Effects of HIIT on Postprandial Metabolism in Males

Introduction

Exposure of the arterial endothelium to lipids and low-density lipoproteins and their remnants can cause an innate immune, local inflammatory response, which drives the formation and development of atherosclerotic lesions and plaques (^{Hansson, Robertson, & Soderberg-Naucler, 2006}). Western diets and dietary habits typically feature substantial fat intake and frequent meal consumption, creating a sustained environment of postprandial lipemia (PPL), which exacerbates and protracts the potentially deleterious effects of endothelial exposure (^{Hyson, Rutledge, & Berglund, 2003}).

Prior moderate-intensity continuous exercise has been shown to reduce both the fasting and postprandial circulating concentrations of lipids and lipoproteins, in a dose-dependent fashion, when performed between 1h and 48h before feeding (^{Gill, Herd, & Hardman, 2002; Plaisance et al.}

, 2008). High-intensity interval training (HIIT) has grown popular in recent years, seemingly offering similar metabolic adaptations to continuous exercise, but with lower energy expenditure and a reduced time commitment. Indeed, acute HIIT appears to lower PPL comparably to continuous exercise, with as little as 120s of intense activity (^{Freese, Levine, Chapman, Hausman, & Cureton, 2011}). At present, however, little is understood about the effects of chronic HIIT on PPL. Previous studies have been limited by reasonably short durations of exercise abstinence (0-48h) following the last bout of training, making it difficult to distinguish between the enduring effects of training and the acute responses to the last training bout.

The aim of this study was to determine whether chronic HIIT confers enduring improvements in PPL and the protein content of associated enzymes and transporters, as well as other biomarkers of metabolic function, in healthy young males.

Methods

Ethical Approval

The study was approved by the University of Nottingham Faculty of Medicine and Health Sciences Research Ethics Committee (reference no. D14112016) in observance of the present regulations laid out by the Declaration of Helsinki, except for registration in a database. Prior to participation, all individuals were comprehensively informed of the nature of the study; thereafter, written informed consent was obtained.

Participants

Eight young recreationally active males were recruited (age 22 ± 3 years, height 1.77 ± 0.07m, body mass 67.7 ± 6.2kg). Medical screenings, including anthropometric measures, blood pressure, a blood sample for routine screening, a 12-lead electrocardiogram, and an International Physical Activity Questionnaire (IPAQ), were undertaken. Eligibility was defined as BMI...

Experimental Overview

Following satisfactory medical screening and subsequent recruitment to the study, participants attended the laboratory for two preliminary visits; once to assess and confirm maximal oxygen uptake (^V̇O2max) and once to undertake an exercise familiarization session (See Fig. 1 for experimental design overview). At least 72h after the familiarization visit, following a 3-day period of exercise abstinence and dietary standardization, participants were provided a set mixed meal before fasting overnight (≥12h). The following morning, participants attended the laboratory to provide a sample of muscle tissue and to undergo a 6h mixed meal tolerance test (main experimental visit 1; a schematic overview of the experimental proceedings of these visits is provided in Fig. 2). The main experimental visit proceedings and control measures were repeated ≥72h after the final HIIT session (main experimental visit 2). Lastly, participants returned to the laboratory one or two days after the second main experimental visit to have their V̇O2max reassessed and confirmed.

Preliminary Visits

Incremental cardiopulmonary exercise tests were performed on an electronically braked cycle ergometer (Excalibur, Lode B.V., Groningen, NL) to determine ^V̇O2max and corresponding heart rates and workloads for the prescription of training intensities. To confirm if ^V̇O2max was achieved, following >30min of rest, a second abbreviated test was performed until a workload ~20W greater than the final stage of the first test was achieved. On a separate day, participants completed a session of HIIT to familiarize themselves with the exercise protocol and to confirm the appropriate difficulty of the calculated workloads. Following a 3min warm-up at 45% ^V̇O2max, 10 intervals of 60s cycling at a workload corresponding to 90% ^V̇O2max, interspersed with 60s cycling at 45% ^V̇O2max were completed. The sessions ended with a 2min cool-down at 45% ^V̇O2max.

