Lectures 13-15 Flashcards
(191 cards)
1
Q
What is the main proposed model for sleep?
A
The Opponent Process Model of Sleep; the homeostatic sleep load vs the circadian drive for wakefulness.
2
Q
Give two examples providing evidence towards the existence of the Opponent Process Model of Sleep.
A
SCN lesions lead to fragmented sleep, suggesting a homeostatic control of sleep without its presence.
If you ignore the desire to sleep, you feel a rhythm of tiredness; when you stay up past the point of falling asleep, you feel awake again - evidence for the circadian drive.
3
Q
Which factors dictate the onset and duration of sleep?
A
Circadian phase and the sleep load.
4
Q
Name the regions of the brain involved in the flip flop model of sleep-wake. Name which part they are involved in.
A
Wake Promoting Areas:
- Tuberomammillary Nucleus (hypothalamus).
- Dorsal Raphe (brainstem).
- Locus Coeruleus (brainstem).
- Laterodorsal Tegmental/Pedunculopontine Tegmental (brainstem).
Sleep Promoting Areas:
- Ventrolateral Preoptic Areas (hypothalamus).
5
Q
What is the main neuropeptide implicated in sleep? Explain briefly.
A
Orexin - it’s one of the main routes by which the circadian rhythm controls wake; it is secreted into CSF and promotes the wake side of rhythms.
6
Q
What is the main nucleoside implicated in sleep? Explain briefly.
A
Adenosine - it stimulates the sleep promoting regions.
7
Q
What are the three main regions the SCN projects to? What area is it via?
A
Via the Sub Paraventricular Zone…
Paraventricular Nucleus (PVN)
Medial Preoptic Nucleus (MPO)
Dorsomedial Hypothalamus (DMH)
8
Q
What is the Medial Preoptic Nucleus (MPO) known for? Why is this implicated in sleep?
A
Steroid production and thermoregulation.
There is a characteristic relationship between body temp and sleep (your body gets colder when you sleep).
9
Q
What is the Paraventricular Nucleus known (PVN) known for? Why is this implicated in sleep?
A
It regulates wakefulness by controlling cortisol and melatonin.
It triggers cortisol release via the HPA axis, promoting alertness, and signals the pineal gland to release melatonin, which supports sleep.
10
Q
What is the Dorsomedial Hypothalamus (DMH) known for? Why is this implicated in sleep?
A
It regulates sleep-wake cycles by integrating circadian and stress signals.
It promotes wakefulness by activating arousal centres (e.g., orexin neurons) and inhibiting sleep-promoting areas (VLPO).
*DMH lesions abolish rhythms in sleep/wake.
11
Q
What are the three main areas that the Dorsomedial Hypothalamus (DMH) outputs?
A
The Paraventricular Nucleus (PVN)
The Lateral Hypothalamus (LH)
The Ventrolateral Preoptic Area (VLPO)
12
Q
What are the three main constituents of the sleep wake cycle?
A
Homeostatic
Circadian
Light
13
Q
Briefly describe the homeostatic control of sleep.
A
Adenosine build up and VLPO activation.
14
Q
Briefly describe the brain structures responsible for circadian control of sleep.
A
SCN outputs to DMH/ Regulation of LH and VLPO neurons.
15
Q
Briefly describe the control of light on sleep.
A
Effects on SCN and sleep/wake regulatory centres; biological influence AND it drives our behaviour (shutting curtains/burrowing in den).
16
Q
What is the underlying cause of delayed/advanced phase sleep syndromes?
A
Involve genetic differences in clockwork.
17
Q
What are the underlying causes of irregular/non-24 hour sleep?
A
Impaired clock function/photoentrainment.
*usually damage present in the pathway between the retina and the brain.
18
Q
What are the underlying causes of insomnia/hypersomnia?
A
Causes are probably unrelated to clock.
19
Q
What disorders are associated with sleep and circadian disruption and how common are they?
A
Sleep and circadian disruptions are linked to many disorders:
- Advanced/Delayed Sleep Phases: Schizophrenia, OCD, Seasonal Affective Disorder.
- Non-24-hour Sleep: Schizophrenia, Non-24-Hour Sleep-Wake Disorder.
- Irregular/Fragmented Sleep: Alzheimer’s, PTSD, Multiple Sclerosis, Depression.
- Hypersomnia: Bipolar Disorder, Schizophrenia, Depression.
- Hyposomnia: Insomnia, Parkinson’s, Autism, Anxiety.
Over 20% of the population suffers severe sleep disturbances, with higher rates in
the elderly, obese, shift workers, and those with neurodegenerative or psychiatric conditions.
20
Q
What is the gold standard of sleep research and what does it contain?
A
Polysomonography.
- Brain Activity (EEG): Tracks electrical activity in the brain to identify sleep stages (e.g., REM, non-REM).
- Eye Movements (EOG): Detects REM (rapid eye movement) sleep and helps differentiate sleep stages.
- Muscle Activity (EMG): Monitors muscle tone, detecting issues like sleep apnoea or restless legs syndrome.
- Heart Rate and Rhythm (ECG): Identifies cardiovascular changes during sleep.
- Breathing Patterns: Measures airflow, respiratory effort, and oxygen levels to detect conditions like obstructive sleep apnoea.
- Body Movements: Tracks limb or body movements that may disrupt sleep.
21
Q
What is a good way of testing circadian rhythms in humans?
A
Melatonin Rhythm.
22
Q
What are the % chronotypes of the population?
A
Morning ‘Lark’: 15-20%
Indifferent: 60-70%
Evening ‘Owl’: 15-20%
23
Q
Outline the Brown et al., (2008) study into human clock properties revealed by skin biopsy.
A
METHODS:
- Aimed to understand the intrinsic properties of the circadian clock.
- Took a skin biopsy, took fibroblast and used virus to introduce BMAL1::luc and track the rhythms in the intrinsic molecular clock in individuals.
