Transcript Lecture: Fasting and gene expression, Part 1

Nutrition and Gene Expression
Lecture, Part 1, Feb 26, 2015
Changes in Gene Expression
During the Fasted State
Effect of Short-Term Fasting and Refeeding on
Transcriptional Regulation of Metabolic Genes in
Human Skeletal Muscle
Henriette Pilegaard, Bengt Saltin,
and P. Darrell Neufer
Where is PDK4 on the genome?
On chromosome 7.
PDK4
THIS PAPER LOOKED PRIMARILY AT THE SYNTHESIS OF RNA
FROM SEVERAL GENES: PDK4 WAS VERY IMPORTANT
But what controls that?
Regulatory proteins (called TRANSCRIPTION FACTORS) have to
bind to the promoter, to start production of the RNA transcript.
MAKE THE INITIAL RNA
PROCESS TO mRNA
R
WHY DO WE NEED TO ACTIVATE PDK-4?
YOUR FASTING BLOOD SUGAR IS TYPICALLY
ABOUT 80-100 mg/100 ml (when you wake up).
If your blood sugar is 80 (or higher) after 6 hours of
sleep, and no food intake, where it that coming from?
The brain and kidneys use about 10 grams/hour.
There is about 20 grams in blood and extracellular
fluid, that would be used up over 2 hours.
WHERE DID THAT GLUCOSE COME FROM?
IN THE MORNING, AFTER AN OVERNIGHT FAST,
METABOLIC ADAPTATIONS ARE NEEDED TO
MAINTAIN A NORMAL LEVEL OF BLOOD GLUCOSE
Since you (or your ancestors!) needed to be alert and
start foraging for food, good levels of blood sugar
were really important in the morning.
HOW DO WE CHANGE OUR METABOLISM, TO
KEEP BLOOD SUGAR AT A NORMAL LEVEL?
Bloodstream
Glucose
3-carbon metabolites
(glycerol-3-phosphate, etc)
High-energy condition:
energy flow toward
Krebs cycle, when there
is excess carbohydrate
Pyruvate
available
PDH
Krebs cycle for
energy generation
Bloodstream
Glucose
3-carbon metabolites
(glycerol-3-phosphate, etc)
Pyruvate
Energy deprivation:
carbon flow
toward glucose, to
maintain blood sugar
at 80 mg/dL.
PDH complex: inactive form
Krebs cycle for
energy generation
If we can stop pyruvate from
entering the TCA cycle,
it can be shunted toward
gluconeogenesis instead.
Other amino acids feed the
TCA at different points: but
their carbon skeletons can
also be used for the
production of glucose.
During the fasted state, we
need to generate about
80 grams of glucose.
Block PDH!
This diagram of changes
in gene expression during
show how energy can flow
toward Acetyl-CoA, to be
used for ATP and fatty
acid synthesis..OR it can
flow back up to glucose.
Energy flow to glucose
is favored if we restrict
transfer of pyruvate
to Acetyl-CoA
LACTATE
EXPORTED
TO THE
BLOODSTREAM
The green arrows show steps
activated by glucagon.
KEY STEP
IN THE LIVER, THE GLUCAGON WHICH IS ACTIVATED BY
FASTING (GLUCAGON IS HIGH IN THE MORNING) ROUTES
METABOLITE FLOW OVER TO GLUCONEOGENESIS
GLYCOLYSIS
Most of these steps are
reversible. An important
DIFFERENT step is the
conversion of pyruvate back
to phosphoenolpyruvate.
This is the what happens
if there are LOTS of
calories available.
IN
But what is there is a shortage
of calories and carbs?
OUT
PDK-4 IS A MAJOR ENZYME THAT
INACTIVATES THE PDH COMPLEX.
This prevents Acetyl-CoA from being
used for ATP. Metabolite flow can
now be reversed toward glucose.
WHERE DOES OUR MORNING GLUCOSE COME FROM?
Some of the glucose comes from stored glycogen.
In the morning, there is also a CORTISOL BURST, which
causes muscle to release 30 grams of amino acids.
In the muscle, various rearrangements occur.
Much of the amino acid from muscle is converted
to ALANINE for export.
CH3
+H N-C-COO3
The alanine, with other amino acids, travels to the liver.
Alanine in the liver is deaminated to pyruvate.
What can now be done with pyruvate?
HOW CAN CHANGES IN THESE PATHWAYS
BE STUDIED IN HUMAN VOLUNTEERS?
The changes in gene expression that we review
today can happen after a standard overnight fast..
If you don’t eat between 10 PM and 6 AM, these
changes are very likely to occur.
The chief issue that needs to be addressed is the
need to maintain blood glucose in the morning.
For alertness (you don’t want to become a meal
for a roving predator!) you want to keep your blood
glucose around 80-90 in the morning.
That helps your brain function, since the brain normally
gets all its energy from glucose.
Nine healthy male subjects ranging in age from 22 to 28 years,
with an average height of 185 cm (range 175–192) and a mean
weight of 81 kgm (range 65–110) participated in the study. The
subjects were habitually physically active and maintained their
normal activity pattern between the two trials.
The subjects were given both oral and written information about
the experimental procedures before they gave their informed
consent. The study was approved by the Copenhagen and
Frederiksberg Ethics Committee (Denmark) and the Human
Investigations Committee (Yale University).
