09 - Gluconeogenesis

TARGET DECK: MED::I::Signaling Pathways in Health and Disease::Metabolic Biochemistry::09 - Gluconeogenesis

Gluconeogenesis — Overview

Why Gluconeogenesis?

• The brain depends on a continuous supply of glucose: it cannot synthesize glucose or store much glycogen.
• All glucose in the bloodstream can supply the brain for no more than one hour → hypoglycemia has rapid neural consequences.
• When dietary sources are unavailable and liver glycogen is exhausted, glucose must be synthesized from non-carbohydrate sources.
• Gluconeogenesis can provide a substantial part of blood glucose just a few hours after eating.

Primary (only) sites of gluconeogenesis: Liver, kidney cortex

Gluconeogenic sources:

• Lactate
• Pyruvate (ending point of glycolysis)
• Glycerol
• Glucogenic amino acids:
  • These can be converted into pyruvate or other intermediates:
  • Gly, Ala, Cys, Ser, Asp, Asn, Glu, Gln, Pro, Arg, His, Val, Met, Thr
  • Partly glucogenic: Trp, Ile, Phe, Tyr

What are the primary sites of gluconeogenesis?

Liver and kidney cortex.

Anki cloze

The brain cannot store significant glycogen, and all blood glucose can supply it for no more than {1:one hour}, making gluconeogenesis essential during fasting.

---

Gluconeogenic Precursors — Metabolic Entry Points

Precursor	Entry Point
Glycerol	Glycerol-3-P → DHAP
Lactate / Alanine	Pyruvate
Glucogenic amino acids	Pyruvate or TCA intermediates → Oxaloacetate
TCA intermediates (from FA oxidation)	Oxaloacetate

Warning

Fatty acids are NOT glucogenic Acetyl-CoA (product of FA oxidation) cannot provide net synthesis of oxaloacetate → fatty acids cannot be converted to glucose.

---

The Cori Cycle

Cori Cycle

Intercellular cycle between muscle and liver:

• Muscle: Glucose → (Glycolysis) → Pyruvate → (LDH) → Lactate + $2\text{NADH}$
• Liver: Lactate → (LDH) → Pyruvate → (Gluconeogenesis) → Glucose (costs $6\text{ATP}$, $2\text{GTP}$)

$\text{Muscle: Glucose}\stackrel{\text{Glycolysis}}{\to }2\text{Pyruvate}\stackrel{\text{LDH}}{\to }2\text{Lactate}$

$\text{Liver: }2\text{Lactate}\stackrel{\text{LDH}}{\to }2\text{Pyruvate}\stackrel{\text{GNG}}{\to }\text{Glucose}$

What is the Cori Cycle?

The metabolic cycle in which lactate produced by anaerobic glycolysis in muscle is transported to the liver, converted back to pyruvate, and used for gluconeogenesis to regenerate glucose.

---

The Alanine Cycle

Alanine Cycle

Parallel to the Cori cycle, but carries nitrogen as well as carbon:

• Muscle: Amino acids → transamination → Alanine + α-ketoglutarate (from pyruvate + ${\text{NH}}_4^{+}$ via glutamate)
• Alanine travels in blood to liver
• Liver: Alanine → pyruvate → gluconeogenesis; nitrogen enters the urea cycle

How does the Alanine Cycle differ from the Cori Cycle?

The Alanine Cycle transports both carbon (as pyruvate) and nitrogen (as an amino group) from muscle to liver, while the Cori Cycle transports only carbon (as lactate). Nitrogen from the Alanine Cycle feeds into the urea cycle in the liver.

---

Gluconeogenesis vs. Glycolysis — General Relationship

Key Principle

Gluconeogenesis follows glycolysis in reverse, except for three irreversible reactions, which are bypassed by three distinct sets of reactions. While glycolysis is entirely cytosolic, the first bypass of gluconeogenesis involves mitochondrial steps.

