*4.4. Diabetes, Obesity, and Metabolic Control*

Normal CLOCK and BMAL-1 activity play a role in defending the body against metabolic disturbances, but CLOCK gene mutations are associated with hyperphagia, hyperlipidemia, hyperinsulinemia, hyperglycaemia, and sleep disorders—all of which are common in metabolic diseases such as diabetes and obesity [109]. Diabetes, especially Type 2 Diabetes (T2D), is connected to disruptions in normal circadian rhythms with shiftwork, light pollution, jet lag, and increased screen time, all acting as potential contributory factors [33,110,111]. Emerging evidence has also identified melatonin, already known for its roles in circadian and redox physiology, respectively, as a potential mediator of glucose levels and insulin production [109] adding further credence to the existence of an important cross-talk between circadian rhythms and redox pathways in the regulation of metabolic control. Underpinning all forms of diabetes is either a decrease in β-cell mass or β-cell function [112–114]. Mechanistically, oxidative stress, potentially stemming from increased mitochondrial O2 production following excessive caloric intake and low activity levels, has also been implicated in diabetes as a key mediator of β-cell dysfunction and insulin resistance [5,115]. This appears to be secondary to circadian dysregulation as β-cells contain critical antioxidant genes that are targets not only of Nrf2, but also BMAL-1 which are vulnerable to circadian disruption in *BMAL-1*−*/*<sup>−</sup> mice [116]. Circadian disruption to these key antioxidants can thus lead to augmented β-cell mitochondrial RONS production in

cells that already have relatively less antioxidant capacity, culminating in the impairment in β-cell function, insulin resistance, and diabetes [111,117].

Alterations to normal circadian control and oxidative stress are understood to be instrumental in the pathogenesis of other metabolic conditions associated with T2D, such as obesity [118]. Adipose tissue is a critical modulator of metabolic health, and oxidative stress can cause adipose tissue dysfunction by stimulating preadipocyte proliferation, adipogenesis, and chronic inflammation, which leads to obesity [119]. The circadian clock controls energy homeostasis by regulating circadian expression and/or activity of enzymes, hormones, and transport systems involved in metabolism with evidence showing that knockout and mutations in clock genes induce disruptions in adipose tissue function, differentiation, and metabolism (see review by Froy and Garaulet [120]). Disruptions to circadian control and oxidative stress may also affect other important metabolic organs. For instance, a relatively recent study found that night shift workers have a higher serum concentration of alanine aminotransferase (ALT) than daytime workers, indicating a potential association between circadian disruption and liver function [121] that could be important in the development of conditions such as non-alcoholic fatty liver disease (NAFLD). Moreover, such conditions proposed to be related to dietary factors (see review by Arrigo et al. [122]), are often accompanied by oxidative stress and seem to favourably respond to antioxidant and circadian therapy in the form of melatonin administration [123]. As alluded to, dietary factors and feeding patterns undoubtedly represent an important metabolic focus for their ability to modulate circadian function and redox control in health and disease, though further scrutiny is beyond the scope of this review.

Far from being fully determined, it does appear that a bidirectional relationship exists between RONS production and circadian rhythms: core clock function appears to be sensitive to changes in redox status and redox homeostasis may conversely be governed by clock machinery [124]. When out of sync, circadian dysregulations, oxidative stress, or both, can manifest. This is an intriguing frontier of research emerging, and the ability to harness these insights, combined with other important drivers of circadian and redox biology, will help to provide more focussed health and lifestyle prescriptions.

#### **5. Exercise—Zeitgeber and Modulator of Redox Homeostasis**

Regular exercise is often regarded as one of the 'best buys' for public health given its multiple health-promoting benefits [125], with the Academy of Medical Royal Colleges [126] describing 'the miracle cure' of performing 30 min of moderate exercise, five times a week, as more powerful than many drugs administered for chronic disease prevention and management. Aerobic exercise training improves risk factors of metabolic syndrome such as glucose intolerance, hyperlipidemia, high blood pressure, low high-density lipoprotein content, and visceral obesity. Improved aerobic fitness increases neurogenesis, enhances memory, and may prevent brain atrophy [127]. Exercise is widely regarded for its substantial health benefits and there is now ample evidence from both observational studies and randomised trials to support that regular exercise is a contributing factor in the prevention of cardiovascular disease, cancer, diabetes, and other chronic conditions, as well as reducing the risk of all-cause mortality [128,129]. As more is understood regarding the molecular pathways and mechanisms through which the beneficial effects of exercise are transmitted, interest continues to grow in strategies to optimise the benefits of such effects and how these can be translated for optimal health and performance purposes (see Figure 4).

