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. 2023 Feb 9;55(7):1160–1171. doi: 10.1249/MSS.0000000000003136

Contribution of High-Intensity Interval Exercise in the Fasted State to Fat Browning: Potential Roles of Lactate and β-Hydroxybutyrate

SUJIN KIM 1, DONG-HO PARK 2,3, SANG-HYUN LEE 2, HYO-BUM KWAK 2,3, JU-HEE KANG 1,3
PMCID: PMC10242519  PMID: 36790381

ABSTRACT

Purpose

Fat browning contributes to energy consumption and may have metabolic benefits against obesity; however, the potential roles of lactate and β-hydroxybutyrate (β-HB) in fat browning remain unclear. We investigated the roles of a single bout of aerobic exercise that increases lactate and β-HB levels in the fasted state on the regulation of fat browning in rats and humans.

Methods

Male Sprague–Dawley rats were exposed to 24-h fasting and/or a single bout moderate-intensity aerobic exercise (40 min): sedentary (CON), exercise (ND-EX), fasting (FAST), and exercise + fasting (F-EX). Adult men (n = 13) were randomly assigned into control with food intake (CON), exercise with intensity at onset of blood lactate accumulation in the fasted state (F-OBLA), and high-intensity interval exercise in the fasted state (F-HIIE) until each participant expended 350 kcal of energy. For evaluating the effects of exercise intensity in rats, we conducted another set of animal experiment, including groups of sedentary fed control, fasting control, and exercise with moderate-intensity or HIIE for 40 min after a 24-h fasting.

Results

Regardless of fasting, single bout of exercise increases the concentration of lactate and β-HB in rats, but the exercise in the fasted state increases the β-HB level more significantly in rats and humans. F-EX-activated fat browning (AMPK–SirT1–PGC1α pathway and PRDM16) and thermogenic factor (UCP1) in white fat of rats. In rats and humans, exercise in the fasted state increased the blood levels of fat browning–related adipomyokines. In particular, compared with F-OBLA, F-HIIE more efficiently increases free fatty acid as well as blood levels of fat browning adipomyokines in humans, which was correlated with blood levels of lactate and β-HB. In rats that performed exercise with different intensity, the higher plasma lactate and β-HB levels, and higher expression of p-AMPK, UCP1, and PRDM16 in white adipose tissue of HIIE group than those of moderate-intensity group, were observed.

Conclusions

A single bout of aerobic exercise in the fasted state significantly induced fat browning–related pathways, free fatty acid, and adipomyokines, particularly F-HIIE in human. Although further evidence for supporting our results is required in humans, aerobic exercise in the fasted state with high intensity that increase lactate and β-HB may be a modality of fat browning.

Key Words: AEROBIC EXERCISE, FASTING, LACTATE, β-HB, FAT BROWNING

A characteristic of brown and beige adipose tissues (BAT) is the dissipation of energy by conversion into heat instead of storage. Pharmacological and nonpharmacological interventions that convert energy-storing white adipose tissue (WAT) to thermogenic BAT (so-called fat browning) may confer beneficial effects on weight reduction and metabolic complications of obesity, although whether the induction of browning in human will be a promising strategy to combat obesity is not clear (1). Furthermore, fat browning strategies may contribute to the maintenance of weight loss and metabolic benefits in the longer term (2,3) compared with dietary restriction for acute weight loss, although the direct evidence of browning of human WAT is lacking (4). Pharmacotherapy and cold exposure have both been suggested as inducers of fat browning. However, adverse drug effects (5) or insufficient metabolic benefits of low-temperature treatment (6) limit their potential, respectively. Therefore, development of a new therapeutic modality supporting the possible beneficial effects of fat browning and increasing BAT mass on obesity is required. For example, aerobic exercise in the fasted state accelerates use of fat instead of using carbohydrates or glycogen, which increases the production of ketone bodies (7) and promotes lipid metabolism (8). Furthermore, aerobic exercise in the fasted state that can induce lactate and ketones (e.g., β-hydroxybutyrate [β-HB]) may have beneficial effects on body weight reduction and induce mitochondrial biogenesis in rodents (9,10) and elevate fat oxidation and lipolysis in human (11) In particular, lactate and β-HB induce thermogenic genes in WAT in a redox-dependent manner (12) and activation of brown fat (13) in rodents, respectively.

In addition to the regulation of energy homeostasis by lactate and/or β-HB (14), these signaling metabolic intermediates can directly and/or indirectly regulate the production of adipomyokines in rodents (15,16). For example, lactate induces irisin production in mice through sirtuin 1 (SirT1)-dependent upregulation of fibronectin type III domain containing 5 (FNDC5), a precursor of irisin (15), and in human, postexercise incremental changes of lactate significantly correlates with changes of postexercise irisin concentrations in an exercise intensity-dependent manner (17). Most adipomyokines secreted from muscle and/or adipose tissues by muscle contraction, pharmacological intervention, or diet may assist in diverse physiological processes such as muscle regeneration and hypertrophy (18), fat browning and thermogenesis (19,20), regulation of immunity (21), and neuroprotection (22). Accordingly, adipomyokines have attracted attention as major targets for the prevention and treatment of metabolic diseases such as obesity and type 2 diabetes (23,24).

Although there is still some debate whether the induction of hyperketonemia by a ketogenic diet has metabolic benefits, numerous studies support the concept of metabolic benefit of lactate and/or β-HB generated by fasting and aerobic exercise (25,26). However, it is not clear whether aerobic exercise in the fasted state augments the upregulation of these metabolic intermediates and fat browning–associated adipomyokines. Furthermore, it remains to be determined whether the association of aerobic exercise in the fasted state with alteration of adipomyokines and metabolic intermediates, if any, is dependent on the type or intensity of aerobic exercise, in both rodents and human.

