Lecture 18: Clocks and Metabolism II

Question 1

Why have such a robust food entrainment system?

Answer 1

food essential to survival.
As such we have many ways of measuring food so it is a robust system.

Food may appear at variable times relative to other environmental cues (light)

Feeding (or conversely fasting) elicits many parallel and mutually reinforcing physiological signals
(thus multiple internal entraining factors which can act on multiple/interacting FEOs)

Question 2

Bmal1 KO mice?

Answer 2

still show entrainment/FAA

Question 3

mice v rats

Answer 3

mice cant be fasted for 3 days.

so hard to get long term responses and show it’s a clock, not just a metabolic cycle.

Question 4

Bmal1 and Per1/2 KO mice?

Answer 4

dont show limits of entrainment, can show 18hr FAA.

does this show it’s not a clock? or limits are huge? ??

Question 5

PER2 mutant mice?

Challet, 2010 Genes, Brain and Behavior 9(5):467-477

Answer 5

still make PER2 but can’t interact with the clock.

don’t show FAA in LD.

show some FAA in DD, but diminished.

suggests PER2 important for sensing energy status.

Question 6

Rev-erb a KO mice with RFS

Answer 6

no FAA in KO.

wt. limit energy expendature as hypocalorific diet.
KO mice don’t show this.

wt. decrease body temp to conserve energy.
KO don’t show this.

wt. also show anticipation in body temp, increases before food.
KO don’t show this.

rev-erb a important for timing and energy sensing.
?

Question 7

signals that may be involved in the entrainment of clocks?

Answer 7

nutrients (sterols, lipids, and/or carbohydrates),

humoral signals (insulin, glucocorticoid, leptin)

vagal efferents that travel from autonomic centers to the liver.

Feeding induced temperature cycles

In cultured cell assays, many of these stimuli can induce or reset circadian clocks through regulation of clock gene expression
(for e.g. Balsalobre et al., 1998, 2000).

Question 8

ClockΔ19 mice?

Answer 8

clock protein doesn’t work properly??

obese.
not arrythmic but have diminished rhythms?

Question 9

clock function and metabolic homeostasis?

Answer 9

Clock function exerts a profound impact on metabolic homeostasis.

most clock KO/mutants are obese, Per2bmrd1 is lean.

Question 10

obesity in hoomans?

Answer 10

Diurnal rhythms in metabolic function are dampened in metabolic syndrome (obesity).

Clock gene polymorphisms have been associated with obesity in humans
(CLOCK, REVERB).

‘Disruption’ of our circadian rhythms impact metabolic health:
Chronic shift work PAPERS

social jet lag

forced desynchrony -

Question 11

Forced desyncrony?

Answer 11

behavioural 28hr, clock 24hr so they desync.
can test metabolic parameters when in or out of phase with SCN.

looked at leptin levels -
comparing aligned to misaligned day.

misaligned reduced circulating leptin.

glucose/insulin post meal - on misaligned day sensitivity to insulin decreased.

Forced desynchrony leads to altered orexigenic drive, exaggerated post-prandial glucose/insulin response, and hypertension… Scheer et al. PNAS 2009;106:4453-4458

Question 12

what is leptin?

Answer 12

white adipose tissue, suppresses food intake.

Question 13

links between circadian disruption and metabolic disease?

Answer 13

Our response to changes in metabolic status can be very different at different times of day.

example in mice:
profound insulin-induced hypoglycemia is observed at night, but not in the day (same animals, same insulin dose)

inject insulin and look at glucose response in the blood.

same animals, same dose, night dose more sensitive (when would be eating).

Question 14

clock effects on rate limiting metabolic regulators?

Answer 14

Many rate-limiting metabolic regulators are directly regulated by clock gene transcriptional control.

examples? in notes

Question 15

describe reverbα

Answer 15

nuclear hormone receptor.

repressor, regulates/interacts with numerous metabolic regulators (e.g. PPARs, GR, HDAC3).

recruits HDAC3/NcoR1 to promotor sites.

