it has become clear in the last 10 years or so that adipose tissue plays an active role in many physiological processes and pathological states (74, 78), and dysfunction within this tissue is characterized by tissue-specific insulin resistance, local inflammation, fibrosis, and the abnormal secretion of adipokines (36). Adipose tissue secretes dozens of mediators including the archetypal adipokines, adiponectin and leptin (16, 38, 46). Adiponectin is exclusively derived from adipose tissue and circulates in high concentrations (10−20 mg/l). In other cases, the quantitative amounts secreted by adipose can be substantial; for example, subcutaneous adipose contributes up to a third of circulating interleukin-6 (IL-6) (45).

Feeding has a pronounced effect on the whole body metabolic responses to exercise and reduces the contribution of fat towards metabolism (3, 23, 77). In addition, feeding influences the skeletal muscle responses to various forms of exercise (9, 10, 13). For example, pyruvate dehydrogenase kinase isozyme 4 (PDK4) is significantly upregulated in muscle with exercise in fasted but not fed conditions (10). Other feeding-related changes in gene expression in muscle after exercise have been reported, including altered expression of glucose transporter type 4 (GLUT4), PDK4, fatty acid translocase/CD36 (FAT/CD36), carnitine palmitoyltransferase 1 (CPT-1), uncoupling protein-3 (UCP3), and AMP-activated protein kinase (AMPK) (9). Collectively, therefore, there is strong evidence that feeding affects the responses to exercise in skeletal muscle. Adipose tissue plays a crucial role during exercise (18), and this led us to speculate that preexercise feeding may also affect the adipose tissue responses to acute exercise.

During moderate-intensity exercise, adipose tissue provides much of the energy for working skeletal muscle through the mobilization of stored triacylglycerol (27). Exercise also initiates a number of other responses in adipose tissue such as increased blood flow and altered expression of various adipokines within abdominal subcutaneous adipose tissue (66). It is possible that these acute exercise-induced changes could be part of the mechanism through which exercise improves health (66). However, all prior studies of adipose tissue responses to exercise in humans have been conducted in the fasted state (8, 19, 25, 31, 32, 39). The effect of feeding status on the response of human adipose tissue to exercise has never been studied. This is despite the fact that feeding has a profound effect on adipose tissue (1, 30) and that we spend the vast majority (~70%) of a 24-h period in a fed state (58). Consequently, the aim of the present study was to investigate whether feeding influences the adipose tissue responses to exercise.

Descriptive data are presented as means ± SD. The variance bars on figures are presented as means with 95% confidence intervals (CI). Time series data were analyzed using a two-way ANOVA (trial × time) with repeated measures irrespective of minor deviations from a normality of distribution ( 43 ) using SPSS version 22 (IBM, Armonk, NY). Where significant interactions (trial × time) were found, post hoc paired t -tests were used to determine changes over time. Analysis of gene and protein expression data were conducted using logged transformed data as previously described ( 72 ). Incremental area under the curve (iAUC) was calculated for insulin, glucose, and NEFA using the trapezoid method ( 75 ) and analyzed using paired t -tests. Statistical significance was set at P ≤ 0.05.

The adipose tissue protein fraction was isolated from the TRIzol phenol-chloroform phase following the manufacturer’s protocol (TRIzol Reagent, Life Technologies). Briefly, 1 ml of organic phase was mixed with 1.5 ml isopropanol. After being mixed, the samples were incubated for 10 min at room temperature, followed by 10 min of centrifugation at 12,000 g at 4°C to pellet the protein. One milliliter of protein pellet was washed using 2 ml of 0.3 M guanidine hydrochloride in 95% ethanol for a 20-min incubation followed by centrifugation at 7,500 g for 5 min at 4°C. This process was repeated three times. After completion of the washing procedure, 2 ml of 100% ethanol was added to the protein pellet for an additional 20-min incubation at room temperature before being centrifuged. The pellet was then left to air dry for 5−10 min. Then, 200 μl of 1% SDS were added to resuspend the pellet. The protein content of the samples was determined using a BCA protein assay kit (Thermo Scientific, Waltham, MA). Proteins (25 μg/lane) were separated by SDS-PAGE and transferred using a semidry electro-transfer method to a nitrocellulose membrane. Western blotting analysis was performed with the following antibodies: RAC-β serine/threonine-protein kinase (Akt2)/PKB-β (Millipore, Billerica, MA) ( 34 ), AMPK (Cell Signaling Technology, Beverly, MA) ( 72 ), GLUT4 ( 26 ), IRS1 (Millipore) ( 35 ), IRS2 (Millipore) ( 47 ), PDK4 (ABGENT, San Diego, CA) ( 29 ), HSL (Cell Signaling Technology) ( 56 ), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Proteintech, Rosemont, IL) ( 62 ). The images were acquired in an EPI Chemi II darkroom (Analytik Jena, Upland, CA), and bands quantified using VisionWorks LS analysis software (Analytik Jena).

