Effect of combined thermal and electrical muscle stimulation on cardiorespiratory fitness and adipose tissue in obese individuals

Abstract

Background

To better understand how prolonged electrical muscle stimulation can improve cardiorespiratory risk markers in obese subjects, we investigated the effect of prolonged combined thermal and electrical muscle stimulation (cTEMS) on peak oxygen consumption (VO_2peak) and body composition with subsequent lipolytic and mitochondrial activity in adipocytes.

Methods and results

Eleven obese (BMI ≥ 30 kg/m²) individuals received cTEMS in three 60-minute sessions per week for 8 weeks. Activity levels and dietary habits were kept unchanged. Before and after the stimulation period, functional capacity was assessed by VO_2peak, and body composition was analysed. Lipolytic activity was determined in abdominal adipose tissue by 24 hours of microdialysis on a sedentary day, and adipose tissue biopsies were taken for the gene expression analysis. Eight weeks of cTEMS significantly increased VO_2peak from 28.9 ± 5.7 to 31.7 ± 6.2 ml/kg/min (p < 0.05), corresponding to an average increase of 1.2% per week. Oxygen uptake and work capacity also increased at the anaerobic threshold. Mean microdialytic glycerol concentration over 24 hours, an index of sedentary lipolytic activity, increased from 238 ± 60 to 306 ± 55 µM (p < 0,0001), but no significant changes in body composition were observed. In addition, PGC-1α and carnitine-palmitoyltransferase-2 mRNAs were significantly upregulated in subcutaneous abdominal adipose tissue.

Conclusions

In obese individuals with unchanged lifestyles, 8 weeks of cTEMS significantly improved functional capacity towards a higher fatigue resistance. This increase also gave rise to elevated lipolytic activity and increased mitochondrial activity in abdominal adipose tissue.

Introduction

Electrical muscle stimulation (EMS) of unloaded muscles improves cardiorespiratory fitness in healthy individuals^1,2 and patients with heart failure,^3,4 but it is unclear whether EMS could serve as an adjunct approach to improve fitness and body composition in obese individuals.

Obesity is associated with an increased risk of numerous comorbidities.^5,6 While targeting obese subjects with sedentary habits and low fitness appears to be an effective strategy in improving the health of this population,⁷ the possible role of prolonged EMS within this strategy remains unexplored. Muscular strength and cardiorespiratory capacity are both inversely associated with metabolic syndrome prevalence,⁸ and the maintenance or improvement of physical fitness may counteract obesity-associated disease risks and lead to health benefits.⁹ In addition to improving physical fitness, exercise training has beneficial effects on lipoprotein profiles,¹⁰ and, even at low intensity, exercise training also tends to increase fat oxidation and lead to positive changes in the expression of genes essential for fat metabolism.¹¹

Obesity may restrict both exercise training practice and performance, which makes alternative methods to promote fitness desirable. However, these same restrictions may also limit the potential benefit of EMS. For example, the electrical current threshold is reported to be higher in obese than in nonobese subjects, and their stimulation tolerance appears to diminish within one stimulation session.¹² Alterations have been proposed to reduce discomfort while maintaining the efficacy of EMS in obese populations,¹³ but the potential benefits have not yet been explored.

This study aimed to investigate the effects of combined thermal and electrical muscle stimulation (cTEMS) on cardiorespiratory fitness in obese subjects as measured by VO_2peak. In addition, we investigated effects of cTEMS on body composition and adipose tissue.

Methods

Subjects

This study recruited 12 obese (body mass index, BMI, ≥ 30 kg/m²) and sedentary subjects ( < 20 minutes of exercise <3 days per week) through an invitation sent to employees at the Haukeland University Hospital in Bergen, Norway. Other inclusion criteria were age between 30 and 70 years and the ability to undergo exercise testing. Individuals with a pacemaker, regular medication, cardiovascular disease, pulmonary disease, extensive dermatological disease, or other primary diseases, pregnant women, and individuals who were abusing alcohol or drugs were excluded from the study. For completion of the study, participation in more than 70% of the cTEMS sessions was mandatory.

Among the 12 volunteers (six males and six females), one female withdrew her consent during the study. This was not related to the study or methods used. Before inclusion, all subjects underwent a clinical examination and provided written informed consent. The study was approved the Regional Committee for Medical and Health Research Ethics (Western Norway ref. 2009/1273) and conformed to the Declaration of Helsinki.