Main Experimental Visit Proceedings

In advance of the first main experimental visit, participants completed a 3-day food diary to assess habitual dietary intake. Dietary analysis was performed using Nutritics (Dublin, IE) software, and individually tailored suggestions were provided, aiming to produce similar macronutrient intake between participants (45% carbohydrate [CHO], 35% fat, 20% protein) for the 3 days prior to each main experimental visit, during which diet diaries were again recorded, to confirm standardization.

Participants arrived at the laboratory at ~08.00am and provided a urine sample before their body mass was measured. Subjects were then asked to rest, semi-supine, in a bed while a fasting muscle biopsy sample was obtained from the vastus lateralis of one leg using the suction-modified Bergstrom biopsy technique (^{Bergstrom, 1975; Evans, Phinney, & Young, 1982}) for the determination of skeletal muscle protein content of lipolytic enzymes and lipid transporters.

Two retrograde cannulas were then inserted; one into a superficial hand vein, which was kept in a monitored hand-warming unit (55°C) to obtain arterialized-venous blood samples (^{Gallen & Macdonald, 1990}), and another was guided using ultrasound into a deep vein of the opposing forearm. The cannulas were kept patent via a saline drip, and samples from each line were simultaneously obtained at baseline (fasted) and then every 20min for the first 3h, and hourly for the following 3h, after consumption of the mixed meal. Brachial artery blood flow (BF; expressed in ml∙min^-1) was assessed using Doppler ultrasound (Aplio 300, Toshiba, TYO, JP) immediately after each blood sample, enabling the determination of rates of substrate (S) uptake (expressed in mmol∙l^-1 or µmol∙l^-1) across the forearm, using the following equation:

^{S_uptake=( [S_arterialized]-[S_venous] )×BF}

Resting substrate oxidation rates and energy expenditure were assessed pre, and 2, 4, and 6h postprandial, via indirect calorimetry using a flow-based dilution canopy hood (Quark RMR, Cosmed, IT). Total serum concentrations of branched-chain amino acids (BCAA) were determined spectrophotometrically as has been previously described (^{Ohshima, Misono, & Soda, 1978}). Urea concentrations were quantified in plasma (from the first and last blood samples) as well as in urine, using a commercially available enzymatic kinetic assay (UR220, Randox, NI) to determine nitrogen urea excretion rates, which were used to correct indirect calorimetry data for protein oxidation rates using equations from ^{Frayn (1983)}.

Discussion

The principal novel finding of this study was that 16 sessions of HIIT significantly increased postprandial FFA uptake across the forearm of healthy male individuals, but did not influence circulating TAG concentrations or their clearance, when measured ≥72h after the final bout of exercise training. This suggests that the lipaemia-lowering effects of the HIIT modality, which has previously been shown to acutely induce marked improvements in postprandial lipid handling (^{Thackray, Barrett, & Tolfrey, 2013}), are primarily transient in nature and do not accumulate with regular exercise training.

In healthy young males, the triglyceridaemia-lowering effects of a single bout of HIIT have been shown to resolve within 48h (^{Gabriel et al., 2013}). In women at risk of the metabolic syndrome, acute HIIT reduced fasting and postprandial TAG when assessed 48h after the final exercise session; however, 6 weeks of training did not magnify this attenuation (^{Eric C. Freese et al., 2015}). From this data, however, it cannot be known if chronic HIIT extends the efficacious duration of TAG reduction beyond 48h. Our study attempts to bridge this gap, having assessed the effects of regular HIIT beyond the period of influence of acute lipaemia co-regulators, such as an exercise-induced energy deficit, and shifts in substrate oxidation and delivery. Expanding upon the work of those before us, we can infer from our findings that regular HIIT does not enhance, in magnitude or duration, the triglyceridaemia-lowering effects of acute HIIT in metabolically healthy individuals, and as such must be performed at least every 48h to maintain its efficacy.