- Related it to the chronotype which was measured by the Horne-Ostberg:
- Low scores are night owls.
- High scores are morning types.
RESULTS:
- Night owls have a slower clock than morning people.
- The difference in clock speed is roughly an hour - this is much smaller than the phase.
- Small changes in clock speed leads to larger differences in clock phase that you are entrained to.
*There is a lot of individual variability.
24
Q
What is the most critical process within the molecular clock?
A
The degradation of cryptochrome and period; without this, the whole cycle would stop and it wouldn’t be able to sustain oscillations.
25
What modulates the circadian clock speed?
The degradation of cryptochrome and period.
26
What are the names of the enzymes that, A, control PER degradation, and B, control CRY degradation?
A) CK1epsilon/delta - PER
B) FBXL3 & FBXL21 (F-Box Proteins) - CRY
27
What are the two variants of PER3?
PER3 4/4
PER3 5/5
28
What does PER3 4/4 associate with?
An evening preference and delayed sleep phase.
(Higher chance of being a night owl)
29
What does PER3 5/5 associate with?
Strongly predictive of extreme morning preference, increased evening sleep drive, higher deep sleep, early arousal and very poor performance is sleep deprive.
(Higher chance of being a morning lark).
30
What percentage (%) of the UK population are thought to have the "long variant" of PER3? Which variant is it?
~10% of the population and PER3 5/5
31
What does FASPS stand for?
Familial Advanced Sleep Phase Syndrome.
32
What is Familial Advanced Sleep Phase Syndrome? What is an associated cause?
It's a heritable (& rare) form of extreme early chronotype.
Sleep at ~7:30pm and wake at ~4:00am.
It's associated with autosomal dominant point mutations.
33
Where do the mutations occur in FASPS?
Per2 (S662G)
CK1delta (T44A)
CK1delta (H46R)
All alter PER protein phosphorylation and speed up the clock.
Very similar to the tau mutation - they have the same effect.
34
How does the altered PER protein stability impact the speed of the circadian clock in FASPS?
In FASPS, mutations in the *PER* gene increase the stability of the *PER* protein, causing it to persist longer.
It's the FASPS mutation that leads to a quicker degradation pathway taht accelerates the circadian feedback loop, shortening the overall circadian cycle.
As a result, individuals with FASPS experience a faster internal clock, leading to earlier sleep onset and wake times, despite the persistance of *PER*.
35
Outline the study into CK1delta/epsilon inhibition and its effect on the clock.
METHODS:
- Tested the effects of CK1 inhibitors (drugs).
- Used PER2::Luc SCN slices.
- Looked ex vivo and in vivo.
RESULTS:
- Inhibiting CK1's slows down the clock in a dose-dependent manner.
- Happens in ex vivo and in vivo.
- Led to animals having a slower clock in the presence of the inhibitor.
36
What is PER2 and why is its phosphorylation important?
PER2 is a core circadian clock protein with 21 phosphorylation sites, each influencing its function.
Phosphorylation regulates nuclear translocation, degradation, and repression of CLOCK/BMAL1 activity, which are essential for maintaining circadian rhythms.
37
How does PER2 phosphorylation affect its nuclear translocation?
Specific phosphorylation sites on PER2 increase its rate of nuclear translocation, allowing it to repress CLOCK/BMAL1 activity, a key process in circadian rhythm regulation.
38
What role does phosphorylation play in PER2 degradation?
Phosphorylation can target PER2 for proteasome-mediated degradation, regulating its stability and ensuring proper circadian cycling.
39
How does PER2 phosphorylation influence CLOCK/BMAL1 repression?
Phosphorylated PER2 enhances its ability to repress CLOCK/BMAL1, which is critical for controlling the expression of circadian rhythm genes.
40
What is the impact of phosphorylation "priming sites" on PER2?
Certain phosphorylation sites on PER2 act as priming sites, influencing subsequent phosphorylation events.
This complex interdependence fine-tunes PER2 stability and activity.
41
Outline the Shanware et al., (2011) study into the degradation of PER2 in WT vs FASPS mimic mutations.
METHODS:
- Took WT Per2, PER2-FASPS mimic and PER2-S662D models and compared their degradation rates.
- Did this via a western blot.
- Introduced a protein synthesis blocker (so you can't make any new protein) allowing measurement at different times, providing data about the degradation rate.
RESULTS:
- PER2 WT, after 8 hours the Per levels were about half.
- In PER2-FASPS mutation the amount at the end was very low (so degradation is a lot quicker).
- If introduced a phosphorylation at this point, it decreased the rate of degradation.
- Showing this region is important in the breakdown of PER2.
42
Outline the Vanselow et al., (2006) study into blocking phosphorylation at the FASPS site.
METHODS:
- Took PER2 (control) and PER2-FASPS models.
- Blocked phosphorylation at the FASPS site.
- Recorded CK1 a late time point in which there was meant to be a high level of degradation.
- Did this via a western blot.
RESULTS:
- Found no CK1, the fast mutation did nothing - same levels in both.
- If CK1e is present, the degradation occurs much quicker.
Therefore, when you block phosphorylation at THIS site, you get more at other sites that leads to degradation a lot quicker.
43
What are two important sites for control of PER2 protein stability by phosphorylation?
FASPS and Beta-TrCP sites.
44
Why does FASPS mutation lead to a quicker clock?
Normally, if you get phosphorylated at FASPS site, you can get phosphorylated across all the sequential period genes, making the process slower.
If this CAN'T occur, you go down an alternate pathway which is much more rapid (Beta-TrCP pathway), thus you get quicker degradation via the alternative pathway.
45
How many paths are there for PER degradation?
Two; FASPS and Beta-TrCP (rapid pathway).
46
What is DSPS?
Delayed Sleep Phase Syndrome.
