Okay, now we’re going to talk about gluconeogenesis.

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Tymoczko • Berg • Gatto • Stryer

CHAPTER 17
Gluconeogenesis

Biochemistry: A
Short Course

Fourth Edition

© 2019 Macmillan Learning

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.]

And we’re not going to go too deeply into this, but we will cover it in as much detail
as we need. When you’re fasting, your brain still needs to get glucose, and so do your
red blood cells – and fasting is something that the American Indians do, it’s a health
practice that’s done across the world – otherwise you’ll die.

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CHAPTER 17
Gluconeogenesis

So the body has a way of creating glucose from noncarbohydrate precursors,

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Chapter 17: Outline

17.1 Glucose Can Be Synthesized from Noncarbohydrate
Precursors

17.2 Gluconeogenesis and Glycolysis Are Reciprocally
Regulated

17.3 Metabolism in Context: Precursors Formed by
Muscle Are Used by Other Organs

such as amino acids and fats – and that process is called gluconeogenesis. Gluco =
glucose, neo = new, genesis = {producing} – producing new glucose. So it mostly
happens in the liver, a little bit in the kidney. But it’s one of the main jobs of the liver,
actually – the liver is like a buffer zone for glucose.

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Gluconeogenesis

• Gluconeogenesis is the synthesis of glucose from
noncarbohydrate precursors (mostly pyruvate).

• The major site of gluconeogenesis is the liver, although
gluconeogenesis can occur in the kidney.

• Gluconeogenesis is especially important during fasting or
starvation, as glucose is the primary fuel for the brain and
the only fuel for red blood cells.

If you don’t have enough in your system, then it will make more. And there are three
or four different ways that you can get the substrates that you need to make glucose.
You can start by using pyruvate, which can come from lactate. And you can also use
amino acids. Or glycerol, which is from the triacylglycerols – that’s just your three-
carbon chain here with a hydroxyl moiety on each one.

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Section 17.1 Glucose Can Be Synthesized
from Noncarbohydrate Precursors

Learning Objective 3: Describe how gluconeogenesis is powered
in the cell.
• The gluconeogenic pathway converts pyruvate into glucose.

• Pyruvate can be formed from muscle-derived lactate in the liver by
lactate dehydrogenase.

• The carbon skeletons of some amino acids can be converted into
gluconeogenic intermediates.

• Glycerol, derived from the hydrolysis of triacylglycerols, can be
converted into dihydroxyacetone phosphate, which can be processed
by gluconeogenesis or glycolysis.

And that can be changed into dihydroxyacetone phosphate and then injected
into the gluconeogenesis pathway. And your book goes into some detail about
how gluconeogenesis is not exactly the reverse of glycolysis, but in most ways
it is. And so we’re just going to review glycolysis here. Glucose goes down to
glyceraldehyde 3-phosphate in these steps. And you can see that some of
these steps – aldolase, and the phosphoglucose isomerase – can go both ways.
But this one, glucose to glucose 6-phosphate, and this one, fructose 6-
phosphate to fructose 1,6-bisphosphate, are one-way reactions. And those
are these two large energy drops here where you need to input ATP. So these
reactions do not happen spontaneously in reverse, but they can be mediated
by other enzymes. So in this case, glucose 6-phosphatase pops that phosphate
right off and makes glucose. And fructose 1,6- bisphosphatase can pop a
phosphate right off and make fructose 6-phosphate. There’s no need to add
ATP or anything, either. It’s kind of a free step there, but it’s just not mediated
by the same molecules, which gives it another opportunity to control it
differently.

The second half of glycolysis, we have, remember, broken into two things.
You’re starting here with your glyceraldehyde 3-phosphate. Your
phosphoglycerate kinase – all of these are bidirectional so you can go both
ways on them. But pay special attention to this one where you got an ATP in
glycolysis – you have to put that back in, in gluconeogenesis. And then you get
down to the end. Pyruvate can be turned back into phosphoenolpyruvate by
changing it to oxaloacetate, and then back into phosphoenolpyruvate.

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Figure 17.1 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 acids are shown.

