The molecular circadian clock and circadian rhythmicity

Question 1

Characteristics of circadian clocks

Answer 1

• a hierarchical network of central + peripheral clocks
• cell autonomous (pacemakers!)
• All clocks: same components, same properties but
  ○ peripheral clocks only affect the respective tissues + organs
• Central clock (SCN):
  ○ entrains peripheral clocks
  ○ takes 4-12 h to synchronise the peripheral clocks
  § If one peripheral clock decided to be disrupted e.g. eating a meal in the middle of the night for several nights = that’s going to throw out the synchrony b/w the peripheral clocks + the body master clock = when eat in middle of night the enzymes for digestion are not there (not meant to be at night) = will really throw things off so there is no synchrony b/w the peripheral + master clocks
  Yamazaki et al, 2000

Question 2

Non-photic zeitgebers (feeding, activity, and stress):

Answer 2

• serve as time cues to peripheral tissue clocks
• can act on peripheral tissues:
  ○ influencing the phase of peripheral tissue clocks w/ the potential for putting them out of alignment w/ the phase set by the SCN (see later)

Question 3

The molecular clock

Answer 3

• clock components regulate tissue function via expression of hundreds or thousands of genes in different organs
• networks of biochemical (transcriptional-translational) feedback loops that generate 24-h rhythms (i.e., a daily program of gene expression)
• During the day you will want to express a lot of genes = once these genes are expressed they produce target proteins e.g. target specific things e.g. when exercising to get fuel + energy need to activate the enzymes that can burn/mobilise carbs + fat to get ATP

Question 4

Circadian rhythms = period

Answer 4

Circadian rhythms
* daily oscillations in gene expressions

PERIOD (per)
* Seymour Benzer (California Institute of Technology) + Ronald Konopka identified the gene PERIOD in fruit flies
* Nobel laureates Jeffrey C Hall, Michael Rosbash (Brandeis University) and Michael W Young (Rockefeller University) discovered the molecular mechanism that control the circadian rhythm
* This molecular mechanisms is the transcriptional-translational feedback loop

Question 5

Transcriptional-translational feedback loops form the basis of the 24-hour rhythm

Answer 5

• –> Requires the process of transcription:
  ○ DNA makes RNA
• –> Requires the process of translation:
  ○ RNA makes proteins

The promoter - a region of DNA where RNA polymerase begins to transcribe a gene

Question 6

The 24-h transcriptional-translational feedback loops

Answer 6

Regulate the 24-h rhythms by linking tightly to:
* The daily light-dark cycle
* Day-night activities
○ Daytime: gene expressions – make target proteins for cellular tasks
○ Night-time: suppression of clock gene expression
§ Allows body to prepare for sleep + recovery (along w/ melatonin secretion + release

Question 7

Transcriptional-translational feedback loop – the basis of the 24-hour rhythm
* Important transcriptional activators:

Answer 7

• Important transcriptional activators:
  ○ “BMAL1:CLOCK activator complex” of clock genes: Cry1, Cry2, Per1, Per2, Per3, CCGs
• the Per gene encodes Period (PER)
• the Cry gene encodes Cryptochrome (CRY)
• PER-CRY dimers act to repress CLOCK:BMAL1 w/ a periodicity of ~24h
  ○ Become very stable when they attach = good as form an inhibitory process that suppress gene expressions at night

Question 8

How circadian rhythms are generated..

Answer 8

• E-box = promoter = start where transcription can take place = for this to happen need the activator which is the complex BMAL1 + CLOCK
• Per + cry proteins in muscle not the same as in the skin or liver

At night turn off entire process
* As PER + CRY are being produced they also diffuse out into the cytoplasm, by nightfall a lot of these proteins are accumulating as well = but don’t want them at night in the cytoplasm = so we can turn off this pathway
* Per + cry can be destroyed by an enzyme known as proteasome = as they accumulate it will come along + chomp them up
* In addition per + cry but particularly the per protein is quite delicate - can easily be destroyed = so nature has it that they form a dimer (pair together) when they are paired they are more stable + is a good thing cause as a dimer they can come back into the nucleus + serve to inhibit the transcriptional-translational process = serve as a negative feedback loop on this system

Question 9

The molecular clock has 3 key features:

