Metabolism Integration

PSW1 or Metabolic Cluedo

Dr Stuart Knight

map
 Plan
• Introduction
• General discussion of experiment 1
• Group discussions experiment 2-4
• General discussion of experiment 2-4
• Solution !
 GLYCOLYSIS- GLYCOLYSIS
Glucose Fructose

(e2) (e1)

Glucose -6-phosphate Fructose -1-phosphate

S IS
Fructose-6-phosphate

GLUCONEOGENESIS
SIS

HE
Fatty Acid
Fructose1,6,bisphosphate

NT
(e22)

GLUCONEOGENE
Acyl(Rn)-ACP

SY
Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate
(e22)

C ID
1,3 bisphosphoglycerate
Acyl(R6)-ACP

YA

Cytoplasm
3- phosphoglycerate (e22)

TT
Acyl(R4)-ACP
2- phosphoglycerate
(e22)

FA
(e18) 2- phosphoenolpyruvate Malonyl-CoA
(e3) (e21)
(e20)
Oxaloacetate pyruvate Lactate Acetyl-CoA Citrate
(e4)
t5 t19
t17
(e6)
pyruvate Acetyl-CoA

(e16) + (e7)
Oxaloacetate Citrate

(e15) TCA (e8)

Mitochondrion
Malate cis-Aconitate
(e14) (e9)

Fumarate Isocitrate
(e13) (e10)

Succinate -Ketoglutarate

(e12) TCA (e11)

Succinyl-CoA
 All the enzymes and
e1 glucokinase/hexokinase transporters are given
e2 glucose 6- phosphatase an e/t number code
e3 pyruvate kinase
e.g. e1, this makes
e4 lactate dehydrogenase
t5 pyruvate transporter
things simpler
e6 pyruvate dehydrogenase
e7 citrate synthase
e8 aconitase
e9 aconitase
e10 isocitrate dehydrogenase
e11 -ketoglutarate dehydrogenase
e12 succinyl-CoA synthetase
e13 succinate dehydrogenase
e14 fumarase
e15 malate dehydrogenase
e16 pyruvate carboxylase
t17 oxaloacetate shuttle
e18 phosphoenolpyruvate carboxykinase (PEPCK)
t19 citrate shuttle
e20 citrate lyase
e21 acetylCoA carboxylase
e22 fatty acid synthase
 The effects of Y on metabolism in rat
epididymal adipose tissue (expt 1)
Control and experimental fat-pads were incubated in flasks containing 10ml of
Krebs’ bicarbonate-buffered medium gassed with O2:CO2 (95:5). 14C-labelled
glucose were present at 10mM and 3.7kBq/ml. Pads were incubated for 30
minutes and then removed, weighed, frozen and ground in liquid N2. Incorporation
of 14C from the glucose into fatty acids; the output of pyruvate and lactate into the
medium; and the tissue concentration of ATP were measured. Results are given in
Table 1 and are the mean  SEM for 4 separate experiments (* = p<0.01,
statistically significant).

Incorporation of Tissue ATP

Additions to the 14C into fatty concentrati
incubation Y Lactate output Pyruvate output
acids on
medium (mM) (mmol.h-1.gm-1) (mmol.h-1.gm-1)
(mmol.h-1.gm-1) (nmol.gm-1)

[U-14C] Glucose 0 4.64  0.20 0.82  0.09 0.16  0.014 140.0  3.0

0.1 3.41  0.18* 1.28  0.06* 0.34  0.015* 146.0  6.0

 GLYCOLYSIS- GLYCOLYSIS
Glucose Fructose

(e2) (e1)

Glucose -6-phosphate Fructose -1-phosphate

S IS
Fructose-6-phosphate

GLUCONEOGENESIS
SIS

HE
Fatty Acid
Fructose1,6,bisphosphate

NT
(e22)

GLUCONEOGENE
Acyl(Rn)-ACP

SY
Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate
(e22)

C ID
1,3 bisphosphoglycerate
Acyl(R6)-ACP

YA

Cytoplasm
3- phosphoglycerate (e22)

TT
Acyl(R4)-ACP
2- phosphoglycerate
(e22)

FA
(e18) 2- phosphoenolpyruvate Malonyl-CoA
(e3) (e21)
(e20)
Oxaloacetate pyruvate Lactate Acetyl-CoA Citrate
(e4)
t5 t19
t17
(e6)
pyruvate Acetyl-CoA

(e16) + (e7)
Oxaloacetate Citrate

(e15) TCA (e8)

Mitochondrion
Malate cis-Aconitate
(e14) (e9)

Fumarate Isocitrate
(e13) (e10)

Succinate -Ketoglutarate

(e12) TCA (e11)

Succinyl-CoA
 Experiment 1

• What is the effect of Y on glucose metabolism?

