Carbohydrate Metabolism III & IV

Home , Carbohydrate metabolism, Gluconeogenesis, Glycolysis

Carbohydrate Metabolism III & IV - Gluconeogenesis - - Anaplerosis and Cataplerosis - - Pentose Phosphate Pathway – - Regulation of Glycolysis & Gluconeogenesis -

FScN4621W Food Science and Nutrition University of Minnesota

Unit III

 Gluconeogenesis  Anaplerosis and Cataplerosis

Glycolysis Glycolysis under anaerobic conditions

C6H12O6 (6 carbon) Cytosol

2X Pyruvate (3 carbon) NADH NAD+ 2X lactate (3 carbon)

1 Glycolysis &TCA Oxidation of glucose or breakdown of glucose

C6H12O6 (6 carbon) Cytosol glycolysis

2X Pyruvate (3 carbon) PDH CO2 2X Acetyl CoA (2 carbon) aerobic conditions TCA O2

6 CO2 + 6H2O + 2ATP + 6NADH & 2FADH Oxidative phosphorylation

Mito ATPs 4

Respiratory Chain and Oxidative Phosphorylation

 Respiratory chain consists of four complexes  Complex I: NADH dehydrogenase  Complex II: Succinate dehydrogenase  Complex III: Cytochrome bc1 complex  Complex IV: Cytochrome c oxidase  Complex I, III, and IV are proton pumps  ATP synthase is an ATP-dependent proton pump  A series of oxidation-reduction reactions make up the electron transport.  Oxidation: a chemical substance takes on oxygen or loses electrons.  Reduction: a chemical substance gives off oxygen or takes on electrons  Electrons pass through four complexes and generate energy  This energy is transferred to ADP for synthesis of ATP by establishing proton gradient concentration across the inner membrane 5

Respiratory Chain

FADH2

NADH → Complex I → Q → Complex III → cytochrome c → Complex IV → O2 ↑ ↑ Complex II 6

2 Glycerol 3-Phosphate Shuttle

Malate- Aspartate Shuttle

Respiratory chain

Gluconeogenesis

 Biosynthesis of glucose from non- carbohydrate precursors

 Maintenance of blood glucose within normal ranges

 It occurs primarily in _____ and _____ (tissues)

 It occurs primarily in ______and ______(intracellular compartments)

 NOT exactly a reversal of glycolysis

3 Common Precursors for Gluconeogenesis

 Pyruvate  Lactate  Glycerol  Propionate

 Glucogenic amino acids

 AA converted to Pyruvate: Ala, Ser, Gly, Thr, Cys, Tryp  AA converted to Oxaloacetate: Asp  AA converted to -Ketoglutarate: Glu, Gln, Pro, His, Arg

Three Irreversible Reactions in Glycolysis

Hexokinase Glucokinase Glucose glucose 6-phosphate

6-Phosphofructo-1-kinase Fructose 6-phosphate Fructose 1,6-bisphosphate

Pyruvate kinase Phospoenolpyruvate pyruvate

Three Bypass Reactions in Gluconeogenesis

Pyruvate kinase Phospoenolpyruvate pyruvate

2 1 Phosphoenolpyruvate Pyruvate carboxykinase carboxylase

6-Phosphofructo-1-kinase Fructose 6-phosphate Fructose 1,6-bisphosphate

Fructose 1,6-biphosphatase

Hexokinase Glucose glucose 6-phosphate

Glucose 6-phosphatase 12

4 Gluconeogenesis

* Occurs in mitochondria and cytosol

Mitochondria: • Generation of PEP • 2 key reactions • 2 key enzymes PEP Cytosol: • Conversion of PEP to glucose • 2 key reactions * • 2 key enzymes

Lactate/pyruvate: 4 reactions PEP Glutamate: 3 reactions * Glycerol: 2 reactions

13 *

Conversion of PEP Pyruvate to PEP 2 2 key reactions 2 key enzymes *

Mitochondrion PEP

1 *

* 14

Gluconeogenesis

Glycogen

Cytosol: • Conversion of PEP to glucose • 2 key reactions • 2 key enzymes

PEP

5 Gluconeogenesis

Conversion of F-1,6-BisP to F-6-P

glycolysis GNG * *

Conversion of G-6-P to Glucose

GNG * * glycolysis

 Catalyzed by Glucose-6-phosphatase  Its presence determines whether a tissue can contribute to circulating glucose

