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The effect of passive heating on heat shock protein 70 and interleukin-6: A possible treatment tool for metabolic diseases?

  1. H. Faulkner e,S J3ckson, G. Fatania, and C. A. Leicht

Nationa Centre for Eports and Exercise Medicine, S:hool of Eport Exercise and 1-Eath 8:ienoos, Loughborough University, Loughborough, Leice:tersiire, Uiited Kingdom

ABSTRACT

Increasing physical activity remains the most widely publicized way of improving health and wellbeing. However, in populations that benefit most from exercise (EX), adherence is often poor and alternatives to EX are important to bring about health improvements. R3cent work suggests a role for passive heating (PH) and heat shock proteins (HEP) in improving cardio-metabolic health. The aim of this study was to investigate the expression of HEP?0 and interleukin-6 in response to either EX or PH and the subsequent effect on glucose control. Fourteen males volunteered and

were categorized lean (BMI 23.5 § 2.2 kg¢n1 2) o”r overweight (29.2 § 2.7 kg¢n1 2) and completed

60 minutes of either moderate cyding at a fiXed rate of metabolic heat production (EX) or warm water immersion in 4 water (PH). Extracellular HEP?0 increased from baseline in both conditions with no differences between PH (0.98 § 1.1 ng¢nL’ 1) or EX (0.84 § 1.0 ng¢nL’ 1, p D 0.814). IL-6 increased following both conditions with a two-fold increase after PH and four-fold after EX. Ehergy expenditure increased by 61.0 § 14.4 kcal¢11 1 (79%) after PH. Peak glucose concentration after a

meal immediately following PH was reduced when compared with EX (6.3 § 1.4 mmol¢..1 1 versus

6.8 § 1.2 mmol¢..1 1; p < 0.05). There was no difference in 24-hour glucose area under the curve (AUG) between conditions. These data indicate the potential for thermal therapy as an alternative

treatment and management strategy for those at risk of developing metabolic disease where adherence, or ability to EX, may be compromised.

Introduction

Overweight and obesity are characterized by chronic inflammation and impairments to insulin sensitivity and glucose control. A multitude of factors contribute to enhancing health and wellbeing; increasing physical activity remains the most widely publicized way of doing so. However, in populations that benefit most from exercise (EX), adherence is often poor, most likely due to medical conditions and disability,1 poor motivation and  a  lack of  convenience2.    There are many factors involved in the development and treatment of insulin resistance, with the roles played  by both thermal therapy and heat shock proteins (HSPs) receiving increased attention.4 7 Furthermore, it has been suggested that HSPs may provide an early indi­cator of the onset of metabolic diseases, such as type 2 diabetes (T2DJvI),8 9 while also acting as a possible bio­ marker for the chronicity of diabetes.10

HSPs are synthesized in response to a number of physiological stressors,l H     3 and are individually char­acterized by their molecular weight. The most widely studied HSP in humans is the 70 kDa family of HSPs which includes both the constitutive HSP73 and inducible HSP72 forms. HSPs have wide-ranging functions including their roles as thermo-protectants and volvement  in protein  synthesis.14  The resting level of extracellular HSP70 (eHSP70) appears to be related to adiposity” and inflammation with elevations reported in obesity and T2DM, whereas resting intra­ cellular HSP70 (iHSP70) is decreased in T2DM.9 A reduction in iHSP70 is an important  component  in the vicious cycle of chronic inflammation and reduced resistance.17 18  Indeed, a reduction  in iHSP70 has been insulin  signalling15 16  and  the development  of insulin reported in T2DM and correlates to insulin resistance and glucose disposal rates.19 2

