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Acute inspiratory muscle exercise effect on glucose levels, glucose variability and autonomic control in patients with type 2 diabetes: A crossover randomized trial

  • Andressa S.O. Schein
    Correspondence
    Corresponding author at: Laboratório de Fisiopatologia do Exercício - Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos 2350, prédio 12, 4° andar, Porto Alegre, RS, Brazil.
    Affiliations
    Postgraduate Program in Cardiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Exercise Pathophysiology Research Laboratory, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
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  • Ana P.S. Corrêa
    Affiliations
    Faculty of Health Sciences, The University of Sydney, Lidcombe, NSW, Australia
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  • Aline C.P. Macedo
    Affiliations
    Postgraduate Program in Endocrinology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Exercise Pathophysiology Research Laboratory, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
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  • Daniela R. Dartora
    Affiliations
    Sainte-Justine University Hospital Research Center, University of Montreal Depatment of pediatrics, Montreal, Quebec, Canada
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  • Anderson Donelli da Silveira
    Affiliations
    Postgraduate Program in Cardiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
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  • Mateus Dornelles Severo
    Affiliations
    Postgraduate Program in Endocrinology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
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  • Karina R. Casali
    Affiliations
    Institute of Science and Technology, Universidade Federal de São Paulo, São Paulo, Brazil
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  • Beatriz D. Schaan
    Affiliations
    Postgraduate Program in Cardiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Postgraduate Program in Endocrinology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Exercise Pathophysiology Research Laboratory, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil

    Department of Internal Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
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Open AccessPublished:March 26, 2020DOI:https://doi.org/10.1016/j.autneu.2020.102669

      Highlights

      • In type 2 diabetes, inspiratory muscle exercise does not improve blood glucose levels, glucose variability and autonomic modulation.
      • Inspiratory muscle exercise with a load of 60% of PImax induced cardiovascular changes.
      • At this time, inspiratory muscle training is not recommended for long-term glucose control in type 2 diabetic patients.

      Abstract

      Inspiratory muscle exercise (IME) can be an alternative to conventional exercise. We aimed to evaluate the effect of IME on glucose, glucose variability, and autonomic cardiovascular control in type 2 diabetes. Fourteen diabetic subjects were randomly assigned to IME with 2% maximal inspiratory pressure (PImax) or 60% PImax wearing a continuous glucose monitoring system for three days. Glucose variability [glucose variance (VAR), glucose coefficient of variation (CV%), glucose standard deviation (SD), and mean amplitude of glycemic excursions (MAGE)] were evaluated. Glucose reduction was observed in 5 min (60% of PImax 33.2% and 2% of PImax 32.0%), 60 min (60% of PImax 29.6% and 2% of PImax 31.4%) and 120 min (60% of PImax 21.4% and 2% of PImax 24.0%) after IME (vs.1 h before the exercise), with no difference between loads. This reduction in glucose levels was observed in all moments of the IME protocol. Glucose variability was reduced after 12 h and 18 h of the IME (ΔCV: P < 0.001, ΔSD: P < 0.001 and ΔVAR: P < 0.001) for both loads. No difference was found in MAGE (P = 0.594) after IME. Mean arterial pressure and heart rate rose during the exercise session with 60% of PImax. Although sufficiently strong to induce cardiovascular changes, an inspiratory muscle exercise session with 60% of PImax in subjects with type 2 diabetes has failed to induce any significant improvement in glucose, glucose variability and autonomic control, compared to the 2% Plmax exercise session.

