Adipose tissue is a sizeable and complex endocrine organ that plays a role in metabolic control in part through the secretion of adipokines such as adiponectin and interleukin 6 (IL-6) (1). Adipose dysfunction is characterized by tissue-specific insulin resistance, local inflammation, fibrosis, and the abnormal secretion of adipokines (2). These adipokines regulate various physiological processes and play a crucial role in the pathophysiology of chronic diseases such as cardiovascular diseases and type 2 diabetes (1). Thus, there is a clear need to develop interventions and strategies that successfully target adipose tissue function.
Regular prolonged exercise improves various aspects of adipose tissue function, including measures of inflammation and insulin sensitivity (3). This form of prolonged physical activity also has demonstrably important effects on various health outcomes, including risk factors for cardiovascular disease and diabetes (4). Unfortunately, many people struggle to undertake such structured exercise, and alternative forms of physical activity could potentially play an important role. In many modern societies, people spend much of their time engaged in sedentary behaviors such as using a computer and watching television. In addition to causing a positive energy balance and weight gain, several studies have shown that sedentary behavior is associated with increased risk of abnormal glucose metabolism, metabolic syndrome, type 2 diabetes, and cardiovascular disease (5). Thus, targeting this sedentary behavior may be important for health.
In support of this suggestion, short bouts of physical activity lasting just a few minutes spread over the course of a day improve postprandial triglyceridemia, glycemia, and insulinemia (6–9). Breaking prolonged sitting with short bouts of physical activity also affects skeletal muscle signaling pathways related to carbohydrate metabolism (8). However, there has been no assessment of whether breaking prolonged sedentary behavior affects on adipose tissue physiology. If this strategy is effective, it would have major implications for the prevention and management of disorders associated with dysfunctional adipose tissue. Thus, this study aims to examine the acute effect of breaking prolonged sedentary behavior (sitting) on adipose tissue.
MATERIALS AND METHODS
Eleven participants (seven men and four postmenopausal women) between 35 and 64 yr of age with increased central adiposity were recruited via local advertisement. An initial screening was followed by two subsequent main trials (prolonged sitting and breaking sitting). Trials were conducted in a randomized, counterbalanced design separated by a 3- to 4-wk washout period. Two identical meals based on participants’ total body mass were provided on the main trial days (morning and lunchtime). Blood was taken regularly during each trial, and adipose tissue biopsies were collected at baseline and at the end of each trial. The study protocol was approved by Bristol Research Ethics Committee (REC reference number: 13/SW/0321) and is registered at ClinicalTrials.gov (ID: NCT02870088). All participants provided written informed consent before taking part.
There are no data regarding the effect of breaking prolonged sitting on adipose tissue. Adipose tissue is highly responsive to insulin (10), and we have therefore determined sample size based on differences in insulin responses to a standard meal. A previous similar study reported breaking prolonged sitting to reduce postprandial insulinemia from 3337 to 2470 IU per 9 h·L−1 (9). Based on an estimated SD of 600 IU per 9 h·L−1, we would require nine participants to show a difference in iAUC for insulin with 95% power and 5% alpha using a two-tailed paired t-test. Eleven participants were recruited to allow for dropout.
Inclusion and Exclusion Criteria
Participants were required to be central overweight with at least a waist circumference of 80 cm for women and 94 cm for men (11) and weight stable (no self-reported change in weight ± 3%) (12) for at least 3 months before taking part in this study. Participants completed a health questionnaire to exclude any existing cardiovascular and metabolic diseases and a Physical Activity Readiness Questionnaire to ensure that participants were able to walk on the treadmill safely. Participants were asked to walk on a treadmill for 2 min to ensure that this would be tolerated. Smokers, premenopausal women, and volunteers who used any medications that could influence metabolic and inflammatory responses were excluded. A summary of participants’ physical characteristics is shown in Table 1.
Physical activity assessment
As part of the pretrial assessments, participants wore a combined heart rate/accelerometer monitor for 7 d to assess habitual physical activity energy expenditure (Actiheart, Cambridge Neurotechnology Ltd., Cambridge, UK). This was attached to the left of the chest via two adhesive ECG pads for 24 h·d−1 except for during showering/bathing/swimming (14).
Body composition analysis
Body mass was assessed using digital scales postvoid (TANITA Corp., Tokyo, Japan). Waist and hip circumferences were assessed based on World Health Organization guidelines (11). Body composition was determined by using dual-energy x-ray absorptiometry (DEXA; Discovery, Hologic, Bedford, UK). Abdominal subcutaneous and visceral adipose tissue mass was estimated from a central region between L1 and L4 (13).