In this study, we investigated the role of a single bout of aerobic exercise in the fasted state that increases lactate and β-HB levels compared with the effect of exercise in the fed state on the regulation of fat browning and adipomyokine expression in WAT in rats. Given that the production of lactate and β-HB is closely associated with exercise intensity, we applied two types of exercise intensity to evaluate the differential effects of exercise with different intensities (i.e., continuous moderate- and high-intensity interval exercise [HIIE]) on fat browning and adipomyokine expression. In addition, we evaluated the effects of aerobic exercise with different intensities in the fasted state on the blood levels of lactate, β-HB, lipids, and adipomyokines in young healthy adults.

MATERIALS AND METHODS

Animal experimental design

All animal experimental procedures were conducted in accordance with the guidelines of the National Institutes of Health and the Korean Academy of Medical Science. This study was approved by the Institutional Animal Care and Use Committee of the Inha University (INHA 181004-597). Eight-week-old (230–250 g) male Sprague–Dawley rats were randomly divided into four groups (n = 9 per group): sedentary control group in the fed state (CON), moderate-intensity aerobic exercise group in the fed state (ND-EX), 24-h fasting sedentary group (FAST), and moderate-intensity aerobic exercise after a 24-h fast (F-EX). Previous studies suggest that the fasted state over 14 h in human or 24 h in rodents upregulates ketone bodies in blood (27,28); therefore, we limited food consumption for 24 h before exercise in the FAST and F-EX groups to increase the level of systemic β-HB. To measure the effect of a single bout of aerobic exercise on the levels of systemic lactate and β-HB, we measured the blood levels of lactate and β-HB before a single bout of aerobic exercise (Pre) (exercise 1: exercise intervention for blood test), immediately after exercise (Post), and at 1 h recovery after exercise (1 h Rec). After 1 wk acclimation, rats performed acute aerobic exercise for 40 min (5-min warm-up [8 m·min−1], 32-min main exercise [20 m·min−1], and a 3-min cooldown [5 m·min−1]) on a motorized, speed-controlled treadmill apparatus (DJ2-242, Dual Treadmill Daejong, Ltd., Seoul, Korea). The exercise intensity was moderate, reaching approximately 60%–75% of V̇O2max (29). Detailed protocols are described in the Supplemental Methods (see Supplemental Digital Content, http://links.lww.com/MSS/C796). One week after the blood test for clearance of the exercise effect, we treated animals with an additional single bout of aerobic exercise with the same protocol (exercise 2: exercise for tissue analysis). Immediately after exercise 2 (see Supplemental Fig. S1A, Supplemental Digital Content, Animal and clinical experiment flow, http://links.lww.com/MSS/C796), rats were anesthetized with an intraperitoneal injection of Zoletil (50 mg·kg−1) and intramuscular Rompun (5–10 mg·kg−1), and blood was collected by cardiac puncture. Periepididymal white adipose tissue (eWAT) was collected, immediately frozen in liquid nitrogen, and stored at −80°C until analysis. Additional experimental protocols for comparing the effects of exercise intensity in the fasted state that is comparable with human study are described in the Supplemental Methods (see Supplemental Digital Content, http://links.lww.com/MSS/C796). Briefly, we measured the maximal aerobic exercise capacity by estimating the speed until exhaustion, then we applied exercise with moderate-intensity (20 m·min−1 for 32 min) or HIIE (8 cycles of 2 min running at 30 m·min−1 followed by 2 min running at 10 m·min−1).

Human participants and exercise intervention

The clinical study followed the Declaration of Helsinki. The Institutional Review Board and Ethical Committee of Inha University (Incheon, Republic of Korea) approved the study (approval no. 19053-4A).

Classification of the groups (n = 13, male volunteers per group) was configured as follows: control group (CON) with a calorie-restricted diet (250 kcal) before the experiment, a group performing aerobic exercise at onset of blood lactate accumulation (OBLA) intensity in the fasted state (F-OBLA), and a group performing HIIE in the fasted state (F-HIIE). Excluding CON, F-OBLA and F-HIIE performed a single bout of aerobic exercise that consumed 350 kcal in the same way. Overnight fasting was maintained for more than 14 h, and only water intake was allowed. The exercise test for determining V̇O2max, lactate threshold (LT), and OBLA was as previously described (28–30). Detailed protocols for diet management and aerobic exercise are described in the Supplemental Methods and Supplementary Figure S1B (see Supplemental Digital Content, http://links.lww.com/MSS/C796). All groups were assigned in a random order every week in a crossover design. Body composition, including lean body mass, fat mass, percent fat, and body mass index, was assessed using a bioelectrical impedance analysis apparatus (Inbody 770; Inbody, Seoul, Korea) according to the manufacturer’s instruction. Participants were restricted to have food intake and exercise before analysis for at least 5 h. The characteristics of the participants are summarized in Table 1.

TABLE 1.

Anthropometric and incremental test results of participants.

Variables Mean ± SD
Age (yr) 23.00 ± 1.96
Height (cm) 177.62 ± 3.12
Body weight (kg) 75.64 ± 8.61
Fat weight (kg) 14.35 ± 7.26
Lean body weight (kg) 34.68 ± 2.44
Fat (%) 18.36 ± 7.05
Body mass index (kg·m−2) 24.20 ± 2.99
V̇O2max (mL·kg−1⋅min−1) 44.60 ± 4.74
%V̇O2max at OBLA intensity 49.07 ± 3.54
%V̇O2max at HIIE intensity Low intensity: 24.38 ± 2.14
High intensity: 96.08 ± 4.20
HRmax (bpm) 194.23 ± 8.03
LAmax (mmol·L−1) 15.19 ± 3.55
Final RPE 18.15 ± 0.69
Final RER 1.24 ± 0.06
Speed at V̇O2max (km·h−1) at 3% grade 10.62 ± 1.08
Speed at OBLA (km·h−1) at 3% grade 6.74 ± 0.61
Speed at LT (km·h−1) at 3% grade 4.80 ± 0.00
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Data are presented as mean and SD. V̇O2max, maximal oxygen consumption; LAmax, maximal lactate concentration; OBLA, onset of blood lactate accumulation (~4.0 mmol·L−1); LT, lactate threshold (~2.0 mmol·L−1).

