Lecture 25: Regulation of carbohydrate metabolism; pentose phosphate pathway

True or False: Fructose 2,6-biphosphate activates glycolysis and inhibits gluconeogenesis via conversion of fructose 6-phosphate to fructose 1,6-biphosphate.

true

What activates PFK (and thus the conversion of fructose 6-phosphate to fructose 1,6-biphosphate)?

• F-2,6-BP

• AMP

What inhibits PFK (and thus the conversion of fructose 6-phosphate to fructose 1,6-biphosphate)?

• ATP

• citrate

• H⁺

What activates fructose 1,6-biphosphatase (and thus the conversion of fructose 1,6-biphosphate to fructose 6-phosphate)?

citrate

What inhibits fructose 1,6-biphospatase (and thus the conversion of fructose 1,6-biphosphate to fructose 6-phosphate)?

• F-2,6-BP

• AMP

What activates pyruvate kinase (and thus the conversion of PEP to pyruvate)?

• F-1,6-BP

What inhibits pyruvate kinase (and thus the conversion of PEP to pyruvate)?

• ATP

• alanine

What activates PEP carboxykinase (and thus the conversion of PEP to OAA)?

nothing!

What inhibits PEP carboxylase (and thus the conversion of PEP to OAA)?

ADP

What activates pyruvate carboxylase (and thus the conversion of pyruvate to OAA)?

acetyl CoA

What inhibits pyruvate carboxylase (and thus the conversion of pyruvate to OAA)?

ADP

What are the key regulatory points of glucose utilization by tissues and glucose biosynthesis by the liver?

• glucose entry

• hexokinase isozymes

• PFK-1 versus fructose 1,6-biphosphatase

• pyruvate kinase versus pyruvate carboxylase/PEP carboxykinase

How is glucose entry regulated?

• in some tissues such as the liver, RBC, and the CNS, glucose transporters are constituvely “on”

• in tissues such as muscle and adipose tissue, glucose transporters are up-regulated by insulin and removed from the plasma membrane in the absence of insulin

What is the process for regulation of glucose transporters?

1. glucose transporters are “stored” within the cell in membrane vesicles

2. when insulin interacts with its receptor, vesicles move to the surface and fuse with the PM—increasing the number of glucose transporters in the PM

3. when insulin level drops, glucose transporters are removed from the PM by endocytosis—forming small vesicles

4. the smaller vesicles fuse with the larger endosome

5. patches of the endosome enriched with glucose transporters bud off to become small vesicles, ready to return to the surface when insulin levels rise again

True or False: Glucose transporters from intracellular pool move to the cell membrane upon insulin action in many cell types such as adipose and muscle cells.

true

What is important to know about the regulating hexokinases?

• hexokinases I-III:

  • low V_max

  • K_m is lower than blood glucose

  • inhibited allosterically by glucose 6-phosphate

  • high affinity for glucose

• hexokinase IV/glucokinase:

  • half saturated at 10 mM glucose

  • the rate of glucose phosphorylation is roughly proportional to blood glucose

  • high V_max

  • liver isozyme

  • low affinity for glucose

  • not allosterically inhibited by glucose 6-phosphate

What does insulin decrease and glucagon increase the expression of?

glucose 6-phosphatase expression

True or False: The K_m of glucokinase (10 mM) is too high to reconvert glucose (4mM) back to G-6-P during gluconeogenesis.

true

What is important to know about the regulation of fructose-2,6-bisphosphate?

• strong positive regulator of PFK-1

• negative regulator of fructose-1,6-biphosphoatase

• F-2,6-BP concentrations are high in the fed state and low in the fasting/starving state

• fructose-2,6-biphosphate increases the affinity of PFK-1 for fructose-6-phosphate

What is the source of F-2,6-BP, and how is it regulated?

