Lecture 9 Gluconeogenesis

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What is gluconeogenesis?

The synthesis of glucose from non carbohydrate precursors

What are the phases of glucose use over time?

Absorptive phase, postabsorptive phase, early starvation, intermediate starvation, then prolonged starvation

The postabsorptive phase is when what pathway is activated?

Glycogenolysis (since the exogenous carbon source is depleted)

What pathway is activated at some point in the early starvation phase?

Gluconeogenesis (since glycogen levels are decreasing)

What pathways are activated in intermediate starvation?

Ketogenesis and beta oxidation of fats

Why is gluconeogenesis activated during starvation?

To keep critical organs functional by making glucose from alternate sources

What is the overall reaction for gluconeogenesis from pyruvate?

2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H2O + 2H+ → Glucose + 2 NAD + 4 ADP + 2 GDP + 6 Pi

Does gluconeogenesis need more energy than the ATP directly produced by glycolysis?

YES

What process gives fuel for gluconeogenesis?

Beta oxidation of fats in the liver

Gluconeogenesis happens in situations where there is no?

Glucose

Any molecule that can be converted to pyruvate can be considered as?

Glucogenic

What are some non carbohydrate carbon sources that can be used for gluconeogenesis?

Pyruvate, lactic acid, oxaloacetate, glycerol, certain amino acids, so on

What are glucogenic molecules?

Molecules that can be used to make glucose (aka can feed into gluconeogenesis)

What enzymes catalyze reactions in gluconeogenesis and glycolysis?

Phosphoglucose isomerase, Aldolase, Triose phosphate isomerase, glyceraldehyde-3-phosphate (G3P) dehydrogenase, Phosphoglycerate kinase (PGK), Phosphoglycerate mutase, and Enolase

What are the bypass steps in gluconeogenesis?

Pyruvate → Oxaloacetate (catalyzed by pyruvate carboxylase)

Oxaloacetate → PEP (catalyzed by PEP carboxykinase)

F1,6-BP → F6P (catalyzed by fructose 1,6-bisphosphatase)

G6P → Glucose (catalyzed by glucose 6-phosphatase)

What are the enzymes involved in bypass steps in glycolysis?

Hexokinase, phosphofructokinase, and pyruvate kinase

What are bypass steps?

Steps that are not reversible between two opposite pathways (ex. glycolysis and gluconeogenesis)

Not reversible, exergonic, unidirectional steps

What enzymes are most likely inhibited when glycolysis is favored over gluconeogenesis?

PEP carboxykinase and pyruvate carboxylase

What is the first bypass reaction in gluconeogenesis?

Pyruvate is converted to oxaloacetate by pyruvate carboxylase using biotin as a cofactor and ATP as an energy source

What step in gluconeogenesis is anaplerotic for the TCA cycle and what molecule does it replenish?

1st step (Pyruvate to OAA) replenishes OAA

What is the cofactor for pyruvate carboxylase?

Biotin

Pyruvate carboxylase is allosterically activated by what molecule?

Acetyl CoA

Where does the first step of gluconeogenesis take place?

In the mitochondrial matrix

Where does the second step of gluconeogenesis take place?

IN the cytosol

What does PEPCK stand for?

PEP carboxykinase

In fasting, the synthesis of what gluconeogenic enzyme increases?

PEPCK

What is step 2 of gluconeogenesis?

Oxaloacetate is phosphorylated and converted into PEP by phosphoenolpyruvate carboxykinase (PEPCK) using a phosphate from either 1 ATP or 1 GTP

What is the source of phosphate groups for PEP carboxykinase?

ATP or GTP

Is the conversion of pyruvate into oxaloacetate during gluconeogenesis metabolically reversible?

NO it isn’t reversible

What is biotinylation?

The covalent attachment of biotin to an enzyme

Carbon is added to a substrate at what site in a carboxylase?

The C acceptor site

What are the steps of gluconeogenesis?