Blood analysis

To evaluate change in blood concentrations of lactate and β-HB in the Pre, Post, and 1 h Rec, capillary blood samples were drawn from the rat’s tail and human fingertips. Lactate and β-HB levels in blood were analyzed using the lactate meter (Lactate Pro 2; ARKRAY, Kyoto, Japan) and the β-HB meter (FreeStyle Optium Neo H; Abbott, Champaign, IL), respectively, via a drop of blood determined by reflectance photometry or colorimetric β-HB assay kit (Cayman Chemical, Ann Arbor, MI). Blood drawn from the heart of rats and the antecubital vein of participants was analyzed for lipid profile and adipomyokines. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and free fatty acid (FFA) were measured with commercial kits (Labtest®, Sao Paulo, Brazil) following the manufacturer’s instructions. Glucose was measured by the glucose oxidase method (Beckman Instruments, Palo Alto, CA). Plasma insulin and leptin were measured with an enzyme-linked immunosorbent assay using commercially available kits (R&D Systems, Minneapolis, MN).

Adipomyokines (plasma irisin, fibroblast growth factor 21 [FGF-21], follistatin-like 1 [FSTL-1], and brain-derived neurotrophic factor [BDNF]) were also measured using the xMAP-Luminex multiplex immunoassay platform (Luminex Co., Austin, TX) with the Multiplex Magnetic Bead panel kit (Millipore, Billerica, MA) following the manufacturer’s instructions. Briefly, assay buffer containing beads conjugated with capture antibodies was pipetted into 96-well plates and incubated for 10 min at room temperature (RT). The sample (25 μL) was added and incubated with agitation on a plate shaker for 16 h at 4°C. After washing three times, 25 μL of detection antibodies was added to each well and incubated with agitation for 2 h at RT. Then streptavidin–phycoerythrin (25 μL) was added and agitated at RT for 30 min. Wells were washed three times by vacuum filtration, and 150 μL of sheath fluid was added. Finally, we read the plate using the Luminex®200™. The xPONENT® software was used to measure the concentration of adipomyokines. Blood analysis for additional experiment was performed by using ELISA kits (see Supplemental Methods, Supplemental Digital Content, http://links.lww.com/MSS/C796).

Western blot analysis

Protein from eWAT was extracted with 50 mM Tris, 150 mM sodium chloride, and 1.0% NP-40 containing both a protease inhibitor and a phosphatase inhibitor cocktail (RIPA buffer; Sigma-Aldrich, St. Louis, MO). Equivalent samples (10–20 μg of protein in each well) were electrophoresed using 8%–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, NY) in transfer buffer (25 mM Tris–HCl, 192 mM glycine, and 20% methanol). After 1 h blocking with 5% (w/v) fat-free milk in TBST buffer, membranes were incubated overnight at 4°C with primary antibodies as appropriate (see Supplemental Table S1, Supplemental Digital Content, Antibodies used for the expression of protein in the eWAT of rats, http://links.lww.com/MSS/C796). Membranes were then incubated for 2 h at RT with horseradish peroxidase–conjugated goat antirabbit IgG or goat antimouse IgG antibodies. Signals were detected with enhanced chemiluminescence reagents (Millipore) and analyzed using a ChemiDoc System (Bio-Rad, Hercules, CA).

Statistical analysis

All statistical analyses in this investigation were conducted using the Statistical Package for the Social Sciences version 26 software, with data expressed as mean and SD. In the animal study, continuous variables were tested for normality using the Shapiro–Wilk test. Then one-way ANOVA and the Kruskal–Wallis test were used to assess group differences in protein expression levels and other measurements according to standard statistical assumptions. After finding significant differences in univariate tests, the Tukey HSD and the Mann–Whitney U test as a post hoc analysis were used to determine a location of significant differences. Differences in groups (CON, FAST, EX, and F-EX) over time (Pre, Post, and 1 h Rec) for lactate and β-HB were identified using a two-way (4 × 3) mixed-measures ANOVA. After finding significant differences in multivariate and univariate tests, the Bonferroni test as a post hoc analysis was used to determine a location of significant differences.

In the clinical study, differences in groups (CON, F-OBLA, and F-HIIE) over time (Pre, Post, and 1 h Rec) among all variables were identified using a two-way (3 × 3) repeated-measures ANOVA in a complete within-subjects design. After finding significant differences in multivariate and univariate tests, the Bonferroni test as a post hoc analysis was used to determine a location of significant differences. Significance was set at an alpha level of 0.05 for all statistical tests.

RESULTS

Effects of aerobic exercise on the levels of blood lactate, β-HB, lipids, and adipomyokines in rats

Changes in the levels of blood lactate, β-HB, and glucose were significantly different among groups at different times (group–time interaction) (Fig. 1A). In the case of lactate levels as the main effect on time, a significant increase was only observed in the exercise groups (ND-EX and F-EX) in the Post compared with the Pre. The rise decreased at 1 h Rec. However, unlike lactate, β-HB levels increased more at 1 h Rec compared with the Post. This phenomenon was more prominent in the F-EX group. Collectively, lactate levels significantly increased upon exercise regardless of food intake, whereas β-HB levels, which were significantly increased by both fasting and exercise, further increased at 1 h Rec. As the main effect on time, glucose levels decreased significantly in the Post and 1 h Rec compared with the Pre only in the moderate-intensity aerobic exercise group in the fed state (ND-EX). In addition, regardless of exercise, the fasting groups (FAST and F-EX) showed significantly lower glucose levels than the normal diet groups (CON and ND-EX).

FIGURE 1.

