Most animals can metabolize a variety of carbon sources to generate energy

1
Gluconeogenesis - the synthesis of glucose from
non-carbohydrate precursors
Most animals can metabolize a variety of carbon
sources to generate energy (triacyglycerols,
sugars, pyruvate, amino acids) BUT the brain
and central nervous system use glucose as their
sole or primary carbon source (also kidney
medulla, testes and erythrocytes) Per day
basis _at_ 160 grams of glucose are used by the
body _at_ 120 of those 160 grams are used by the
brain _at_ 190 grams of glucose can be generated
from the bodies glycogen reserves _at_ 20 grams of
glucose are in body fluids Therefore readily
available glucose reserves in the body would last
one day Fasting for more than one day or intense
exertion would deplete glucose reserves The
process of creating new glucose is called
gluconeogenesis (from three and four carbon
precursors generally not carbohydrates)
121
2
Q Where in a cell does gluconeogenesis
occur? A In the cytosol (primarily) some
precursors are generated in the mitochondria and
must be transported to the cytosol for
utilization Q What organs does gluconeogenesis
occur in? A Liver (primarily) and kidney
cortex Note gluconeogenesis is not just used
to generate glucose for immediate catabolism or
storage as glycogen glucose is the primary
structural precursor for all other
carbohydrates (amino sugars, polysaccharides,
glycoproteins and glycolipids)
122
3
Gluconeogenesis is not a simple reversal of
glycolysis
?G0 of an overall pathway must be negative for
that pathway to proceed in the direction
written glycolysis from glucose to pyruvate
?G0 of _at_ -96 kJ/mol (exergonic) How can
pyruvate to glucose be made exergonic?

• There are three steps in glycolysis that are so
  exergonic that they are essentially irreversible
• hexokinase
• phosphofructokinase
• pyruvate kinase

In gluconeogenesis, different enzymes are used at
each of these steps so that these irreversible
reactions are bypassed by using enzymes specific
for gluconeogenesis These reactions run very
strongly in the direction of glucose synthesis
(but energy must be expended) The other seven
reactions use the same enzymes as glycolysis
(These enzymes catalyze reversible reactions
that are driven in either direction )
123
4
1 Conversion of pyruvate to phosphoenolpyruvate
(by-passing pyruvate kinase) involves
two reactions
pyruvate carboxylase catalyzes the ATP and
biotin-dependant conversion of pyruvate to
oxaloacetate in the mitochondrial matrix
this enzyme requires acetyl-CoA as an allosteric
activator
Biotin is a cofactor in most carboxylation
reactions involving CO2
Biotin is linked to an lysine residue in the
enzyme active site The first step is an ATP
dependent carboxylation to yield
N-carboxybiotin (a nitrogen atom of biotin makes
a nucleophilic attack on a bicarbonate ion) (at
the same time, an oxygen atom of bicarb attacks
the terminal phosphate of ATP displacing
Pi) Pyruvate makes a nucleophilic attack on the
activated CO2, displacing the biotinylated enzyme
forming oxacolacetate
125
5
this is one of the anapleurotic reactions used to
maintain levels of citric acid cycle
intermediates, oxaloacetate can be oxidized in
the citric acid cycle
For oxaloacetate to be used for gluconeogenesis,
it must move out of the mitochondrial matrix
124
6
For oxaloacetate to be used for gluconeogenesis,
it must move out of the mitochondrial matrix
The mitochondrial membrane does not have an
effective transporter for oxaloacetate Therefore
oxaloacetate is reduced by mitochondrial malate
dehydrogenase to malate
malate is transported into the cytosol in
exchange for orthophosphate in the cytosol,
malate is reoxidized to oxaloacetate by malate
dehydrogenase phosphoenolpyruvate carboxykinase
(PEPCK) uses GTP to convert oxaloacetate to
phosphoenolpyruvate by phosphorylation
126
7
1 Conversion of pyruvate to phosphoenolpyruvate
(by-passing pyruvate kinase) involves
two reactions
In the cytosol, oxaloacetate is converted to
phosphoenolpyruvate by phosphoenolpyruvate
carboxykinase (PEPCK)
The CO2 incorporated by the pyruvate carboxylase
reaction is lost here as CO2 therefore no net
fixation of C02 occurs The decarboxylation
leads to a rearrangement of electrons that
facilitates attack of the carbonyl oxygen of the
pyruvate moiety on the g phosphate of GTP
127
8
There are cytosolic and mitochondrial forms of
PEPCK
Why have two pathways to generate oxaloacetate
from pyruvate?
128
9
Summary reaction one of gluconeogenesis
the ?G0 for the two reactions combined is
slightly positive under intracellular conditions
however the ?G -25 kJ/mol Bottom line of this
reaction two high-energy phosphate were invested
to generate one super-high-energy
phosphoenolpyruvate In the next series of
reactions phosphoenolpyruvate is converted to
fructose-1,6-bisphosphate by glycolytic enzymes
acting in reverse
129
10
2 Conversion of fructose-1,6-bisphosphate to
fructose-6-phosphate (which bypasses
phosphofructokinase)
the phosphofructokinase reaction in glycolysis is
essentially irreversible because it is driven by
a phosphate transfer from ATP the bypass
reaction is a simple hydrolytic cleavage,
catalyzed by fructose-1,6-bisphosphatase the
enzyme requires Mg2 this is one of the major
control sites regulating gluconeogenesis
the next reaction involves isomerization of
fructose-6-phosphate to glucose-6-phosphate in a
reaction catalyzed by phosphoglucoisomerase
3 Conversion of glucose-6-phosphate to glucose
(by passing hexokinase and glucokinase)

