Download 16 Gluconeogenesis

CHAPTER
16
Gluconeogenesis
16.1 Glucose Can Be Synthesized from
Noncarbohydrate Precursors
16.2 Gluconeogenesis and Glycolysis
Are Reciprocally Regulated
16.3 Metabolism in Context:
Precursors Formed by Muscle Are
Used by Other Organs
16.4 Glycolysis and Gluconeogenesis
Are Evolutionarily Intertwined
Fasting is a part of many cultures and religions, including those of the Teton Sioux.
Fasting is believed to cleanse the body and soul and to foster spiritual awakening.
Gluconeogenesis is an important metabolic pathway during times of fasting
because it supplies glucose to the brain and red blood cells, tissues that depend
on this vital fuel. [Edward S. Curtis Collection, ”Fasting Indians,”Library of Congress.]
W
e now turn to the synthesis of glucose from noncarbohydrate precursors, a
process called gluconeogenesis. Maintaining levels of glucose is important
because the brain depends on glucose as its primary fuel and red blood cells use
glucose as their only fuel. The daily glucose requirement of the brain in a typical
adult human being is about 120 g, which accounts for most of the 160 g of glucose
needed daily by the whole body. The amount of glucose present in body fluids is
about 20 g, and that readily available from glycogen, the storage form of glucose,
is approximately 190 g. Thus, the direct glucose reserves are sufficient to meet
glucose needs for about a day. Gluconeogenesis is especially important during a
longer period of fasting or starvation.
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16 Gluconeogenesis
The major site of gluconeogenesis is the liver, with a small amount also taking place in the kidney. Little gluconeogenesis takes place in the brain, skeletal
muscle, or heart muscle. Rather, gluconeogenesis in the liver and kidney helps to
maintain the glucose level in the blood where it can be extracted by the brain and
muscle to meet their metabolic demands.
In this chapter, we begin by examining the reactions that constitute the gluconeogenic pathway. We then investigate the reciprocal regulation of gluconeogenesis and glycolysis, and end the chapter with a look at how gluconeogenesis and
glycolysis are coordinated between tissues.
16.1 Glucose Can Be Synthesized
from Noncarbohydrate Precursors
Although glucose is usually available in the environment of most organisms, this
molecule is so important biochemically that a pathway exists in virtually all forms of
life to synthesize it from simple precursors. The gluconeogenic pathway converts pyruvate into glucose. Noncarbohydrate precursors of glucose are first converted into
pyruvate or enter the pathway at later intermediates (Figure 16.1). The major noncarbohydrate precursors are lactate, amino acids, and glycerol. Lactate is formed by
active skeletal muscle through lactic acid fermentation when the rate of glycolysis
exceeds the rate at which muscle can process pyruvate aerobically (p. 235). Lactate is
readily converted into pyruvate in the liver by the action of lactate dehydrogenase.
Amino acids are derived from proteins in the diet and, during starvation, from the
breakdown of proteins in skeletal muscle (p. 483). The hydrolysis of triacylglycerols
(p. 404) in fat cells yields glycerol and fatty acids. Glycerol is a precursor of glucose,
but animals cannot convert fatty acids into glucose, for reasons that will be given later
(p. 413). Unlike lactate and amino acids, glycerol can be metabolized by glycolysis and
can be converted into glucose by gluconeogenesis. Glycerol may enter either the gluconeogenic or the glycolytic pathway at dihydroxyacetone phosphate.
CH2OH
HO
C
H
CH2OH
ATP
ADP
+ H+
Glycerol
kinase
CH2OH
HO
C
H
CH2OPO32–
NAD+
Glycerol
phosphate
dehydrogenase
Glycerol
phosphate
Glycerol
NADH
+ H+
CH2OH
O
C
CH2OPO32–
Dihydroxyacetone
phosphate
Gluconeogenesis Is Not a Reversal of Glycolysis
In glycolysis, glucose is converted into pyruvate; in gluconeogenesis, pyruvate is
converted into glucose. However, gluconeogenesis is not a reversal of glycolysis. Several reactions must differ because the equilibrium of glycolysis lies far on the side
of pyruvate formation. The actual ⌬G for the formation of pyruvate from glucose
is about ⫺84 kJ mol⫺1 (⫺20 kcal mol⫺1) under typical cellular conditions. Most
of the decrease in free energy in glycolysis takes place in the three essentially irreversible steps catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.
