Infrafrontier-I3 meeting 2016 · 2020-05-26 · Body weight o Horse (500kg) vs. Rat (500g) o The...

Infrafrontier-I3 meeting 2016

Energy Expenditure and Metabolic Phenotyping in Mice

13.10.2016

Timo D. Müller, PhD Paul T. Pfluger, PhD Carola Meyer, PhD

Harry Knot, PharmD PhD Patrick Hofmann

Literature

Aims of the Session

At the end of this session, you should be able to:

explain how food is converted to metabolizable energy

to explain why O2 is consumed and CO2 is produced to generate ATP and H2O

How we can conclude from O2 and CO2 values which substrate was used for

metabolism

What the difference is between the physical and physiological calorific value

How we can measure the assimilated and metabolizable energy

Aims of the Session

How we can measure energy expenditure using direct calorimetry

How we can measure energy expenditure using indirect calorimetry

Which parameter affect energy expenditure

What the basal and resting metabolic rate is

At the end of this session, you should be able to:

The theoretical basics of energy metabolism

The theoretical basics of energy metabolism

The first law of thermodynamics (1841)

Julius Robert v. Mayer

Julius Robert v. Mayer (1814 – 1878)

German Physician and Physicist.

studied medicine in Tübingen

1841: ‘the law of conversation of energy’

1842: ‘oxidation is the primary source of energy for any living creature’

1842: plants convert light energy into chemical energy

ignored by the scientific community

suicide attempt mental hospital

187: Copley-Medal by the Royal Society

Light energy Electric energy


Energy (gasoline) Kinetic energy (movement) + thermal energy (heat)

Julius Robert v. Mayer

Light energy Organically bound Energy (Carbohydrates, Lipids, Proteins)

O2 H2O + CO2

Energy can’t be destroyed, just transformed from

one form into another

Energy intake (kcal) = Energy expenditure (kcal) weight stable

Energy intake (kcal) < Energy expenditure (kcal) weight loss

Energy intake (kcal) > Energy expenditure (kcal) weight gain

growth, maintenance, and storage

Carbohydrates Lipids, Protein

ATP

ATP


ATP - the metabolic currency of energy metabolism

what does oxidation mean?

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Oxidation

A chemical reaction where a molecule (A) transfers electrons (to another molecule B)

A A+ + e- Molecule A is then oxidized, molecule B is reduced

Historically, oxidation was defined as a chemical reaction that involved oxygen

C + O2 CO2

2H2 + O2 2H2O

Carbon is the electron donor and gets oxidized

Oxygen is the electron acceptor and gets reduced

Hydrogen is the electron donor and gets oxidized

Oxygen is the electron acceptor and gets reduced

What does it now mean, oxidation of nutrients?

Cell level: dCO2/dO2 Respiratory Quotient = RQ

The Respiratory Exchange Ratio (RER)

The amount of consumed O2 and produced CO2 is not a fixed value and

depends on the macronutrients that are oxidized

Macronutrients: Carbohydrates

Lipids

Proteins

During oxidation of nutrients (oxidative phosphorylation) we convert

food and O2 into CO2 and energy



comprise of carbon (C), hydrogen (H) and oxygen (O) atoms in the ratio Cn(H2O)m

The Ratio between hydrogen (H) and oxygen (O) is always 2:1

Oxidation of carbohydrates requires 1 molecule of O2 to produce 1 molecule of CO2

Carbohydrates

glucose, fructose, galactose

C6H12O6


Fatty acids contain much more hydrogen (H) as oxygen (O) atoms

A lot more O2 molecules are needed for oxidation of fatty acids

Palmitic acid

Fatty Acids


Only very small part of the proteins is used for substrate utilization

Vast majority of proteins is used for biosynthesis of new proteins

That’s why proteins are ignored in the equation

Proteins


Summary

RQ = 1 for pure carbohydrates

RQ = 0.7 for pure lipids

RER (RQ) is a marker for which substrate is used for fuel utilization


Chow diet HFD

Same mice (N=8 each panel) used in panel A and B

A B

How do you interpret these data?

Oxidation of nutrients

The amount of O2 consumed is directly related to the amount of energy made

available for metabolism


What else do we learn from this formula?

