during intense short-term exercise, glycogen breakdown is induced to provide the substrate for activating anaerobic glycolysis, resulting in the accumulation of pyruvate and lactate in plasma. Recently, Lewis et al
Our analysis demonstrated a significant increase in TCAIs (malate, citrate, succinate, fumarate and α-ketoglutarate) in the control subjects in response to exercise. These results are in accordance with a previous metabolomics-based study investigating the plasma signature of exercise
Concerning lipolysis, our data demonstrated that the healthy controls showed an increase in free fatty acids and glycerol in response to exercise, however, this response was attenuated in the T1D group. The increase in lipolysis after acute exercise had been previously reported in healthy individuals
Our results demonstrated that control exercisers presented a significant reduction in leucine levels. It is well established that exercise increases energy expenditure, resulting in the promotion of amino acid catabolism in general and, in particular, in the oxidation of branched chain amino acids, mainly leucine
Of special note, we detected an increase in insulin serum levels in all the T1D patients after 30-minute of acute exercise. This increase may explain in part the attenuation in the metabolomic response of all energetic substrates. Pharmacokinetic studies have shown that the peak action of insulin glargine is usually within the first 3 or 4 hours after injection
Although we speculate that high insulin levels induced by exogenous treatment may be responsible for the attenuated response of metabolites to acute exercise, alternative explanations must also be considered. For example, the possible presence of insulin resistance (IR), an important condition described in T1D patients, cannot be ignored. Some authors have considered that supra-physiologic levels of exogenous insulin
Metabolic flexibility defined as the ability to switch from fat to carbohydrate oxidation is usually impaired during a hyperinsulinemic clamp in insulin-resistant subjects. Thus, the phenomena of metabolic inflexibility mainly described in T2D and other insulin-resistant states could explain some of the alterations occurring in the machinery of lipid and glucose metabolism
Several studies involving T2D patients and their offspring have demonstrated the presence of mitochondrial dysfunction
Although our study was not designed to analyze IR or metabolic flexibility, these factors might have influenced the metabolomic spectrum described in our T1D patients. In addition, the presence of hyperglucagonemia and impaired glucagon counterregulation, as reported in several studies
In summary, we report that T1D patients have an attenuated metabolic response as compared to their healthy control counterparts after a short period of acute, intense exercise. We speculate that exercise could mobilize the subcutaneous exogenous insulin depot in adipose tissue. Furthermore, our data suggest that high insulinemia levels might play a role in the attenuated response in lipolysis, proteolysis, glycogenolysis, and oxidative metabolism observed in T1D patients following exercise. Whether the attenuation of metabolic response to acute and intense exercise might interfere in the training performance of T1D patients remains to be elucidated, and additional studies are required.
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We are grateful to Kimberly Katte for the editorial assistance.