Breaking up prolonged sitting alters the postprandial ......breaking up prolonged sitting time may impart beneficial effects on the postprandial plasma lipidome of adults with T2D

Breaking up prolonged sitting alters the postprandial plasma lipidomic profile of adults with Type 2 Diabetes

Megan S. Grace, Paddy C. Dempsey, Parneet Sethi, Piyushkumar A. Mundra, Natalie A. Mellett, Jacquelyn M. Weir, Neville Owen, David W. Dunstan, Peter J. Meikle, Bronwyn A. Kingwell

The Journal of Clinical Endocrinology & Metabolism Endocrine Society Submitted: December 13, 2016 Accepted: March 08, 2017 First Online: March 13, 2017

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The Journal of Clinical Endocrinology & Metabolism; Copyright 2017 DOI: 10.1210/jc.2016-3926

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Breaking up sitting alters postprandial lipidome

Breaking up prolonged sitting alters the postprandial plasma lipidomic profile of adults with Type 2 Diabetes

Megan S. Grace1,2, Paddy C. Dempsey1,2, Parneet Sethi1, Piyushkumar A. Mundra1, Natalie A. Mellett1, Jacquelyn M. Weir1, Neville Owen1,3, David W. Dunstan1,4,5, Peter J. Meikle1,6, Bronwyn A. Kingwell1 1Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia 2Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Victoria, Australia 3Swinburne University of Technology, Melbourne, Victoria, Australia 4Centre of Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Burwood, Victoria, Australia 5Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, Victoria, Australia 6Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia Received 13 December 2016. Accepted 08 March 2017.

Context: Postprandial dysmetabolism in type 2 diabetes (T2D) is exacerbated by prolonged sitting, and may trigger inflammation and oxidative stress. It is unknown what impact countermeasures to prolonged sitting have on the postprandial lipidome. Objective: In this study, we investigated the effects of regular interruptions to sitting, compared to prolonged sitting, on the postprandial plasma lipidome. Design: Randomized cross-over experimental trial. Setting: Participants underwent three 7h conditions: uninterrupted sitting (SIT); light-intensity walking interruptions (LW); and simple resistance activity interruptions (SRA). Participants and Samples: Baseline (fasting) and 7h (postprandial) plasma samples from 21 inactive overweight/obese adults with T2D were analyzed for 338 lipid species using mass spectrometry. Main Outcome Measures: Using mixed model analysis (controlling for baseline outcome variable, gender, BMI and condition order), the percentage change in lipid species (baseline to 7h) was compared between conditions with Benjamini-Hochberg correction. Results: Thirty-seven lipids were different between conditions (p<0.05). Compared to SIT, postprandial elevations in diacylglycerols, triacylglycerols and phosphatidylethanolamines were attenuated in LW and SRA. Plasmalogens and lysoalkylphosphatidylcholines were reduced in SIT, compared to attenuated reductions or elevations in LW and SRA. Phosphatidylserines were elevated with LW, compared to reductions in SIT and SRA. Conclusion: Compared to SIT, LW and SRA were associated with reductions in lipids associated with inflammation; increased concentrations of lipids associated with antioxidant capacity; and differential changes in species associated with platelet activation. Acutely breaking up prolonged sitting time may impart beneficial effects on the postprandial plasma lipidome of adults with T2D. Evidence on longer-term intervention is needed.

We investigated the effects of breaks in sitting on the postprandial plasma lipidome in adults with T2D. Compared to prolonged sitting, activity breaks reduced pro-inflammatory and augmented antioxidant plasma lipid levels.

Introduction

Lifestyle interventions are recommended as front-line therapy for the management of type 2 diabetes (T2D), including moderate-to-vigorous aerobic and resistance exercises 1. Despite

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this, many with T2D remain inactive, and spend a large proportion of their waking hours in sedentary behaviours (sitting) 2,3. Detrimental associations of sedentary behaviours with increased risk of cardiometabolic diseases, including T2D, have been consistently observed 4,5. Importantly, the manner in which sedentary time is accumulated seems to be important, where frequent interruptions in sitting is beneficially associated with biological markers of metabolic risk, compared to an equal volume of sitting accumulated in prolonged uninterrupted bouts 6.

