Gluconeogenesis (making glucose from non-carbohydrate sources)

Glycolysis is the reverse of gluconeogenesis.

pg 7, pg. 8, pg. 9, pg. 10 in biochemistry notebook

gluconeogenesis is the metabolic pathway in the synthesis of glucose from non-carbohydrate precursors:

1. lactic acid

2. amino acids

3. glycerol

4. propionate

gluconeogenesis occurs in the liver and kidney

the reason why gluconeogenesis occurs in the liver and kidney is because the liver and kidney contain all the enzymes for gluconeogenesis.

-gluconeogenesis does not occur in skeletal muscles due to deficiency of glucose-6-phosphatase.

gluconeogenesis does not occur in heart muscle, smooth muscle and adipose tissue due to deficiency of fructose 1-6 diphosphatase.

gluconeogenic precursors

-non-carbohydrates: lactate, glycerol, and (alanine?)

what is a carbohydrate? carbohydrates are organic molecules that contain an aldehyde or ketone group, and 2 hydroxyl groups or more.

non-carbohydrate pre-cursors

in this slide, you will notice a few examples of non-carbohydrate pre-cursors such as glycerol (far left).

glycerol is a three carbon molecule with three hydroxyl groups on each carbon.

in the middle is lactic acid, a three carbon molecule with only one hydroxyl group and one carboxylic acid group.

because lactic acid is a weak acid, when we deprotonate the carboxyl, we obtain a conjugated base.

-when lactic acid combines with sodium, it forms a salt formed in the right figure (sodium lactate).

This table makes reference to the amino acids that are used for gluconeogenesis. The amino acids are referred to as glucogenic amino acids.

in this table, each amino acid is group based on the glycolytic intermediate on which it enters gluconeogenesis.

as you examine this table, you will see that some intermediates fall within a number of carbons in a structure. take for example, a-ketoglutarate, a 5-carbon alpha keto acid and glycolytic precursor that can be obtained from 5-carbon amino acids.

kreb cycle intermediates, metabolites, are gluconeogenic

-an important source of non-carbohydrate precursors is oxaloacetate from the kreb’s cycle.

-the other kreb cycle intermediates helps sustain the pool of oxaloacetate to keep the cycle going.

is gluconeogenesis the exact reverse of glycolysis?

all metabolic pathways are irreversible (highly spontaneous). For instance, the delta G total for glycolysis is -21.9 Kcal/mol. Therefore, going backwards would be expensive.

why is glycolysis irreversible?

keep in mind that you have certain steps in which reactions are directional/ go one route.

-hexokinase (first example)

-phosphofructokinase (the regulating step of glycolysis)

-pyruvate kinase (with the formation of PEP and pyruvate)

These enzymes catalyze reversible steps in glycolysis, Thus, in order to revert these steps, different enzymes are needed.

gluconeogenesis

-most of the enzymes used in gluconeogenesis are those using glycolysis EXCEPT at irreversible steps.

gluconeogenesis

-glucose-6-phosphatase reverts the reaction catalyzed by glucokinase in liver

-fructose-1,6-biphosphatase reverts phosphofructokinase (PFK).

fructose-1,6-biphosphatase takes a phosphate OFF of fructose.

PFK puts a phosphate on fructose-6-phosphate.

gluconeogenesis

-the enzyme pyruvate carboxylase (in the mitochondria) and the enzyme phosphoenol pyruvate carboxylkinase reverts pyruvate kinase.

background

1. What pyruvate kinase does in glycolysis

In glycolysis, the enzyme pyruvate kinase catalyzes the last step:

Phosphoenolpyruvate (PEP)→Pyruvate

Reaction: PEP+ ADP→ Pyruvate + ATP

2. Gluconeogenesis uses two enzymes to bypass it

Instead of reversing pyruvate kinase, the body uses two enzymes:

1⃣ Pyruvate carboxylase (mitochondria)
2⃣ Phosphoenolpyruvate carboxykinase (PEPCK)

Together they convert: Pyruvate → PEP

Step 1 — Pyruvate carboxylase

Pyruvate carboxylase

Location: Mitochondria

Pyruvate+ CO2​+ ATP → Oxaloacetate (OAA)

Key facts:

• Requires biotin (vitamin B7)
• Uses ATP
• Adds CO₂ to pyruvate

So the molecule goes: 3C (pyruvate)→4C (oxaloacetate)

This step is called carboxylation.