Experimental set up

The study was performed in a designated laboratory in the Haukeland University Hospital, Bergen, Norway. Prior to the baseline visit, all subjects had one session of cTEMS adaptation and tolerance testing. Prior to the baseline and follow-up examinations, the participants fasted overnight but were allowed to drink water.

Blood samples were collected in the fasting state, and the body composition was measured. Lipolytic activity in subcutaneous abdominal adipose tissue (SCAAT) was analysed for the following 24 hours using microdialysis. We also performed exercise testing and collected a SCAAT biopsy to analyse mitochondrial and lipolytic-related mRNAs. To avoid interactions, we separated the examinations (microdialysis, exercise testing, and biopsy) by at least 48 hours.

Following the baseline examinations, all subjects underwent an 8-week intervention period with three cTEMS sessions per week, followed by follow-up examinations. During the study, all participants were instructed to maintain their normal dietary habits and physical activity levels. Possible changes in lifestyle were controlled for with a nutritional questionnaire and with accelerometer.

Combined thermal and electrical muscle stimulation

In this study, we used cTEMS, a combination of applied superficial heat and electrical muscle stimulation. cTEMS was applied using a stationary stimulator (TEI System; RÖS’S Estética, Barcelona Spain).¹⁴ The system consists of an electrical power source connected to ten silicone pads with two different sizes (24 × 26 cm, with maximum heating capacity 25 W, and 24 × 17 cm, with maximum heating capacity 15 W). Within the silicone pads, both electrode bands for the delivery of electrical current and heating elements are incorporated.

At each stimulation session, ten electrodes were attached to the designated muscle groups (two electrodes each to quadriceps, hamstrings, glutei, and obliques, and one electrode each to rectus abdominis and lower lumbar muscles) with elastic bands. Optimal skin adhesion was secured by standard ultrasound gel.

Stimulation protocol

cTEMS was delivered three times a week for 60 minutes at each individual’s stimulation threshold. To secure compliance throughout the intervention period, we varied the electrical pulse types during the sessions using 2–4 different pulse types at each stimulation visit. Heat stimulation was set at 40% of the maximum heating capacity. The detailed stimulation protocols are given in Table 1.

Biopsy from abdominal adipose tissue

Abdominal adipose tissue was collected using a 14-gauge needle connected to a 10-ml syringe with a locking member to create vacuum (Hepafix; B Braun Melsungen, Germany). While maintaining vacuum in the syringe, 4-6 ml of fat and fluid were aspirated, cleansed in saline, frozen in liquid nitrogen, and kept at −80°C until further analysis.

Gene expression

Tissue samples were homogenized and total cellular RNA was purified and quantified spectrophotometrically. RNA quality was evaluated by capillary electrophoresis. One microgram of total RNA was reverse transcribed in 100 µl reactions using TaqMan reverse transcription reagents with RNase inhibitor (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed with 384-well Multiply-PCR Plates (Sarstedt, Newton, NC, USA) on the following genes using probes and primers from Applied Biosystems: carnitine palmitoyltransferase 1a (CPT-1a, Hs00157079), CPT-2 (Hs00264677), hormone-sensitive lipase (LIPE, Sh00943410), peroxisome proliferator-activated receptor gamma (PPARγ, Hs00174128), PPARγ coactivator 1 (PPARGC-1a (PGC-1a), Hs00173304), resistin (RETN, Hs00982492), uncoupling protein 1 (UCP-1, Hs00222453), UCP-2 (Hs00163349), and UCP-3 (Hs00243297). Three reference genes were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs99999905) and ribosomal protein large P0 (RPLP0, 4333761T) from Applied Biosystems and 18S MP (Kit-FAM-TAMRA, RT-CKFT-18s) from Eurogentec (Seraing, Belgium). We used the Normfinder algorithm¹⁵ to assess the stability of the measured reference genes. RPLP0 showed the best results, with a stability value of 0.039, and was subsequently used to normalize the target gene values. We calculated the relative expression values as the ratio of target gene expression to RPLP0.

Microdialysis

Lipolysis in adipose tissue was assessed with microdialysis, a technique previously described by Arner et al.¹⁶ After light epidermal anaesthesia (Emla patch 5%), one microdialysis probe (CMA 63, 20-kDa molecular-weight cut-off; CMA Microdialysis, Sweden) was inserted percutaneously into the SCAAT, 8–10 cm lateral to the umbilicus. The probe was connected to a microdialysis pump (CMA 107, CMA Microdialysis) and continuously perfused with sterile Ringer′s solution (154 mM Na⁺, 6 mM K⁺, 2,5 mM Ca²⁺, 160 mM Cl⁻). The perfusion was set at a standard flow rate of 2 µl/min, and fractions were collected every 2 hours following a 30-minute equilibration period. The microdialysate was kept at 4°C until it was analysed in a microdialysate analyser (Iscus Flex; CMA Microdialysis). The lipolytic activity in SCAAT was expressed as the dialysate glycerol.

