## Introduction

Prolonged uninterrupted sitting is positively associated with cardiometabolic risk biomarkers and premature mortality, independent of moderate-to-vigorous intensity physical activity1. There is emerging interest in the underlying biological responses to prolonged sitting that may drive disease pathophysiology, and how breaking up sitting with short, regular bouts of physical activity can potentially act to mitigate these adverse effects. Studies examining beneficial effects of breaking up sedentary time have implicated regulation of postprandial glucose, insulin and lipid metabolism2,3,4, endothelium-mediated arterial vasodilation5,6,7, and anti-inflammatory mechanisms8,9,10,11.

The mechanistic underpinnings of the beneficial effects of breaking up prolonged sitting are likely to be multifactorial, and involve peripheral organs that are known to play a key role in metabolism, such as skeletal muscle, liver and adipose tissue. Our group has observed favourable changes in skeletal muscle gene and protein expression that likely contribute to the improved glucose control associated with breaking up of prolonged sitting via light- or moderate-intensity activities. These changes include some which align with, and others which may be distinct from, the effects of continuous acute exercise12,13.

Subcutaneous white adipose tissue is another important metabolic regulatory tissue that may be a central mediator of the cardiometabolic effects of breaking up prolonged sitting time. In addition to its key role in lipid storage, factors secreted from white adipose tissue play pivotal roles in appetite regulation, energy homeostasis, insulin sensitivity, inflammation, and immunological responses14,15. Excessive accumulation of visceral adipose tissue positively associates with increased cardiometabolic disease risk16. While the role of subcutaneous adipose tissue in disease pathophysiology is less clear, it has also been associated with increased disease risk17,18,19,20,21,22,23.

Storage of excess lipid in subcutaneous adipocytes has been driven by the vital physiological roles of lipids; including as structural elements in plasma membranes, second messengers and energy substrates. However, upon exceeding storage capacity of subcutaneous adipocytes, lipid accumulates in visceral adipose and eventually ectopically in metabolic regulatory organs17. Ectopic lipid accumulation has been well established as a contributor to low-grade inflammation, endoplasmic reticulum stress and tissue insulin resistance17. In adipose tissue, insulin resistance is characterized by reduced glucose uptake, impairment in both lipogenesis and insulin-stimulated inhibition of lipolysis14,24. Thus, insulin resistance increases lipid availability to visceral and ectopic sites, contributing to increased cardiometabolic risk14.

Acute exercise protects against ectopic lipid accumulation by oxidising fatty acids for ATP production and enhancing insulin sensitivity in multiple tissues. In adipose tissue, exercise training also reduces inflammation and increases glucose uptake through pathways which have been well characterized25. To date, studies have typically focused on understanding the impact of continuous exercise bouts on gene and protein expression. Few studies have investigated whether regular active breaks from prolonged sitting modulate similar pathways13,26, and the effects of breaks from prolonged sitting on adipose tissue are unknown. In a previous study we showed that, compared to uninterrupted sitting, frequent brief bouts of either light- or moderate-intensity walking lowered acute postprandial glucose and insulin responses2. An ancillary analysis of vastus lateralis muscle collected from 8 participants in the main study showed that those brief interruptions to sitting time resulted in upregulation of genes involved in cell development, glucose uptake, and anti-inflammatory pathways; and, downregulation of genes associated with protein degradation and muscle atrophy13.

In this investigation, we now aim to define the acute transcriptional events induced in subcutaneous adipose tissue by regular brief active interruptions to prolonged sitting time, using subcutaneous abdominal adipose tissue collected from the same subset of 8 participants in the skeletal muscle-tissue study described above2,13. We hypothesized that interrupting sitting time with brief, intermittent activity bouts of either light or moderate intensity would change expression of genes involved in substrate metabolism and inflammation, relative to prolonged sitting.

## Methods

### Study Overview

This randomized, three-intervention crossover trial was conducted in accordance with the Declaration of Helsinki and approved by the Alfred Health Human Research Ethics Committee (Melbourne, Australia). The study is registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12609000656235, 04/08/2009) and participants provided written, informed consent.