Answer 9

1. Entrainment
2. Endogenous pacemaker
3. Temp compensation

Question 10

1. Entrainment:

Answer 10

• The clock is entrained to the light-dark cycle
• Under light-dark cycle entrainment: –> Zeitgeber period T = 24h
• Zeitgebers can advance or delay the circadian clock

Actogram - activity-rest data in entrained organisms
* Diurnal – wakeful + active period occurs during the day
* Nocturnal – wakeful + active period occurs during the night
- eyes specialized for night vision

Question 11

2. Endogenous pacemaker

Answer 11

Endogenous:
* clock is internal to the organism
* clock keeps track of time persistently even w/o external cues
* daily rhythms have a natural period close to a solar day w/ a natural rhythm τ ~24.2h

Question 12

What is a free-running rhythm?

Answer 12

• Rhythms under free-running conditions
  ○ –> absence of zeitgebers (endogenous circadian period, tau τ)
  ○ i.e., rhythms not synced to any env stimuli (light/dark, temp) or behaviour (rest/activity)
  ○ e.g., Mimosa plant placed in constant darkness
• Free-running rhythm: an exclusive feature of the endogenous circadian clock

Question 13

The cave experiment 1938

Answer 13

Findings:
* Study identified the endogenous circadian rhythm
* the 24-hr core body temp continued to fluctuate in an ~24-h cycle in the absence of external cues
* sleepiness was in sync w/ the temp cycle –> phase r/s
* Bedtime shifted 4h later each day during the Mammoth Cave study ∵ the circadian cycle was longer than 24-h (clock not reset)

Question 14

Biological day and night

Answer 14

• Diurnal organism under entrained conditions of light-dark cycle
  ○ Beginning of biological day anchored to the onset of light
• Subjective day and night
  ○ Diurnal organism under free-running conditions:
  § Beginning of subjective day anchored to the onset of activity

Question 15

3. The clock is temperature compensated

Answer 15

• The clock is not affected by external changes in temp –> a robust clock keeps time + its period length)
• seen in Drosophila, light-sensitive cyanobacteria, homoesthermic mammals
• Pittendrigh: studied Drosophila rhythm of eclosion over a temp range of 16–26°C.

The clock is temperature compensated
* Pittendrigh:
○ Drosophila rhythm of eclosion retained a 24 -h rhythmicity in total darkness over a temp 16 –26 ° C

Question 16

Terminology in chronobiology

Answer 16

• Actogram - A graphical representation of an organism’s phases of activity + rest over the course of a day
• Temp compensation - The ability of circadian clocks to maintain a relatively constant free-running period despite fluctuations in environmental temp
• Endogenous - Growing or working from within an organism; intrinsic.
• Entrainment - The coupling of an observable rhythm in an organism to a Zeitgeber resulting in shared period, where (in contrast to masking) this change is caused by an alteration of the endogenous clock that schedules the observable rhythm. ‘entrain’ means ‘synchronise’

Question 17

Circadian misalignment

Answer 17

• An inappropriate timed rhythm in relation to another rhythm e.g., the shifting of the body clock time w/ respect to the sleep-wake cycle = two rhythms are out of sync w/ each other
• Two types of misalignment:
  ○ phase advance = an event occurring much earlier = go to bed earlier
  ○ phase delay = go to bed at a later time

Question 18

Misalignment of sleep-wake rhythms (with your light-dark cycle will cause sleep disturbances)

Answer 18

Negative impact:
* sleep disturbance
○ Difficulty in falling asleep
○ difficulty waking in the morning
○ –> poor sleep quality
○ –> ↑ health risks
* daytime sleepiness
○ –> impact on perf, errors, accidents

Question 19

Assessing timing .. (circadian misalignment measurements)

Answer 19

• Assessing body clock time: dim-light melatonin onset (DLMO = onset release of melatonin is directly linked to the presence of a light source + therefore it can be measured quite readily even though it is quite a rigorous protocol to measure) = can also look at core body temp, cortisol rhythms but more difficult to assess
  ○ For example w/ body temp = because we are so active w/ different metabolic rates, that can alter your core temp somewhat + as a result you will have more variables to control for if want to measure core body temp

Assessing the timing of the sleep-wake cycle: mid-point of sleep bout

Question 20

How is circadian misalignment measured?

