Heat exposure increases circulating fatty acids but not lipid oxidation at rest and during exercise

Journal of Thermal Biology 55 (2016) 39–46

Contents lists available at ScienceDirect

Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio

Heat exposure increases circulating fatty acids but not lipid oxidation at rest and during exercise Katharine O’Hearn a, Hans Christian Tingelstad a, Denis Blondin b, Vera Tang c, Lionel G. Filion d, François Haman a,n a

School of Human Kinetics, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5 Department of Medicine, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Universite de Sherbrooke, Quebec, Canada J1H 5N4 c University of Ottawa, Flow Cytometry Core Facility, Canada d Department of Biochemistry Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa Ontario, Canada K1H 8M5 b

art ic l e i nf o

a b s t r a c t

Article history: Received 2 July 2015 Accepted 2 November 2015 Available online 28 November 2015

Alterations in lipid oxidation during exercise have been well studied, but limited data exists on the effects of passive heat exposure and exercise in the heat on changes in lipid oxidation. This study was designed to examine: (1) the effects of heat exposure on lipid metabolism during passive heating and subsequent exercise in the heat by focusing on changes in whole-body lipid oxidation and plasma lipid concentrations, and (2) the effects of extended passive pre-heating on exercise performance in the heat. Male participants (n ¼ 8) were passively heated for 120 min at 42 °C, then exercised on a treadmill in the same temperature at 50% V̇ O2max for 30 min (HEAT). This same procedure was followed on a separate occasion at 23 °C (CON). Results showed that lipid oxidation rates were not different between HEAT and CON during passive heating or exercise. However, non-esterified fatty acid (NEFA) concentrations were significantly higher following passive heating (618 mM, 95% CI: 479–757) compared to CON (391mM, 95% CI: 270–511). The same trend was observed following exercise (2036 mM, 95% CI: 1604–2469 for HEAT and 1351 mM, 95% CI: 1002–1699). Triacylglycerol, phospholipid and cholesterol levels were not different between HEAT and CON at any point. Four of 8 participants could not complete 30 min of exercise in HEAT, resulting in a 14% decline in total external work. Rate of perceived exertion over the final 5 min of exercise was higher in HEAT (9.5) than CON (5). We conclude that: (1) heat exposure results in increased circulating NEFA at rest and during exercise without changes in whole-body lipid utilization, and (2) passive pre-heating reduces work capacity during exercise in the heat and increases the perceived intensity of a given workload. & 2015 Elsevier Ltd. All rights reserved.

Keywords: Non-esterified fatty acids Hyperthermia Fuel selection Exercise performance

1. Introduction Research investigating heat-induced changes in metabolism has generally been focused on changes occurring during exercise, specifically with regards to carbohydrate (CHO) metabolism. This focus on CHO metabolism is not surprising as this fuel plays an important role in sustaining energy demands under various conditions while representing only  1% of total reserves (Weber and Haman, 2005). In contrast to CHO, lipids can account for as much as  95% to 98% of total energy reserves and play a substantial role in sustaining ATP production during prolonged aerobic activity (Jeukendrup, 2003). Despite the important role of both of these fuels, very little is known about changes in lipid metabolism n Correspondence to: Faculty of Health Sciences, University of Ottawa, 125 University St., Ottawa, Ontario, Canada K1N 6N5. E-mail address: [email protected] (F. Haman).

http://dx.doi.org/10.1016/j.jtherbio.2015.11.002 0306-4565/& 2015 Elsevier Ltd. All rights reserved.

during passive heating or exercise in hot conditions. During passive heat exposure, non-esterified fatty acids (NEFA) have been shown to increase above baseline values or thermoneutral conditions, suggesting that lipid metabolism is altered (Eddy et al., 1976; Yamamoto et al., 2003). Exercise in hot conditions have been demonstrated to lead to an increased reliance on CHO oxidation, based on repeated observations of accelerated muscle glycogen utilization (Febbraio et al., 1994; Fink et al., 1975; Jentjens et al., 2002; Starkie et al., 1999), higher plasma and skeletal muscle lactate concentrations (Dolny and Lemon, 1988; Febbraio et al., 1994; Fink et al., 1975; Hargreaves et al., 1996), and a higher respiratory exchange ratio (Dolny and Lemon, 1988; Febbraio et al., 1994; Hargreaves et al., 1996; Young et al., 1985). In addition, it has been suggested that this increase in CHO metabolism during exercise is associated with a decrease in the contribution of lipid oxidation (Febbraio, 2000; Fink et al., 1975). Therefore, during prolonged passive heat exposure and subsequent exercise, the purpose of this study was to determine the effects of heat

40

K. O’Hearn et al. / Journal of Thermal Biology 55 (2016) 39–46

exposure on changes in oxidative fuel selection and plasma lipid concentrations. More specifically, this study sought to quantify whole body lipid, CHO and protein oxidation rates, as well as triglyceride (TG), NEFA, phospholipid (PL) and cholesterol (CHOL) levels in non-heat acclimatized men exposed passively to 42 °C for 2 h before exercising at the same temperature for 30 min at 50% V̇ O2 max. Based on non-published preliminary experiments from our laboratory), we predicted that heat exposure would lead to an increase in CHO contribution while levels of circulating lipids would be increased in comparison to levels observed under normothermic conditions. In addition, as a secondary objective, this study will assess the effects of 2 h of passive heat exposure on exercise performance. It is established that performance during endurance exercise at a specific intensity is reduced during heat exposure (Arngrimsson et al., 2004; MacDougall et al., 1974; Morris et al., 2005). In addition, active or passive pre-heating to a core temperature of 37.8 to 38.0 °C prior to exercise also reduces time to exhaustion (Arngrimsson et al., 2004; Gregson et al., 2002). Here, we anticipated that heat exposure (passive and active) will impede the completion of a 30 min run at 50% VO2 max even if water loss is compensated.