And there is energy required for both of these steps. So we will discuss those
in more detail. So we have here on the left glycolysis, and up here on the right
gluconeogenesis. And you can see that lactate and some amino acids can start
at pyruvate. There’s, of course, some steps involved to transform them into
pyruvate. Some amino acids can be transformed directly into oxaloacetate.
And we’ll discuss oxaloacetate in more detail when we discuss the citric acid
cycle in the following chapters.

Figure 17.1 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 acids are shown.

And then glycerol from your fats can come in in the middle here and continue
down the list.

Figure 17.1 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 acids are shown.

Alright, so now we’re going to continue with the last part of gluconeogenesis – and a
comparison here to the first part of glycolysis. It is a reversal in the sense that you are
creating the same substrates, but it is using two new enzymes that we will talk about in a
second.

So they make a big deal in the book of saying that “gluconeogenesis is not a reversal
of glycolysis”. Well, it mostly is – but there are three points that need to be bypassed.
Those three irreversible steps in glycolysis. And you can see them here where the
blue arrows are. That is the conversion of glucose to glucose-6-phosphate. The action
of phosphofructokinase, which we’ve discussed, has a very important control point.
And then the final step of pyruvate kinase, which is important for creating pyruvate,
which is the substrate for the activity inside the mitochondria.

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Gluconeogenesis Is Not a Complete
Reversal of Glycolysis

The three irreversible steps in glycolysis must be bypassed
in gluconeogenesis.

So you start by going from pyruvate to phosphoenolpyruvate. And that is a big
energy step here. So it’s done in two steps. And it’s done first by converting
pyruvate to oxaloacetate by pyruvate carboxylase. And then by taking that
oxaloacetate and converting it to phosphoenolpyruvate out in the cytoplasm.
So the overall equation here is you’re putting in an ATP and a GTP. And so
you’re using up 2 high energy molecules there. So it does chew up quite a bit
of energy.

Figure 17.2 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.]

And we have here the carboxylation taking place in three steps. And this takes place
with the pyruvate carboxylase. The first thing is the activation of your carbon dioxide
here, which they pull from this bicarbonate ion. And that gets attached to the enzyme
and that’s at a cost of one ATP – and that is actually an important step. That’s where
the energy gets consumed. In this step. After that, it just gets passed along to biotin.
And then the CO2 gets passed to pyruvate to create oxaloacetate. Now, a couple of
thoughts here – biotin is one of those vitamins that you need to have in your system.
And you can get it from several sources. You can also just buy it on the shelf in
bottles. And that biotin gets attached to the end of a lysine residue in the enzyme.
And then it can be used. This structure here is very well-suited to taking a carbon
dioxide here and transferring it to the target.

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The conversion of pyruvate into phosphoenolpyruvate
begins with the formation of oxaloacetate.

• The formation of oxaloacetate by pyruvate carboxylase
occurs in three stages.

Figure 17.2b (second edition) The structure of carboxybiotin. 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.]

So here’s a picture of that biotin in the immediate context of the enzyme that it’s in.
Here’s the domain they call the “biotin carboxyl carrier” domain, which makes sense.

Figure 17.3 A subunit of pyruvate carboxylase. Biotin, covalently attached to the
biotin carboxyl carrier domain, transports CO2 from the biotin carboxylase active site
to the pyruvate carboxylase active site of an adjacent subunit. [Based on G. Lasso,
L.P.C. Yu, D. Gil, S. Xiang, L. Tong, and M. Valle, Structure 18:1300–1310, 2010.]

In the context of the rest of the enzyme – this is a subunit, this is the domain, and it
gets the carboxyl group from this domain. This is the biotin carboxylase domain,
which attaches the carbon dioxide to the biotin. And then, you can see, it’s on this big
floppy arm. It flops around and goes to this other domain, pyruvate carboxylase,
which then takes the carboxyl group and attaches it to the pyruvate. And this domain
is to create a tetramer,

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Structure of a Subunit of
Pyruvate Carboxylase

which is what it exists as in the the biological context. And it’s a little bit difficult to see
perhaps, but in here (let’s just look at this blue subunit of the tetramer) this is the biotin
carboxyl carrier protein domain, and it actually flops from one domain of its own subunit
to a domain of a different subunit. So it flops around in here, but it’s tethered. So it keeps
it within the context of the molecule. And that helps very much to accelerate the
synthesis of the oxaloacetate, in this context. Very similar also to the activity of pyruvate
dehydrogenase. You might imagine. Same concept.