Answer 20

• By assessing the phase angle of entrainment:
  ○ the time interval b/w the timing of the body clock + the timing of an external time cue, e.g.,
  ○ the time b/w DLMO (dim-light melatonin onset) + bedtime, sleep onset, sleep offset, mid-sleep or timing of light exposure
  ○ Need two lots of rhythms = the timing of the DLMO + the timing of the sleep-wake rhythm

Question 21

Phase angle of entrainment: chronotypes

Answer 21

• For morning type the DLMO occurs just before 7:00pm
• For evening types it’s nearly at midnight
  ○ If you are a late chronotype the release of melatonin will be a lot later
• For mid-sleep time for morning type = around 2:00am but for the late time its almost in the morning at around 5:15am
• Linking DLMO to the mid-sleep time
  ○ For the morning type = notice that the DLMO occurs early + mid-sleep time is there (as seen in image) = so the phase angle b/w DLMO and mid-sleep time is a lot longer compared to the phase angle b/w DLMO + mid-sleep for late chronotypes = a lot shorter

evening type:
- shorter phase angle
- longer tau

Question 22

Morning/Evening type

Answer 22

• Inappropriate timing of sleep + wake behaviour to the light-dark cycle –> circadian misalignment

Compared to the Morning type, the Evening type:
* demonstrates a phase delay (go to bed later, wake up later); a shorter phase angle difference
* is predisposed to shorter sleep duration on weekdays, catch up on weekends
* 2.5x more likely to report poor/fair general health (2.5x ↑ in Type II diabetes, 1.3x ↑ hypertension prevalence)
* poorer lifestyle choices e.g. alcohol consumption, exercise

Question 23

Circadian rhythm disorders

Answer 23

Examples of a normal shift in one’s internal clock
* Delayed sleep phase disorder (DSPD) in adolescents
* Advanced sleep phase disorder (ASPD) in older adults

Question 24

DSPD

Answer 24

DSPD occurs during puberty due to
* a biological delay in the circadian rhythm = naturally occurring, not much we can do
* slow build-up of sleep pressure
○ difficulty in falling asleep (at a socially acceptable time of night)
○ difficulty waking in the morning
○ –>daytime sleepiness, poor school perf –> behavioural problems
* Thorpy et al, 1988

SPD Treatment: morning bright light exposure strengthens the sleep-wake cycle + advances circadian rhythms = to shift bed-time to an earlier time

Question 25

Advanced sleep phase disorder (ASPD)

Answer 25

• circadian timing being early relative to the desired + conventional bedtime (Malkani and Zee, 2014)
  ○ As we grow older, the circadian clocks + all other body systems = they all age w/ time –> part of the aging process, the body clock ages as well + consequently the circadian timing is altered
• feel sleepy early evening; early awakening
• a natural, expected shift in rhythm

APSD Treatment: evening bright light exposure strengthens the sleep-wake cycle + delays circadian rhythms = to shift bed-time to a later time = will suppress melatonin secretion

Question 26

Time-zone travel and jet lag
Common problems:

Answer 26

Common problems:
* Travel fatigue
○ difficulties associated w/ long haul flight
§ boredom, in a cramped posture, dry cabin air
§ sleep deprivation
* Jet lag
○ a rhythm disturbance due to crossing many time zones
○ a discrepancy b/w ‘body clock time’ + the ‘real time’ in the new env

Question 27

Jet lag + symptoms

Answer 27

Jet lag
* body rhythms occurring at inappropriate times affecting alertness, sleepiness, hunger, micturition, bowel movts = some people become constipated = because intestinal system is all disrupted

Symptoms
* sleep disruption, daytime fatigue, loss of appetite, bowel irregularities, generalised malaise
* Feeling tired but unable to sleep; loss of concentration + motivation to train, impaired perf

Question 28

Severity of jet lag determined by

Answer 28

Severity of jet lag determined by
* Travel directions
* It is harder:
○ to travel E than traveling W: East 65%; West 19%; Neither 15%
§ Day is shortened by travelling east = harder to adjust to a shorter day, compared to if you are trying to adjust to a longer day = for a longer day, you just delay your bedtime = so much easier to adapt to
* Number of time zones crossed + duration of flight
* Individual differences:
○ ‘larks’, ‘owls’, solo vs group travellers, extroverts vs introverts
§ Group travellers = tend to do more things/activities = easier to adapt, same for extroverts tend to adjust to jetlag far easier than introverts who may be indoors most of the time
* ** Not all rhythms adjust quite quickly
○ pattern of eating meals + digestive function adapt sooner
○ sleep-wake rhythm + perf
§ ~3 days travel W, ~7 days travel E

Question 29

Shift work

Answer 29

• Work outside hrs of ~ 7AM to 6PM
• Nightwork + early morning start times:
  ○ associated w/ the most sleep curtailment = meaning very restricted
  § –> a major disruption to metabolic health + sleep disruption.
• Shift worker – poor health outcomes
  ○ Truncal obesity, dyslipidemia, hypertension, cardiovascular disease, gastrointestinal disorders, cancers

Question 30

Shift worker – poor health outcomes
Why?

