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Glycolysis and gluconeogenesis

Dr. Lieberman
lieberma@ucmail.uc.edu
MEDS3020
Chapters 19 and 26
November 10, 15, 2016

Diabetes a disorder of carbohydrate


and lipid metabolism
Type 1 -cells of pancreas no longer produce
insulin (auto-immune disease, usually occurs in
juvenile years)
Type 2 tissues no longer respond as well to insulin
(takes many years to develop, although there are
some inherited forms)
As we go through carbohydrate metabolism, focus on
the effects of insulin, and think of how diabetes
affects the metabolism of these sugars in the
absence of insulin, and the consequences of this
altered metabolism

What are Carbohydrates?


CnH2nOn general formula
Glucose - C6H12O6
Straight chain vs ring (alpha vs beta)
Hexose (pyranose ring, Cs 1 and 5 joined)
Pentose (furanose ring, Cs 2 and 5 joined)

Aldose vs ketose
Epimers (altered conformation at one
assymetric carbon - diastereoisomer)
Stereoisomers (D vs L forms)

Diabetes type1
A nine-year old girl complains to her parents that she is
tired all the time, and at times her eyesight is blurry. Her
parents have noticed that she has lost some weight
recently, despite a good appetite. She has been drinking
much more than usual, and urinating much more
frequently. She often complains of being thirsty. Her
parents take her to the pediatrician where it is determined
that her blood glucose level is 425 mg/ml (non-fasting
levels should be around 100 mg/dl). Examination of a
blood sample with a pH strip indicates an acidic pH.
Further examination of the hemoglobin in the red blood
cells demonstrated an elevated level of HbA1c, greater
than 7% of the total hemoglobin in her blood.

Questions you will be able to


answer after this set of lectures
Why are blood glucose levels elevated?
Why is her vision blurry?
Why does the patient always feel thirsty,
and experience frequent urination?
Why is the patient losing weight?
What does an elevated HbA1c
indicate?

Glycolysis - Major Metabolic Roles


Entry point of all carbohydrates into
metabolism
Generate energy (ATP) from carbohydrates
Supply precursors for amino acid, large lipid,
and purine/pyrimidine biosynthesis
In times of energy excess, generate
precursors for triglyceride and glycogen
biosynthesis (the energy storage molecules)

GLUT transporters
GLUT1 RBC, endothelial cells of
blood-brain barrier (high affinity)
GLUT2 liver, pancreas, renal tubular
cells, allows bidirectional transport (low
affinity)
GLUT3 neurons (high affinity)
GLUT4 adipose tissue, muscles,
insulin-responsive (high affinity)
GLUT5 fructose transport

GLUT1 deficiency syndrome


Classic presentation infantile seizures,
developmental delay, acquired microcephaly,
hypotonia, spasticity, movement disorder
Leads to hypoglycorrhachia (low glucose levels
in the cerebrospinal fluid)
Confirm diagnosis by molecular techniques
SLC2A1 gene (Glut1) is altered
Rare disorder 1/90,000 in Australia, but only 500
cases worldwide (underdiagnosed)
Autosomal dominant (90% of cases new mutations)

Treat with ketogenic diet (force brain to use


ketone bodies instead of glucose as an energy
source)

Glycolysis in Muscle and Liver


Tissues perform different tasks, so the role of
pathways can be different between tissues;
regulation of pathways will be tissue-specific
Muscle - use energy from glycolysis to do work
(contraction); generate energy in absence of
oxygen (anaerobic metabolism)
Liver use carbons and energy from
carbohydrates to make fat, serum proteins, and
other biosynthetic precursors
Regulation hormonal (whole body) and
allosteric (cell-specific); tissue specific
differences in regulation are common

Glycogen

Glucose 1 - P

CH - O - P
CH OH
GLUCOKINASE
2
CH - O - P
2O
HEXOKINASE
2 O
O
CH OH
2+
H H
H
2
PHOSPHOHEXOSE
H
H
Mg
H
H
HO
OH H
H
OH
OH H
OH
OH
OH
OH
ISOMERASE
ADP
OH
H
ATP
H
OH
H
OH

