Pharmaceutical-grade circadian manipulation.

BMAL1 and CLOCK, ‘positive’ regulators of circadian gene expression, activate transcription of the negative regulators Per, Cry, and Rev-erb.  PER and CRY inhibit BMAL1 and CLOCK, whereas Rev-erb inhibits Bmal1.  It is said that Rev-erb is “an important link between the positive and negative loops of the circadian clock.”  You don’t really need to know any of that to follow this blog post.

circadian genes

Background: Liver-specific Rev-erb knockout mice (Bugge et al., 2012)

There are two Rev-erbs (alpha and beta) which have overlapping functions – deleting either one of them fails to affect Bmal1 rhythmicity (left), but deleting both does the trick (right):

Bmal1 in KO

Normal circadian Rev-erb expression looks like this:

Rev-erb

Mice lacking both Rev-erbs in liver are mildly hypoglycemic with elevated free fatty acids:

KO hypoglycemia

…now, on to more pressing matters.  The token indicator of circadian arrhythmia is disrupted activity patterns, which is apparent in the mice lacking Rev-erb globally (Cho et al., 2012):

KO activity

When exposed to proper 12 hour light-dark cycles, the wild-type and single knockouts exhibit normal activity patterns (“LD;” yellow portion in the figures above indicates daytime, when they sleep).  And when in constant darkness, phase gradually shifts earlier and earlier but remains intact in the normal mice.  This is bollixed in Rev-erb knockout mice (iDKO), confirming circadian arrhythmia.  Also, in contrast to liver-specific knockouts, glucose levels are elevated in these mice.

So timing is off and a bit of metabolic disregulation, but no other gross abnormalities, which is why I found this study pleasantly surprising:

Regulation of circadian behavior and metabolism by synthetic Rev-erb agonists (Solt et al., 2012)

drogas

In brief, these compounds exhibited good selectivity and were able to repress Bmal1 in in vitro studies, suggesting Rev-erb agonism.  And when the drug was injected just once (see large arrow in figure below), rhythmicity in activity patterns is completely lost for about 24 hours (see the line just below the arrow), but is restored the following day, when the drug has presumably been cleared.

drug vs activity

Both genetic ablation of Rev-erb and pharmacologically increased Rev-erb activity alters circadian rhythmicity.  Further, the agonist exhibits all of the predicted effects on gene expression:

drug vs genes

 

cheat sheet

cheat sheet

 

Treatment for 12 days caused weight loss despite no reduction in food intake.  Or, to be more accurate, the mice ate about 10% more when they were supposed to (at night) and slightly less when they weren’t (day); total food intake was actually elevated (albeit statistically non-significantly).  Pharmaceutical-grade circadian augmentation = unwittingly better-timed meals? Improved body composition?

BW and FI 

Further analysis showed that drug treatment increased energy expenditure, but they weren’t exercising more; total physical activity was reduced by ~15%.

TPMC

…they were eating more and moving less.

Circadian rhythms: 2
CICO: 0

One possible mechanistic explanation comes from mice lacking Rev-erb in skeletal muscle (Woldt et al., 2013).  Mitochondrial biogenesis was impaired and exercise tolerance was reduced in these mice.  Spontaneous physical activity was lower; thus, it stands to reason that Rev-erb agonism may improve skeletal muscle mitochondrial function.  This isn’t entirely consistent with the drug study, but pretty close.

I think these findings are somewhat surprising because there weren’t many hints from the knockout studies, and we (I) would’ve expected any major tampering with circadian rhythms to wreak metabolic mayhem.

…one good thing about the drug is that there were no lasting effects; activity patterns returned to normal after the drug was withdrawn.  This is a somewhat mandatory “regulatory” feature for most drugs, even appetite suppressants: residual lack of appetite upon drug withdrawal suggests permanent, potentially neurological changes, which can be a nightmare for drug companies.  [insert conspiracy theory here].

Some things remain to be fully elucidated, such as: free fatty acids are increased in the liver knockouts and, as expected, decreased with drug treatment (but are also decreased in Rev-erb whole body knockouts [Cho et al., 2012]); and glucose levels decline in liver knockouts and drug-treated mice, but are elevated in Rev-erb global knockouts (Cho et al., 2012).  In a perfect world, agonists and antagonists/knockouts have opposing effects.

Disclaimer: “new” drugs suck.  Just about all of them.  YEARS after their discovery and initial characterization, off-site targets are discovered (eg, resveratrol), weird side effects pop up (which may or may not be due to non-specificity), etc.  So for the time being, best stick with morning bright light exposure, blue blockers (if necessary), and ‘normal’ meal timing.  And be sure to catch your Zzzzs.

Speaking of resveratrol, it reversed the changes in adipocyte Rev-erb expression in diet-induced obese mice (Miranda et al., 2013).  In this model, resveratrol is a bona fide miracle drug.  Is normalization of adipocyte Rev-erb why?  I don’t know, but I’d bet mice with adipocyte-specific Rev-erb deletion are in the works.