In the 72 h before each main trial, participants were asked to refrain from performing any vigorous-intensity exercise. In the 48 h before each main trial, consuming alcohol/caffeine was not allowed, and a weighed food and fluid record was completed. Participants were asked to replicate this diet before their second main trial. In addition, in the 48 h before each main trial, participants were asked to wear a pedometer (Yamax, Japan) to restrict their step counts to under 4000 steps per day to mimic a sedentary lifestyle (15) and eliminate any acute effects from recent physical activity.
A 12-h fast was performed before arriving at the laboratory on main trial days between 8:00 and 9:00 AM. After anthropometric measurements, participants rested on a bed for 15 min, followed by two 5-min expired gas sample collections using Douglas bags (Hans Rudolph, MO) to determine resting metabolic rate (RMR) (16) from substrate oxidation as described (17).
After RMR assessment, a 20-gauge cannula (BD VenflonTM Pro) was inserted into an antecubital forearm vein, and a 10-mL baseline venous blood sample was collected and allocated into tubes with either EDTA or serum separation beads (Sarstedt Ltd., Leicester, UK). Plasma samples were centrifuged immediately at 3465g at 4°C for 10 min. Serum samples were left for 45 min to clot before centrifugation. Subcutaneous adipose tissue samples (~1 g) were subsequently collected under local anesthetic (1% lidocaine) from the area around the waist approximately 5 cm lateral to the umbilicus with a 14-G needle using an aspiration technique (18) followed by adipose tissue cleaning and processing as previously described (19).
Two identical meals (breakfast and lunch) were provided and consumed within a 15-min period during each main trial. The first meal (breakfast) was consumed after taking the baseline adipose and blood sample. The second meal (lunch) was consumed 3 h after the first. The test meal was prescribed according to total body mass and provided 0.35 g fat, 1.17 g carbohydrate, 0.29 g protein, and 37 kJ energy per kilogram body mass (6). The percentage of energy from macronutrients was 35% fat, 52% carbohydrate, and 13% protein (6). The meal comprised white bread (Hovis; soft white bread, medium sliced), sliced cheese (Sainsbury; cheese slices, basic), butter (Unilever; I can’t believe it’s not butter), mayonnaise (Hellmann; light mayonnaise), lettuce (Sainsbury; Iceberg lettuce), tomato (Sainsbury; tomatoes, basics), ham (Sainsbury; British honey roast), whole milk (Sainsbury; British), cocoa powder (Nesquik; cocoa powder), and yoghurt (Müller; fruit corner strawberry).
Prolonged Sitting and Breaking Prolonged Sitting
The study protocol is shown in Figure 1. In the prolonged sitting trial, participants sat on a chair for the whole period. In the breaking sitting trial, after consuming the first meal, participants walked 2 min on a treadmill at 6.4 km·h−1 speed (7) every 20 min for the following 180 min. After finishing the second meal, participants continued 2 min of breaking prolonged sitting physical activity at the same speed every 20 min for the following 120 min. For the remainder of the time, participants sat on a chair. Thus, in total, participants performed 15 two-min bouts of walking throughout the trial (i.e., 30 min of physical activity over 300 min).
RPE and heart rate were collected in the last 30 s of each 2-min bout of walking during the breaking sitting trial. Two 1-min expired air samples were collected during the last minute of walking during the breaking sitting trial to estimate energy expenditure and substrate use. In addition, expired air samples were taken using Douglas bags (Hans Rudolph, MO) during two 5-min periods of sitting to calculate total energy expenditure under resting conditions. While sitting, participants were only allowed to read, use a laptop, or watch television but were otherwise asked to keep as still as possible throughout (including specific instructions to avoid fidgeting). In the first trial, participants were allowed to consume water ad libitum, and the volume ingested was replicated for the second trial. In each main trial, baseline blood samples were collected before the first meal and hourly for the following 5 h. Additional blood samples were collected every 15 min for the first hour after each meal. A total of 14 blood samples were collected for each trial (Fig. 1).