An RNeasy Mini Kit (Qiagen, Crawley, UK) was used to extract RNA from adipose tissue as described ( 72 ). Tissue samples were quantified using a Qubit 2.0 fluorimeter (Life Technologies, Paisley, UK). RNA was reversed transcribed (1 μg) to cDNA using a High-Capacity Reverse Transcription Kit (Applied Biosystems, Warrington, UK). Organic phenol-chloroform phase from the RNA extraction was kept for further protein analysis. Real-time PCR was performed using a StepOne (Applied Biosystems). Predesigned primers and probes were obtained from Applied Biosystems for the measurement of expression of interleukin 6 (IL-6) (Hs00985639_m1), adiponectin (Hs00605917_m1), leptin (Hs00174877_m1), IL-18 (Hs00155517_m1), tumor necrosis factor-α (TNF-α) (Hs99999043_m1), monocyte chemoattractant protein-1 (MCP-1) (Hs00234140_m1), AMPK (Hs01562315_m1 and Hs00178903_m1 combined), GLUT4 (Hs00168966_m1), hormone-sensitive lipase (HSL) (Hs00193510_m1), insulin receptor substrate 1 (IRS1) (Hs00178563_m1), IRS2 (Hs00275843_s1), sterol regulatory element binding protein 1c (SREBP-1c) (Hs01088691_m1), PDK4 (Hs00176875_m1), peroxisome proliferator-activated receptor γ (Hs01115513_m1), peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) (Hs01016719_m1), RAC-α serine/threonine-protein kinase (Akt1) (Hs00178289_m1), adipose triglyceride lipase (ATGL) (Hs00386101_m1), FAT/CD36 (Hs00169627_m1), forkhead box protein O1 (Hs01054576_m1), hexokinase 2 (HK2) (Hs00606086_m1), PI3K-85α (PIK3R1) (Hs00933163_m1), and CPT-1B (Hs03046298_s1). The G0/G1 switch gene 2 (G0S2) (Hs00605971_m1), peptidylpropyl isomerase A (PPIA), was used as an endogenous control ( 48 ). The comparative threshold cycle (C t ) method was used to process data, where ΔC t = C t target gene − C t PPIA. C t target genes were normalized to an internal calibrator (lowest ΔC t for each target gene) and baseline. The C t values for IL-6 (31 out of 40 samples), TNF-α (16 out of 40 samples), and IL-18 (37 out of 40 samples) were frequently over 35, and thus these results are not included.

After we cleaned and minced the adipose tissue biopsy sample, one portion of adipose tissue (~200 mg) was immediately homogenized in 5 ml TRIzol (Invitrogen, Paisley, UK) in an RNase/DNase-free sterile tube (Invitrogen) and stored at −80°C before mRNA gene expression and protein analysis. The remaining adipose tissue was used for culture, and four ~100-mg portions were placed in sterile culture plates (Nunc, Roskilde, Denmark) with endothelial cell basal media (ECBM) (Promocell, Heidelberg, Germany) containing 0.1% fatty acid-free bovine serum albumin, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma-Aldrich, Gillingham, UK). Adipose tissue was incubated with a final proportion of 100 mg tissue per 1 ml ECBM media for 3 h ( 68 ) at 37°C in a 5% CO 2 and 95 ± 5% relative humidity incubator (MCO-18A1C CO 2 incubator; Sanyo, Osaka, Japan). After the 3-h incubation, media was transferred to sterile tubes and stored at −80°C. Adipokine secretion from adipose explants was normalized to explant adipose mass and then L1-L4 fat mass as described ( 67 ).