What compound do you start with?, what do you end up with? according to
the data provided
• What is the effect of Y on [ATP]?
Look for the (*) – this indicates statistically significance, according to the text
There is no change to the [ATP]
Is the ATP generated by glycolysis or Krebs cycle? This is ambiguous, assume that most
comes from Krebs but we do not know, it is not possible to exclude the Krebs cycle
based on this assumption

• What are the possible sites of action of Y based on

the data in Table1?
Use the e/t number code to list the options
 What is the impact of Y
on the process?

Glucose Fatty Acids

Pyruvate FA

Pyruvate increases –
this can’t be caused by
an inhibitor, pyruvate to
FA is not inhibited
This means that we can
exclude some of the
enzymes and
transporters
 Effect of Y on pyruvate oxidation by rat heart
mitochondria Experiment 2
• Rat heart mitochondria were suspended in 1ml of
medium (KCl 125mM, Tris-HCl 20mM pH 7.4) at 30oC
in an oxygen electrode to measure oxygen utilisation.
The medium also contained Phosphate (2mM),
malate (0.5mM) and pyruvate (2.5mM). Other
additions were made as follows:
• A ADP (2mmol) B Inhibitor Y (25mmol)
C α-Ketoglutarate {2 oxoglutarate} (5mmol)
 A
0.5

0.45
B

oxygen in medium (ug.atom.ml-1)

0.4

0.35

0.3
C
0.25

0.2

0.15

0.1

0.05

0
0 2 4
Time (minutes)
 Effect of Y on pyruvate oxidation by rat heart
mitochondria Experiment 2
• Rat heart mitochondria were suspended in 1ml of
medium (KCl 125mM, Tris-HCl 20mM pH 7.4) at 30oC
in an oxygen electrode to measure oxygen utilisation.
The medium also contained Phosphate (2mM),
malate (0.5mM) and pyruvate (2.5mM). Other
additions were made as follows:
• A ADP (2mmol) B Inhibitor Y (25mmol)
C α-Ketoglutarate {2 oxoglutarate} (5mmol)
 Experiment 2
2.1 Write an equation for the overall reaction that occurs in
Experiment 2
Pyruvate + oxygen = carbon dioxide
2.2 What is the effect on Oxygen consumption by the addition of ADP?
Rate of oxygen use increases, the reaction is coupled
2.3 What is the effect of adding Y?
The rate of oxygen use remains unchanged
Y does not inhibit this process
2.4 Why were pyruvate, malate and phosphate present in the reaction
medium?
Substrate for the reaction; to provide the oxaloacetate; to make use the reaction is
coupled
2.5 Based on this data what is the impact of Y on the Krebs cycle?
Non of the enzymes of the Krebs cycle are inhibited as Y has no affect
Pyruvate CO2

Nothing is inhibited
in this experiment –
this tells us what is
working

The data tells us

nothing about what
is NOT working
(what is inhibited)
 Effect of Y on gluconeogenesis from various
substrates by rat kidney cortex slices (expt 3)

Slices of rat kidney cortex were incubated in 4ml of Kreb’s-bicarbonate-

buffered medium gassed with O2:CO2 (95:5) for 1 hour at 37oC with the
appropriate gluconeogenic substrate. After incubation the slices were
removed, freeze-dried and weighed. The medium was acidified with 0.4ml 4M
HCl before assay of glucose using the glucose oxidase method. Results are
given in Table 3 and are the means  SEM of 3 observations.
Effects of x compared with the control (* = P< 0.01)
Table 3

Glucose production
(mmol.h-1.gm-1)
Substrate added (mM) CONTROL 0.1 mM Y
None 11.4  0.7 7.56  0.6*
Pyruvate (10mM) 201.3  22.1 32.4  2.0*
Lactate (10mM) 58.1  12.2 13.1  4.9*
Succinate (10mM) 114.8  4.4 107.7  9.7
 Gluconeogenesis from pyruvate/succinate
Glucose

Glucose -6-phosphate

Fructose-6-phosphate
Fatty Acid
Fructose1,6,bisphosphate

Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate Acyl(Rn)-ACP

1,3 bisphosphoglycerate
Acyl(R6)-ACP
3- phosphoglycerate
Acyl(R4)-ACP
2- phosphoglycerate

2- phosphoenolpyruvate Malonyl-CoA

Acetyl-CoA Citrate
Oxaloacetate pyruvate Lactate
T T T T

pyruvate Acetyl-CoA
+
Fatty acyl CoA Oxaloacetate Citrate

Malate cis-Aconitate

Fumarate Isocitrate

Succinate -Ketoglutarate

Succinyl-CoA
SWK 11/00
 Experiment 3

3.1What is the effect of Y on gluconeogenesis?