Hexokinase
Glucose + ATP 9999: glucose 6-phosphate + ADP
¢G = - 33 kJ mol - 1 ( - 8.0 kcal mol - 1)
Phosphofructokinase
Fructose 6-phosphate + ATP 999999:
fructose 1,6-bisphosphate + ADP
¢G = - 22 kJ mol - 1 ( - 5.3 kcal mol - 1)
Pyruvate kinase
Phosphoenolpyruvate + ADP 99999: pyruvate + ATP
¢G = - 17 kJ mol - 1 ( - 4.0 kcal mol - 1)
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16.1 Gluconeogenesis
CH2OH
O
Glucose
OH
Glucose
6-phosphatase
OH
HO
Pi
OH
CH2OPO32–
H2O
O
Glucose 6-phosphate
OH
HO
Phosphoglucose
isomerase
OH
2–O
3POH2C
O
Fructose 6-phosphate
OH
CH2OH
HO
OH
Pi
Fructose
1, 6-bisphosphatase
HO
2– O
3POH2C
H2O
CH2OPO32–
O
HO
Fructose 1,6-bisphosphate
Glycerol
OH
OH
Aldolase
Dihydroxyacetone
phosphate
Triose phosphate
isomerase
Glyceraldehyde
3-phosphate
CH2OH
O
C
CH2OPO32–
Glyceraldehyde
3-phosphate
dehydrogenase
H
NADH
2–
C
H
C
H
2-Phosphoglycerate
H
OH
OPO32–
C
CH2OH
H2O
Phosphoenolpyruvate
O
–
GTP
O
C
C
H
–
H
O
H2
C
C
O
C
ADP + Pi
ATP, HCO3–
OPO32–
C
GDP, CO2
Oxaloacetate
Pyruvate
C
CH2OPO32–
O – O
C
Phosphoglycerate
mutase
Lactate
Some amino acids
OH
CH2OPO32–
O – O
C
3-Phosphoglycerate
Pyruvate
carboxylase
O
O3PO
ATP
Some amino acids
OH
CH2OPO32–
ADP
Phosphoenolpyruvate
carboxykinase
C
Pi, NAD
Phosphoglycerate
kinase
Enolase
C
+
1,3-Bisphosphoglycerate
2X
O
H
O
–
O
O
C
O
O
C
O
C
CH3
–
Figure 16.1 The pathway of
gluconeogenesis. The distinctive
reactions and enzymes of this pathway
are shown in red. The other reactions
are common to glycolysis. The enzymes
for gluconeogenesis are located in the
cytoplasm, except for pyruvate
carboxylase (in the mitochondria) and
glucose 6-phosphatase (membrane
bound in the endoplasmic reticulum).
The entry points for lactate, glycerol,
and amino acidsp are shown.
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16 Gluconeogenesis
In gluconeogenesis, the following new steps bypass these virtually irreversible
reactions of glycolysis:
1. Phosphoenolpyruvate is formed from pyruvate by way of oxaloacetate through the
action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase.
Pyruvate carboxylase
Pyruvate + CO2 + ATP + H2O 9999999:
oxaloacetate + ADP + Pi + 2 H +
Phosphoenolpyruvate carboxykinase
Oxaloacetate + GTP 99999999999:
phosphoenolpyruvate + GDP + CO2
2. Fructose 6-phosphate is formed from fructose 1,6-bisphosphate by the hydrolysis of the phosphate. Fructose 1,6-bisphosphatase catalyzes this exergonic
hydrolysis.
Fructose 1,6-bisphosphate + H2O ¡ fructose 6-phosphate + Pi
3. Glucose is formed by the hydrolysis of glucose 6-phosphate in a reaction catalyzed
by glucose 6-phosphatase.
Glucose 6-phosphate + H2O ¡ glucose + Pi
We will examine each of these steps in turn.
The Conversion of Pyruvate into Phosphoenolpyruvate
Begins with the Formation of Oxaloacetate
The first step in gluconeogenesis takes place inside the mitochondria. In this step,
pyruvate is carboxylated by pyruvate carboxylase to form oxaloacetate at the
expense of a molecule of ATP.
COO–
O
O
C
Pyruvate
carboxylase
–
O + CO2 + ATP + H2O
C
C
O
H
C
H + ADP + Pi + 2H+
COO–
CH3
Pyruvate
Oxaloacetate
Then, oxaloacetate is decarboxylated and phosphorylated by phosphoenolpyruvate carboxylase to yield phosphoenolpyruvate, at the expense of the high
phosphoryl-transfer potential of GTP.
COO–
O
Phosphoenolpyruvate
carboxylase
C
H
C
H + GTP
O
–
O
+ GDP + CO2
C
COO–
C
H
Oxaloacetate
OPO32–
C
H
Phosphoenolpyruvate
The sum of these reactions is
Pyruvate + ATP + GTP + H2O ¡
phosphoenolpyruvate + ADP + GDP + Pi + 2 H +
(B)
(A)
Activated
CO2
Lysine
Biotin
Lysine
Figure 16.2 The structure of carboxybiotin. (A) Biotin is shown with CO2 attached.
(B) The biotin-binding domain of pyruvate carboxylase shows that biotin is on a
flexible tether, allowing it to move between the ATP-bicarbonate site and the pyruvate site.
[(B) Drawn from 1BDO.pdb.]
Pyruvate carboxylase requires the vitamin cofactor biotin, a covalently attached
prosthetic group that serves as a carrier of activated CO2 in that it readily donates
the CO2 to other molecules. The carboxylate group of biotin is linked to the side
chain of a specific lysine residue by an amide bond (Figure 16.2). Note that biotin
is attached to pyruvate carboxylase by a long, flexible chain.
The carboxylation of pyruvate takes place in three stages:
HCO3- + ATP Δ HOCO2-PO32- + ADP
Biotin A vitamin used in CO2 transfer and
carboxylation reactions. Biotin deficiency is
characterized by muscle pain, lethargy,
anorexia, and depression. This vitamin is
synthesized by microflora in the intestinal
tract and can be obtained in the diet from
liver, soybeans, nuts, and many other
sources.
Biotin–enzyme + HOCO2-PO32- Δ CO2 - biotin–enzyme + Pi
CO2 - biotin–enzyme + pyruvate Δ biotin–enzyme + oxaloacetate
In aqueous solutions, CO2 exists primarily as HCO3⫺, which is activated to
carboxyphosphate by the addition of a phosphate group from ATP. This activated
CO2 is subsequently bonded to the biotin ring attached to pyruvate carboxylase
to form the carboxybiotin–enzyme intermediate (see Figure 16.2). However, this
step can take place only in the presence of acetyl CoA. Biotin is not carboxylated
unless acetyl CoA is bound to the enzyme. The allosteric activation of pyruvate carboxylase by acetyl CoA is an important physiological control mechanism that
will be discussed on page 289. The CO2 attached to biotin is activated. The ⌬G°⬘
for its cleavage
[Charles Brutlag/FeaturesPics.]