From a mechanistic perspective, inflammation provides a potential link between sedentary time and chronic diseases, as high overall sitting time and television viewing time are detrimentally associated with key inflammatory markers 7-10. A likely mechanism by which exercise and active breaks in sitting may act to reduce inflammation and disease risk is by lowering postprandial glucose excursions, and altering postprandial lipid responses.

Plasma lipids for cardiovascular risk prediction are routinely measured in the fasting state 11. However, except in the early morning, typical Western lifestyles are mostly spent in a postprandial state. Transient postprandial glucose and lipid excursions (postprandial dysmetabolism) promote oxidative stress and inflammation, which create an environment conducive to the development of metabolic and cardiovascular disease 12. It is, therefore, important to understand factors that influence the duration and magnitude of postprandial hyperlipidemia. Furthermore, lifestyle modifications that can practically and effectively attenuate postprandial lipid excursions are likely to be important in preventing and managing disease.

There are over 500 lipid species present in plasma which, in addition to their role as energy substrates, are important inflammatory and signalling mediators 13. Patients with T2D exhibit an altered fasting plasma lipidome compared to healthy controls, demonstrating dysregulation of several lipid classes which are mechanistically linked to oxidative stress and inflammation, and which may be both a cause and a consequence of insulin resistance 14-16. Recent studies have demonstrated that meal composition, and an acute exercise bout can modulate plasma and tissue lipidome profiles 15,17,18. However, the potential benefits of breaking up prolonged sitting on the postprandial lipidome have yet to be explored.

This ancillary study was conducted on a subset of participants from a larger trial that has been previously described 19. The main study showed that interrupting sitting time with light-intensity activities lowered postprandial glucose, insulin and triglyceride levels, compared to uninterrupted sitting 19. The aim of the present study was to determine the effects of light-intensity breaks in sitting on the postprandial plasma lipidome in adults with T2D. We hypothesised that interrupting sitting time would differentially alter postprandial plasma lipidome profiles, in comparison to uninterrupted sitting.

Materials and Methods

Study Overview This trial was approved by the Alfred Hospital Human Ethics committee, and was carried out in accordance with the Declaration of Helsinki. Participants provided signed informed consent. The study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12613000576729).

Participant characteristics, screening and testing procedures for the main study have been described in Supplemental Material, and previously 19. Of the 24 participants in the main study, 21 (13 men, 8 women) overweight/obese adults with T2D were included in the current investigation. Three participants from the main study were excluded due to lack of available plasma samples for the baseline or 7h time-points. The resulting unbalanced order of intervention was adjusted for in the statistical model.

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Trial Conditions Details of the trial conditions have been described in Supplemental Material, and previously 19. Each experimental condition commenced upon starting the breakfast meal (0h), participants consumed lunch at 3.5h. Fasting (-1h) and postprandial (7h) blood samples were analysed. The three trial conditions were as follows:

SIT. Participants sat upright in a comfortable chair throughout the experimental period, and were instructed to minimise excessive movement, only rising to attend the lavatory.

LW. Participants rose from the seated position every 30 min and completed a 3 min bout of light walking on a treadmill (3.2 km/h, zero gradient), and then returned to the seated position. This procedure was undertaken on 12 occasions (no break was undertaken at lunch or at 7h), totalling 36 min of activity.

SRA. Identical to the LW condition, except with bouts of simple resistance activities, totalling 36 min. Each 3 min was divided into a total of nine 20-sec movement segments, alternating between body weight resisted half-squats, calf raises, and knee raises with a gluteal contraction.

Lipidomic Analysis The lipidomic analysis method used was semi-quantitative, performed as previously described 20 with some minor adjustments (see Supplemental Material). The nomenclature used when referring to individual lipid species is based on LIPID MAPS 21, with recent revisions 22. For a number of the lipids which contain two fatty acid chains, the MS-based measurements do not directly determine the constituent double bonds, but rather the sum of the number of carbons and the sum of the number of double bonds across both fatty acids. These species are denoted as the combined length and number of double bonds (e.g. 36:4).