FIGURE 1

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Effects of aerobic exercise in the fed and fasted state on lactate, β-HB, lipids, insulin, and adipomyokines in rats. A, Responses of blood lactate, β-HB, glucose, and insulin levels according to the presence or absence of aerobic exercise after normal diet or fasting. A significant time–group interaction for the lactate, β-HB, and glucose levels was observed (P < 0.05, P < 0.001, P < 0.05, respectively). $P < 0.05, $$P < 0.01, $$$P < 0.001 vs Pre; &P < 0.05, &&&P < 0.001 vs Post (see the Supplementary Table S2 in details, Supplemental Digital Content, http://links.lww.com/MSS/C796). For insulin and β-HB at 1 h after exercise recovery (1 h Rec), the levels in FAST or F-EX group were significantly different with those in CON or ND-EX. ***P < 0.001 vs CON; ###P < 0.001 vs ND-EX (one-way ANOVA with Tukey multiple comparisons). B, Responses of blood lipid profiles according to the presence or absence of aerobic exercise after normal diet or fasting. *P < 0.05, **P < 0.01, ***P < 0.001 vs CON; #P < 0.05 vs ND-EX. C, Responses of blood adipomyokines according to the presence or absence of aerobic exercise after normal diet or fasting. * P < 0.05, **P < 0.01, ***P < 0.001 vs CON; ##P < 0.01, ###P < 0.001 vs ND-EX; and †P < 0.05, †††P < 0.001 vs FAST (one-way ANOVA with Tukey multiple comparisons). Values are expressed as mean and individual values (n = 9 per group). Pre, preexercise; Post, postexercise; 1 h Rec, 1 h recovery after exercise; CON, control group in the fed state (normal diet, ND); ND-EX, moderate-intensity aerobic exercise group in the fed state; FAST, 24-h fasting sedentary group; F-EX, moderate-intensity aerobic exercise after 24-h fasting.

In the lipid profile, LDL-C, TG, and FFA were altered by fasting and/or exercise, which was particularly noticeable during exercise in the fasted state (Fig. 1B). In the FAST and F-EX groups, insulin and leptin levels were significantly lower than those in CON and ND-EX groups. To evaluate the effects of aerobic exercise in the fasted state on the level of adipomyokines, which directly and/or indirectly regulate fat browning, we measured the plasma levels of several adipomyokines. The level of FGF-21, a fat browning marker for lipid oxidation and regulator of the expression of uncoupling protein 1 (UCP1) in adipocytes (30), was significantly increased by exercise in the fasted state. In both the ND-EX and the FAST groups, the levels of FGF-21 were higher than those in the CON, but not significantly. By contrast, irisin levels were upregulated by exercise regardless of food intake. Levels of FSTL-1, which activate WAT browning (31), showed lower levels in the FAST group (P < 0.05) than that in the EX group. Levels of BDNF, an exercise-inducible myokine involved in fatty acid metabolism and mitochondrial biogenesis (32,33), were higher in the F-EX group compared with the CON (P < 0.05) and FAST groups (P < 0.05) (Fig. 1C). Detailed results of statistical analysis were presented in Supplemental Tables S2 and S3 (see Supplemental Digital Content, Blood lactate, β-HB, and glucose concentrations by group in rats, and Effects of aerobic exercise in the fed and fasted state on lipid profiles and adipomyokines in rats, http://links.lww.com/MSS/C796).

Effects of aerobic exercise on the expression of fat browning–related markers in the eWAT of rats

Adenosine monophosphate–activated protein kinase (AMPK) has been demonstrated to be a crucial sensor of cellular energy mechanism and plays an important role in the development and maintenance of BAT in a SirT1–PGC1α-dependent manner (34). Therefore, we evaluated the effects of a single bout of aerobic exercise in both the fed and the fasted states on a pathway related to mitochondrial biogenesis (AMPK–SirT1–PGC1α pathway) and on the expression of fat browning–related and thermogenesis-related proteins (PRDM16 and UCP-1) in eWAT (35). As shown in Figure 2A, the levels of phosphorylated AMPK and its downstream effectors ACC, SirT1, and PGC1α were significantly higher in the eWAT of ND-EX, FAST, and F-EX groups compared with the CON. PR domain containing 16 (PRDM16) is a transcription factor that constitutes a molecular switch that determines the fate of progenitor cells becoming muscle cells or brown fat cells (36). It strongly induces the expression of fat browning factors including UCP1 through binding with PGC1α (35). In our experiments, the levels of PRDM16 and UCP1 in the eWAT of the F-EX group but not others were significantly higher compared with the CON.

FIGURE 2.

FIGURE 2

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Effects of aerobic exercise in the fed and fasted state on the expression of protein-inducing fat browning in the eWAT of rats. A, In rats exercising in the fasted state (F-EX), levels of proteins related to mitochondrial biogenesis (AMPK–SirT1–PGC1α pathway) or fat browning–related transcriptional activation (PRDM16 and UCP1) were significantly higher than controls (CON). Exercise in the fed state (ND-EX) also activated the AMPK–SirT1–PGC1α pathway but did not induce PRDM16 and UCP1. B, Regardless of food intake, FGF-21 levels in groups with exercise were higher than controls. Level of FNDC5 in the ND-EX was significantly higher than that in the CON, whereas that in the F-EX showed a trend of upregulation. Values are expressed as mean and SEM (n = 7–8 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs CON; #P < 0.05 vs ND-EX (one-way ANOVA or Kruskal–Wallis test with multiple comparisons). CON, control group in the fed state (normal diet, ND); ND-EX, moderate-intensity aerobic exercise group in the fed state; FAST, 24-h fasting sedentary group; F-EX, moderate-intensity aerobic exercise after 24-h fasting; p-AMPK, phosphorylated AMPK; p-ACC, phosphorylated acetyl-CoA carboxylase; PGC1-α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; FNDC5, fibronectin type III domain containing protein 5.

Given that FGF-21 and irisin are adipomyokines involved in fat browning and activate AMPK-mediated energy metabolism (37,38), we evaluated the expression of FGF-21 and FNDC5 (precursor of irisin) in eWAT. Consistent with the blood level of FGF-21, we found that the expression levels of FGF-21 in the eWAT of ND-EX, FAST, and F-EX groups were significantly higher compared with the CON, whereas the protein levels of FNDC5 in the eWAT of the ND-EX group were significantly higher compared with the CON. The protein levels of FNDC5 in the F-EX group showed a similar trend (Fig. 2B).

