Circadian Rhythmicity - Intermittent Fasting (Re-)Sets the Peripheral Clock: Macros, Body & Liver Fat, AMPK & More

Fridge raiding in the middle of the night is just a side issue in today's installment of the Circadian Rhtythmicity Series that revolves around Intermittent Fasting with different macronutrient ratios and its effects on body weight, body fat, liver fat and the expression of circadian clock genes.

Everyone who's also following my posts on facebook will probably be aware that (a) I don't miss a single opportunity to kiddingly refer to scientists and journalists reading this series, whenever I point you towards a new paper or news item about circadian rhythmicity. He (or she) will yet also be aware that (b) one of the studies, I actually postponed to this installment of the series, made the rounds as of late, as the ScienceDaily piece on it - which was basically a "copy & paste job" of the EurekaAlert! press release - gave the impression that a "time-restricted" (others would call it "intermittent fasting") high fat diet would be superior to the carbohydrate-laden standard rodent chow, as far as the body compositional outcomes are concerned. I wrote and talked about this on facebook, as well as the SuppVersity Science News Roundup on Super Human Radio, last Thursday. Anyways,... today, I am going to deliver on my promise from two weeks ago and will eventually  discuss the results of the Sherman study (Sherman. 2013) and the influence of nutrient timing and composition on the  mammalian circadian rhythm in detail.

To fast or not to fast, this is not a question, anymore

As I have already pointed out in the "Break-Fast Installment" of this series, it is, at least from a circadian perspective, actually not a question of whether or not you should "fast", but more or less only one of how long this fast should last. An exact answer to this question has not yet been found, though, and I would even bet money that it never will, simply because it depends on too many confounding and highly individual (epi-)genetic and lifestyle factors. And still, based on what we know now, one thing can be said for sure: If you are standing up in the middle of the night either voluntarily or because whatever pathology may be driving you and have a protein shake or ransack the fridge, you have a problem. A psychological one in the first case, a physiological and probably clock-gene related one in the latter case.

*Always keep in mind: Mice are nocturnal animals, they start to party with pubertal humans (though this is meant as a joke, I suggest you take a look at my additions to figure 5, before you totally discard it as insignificant), when the lights go out. It is therefore only natural to restricting their food-intake to the so-called dark-phase - just as natural as not ransacking the fridge in the middle of the night would be for a normal human being!

That said, I don't think that the ad libitum (=whenever they wanted) fed mice in the Sherman study did have a fridge.  What's certain though is that the simple restriction of their food-access to a 4h time window that opened with the light being switched off* did have profound effects on the body composition of the mice in both, the low fat as well as the high fat groups. It did not, however, totally blunt the negative effects the above all energetically dense "high fat diet" had on the gynoid fat pads and the hepatic lipid content and thus sooner or later the liver function of the four-week-old male C57BL/6 mice (Sherman. 2013):

"[...] although body weight and epididymal fat mass were 20 and 48% lower, respectively, in the RF-HF group than in the AL-HF group, liver lipid content was not significantly different between these groups."

In view of the role a "fatty" and thus malfunctioning liver plays in the etiology of the metabolic syndrome, it can therefore not be said that the high fat diet, respectively the incarnation of the latter that was used in the study at hand, is either healthy or anti-obesogenic. After all, the increase in visceral and liver fat, of which only the former was slightly blunted in the study at hand, usually precede the development of the other classic features of a conglomerate of metabolic ailments, we usually refer to as "metabolic syndrome".

Less body weight, more liver fat - a pretty poor trade, I would say

Against that background, it is of questionable value that the overall body weight of the "intermittendly fasted" (=restricted feeding, short RF) rodents on the intentionally fattening "high fat diet" (HF) that contained 42% of the energy in the form of soybean oil and palm sterate, was lower than that of their peers on the ad-libitum (AL) high or low fat diets (AL-LF, AL-HF).

Figure 1: Comparison of gynoid (=epididymal) and liver fat (left) and body weight development (right) of male mice on ad-libitum (AL) or (time-)restricted (RF) low (LF) and high fat (HF) diets (based on Sherman. 2013)

Moreover, the significance of these allegedly "beneficial" effects, of which the author of the ScienceDaily article falsely states that they would also entail that the mice "on the scheduled high-fat diet [...] had a lower final body weight than the mice that ate an unscheduled low-fat diet" (Hebrew University. 2013), become even more questionable if we also take into account that ...