2. Methods 2.1. Participants Eight healthy, non-heat acclimatized active males volunteered for this study, which conformed to the standards set by the latest revision of the Declaration of Helsinki and was approved by the Health Sciences Ethics Committee of the University of Ottawa. Written informed consent was obtained from all participants. Exclusion criteria were; heat acclimatized (e.g. outdoor workers), smokers, and/or physically active less than 3 days/week. Anthropometric measurements (height, weight, percent body fat) and maximal oxygen consumption (Bruce Ramp treadmill protocol) were obtained prior to the first experimental session (Table 1). 2.2. Experimental Protocol Each subject participated in two experimental trials, separated by at least 7 days. The trials consisted of a 90 min baseline period in ambient temperature, followed by 120 min at rest and 30 min exercise at 50% V̇ O2max at either 42 °C (HEAT) or 23 °C (CON). The order of the trials was randomly assigned in a balanced, cross-over design. Experiments were conducted between 8 h00 and 13 h00. Participants were asked to refrain from consuming caffeine or alcohol for 12 h, and to avoid heavy physical activity for 48 h prior to the experiments. The last evening meal was standardized (  900 kcal,  51% CHO,  27% lipids,  22% proteins). Participants were instructed to drink at least 1 L of water the evening before the trial, and to continue drinking water the morning of the trial to ensure they were well hydrated prior to the start of heat exposure. Participants reported to the laboratory at 8 h00 after a 12–14 h fast. Care was taken to minimize thermal stress between awakening and the start of the experiment (avoiding exposure to heat or cold, only very low-intensity exercise when traveling from home to the laboratory). Upon arrival at the laboratory, participants were instrumented with skin temperature transducers, an esophageal thermocouple and heart rate monitor while wearing shorts and a t-shirt. Participants were then asked to void their bladder. To start the experimental trial, participants sat quietly for 90 min at ambient temperature (24 70.5 °C, 36 74% RH) for baseline measurements. At the end of the baseline period, participants again voided their bladder and nude weight was recorded. Participants were then

Table 1 Physical characteristics of subjects (n ¼8) Age (years)

25 (23–27)

Body mass (kg) 76.2 (71.1–81.4) Height (cm) 181 (177–185) Body surface area (m2) 1.96 (1.86–2.05) a Percent body fat (%) 12 (10–15) VO2max (ml  min  1  kg  1)b 56 (52–60) Values are means with 95% confidence interval (CI) in parentheses. a b

Underwater weighing. Bruce Ramp treadmill protocol.

transferred for 120 min to a thermal chamber (t¼ 0) at either 4270.3 °C, 2473% RH (HEAT) or at 23 70.4 °C, 3575% RH (CON). During HEAT, participants also donned a sauna suit with elastic waist, wrists, neck and ankles (Training Sauna Suit, TKO Sports Group, Houston, TX, USA) to minimize evaporative heat loss. Throughout the passive period, participants consumed 1.5 L of water to replenish fluids lost through sweating. Heart rate and thermal responses were measured throughout the baseline and passive periods. Metabolic and ventilation measurements were recorded every 30 min. Blood samples were drawn prior to (Baseline), midway (T60) and after (T120) the passive period to determine changes in NEFA, TG, PL and CHOL concentrations. After 120 min, participants removed the sauna suit and toweled off, then exited the thermal chamber for a maximum of 5 min while nude weight was recorded. Participants then returned to the thermal chamber and walked on a treadmill for 30 min or until exhaustion. Speed was set at 3.5 miles per hour and the treadmill incline was adjusted to a pre-determined level equivalent to 50% of the participant’s V̇ O2max. Metabolic data, heart rate, thermal response and ventilation were recorded throughout the exercise period and a 10-point category scale (Borg, 1982) was used every 5 min to determine the participants’ rate of perceived exertion (RPE). At the end of exercise, nude weight was again recorded and a final blood sample was drawn. 2.3. Thermal response Changes in heat production (Ḣ ) were calculated by indirect calorimetry and corrected for protein oxidation (see below). Esophageal ( T ̅ es) and mean skin temperature ( T ̅ skin) were monitored continuously throughout the baseline period and experimental trial. Core temperature was measured using a pediatric probe (Mon-a-therm general purpose, Mallinckrodt Medical Inc., St. Louis, MO, USA) and skin temperature was measured using skin transducers (Concept Engineering, Old Saybrook, CT). Measures of skin temperature was collected from 12 skin sites, and average skin temperature was calculated based on the following proportions: forehead 7%, chest 9.5%, biceps 9%, forearm 7%, abdomen 9.5%, lower back 9.5%, upper back 9.5%, front calf 8.5%, back calf 7.5%, hamstrings 9.5% and hand 4% (Hardy and Dubois, 1938). The esophageal probe was inserted through the nose and the tip of the thermocouple placed at the level of the left atrium, or to a depth of one-quarter the standing height of the subject (Mekjavic and Rempel, 1990). 2.4. Cardiorespiratory response Heart rate (HR) was measured using a Polar heart rate monitor (Polar FS2C Fitness Heart Rate Monitor System, Polar USA, Lake Success, NY, USA) and was recorded every 5 min during the baseline and passive periods, and every 1 min during exercise.

K. O’Hearn et al. / Journal of Thermal Biology 55 (2016) 39–46

41

Ventilation was measured using an automated metabolic cart (MOXUS, Applied Electrochemistry Inc., Pittsburgh, PA, USA) and expressed in STPD. Measurements were taken every 30 min during the baseline, passive periods and continuously during exercise.

available enzymatic assay reagents from Wako Diagnostics (Wako Chemicals, Richmond, VA, USA).