Statistical Analyses Normality of the outcome variables was checked using histograms and the number of severe outliers. A severe outlier was defined as value less than 25th percentile minus 3 inter quartile range (IQR) or greater than 75th percentile plus 3IQR 23. All models were adjusted for potentially important covariates explaining residual outcome variance (sex and BMI), baseline outcome values, and period effects (order of condition). Sensitivity analyses including additional covariates (statin use, waist:hip ratio) were performed to test the robustness of the results. Plots of residuals versus predicted values indicated that the data were normally distributed and homoschedastic.

Linear mixed models accounting for dependency in the data (repeated measures) were used to evaluate the differential effects of the trial conditions on percentage change in lipid species and lipid classes from baseline to post intervention.

All p-values obtained were corrected for multiple comparisons using the false discovery rate method of Benjamini-Hochberg (BH) approach 24. Post-hoc, pair wise analyses were performed using post estimation commands of the linear mixed model, and the resultant pair wise 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 LP, College Station, TX).

Results

The 21 participants included in the current analysis had a mean (±SD) age of 63±6 years, and body mass index 32.7±3.4 kg/m2; 20 participants were taking metformin medication. Detailed participant characteristics are provided in Supplemental Material, and previously 19. RER for SIT (0.85±0.02), LW (0.83±0.01) and SRA (0.81±0.01) indicate increased fat oxidation in SRA and LW compared to SIT, and SRA compared to LW (p-value for difference = 0.049). Controlling for treatment order, no significant difference was observed in fasting lipid concentrations between conditions for any lipid species (p>0.05), indicating reproducibility of the lipidomic profile at baseline. Before correction for multiple comparisons, the percent

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change from fasting (baseline) to 7h postprandial plasma concentrations of 4 of 24 lipid classes and subclasses (diacylglycerol, triacylglycerol, alkenylphosphatidylcholine and phosphatidylserine) were significantly different between conditions (Supplemental Table 1). Significance was lost after BH correction, but strong trends remained for triacylglycerols (p=0.06), phosphatidylserines (p=0.06), and alkenylphosphatidylcholines (p=0.08; Figure 1). Before correction, 85 of 338 lipid species were significantly different between conditions, and 37 remained significant following BH correction (Supplemental Table 2). These included species in the diacylglycerol, triacylglycerol, alkenylphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and lysoalkylphosphatidylcholine lipid classes and subclasses.

Diacylglycerol and Triacylglycerol All postprandial diacyl- and triacylglycerol species were elevated over the day compared to baseline fasting values (Figures 2 and 3). Of the 21 diacylglycerol and 42 triacylglycerol species, 9 and 16 species, respectively, were significantly different between conditions following BH correction. Post-hoc pair wise comparisons showed attenuated elevations for 9 and 16 species in LW, and 8 and 16 species in SRA, for diacyl- and triacylglycerol species respectively, compared to SIT. No significant differences were observed between LW and SRA.

The difference between the activity and SIT conditions appeared to be largely driven by lipids containing saturated fatty acids (SFAs), particularly palmitic acid (16:0). All 9 diacyl- and 16 triacylglycerols that were significantly different between conditions contained at least one SFA; 5 diacylglycerols and 10 triacylglycerols contained at least one 16:0.

Alkenylphosphatidylcholine Postprandial alkenylphosphatidylcholine (plasmalogen) species were reduced in SIT, compared to baseline (Figure 4). By contrast, alkenylphosphatidylcholine species were generally elevated or unchanged in LW; and elevated or reduced to a lesser extent (compared to SIT) in SRA. Of the 13 alkenylphosphatidylcholine species detected, PC(P) 30:0, 32:1, 34:1, 34:2 and 36:2 were significantly different between conditions following BH correction. Pair wise, all 5 species were significantly different in LW, and PC(P) 34:1 and 36:2 in SRA, compared to SIT. No significant differences were observed between LW and SRA.