• F-2,6-BP is synthesized from F-6-P by PFK-2

• F-2,6-BP is converted back into F-6-P b FBPase-2

• a single bifunctional protein has both PFK-2 and FBPase-2 enzymatic activities, but enzymatic activities are reciprocally regulated 

What is the process for PFK2/FBP2 regulation?

1. high glucagon to insulin ration causes elevated cAMP and increased levels of active protein kinase A

2. increased protein kinase A activity favors the phosphorylated form of the bifunctional PFK-2/FBP-2

3. phosphorylation of the PFK-2 domain inactivates it allowing the FBP-2 domain to be active

4. decreased levels of F-2,6-BP decreases the inhibition of FBP-1, which leads to an increased rate of gluconeogenesis

True or False: Glucagon induces PEPCK expression to stimulate gluconeogenesis—leading to increased transcription and the stabilization the PEPCK mRNA.

true

What are other names for the pentose phosphate pathway?

• hexose monophosphate shunt

• 6-phosphogluconate pathway

True or False: No ATP is consumed or produced in the pentose phosphate pathway; the pathway is energetically neutral.

true

What is the starting point of the pentose phosphate pathway?

glucose 6-phosphate

What are the goals of the pentose phosphate pathway?

• generate NADHP (which is necessary for reductive biosynthesis)

• generate ribose-5-phosphate (which is necessary for nucleotide biosynthesis)

• interconversion of sugars

True or False: A sudden increase in the pentose phosphate pathway activity is the fastest known cellular response to oxidative stress by generating NADPH.

true

What are the two stages of the PPP?

• oxidative reactions (irreversible)

• nonoxidative reactions (reversible)

What is important to know about the first step in the PPP?

• substrate(s): a-D-glucose-phosphate

• coactivator(s): NADP⁺

• enzyme: glucose-6-P dehydrogenase (G6PD)

• product(s): 6-phospho-D-gluconolacetone + NADPH + H⁺

• notes: rate limiting step, irreversible, NADPH inhibits G6PD

What is important to know about step 2 of the PPP?

• substrate(s): 6-phospho-D-gluconoalctone

• coactivator(s): H₂O and Mg²⁺

• enzyme: lactonase

• product(s): 6-phospho-gluconate

• notes: NOT rate-limiting but is irreversible

What is important to know about step 3 of the PPP?

• substrate(s): 6-phosphogluconate

• coactivator(s): NADP⁺ and Mg²⁺

• enzyme: 6-phosphogluconate dehydrogenase

• product(s): D-ribulose-5-phosphate + NADPH + H⁺ + CO₂

• notes: irreversible, oxidative decarboxylation reaction

How does NADP⁺ differ from NAD⁺?

by a single phosphate on one of the riboses

What is NAPDH important for?

• reductive biosynthesis (fatty acid and steroids)

• keeping glutathione reduced

• generating NO

What is the approximate ration of NADPH to NADP⁺ in the cytosol of hepatocytes?

10:1

Why is NADPH kept mostly in the reduced state in the cytosol?

to participate as electron donors in reductive biosynthesis

What is the ration of NADH to NAD⁺ in the cytosol of heptaocytes?

1:1000

Why is NAD⁺ kept mostly in th?

to serve as an electron acceptor in catabolic reactions

What is important to know about glutathione?

• functions:

  • acts as a redox buffer

  • keeps cysteines of cytosolic proteins in the reduced state

  • keeps heme iron in the Fe²⁺ state

  • detoxifies reactive oxygen species

  • serves as the reducing agent in some biosynthetic reactions

  • protects cells from reactive oxygen species (when glutathione is reduced)

What keeps glutathione in the reduce state?

NADPH

True or false: The PPP is the only that red blood cells can make NADPH.

true

What are the two other uses of NADPH?

• cytochrome P450

• synthesis of NO

What is important to know about cytochrome P450?