The circadian clock can be synchronised by photic and non-photic stimuli (temperature, physical activity, and food intake). Given that exercise represents a major challenge to whole-body homeostasis, provoking widespread perturbations in cells, tissues, and organs [130], modern theories have begun to probe the connections between exercise and circadian rhythms for both performance and health purposes. An extensive body of literature has established that exercise can influence the circadian system in rodents (see [1]) and emerging human evidence shows exercise can elicit phase-shifting effects which may

be dependent on chronotype [34]. Exercise appears to be a potent entrainment factor for central as well as peripheral clocks, including those in muscle. For instance, the average core-clock gene expression (*BMAL1*, *ROR-α*, *CRY1*, *PER2*, *PER1*, and *NR1D1*) in male rugby players is significantly higher compared to sedentary males [131]. Therefore, the possibility of (a) exercise being able to attenuate the negative health effects of circadian misalignment and (b) the existence of an optimal time to exercise to maximise its therapeutic effects have become increasingly attractive to researchers and clinicians.

**Figure 4.** The interplay between Circadian and Redox Biology: Exercise as a zeitgeber and source of RONS. Exercise is a potent stimulus to entrain dysregulated circadian systems and positively affect the core molecular clock and subsequent expression of CCGs throughout the body. At the same time, exercise-induced RONS are integral to several recognised physiological responses and adaptations via the activation of redox-sensitive transcription factors (e.g., up-regulation of immunity, antioxidant enzymatic activity, etc.). It is possible that both circadian and redox signalling are inter-connected and operate synergistically to confer protective health effects following regular exercise.

Of all the peripheral tissues, skeletal muscle represents a major downstream target for clock activity. Among other functions, elegant studies have shown how the molecular clock governs glucose metabolism in skeletal muscle. In one such study, *BMAL-1* deletion in mouse skeletal muscle manifested in impaired glucose uptake, reduced GLUT-4, and disrupted the activity of key glycolytic enzymes [132]. Thus, impairments in the muscle molecular clock appear to have important implications for the development of metabolic diseases such as T2D. As exercise is recommended for the prevention and treatment of T2D [9], it now appears that the beneficial metabolic effects exercise confers are, at least partly, achieved through actions on the muscle molecular clock to restore local circadian regulation [7]. For instance, skeletal muscle gene and protein expression of *BMAL-1* and *PER2* were increased in adults with obesity and pre-diabetes following 12 weeks of exercise training and this was accompanied by improvements in body composition, peripheral insulin sensitivity (glucose disposal rate), and maximal oxygen consumption. Specifically, *BMAL-1* gene expression correlated with glucose disposal rate [133].

While exercise undoubtedly elicits favourable, modulating effects in skeletal muscle metabolism, relatively little is known about the potency of these effects at different times of the day. This is important as the biological clock seems to drive patent rhythms in human skeletal muscle metabolism whereby mitochondrial oxidative capacity follows a day-night rhythm, peaking in the late evening and being lowest in the early afternoon [134]. As oxidative capacity is a vital determinant of exercise performance, it is unsurprising that studies report clear time of day effects (~10%) for exercise performance, capacity [8], and/or strength measures in human volunteers, which in one recent paper were correlated with *PER2* daily profiles [135]. The underlying mechanisms for such phenomena are complex but are thought to involve circadian fluctuations in core temperature, endocrine hormones, neuromuscular function, and metabolic flux [8,9]. One of the key contributing factors in these performance fluctuations is body temperature which can exert a myriad of regulatory effects on neuromuscular and metabolic activity and appears to peak in the late afternoon. As thermoregulation during exercise itself appears to follow a circadian rhythm, this may also explain the variability of fatigue onset when the same activity is performed at different times across the daily cycle, especially in longer, endurance-type exercise bouts [136]. Variations in human exercise efficiency (improved in Late vs. Early) have been ascribed to clock-driven fluctuations in metabolic control such as carbohydrate metabolism that requires lower oxygen consumption, a lower heart rate, and a lower rate of perceived exertion [8]. In competitive and elite sporting settings, this connection between exercise capacity and the molecular clock may be useful, to some extent, when planning to optimise training and competition schedules, but this is not an entirely new concept (see [137]).

Syncing exercise bouts, alongside other interventions not scrutinised in the current review (e.g., time-restricted eating), to align with circadian rhythms is an appealing paradigm to consider when trying to maximise the metabolic and health effects of exercise. In a crossover study involving 2 weeks of high-intensity interval training (HIIT), afternoon HIIT was more efficacious than morning HIIT at improving blood glucose in *n* = 11 men with T2D [138]. Such data indicate that the timing of exercise should be considered when prescribed for the management of T2D. However, in a larger study investigating the effects of exercise timing on glycaemic control in those with and without T2D, no distinct glycaemic benefits or alterations in circadian rhythm (as assessed by skin temperature) were detected between morning versus evening exercise [139].

