Hexokinase: A central player in the synergism of high-intensity intermittent exercise and every-other-day intermittent fasting regimen on energy metabolism adaptations

Visceral lipid accumulation, organ hypertrophy and a reduction in skeletal muscle strength are all signs associated with the severity of obesity related disease. Intermittent fasting (IF) and high-intensity intermittent exercise (HIIE) are natural strategies that, individually, can prevent and ameliorate obesity along with metabolic syndrome and its associated diseases. However, the combinatorial effect of IF and HIIF on energetic metabolism is currently not well understood. We hypothesized that their combination could have a potential for more than strictly additive benefits. Here, we show that two months of every-other-day intermittent fasting regimen combined with a high-intensity intermittent exercise protocol (IF/HIIE) produce a synergetic effect, preventing fat accumulation, enhancing physical performance and optimizing energy production. The IF/HIIE group presented increased glucose uptake, lower levels of serum insulin and a global activation of hexokinases in skeletal muscle, heart and liver comparing to control, IF and HIIE groups. IF/HIIE synergism led to activation of the FoF1 ATP synthase and promoted a more oxidative profile of mitochondria in observed skeletal muscle. Additionally, high-resolution respirometry of muscle fibers showed that animals in the IF/HIIE group presented characteristics suggestive of augmented mitochondrial mass and efficiency. Finally, an important reduction in serum oxidative stress markers were observed in IF/HIIE group. These findings provide new insights for the implementation of non-pharmaceutical strategies to prevent/treat metabolic syndrome and associated diseases.

1 hexokinase, as this enzyme is known to be essential to overcome the rate-limiting step of 2 the glucose metabolism (22) and the rate-limiting step of the oxidative phosphorylation 3 (23). 4 Material and methods 5 Animals and intermittent fasting protocol 6 All animal procedures performed received prior approval from the Animal Use  To determine the adaptive changes on energetic metabolism and physical 20 performance induced by IF, HIIE, and their combination, the three regimens were imposed 21 on age-matched young adult Wister rats over 8 weeks (Fig 1). Over the course of the 22 experimental conditions, the weight of the animals was tracked weekly and plotted in a 23 curve (Fig 2A). The cumulative increase in the weight of animals on an ad libitum diet (C 24 and HIIE groups) is very apparent and the prevention of excessive weight gain in animals 1 Serum glucose uptake is integrated with hexokinase activity in IF/HIIE 2 group 3 To investigate how IF and HIIE could impact the systemic glucose availability and 4 metabolism, we measured the fasting blood glucose levels, rate of glucose uptake and 5 fasting serum insulin levels (Fig 3). All groups presented similar values for fasting blood 6 glucose (Fig 3A). Initially, the glucose tolerance test also showed similarities between 7 groups (Fig 3B). However, an analysis of the area under the curve (AUC) revealed that the 8 IF/HIIE group presented a significantly faster glucose uptake (Fig 3C). Glucose uptake 9 allied to fasting serum insulin levels that could present a predictive factor of insulin 10 sensitivity (32, 33). For this reason, we measured the fasting insulin level of the different 11 groups (Fig 3D) and we observed an important reduction for the IF/HIIE group followed 12 by the IF group.