Rev-erb-alpha circadian gene variant associates with obesity in two independent populations: Mediterranean and North American (Garaulet et al., 2014)

calories proper

Food for thought: an endogenous ligand of Rev-erb is heme (the iron-binding element in red blood cells).  Heme is degraded into bilirubin.  Elevated levels of bilirubin cause jaundice.  A treatment of neonatal jaundice is exposure to blue light.  Blue light is a major regulator of circadian rhythms and Rev-erb is an executive-level player in this game.  The primary mechanisms of blue light appear unrelated in these two models (melanopsin activation vs. bilirubin photoisomerization), but seem intertwined, because heme activates Rev-erb.  Cool.

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  • Marion Boulden

    you said:
    “I think these findings are somewhat surprising because there weren’t many hints from the knockout studies, and we (I) would’ve expected any major tampering with circadian rhythms to wreak metabolic mayhem.”

    Albert H. Meier, did studies back in the seventies at LSU that suggested that the temporal phase relationship between the circadian patterns of corticosteroids and prolactin affected whether test subjects gained or lost fat, without changes to diet or activity. (Advances in Experimental Medicine and Biology Volume 80, 1977, pp 153-171). Before that (’60′s?) he showed that song sparrows could be induced to lose or gain fat by photoperiod manipulation alone (sorry, no reference for that… something he mentioned in class… I’ll look and see if I can find the paper(s) later, but I’m at work now.). So the idea that circadien manipulations induce changes in metabolism has been around for a while…

    • http://www.caloriesproper.com/ William Lagakos

      Thanks for the info. Very interesting.

      “So the idea that circadien manipulations induce changes in metabolism has been around for a while…”

      I agree 100%! and have written as much, eg, http://caloriesproper.com/?p=4559.

  • http://www.caloriesproper.com/ William Lagakos

    “Drug SR9009 Mimics Exercise Boosting Metabolic Activity And Running Capacity”

    http://www.huffingtonpost.ca/2013/08/26/drug-mimics-exercise-sr9009-_n_3817301.html

  • http://nigeepoo.blogspot.com/ Nigel Kinbrum

    “Treatment for 12 days caused weight loss despite no reduction in food intake.”
    “…drug treatment increased energy expenditure…”
    In a ~30g mouse, energy expenditure due to heat loss >> energy expenditure due to physical activity.
    Circadian rhythms: 2
    CICO: 1
    CICO denialists: 0 :-)

    • http://www.caloriesproper.com/ William Lagakos

      Nigel’s back!

  • Wenchypoo

    Speaking of weird side-effects, isn’t that how we got Viagra?

    • http://www.caloriesproper.com/ William Lagakos

      good point!

  • http://www.caloriesproper.com/ William Lagakos

    “REV-ERB and ROR nuclear receptors as drug targets.”

    http://www.ncbi.nlm.nih.gov/pubmed/24577401

  • Gerard Pinzone

    This might be an explanation to the conflicting results of meal timing and weight loss. Some studies show a minor benefit and others show none. Perhaps it’s the “circadian rhythm” wagging the “meal timing” tail and not the other way around?

    • http://www.caloriesproper.com/ William Lagakos

      …and all the more reason why macronutrients alone are far from the sole contributor to health.

      • Gerard Pinzone

        Well, yes, but which are the first order terms of the health equation and which are nth order ones?

        • http://www.caloriesproper.com/ William Lagakos

          I don’t know! (it’s complicated?)… meal timing & light exposure are two major circadian zeitgebers… but I think I see your point: Rev-erb agonism *caused* “better” meal timing (seems weird to phrase it this way); thus, circadian manipulation was upstream of the “zeitgeber” in this case.

          • Jack Kruse

            Circadian biology and then electrons. This is why our species has 3 pounds of DHA in our brain. It is designed to collect and catch electrons best. 600 million yrs of evolution and not one lipid has proven to be a better catcher of electrons.

          • http://www.caloriesproper.com/ William Lagakos

            This ? “A quantum theory for the irreplaceable role of docosahexaenoic acid in neural cell signalling throughout evolution.”

            http://www.ncbi.nlm.nih.gov/pubmed/23206328

            http://bit.ly/1o86ZBW ? pdf

      • Jack Kruse

        BOOM

        • Carol

          GASP

  • http://www.caloriesproper.com/ William Lagakos

    “Rev-erb-? modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy.”

    http://www.ncbi.nlm.nih.gov/pubmed/23852339

  • http://www.caloriesproper.com/ William Lagakos

    Impaired glucocorticoid production and response to stress in BMAL1 KO mice.

    http://www.ncbi.nlm.nih.gov/pubmed/24189141

  • Primal Jen

    Curious what this could mean for those of us who have had our circadian rhythms demolished by working nightshift for years. To say that the effects on my body have been devastating is an understatement. Tanked thyroid, adrenals, etc, as expected.

    • http://www.caloriesproper.com/ William Lagakos

      Great point. I’ve seen some studies showing recovery of the circadian secretion pattern of certain hormones (eg, cortisol & melatonin) rather quickly after shifters switched back to diurnal life, which would be in line with how soon we recover from jet lag (1 day per hour of flight?).
      As to the long-term implications, I really don’t know; there are probably some biological systems that require much more time to recover.