---

The Three Bypasses of Gluconeogenesis

First Bypass — Pyruvate → Phosphoenolpyruvate

Glycolytic reaction (irreversible): $\text{Pyruvate kinase:}\text{PEP}+\text{ADP}\to \text{Pyruvate}+\text{ATP}$

Bypass reactions:

1. Pyruvate carboxylase (mitochondrial): $\text{Pyruvate}+{\text{HCO}}_3^{-}+\text{ATP}\to \text{Oxaloacetate}+\text{ADP}+P_i$

2. PEP carboxykinase (PEPCK): $\text{Oxaloacetate}+\text{GTP}\to \text{PEP}+{\text{CO}}_2+\text{GDP}$

Energetics of First Bypass

The overall free energy change is strongly negative ($\Delta G=-25\text{kJ/mol}$), making this bypass effectively irreversible.

What two enzymes bypass the pyruvate kinase reaction in gluconeogenesis?

Pyruvate carboxylase (pyruvate → oxaloacetate) and PEP carboxykinase (oxaloacetate → PEP).

Anki cloze

Pyruvate carboxylase converts pyruvate to {1:oxaloacetate} using {2:${\text{HCO}}_3^{-}$} and {3:ATP}, and is exclusively {4:mitochondrial}.

---

Second Bypass — Fructose-1,6-bisphosphate → Fructose-6-phosphate

Glycolytic reaction (irreversible): $\text{PFK-1:}\text{Fructose-6-P}+\text{ATP}\to \text{Fructose-1,6-bisP}+\text{ADP}$

Bypass: $\text{FBPase-1:}\text{Fructose-1,6-bisP}+{\text{H}}_2\text{O}\to \text{Fructose-6-P}+P_i$

---

Third Bypass — Glucose-6-phosphate → Glucose

Glycolytic reaction (irreversible): $\text{Hexokinase/Glucokinase:}\text{Glucose}+\text{ATP}\to \text{Glucose-6-P}+\text{ADP}$

Bypass: $\text{Glucose-6-phosphatase:}\text{Glucose-6-P}+{\text{H}}_2\text{O}\to \text{Glucose}+P_i$

What are the three irreversible glycolytic reactions that are bypassed in gluconeogenesis, and what enzymes perform the bypass?

1. Pyruvate kinase → bypassed by Pyruvate carboxylase + PEPCK
2. PFK-1 → bypassed by FBPase-1
3. Hexokinase/Glucokinase → bypassed by Glucose-6-phosphatase

---

Pyruvate Carboxylase — Structure and Mechanism

Pyruvate Carboxylase

• Exclusively mitochondrial
• Four subunits, each with:
  • ATP + ${\text{HCO}}_3^{-}$ binding site
  • Biotin-binding site (mobile arm — Vitamin B7/H)
  • Pyruvate-binding site
  • Allosteric site for acetyl-CoA
• Positively regulated by glucagon
• Negatively regulated by insulin

Mechanism: $\text{Step 1: }{\text{HCO}}_3^{-}+\text{ATP}\stackrel{\text{biotin}}{\to }\text{Carboxybiotinyl-enzyme}+\text{ADP}+P_i$ $\text{Step 2: Carboxybiotinyl-enzyme}+\text{Pyruvate (enolate)}\to \text{Oxaloacetate}+\text{Biotinyl-enzyme}$

Biotin is Vitamin B7 (also called Vitamin H) — water-soluble. It acts as a mobile carboxyl carrier on a long flexible arm.

What is the allosteric activator of pyruvate carboxylase?

Acetyl-CoA.

Anki cloze

Pyruvate carboxylase is {1:exclusively mitochondrial} and uses {2:biotin} as a cofactor (mobile arm) to transfer a carboxyl group from {3:${\text{HCO}}_3^{-}$} to pyruvate, forming {4:oxaloacetate}.

---

PEP Carboxykinase (PEPCK)

PEPCK

• Located in both mitochondria and cytosol
• Catalyzes phosphorylation from GTP and decarboxylation: $\text{Oxaloacetate}+\text{GTP}\to \text{PEP}+{\text{CO}}_2+\text{GDP}$
• The ${\text{CO}}_2$ removed is the same carbon previously added by pyruvate carboxylase

Where is PEP carboxykinase (PEPCK) located?