We now learned that the ratio between CO2 / O2 tells us something about the

oxidized substrate


Combustion of Methan (CH4) in the flame

Oxidation of nutrients equals a combustion

Both consume O2 and produce CO2 and H2O and energy

Bioenergetically, oxidation of nutrients equals a combustion reaction


The energy made available is the same

It doesn’t matter if you eat or burn the burger


Antoine de Lavoisier

French Chemist (1743 – 1794) Co-discovered oxygen

Joseph Priestley

English Chemist (1733 – 1804) Co-discovered oxygen

Experiment 1:

• the mouse having a plant in the glass jar lives longer than the mouse without a plant


Antoine de Lavoisier

Joseph Priestley

Experiment 2:

• the mouse dies exactly at the time where the flame of the candle goes out

• having a plant in the jar makes the flame to burn longer and the mouse to live longer

Conclusion

The flame and the mouse both consume a gas from the air

This gas is produced by the plant and is released into the environment

The respiration process equals a combustion reaction

‘La respiration et donc une combustion’


physiological calorific value = physical calorific value

Energy from nutrient oxidation

Energy from combustion in a flame

Reason: some steps of nutrient digestion require ATP

How is this possible since energy can neither be created nor being destroyed!

But in reality: physilogical calorific value < physical calorific value

Burning the food gives more net energy than eating the food

It doesn’t matter if you eat or burn the burger


Combustion of Methane in the flame

Take home message

There is a direct relation between the amount of O2 consumed

and the amount of energy that is made available for metabolism

If we know an animals O2 consumption, we can thus calculate the energy expenditure

Principle of measuring energy expenditure

Direct vs. indirect calorimetry

Measuring energy expenditure via direct calorimetry

Using direct calorimetry, an individuals energy expenditure is calculated by the

direct measurement of the bodies heat production

In a steady state, all energy from nutrient oxidation is released as heat and

heat production directly equals energy expenditure

First direct calorimeter (ice-calorimeter) to measure heat

production was used in 1782 by Antoine Lavoisier and

Pierre-Simon Laplace

Ice-calorimeter by Lavoisier

Measuring energy expenditure via direct calorimetry

Pros

Highly reproducible Only 1-3% errors

Cons

High maintenance costs Slow response time No information of oxidized substrate Works only at 4°C Measurement not in home cage environment


Metabolizable Energy using indirect calorimetry (bomb calorimeter)

Metabolizable Energy using indirect calorimetry (bomb calorimeter)

Bomb calorimeter


Measuring energy expenditure by double-labeled water

Isotope elimination technique (1960’s Nathan Lifson)

Mouse injected with known volume of 2H218O

Isotopes diffuse through the body fluid

Disappearance rate is measured at different time points


18O is eliminated through both H2O excretion and CO2 production

2H is eliminated through CO2 only

CO2 production calculated by difference between 2H and O2 clearance and

converted to energy expenditure


Cons

Isotopes are expensive

No conclusion about the oxidized substrate

Not possible to measure diurnal changes in energy expenditure

Ventilated open circuit calorimeter

Measurement of energy expenditure from respiratory gases


Pros

Cheaper as direct calorimetry

Home cage environment

Climate controlled cabinets (4°C – 31°C)

Automatic measurement of food and water intake

Locomoter activity

Body core temperature, heart rate, blood pressure

Direct effects of drug treatment

Longitudinal measurement allows measurement of basal metabolic rate

Information of utilized substrate via RER

Cons

System requires quite a good understanding of the principle

Quite intense in maintenance, it’s not just switch on and measure

Ventilated open circuit calorimeter 1 kcal/h = 0,00116 kW 1 kW = 860 kcal/h 1 kcal = 4.184 kJ

How does the O2 consumption relate to the energy expenditure?

The Energy expenditure is independent of the oxidized substrate

always around 20 kJ per liter O2


Based on the O2 consumption we can now calculate the energy expenditure

For every liter oxygen we make 20 kJ available for metabolism. This is independent of the

oxidized macronutrient.

But the amount of O2 that is required to oxidize a substrate varies substantially between

the macronutrients.


Glucose has an enthalpy of ΔH = -2,805 kJ mol-1. That’s the energy in 1 mol of glucose

MW of glucose is 180.16 g mol-1

Energy per gram of glucose is 2,805 kJ mol-1 / 180.16 g mol-1 = 15.57 kJ g-1

Assuming that the energy expenditure is 20 kJ per liter O2,

we thus need 1 liter / 20 kJ x 15.57 kJ g-1 = 0.78 liter O2 per gram glucose

Summary

Oxidation of 1 gram of glucose requires 0.78 liter oxygen

and makes 15.57 kJ available for metabolism

Example 1: Glucose (C6H12O6)

Ventilated open circuit calorimeter Example 2: Palmitate (C16H32O2)

Palmitate has an enthalpy of ΔH = -9957.92 kJ mol-1. That’s the energy in 1 mol of palmitate

MW of palmitate is 256.42 g mol-1

Energy per gram of palmitate is 9957.92 kJ mol-1 / 256.42 g mol-1 = 38.83 kJ g-1

Assuming that the energy expenditure is 20 kJ per liter O2,

we thus need 1 liter / 20 kJ x 38.83 kJ g-1 = 1.96 liter O2 per gram palmitate

Summary

Oxidation of 1 gram of palmitate requires 1.96 liter oxygen

and makes 38.83 kJ available for metabolism


Summary:

Oxidation of 1g fat requires more than twice the amount of O2 as compared to

oxidation of 1g glucose

More than twice the amount of energy is stored in 1g fat as compared to 1g glucose


Why do we then store not all energy in fat? For what do we need glucose and glycogen?