It's a sleep onset insomnia which results in an inability to wake up at conventional times.
47
What is the genetic basis of DSPS? Name Three.
Having PER3 4/4 increases the chance of having DSPS
Missense mutation (V647G) in Per3 increases risk of developing DSPS.
Mutation in CK1epsilon (S408N) gene **decreases** risk of developing DSPS
48
Name two point mutations that have been shown to dramatically lengthen the clock period in mice.
Afterhours (C358S) and Overtime (I364T) mutations in FBXL3.
They block FXBL binding to CRY and the subsequent targeting for degradation.
49
What sleep phenotype is linked to the Per1 gene?
Advanced sleep phase.
50
What is the Per2 gene associated with in sleep phase?
Associated with advanced sleep phase.
51
How does the Per3 gene affect sleep phenotypes?
Long Per3 allele: Advanced sleep phase.
Short Per3 allele: Delayed sleep phase.
52
What is the sleep phenotype linked to CK1 delta?
Advanced sleep phase.
53
How does the CK1 epsilon gene influence sleep?
It is linked to advanced sleep phase.
54
What is the disputed associated phase and illness of the Clock gene in sleep?
Associated with delayed sleep phase, hyposomnia (3111C allele).
55
What sleep phenotype is linked to the Cry1 gene?
Advanced sleep phase.
56
What is predicted about how the Cry2 gene affects sleep based on animal studies?
Delayed sleep phase.
Altered NREM bout duration and EEG activity.
57
What is predicted about the sleep phenotype is associated with the FBXL3 gene based on animal studies?
Delayed sleep phase.
58
What is predicted about the effect of the Bmal1 gene on sleep based on animal studies?
Irregular sleep timing.
BMAL1 is critical for overall clock so LoF would lead to this.
59
Name three methods to fix circadian-related sleep disorders.
Light
Melatonin
Pharmaceuticals
60
How can blue light be used to fix circadian-related sleep disorders?
Up to 2 hours after desired wake-up time:
- Maximise activation of melanopsin (whilst making colour resemble daylight i.e. white).
At least 2 hours before desired sleep time:
- Minimise activation of melanopsin (whilst making colour resemble 'night' i.e. dim blue light)
61
What is an example of a melatonin agonist and how can it be used to fix circadian-related sleep disorders?
Tasimelteon.
As it's a key output of the clock and provides feedback to the central clock, dosing it just before you plan to sleep will help entrain a rhythm to that time.
62
What are the three main types of pharmacological solutions we have to target the clock? What do they do?
Reverb Ligands:
- Reset Phase, Target Metabolism.
CK1 Inhibitors:
- Control Period, Entrainment.
CRY Activators:
- Control Period, Entrainment.
*None of these are used clinically yet but they are being tested and show promising results.
63
Provide a broad definition of metabolism.
Metabolism is every chemical reaction and transformation which occurs within an organism.
The overall balance of these processes determines metabolic rate.
64
What are the two key processes in metabolism?
Anabolism:
The set of metabolic processes that build complex molecules (e.g., proteins, lipids) from simpler ones, requiring energy.
Catabolism:
The set of metabolic processes that break down complex molecules (e.g., glucose, fats) into simpler ones, releasing energy.
65
Name 7 Metabolic Pathways in Humans.
1. Glycolysis/Gluconeogenesis
2. TCA cycle
3. Lipid Metabolism
4. FA synthesis
5. FA oxidation
6. Urea Cycle
7. Steroid and Cholesterol metabolism
66
Name three levels of energy metabolism.
Cellular Level
Tissue/System Level
Organism Level
67
Provide 3 examples of cellular level energy metabolisms.
Anabolic/catabolic pathways
ATP/AMP (energy) levels
Redox status (NAD+/NADH, NADP/NADPH...)
68
Provide an example of Tissue/System level energy metabolism.
Glucose homeostasis (glucose production/storage; release/uptake).
69
Provide 3 examples of organism level energy metabolism.
Metabolic Rate (VO2, VCO2, RQ).
Thermogenesis and body temperature.
Feeding behaviour.
70
What is an external cue besides light that is a strong zeitgeber, influencing our clock?
Food - the timing of when we eat can have a big effect on clocks.
71
What external cues influence the circadian system?
Light: Regulates the brain’s SCN (suprachiasmatic nucleus) and extra-SCN oscillators.
Food: Influences peripheral tissues through endocrine and autonomic signals.
72
How does the central nervous system (CNS) circadian clock affect food intake?
Food intake follows a circadian rhythm.
High-fat diets reduce rhythmic feeding behaviour in mice.
73
What are the outputs of the peripheral clock in metabolism?
Glucose uptake, insulin sensitivity and liver glucose output.
Ensures circadian alignment with post-meal glucose surges.
74
What a piece of evidence for rhythms in cell metabolism?
Cellular metabolic rhythms (e.g., CO₂ production, NAD⁺ cycling) persist even in isolated cells.
Reflect circadian regulation of energy metabolism.
75
How are the CNS and peripheral clocks connected?
The SCN (central clock) coordinates peripheral clocks via endocrine and autonomic pathways, aligning metabolism with environmental cues like light and food.
76
What is RF?
Restricted Feeding Schedule:
Food access is restricted in time and/or quantity.
77
What is FAA?
Food Anticipatory Activity:
Increased activity leading up to expected mealtime (can also be seen in body temperature).
78
What is an FEO?
Food Entrainable Oscillator:
Clock structure(s) which time feeding schedules.
79
Name three alternative theories for the circadian clock in relation to driving food entrainment.
Metabolic 'Hour-Glass'
Conditioned Response
Unknown Timing System
80
Outline the Petersen et al. (2014) study into restricted feeding paradigm.
METHODS:
- Rats were housed under either L/D or L/L cycles.
- Used restricted feeding timing paradigm by training rats to press a lever to get food out.