eHSP70 is increased following acute EX13 with the magnitude of change dependent on both the duration and intensity.21,22 A  core temperature  (Tc) threshold for elevations in eHSP70 has been suggested, with a 0.8-1.5 increase in Tc being required in order to elevate  eH SP70.,23 24 Recently,  thermal  therapy has been demonstrated to stimulate HSP70 production, although to a lesser extent than following EX,4’24 and may offer  an  alternative way by which the  positive effects ofHSP  may be induced.9 The influence  of thermal therapy on  glycemic regu­lation and metabolic disease has primarily been inves­ tigated  using rodent  models.17’18’25   7. However, there are human data to suggest that local heating at the site of  insulin   infusion   improves  postprandial insulin action28·29 and may benefit long-term glycemic con­ trol29·30  and  cardiovascular health in hum ans.31 How­ever, the mechanisms by which such an effect may occur in humans remain to be elucidated. Chung et al. were the first  to  demonstrate  that  passive  heating (PH) of mice resulted in an elevation  in iHSP70 which was protective against the deleterious effects of con­ suming a high-fat  diet.18 They report  that  in response to a high-fat diet there was an increase in the phos­ phorylation of c-Jun N-terminal kinase (JNK). Such an increase has been linked to impaired insulin sensi­ tivity due to JNK binding with the insulin receptor substrate, thus preventing the initiation of the insulin signaling cascade and resulting in an elevation in plasma glucose (For review, the reader is directed to ref [32]). Importantly, Chung et al. demonstrated that PH attenuated hyperinsulinemia, glucose intolerance and insulin resistance and as a direct consequence of elevations in HSP70 and was closely associated with prevention of JNK. phosphorylation. Therefore, there is a need to investigate how well data from these rodent models translate into human participants, with a specific focus on the ability of thermal therapy to ele­ vate HSP70 and improve glycemic control.

HSPs may impact on the inflammatory state by directly inducing pro-inflammatory cytokines. Regular EX has been demonstrated to have a positive effect on the inflammatory profile that is suggested to occur asa consequence of the repeated exposure to an anti­ inflammatory environment following each EX bout.33This may occur as a consequence of secretion of pro­ inflammatory cytokines, including interleukin-6 (IL-6) that may in turn up-regulate anti-inflammatory cytokines.34  Importantly,   it has been shown that HSP70 independently induces pro-inflammatory cytokines.35·36 Therefore, thermal therapy may be able to replicate some of the anti-inflammatory benefits of EX, which could help reduce the level of inflammation evident in many types of chronic disease. While EX has significant benefits to improving health, individuals  with many types  of chronic disease often  experience low EX tolerance and  poor rates of adherence.1·37 Based on the above animal literature, there is evidence to suggest that thermal therapy may replicate some of the health benefits of EX and allevi­ ate some of the comorbidities often associated with chronic diseases such as T2DM.38 If successful, the implementation of thermal therapy may help to lessen some of the financial burden of treating chronic by potentially reducing the dependence on pharmacolog­ical interventions. Thermal therapy could offer a simple home-based intervention that may appeal to individuals unable or unwilling to participate in regu­lar EX

The primary aim of this study was to  investigate the expression of eHSP70 in response to either  EX or thermal therapy in the form  of  PH  via  warm water immersion. A secondary aim was  to  investi­ gate the effect of body composition on the eHSP70 response. Finally, we wished to investigate the potential effect of PH on glycemic  control  in response to replicated dietary intake.  It was hypothesized that both EX and PH would elevate eHSP70, with a larger magnitude of change follow­ ing EX. It was further  hypothesized that  there would be a differential response in eHSP70 between lean and overweight participant groups.

Methods

Participants

A total of 14 males volunteered  to  participate  in the study and were split into two groups: (i) Lean  (LEAN, n  D  7; BMI  <  25  kg¢n1 2,  body fat  <  15% and fat mass <  12 kg) and  (ii) Overweight  (OW, n  D 7; BMI >   27.0  kg¢n 1 2,  body  fat  >   20%  and  fat  mass > 25  kg).  Participant  characteristics  are  provided  in Table 1. All  participants  were  healthy  non-smokers with no history of cardiovascular, hematological or metabolic  disorders  and  had been weight stable for [ID 3 months. Participants were habitually inactive; per­ forming   less  than   1.5  hours   of  structured  physical activity  per  week  and  had  avoided  any  hot weather

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exposure in the previous two months, including fre­ quent sauna or spa use.

Ethical approval

Full ethical approval was granted by the Loughbor­ ough University Ethical Advisory Committee. All pro­ cedures  conformed  to the principles  defined  in the Declaration of  Helsinki.  Participants        were  fully informed   of  the  experimental   protocols   and any potential risks were identified before they provided the:ir written consent to participate.