The Glycolytic
Pathway

COOH
H C OH

C O~P
H C OH

CH - O - P
2
ATP

3 - Phosphoglycerate

GLYCERALDEHYDE
3-PHOSPHATE
DEHYDROGENASE

PHOSPHOGLYCERATE
KINASE

CH - O - P
2

ADP

NADH + H +

1, 3 - Bisphospho-

NAD

ADP

CH O P
2
ALDOLASE
Glyceraldehyde 3-P

Pi

TRIOSE PHOSPHATE ISOMERASE

ENOLASE

COOH
C O~ P

NADH + H +
PYRUVATE
KINASE

CH2
Phosphoenolpyruvate

ADP

ATP

COOH

COOH

CH

OH
SPONTANEOUS

(ENOL)
PYRUVATE

CH

NAD

3
(KETO)
PYRUVATE

COOH
HO C

O
LACTATE
DEHYDROGENASE

D - Fructose 1, 6 - Bisphosphate

CH OH
2
Dihydroxyacetone - P

H O
2

CH - O - P
2
O
* O- P
CH
2
H HO
H
OH
HO

*
CH O - P
2
C=O

2 - Phosphoglycerate

CH OH
2

PHOSPHOFRUCTO KINASE

H C OH

COOH
P

ATP

H C O

glycerate

PHOSPHOGLYCERATE
MUTASE

H C O

D - Fructose 6-P

- D - Glucose 6-P

- D - Glucose

CH
3
L(+)-LACTATE

The Hexokinase Reaction


ATP

H
H CO H
H C
H C OH
H C OH
H C OH
H C
OH
Glucose

1.
2.
3.
4.
5.

Adenine

O +

Ribose
Hexokinase
O

H C O P OOH C
H C OH

ADP
Adenine
Ribose
O

H C OH O
O P O
+ O P O
Glucokinase H C OH
O
O
(liver
and
H C
-O P O
O P O pancreas
OH
O
OSpecific)
-O P O
Glucose-6
Phosphate
O-

Group Transfer Reaction


Irreversible (energy loss as ATP is hydrolyzed)
Activate glucose
Trap glucose inside the cell as G6P
Glucokinase and hexokinase are isozymes

The Phosphofructokinase-1 Reaction


ATP
Fructose-6-Phosphate

Fructose 1,6 Bisphosphate


ADP
PFK-1

1.
2.
3.
4.

Second activation step, lose energy


Mechanism same as HK step
Major regulated step of glycolysis
Sugars are now committed to the
glycolytic pathway

H C O P OC O OHO C H
H C OH
H C OH O H C O P O
OH

Bisphosphate vs Diphosphate
Bis refers to the same functional group on
different atoms within the same molecule (F
1,6 BP)
Di refers to two of the same functional groups
attached to the same atom of a molecule, or
linked together and attached to just one atom
of the molecule (ie ADP, has the two
phosphates attached to each other, which are
then linked to the 5-carbon of ribose)

The aldolase reaction


Splits Fructose 1,6 bisphosphate to
dihydroxyacetone phosphate (DHAP)
and glyceraldehyde 3-phosphate (G3P)
Requires an active amino group at the
active site which comes from the amino
acid lysine
Triose phosphate isomerase then
catalyzes the conversion of DHAP and
G3P

Generation of a high-energy bond


Glyceraldehyde3-phosphate
Dehydrogenase

O
C H
H C OH
O

+ NAD

H C O P O
H

Glyceraldehyde-3
Phosphate (G3P)

Pi

O O
C O

O
O

NADH + H C OH
H C O
H O

O
O

1, 3 BisphosphoGlycerate (1,3 BPG)

1. Carbon 1 of G3P is oxidized (aldehyde to acid)


2. NAD+ is reduced (gains a proton and two electrons to form NADH)
3. The formation of a phospho-anhydride bond generates a high
energy bond
4. The enzyme requires an active sulfhydryl group (from cysteine)