      Keywords

      1. Introduction

      Diabetes mellitus (DM) is a chronic disease with a worldwide prevalence. Its management relies on strict glucose control, which is monitored through glycated hemoglobin (HbA1c) levels (
      • America Diabetes Association
      Glycemic targets: standards of medical care in diabetes.
      ). Beyond HbA1c, glucose fluctuations can also be involved in the pathogenesis of chronic complications of the disease (
      • Giacco F.
      • Du X.
      • Carratú A.
      • Gerfen G.J.
      • D’Apolito M.
      • Giardino I.
      • Rasola A.
      • Marin O.
      • Divakaruni A.S.
      • Murphy A.N.
      • Shah M.S.
      • Brownlee M.
      GLP-1 cleavage product reverses persistent ROS generation after transient hyperglycemia by disrupting an ROS-generating feedback loop.
      ;
      • Xu F.
      • Zhao L.H.
      • Su J.B.
      • Chen T.
      • Wang X.Q.
      • Chen J.F.
      • Wu G.
      • Jin Y.
      • Wang X.H.
      The relationship between glycemic variability and diabetic peripheral neuropathy in type 2 diabetes with well-controlled HbA1c.
      ), but not all reports agree that blood glucose variability is associated with microvascular (
      • Kilpatrick E.S.
      • Rigby A.S.
      • Atkin S.L.
      The effect of glucose variability on the risk of microvascular complications in type 1 diabetes.
      ) or macrovascular (
      • Siegelaar S.E.
      • Barwari T.
      • Kulik W.
      • Hoekstra J.B.
      • DeVries J.H.
      No relevant relationship between glucose variability and oxidative stress in well-regulated type 2 diabetes patients.
      ) complications.
      Exercise training (aerobic exercise, resistance exercise, or a combination of both) is useful for reducing HbA1c in type 2 DM (
      • Umpierre D.
      • Ribeiro P.A.
      • Kramer C.K.
      • Leitão C.B.
      • Zucatti A.T.
      • Azevedo M.J.
      • Gross J.L.
      • Ribeiro J.P.
      • Schaan B.D.
      Physical activity advice only or structured exercise training and association with HbA1c levels in type 2 diabetes: a systematic review and meta-analysis.
      ). Other benefits, such as blood pressure control (
      • Figueira F.R.
      • Umpierre D.
      • Cureau F.V.
      • Zucatti A.T.
      • Dalzochio M.B.
      • Leitão C.B.
      • Schaan B.D.
      Association between physical activity advice only or structured exercise training with blood pressure levels in patients with type 2 diabetes: a systematic review and meta-analysis.
      ), and improved cardiovascular autonomic function (
      • Pagkalos M.
      • Koutlianos N.
      • Kouidi E.
      • Pagkalos E.
      • Mandroukas K.
      • Deligiannis A.
      Heart rate variability modifications following exercise training in type 2 diabetic patients with definite cardiac autonomic neuropathy.
      ) are expected. Some studies have shown that aerobic or resistance exercise are associated with lower glucose variability (
      • Figueira F.R.
      • Umpierre D.
      • Casali K.R.
      • Tetelbom P.S.
      • Henn N.T.
      • Ribeiro J.P.
      • Schaan B.D.
      Aerobic and combined exercise sessions reduce glucose variability in type 2 diabetes: crossover randomized trial.
      ;
      • Van Dijk J.W.
      • Manders R.J.
      • Canfora E.E.
      • Mechelen W.V.
      • Hartgens F.
      • Stehouwer C.D.
      • Van Loon L.J.
      Exercise and 24-h glycemic control: equal effects for all type 2 diabetes patients?.
      ) which could be beneficial for these patients.
      However, not all patients tolerate conventional exercise, and most present difficulty in adhering to exercise programs (
      • Baldi J.C.
      • Wilson G.A.
      • Wilson L.C.
      • Wilkins G.T.
      • Lamberts R.R.
      The type 2 diabetic heart: its role in exercise intolerance and the challenge to find effective exercise interventions.
      ). Inspiratory muscle training is an exercise alternative that increases strength and endurance of inspiratory muscles in DM (
      • Kaminski D.M.
      • Schaan B.D.
      • da Silva A.M.
      • Soares P.P.
      • Lago P.D.
      Inspiratory muscle training in patients with diabetic autonomic neuropathy: a randomized clinical trial.
      ).
      • Ahmad M.A.
      • Abdelsalam H.M.
      • Lotfy A.O.
      Effect of inspiratory muscle training on blood glucose levels and serum lipids in female patients with type 2 diabetes.
      showed that glucose levels did not change after inspiratory muscle training with 30% of maximal inspiratory pressure (PImax), but other authors have shown that using an inspiratory load of 40% of PImax can promote decreased fasting glucose in older adults with hyperglycemia (
      • Dos Santos Silva M.
      • Ramos L.R.
      • Tufik S.
      • Togeiro S.M.
      • Lopes G.S.
      Influence of inspiratory muscle training on changes in fasting hyperglycemia in the older adult: the Epidoso project.
      ). Preliminary data from our group indicated that acute inspiratory muscle exercise (IME) with a load of 60% of PImax decreases glucose levels and glucose variability in a similar magnitude as aerobic exercise in type 2 DM (
      • Corrêa A.P.
      • Figueira F.R.
      • Umpierre D.
      • Casali K.R.
      • Schaan B.D.
      Inspiratory muscle loading: a new approach for lowering glucose levels and glucose variability in patients with type 2 diabetes.
      ). We hypothesized that IME would reduce glycemia in DM, and that this effect may be linked to changes in cardiovascular autonomic control induced by exercise. Therefore, the purpose of this study is to evaluate the effect of acute IME on glucose levels, glucose variability, and autonomic cardiovascular control in patients with type 2 DM.

      2. Methods

      2.1 Study design/participants

      This is a double blind crossover randomized clinical trial previously described in detail (
      • Schein A.
      • Correa A.
      • Casali K.R.
      • Schaan B.D.
      Are glucose levels, glucose variability and autonomic control influenced by inspiratory muscle exercise in patients with type 2 diabetes? Study protocol for a randomized controlled trial.
      ). Patients with type 2 DM were recruited from the Endocrinology Outpatient Clinic at Hospital de Clínicas de Porto Alegre (HCPA), Brazil and through website posting. Subjects were included if they were 30 years-old or more, and had an HbA1c higher than 7.5% but lower than 10%. Exclusion criteria included pulmonary disease, cardiac arrhythmias, chronic kidney disease (glomerular filtration rate < 30 mL/min), insulin treatment, pregnancy, exclusive use of beta-blockers as antihypertensive therapy, current smoking, varicose veins, and musculoskeletal conditions that would hinder the safe completion of the proposed exercise protocols. The study was approved by the Ethics Committee of HCPA, and was registered at clinicalTrials.gov under the identifier NCT02292810. All patients signed an informed consent before beginning the study.

      2.2 Data collection

      Participants were submitted to baseline evaluations on three different days. Clinical characteristics, Ewing tests, usual physical activity (International Physical Activity Questionnaire IPAQ), fasting blood samples (HbA1c, glucose and creatinine) and urine collection (albuminuria) were done on first day: HbA1c was analyzed by ion-exchange high performance liquid chromatography (HPLC) (Merck-Hitachi L-9100 HbA1c analyzer; Merck, Darmstadt, Germany) and plasma glucose was analyzed by the glucose oxidase method. Glomerular filtration rate was calculated using the Modification of Diet in Renal Disease equation (MDRD), and serum creatinine by Jaffé's reaction. Urinary albumin excretion was measured by immunoturbidimetry (MICROALB-AMES Kit, CA, USA). Elevated albuminuria was defined as values higher than 17 mg/L of albumin in urine. On the second day, resting electrocardiogram, inspiratory muscle function test and pulmonary function test were performed, with the cardiopulmonary exercise testing being performed on day three.