Experimental design. The subjects completed two trials
(separated by 2–3 weeks), each consisting of 20 h of fasting
followed by intake of a standardized refeeding meal, which in
one trial was a carbohydrate-rich meal (CHO trial) and in the
other a low-carbohydrate/high-fat meal (FAT trial).
Muscle biopsies were obtained from the
middle portion of the vastus lateralis muscle
using the percutaneous needle biopsy
technique with suction:
- 3 h after a light standardized meal (control)
- after 20 h of fasting
- 1 h after finishing the refeeding meal.
FOR STUDIES OF METABOLISM IN HUMANS,
BLOOD SAMPLES AND MUSCLE BIOPSIES
ARE THE USUAL LIMITS.
We need to consider just which genes they examined.
These genes play a role in the catabolism of fat for energy,
since they produce the following proteins:
Lipoprotein lipase (LPL) allows the cell to oxidize circulating
triglycerides, thereby obtaining free fatty acids for energy.
Carnitine palmitoyltransferase (CPT1) helps move fatty acids
into the mitochondria, where they are degraded for energy
during beta-oxidation.
Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes a
step in the beta-oxidation of long-chain fatty acids.
Pyruvate dehydrogenase kinase 4 (PDK4) suppresses pyruvate
dehydrogenase, and blocks the routing of glucose into
the citric acid cycle: this conserves glucose for other tasks.
EPINEPHRINE AND GLUCAGON = ELEVATED DURING FASTING
Hormone-sensitive
lipase (inactive)
EVENTS WITHIN
THE FAT CELL:
Plasma epinephrine and glucagon
increases are part of the changes
that occur during fasting.
Hormone-sensitive
lipase (active)
Free fatty
acids to
muscle
Lipoprotein lipase (LPL) acts to convert
triglycerides to glycerol and free fatty acids, which
are then transported into the cell.
0H
H
Carnitine
Fatty acid (acyl-CoA also
needed here)
OUTSIDE THE
MITOCHONDRIA
INSIDE THE
MITOCHONDRIA
Enzyme: CPT-1
The carnitine derivative can cross
the mitochondrial membrane!
H
0H
ATP
Into mitochondria: the fatty acid is released, and
then used for beta-oxidation and ATP production
To another
round of
beta-oxidation
ACETYL-CoA
Make ATP
Bloodstream
Glucose
Energy deprivation:
carbon flow
toward glucose.
3-carbon metabolites
(glycerol-3-phosphate, etc)
Pyruvate
PDH-PO4 : inactive form
Acetyl-CoA from
beta-oxidation
Krebs cycle for
energy generation
The promoter for human PDK-4 contains a binding site for
the Glucocorticoid Response Element (GRE), a transcription
factor activated by cortisol.
Since cortisol is activated in the AM, after an overnight fast,
cortisol plays a part in activation of PDK-4 expression.
Other details of PDK-4 activation will be discussed.
We need to consider just which genes they examined.
These genes play a role in the catabolism of fat for energy,
since they produce the following proteins:
Lipoprotein lipase (LPL) allows the cell to oxidize circulating
triglycerides, thereby obtaining free fatty acids for energy.
Carnitine palmitoyltransferase (CPT1) helps move fatty acids
into the mitochondria, where they are degraded for energy
during beta-oxidation.
Uncoupling protein-3 (UCP-3): controls membrane potential
in the mitochondria, and rate of ATP-production
Pyruvate dehydrogenase kinase 4 (PDK4) suppresses pyruvate
dehydrogenase, and blocks the routing of glucose into
the citric acid cycle: this conserves glucose for other tasks.
FASTING LED TO A SUSTAINED
TRANSCRIPTION OF THE GENES
IN MUSCLE FOR:
PDK4
Lipoprotein lipase
This is consistent with the switch
to burning fatty acids for
energy in muscle
The CONTINUED increase after
refeeding is not yet explained!
THE AUTHORS EXAMINED GENE EXPRESSION AT
TWO LEVELS:
-primary transcript (the RNA that was read directly off
the DNA)
-the mRNA (the complete RNA after processing, when
it was prepared to be read into protein)
-later this semester, we have further discussion
of how the RNA was measured
THE INCREASE IN MUSCLE
PDK-4 AND LPL WAS MUCH
GREATER FOR TWO SUBJECTS,
#7 and #9.
These two probably did a
better job of switching over
to oxidizing fat in muscle
for energy, after a fast.
HOW COULD WE LOOK AT
THAT AMONG OURSELVES,
WITHOUT TAKING A
MUSCLE BIOPSY?
UNCOUPLING-PROTEIN 3
ALSO INCREASED: Why?
CPT-1 INCREASED, TO
HELP OXIDIZE MORE
FATTY ACIDS FOR ENERGY
LEVELS OF mRNA SHOWED SAME DIRECTION AS CHANGES IN
PRIMARY TRANSCRIPT, BUT EFFECTS WERE LESS DRAMATIC
The Pilegaard study adds to our knowledge of how
fasting changes the physiology of energy metabolism
in skeletal muscle, including changes in gene
expression in muscle.
Carbohydrate intake blunts these effects, and
actually moves the dynamic toward MAKING fat
instead of BURNING fat. We will discuss that in
part 2 of today’s lecture.