Mechanism of G3P DH
H C

S-Enz
H C OH

H C OH
CH

H C OH
CH O
2

Glyceraldehyde 3-P

+
NAD
P

Enzyme-substrate complex
HS-Enz

Bound
Coenzyme

+
NAD

O~ P
C O
H C OH
CH2 O

Pi

1, 3-Bisphosphoglycerate

C O

C O

NAD+

H C OH
CH O
2

Substrate
oxidation

S-Enz

S-Enz

Formation of thioester
bond leads to the
formation
of the high energy
bond

The enzyme
has a
higher
affinity for
NAD+ than
NADH

NADH
+H+

H C OH
+
NADH+H+ NAD

CH O
2

Pellagra
Lack of niacin in the diet results in pellagra:
Four Ds

Dermatitis (photosensitive)
Diarrhea
Dementia
Death

Poor diet, or from use of drug isoniazid (used to


treat tuberculosis; prevents mycolic acid
production; niacin analog)

Obtained from many sources in the diet: tuna,


chicken, turkey, salmon, lamb, beef, sardines,
peanuts, shrimp, brown rice

The phosphoglycerate kinase reaction


O

O
O
CO P O
-O P O
O+
H C OH
O
O
HCO P O
H
O1,3 BPG

Phosphoglycerate
Adenine kinase

P O Ribose
O(ADP)

O
C OH
ATP + H C OH
O
H C O P O
O
H
3 PhosphoGlycerate (3PG)

1. Phosphoryl group transfer, and the high energy of the phosphoanhydride bond is conserved as a high energy bond of ATP
2. Reversible reaction; substrate-level phosphorylation
3. Since 1 glucose gives rise to 2 molecules of 1,3 BPG, this step
generates 2 ATP molecules per glucose, but the overall
yield of glycolysis is zero at this point (due to the two
activation steps).

Formation of ATP by Pyruvate Kinase


Adenine

ENOLASE O
C OH O
C OH O
H C O P O
C O P OOOCH OH
HOH
C
2
H H

2-PG

PEP

Ribose
+

O
O
O P O P O
-O
-O

ADP
PYRUVATE KINASE

ATP
O

C OH

C OH

C OH

C O

CH2

CH3

Enol-Pyruvate

Pyruvate

PEP CANNOT isomerize to form PHOSPHOPYRUVATE


due to the lack of a hydroxyl on C2. This renders PEP a
high energy phosphate donor. Enolase is inhibited by
fluoride

Fates of Pyruvate, NADH


Pyruvate to lactate, anaerobic metabolism
(always in the red blood cells)
Pyruvate to oxaloacetate (pyruvate
carboxylase, anaplerotic reaction)
Pyruvate to alanine (transamination)
NADH in the cytosol needs to enter the
mitochondria, but there is no transport system
for NADH, so have to use a shuttle system

What About Cytosolic NADH?


Glycerol-Phosphate
Shuttle

No compounds transported into the mitochondria


NADH goes to FADH2 goes to 1.5 ATP (loss of 1 ATP)
Skeletal muscle and brain mitochondria use this shuttle

The Malate Aspartate Shuttle


Matrix
Cytoplasm

NAD+

Malate
-ketoglutarate
transporter

Malate

Malate

Malate
Dehydrogenase

NAD+

Malate
Dehydrogenase

NADH
Oxaloacetate

Glutamate

-ketoglutarate

Aspartate
Aminotransferase

Aspartate

Overall equation: NADH + NAD +


out
in

NADH
Oxaloacetate
Glutamate
-ketoglutarate
Aspartate
Glutamate
Aspartate
Transporter

NAD+ + NADH
out
in

Glutamate
Aminotransferase

Energy yield anaerobic


glycolysis
Glucose to pyruvate to lactate
Regenerate NADH for the glyceraldehyde
3-phosphate dehydrogenase step
Occurs in Red Blood cells and exercising
muscle

2 ATP per glucose molecule through


anaerobic glycolysis generated at a
very rapid rate

Energy Yield From Glucose Oxidation


Glycolysis
1 glucose to 2 pyruvate
Generate 2 NADH at the G3PDH step
Gives rise to 3 ATP if use the glycerol phosphate shuttle
Gives rise to 5 ATP is use the malate aspartate shuttle