The meal in the fed trial was the same as previously described in detail ( 7 ). The composition of the meal was selected to reflect typical breakfasts in the UK ( 7 ). The amount was normalized to resting energy requirements ( 7 ). Briefly, the total energy provided was 648 ± 115 kcal (carbohydrate 120.1 ± 21.3 g, fat 12.7 ± 2.3 g, and protein 20.9 ± 3.7 g). The meal included white bread (Brace’s thick white), cornflakes (Kellogg’s cornflakes), semiskimmed milk (Sainsbury’s British semi-skimmed milk), orange juice (Sainsbury’s 100% pure squeezed smooth orange juice), spread (Unilever; I Can’t Believe It’s Not Butter), jam (Sainsbury’s strawberry jam), and sugar (Sainsbury’s British white granulated sugar). Participants were asked to consume the meal within 15 min. In the fasted trial, participants sat quietly for a 15-min period.

Participants then either consumed a meal (fed) or remained fasting (fasted), and cannula-derived venous blood samples were taken every 15 min for the following 60 min. A further blood sample was collected at 120 min immediately before the walking protocol. In both the fasted and fed treatments, participants walked on the treadmill at 60% V̇o 2max for 60 min, and 1 min of expired air samples, RPE, and heart rate were collected at 5, 20, 40, and 60 min. After completion of exercise, another blood sample was immediately collected, and participants then rested for a further 60 min. At this point, a second adipose tissue and final blood sample were taken.

After RMR assessment, a cannula was inserted into an antecubital forearm vein, and a baseline venous blood sample was taken and allocated into tubes with either EDTA or serum separation beads (Sarstedt, Leicester, UK). Plasma samples were centrifuged immediately at 3,465 g at 4°C for 10 min. Serum samples were left for 45 min to coagulate before centrifugation. Subcutaneous adipose tissue samples (~1 g) were taken under local anesthetic (1% lidocaine) from the area around the waist ~5 cm lateral to the umbilicus with a 14-gauge needle using an aspiration technique ( 72 ) followed by adipose tissue cleaning and processing as described previously ( 67 ).

On main trial days, participants arrived at the laboratory between 8 and 9 AM following a 12-h fast. After anthropometric measurements, participants rested on a bed for 15 min, followed by four 5-min expired gas sample collections to determine resting metabolic rate (RMR) ( 5 , 11 ) using substrate oxidation under resting conditions ( 17 ). During exercise, alternative equations were used ( 28 ).

In the 72 h before each main trial, participants were asked to refrain from performing any strenuous physical activity and from consuming alcohol/caffeine for 48 h before the main trials. A dietary record was completed 48 h before the first main trial, and participants replicated this diet before their second main trial.

An incremental continuous treadmill test until the point of volitional fatigue was used to determine V̇o 2max . For most participants, a treadmill speed of 4 km/h and gradient of 8.5% were appropriate. This grade was maintained, and the speed was increased by 1 km/h after every 3-min stage. One minute expired air samples were collected into Douglas bags (Hans Rudolph, Shawnee, KS), and rate of perceived exertion (RPE) and heart rate were measured in the final minute of each stage and also at the point of volitional fatigue, defined as when the participant indicated that only 1 min remained until fatigue. Samples were analyzed for relative expired fractions of oxygen and carbon dioxide (Servomex, Crowborough, UK), and the total volume within the bag was measured using a dry gas meter (Harvard Apparatus, Kent, UK).

To be eligible to take part, participants were required to be overweight with a waist circumference of 94−128 cm ( 76 ). Participants were also required to be weight stable ( 63 ) for at least 3 mo (mass stable ± 3%). Participants completed a health questionnaire to exclude any existing cardiovascular and metabolic diseases and a Physical Activity Readiness Questionnaire (PAR-Q) to make sure that participants were able to exercise safely. Individuals taking any medications known to influence lipid/carbohydrate metabolism or immune function and smokers were excluded. A summary of participants’ physical characteristics is shown in Table 1 .