A detailed description of the participant characteristics, screening and testing procedures for the full study have been previously described2 and the relevant aspects of the study design and protocol are summarised in Fig. 1. Of the 19 participants in the main study, eight (7 male, 1 female) consented to subcutaneous adipose biopsies, and are included in the current investigation. The inclusion criteria were: age 45–65 years, body mass index (BMI) 25–45 kg/m2. Participants were excluded if they had a diagnosis of diabetes, were taking glucose and/or lipid-lowering medication, or were regularly engaged in moderate intensity exercise ≥150 minutes/week for at least 3 months. They attended three study visits at the Baker Heart and Diabetes Institute to complete the study conditions in a randomized order2. Study conditions were as follows:

1. (1)

Uninterrupted sitting. Participants remained seated throughout the experimental period and were instructed to minimize excessive movement, only rising from the chair to void.

2. (2)

Sitting interrupted with light intensity walking breaks. Participants rose from the seated position every 20 minutes throughout the experimental period (3 breaks per hour) and completed a 2-minute bout of light intensity walking (3.2 km/h) on a motorized treadmill on a level surface. The participants then returned to the seated position. This procedure was undertaken on 14 occasions, providing a total of 28 minutes of light-intensity activity.

3. (3)

Sitting interrupted with moderate intensity walking breaks. Identical to the light intensity walking breaks condition, but participants completed 2 minute bouts of moderate-intensity walking (5.8–6.4 km/h) on the treadmill, providing a total of 28 minutes of moderate-intensity activity. The speed of walking for this condition was based on individual perception of activity intensity, determined at a familiarisation session, and based on a Borg Rating of Perceived Exertion between 12 and 142.

A minimum washout period of 6 days between each condition was imposed to avoid potential confounding effects since an acute bout of activity may enhance insulin sensitivity for up to 72 hours. Participants were instructed to refrain from any structured moderate-vigorous exercise, alcohol and caffeine in the 48 hours prior to each of the trial conditions. During this time, physical activity was monitored using an Actigraph GT1M accelerometer (Actigraph, Pensacola, FL), which was worn around the hip during waking hours.

Participants reported to the laboratory between 0700 and 0800 hours, having fasted from at least 2200 hours the night before. A cannula was inserted into an antecubital vein for hourly blood sampling. For all of the three experimental conditions, and following the initial blood collection (time point: −2 hours), they remained seated for 2 hours to achieve a steady state before consumption of a standardized test drink (time point: 0 hours). The 200 mL test drink consisted of 75 g carbohydrate (100% corn maltodextrin powder, Natural Health) and 50 g fat (Calogen, Nuticia). The specific nutritional components were as follows: energy: 3,195 kJ; total fat: 50 g; saturated fat: 5 g; monounsaturated fat: 30.4 g; polyunsaturated fat: 14.3 g; total carbohydrate: 75 g; total sugars: 12.8 g; protein nil; fiber <1 g; sodium: 46.9 mg; and water: 90 g. Blood was sampled at baseline before drink consumption and hourly post drink consumption. The incremental area under the glucose-time, insulin-time and insulin/glucose-time curves have been previously presented for the whole cohort2, and the 8 participants included in this sub-study13.

Abdominal subcutaneous adipose tissue biopsies were obtained using standard aseptic technique and local anaesthesia (lignocaine) approximately 40–50 minutes after the last activity bout, and 5 hours after the drink ingestion (Fig. 1). During the first intervention visit, a 0.5–1 cm skin incision was made ~5 cm lateral to the navel/umbilicus, and a Bergstrom biopsy needle passed through to obtain approximately 1–2 cm3 of subcutaneous adipose tissue under suction. Biopsies taken at the second and third intervention trials were obtained from the side opposite that of the preceding trial and at the third trial >5 cm superior or inferior to the first to avoid the potential for sampling prior injured tissue. All biopsies were rinsed of blood in ice-cold sterile saline, the connective tissue removed and cleaned adipose tissue was snap frozen in liquid nitrogen for subsequent storage at −80 °C until further analysis.