Generate 4 ATP by substrate level phosphorylation


1,3 BPG to 3 PG
PEP to Pyruvate

Must deduct 2 ATP for six carbon sugar activation


Yield from aerobic glycolysis is 5 or 7 ATP depending
on the shuttle system used

Energy Yield, Continued


From Pyruvate Dehydrogenase and the TCA
cycle
8 NADH gives rise to 20 ATP

PDH, isocitrate-DH, kg-DH, malate-DH each give rise to 2


NADH per starting glucose molecule

2 FADH2 gives rise to 3 ATP (succinate-DH step)


2 ATP from substrate level phosphorylation (succinate
thiokinase step)

Overall yield from PDH/TCA is 25 ATP from 2


molecules of pyruvate (1 molecule of glucose)
Thus, glucose to carbon dioxide and water will
yield either 30 (glycerol-phosphate shuttle) or 32
(malate-aspartate shuttle) molecules of ATP
(compare to 2 ATP and 2 NADH from anaerobic
glycolysis)

Three Irreversible Steps


Hexokinase (Glucokinase in liver, pancreas)
Glucose + ATP yields G6P + ADP
PFK-1
F6P + ATP yields F 1,6BP + ADP
Pyruvate Kinase
PEP + ADP yields pyruvate + ATP
All of these steps involve large energy changes as
the reactant is converted to the product. Why is
this not the case with the other ATP generating step
in glycolysis?

PFK-1
F6P + ATP

F 1,6 BP + ADP

Second irreversible step of glycolysis, and the


MAJOR REGULATORY ENZYME of glycolysis
Allosteric regulation: binding of small molecules
outside the active site alters kinetic behavior of the
enzyme
Remember the roles of glycolysis:
Derive energy from glucose
Provide carbon skeletons for biosynthesis
Therefore, if energy levels are high, and there is an
adequate supply of carbon skeletons, glycolysis is
inhibited (use glucose to store energy as glycogen or
fat)

PFK-1 (continued)
Inhibitors of the enzyme are
ATP (high energy available; it is also a substrate
for the reaction)
citrate (from the Krebs tricarboxylic acid cycle, and
signifies adequate carbon skeletons for
biosynthesis)
Activators of the enzyme are
Fructose 2,6 Bisphosphate (F 2,6 BP), which links
the regulation of glycolysis to hormonal signals (in
the liver only)
AMP, indicating low energy levels

Where does F2,6 BP come from?


The reaction is F6P + ATP yields F2,6 BP + ADP
The enzyme is phosphofructokinase-2 (PFK-2) has
two activities
A kinase which creates F2,6BP
A phosphatase which converts F2,6BP to F6P + Pi
PFK-2 is in all tissues regulation between tissues
differs
Hormonal regulation in liver
Allosteric regulation in other tissues

Why AMP as an indicator of low energy


levels?
The ratio of [AMP]/[ATP] increases more
rapidly than the [ADP]/[ATP] ratio
The [AMP]/[ATP] ratio is proportional to the
square of the [ADP]/[ATP] ratio
2 ADP AMP + ATP (myokinase, adenylate
kinase)
K = ([AMP][ATP])/([ADP])2
[AMP] = ([ADP]2/[ATP])K; thus
([AMP]/[ATP] = ([ADP]/[ATP])2K
So an increase in ADP which leads to a 2:1 ADP to
ATP ratio is proportional to an AMP to ATP ratio of
4:1

Activity Profile of PFK-1


Low [ATP]

1 M F 2,6 BP

1 M F 2,6 BP

Velocity

0.1 M

0.1 M
High [ATP]

[Fructose 6-phosphate]

0
0

[Fructose 6-phosphate]

[ATP]

( M)

Velocity: the rate at which the PRODUCT of the reaction is formed


F2,6 BP: reduces substrate concentration required for the enzyme to
reach one-half maximal velocity. The maximal velocity does not change,
but the amount of substrate required to reach that velocity is decreased.