• the hexokinase reaction is another essentially
  irreversible phosphate transfer
• the bypass reaction is again a simple hydrolysis
  catalyzed by glucose-6-phosphatase
• this enzyme also requires Mg2

1210
11
overall conversion of 2 moles of pyruvate to 1
mole of glucose is exergonic _at_ -47.6 kJ/mol but
the synthesis of glucose costs the cell energy
6 high energy phosphate groups are consumed (four
ATPs, 2 GTPs) 2 moles of NADH are also
consumed (if this NADH were used in oxidative
phosphorylation then _at_ 5 moles of ATP would be
produced) But if you followed glycolysis in the
reverse direction without making the changes in
gluconeogenesis the ?G0 would be 73.3
kJ/mol Therefore the investment of the extra
four high energy phosphate bonds is necessary for
the net synthesis of glucose to occur as an
irreversible process
1211
12
1212
13
Lactate quantitatively, the most significant
gluconeogenic precursor
Where does the lactate used for gluconeogenesis
come from? produced primarily from glycolysis in
skeletal muscle and erythrocytes
In muscle cells, when oxygen is insufficient, the
pyruvate generated by glycolysis cannot be
further metabolized by the pyruvate dehydrogenase
complex lactate dehydrogenase reduces pyruvate
to lactate which is released into the blood from
the bloodstream lactate has a number of fates

• used by the heart and oxidized as fuel
• re-oxidized in the liver to pyruvate (which can
  then undergoe gluconeogenesis to glucose and
  returned to the bloodstream where muscle cells
  can convert the glucose to glycogen Cori-cycle

