• Introduction• Basic mechanisms – intersections of pathways• Physiological questions

• Major sites of regulation• Regulation in liver• Regulation in cardiac muscle

• Alcohol metabolism• Pathways• Clinical correlations • Clinical scenario: Chronic alcohol consumption

Lecture 5-3: Regulation of GlycolysisGlycolysis is a central pathway of glucose metabolism and therefore of carbohydrate metabolism. Its regulation is central to energy management.

All images are from “Marks et al., 2nd Edition, Copyright © 2005 Lippincott & Williams, A Wolters Kluwer Co., All rights reserved” unless otherwise noted.

NADPH

(storage)

Complexcarbohydrates

ATP

TCA Cycle

Glycolysis is central to both catabolic and anabolic metabolism

Glucose-6-P

Glycolysis

Figure 2 of the Section Five Introduction, Mark’s et al.

Note: G6P is not transported back across the plasma membrane

• Catabolic reactions: •Source of ATP

• Anabolic reactions: •Pyruvate is precursor for fatty acid biosynthesis and alanine

•Pentose phosphates precursor of nucleotides.

Major pathways of glucose metabolism

NADPH

(storage)

Complexcarbohydrates

ATP

TCA Cycle

Glucose-6-P

Glycolysis

Figure 2 of the Section Five Introduction, Mark’s et al.

Glucose

Lactate

AnaerobicGlycolysis

Alternate fates of pyruvate

Figure 22.5: glycolysis occurs exclusively in the cytosol

Glucose + 2 ADP + 2 Pi →2 Lacate + 2 ATP + 2 H2O + 2 H+

The overall ATP yield for the complete oxidation of one mole of glucose to CO2 is 36 – 38 moles

x2

x2

Fig. 22.9

A brief note about ‘shuttles’

Fig. 22.8: Malate - aspartate shuttle Fig. 22.7: Glycerol 3-phosphate shuttle

Review of Unit 4…

Regulation of enzymatic activity – A pathway is only as fast as its slowest step

• Enzymes may be activated (+) or inhibited (-)• The concentration of enzymes may be increased or decreased

through the regulation of transcription or translationInduction (+) or repression (-)

• Mechanisms of regulation includeAllostericHormonal

Physiological roles• Tissue specific responses in glucose homeostasis

Liver is a “maintainer organ” – its principal role is to maintain homeostasisMuscle is a “consumer organ” – its principal role is to convert chemical to mechanical energy

PiGlucose 6-

phosphatase

The major sites of glycolysis regulation in the liver

Figure 36.1: Regulation of glucokinase, PFK-1, and pyruvate kinase in the liver.

↑ Insulin : glucagon ratio –positively regulates PFK-1 and PK

Synthesis of Glucokinase is induced by insulin

The major sites of glycolysis regulation in the skeletal muscle

Figure 22.12: Regulation of hexokinaseand PFK-1 in skeletal muscle.

Product inhibition

Regulatory considerations with regard to glucokinase• Km (S0.5) ≈ 7 - 10 mM - physiological blood glucose is

5 - 10 mM (80-160 mg/dl)• Glucokinase enzyme induced by increased ratio of insulin : glucagon• Balance of glucokinase and glucose 6-phosphatase activities

(G 6-P → glucose + Pi)

Fig. 31.14 -Differences in Kmbetween the liver enzyme glucokinaseand hexokinase, the enzyme that is found in other tissues and catalyzes the same reaction

PiGlucose 6-

phosphatase

The major sites of glycolysis regulation in the liver

Figure 36.1: Regulation of glucokinase, PFK-1, and pyruvate kinase in the liver.

↑ Insulin : glucagon ratio –positively regulates PFK-1 and PK

Regulation of Phosphofructokinase-1 (PFK-1)PFK-1 catalyzes Fructose 6-Phosphate → Fructose 1,6 Bisphosphate

• Allosteric regulation of PFK-1 by AMP to a lower Km (higher affinity)

Fig. 22.14 Regulation of PFK-1 by AMP, ATP and fructose 2,6-bisphosphate.

+AMP

Regulation of glycolysis in cardiac muscle by AMP

Marks Figure 22.13: Changes in ATP, ADP and AMP concentrations in skeletal muscle during exercise.

Rest

Exercise~ 20%

~ 300%2 ADP ←⎯⎯→ AMP + ATP

adenylate kinase

Regulation of Phosphofructokinase-1 (PFK-1)

• Allosteric regulation of PFK-1 by F2,6-BP to a lower Km (higher affinity)

• F2,6-BP is more effective activator of PFK-1, mole for mole, than AMP

Fig. 22.14 Regulation of PFK-1 by AMP, ATP and fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate (F2,6-BP) regulates PFK-1• Fuctose 2,6-BP is synthesized by Phosphofructokinase-2 (PFK-2)• The level of F 2,6-BP is controlled by phosphorylation of PFK-2

• F2,6-BP is not an glycolysis intermediate

• F2,6-BP regulates PFK-1 in both liver and adipose tissue

• [F2,6-BP] increases when the insulin : glucagon ratio increases since glycolysis in these tissues is the carbon source for the synthesis of triacylglycerols

PFK-2 has both of these activities

Phosphofructokinase-2 (PFK-2) is a bifunctional enzyme. The ratio of activities is changed by phosphorylation and dephosphorylation of PFK-2.