CO2 - biotin–enzyme + H + ¡ CO2 + biotin- enzyme
is ⫺20 kJ mol⫺1 (⫺4.7 kcal mol⫺1). This negative ⌬G°⬘ indicates that carboxybiotin is able to transfer CO2 to acceptors without the input of additional free
energy.
The long, flexible link between biotin and the enzyme enables the carboxybiotin to rotate from one active site of the enzyme (the ATP-bicarbonate site) to the
other (the pyruvate site). The activated carboxyl group is then transferred from
carboxybiotin to pyruvate to form oxaloacetate.
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Oxaloacetate Is Shuttled into the Cytoplasm
and Converted into Phosphoenolpyruvate
Cytoplasm
Matrix
Pyruvate
CO2 + ATP
ADP + Pi
Oxaloacetate
NADH + H+
NAD+
Malate
Malate
NAD+
Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of
gluconeogenesis are present primarily in the cytoplasm. Oxaloacetate, the product of the pyruvate carboxylase reaction, must thus be transported to the cytoplasm to complete the pathway. Oxaloacetate is transported from a
mitochondrion in the form of malate: oxaloacetate is reduced to malate inside the
mitochondrion by an NADH-linked malate dehydrogenase. After malate has been
transported across the mitochondrial membrane, it is reoxidized to oxaloacetate
by an NAD⫹-linked malate dehydrogenase in the cytoplasm (Figure 16.3). The
formation of oxaloacetate from malate also provides NADH for use in subsequent
steps in gluconeogenesis. Finally, oxaloacetate is simultaneously decarboxylated
and phosphorylated by phosphoenolpyruvate carboxykinase to generate phosphoenolpyruvate. The phosphoryl donor is GTP. The CO2 that was added to pyruvate
by pyruvate carboxylase comes off in this step.
Why are a carboxylation and a decarboxylation required to form phosphoenolpyruvate from pyruvate? Recall that, in glycolysis, the presence of a phosphoryl group traps the unstable enol isomer of pyruvate as phosphoenolpyruvate
(p. 233). However, the addition of a phosphoryl group to pyruvate is a highly
unfavorable reaction: the ⌬G°⬘ of the reverse of the glycolytic reaction catalyzed
by pyruvate kinase is ⫹31 kJ mol⫺1 (⫹7.5 kcal mol⫺1). In gluconeogenesis, the
use of the carboxylation and decarboxylation steps results in a much more favorable ⌬G°⬘. The formation of phosphoenolpyruvate from pyruvate in the gluconeogenic pathway has a ⌬G°⬘ of ⫹0.8 kJ mol⫺1 (⫹0.2 kcal mol⫺1). Instead, a
molecule of ATP is used to power the addition of a molecule of CO2 to pyruvate
in the carboxylation step. That molecule of CO2 is then removed from oxaloacetate to power the formation of phosphoenolpyruvate in the decarboxylation step.
Decarboxylations often drive reactions that are otherwise highly endergonic.
NADH + H+
Oxaloacetate
Figure 16.3 Compartmental cooperation.
Oxaloacetate used in the cytoplasm for
gluconeogenesis is formed in the
mitochondrial matrix by the carboxylation
of pyruvate. Oxaloacetate leaves the
mitochondrion by a specific transport
system (not shown) in the form of malate,
which is reoxidized to oxaloacetate in the
cytoplasm.
The Conversion of Fructose 1,6-bisphosphate into Fructose 6-phosphate
and Orthophosphate Is an Irreversible Step
On formation, phosphoenolpyruvate is metabolized by the enzymes of glycolysis
but in the reverse direction. These reactions are near equilibrium under intracellular conditions; so, when conditions favor gluconeogenesis, the reverse reactions
will take place until the next irreversible step is reached. This step is the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pi.
2–O
3POH2C
O
CH2OPO32–
+ H2O
HO
Fructose
1,6-bisphosphatase
2–O
3POH2C
O
HO
OH
OH
Fructose 1,6-bisphosphate
CH2OH
+ Pi
OH
OH
Fructose 6-phosphate
The enzyme responsible for this step is fructose 1,6-bisphosphatase. Like its glycolytic counterpart, it is an allosteric enzyme that participates in regulation—in
this case, of gluconeogenesis. We will return to its regulatory properties later in
the chapter.
The Generation of Free Glucose Is an Important Control Point
The fructose 6-phosphate generated by fructose 1,6-bisphosphatase is readily
converted into glucose 6-phosphate. In most tissues, gluconeogenesis ends here.
Free glucose is not generated; rather glucose 6-phosphate is commonly converted
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Ca2+-binding protein
16.1 Gluconeogenesis
Cytoplasmic side
Glucose 6phosphatase
H2O +
glucose
6-phosphate
Pi + glucose
ER lumen
Figure 16.4 The generation of glucose from glucose 6-phosphate. Several endoplasmic
reticulum (ER) proteins play a role in the generation of glucose from glucose 6-phosphate.
