BCH 351: PRIMARY METABOLIC PATHWAY I

GLUCONEOGENESIS
Introduction
Glucose occupies a very important position in the metabolism of many organisms
including mammals. It not only serves as an excellent energy source, but is also a versatile
precursor for many biosynthetic reactions. In mammals, circulating glucose from blood is
the sole or major source of energy for certain cells/tissues such as the brain and nervous
system, testes, red blood cells, etc. Dietary carbohydrate ingestion is the main source of
glucose for the body, however, during long fasts, after vigorous exercise, or sometimes
between meals, glycogen stores are depleted. During these times, the liver is able to
synthesize glucose from non-carbohydrate sources to maintain blood glucose via the
process of gluconeogenesis (new formation of sugar/glucose). Gluconeogenesis is a
metabolic process that generates glucose form non-carbohydrate substrates. This process
ensures the maintenance of appropriate blood glucose levels when the liver glycogen is
almost depleted and no carbohydrates are ingested. In mammals, gluconeogenesis takes
place mainly in the liver and, to a lesser extent, in the renal cortex.

Reactions of gluconeogenesis
Although it shares several steps with the glycolytic pathway, gluconeogenesis and
glycolysis are not identical pathways running in opposite directions. In other words,
gluconeogenesis is not the reversal of glycolysis even though seven (7) of the ten (10)
enzymatic steps of gluconeogenesis are the reversal of the reactions of glycolysis. In
glycolysis, there are three (3) irreversible steps that cannot be utilized in gluconeogenesis
and therefore need to be bypassed by separate set of enzymes. These reactions include:
a. The conversion of glucose to glucose-6-phosphate by hexokinase
b. Phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by
phosphofructokinase-I
c. Conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase.

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1. Conversion of pyruvate to phosphoenolpyruvate
In the first reaction of gluconeogenesis which is the first bypass reaction, pyruvate is
converted (via phosphorylation) to phosphoenolpyruvate (PEP). This reaction is not a
simple reversal of the pyruvate kinase reaction of glycolysis. Instead, pyruvate is first
converted to oxaloacetate by the enzyme pyruvate carboxylase in the mitochondrial
matrix in a reaction that requires biotin as a coenzyme.
To proceed in the gluconeogenic pathway, the oxaloacetate formed must be transferred
back into the cytosol. However, mitochondria lack an efficient transporter for
oxaloacetate, therefore, oxaloacetate is reduced to malate by malate dehydrogenase which
converts one molecule of NADH to NAD+. Malate is then transported out of the
mitochondria and re-oxidized to oxaloacetate, regenerating NADH from NAD + in the
cytosol. In the cytosol, a second enzyme, phosphoenolpyruvate carboxykinase
catalyzes the decarboxylation and phosphorylation of oxaloacetate to
phosphoenolpyruvate (PEP) using GTP (from TCA cycle) as the phosphate group donor.

Note* - The glucogenic precursor determines whether this reaction occurs entirely
in the mitochondrion or partially in both the mitochondrion and the cytosol.

When pyruvate is the glucogenic precursor, it is first transported from the cytosol into
the mitochondrion or generated from alanine via transamination reaction in the
mitochondrion. The pyruvate then undergoes the pyruvate carboxylate reaction to yield
oxaloacetate which is reduced to malate and transported out of the mitochondrion into
the cytosol. In the cytosol, malate is re-oxidized back to oxaloacetate by malate
dehydrogenase with a concomitant production of NADH. The oxaloacetate is then
simultaneously decarboxylated and phosphorylated to phosphoenolpyruvate (PEP) by
phosphoenolpyruvate carboxykinase. This reaction requires Mg 2+ and GTP and the
phosphoryl group donor.
*** The oxaloacetate-malate shuttling allows the transport of reducing equivalents
(NADH) into the cytosol where their concentration is relatively low. This reaction is
important because gluconeogenesis cannot proceed without the availability of NADH.