Experimental overview

Participants visited the laboratory on three occasions. The preliminary visit to the laboratory consisted of initial measures including a blood profile, body com­ position and both submaximal and maximal oxygen uptake tests (Y.02 max test). Visits two and three com­ prised the experimental trials which consisted of either 60 minutes of PH via warm water immersion (PH), or 60 minutes of EX at a fixed rate of metabolic heat pro­duction (EX) which were completed in a counterbal­ anced  order  and  were  matched  for  DTc,  On  arrival, participants were asked to void the:ir bladder and were weighed in minimal clothing and then fully instru­ mented. The first blood sample was then collected and muscle temperature measured. Participants then com­ pleted either PH or EX as detailed below. On comple­ tion of each trial, a second blood sample and muscle temperature measure were obtained, with a final blood sample being  taken  2  hours  after  completion (Figure 1). Experimental  trials  were separated  by a minimum of 5 days to reduce any acclimation effect 39 and to minimize any effect of prior EX or heating upon insulin sensitivtiy.40 All trials were completed at the same time of day to minimize effects of c:ircadian variation.

 

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Preliminary visit

Participants arrived in a fasted state and a capillary blood sample was drawn for assessment of total cholesterol, high-density lipoprotein cholesterol (HDL-C),  low-density  lipoprotein  cholesterol  (illL­ C), glucose and triglycerides (TG)  (Cardiochek, Polymer Technology Systems, Indianapolis, IN). Participants’ height and weight were  recorded  (Seca 360, Birmingham, UK), and BM[ calculated. Body composition was determined with skinfold callipers (Harpenden,   Warwickshire,   UK)   using   the  seven­ site  formula. 41  Participants  then  completed  a sub­ maximal EX test on an electronically braked cycle ergometer (Lode Excalibur Sport, Groningen, Neth­ erlands) with an online gas analyzer (Metamax 3B, Cortex Biophysik GmbH, Germany). The test com­pised   four   stages,   increasing   by·  20   W every 4 minutes.  Y…02 and‘ \l .C0 2   were determined  over a 30-second rolling average from the final minute of each stage. On completion of the submaximal test, participants were given a minimum of a 10-minute recovery period prior to commencing the YO2 max test. This test used a continual ramp  protocol  at a rate of 20 W¢nin1 1 until the participant reached volitional exhaustion. YO2 max was determined over a 30-second rolling average from the final minute  of the test.

Experimental trials

Seventy-two hours prior to the start of each experi­ mental trial, participants reported to the laboratory where they were fitted with a continuous glucose monitor device (CGM; Freestyle Libre, Abbott Lab­ oratories, Berkshire). The sensor  was  inserted  on the posterior aspect of the upper arm, with intersti­ tial glucose measured  every 15 minutes.  CGM data were analyzed for 2-hours post meal glucose AUC and peak glucose concentration.42 Participants were provided   with   scales   (HoMedics   Group   Salter, Kent) and detailed instruction on how to measure dietary intake in order to accurately  complete  a 3- day diet diary during the first trial, which com­ menced 24 hours before and ended 24 hours after completion of  trial  1.  Participants  were  instructed to consume the first meal after each condition fol­ lowing a standardized 3-hour time period. Partici­ pants were required to replicate their dietary intake and timing of meals for the second trial.

Exercise trial

Participants performed 1-hour cycling at a fixed rate of metabolic heat production (1:1..proo) equivalent to 7 W/kg in an environmental chamber (25.6 §  0.7Ue; 49.8 § 3.8% relative humidity, rh). The  initial  work­ load was determined from the submaximal test and external  workload  was  manipulated  throughout   the 60 minutes in order to maintain 1:1..proo at 7 W/kg. Par­ ticipants  completed  the  EX at a self-selected cadence. Y 02 and YCO2 were recorded throughout using an online gas analyzer. Airflow was provided by three fans stacked vertically, positioned 2.5 min front of the ergometer at an air speed of 1.5 m¢;1 1 .

Passive heating trial

Participants  were  seated  in  a  water  bath  (40.2  §0.2 ) and immersed up  to the  waist  for  1 hour. Water was circulated throughout to ensure main­ tenance of water temperature. V.D2 and  V….CO2 were recorded  throughout. Ambient  conditions dur­ing the trial were 23.9 § 0.9 Ue; 49.9 § 4.4% rh. Owing to the effect of temperature and duration on HSP70 and IL-6 expression, we attempted to match the change in core temperature between conditions by eliciting a 1 increase in core temperature. Further­ more, the duration  of both EX and PH  was matched to limit the effect of intervention  exposure time on key outcome measures.