2.5. Body weight changes

Data are expressed as mean 795% confidence interval (CI) or median (interquartile range) in case of non-normal data distribution. Appropriate transformations of variables were performed when normal distribution was not observed for parametric statistical testing. T ̅ skin, T ̅ es, HR, V̇ O2, V̇ E, Ḣ , fuel oxidation and circulating lipids were analyzed using a two-way repeated measure ANOVA to assess changes across conditions and over time (SPSS version 15.0). Significant differences were followed up with Bonferroni's multiple-comparisons post-hoc tests. Absolute protein oxidation, quantified only during the baseline period, was analyzed using a paired Student's t test. The threshold for significance was set at pr 0.05. All values are presented as mean 7standard error (SE).

Change in body weight throughout the trial were determined from the nude weight recorded at baseline, following passive heating and following exercise and are presented in kilograms (kg). 2.6. Total external work, exercise duration and rate of perceived exertion Total external work performed was calculated for the exercise portion of the trial and presented in kilojoules (kJ). Exercise duration was determined as the total amount of time, in minutes, of exercise completed. Rate of perceived exertion (RPE) was obtained during exercise using a 10-point category scale (Borg, 1982). Participants were shown the 10-point scale and asked to give their RPE every 5 min.

2.9. Statistical analysis

3. Results 3.1. Thermal Response

2.7. Fuel selection. Oxygen consumption ( V̇ O2) and carbon dioxide production ( V̇ CO2) were measured using the MOXUS automated metabolic cart and expressed in STPD. Total protein (RPox), CHO (RGox), and lipid (RFox) oxidation rates (in g min  1) were calculated as described previously (Livesey and Elia, 1988).

(

)

(

)

RPox g min−1 = 2.9 × UREAurine gmin−1

(1)

RGox g min−1 = 4.59 VCO2 l min−1 –3.23 V̇ O2 l min−1

(

)

(

)

(

)

(2)

RFox g min−1 = −1.70 VCO2 l min−1 + 1.7 V̇ O2 lmin−1

(

)

(

)

(

)

(3)

where V̇ CO2 (l min  1) and V̇ O2 (l min  1) were corrected for the volumes of O2 and CO2 corresponding to protein oxidation (1.010 and 0.843 g l  1, respectively). RPox was estimated from urinary urea excretion (UREAurine) in urine collected for 90 min during the baseline period and this value was used for the duration of the trial. Urine urea concentration was determined using a commercial urine assay kit (BioAssay Systems, CA, USA). Energy potentials of 16.3 kJ g  1 (CHO), 40.8 kJ g  1 (lipids), and 19.7 kJ g  1 (proteins) were used to calculate the relative contributions of each fuel to total heat production (Elia, 1991; Peronnet and Massicotte, 1991). Area under the curve (AUC) was used to calculate total oxidation of CHO, lipids and proteins using the Trapezoidal rule method of Riemann summation

AUC Fuelox = Σ Fuelox ⎡⎣ ( y1 +yi )/2 × Dt⎤⎦

(4)

where Fuelox represents the oxidation of CHO, lipids or proteins in mg min  1, and Dt is the time interval of 30 min. Total fuel oxidation is presented as a function of time to give the rate of utilization. 2.8. Plasma lipids Blood samples were collected in sodium EDTA tubes at baseline, midway through the passive heating period (T60), at the end of the passive period (T120) and post exercise. Upon collection, blood samples were placed on ice and spun in a centrifuge. Plasma was separated and stored at  80 °C until analyzed. Plasma NEFA, TG, PL and CHOL concentrations were assessed using commercially

Changes in T ̅ es, T ̅ skin and Ḣ measured during baseline, passive heating and exercise are presented in Fig. 1. T ̅ skin was similar between conditions at baseline (32.6 °C, 95% CI: 32.3–32.9 °C for HEAT and 32.7 °C, 95% CI: 32.3–33.3 °C for CON), but increased during the passive period for HEAT and was significantly higher than CON from 30 min (35.8 °C, 95% CI: 35.5–36.0 °C vs. 32.1 °C, 95% CI: 31.6–32.6 °C) to the end of passive heating (36.1 °C, 95% CI: 35.8–36.4 °C vs. 32.2 °C, 95% CI: 31.7–32.7 °C). T ̅ skin increased during exercise for both conditions and was significantly higher in HEAT (37.1 °C, 95% CI: 37.1–37.6 °C) than CON (33.7 °C, 95% CI: 33.4–34.0 °C) at termination of exercise (po 0.001). T ̅ es was not different between conditions at baseline (36.7 °C, 95% CI: 36.6– 36.8 °C for HEAT and 36.7 °C, 95% CI: 36.6–36.7 °C for CON) and for the first 60 min of passive heat exposure. T ̅ es increased for HEAT over the last 60 min of passive and was significantly higher than CON at 90 min (37.2 °C, 95% CI: 37.0–37.5 °C vs. 36.7 °C, 95% CI: 36.5–36.9 °C) and at 120 min (37.4 °C, 95% CI: 37.1–37.7 °C vs. 36.7 °C, 95% CI: 36.5–36.9 °C; p o0.001). During exercise, T ̅ es increased for both conditions and was significantly higher for HEAT (39.0 °C, 95% CI: 38.6–39.4 °C) than CON (37.6 °C, 95% CI: 37.4– 37.8 °C) at termination of exercise (p o0.001). Ḣ was not different between conditions during baseline or passive (p ¼0.433). Ḣ increased during exercise from 6.2 kJ min  1 (95% CI: 5.7–6.8, HEAT) and 5.7 kJ min  1 (95% CI: 5.1–6.3, CON) at the end of the passive period to 59.4 kJ min  1 (95% CI: 52.5–66.3, HEAT) and 56.5 kJ min  1 (95% CI: 50.6–62.4, CON) at the termination of exercise with no difference between conditions (p ¼0.08). 3.2. Cardiorespiratory response Changes in V̇ E and HR are presented in Fig. 2. HR was similar between conditions at baseline (65 bpm, interquartile range: 58– 75 for HEAT and 63 bpm, interquartile range: 58–73 for CON) then increased for HEAT and was significantly higher than CON from 30 min (68 bpm, interquartile range: 66–77 vs. 61 bpm, interquartile range: 54–66) until the end of the passive period (79 bpm, interquartile range: 77–85 vs. 62 bpm, interquartile range: 54–67). HR increased during exercise in both conditions, and was significantly higher in HEAT (181 bpm, interquartile range: 178–183) compared to CON (162 bpm, interquartile range: 155 to 168) at the termination of exercise (po 0.01). V̇ E was not different between conditions at baseline (8.2 l min  1 95% CI: 7.4–9.0 for HEAT and