Hormonal Regulation of PFK-2 (liver)


Hormone
Cell Membrane

Receptor

ATP

PFK-2

(ACTIVE kinase)
ATP

Adenylate
Cyclase
cAMP
cAMP-Dependent
protein kinase R2C2

ADP
PFK-2-P

(INACTIVE kinase)

+
R-cAMP

1. Activators of the cAMP system tend to signal a need for energy (hormones
epinephrine and glucagon)
2. Liver stops using glucose so glucose can be exported
3. To stop the liver from using glucose, glycolysis is slowed down by reducing F 2,6
BP levels, which reduces PFK-1 activity (liver specific response to glucagon)
4. Muscle will still use glucose, as skeletal muscle PFK-2 is not phosphorylated
(different isozyme but heart is an exception)

More on PFK-2
PFK-2 is a bifunctional enzyme
Contains a kinase activity which produces fructose 2,6
bisphosphate
Contains a phosphatase activity which converts F 2,6 BP to
fructose 6-phosphate and Pi

Phosphorylation of only the liver isozyme activates the


phosphatase activity, and inhibits the kinase activity
So when glucagon is released, the phosphatase is active,
F2,6 BP levels drop, and PFK-1 activity is reduced

In skeletal muscle, F6P activates the kinase, and


inhibits the phosphatase activity the skeletal muscle
isozyme is not a substrate for protein kinase A;
additionally, skeletal muscle lacks glucagon receptors.

PFK-2 Regulation: Liver vs Muscle


Liver
Job: export glucose when blood glucose levels drop
When blood glucose levels drop glucagon is released,
the cAMP cascade is activated, PFK-2 is
phosphorylated, PFK-1 less active, glycolysis is slowed,
and glucose export can occur
Skeletal Muscle:
Job: to do work, using glucose or fat for energy
Blood glucose levels drop, muscle takes glucose from
blood (special conditions) or uses its own glucose;
PFK-2 in muscle not phosphorylated, so activity not
altered under these conditions

Heart PFK-2
Cardiac PFK-2 is regulated differently
than liver or skeletal muscle PFK-2
Will be phosphorylated by an insulinstimulated protein kinase (protein kinase
B), or an AMP-activated protein kinase
When phosphorylated its kinase activity is
active (opposite of liver PFK-2)
This allows for glycolysis to be activated under
low energy conditions (high AMP) and when
glucose levels are high (insulin release)

Regulation of HK (muscle) and GK (liver)


Glucose

As PFK-1 is inhibited (by either


ATP, or lack of F 2,6 BP), the
levels of F6P increase. This
increases the levels of G6P, which
will feedback and inhibit the
activity of HK (not liver).

ATP
HK
ADP
Glucose-6-P

Fructose-6-P
ATP

PFK-I

ADP
Fructose 1,6 BP

ATP

Glucokinase is not inhibited by


G6P; under conditions of high
blood glucose levels GK will
be active, and can force liver
glycolysis to proceed in order
to store energy as fat. GK is
sequestered in the nucleus by
a regulatory protein under low
blood glucose conditions.

Regulation of Pyruvate Kinase


Three forms
L (liver)
M1/M2 (muscle and other tissues)
R (red blood cells)
All three forms catalyze the same reaction,
but the regulation of the three forms differ
PEP + ADP gives rise to pyruvate + ATP

Pyruvate Kinase (continued)


L form
activated by compounds which indicate a high level of
glycolytic intermediates
PEP
F 1,6 BP
inhibited by compounds indicating high energy or
adequate carbon skeleton precursors
ATP
alanine
Also regulated by COVALENT MODIFICATION by the cAMP
dependent protein kinase (phosphorylation inhibits
activity)
M1 form (brain, heart, muscle) is not subject to covalent
modification nor to the allosteric controls exhibited by the L
form

Role of cAMP dependent protein kinase in


liver

Hormone

Receptor

Cell Membrane

ATP

PFK-2

(ACTIVE)
ATP

Adenylate
Cyclase
cAMP

ADP

PFK-2-P

(INACTIVE)

+
cAMP-Dependent
protein kinase R2C2

PK

R-cAMP

1. Inhibition of PFK-2 leads to an increase in


[F6P], which increases [G6P], and can lead to
glucose export
2. Above events, coupled with the inhibition of PK,
shuts off liver, but not muscle, glycolysis.
3. Liver switches to glucose export and glucose
production via gluconeogenesis.

