Download L26_Adv06

‘40 minute famine’®
CAT
FOOD!
Gareth
Presentation and history:
• A 3-year old boy, Mark, suffering from stupor and
very rapid breathing (hyperpnoea).
• His mother reports he has sweats, trembling and
occasionally convulsions after more than a few
hours without food.
• enlarged abdomen and hepatomegaly (an
enlarged liver).
• xanthomas (nodules of lipid) around his body.
• An 'instant' blood glucose reading = only 2 mM.
• After i.v. glucose, Mark's condition stabilised 
further tests
What is the likely cause of the
sweats, convulsions, etc?
A
B
C
D
Hypoglycaemia ‘instant’ BSL = 2mM
Hyperglycaemia
Hypoinsulinaemia
Hyperlipidaemia
Initial blood tests
Control
Metabolite/Enzyme
Mark
(range)
Glucose (mM) 
4.5 - 5.5
1.9
Triglycerides (mM) 
0.6 - 3.2
8.1
Fatty acids (mM) 
0.2 - 0.8
4.0
Lactate (mM) 
0.6 - 1.8 10.5
Uric Acid (mM) 
0.2 - 0.4
2.9
Alanine aminotransferase (mU ml-1) 10 - 40
170
Bilirubin (µM) 
3 - 15
70
pH 
7.35 - 7.45 7.18
Ketone bodies (mM) 
<0.5
0.6
Where does bilirubin come from?
A The breakdown of bile salts
B The breakdown of cholesterol
C The breakdown of red blood cells
D Artificial food colourings in the diet
E None of the above RBC can only use glucose
What normally happens to bilirubin?
A Excreted in urine
B Sent from liver to gall bladder
C Oxidised to carbon dioxide
What is a high blood [uric acid] indicative of?
A A low cellular 'energy charge'
B An increased rate of deamination of adenosine
C A decrease in ATP
D A marked rise in AMP
E All of the above
Alanine aminotransferase (ALT) is not just found in the
liver. How could we be sure that the ALT measured here
was from the liver?
A Measure the Km in an enzyme dependent assay
B Measure the Vmax in an enzyme dependent assay
C Look at the isoenzyme banding pattern on gel
D We couldn't be sure
How could triglycerides be in the blood?
A High rate of VLDL release from liver
B High rate of lipolysis in adipose tissue
C Failure to clear LDL
VLDL = high in TGs
When are fatty acids released into the
bloodstream?
A When insulin levels are high
B When glucagon levels are low
C When insulin levels are low
BSL   insulin   lipolysis   FA +  glycerol
What processes can lead to
increased blood lactate?
A  liver gluconeogenesis lactate is a substrate
B liver glycolysis
lactate is a product of rapid
glycolysis
C muscle glycolysis
D  muscle PDH activity lactate is a substrate
E All of the above
Which processes are normally functioning
after a few hours of fasting?
aim to maintain blood glucose
A.
B.
C.
D.
E.
F.
G.
release glucose from store
Glycogenolysis
Glycogen synthesis
Lipolysis  insulin   lipolysis   FA +  glycerol
Lipogenesis
Gluconeogenesis glucose req’d for brain, RBCs
Insulin secretion
Glucagon secretion BSL  glucagon from pancreas
Mark’s problem could be with one of these processes
Initial blood tests
Hormone
Insulin (pM)
Glucagon (pM)
Control (range)
Mark
20 - 100
20 - 50
2
250
So, Mark is secreting glucagon: are his cells responding?
Glucagon tolerance test
This involved infusing Mark with intravenous glucagon
Mark is not making
glucose:
what would happen
in a normal person?
normal
Mark
What process does NOT   glycogenolysis
in response to glucagon?
A
B
C
D
cAMP production
Activation of Protein Phosphatase I
Activation of Protein Kinase A
Phosphorylation of phosporylase
N
low blood glucose
Glucagon
LIVER
a
GTP
GPCR
C
ATP
cAMP
cAMP cAMP
cAMP
cAMP
cAMP
cAMP
cAMP
Protein Kinase A
cAMP
ACTIVE
 blood [glucose]  GLUCAGON   cAMP PKA
cAMP
pyruvate
kinase
glycolysis
glycogen
synthase
glycogen
synthesis
cAMP
glycogen
phosphorylase
glycogenolysis
fructose 1,
6- bis
phosphatas
e
gluconeogenesis
 blood [glucose]  GLUCAGON   cAMP PKA
cAMP
P
pyruvate
kinase
glycolysis
P
glycogen
synthase
glycogen
synthesis
cAMP
P
glycogen
phosphorylase
glycogenolysis
P
fructose 1,
6- bis
phosphatas
e
gluconeogenesis
blood [glucose]  GLUCAGON   cAMP
protein kinase A
cAMP
P
pyruvate
kinase
glycolysis
P
glycogen
synthase
glycogen
synthesis
cAMP
P
glycogen
phosphorylase
glycogenolysis
 blood GLUCOSE
P
fructose 1,
6- bis
phosphatas
e
gluconeogenesis
blood [glucose]  GLUCAGON   cAMP
protein kinase A
cAMP
P
pyruvate
kinase
glycolysis
P
glycogen
synthase
glycogen
synthesis
cAMP
P
glycogen
phosphorylase
glycogenolysis
P
fructose 1,
6- bis
phosphatas
e
gluconeogenesis
CO-ORDINATED  Available glucose
glycogen (n)
debranching enzyme
glycogen
phosphorylase
P
glucose 1-P
glycogen (n-1)
phosphogluco
mutase
P
glucose
Glucose 6phosphatase
glucose 6-P
glycolysis
glycolysis
FA oxidatn AcCoA + Pyr Carboxylase, - PDH
glucagen (stimulates F16bP’tase)
gluconeogenesis
substrates (lactate, glycerol, certain amino acids
Gluconegenesis stimulated by
stim by glucagon
Which one of these does not participate in a
pathway that allows glucose production in
response to glucagon?