42

K. O’Hearn et al. / Journal of Thermal Biology 55 (2016) 39–46

Passive

Baseline

Exercise

Passive

Baseline

Exercise

40

*

n=7

n=6

100

n=4

CON

-1

Ventilation (l min )

T sk ( C)

38 36 34 32

40

38 200

34

80

n=7

n=6

n=4

60

H (kJ min-1)

n=7

-1

36

.

n=4

*

0

n=7

HEAT

*

n=6

40 20

n=4

Heart rate (beats min )

Tes ( C)

n=6

n=4

60

30 CON

n=6 n=7

HEAT

80

160

120

*

80

40 -60

- 30

0

30

60

90

120

125

130

135

140

145

150

Time (min)

40

Fig. 2. Changes in heart rate (A) and ventilation (B) measured in men during passive heat exposure and subsequent exercise in the heat (●) compared to control (○). Values are means 7 SE. *Significantly higher than CON, p o 0.05.

20

0 -60

-30

0

30

60

90

120

125

130

135

140

145

150

Time (min) Fig. 1. Changes in mean skin ( T ̅ skin) (A) and esophageal temperatures ( T ̅ es) (B) as well as rates of heat production (Ḣ ) (C) measured in men during passive heat exposure and subsequent exercise in the heat (●) compared to control (○). Values are means 7 SE. *Significantly higher than CON, po 0.05.

8.3 l min  1 95% CI: 7.8–8.7 for CON) and for the first 90 min of passive. Over the last 30 min of passive, V̇ E increased for HEAT and was significantly higher than CON at 120 min (9.1 l min  1 , 95% CI: 8.5–9.7 vs. 8.5 l min  1 , 95% CI: 7.7–9.2; p o0.05). V̇ E also increased throughout exercise for both HEAT and CON, and was significantly higher for HEAT (71.1 l min  1, 95% CI: 63.4–78.7) than for CON (56.6 l min  1, 95% CI: 52.3–60.8) at the termination of exercise (p o0.01). 3.3. Changes in weight Body mass was significantly lower after exercise compared to baseline values (p o0.05) with reductions of 1.0% (95% CI: 0.2–1.7) for HEAT and 0.8% (95% CI: 0.3–1.4) for CON. However, there were no significant differences between conditions (p ¼ 0.76). 3.4. Total external work, exercise duration and rate of perceived exertion Heat exposure resulted in a significant decrease in external work from 265.1 kJ (95%CI: 246.7–283.4) for CON to 227.8 kJ (95% CI: 187.7–267.8) for HEAT (p o0.05) during exercise. Four participants were unable to complete 30 min of exercise in HEAT. As a result, mean exercise duration was lower for HEAT (26 min, 95%CI: 22–30) than CON (30 min, 95%CI: 30–30) although this difference

did not reach statistical significance (p ¼0.054). At termination of exercise, RPE was significantly higher for HEAT 9.5 (95%CI: 8.5– 10.0) than for CON 5.0 (95%CI: 4.3–5.7) (p o0.01). 3.5. Fuel selection The degree of thermal stress, combined with exercise, caused two participants to hyperventilate frequently during exercise and made fuel oxidation calculations for these participants impossible. Therefore, the results presented for fuel selection are based on six participants. Absolute rates of CHO, lipid, and protein oxidation are presented in Fig. 3A. Rates of CHO (p ¼0.24) and lipid (p ¼0.846) oxidation were not different between conditions. Absolute rates of CHO oxidation were 69 mg min  1 (95% CI: 31–106) and 95 mg min  1 (95% CI: 43–147) during baseline, and 109 mg min  1 (95% CI: 72–146) and 85 mg min  1 (95% CI: 40–131) during passive, and absolute rates of lipid oxidation were 85 mg min  1 (95% CI: 61–110) and 73 mg min  1 (95% CI: 50–95) at baseline and 74 mg min  1 (95% CI: 57–92) and 74 mg min  1 (95% CI: 52–96) during passive for HEAT and CON respectively. CHO oxidation increased during exercise averaging 2573 mg min  1 (95% CI: 2229– 2916) and 2393 mg min  1 (95% CI: 2089–2696) for HEAT and CON, respectively. Lipid oxidation increased during exercise for both HEAT (244 mg min  1, 95% CI: 166–323) and CON (268 mg min  1, 95% CI: 162–374). Absolute protein oxidation (measured following the baseline period) was similar between conditions (69 mg min  1, 95% CI: 31–106 for HEAT and 73 mg min  1, 95% CI: 60–85 for CON; p¼ 0.549). Changes in relative contributions of CHO, lipids and proteins to total Ḣ are presented in Fig. 3B. Heat production and absolute and relative fuel oxidation are summarized in Table 2. There was no effect of condition for CHO (p ¼0.984) or lipid (p¼ 0.777) contribution to total heat production. Values for relative CHO oxidation were 19 % (95% CI: 8–30) and 26 % (95% CI: 12–41) during

K. O’Hearn et al. / Journal of Thermal Biology 55 (2016) 39–46

43

Fig. 3. Changes in absolute rates (A) and relative contribution (B) of CHO (i), lipid (ii), and protein oxidation (iii) measured in men during passive heat exposure and subsequent exercise in the heat (●) compared to control (○). Values are means 7 SE.

baseline; and 29 % (95% CI: 19–38) and 24 % (95% CI: 11–37) during passive for HEAT and CON respectively. Contributions of CHO increased during exercise for both HEAT (79 %, 95% CI: 73–84) and for CON (76 %, 95% CI: 69–82). Lipids contributed 58 % (95% CI: 46– 70) and 50 % (95% CI: 38–61) during baseline and 49 % (95% CI: 39– 59) and 51 % (95% CI: 40–63) during passive for HEAT and for CON. Relative contribution of lipids decreased during exercise to 19 (95% CI: 13–24) for HEAT and 21 (95% CI: 14–28) for CON. Protein contribution was not different between conditions (p ¼0.569).