• the mice in the AL-LF (ad libitum, low fat) group consumed 6% more energy than their peers in the RF-HF group (restricted feeding, high fat) and
• the overall reduced body weight is unlikely to be solely the resul of lower body fat levels - specifically in the RF-HF group, where the fat is already beginning to clog the liver

And while the macronutrient and fatty acid composition of the diets does not allow for any definitive statements assertions about the value of real low carb diets with ~80% of the energy from fat, the data in figure 2 illustrates a couple of metabolically relevant differences that arose as a result of the different macronutrient composition of the diets (please remember that I plotted only values with significant inter-group differences between low and high fat groups, yet not those difference, which are attributable solely to the intermittent feeding / fasting regimen and have no relation to the macronutrient composition).

Figure 2: Metabolically relevant serum markers with statistically significant differences that can be attributed to the differences in macronutrient composition of the diets; arrows indicate changes that are more or less characteristic for "fasting" as in "not eating" or being in a energy deficit (data expressed rleative to AL-LF group; based on Sherman. 2013)

If you take a closer look at the data in figure 2 you will probably realize that the classic effects of fasting, i.e. a reduction in leptin, a reduction in insulin, a reduction in glucose, a reduction in triglycerides and increases in glucocorticoids (to squeeze the fat out of the cells and catabolize amino acids in order to stabilize the blood sugar via gluconeogenesis) and the "hunger hormone" ghrelin are either mitigated or almost blunted by the "high fat" approach to intermittent fasting (I repeat this once more: the "high fat" diet in this study has more in common with the standard American diet than with a ketogenic diet). This raises the question: Is this a good or a bad thing? Or, put another way: If this does not look like fasting does it have the same effects on the circadian clock as "real" fasting would? The study results suggest that this is the case - albeit with a small, but potentially relevant quantitative difference in the fasting induced phase shifts:

Figure 3: The RF-HF mice were the almost twice as active as the AL-HF mice and 42% more active than the RF-HF animals (of whom Sherman et al. write that they were about as active as the AL-LF mice)

• the casein kinase Iε (CkIε) oszillated robustly only in the low fat groups 
• "high fat" fasting reversed the phase advance of the circadian expression of Per1 and produced a phase advance of the previously phase-delayed expression of the Cry1 and Ror genes
• "low fat" fasting induced a phase advance in all measured clock genes
• compared to the high fat restricted feeding regimen the low fat restricted feeding group exhibited a phase delay with respect to the Bmal1 and Cry 1 genes

While the overall image that emerges here, i.e. the ability of a timed feeding regimen (aka intermittent fasting) to rectify and/or advance the peripheral (here, hepatic) shifts induced by the HF diet, is pretty obvious, our understanding of the interactions of the individual clock genes is still too premature to say something definitive about the implications of the registered changes.

"So what's that supposed to mean, now? Is fasting good? Is fat bad? ... or what?"

No effect of macros? In as much as these observations support that restricted feeding aka intermittent fasting alone can reverse the majority of negative effects on the peripheral expression of clock genes, it says nothing about their central expression (all values here were measured in the liver!) and the effects on body composition. The increase in pAMPK, for example speaks in favor of the low fat diet. The same is true for the potential longevity effects. Plus, it is essentially pointless to speculate about the best macronutrient ratios, as long as a clone of the fattening standard American diet, of which I do simply assume no one of you will truly believe that it would deliver better results than a low fat diet in a rodent model, is all we can compare the low fat approach to intermittent fasting to (read more on the practical side of things in the next installment).

If we use the results of Um et al., who found in 2007, already, that the expression of the aforementioned casein kinase Iε (CKIε) gene and its negative feedback mechanism on another clock gene, namely mPER2, are triggered by the AMPK promoting effects of the "wonder-drug" metformin (Um. 2007), the following observations Sherman made with respect to the expression of AMPK and other proteins that are involved in the fatty acid and glucose metabolism begin to make some sense:

• The RF-LF diet led to increased levels of pAMPK and pACC, indicating intracellular low energy levels, inhibition of fatty acid synthesis and increased fatty acid oxidation. 
• The AL-HF diet down-regulated AMPK, ACC and SIRT1 daily protein levels by 50% compared with AL-LF mice.
• The timed HF diet led to 37% lower levels of pAMPK than those in the RF-LF group and 62% increased pACC levels compared with the AL-LF group, indicating adequate energy levels but reduced fatty acid synthesis .
• The RF-HF diet also increased daily levels of PPARα mRNA.