      2.2.1 Autonomic neuropathy evaluation (Ewing tests)

      Diabetic cardiovascular autonomic neuropathy was assessed by five noninvasive cardiovascular reflex tests: deep breathing, stand up (30:15 ratio), Valsalva maneuver, orthostatic hypotension and sustained handgrip, as proposed by Ewing and standardized in our institution. All tests were evaluated using a digital electrocardiograph with the software VNS-Rhythm Neurosoft (Ivanovo, Russia) and diagnosis of cardiovascular autonomic neuropathy was defined when two or more abnormal tests were found (
      • Neumann C.
      • Schmid H.
      Standardization of a computerized method for calculating autonomic function test responses in healthy subjects and patients with diabetes mellitus.
      ).

      2.2.2 Inspiratory muscle testing

      The PImax was assessed using a pressure transducer during deep breath from residual volume against an occluded airway with a minimum air lack (2 mm) to relieve facial muscle pressure. The highest value among six measurements with <10% variation was considered (
      • Dall’Ago P.
      • Chiappa G.R.
      • Guths H.
      • Stein R.
      • Ribeiro J.P.
      Inspiratory muscle training in patients with heart failure and inspiratory muscle weakness: a randomized trial.
      ).

      2.2.3 Pulmonary function

      Pulmonary function was measured with a computerized spirometer (Eric Jaeger GmbH, Würzburg, Germany); the measurement of forced vital capacity (FVC), vital capacity (VC), forced expiratory volume in 1 s (FEV1) and maximal voluntary ventilation (MVV) were obtained according to the recommendations of the American Thoracic Society (
      • Miller M.R.
      • Hankinson J.
      • Brusasco V.
      • Burgos F.
      • Casaburi R.
      • Coates A.
      • Crapo R.
      • Enright P.
      • van der Grinten C.P.
      • Gustafsson P.
      • Jensen R.
      • Johnson D.C.
      • MacIntyre N.
      • McKay R.
      • Navajas D.
      • Pedersen O.F.
      • Pellegrino R.
      • Viegi G.
      • Wanger J.
      • ATS/ERS Task Force
      Standardisation of spirometry.
      ).

      2.2.4 Cardiopulmonary exercise testing

      Maximal functional capacity was evaluated on a treadmill (General Electric T-2100, GE Healthcare, USA) with incremental exercise test using a ramp protocol. The electrocardiogram (Nihon Khoden Corp, Tokyo, Japan) was continuously assessed, and blood pressure values were obtained every two minutes. Gas exchange variables were assessed breath-by-breath by a previously validated system (Metalyzer 3B, CPX System; Cortex, Leipzig, Germany) (
      • Dall’Ago P.
      • Chiappa G.R.
      • Guths H.
      • Stein R.
      • Ribeiro J.P.
      Inspiratory muscle training in patients with heart failure and inspiratory muscle weakness: a randomized trial.
      ).

      2.3 Study intervention

      The intervention was performed during three consecutive days on the first week, and it was repeated in the following week, as follows: day 1: placement of the glucose sensor to start evaluation at approximately 9:00 a.m. (continuous glucose monitoring system, CGMS), day 2: IME with 60% of PImax (experimental load) or 2% of PImax (placebo load, without inspiratory threshold pressure valve diaphragm) to start at approximately 9 a.m. and day 3: glucose sensor removal. The 2% of PImax was arbitrarily chosen as it represents the lowest possible inspiratory pressure provided by the device.
      The protocol began with a 10 min rest. Then, patients started controlled ventilation for 10 min and they breathed at 15 breaths per min with a duty cycle (TI/TTot) of 0.7. Shortly after, there was a 40-min interval, when the individuals started exercise breathing in a two-way valve (Hans Rudolph, 2600 series, Shawnee, KS, USA) against a 2% of PImax through a threshold inspiratory muscle trainer (DHDInspiratory Muscle Trainer, Chicago, IL) for 10 min (placebo load) or 60% of PImax through a POWERbreathe® inspiratory muscle trainer (Southam, UK) until task failure (approximately 7.5 min, experimental load). During both exercise sessions, patients maintained their breathing pattern (15 breath per min and TI/TTot: 0.7) and were guided by audio and visual feedback. At the end of the protocol, a 10-min recovery record was obtained. Respiratory and hemodynamics parameters were evaluated during the entire intervention; measurement of arterial oxygen saturation (SpO2), respiratory rate (fb), heart rate and end-tidal partial pressure of CO2 (PetCO2) were obtained through oxycapnography (Takaoka Oxicap, São Paulo, Brazil). Glucose levels, continuous blood pressure, mean arterial blood pressure (MAP) (Dinamap, USA) and calf blood flow (CBF) were also evaluated (venous occlusion plethysmography; Hokanson, TL-400, Bellevue, WA,USA). Calf blood resistance (CVR) was calculated as MAP/CBF.

      2.3.1 Glucose variability evaluation

      All patients were monitored by the CGMS (Medtronic MiniMed, Northridge, CA 91325, USA) for three days; they did not know their glucose values during the evaluation. Individuals were instructed to accomplish at least four capillary blood glucose measurements per day for calibration. Glucose variability was assessed using conventional analyses, which included standard deviation of glucose (SD), variance of glucose (VAR), coefficient of variation of glucose (CV%) and mean amplitude of glucose excursion (MAGE) (
      • Hill N.R.
      • Oliver N.S.
      • Choudhary P.
      • Levy J.C.
      • Hindmarsh P.
      • Matthews D.R.
      Normal reference range for mean tissue glucose and glycemic variability derived from continuous glucose monitoring for subjects without diabetes in different ethnic groups.
      ).