3.6. Plasma Lipids Changes in plasma concentrations of NEFA, TG, PL and CHOL are presented in Table 3. NEFA concentrations were not different between HEAT and CON at baseline or midway through passive heating (T60), but increased over time for HEAT and were significantly higher than CON at the end of passive heating (618 mM, 95% CI: 479–757 and 391 mM, 95% CI: 270–511) and at the termination of exercise (2036 mM, 95% CI: 1604–2469 and 1351 mM, 95% CI: 1002–1699; po 0.01). TG (p ¼0.877), PL (p ¼0.694) and CHOL (p ¼0.401) concentrations were not different between conditions.

Table 2 Absolute oxidation rates and relative contributions of lipids, CHO and proteins to total heat production before heat exposure (23 °C), during passive heat exposure (42 °C) and during exercise in the heat (42 °C) compared to control (CON; 23 °C). CON

Ḣ kJ min  1

HEAT

Baseline 23 °C

Passive 23 °C

6.0 (5.2–6.7)

5.8 (5.1–6.5)

Exercise 23 °C

Baseline 23 °C

Passive 42 °C

Exercise 42 °C

51.6 (45.5–57.7)

6.0 (5.1–6.8)

6.2 (5.6–6.7)

53.3 (46.8–59.7)

Lipids mg min  1 %Ḣ

73 (50–95) 49.5 (37.8–61.2)

74 (52–96) 51.4 (39.8–63.0)

268 (162–374) 20.9 (13.8–27.9)

85 (61–110) 57.8 (46–69.6)

74 (57–92) 49.0 (39.4–58.6)

244 (166–323) 18.6 (13.4–23.9)

CHO mg min  1 %Ḣ

95 (43–147) 26.4 (11.8–40.9)

85 (40–131) 24.2 (11.1–37.3)

2393 (2089–2696) 75.8 (69.2–82.3)

69 (31–106) 19.0 (8.4–29.6)

109 (72–146) 28.8 (19.4–38.2)

2573 (2229–2916) 78.8 (73.3–84.3)

Proteins mg min  1 %Ḣ

73 (60–85) 24.2 (20.2–28.1)

24.5 (21.4–27.6)

69 (50–88) 23.2 (15.8–30.5)

22.2 (15.5–28.9)

2.8 (2.4–3.2)

Values are means with 95% confidence interval (CI) in parentheses. n ¼6 subjects (exercise). Ḣ , total heat production; CHO, carbohydrates.

2.6 (1.8–3.4)

K. O’Hearn et al. / Journal of Thermal Biology 55 (2016) 39–46

(1604–2469)* (343–1147) (1.35–1.83) (3607–4808)

4. Discussion

(425–772) (363–1127) (1.22–1.68) (3203–4505)

618 784 1.56 3744

(479–757)* (363–1205) (1.40–1.73) (3193–4295)

2036 745 1.59 4208

This study quantifies alterations in in whole-body lipid utilization and circulating plasma lipids during prolonged passive heat exposure and subsequent exercise in the heat. Changes in absolute rates of utilization and relative contributions of lipids, CHO and proteins remained unchanged during heat exposure when compared to the control condition. In addition, of all circulating lipid fractions measured, only NEFAs concentrations were found to be higher (  37%) following prolonged passive heat exposure and higher by the end of 30 min exercise at 50% V̇ O2 max (  34%) (Table 3). These increases in plasma NEFA occurred without any modifications in whole body lipid oxidation suggesting an increase in lipolysis and fatty acid mobilization in the heat (Fig. 3A and B). Finally, our assessment of exercise performance in the heat also showed that half of the participants (4 of 8) were unable to complete the exercise portion during heat exposure but exact reasons remain uncertain.

(270–511) (363–1152) (1.42–1.83) (3158–4742)

1351 719 1.60 4334

(1002–1699) (377–1061) (1.41–1.80) (3510–5157)

420 774 1.54 4025

(238–602) (408–1140) (1.33–1.76) (3199–4851)

598 745 1.45 3854

4.1. Passive heating

Significantly higher than CON, po 0.05. *

Values are means with 95% confidence interval (CI) in parentheses.

391 758 1.63 3950 (352–500) (430–1044) (1.42–1.62) (3255–4717) 426 738 1.52 3986 (193–360) (428–1124) (1.31–1.66) (3670–4812) 277 776 1.48 4241 NEFA (mM) TG (mM) PL (g/L) CHOL (mM)

Passive T120 Passive T60 Baseline T0 Baseline T0

Passive T60

Passive T120

Post Ex

HEAT CON

Table 3 Plasma NEFA, TG, PL and cholesterol concentrations before (Baseline), during (T60), and after (T120) passive heat exposure and after exercise in the heat (Post Ex) compared to control (CON).