The overall effect on the expression of AMPK, Sirt1 and ACC, i.e. the reversal of most of the negative effects of the high fat diet in response to the intermittent feeding regimen, is thus very similar to what we have seen in the case of the the clock genes (see previous list). Reason enough for Sherman et al. to conclude that "[t]hese results demonstrate again the dominance of the timed feeding over the HF diet." (Sherman. 2013) - or, as a take home message for you:

At least with respect to the circadian rhythm, it appears that...
when you eat is of greater importance, than what you eat!

Central vs. peripheral, master vs. slave, light vs. food - every orchestrate needs a director

Now that we have gained insight into the priority of when you eat over what you eat as far as their ability to correct, readjust and resynchronize the peripheral circadian clock are concerned, we are facing another, a follow up question that reads "Is when you eat also more important than what the light cues are telling your suprachiasmatic nucleus?" [(re-)read Part I & Part II of this series to learn all about the importance of light exposure]
 

Figure 5: Regardless of the model you prefer, the central clock and with it its exclusive direct regulator will always be the most important factor in the circadian master/slave or orchestrate system (based on Richard. 2013. Fig. 3)

In one of the most recent reviews of the currently available literature on circadian rhythmicity, Richard & Gymz write about the relation of central (=brain) and peripheral (=organs) aspects:

On a basic level, the circadian clock can be divided into 2 parts: the central clock, residing in the suprachiasmatic nucleus (SCN) of the brain, and the peripheral clocks that are present in nearly every tissue and organ system tested. Light enters through the retina of the eye, causing electrical signals to pass through the retinal hypothalamic tract, which are converted to chemical signals in the SCN. Light signals and other physiological factors, such as feeding cues, entrain the central circadian clock. There has been much debate among chronobiologists about the relationship between the central clock and the peripheral clock with 2 major theories emerging.

These two theories scientists have come up with do explain the complex interrelation between the mostly light-driven central and the various peripheral clocks based on either a ...

• "master-slave" model which gives complete synchronization power to the central clock and thus assumes that all peripheral clocks are centrally synchronized in the brain, or an
• "orchestra" model according to which the multiple peripheral clocks are like the members of an orchestra, with each of them playing its own "instrument"

If you don't want the metabolic concert of your body to end up as a cacophonous cat's concert as in "David Teniers the Younger Cat concert" (see image), you better make sure that all the member's of it's orchestrate are in time with the director, the central and light dependent circadian clock in the suprachiasmatic nucleus of your hypothalamus.

The "orchestra" model has the beauty of ascribing greater importance to the previously discussed effects of feeding-times and (macro-)nutrient composition - a position that is backed up by the majority of recent studies, by the way (Richard. 2013).

"Thus, each peripheral clock can adapt to its own external and internal stimuli, such as feeding cues for the liver, kidney, and pancreas, but is "conducted" by the light-dark cues sensed by the central clock." (Richard. 2013, my emphases)

In other words, while your "peripheral clock", e.g. the clock of your fatty acid metabolism in your liver can go wrong, you better make sure that it does not, because in the end, it does not really matter how "independent" each of the members of an orchestra may be. When they start playing, their synchronization by the director, or, analogously, appropriate light cues determines whether the overall outcome of this intricate metabolic concert is a symphony and their concert hall, i.e. you(!) lean & healthy or a cacophonous mess that's making you fat & sick!

Light over Food Timing & Food Timing over Food Types.
On that note: Screens out for today, folks!