1. Pyruvate is reduced to OAA by Pyruvate carboxylase (using 1 ATP for energy and biotin as cofactor)

2. OAA is phosphorylated to get PEP by PEP carboxykinase (using 1 ATP or 1 GTP as phosphate source, releasing 1 CO2)

3. PEP undergoes hydration to get 2PG by Enolase

4. 2PG is isomerized to 3PG by Phosphoglycerate mutase

5. 3PG is phosphorylated to get 1,3BPG by Phosphoglycerate kinase

6. 1,3BPG is reduced to G3P by G3P dehydrogenase

7. DHAP from glycerol processing is converted into G3P by Triose phosphate isomerase

8. 2 molecules of G3P are rearranged into F1,6BP by Aldolase

9. F1,6BP is dephosphorylated to get F6P by Fructose 1,6-bisphosphatase

10. F6P is isomerized to G6P by phosphoglucose isomerase

11. G6P is dephosphorylated to get Glucose by glucose 6-phosphatase

What is dephosphorylation?

The hydrolysis of a phosphate group to remove said phosphate group from a substrate (usually done by phosphatases)

What enzyme converts G6P to glucose?

Glucose-6-phosphatase

What enzyme converts F6P to G6P?

Phosphoglucose isomerase

What enzyme converts F1,6BP to F6P?

Fructose 1,6-bisphosphatase

What enzyme converts G3P to F1,6BP?

Aldolase

What enzyme converts DHAP to G3P?

Triose phosphate isomerase

What enzyme converts 1,3BPG to G3P?

G3P dehydrogenase

What enzyme converts 3PG to 1,3BPG?

Phosphoglycerate kinase (PGK)

What enzyme converts 2PG to 3PG?

Phosphoglycerate mutase

What does PGK stand for?

Phosphoglycerate kinase

What molecule does pyruvate carboxylase use as an energy source?

ATP

What enzyme converts PEP to 2PG?

Enolase

What enzyme converts oxaloacetate to PEP?

PEPCK (PEP carboxykinase)

What enzyme converts pyruvate to oxaloacetate?

Pyruvate carboxylase

Name the major reactants, intermediates, and products of the gluconeogenesis of pyruvate?

Pyruvate + ATP + HCO3- or CO2 → Oxaloacetate + ADP + Pi

Oxaloacetate + ATP or GTP → PEP + ADP or GDP + CO2

PEP + H2O → 2PG

2PG → 3PG

3PG + ATP → 1,3BPG + ADP

1,3BPG + NADH → G3P + Pi + NAD+

DHAP → G3P

2x G3P → F1,6BP

F1,6BP + H2O → F6P + Pi

F6P → G6P

G6P + H2O → Glucose + Pi

What step in gluconeogenesis releases 1 CO2?

2nd step: OAA to PEP catalyzed by PEPCK

Where does gluconeogenesis happen?

Trick question: it happens in the mitochondria (step 1) then the cytosol (rest of the steps)

Can oxaloacetate exit the mitochondria?

NO

Is the a mitochondrial oxaloacetate transporter?

NOPE

Oxaloacetate must be converted into what molecule to exit the mitochondria?

Malate

Is there a malate shuttle in the mitochondria?

YES

How does oxaloacetate exit the mitochondria during gluconeogenesis?

Oxaloacetate is reduced to malate by malate dehydrogenase

Malate leaves the mitochondria via a malate transporter

Malate is oxidized to oxaloacetate by malate dehydrogenase

Is oxaloacetate reduced or oxidized to malate?

REDUCED

What enzyme reduces oxaloacetate to malate?

Malate dehydrogenase (Malate DH)

What enzyme oxidizes malate to oxaloacetate?

Malate dehydrogenase

Malate dehydrogenase is found where in the cell?

Trick question: It’s in the cytosol and the mitochondria

Is the dephosphorylation of F1,6BP metabolically reversible?

NO!

What does F1,6BPase stand for?

Fructose 2,6-bisphosphatase

What molecules allosterically inhibit F1,6BPase?