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When lactate (from erythrocytes and vigorously exercising muscles) is the glucogenic
precursor, it is first converted to pyruvate by the lactate dehydrogenase reaction to yield
pyruvate and a molecule of NADH. The pyruvate is then transported into the
mitochondrion where it is converted to oxaloacetate. The oxaloacetate produced is not
however, transported out of the mitochondrion. Rather, it is converted directly to PEP by
the mitochondrial isozyme of PEP carboxykinase. The PEP is then transported out of the
mitochondrion to continue in the gluconeogenesis pathway.
*** Oxaloacetate is not shuttled out (as malate) in this reaction because NADH is already
generated in the cytosol during the lactate dehydrogenase reaction and does not need to
be shuttled out of the mitochondrion.

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2. Conversion of fructose 1,6-bisphosphate to fructose 6-phosphate
The second step of gluconeogenesis is the bypass of the reaction catalyzed by
phosphofructokinase-1 (PFK-1) in the glycolytic pathway. This reaction involves the
dephosphorylation of fructose 1,6-bisphosphate to fructose 6-phosphate catalyzed by
fructose 1,6-bisphosphatase, a Mg2+-dependent enzyme located in the cytosol, leading
to the hydrolysis of the C-1 phosphate of fructose 1,6-bisphosphate, without production of
ATP.

3. Conversion of glucose 6-phosphate to glucose

The third step of gluconeogenesis is the bypass of the reaction catalyzed by hexokinase in
glycolysis. This involves the dephosphorylation of glucose 6-phosphate to glucose
catalyzed by glucose 6-phosphatase. Glucose 6-phosphatase is found in the lumen of
the endoplasmic reticulum (ER) rather than in the cytoplasm. Thus, for the final step of
gluconeogenesis, glucose 6-phosphate must be transported into the ER where the
phosphate is cleaved off, and then glucose and phosphate are transported back out.
**This enzyme is absent in skeletal muscle and adipose tissue and that is why
gluconeogenesis cannot take place in these tissues.

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Summary of the Reactions of Gluconeogenesis

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Substrates for gluconeogenesis
In addition to pyruvate and lactate, other non-carbohydrate precursors can be used as
substrates for gluconeogenesis in animals. These include most of the amino acids, as well

as glycerol and all the TCA cycle intermediates.

1. Lactate is produced by exercising skeletal muscle and in cells that lack mitochondria
(e.g. erythrocytes) and transported back to the liver where it is converted to pyruvate
by lactate dehydrogenase and used for gluconeogenesis.

2. Amino acids generated from dietary protein or from the breakdown of muscle
protein during starvation, also undergo transamination or deamination in the
mitochondrial matrix to yield pyruvate or intermediates of the tricarboxylic acid
(TCA) cycle.

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3. Glycerol released from lipolysis of adipose tissue triacylglycerol and
glycerophospholipids are also utilized for gluconeogenesis. Glycerol enters
gluconeogenesis, or glycolysis depending on the cellular energy charge, as
dihydroxyacetone phosphate (DHAP). It is first phosphorylated to glycerol 3-
phosphate by glycerol kinase, which utilizes one ATP molecule for phosphorylation
of glycerol. Glycerol 3-phosphate is then oxidized to dihydroxyacetone phosphate by
glycerol 3-phosphate dehydrogenase. In this reaction NAD+ is reduced to NADH.
During prolonged fasting, glycerol is the major gluconeogenic precursor, accounting
for about 20% of glucose production.

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Regulation of Gluconeogenesis
Glycolysis and gluconeogenesis are reciprocally regulated to prevent wasteful operation of
both pathways at the same time. The points of regulation of gluconeogenesis are
highlighted below.

1. Conversion of pyruvate to phosphoenolpyruvate

Pyruvate carboxylase, the enzyme that catalyzes the first step in gluconeogenesis, is
activated by acetyl-CoA and inhibited by ADP which is also an inhibitor of
phosphoenolpyruvate carboxykinase. Acetyl-CoA can be seen as a reciprocal regulator of
glycolysis and gluconeogenesis since it acts on the enzymes that interconvert pyruvate
and phosphoenolpyruvate. It is an activator of pyruvate carboxylase and an inhibitor of
pyruvate kinase and the pyruvate dehydrogenase complex. When its levels rises, it signals
the availability of substrates for the TCA cycle, allowing more carbon to be shuttled into
gluconeogenesis and ultimately stored as glycogen.