Phosphatidylethanolamine Postprandial phosphatidylethanolamine species were generally elevated or unchanged in all conditions, compared to baseline (Figure 4). Of the 15 phosphatidylethanolamine species detected, PE 32:1, 34:2, 36:2 and 36:3 were significantly different between conditions following BH correction. Pair wise, PE 34:2, 36:2 and 36:3 showed significantly attenuated elevations in LW, and all 4 were attenuated in SRA, compared to SIT. No significant differences were observed between LW and SRA.

Phosphatidylserine Postprandial phosphatidylserine species were elevated in LW, compared to a reduction in SIT and SRA (Figure 4). Of the 3 phosphatidylserine species detected, only PS 38:4 was significantly different between conditions following BH correction, though PS 36:1 and PS 40:6 showed strong trends (p<0.08, Supplemental Table 2). Pair wise, PS 38:4 was significantly elevated in LW, compared to reduced postprandial concentrations in SIT and SRA. No significant differences were observed between SIT and SRA.

Lysoalkylphosphatidylcholine Lysoalkylphosphatidylcholine species were reduced in SIT, compared to being either elevated or unchanged in LW, or reduced to a lesser extent in SRA (Figure 4). Of the 9 lysoalkylphosphatidylcholine species detected, only LPC(O) 18:1 was significantly different between conditions following BH correction, though 5 others (16:0, 18:0, 22:1, 24:2 and

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24:1) showed moderate to strong trends (p<0.20, Supplemental Table 2). Pair wise, LPC(O) 18:1 was unchanged in LW, compared to reduced postprandial concentrations in SIT. No significant differences were observed between SIT and SRA, or LW and SRA.

Sensitivity Analyses Waist:hip ratio was added as a covariate in the statistical model as an alternative method of assessing fat mass, in addition to BMI. Waist:hip ratio and BMI were moderately correlated (r=-0.42). Addition of waist:hip ratio to the model identified the same lipid species as different between conditions as the original model, as well as three additional species (following BH correction). These additional species included THC 22:0 and PS 36:1, which were significantly elevated in LW, compared to reductions in SIT and SRA; and the postprandial elevation in DG 16:0/22:6 was significantly attenuated in LW and SRA, compared to SIT. Including statin use as a covariate in the model additionally identified THC 22:0, but moderated CE 18:1 and PC(P) 36:2 to insignificance following BH correction.

Discussion

This study demonstrates that, compared to prolonged sitting, light-intensity activity interruptions are associated with changes in the postprandial plasma lipidome in T2D patients. Following correction for multiple comparisons, lipid species in the diacylglycerol, triacylglycerol, alkenylphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and lysoalkylphosphatidylcholine lipid classes and subclasses were significantly different between conditions. The type of activity that was performed during each break (LW versus SRA) appeared to induce differential postprandial effects, including lower RER (higher fat oxidation) in the SRA condition. This study extends upon previous experimental trials, which have shown benefits for traditional cardio-metabolic risk biomarkers with breaks in sitting time. Our findings provide new insight into the potential candidate mechanisms driving the benefits of interrupting prolonged sitting, and support this approach as a lifestyle-based treatment strategy for patients with T2D.

Diacylglycerol and Triacylglycerol Previous studies have demonstrated that frequent active breaks from sitting are as or more effective at reducing postprandial plasma triglyceride levels, compared to a continuous bout of exercise 25,26. Taken together, these previous studies and our current results suggest that the type, intensity, frequency and duration of activity breaks, and their timing relative to meals, may be important in attenuating postprandial triglyceride responses. In the current study, the significant between-condition effect for triacylglycerols as a lipid class (p=0.004), was attenuated after correction for multiple comparisons (p=0.057). In pair-wise comparisons, triacylglycerols were reduced in LW and SRA, compared to SIT (p<0.05). This contrasts somewhat with the findings from the main trial, where SRA, but not LW, breaks significantly lowered postprandial plasma triglyceride responses in comparison to SIT 19. These differences between our current and previously reported results are likely due to the different statistical and analytical methods used to assess plasma triacylglycerol content (see Supplemental Material).