4. Step 2 — PEP carboxykinase (PEPCK)

Reaction: Oxaloacetate + GTP→ PEP + CO2

What happens here:

• CO₂ is removed
• GTP provides energy

So the molecule goes:

4C (OAA)→3C (PEP)

This step is decarboxylation + phosphorylation.

pyruvate can go up and down depending on energy needs

pyruvate (the end result of glycolysis) can go up and down depending on energy needs.

-the diagram on the left shows the end point of the flow of glucose synthesis.

-multiple precursors are able to enter at different sites, or entry points, all of which converge in the formation of glucose-6-phosphate.

-however, only in the liver and kidney can you go from glucose-6-phosphate to free glucose.

gluconeogenesis

-starting from , pyruvate is formed by redox reactions.

-pyruvate is formed when the hydroxyl group in carbon 2 is oxidized to form the keto group. (alcohol → ketone (C=O)

pyruvate <lactate dehydrogenase> lactate.

-NAD+ → NADH +H (the hydrogen from the OH is attached to the NADH, the hydride that is highlighted in pink is released into the solution)

-for this redox to occur, the oxidized form of NADH (nicotineamine adenine dinucleotide, or NAD+) is the coenzyme that accepts hydrogens.

the result is an NADH and also a proton ion, also the carbonyl is now a ketone

-lactate dehydrogenase is an intracelluar that mediates this reversible reaction.

-note that depending on which direction this reaction is going, this reaction will depend on the reduced or oxidized form of the co-enzyme (NADH). This fact is high yield because it’s at this step that alcohol inhibits gluconeogenesis AND can induce hypoglycemia under starvation conditions.

2 directions

lactate dehydrogenase (takes the hydrogen from NADH and gives it to lactate)

lactate dehydrogenase (takes two hydrogens (oxidizes) lactate to form pyruvate) (this is the one we are looking at).

this is for context on the last flashcard

Acetyl-CoA is a positive allosteric effector in this reaction. (lots of Acetyl-CoA means to proceed with the reaction because we are going backwards).

since two pyruvates are required, this step consumes two ATPs.

this is the reaction mechanism where pyruvate (3-carbon precursor) becomes oxaloacetate (4 carbon pre-cursor).

-this step requires ATP and uses biotin as a co-enzyme.

in this slide, we have the phosphoenolpyruvate carboxykinase.

This is a cytoplasmic enzyme.

once oxaloacetate is formed in the mitochondria, it is moved into the cytoplasm.

in the cytoplasm, the enzyme phosphoenolpyruvate carboxy kinase converts oxaloacetate → phosphoenol pyruvate.

keep in mind that because 2 oxaloacetates are needed, 2 GTPs are used. The resulting 2 GDPs are converting them back into 2 GTPs by a nucleotide diphosphate kinase using 2 ATPs.

here, you can appreciate the mechanism of action, depicting the use of GDP.

carbon dioxide is released as a biproduct.

in this step, the reaction is irreversible, opposing the direction of the pyruvate kinase from glycolysis.

in gluconeogenesis, this step is required to circumvent the energy demands of going from pyruvate to phosphoenolpyruvate.

the next irreversible step in gluconeogenesis is a rate limiting step. The pacemaker enzyme of this gluconeogenesis pathway is the fructose 1,6 biphosphatase.

the substrate is fructose-1,6-biphosphate and the product of this reaction is the fructose-6-phosphate.

in the liver and in some parts of the kidney, expression of glucose-6-phosphate carries out the last step of gluconeogenesis.

this enzyme opposes the action of glucokinase in the liver and its product is the free glucose that will supply the blood glucose.