Instrumentation

Core temperature (Tc) was measured using a rectal thermistor (Grant Instruments Ltd, Cambridge) inserted 10 cm beyond the anal sphincter. Wireless skin sensors (iButtons, DS1922, California, USA) were applied and secured by Medipore tape (3M, Berkshire, UK.). Mean skin temperature (Ts<./ was calculated according to the formula of Ramanathan.43 Partici­ pants  wore a heart  rate monitor  throughout (RS800,Kempele, Polar, Finland).

Muscle temperature

Muscle temperature (Tm) was measured in the right vas:us lateralis using a solid needle probe (MKA08050A275TS Ellab, Copenhagen, Denmark) immediately pre- and post-trial. Following standard sterile procedure,  the  needle  probe  was first  inserted to  an  initial depth  of 3  cm  beyond  the  muscle fascia where the temperature was allowed to stabilize before the probe was withdrawn to 2 cm and  then  1cm depths, with the  temperature recorded  at  each depth.

Blood sampling

Venous blood samples were collected byvenepuncture from an antecubital vein in the right arm into a 10 mL Vacutainer that had been pre-cooled  and  pre-treated with K3EDTA (BD Biosciences, San Diego, USA). Samples were obtained before and on completion  of each  heating  protocol  with  a   final   sample   taken 120 minutes later (Figure  1). Samples  were stored  on ice  until  they  were   centrifuged   at   3,500   rpm   for 10 min at 4Ue and the plasma stored at i 80Lle until subsequent analysis.

Enzyme-linked immunosorbent assays Plasma  HSP70  (ENZ-101, AMP’D6  hs  HSP70, Enzo Life Sciences, Exeter, UK) and IL-6 (HS600B, Quantikine6 HS IL-6, R&D Systems, Abingdon, UK) concentrations were analyzed using enzyme-linked immunosorbent sandwich assays  (EilSAs)  according to the manufacturer’s instructions. Plasma HSP70 and IL-6 concentrations were determined in relation to a four-parameter standard curve (version 6.0 GraphPad Software, La Jolla, CA, USA). All samples were ana­ lyzed in duplicate  with a mean intra plate coefficient of variation  (CV) of 4.5% for HSP70 and  5.8% for IL-

The interplate CV was 6.3% for HSP70 and 6.1% for IL-6.

Calculations

Energy expenditure during PH and EX was calculated via indirect calorimetry. Heat balance parameters were estimated via partitional calorimetry and are pre­sented as the mean value for each condition. All parameters were calculated in W/m 2 but  presented as W/kg where appropriate. The rate of metabolic energy expenditure was estimated as where  RER  is  the  respiratory  exchange  ratio  and  Eb and    Ej, represent the  energy equivalent  of carbohydrate (21.13 kJ) and fat (19.69 kJ) respectively per liter of 0 2 consumed per minute (L¢nin1 1 ) . tlpr00 was deter­ mined  as the  difference  between  M and  the  external work rate (W)

Mean body ternperature (DTb) was estimated using the three-compartment model as follows: D1\ D . 0 :63 D Tc/ C . 0 :24 DT 9/  C . 0 :13 D T ml (3) where DTc represents the change in core temperature, DTfl< the change in mean  skin temperature and  DTm the change in muscle temperature at a depth of3 cm.44 AUC was calculated  using the trapezoid method. S:atistical analysis The normality  and  distribution  of  data  was  assessed using  the  Shapiro-Wilk  normality  test.   Where   data failed to  meet  the  criteria  of  normal  distribution,  the data were log transformed. Mean participant  charac­ teristics were  compared  using  independent  samples  t­ tests. Tc, T fl<, Tm, HSP70, IL-6 were analyzed using repeated measures ANOVA. Where significance was obtained  post  hoc  tests  were  completed  using  Sidak’s test for multiple comparisons. l:i..proct, DTb and energy expenditure  were  analyzed  using  one-way   ANOVA with  Sidak’s  test  for  multiple  comparisons.  Correla­ tional analysis was done using Pearson’s correlation coefficient. Linear regression was used to analyze the relative contributions of DTc, DTsk, DTm and DTb to DHSP70  and  DIL-6. The value was employed  to determine the variance explained by  each  predictor variabie.  Where  reported,  the  adjusted       value is pro­ vided  for  multiple  linear  regression   in   order   to account for the number of  predictor  variables  in  the model. All statistical analyses were performed using GraphPad  Prism   (version   6.0  GraphPad   Software, La Jolla, CA, USA). All data are presented as mean § SD unless otherwise stated. p values illb.05 were consid­ ered statistically significant. Effect sizes (ES) corrected for bias using Hedge’s g were calculated  as the  ratio of the mean difference to  the  pooled  standard  deviation of the difference, with 95% confidence intervals (95% CI) for differences also presented.  The  magnitude  of the  ES was  classed  as trivial (<  0.2), small (0.2-0.6), moderate   (0.6-1.2),   large   (1.2-2.0)   and   very large crn:h.o).45

