Inflammation, Vascular Repair, Injury After Exercise in T1D
Inflammation, Vascular Repair, Injury After Exercise in T1D
Eligibility criteria for type 1 diabetes patients consisted of being aged between 18 and 35 years, a duration of diabetes > 2 years, and an HbA1c < 8.0 % (64 mmol/mol). In addition, patients were required to be free of all diabetes-related complications including impaired awareness of hypoglycaemia (assessed via the Clarke method) and not receiving any medication other than insulin. Participants in both the Type 1 diabetes and control group had to be regularly and consistently undertaking exercise (participating in aerobic based exercise for a minimum of 30 min at a time, at least three times per week, for > 12 months). Smokers were excluded from both groups. Ten males with type 1 diabetes and nine non-diabetic male controls, matched for age, anthropometry, and fitness were recruited for this study (Table 1). Patients were treated with a basal-bolus regimen composed of long-acting insulins glargine (n = 8) or detemir (n = 2), and rapid-acting insulin aspart. All patients were stable on their respective insulin regimen for a minimum of 1 year, and were familiar with carbohydrate counting, administering 1.0 ± 0.2 units of insulin aspart (IU) per 10 g of carbohydrate.
Fully informed written consent was obtained from all participants following the study's approval from local National Health Service Research Ethics Committee (13/NE/0016). Type 1 diabetes participants attended the Newcastle National Institute for Health Research Clinical Research Facility exercise laboratory for a preliminary screening visit as described previously in detail, before returning to establish peak cardio-respiratory parameters during the completion of an incremental-maximal treadmill running protocol as per the methods of Campbell et al, to determine the individual speed participants would run at during the experimental trial. The control group completed the same preliminary tests at the Exercise Physiology laboratory at Northumbria University.
Type 1 diabetes patients were fitted with a real-time continuous glucose monitor (Paradigm Veo, Medtronic diabetes, Northridge, CA, USA) >24 h prior to the laboratory visit with high and low alerts set to help maintain glycaemia within normal ranges prior to the experimental session. Participants did not exercise within 96 h of the experimental visit.
On the experimental day participants were provided with standardised cereal-based breakfast and pasta-based lunch; prescribed by the research team. Participants arrived to the laboratory at 17:00 h. Following a resting blood sample participants consumed a pre-exercise carbohydrate bolus (corn flakes, peaches, semi-skimmed milk; 1738 ± 71 kJ/415 ± 17 kcal) equating to 1.0 g.carbohydrate.kg body mass. With this meal, patients self-administered a 25 % (2.0 ± 0.5 IU) dose (i.e. a 75 % reduction) of rapid-acting insulin into the abdomen. Following this bolus, participants remained rested for 60 min, before commencing 45 min of treadmill running at a speed calculated to elicit 70 % of VO2max. This exercise intensity falls within current recommendations of the American College of Sports Medicine. All participants completed the exercise protocol and there were no hypoglycemic episodes within the Type 1 diabetes group.
After exercise, participants remained at rest for 60 min before a further blood sample was collected. Participants were then discharged from the laboratory. On the following morning, participants returned to the laboratory at 08:00 h for a further resting, fasted blood sample.
Venepuncture technique was used to collect 10 ml of whole blood at each respective sample point. After discarding the first four ml of collected blood, samples were evenly dispersed into a K2EDTA and lithium heparin tube. The lithium heparin tube was centrifuged for 15 min at 3000 rpm (4 °C) and stored at -80 °C for retrospective analysis of plasma Human TNF-α (Quantikine ELISA, R&D Systems, Roche Diagnostics, West Sussex, UK). The K2EDTA was sent for analysis of cEPCs and cECs immediately and was processed within 2 h.
Flow Cytometry. 100 μl of whole blood was incubated with 5 μl of V500 CD45 (BD Biosciences, Oxford, UK), 20 μl of PerCP-Cy5.5 CD34 (BD Biosciences, Oxford, UK), 5 μl of PE VEGFR-2+ (R&D Systems, Roche Diagnostics, West Sussex, UK), 5 μl APC CD133 (BD Biosciences, Oxford, UK), 10 μl FITC CD144 (BD Biosciences, Oxford, UK) for 30 min. Subsequently, 2 ml of pharmlyse (BD Biosciences, Oxford, UK) was used to lyse the red cells. The sample was then analysed by flow cytometry on BD FACS Canto™ II system and samples were run to approximately 500,000 total events. Analysis was carried out using BD FACSDiva™ software. On average 440,000 events were counted. CEPCs events were defined using the most recent definition of CD45CD34 VEGFR2 (KDR), as recommended by Van Craenenbroeck et al.. Intra-assay variation (CD45CD34VEGFR-2) was less than 8 %. The results were expressed as % leukocytes. CECs events were defined as CD45CD133CD34CD144.
Statistical Analysis. Statistical analysis was performed using PASW Statistics 18 software (IBM, Armonk, NY) with significance set at p ≤ 0.05. Plasma TNF-α responses were analysed using mixed model ANOVA (group*time) and are presented as mean ± SEM. CEPCs and CECs responses were assessed using two-way Friedman's analysis and are presented as median ± IQR. Between group differences, and comparisons of the delta-change in cEPCs and cECs counts, were assessed using Mann-Whitney U test. Relationships were assessed with Pearsons product moment correlation coefficient or Spearman's rank order correlation coefficient.