Comparisons of responses to two different intensities of exercise in the fasted state (F-OBLA vs F-HIIE) in humans

As mentioned above, each participant’s V̇O2max was determined while using a continuous, incremental, fixed grade (3%) treadmill protocol to determine the V̇O2, heart rate (HR), and velocity of treadmill associated with LT, OBLA, and V̇O2max. In addition, the V̇O2 values at V̇O2-LT, V̇O2-OBLA, and V̇O2-V̇O2max intensities were used as a reference to calculate caloric expenditure during exercise trials in both OBLA (continuously moderate exercise) and HIIE. Based on these data, the differences in metabolic and physiological responses between the groups (F-OBLA vs F-HIIE) under the same conditions of consuming about 350 kcal were calculated (Table 2).

TABLE 2.

Average exercise duration, HR, and concentrations of lactate and β-HB in two exercise intensities under the same condition, fasting, and consuming 350 calories in clinical trials.

Variables in Exercise Trial F-OBLA F-HIIE
Treadmill exercise time (min) 41.40 ± 8.74 34.85 ± 2.61*
Energy expenditure (kcal) 351.46 ± 3.43 347.83 ± 6.92
Average HR (bpm) 133.67 ± 12.33 161.33 ± 8.97***
Average lactate level (mmol·L−1) IAE 5.52 ± 0.63 16.85 ± 3.06***
Average β-HB level (mmol·L−1) IAE 0.37 ± 0.30 0.22 ± 0.15
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*P < 0.05.

***P < 0.001.

IAE, immediately after exercise; F-OBLA, aerobic exercise at OBLA intensity in the fasted state; F-HIIE, aerobic exercise at HIIE (1-min V̇O2max intensity + 2-min LT intensity) in the fasted state.

Treadmill exercise time in F-HIIE was significantly shortened compared with F-OBLA, whereas the HR and the lactate concentration in F-HIIE were higher than those in F-OBLA. When the exercise intensity in each group was analyzed based on HRmax, the average HR of F-OBLA was equivalent to 69% of HRmax (133.7 bpm), whereas that of F-HIIE corresponded to 83% of HRmax (161.3 bpm). Although the difference in average HR was about 20%, the blood lactate level in F-HIIE (16.85 ± 3.06 mmol·L−1) was about three times higher than that in F-OBLA (5.52 ± 0.63 mmol·L−1), indicating that F-HIIE increased blood lactate more dramatically than F-OBLA. Notably, there was no significant difference between exercise intensities in the level of β-HB after aerobic exercise in the fasted state with the same energy consumption (~350 kcal) (Table 2).

Differential responses of blood lactate, β-HB, glucose, insulin, lipid profiles, and adipomyokines according to the intensity of exercise in fasted state in humans

Under different intensities of aerobic exercise in the fasted state, we measured the blood levels of lactate, β-HB, glucose, and insulin in Pre, Post, and 1 h Rec in humans to compare with the effect of aerobic exercise in the fasted state in rats. Blood lactate levels were dramatically increased after aerobic exercise in the fasted state regardless of exercise intensity (interaction effect; P < 0.001) compared with the CON. After 1 h Rec, the increased blood lactate levels by both exercises in the fasted state were reduced (P < 0.001), while the levels were still higher than the levels in the Pre (P < 0.001). In comparison with F-OBLA, F-HIIE showed significantly higher lactate levels in the Post (P < 0.001) (Fig. 3A). Regardless of exercise intensity, β-HB levels were significantly increased by exercise in the fasted state (P < 0.05), which further increased for at least 1 h Rec (P < 0.05). Interestingly, unlike lactate levels that were decreased after 1 h Rec, β-HB levels of both exercise intensities at 1 h Rec were approximately 1.5- to 1.6-fold higher than the levels in the Pre (from 0.37 to 0.60 mM for F-OBLA and from 0.40 to 0.59 mM for F-HIIE) (Fig. 3A). Blood glucose levels after exercise in F-HIIE showed no significant difference from those in the Pre, whereas the levels in F-OBLA group at 1 h Rec (77.0 ± 6.8 mg·dL−1, P < 0.05) were significantly lower than the Pre (82.6 ± 6.7 mg·dL−1). Insulin levels were also significantly reduced in F-OBLA at 1 h Rec (4.14 ± 1.75 μU·mL−1, P < 0.01) but not in F-HIIE compared with the Pre (6.28 ± 3.06 μU·mL−1) (Fig. 3A).

FIGURE 3.

FIGURE 3

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Effects of exercise intensities in the fasted state on the levels of blood lactate, β-HB, glucose, insulin, lipid profiles, and adipomyokines in humans. A, Significant time–group interaction effects were observed between groups (CON vs F-OBLA, CON vs F-HIIE, and F-OBLA vs F-HIIE) at lactate level, but not β-HB, glucose, and insulin levels. All of these variables showed significant time differences as the main effect on time. In the figure, only the main effect on time is presented (see the Supplementary Table 4 in details, Supplemental Digital Content, http://links.lww.com/MSS/C796). B, All lipid profiles except FFA showed significant interaction effects. Only the FFA level in HIIE was significantly increased in the Post compared with the Pre (see the Supplementary Table 4 in details, Supplemental Digital Content, http://links.lww.com/MSS/C796). C, Only the level of IL-6 showed significant interaction effects. The pattern difference of irisin, BDNF, and IL-6 according to the time period between the groups was notable in F-HIIE, unlike the CON and the F-OBLA groups (see the Supplementary Table 4 in details, Supplemental Digital Content, http://links.lww.com/MSS/C796). Data are expressed as mean and individual values (n = 12–13 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs Pre; and #P < 0.05, ##P < 0.01, ###P < 0.001 vs Post. Pre, preexercise; Post, postexercise; 1 h Rec, 1 h recovery after exercise; CON, control group without exercise; F-OBLA, aerobic exercise at OBLA intensity in the fasted state; F-HIIE, aerobic exercise at high intensity with interval in the fasted state; IL-6, interleukin 6; LT, lactate threshold (~2.0 mmol·L−1 of lactate).

The significant time–group interaction in the levels of TC, HDL-C, LDL-C, and TG between CON and F-OBLA as well as F-HIIE was observed. Significant interactions were observed between the F-OBLA and the F-HIIE in lipid profiles except for FFA. This suggests that the difference in lipid profiles may depend on the intensity of single bout aerobic exercise. By contrast, the level of FFA showed a significant main effect only in F-HIIE, and this significantly increased in the Post compared with the Pre and then decreased to the Pre level at 1 h Rec (Fig. 3B).