2. Interconversion of fructose-6-phosphate and fructose-1,6-

bisphosphate.
Fructose 1,6-bisphosphatase is inhibited by adenosine monophosphate (AMP) and
activated by citrate. Adenosine monophosphate is a product of ATP breakdown, and a
rise in its concentration indicates a low energy charge in the cell and signals the need for
ATP. This stimulates the glycolytic pathway by activating phosphofructokinase-1. On the
other hand, high levels of ATP and citrate indicate high energy charge and the abundance
of biosynthetic intermediates. Under this condition (high energy levels), glycolysis is
greatly slowed down and gluconeogenesis is accelerated. Thus AMP levels indicate the
energy state of the cell, consequently regulating the activity of PFK-1 in response
to cellular energy needs.
Phosphofructokinase-1 and Fructose 1,6-bisphosphatase are also reciprocally controlled by
Fructose 2,6-bisphosphate (F 2,6-BP). This molecule is structurally related to fructose
1,6-bisphosphate, but is not an intermediate in glycolysis or gluconeogenesis. The level of
F 2,6-BP is high in fed state and low during starvation. Therefore, at high F 2,6-BP levels,
gluconeogenesis in inhibited while the reaction is activated at low F 2,6-BP levels.

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3. Regulation by the action of Glucagon/Insulin
Two hormones, namely glucagon and insulin, are also involved in the regulation of
glycolysis and gluconeogenesis. These hormones act intracellularly through fructose 2,6-
bisphosphate, an allosteric effector of Phosphofructokinase-1 (PFK-1) and Fructose 1,6-
bisphosphatase-1. By binding to the allosteric site on PFK-1, fructose 2,6-bisphosphate
reduces the affinity of the enzyme for ATP and citrate (allosteric inhibitors) and at the
same time increases the affinity of the enzyme for fructose 6-phosphate (its
substrate). Therefore, in the absence of fructose 2,6-bisphosphate, and in the presence of
physiological concentrations of ATP, fructose 6-phosphate, and of allosteric effectors
AMP and citrate, PFK-1 is practically inactive. Summarily, high concentration of fructose
2,6-bisphosphate activates PFK-1, thus stimulating glycolysis in the liver, while at the
same time slowing down gluconeogenesis by inhibiting fructose 1,6-bisphosphatase.

During starvation or fasting, glucagon is released into the circulation in response to low
blood glucose signaling the liver to reduce glucose consumption for its own needs and to
increase de novo synthesis of glucose and its release from glycogen stores. By binding to
specific membrane receptors, glucagon stimulates hepatic adenylate cyclase to synthesize
3′,5′-cyclic AMP (cAMP), which activates cAMP-dependent protein kinase or protein
kinase A (PKA). The kinase catalyzes the phosphorylation of PFK-2/FBPase-2 increasing
phosphatase activity while decreasing kinase activity. This causes a decrease in the levels

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of fructose 2,6-bisphosphate, that, in turn, inhibits glycolysis and stimulates
gluconeogenesis. Therefore, in response to glucagon, hepatic production of glucose
increases, enabling the organ to counteract the fall in blood glucose levels. In activating
gluconeogenesis, glucagon simultaneously stimulates the release of fatty acids from
adipose tissue into the liver, increasing fatty acid oxidation and a consequent increase in
acetyl-CoA production.
On the other hand, insulin released into circulation after a carbohydrate-rich meal binds
to its specific membrane receptors and activates a protein phosphatase (phosphoprotein
phosphatase 2A or PP2A) that catalyzes the removal of the phosphate group from PFK-
2/FBPase-2, thus increasing PFK-2 activity and decreasing FBPase-2 activity. At the same
time, insulin also stimulates a cAMP phosphodiesterase that hydrolyzes cAMP to AMP.
This increases the level of fructose 2,6-bisphosphate, that, in turn, inhibits
gluconeogenesis and stimulates glycolysis. In addition, fructose 6-phosphate
allosterically inhibits FBPase-2, and activates PFK-2.

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