The diacyl- and triacylglycerol responses observed in the current analysis are likely to be driven by the major constituents of the standardised meals. Differences between the LW and SRA conditions, compared to SIT, could indicate differences in both diacyl- and triacylglycerol entry into the circulation and how quickly they are removed. As little as one day of inactivity with high levels of sitting time can reduce whole-body insulin action, even when energy intake is reduced to match expenditure 27. Therefore, increased entry of lipids into the plasma in the SIT condition may result from an acute increase in insulin resistance of the adipose tissue, which would impair insulin-mediated inhibition of lipolysis. Increased

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levels of fatty acids in the circulation, in combination with acute insulin resistance in the liver, could act to increase hepatic triacylglycerol synthesis and release of triacylglycerol-rich VLDL 16. Moreover, lack of muscle activity in the SIT condition may lead to a reduction in lipoprotein lipase activity, thus reducing muscle-mediated uptake of fatty acids for ATP production, and adipose-mediated uptake for storage 28.

The constituent fatty acids (FAs) incorporated into circulating diacyl- and triacylglycerol species have important implications for cardiometabolic health. Several of the plasma diacylglycerols and triacylglycerols that have previously been shown to be positively associated with insulin resistance and excess weight 29 were attenuated in the LW and SRA conditions in the present study, compared to SIT. We also observed that several diacyl- and triacylglycerol species that were differentially altered in the activity conditions contained the SFA palmitic acid (16:0). Emerging evidence suggests that individual FAs within a given class have distinct associations with inflammation and oxidative stress 30. A recent study showed that fasting levels of 14:0 and 16:0 SFAs were positively associated with markers of inflammation, possibly due to stimulation of toll like receptors and activation of pro-inflammatory NFκB pathways. In contrast 18:0 SFA was inversely associated with markers of inflammation 30. Therefore, the attenuated increase in 16:0 SFAs observed in the activity conditions in this study could indicate reduced postprandial inflammation (Table 1).

Alkenylphosphatidylcholine Plasmalogens are thought to act as endogenous antioxidants, and a decrease in concentration of these species is suggested to relate to higher oxidative stress 31,32. Indeed, plasmalogens are negatively associated with T2D and CVD 14,15,31,33. Postprandial dysmetabolism leading to high plasma lipids may promote oxidative stress and reduce the antioxidative potential of the phospholipid pool 12,15. In the present analysis, postprandial alkenylphosphatidylcholine species were generally reduced in the SIT condition; elevated or unchanged in LW; and were elevated or reduced to a lesser extent (compared to SIT) in SRA. Statin use has been shown to normalise plasma plasmalogen levels in patients with metabolic syndrome, however, additionally controlling for statin use in the statistical model only attenuated the effect for one plasmalogen species - PC(P) 36:2 15. These results suggest a potentially beneficial effect of active breaks in sitting for antioxidative capacity in the postprandial state (Table 1). The mechanisms behind this benefit are not clear, but could relate to reduced breakdown of alkenylphosphatidylcholine plasmalogens due to greater clearance of pro-inflammatory SFAs in the activity conditions, and therefore lower levels of oxidative stress. These findings suggest that some of the benefit from breaking up prolonged sitting in those with T2D may be via improved antioxidant capacity.

Phosphatidylethanolamine Several phosphatidylethanolamine species showed attenuated elevations in the LW and SRA conditions, relative to SIT. High plasma concentrations of phosphatidylethanolamine and a decreased phosphatidylcholine:phosphatidylethanolamine ratio are positively associated with obesity, prediabetes, and T2D 14,20. However, plasma phosphatidylethanolamines were not reported to be significantly associated with insulin resistance or overweight/obesity 29. Phosphatidylethanolamine may also play a role in thrombus formation, where it can act both synergistically with and independently of phosphatidylserine to catalyse the formation of thrombin from pro-thrombin 34 (Table 1).