A Phosphorylase
B Glucose 6 phosphatase
C Glucokinase glucose  g6P in liver: high Km, so active
only when  [glucose]  glycogen
D GLUT-2
What is NOT an effect of glucagon?
A An increase in WAT lipolysis
B Stimulation of glucose release from the liver
C Production of glucose from lactate by the liver
D Breakdown of muscle glycogen
glycogenolysis in response to
glucagon in liver, not muscle
2
2
4
GLUT-2 in liver (high Km)
active only when glucose high,
gets last dibs on glucose (1st brain, muscle)
GLUT-4 in muscle
What is NOT an effect of glucagon?
A An increase in WAT lipolysis
B Stimulation of glucose release from
liver
C Production of glucose from lactate by
liver
D Breakdown of muscle glycogen
muscle glycogen breakdown in response to adrenaline,
no glucagon receptors
Can Mark make glucose in response to
glucagon?
A
B
C
Yeah
Nope
Can’t tell
see glucagon TT
Can Mark enter glycolysis in response to
glucagon?
Probably:
A Yeah
glucagon TT   lactate
B Nope
C Can’t tell
Can Mark breakdown glycogen in response to glucagon?
A Yeah
glucagon TT   lactate
B Nope
C Can’t tell
Glucagon tolerance test
Mark can break down glycogen  lactate from glycolysis
normal
Mark
but is not making glucose…….?
Marks main problem is low
blood glucose:
What other sources of glucose
should he be able to access?
Galactose tolerance test
This involved infusing Mark with 200 mg/kg galactose via one of his veins.
What is the GI of galactose?
A negligible - very low
B medium (50-60 ish)
C 100 (same as pure glucose)
normal
Mark
Does Mark have a problem clearing
galactose from the blood?
A Yes
B No
C Can’t tell
galactose  lactate
galactose  glycolysis
Galactose
Metabolism
What do you think is the next step in
"Normal Glucose Metabolism"?
A Fructose 6-P
B UDP-Glucose
C Glucose 6-P
D Glucose
galactokinase
galactose
galactose 1- P
UDP glucose
transferase
epimerase
UDP galactose
glucose 1- P
mutase
glucose 6- P
glucose
G6 phosphatase
glycolysis
What would you expect the main route of disposal
of galatose to be?
A  carbon dioxide in muscle
B  glucose in liver
C  fat in adipose tissue
only the liver has
galactose enzymes
What do you notice about Mark's Lactate
response?
A Even at time zero, he has hyperlactemia
B Galactose causes a rise in blood lactate
C Glucagon causes a rise in blood lactate
D All of the above
What processes can lead to
increased blood lactate?
A  liver gluconeogenesis lactate is a substrate
B liver glycolysis
lactate is a product of rapid
glycolysis
C muscle glycolysis
D  muscle PDH activity lactate is a substrate
E All of the above
Can Mark make glucose from galactose?
A Yes
see galactose tolerance test
B No
Can Mark enter glycolysis from galactose?
A Yes
galactose  lactate
B No
Can Mark make glucose in response to glucagon?
A Yes
see glucagon tolerance test
B No
Can Mark enter glycolysis in response to glucagon?
A Yes
glucagon  glycogenolysis 
B No
lactate
so….
galactose
LACTATE in
response to
glucagon
(gluconeogenesis
and
glycogenolysis)
and to galactose
BUT cannot make
glucose
Which is the ‘branch point’ between
glycolysis and glucose common to
glycogenolysis and galactose catabolism?
A
B
C
D
fructose 6 phosphate
Fructose 1,6 bisphosphate
glucose 1 phosphate
Glucose 6-phosphate
Which enzyme is common to glycogenolysis
and galactose catabolism?
A
B
C
D
Lactate dehydrogenase
Fructose 1,6 bisphosphatase
Glucose 1 phosphatase
Glucose 6-phosphatase
In what other process is g6p’tase?
A
B
C
D
glycolysis
lipolysis
gluconeogenesis
glycogen synthesis
Dual-labelled glucose infusion
This involved infusing Mark with intravenous glucose which is labelled
with 14C on every carbon atom but with 3H (tritium) on C2.
Which glucose is [U14C] labelled (red dot = 14C)
A
B
C
D
Which glucose is [3H] labeled on C2 (red dot = 3H)?