Answer 30

• Why?
  ○ circadian disruptions
  ○ altered sleeping pattern e.g. meant to be asleep, but are awake to do work
  ○ altered feeding pattern
  ○ poor health behaviour e.g. despite night shift, can eat healthy (don’t eat fatty food, sugary food), exercise, eat lots of fruit + veg = can avoid having poor health outcomes

Question 31

Nightshift work reduces total daily energy expenditure

Answer 31

• One of the reasons they put on weight is that night shift workers tend to expend less energy
• Grey line = day-shift = expend a lot of energy during the day + by nightfall energy expenditure is very low
• 3 night-shifts:
  ○ Black line:
  ○ Red + blue lines: relatively the same in expenditure = after the first night-shift (the black line) they stay in bed, daytime sleep = but there sleep isn’t usually that consolidated = as most people sleep in a room that is not complete pitch black/dark
• Can see that overall doing night-shift work = they tend to expend less energy compared to someone who did day-shift

Question 32

Shift work and feeding pattern

Answer 32

• Dietary intake:
  ○ macronutrient types or total energy intake per 24 h not significantly dif b/w day + rotating shifts (Pasqua + Moreno 2004; Tepas, 1990; Romon-Rousseaux et al, 1985; Reinberg, 1983)
• Altered eating habits
  ○ higher intake of snacks = maybe to keep awake
  ○ lower intake of large hot meals = as canteen is closed
  ○ eat fewer meals
  ○ **eat at different times of the day

Question 33

Postprandial (after a meal) metabolic profiles

Answer 33

Insulin:
* During the day when consume carbs = there is a spike in insulin = good thing (normal spike) because insulin is required to take out glucose from the blood into the muscles, into the liver and into other organs
* Insulin at night = around 2:00am = insulin response is attenuate = blunted/reduced

Effect of glucose:
* For the night shift because insulin release is low, glucose is not being taken up by the tissues, instead it is left behind in the blood, so we see there is glucose intolerance on night shift
* Whereas on day shift = no problem there = because have high insulin, glucose is taken up = what is left behind in the blood is normal

Question 34

Postprandial metabolic profiles = high fat snack

Answer 34

Snack that was taken = contains a lot of fat
* Was taken at 4:00pm for day-shift workers
* Was taken at 4:00am for night-shift workers
○ Notice difference here: an hour later the triglyceride changes was quite significant = big difference in triglyceride level
○ Night snack = led to a higher triglyceride level in the blood = explanation is that normally with insulin you stimulate the release of lipoprotein lipase, an enzyme that breaks down triglyceride into free fatty acids and glycerol but because during the night insulin release is blunted = therefore triglyceride is not broken down readily and therefore people on night-shift, when they took a snack there triglyceride levels is very much higher, therefore these people suffered from dyslipidaemia

Question 35

Nocturnal feeding: incompatible with circadian timing = body not built to consume food in middle of the night

Answer 35

• Nocturnal loading of nutrients + energy not expected
  ○ gastric emptying rate, intestinal blood flow, kidney + liver activity = ALL LOWER/SLOWER AT NIGHT –> all the enzymes that are used to digest fat + process glucose = not meant to be active during the night
• Unfavourable hormonal + biochemical responses
• Foods eaten b/w 00:00 - 02:00h: persistent relative insulin + glucose intolerance throughout the night compared to daytime consumption of the same foods = not well processed at night
• Night-eaters showed attenuation (slow down or blunting) in the nocturnal rise in plasma melatonin –> pathogenesis of gastrointestinal disorders (Motilva et al 2001) = why some get bowel cancers, others constipation or even diarrhoea
  ○ Melatonin in intestine not same as melatonin secreted by pineal gland in the brain = directly secreted by mucosa cells in intestine