      2.3.2 Cardiovascular autonomic control evaluation

      Participants were given specific information about testing and were asked to avoid consuming beverages containing caffeine, smoking, and performing physical and vigorous activities 24 h before the evaluation; room's light and temperature were controlled.
      Cardiovascular autonomic control was assessed using a finger cuff connected to a noninvasive blood pressure device (NIBP100D system; Biopac, Santa Barbara, CA, USA) calibrated and connected to an amplifier (DA100; Biopac) during all the intervention protocol. Four different conditions were evaluated: rest, controlled ventilation, IME and recovery. Temporal series of beat-to-beat systolic blood pressure and pulse interval were obtained from continuous blood pressure signal (sample 1000 Hz) for the analysis of blood pressure variability (BPV) and heart rate variability (HRV). Frequency domain analysis was assessed through spectral decomposition by autoregressive modeling, which provided data on very low frequency bands (VLF: 0.0 to 0.04 Hz), low frequency bands (LF: 0.04 to 0.15 Hz), and high frequency bands (HF: 0.15 to 0.40 Hz). Power spectrum was quantified in absolute values (mmHg2 and ms2) and in normalized units (nu). Normalization was calculated by the division of the absolute value of the spectral power of each component, LF or HF, by the total power (without the value of the VLF component), multiplied by 100 (
      • Malliani A.
      • Pagani M.
      • Lombardi F.
      • Cerutti S.
      Cardiovascular neural regulation explored in the frequency domain.
      ).

      2.4 Randomization and statistical analysis

      Patients were randomized to IME with experimental or placebo load, which were separated by at least seven days. A randomization sequence was generated by software R (x64 version 3.1.1) by an independent investigator. Data was expressed as mean ± SD or medians and interquartile ranges for nonparametric variables. Categorical variables were expressed as number and percentage. Data normality was assessed by Shapiro-Wilk test. The effect of both IME (placebo or experimental loads) was estimated by generalized estimating equations (GEE) followed by Bonferroni's post-hoc test. Statistical significance was considered for p < 0.05. All data was analyzed using the Statistical Package for Social Sciences (SPSS- version 18.0).