Results

l::iprod· There were differences  in  l::ipr00 between PH (1.9 §  0.2 W/kg)  and EX (7.3 §  0.5 W/kg,  p <  0.001). l::iprod was  successfully  matched  between  groups  for both  PH  (LEAN  2.0 §   0.2  W/kg,  OW  D  1.8 §   0.2  W/ kg, p D 0.433, CI D i 0.03 to 0.43, ES  D  0.9)  and  EX (LEAN  7.3  §   0.4,  OW  7.2  §   0.5  W/kg;  p  D 0.763, CI D i 0.63 to 0.43, ES D 0.21).  However,  when  lean body mass is accounted for, differences in l::iprod were evident (IEAN 8.4 §   0.4 W /k g/LBM1  1; OW 9.8 § 1.6 W/kg!IBM1 1, p <  0.05, CID   0.04 to 2.76, ES D 1.1).eHs:170. There was a main effect of time (p  < 0.005) but not condition (p D 0.887) on the eHSP70 response (Figure 2A). The increase in eHSP70 follow­ ing PH and EX was 23% ·and 24%,  respectively (Figure 2B). Across conditions DeHSP70  correlated with mean Tm (r D 0.485, p < 0.05). DeHSP70 was negatively correlated to body mass (r D i 0.400, p < 0.05). Of the thermal variables, DTm provided the best predictor of DHSP70, with the DTm explaining 24.7% of the total variance in DHSP70 (r  D  0.498,  p < 0.01) Body mass also predicted DHSP70, explaining 16.4% of the total variance (r D i 0.405, p < 0.05). When combined, DTm and body mass provided the strongest predictor, accounting for 28% of the variance in DHSP70 (Adjusted D 0.280, r D 0.585, p < 0.01). IL-6. There was a main effect of time (P < 0.001), condition (P < 0.05) and an  interaction  effect  (P  < 0.01, Figure 2C) on IL-6 concentration. IL-6 was  higher    immediately    following   EX   (1.74   §   1.79 pg¢nL1  1 )  compared to PH (0.97 §  0.86 pg¢n L1 1, p <0.0001, ES D 0.5) and equated to an increase of346% after EX and 118% following PH (Figure 2D) from baseline  values.  IL-6  had   returned  to  baseline  at 2-hours  post  for  PH  (0.58  §  0.47  pg¢nL’ 1    and EX (0.73 § 0.79 pg¢n L1 1 ) . When considering differences between IEAN and OW, there was a  main  effect  of time (p < 0.05) but not group (p D 0.533) on the IL-6 response. None of the thermal  variables,  nor  body mass, provided an effective model for predicting the change in IL-6.

Energy expenditure

There were no differences in resting metabolic  rate between  IEAN  (1811.2  §   82.3  kca1¢iay1   1 )   and OW (1873.9  §   140.7  kcal¢iay1 1; p  D  0.330,  ES  D  0.5).p3There was an overall effect of condition on energy expenditure during heating (p < 0.0001) and an inter­ action effect (p < 0.05). PH increased energy expenditure by 61.0 § 14.4 kcal¢i.1 1 compared to rest (p < 0.0001, ES D 4.8), which equates to a 79.5% increase. EX resulted  in  an  additional  energy expenditure of 556.3 § 92.0 kcal¢i.1 1 compared to rest  (p <  0.0001, ES  D  7.1),  equating  to  a  721% increase.  EX energy expenditure was greater for OW compared to IBAN (11.3 § 1.9 kcal¢nin1 1 versus 9.8 § 0.9 kcal¢nin1 1, respectively,  p <  0.05, ES D 1.0), resulting in an addi­tional  caloric   expenditure   from   rest   of  597.7 §107.9 kcal for OW (748%), and 515 § 52.1 kcal for IBAN (681%). During PH, there was no difference in the increase in energy expenditure between IBAN and OW (2.3 § 0.3 kcal¢nin1 1 versus 2.3 § 0.3 kcal¢nin1 1, respectively, p D 0.997, ES D 0.0). Caloric expenditure increased  from  rest  by  63.4  §  16.6 kcal  (84%)  and 58.5   §   12.7  kcal  (74%)   kcal  for  IBAN   and OW, respectively although there was no difference between groups (p D 0.623, ES D 0.3).