Phosphatidylserine When platelets are activated, they release platelet derived microparticles (PDMPs), which are important in inflammatory processes, and play a central role in thrombosis in part due to the function of phosphatidylserine, which are contained on their cell surface 35. It has been suggested that a sedentary lifestyle may affect exercise-induced platelet function in a pro-

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thrombotic manner 36,37. Compared to decreases in SIT and SRA, we observed a consistent increase in plasma phosphatidylserine species in the LW condition, possibly due to greater shear stress mediated platelet activation and release of phosphatidylserine-containing PDMPs (Table 1). This is a novel finding, however, haemostasis and thrombus formation are regulated via complex signalling pathways, and thus the physiological outcome of an increase in phosphatidylserine species in the LW condition is unclear.

Lysoalkylphosphatidylcholine Platelet activating factor (PAF) is a pro-inflammatory, pro-thrombotic signalling lipid. Whereas, lysoalkylphosphatidylcholine (lyso-PAF) opposes the activities of PAF 38. Several lysoalkylphosphatidylcholine species are negatively associated with obesity, insulin resistance and T2D 14,29,39. In the current analysis, only LPC(O-18:1) remained significant following BH correction, however similar trends were observed for almost all species within this lipid subclass. Lysoalkylphosphatidylcholine species tended to decrease or remain unchanged postprandially in SIT and SRA, whereas several species tended to increase or remain unchanged in the LW condition. Though no strong conclusions can be drawn from these data, the overall trends suggest a potentially beneficial effect of LW breaks on lysoalkylphosphatidylcholine species, indicating a possible anti-inflammatory and anti-thrombotic effect (Table 1).

Strengths and Limitations Key strengths of our study include the focus on men and women with overt T2D, who are at high risk of postprandial dysmetabolism, and could derive substantial benefits from lifestyle interventions that help to manage exaggerated postprandial lipid excursions. Participants were their own controls, which reduced variance and permitted a smaller sample size. We also incorporated standardised trial condition lead-in periods, strict behavioural supervision, and standardised feeding of a typical Western diet during experimental conditions. False positives were minimised by the use of mixed model analysis that accounted for repeated measures and adjusted for potential confounders, and correction for multiple testing with the false discovery rate method by Benjamini and Hochberg 24. Limitations of the study include the acute nature of the experimental protocol. Further experimental insights into the chronic effects of breaking up sedentary time on the postprandial plasma lipidome are required. The size of the cohort, though sufficient to detect significant differences between conditions, was relatively small. A larger cohort may uncover a greater number of differentially regulated lipid classes, subclasses and species, and would allow for more in depth analysis of the potential effects of other covariates (such as ethnicity, visceral adiposity, and presence of the metabolic syndrome or its components). The results from this study may not be generalizable to other populations e.g. non-diabetic, non-obese, less well-controlled T2D (insulin-dependent), those with diabetic complications or other comorbidities, and those of non-Caucasian ethnicity. Moreover, the generalizability of this highly controlled laboratory-based trial to free-living settings is not certain, and the behaviours implemented in this study may not reflect habitual behaviours. For example, the SIT condition is unlikely to represent normal daily sitting patterns, and exaggerate prolonged uninterrupted sitting. The standardised meals reflecting a Western diet will inevitably vary in daily life settings, and how such variations interact with physical activity are also unknown. Finally, future studies should incorporate concurrent measures of inflammatory and oxidative stress markers, which could help to clarify the mechanisms by which prolonged sitting may affect health outcomes via alterations in the postprandial plasma lipidome.