Ten men aged 18-35 yr with increased central adiposity were recruited via local advertisement. Participants attended the laboratory on three occasions for initial assessment of maximum oxygen uptake (V̇o 2max ) and two subsequent main trials. The trial days involved walking for 60 min at 60% V̇o 2max under either fasted or fed conditions in a randomized, counterbalanced design separated by a 3- to 4-wk washout period. This intensity and duration of exercise were selected because they rely heavily on fatty acids mobilized from adipose tissue and also because this is the type of exercise recommended in recent position stands ( 20 , 27 ). Blood and adipose tissue were sampled at baseline and after exercise to examine the impact of prior feeding. There are no data regarding how different dietary status (fasted vs. fed) affects the adipose responses to exercise. However, a previous study using a similar meal showed that feeding had an enormous effect on the use of lipid during exercise, and thus this indicates that the role of adipose tissue during exercise would be potentially very different ( 3 , 77 ). On the basis of these results, to see an effect on lipid oxidation during exercise with 95% power and 5% alpha, we would require between six and eight participants. We recruited 10 men to account for greater variability in other outcome measures.

Serum IL-6 increased ( Fig. 4 A ) and serum leptin decreased ( Fig. 4 C ) in both trials ( P < 0.05). There was a modest time effect for serum adiponectin ( Fig. 4 E ) although no interaction effects were identified for serum IL-6, leptin, and adiponectin. Furthermore, no time or time × trial interactions were identified for ex vivo adipose explant secretion of IL-6, leptin, and adiponectin in response to exercise under fasted and fed conditions ( Fig. 4 ).

Fig. 3. Fold changes in relative protein content in adipose tissue under fasted and fed trials (all n = 8 due to lack of sufficient protein for 2 participants) ( A ). The dashed line indicates baseline. Data were normalized to GAPDH. Values are means ± 95% CI. #Significant interaction effect between fasted and fed trials using 2-way ANOVA with repeated measures ( P ≤ 0.05). B : representative images of Western blots in adipose tissue under fasted and fed trials. Insulin receptor substrate 1/2 (IRS1/2), RAC-β serine/threonine-protein kinase (Akt2), and AMP-activated protein kinase (AMPK) represent participants 2 and 8. Glucose transporter 4 (GLUT4), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and hormone-sensitive lipase (HSL) represent participants 3 and 4.

There was an interaction effect for IRS2 protein expression between trials ( P ≤ 0.05), showing an approximately twofold increase in fasted conditions and no change in fed conditions ( Fig. 3 A ). The change in the expression of the remaining proteins was not statistically different between fasted and fed conditions. Representative Western blots are shown in Fig. 3 B .

Fig. 2. Fold changes in relative gene expression in adipose tissue under fasted and fed trials (all n = 10, except for HSL and G0S2 , n = 9 owing to Ct > 35 for 1 participant). The dashed line indicates baseline. Data normalized to peptidylpropyl isomerase A, internal calibrator, and baseline. Samples that exceeded the detectable limit (Ct > 35) were excluded from the analysis. Values are means ± 95% CI. #Significant interaction effect between fasted and fed trials using 2-way ANOVA with repeated measures ( P ≤ 0.05). †Time effect ( P ≤ 0.05). *Comparison between baseline and 1 h postexercise by paired t -tests ( P ≤ 0.05).

There was an interaction effect for adipose tissue and PDK4 , ATGL , HSL , FAT/CD36 , GLUT4 , and IRS2 (all P ≤ 0.05) after exercising under fasted versus fed conditions ( Fig. 2 ). These interaction effects are explained by divergent responses between fasted and fed trials with a larger increase in fasted conditions when compared with either a smaller change or a modest decrease in fed conditions ( Fig. 2 ). There was a time effect for HK2 , MCP-1 , and PGC-1α ( Fig. 2 ). The expression of the remaining genes was not significantly different between trials or over time ( Fig. 2 ).

Fig. 1. Glucose ( A ), insulin ( C ), and nonesterified fatty acids (NEFA) ( E ) concentrations in fasted and fed trials and incremental area under the curve (iAUC) for glucose ( B ), insulin ( D ), and NEFA ( F ); n = 9 in both fasted and fed trials (attributable to difficulty in cannulating 1 participant). Values are means ± 95% CI. #Significant interaction effect between fasted and fed trials by 2-way ANOVA with repeated measures ( P ≤ 0.05). *Significantly different between fasted vs. fed trials using paired t -tests ( P ≤ 0.05). The shaded box in A , C , and E denotes meal time.