### RNA extraction

RNA isolation from 100 mg of adipose tissue was performed using TRIzol Reagent as per manufacturer’s instructions (ThermoFisher Scientific, Massachusetts, USA). The RNA phase (the clear upper aqueous layer) was transferred to a new tube without disturbing the interphase. Then 1.5 volumes of 100% ethanol was added to the samples and loaded onto the RNeasy mini spin column (Qiagen, Hilden, Germany). RNA was extracted according to the manufacturer’s protocol. The RNA was quantified using a Nanodrop spectrophotometer (ThermoFisher Scientific, Massachusetts, USA) and Qubit fluorometer (ThermoFisher Scientific, Massachusetts, USA). The integrity of the RNA was assessed using a MultiNA Microchip Electrophoresis System (Shimadzu, Kyoto, Japan).

### Gene Expression Profiling/Microarrays

The amplification and labelling of RNA was performed using the TotalPrep amplification kit according to manufacturer’s instructions (Ambion; ThermoFisher Scientific Massachusetts, USA). The labelled samples were hybridized to the Human HT-12 v4.0 Expression BeadChip as per the manufacturer’s protocol (47,231 probes; Illumina, California, USA). The BeadChips were scanned using the iScan microarray BeadStudio platform (Illumina, California, USA). Quality standards for hybridization, labelling, staining, background signal, and basal level of housekeeping gene expression for each chip were verified. After scanning on the Illumina iScan, the resulting images were background subtracted, quantile normalized and processed using the GenomeStudio software (Illumina, California, USA). Data files were deposited into the National Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE115645).

An Illumina detection P-value of <0.05 was used to determine presence or absence of a probe in each tissue sample. Call rates for each of the three study conditions were then calculated for each gene, per condition using equation 1:

$$Call\,rate=\frac{number\,of\,adipose\,samples\,with\,a\,detection\,p\,value < 0.05}{total\,number\,of\,adipose\,samples}$$
(1)

A gene was included in the subsequent analysis if a call rate of ≥0.5 was calculated for at least one condition. Analysis of the effects of activity breaks versus prolonged sitting was performed in Stata (StataCorp, College Station, TX). To identify genes with the most marked changes in expression level, we applied an absolute fold change threshold of ≥0.5 (in either direction from 1) for any of the three experimental condition pairs (light intensity breaks versus uninterrupted sitting; moderate intensity breaks versus uninterrupted sitting; moderate versus light intensity breaks), where a fold change <1 refers to downregulation and >1 refers to upregulation of the gene relative to the comparator condition. This approach has been previously validated27, showing close qualitative and quantitative relationships to qPCR, including in skeletal muscle from this same study13.

### Statistical Analysis

Linear mixed models accounting for dependency in the data (repeated measures) were used to evaluate the differential effects of the trial conditions on expression of genes. All models were adjusted for age and BMI. P-values for the overall condition variable were obtained from post-hoc Wald tests and corrected for multiple comparisons using the False Discovery Rate (FDR) method of Benjamini-Hochberg28. Post hoc, pairwise analyses were performed using post-estimation commands of the linear mixed model, and the resultant pairwise P-values were corrected by the Dunn-Sidak approach. A corrected P-value of <0.05 was considered to be statistically significant. All statistical analyses were performed using Stata 14.1 for Windows (StataCorp, College Station, TX).

### Pathway analysis

Genes were ranked using a signed log10-transformed P-value (from the linear mixed model analysis) with the sign denoting the direction of change, positive for increasing and negative for decreasing29. The rank score for genes that were represented by more than one transcript, was calculated for the transcript with the lowest P-value, and the others were disregarded. The ranked gene list was inputted into GSEA 3.0 software (Broad Institute, Cambridge, MA). A normalized enrichment score (NES), the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genes, was calculated30. NES scores greater than zero indicate upregulation of a pathway, whereas NES values of less than zero represent down regulation.

Gene sets with a P-value ≤ 0.1, following false discovery rate (FDR) correction, were considered likely to generate valid hypotheses and drive further research. Leading edge analysis was used to examine the genes that were in the leading-edge subsets of the enriched gene sets. The Reactome pathway database (www.reactome.org) was used to categorize genes by pathways to facilitate biological interpretation.

All analyses were performed in a blinded manner.