8Abstrate oxidation

Total EX carbohydrate oxidation was higher for OW (125.3 §  20.8 g) compared to IBAN  (92.5 §  28.2 g; p < 0.05, ES D 1.3). There was a moderate effect of body mass on total carbohydrate oxidation in PH between OW (7.5 § 4.2 g) and IBAN (13.2 § 8.3 g, ES D 0.83) although  this did not reach  significance (p D 0.13). There was no difference between OW and IBAN in total fat oxidation during EX (p D 0.46). There was a tendency for OW to display a greater total fat oxidation than IBAN in PH, although this did not reach statistical significance (10.6 § 1.54 g versus 9.0

  • 3.1 g; ES 0.7; p D 0.13).

Continuous  glucose monitoring

Owing to instances of poor compliance to diet replica­ tion and sensor malfunction and drop out, CGM anal­ ysis was only completed on eight participants, with an unequal  split  between  IBAN  (n   D  3)  and   OW (n D 5). Therefore, it was only possible to conduct analysis on PH compared to EX Peak glucose concen­ tration  in response to the meal following PH  (6.3  §1.4 mmol¢:.} 1) was lower compared to the same meal after EX (6.8 § 1.2 mmol9t) 1; p < 0.05, CID j 0.9 to 1.9, ES D 0.4, Figure 3A). There was no difference in glucose AUC for PH compared to EX following this meal  (p  D   0.875,  CI  D   i  11.2  to   12.4,  ES  D 0.1 Figure 3B). There were no differences in either peak glucose (meal 2  p D  0.168, meal 3 p D  0.266) or AUC (meal 2 p D 0.070, meal 3 p D 0.183) consumed in the subsequent 24-hour period. There were also no differ­ ences in total  24-hour  AUC  between  PH  and  EX (p D 0.168)

Core temperature

There was a main effect of time (p < 0.0001) and an interaction effect (P < 0.0001), but no main effect of condition on DTc (p D 0.112, Figure 4a). PH DTc was lower  in  OW  compared  to  IBAN  from  35  minutes (p < 0.05), although by 60 minutes there was no dif­ ference in DTc (1.0 § o.2Ue versus 0.9 § 0.2 , LEAN versus OW, respectively p D  0.089,  CI  D  j  0.13  to 0.33, ES D 0.5). There were no differences in DTc between  OW  and IBAN during EX at  any time point, with DTc at 60 minutes being 0.8 § 0.1Lle versus 0.8 §o.2Ue for LEAN and OW, respectively (p D 0.999). S<in temperature There were main effects of time (p < 0.0001), condi­ tion (p < 0.0001) and an interaction effect (p < 0.0001) on DT s< (Figure 4B). There were no differen­ ces in DTs< between OW and LEAN following EX (LEAN 1.7 § 0.7U@; versus OW 1.7 § l.6Ue;, p D 0.999, ES D 0.0) or PH (LEAN 4.2 § o.4Ue; versus OW 4.7 § 0.6 , pD 0.095, ES D 0.9). Muscle temperature There was an effect of depth on DTm (p < 0.001), but no effect of condition (p D 0.285) or interaction (p D 0.111, Figure 4C). Following PH, the  DTm    was greater for LEAN compared to OW at a depth oflcm (LEAN, 2.5 § 0.8Ue; OW 1.9 § 0.6Lie;, p < 0.0005, CI D i 0.2 to 1.4, ES D 0.8) and 3cm (LEAN, 2.3 § 0.6U@;; OW 1.9 § 0.7, p < 0.005, CID  i 0.4 to 1.2, ES D 0.6). After EX,  DTm  was  only  different  at  1cm  (LEAN,  2.3 § 0.6Ue;; OW  2.0  §  0.7,  p <  0.005,  CI  D  i  0.5 to  1.1, ES D 0.4).