Post Ex

44

The 42 °C (  24.6%RH) thermal heat stress used in this study resulted in significant increases in heart rate, ventilation, skin temperature and core temperature by the end of the passive period (Fig. 1). These changes indicate that the heat exposure was sufficient to increase core temperature by 0.7 °C by the end of 120 min, without modifying heat production or fuel selection (Fig. 1 and Table 2). This is in contrast to our hypothesis, were we expected that any changes in plasma NEFA would be associated closely to changes in lipid oxidation. Previous research has shown that increases in NEFA levels are generally matched by an increase in lipid oxidation and a concomitant decrease in glucose uptake and oxidation (Ferrannini et al., 1983; Groop et al., 1991). Conversely, we observed no differences in either the relative or absolute contributions of lipids to total energy production, in spite an almost 40% greater increase in plasma NEFA concentrations by the end of passive heat stress compared to control condition. The higher NEFA concentrations following passive heating is consistent with observations made in previous studies (Eddy et al., 1976; Yamamoto et al., 2003) were increased circulating NEFA following exposure to heat at rest has been reported. Lipid oxidation was not measured in either of these studies. A number of mechanisms could explain this increase in NEFA levels. Because plasma NEFA concentrations represent the balance between the rate of appearance (Ra) and the rate of disappearance (Rd) of NEFA into or out of the circulation, any heat stimulated factor that could modify NEFA Ra (i.e. lipolysis) or NEFA Rd (i.e. lipid oxidation, re-esterification of NEFA into TG) could ultimately be responsible for the higher NEFA concentrations. The large majority of circulating NEFA comes from lipolysis in adipose tissue (Jensen, 2003), and catecholamines are important regulators of this process. Although not measured in this study, several authors have confirmed higher circulating levels of both catecholamines (epinephrine and norepinephrine) following passive heat exposure (Iguchi et al., 2012; Powers et al., 1982). An increase in catecholamine release could be responsible for the increase in concentration via an increase in NEFA Ra. During rest, a small infusion of epinephrine, 3-fold above resting values, has been show to increase plasma NEFA (Galster et al., 1981). Taken together, these findings indicate that catecholamines could explain the increase in NEFA concentrations observed in this study during passive heat exposure. While this supports the idea that an increase in Ra is, at least in part, responsible for the increase in circulating NEFA, one must also consider Rd when examining plasma concentrations. The Rd of NEFA is largely dependent on two processes – NEFA taken up from the plasma can either be oxidized for

K. O’Hearn et al. / Journal of Thermal Biology 55 (2016) 39–46

energy or re-esterified back into TG (Jensen, 2003). The effects of heat exposure on NEFA re-esterification have not been previously determined; therefore it is unclear if NEFA re-esterification was altered in the current study. Our results show that the relative and absolute contribution of lipids to whole body lipid oxidation is not changed during passive heat stress. This suggests that Rd of NEFA may not be affected by heat exposure and that an increase in lipolysis is a contributing factor to the higher NEFA concentrations observed. 4.2. Exercise in the heat following passive pre-heating Following the passive heating period, heart rate, ventilation, skin and core temperature were higher compared to control conditions, resulting in a different pre-exercise baseline. These same measures were higher following exercise in the heat compared to control conditions. The degree of hyperthermia achieved from preheating and exercise in the heat was enough to significantly increase the perceived workload of the exercise portion and induce hyperventilation in two participants. As with passive heat exposure, there were no significant changes in heat production or fuel selection. In spite of the increase in circulating NEFA (  34% higher than CON), both relative and absolute whole-body lipid oxidation were not affected. Our observation of higher NEFA concentrations following exercise in the heat conflicts with the findings of previous researchers who have not found significant differences in NEFA concentrations compared to control conditions (Fink et al., 1975; Gregson et al., 2002; Jentjens et al., 2002; Nielsen et al., 1990). An examination of the methodology used in these studies could explain the discrepancy with our results. Previous authors have used a counterbalanced study design (Nielsen et al., 1990); administered a glucose solution to participants prior to and during exercise (Jentjens et al., 2002); or used higher exercise intensities of 70–85% V̇ O2max (Fink et al., 1975; Gregson et al., 2002). Consumption of CHO prior to exercise (Coyle et al., 1997), higher exercise intensities (Romijn et al., 1993); and increased circulating lactate associated with higher exercise intensities (Boyd et al., 1974; Green et al., 1979) have all been shown to inhibit lipolysis and could have attenuated a heat-induced increase in circulating NEFA. This study shows that increases in NEFA were not related to changes in whole-body lipid oxidation, as lipid utilization was not altered during exercise in the heat. However, while circulating NEFA are the main energy source for lipid oxidation, intramuscular triglyceride (IMTG) can also contribute, particularly in trained subjects (Brooks and Fahey, 2005) and at higher exercise intensities (Romijn et al., 1993). The indirect calorimetry used in this study measures whole-body lipid oxidation, and does not distinguish between oxidation of NEFA or IMTG. It is therefore not known if the relative proportions of these fuel sources are affected by heat stress. A change in the proportions of NEFA and IMTG being oxidized could alter the amount of NEFA being oxidized without a measurable change in total lipid oxidation. The present study is unique in that an extended passive preheating period was employed prior to exercise in the heat. The degree of heat stress during passive heating resulted in elevated NEFA concentrations, possibly in response to increased catecholamines. As a result NEFA concentrations were already higher in HEAT prior to the start of exercise. It is not known whether the heat stress during exercise contributed to an increase in NEFA, or whether elevated NEFA levels were simply maintained during exercise in the heat, resulting in greater concentrations in the heat compared to control at termination of exercise. Given the extreme importance of lipids to providing and sustaining ATP production, further research should be conducted to establish the mechanisms that contribute to the increase in circulating NEFA following heat