- Food was scheduled at a regular time everyday and the lever would only release food in this window.
RESULTS:
- Restricting food timing works; rats get very good at pressing the lever only when it's nearly time for food to come out.
- Doesn't matter what phase the food timing is provided in relation to light/dark cycle, the rats will entrain to it.
- Even when in constant light and free running, the rats entrain to food.
Shows strong entrainment effect as FFA is not effected by the light/dark cycles.
81
What is the relationship between food entrainment and SCN rhythms?
Food entrainment is independent of the SCN.
Even when the SCN is controlling the free running behaviour, there will be grouped activity over a series of day by a restricted feeding window if presented consistently.
This shows it has a strong effect.
82
What are the 4 main properties of the clock that we can test?
Maintenance in the absence of the zeitgeber.
Slow adaptation to a shift in zeitgeber timing.
Limits of entrainment.
Timing of clock gene expression.
83
Outline the Mistlberger lab study into SCN ablation and restricted feeding.
METHODS:
- Monitored rats' activity under food ad libitum (unrestricted) and restricted feeding (RF) schedules.
- Included both SCN-intact and SCN-ablated rats to assess the role of the SCN in food entrainment.
- Imposed fasting (e.g., 72 hours) to observe the persistence of anticipatory behaviour.
RESULTS:
- SCN-Intact Rats: Anticipatory activity was observed before restricted feeding times, even after 72 hours of food deprivation.
- SCN-Ablated Rats: Rats still entrained to food schedules, showing anticipatory activity despite the absence of the SCN, indicating food entrainment does not require the SCN.
84
Outline the Yoshihara (1997) study into SCN lesions and food entrainment.
METHODS:
- SCN-ablated rats cycled between food restriction, ad libitum feeding and fasting to test memory or timing mechanisms.
- Observed rhythmicity under different feeding conditions and its persistence after fasting.
RESULTS:
- SCN-ablated rats remembered and maintained anticipation of restricted feeding times after fasting, showing evidence of a "food-entrainable oscillator" independent of the SCN.
- When food was given ad libitum, rhythmic behaviour became arrhythmic but re-emerged with fasting and restricted feeding.
85
What do studies into non-24 hour feeding schedules suggest about the hourglass model?
Non-24 hour feeding schedules (e.g., >24 hours) still work to entrain rhythms.
This disproves the hourglass model as if it was the case, wouldn't the energy run out and the animal be unable to maintain activity?
86
What is the hourglass model of food entrainment?
The hourglass model proposes that food anticipation is driven by a resettable countdown mechanism:
- Resets with food availability and begins a countdown to the next expected feeding.
- Builds anticipation as the countdown progresses, peaking before food time.
- Operates via a food-entrainable oscillator (FEO), independent of the SCN.
- Retains "memory" of prior feeding schedules, even after fasting or schedule changes.
87
What does limits of entrainment mean?
The highest and lowest frequency cycle to which the clock can entrain.
88
What did Stephan and Becker (1989) reveal about FFA behaviour?
Food Anticipatory Activity can be entrained ONLY if food is presented within the circadian range (22.5-29 hours).
89
Outline the Damiola et al (2000) study into food entrainment in the SCN compared to peripheral oscillators.
METHODS:
- Allows rodents to have food in the night-time or daytime.
- Took samples from the rodents and performed western blots to observe the levels of clock gene expression differences between these two feeding types.
RESULTS:
- In the SCN, whether the feeding was in the night or day did not matter as the phases didn't change - it remained entrained to the light/dark phases.
- In the Liver, there were changes in the phases of clock gene expression, as the rhythms reverse themselves when in the different feeding periods.
Thus, clock gene rhythms are entrained to food in the peripheral oscillators but not in the SCN.
90
What did Damiola et al (2000) discover about peripheral oscillators and the influence of food and light?
Most peripheral tissues in the body, and many brain sites, will entrain to food over light.
91
Outline the importance of food intake and its relation to the SCN and peripheral clocks.
Food intake is very influential for regulating peripheral clocks.
Thus, the SCN uses it to set our feeding behaviours to control downstream peripheral clocks.
When we IMPOSE feeding schedules, this has a strong effect on our peripheral clocks.
92
Summarise the key information about Food Anticipatory Activity.
It's independent of the SCN and Light/Dark cycle.
It persists after removal of zeitgeber.
It can 'free-run' with a similar period to a previously experienced restricted feeding schedule (RFS).
RFS is capable of entraining the molecular clocks outside the SCN and is more powerful than light for entrainment of peripheral clocks.
93
What are the two continuums of food intake?
Hunger <-> Satiety
Reward <-> Nausea
94
What are the two continuums of energy expenditure?
Energy use <-> Storage
Activity <-> Thermogenesis
95
What are the three main inputs into the neural control of energy balance? What is the area that mediates this? What behaviours does it drive?
INPUTS
- Neural inputs (e.g., vagal inputs).
- Nutrient signals (e.g., glucose).
- Hormone signals (e.g., leptin).
AREA
- Hypothalamus.
BEHAVIOUR:
- Hunger
- Foraging
- Feeding
96
What are the outputs of the Hypothalamus's neural control of energy balance? (4)
Cognitive Processes (motivation, hedonic drives)
Neural Outputs (ANS)
Hormone Signals (e.g., ACTH, TSH)
Behaviours (Hunger, Foraging, Feeding).
97
What are the components involved in the neural control of energy balance and what areas might serve as a food entrainable oscillator (FEO)?
There are semi-autonomous oscillators and slave oscillators.
Systemic signals tell us about food intake.
Then outputs to control this information.
Hypothalamus and the reward centres are suggested areas for potential FEO
98
What is a likely target for the neural control of energy balance? Why?
Arcuate Nucleus (Hypothalamus) as it is very strongly involved in controlling food intake.