References:

• Hebrew University of Jerusalem. "A carefully scheduled high-fat diet resets metabolism and prevents obesity, researchers find." ScienceDaily, 12 Sep. 2013. Web. 16 Sep. 2013.
• Richards J, Gumz ML. Advances in understanding the peripheral circadian clocks. FASEB J. 2013 Sep;26(9):3602-13. Epub 2013 Jun 1.
• Sherman H, Genzer Y, Cohen R, Chapnik N, Madar Z, Froy O. Timed high-fat diet resets circadian metabolism and prevents obesity. FASEB J. 2013 Aug;26(8):3493-502.
• Um, J. H., Yang, S., Yamazaki, S., Kang, H., Viollet, B., Foretz, M., and Chung, J. H. (2007) Activation of 5=-AMP-activated kinase with diabetes drug metformin induces casein kinase Iε
  (CKIε)-dependent degradation of clock protein mPER2. J. Biol. hem. 282, 20794–20798

Circadian Rhythmicity: Retinol (Vitamin A) & Caffeine and Their Effects on the Central & Peripheral Clocks of the Body

Image 1: Is it a bad idea to "wake yourself up" with a pot of coffee in the morning, I mean from a circadian rhythm perspective?

In the last installment of this series we have been dealing with breakfast. Now, if you are following the mainstream advice neither of the two subjects of this installment should actually be a staple of it. Vitamin A, in its active form, retinol, is "bad and dangerous" and only present in such "evil cholesterol laden foods" such as eggs. And since coffee will sure give you a heart attack, you better stick to your calcium fortified orange juice, a minimal amount of white water, ah.. I mean low fat "milk" (learn more about the difference between white water and milk in "Mutant Milk!? New Research Fuels the Flames on Hushed Up Concerns About Ill Health Effects of Homogenized Milk") and - of course - "healthy cereals". And while you will hardly be able to argue that skipping a breakfast like that is probably the best you can do for your health, this was the topic of the last installment, while vitamin A and caffeine, will be what this episode of the Circadian Rhythmicity Series will be all about.

Vitamin A the circadian vitamin?

Only recently (officially, at least; preliminary results have been published ahead of print in March 2013, already; cf. Golini. 2013) a group of researchers from the Multidisciplinary Institute of Biological Research San Luis (IMIBIO-SL), at the National University of San Luis in Argentina found that contrary to the peripheral clock gene expression in the liver, which does not appear to be disturbed by vitamin A deficiency (Shirai. 2006), the superordinate (=master) clock gene expression in the hippocampus of rats housed at a regular 12h-light/dark interval gets profoundly compromised, when the rodents are fed a vitamin A (retinol, not beta carotene!) deficient diet (Navigatore-Fonzo. 2013). According to Navigatore-Fonzo et al. the effects are mediated by modified temporal patterns of the retinoic acid receptor in the hippocampus, which plays an essential role in the activation of a whole set of clock-genes that, in turn, have been implicated - among others in the anti-cancer effects of vitamin A, you've read about at the SuppVersity not too long ago!

In the light of these recent results many previously observed, but not fully understood effects of vitamin A deficiency, such as the permanent memory impairments (Etchamendy. 2003) and its repeatedly suggested involvement as a signaling molecule (and as it now turns out potential zeitgeber) in physiological (synaptic plasticity, learning and memory, sleep), as well as pathological (schizophrenia, depression, Parkinson disease, and Alzheimer disease) neurological conditions (cf. Tafti. 2007).

The fact retinol availability is so tightly regulated alone tells us something about its importance

Is there a fluctuation in serum retinol levels as well or is vitamin A only a prerequesite for the circadian rhythm to function normally? With the activity level of vitamin A depending on both the availability as well as the release and binding of retinoic acid from the stores (mostly) in the liver and to the respective binding proteins, which are also produced in the liver, it is obvious that the liver is the most important regulator of vitamin A metabolism (Buzio. 1989). Maybe this is also why it is protected against circadian disturbances subsequent to vitamin A deficiency.

Unfortunately, our understanding of the exact function of the retinol binding proteins is still very limited, what we do know, however, is that their release and renal clearance show a distinct circadian rhythm which is synchronized to meal ingestion and the excretion of (Buzio. 1989). Our understanding of these mechanisms is yet still too preliminary to make any supplement recommendations besides "don't avoid the full-fat vitamin A rich foods, we have been eating for ages!". This is all the more true, since the range, within which beneficial effects can be seen is not just very narrow, but will also depend on (a) your baseline vitamin A status and (b) the way your body metabolizes dietary and supplemental vitamin A, the latter of which usually comes in the form of retinyl palmitate.