Regular physical activity and exercise programmes have been shown to reduce the symptoms of patients with established CVD; additionally, prospective epidemiological studies of occupational and leisure-time physical activity have consistently documented a reduced incidence of CVD in the more physically active and fit individuals [140–142]. Consequently, exercise is regarded as an important intervention in tackling the burden imposed by CVD. Yet, the precise mechanisms by which exercise exhibits its ameliorating effects on the cardiovascular system still require elucidation. For instance, relatively little is known about the potential for exercise to entrain central and peripheral clocks and how this might influence cardiovascular health. It is conceivable that exercise may regulate circadian factors to partially influence cardiovascular health through the documented effects on skeletal muscle—given the connections between metabolic and cardiovascular health [5,7]. Studies have begun to explore this theme as well as the possible existence of diurnal effects of exercise on cardiovascular risk markers. Elevated arterial blood pressure, or hypertension, is a traditional cardiovascular risk factor that also forms one of the main constituents of metabolic syndrome [143,144]. Acute exercise can induce a transient reduction in blood pressure or post-exercise hypotension (PEH) [145]. This reduction is largely thought to be a result of increased blood flow to vascular beds and the subsequent decrease in the total peripheral resistance associated with exercise. The prolonged hypotensive effects of regular aerobic exercise may thus be due to repeated instances of PEH [146]. Despite this, BP reductions after aerobic training vary across studies and some factors, such as higher initial BP, moderate to high training intensities, and concomitant diet-induced weight loss, have been identified as promoters of a greater BP decrease [147]. Moreover, alterations to dietary composition (reduced fat and increased fibre) alongside exercise training have also been reported to decrease BP and oxidative stress markers in the absence of weight loss [148]. It is also possible that the time of

day when aerobic training is performed may influence the extent of BP reductions after training. As most aspects of cardiovascular regulation demonstrate a circadian or diurnal pattern [149], it is plausible that the mechanisms driving sustained PEH are impacted by time of day. Some studies have tested this hypothesis with mixed results. In one study, the acute hypotensive effects following 30 min of steady state exercise were less marked in the morning versus the afternoon [150], but this may be masked due to the circadian effect on morning blood pressure [7]. While de Brito et al. [151] demonstrated that aerobic training performed in the evening decreased clinic and ambulatory BP when compared to morning training in hypertensive men, this finding may be explained by the use of antihypertensive medications in the morning group. In contrast, while aerobic exercise performed both in the morning and the afternoon/evening contributed to PEH when circadian influences of morning blood pressure were considered, PEH was greater following morning exercise rather than evening exercise in a separate study [152]. The influence of exercise intensity and other zeitgebers like food ingestion, light exposure, and sleeping patterns are complex confounders in such studies, but exercise may induce re-alignment of the circadian clock and better cardiovascular outcomes by modulating hormonal responses and heart rate (see review by [7]).

It is well established that aerobic exercise can improve endothelial function and flowmediated dilation (FMD) responses [153], particularly in those with cardiovascular disease and related risk factors [154]. Despite this link, the diurnal effects of exercise on FMD have received relatively little attention, even though FMD has been shown previously to vary with time of day [155]. A handful of studies have nevertheless provided useful insights to illustrate the apparent diurnal effects of exercise on FMD in some [156,157] but not all instances [152]. Although beyond the scope of this paper, the equivocal findings in such studies may be partially explained by variations in the exercise stimuli used e.g., whole body versus isolated limbs.

Endurance exercise training elicits a number of benefits such as increased skeletal muscle mitochondrial number and volume density leading to improved oxidative capacity [158]. An important development in unraveling the cellular events that promote mitochondrial biogenesis and other beneficial effects such as angiogenesis, improved antioxidant defences, and enhanced fat metabolism [11] was the discovery of the transcriptional coactivator PGC-1α, thought to be the master regulator of the process. Recently, this exercise-stimulated pathway has been identified as being downstream of the molecular clock, providing a molecular mechanism through which circadian timing can influence exercise responses [12,159]. In recent years, the understanding of this mechanism has grown to the extent that the pathway relies on redox signals, whereby exercise-induced RONS appear to potentiate PGC-1α and NF-kB [48]. Evidence to support this supposition comes from studies such as that from Ristow and Colleagues [53] who showed the use of a vitamin C and vitamin E antioxidant regime blunted mRNA responses in several markers of mitochondrial biogenesis, including PGC-1α, in healthy young men completing a training intervention. Mechanistically, PGC-1α regulation at rest and during exercise depends on several factors with evidence suggesting that AMPK, Nrf2, and p38 serve as the principal intermediate molecules connecting RONS with PGC-1α activation and/or mitochondrial biogenesis markers [57]. Redox activation of Nrf2 has already been identified in the priming of the antioxidant network [83] and in the regulation of core clock function through its activation of clock machinery [85]. Moreover, exercise-induced RONS and redox-sensitive genes and transcription factors, including AMPK, HIF-1α, and PGC-1α, may influence the expression of core molecular clock genes [11,12]. Given the cross-talk between HIF pathways and circadian clock control [27], and that RONS are thought to be instrumental in the exercise-induced increases in HIF-1α [57], HIF-1α may be a crucial mediator connecting circadian control, redox homeostasis, and exercise.