If you stimulate neurons here they either drive feeding or inhibit feeding.
99
What inputs the Arcuate Nucleus and what does it output?
Input from the SCN and outputs the lateral hypothalamus.
100
What did the study into mPER2::Luc expression in the Arcuate Nucleus show?
There is an oscillation in gene expression and electrical activity in the mediobasal hypothalamus.
Showing it's a very strong clock.
101
What are the two main types of neurons in the Arcuate nucleus and what are the subtypes of each?
Orexigenic:
- Neuropeptide Y (NPY)
- Agouti-Related Peptide (AGRP)
Anorexigenic:
- Pro-Opiomelanocortin (POMC)
102
What are the two main characteristics of orexigenic neurons?
Increase food intake
Decrease energy expenditure
103
What are the two main characteristics of anorexigenic neurons?
Decrease food intake
Increase energy expenditure
104
What are the three main strategies for locating the FEO?
1. Examine rhythms in clock gene expression and/or neuronal activity which match FAA meal timing.
2. Lesion discrete brain regions and assess food entrainment.
3. Remove clock genes in discrete brain regions.
105
What do ALF and RF in relation to food studies?
Ad Libitum Feeding (free access).
Restricted Feeding.
106
When looking for Per1 expression associated with FFA, which two areas stood out?
Dorsomedial Hypothalamus (DMH)
Nucleus of the Solitary Tract (NTS)
Because under a RF you start to see gene expression within the areas that it's time for food.
107
Why is c-fos useful in studying circadian rhythms?
It's expressed in neurons that are highly active, thus if this is present to a high level in areas associated with circadian behaviour, you can begin to measure the association.
108
What did the Angeles-Castellanos et al (2004) c-fos tracking study show about activity in the DMH during Ab libitum vs Restricted feeding schedules?
The DMH becomes really activated when food is presented; it doesn't when there is ab libitum presentation of food.
109
Outline the Landry et al (2006) study into DMH lesioning.
METHODS:
- Took brain sections from rats with no lesion, partial DMH lesions and total DMH ablation.
- Looked to see if there was any difference in the ability to entrain to food.
RESULTS:
- There was NO difference in food entrainment despite the lesions; this area is not the cause of food entrainment.
110
What do lesion studies suggest about the organisation of the food entrainable oscillator (FEO)?
Lesion studies suggest that the FEO has a distributed organisation, involving both central and peripheral components, rather than being localised to a single discrete site.
111
How do the SCN and DMH interact in food entrainment according to lesion studies?
Reciprocal feedback between the SCN (suprachiasmatic nucleus) and DMH (dorsomedial hypothalamus) integrates food entrainment signals.
The DMH can inhibit the SCN to facilitate food-anticipatory activity (FAA).
112
What effect does a DMH lesion have on food-anticipatory activity (FAA)?
A DMH lesion reduces FAA, but full expression of FAA can be restored by subsequently lesioning the SCN, indicating an inhibitory role of the DMH on the SCN.
113
What role does the SCN play in food-anticipatory activity under restricted feeding schedules (RFS)?
The SCN normally suppresses daytime FAA. Lesioning the SCN can remove this suppression, allowing FAA to be expressed even if the DMH is lesioned.
114
What do tract-tracing studies reveal about SCN-DMH communication?
Tract-tracing studies reveal efferent and afferent connections between the SCN and DMH, supporting their role in coordinated food entrainment and FAA.
115
What is the effect of SCN lesioning on food entrainment after DMH lesioning?
SCN lesioning restores FAA that is diminished by DMH lesions, showing the DMH inhibits the SCN to facilitate food entrainment.
116
Summarise the current understanding of a food entrainable oscillator (FEO).
Studies show that multiple sites in the brain and periphery 'time' rhythmic food intake.
Simple lesion studies have failed to locate a single area for an FEO; almost certainly an interconnected network responding to many peripheral energy signals.
(E.g., reciprocal inhibition between the SCN and DMH).
117
Give 3 reasons why there are such robust food entrainment systems
Energy is essential for survival.
Food may appear at variable times relative to other environmental cues (light)
Feeding (or conversely fasting) elicits many parallel and mutually reinforcing physiological signals.
118
How does the absence of Bmal1 affect food entrainment in mice?
Despite being arrhythmic, Bmal1 knockout mice can still entrain to a restricted feeding schedule, showing that food entrainment does not strictly require canonical circadian clock genes.
119
What is the phenotype of food anticipatory activity (FAA) in Per2 mutant mice?
Per2 knockout mice maintain FAA, but FAA is absent in Per2 mutant mice with the Per2Brdm1 mutation, suggesting some mutations impact FAA differently.
120
How do Cry1 and Cry2 knockout mice affect food entrainment?
Cry1 and Cry2 double-knockout mice are arrhythmic, but they still show FAA, although it is less stable compared to wild-type mice.
121
What does the FAA phenotype in Clock mutant mice indicate about food entrainment?
Clock mutant mice with arrhythmic or longer circadian periods still show normal FAA, demonstrating that canonical circadian timing mechanisms are not essential for food-anticipatory activity.
122
What do transgenic studies reveal about canonical clock genes and food entrainment?
Many canonical circadian clock gene knockouts, including Bmal1, Cry1/Cry2, and Clock, still exhibit food entrainment, suggesting that food-anticipatory activity is regulated by mechanisms independent of the canonical clock.
123
What does the ability of knockout mice to entrain to food suggest about food-anticipatory mechanisms?
It suggests that food entrainment relies on non-canonical pathways and distributed mechanisms rather than strictly on the circadian clock genes.
124
What are the limits to food entrainment in a BMAL1-/- mouse?
Usually they are limited to about 20-24 hours for the entrainment of FFA.
However, in BMAL1-/- can entrain no matter the length
125
What was shown about Rev-Erbalpha-/-, Per2brdm1-/- and the ability to entrain to food?