      3. Results

      Seventeen patients participated in the study and fourteen finished both protocols. Three patients withdrew consent by personal reasons (Fig. 1). Clinical characteristics of the participants are shown in Table 1. They were 53.6 ± 1.9 years-old, predominantly men (57.1%), being overweight (29.6 ± 0.9) and with a median duration of DM of 6.5 (2.7–11.0) years. Only one patient was diagnosed with cardiovascular autonomic neuropathy. Glucose control was not good (HbA1c: 8.8 ± 0.2%, fasting plasma glucose: 196.8 ± 9.6 mg/dL), albuminuria was 12.3 (8.2–35.0) mg/L, creatinine was 0.78 ± 0.05 mg/dL, and pulmonary function and inspiratory muscle strength were normal. Most of the patients were sufficiently active (57.1%), as defined by the IPAQ questionnaire, with a reduced peak oxygen consumption (VO2 peak) [21.7 (19.5–33.0) (mL/Kg/min)].
      Fig. 1
      Fig. 1Flow diagram of patient's selection. IPAQ, International Physical Activity Questionnaire; PImax, maximal inspiratory pressure; HbA1c, glycated hemoglobina; CGMS, continuous glucose monitoring system.
      Table 1Clinical characteristics of the participants studied.
      CharacteristicsTotal sample (n = 14)
      Age53.6 ± 7.4
      Male gender8 (57.1)
      Duration of diabetes (years)6.5 (2.7–11.0)
      BMI (Kg/m2)29.6 ± 3.7
      Office SBP (mmHg)131.6 ± 11.0
      Office DBP (mmHg)83.3 ± 10.0
      HbA1c (%)8.8 ± 0.9
      Fasting plasma glucose (mg/dL)196.8 ± 35.8
      Albuminuria12.3 (8–35)
      Creatinine (mg/dL)0.78 ± 0.2
      GFR (mL/min/1.73 m2)96.3 ± 14.2
      Pulmonary function
      FEV1, % predicted101.9 ± 16.8
      FVC, % predicted102.8 ± 13.7
      MVV, % predicted111.4 ± 26.3
      Inspiratory muscle function
      PImax, cmH2O94.1 ± 38.8
      PImax, % predicted106.9 (82.8–115.5)
      Physical activity level (IPAQ)
      Insufficiently active1 (7.1)
      Sufficiently active8 (57.1)
      Very active5 (35.7)
      Cardiopulmonary exercise test
      VO2 peak (mL.Kg.min−1)21.7 (20−33)
      Heart rate peak (bpm)163 (154.2–174.2)
      RER peak1.2 (1.1–1.2)
      VE/VCO2 slope32.0 ± 4.9
      OUES1.9 ± 0.7
      Heart rate recovery (bpm)20.0 ± 7.9
      Medications
      Metformin14 (100)
      Sulfonylureas6 (42.9)
      Other antidiabetic drugs2 (14.3)
      Calcium channel blockers2 (14.3)
      Angiotensin II receptor blockers4 (28.6)
      Angiotensin-converting enzyme inhibitors3 (21.4)
      Diuretics6 (42.9)
      β-blockers3 (21.4)
      Antiplatelet1 (7.1)
      Statins3 (21.4)
      Continuous variables are expressed as mean ± SD or median (interquartile range (p25-p75)). Categorical variables are expressed as number (%).
      BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HbA1c, glycated hemoglobin; GFR, estimated glomerular filtration rate calculated by the MDRD equation; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; MVV, maximal voluntary ventilation; PImax, maximal inspiratory pressure; VO2 peak, peak oxygen uptake; RER, respiratory exchange ratio; VE/VCO2 slope, minute ventilation/carbon dioxide production slope; OUES, oxygen uptake efficiency slope; IPAQ, International Physical Activity Questionnaire.
      In Fig. 2, panel A shows glucose levels 4 h before and 6 h after IME (placebo load or experimental load). There were reductions in glucose levels only in relation to time (p < 0.001), but there was no difference between the groups (p = 0.741). After 5 min, glucose decreased 32.0% and 33.2%, after placebo or experimental load, respectively (vs. 1 h before exercise). Sixty minutes after IME, placebo and experimental loads determined reductions of 31.4% and 29.6%, respectively (vs. 1 h before exercise). Two hours after IME, placebo load and experimental load determined reductions of 24.0% and 21.4%, respectively (vs. 1 h before exercise). The same results were shown when comparing glucose levels between the different loads during the protocol (Fig. 2, Panel B). Glucose levels decreased during controlled ventilation with placebo load and experimental load (2.9% and 2.7%), during the interval (14.5% and 12.1%), exercise (17.6% and 17.2%) and recovery (18.2% and 18.5%) when compared to baseline, with no difference between loads applied (time p < 0.001 and group p = 0.912).
      Fig. 2
      Fig. 2Panel A Glucose levels 4 h before and 6 h after acute inspiratory muscle exercise protocol with placebo load (2% of PImax) or experimental load (60% of PImax). Pimáx, maximal inspiratory pressure. Data are expressed as mean ± SEM. Statistics: Generalized estimating equations (GEE). Time (p < 0.001), group (p = 0.741) and interaction (p = 0.343). *p < 0.05 vs 1 h before exercise. Panel B: Detail from intervention (baseline, controlled ventilation, interval, inspiratory muscle exercise, recovery). Data are expressed as mean ± SD. Statistics: generalized estimating equations (GEE). Time (p < 0.001), load (p = 0.912) and interaction (p = 0.177). *p < 0.05 vs 5 min baseline.
      The comparison of patients using only metformin vs. those using metformin and sulfonylureas showed no differences between glucose reductions (lowering during the protocols (P < 0.001), with no differences between these subgroups (P = 0.717) (data not shown).
