Gluconeogenesis

How can glucose be synthesized from lactate? (i.e. gluconeogenesis)

lactate --> pyruvate --> undergoes a series of reverse glycolytic steps to produce glucose

Where does gluconeogenesis take place? What parts of the cell participate in gluconeogenesis?

occurs mainly in liver & kidneys
occurs in mitochondria & cytoplasm

Name the most common precursors for gluconeogenesis. Where do these precursors for gluconeogenesis come from?

lactate (from RBCs & exercising muscle)
glycerol (breakdown of triglycerides)
amino acids (breakdown of protein)
propionate (odd # fatty acids, some AAs)
pentoses (PPP)
hexoses

Name the 3 irreversible steps in glycolysis. Name the enzymes.

Glucose --> G-6-P (hexokinase)
F-6-P --> F-1,6-BisP (phosphofructokinase)
PEP --> pyruvate (pyruvate kinase)

How are these steps bypassed in gluconeogenesis? Name the enzymes.

G-6-P --> G + Pi (glucose-6-phosphatase)
F-1,6-BisP --> F-6-P + Pi (fructose-1,6-Bisphosphatase)
Pyr --> PEP (3 steps)
1. Pyr + CO2 + ATP --> oxaloacetate + ADP (occurs in mitochondria; pyruvate carboxylase - contains biotin & requires acetyl CoA as positive modulator)
2. TRANSPORT: (2 rxns)
oxaloacetate + NADH --> malate + NAD+ (in mitochondria; malate dehydrogenase)
malate + NAD+ --> NADH + oxaloacetate (in cytoplasm; malate dehydrogenase)
(goal: moving oxaloacetate out to cytoplasm)
3. oxaloactetate + GTP --> PEP + CO2 + GDP (phosphoenolpyruvate carboxykinase - PEPCK)
OVERVIEW:
Mitochondria: Pyr --> oxac --> malate
Cytoplasm: malate --> oxac --> PEP + CO2

What is the role of pyruvate carboxylase in gluconeogenesis & anaplerosis? How is it regulated?

gluconeogenesis: pyruvate carboxylase adds CO2 to pyruvate --> oxaloacetate in mitochondria
anaplerosis: forms catalytic amounts of oxaloactetate in ALL mitochondria - produces oxaloacetate (when lacking) when stimulated by acetyl CoA, also requires ATP.
Pyruvate carboxylase = regulatory enzyme (requires biotin as coenzyme)
Key regulator: acetyl CoA = positive modulator (EXAM)
(acetyl CoA comes from beta-oxidation)

Write the sequence of steps involved in the conversion of pyruvate to phosphoenol pyruvate.

Pyr --> PEP (3 steps)
1. Pyr + CO2 + ATP --> oxaloacetate + ADP (occurs in mitochondria; pyruvate carboxylase - contains biotin & requires acetyl CoA as positive modulator)
2. TRANSPORT: (2 rxns)
oxaloacetate + NADH --> malate + NAD+ (in mitochondria; malate dehydrogenase)
malate + NAD+ --> NADH + oxaloacetate (in cytoplasm; malate dehydrogenase)
(goal: moving oxaloacetate out to cytoplasm)
3. oxaloactetate + GTP --> PEP + CO2 + GDP (phosphoenolpyruvate carboxykinase - PEPCK)
OVERVIEW:
Mitochondria: Pyr --> oxac --> malate
Cytoplasm: malate --> oxac --> PEP + CO2

Explain why acetyl CoA cannot serve as a precursor for gluconeogenesis.

acetyl CoA comes from beta-oxidation & from pyruvate (pyruvate dehydrogenase)
PDH is IRREVERSIBLE
pyr + CoA + NAD+ --> Acetyl CoA + NADH + CO2 (pyruvate dehydrogenase)

What key role does acetyl CoA play in gluconeogenesis?

It is a positive modulator of pyruvate carboxylase
Without it, the enzyme would not be able to bypass the conversion of pyruvate to PEP
EXAM

What is the role of biotin in gluconeogenesis? Recognize the structure of biotin. What is the nature of the biotin-enzyme linkage?

Acts as a coenzyme in carboxylation reactions, where it is a carrier of CO2 (find specific role)
With pyruvate carboxylase, phosphate from ATP activates the imidizole ring of biotin, allowing PC to add CO2 to pyruvate to produce oxaloacetate
Biotin-enzyme linkage: AMIDE linkage
CO2 binds to a nitrogen in imidizole ring

What kind of rxn is biotin known to participate in? Give the names of 3 such rxns.