One transporter brings glucose 6-phosphate into the lumen of the ER, whereas separate
transporters carry Pi and glucose back into the cytoplasm. Glucose 6-phosphatase is
stabilized by a Ca2⫹-binding protein. [After A. Buchell and I. D. Waddel. Biochem. Biophys. Acta
1092:129–137, 1991.]
into glycogen, the storage form of glucose. The final step in the generation of free
glucose takes place primarily in the liver, a tissue whose metabolic duty is to
maintain adequate levels of glucose in the blood for use by other tissues. Free glucose is not formed in the cytoplasm. Rather, glucose 6-phosphate is transported
into the lumen of the endoplasmic reticulum, where it is hydrolyzed to glucose
by glucose 6-phosphatase, which is bound to the ER membrane (Figure 16.4).
Glucose and Pi are then shuttled back to the cytoplasm by a pair of transporters.
Six High-Transfer-Potential Phosphoryl Groups
Are Spent in Synthesizing Glucose from Pyruvate
As we have seen in the preceding sets of reactions, the formation of glucose from
pyruvate is energetically unfavorable unless it is coupled to reactions that are
favorable. Compare the stoichiometry of gluconeogenesis with that of the reverse
of glycolysis. The stoichiometry of gluconeogenesis is
Pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H2O ¡
glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD + + 2 H +
¢G°¿ = - 38 kJ mol - 1 ( - 9 kcal mol - 1)
In contrast, the stoichiometry for the reversal of glycolysis is
Pyruvate + 2 ATP + 2 NADH + 2 H2O ¡
glucose + 2 ADP + 2 Pi + 2 NAD + + 2 H +
¢G°¿ = + 84 kJ mol - 1 ( + 20 kcal mol - 1)
Note that six nucleoside triphosphate molecules are hydrolyzed to synthesize glucose from pyruvate in gluconeogenesis, whereas only two molecules of ATP are
generated in glycolysis in the conversion of glucose into pyruvate. Thus, the extra
cost of gluconeogenesis is four high-phosphoryl-transfer-potential molecules for
each molecule of glucose synthesized from pyruvate. The four additional highphosphoryl-transfer-potential molecules are needed to turn an energetically unfavorable process (the reversal of glycolysis) into a favorable one (gluconeogenesis).
Here, we have a clear example of the coupling of reactions: NTP hydrolysis is used
to power an energetically unfavorable reaction.
QUICK QUIZ 1 What barrier
prevents glycolysis from simply
running in reverse to synthesize glucose?
How is this barrier overcome in
gluconeogenesis?
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16 Gluconeogenesis
16.2 Gluconeogenesis and Glycolysis
Are Reciprocally Regulated
Gluconeogenesis and glycolysis are coordinated so that, within a cell, one
pathway is relatively inactive while the other is highly active. If both sets of reactions were highly active at the same time, the net result would be the hydrolysis
of four nucleoside triphosphates (two ATP molecules plus two GTP molecules)
per reaction cycle. Both glycolysis and gluconeogenesis are highly exergonic
under cellular conditions, and so there is no thermodynamic barrier to such
simultaneous activity. However, the activities of the distinctive enzymes of each
pathway are controlled so that both pathways are not highly active at the same
time. The rate of glycolysis is also determined by the concentration of glucose,
and the rate of gluconeogenesis by the concentrations of lactate and other precursors of glucose. The basic premise of the reciprocal regulation is that, when
glucose is abundant, glycolysis will predominate. When glucose is scarce, gluconeogenesis will take over.
Energy Charge Determines Whether Glycolysis
or Gluconeogenesis Will Be Most Active
The first important regulation site in the gluconeogenesis pathway is the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate (Figure 16.5).
Consider first a situation in which energy is needed. In this case, the concentration of AMP is high. Under this condition, AMP stimulates phosphofructokinase
Glucose
GLYCOLYSIS
GLUCONEOGENESIS
Fructose 6-phosphate
F-2,6-BP +
AMP +
ATP −
Citrate −
Phosphofructokinase
Fructose
1, 6-bisphosphatase
− F-2,6-BP
− AMP
+ Citrate
H+ −
Fructose 1,6-bisphosphate
Several steps
Phosphoenolpyruvate
F-1,6-BP +
ATP −
Phosphoenolpyruvate
carboxykinase
Pyruvate
kinase
− ADP
Oxaloacetate
Alanine −
Pyruvate
carboxylase
Pyruvate
+ Acetyl CoA
− ADP
Figure 16.5 The reciprocal regulation of gluconeogenesis and glycolysis in the liver. The
level of fructose 2,6-bisphosphate (F-2,6-BP) is high in the fed state and low in starvation.
Another important control is the inhibition of pyruvate kinase by phosphorylation during
starvation.
but inhibits fructose 1,6-bisphosphatase. Thus, glycolysis is turned on and
gluconeogenesis is inhibited. Conversely, high levels of ATP and citrate indicate
that the energy charge is high and that biosynthetic intermediates are abundant.
ATP and citrate inhibit phosphofructokinase, whereas citrate activates fructose
1,6-bisphosphatase. Under these conditions, glycolysis is nearly switched off and
gluconeogenesis is promoted. Why does citrate take part in this regulatory
scheme? As we will see in Chapter 18, citrate reports on the status of the citric
acid cycle, the primary pathway for oxidizing fuels in the presence of oxygen.
High levels of citrate indicate an energy-rich situation and the presence of
precursors for biosynthesis.