The 24 hours of registration the subjects spent in their habitual surroundings. At baseline, the subjects registered their food intake and were instructed to follow exactly the same dietary intake on the follow-up registration.

Exercise testing

The cardiorespiratory fitness before and after the 8-week intervention period with cTEMS was evaluated by ergospirometry during a standard treadmill test while using a modified Balke protocol. The subjects were acclimated to the treadmill before the baseline test was performed. At a speed of 5.5 km/h, we increased the elevation by 2° every 2 minutes, during which ventilatory gas exchange was measured by an ergospirometer with a breath-to-breath analyser (Cardiovit CS-200 Ergo-Spiro/13 Ganzhorn Power Cube; Schiller).

Peak oxygen consumption (VO_2peak) was considered to have been reached when all of the following criteria were met: the maximal heart rate measured at exhaustion was higher than 90% of the age-predicted maximal heart rate; the respiratory exchange ratio (RER) measured at exhaustion was greater than 1.1; and the subject was not able to sustain sufficient speed on the treadmill.

Anaerobic threshold was determined using the V-slope method.¹⁷

Body composition

Dual-energy X-ray absorptiometry (Lunar Prodigy DF; GE Medical Systems) was used to analyse body composition, while body weight and visceral fat area were measured using bioelectrical impedance analysis (InBody 720; BioSpace, Seoul, Korea).¹⁸

Lifestyle monitoring

Subjects were encouraged to maintain their sedentary behaviour, and activity levels were measured before and at the end of the 8-week treatment period. For 5 consecutive days, the participants wore a triaxial accelerometer (GT3X; The Actigraph, Fort Walton Beach, FL, USA) on their hip while awake. The data were processed by ActiLife software (The Actigraph), and the total physical activity time spent in sedentary, light, moderate, vigorous, and moderate to vigorous physical activity modes was calculated and converted into the percentage of total time spent in each activity mode. We used a 15-item SmartDiet self-administered questionnaire to monitor possible dietary habit changes.¹⁹

Serum lipid analysis

Serum lipids were measured enzymaticly on a Hitachi 917 system (Roche Diagnostics, Mannheim, Germany) using the triacylglycerol (GPO-PAP), cholesterol (CHOD-PAP), HDL cholesterol, and LDL cholesterol kits from Roche (Roche Diagnostics). The phospholipids FS, NEFA FS (nonesterified fatty acids), and free cholesterol FS kits were from Diagnostic Systems (Holzheim, Germany).

Statistical analysis

Baseline and follow-up measurements were presented as mean ± standard deviation and compared using the nonparametric Wilcoxon signed-rank test.

Mean glycerol dialysate levels were estimated over time. Relative gene expression values at baseline and follow-up were estimated with a random-intercept linear mixed-effects model in a two-way repeated measure configuration.

Statistical analyses were conducted with R version 2.15.2 (R Foundation for Statistical Computing, Vienna, Austria) and linear mixed-effects models were analysed using package nlme-3.1–107.

Results

Eleven subjects (five female; six male) with a mean age of 44.6 ± 5.9 years completed the study and were included in the analysis. We observed no notable complications related to the cTEMS intervention.

Exercise testing

All subjects completed the exercise testing (Table 2). After 8 weeks of cTEMS intervention, we observed a 9.6% increase in VO_2peak (p = 0.014) and a 10.8% increase in peak work capacity (p = 0.014). The higher VO_2peak and workload were accompanied by a significantly lower RER. At the anaerobic threshold, both total VO₂ and work capacity increased significantly, and RER decreased (p = 0.022). The decrease in RER was also found at rest when analysing true resting values (from 0.87 ± 0.06 to 0,79 ± 0,06; p = 0.028).

Body weight and body composition

As shown in Table 2, we observed no significant changes in body weight or body composition between baseline and follow up.

Cholesterol and lipids

During 8 weeks of cTEMS, the ratio between the total and HDL cholesterol was unchanged despite significant reductions of total cholesterol from 5.60 ± 1.37 to 5.21 ± 1.14 mmol (p = 0.042). Both LDL and HDL cholesterol were nonsignificantly decreased. The other lipid biomarkers remained unchanged (Table 3).