      Glucose variability was evaluated 0–6 h, 6–12 h and 12–18 h after IME (Fig. 3). Deltas of CV, SD and VAR were calculated from −0 to −6 h before IME. These indexes were lower 6–12 h after the IME, regardless of the load used: ΔCV: −4.21%, ΔSD: −8.39 mg/dL and ΔVAR: −657.95 (mg/dL)2 (vs. after 0-6 h IME). The same was observed 12–18 h after the IME: ΔCV: −5.65%, ΔSD: −14.37 mg/dL and ΔVAR: −872.35 (mg/dL)2 (vs. after 0–6 h IME). When comparing groups, regardless of time, delta of CV was 7.6 times higher in experimental load than placebo load group, delta of SD was 8.6 times higher than in placebo load group and delta of VAR was 4.5 times higher in experimental load group. After IME, MAGE was evaluated in placebo load or experimental load and no difference was observed in MAGE in both loads [placebo load: 103.1 (61.9–121.4) mg/dL and experimental load: 97.3 (61.3–160.1) mg/dL].
      Fig. 3
      Fig. 3Delta of glucose variability (all values Δ from −0 to −6 h before exercise) assessed 0–6 h, 6–12 h and 12–18 h after acute inspiratory muscle exercise protocol with placebo load (2% of PImax) or experimental load (60% of PImax). (A): glucose coefficient of variation, (B): glucose standard deviation, (C): glucose variance, PImax: maximal inspiratory pressure. Data expressed as medians and interquartile ranges. Statistics: generalized estimating equations (GEE). *p < 0.05 vs 0 h–6 h.
      Cardiovascular autonomic control evaluation data is shown in Table 2. Four patients were excluded because continuous blood pressure signal could not be analyzed due to artifacts. Heart rate variability was similar during and after IME with both loads, as well as power spectral components of HRV (p = 0.590), except for HF(nu), which was lower during the IME with experimental load as compared to placebo load (p = 0.010). Blood pressure variability was higher during the IME with the experimental load as compared to that with the placebo load (p = 0.019). Although the LF component of BPV was similar between loads applied (p = 0.116), the HF component of BPV spectrum was higher during the exercise with the experimental load (p = 0.001).
      Table 2Spectral analysis of heart rate variability components and blood pressure variability components during acute inspiratory muscle exercise protocol with placebo load (2% of PImax) or experimental load (60% of PImax).
      Acute inspiratory muscle exercise protocol with placebo load (2% of PImax) (n = 10)Acute inspiratory muscle exercise protocol with experimental load (60% of PImax) (n = 10)P interaction
      BaselineControled ventilationInspiratory exerciseRecoveryBaselineControled ventilationInspiratory exerciseRecovery
      HRV (ms2)1605.0 ± 1618.51592.7 ± 1295.61059.7 ± 531.21495.0 ± 809.61570.0 ± 1126.21292.5 ± 853.11158.2 ± 695.71862.6 ± 1888.60.590
      LF (nu)29.9 ± 25.225.9 ± 17.016.5 ± 13.725.8 ± 21.535.7 ± 27.219.3 ± 13.624.9 ± 18.324.4 ± 18.80.073
      LF (ms2)722.6 ± 1274.0400.2 ± 522.5183.2 ± 227.6485.1 ± 536.2548.4 ± 668.1244.2 ± 364.1299.8 ± 380.4469.1 ± 635.80.351
      HF (nu)31.5 ± 23.134.1 ± 17.244.8 ± 22.433.1 ± 12.826.4 ± 15.331.4 ± 12.933.0 ± 12.6
      P < 0.05 inspiratory exercise 60%PImax vs inspiratory exercise 2%PImax.
      36.3 ± 13.50.010
      HF (ms2)390.6 ± 392.9473.6 ± 475.5399.4 ± 320.0464.5 ± 306.5296.0 ± 213.9401.5 ± 398.3301.4 ± 135.5487.4 ± 392.70.952
      LF/HF1.7 ± 2.01.0 ± 0.90.5 ± 0.60.8 ± 0.72.1 ± 2.10.8 ± 0.81.0 ± 0.90.7 ± 0.60.481
      BPV (mmHg2)9.9 ± 6.211.8 ± 5.814.0 ± 10.28.4 ± 6.115.3 ± 9.918.0 ± 17.880.6 ± 132.9
      P < 0.05 inspiratory exercise 60%PImax vs inspiratory exercise 2%PImax.
      12.0 ± 9.00.019
      LF BPV (mmHg2)1.7 ± 1.52.2 ± 3.02.1 ± 2.70.8 ± 0.83.9 ± 3.82.2 ± 2.56.1 ± 5.72.0 ± 3.10.116
      HF BPV(mmHg2)2.6 ± 1.95.1 ± 2.96.2 ± 5.23.5 ± 2.95.2 ± 7.99.2 ± 11.957.6 ± 106.9
      P < 0.05 inspiratory exercise 60%PImax vs inspiratory exercise 2%PImax.
      4.7 ± 5.00.001
      HRV; Heart rate variability, LFms2; power spectrum of low frequency band in absolute value, HFms2; power spectrum of high frequency band in absolute value, LFnu; power spectrum of low frequency band in normalized units, HFnu; power spectrum of high frequency band in normalized units, LF/HF; sympathetic–vagal balance; BPV; Blood pressure variability, LF BPVmmHg2; power spectrum of low frequency band in absolute value, HF BPVmmHg2; power spectrum of high frequency band in absolute value. Data are expressed as mean ± SD. Results of generalized estimating equations (GEE) for repeated measures.
      low asterisk P < 0.05 inspiratory exercise 60%PImax vs inspiratory exercise 2%PImax.
      Hemodynamic and respiratory variables during and after the IME with both loads are shown in Fig. 4. The experimental load determined higher HR as compared to the results obtained with the placebo load, at the first minute (22.2% higher, p < 0.001), higher MAP and HR at the second minute [10.8% (p = 0.016), and 26.2% (p < 0.001) higher, respectively) and also in the last minute of the exercise [20.7% (p < 0.001) and 22.5% (p < 0.001) higher, respectively]. No differences were observed in CBF (P = 0.921), CRV (p = 0.116), SpO2 (P = 0.431) and fb (P = 0.124) between loads. However, PetCO2 was higher after the experimental load in comparison to the placebo load.
      Fig. 4
      Fig. 4Hemodynamic effects and respiratory variables in baseline and during acute inspiratory muscle exercise protocol with placebo load (2% of PImax) or experimental load (60% of PImax). MAP, mean arterial pressure; HR, heart rate; CBF, calf blood flow; CVR, calf blood resistance. Data are expressed as mean ± SD. Statistics: Generalized estimating equations (GEE), followed by Bonferroni's post hoc test.*p < 0.05 vs. 2%PImax.