These KO's show no entrainment to food.
This is likely due to the whole unwiring of the clock so the rats probably don't know that they need to eat.
126
What are some of the signals that arise from feeding that may entrain the liver clock and other peripheral oscillators?
Nutrients (sterols, lipids and/or carbohydrates).
Humoral signals (insulin, glucocorticoid, leptin).
Vagal efferent that travel from autonomic centres to the liver.
Feeding induced temperature cycles.
127
Provide an overview of the interface between the clock and metabolism.
The clock is coupled to energy state and thus it controls many of metabolic cycles.
Feeding and fasting will input into the metabolic cycles (active and eating during day, fasting and not eating during night) as this is where food comes in and is processed into energy (ATP)
This loops back in the cycle to the clock as you it receives information about energy state from the metabolic cycle, modulating the circadian clock.
128
What did the Turek et al (2005) study show about Clockdelta19 mice.
These mice, that had a weak rhythm, put on a high fat diet obtained higher levels of obesity when compared to their WT counterparts.
Thus, clock function exerts a profound impact on metabolic homeostasis.
129
What is the current understanding of the result of circadian disruption on health?
It impacts metabolic health negatively.
130
What did Lu et al., (2014) show about chronic shift work? Provide a brief overview of what they did to show this.
It increases the incidence of Type 2 diabetes and obesity.
Meta analysis of 220K people; rotating shift work -> 42% increase in relative risk.
131
State in one sentence what Scheer et al., (2009) show about forced desynchrony and sleep restriction and its result on insulin.
It causes a decrease in insulin sensitivity.
132
What *specifically* has been shown about the impact social jet lag can have on health?
It can increase severity of obesity and insulin resistance.
133
What did Shi et al., (2013) discover in relation to insulin in mice when you disrupt their circadian rhythm?
A decrease in insulin sensitivity.
134
What a good methodology to look at the effect of disturbing/misaligning the clock from our behaviour?
Forced desynchrony studies.
135
Outline the Scheer et al., (2009) study into the links between circadian disruption and metabolic disease.
METHODS:
- Participants underwent a forced desynchrony protocol, alternating between circadian alignment and misalignment.
- Researchers measured leptin levels (a hormone signalling satiety) and glucose/insulin responses after meals under both conditions.
RESULTS:
**Leptin**:
- Diurnal rhythm persisted during misalignment but with reduced amplitude.
- Lower leptin levels during misalignment resulted in decreased satiety signalling.
**Glucose and Insulin**:
- Misalignment caused exaggerated postprandial glucose spikes and required higher insulin levels to regulate blood sugar.
- The impact was more pronounced with repeated misalignment, potentially worsening over time (e.g., shift work).
136
Outline the study into mice and the circadian relationship to hypoglycemia.
METHODS:
- Blood glucose was measured starting from resting levels.
- Two wild-type (WT) mice received insulin injections either at ZT6 (daytime) or ZT18 (night time), with a 1-week interval between experiments.
RESULTS:
- A significant difference in glucose uptake was observed between ZT6 and ZT18.
- At ZT18 (night), tissues are less sensitive to insulin, as this is the "resting" phase when the body is not primed for glucose regulation.
- At ZT6 (day), insulin sensitivity is high because this is the "active" phase when the body anticipates food intake.
- Eating during the "resting" phase (e.g., ZT18) leads to hypoglycaemia due to lower insulin sensitivity.
- No hypoglycaemia was observed at ZT6, as the body efficiently used insulin during its active phase.
137
What is ZT time? Give two key examples and how they relate to each other.
Zeitgeber Time:
ZT 0: Start of the light phase (e.g., sunrise or lights on).
ZT 12: Start of the dark phase (e.g., sunset or lights off).
138
The main genes involved in the circadian clock are highly influential transcriptional factors.
They don't only act in within the clock, so provide 3 ways in which they can control gene expression and ultimately cell processes, leading to behavioural changes.
1. Feed out directly.
2. Indirectly via chromatin dynamics (One of the key ways it does).
3. Protein-protein interactions.
139
How does the circadian clock drive rhythmic expression of metabolic genes **directly**?
Via E-Box, RORE and D-box sites.
140
How does the circadian clock drive rhythmic expression of metabolic genes **indirectly**?
Via Chromatin structuring (e.g., via HAT/HDAC activity).
141
How does the circadian clock drive rhythmic expression of metabolic genes protein-protein interactions?
Via direct protein-protein interactions with metabolic regulators (e.g., nuclear hormone receptors such as PPAR and GR).
142
Name 5 metabolic pathways influenced by circadian outputs.
1. Metabolism
2. Energy Balance.
3. Hormone Secretion.
4. Cellular Homeostasis
5. Biological Pathways
143
What have ALL transcription or proteomic array studies shown about metabolic pathways?
They are highly rhythmic.
144
What role do clock genes play in regulating metabolic pathways?
Clock genes directly regulate **rate-limiting metabolic enzymes**, aligning metabolic activity with the circadian rhythm.
- Enzymes (e.g., those in glycolysis and gluconeogenesis) show rhythmic gene expression, optimising energy use during the active phase.
145
How does circadian rhythm influence mitochondrial function?
The mitochondrial **electron transport chain** is highly rhythmic, with at least 7 components regulated by the circadian clock.
- This ensures energy production aligns with activity levels across the day-night cycle.
146
Why are statins more effective when taken at night?
Cholesterol synthesis peaks at night due to circadian regulation of enzymes like **HMG-CoA reductase**.
- Taking statins at night aligns with this rhythm, maximising their cholesterol-lowering effect.
147
What metabolic pathways are directly controlled by circadian rhythms?
Key pathways include:
- **Glycolysis**: Generates energy during the active phase.
- **Gluconeogenesis**: Conserves energy during the resting phase.