Glycolysis and gluconeogenesis are also reciprocally regulated at the interconversion of phosphoenolpyruvate and pyruvate in the liver. The glycolytic
enzyme pyruvate kinase is inhibited by allosteric effectors ATP and alanine,
which signal that the energy charge is high and that building blocks are abundant. Conversely, pyruvate carboxylase, which catalyzes the first step in gluconeogenesis from pyruvate, is inhibited by ADP. Likewise, ADP inhibits
phosphoenolpyruvate carboxykinase. Pyruvate carboxylase is activated by acetyl
CoA, which, like citrate, indicates that the citric acid cycle is producing energy
and biosynthetic intermediates (Chapter 18). Hence, gluconeogenesis is favored
when the cell is rich in biosynthetic precursors and ATP.
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16.2 Regulation of Glycolysis and
Gluconeogenesis
QUICK QUIZ 2 What are the
regulatory means that prevent high
levels of activity in glycolysis and
gluconeogenesis simultaneously? What
would be the result if both pathways
functioned rapidly at the same time?
The Balance Between Glycolysis and Gluconeogenesis
in the Liver Is Sensitive to Blood-Glucose Concentration
In the liver, rates of glycolysis and gluconeogenesis are adjusted to maintain
blood-glucose levels. Recall that fructose 2,6-bisphosphate is a potent activator
of phosphofructokinase (PFK), the primary regulatory step in glycolysis (p. 241).
Fructose 2,6-bisphosphate is also an inhibitor of fructose 1,6-bisphosphatase.
When blood glucose is low, fructose 2,6-bisphosphate loses a phosphoryl group
to form fructose 6-phosphate, which no longer binds to PFK. How is the concentration of fructose 2,6-bisphosphate controlled to rise and fall with bloodglucose levels? Two enzymes regulate the concentration of this molecule: one
phosphorylates fructose 6-phosphate and the other dephosphorylates fructose
2,6-bisphosphate. Fructose 2,6-bisphosphate is formed from fructose 6-phosphate
in a reaction catalyzed by phosphofructokinase 2 (PFK2), a different enzyme from
phosphofructokinase. In the reverse direction, fructose 6-phosphate is formed
through hydrolysis of fructose 2,6-bisphosphate by a specific phosphatase, fructose
bisphosphatase 2 (FBPase2). The striking finding is that both PFK2 and FBPase2 are
present in a single 55-kd polypeptide chain (Figure 16.6). This bifunctional enzyme
contains an N-terminal regulatory domain, followed by a kinase domain and a
phosphatase domain.
Kinase domain
Regulatory
domain
Phosphatase domain
Figure 16.6 The domain structure
of the bifunctional regulatory
enzyme phosphofructokinase 2/fructose
2,6-bisphosphatase. The kinase domain
(purple) is fused to the phosphatase
domain (red). The bar represents the
amino acid sequence of the enzyme.
[Drawn from 1BIF.pdb.]
Glucagon stimulates PKA
when blood glucose is scarce.
FBPase 2 is activcated.
Glycolysis is inhibited, and
gluconeogenesis is stimulated.
GLUCOSE ABUNDANT
(glycolysis active)
Fructose 2,6-bisphosphate
(stimulates PFK)
GLUCOSE SCARCE
(glycolysis inactive)
Protein kinase A
ADP
ATP
PFK2
Pi
ADP
FBPase2
PFK2
Fructose 6-phosphate
(no PFK stimulation)
FBPase2
Pi
PFK
more active
ATP
Pi
Fructose 6-phosphate
+
H2O
Phosphoprotein
phosphatase
H2O
Fructose
2,6-bisphosphate
High levels of fructose 6-phosphate
stimulate phosphoprotein phosphatase.
PFK2 is activated.
Glycolysis is stimulated, and
gluconeogenesis is inhibited.
Figure 16.7 Control of the synthesis and
degradation of fructose 2,6-bisphosphate.
A low blood-glucose level as signaled by
glucagon leads to the phosphorylation of
the bifunctional enzyme and, hence, to a
lower level of fructose 2,6-bisphosphate,
slowing glycolysis. High levels of fructose
6-phosphate accelerate the formation of
fructose 2,6-bisphosphate by facilitating
the dephosphorylation of the bifunctional
enzyme.
What controls whether PFK2 or FBPase2 dominates the bifunctional
enzyme’s activities in the liver? The activities of PFK2 and FBPase2 are reciprocally controlled by the phosphorylation of a single serine residue. When glucose is
scarce, as it is during a night’s fast, a rise in the blood level of the hormone
glucagon triggers a cyclic AMP signal cascade (p. 177), leading to the phosphorylation of this bifunctional enzyme by protein kinase A (Figure 16.7). This covalent modification activates FBPase2 and inhibits PFK2, lowering the level of
F-2,6-BP. Gluconeogenesis predominates. Glucose formed by the liver under these
conditions is essential for the viability of the brain. Glucagon stimulation of
protein kinase A also inactivates pyruvate kinase in the liver (p. 244).
Conversely, when blood-glucose is abundant, as it is after a meal, glucagon concentration in the blood falls and insulin levels in the blood rise. Now,
gluconeogenesis is not needed and the phosphoryl group is removed from the bifunctional enzyme. This covalent modification activates PFK2 and inhibits FBPase2. The
resulting rise in the level of F-2,6-BP accelerates glycolysis. The coordinated control of
glycolysis and gluconeogenesis is facilitated by the location of the kinase and phosphatase domains on the same polypeptide chain as the regulatory domain.