## Results

Participant demographics and biochemical analyses for the eight participants recruited for this sub-study have been reported previously13. Those recruited for the present sub-study had a mean age of 55 ± 6 years and BMI 30.9 ± 2.9 kg/m2 (mean ± SD). In the main study (n = 19), interrupting prolonged sitting with both light- and moderate-intensity walking significantly reduced postprandial glucose (by 24% and 30%, respectively) and insulin (by 23% for both conditions) incremental area under the curve (iAUC), relative to uninterrupted sitting2. In the current subset of eight participants, there was a trend toward a decrease in glucose iAUC (−22% and −20%; P = 0.1) and a significant decrease in insulin iAUC (−25% and −23%; P < 0.05) in the light-intensity and moderate-intensity breaks conditions, respectively, relative to uninterrupted sitting13.

The microarray analysis identified 18,844 transcripts expressed in the adipose tissue. Of these, 469 satisfied the 0.5-fold change criteria (described in the Methods) in at least one out of the three comparisons between the experimental condition pairs. Thirty-six genes were significantly differentially expressed (FDR-adjusted P < 0.05; Table 1, Fig. 2). Of these, 7/36 genes were upregulated and 2/36 genes were downregulated in the light-intensity breaks condition, compared to uninterrupted sitting; 1/36 genes was upregulated and none were downregulated in the moderate-intensity breaks condition compared to uninterrupted sitting; and, 29/36 genes were upregulated and none were downregulated in the moderate-intensity breaks condition compared to the light-intensity breaks (Table 1).

Analysis of the ranked gene list identified 102 differentially (up- or down-) regulated pathways, with an FDR-adjusted P ≤ 0.10 (Fig. 3, Supplementary Table 1). In the light-intensity breaks condition, 64/102 pathways were upregulated and 19/102 were downregulated, compared to uninterrupted sitting. In the moderate-intensity breaks condition, 11/102 pathways were upregulated and 0/102 were downregulated, compared to uninterrupted sitting; and, 23/102 pathways were upregulated and 42/102 were downregulated, compared to light-intensity breaks. Among the regulated pathways were those involved with metabolism of macronutrients, adenosine triphosphate (ATP) synthesis, immune function, signal transduction, extracellular matrix organisation and cell cycle.

## Discussion

The major novel finding of this study is that actively breaking up prolonged sitting time in the postprandial state was associated with changes in the expression of subcutaneous abdominal adipose tissue genes and pathways involved in macronutrient metabolism and ATP synthesis, immune function, signal transduction, and cell cycle regulation. These changes are likely induced by systemic physiological drivers associated with physical activity and known to modulate adipose tissue function through multiple mechanisms including via the sympathetic nervous system, increased adipose tissue blood flow and changes in circulating factors including adrenaline, insulin and glucagon31. Interestingly, the majority of the differences were between the light-intensity activity, relative to both the uninterrupted sitting and the moderate-intensity breaks conditions. This may relate to the fact that of all the interventions studied, light intensity breaks would be most dependent on fat metabolism (adipose tissue)32,33. By contrast, moderate intensity breaks which would have been more reliant on glucose metabolism, had relatively few differentially regulated genes and pathways in comparison to prolonged uninterrupted sitting.

### Active breaks during prolonged sitting alter adipose expression of metabolic regulatory networks controlling ATP synthesis

Consistent with fat oxidation being the major pathway for ATP production induced by low-intensity activity, upregulation of genes and pathways associated with enhanced capacity for lipid oxidation were observed for the light-intensity breaks condition in comparison to both the uninterrupted sitting and moderate-intensity breaks conditions (Figs 2 and 3). Mitochondrial (tricarboxylic acid cycle and respiratory electron transport) and peroxisomal β-oxidation pathways were upregulated in the light-intensity breaks condition. The key functional difference between these compartmental processes is that mitochondrial β-oxidation generates acetyl-CoA and reduced cofactors via degradation of energy substrates, primarily long-chain fatty acids, which is coupled to oxidative phosphorylation; whereas, peroxisomes degrade a wide variety of lipophilic compounds that have important functions not involved in ATP resynthesis34. Although it did not reach the fold-change cut-off for the main analysis (1.45 fold change compared to uninterrupted sitting), upregulation of the NDUFAB1 (NADH:Ubiquinone Oxidoreductase Subunit AB1) gene was identified in the pathway analysis for the light-intensity breaks condition. This non-catalytic accessory subunit of the mitochondrial membrane Complex I functions in the transfer of electrons from NADH to the respiratory chain, indicating an increased capacity for substrate oxidation. Significantly lower expression of ID1 and NOS3 (endothelial nitric oxide synthase, eNOS) in the light-intensity breaks condition, compared to the uninterrupted sitting and moderate-intensity breaks conditions, is also consistent with higher lipid oxidation. Deletion of the ID1 gene (a transcriptional regulator of helix-loop-helix transcription factors that control cell type-specific gene expression) in mice has been implicated in numerous physiological effects with potential metabolic benefit. It was found that loss of the ID1 gene leads to increases in expression of genes which promote fatty acid oxidation, with id1−/− mice also exhibiting reduced insulin resistance and fat mass while on a high fat diet, and increased oxygen consumption compared to wild type mice35,36. Adipose eNOS expression is increased in obese adults, and has been implicated in the inhibition of lipolysis via downregulation of lipolytic pathways37,38. Downregulation of eNOS (NOS3) expression in the light-intensity breaks condition may therefore also indicate an increase in lipolysis. The differing expression patterns between the two break conditions for genes regulating lipolysis occurred despite similarity in blood glucose and insulin levels, suggesting that mechanisms associated with activity break intensity such as adrenergic pathways may have been the predominant influence.