(ACTIVE)

ATP
ADP

PK-P
(INACTIVE)

Regulation Summary (to date)


Allosteric vs covalent modification
Allosteric controls related to the ENERGY STATE of the cell
Covalent modification (phosphorylation) linked to hormonal
signals, which monitor blood glucose levels
Liver
Supply other tissues with glucose when blood glucose levels
are low
When blood glucose levels are high, convert excess glucose
to energy storage forms (glycogen or triglyceride)
Skeletal Muscle
When blood glucose levels low, use glucose from own stores,
or from the blood under special conditions
When blood glucose levels high, store as glycogen in the
muscle

Differential Regulation
Low blood glucose levels
muscle glycolysis can be active
liver glycolysis inhibited
accomplished by hormonal regulation
PFK-II (kinase activity), PK inhibited in liver
PFK-II, PK active in muscle

High blood glucose levels


glucokinase allows liver glycolysis to
continue so triglyceride and anabolic
precursors can be made
Allosteric controls still effective in muscle

Cori Cycle
Glucose

Glucose

Pyruvate

Pyruvate
NADH
NAD+

NADH
NAD+
lactate
H
CH

lactate

C C
OH OH

MUSCLE

BLOOD

LIVER

1.Forming lactate in
the muscle
regenerates NAD+,
which allows
glycolysis to
continue.
2.Carbons from
lactate, once they
reach the liver, are
used for glucose
production
3.The liver exports the
newly synthesized
glucose into the
blood for use by the
muscle.

Lactic acidosis (acidemia)


Lactate production is normal, but too
much can overcome the buffering
capacity of the blood
Increased NADH/NAD+ ratio in tissues
Hypoxia (lack of oxygen)
Genetic defects (OXPHOS diseases)
ETC problems
PDC, PC mutations leading to reduced
activity
Thiamin deficiency

Fructose Metabolism (liver)


Fructose
ATP

Fructokinase

ADP

Glycolysis

F-1-P
Aldolase
B

TPI
DHAP

G3P

Glyceraldehyde

ATP

ADP

Triose Kinase

Fructokinase very
active (liver)
Aldolase B cleaves
both F1P and F 1,6
BP; expressed
primarily in liver
High fructose load
leads to decrease in
ATP levels due to
rapid phosphorylation
HK can phosphorylate
fructose, but the Km is
very high

Essential Fructosuria
Elevated levels of fructose in the urine
Defect in fructokinase
Fructose enters the liver cell, but cannot be
phosphorylated, so it leaves and enters the
circulation
The high fructose ends up in the urine

Benign condition; levels of fructose in


urine are dependent on the diet
Positive result in reducing sugar test of
the urine

Hereditary Fructose Intolerance


Defect in Aldolase B (<15% activity)
Aldolase B; primarily in liver and kidney
Aldolase A, C in other tissues
Liver still splits F 1,6 BP with residual B activity and low
levels of aldolase A (A form will not split F1P)
F1P accumulates due to speed of fructokinase reaction
Hypoglycemia results from increased F1P
Glycogen degradation inhibited due to F1P inhibition of
glycogen phosphorylase (normal product is G1P), and
sequesteration of phosphate in F1P (the enzyme requires
Pi to work)
Gluconeogenesis impaired due to low activity of aldolase
Lactic acidosis occurs (due to inhibition of gluconeogenesis,
lack of ATP due to fructokinase reaction, and uric acid [due to
increase in AMP levels] blocking lactate uptake from the
blood into the kidney)
Patients avoid fructose and are fine

Galactose Metabolism
Galactose
ATP

Galactokinase

ADP

Gal-1-P

UDP-glucose
Gal-1P uridylyl
transferase

Glu- 1- P
Phosphoglucomutase
G6P

UDP-galactose
C4-epimerase
UDP-glucose

C4-epimerase only recognizes UDP-gal (not free gal)

Galactosemia
Elevated levels of galactose in blood; elevated levels
in urine known as galactosuria
Classical Type
Lack of gal-1P uridylyl transferase; get accumulation of
galactose and the alcohol form of galactose (galactitol), which
leads to cataract formation. The accumulation of gal 1-P in the
nervous system leads to irreversible mental retardation.
Hypoglycemia also results from galactose-1-phosphate
inhibition of phosphoglucomutase (normal substrate Glu-1-P).