Some organs prefer glucose as energy substrate (brain, muscles etc.) Mobilization of glucose from glycogen is faster as β-oxidation of lipids

Glycolysis generates ATP without consuming O2 (anaerobic ATP production)

Carbohydrate metabolism delivers metabolites of citric acid cycle (e.g.

oxalacetate). Without glucose utilization citric acid cycle would collapse

Availability of glucose is so important that the liver can generate glucose - gluconeogenesis

sleep/wake rhythm

locomoter activity

Group size (thermal conduction)

Food intake (thermic effect of food)

Body weight

Body composition (muscles)

Body size (Bergman’s rule)

Environmental temperature

Parameters affecting energy expenditure

What Parameters affect energy expenditure?


Food intake (thermic effect of food)

Dog eats 1.2kg meat

o Digestion requires ATP, in particular

deamination of proteins requires a lot of energy

o Mice show only very little changes in

energy expenditure after food intake

o Thermic effect of food depends on the meal size

and meal composition and is species dependent


Body weight

VO2 (ml h-1) = 3.9 x BW (g)0.75


Body weight

o Metabolic rate (ml O2 h-1)

increases with body weight

o A horse of 500kg has a greater

metabolic rate than a rat of 500g

o Metabolic rate does not increase

in direct linear proportion to

body weight

VO2 (ml h-1) = 3.9 x BW (g)0.75


Body weight

o Mass-specific metabolic rate

(ml O2 g-1 h-1) decreases with body

weight

o Small animals consume more O2 per gram

body weight as big animals


VO2 (ml h-1) = 3.9 x BW (g)0.75

Body weight

o Horse (500kg) vs. Rat (500g)

o The horse is 1000 x heavier than the rat

Rat: 3.9 x 5000.75 = 0.41 liter O2 h-1

Horse: 3.9 x 500,0000.75 = 73.33 liter O2 h-1

The MR of the horse is 178 times greater as the MR of the rat

We can’t correct energy expenditure by body weight

The metabolic factor of BW0.75 is only for comparison of different species and the intercept does not go through zero

We can’t correct energy expenditure by BW0.75


Environmental temperature

Energy expenditure increases with decreasing environmental temperture


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Basal Metabolic Rate (BMR)

BMR: lowest energy expenditure of a post-absorptive animal at thermoneutrality

RMR: lowest energy expenditure of resting animal at a given temperature

o Bigger animals have a relatively smaller

body surface and thus loos relatively to

their body size a lower amount of energy

over their surface

o Body surface changes with 2/3 power

to the volume


Body size


Body composition

Tissues differentially contribute to whole body energy expenditure

In mammals, 90% of O2 is consumed by the mitochondria

Protein biosynthesis (25 – 30%)

Na+/K+ ATPase (19 – 28%)

Gluconeogenesis (7 – 10%)

Ca2+-ATPase (4 – 8%)

Most energy consuming processes Most energy consuming organs

Muscles (25%)

Liver (17-20%)

GI-tract (5-10%

Kidney (6-7%)

Lung (1-4%)

Brain (3-20%)

Heart (3-11%)

Aims of the Session

Now, you should be able to:

explain how food is converted to metabolizable energy (ATP) oxidative phosphorylation

to explain why O2 is consumed and CO2 is produced to generate ATP and H2O CO2 in citrate acid cycle, O2 is electron acceptor and takes electrons from NADH2 and FADH2

How we can conclude from O2 and CO2 values which substrate was used for

metabolism

RER (animal, 0.7-1.4) based vs. RQ (cell, 0.7-1.0) based

Aims of the Session

What the difference is between the physical and physiological calorific value

physiological calorific value is somewhat lower

How we can measure the assimilated and metabolizable energy

bomb calorimetry

How we can measure energy expenditure using direct calorimetry

directly over heat production

Aims of the Session

How we can measure energy expenditure using indirect calorimetry

ventilated open circuit calorimeter

Which parameters affect energy expenditure

a lot !

What the basal and resting metabolic rate is

BMR: lowest EE of a post-absorptive animal at rest at thermoneutrality RMR: lowest EE of an animal at given temperature

Questions?

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Infrafrontier-I3 meeting 2016 · 2020-05-26 · Body weight o Horse (500kg) vs. Rat (500g) o The...

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