Question 16

describe HDAC3

Answer 16

HDAC3 (histone de-acetylase 3) plays a major role in regulating lipid and glucose metabolism (among other functions).

chromatin usually tightly wound around histones and hard to access.
need to open for gene transcription to occur, can do this by modifying the histones.

acetylation expresses gene more.
deacetylation (ie HDAC) represses gene expression.

controls rhythm and binding of HDAC

Question 17

Where do Reverbα and HDAC3 bind?

Answer 17

Reverbα and HDAC3 exhibit wide-spread co-binding across the genome
(especially at metabolic genes).

look at binding of HDAC3/reverba to promoter elements in genome.

ZT10 high reverba, binds lots of HDAC3.
ZT22 low reverb low HDAC.

this influences metabolic processes most?

Question 18

What is required for rhythmic HDAC3 binding to DNA?

Answer 18

Reverba.

ZT10 reverba KO decreased HDAC3 binding and lose rhythm.

Question 19

reverba and lipogenesis

Answer 19

during fasting want to suppress lipogenesis, don’t want to be storing fat.

reverba suppresses gene metabolism of what?

Question 20

HDAC3 KO in liver mice?

Answer 20

lipid accumulation - obese.

Question 21

reverba KO mice?

Answer 21

obese normally, if given high fat diet become huuuuuuge

lipogenesis increases.

Question 22

sensors of cellular energy metabolism?

Answer 22

AMP kinase - AMP/ATP levels.

SIRT1/3 - sensitive to redox states.

Question 23

what is a nuclear hormone receptor?

Answer 23

don’t sit on the cell surface. sit in cytoplasm or in nucleus and respond to things coming into the cell.

Question 24

What is PPAR?

Answer 24

peroxisome proliferator-activated receptors
(nuclear hormone receptors)

respond to fatty acids, metabolic sensors, influence lipid metabolism.

directly regulate gene expression (reverba + BMAL1).
Also drive transcription of reverb/BMAL.

reciprocal circuit, form an auxiliary loop.

PPAR a/d/y all show cycles/clock regulated.

Question 25

PPARα

Answer 25

PPARα expression is regulated by CLOCK and BMAL1 proteins via intronic cis elements

Question 26

PPARγ

Answer 26

PPARγ is regulated by Reverba, DBP and E4BP4 (Takahashi et al. 2010).

Question 27

PER2 and PPAR?

Answer 27

PER2 interacts with PPARa/y.

PER2 linked to metabolic control, not a nuclear hormone receptor but has sites in it allowing to interact with other nuclear homones.

PER2 interacts with nuclear receptors involved in lipid metabolism and thermogenesis.
PER2 inhibits PPAR activity.
If mutate so PPAR so can’t interact with PER2, no effect, so physical interaction occurring.

Co-immunoprecipitation and Western blot showing NRs with a physical interaction with PER2.

Question 28

PER2 KO and PPARy adipose tissue

Answer 28

not sure what animal, huge increase of PPARy targets since less repressed.

Question 29

Ucp1

Answer 29

uncoupling protein 1 is a PPAR target.
Thermogenic uncoupler, mitochondria produce heat rather than ATP.

huge increase in expression in PER2 KO, since less inhibition, leads to “lean” mice since burn so much energy.

Question 30

food restriction and clock stuffs

Answer 30

liver, look at food restriction.

food in day, none at night - reversed.

FIRST DAY:
First day of inversed feeding leads aberrant peak in Per1/2 and Reverbα during the day.
PPARa also shows it.

PPARa is binding to reverba, if knock out PPARa lose response of reverb to food switch.

No induction of RevErbα without PPARα.

Looked at genes every 4 hours for 5 days after reversing food:

Reverb switches by 2/3rd day.
Bmal day longer, per1 takes 4 days.

BMAL binding to reverb:

PPAR binding to reverb:

in first 2 days, PPAR binds at feeding time as responds to feeding signals.
Drives reverb, as reverb feeds back on clock, changes bmal and clock shifts with it.

Reverb KO clock genes dont respond/ no food entrainment