Adipose Tissue Culture and Gene Expression
After cleaning and mincing the adipose tissue biopsy sample, a 200-mg portion of adipose tissue was homogenized in 5 mL TRIzol (Invitrogen, Paisley, UK) in a 15-mL centrifuge RNase/DNase-free sterile tube (Invitrogen) and stored at −80°C for gene expression analysis. The remaining adipose tissue was directly placed in sterile culture plates in duplicate (Nunc, Roskilde, Denmark) with endothelial cell basal media (Promocell, Germany) containing 0.1% fatty acid-free bovine serum albumin, 100 U·mL−1 penicillin and 0.1 mg·mL−1 streptomycin (Sigma-Aldrich, Gillingham, UK). Adipose tissue was incubated with a final ratio of 100 mg tissue per 1 mL endothelial cell basal media for 3 h (19). A 37°C, 5% CO2, and 95% ± 5% relative humidity incubator was used (MCO-18A1C CO2 incubator; Sanyo, Osaka, Japan). After the 3-h incubation, media were transferred to sterile Eppendorfs and stored at −80°C. Adipokine secretion from cultured adipose explants was normalized to explant adipose mass and then L1–L4 fat mass as described previously (20).
An RNeasy Mini Kit (Qiagen, Crawley, UK) was used to extract RNA from TRIzol-digested adipose tissue as described (18). The amount of RNA was 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). 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 IL-6 (Hs00985639_m1), adiponectin (Hs00605917_m1), leptin (Hs00174877_m1), IL-18 (Hs00155517_m1), tumor necrosis factor alpha (Hs99999043_m1), monocyte chemoattractant protein-1 (MCP-1) (Hs00234140_m1), 5′ AMP-activated protein kinase (Hs01562315_m1 and Hs00178903_m1 combined), glucose transporter type 4 (Hs00168966_m1), hormone-sensitive lipase (HSL) (Hs00193510_m1), insulin receptor substrate 1 (IRS-1) (Hs00178563_m1), insulin receptor substrate 2 (IRS-2) (Hs00275843_s1), sterol regulatory element binding protein 1c (SREBP1c) (Hs01088691_m1), pyruvate dehydrogenase kinase isozyme 4 (Hs00176875_m1), peroxisome proliferator-activated receptor γ (Hs01115513_m1), peroxisome proliferator-activated receptor-gamma coactivator 1α (Hs01016719_m1), RAC-alpha serine/threonine-protein kinase (Hs00178289_m1), adipose triglyceride lipase (Hs00386101_m1), fatty acid translocase/CD36 (Hs00169627_m1), forkhead box protein O1 (Hs01054576_m1), hexokinase 2 (Hs00606086_m1), phosphoinositide 3-kinase 85α (Hs00933163_m1), and fatty acid synthase (Hs00188012_m1), and peptidylpropyl isomerase A (PPIA) was used as an endogenous control (21). The comparative Ct method was used to process data, where ΔCt = Ct target gene − Ct PPIA. Ct target genes were normalized to an internal calibrator (lowest ΔCt for each target gene) and baseline.
Concentrations of plasma glucose, plasma triglycerides, and plasma nonesterified fatty acids were analyzed using commercially available assay kits and analyzer (Daytona Rx; Randox, Crumlin, UK). Serum insulin (Mercodia, Uppsala, Sweden) and both serum and adipose explant secretion of IL-6, leptin, and adiponectin (R&D systems) were measured using enzyme-linked immunosorbent assay.
Descriptive data are presented in text and tables as mean ± SD; variance bars on figures are presented as mean and SEM. Time series data were examined using a two-way repeated-measures ANOVA (trial–time) using SPSS version 22 (IBM, Armonk, NY). Greenhouse–Geisser corrections were applied to intraindividual contrasts where [Latin Small Letter Open E] < 0.75; however, for less severe asphericity, the Huynh–Feldt correction was selected (22). Incremental area under curve (iAUC) was calculated using the trapezoid method (23), and the differences in summative scores between trials were analyzed using paired t-tests. Data for iAUC represent the period from the consumption of the first meal to the conclusion of the second meal (330 min). iAUC analysis and gene expression data were conducted using logged transformed data as previously described (18). Statistical significance was set at P ≤ 0.05.
Blood glucose, insulin, triglyceride, and NEFA concentrations in prolonged and breaking sitting trials
Trial–time interaction effects were found for blood glucose and insulin between prolonged and breaking sitting trials (all P ≤ 0.05; Fig. 2A and C). The glucose and insulin iAUC values were attenuated significantly by breaking sitting compared with prolonged sitting (359 ± 117 vs 697 ± 218 mmol per 330 min·L−1, P = 0.001, and 202 ± 71 vs 346 ± 150 nmol per 330 min·L−1, P = 0.001, respectively; Fig. 2B and D). There was no interaction effect for blood triglyceride and NEFA (Fig. 2E and G). Triglyceride iAUC was significantly higher in breaking sitting compared with prolonged sitting trial (283 ± 36 vs 232 ± 32 mmol per 330 min·L−1, P = 0.002, respectively; Fig. 2F).