Clinical Insight
Insulin Fails to Inhibit Gluconeogenesis in Type 2 Diabetes
Insulin, the hormone that signifies the presence of fuels in the blood or the fed
state, normally inhibits gluconeogenesis. When glucose is present in the blood
after a meal, as signaled by the appearance of insulin, there is no need to synthesize glucose. Recall from Chapter 12 that insulin normally starts a signaling cascade that increases the expression of genes that encode the proteins of glycolysis.
Insulin also turns off the gene that encodes phosphenolpyruvate carboxykinase
(PEPCK), thereby inhibiting gluconeogenesis. However, in type 2 diabetes (or
non-insulin-dependent diabetes), insulin, although present, fails to inhibit the
expression of the gene that encodes PEPCK and other genes of gluconeogenesis.
This metabolic circumstance is called insulin resistance and is the defining feature
of type 2 diabetes. The higher-than-normal concentration of PEPCK results in an
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increased output of glucose by the liver even when glucose
from the diet is present. Blood glucose rises to abnormally
high levels (hyperglycemia). High levels of blood glucose
result in excessive thirst, frequent urination, blurred vision,
fatigue, and frequent or slow-healing infections. The cause of
type 2 diabetes, the most common metabolic disease in the
world, is unknown, although obesity may be a contributing
factor. Untreated, type 2 diabetes can progress to type 1, or
insulin-dependent, diabetes. The treatment of type 2 diabetes
includes weight loss, a healthy diet, exercise, and drug treatment to enhance sensitivity to insulin (Figure 16.8). ■
Substrate Cycles Amplify Metabolic Signals
A pair of reactions such as the phosphorylation of fructose 6Figure 16.8 Diet can help to prevent the development of type 2
phosphate to fructose 1,6-bisphosphate in the glycolytic pathdiabetes. A healthy diet, one rich in fruits and vegetables, is an
way and its hydrolysis back to fructose 6-phosphate in the
important step in preventing or treating type 2 diabetes.
gluconeogenic pathway is called a substrate cycle. As already
[Photodisc/Getty Images.]
mentioned, both reactions are not simultaneously fully active
in most cells, because of reciprocal allosteric controls. However, the results of isoATP
ADP
tope-labeling studies have shown that some fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate even in gluconeogenesis. There is also a limited
100
degree of cycling in other pairs of opposed irreversible reactions. This cycling was
A
B
regarded as an imperfection in metabolic control, and so substrate cycles have
90
sometimes been called futile cycles. Indeed, there are pathological conditions, such
as malignant hyperthermia, in which control is lost and both pathways proceed
H2O
Pi
rapidly. In malignant hyperthermia, there is rapid, uncontrolled hydrolysis of ATP,
Net flux of B = 10
which generates heat and can raise body temperature to 44 °C (111 °F). Muscles
may become rigid and destroyed as well.
ATP
ADP
Despite such extraordinary circumstances, substrate cycles now seem likely
to be biologically important. One possibility is that substrate cycles amplify meta120
bolic signals. Suppose that the rate of conversion of A into B is 100 and of B into
A
B
A is 90, giving an initial net flux of 10. Assume that an allosteric effector
72
increases the A S B rate by 20% to 120 and reciprocally decreases the B S A
rate by 20% to 72. The new net flux is 48, and so a 20% change in the rates of
H2O
Pi
the opposing reactions has led to a 380% increase in the net flux. In the examNet flux of B = 48
ple shown in Figure 16.9, this amplification is made possible by the rapid
hydrolysis of ATP. The flux of each step of the glycolytic pathway has been sugFigure 16.9 A substrate cycle. This ATPgested to increase as much as 1000-fold at the initiation of intense exercise,
driven cycle operates at two different rates.
when a lot of ATP is needed. Because the allosteric activation of enzymes alone
A small change in the rates of the two
seems unlikely to explain this increased flux, the existence of substrate cycles
opposing reactions results in a large
may partly account for the rapid rise in the rate of glycolysis.
change in the net flux of product B.
16.3 Metabolism in Context: Precursors Formed
by Muscle Are Used by Other Organs
Lactate produced by active skeletal muscle and red blood cells is a source of
energy for other organs. Red blood cells lack mitochondria and can never oxidize glucose completely. Recall that, in contracting skeletal muscle during vigorous exercise, the rate at which glycolysis produces pyruvate exceeds the rate
at which the citric acid cycle oxidizes it. In these cells, lactate dehydrogenase
reduces excess pyruvate to lactate to restore redox balance (p. 235). However,
lactate is a dead end in metabolism. It must be converted back into pyruvate
before it can be metabolized. Both pyruvate and lactate diffuse out of these cells
through carriers into the blood. In contracting skeletal muscle, the formation and
261
262
16 Gluconeogenesis
IN LIVER
GLUCONEOGENESIS
Glucose
GLYCOLYSIS
B
Glucose
L
6 ~P
Figure 16.10 The Cori cycle. Lactate
formed by active muscle is converted into
glucose by the liver. This cycle shifts part
of the metabolic burden of active muscle
to the liver. The symbol ⬃P represents
nucleoside triphosphates.
IN MUSCLE
Pyruvate
O
2 ~P
Pyruvate
O
Lactate
D
Lactate
release of lactate lets the muscle generate ATP in the absence of oxygen and shifts
the burden of metabolizing lactate from muscle to other organs. The pyruvate and
lactate in the bloodstream have two fates. In one fate, the plasma membranes
of some cells, particularly cells in cardiac muscle, contain carriers that make
the cells highly permeable to lactate and pyruvate. These molecules diffuse
from the blood into such permeable cells. Inside these well-oxygenated cells,
lactate can be reverted back to pyruvate and metabolized through the citric
acid cycle and oxidative phosphorylation to generate ATP. The use of lactate in
place of glucose by these cells makes more circulating glucose available to the
active muscle cells. In the other fate, excess lactate enters the liver and is converted first into pyruvate and then into glucose by the gluconeogenic pathway.