Microdialysis

Lipolytic activity in the abdominal adipose tissue was estimated by the mean glycerol concentration over 24 hours on a sedentary day. We observed a significant increase from 239 ± 60 µM at baseline to 304 ± 55 µM at follow up (p = 0.001) after 8 weeks of cTEMS, as shown in Figure 1. Further, we also observed an increase in mean 24-hour microdialytic lactate from 2.85 ± 0.27 mM at baseline to 3.73 ± 0.58 mM at the follow-up examination (p = 0.002).

Adipose tissue analysis

The gene expression in SCAAT is presented as the mean ± standard error relative expression. Resistin and UCP-1 mRNAs were not expressed in the SCAAT specimen and thus not included in the analysis.

Eight weeks of cTEMS significantly increased PGC-1α expression in the adipose tissue from 8.10 ± 1.08 to 10.00 ± 1.56 (p < 0.05). Further, we observed an increase in CPT-2 from 7.89 ± 0.24 at baseline to 8.82 ± 0.35 at follow up (p < 0.001). CPT-1a (4.43 ± 0.94 to 4.73 ± 0.67; p = 0.445), LIPE (3.04 ± 0.35 to 3.15 ± 0.50; p = 0.641), PPARγ (75.30 ± 5.53 to 77.21 ± 7.31; p = 0.518), UCP-2 (5.53 ± 1.09 to 5.58 ± 0.79; p = 0.908), and UCP-3 (41.25 ± 3.24 to 48.21 ± 3.85; p = 0.518) all showed slight but nonsignificant increases in mRNA expression.

Lifestyle monitoring

Dietary habits remained unchanged, as indicated by a SmartDiet score of 29.27 ± 5.16 at baseline and 29.36 ± 4.15 at follow up (p = 1.000). Physical habits, as measured with an accelerometer, also remained unchanged. The percentage of total time spent in the sedentary state over 5 consecutive days was 78.8 ± 7.7% at baseline and 80.0 ± 6.7% at follow up (p = 0.232). Additionally, 18.9 ± 7.8% of their time was spent in light physical activity at baseline vs. 18.3 ± 6.8% at follow up (p = 0.375), and we observed a slight decrease in time spent in moderate activity (2.3 ± 0.9 vs. 1.7 ± 0.7%; p = 0.027). The participants barely performed heavy physical activity (0.01 ± 0.03 vs. 0.02 ± 0.04%; p = 1.000), and no time was spent with very heavy physical activity.

Discussion

This study provides new insights into the effects of prolonged electrical muscle stimulation. In obese sedentary individuals with unchanged physical activity levels and nutritional habits, 8 weeks of cTEMS resulted in significant increases in peak aerobic capacity, as expressed by VO_2peak. The improved work capacity, towards a higher fatigue resistance, was both reflected in an increased maximal workload during the exercise testing and secondary improvements in sedentary lipolytic activity and adipose tissue mitochondrial activity. To our knowledge, this is the first time such a training effect has been observed in obese individuals using EMS as an exercise training modality.

The observed changes in VO_2peak from baseline to follow up represented a 9.6% increase, with an average increase of 1.2% per week of cTEMS intervention. In comparison, when investigating the training effects in individuals with metabolic syndrome, Tjønna et al. observed an increase of 1.0% per week with continuous moderate exercise at 70% of the maximal heart rate and 2.2% per week with aerobic interval training at 90% of the maximal heart rate.²⁰ Exercise capacity is a more powerful predictor of mortality than many other established cardiovascular risk factors,²¹ thus our finding may prove to have future clinical relevance for obese individuals not able or willing to participate in regular exercise training.

In healthy individuals, a rise in the maximal oxygen uptake is caused by an increase in the cardiac output, increased arteriovenous oxygen extraction, or both.²² We have previously shown that cTEMS resulted in a 3.2-fold increase in the resting VO₂, corresponding to 2.8 metabolic equivalents,¹⁴ an increase less likely to give a training effect on the heart. In the present study there was no change in resting blood pressure or heart rate, indicating that stroke volume and cardiac output was unaffected. The increased oxygen consumption caused by cTEMS is therefore likely to be associated with increased arteriovenous oxygen extraction by the stimulated muscle groups. This would also be in agreement with the finding of Nuhr et al.,²³ who previously showed that EMS in healthy individuals induced changes both in skeletal muscle fibre composition and energy metabolism.