Non-classical
Loss of galactokinase
Cataract formation occurs due to galactitol accumulation, but
not hypoglycemia or mental retardation.

Treat by removing lactose from the diet.

Lactose intolerance
In certain populations, the expression of lactase
decreases with age
Lactose not absorbed by intestinal epithelial cells
Bacteria in large intestine metabolize the lactose,
generating gases and acids (osmotic imbalance)

Damage to the intestinal epithelial cells can lead to


temporary lactose intolerance (viral
gastroenteritis)

Glycolysis and Diabetes


Glucose Transport
Insulin stimulates glucose transport into non-hepatic cells
(primarily muscle and adipose tissue)
Insulin recruits pre-existing transporters from the
endoplasmic reticulum to the plasma membrane (Glut4)
In the absence of insulin, glucose uptake into non-hepatic
cells is low, and it remains in circulation (contributes to the
observed hyperglycemia)
Glucose transport into the liver and brain is still high
Glycolysis in the liver
Glucagon predominates, so PFK-2 and PK are
phosphorylated and inhibited
Gluconeogenesis is stimulated and glucose is exported
Glucokinase forces glycolysis forward, liver makes fat from
glucose (due to high blood glucose levels; non-alcoholic
steatohepatitis- NASH)
Liver also metabolizes fat (liver confused)

Glucose and Diabetes


Elevated blood glucose can lead to the
following:
Nonenzymatic glycosylation of proteins (can be
measured by HbA1c content of RBC)
Leads to neuronal damage (diabetic neuropathy)

Reduction of glucose to sorbitol via aldose


reductase (requires NADPH)
Buildup of sorbitol in lens of eye leads to cataract
formation (similar to classical galactosemia and
galactitol)

Where does glucose come from?


Diet (all absorbed in intestinal lumen;
amylase from pancreas digests starch, all
other enzymes on enterocyte membrane)
Glycogen (stored glucose polymer)
Synthesis (gluconeogenesis)
Lactate (muscle and red blood cells)
Glycerol (fat cells)
Amino acids (muscle)

Gluconeogenesis
The synthesis of glucose
Need to reverse three reactions of
glycolysis
Pyruvate kinase (how can pyruvate be
converted to PEP?)
PFK-1: how can F 1,6 BP be converted to
F6P?
Hexokinase: how is G6P converted to
glucose?

Reactions to Reverse Glycolysis


Two reactions are required to reverse the pyruvate kinase
reaction.
Pyruvate Carboxylase:
O

CH 3 C

OH + ATP + CO

C
-O

CH2

2 ADP, P i

CH2

-O

OH
(Oxaloacetate)

PEP Carboxykinase:
O

Biotin

H
+ GTP
OH

CO + GDP +
2

O
C

C
O

O-

O-

(PEP)

O-

More reactions to reverse glycolysis


Fructose 1,6 Bisphosphatase:
Pi
F 1,6 BP

F6P

(this reaction reverses PFK-1)

Glucose - 6 - Phosphatase:
Pi
G6P

Glucose

(Glucose-6-phosphatase is found only


in the liver and kidney)

Glycerol to glucose
Glycerol to glycerol 3-phosphate (liver
only has the enzyme glycerol kinase)
Glycerol 3-phosphate is oxidized to
DHAP (NAD+ is converted to NADH
glycerol 3-phosphate dehydrogenase)

Compartmentation Issues
The enzymes of Gluconeogenesis are split between
the mitochondria and cytoplasm
Pyruvate carboxylase is only located in the
mitochondria
PEP carboxykinase, F 1,6 Bisphosphatase, and
glucose-6-phosphatase are cytoplasmic
Transport mechanisms exist to get pyruvate inside
the mitochondria to be converted to oxaloacetate,
and for OAA equivalents to leave the mitochondria