While the majority of redox-focussed exercise studies have examined aerobic/endurance training, a growing appreciation for the role of RONS signalling in resistance trainingmediated adaptations have been outlined. In particular, the mechanistic target of rapamycin

complex 1 (mTORC1) activation, which stimulates protein synthesis via increased translation of contractile protein mRNA central to muscular hypertrophy, has shown to be redox-sensitive. For example, and as previously highlighted, ONOO− has been implicated in the signal transduction pathways for mTORC1 that lead to hypertrophy [67]. As mTORC1 appears to exhibit diurnal oscillations—current evidence has highlighted the suppressive effect of PER2 on mTORC1 [160], the extent to which these are influenced by RONS signalling and if/how this might be integral to diurnal exercise responses remains uncovered and ripe for investigation.

#### **6. Future Directions**

Contemporary research has identified circadian disruptions to augment oxidative stress and subsequently aberrate adipose tissue function and metabolism. Therefore, circadian machinery in the adipose tissue may be a novel therapeutic target for the prevention and treatment of metabolic and cardiovascular diseases [119]. We believe that exploratory exercise studies connecting circadian principles to adipose tissue and redox metabolism may represent one such approach. Based on the existing literature, there appears to be scope for mechanistic and maybe even clinical progress, but it is imperative that future investigations are carefully designed to control for confounding factors including participant chronotype and the influence of other zeitgebers [34] and testing of insufficient numbers of volunteers in a narrow range of times across the 24 h day [1]. Importantly, new studies could also benefit from the careful selection of appropriate methods to measure and assess the impact of potential interventions on circadian rhythms [161]. Exercise stimuli and protocols such as intensity, mode, etc. should also be carefully chosen when considering the potential implications for biological rhythms and RONS. Some of the equivocal findings on the health/physiological effects of exercise timing described in the review may be due to the heterogeneity of the volunteers. More parallel investigations are warranted for this potentially promising realm of exercise prescription research, such as those that incorporate larger sample sizes, crossover study designs, and, crucially, chronic exercise interventions that assay a range of clinical and mechanistic redox markers. Of course, as alterations in feeding patterns and diet can affect circadian rhythms and redox markers [119,162], further mechanistic investigations are also welcome that seek to simultaneously explore dietary manipulations with exercise to aid in the development of targeted treatment strategies to improve health in those with/or at risk from chronic disease.

#### **7. Conclusions**

Roenneberg and Merrow [19] offer a plan on how to implement chronobiological principles into medicine through the identification of a mechanism that determines the circadian clocks of an individual and their ability to be synchronised. This knowledge could inform the prescription of specific exposures of zeitgebers to target the circadian system and personalise the therapeutic schedules for any individual [109]. Targeting circadian health would be especially pertinent to those at risk from circadian misalignment from shift work, frequent bright light exposure, sleeping disorders, and frequent travel across multiple time zones. We believe that exercise might represent one such zeitgeber given its ability to potentially entrain the human circadian system; additionally, careful interrogation of how it may modulate the cross-talk between the molecular clock and redox biology could yield further insights into the salient mechanistic pathways such as those that control antioxidant defences and DNA repair. At present, and despite the promise of studies showing diurnal variations in exercise responses/adaptations, much more work is needed to comprehensively identify whether an optimal exercise time exists. As it stands, exercise prescription should be personalised based on several variables such as the individual's health and fitness status (i.e., the absence or presence of CVD risk factors), chronotype, work and domestic patterns (including mealtimes and work schedules), and preferences. Given the recent advances in redox and molecular biology, informed by advancements in analytical techniques, exercise studies positioned at the emerging nexus between circadian

rhythms and the redox milieu represent a fascinating area for exploration, especially for those that advocate, or wish to scrutinise, exercise prescription for health.

**Funding:** This research received no external funding.

**Acknowledgments:** All figures contained within this manuscript were created with BioRender.com.

**Conflicts of Interest:** The authors declare no conflict of interest.