Both in the mitochondria and cytosol (unlike pyruvate carboxylase, which is exclusively mitochondrial).

---

Reducing Power for Gluconeogenesis

Cytosolic NADH Requirement

Gluconeogenesis requires cytosolic NADH for the reduction of 1,3-BPG to glyceraldehyde-3-phosphate.

Source depends on gluconeogenic precursor:

Carbon Source	How Cytosolic NADH is Generated
Lactate	$\text{Lactate}+{\text{NAD}}^{+}\to \text{Pyruvate}+\text{NADH}+{\text{H}}^{+}$ (cytosolic LDH)
Amino acids	Malate exported from mitochondrion → oxidized in cytosol: $\text{Malate}+{\text{NAD}}^{+}\to \text{OAA}+\text{NADH}+{\text{H}}^{+}$

$\Delta G_{\text{Malate}\to \text{OAA}}^{\prime \circ }=+29.7\text{kJ/mol}$

Why is the malate shuttle used during gluconeogenesis from amino acids?

Oxaloacetate cannot cross the mitochondrial membrane, so it is first reduced to malate (which can cross), then re-oxidized to oxaloacetate in the cytosol — simultaneously generating the cytosolic NADH needed for gluconeogenesis.

---

Gluconeogenesis from Amino Acids

Entry Points into the Pathway

Entry Point	Amino Acids
Pyruvate	Ala, Cys, Gly, Ser, Trp*
α-Ketoglutarate	Arg, Glu, Gln, His, Pro
Succinyl-CoA	Ile*, Met, Thr, Val
Fumarate	Phe*, Tyr*
Oxaloacetate	Asn, Asp

*Also ketogenic

Purely Ketogenic Amino Acids (NOT glucogenic)

Leucine and Lysine — broken down only to acetyl-CoA and/or acetoacetyl-CoA; they cannot furnish net carbon for glucose synthesis.

Mnemonic — "Leu and Lys are the Lonely Ketones"

Leucine and Lysine = the only Locked-out amino acids (purely ketogenic, never glucogenic).

Specific metabolic routes:

• Alanine → Pyruvate → Oxaloacetate → Malate → cytosol
• Aspartate → Oxaloacetate → Malate → cytosol
• Glutamate → α-Ketoglutarate → Succinyl-CoA → … → Malate → cytosol
• Valine → Succinyl-CoA → … → Malate → cytosol

Which two amino acids are purely ketogenic and cannot contribute to gluconeogenesis?

Leucine (Leu) and Lysine (Lys).

Anki cloze

Aspartate enters gluconeogenesis at the level of {1:oxaloacetate}, while alanine first becomes {2:pyruvate} before being carboxylated to oxaloacetate.

---

Routes for Cytosolic OAA — Two Variants of the First Bypass

When lactate is the precursor

• Lactate → pyruvate (cytosolic LDH, generating NADH)
• Pyruvate enters mitochondria → carboxylated to OAA by pyruvate carboxylase
• Cytosolic PEPCK converts OAA → PEP directly in cytosol
• Malate shuttle not primarily needed (NADH already provided by LDH)

When amino acids are the precursor

• OAA in mitochondria → reduced to malate → exported
• Malate → OAA in cytosol (generates cytosolic NADH)
• Cytosolic PEPCK converts OAA → PEP

---

Overall Stoichiometry of Gluconeogenesis

$2\text{Pyruvate}+4\text{ATP}+2\text{GTP}+2\text{NADH}+2{\text{H}}^{+}+4{\text{H}}_2\text{O}\to \text{Glucose}+4\text{ADP}+2\text{GDP}+6P_i+2{\text{NAD}}^{+}$

Sequential reactions from pyruvate:

Reaction	Enzyme	×n
Pyruvate + ${\text{HCO}}_3^{-}$ + ATP → OAA + ADP + $P_i$	Pyruvate carboxylase	×2
OAA + GTP → PEP + ${\text{CO}}_2$ + GDP	PEPCK	×2
PEP + ${\text{H}}_2\text{O}$ → 2-phosphoglycerate	Enolase	×2
2-PG → 3-PG	Phosphoglycerate mutase	×2
3-PG + ATP → 1,3-BPG + ADP	Phosphoglycerate kinase	×2
1,3-BPG + NADH → G3P + NAD${}^{+}$ + $P_i$	GAPDH	×2
G3P → DHAP	Triose phosphate isomerase	×1
G3P + DHAP → Fructose-1,6-bisP	Aldolase	×1
Fructose-1,6-bisP + ${\text{H}}_2\text{O}$ → Fructose-6-P + $P_i$	FBPase-1 (bypass)	×1
Fructose-6-P → Glucose-6-P	Phosphoglucose isomerase	×1
Glucose-6-P + ${\text{H}}_2\text{O}$ → Glucose + $P_i$	Glucose-6-phosphatase (bypass)	×1

Anki cloze

Gluconeogenesis from 2 pyruvate costs {1:4 ATP}, {2:2 GTP}, and {3:2 NADH} — significantly more energy than is gained by glycolysis ({4:2 ATP net}).

---

Physiological Role of Gluconeogenesis

Why gluconeogenesis is necessary Brain, nervous system, and red blood cells generate ATP mainly from glucose. Gluconeogenesis:

• Allows generation of glucose when glycogen stores are depleted (starvation, vigorous exercise)
• Can generate glucose from many amino acids, but not from fatty acids

During prolonged starvation

• Initial fuel: amino acids (gluconeogenic)
• Gradually, body shifts to protect critical proteins and enzymes
• Brain shifts from all-glucose to glucose + ketone bodies (acetone, acetoacetate, β-hydroxybutyrate) derived from FA oxidation

---

Gluconeogenesis from Glycerol

Glycerol is the only glucogenic part of lipids (the glycerol backbone of triacylglycerols):

$\text{Glycerol}\stackrel{\text{glycerol kinase (ATP)}}{\to }\text{Glycerol-3-phosphate}\stackrel{{\text{glycerol-3-P dehydrogenase (NAD}}^{+}\text{)}}{\to }\text{DHAP}\to \text{gluconeogenesis}$

OCC(O)CO

(Glycerol)

What part of a lipid molecule can be used for gluconeogenesis?

The glycerol backbone (only). Fatty acids cannot contribute to net glucose synthesis.

---

What CANNOT Be Used for Gluconeogenesis

Not fuel for gluconeogenesis

• Acetyl-CoA
• Fatty acids (broken down to acetyl-CoA)
• Leucine and Lysine (broken down only to acetyl-CoA / acetoacetyl-CoA)

There is no path for the NET synthesis of oxaloacetate from acetyl-CoA.

However, fatty acid oxidation provides much of the ATP that fuels gluconeogenesis.

---

Acetyl-CoA as a Metabolic Signal

Acetyl-CoA signals that further glucose oxidation is not needed:

• Allosterically stimulates pyruvate carboxylase → promotes gluconeogenesis
• Allosterically inhibits pyruvate dehydrogenase complex → prevents further pyruvate → acetyl-CoA

How does acetyl-CoA coordinate gluconeogenesis and glucose oxidation?

Acetyl-CoA allosterically activates pyruvate carboxylase (promoting gluconeogenesis) and allosterically inhibits pyruvate dehydrogenase (blocking further oxidation of pyruvate).

---

Regulation of Gluconeogenesis in the Liver

Hormonal Regulation

Hormone	Effect on Gluconeogenesis	Effect on Glycolysis
Glucagon	↑ (stimulates)	↓ (inhibits)
Insulin	↓ (inhibits)	↑ (stimulates)
Cortisol	↑ (stimulates)	—

Anki cloze

The major regulatory step in gluconeogenesis is at the level of {1:PFK-1} (glycolysis) and {2:FBPase-1} (gluconeogenesis), at the interconversion of {3:fructose-6-phosphate} and {4:fructose-1,6-bisphosphate}.