Acts as a coenzyme in carboxylation reactions, where it is a carrier of CO2 (carboxyl groups bound to lysine residue of enzyme)
acetyl-CoA carboxylase: acetyl CoA --> malonyl CoA (ACC in FA synthesis)
pyruvate carboxylase: pyruvate --> oxaloacetate
(in mitochondria, gluconeogenesis)
propionyl CoA carboxylase (3 steps):
propionyl CoA --> succinyl CoA (TCA cycle)
ATP IS REQUIRED TO FORM CARBOXY BIOTIN-ENZYME

State the steps involved in the transport of oxaloacetate from mitochondria to cytoplasm. What role does malate play in this process?

mitochondria:
oxaloacetate + NADH --> NAD+ + malate (MDH)
Cytoplasm:
malate + NAD+ --> NADH + oxaloacetate (MDH)
oxaloacetate + GTP --> PEP (PEPCK)
role? malate transports oxaloacetate to cytoplasm

Describe why the glycerol-moiety of triglycerides and not the even-numbered fatty acids can be converted to glucose. Name the first glycolytic intermediate formed from glycerol.

Glycerol can be converted to glucose starting via glycerol kinase:
glycerol + ATP --> glycerol-3-P + ADP
glycerol-3-P + NAD+--> DHAP + NADH (glycerol-3-P-dehydrogenase)
First glycolytic intermediate: DHAP
Even-numbered fatty acids cannot form glucose because they are catabolized to acetyl CoA units (which cannot make pyruvate because PDH is irreversible)

Explain why muscle and brain cannot synthesize glucose from lactate.

brain & muscle are lacking in glucose-6-phosphatase
This allows the organs to use glucose solely for energy (none exported to liver)

How and where are (a) lactate and (b) alanine (produced by active skeletal muscle) converted to glucose?

(a) lactate from muscle is converted in the liver
(b) alanine from muscle is converted primarily in the liver (sometimes in kidney)
Both are converted to pyruvate --> eventually synthesizes new glucose

Why does adipose tissue triglyceride biosynthesis depend on the availability of glucose?

For triglyceride synthesis, glucose is required to make the backbone. However, in adipose tissue GLYCEROL KINASE is NOT present, so glycerol can't be phosphorylated --> DHAP not synthesized. Therefore, glucose is needed to synthesize DHAP, which can be converted into glycerol-3-P.
GLYCEROL KINASE IS MISSING IN ADIPOSE TISSUE

Explain how amino acids can serve as precursors for synthesis of glucose.

All feed directly or indirectly into TCA cycle intermediates...
Amino acids can be converted to TCA cycle intermediates by removal of amino group. Eventually, all of the TCA cycle intermediates will generate oxaloacetate --> transported out of mitochondria --> malate --> PEP --> gluconeogenic pathway

State the primary and secondary control steps in the synthesis of glucose from pyruvate.

1. acetyl CoA as positive modulator of pyruvate carboxylase
2. fructose-1,6-bisphosphatase (stimulated by ATP & citrate; inhibited by fructose-2,6-bisphosphate, fructose-1,6, bisphosphate, AMP, glucagon)
Gluconeogenesis and glycolysis are reciprocally regulated

How much ATP, GTP and NADH are required to synthesize 1 molecule of glucose from pyruvate in liver?

2 pyr + 4 ATP + 2 GTP + 2 NADH + 6 H2O --> glucose + 4 ADP + 2 GDP + 6Pi + 2NAD+
2 ATP @ pyr --> oxaloacetate
2 ATP @ 3-PG --> 1,3-BisPG
2 GTP @ oxaloacetate --> PEP
2 NADH @ 1,3 BisPG --> G-3-P

How does ethanol inhibit gluconeogenesis?

ethanol + NAD+ --> acetaldehyde + NADH+ + H+
(alcohol dehydrogenase)
acetaldehyde + NAD+ --> acetate + NADH
(acetaldehyde dehydrogenase)
Depletion of NAD+ = bad
Required to convert lactate to pyruvate. Inhibition of lactate dehydrogenase (due to NAD+ depletion) can lead to lactic acidosis, hypoglycemia, coma.