Fructose 2,6-Bisphosphate is synthesized and degraded by a single enzyme, PFK-2

Coordinate regulation of liver PFK-2

↑ cAMP

Higher PFK-2 kinase activity

Higher PFK-2 phosphataseactivity

results in more F 2,6-BP

results in lessF 2,6-BP

• The two activities of PFK-2 are controlled by Protein Kinase A and phosphoprotein phosphatase

Activation of glycolysis by Fructose 2,6-Bisphosphate

• In liver and adipose tissue after a high carbohydrate meal (↑ Insulin : Glucagon)

• PFK-2 is dephosphorylatedby protein phosphorylase

• The kinase activity of PFK-2 increases

• Therefore, levels of F2,6-BP increase

• F2,6-BP allostericallyactivates PFK-1

• Glycolysis is stimulated allowing glucose to be converted to triacylglycerols for storage

P

PFK-2 PFK-2phosphorylase

F-6-PF-2,6-BP

PFK-1F-6-P F-1,6-P

Inhibition of glycolysis in the LIVER by glucagon or epinephrine via a signal transduction cascade

The steps of a signaling cascade…activation of protein kinase A (PKA) →phosphorylation of phosphofructokinase-2 (PFK-2) →increase in the fructose 2,6-bisphosphatase activity →less fructose 2,6-bisphosphate (F 2,6-BP) →phosphofructokinase-1 (PFK-1) activity decreases →

…resulting in less glycolysis in the liver

From Figure 28.8:

↓ Insulin : Glucagon

The response of PFK-2 to hormones is tissue specific

For example, the hormone epinephrine mobilizes fuels during acute stress -

• Liver: A ‘maintainer’ organ, provides plasma glucose by limiting glycolysis: Epinephrine → elevated cAMP, phosphorylates bifunctional enzyme → increases PFK-2 phosphatase activity → less F 2,6 BP

• Cardiac muscle: A ‘consumer’ organ, increases glycolysis to produce pyruvate for energy production:Epinephrine → elevated cAMP, phosphorylates bifunctional enzyme → increases PFK-2 kinase activity → more F 2,6 BP

LiverPFK-2

Liver phosphoryation favors phosphatase activity

CardiacMusclePFK-2 Muscle phosphoryation favors kinase activity

Regulation of liver pyruvate kinase by phosphorylation status. P = a serine residue phosphorylated by protein kinase A. (Alternative splicing yields different proteins.)

Tissue specific enzyme isozymes

Liver:Pyruvate kinase

Liver:Pyruvate kinase

Alcohol (Ethanol) is a fuel

Ethanol is principally metabolized in the liver providing as many as 13 moles of ATP per mole of ethanol

Alcohol (Ethanol) is a fuel

Use of ethanol as a motor fuel on a large scale raises environmental and food supply concerns

Alcohol metabolismAlcohol dehydrogenase(Cytosolic enzyme)

Aldehyde dehydrogenase(Mitochondrial enzyme)

Fig. 25.1

(Toxic)

(Nontoxic)Fig. 25.2

Alcohol metabolism

TCA cycleFatty Acid Synthesis

Clinical scenario: Metabolic effects of chronic ethanol consumption

• Summary of metabolic pathways• Ethanol is both lipid and water soluble and thus is

readily absorbed by passive diffusion • Ethanol is principally metabolized in the liver with as

many as 13 ATP synthesized per molecule• Products are acetaldehyde, acetate and acetyl CoA• Acetyl CoA →TCA cycle or fatty acid synthesis in the liver • Acetaldehyde is toxic• Reducing equivalent NADH and H+ produced

• Nutritional Effects• Chronic alcohol consumption results in decreased

adsorption of vitamins B1 (thiamin) and B12

• Behavioral Effects – (Acetaldehyde)

Nutritional effects?

?Source: Carlsberg brewery website

• The enzymes of alcohol metabolism are a family of isozymes• Variation in the isozyme proportions affects

• rate of alcohol clearance, • degree of inebriation, • side effects of alcohol consumption and• susceptibility to alcohol-induced liver disease

• Points 2 – 4 are directly related to the concentration of the acetaldehyde intermediate

• The common allele of ALDH a low Km resulting in a high affinity for its substrate acetaldehyde

• Since even low concentrations of acetaldehyde (toxic) are converted to acetate (nontoxic), the concentration of the intermediate rarely exceeds 20 μM even during intoxication.

• An ALDH isozyme present with ~ 40% frequency in East Asian populations has a high Km resulting in low affinity for its substrate acetaldehyde

• High acetaldehyde concentrations result from minimal alcohol consumption

• Accumulation of toxic acetaldehyde results in facial flushing, nausea and vomiting

• Possession of this isozyme is associated with protection against alcoholism

Genetic variation of aldehydedehydrogenase (ALDH)

• Isozymes of alcohol dehydrogenase that have a low Km result in metabolic changes similar to an acetaldehyde dehydrogenase isozyme with high Km and thus low affinity.

• The concentration of a reaction intermediate is the result of its influx and efflux…

Genetic variation of alcohol dehydrogenase (ADH)

Why isn’t ethanol a ‘good’ fuel?

The products of its metabolism alter the flow of metabolites…

Alcohol metabolism favors ketosis and triglyceride synthesis through the excessive production of NADH