A
B
C
D
glycolysis
H
P-
exchange of H+ with medium
*
*
H
*
Pglucose
P-
glucose 6-P
P-
fructose 6-P
fructose
1,6-bis P
hexokinase
isomerase
PFK
gluconeogenesis
H
P-
H
Pglucose
G 6- P’tase
glucose 6-P
P-
P-
fructose 6-P
fructose 1,6-bis P
F 1,6 b iP’tase
isomerase
+
glucagon
21. At which step is 14C lost from U14C-glucose?
A Glucose --> G6P
B G6P ---> F6P
all C are retained
C F6P ---> F16BP
D F16PB ---> DHAP + glyceraldehyde 3P
E None of the above
22. At which step is 3H lost from the C2 on glucose?
A Glucose --> G6P
B G6P ---> F6P
exchange with H+ from medium
C F6P ---> F16BP
D F16PB ---> DHAP + glyceraldehyde 3P
E None of the above
23. Normally, can F6P be made into glucose?
A Yes
B No
What happens to the 3H and 14C content
of [2-3H, U-14C] glucose when the glucose
goes to F16BP and back again?
A Both the 3H and 14C content goes down
B Both the 3H and 14C content goes up
C The 3H goes up and 14C stays the
same
D The 3H goes down and 14C stays the
same the RATIO of 3H:14C goes DOWN
E The 3H stays the same and 14C goes
down
What happens to the 3H and 14C content
of [2-3H, U-14C] glucose when
gluconeogenesis is faulty?
A The 3H : 14C ratio goes down
B The 3H : 14C ratio goes up
C The ratio does not alter
What happens to the 3H and 14C content of
[2-3H, U-14C] glucose in Mark?
A The 3H : 14C ratio goes down
B The 3H : 14C ratio goes up
C The ratio does not alter
Mark also has a fault in gluconeogenesis
What is needed to allow glucose to go to F6P
and back again?
A Phosphofructokinase
B Fructose 1,6 bisphosphatase
C GLUT-2
D Glucose 6 phosphatase
If we took a liver biospy from Mark and
measured the rate of glycolysis from glucose in
isolated hepatocytes, would it be:
A Higher than normal
B Normal
C Lower than normal
Which is the ‘branch point’ between
glycolysis and glucose
common to glycogenolysis, gluconeogenesis
and galactose catabolism?
A
B
C
D
fructose 6 phosphate
Fructose 1,6 bisphosphate
glucose 1 phosphate
Glucose 6-phosphate
galactose
trace which bits
Mark can and can’t
do: where is the
road block?
Which is the ‘branch point’ between
glycolysis and glucose
common to glycogenolysis, gluconeogenesis
and galactose catabolism?
A
B
C
D
fructose 6 phosphate
Fructose 1,6 bisphosphate
glucose 1 phosphate
Glucose 6-phosphate
Which enzyme is common to
glycogenolysis, gluconeogenesis and
galactose catabolism?
A
B
C
D
Lactate dehydrogenase
Fructose 1,6 bisphosphatase
Glucose 1 phosphatase
Glucose 6-phosphatase
So what’s the defect??
Glucose 6-phosphatase?
as we love invasive,
expensive tests….
let’s do a liver biopsy…
Enzyme/metabolic flux
Control
(range)
Mark
6-9
22.2
Glycogen (mg/(g wet weight
tissue))
Why is glycogen high when Mark can break it down?
His mum has noticed how poorly he gets if he doesn’t eat, so she ensures
Glucose
6-phosphatase
he never goes
too long without a little snack! Thus, he never gets a chance
/(g
(µmol/min
tissue))
to
start breaking
down
his glycogen. Aha! Did you work this out?
using fresh tissue
8 - 12
0.1
in extracts of frozen tissue
8 - 12
10
G6Pase activity low in fresh, live liver cells
But seems to have normal G6Pase activity.
in tissue that is frozen, then thawed:
What does this mean?
A Mark makes normal G6Pase
B Mark has a G6Pase that only works in cold
C The defect is G6Pase in muscle only
So what’s the defect??
What does freezing/thawing do?
A Breaks up all internal membrane
structures
B Breaks the plasma membrane only
C Destroys G6Pase activity
glucose 6-P
glucose
GLUT-9
G-6Phosphatase
glucose 6-P
glucose
G6P’tase is bound inside membrane of the endoplasmic reticulum
(Will you always call it a ranunculum now?!!) G6P must get in, and
glucose must get out for the blood glucose level to rise.
If the transporters are not functioning, this would have a similar effect
to a deficiency of G6P’tase. Freeze/thaw would release the enzyme.
Exam hints!
• Bioenergetics
–
–
–
–
given equations: no plug and chug!
Implication of terms (+, -, , )
How, when and why of coupling
Why is ATP so useful
• Enzymes
– functions, mechanisms: compare and contrast
• Complex IV
– where and when of electrons
• Case Studies
– understand how lab results arose for cases given
– AND be able to apply to new pathways (given)
• No macro, no ELMA simulation (BUT Gareth is
likely to cover ELMA in theory of prac)