---

Fructose-2,6-Bisphosphate (F-2,6-bP) — Key Allosteric Regulator

F-2,6-bP is NOT a glycolytic intermediate — it is a regulatory molecule only

Effect of F-2,6-bP	Target	Pathway
Allosteric activation	PFK-1	Glycolysis ↑
Allosteric inhibition	FBPase-1	Gluconeogenesis ↓

$\Delta G_{\text{F6P}\to \text{F1,6bP}}^{\prime \circ }=-16.3\text{kJ/mol}$

What is the role of fructose-2,6-bisphosphate in metabolic regulation?

It allosterically activates PFK-1 (promoting glycolysis) and inhibits FBPase-1 (suppressing gluconeogenesis). It is a regulatory molecule, not a metabolic intermediate.

---

The Tandem Enzyme PFK-2/FBPase-2

PFK-2 and FBPase-2 are contained in the same bifunctional protein

Regulation by phosphorylation state:

State	Active Domain	F-2,6-bP Level	Metabolic Effect
Dephosphorylated	PFK-2 (kinase)	↑ High	Stimulates glycolysis, inhibits gluconeogenesis
Phosphorylated	FBPase-2 (phosphatase)	↓ Low	Inhibits glycolysis, stimulates gluconeogenesis

Hormonal control:

$\text{Glucagon}\to \uparrow [\text{cAMP}]\to \text{PKA}\to \text{Phosphorylates tandem enzyme}\to \text{FBPase-2 active}\to \downarrow [\text{F-2,6-bP}]\to \text{Gluconeogenesis}\uparrow$

$\text{Insulin}\to \text{PP2A}\to \text{Dephosphorylates tandem enzyme}\to \text{PFK-2 active}\to \uparrow [\text{F-2,6-bP}]\to \text{Glycolysis}\uparrow$

PP2A (Protein Phosphatase 2A) is also activated by xylulose-5-phosphate.

What happens to the PFK-2/FBPase-2 tandem enzyme when glucagon levels rise?

Glucagon → cAMP → PKA phosphorylates the tandem enzyme → FBPase-2 becomes active (PFK-2 inactive) → F-2,6-bP falls → PFK-1 inhibited, FBPase-1 activated → gluconeogenesis increases.

Anki cloze

Phosphorylation of the PFK-2/FBPase-2 bifunctional enzyme activates {1:FBPase-2}, while dephosphorylation activates {2:PFK-2}. Glucagon promotes {3:phosphorylation} via {4:PKA}.

---

Additional Allosteric Regulation of PFK-1/FBPase-1

Effector	Effect on PFK-1	Effect on FBPase-1
F-2,6-bP	Activates ↑	Inhibits ↓
AMP, ADP	Activates ↑	—
ATP	Inhibits ↓ (negative effector AND substrate)	—
Citrate	Inhibits ↓	—
AMP	—	Inhibits ↓

What is the dual role of ATP at PFK-1?

ATP is both a substrate for PFK-1 and a negative allosteric effector — at high concentrations it inhibits PFK-1, slowing glycolysis when energy charge is high.

---

Transcriptional Regulation of Gluconeogenesis

Transcription Factor	Activator	Effect
CREB	Glucagon → cAMP	Induces gluconeogenic enzymes
ChREBP	Insulin → PP2A	Induces glycolytic and insulin-dependent enzymes
SREBP	Insulin	Induces glycolytic enzymes; represses glucogenic enzymes
FOXO1	(constitutive; inhibited by insulin)	Stimulates gluconeogenic enzymes

FOXO1 and Insulin Insulin → PKB (Akt) → phosphorylates FOXO1 → FOXO1 undergoes ubiquitin-mediated degradation → gluconeogenic gene expression ↓

How does insulin suppress gluconeogenic gene expression via FOXO1?

Insulin activates PKB (Akt), which phosphorylates FOXO1, targeting it for ubiquitin-mediated degradation, thereby reducing transcription of gluconeogenic enzymes.

---

Nutritional Sensors of Metabolism

Sensors of Low Energy / Low Nutritional Intake

(Activate catabolic pathways, gluconeogenesis, mitochondrial function)

• AMPK — AMP-dependent protein kinase
• Sirtuins — protein deacetylases
• PGC-1α (PPAR-γ Coactivator 1α) — activates mitochondrial biogenesis
• FOXO — transcription factor

Sensors of High Nutritional State

(Activate anabolic pathways, protein synthesis, growth)

• Insulin
• IGF — Insulin-like Growth Factor
• mTOR (Target of Rapamycin) — activated by PKB

What does AMPK sense and what pathways does it activate?

