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 
confirmed these results by using LC-MS-based metabolomics in a non-diabetic population. Our data, using a different metabolomic approach based on 1
H-NMR and GC-MS techniques, showed that the non-diabetic group presented an increase in serum lactate and pyruvate concentration after exercise, indicating that glycolysis was activated, as previously reported by other groups 
. However, the T1D patients elicited only an increase in lactate levels having blunted the pyruvate response, suggesting that exercise induces a reduced activation of glycogenolysis and glycolysis in the T1D group as compared to the control group. Given the inhibitory effect of insulin on glycogen breakdown, the higher insulin levels observed in the T1D group could explain in part the attenuated glycogenolytic response.
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 
, in which accumulated levels of malate, fumarate and succinate were reported following 60 minutes of exercise in a healthy cohort. Other studies have demonstrated that an increase in the total concentration of TCAIs is necessary to enhance and maintain TCA cycle flux during strenuous exercise 
. In addition, a positive correlation has been demonstrated between the total concentration of TCAIs and the estimated TCA cycle flux in human skeletal muscle taken from muscle biopsies during exercise 
. In the case of patients with T1D, our results also pointed to a less significant accumulation of TCAIs after exercise, which might also compromise the TCA flux rates. Given that many of the reactions that lead to a net influx of TCAIs are directly or indirectly dependent on the level of pyruvate and that elevated concentrations of pyruvate appear to be necessary for the anaplerosis pathway 
, we suggest that the insufficient increase in pyruvate concentrations in T1D subjects might have an impact on TCA cycle replenishment. In this sense, our findings of an attenuated enrichment of TCAIs in the serum of T1D patients in response to a short period of intense exercise indicate that the activation of the TCA cycle flux rates might be affected. This suggests that the T1D group has a compromised oxidative aerobic system as compared to the control group, as the T1D group did not show a significant turnover of TCA metabolites.
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 
. Under conditions of hyperinsulinemia induced by clamp techniques, a suppression of the intramuscular and subcutaneous adipose tissue lipolysis was demonstrated in healthy volunteers 
. In another study performed in obese men, a delay in the lipolytic activation was observed together with an increase in plasma insulin levels following 30 minutes of acute resistance exercise, a finding that the authors attributed to an increase in insulin levels 
. The increased insulin levels found in the T1D group in our study could have a role in the attenuated lipolytic action observed following exercise.
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 
. In parallel to the reduction of leucine, we observed an increase in circulating levels of 2-KIC, which is the first step in leucine degradation. This increase in 2-KIC is in concordance with a previous reported study on the metabolomic profiling performed on urine samples of men following acute exercise 
. Our results suggest that the effect induced by exercise on protein catabolism was attenuated in the diabetic patients, in agreement with other authors who have previously demonstrated a decrease in protein catabolism in T1D patients 
. Moreover, there are evidences confirming that elevated insulin levels induced by infusion in healthy men promote muscle protein anabolism by inhibiting protein breakdown 
and elicit the ability to stimulate glucose uptake and alanine transport, thus suppressing protein degradation in skeletal muscle 
. Taking all these data into consideration, we propose that the high insulinemia levels induced by exogenous insulin administration in our T1D group could be the cause of reduced protein breakdown, as demonstrated by the minor alterations observed in the levels of leucine and 2-KIC following exercise.
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 
, and in our protocol, samples were taken 10 hours later. One possible explanation for the increased insulin concentration detected in the serum of our T1D group could be that intense exercise induces the lipolysis of subcutaneous adipose tissue where insulin is stored and rapidly released into the circulation. In line with this hypothesis, Davison et al 
previously reported an exercise-induced lipolysis effect by monitoring the release of liposoluble vitamins from the subcutaneous tissue into the bloodstream, an effect which may be responsible for the increased insulin levels observed in our T1D patients.
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 
and hyperglycemia per se 
could be responsible for IR in T1D patients. Insulin, not the rate of glucose disposal per se, regulates glycogen synthesis, meaning that the low level of glycogen synthase activity found in insulin-resistant states is a consequence of impaired insulin action, rather than reduced glucose disposal. Nevertheless, this assumption has been challenged in a more recent study in which adult patients with T1D exhibited both impaired glucose utilization and impaired insulin-induced non-esterified fatty acid suppression 
. In addition, these patients showed IR in hepatic and skeletal muscle tissue, despite good glycemic control 
. Another report 
demonstrated that T1D adolescents had significantly impaired functional exercise capacity and decreased insulin sensitivity as compared to non-diabetic adolescents. In spite of their IR and reduced cardiovascular fitness, T1D youth showed paradoxically normal intramyocellular lipid content (IMCL). This finding contradicts the previously well- established theory that IMCL accumulation is a marker for insulin resistance in both T1D and T2D 
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 
. In addition, the inability to modify fuel oxidation in response to changes in nutrient availability has been implicated in the accumulation of intramyocellular lipids as well as in insulin resistance. Exercise represents a paradigm which requires a highly regulated coordination between fuel supply and oxidative machinery. The assumption that metabolic inflexibility may affect energy metabolism under exercise conditions in T1D patients remains to be elucidated.
Several studies involving T2D patients and their offspring have demonstrated the presence of mitochondrial dysfunction 
. In T1D patients, mitochondrial dysfunction has also been described, contributing to abnormalities in the TCA cycle and fatty acid metabolism. A recent report 
verified that the mitochondrial capacity of untrained women with T1D correlates positively with glycemic control. The hypothesis that mitochondrial abnormalities may be a primary cause of metabolic inflexibility and insulin resistance has been offered. Significant differences in mitochondrial number, structure and function have been described between insulin-resistant and insulin-sensitive subjects, but the causal link still remains unknown 
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 
, could affect lipolysis, gluconeogenesis and protein metabolism. Finally, other situations not present in our patients should be taken into account, such as autonomic dysfunction, which influences lypolitic responsiveness 
, cardiac dysfunction, which has been documented in T1D patients as affecting exercise responses 
, and abnormal blood flow during exercise, which could affect muscular vascular function, contributing toward metabolic disturbances 
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.