In animal experiments, aerobic exercise induced a significant increase in irisin levels regardless of dietary intake. The level of leptin was significantly decreased only in FAST and F-EX groups, whereas the level of BDNF increased significantly only in the F-EX group compared with the CON. Moreover, the level of FFA in the FAST and F-EX groups was significantly higher than that in the CON and ND-EX groups. These results can be inferred as evidence that aerobic exercise in the fasted state can increase the use of fat as an energy source and be more advantageous as a condition for fat browning than aerobic exercise in the fed state. However, because the response of adipomyokines according to the intensity of aerobic exercise (i.e., OBLA vs HIIE) could not be confirmed in animals, we evaluated the change in the levels of adipomyokines according to exercise intensity. Among myokines, the interaction was found only at IL-6 concentration, which was induced by the dramatic increase in IL-6 in the Post only in F-HIIE (F-HIIE vs CON and F-HIIE vs F-OBLA). Significant main effects of leptin, BDNF, and irisin in the time period were found in exercise groups. Leptin levels were significantly decreased at 1 h Rec compared with the Pre and Post in both exercise groups regardless of exercise intensity. However, the concentrations of irisin and BDNF were significantly increased only in F-HIIE in the Post and 1 h Rec compared with the Pre. By contrast, the concentrations of FSTL-1 and FGF-21 did not show any statistically significant difference (Fig. 3C). Detailed results of statistical analysis were presented in Supplemental Tables S4 and S5 (see Supplemental Digital Content, Effects of exercise types in the fasted state on the levels of blood lactate, β-HB, glucose, insulin, and lipid profiles in humans, and Effects of exercise types in the fasted state on the levels of adipomyokines in humans, http://links.lww.com/MSS/C796).

In previous studies (27,28), lactate and β-HB are proposed as intermediate metabolites that induce fat browning, so the association with adipomyokines according to the increase of these metabolites was evaluated. As lactate concentration reached the highest level in the Post while β-HB did at 1 h Rec, we evaluated the correlation between the level of lactate or β-HB and the blood variables at that time. The concentration of lactate in the Post showed a significant correlation of positive direction with FFA, IL-6, BDNF, FGF-21, and irisin at that time (Post) but an inverse correlation with glucose and insulin, which was weakening at 1 h Rec. The concentration of β-HB at postexercise or 1 h Rec also showed a significant correlation of positive direction with irisin, FGF-21, and FFA and an inverse correlation with glucose at both postexercise and 1 h Rec, but levels of leptin and insulin showed a significant correlation with the level of β-HB at 1 h Rec and at postexercise, respectively (Fig. 4 and Supplemental Fig. S2, Supplemental Digital Content, Correlation of plasma levels of lactate and β-HB with plasma glucose and insulin after single bout of exercise, http://links.lww.com/MSS/C796).

FIGURE 4.

FIGURE 4

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All blood variables showing significant correlation according to changes in lactate or β-HB immediately after exercise (Post) and 1 h Rec periods in human. A, Blood variables correlated with changes in lactate concentrations in the Post and 1 h Rec. As lactate increased immediately after exercise (Post), both glucose and insulin showed inverse correlations, whereas FFA, IL-6, BDNF, irisin, and FGF-21 showed a positive correlation. B, Levels of blood variables including glucose, irisin, FGF-21, IL-6, FFA, and insulin at immediately after exercise (Post) significantly and the levels of glucose, irisin, FGF-21, leptin, and FFA at 1 h Rec correlated with levels of β-HB in the Post and 1 h Rec, respectively. The Pearson correlation coefficient method is applied. Post, immediately after exercise; 1 h Rec, 1 h recovery after exercise; FNDC5, fibronectin type III domain containing protein 5; IL-6, interleukin 6.

Effects of exercise with different intensity in the fasted state on fat browning–related markers in eWAT, and lactate, β-HB, lipids, and adipomyokines in blood of rats

As we observed the differential effects of exercise in the fasted state on several blood markers according to the exercise intensity (i.e., OBLA intensity and HIIE) in human, we conducted another animal experiments with design similar to the clinical study (moderate-intensity and HIIE; see details in Methods and Supplemental Methods, Supplemental Digital Content, http://links.lww.com/MSS/C796). In eWAT, both intensities of exercise in the fasted state significantly activated AMPK–SirT1–PGC1α pathway and upregulated the fat browning (PRDM16) and thermogenic (UCP1) factors comparing the fed control group. In addition, we found that the HIIE, but not exercise of moderate intensity, upregulated the levels of p-AMPK, PRDM16, and UCP1 expression in eWAT as compared with those of FAST group (Fig. 5A). Protein levels of FGF-21 and FNDC5 in the eWAT of both exercise groups were higher than those of fed control group, without differential effects according to the intensity (Fig. 5B). In the analysis of blood collected immediately after exercise, we found that plasma levels of lactate and FFA in HIIE group were significantly higher than those in exercise group of moderate intensity, and β-HB showed a higher tendency in HIIE than moderate-intensity group, consistent with findings in human study. In addition, the levels of FGF-21, irisin, and BDNF in HIIE were higher than control groups. Levels of glucose, insulin, TC, LDL-C and TG in both exercise groups were lower than those in the fed control group (Supplemental Fig. S3, Supplemental Digital Content, Effects of exercise types in the fasted state on the levels of blood lactate, β-HB, glucose, insulin, lipid profiles, and adipomyokines in rats, http://links.lww.com/MSS/C796). Combined, overall findings in the animal study for evaluating effects of exercise intensity might support the clinical findings.

FIGURE 5.