Thus, the liver restores the level of glucose necessary for active muscle cells, which
derive ATP from the glycolytic conversion of glucose into lactate. These reactions
constitute the Cori cycle (Figure 16.10).
Studies have shown that alanine, like lactate, is a major precursor of glucose in
the liver. The alanine is generated in muscle when the carbon skeletons of some
amino acids are used as fuels. The nitrogens from these amino acids are transferred to pyruvate to form alanine (p. 458); the reverse reaction takes place in
the liver. This process also helps to maintain nitrogen balance. The interplay
between glycolysis and gluconeogenesis is summarized in Figure 16.10, which
shows how these pathways help meet the energy needs of different cell types.
16.4 Glycolysis and Gluconeogenesis
Are Evolutionarily Intertwined
The metabolism of glucose has ancient origins. Organisms living in the early biosphere depended on the anaerobic generation of energy until significant amounts
of oxygen began to accumulate 2 billion years ago.
We can speculate on the evolutionary relationship between glycolysis and gluconeogenesis if we think of glycolysis as consisting of two segments: the metabolism of hexoses (stages 1 and 2, see Figure 15.1) and the metabolism of trioses
(stage 3). The enzymes of stages 1 and 2 are different in some species and are missing entirely in some archaea, whereas the enzymes of stage 3 are quite conserved.
In fact, four enzymes of the lower segment are present in all species. This lower
part of the pathway is common to glycolysis and gluconeogenesis. This common part
of the two pathways may be the oldest part, constituting the core to which the
other steps were added. The upper part would have varied according to the sugars that were available to evolving organisms in particular niches. Interestingly,
this core part of carbohydrate metabolism can generate triose precursors for
ribose sugars, a component of RNA and a critical requirement for the RNA world.
Thus, we are left with the unanswered question, was the original core pathway
used for energy conversion or biosynthesis?
263
SUMMARY
Key Terms
16.1 Glucose Can Be Synthesized from Noncarbohydrate Precursors
Gluconeogenesis is the synthesis of glucose from noncarbohydrate
sources, such as lactate, amino acids, and glycerol. Several of the reactions
that convert pyruvate into glucose are common to glycolysis. Gluconeogenesis, however, requires four new reactions to bypass the essential irreversibility of three reactions in glycolysis. In two of the new reactions,
pyruvate is carboxylated in mitochondria to oxaloacetate, which, in turn,
is decarboxylated and phosphorylated in the cytoplasm to phosphoenolpyruvate. Two molecules having high phosphoryl-transfer potential
are consumed in these reactions, which are catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Pyruvate carboxylase
contains a biotin prosthetic group. The other distinctive reactions of gluconeogenesis are the hydrolyses of fructose 1,6-bisphosphate and
glucose 6-phosphate, which are catalyzed by specific phosphatases. The
major raw materials for gluconeogenesis by the liver are lactate and alanine
produced from pyruvate by active skeletal muscle. The formation of
lactate during intense muscular activity buys time and shifts part of the
metabolic burden from muscle to the liver.
16.2 Gluconeogenesis and Glycolysis Are Reciprocally Regulated
Gluconeogenesis and glycolysis are reciprocally regulated so that one pathway is relatively inactive while the other is highly active. Phosphofructokinase and fructose 1,6-bisphosphatase are key control points. Fructose
2,6-bisphosphate, an intracellular signal molecule present at higher levels
when glucose is abundant, activates glycolysis and inhibits gluconeogenesis
by regulating these enzymes. Pyruvate kinase and pyruvate carboxylase are
regulated by other effectors so that both are not maximally active at the
same time. Allosteric regulation and reversible phosphorylation, which are
rapid, are complemented by transcriptional control, which takes place in
hours or days.
16.3 Metabolism in Context: Precursors Formed by Muscle
Are Used by Other Organs
Lactate that is generated by glycolysis in contracting muscle is released into
the bloodstream. This lactate is removed from the blood by the liver and is
converted into glucose by gluconeogenesis. This metabolic cooperation
between muscle and liver is called the Cori cycle. Alanine is used to transport nitrogen as well as carbon skeletons from muscle to the liver.
16.4 Glycolysis and Gluconeogenesis Are Evolutionarily Intertwined
Parts of glycolysis and gluconeogenesis are ancient pathways. In particular, the metabolism of trioses that is common to both pathways may be
the oldest part. This common set of reactions formed the basis of carbohydrate metabolism from which glycolysis and gluconeogenesis evolved.
Key Terms
gluconeogenesis (p. 251)
pyruvate carboxylase (p. 254)
biotin (p. 255)
glucose 6-phosphatase (p. 257)
bifunctional enzyme (p. 259)
substrate cycle (p. 261)
Cori cycle (p. 262)
264
16 Gluconeogenesis
Answers to QUICK QUIZZES
1. The reverse of glycolysis is highly endergonic under
cellular conditions. The expenditure of 6 NTP molecules in
gluconeogenesis renders gluconeogenesis exergonic.
2. Reciprocal regulation at the key allosteric enzymes
in the two pathways. For instance, PFK is stimulated by
fructose 2,6-bisphosphate and AMP. The effect of these signals is opposite that of fructose 1,6-bisphosphatase. If both
pathways were operating simultaneously, a futile cycle
would result. ATP would be hydrolyzed, yielding only heat.