Mean body temperature

There was  a  difference  in  DTb  between  conditions (p < 0.0001, Figure 4D). There were  differences between LEAN PH and LEAN EX (1.7 § 0.3iJe: versus 1.1 §  0.3iJe:,  p <  0.005,  CI  D  0.3 to  1.0, ES D 1.9); OW PH and OW EX (1.8 § o.21J@: versus 1.0 § 0.4Lle; p < 0.001, CI D 0.4 to 1.2, ES D 2.4). There were no differences between LEAN and OW within the same condition  (both  p > 0.9).

Heart rate

There were significant effects of time, condition and interaction effect of heart rate (all p < 0.0001). Mean percentage HRmax EX was 78 § 8% compared to 54 § 7% in PH (p < 0.0001). There were no  differences  in the mean percentage of HRmax between groups for either  PH  (LEAN  D  54.0 §  6.0% versus OW  D 55.2

  • 6.2%, p D 0.719) or EX (LEAN D 78.5 § 7.3%; OW

D 81.7 § 7.4%, p D 0.428).

Discussion

The primary outcome of the present study was that the concentration of eHSP70 in the plasma was similar between EX and PH when both conditions were matched for DTc, with DTm being the best individual predictor of DeHSP7O, explaining » 25% of the total variance. This suggests that changes to Tm may in part determine the appearance of eHSP7O. An important secondary outcome is the increase in energy expendi­ ture evident as a consequence of PH. This has impor­ tant implications for the use of PH as a future intervention that may help to moderate body mass, particularly for individuals unable or unwilling to complete regular physical activity. However, we recog­ nize that PH should be coupled with dietary manipu­ lation to maximize any potential benefit to body composition. Finally, the reduction in peak glucose in the meal following PH compared to EX suggests that PH may have a beneficial effect on glycemic regulation and provide an alternative or augmentative interven­ tion by which individuals with metabolic diseases may be able to improve their glycemic control. However, additional research needs to be conducted to confirm these data in larger cohorts over a prolonged period of time.

The HSP7O response may be hindered by excess body mass. Regression analysis demonstrated a link between body mass and the DeHSP7O. A greater body mass was associated with an impaired eHSP7O response to heating, via either EX or PH, explaining 16% of the total variance. Such variation may be indic­ ative of an impaired HSP response in overweight indi­viduals in the face of physiological stress and would likely be compounded by low aerobic capacity.46 The implications  for  such  a deficiency are wide ranging and may increase susceptibility to metabolic disease,46,47 impaired vascular function48 and even con­tribute to the impaired thermoregulatory control evident in individuals with diabetes.49 While there were no clear differences in the HSP7O response between either PH or EX, there were clear differences  in  the  IL-6  response.  Previous  work has shown that temperature increases independently ele­ vate IL-6.50However, the present data do not support the view that skeletal muscle may act as a “heat stress sensor” triggering a subsequent cytokine response, as none of the thermal measures taken during the trials were effective predictors of DIL-6 and could not account for a significant proportion of the variation in IL-6 following heating. Our data also indicate that the difference in IL-6 between PH and EX is not due to HSP7O induction of IL-6,35’36 as DHSP7O was similar following PH and EX This suggests the IL-6 response to EX occurs independently ofHSP7O expression and is more dependent on intensity and duration of EX,51 than exposure to heat stress. IL-6 combined with its receptor (IL-6R), may also have a positive effect on insulin independent glucose uptake at rest.52 Should chronic  PH enhance  IL-6R expression  as is evident following EX training, 53 interventions capable of increasing both IL-6 and IL-6R, may provide a thera­ peutic  basis  for  improving  glycemic  control.  More­over, it has recently been shown that acute PH elevates the anti-inflammatory cytokines IL-lra and IL-10,54 suggesting that chronic PH may benefit the inflammatory profile in a similar way to EX However, long-term investigation of PH is required to corrobo­ rate this hypothesis.