Reflecting the preponderance of lipid over carbohydrate metabolism for provision of ATP during light-intensity activity physical activity32,33, pathways linked to carbohydrate metabolism were downregulated in comparison to both uninterrupted sitting and moderate-intensity breaks. Here, significantly lower adipose tissue NOS3 and MYO1C gene expression in the light-intensity breaks condition may indicate reduced insulin-dependent and -independent GLUT-4 translocation and subsequent glucose uptake39,40. In contrast, significantly higher expression of PFKFB3 was observed in the moderate-intensity breaks condition compared to both the uninterrupted sitting and light-intensity breaks conditions. PFKFB3 is an enzyme whose product (fructose 2,6-biphosphate) is a powerful activator of the glycolytic enzyme 6-phosphofructo-1-kinase, the rate-limiting step in glycolysis41,42, indicating a greater capacity for glucose oxidation in the moderate-intensity breaks condition (Fig. 3).

### Active breaks in prolonged sitting regulate anti-inflammatory and anti-oxidative stress pathways, and improve insulin sensitivity

Inflammatory and metabolic signaling are closely interlinked, such that chronic disturbance of metabolic homeostasis can lead to aberrant immune responses and inflammation; and, vice versa, where chronic inflammation can induce insulin resistance and metabolic dysfunction43. While a continuous moderate-to-high intensity exercise bout has been shown to activate inflammatory and oxidative pathways44,45, we found little evidence for modulation of immune function or inflammatory status with the brief moderate-intensity breaks from sitting in the current study, compared to uninterrupted sitting (Fig. 3). In contrast, light intensity breaks from sitting appeared to upregulate immune function and downregulate inflammatory signals compared to the uninterrupted sitting and moderate-intensity breaks conditions.

Tumour necrosis factor alpha (TNFα), derived primarily from resident macrophages, is known to be overexpressed in adipose tissue of obese humans and mice46,47,48. TNFα is a pro-inflammatory cytokine that activates various signal transduction cascades, including pathways involved in inhibiting insulin action43,49,50. TNFα and other inflammatory cytokines impair insulin action by post-translational modification of insulin receptor substrate 1 through serine phosphorylation, which interferes with the ability of this protein to engage in insulin-receptor signaling43. The ID1 gene has been suggested to be important in mediating the increased expression of TNFα in adipose tissue following a high-fat diet in mice36; the ID1 gene was downregulated in the light-intensity breaks compared to the moderate-intensity breaks condition. Receptors for TNFα play an important role in its downstream effects. Here, we also observed lower expression of the receptor TNFRSF25 in the light-intensity breaks condition; TNFRSF25 is known to stimulate NF-κB activity51. NF-κB inflammatory pathways contribute to the pathology of metabolic disorders52. Indeed, clinical trials have shown amelioration of insulin resistance and improved glucose homeostasis in type 2 diabetes patients treated with salicylates, which inhibit NF-κB activation53. Significantly lower expression of ID1 and TNFRSF25 in the light-intensity breaks condition compared to moderate-intensity breaks is suggestive of reduced production of TNFα and its downstream signaling. Despite not reaching the 1.5 fold change threshold, both the ID1 and TNFRSF25 genes were also significantly downregulated in the light-intensity breaks condition compared to uninterrupted sitting.