45

stress. 4.3. Effects of heat exposure on performance Four out of eight participants were unable to complete the 30 min exercise in the heat. No difference in heat production, skin or core temperature was observed between finishers and nonfinishers. Blood samples were analyzed for stress and inflammatory markers (data not presented), but no difference could be observed between finishers and non-finishers. Previous studies have reported increased levels of circulating pro-inflammatory markers during heat exposure (Barberio et al., 2015; Fortes et al., 2013; Walker et al., 2015; Wright-Beatty et al., 2014), and increased levels of pro-inflammatory markers have been suggested to have a detrimental effect on physical performance (Brinkley et al., 2009; Cesari et al., 2004; Robson-Ansley et al., 2004), but no difference in either stress or inflammatory markers were observed between finishers and non-finishers in this study (data analysed but not presented). We could speculate in differences in training status having an effect on the participant’s ability to complete the 30 min exercise in the heat (Cheung and McLellan, 1998), but none of our measured data are able to explain why four out of eight participants could not complete exercise in the heat. It is important to note that the small sample sized used in this study could limit ability to generalize the results from this study. Even though eight subjects were recruited for this study, heat exposure during exercise was sufficient to result in four subject being unable to complete the 30 min at 50%VO2max. This remains an important finding in this study as it clearly indicates that passive heat exposure for 2 h and subsequent exercise bout has major effects on performance. As result, the reduce sample size decrease to some extent our statistical power for determining changes in lipid metabolism. However, the effects on plasma NEFA concentration remained substantial which was determined using nonparametric statistical test.

5. Conclusion In summary, this study shows that plasma NEFA concentrations are elevated by heat exposure at rest. This change is not matched by an increase in whole-body lipid oxidation. During exercise in the heat subsequent to pre-heating, this increase in NEFA is maintained and whole-body lipid oxidation remains unchanged. In addition, passive heat stress combined with heat exposure during exercise reduces work capacity and increases the perceived exertion of a given exercise intensity.

Grants This study was supported by the Natural Sciences and Engineering Research Council of Canada to F. Haman and Canadian Institutes of Health Research graduate scholarship to K O’Hearn.

Disclosures No conflicts of interest, financial or otherwise, are declared by the authors.

Funding Natural Sciences and Engineering Research Council of Canada (326907-2011) to F. Haman and Canadian Institutes of Health

46

K. O’Hearn et al. / Journal of Thermal Biology 55 (2016) 39–46

Research (98256) graduate scholarship to K O’Hearn.

Acknowledgments The authors would like to acknowledge the contribution and assistance provided by Denis Blondin, Jean-François Mauger and Bernard Pinet. We would also like to thank the participants for their collaboration. A special thanks to Elizabeth Lavoie and Mathieu Lebreton for their assistance with blood sampling, and to Dr. Nathalie Chapados and Dr. Tara Reilly for reviewing the manuscript. This work was funded by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC Canada) to F. Haman, and a scholarship from the Canadian Institutes of Health Research (CIHR) to K. O’Hearn as well as from the Ontario Graduate Scholarship (OGS). The results of the present study do not constitute endorsement by the American College of Sports Medicine.

References Arngrimsson, S.A., Petitt, D.S., Borrani, F., Skinner, K.A., Cureton, K.J., 2004. Hyperthermia and maximal oxygen uptake in men and women. Eur. J. Appl. Physiol. 92, 524–532. Barberio, M.D., Elmer, D.J., Laird, R.H., Lee, K.A., Gladden, B., Pascoe, D.D., 2015. Systemic LPS and inflammatory response during consecutive days of exercise in heat. Int. J. Sport. Med. 36, 262–270. Borg, G.A., 1982. Psychophysical bases of perceived exertion. Med. Sci. Sports Exerc. 14, 377–381. Boyd 3rd, A.E., Giamber, S.R., Mager, M., Lebovitz, H.E., 1974. Lactate inhibition of lipolysis in exercising man. Metab.: Clin. Exp. 23, 531–542. Brinkley, T.E., Leng, X., Miller, M.E., Kitzman, D.W., Pahor, M., Berry, M.J., Marsh, A.P., Kritchevsky, S.B., Nicklas, B.J., 2009. Chronic inflammation is associated with low physical function in older adults across multiple comorbidities. J. Gerontol. Ser. A: Biol. Sci. Med. Sci. 64A, 455–461. Brooks, G., Fahey, T., 2005. Exercise Physiology: Human Bioenergetics and its Applications. McGraw-Hill Companies, New York, NY. Cesari, M., Penninx, B.W., Pahor, M., Lauretani, F., Corsi, A.M., Rhys Williams, G., Guralnik, J.M., Ferrucci, L., 2004. Inflammatory markers and physical performance in older persons: the InCHIANTI study. The journals of gerontology. J. Gerontol. Ser. A: Biol. Sci. Med. Sci. 59, 242–248. Cheung, S.S., McLellan, T.M., 1998. Heat Acclimation, Aerobic Fitness, and Hydration Effects on Tolerance During Uncompensable Heat Stress. Coyle, E.F., Jeukendrup, A.E., Wagenmakers, A.J., Saris, W.H., 1997. Fatty acid oxidation is directly regulated by carbohydrate metabolism during exercise. Am. J. Physiol. 273, E268–275. Dolny, D.G., Lemon, P.W., 1988. Effect of ambient temperature on protein breakdown during prolonged exercise. J. Appl. Physiol. 64, 550–555. Eddy, D.O., Sparks, K.E., Turner, C.L., 1976. The adipokinetic effect of hyperthermic stress in man. Eur. J. Appl. Physiol. Occup. Physiol. 35, 103–110. Elia, M., 1991. Energy equivalents of CO2 and their importance in assessing energy expenditure when using tracer techniques. Am. J. Physiol. 260, E75–88. Febbraio, M.A., 2000. Does muscle function and metabolism affect exercise performance in the heat? Exerc. Sport Sci. Rev. 28, 171–176. Febbraio, M.A., Snow, R.J., Stathis, C.G., Hargreaves, M., Carey, M.F., 1994. Effect of heat stress on muscle energy metabolism during exercise. J. Appl. Physiol. 77, 2827–2831. Ferrannini, E., Barrett, E.J., Bevilacqua, S., DeFronzo, R.A., 1983. Effect of fatty acids on glucose production and utilization in man. J. Clin. Investig. 72, 1737–1747. Fink, W.J., Costill, D.L., Van Handel, P.J., 1975. Leg muscle metabolism during exercise in the heat and cold. Eur. J. Appl. Physiol. Occup. Physiol. 34, 183–190. Fortes, M.B., Di Felice, U., Dolci, A., Junglee, N.A., Crockford, M.J., West, L., HillierSmith, R., Macdonald, J.H., Walsh, N.P., 2013. Muscle-damaging exercise increases heat strain during subsequent exercise heat stress. Med. Sci. Sport.