At doses in the <10,000IU/day range vitamin A is regarded as totally benign, but even doubling that dosage, which was basically what Behr et al. did for their recently published paper on the potential anti-oxidant effects of vitamin A on menopausal increases in oxidative brain damage, when they  supplemented the diets of ovariectomized rats with 1,500IU /kg retinol palmitate (human equivalent ~20,000IU) per day, can result in profound increases in cerebral oxidative damage (Behr. 2013 Jul).

In conjunction with vitamin A's beneficial effect on serum markers of oxidative damage Behr et al. had observed in a previous trial with 500IU/kg and 1,500IU/kg per day (human equivalent ~6,700IU / ~20,000IU) in the same ovariectomized rodent model of menopause (Behr. 2013 Apr), the latest results from the laboratories of the Center of Oxidative Stress Research, at the Federal University of Rio Grande do Sul in Rio Grande do Sul, Brazil, only contribute to the emerging image of the hitherto hardly understood "Dr. Jekyll and Mr. Hide nature" of the (imho) most underrated vitamin there is (sorry, for the rant, but I won't get tired of raising the awareness that retinoic acid is, contrary to its overrated cousin, "vitamin D", a "real vitamin", in the sense that it is a substance we must necessarily get from our diet, while "vitamin D" is nothing but a cholesterol metabolite we should actually be able to produce ourselves, if we just got enough dietary cholesterol and sun exposure).

Figure 1: The profound loss of the rhythmicity of clock gene expression (BMAL1, PER1, top)  subsequent to three months of a virtually retinol free diet could not be restored after only 15 days on the regular rodent chow (same as control). These changes coincide with a similar loss of / shift in the expression of the antioxidant enzymatic cascade (shown here is the GPx activity) and subsequent increases shifts (deficiency) and increases in malondeyaldehyde expression (vitamin A refed group; bottom right - based on Fonzo. 2009

A closer analysis of the expression of selected markers of antioxidant activity and oxidative damage in the brain of vitamin A deficient rodents (3 months on a virtually retinol free diet) and vitamin A replete animals, who were fed the control chow for only 15 days after the depletion phase appears to confirm some of these results (Fonzo. 2009):

As expected, temporal patterns of CAT and GPx activities observed in the rat hippocampus were consistent with the rhythm of lipoperoxidation. While the lowest CAT activity occurs during the light period and, at least in part, brings lipid peroxidation into the maximal level, highest CAT and GPx activities, practically concur with the nocturnal peak of lipoperoxidation. Thus, antioxidant enzymes would have a complementary and proper timing for protecting hippocampus against peroxides, maintaining lipoperoxidation at controlled fluctuating levels, with the lowest MDA concentration occurring during the diurnal, anabolic, period in rats [...] the location of enzymes activity peaks during the night-feeding-period, may suggest the influence of feeding cycle, and macro or micronutrients, such as proteins, carbohydrates, aspartate, glutamate or some vitamins, on those rhythms, [...] the nocturnal peaks of CAT and GPx antioxidant activity seen in the hippocampus of our control rats would be in phase with the best time for performing learning and memory tests."

In this context it is interesting to see that the peak of CAT and GPX (in figure 1, only GPx is shown) does still coincide with the nightly (remember, rats eat during the dark period!) drop in GPX activity. The daily (=sleep / low activity phase) steady decline of which Fonzo et al. state that in coincides with the variation in the expression and activity of the BMAL1:CLOCK and the PER1 protein activity with

• a peak in GPx and Cat activity following the the BMAL1 protein peak at the end-of-the-night/beginning-of-the-day in the control rats, and 
• a trough of the Cat and GPx experssion after the negative regulator, PER1 protein peaks at the end of the activity phase during the day,

on the other hand, is profoundly disturbed in the vitamin A deficient animals that present with a complete loss of the BMAL1 and PER1 rhythm (figure 1, top). It does therefore appear obvious that we are (once more) dealing with two controlling mechanism:

1. an "externally" modulated, food (in the widest sense) induced regulatory mechanism and 
2. a fundamental, time- or rather light-dependent, centrally mediated circadian rhythm 

And while the latter of the two can be partly restored by vitamin A repletion. The 15-day repletion phase in the study at hand was obviously not long enough for the GPx and lipid peroxidation levels (as measured in malondyaldehyde TBARs) to return to their pre-intervention levels. If this is, as the scientists argue a result of transcriptional changes in the vitamin A receptor (RXR) "sensitivity", it is however likely that both the GPx peak activity (which should increase) and the closely related formation of lipid oxidation byproducts (MDA) should return to baseline, as soon as the stores are fully replete and the RXR levels have recovered.

