Changes in Gene ExpressionDuring the Fasted State

Effect of Short-Term Fasting and Refeeding onTranscriptional Regulation of Metabolic Genes in

Human Skeletal Muscle

Henriette Pilegaard, Bengt Saltin, and P. Darrell Neufer

Nutrition and Gene Expression

Lecture, Part 1, Feb 26, 2015

Where is PDK4 on the genome?On chromosome 7.

PDK4

R

MAKE THE INITIAL RNA

PROCESS TO mRNA

THIS PAPER LOOKED PRIMARILY AT THE SYNTHESIS OF RNAFROM 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.

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?

3-carbon metabolites(glycerol-3-phosphate, etc)

Pyruvate

Krebs cycle forenergy generation

PDH

Glucose

Bloodstream

High-energy condition: energy flow toward

Krebs cycle, when there is excess carbohydrate

available

3-carbon metabolites(glycerol-3-phosphate, etc)

Pyruvate

Krebs cycle forenergy generation

PDH complex: inactive form

Glucose

Bloodstream

Energy deprivation: carbon flow

toward glucose, tomaintain blood sugar

at 80 mg/dL.

Block PDH!

If we can stop pyruvate fromentering the TCA cycle,it can be shunted towardgluconeogenesis instead.

Other amino acids feed theTCA at different points: buttheir carbon skeletons canalso be used for theproduction of glucose.

During the fasted state, weneed to generate about80 grams of glucose.

This diagram of changes in gene expression duringshow how energy can flowtoward Acetyl-CoA, to beused for ATP and fattyacid synthesis..OR it canflow back up to glucose.

LACTATE

Energy flow to glucoseis favored if we restrict transfer of pyruvate to Acetyl-CoA

IN THE LIVER, THE GLUCAGON WHICH IS ACTIVATED BYFASTING (GLUCAGON IS HIGH IN THE MORNING) ROUTES METABOLITE FLOW OVER TO GLUCONEOGENESIS

The green arrows show stepsactivated by glucagon. KEY STEP

EXPORTEDTO THEBLOODSTREAM

Most of these steps arereversible. An importantDIFFERENT step is theconversion of pyruvate backto phosphoenolpyruvate.

GLYCOLYSIS

IN

OUT

This is the what happensif there are LOTS ofcalories available.

But what is there is a shortageof calories and carbs?

PDK-4 IS A MAJOR ENZYME THATINACTIVATES THE PDH COMPLEX.This prevents Acetyl-CoA from beingused for ATP. Metabolite flow cannow 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 convertedto ALANINE for export.

CH3

+H3N-C-COO-

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 reviewtoday can happen after a standard overnight fast..

If you don’t eat between 10 PM and 6 AM, thesechanges are very likely to occur.

The chief issue that needs to be addressed is theneed to maintain blood glucose in the morning.For alertness (you don’t want to become a mealfor a roving predator!) you want to keep your bloodglucose around 80-90 in the morning.

That helps your brain function, since the brain normallygets 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-sensitivelipase (inactive)

Hormone-sensitivelipase (active)

Free fattyacids tomuscle

EVENTS WITHINTHE FAT CELL:

Plasma epinephrine and glucagon increases are part of the changes that occur during fasting.

Lipoprotein lipase (LPL) acts to convert triglycerides to glycerol and free fatty acids, which are then transported into the cell.

0H H

Fatty acid (acyl-CoA also needed here)

The carnitine derivative can crossthe mitochondrial membrane!

Carnitine

Enzyme: CPT-1OUTSIDE THEMITOCHONDRIA

INSIDE THEMITOCHONDRIA

0H

H

Into mitochondria: the fatty acid is released, andthen used for beta-oxidation and ATP production

ATP

Make ATP

To anotherround ofbeta-oxidation

ACETYL-CoA

3-carbon metabolites(glycerol-3-phosphate, etc)

Pyruvate

Krebs cycle forenergy generation

PDH-PO4 : inactive form

Glucose

Bloodstream

Energy deprivation: carbon flow

toward glucose.

Acetyl-CoA frombeta-oxidation

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 switchto burning fatty acids forenergy in muscle

The CONTINUED increase afterrefeeding is not yet explained!

THE AUTHORS EXAMINED GENE EXPRESSION ATTWO 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 discussionof 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 abetter job of switching overto oxidizing fat in musclefor energy, after a fast.

HOW COULD WE LOOK ATTHAT AMONG OURSELVES,WITHOUT TAKING AMUSCLE BIOPSY?

UNCOUPLING-PROTEIN 3ALSO INCREASED: Why?

CPT-1 INCREASED, TOHELP 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 metabolismin 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 inpart 2 of today’s lecture.