AMP and F2,6BP

The third bypass reaction in gluconeogenesis is catalyzed by?

F1,6BPase

What gluconeogenic enzyme opposes the activity of PFK1?

F1,6BPase

What do phosphatases do to their substrate?

They dephosphorylate their substrate using water to hydrolyze the substrate phosphate group bond

If F1,6BPase is active, what enzyme must be inhibited?

PFK1

Is the hydrolysis of G6P to Glucose metabolically reversible?

NOPE

Glucose-6-phosphatase is only found in what organs?

Main ex: liver, kidney

Possible ex: pancreas, and small intestine

Tissues from what organs are the only ones that can convert G6P to glucose?

Mainly: Liver, kidney

Possibly: pancreas, and small intestine

What is the final bypass step in gluconeogenesis?

G6P → Glucose (hydrolysis by glucose-6-phosphatase)

What is G6P a precursor for?

Glycogen and glucose synthesis

Glucose-6-phosphatase is highly regulated in what organ?

Liver

Is gluconeogenesis endergonic or exergonic?

ENDERGONIC

Is an input of energy needed to synthesize glucose?

YES, an input of energy is needed

What are the 4 mechanisms controlling flux through glycolysis/gluconeogenesis?

Allosteric control

Covalent modifications

Substrate cycles aka Futile cycles

Genetic control via regulation of enzyme concentrations (control transcription/translation)

Flux through a pathway is controlled at what type of steps?

Rate limiting steps

In the liver, what two pathways are reciprocally regulated?

Glycolysis and gluconeogenesis

What pathway does insulin stimulate in the liver?

Glycolysis

What pathway does glucagon stimulate in the liver?

Gluconeogenesis

High energy molecules generally stimulate what pathway in the liver?

Gluconeogenesis

Low energy state indicators generally inhibit what pathway in the liver?

Gluconeogenesis

What molecules are indicators of a high energy state in the liver?

TCA cycle intermediates (ex. citrate, acetyl CoA), high ATP concentrations

What molecules are indicators of a low energy state in the liver?

AMP, ADP, F2,6BP

What is a futile cycle?

Two opposing steps in two pathways (that, if both pathways are running, result in a cyclic process between the two opposing steps)

What are the enzymes in the futile cycle example for glycolysis and gluconeogenesis?

PFK1 and FBPase-1

What does a 1 after the enzyme name (ex. enzyme-1) stand for?

That the enzyme catalyzes a metabolic step

What does a 2 after the enzyme name (ex. enzyme-2) stand for?

That the enzyme regulates the metabolic enzymes

What is the isomeric form of F1,6BP?

F2,6BP

Is F2,6BP a metabolically intermediate in glycolysis or gluconeogenesis?

NONE, it ain’t in either pathway

What pathway is favored if F2,6BP levels are high?

Glycolysis

What enzyme does F2,6BP activate?

PFK1

What enzyme does F2,6BP inhibit?

FBPase-1

F2,6BP is what type of regulator?

Allosteric regulator

What enzymes regulate F2,6BP levels?

PFK2 and FBPase2

What effect does PFK2 have on F2,6BP levels?

Favors forming more F2,6BP so increases F2,6BP levels?

What effect does PFK2 have on PFK1 and FBPase1?

Increases F2,6BP levels, which promotes PFK1 activation and inhibition of FBPase1

Does PFK2 favor glycolysis or gluconeogenesis?

Glycolysis

What effect does FBPase2 have on F2,6BP levels?

FBPase2 reduces F2,6BP levels by dephosphorylating F2,6BP

What effect does FBPase2 have on FBPase1 and PFK1?

Decreases F2,6BP levels by dephosphorylating F2,6BP to make F6P, which decreases promotion of PFK1 activity and decreases inhibition of FBPase1

Does FBPase2 favor glycolysis or gluconeogenesis?

Gluconeogenesis

What enzyme phosphorylates FBPase2 and PFK2?

PKA