The observed increase in FFA uptake appears to represent trends towards both increased FFA arterio-venous differences and increased forearm blood flow between postprandial hours 4-6. Consistent with our findings, it has previously been reported in obese adults that 8 weeks of 10x60s HIIT did not alter resting brachial artery diameter ~72h after exercise, but did increase flow-mediated dilatation, despite no significant changes in insulin concentration nor markers associated with vessel responsiveness (^{Sawyer et al., 2016}). Further examination of our results revealed strong negative correlations between circulating arterialized FFA and insulin concentrations both pre- and post-training (r = -0.931 and r = -0.963, respectively). Interestingly, however, circulating insulin concentration and indices of insulin sensitivity remained unchanged with training, suggesting that the observed increase in FFA uptake is insulin-independent. Similarly, no improvements in fasting or postprandial glucose concentrations, uptake, nor AUCs, were observed following training. Our findings contrast those of Babraj et al. (2009) who reported reduced tAUC for plasma glucose, insulin, and FFA in response to an oral glucose tolerance test, without changes in their fasting concentrations when assessed 2-3 days after 6 sessions of HIIT.

Seemingly incongruously, increased FFA uptake has been associated with the insulin-resistant state for its role in the accumulation of intracellular lipids, while impaired FFA uptake has been observed in individuals with impaired glucose tolerance (^{Turcotte & Fisher, 2008; Turpeinen et al., 1999}). It is important to note, however, that the deleterious associations of FFA uptake and metabolic health are observed in physically inactive, but not trained, individuals. Fasted skeletal muscle FFA uptake and oxidation are greater in lean than obese individuals, and regular exercise training increases muscle lipid oxidative capacity and intramyocellular triglyceride content (IMTG) (^{Colberg, Simoneau, Thaete, & Kelley, 1995; Pruchnic et al., 2004}). Training-induced increases in FFA uptake may, therefore, increase the potential for lipid oxidation while also increasing re-esterification of circulating fatty acids into the intramyocellular lipid pool. However, the rise in FFA uptake reported in the present study was not accompanied by a corresponding increase in whole-body fasted fat oxidation, nor in response to feeding, which is consistent with previous findings of increased fat and decreased CHO oxidation at rest 24h, but not 72h, after the final bout of two weeks of HIIT comprising repeated 30s maximal effort cycling sprints (^{Whyte, Gill, & Cathcart, 2010}). Collectively, our observations might be indicative of greater FFA-derived IMTG accumulation in response to regular HIIT. It has been postulated that IMTG accrual in response to endurance training is an adaptive response, enabling a greater contribution of locally stored lipids as a substrate during exercise (^{van Loon & Goodpaster, 2006}).

We reported a modest but significant reduction in the protein content of skeletal muscle ATGL at rest after chronic training, as well as a tendency for FATP1 content to decline in a similar fashion, but found no change in that of CD36 or LPL. ATGL is principally responsible for catalyzing the initial step in TAG hydrolysis (^{Zimmermann et al., 2004}). Inactivation of the Atgl gene in mice has been shown to increase IMTG content as well as to decrease plasma FFA concentration in both the fed and fasted state (^{Haemmerle et al., 2006}). The effect of exercise training on skeletal muscle ATGL is currently poorly understood. It has been reported in both normal-weight and obese males that skeletal muscle ATGL protein content is increased approximately two-fold 48h after cessation of 8 weeks of moderate-intensity exercise training and is accompanied by a substantial reduction (28 - 42%) in IMTG content (^{Alsted et al., 2009; Louche et al., 2013}). In contrast to our findings, it has been shown in rat muscle that ATGL protein content and lipase activity peak 10h after acute HIIT, and remain elevated 72h post-exercise following 5 weeks of HIIT (^{Nikooie & Samaneh, 2016}).

Divergent signaling responses between muscle fiber phenotypes have been reported in response to HIIT and moderate-intensity exercise (^{Kristensen et al., 2015}), and inter-individual differences in muscle phenotype may explain some of the variation in lipid-handling responses to training. Greater contractile intensity, such as in HIIT, increases recruitment of fast fibers, the local activity of which has been suggested to be associated with increased LPL activation and immunoreactive mass (^{Gabriel et al., 2013; Hamilton, Etienne, McClure, Pavey, & Holloway, 1998; Sale, 1987}). ATGL is purportedly expressed solely in type 1 muscle fibers (^{Jocken et al., 2008}). Since vastus lateralis muscle comprises a mixed distribution of fast and slow type fibers (^{Staron et al., 2000}), and the distribution of myosin heavy chain isoforms varies not only along the length of a muscle but within individual fibers, this might explain some of the disparity in findings where biopsy sampling is employed.