Maximal oxygen uptake has been positively correlated with metabolic flexibility, i.e. the ability of the skeletal muscle to switch between fat and carbohydrate oxidation.²⁴ Furthermore, exercise increases the reliance on fat oxidation and the capacity for free fatty acid mobilization and oxidation during exercise at a given intensity level.^25,26 An upregulation of the anaerobic threshold due to cTEMS and the corresponding increase in metabolic flexibility is the most reasonable explanation for the observed increase in the lipolytic activity measured on sedentary days. Compared with baseline values, we found a significant increase in the 24-hour mean microdialysate concentration of glycerol at follow up, which is an indication of a rise in the mobilization of triacylglycerol and the following conversion into free fatty acids and glycerol.²⁷ Subsequent to the increase in interstitial glycerol, we also found a significant increase in adipose tissue lactate. This pathway remains a major source of energy for the working muscle²⁸ and thus the increased lipolysis provides fuel for the increased muscular utilization of fatty acids.

It is unlikely that changes in microdialytic glycerol levels resulted from blood flow alterations in adipose tissue. Central haemodynamics remained unchanged, and microdialytic examinations were performed during a sedentary day under controlled diet. A physiological pulse of growth hormone may also activate lipolysis²⁹ in adipose tissue. We have previously found an acute increase in growth hormone due to cTEMS set off by both the electrical and the heat stimulation,¹⁴ but the long-term effect of cTEMS and EMS on growth hormone is unknown and was not in focus in this study.

The effect of exercise training on gene expression in human adipocytes remains unexplored. In rats, however, 4 weeks of daily swim training resulted in a significant increase in PGC-1α, a key regulator of mitochondrial biogenesis.³⁰ When analysing key genes related to fatty acid oxidation in SCAAT, we found an increase in PGC-1α, essential in mitochondrial function and in regulating the genes involved in fatty acid oxidation.³¹ We interpreted these findings as early adaptions to the increase in the energy-demanding lipolysis process, as shown by the increase in microdialytic glycerol in SCAAT. The significant increase in CPT-2 and the relative, but not significant, increase in the other investigated genes may support this interpretation.

Despite the observed increase in adipose tissue lipolysis, 8weeks of cTEMS did not significantly influence body composition. When investigating the effect of EMS on the body mass index of sedentary individuals, Banerjee et al.² found similar results. The type of exercise conferred by cTEMS may explain this, as regular steady-state exercise at moderate intensity does not seem to induce significant changes in body composition.³²

Previously, a connection between the fat layer thickness and electrical current has been proposed,³³ and higher currents, associated with increased discomfort, are necessary to evoke muscle activation in obese subjects.¹³ The stimulation modality used in this study, with relatively large electrodes, as suggested by others,^13,34 and applied heat, appear to be alterations making it possible to overcome these limitations. Superficial heat is known to reduce pain³⁵ and thus seem to improve tolerance to afferent electrical stimulation necessary to evoke muscular contraction.¹⁴ In this study, cTEMS was well tolerated and administered without any significant complications or discomfort. After the end of the intervention period, an anonymous questionnaire was sent by mail to the 11 study participants, and nine responded. Only two of nine responders associated the stimulation sessions with light or moderate discomfort. Another two subjects reported being neutral, while the remaining five described light (one person), moderate (one person), or great comfort (three persons) related to cTEMS. If available, eight of the nine responders reported that they would consider cTEMS as a regular part of their daily routines.

Some limitations in our study may be considered in the evaluation of the findings. The main weaknesses are the nonrandomized design and a relatively small study population. Both diet and everyday activity may influence the results; however, when controlled, we did not find any changes in dietary habits or physical activity levels. Further, the fact that the training effect from cTEMS was observed in combination with positive metabolic and genetic findings, we consider a strength of our study.

In conclusion, we found that 8 weeks of prolonged electrical muscle stimulation with added heat in obese sedentary subjects significantly improved the functional capacity towards higher fatigue resistance. Both at peak exercise and at the anaerobic threshold, we observed increased VO₂, most likely caused by increased muscular arteriovenous O₂ extraction. Secondary to these training-effects, we found increased lipolytic activity and increased level of mitochondrial activity in adipose tissue. During this relatively short stimulation period, the body composition and visceral fat area remained unchanged. Further studies are needed to determine the possible role for cTEMS as an adjuvant in the treatment of obese individuals.

Acknowledgements

We would like to thank project nurses Irene Drotningsvik, Anita Isaksen, Brita Kolstad, and Mona Lavik for their work, Clara Gjesdahl, MD, PhD for assistance with the body composition analysis, and Kari Williams for excellent technical assistance.

Funding

This work was supported by the Mohn Research Fund (Grant number 08-09) and Department of Heart Disease, Haukeland University Hospital.

References