From vitamins to ergogenics, from chronic to acute, from retinol to caffeine

Contrary to the effects of vitamin A which can be stored and released whenever our bodies deem it necessary, the impact of caffeine on the circadian rhythm is by the very nature of its metabolism acute and relatively short lived. This is at least true as long as the caffeine-induced circadian shifts do not lead to permanent deteriorations of the circadian rhythm. Intuitively, we all believe that caffeine can effect the circadian rhythm (or what our mainstream understanding is telling us, the circadian rhythm would be). It's not by chance that millions (ab?)use coffee and caffeine beverages on a regular basis to get going in the morning or keep going in the evening - times when our natural, undisturbed circadian rhythm should be telling us that our bed is the place our body would prefer to be, now.

One of the more exercise specific studies on this matter comes from the Exercise Physiology Laboratory at the University of Castilla-La Mancha in Toledo, Spain, where Mora-Rodríguez and his colleagues investigated the effects of a standardized caffeine containing (6mg/kg) or caffeine-free breakfast (ingested at 9:15AM) on early morning (10:00AM) or late afternoon (18:00PM) workout performance.

Figure 2: Hormone levels, performance and catecholamine levels on AM during AM and PM training sessions with or without caffeine containing breakfast (red = AM breakfast contained 3mg/kg caffeine); * indicates significant difference to AM (Placebo), PM trials were always performed on separate days with regular breakfast (based on Mora-Rodríguez. 2013)

As the data in figure 2 goes to show the whopping dose of 225mg of caffeine (note: in the graphical summary the scientists write 6mg/kg, if this is correct and the 3mg/kg that are repeatedly being mentioned in the text, then the dosage would have been 450mg) the twelve highly resistance trained men (75kg body weight; age 20; body fat 11%) did compensate for the "early morning weakness" of the participants and increased their bench press and squat performance as well as their isokinetic leg extensor strength (not shown in figure 2) to late afternoon levels, without inducing statistically significant changes in any of the measured hormonal parameters (growth hormone, testosterone, cortisol) compared to the placebo trial.

Short-term stimulation is not (yet?) equivalent to changes in circadian rhythmicity

Hack your training, not your rhythm? If the chronic use of caffeine and other stims to increase your performance at times of the day, where your circadian rhythm does not allow for maximal performance, entails possible negative downstream effects on the regular expression of your clock-genes, why don't you just train by the clock, then? Basically this is also what Hayes et al. suggested in their 2010 paper in Chronobiology International, where they state that despite the higher testosterone levels in the morning "an increased resistance exercise-induced T response [...] in the late afternoon [would suggest a] greater responsiveness of the hypothalamo-pituitary-testicular axis" later in the day - that this is bullshit, is something you should be aware by now, as the increased expression of testosterone has, as Hayes et al. have to coincide little to no influence on the hypertrophy response to training. Rather than that, they do therefore suggest to obey to the "individual responsiveness" and train whenever you feel you perform best (without the use of stims).

At times, when this is not possible, the use of stims (esp. caffeine, which is still among the "less damaging" stimulants on the OTC market), can provide temporary relief - as soon as even  3 cups of coffee only make you sleepy it is more than high time to take a break from caffeine and high intensity training (see "Tapering & Detraining - When and How to Take a Break")

For Mora-Rodríguez et al. these observations are a clear-cut sign of "circadian rhythm effects", but are they really related to changes in circadian rhythmicity? They blunt the morning reduction in muscle performance due to circadian rhythm - there is no debating that, but the study does not provide convincing evidence that this is due to changes in the expression of zeitgeber proteins and thus a direct consequence of a shift in circadian rhythmicity. If we take another look at figure 2, we would thus expect to see similar hormonal expressions, as well. After all, both the spike in cortisol in the morning as well as the steady decline of testosterone and even steeper decline in cortisol that occurs in the course of the day are both mediated by the circadian rhythm. The adrenaline spike in response to the ingestion of caffeine, which is unquestionably responsible for the observed performance enhancing effects in the study at hand, on the other hand, has nothing to do with circadian rhythmicity.