• monooxygenase (mixed function oxidase)

• superfamily of hemeproteins that catalyze a variety of oxidation reactions

• some P450s involved in biosynthesis of steroid hormones, bile acids, and other molecules

• some P450s detoxify foreign compounds

• R-H + O₂ + NADPH + H⁺ → R-OH + H₂O + NADP⁺

What are some of the functions of NO?

• potent vasodilator

• acts as a neurotransmitter

• decreases platelet aggregation

• role in macrophage function

What is NO synthesized by?

NO synthase from arginine, O₂, and NADPH

How does NO act as a vasodilator?

relaxes vascular smooth muscle by stimulating cGMP production (guanylyl cyclase is an NO receptor and effector)

What does NO synthase activate?

cGMP-dependent protein kinase—leading to myosin light chain dephosphorylation

Which molecule produces NO?

nitroglycerin

What is nitroglycerin used to treated?

Angina pectoris

What is important to know about a glucose 6-P dehydrogenase (G6PD) deficiency?

• most common disease-producing enzyme abnormality in humans

• compromises ability of RBCs to generate NADPH

• results in hemolytic anemia, which is due to the inability to detoxify oxidizing agents (owing to insufficient reduced glutathione in RBCs)

• X-linked

• highest prevalence in Middle East, Mediterranean, tropical Africa, and Asia

• female carriers have increased resistance to falciparum malaria

What are the precipitating factors of hemolytic anemia in G6PD deficiency?

• oxidant drugs

• favism (from oxidizing agents in food such as fava beans)

• infection (from oxidants produced during inflammation)

True or False: G6PD deficiency is associated with increased incidence of neonatal jaundice.

true

True or false: The activity of G6PD declines as red cells age, but even the oldest cells have a sufficient level of activity to provide protection against oxidative damage and hemolysis.

true

True or False: Very few G6PD Mediterranean red cells have sufficient enzyme activity to prevent oxidative damage, whereas a substantial fraction of young G6PD A^- red cells are able to provide protection.

true

What is important to know about the non-oxidative (reversible) reactions in the PPP?

• the pathway catalyzes the interconversion of 3-, 4-, 5-, 6-, and 7- carbon sugars

• allows conversion of ribulose 5-P to ribose 5-P or glycolysis intermediates

• six 5-cabon sugars interconvert with five 6-carbon sugars

• all reactions are fully reversible (direction of metabolite flow depends on the cell’s metabolic needs)

What is important to know about the production of ribose 5-phosphate through the PPP?

• substrate(s): D-ribulose-5-phosphate

• coactivator(s): none

• enzyme: phosphopentose isomerase

• product(s): D-ribose-5-phosphate

• notes: ketose-aldose isomerization reaction

What is the starting point for nucleotide biosynthesis?

D-ribose-5-P

What is important to know about the production of xylulose 5-phosphate through the PPP?

• substrate(s): ribulose 5-phosphate

• coactivator(s): none

• enzyme: ribulose 5-phosphate epimerase

• product(s): xylulose 5-phosphate

What is xylulose 5-P a key regulaor of?

carbohydrate and fat metabolism

How does the PPP create NADPH instead of ribose?

transketolase (shuffles 2 carbons) and trans aldolase (shuffles 3 carbons) can convert xylulose-5-P into glyceraldehyde-3-P and fructose-6-P

True or False: For tissues (like RBC) that need to make NADPH but don’t need to make nucleotide precursors, ribulose 5-P can be recycled back to glucose 6-P.

true

What is important to know about the chemistry of the carbon shuffle?

• in both the transketolase and aldolase reactions, a carbanion nucleophile is resonance-stabilized

• transketolase uses TPP as the cofactor

• transaldolase is protonated by a Schiff base

• the TPP ring stabilizes the carbanion in the dihydroxy-ethyl group carried by transketolase

How does the PPP support rapid cell growth?

to create ribose, the non-oxidative reactions can synthesize ribose 5-P from glyceraldehyde 3-P and fructose 6-P (can bypass oxidative reactions)