Exerc. 45, 1915–1924. Galster, A.D., Clutter, W.E., Cryer, P.E., Collins, J.A., Bier, D.M., 1981. Epinephrine plasma thresholds for lipolytic effects in man: measurements of fatty acid transport with [l-13C]palmitic acid. J. Clin. Investig. 67, 1729–1738. Green, H.J., Houston, M.E., Thomson, J.A., Sutton, J.R., Gollnick, P.D., 1979. Metabolic consequences of supramaximal arm work performed during prolonged submaximal leg work. J. Appl. Physiol. 46, 249–255. Gregson, W.A., Drust, B., Batterham, A., Cable, N.T., 2002. The effects of prewarming on the metabolic and thermoregulatory responses to prolonged submaximal exercise in moderate ambient temperatures. Eur. J. Appl. Physiol. 86, 526–533. Groop, L.C., Bonadonna, R.C., Shank, M., Petrides, A.S., DeFronzo, R.A., 1991. Role of free fatty acids and insulin in determining free fatty acid and lipid oxidation in man. J. Clin. Investig. 87, 83–89. Hardy, J., Dubois, E.F., 1938. The technique of measuring radiation and convection. J. Nutr. 15, 461–475. Hargreaves, M., Angus, D., Howlett, K., Conus, N.M., Febbraio, M., 1996. Effect of heat stress on glucose kinetics during exercise. J. Appl. Physiol. 81, 1594–1597. Iguchi, M., Littmann, A.E., Chang, S.H., Wester, L.A., Knipper, J.S., Shields, R.K., 2012. Heat stress and cardiovascular, hormonal, and heat shock proteins in humans. J. Athl. Train. 47, 184–190. Jensen, M.D., 2003. Fate of fatty acids at rest and during exercise: regulatory mechanisms. Acta Physiol. Scand. 178, 385–390. Jentjens, R.L., Wagenmakers, A.J., Jeukendrup, A.E., 2002. Heat stress increases muscle glycogen use but reduces the oxidation of ingested carbohydrates during exercise. J. Appl. Physiol. 92, 1562–1572. Jeukendrup, A.E., 2003. Modulation of carbohydrate and fat utilization by diet, exercise and environment. Biochem. Soc. Trans. 31, 1270–1273. Livesey, G., Elia, M., 1988. Estimation of energy expenditure, net carbohydrate utilization, and net fat oxidation and synthesis by indirect calorimetry: evaluation of errors with special reference to the detailed composition of fuels. Am. J. Clin. Nutr. 47, 608–628. MacDougall, J.D., Reddan, W.G., Layton, C.R., Dempsey, J.A., 1974. Effects of metabolic hyperthermia on performance during heavy prolonged exercise. J. Appl. Physiol. 36, 538–544. Mekjavic, I.B., Rempel, M.E., 1990. Determination of esophageal probe insertion length based on standing and sitting height. J. Appl. Physiol. 69, 376–379. Morris, J.G., Nevill, M.E., Boobis, L.H., Macdonald, I.A., Williams, C., 2005. Muscle metabolism, temperature, and function during prolonged, intermittent, highintensity running in air temperatures of 33 degrees and 17 degrees C. Int. J. Sport. Med. 26, 805–814. Nielsen, B., Savard, G., Richter, E.A., Hargreaves, M., Saltin, B., 1990. Muscle blood flow and muscle metabolism during exercise and heat stress. J. Appl. Physiol. 69, 1040–1046. Peronnet, F., Massicotte, D., 1991. Table of nonprotein respiratory quotient: an update. Can. J. Sport Sci. 16, 23–29. Powers, S.K., Howley, E.T., Cox, R., 1982. A differential catecholamine response during prolonged exercise and passive heating. Med. Sci. Sport. Exerc. 14, 435–439. Robson-Ansley, P.J., De Milander, L., Collins, M., Noakes, T.D., 2004. Acute Interleukin-6 administration impairs athletic performance in healthy, trained male runners. Can. J. Appl. Physiol. 29, 411–418. Romijn, J.A., Coyle, E.F., Sidossis, L.S., Gastaldelli, A., Horowitz, J.F., Endert, E., Wolfe, R.R., 1993. Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. Am. J. Physiol. 265, E380–391. Starkie, R.L., Hargreaves, M., Lambert, D.L., Proietto, J., Febbraio, M.A., 1999. Effect of temperature on muscle metabolism during submaximal exercise in humans. Exp. Physiol. 84, 775–784. Walker, A., Keene, T., Argus, C., Driller, M., Guy, J.H., Rattray, B., 2015. Immune and inflammatory responses of Australian firefighters after repeated exposures to the heat. Ergonomics, 1–8. Weber, J.M., Haman, F., 2005. Fuel selection in shivering humans. Acta Physiol. Scand. 184, 319–329. Wright-Beatty, H.E., McLellan, T.M., Larose, J., Sigal, R.J., Boulay, P., Kenny, G.P., 2014. Inflammatory responses of older Firefighters to intermittent exercise in the heat. Eur. J. Appl. Physiol. 114, 1163–1174. Yamamoto, H., Zheng, K.C., Ariizumi, M., 2003. Influence of heat exposure on serum lipid and lipoprotein cholesterol in young male subjects. Ind. Health 41, 1–7. Young, A.J., Sawka, M.N., Levine, L., Cadarette, B.S., Pandolf, K.B., 1985. Skeletal muscle metabolism during exercise is influenced by heat acclimation. J. Appl. Physiol. 59, 1929–1935.