3.2 How does the effect of Y on the conversion of
succinate to glucose, help in narrowing the possible
sites of action?
Use the metabolic map provide to trace the pathway of
conversion of succinate to glucose
3.3 What are the two possibilities for the site of action
of Y?
•
•
 Expt 3: Gluconeogenesis in presence of Y
Pyruvate Glucose
Succinate Glucose

• List the enzymes/transporters succinate to glucose

All the e/t on succinate to glucose are not inhibited

• List the enzymes/transporters pyruvate to glucose

• Which enzyme or transporter are unique to the pathway pyruvate

to glucose?

• t5 (pyruvate transporter)
• E16 (pyruvate carboxylase)
 Gluconeogenesis from pyruvate/succinate
Glucose

Glucose -6-phosphate

Fructose-6-phosphate
Fatty Acid
Fructose1,6,bisphosphate

Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate Acyl(Rn)-ACP

1,3 bisphosphoglycerate
Acyl(R6)-ACP
3- phosphoglycerate
Acyl(R4)-ACP
2- phosphoglycerate

2- phosphoenolpyruvate Malonyl-CoA

Acetyl-CoA Citrate
Oxaloacetate pyruvate Lactate
T T T T

pyruvate Acetyl-CoA
+
Fatty acyl CoA Oxaloacetate Citrate

Malate cis-Aconitate

Fumarate Isocitrate

Succinate -Ketoglutarate

Succinyl-CoA
SWK 11/00
Pyruvate glucose

Succinate glucose

We can now
exclude all the
parts of pathways
that are NOT
uniquely covered
in the conversion
of pyruvate to
glucose
Effect of Y on carboxylation of pyruvate by intact
mitochondria (Experiment 4)
• Liver mitochondria were incubated for 10 minutes in 0.5ml of
medium containing KCl (110mM), potassium phosphate
(10mM), H14CO3 (10mM, 3.7kBq/ml), Tris-HCl (5mM) and
pyruvate (5mM). The assay was started by the addition of
mitochondria and ended by the addition of 0.1ml 5M HCl. The
acidified medium was centrifuged for 1 minute and the
supernatant transferred to a scintillation vial. The supernatant
was evaporated to dryness at 100oC, and the residue
dissolved in water (0.2ml). Acid-stable counted in a liquid
scintillation counter. Control incubations without pyruvate
were carried out to determine pyruvate-independent
carboxylation and the results in the figure corrected for this.
Controls also showed that the incorporation of H14CO3- into
acid-stable material was linear for at least 10 minutes.
 pyruvate carboxylation
(nmol.min-1.ug protein-1)
10
12
14

0
2
4
6
8

0
0.05
[Y] mM
0.1
1.0
0.15
Effect of Y on carboxylation of pyruvate by intact
mitochondria (Experiment 4)
• Which pathways involves carboxylation of pyruvate?

• Pyruvate + “CO2” = Oxaloacetate

• This has two functions:
– Anaplerotic pathway – creating more oxaloacetate for the
start of the Krebs cycle
– Oxaloacetate is also key stage in gluconeogenesis
 Experiment 4

4.1 What is the effect of Y on the process of pyruvate

carboxylation?
There is a dose dependent increase in the rate of
inhibition as Y is added
4.2 What are the two possible sites of actions based on
this data?
• t5
• e16
• This data tells us that that the inhibitor is
specific for an individual enzyme or
transporter
 Experiment 4 / Solution

4.3 How does the data from Experiment 2 allow you to

choose between them?
In experiment 2 pyruvate oxidation is not inhibited, this
means that the pyruvate has entered the
mitochondria via t5
4.4 What is the most likely target of action of
compound Y?
E16 : pyruvate carboxylase
e1 glucokinase/hexokinase
e2 glucose 6- phosphatase
e3 pyruvate kinase
e4 lactate dehydrogenase
t5 pyruvate transporter
e6 pyruvate dehydrogenase
e7 citrate synthase
e8 aconitase
e9 aconitase
e10 isocitrate dehydrogenase
e11 -ketoglutarate dehydrogenase
e12 succinyl-CoA synthetase
e13 succinate dehydrogenase
e14 fumarase
e15 malate dehydrogenase
e16 pyruvate carboxylase
t17 oxaloacetate shuttle
e18 phosphoenolpyruvate carboxykinase (PEPCK)
t19 citrate shuttle
e20 citrate lyase
e21 acetylCoA carboxylase
e22 fatty acid synthase