FIGURE 5

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Effects of aerobic exercise with different intensity in the fasted state on the expression of proteins in the eWAT of rats. A, In rats exercising in the fasted state with moderate-intensity (F-EX(M)) or high-intensity interval (F-EX(HIIE)) protocol, levels of proteins related to mitochondrial biogenesis (AMPK–SirT1–PGC1α pathway) and fat browning transcriptional activation (PRDM16 and UCP1) were significantly higher than fed (CON) or fasted (FAST) sedentary controls. B, Regardless of exercise intensity, FGF-21 levels in groups with exercise were significantly higher than fed controls (CON). Levels of FNDC5, precursor protein of irisin, showed a higher tendency in both exercise groups than those in control groups. (Graphs in A and B) *P < 0.05, **P < 0.01, ***P < 0.001 vs CON; and #P < 0.05, ##P < 0.01 vs FAST (Kruskal–Wallis test with post hoc Dunn’s multiple comparison). Values are expressed as mean and SEM (n = 7–8 per group). CON, sedentary control group in the fed state; FAST, 24-h fasting sedentary group; F-EX(M), moderate-intensity aerobic exercise after 24-h fasting; F-EX(HIIE), HIIE after 24-h fasting.

DISCUSSION

Aerobic exercise in the fasted state is a strategy to increase lipolysis instead of glycogen as an energy source during exercise in adipose and muscle tissue, which may help weight loss (39). Although it has been suggested that aerobic exercise in the fasted state is effective for weight reduction (40), the effective exercise protocols (e.g., intensity or type of exercise) after fasting are variable across the studies (39). Furthermore, the molecular links between aerobic exercise in the fasted state and body fat reduction are not fully elucidated, which hinders researchers from establishing a clinical intervention. In this study, we demonstrated several main findings. First, exercise after fasting increased the blood concentration of lactate and β-HB in rats and humans. Second, aerobic exercise in the fasted state activated fat browning (AMPK/SirT1/PGC1α pathway and PRDM16) and thermogenic factors (UCP1) in the eWAT of rats. Third, aerobic exercise in the fasted state contributed to the upregulation of adipomyokines related to fat browning in the blood and adipose tissue of rats. Finally, although we did not confirm the expression of fat browning–related markers in human adipose tissue, we observed that aerobic exercise in the fasted state, F-HIIE in particular, upregulated circulating lactate, β-HB, fat browning–related adipomyokines (i.e., BDNF and irisin), and FFA in humans. Collectively, the benefits of aerobic exercise in the fasted state that induces the upregulation of lactate and β-HB may, at least in part, result from the activation of lipolysis and fat browning–related factors, the effects of F-HIIE being stronger than the F-OBLA.

It has been reported that lactate and β-HB stimulate thermogenesis in white adipocytes through redox-dependent UCP1 upregulation (12) or AMPK-dependent manner (41). We consistently observed that a single bout of exercise, a condition of acute increase in circulating lactate and β-HB, activated the AMPK pathways and upregulated fat browning–associated adipokines (FGF-21 and FNDC5) in the eWAT of rats, regardless of food intake. AMPK and SirT1, through the reciprocal activation, are known to activate PGC1α, which is involved in mitochondrial biogenesis (42–44) and lipolysis during aerobic exercise (45). Because activation of PGC1α is dependent on AMPK activation (42), the activation of the AMPK–SirT1–PGC1α pathways by aerobic exercise in rat eWAT is likely to increase mitochondrial biogenesis. Interestingly, we observed that the expression of thermogenic genes (UCP1 and PRDM16) was also upregulated by a single bout of aerobic exercise in the fasted state, but not by exercise with a normal diet. SirT1 deacetylates PPAR-γ to promote binding with PRDM16, which stimulates fat browning–related gene expression in WAT (46). In addition, Carriere et al. (12) reported that lactate- or β-HB-mediated fat browning, and thermogenesis are dependent on a PPAR-γ transduction pathway. Therefore, our results suggest that aerobic exercise in the fed state activates AMPK-mediated upregulation of mitochondrial biogenesis and lipolysis, whereas aerobic exercise in the fasted state stimulates additional mechanisms that increase thermogenesis in WAT and fat browning. Considering the intensity of exercise, we observed the higher levels of blood lactate and β-HB in HIIE group than moderate-intensity group. Therefore, the stronger upregulation of p-AMPK and PRDM16 by exercise with high intensity in the fasted state, compared with moderate intensity, may be associated with the higher upregulation of lactate and/or β-HB, although the differential regulation of signaling pathways by these metabolic intermediates should be evaluated. However, it should be noted that the browning of human omental WAT with little propensity to acquire a brown phenotype is rarely induced unless WAT is exposed to extreme condition (4,47). Although we observed the induction of PRDM16 in rat WAT by exercise in the fasted state and significant correlation between the plasma levels of lactate/β-HB and fat browning–related adipomyokines, further direct evidence for phenotype conversion of WAT by lactate/β-HB induction in human is required.

Irisin is a myokine produced by proteolysis of FNDC5. Acute and chronic exercises induce irisin production in a PGC1α-dependent manner in skeletal muscle (17,19), which contributes to fat browning, thermogenesis, and alteration of lipid metabolism (48). In addition, irisin is produced by WAT (49), which is upregulated by exercise (19). Consistent with previous studies (49,50), we observed that exercise increased the expression of irisin in eWAT and plasma of rats, regardless of food intake. AMPK activation in WAT and skeletal muscle upregulates irisin expression (51), and reciprocally irisin activates the AMPK pathway (19). Therefore, the activation of the AMPK/SirT1/PGC1α pathway in the eWAT of exercised rats is likely associated with increased levels of irisin and lipolysis (52). In addition, circulating levels of irisin in humans were increased by F-HIIE, but not F-OBLA, indicating that irisin upregulation may depend on exercise intensity in humans. Furthermore, we observed a significant correlation between the exercise-induced elevation of lactate and the elevation of adipomyokines including irisin, BDNF, FGF-21, and IL-6, which may further support the role of lactate in the regulation of fat browning adipomyokine. Unlike lactate, β-HB showed only a weak correlation with irisin but not with others, so it was somewhat difficult to determine the effect of β-HB on adipomyokines.