Refer to this diagram that shows the overall picture of how glycolysis and gluconeogenesis are metabolically intertwined with common intermediates.

starting from lactate, gluconeogenesis in the liver will require 6 ATPs

-what is the source of ATP to support gluconeogenesis?

answer: Beta-oxidation of fatty acids (beta oxidation of fatty acids takes place under starvation.

control of gluconeogenesis

globally, gluconeogenic enzymes are controlled by hormone induction

-for example, we have epinephrine, glucagon, and cortisol.

control of gluconeogenesis

-the pacemaker enzyme catalyzes the rate-limiting step

-this enzyme is known as the fructose-1,6-biphosphatase and has a positive allosteric effector including ATP and citrate.

control of gluconeogenesis

when we talk about negative allosteric effectors for the fructose-2,6-biphosphate, we can think of ADP.

ADP is a marker of low energy.

ethanol INHIBITS gluconeogenesis from lactate

this slide goes back to the step of pyruvate from lactate. Alcohol can lead to hypoglycemia, a serious and life threating drop in blood sugar levels.

A coenzyme needed for this reaction is the oxidized form of nicotine adenine dinucleotide, or NAD+.

this exact coenzyme is used in a different reaction mediated by the alcohol dehydrogenase to convert the ethanol to acetaldehyde.

keep in mind that ethanol does not inhibit lactate dehydrogenase directly, but rather impairs its activity by capturing a much needed enzyme.

gestational diabetes and gluconeogenesis

the clinical correlation for gluconeogesis is gestational diabetes

when mothers who are pregnant have high blood glucose levels, this can cause severe consequences for the fetus.

high blood glucose levels from the mothers blood means a fed state in the uterine environment of the fetus (extra glucose).

as a result, upon birth, the environment drastically changes for the neonate, and is now not receiving the levels of glucose flowing from the mothers blood.

under starving conditions, the neonate will need to start gluconeogenesis, because of this process maybe delayed in the neonate who has been enriched with high blood glucose levels during term, it can result in hypoglycemia.

in short, the fetus does not use gluconeogenesis, as glucose is obtained from the mother.

gluconeogenesis starts immediately after birth.

babies from diabetic mothers experience a lasting fed state and gluconeogenesis is delayed.

severe hypoglycemia

the treatment involves cortisol administration and low concentrations of glucose

gestational diabetes

Gestational diabetes mellitus (GDM) is a type of diabetes that develops during pregnancy, usually in the second or third trimester, because pregnancy hormones make the mother’s tissues resistant to insulin.

Let’s break it down step-by-step so the physiology makes sense.

1. Etymology

gestatio = to carry or bear (a child)

Diabetes

• to pass through (referring to excessive urination)

Mellitus

• Latin mel = honey/sweet

So the full meaning is:

“Sweet urine condition occurring during pregnancy.”

2. What happens physiologically

During pregnancy the placenta produces hormones that oppose insulin.

Major hormones:

• Human placental lactogen (hPL)
• Progesterone
• Estrogen
• Cortisol
• Growth hormone (placental GH)

These hormones cause: Insulin resistance

This ensures that more glucose stays in the mother’s blood, so the fetus has enough fuel.

3. Why diabetes develops

Normally the mother compensates by producing more insulin.

Pancreatic β-cells increase insulin secretion.

However, IF the pancreas cannot produce enough insulin, blood glucose rises.

Insulin resistance + insufficient insulin → gestational diabetes

4. Timeline

Gestational diabetes usually appears: 24–28 weeks of pregnancy

This is when placental hormones become strongest.

That is why screening tests are done at this time.

5. How it affects the fetus

1. Maternal glucose crosses the placenta, but maternal insulin does not.

2. So the fetus receives high glucose levels.

3. The fetal pancreas responds by producing large amounts of insulin.

Result: Fetal hyperinsulinemia

Insulin is an anabolic hormone, so the baby grows excessively.

6. Major fetal complications

1. Macrosomia: Large baby (>4 kg)

• insulin stimulates fat deposition
• increases body growth

2. Neonatal hypoglycemia (after birth)

Before birth: mother provides glucose.

After birth: glucose supply stops but the baby still has high insulin.

Result: severe hypoglycemia