      4. Discussion

      To our knowledge, this is the first randomized clinical trial assessing the effect of acute IME on glucose levels and glucose variability in patients with type 2 DM. Inspiratory muscle exercise with an experimental load of 60% of PImax did not reduce glucose levels and glucose variability, when compared with a placebo load. The experimental load, however, caused elevation of blood pressure and heart rate levels, and induced expected cardiovascular autonomic changes during the exercise.
      Reduction of glucose levels after IME in approximately 30% (1 h) and 22% (2 h) was observed, with no differences between placebo and experimental loads. As expected, higher glucose levels (1 h before IME) were observed immediately after breakfast, followed by a gradual reduction. In contrast,
      • Erickson M.L.
      • Little J.P.
      • Gay J.L.
      • McCully K.K.
      • Jenkins N.T.
      Effects of postmeal exercise on postprandial glucose excursions in people with type 2 diabetes treated with add-on hypoglycemic agents.
      reported post-breakfast exercise lowered glucose during the exercise bout, although this effect was not sustained at later meals. Clearly, the difference between those results and ours was the higher intensity and duration of exercise and also the type (treadmill walking for 30 min at 50% of estimated VO2 peak).
      The decrease in glucose probably occurred in response to raising insulin levels induced by the meal, and also because of the effect of antidiabetic agents that patients have taken. However, previous data in patients with type 2 DM showed lower reductions of glucose after ingestion of similar meals (
      • Bell K.J.
      • Bao J.
      • Petocz P.
      • Colagiuri S.
      • Brand-Miller J.C.
      Validation of the food insulin index in lean, young, healthy individuals, and type 2 diabetes in the context of mixed meals: an acute randomized crossover trial.
      ). We hypothesize that controlled ventilation, applied before both experimental loads (2% and 60% of PImax), would be the determinant of glucose reduction. Controlled ventilation causes vagal stimulation, insulin secretion and glucose reduction, in accordance with reports that showed that parasympathetic activation reduces hepatic glucose release and increases insulin secretion in hyperglycemic conditions (
      • Tanida M.
      • Yamamoto N.
      • Morgan D.A.
      • Kurata Y.
      • Shibamoto T.
      • Rahmouni K.
      Leptin receptor signaling in the hypothalamus regulates hepatic autonomic nerve activity via phosphatidylinositol 3-kinase and AMP-activated protein kinase.
      ). Other authors demonstrated that afferent vagal nerve stimulation causes a sustained increase in glucose, partly mediated by suppression of pancreatic insulin secretion, and in contrast, efferent vagal nerve stimulation determines pancreatic glucagon secretion that appears to be antagonized by insulin secretion when selective efferent vagal nerve stimulation occurs (
      • Meyers E.E.
      • Kronemberger A.
      • Lira V.
      • Rahmouni K.
      • Stauss H.M.
      Contrasting effects of afferent and efferent vagal nerve stimulation on insulin secretion and blood glucose regulation.
      ).
      The present results are not in accordance with preliminary data in six patients that we have published (
      • Corrêa A.P.
      • Antunes C.F.
      • Figueira F.R.
      • de Castro M.A.
      • Ribeiro J.P.
      • Schaan B.D.
      Effect of acute inspiratory muscle exercise on blood flow of resting and exercising limbs and glucose levels in type 2 diabetes.
      ). Possible explanations for this discrepancy could be the small sample size evaluated previously, and different protocols, particularly the inclusion, in the present protocol, of controlled ventilation before IME (before both loads tested, 2% and 60% of PImax). Moreover, in the previous report most patients that we included had cardiovascular autonomic neuropathy (
      • Corrêa A.P.
      • Antunes C.F.
      • Figueira F.R.
      • de Castro M.A.
      • Ribeiro J.P.
      • Schaan B.D.
      Effect of acute inspiratory muscle exercise on blood flow of resting and exercising limbs and glucose levels in type 2 diabetes.
      ), which is frequently associated with inspiratory muscle weakness (
      • Kaminski D.M.
      • Schaan B.D.
      • da Silva A.M.
      • Soares P.P.
      • Plentz R.D.
      • Dall’Ago P.
      Inspiratory muscle weakness is associated with autonomic cardiovascular dysfunction in patients with type 2 diabetes mellitus.
      ). Challenging weaker muscles could result in more important glucose reductions than inspiratory loading applied to healthy/strong muscles.
      In addition, reduction of glucose variability was observed after IME, with no differences detected between placebo load and experimental load. Glucose variability could change because of food ingestion behavior during the day, which is probably not what occurs during the night, or in response to insulin secretion and to antidiabetics effect (
      • Guerci B.
      • Monnier L.
      • Serusclat P.
      • Petit C.
      • Valensi P.
      • Huet D.
      • Raccah D.
      • Colette C.
      • Quéré S.
      • Dejager S.
      Continuous glucose profiles with vildagliptin versus sitagliptin in add-on to metformin: results from the randomized optima study.
      ;
      • Vianna A.G.D.
      • Lacerda C.S.
      • Pechmann L.M.
      • Polesel M.G.
      • Marino E.C.
      • Faria-Neto J.R.
      A randomized controlled trial to compare the effects of sulphonylurea gliclazide MR (modified release) and the DPP-4 inhibitor vildagliptin on glycemic variability and control measured by continuous glucose monitoring (CGM) in Brazilian women with type 2 diabetes.
      ). On the other hand, maybe glucose variability could have a circadian rhythm. Considering this, we compared day and night glucose variability, but no differences were observed (data not shown). Other studies have reported reduction in glucose variability after aerobic exercise (
      • Van Dijk J.W.
      • Manders R.J.
      • Canfora E.E.
      • Mechelen W.V.
      • Hartgens F.
      • Stehouwer C.D.
      • Van Loon L.J.
      Exercise and 24-h glycemic control: equal effects for all type 2 diabetes patients?.
      ;
      • Karstoft K.
      • Clark M.A.
      • Jakobsen I.
      • Müller I.A.
      • Pedersen B.K.
      • Solomon T.P.
      • Ried-Larsen M.
      The effects of 2 weeks of interval vs continuous walking training on glycaemic control and whole-body oxidative stress in individuals with type 2 diabetes: a controlled, randomised, crossover trial.
      ) and combined exercise (
      • Figueira F.R.
      • Umpierre D.
      • Casali K.R.
      • Tetelbom P.S.
      • Henn N.T.
      • Ribeiro J.P.