Did I mention that results from in-vitro studies suggest that cortisol spikes, esp. the huge spike in the morning, could act as a "reset switch" for the circadian clock? (cf. Balsalobre. 2000)

If anything, we could - based on the acute catecholamine response in the Mora-Rodíguez study, that chronic morning caffeine consumption could lead to subsequent downstream changes in the expression of zeitgeber genes, which would in turn trigger a 12h shift in circadian rhythmicity with low morning and high evening cortisol levels that would basically reverse the natural pattern as it was observed in the AM/PM(Placebo) trials.That this would entail a whole host of negative health effects is something you should by now be familiar and renders the (long-term) use of caffeine to "avoid the morning reduction in muscle performance due to circadian rhythm" at least highly questionable, as it would go- in the most fundamental sense of the word - against our nature. If chronic caffeine consumption did actually induce the aforementioned changes in circadian rhythmicity. So, the next question would be...

Are the effects of caffeine even of circadian origin / does it affect circadian rhythms?

The answer to this question is not exactly easy to find, as most studies follow the flawed assumption that "being more awake" would equal "being able to hack the circadian rhythm", when it could just as well be nothing more (and nothing less) than a highly effective way to outwit the latter. Against that background it's strange that Oike et al. were the only scientists I found that explicitly mention that it "remains unknown" "whether or not [caffeine] affects mammalian circadian clocks remains unknown" (Oike. 2011).

Figure 3: The in-vitro exposure of human osteosarcoma cells (a common model used in gene essays) messes with the previously mentioned clock genes Per2 and  Bmal1 genes (left) and the in vivo ingestion of coffee / administration of caffeine in drinking water did increase the locomotor activity period length of mice after normal lighting conditions (first two weeks lower panel) and constant darkness (upper panel, right; based on Oike. 2011).

Luckily Oike at el. did not just nag at the absence of reliable evidence for / against the effects of caffeine on circadian rhythmicity, but also conducted a couple of in vitro and in vivo studies, in the course of which they were able to show that notwithstanding it's disturbing effects in on clock gene expression in the petri dish (figure 3, left), the "real-world" test with coffee and caffeine did

• lengthen the circadian rhythm of reporter gene expression in liver explants of the rodents, without affecting the time of the rhythm peak in the liver explants (not shown), while
• left the period length in the likewise explanted suprachiasmatic nuclei unchanged, but delayed the peak time of the rhythm

the real world results of these somewhat schizophrenic modulatory effect of caffeine on the peripheral (liver) and central (suprachiasmatic nucleus) rhythm is an increased length of the circadian pattern in dark-exposed (=constant day for mice!) mice, without affecting the locomotor activity in the presence of appropriate light cues!

In other words: The effects of caffeine will only mess with your circadian rhythm if they are not overridden by appropriate light cues!

Similar results have been reported by Sherman et al. who made an even more complex experiment which the results of which will be part of the next installment of this series, as the inclusion of a restricted feeding regimen a la intermittent fasting with a minimalist 3h feeding window segues quite nicely into the discussion of the metabolic implications of caffeine and nutrient (esp. glucose) availability, we will take up in the next installment of the Circadian Rhythmicity Series.

Image 2: I admit that all this is not easy to understand and many of the implications on our everyday lives are yet not clear, either. I still hope you don't feel you have wasted your valuable time with this post.

Before I let you go, I do yet still want to give you the elevator pitch on this long and allegedly very complicated post. While much of what we have been studying today must still be considered preliminary (also on the expert level) there are three important and theoretically, as well as experimentally relatively well certain take home messages. The first pertains to the importance of light cues as the main regulators of the central clock gene expression in the brain, the second relates to the vital, hence "vitamin", importance of vitamin A for the integrity of the central clock, and the third relates to the modulatory effect certain molecules, such as caffeine, can have on the peripheral clocks.