Although the sample size for analysis of the glyce­ mic response to PH and EX was somewhat limited, the fact that comparable glycemic profiles were evi­ dent  between  PH  and  EX is interesting.  Given the acute effect of EX on improving glucose control as a consequence of improved insulin sensitivity5,5this is a somewhat surprising result. However, it has recently been shown that heat stress activates insulin indepen­ dent glucose transport via s0 adenosine monophos­ phate-activated protein kinase (AMPK) activation. 56 In response to the heat stress stimulus, a reduction in muscle adenosine triphosphate, phosphocreatine and glycogen was reported, indicating a significant energy cost to the heating protocol and hence activation of AMPK This suggests that, acutely at least, heat stress may improve glucose uptake due to increased energy expenditure. Further work needs to be conducted to establish the exact mechanisms by which PH may exert such an effect. In addition to benefits to metabolic regulation, PH has been shown to benefit cardiovascular health. In a recent meta-analysis, Laukkanen et al. report that life­ long sauna  use reduced  cardiovascular and  all cause mortality, with the largest benefits associated with more frequent sauna use.57 Although this relationship does not establish causality,31’58 it is nonetheless an interesting link that is supported by recent investiga­ tions.59-61 PH appears to improve vascular : function, with  improvements  to  flow-mediated  dilatation, arte­rial stiffuess and blood pressure reported after  8 weeks of PH.59 Moreover, Tho as et al.  have shown  that 30 ;minutes of PH can induce a significant shear stress response and a reduction  in mean arterial pressure in patients with peripheral arterial disease.62 Further­ more, in a healthy cohort who underwent matched duration PH or EX, the PH group demonstrated a greater shear stress response compared to that in response to EX60 Based on heart rate responses, the cardiovascular strain in the present study was compa­ rable to that  of Thomas et al., which elicited » 50% HRmax during their immersion protocol.60 These recent findings  indicate that  PH has the potential to induce significant systemic benefits, which in some instances may be comparable to or even  greater  than the effects evident following EX. There are a few limitations with the present investi­ gation. First, the omission of a measure of iHSP7O, either derived from skeletal muscle or leukocytes malces it difficult to draw firm conclusions on the potential intracellular effect of PH and glycemic regu­ lation. Without direct measures ofiHSP7O, in addition to  JNK and other molecules in the  insulin-signaling cascade, it is difficult to derive the exact effect of PH on acute glycemic control. Therefore, we are limited to discuss the relationship between eHSP7O and iHSP7O and that an increase in the extracellular com­ ponent is reflective of changes in the intracellular compartment. Future investigation should employ techniques to measure both iHSP7O and eHSP7O to enable greater understanding of the effect of PH  on the intracellular to extracellular ratio. Second, the rela­ tively small sample size of this study, which may limit the statistical power of our experiments. We acknowl­ edge that further experiments with larger cohorts should be conducted in order to replicate the present data.

In summary, the data presented here demonstrate that both acute PH and EX result in comparable increases in HSP7O. It is suggested  that DHSP7O is sensitive to the DTm· The elevations in both HSP7O and IL-6 may promote an anti-inflammatory milieu and help combat the chronic inflam­mation associated with many disease states. The increase in energy expenditure following PH dem­onstrates a systemic effect of PH, which has the ability to reduce adipose tissue and further contrib­ute to reducing inflammation.  However,  it should be noted that EX still offers the greatest  overall benefit to weight control and metabolic health. Finally, in agreement with that of  other authors, the present data indicate that PH might provide an alternative to  EX in some individuals  who are  too physically impaired to undertake prolonged aerobic activity to improve their cardio-metabolic health. The possible therapeutic benefits of  PH  require more extensive investigation.

Abbreviations

AM:PK    s0 adenosine monophosphate-activated protein kinase Area under the curve Body mass index blood pressure body surface area Continuous glucose monitor confidence interval coefficient of variation extracellular  heat shock protein enzyme-linked immunosorbent sandwich assay effect size Exercise trial High-density  lipoprotein cholesterol Metabolic heat production Heat  shock protein Intracellular heat shock protein Interleukin 1 receptor antagonist Interleukin 6 Interleukin 10 c-Jun  N-terminal kinase Low density lipoprotein cholesterol Lean participant group Overweight participant group Passive heating trial Standard deviation Standard error of mean Type 2 diabetes mellitus body temperature core temperature muscle temperature skin temperature Triglyceride carbon dioxide production oxygen uptake Maximum volume of oxygen uptake change in temperature Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to thank; Prof George Havenith for granting access to the Environmental Ergonomics Research Centre and Dr Caroline Smith for her insightful discussion and comments during the preparation of the manuscript.

Funding

The research was also partly supported by the National Insti­ tute for Health Research (NIHR) Diet, Lifestyle & Physical Activity Biomedical Research Unit based at University Hospitals of Leicester and Loughborough University. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

ORCID

  1. H. Faulkner http://orcid.org/0000-0003-4688-7252 C. A. Leicht http:l/orcid.org/0000-0002-3539-8480

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