- **Cholesterol metabolism**: Peaks at night, aligning with enzyme activity.
148
Why are metabolic pathways among the most rhythmic processes in the body?
Studies show that transcriptional and proteomic activity in metabolic pathways follow circadian rhythms.
- Examples: Cholesterol metabolism, membrane channel activity, mitochondrial function.
- This rhythmicity has major implications for drug efficacy and timing (chronotherapy)
149
What is ReverbAlpha and what does it bind to?
It's a nuclear hormone receptor and a transcriptional repressor, which binds to RORE and RevDR2 sites
150
What are Reverbalpha and ROR independently and what is their relationship?
Reverbalpha - is a negative regulator.
ROR - is a positive regulator.
Reverbalpha and ROR compete at the ROR elements (RORE).
Reverbalpha ALSO acts still at these sites or other elements that it binds to in DNA.
They bring over a repressive complex that includes HDAC3 - histone deacetylase 3.
151
What does Reverbalpha-mediate repression of gene expression involve?
The recruitment of HDAC3/NcoR1 to promoter sites.
152
Outline how transcriptional regulators work.
DNA ss twirled around Histones.
For transcription factors to get onto promoters to drive gene expression you need open chromatin,
You need to aceylate the histones to open up genes to be active.
This opening and closing can be controlled by:
Histone DE-Acetylase (decrease acetylation and thus closing).
Histone Acetylase (increase acetylation and thus opening).
Reverb is really important in bringing these enzymes in.
153
What does HDAC3 stand for and why is it important?
Histone de-acetylase 3 - it plays a major role in regulating lipid glucose metabolism (among other functions).
154
Outline the Feng et al., (2011) study into Reverbalpha and HDAC3.
**METHODS:**
- Use an antibody against reverb and HDAC3 to pull down EVERY gene that it binds to.
- Check at both ZT10 (late light phase) and ZT22 (late dark phase)
**RESULTS:**
- At ZT10 reverb is the highest and is at a lot of its sites.
- At ZT22 there is very little reverb.
- There is a wide-spread co-binding across the genome of Reverbalpha and HDAC3 (especially at metabolic genes).
The vast majority of processes it's involved in are metabolic.
155
Outline the Hand et al (2015) reverbalpha-/- study.
METHODS:
- Take WT and Reverba-/- mice and feed them on normal food or high fat diet (HFD)
- Record body weight over a series of 8 weeks and see what occurs.
RESULTS:
- Reverb KO mice are better at storing fat (top chart).
- Those on a high fat diet balloon up like crazy.
- They are so efficient at storing energy as fat and get huge as a result.
- This is due to there being no break in lipogenesis.
Thus we have proof that reverb binding inhibits lipogenesis.
156
What are PPARs?
Peroxisome proliferator-activated receptors - Nuclear hormone receptors.
157
Is activation of PPAR metabolically healthy or unhealthy?
Healthy.
158
What are the natural ligands of PPARs?
Poly-unsaturated Fatty Acids, oxidised FAs, phospholipids.
(FAs: Linoleic, linolenic, arachidonic and rosiglitazone).
159
What do PPARs do?
Bind to promoter elements in the DNA and drive transcription.
They also directly regulate Reverba and BMAL1 clock gene expression in the presence of free fatty acid (FFA).
160
Why are feeding signals important in metabolic transcription factors?
They respond directly to metabolites (nutrients from diet).
161
What regulates PPARalpha expression?
CLOCK and BMAL1 proteins via intronic cis elements.
162
What regulates PPARgamma expression?
Reverba, DBP and E4BP4.
163
Name two PPARs.
PPARalpha
PPARgamma
164
What is the relationship between PPARs and PER2 in white adipose tissue?
Of the 34% of genes upregulated by PER2, 71% are PPARgamma targets.
165
What is the relationship structurally between PER2 and NHRs?
There is a conserved LXXLL sequence conserved across nuclear hormone receptors that allows binding to occur.
166
What did the Grimaldi et al., (2010) coimmunoprecipitation study show?
When coimmunoprecipitation was used via antibodies specific to PPAR, it was found that PER2 comes with it, showing they physically interact.
167
What are PPREs?
They are the response elements in genes that PPAR bind to and drive transcription through.
168
Which two genes appear to be important for food entrainment?
Reverba and Per2
169
What two things are entrained to food?
Behavioural rhythms and clock gene rhythms in peripheral oscillators.
170
Outline the *PART 1** Crosby et al., (2019) study into Insulin/IGF1 modulation of the clock via Per2.
METHODS:
- Used PER2::Luc mouse models.
- Introduced Insulin or the vehicle (control) in primary fibroblasts, cortical neurons, organoids and whole mice in vivo.
- Measured the levels of Per2 as a result.
RESULTS:
- You get a major increase in Per2 in response to insulin.
- Insulin treatment was also sufficient for alteration of clock phase and amplitude.
- Suggests it plays a role as a food associated cue that acts on clocks across the body.
171
Outline the study into Insulin/IGF1 modulation of the clock via Per2 in a flipped food cycle.
**METHODS**:
- Used PER2::Luc mouse model.
- Entrained mice to a food cycle and then flipped it by 12 hours after 4 days.
- Measured activity and recorded it in actograms.
- Insulin inhibitor to see how this effected the shift to food.
**RESULTS**:
- In normal mice, PER2 rhythm shifted quite quickly over time when insulin was not inhibited.
- In Insulin inhibitor, the shift of the PER2 rhythm was slowed, showing good evidence that insulin is key in regulating the clock.
172
Outline the **PART 1** Mukherji et al (2015) study into reversal of feeding and its influence on the core clock.
**METHODS:**
- Mice were subjected to a reversed feeding schedule, shifting food availability to their usual rest period.
- Liver clock gene expression was measured at multiple time points.
- Chromatin immunoprecipitation (ChIP) was performed to examine PPARα binding at the RevErbα promoter.