Our findings contrast some of the existing literature; however, synthesizing previous observations with our own and our additional finding of increased FFA uptake, it could be speculated that the observed downregulation of ATGL in this study also reflects a shift towards IMTG accumulation allowing for greater local substrate oxidation during exercise, as previously reported (^{Shepherd et al., 2013}).

Such a notion, however, challenges our understanding of the role and importance of FATP1 in IMTG accumulation, since it tended to be downregulated by a similar magnitude; however, variation between individual responses was substantial. FATP1 is an integral membrane protein that is highly expressed in skeletal muscle and plays a role in mediating fatty acid import into skeletal myocytes (^{Lewis, Listenberger, Ory, & Schaffer, 2001}). It has been shown in cultured human skeletal muscle cells that FATP1 stimulates transport and consumption of fatty acids and channels them away from oxidation and towards intramyocellular TAG synthesis (^{García-Martínez et al., 2005}). In contrast, FATP1 overexpression was shown to induce greater fatty acid oxidation, but not TAG esterification in rats, while enhanced disposal of both circulating fatty acids and IMTG has been reported in mice (^{Guitart et al., 2014; Holloway et al., 2011}).

Conclusion

The effects of exercise training on FATP1 are poorly understood. Consistent with our findings, 8 weeks of aerobic training, eliciting a 15% increase in ^V̇O2max, induced a 20% reduction in FATP1 protein content, but did not alter CD36 content in comparable participants (^{Jeppesen et al., 2012}). Furthermore, 16 weeks of aerobic interval training in metabolic syndrome patients had no effect on FATP1 protein content in vastus lateralis muscle but was reduced 3- to 4-fold in adipose tissue (^{Tjønna et al., 2008}). Even in habitually active individuals, FATP1 and CD36 mRNA and protein expression have been shown not to differ between age-matched, sedentary, normoglycaemic individuals and type 2 diabetics (^{Pelsers et al., 2007}). Clearly, further research into the roles of fatty acid transport proteins and lipases, with regards to adaptations to exercise training, is warranted in order to better understand their significance in metabolic health.

In the current study, HIIT had no effect on insulin measures or indices, and as might be expected therefore, did not alter glycaemic control. Interval training has been shown to improve HOMA-IR score 72h post-exercise in men at risk for insulin resistance; however, this was accompanied by a modest reduction in body mass (^{Earnest et al., 2013}). Given that energy-restriction-induced weight-loss alone is sufficient to improve TAG and glucose responses to feeding (^{Dallongeville et al., 2007; Dengel, Galecki, Hagberg, & Pratley, 1998}), it cannot be ignored that weight-loss may be the primary driver of such sustained improvements. It is important to recall the good metabolic health of the participants in this study prior to the training intervention, leaving a smaller window for improvement compared to metabolically at-risk populations. Nevertheless, despite the well-established positive associations between habitual physical activity level and glycaemic control, the case for HIIT interventions per se as a means to improve glycaemic control remains to be supported by compelling evidence when looking beyond the period of prior exercise influence. Indeed, even endurance trained athletes with twice the glucose clearance rates of sedentary individuals no longer exhibited significantly greater glucose disposal after just 60h of exercise abstinence (^{Burstein et al., 1985}).

In conclusion, four weeks of high-intensity interval training without weight-loss increases the postprandial uptake of circulating FFA, with concurrent reduction in the protein content of skeletal muscle ATGL, but does not affect conventional markers of fasting or postprandial lipaemia or glycaemia when assessed ≥72h after the last bout of exercise. This finding highlights the transience of the effects of HIIT on postprandial metabolism and emphasizes the importance of regular exercise (at least every 48h) for the maintenance of lipaemic and glycaemic control.