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Summary

Much of what is known about the link between the plasma lipidome and disease is understood within the context of the fasted state. However, increasing evidence suggests that measurement of postprandial glucose, lipids, inflammation and oxidative stress may provide important clinical information concerning susceptibility to disease onset and progression 40. Patients with T2D are vulnerable to postprandial oxidative stress, and may benefit from lifestyle interventions aimed at improving antioxidant capacity, and attenuating postprandial dysmetabolism. This study builds on recent findings that the postprandial lipidome can be modified by alterations in diet and exercise, and it poses a number of questions for future research. We have shown that regular active interruptions in sitting may be of benefit in reducing postprandial pro-inflammatory lipids, and increasing concentrations of lipids with antioxidant capacity in adults with well controlled non-insulin dependent T2D. Moreover, we have observed differential changes in lipid species linked to platelet activation. The majority (n=20) of participants in this study were taking metformin, and 15 were on statins, indicating a potentially beneficial effect of breaks in sitting in addition to medication. These areas of investigation will be important to target in future studies, and could further elucidate the mechanisms driving the cardiometabolic effects linked to reducing and breaking up prolonged sitting.

Corresponding author and person to whom reprint requests should be addressed: Megan S. Grace, Baker Heart and Diabetes Institute, Level 4 Alfred Centre, 99 Commercial Road, Melbourne, VIC 3004, Australia, e-mail: [email protected]

Funding

This research was supported by a NHMRC project grant (no. 1081734) and the Victorian Government OIS scheme. PCD is supported by an Australian Postgraduate Award and a Baker Bright Sparks top up scholarship. BAK, PJM, NO and DWD are supported by the NHMRC Fellowships scheme.

Clinical Trial Registration Number: ACTRN12613000576729, www.anzctr.org.au.

Disclosure Summary: The authors have nothing to disclose

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Figure 1. Postprandial plasma lipid class and subclass % change from fasting baseline. Data are presented as mean ± SEM. Following Benjamini-Hochberg correction, no significant differences (p<0.05) were observed across treatment conditions (linear mixed model analysis). However three lipid classes showed strong trends, as indicated.

Figure 2. Postprandial plasma diacylglycerol lipid species % change from fasting baseline. Data are presented as mean ± SEM for diacylglycerol species that were significantly different (p<0.05) across treatment conditions in the linear mixed model analysis, following Benjamini-Hochberg correction. * Significant compared to SIT (post-hoc pairwise analysis with Dunn-Sidak correction; p<0.05). Mean ± SEM fasting concentration for each diacylglycerol species is indicated on the right (µM).

Figure 3. Postprandial plasma triacylglycerol lipid species % change from fasting baseline. Data are presented as mean ± SEM for triacylglycerol species that were significantly different (p<0.05) across treatment conditions in the linear mixed model analysis, following Benjamini-Hochberg correction. * Significant compared to SIT (post-hoc pairwise analysis with Dunn-Sidak correction; p<0.05). Mean ± SEM fasting concentration for each triacylglycerol species is indicated on the right (µM).

Figure 4. Postprandial plasma alkenylphosphatidylcholine (PC(P)), phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysoalkylphosphatidylcholine (LPC(O)) lipid species % change from fasting baseline. Data are presented as mean ± SEM for species that were significantly different (p<0.05) across treatment conditions in the linear mixed model analysis, following Benjamini-Hochberg correction. * Significant compared to SIT; # significant compared to SRA (post-hoc pairwise analysis with Dunn-Sidak correction; p<0.05). Mean ± SEM fasting concentration for each lipid species is indicated on the right (µM).

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Table 1. Summary of changes in the postprandial lipidome, compared to fasting, in the prolonged sitting and frequent activity conditions.

Pathways Key lipid class/subclass SIT LW SRA Diacylglycerol ↑↑ ↑ ↑ Triacylglycerol ↑↑ ↑ ↑

Pro-inflammatory

Phosphatidylethanolamine ↑↑ ↑ ↑ Anti-inflammatory Lysoalkylphosphatidylcholine ↓ ↑/↔ ↓ Anti-oxidant capacity Alkenylphosphatidylcholine ↓ ↑/↔ ↔

Phosphatidylserine ↓ ↑ ↓ Phosphatidylethanolamine ↑↑ ↑ ↑

Platelet activation

Lysoalkylphosphatidylcholine ↓ ↑/↔ ↓

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Breaking up prolonged sitting alters the postprandial ......breaking up prolonged sitting time may impart beneficial effects on the postprandial plasma lipidome of adults with T2D