- PPARα knockout mice were used to assess the necessity of PPARα in feeding-induced clock shifts.
**RESULTS:**
- Reversed feeding rapidly altered the daily expression patterns of Per1/2 and RevErbα in the liver, while Bmal1 and Cry1 remained stable.
- PPARα directly bound to the RevErbα promoter when feeding was shifted.
- In PPARα-deficient mice, RevErbα induction failed under reversed feeding conditions, demonstrating that PPARα is essential for translating feeding time cues into changes in the hepatic clock.
173
Outline the **PART 2** Mukherji et al (2015) study into reversal of feeding and its influence on the liver’s circadian clock.
**METHODS:**
- Mice were fed during their usual rest phase.
- Measurements of liver clock genes were performed.
- Chromatin Immunoprecipitation (ChIP) examined BMAL1 and PPARα binding to RevErbα’s promoter.
- RevErbα-deficient mice were tested to determine RevErbα’s necessity for resetting.
**RESULTS:**
- Reversed feeding triggered a rapid, earlier shift in RevErbα expression, making it the “leader” of the clock reset.
- PPARα bound to the RevErbα promoter early in the process, aiding re-entrainment.
- Without RevErbα, the liver’s circadian clock failed to realign to the new feeding times.
174
What effect does fasting have on the clock?
Improves rhythms in clock genes and metabolism.
175
Outline what Mukherji et al (2015) showed about the roles of PPARa and Reverba in the RF-induced core-clock shift.
FASTING:
- It 'primes' the clock by increasing the response of PER and Reverba.
RE-FEEDING: (DAY 1)
- Increases PPARa activity (FFA)
- Activated PPARa then bings to Reverba prompoter.
- This increases Reverba expression (at the new time of feeding)
- Increased Reverba inhibits BMAL1 expression (starts to shift the clock).
FEEDING: (DAY 2)
- Activated PPARa reinforces new timing of Reverba expression (doesn't fight with BMAL1 due to day1 disruption)
- Reverba reinforces new BMAL1 timing
- THUS clock shifts over to feeding time.
176
What does AMPK stand for?
5' AMP-activated protein kinase
177
Provide an overview of AMPK.
It's a kinase that is directly affected by the amount of energy in a cell (AMP).
It binds ATP, ADP and AMPs.
It's maximally active when there are no ATP bound and only ADP/AMP.
**Key point is that it has a STRONG response to energy state.
178
What did Lamia (2009) show about molecular connections between the clock and metabolic pathways?
AMPK is known to phosphorylate CK1 and increase its activity and it can also phosphorylate CRY and speed up its degradation.
AICAR is a chemical that increases the activity of AMPK.
Increased AMPK decreases the levels of CRY.
As ATP levels drop and AMPK gets more active it can modulate the clock (reducing the levels of PER).
179
Name 8 pathways that time restricted feeding (TRF) impacts.
1. Lipid Metabolism
2. Glucose Metabolism
3. Immune Function
4. Inflammation
5. Insulin Resistance
6. Gut Microbiome
7. Autophagy
8. Adipocyte thermogenesis
180
Outline the relationship between feeding windows and the effect it has on the clock.
The tighter the feeding window, the bigger the emphasis it will have on the clock
181
What are the effects when feeding aligns with natural active periods? (3)
Healthy circadian clock and metabolism alignment
Normal metabolism, cardiovascular function, and immune responses
Healthy aging and lower obesity risk
182
What occurs when feeding times are disrupted and not aligned with the circadian clock?
Disturbed clock gene function
Metabolic disorders, cardiovascular dysfunction, immune problems
Accelerated aging and increased obesity risk
183
How does time-restricted feeding restore and improve circadian rhythms and health?
Enhances synchronization between feeding and active periods
Improves metabolic health, cardiovascular function, and immune response
Delays aging and reduces obesity risk
184
Give a one sentence summary about the findings of Hatori et al (2012) on TRF.
Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed on a high-fat diet.
185
Give a one sentence summary about the findings of Vujovic et al (2022) on TRF.
Late isocaloric eating increases hunger, decreases energy expenditure and modifies metabolic pathways in adults with obesity.
186
How does the circadian clock affect gluconeogenesis in the liver?
The clock regulates key gluconeogenic enzymes (like Pepck), increasing their expression during the sleep/fasting phase (when glucose is low) and repressing them during the active/feeding phase, thereby synchronising glucose production with daily energy demands.
187
What role does AMPK play in linking metabolism to the circadian clock?
AMPK (5' AMP-activated protein kinase) acts as an energy sensor that helps align metabolic pathways with the body’s energy status and circadian signals.
By responding to ATP/AMP ratios, it influences gluconeogenesis and other metabolic processes to match the time of day.
188
How do core clock components like CRY interact with gluconeogenic signalling pathways?
CRY proteins, part of the core circadian clock machinery, can inhibit CREB-driven transcription of gluconeogenic genes.
This action prevents excessive glucose production at times when it’s not needed, ensuring metabolic processes remain time-structured and efficient.
189
How does AMPK regulate CRY and its role in glucose metabolism?
AMPK activation (e.g., during fasting) phosphorylates CRY, leading to its degradation via the FBXL3 pathway.
This reduces CRY-mediated inhibition of gluconeogenesis, allowing glucose production irrespective of the time of day.
190
How does CRY inhibit gluconeogenesis, and what pathways are involved?
CRY inhibits CREB-mediated transcription of gluconeogenic genes (Pck1, G6pc) by interacting with the circadian clock and metabolic pathways.
This action modulates glucose production based on circadian timing.
191
What role does AMPK play in fasting-induced glucose regulation?
During fasting, AMPK is activated and reduces CRY’s inhibitory effects on gluconeogenesis by promoting its degradation.
This ensures glucose production aligns with metabolic demands.