1213
14
Amino acids as gluconeogenic precursors
generated from dietary protein or from the
breakdown of muscle protein during starvation
pyruvate in peripheral tissue can be
transaminated to alanine and the alanine returned
to the liver for use in gluconeogenesis
glucose-alanine cycle (discussed later in course)
Glucogenic (gluconeogenic) amino acids amino
acids besides alanine can be converted to
glucose (generally through degradative pathways
that generate citric acid cycle intermediates)
when insufficient carbohydrate is ingested in the
diet, the catabolism of muscle protein is the
major source of intermediates for maintenance of
normal blood glucose levels (this is also the
case in diabetes mellitus)
1214
15
Glucogenic amino acids
1215
16
Glycerol as a gluconeogenic precursors
Glycerol in general a poor gluconeogenic
precursor catabolism of triacylglyerols yields
fatty acids and glycerol fatty acids undergo
?-oxidation to yield acetyl-CoA in plants and
bacteria, acetyl-CoA can be incorporated into
carbohydrate via the glyoxylate cycle in animals
acetyl-CoA cannot be converted to pyruvate or any
other gluconeogenic precursor
remember although two-carbon units from
acetyl-CoA can proceed to oxaloacetate in the
citric acid cycle there is no net conversion
because two carbon atoms are lost in each turn of
the cycle
The only fat breakdown product that can enter
gluconeogenesis is glycerol (although the
exception is odd-chain fatty acids)
1216
17
Glycerol enters gluconeogenesis At the level of
DHAP
note the fatty acids released during lipid
breakdown are mostly converted to acetyl-CoA and
cannot be used for net carbohydrate synthesis
(except in organisms that have a functioning
glyoxylate cycle)
1217
18
Ethanol consumption and gluconeogenesis
Ethanol consumption elevates the NADH/NAD ration
in liver cytosol (and glycolysis is inhibited
because the glyceraldehyde 3 phosphate
dehydrogenase reaction needs NAD Lactate
dehydrogenase is also inhibited
Shifts the equilibrium of cytosolic malate
dehydrogenase and cytosolic oxaloacetate tends
to be reduced to malate Oxaloacetate is
therefore unavailable for gluconeogenesis This
can result in hypoglycemia and hypoglycemia can
lower body temperature
If youre cold and wet and starving and you drink
alcohol the feeling of warmth is due to
vasodilation, which causes further heat loss
from a metabolic standpoint, glucose would be
more effective at raising temperature
1218
19
Regulation of gluconeogenesis
gluconeogenesis and glycolysis are controlled in
a reciprocal fashion (what activates one pathway
tends to inhibit the other) ATP vs ADP levels
important (also AMP) Q Would the rate
controlling steps in glycolysis be activated or
inhibited by high levels of ADP? Q Would the
rate controlling steps in gluconeogenesis be
activated or inhibited by high levels of
ADP? Fructose-2,6-bisphosphate is the most
important regulator of gluconeogeneis (figure
16.7 )
1219
20
Glucose homeostasis
An increase in glucose in the bloodstream
following a carbohydrate rich meal stimulates the
secretion of insulin by the pancreas Insulin
binds to the insulin receptor and results in a
variety of signals transmitted to the cell Among
those signals are the recruitment of glucose
transport proteins from the cytosol to the
membrane in muscle cells
This results in an increase in the amount of
glucose entering a cell As a result, blood
glucose levels fall to normal, slowing insulin
release from the pancrease
1220
21
Glucose homeostasis
1221
22
Fructose 2,6-bisphosphate is an allosteric
regulator of glycolysis and gluconeogenesis
Although structurally related to fructose
1,6-bisphosphate , fructose 2,6-bisphosphate is
NOT an intermediate in gluconeogenesis or
glycolysis It is a regulator whose cellular
level reflects the level of glucagon in the blood
(which rises when blood glucose falls) Glucagon
lowers the level of fructose 2,6-bisphosphate
slowing the consumption of glucose by glycolysis
and stimulating the production of glucose by
gluconeogeneisis The liver releases the glucose
into the blood
Increased levels of fructose 2,6-bisphosphate
result in its binding to its allosteric site on
PFK-1 where it increases that enzymes affinity
for its substrate fructose 6-phosphate and
reduces its affinity for the allosteric
inhibitors ATP and citrate PFK-1 one
is therefore activated and glycolysis is
stimulated At the same time fructose
2,6-bisphosphate inhibits FBPase-1, thereby
slowing gluconeogenesis
1222
23
Fructose 2,6-bisphosphate is formed by
phosphorylation of fructose 6-phosphate via
phosphofructokinase-2
Broken down by dephosphorylation by fructose
2,6-bisphosphatase-2 (note that these enzymes
are distinct from PFK-1 and FBPase-1 which
catalyze the formation and breakdown of fructose
1,6 bisphosphate)
1223