Problems
1. Road blocks. What reactions of glycolysis are not readily reversible under intracellular conditions? How are these
reactions bypassed in gluconeogenesis?
9. Metabolic mutants. What would be the effect on an
organism’s ability to use glucose as an energy source if a
mutation inactivated glucose 6-phosphatase in the liver?
2. Waste not, want not. Why is it in an organism’s best
interest to convert lactic acid from the blood into glucose in
the liver?
10. Never let me go. Why does the lack of glucose 6-phosphatase activity in the brain and muscle make good physiological sense?
3. Metabolic mutant. What are the likely consequences
of a genetic disorder rendering fructose 1,6-bisphosphatase in the liver less sensitive to regulation by fructose
2,6-bisphosphate?
11. Match ’em 1. The following sequence is a part of the
sequence of reactions in gluconeogenesis.
4. Biotin snatcher. Avidin, a 70-kd protein in egg white, has
very high affinity for biotin. In fact, it is a highly specific
inhibitor of biotin enzymes. Which of the following conversions would be blocked by the addition of avidin to a cell
homogenate?
(a) Glucose S pyruvate
(b) Pyruvate S glucose
(c) Oxaloacetate S glucose
(d) Malate S oxaloacetate
(e) Pyruvate S oxaloacetate
(f) Glyceraldehyde 3-phosphate S fructose 1,6-bisphosphate
5. Tracing carbon atoms. If cells synthesizing glucose
from lactate are exposed to CO2 labeled with 14C, what
will be the distribution of label in the newly synthesized
glucose?
6. Working at cross-purposes? Gluconeogenesis takes place
during intense exercise, which seems counterintuitive. Why
would an organism synthesize glucose and, at the same
time, use glucose to generate energy?
7. Powering pathways. Compare the stoichiometries of
glycolysis and gluconeogenesis. Recall that the input of one
ATP equivalent changes the equilibrium constant of a reaction by a factor of about 108 (p. 207). By what factor do the
additional high-phosphoryl-transfer compounds alter the
equilibrium constant of gluconeogenesis?
8. Different needs. Liver is primarily a gluconeogenic tissue, whereas muscle is primarily glycolytic. Why does this
division of labor make good physiological sense?
Pyruvate ¡ Oxaloacetate ¡ Malate ¡
A
B
C
oxaloacetate ¡ Phosphoenolpyruvate
D
Match the capital letters representing the reaction in the
gluconeogenic pathway with parts a, b, c, etc.
(a) takes place in the mitochondria.
(b) takes place in the cytoplasm.
(c) produces CO2.
(d) consumes CO2.
(e) requires NADH.
(f) produces NADH.
(g) requires ATP.
(h) requires GTP.
(i) requires thiamine.
(j) requires biotin.
(k) is regulated by acetyl CoA.
12. Salvaging resources. In starvation, protein degradation
takes place in muscle. Explain how this degradation might
affect gluconeogenesis in the liver.
13. Counting high-energy compounds 1. How many NTP
molecules are required for the synthesis of one molecule of
glucose from two molecules of pyruvate? How many NADH
molecules?
14. Counting high-energy compounds 2. How many NTP
molecules are required to synthesize glucose from each of
the following compounds?
(a) Glucose 6-phosphate
(b) Fructose 1,6-bisphosphate
(c) Two molecules of oxaloacetate
(d) Two molecules of dihydroxyacetone phosphate
265
Problems
16. Lending a hand. How might enzymes that remove
amino groups from alanine and aspartate contribute to
gluconeogenesis?
(a) What was the rationale for comparing the activities of
these two enzymes?
(b) The following data show the activites of both enzymes
for a variety of bumblebee species (genera Bombus and
Psithyrus). Do these results support the notion that bumblebees use futile cycles to generate heat? Explain.
Enzyme activity (units g−1 thorax)
Chapter Integration Problems
15. Match ’em 2. Indicate which of the conditions listed in
the right-hand column increase the activity of the glycolytic
and gluconeogenic pathways.
(a) glycolysis
1. increase in ATP
(b) gluconeogenesis
2. increase in AMP
3. increase in fructose
2,6-bisphosphate
4. increase in citrate
5. increase in acetyl CoA
6. increase in insulin
7. increase in glucagon
8. fasting
9. fed
PFK
FBPase
120
100
80
60
40
20
us
trin
llis
P.
ci
seo
co
tus
inc
gri
B.
oc
B.
ruf
rpl
ex
us
ns
pe
ga
B.
ns
va
B.
tie
tus
B.
im
pa
s
ula
ini
ac
aff
bim
B.
B.
res
ter
B.
Data Interpretation Problem
17. Cool bees. In principle, a futile cycle that includes phosphofructokinase and fructose 2,6-bisphosphatase could be used to
generate heat. The heat could be used to warm tissues. For
instance, certain bumblebees have been reported to use such a
futile cycle to warm their flight muscles on cool mornings.
Scientists undertook a series of experiments to determine if a number of species of bumblebee use this futile
cycle. Their approach was to measure the activity of PFK
and F-1,6-BPase in flight muscle.
tris
0
[After J. F. Staples, E. L. Koen, and T. M. Laverty, J. Exp. Biol. 207:749–754,
2004, p. 751.]
(c) In which species might futile cycling take place? Explain
your reasoning.
(d) Do these results prove that futile cycling does not participate in heat generation?
Selected readings for this chapter can be found online at www.whfreeman.com/Tymoczko