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According to the literature, sugar transport has been integrated with hexokinase (HK) 19 activity in a cellular model (34). To test if this mechanism could explain observations in a 20 more complex system, we hypothesized that increased blood glucose uptake could be 21 integrated to an increased HK activity in multiple organs (Fig 4). In the liver, we observed 22 that the IF/HIIE group had the highest HK activity followed by HIIE and IF groups, which 23 showed similar activity values (Fig 4A). In heart, both IF and IF/HIIE groups showed 24 increased HK activity (Fig 4B). In skeletal muscle, the IF/HIIE group had the highest HK 25 activity (6-fold increase) followed by HIIE group (3-fold increase). Taken together, the HK 1 activity data from all analyzed organs suggest that IF/HIIE group possess the highest values 2 for HK activity.  To investigate how the skeletal muscle mitochondrial respiratory complexes could 4 be affected by IF and/or HIIE protocols, we measured the different respiratory states and 5 the mitochondria respiratory control rate (RCR) (Fig 6). RCR is an reliable indicator of 6 mitochondria function, as high RCR usually indicates healthy mitochondria and low 7 RCR usually indicates mitochondrial dysfunction (42). Initially, we analyzed muscle 8 fiber mitochondria oxygen consumption in the presence of complex I substrate, complex 9 II substrate, and ADP (State 3). We observed a higher O 2 flux rate related to ATP 10 production in IF/HIIE group, followed by both HIIE and IF groups individually, in 11 comparison to the C group (Fig 6A). In the sequence, we added ATP synthase inhibitor 12 (State 4o). HIIE group presented a higher O 2 flux (Fig 6B),  Initially, we observed that the IF fasting protocol promoted a pronounced 23 protection against the weight gain observed in groups with food offered ad libitum ( Fig   24 2A). According to the literature, IF promotes activation of brown adipose tissue (12), 25 however, no noticeable difference in size or weight of brown adipose tissue was observed 26 in any of the groups (Fig 2B). In contrast, IF and IF/HIIE groups presented an 1 expressively low visceral fat mass (Fig 2C). We also weighed the liver and heart of the 2 animals, as these organs can suffer lipid accumulation in some diseases (50). Animals in 3 both the IF and IF/HIIE groups had smaller livers ( Fig 2D) and hearts (Fig 2E) suggesting 4 that the normal diet offered ad libitum can induce aspects of obesity in young adult rats.
5 To investigate the possibility of muscular atrophy promoted by lack of physical activity 6 combined with reduced food offer in IF group, the weight ( Fig 2F) and the CSA of 7 gastrocnemius were evaluated (Figs 2G and 2H). We observed lower weight and CSA of 8 gastrocnemius from IF group comparing to C group, however, the IF group presented 9 similar results to the C group in the physical tests ( Fig 5A). Surprisingly, the IF/HIIE 10 group showed an increase in myofibers CSA (Fig 2H), statistically higher than CSA

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To evaluate the effects of IF and HIIE protocols on glucose metabolism, core of 22 energetic processes (55), we started measuring the fasting blood glucose (Fig 3A), which 23 showed similar values for all groups. Next, we measured glucose uptake via glucose 24 tolerance test (Fig 3B) with AUC analysis (Fig 3C) and we detected a significantly faster 25 uptake of glucose in IF/HIIE group, similarly to observed by other groups (20,37).
26 Additionally, we measured the fasting serum insulin levels and we observed a significant 27 decrease of insulin levels in IF/HIIE group (53% reduction), followed by a smaller 1 reduction in insulin levels in IF group (37 % reduction). Lower levels of circulating 2 insulin were also observed in mice over-expressing follistatin-like 3 (56), a natural 3 blocker of activin/myostatin signaling, and possibly these groups (IF and IF/HIIE) may 4 have higher levels of follistatin-like 3, that could corroborate the effect proposed above 5 for IF in muscle hypertrophy through activin/myostatin signaling modulation. 6 To investigate the consequence of the observed increased in glucose uptake 7 combined with low levels of circulating insulin in the downstream of glucose metabolism 8 within the cellular milieu, we measured the activity of the first enzyme of the glycolysis, 9 HK (Fig 4), whose activity is integrated with sugar transport (34). We observed a 10 synergic effect from the combination of IF with HIIE protocols in liver HK with an 11~130% increase versus the ~90% observed for both IF and HIIE alone ( Fig 4A). In 12 skeletal muscle HK, we observed a 3-fold greater HK activity in HIIE group compared 13 to controls that increased to 6-fold with the combination of HIIE with IF ( Fig 4C).
14 Remarkably, the IF protocol alone showed no promotion of any adaptations in muscle 15 HK activity, yet its combination with HIIE doubled the HK activity in the IF/HIIE group.
16 In contrast, the activation of HK activity in the heart showed an association only with the 17 inclusion of the IF protocol (IF and IF/HIIE groups; Fig 4B). This probably reflects an 18 effect of high acetate serum levels as described by Li, Xie (12) in work that used a similar 19 IF protocol. Furthermore, they also observed an increased level of circulating lactate 20 however we discard the influence of intracellular lactate since lactate were demonstrated 21 to reduce hexokinase activity (57).

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The HK activity is closely connected with mitochondrial activity, since the 23 muscular isoform of this enzyme can bind to the mitochondrial membrane through 24 VDAC (58) that can positively modulate the activities of both (59). To identify a possible 25 consequence from the strong activation of HK in muscle of the IF/HIIE group, we 26 initially compared the results of the physical endurance test (Fig 5A)