Trial × time interaction effects were found for blood glucose, insulin, and NEFA between fasted and fed trials ( P = 0.026, P = 0.003, and P = 0.001, respectively). As expected, iAUCs for glucose and insulin were elevated for all parameters in the fed trial ( P = 0.02 and P = 0.03, respectively) ( Fig. 1 , B and D ). Peak glucose and insulin concentrations in the fed trial were reached 15 min after the meal (7.1 ± 0.6 mmol/l and 370 ± 185 pmol/l, Fig. 1 , A and C , respectively). NEFA concentrations were lower at most time points in the fed trial ( Fig. 1 E ).

DISCUSSION

This study presents the first evidence that feeding status alters the human adipose tissue response to acute exercise, and thus feeding status has the potential to influence the long-term adaptation of adipose tissue to regular exercise.

In the present study, our feeding protocol successfully manipulated systemic concentrations of glucose and insulin. In the 2 h before exercise, there was a 168-fold and 26-fold difference in insulin and glucose iAUC, respectively. As anticipated with this study design, prior feeding increased relative carbohydrate utilization and decreased fat oxidation during exercise (3, 23, 77). Thus exercise in fed and fasted trials was performed in a very different physiological state.

At the gene expression level, we found that adipose tissue responded differently to moderate-intensity exercise under fasted versus fed conditions. When compared with the changes in fasted exercise conditions, feeding led to lower changes or a decrease in PDK4, ATGL, HSL, FAT/CD36, GLUT4, and IRS2 mRNA as demonstrated by interaction effects for these outcomes. Over time, acute differences in skeletal muscle gene expression with exercise conducted in the fasted vs. the fed state have been proposed to contribute to diverse physiological adaptations (12, 70). Our data demonstrate that feeding status also alters adipose tissue responses to an acute bout of exercise. We have previously shown that weight loss leads to a large increase in genes such as PDK4 and HSL in adipose tissue (71), whereas overfeeding leads to a profound decrease in both PDK4 and HSL in adipose tissue (72). Thus feeding has the potential to affect the acute adipose tissue responses to exercise, and, given the important role of adipose tissue in health and the nature of these changes (66, 71, 72), we propose that feeding before exercise blunts some of the health-related changes induced by exercise training.

We found a difference in adipose IRS2 protein content between fasted and fed exercise conditions although the other measured proteins were not affected. We should highlight that our protein measurements represent only total protein content. Repeated small changes in adipose protein synthesis likely lead to accumulated differences and functional changes in adipose phenotype over time (6, 50). Clearly, the only way to know whether the acute changes observed in the present study translate into long-term differences in protein content in adipose is to examine whether chronic training conducted in the fasted vs. postprandial state leads to divergent adaptations.