Regulation of Gluconeogenesis
Fructose-6-Phosphate
AMP

ATP

F2, 6BP

F2, 6BP

citrate

AMP

ATP

citrate

PFK-l

Phosphatase

F, 16BP

Reciprocal regulation; activators of PFK-1 inhibit F 1,6 bisphosphatase


such that a futile cycle is not generated.
Pyruvate carboxylase and pyruvate dehydrogenase are also
reciprocally regulated by acetyl-CoA Pyruvate carboxylase is
activated by acetyl-CoA, while pyruvate dehydrogenase is inhibited by
acetyl -CoA

Pi

Gluconeo
-genesis

Glucose

ATP

Hexokinase
Glucokinase

Glucose 6 - Phosphatase
H2O

Glucose-6-P

ADP

Fructose-6-P

ATP

Glycogen
Pi

Fructose 1, 6Bisphosphatase

Notes:
1. Enzymes in black
specific to glycolysis
2. Enzymes in red used
for gluconeogenesis
3. Lines in red reflect the
reactions required to
reverse glycolysis

PFK-1
Fructose 1,6
Bisphosphate

H2O

ADP
+

Dihydroxyacetone-P
Glyceraldehyde-3-P
NAD

H
D
A
N

Glycerol 3Phosphate
dehydrogenase
+

D
NA

Pi

NADH + H+
1,3 - Bisphosphoglycerate

Glycerol 3-P

ADP

ADP

ATP

Glycerokinase
2 - Phosphoglycerate

3-Phosphoglycerate

ATP

Glycerol
Phosphoenolpyruvate
ADP
GDP + CO2

Phosphoenolpyruvate
GTP Carboxykinase

Oxaloacetate

Pyruvate Kinase
ATP
Pyruvate

Lactate
NADH + H+ NAD+

Pyruvate
ADP
Pyruvate
Carboxylase
ATP
Oxaloacetate

Lactate
Dehydrogenase

Precursors of glucose for


gluconeogenesis
Lactate - goes to pyruvate, then OAA
Glycerol - gets converted to glycerol 3phosphate, then DHAP, then G3P, then
to G6P
Amino acids - depending on the amino
acid the carbons give rise to pyruvate,
OAA, succinyl-CoA, fumarate, ketoglutarate

Regulation Example
Glucose levels high in the blood
Insulin high, glucagon low
Low glucagon leads to low cAMP, thus protein kinase A inactive
PFK-2 kinase active (not phosphorylated), and F 2,6 BP
accumulates
F 2,6 BP stimulates PFK-1, and inhibits F 1,6 bisphosphatase,
allowing glycolysis to proceed, and to store energy as fat or to
provide biosynthetic building blocks from glycolytic intermediates

Glucose levels low in blood


Glucagon released, cAMP increases, protein kinase A active
PFK-2 phosphorylated and kinase inactive, but phosphatase
activity active
F 2,6 BP levels drop, lose inhibitor of F 1,6 bisphosphatase, and
activator of PFK-1
Gluconeogenesis favored in the liver

Summary - Gluconeogenesis
Synthesize glucose from lactate, glycerol and amino
acids under fasting and exercising conditions
(glucagon and epinephrine release stimulate liver
gluconeogenesis)
Use the reversible glycolytic enzymes, plus four
enzymes to reverse the three irreversible steps:

Pyruvate carboxylase
PEP carboxykinase
Fructose 1,6 bisphosphatase
Glucose 6 phosphatase (liver only)

Reciprocal regulation from glycolysis (PFK-1 vs


fructose 1,6 bisphosphatase and pyruvate
carboxylase vs pyruvate dehydrogenase);
transcriptional control, mediated via PKA, of PEPCK

Diabetes Revisited
Type I : no insulin is produced. Glucose has trouble
entering cells, body thinks starvation conditions exist
Type 2 : insulin levels normal, but responses to
insulin do not occur ( previously called NIDDM)
Receptor defects
Processing
Reduced Kinase Activity

Immune system defects


Type A : antibodies to insulin. No free insulin available to bind
to receptor
Type B : antibodies to insulin receptor, such that either insulin
binding is blocked, or the antibody itself is stimulatory

Obesity related diabetes (down-regulation of insulin


receptors; related to metabolic syndrome [obesity, high
glucose levels, high triglyceride levels, high LDL levels,
elevated blood pressure])