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So oxaloacetate does not naturally leave the mitochondria. This is a very carefully
regulated step, the leaving of the oxaloacetate. And you don’t want to just convert
oxaloacetate into phosphoenolpyruvate inside there – that would be not very helpful.
So the control step here is this malate – and this, it turns the oxaloacetate into a
malate, and then shuttles the malate out into the cytoplasm. It costs one NADH to
make that conversion, but you get that back outside, and you end up using this later
in the process of gluconeogenesis. So very important. Also oxaloacetate gets
regenerated from pyruvate for other reasons as well. Oxaloacetate is a very
important part of the citric acid cycle – but we will talk about that in a little more
detail later.

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Oxaloacetate Is Shuttled into the
Cytoplasm and Converted into

Phosphoenolpyruvate
• The formation of oxaloacetate by pyruvate

carboxylase occurs in the mitochondria.

• Oxaloacetate is reduced to malate and
transported into the cytoplasm, where it is
reoxidized to oxaloacetate with the generation
of cytoplasmic NADH.

• PEP is then synthesized from oxaloacetate by
phosphoenolpyruvate carboxykinase.

Now you’ve gotten to phosphoenolpyruvate. At this point, it just kind of drifts through
backwards, the glycolytic pathway. You notice all of these steps here are reversible, and
all of these intermediates have basically the same energy. So phosphoenolpyruvate can
just sort of pop around until it gets here to fructose bisphosphate. And at that point, it
needs to overcome this energetic barrier.

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So that is catalyzed by fructose 1,6- bisphosphatase. And this is an irreversible step
because it releases this phosphate. And that would take a great deal of energy to
reattach it.

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The Conversion of Fructose 1,6-Bisphosphate
into Fructose 6-Phosphate and

Orthophosphate Is an Irreversible Step
• Phosphoenolpyruvate is metabolized by the enzymes of

glycolysis in the reverse direction until the next irreversible
step, the hydrolysis of fructose 1,6-bisphosphate.

• The enzyme catalyzing this reaction is fructose 1,6-
bisphosphatase, an allosteric enzyme.

Figure 17.5 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.]

And then finally, the generation of free glucose, which would be the final step in
gluconeogenesis, basically only happens in the liver. And it doesn’t happen in the
cytoplasm. The glucose 6-phosphate has to get transported into the endoplasmic
reticulum, and inside there it gets turned into free glucose – by this glucose 6-
phosphatase – which then gets exported into the cytoplasm and is able to be
distributed to the rest of the body. And otherwise, glucose 6-phosphate generally just
gets turned into glycogen. If there’s enough energy in the cell already, then it gets
shunted off into that pathway.

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The Generation of Free Glucose Is an
Important Control Point

• The generation of free glucose, which occurs essentially
only in the liver, is the final step in gluconeogenesis.

• Glucose 6-phosphate is transported into the lumen of the
endoplasmic reticulum.

• Glucose 6-phosphatase, an integral membrane on the
inner surface of the endoplasmic reticulum, catalyzes the
formation of glucose from glucose 6-phosphate.

So you can make glucose from these noncarbohydrate precursors, but it costs you.
You have to put a lot of energy into it. Remember, in glycolysis you got a net 2 ATP out
of glycolysis. Well, you have to put about six ATP equivalents back into this to put it
back into glucose. And that is in the form of 4 ATP, 2 GTP, and also some NADH.

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Six High-Transfer-Potential Phosphoryl
Groups Are Spent in Synthesizing Glucose

from Pyruvate

So you don’t want to run these things simultaneously. And they are regulated so that
they don’t run simultaneously. The molecules are allosterically regulated in a
reciprocal fashion, so that if you have a high energy load in the cell, then it will favor
the reverse process, gluconeogenesis. If you have a low energy load, then it’ll favor
glycolysis.

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Section 17.2 Gluconeogenesis and
Glycolysis Are Reciprocally Regulated

Learning objective 4: Describe the coordinated
regulation of glycolysis and gluconeogenesis.