The practical implications of these insights, on the other hand are pretty straight forward and for most of you probably no real news, anyway:

1. stick to the "natural" dark/light cycle - reread episodes one and two of the series for tips on how you can make do so in our "light polluted" world
2. get adequate amounts of vitamin A in your diet - there is no need to supplement, your body manges the levels of vitamin A very effectively, so that a piece of liver once in a while is a way better choice than a vitamin pill every day
3. don't be scared of coffee - as long as you still stick to the natural cycle (see first point), your circadian rhythm may exhibit slight shift, it will yet only break if you use caffeine + light as in popping a caffeine pill and surfing on the Internet with your melatonin suppressing iPad (see episode I) in the middle of the night

Now, before you switch off your iPad and go to bed today, I suggest you check out the SuppVersity Facebook Wall, for the latest news - it is no coincidence that an item about the -57% reduced Parkinson's risk in habitual coffee drinkers who consume at least three or more cups per day, as well as a reference to the latest confirmation of the liver protecting effects of coffee there... ah, and by the way, it could be that we will be able to track those back to circadian gene expression (peripherally, obviously ;-) in the next installment, as well...

References:

• Balsalobre A, Brown SA, Marcacci L, Tronche F, Kellendonk C, Reichardt HM, et al. Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 2000;289:2344–7
• Behr GA, Schnorr CE, Moreira JC. Increased blood oxidative stress in experimental menopause rat model: the effects of vitamin A low-dose supplementation upon antioxidant status in bilateral ovariectomized rats. Fundam Clin Pharmacol. 2013 Apr;26(2):235-49.
• Behr GA, Schnorr CE, Simões-Pires A, da Motta LL, Frey BN, Moreira JC. Increased cerebral oxidative damage and decreased antioxidant defenses in ovariectomized and sham-operated rats supplemented with vitamin A. Cell Biol Toxicol. 2013 Jul 18.  
• Buzio C, Mutti A, Capani F, Andrulli S, Perazzoli F, Alinovi R, Negro A, Rustichelli R. Circadian rhythm of proteinuria: effects of an evening meat meal. Nephrol Dial Transplant. 1989;4(4):266-70.
• Fonzo LS, Golini RS, Delgado SM, Ponce IT, Bonomi MR, Rezza IG, Gimenez MS, Anzulovich AC. Temporal patterns of lipoperoxidation and antioxidant enzymes are modified in the hippocampus of vitamin A-deficient rats. Hippocampus. 2009 Sep;19(9):869-80.
• Golini RS, Delgado SM, Navigatore Fonzo LS, Ponce IT, Lacoste MG, Anzulovich AC. Daily patterns of clock and cognition-related factors are modified in the hippocampus of vitamin A-deficient rats. Hippocampus. 2013 Aug;22(8):1720-32. 
• Hammouda O, Chtourou H, Chahed H, Ferchichi S, Chaouachi A, Kallel C, Miled A, Chamari K, Souissi N. High Intensity Exercise Affects Diurnal Variation of Some Biological Markers in Trained Subjects. Int J Sports Med. 2013 Jul 12.
• Mora-Rodríguez R, García Pallarés J, López-Samanes Á, Ortega JF, Fernández-Elías VE. Caffeine ingestion reverses the circadian rhythm effects on neuromuscular performance in highly resistance-trained men. PLoS One. 2013;7(4):e33807. Epub 2013 Apr 4. 
• Navigatore-Fonzo LS, Golini RL, Ponce IT, Delgado SM, Plateo-Pignatari MG, Gimenez MS, Anzulovich AC. Retinoic acid receptors move in time with the clock in the hippocampus. Effect of a vitamin-A-deficient diet. J Nutr Biochem. 2013 Aug 16.
• Oike H, Kobori M, Suzuki T, Ishida N. Caffeine lengthens circadian rhythms in mice. Biochem Biophys Res Commun. 2011 Jul 8;410(3):654-8. Epub 2011 Jun 13.
• Shirai H, Oishi K, Ishida N. Circadian expression of clock genes is maintained in the liver of Vitamin A-deficient mice. Neurosci Lett. 2006 May 1;398(1-2):69-72.
• Tafti M, Ghyselinck NB. Functional implication of the vitamin A signaling pathway in the brain. Arch Neurol. 2007 Dec;64(12):1706-11.