Adipose tissue mRNA gene expression
There was no interaction effect (trial–time) in adipose tissue mRNA gene expression between breaking sitting and prolonged sitting trials for any of the genes analyzed (Fig. 3). However, there were time effects for IL-6, MCP-1, PDK4, IRS-1 and IRS-2, phosphoinositide 3-kinase 85α, HSL, and SREBP1c (all P ≤ 0.05; Fig. 3).
Serum adipokine concentrations and adipose tissue adipokine secretion ex vivo
There was no difference between prolonged sitting and breaking sitting in serum IL-6, leptin, and adiponectin (Fig. 4A, C, and E) or the secretion ex vivo of IL-6, leptin, and adiponectin (Fig. 4B, D, and F). There was a time effect for serum IL-6 and leptin (P ≤ 0.05) (Fig. 4).
Pretrial physical activity and RMR measures
The average of 48 h step counts before prolonged and breaking sitting trials was not different (3868 ± 1304 vs 3669 ± 1090 steps per day, respectively), and there was no difference in prefed RMR on the trial day between trials (1882 ± 303 vs 1833 ± 338 kcal·d−1, respectively).
Physiological response during trials
During the 15 bouts of 2-min walking, the average heart rate was 135 ± 12 bpm with an RPE (6–20 scale) of 10 ± 2. The difference in energy expenditure between trials was 216 kcal over the 5.5-h period (Table 2).
The present study investigated whether breaking sitting with regular short bouts of walking influences adipose tissue gene expression and secretion of selected proteins from adipose tissue ex vivo. We found that regularly participating in short bouts of walking reduced postprandial glycemic and insulinemic responses by 48% and 42%, respectively. However, despite the profound difference in postprandial insulin and glucose concentrations, changes in subcutaneous adipose gene expression were similar in both trials. There was also no difference in adipose tissue explant secretion and circulating concentrations of IL-6, adiponectin, or leptin.
The effect of breaking sitting on postprandial glycemia and insulinemia
The extent of postprandial glucose and insulin excursions is associated with cardiovascular disease (24,25). Therefore, strategies to attenuate postprandial glucose and insulin responses would be extremely useful. Breaking prolonged sitting has been shown to reduce postprandial glucose and insulin levels in most (7,9,26) but not all studies (27). In the present study, we found that participating in regular short bouts of walking attenuated postprandial glucose and insulin concentrations in sedentary middle-age central overweight men and women (glucose and insulin iAUC values were attenuated by 48% and 42%, respectively). The additional energy cost of walking during the breaking sitting trial was supported by carbohydrate oxidation, and thus we propose that oxidation of carbohydrate may be partly responsible for the relatively lower blood glucose observed in this trial. Thus, breaking prolonged sitting is very effective at reducing postprandial glycemia and insulinemia.
Adipose tissue responses to breaking prolonged sitting
In skeletal muscle, breaking sitting with either light or moderate-intensity walking increases the expression of genes involved in glucose metabolism, including PDK4 (8). In the present study, we did not find any differences between prolonged sitting and breaking sitting trials in the expression of these genes in adipose although our walking speed, frequency, and duration was very similar to that previous study (8). Thus, it appears that acutely breaking prolonged sitting with short bouts of walking reduces postprandial glycemia/insulinemia and leads to corresponding changes in key pathways in skeletal muscle, whereas a similar effect on postprandial glycemia/insulinemia is not associated with corresponding changes within adipose tissue. These divergent effects are likely to reflect the different roles of these tissues. Skeletal muscle plays a major role in the disposal of exogenously ingested glucose (especially during physical activity), whereas adipose tissue plays a relatively minor role in the acute regulation of blood glucose concentrations (28). Instead, during the postprandial period, adipose tissue plays a major role in responding to feeding and storing ingested lipids (29). Given the effect of glucose and insulin infusion on adipose tissue gene expression (10,30), it is possible that the magnitude of postprandial glycemia and insulinemia in the present study was a more-than-adequate stimulus for adipose tissue, even during the breaking sitting trial. We have recently shown that feeding exerts a potent effect on adipose tissue and can blunt the normal responses to prolonged structured exercise (20). Thus, we propose that the effect of feeding is likely to overcome the effect of physical activity on adipose tissue, whether this is during repeated short bouts of physical activity to break prolonged sitting or during more prolonged structured exercise.