FGF-21 is mainly secreted in the liver and fat tissues and is known to be secreted by fasting and exercises (53). SirT1 regulates the expression level of FGF-21 in liver and fat tissues (37), so it is plausible that the exercise-induced upregulation of SirT1 in the eWAT of rats may contribute to the induction of FGF-21. However, in humans, the circulating level of FGF-21 after exercise, regardless of exercise intensities, was not significantly upregulated. WAT lipolysis and thermogenesis may be regulated by hepatic FGF-21-dependent and -independent mechanisms (54), which warrants further research on the molecular link between hepatic and adipose FGF-21 regulation by aerobic exercise in the fasted state.

BDNF is another myokine known to be associated with fat browning through AMPK activation (55); therefore, the increased levels of circulating BDNF by exercise in the fasted state in both rats and humans may have metabolic benefits. Both F-OBLA and F-HIIE increased the levels of circulating lactate and β-HB, while circulating BDNF was increased by F-HIIE but not by F-OBLA. Therefore, the association between the intensity of aerobic exercise in the fasted state and the BDNF expression in humans should be further investigated.

Meanwhile, the effects of aerobic exercise in the fasted state according to exercise intensity on blood lipids in humans were variable. When compared with the CON, both exercise groups showed a decrease in blood lipids (TC, HDL-C, LDL-C, and TG), but there was also a marked difference in patterns between the two exercise groups (F-OBLA vs F-HIIE). Furthermore, in F-HIIE, FFA levels were significantly increased immediately after exercise compared with preexercise. A single bout of exercise intervention increased lipolysis (56); therefore, decreased levels of lipids after exercise were expected. Nevertheless, our results suggest that the F-HIIE may activate lipolysis more than the F-OBLA. Furthermore, the moderate to strong correlation between lactate level and postexercise FFA level may support the significant lipolysis by the F-HIIE-induced elevation of lactate, although we did not directly measure the lipolysis.

Combined, our study provides evidence that aerobic exercise in the fasted state, F-HIIE in particular, may induce browning of WAT through the induction of thermogenic factors and fat browning adipomyokines. Although further evidence for supporting a direct relationship between upregulation of lactate and β-HB by aerobic exercise in the fasted state and fat browning in humans is required, aerobic exercise in the fasted state for more than 14 h may be a modality of the “fat browning exercise.”

Our study has several limitations. First, the molecular changes in the eWAT of rats were not confirmed in human adipose tissue, which may limit the interpretation of the changes in human adipomyokines after aerobic exercise in the fasted state. In particular, whether differential effects of exercise with different intensities on fat browning–related benefits are associated with intensity-dependent regulation of signaling pathways in adipose tissue should be further elucidated. Although we observed a correlation between postexercise levels of adipomyokines and the changes in postexercise levels of lactate or β-HB, our results do not include any direct evidence supporting the cause–effect relationship between lactate or β-HB and postexercise changes in adipomyokine levels and fat browning. Because previous studies supported that lactate and β-HB induce fat browning through increase in thermogenic adaptation (12), further studies are required to evaluate the effects of these metabolites on interorgan communication and whole-body metabolism. Second, we evaluated limited signaling pathways involved in fat browning by aerobic exercise in the fasted state. Transition from energy-storing white adipocytes to metabolically active brown adipocytes (beige adipocytes) is controlled by complicated signaling pathways and transcription factors (35). Although our results could not search through the extensive pathways regulating the fat browning, the reproducible findings from clinical intervention study for results from animal may strengthen the significance. Third, our study did not include female animal and human to exclude the probable hormonal effects on the results, which warrant further studies to evaluate the gender difference. Finally, the design of the clinical study does not completely align with animal study. We did not include fasted control and fed exercise groups in the clinical study to evaluate the effects of fasting itself on fat browning or to compare the effects of HIIE in the fasted with fed state. Furthermore, we did not design the clinical study with equivalence between calorie intake and energy expenditure. In addition, the possible gender differences in the effects of aerobic exercise in the fasted state on fat browning should be of concern, which warrants further clinical investigation.

CONCLUSIONS

Our results clearly demonstrate that a single bout of aerobic exercise in the fasted state significantly induces fat browning–related markers in eWAT and lipolysis and upregulates the expression of adipomyokines contributing to fat browning in rats. The changes induced by aerobic exercise in the fasted state may be associated with an alteration in the levels of adipomyokines and in the AMPK–SirT1–PGC1α-mediated pathways. More importantly, the exercise induced changes in favor of fat browning, but not all are significantly correlated with the upregulation of lactate and β-HB in human. Although further clinical research to confirm the acute or chronic effects of aerobic exercise in the fasted state on fat browning and to elucidate the underlying molecular mechanisms is required, our results suggest that a single bout of aerobic exercise (consuming ~350 kcal) in the fasted state may activate fat browning–related pathways and have clinical metabolic benefits.

Acknowledgments

The authors declare no competing financial interests. This work was supported by the Medical Research Center Program (2021R1A5A2031612), Basic Research Program (2017R1D1A1B03032860 and 2020R1F1A1073415) and Mid-Career Research Program (2018R1A2A3074577) through the National Research Foundation of Korea (NRF) funded by the Korean government, and the NRF funded by the Ministry of Education, Republic of Korea (2022S1A5C2A03092407 and 2020S1A5B5A17089784).

The authors thank Ms. Seung-Hee Kang for her elaborate illustration of Supplemental Figure S1 (Supplemental Digital Content, http://links.lww.com/MSS/C796). The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The results of the present study do not constitute endorsement by the American College of Sports Medicine.

Sujin Kim: data curation, investigation, writing—original draft. Sang-Hyun Lee: data curation, methodology. Hyo-Bum Kwak: investigation, writing—review and editing. Dong-Ho Park and Ju-Hee Kang: conceptualization, supervision, project administration, writing—review and editing.

Footnotes

S. K. and D.-H. P. contributed equally as first authors.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.acsm-msse.org).

Contributor Information

SUJIN KIM, Email: sujin2419@hanmail.net.

DONG-HO PARK, Email: dparkosu@inha.ac.kr.

SANG-HYUN LEE, Email: jame0409@gmail.com.

HYO-BUM KWAK, Email: kwakhb@inha.ac.kr.

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