Methods
Participants
Eligibility criteria for type 1 diabetes patients consisted of being aged between 18 and 35 years, a duration of diabetes > 2 years, and an HbA1c < 8.0 % (64 mmol/mol). In addition, patients were required to be free of all diabetes-related complications including impaired awareness of hypoglycaemia (assessed via the Clarke method) and not receiving any medication other than insulin. Participants in both the Type 1 diabetes and control group had to be regularly and consistently undertaking exercise (participating in aerobic based exercise for a minimum of 30 min at a time, at least three times per week, for > 12 months). Smokers were excluded from both groups. Ten males with type 1 diabetes and nine non-diabetic male controls, matched for age, anthropometry, and fitness were recruited for this study (Table 1). Patients were treated with a basal-bolus regimen composed of long-acting insulins glargine (n = 8) or detemir (n = 2), and rapid-acting insulin aspart. All patients were stable on their respective insulin regimen for a minimum of 1 year, and were familiar with carbohydrate counting, administering 1.0 ± 0.2 units of insulin aspart (IU) per 10 g of carbohydrate.
Preliminary Testing
Fully informed written consent was obtained from all participants following the study's approval from local National Health Service Research Ethics Committee (13/NE/0016). Type 1 diabetes participants attended the Newcastle National Institute for Health Research Clinical Research Facility exercise laboratory for a preliminary screening visit as described previously in detail, before returning to establish peak cardio-respiratory parameters during the completion of an incremental-maximal treadmill running protocol as per the methods of Campbell et al, to determine the individual speed participants would run at during the experimental trial. The control group completed the same preliminary tests at the Exercise Physiology laboratory at Northumbria University.
Experimental Design
Type 1 diabetes patients were fitted with a real-time continuous glucose monitor (Paradigm Veo, Medtronic diabetes, Northridge, CA, USA) >24 h prior to the laboratory visit with high and low alerts set to help maintain glycaemia within normal ranges prior to the experimental session. Participants did not exercise within 96 h of the experimental visit.
On the experimental day participants were provided with standardised cereal-based breakfast and pasta-based lunch; prescribed by the research team. Participants arrived to the laboratory at 17:00 h. Following a resting blood sample participants consumed a pre-exercise carbohydrate bolus (corn flakes, peaches, semi-skimmed milk; 1738 ± 71 kJ/415 ± 17 kcal) equating to 1.0 g.carbohydrate.kg body mass. With this meal, patients self-administered a 25 % (2.0 ± 0.5 IU) dose (i.e. a 75 % reduction) of rapid-acting insulin into the abdomen. Following this bolus, participants remained rested for 60 min, before commencing 45 min of treadmill running at a speed calculated to elicit 70 % of VO2max. This exercise intensity falls within current recommendations of the American College of Sports Medicine. All participants completed the exercise protocol and there were no hypoglycemic episodes within the Type 1 diabetes group.
After exercise, participants remained at rest for 60 min before a further blood sample was collected. Participants were then discharged from the laboratory. On the following morning, participants returned to the laboratory at 08:00 h for a further resting, fasted blood sample.
Blood Sampling and Data Analysis
Venepuncture technique was used to collect 10 ml of whole blood at each respective sample point. After discarding the first four ml of collected blood, samples were evenly dispersed into a K2EDTA and lithium heparin tube. The lithium heparin tube was centrifuged for 15 min at 3000 rpm (4 °C) and stored at -80 °C for retrospective analysis of plasma Human TNF-α (Quantikine ELISA, R&D Systems, Roche Diagnostics, West Sussex, UK). The K2EDTA was sent for analysis of cEPCs and cECs immediately and was processed within 2 h.
Circulating Endothelial Progenitor Cells and Circulating Endothelial Cells
Flow Cytometry. 100 μl of whole blood was incubated with 5 μl of V500 CD45 (BD Biosciences, Oxford, UK), 20 μl of PerCP-Cy5.5 CD34 (BD Biosciences, Oxford, UK), 5 μl of PE VEGFR-2+ (R&D Systems, Roche Diagnostics, West Sussex, UK), 5 μl APC CD133 (BD Biosciences, Oxford, UK), 10 μl FITC CD144 (BD Biosciences, Oxford, UK) for 30 min. Subsequently, 2 ml of pharmlyse (BD Biosciences, Oxford, UK) was used to lyse the red cells. The sample was then analysed by flow cytometry on BD FACS Canto™ II system and samples were run to approximately 500,000 total events. Analysis was carried out using BD FACSDiva™ software. On average 440,000 events were counted. CEPCs events were defined using the most recent definition of CD45CD34 VEGFR2 (KDR), as recommended by Van Craenenbroeck et al.. Intra-assay variation (CD45CD34VEGFR-2) was less than 8 %. The results were expressed as % leukocytes. CECs events were defined as CD45CD133CD34CD144.
Statistical Analysis. Statistical analysis was performed using PASW Statistics 18 software (IBM, Armonk, NY) with significance set at p ≤ 0.05. Plasma TNF-α responses were analysed using mixed model ANOVA (group*time) and are presented as mean ± SEM. CEPCs and CECs responses were assessed using two-way Friedman's analysis and are presented as median ± IQR. Between group differences, and comparisons of the delta-change in cEPCs and cECs counts, were assessed using Mann-Whitney U test. Relationships were assessed with Pearsons product moment correlation coefficient or Spearman's rank order correlation coefficient.
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