• Gluconeogenesis and glycolysis are regulated so that
within a cell, one pathway is relatively inactive while the
other is highly active.

• The rationale for reciprocal regulation is that glycolysis will
predominate when glucose is abundant and that
gluconeogenesis will be highly active when glucose is
scarce.

(Or energy charge, I guess they call it here.) Some of the key elements of that are
fructose 1,6- bisphosphate and fructose 6-phosphate. … The interconversion between
the two of those is the key regulatory step in this process. Very similar to
phosphofructokinase in glycolysis.

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Energy Charge Determines Whether
Glycolysis or Gluconeogenesis Will Be

More Active

• The interconversion of fructose 1,6-bisphosphate and
fructose 6-phosphate is a key regulatory site.

• Additionally, glycolysis and gluconeogenesis are
reciprocally regulated at the interconversion of
phosphoenolpyruvate and pyruvate.

• If ATP is needed, glycolysis predominates. If glucose is
needed, gluconeogenesis is favored.

Figure 17.6 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.

And you can see here phosphofructokinase. You have the substrates that promote
phosphofructokinase and the ones that inhibit phosphofructokinase. These are the
ones that are present in the low energy charge, and these are the ones that are
present in the high energy charge. And you can see, similarly, the same things – AMP
promotes phosphofructokinase, but it inhibits fructose 1,6-bisphosphatase. So
they’re inverted. There’s a reciprocal relationship between these two pathways. Same
down here – this is the other major control site, phosphoenolpyruvate, to pyruvate.
And ATP will inhibit your pyruvate kinase, but ADP will inhibit the reverse reaction.

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Diagram of the Reciprocal Regulation of
Gluconeogenesis and Glycolysis in the Liver

Figure 17.9 Diet can help to prevent the development of type 2 diabetes. A healthy
diet, one rich in fruits and vegetables, is an important step in preventing or treating
type 2 diabetes. [Photodisc/Getty Images.]

Now, normally insulin will inhibit gluconeogenesis. Which makes a lot of sense
because if you have a lot of sugar in your blood, then you don’t need to do
gluconeogenesis. But in type two diabetes, then the insulin does not inhibit
gluconeogenesis, and that’s called insulin resistance. And the enzymes are very active
in gluconeogenesis in type two diabetes, so producing high levels of blood glucose –
which can be mediated by exercise and diet. But of course, it’s a major health
concern in the world these days.

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Clinical Insight: Insulin Fails to Inhibit
Gluconeogenesis in Type 2 Diabetes

• Insulin normally inhibits
gluconeogenesis. In type 2 diabetes,
insulin fails to act, a condition called
insulin resistance.

• The enzymes of gluconeogenesis,
especially PEPCK, are active, leading
to abnormally high levels of blood
glucose.

• The treatment of type 2 diabetes
includes weight loss, a healthy diet,
exercise, and drug treatment to
enhance sensitivity to insulin.

And remember, most of this stuff happens in the liver, as far as the gluconeogenesis
goes. And most of the breakdown of glucose, well, a lot of it, happens in the muscle.
Especially if you’re going to be doing long-term exercise. The muscle can break down
glucose into pyruvate so fast that it’s not able to go through oxidative
phosphorylation, and therefore it gets shunted off and becomes lactate. Lactate can
circulate through the blood … and it gets absorbed by the liver and then converted
back into glucose, which releases back into the blood – which is an example of what
we call the Cori cycle.

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Section 17.3 Metabolism in Context:
Precursors Formed by Muscle Are Used

by Other Organs

• Muscle and liver display interorgan cooperation in a series
of reactions called the Cori cycle.

• Lactate produced by muscle during contraction is released
into the blood.

• Liver removes the lactate and converts it into glucose,
which can be released into the blood.

Figure 17.11 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.

So here’s just a diagram of that happening. You have here your muscle, breaking
down glucose, sending out lactate into the blood. And in the liver, it’s getting turned
back into glucose and sent back to the muscle. So you’re getting a little bit of energy
here. You’re putting a lot of energy in here. It’s not the greatest, as far as efficiency
goes, but it is faster than oxidative phosphorylation, and that’s why it’s an important
mechanism for getting the energy to your muscles.

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Diagram of the Cori Cycle