There was an apparent increase in triglyceride iAUC with breaking sitting trial and this may (ostensibly) appear to be a negative response. However, this is likely explained by an enhanced lipid mobilization in the breaking sitting trial combined with technical considerations related to the assay used for the assessment of plasma triglyceride. Our method for the assessment of plasma triglyceride does not discriminate between free glycerol, bound glycerol in chylomicrons, and very-low-density lipoprotein; thus, it is likely that the greater triglyceride iAUC in the breaking sitting trial reflects an increase in circulating free glycerol due to adipose tissue lipolysis.
Temporal changes in adipose tissue gene expression after feeding
Although we did not observe any differences in gene expression between breaking and prolonged sitting trials, there were temporal changes in both trials that could reflect the effect of feeding per se. The expression of adipose SREBP1c was increased in both trials, and this response is probably indicative of lipid synthesis after feeding (31). Moreover, there was a decrease in HSL and PDK4 in both trials, which may reflect a reduction in lipid mobilization and oxidation. In addition, insulin receptor and IRS-1 and IRS-2 gene expression were consistently upregulated and downregulated, respectively, at the end of both trials. IRS-1 plays a central role as an insulin signaling receptor (32), and similar to the present study, adipose IRS-2 mRNA expression was downregulated in lean, overweight, and obese populations after feeding (33). Taken together, the changes in SREBP1c, HSL, PDK4, and IRS-1 and IRS-2 in both trials probably reflect the normal physiological and metabolic response to feeding.
We also observed an increase in adipose IL-6 and MCP-1 in both trials. Other studies have shown that adipose IL-6 and MCP-1 gene expression were upregulated after meal consumption in lean, overweight, and obese men (33), in people with metabolic syndrome (34), in people with type 2 diabetes (35), and in healthy lean populations (36). A meal-induced upregulation of adipose IL-6 and MCP-1 gene expression might support the notion that feeding induces acute transient adipose tissue inflammation. However, we cannot discount the possibility that these changes could also reflect an adipose circadian rhythm (37).
Experimental design considerations
In the present study, only two adipose biopsies were collected, and therefore we do not have a complete time course of whether the temporal changes observed in both trials was due to meal consumption and/or an entrained biological rhythm. Repeated adipose biopsies might help resolve this issue, but this could be problematic because of potential confounding from local inflammation (36). We should also emphasize that each condition (trial arm) was conducted over the course of a single day. Given the large reduction in serum insulin concentrations in the breaking sitting trial and the powerful effect of insulin on adipose tissue (10,38), it is possible that if this intervention was repeated for multiple days and/or months, then this difference in postprandial insulin would lead to chronic changes in adipose in the longer term. We also do not know how the present results would compare to the same duration of exercise but performed in a different way (e.g., a single bout of 30 min or three bouts of 10 min). In the present study, we provided two mixed meals with a total ~1725 kcal, which is more than twofold the energy intake in the single meal provided by Latouche et al. (8). Given the extended timescale, ~1725 kcal is not an unreasonable amount of energy to consume (39), but it is possible that the physiological pressure of processing and storing this amount of energy might outweigh the effect of small bouts of physical activity in adipose tissue. Had we provided smaller meals, it is possible that physical activity could have had a moderating effect on adipose tissue-related outcomes.
Although breaking prolonged sitting is an effective strategy for acutely managing postprandial glycemia and insulinemia, this does not elicit corresponding temporal changes in adipose tissue.
This project was funded by the Ministry of Education (Taiwan) and the University of Bath. The authors thank all the participants for their time and effort to take part in this project.
The authors declare no competing interests. The results of the present study do not constitute endorsement by the American College of Sports Medicine. The results of this study are presented clearly, honestly, and without fabrications, falsification, or inappropriate data manipulation.
Yung-Chih Chen was responsible for study design and conduct, data collection, data analysis, data interpretation, statistical analysis, and manuscript revision. James Betts was responsible for study design and manuscript revision. Jean-Philippe Walhin assisted with adipose tissue biopsies and manuscript revision. Dylan Thompson was responsible for funding, study design, data interpretation, and manuscript revision.
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