Carbohydrate and Lipid Metabolism in Adipose Tissue A primary function of PDK4 is to regulate glucose metabolism by inhibiting pyruvate dehydrogenase complex activity. Fasting and exercise increase PDK4 mRNA expression in skeletal muscle (52, 53), and insulin suppresses PDK4 mRNA and protein content in skeletal muscle (37, 40). It is possible that insulin could be responsible for the lower adipose PDK4 mRNA response in the fed trial although higher NEFA in the fasted trial could also have increased PDK4 expression (2, 33). Other feeding-related studies have shown that muscle PDK4 mRNA expression remained unchanged 1−4 h after exercise in the fed state (with lower NEFA concentrations) compared with exercise in the fasted state (9, 10). Thus exercise in the fasted state appears to increase PDK4 expression in both muscle and adipose, whereas exercise in the fed state does not. ATGL and HSL mobilize stored fat and release it into the circulation (79). In the present study, gene expression of ATGL, HSL, and FAT/CD36 in adipose were all differentially expressed in fasted and fed exercise conditions. These interaction effects are explained by responses in the fed trial being either lower or in the opposite direction to the fasted trial. There was also greater fat oxidation during exercise in the fasted trial. Other studies have also shown similar responses in fasted exercise with higher fat oxidation and increased skeletal muscle FAT/CD36 gene expression (9). Moreover, these findings are consistent with the previously observed increase in adipose HSL activity reported during cycling exercise, which is blunted with nicotinic acid ingestion (73). Thus, given the nature and direction of these changes, we propose that feeding blunts at least some of the exercise-induced stimulus on adipose tissue. We found that GLUT4 mRNA, IRS2 mRNA, and IRS2 protein were also differentially expressed in adipose in fasted and fed exercise trials. These effects were subtle but consistent, and this also seems to indicate that exercise in a fed state will not generate the same change in pathways involved in glucose metabolism and signaling within adipose tissue as fasted exercise. Previous findings in skeletal muscle have found an increase in GLUT4 mRNA in fasted but not fed conditions after exercise (9, 10). PGC-1α mRNA is a transcriptional coactivator involved in mitochondrial biogenesis (60). Acute exercise increases PGC-1α mRNA expression in human skeletal muscle (21, 54) and in rodent white adipose tissue (64). However, the impact of feeding status and/or carbohydrate variability before exercise on skeletal muscle PGC-1α mRNA is controversial. Some studies indicate that PGC-1α mRNA expression is upregulated after exercise whether acute exercise is performed in fed or fasted conditions (10, 42), but other studies show that higher carbohydrate availability before exercise blunts PGC-1α mRNA expression in skeletal muscle both at rest and after exercise (4, 55). In the present study, the increase in adipose PGC-1α mRNA expression after exercise was unaffected by feeding status. Chronic training studies indicate that adipose PGC-1α mRNA is increased in humans (59) and rodents (64, 69), and this has also been reported to increase PGC-1α protein content and mitochondrial biogenesis in rodents (69). Whether the present results indicate an acute exercise-induced increase in mitochondrial biogenesis in human adipose tissue is plausible but unclear at the present time.

Adipokine Response to Exercise and Impact of Feeding Status Previous studies have shown that circulating adipokine concentrations are affected by acute exercise (8) and that energy consumption during exercise alters these systemic responses (57, 61). However, evidence from studies that have manipulated feeding status before exercise is scarce. Zoladz et al. (80) found no difference in circulating IL-6 and leptin after a single bout of exercise, in a fed or fasted state. However, the duration of exercise lasted only 12 min, and this might be insufficient to examine the notion that feeding status influences circulating adipokines. The exercise in the present study was 60 min, and we too found no evidence that preexercise feeding affects circulating IL-6, leptin, and adiponectin. This may be due to the fact that moderate-intensity exercise has only a modest effect on many of these parameters, and thus there is little potential for feeding to interact with exercise and exert an effect (41). Serum IL-6 was increased over time in both fasted and fed trials in the present study, which could be partly due to the effect of exercise (51). As we did not observe changes of adipose IL-6 secretion ex vivo, the increase in serum IL-6 might be caused by release from skeletal muscle during exercise (49), but it is also possible that this reflects local production of IL-6 attributable to prolonged cannulation (14).

Temporal and Population-Specific Considerations This study is the first to examine the impact of feeding on adipose tissue responses to exercise. We recruited overweight participants, and this focus is a strength given that ~62% of the UK population are overweight (24). Increased adiposity has a profound effect on adipose tissue function [e.g., a downregulation of GLUT4 mRNA (67) and reduction in postprandial adipose tissue blood flow (44)]. These could be important considerations when interpreting our findings. We should also highlight that, in the present study, we were limited to only two adipose tissue biopsies because of a concern over potential interference from repeated sampling (15), so we do not have a full and complete time course. For the first study of this kind and with limited sampling opportunities, a second biopsy 60 min after exercise was considered to balance the requirement to capture pathways that rapidly change and those that are slower to respond. Subsequent studies should consider more frequent adipose sampling and/or the inclusion of additional resting trial(s). Depending on the kinetics of each response, our sample timing framework will be appropriate for some outcomes and less appropriate for others. Furthermore, in the absence of an adipose biopsy immediately before exercise, it is hard to establish whether some effects in the fasted trial are due to the modestly extended fasting period or due to exercise (or the interaction between fasting and exercise). What is very apparent, however, is that exercise in fed conditions does not lead to the same changes as exercise in fasted conditions.