AMPK senses a low energy state (high AMP:ATP ratio) and activates catabolic pathways including gluconeogenesis and mitochondrial function.

---

Clinical Disorders of Gluconeogenesis

Disorder	Prevalence	Key Features
Glucose-6-phosphatase deficiency	~1/100,000	Glycogen storage disease
Pyruvate carboxylase deficiency	<1/250,000	No survival beyond infancy
PEPCK deficiency	Very rare (5–6 cases)	No survival beyond infancy
FBPase-1 deficiency	~1/500,000	Rapid-onset hypoglycemia on fasting; lactic acidosis (NADH buildup); managed by avoiding fructose/sucrose and glucose infusion

FBPase-1 Deficiency

• Presents with rapid-onset hypoglycemia during fasting
• Lactic acidosis due to NADH buildup (nausea, vomiting, weakness)
• Clinical management: avoid fructose and sucrose; glucose infusion

Why does FBPase-1 deficiency cause lactic acidosis?

Without FBPase-1, gluconeogenesis is blocked, so lactate cannot be converted to glucose. Lactate accumulates, and NADH builds up, causing lactic acidosis.

---

TLDR - 09 - Gluconeogenesis

Gluconeogenesis — Comprehensive Summary

• Definition: Synthesis of glucose from non-carbohydrate precursors; occurs mainly in liver and kidney cortex
• Gluconeogenic precursors: Lactate, pyruvate, glycerol, and most amino acids (all except Leu and Lys)
• Fatty acids are NOT glucogenic — acetyl-CoA cannot provide net OAA synthesis; only glycerol(from TAG) can
• Three irreversible glycolytic steps are bypassed by unique enzymes:
  1. Pyruvate kinase → Pyruvate carboxylase (mito) + PEPCK (mito + cyto)
  2. PFK-1 → FBPase-1
  3. Hexokinase → Glucose-6-phosphatase
• Pyruvate carboxylase: mitochondrial only; requires biotin (B7); activated by acetyl-CoA; stimulated by glucagon, inhibited by insulin
• PEPCK: both mitochondrial and cytosolic; removes the same CO₂ added by pyruvate carboxylase
• Cytosolic NADH needed for 1,3-BPG → G3P; provided by LDH (lactate source) or malate shuttle (amino acid source)
• Overall cost: 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH → Glucose + 6 Pᵢ + 2 NAD⁺
• Cori Cycle: muscle lactate → liver gluconeogenesis → glucose back to muscle
• Alanine Cycle: muscle amino acids → alanine → liver gluconeogenesis + urea cycle
• Key regulatory step: PFK-1 (glycolysis) vs. FBPase-1 (gluconeogenesis), regulated by F-2,6-bP
• F-2,6-bP: activates PFK-1 (glycolysis ↑); inhibits FBPase-1 (gluconeogenesis ↓); controlled by bifunctional PFK-2/FBPase-2
• Hormonal regulation: Glucagon → PKA → phosphorylates PFK-2/FBPase-2 → FBPase-2 active → F-2,6-bP ↓ → gluconeogenesis ↑; Insulin → reverse; Cortisol → gluconeogenesis ↑
• Transcriptional regulation: CREB (glucagon/cAMP), ChREBP & SREBP (insulin), FOXO1 (pro-gluconeogenic; degraded by insulin/PKB)
• Nutritional sensors — low energy: AMPK, Sirtuins, PGC-1α, FOXO → activate catabolism & gluconeogenesis
• Nutritional sensors — high energy: Insulin, IGF, mTOR (via PKB) → activate anabolism
• Acetyl-CoA: dual signal — activates pyruvate carboxylase (GNG ↑) and inhibits pyruvate dehydrogenase (glycolysis ↓)
• Clinical disorders: Deficiencies of G6Pase, pyruvate carboxylase, PEPCK (lethal in infancy); FBPase deficiency causes hypoglycemia + lactic acidosis