      • Schaan B.D.
      Aerobic and combined exercise sessions reduce glucose variability in type 2 diabetes: crossover randomized trial.
      ) in patients with DM. Additionally, in the preliminary data of our group, IME with a load of 60% of PImax and aerobic exercise showed comparable reductions in glucose variability not observed in control groups (
      • Corrêa A.P.
      • Figueira F.R.
      • Umpierre D.
      • Casali K.R.
      • Schaan B.D.
      Inspiratory muscle loading: a new approach for lowering glucose levels and glucose variability in patients with type 2 diabetes.
      ).
      The higher HR and MAP were observed during the experimental session (IME with a load of 60% of PImax), but not with the placebo load. It is well known that inspiratory metaboreflex activation during strenuous exercise increases MAP primarily by increases in cardiac output. The increases in cardiac output occur via the combination of tachycardia and increased ventricular contractility and central blood volume mobilization. This combination maintains or slightly increases stroke volume (
      • Crisafulli A.
      • Salis E.
      • Tocco F.
      • Melis F.
      • Milia R.
      • Pittau G.
      • Caria M.A.
      • Solinas R.
      • Meloni L.
      • Pagliaro P.
      • Concu A.
      Impaired central hemodynamic response and exaggerated vasoconstriction during muscle metaboreflex activation in heart failure patients.
      ;
      • Sala-Mercado J.A.
      • Hammond R.L.
      • Kim J.K.
      • Rossi N.F.
      • Stephenson L.W.
      • O’Leary D.S.
      Muscle metaboreflex control of ventricular contractility during dynamic exercise.
      ;
      • Spranger M.D.
      • Sala-Mercado J.A.
      • Coutsos M.
      • Kaur J.
      • Stayer D.
      • Augustyniak R.A.
      • O'Leary D.S.
      Role of cardiac output versus peripheral vasoconstriction in mediating muscle metaboreflex pressor responses: dynamic exercise versus postexercise muscle ischemia.
      ). During vigorous exercise, cardiac output is reduced, while vascular conductance and blood flow to the exercising legs are increased, leading to a whole-body exercise performance rise (
      • Harms C.A.
      • Babcock M.A.
      • McClaran S.R.
      • Pegelow D.F.
      • Nickele G.A.
      • Nelson W.B.
      • Dempsey J.A.
      1997a. Respiratory muscle work compromises leg blood flow during maximal exercise.
      ,
      • Harms C.A.
      • Wetter T.J.
      • St Croix C.M.
      • Pegelow D.F.
      • Dempsey J.A.
      2000b. Effects of respiratory muscle work on exercise performance.
      ). It seems that accumulation of metabolites in the inspiratory muscles when respiratory muscles are fatigued increases sympathetic vasoconstrictor activity, thus being the most likely mechanism that affects cardiovascular indices and baroreflex function. As expected, very low resistances near 0 cmH2O (
      • Convertino V.A.
      • Ratliff D.A.
      • Ryan K.L.
      • Doerr D.F.
      • Ludwig D.A.
      • Muniz G.W.
      • Britton D.L.
      • Clah S.D.
      • Fernald K.B.
      • Ruiz A.F.
      • Lurie K.G.
      • Idris A.H.
      Hemodynamics associated with breathing through an inspiratory impedance threshold device in human volunteers.
      ) or a little flow resistance of about 1.1–0.6 cmH2O (
      • Lurie K.G.
      • Zielinski T.
      • Voelckel W.
      • McKnite S.
      • Plaisance P.
      Augmentation of ventricular preload during treatment of cardiovascular collapse and cardiac arrest.
      ) are not associated with changes in cardiac output and in arterial blood pressure as physiological responses to the effort performed. Our results were similar to those of
      • McConnell A.K.
      • Griffiths L.A.
      Acute cardiorespiratory responses to inspiratory pressure threshold loading.
      who studied acute cardiorespiratory responses to different inspiratory loadings and reported that the load of 60% of PImax determined a sustained increase in HR and MAP. Although an exacerbation of the inspiratory muscle metaboreflex was previously observed in diabetic patients (
      • Corrêa A.P.
      • Antunes C.F.
      • Figueira F.R.
      • de Castro M.A.
      • Ribeiro J.P.
      • Schaan B.D.
      Effect of acute inspiratory muscle exercise on blood flow of resting and exercising limbs and glucose levels in type 2 diabetes.
      ), the present data did not disclose changes in CBF and CVR.
      Decreased vagal modulation, increased BPV and increased HF component of BPV were observed during IME performed with the experimental load (60% of PImax), but not with the placebo load. These findings were also expected (
      • Archiza B.
      • Simões R.P.
      • Mendes R.G.
      • Fregonezi G.A.
      • Catai A.M.
      • Borghi-Silva A.
      Acute effects of different inspiratory resistive loading on heart rate variability in healthy elderly patients.
      ), implying that subjects in fact were submitted to a significant exercise load. However, these changes were not sustained after the exercise ceased. Importantly, these indices were calculated during stable blood pressure levels (
      • Parati G.
      • Saul J.P.
      • Di Rienzo M.
      • Mancia G.
      Spectral analysis of blood pressure and heart rate variability in evaluating cardiovascular regulation. A critical appraisal.
      ). In addition, inspiratory muscle training for 11 weeks with the same load used in our study (60% of PImax) determined improved cardiac vagal control in amateur cyclists (
      • Martins de Abreu R.
      • Porta A.
      • Rehder-Santos P.
      • Cairo B.
      • Donisete da Silva C.
      • De Favari Signini É.
      • Sakaguchi C.A.
      • Catai A.M.
      Effects of inspiratory muscle-training intensity on cardiovascular control in amateur cyclists.
      ).
      The present study has some limitations that should be acknowledged. Firstly, we evaluated CBF by venous occlusion plethysmography instead of doppler ultrasonography, which is the gold standard method. Secondly, the small sample size evaluated could not disclose some results, as it was calculated for the main outcome (glucose, CGMS). Third, there was no standardization of nutrient intake, although patients were instructed to maintain their usual diet.

      5. Conclusion

      In conclusion, although sufficiently strong to induce cardiovascular changes, an inspiratory muscle exercise session with 60% of PImax, in subjects with type 2 DM has failed to induce any significant improvement in glucose, glucose variability and autonomic control, compared to the 2% Plmax exercise session.

      Declaration of competing interest

      None.

      Acknowledgments

      None.

      Funding

      This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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