The potent pro-inflammatory molecule LTB4 is highly expressed in obesity, and has recently been shown to directly induce insulin resistance at least partially through its receptor, LTB4R54,55,56. Here, we observed significantly lower expression of the LTB4R gene in the light-intensity breaks, compared to the moderate-intensity breaks condition. LTB4R was also statistically significantly downregulated in the light-intensity breaks condition compared to uninterrupted sitting though the 0.5 fold change threshold was not reached (0.69 fold change). Furthermore, the Notch signaling pathway was significantly downregulated in the light-intensity breaks condition, compared to uninterrupted sitting and moderate-intensity breaks conditions. Notch signaling is highly conserved in mammals, and rodent studies indicate that inhibition of this pathway improves adipose tissue insulin sensitivity57. Together, these results suggest that downregulation of inflammatory pathways involving TNFα, NF-κB, lipoxygenase and Notch signaling could contribute to the beneficial effects of breaking up sedentary time with light-intensity activities on metabolism by positively influencing adipose tissue insulin sensitivity.

Reactive oxygen species (ROS) are continuously produced as by-products of aerobic metabolism. ROS can act as important signaling molecules, but can also cause oxidative damage to cells and tissues, and their accumulation is regulated by a complex system of anti-oxidative defences. In addition to β-oxidation, the peroxisomal lipid metabolism pathway is involved in the dynamic response to cellular stress. Peroxisomes contain a number of antioxidant defences involved in the regulation of ROS, including the synthesis of the ether phospholipid species plasmalogens58,59. We have previously shown that light-intensity breaks during prolonged sitting in adults with type 2 diabetes ameliorates the postprandial reduction in plasma plasmalogen species, compared to prolonged uninterrupted sitting4. The upregulation of peroxisomal lipid metabolism in the light-intensity breaks condition in the current study suggests that this may be partially due to increased biosynthesis of plasmalogen lipid species in the adipose tissue. This finding may provide an important mechanistic link between the protective effects of light-intensity breaks during prolonged sitting and risk of cardiometabolic diseases, as plasmalogens have been suggested to be protective against atherosclerosis60,61.

### Active breaks in prolonged sitting regulate adipose tissue cell cycle pathways

Coupling of cell cycle progression and programmed cell death pathways are regulated to maintain tissue homeostasis. The cell cycle involves a cascade of events that leads to cell division and DNA replication. Progression of the cell cycle is orchestrated by regulatory (cyclins) and catalytic subunits (cyclin-dependent kinases, CDKs), as well as ubiquitin ligases (such as anaphase promoting complex, APC) which mark cell cycle proteins for degradation62,63. Here, we observed upregulation of several pathways related to cell cycle and DNA replication, concurrently with upregulation of apoptosis pathways, in response to light-intensity breaks from sitting versus uninterrupted sitting. These data suggest adaptation of adipocyte cell cycle in the light-intensity breaks condition. Cell cycle and DNA replication pathways also tended to be upregulated in the moderate-intensity breaks condition, in comparison to uninterrupted sitting, but in most cases did not reach statistical significance (P > 0.1, Table 2). Upregulation of the M, G1, and S phases of cell cycle involved in cell growth and proliferation, as well as pathways involved in the transition between these phases, indicates acute stimulation of adipogenesis pathways. Upregulation of checkpoints also suggests greater quality control to prevent unhealthy cells from proliferating62.

Adipose cells dynamically adapt their growth and metabolism to current requirements, and crosstalk between these two biological responses has recently been identified64. Although relatively few studies have been conducted in humans, animal models provide strong evidence that, in addition to the control of cell proliferation and death, cell cycle mediators also play key roles in the biological function of adipocytes such as insulin sensitivity, lipolysis and glucose transport62,63. Therefore, acute upregulation of cell cycle pathways with active breaks in prolonged sitting (independently of break intensity) may indicate a role for these effectors in metabolic adaptation of adipose tissue to physical activity. This is also characteristic of other general cellular function pathways (e.g. metabolism of RNA and proteins, DNA replication) which were also upregulated with breaks at both intensities.