 In which tissues is this enzyme expressed? 16

Cori Cycle

Specific gluconeogenic pathway involving de novo synthesis of glucose from Lactate

 Production of lactate mainly by muscle and red blood cells Lactate lactate glucose

 Transport to the liver

 Typical gluconeogenic pathway – four key reactions/enzymes are invovled

 Glucose into the circulation Red blood cell

Muscle

Alanine Cycle

 Specific gluconeogenic pathway involving de novo synthesis of glucose from Alanine

 Alanine is released to the circulation by skeletal muscle  In muscle it is derived by transamination of pyruvate  In liver, deamination of alanine generates pyruvate  glucose  circulation  muscle

Alanine Alanine Alanine

deamination transamination Glucose

Pyruvate Glucose Pyruvate

 Gluconeogenesis involves four key reactions/enzymes

6 Glutamate as a precursor

 Gluconeogenic pathway involving TCA cycle and bypassing the reaction by pyruvate carboxylase  Glutamate is converted to Oxaloacetate via TCA cycle  GNG using Glu as a precursor bypasses pyruvate kinase catalyzed reaction

PEP

Glucose Fatty acid

TCA Cycle

Amino acid

Harper’s Chapter 16, Figure 16- 3 Glutamate 20

Glycerol as a precursor

 Gluconeogenic pathway only in cytosol  Two last key reactions/enzymes

7 Energy Utilization in GNG

 6 molecules of ATP are utilized to produce 1 molecule of glucose from 2 molecules of pyruvate

 In liver, this energy required for de novo synthesis of glucose comes from fatty acid oxidation or partial amino acid oxidation

Gluconeogenesis during the starvation

Glucose goes to the partial oxidation in the liver and muscle for sparing carbon backbone Brain (pyruvate and lactate) for Red blood cells gluconeogensis in the liver

Glucose

Cori cycle Lactate Lactate

GNG OXA X X

Alanine

Liver Alanine cycle Muscle

Clinical Correlation

During fasting  Defects in PEPCK  What happens to blood glucose control?

  What happens to blood lactate levels?

  What happens to glycogen store? 

PEPCK: phosphoenolpyruvate carboxykinase

8 Clinical Correlation

 Defects in Glucose-6-phosphatase  What happens to blood glucose control?

  What happens to blood lactate levels?

  What happens to glycogen store? 

Glucose-6-phosphatase

Gluconeogenesis in the Kidney

 It occurs during prolonged starvation  Contributes 10% of gluconeogenically derived glucose  After 14-16h fasting, 20-25% of gluconeogenically derived glucose  provides ~50% of the net glucose synthesis during prolonged starvation

 Precursors: mostly glucogenic amino acids  Glutamine mainly released from muscle

 Gluconeogenic pathway:  It involves anaplerotic and cataplerotic reactions

9 Anaplerosis and Cataplerosis

 Series of enzymatic reactions or pathways that replenish the pools of metabolic intermediates in the TCA cycle.

 Keep the pool size of TCA intermediates not to be largely changed during high energy consumption (e.g. exercise) or during lower energy consumption (e.g. fasting).

 Anaplerosis – Entry of 4- and 5-carbon intermediates into the TCA cycle

 Cataplerosis – Exit of 4- and 5-carbon intermediates from the TCA cycle

 Anaplerosis and cataplerosis need to be balanced to avoid the accumulation of intermediates and to remain the normal function of TCA cycle

Anaplerosis and cataplerosis in the TCA cycle

Owen, O. E. et al. J. Biol. Chem. 2002;277:30409-30412 29

Role of TCA Cycle in Gluconeogenesis

Owen, O. E. et al. J. Biol. Chem. 2002;277:30409-30412 30

10 Role of Anaplerosis and Cataplerosis in Glutamine Metabolism in the small intestine

Glutamine is metabolized for energy in the small intestine.

PEPCK Cytosol

Mito

Owen, O. E. et al. J. Biol. Chem. 2002;277:30409-30412 31

Role of Anaplerosis and Cataplerosis in Glyceroneogenesis in Adipose Tissue

Amino acids Glucose Provides glyceride- glycolysis glycerol from non-sugar precursor Glycerol-P

pyruvate

Anaplerosis

Glycerol-P Cataplerosis

32 Owen, O. E. et al. J. Biol. Chem. 2002;277:30409-30412

Unit IV

 Pentose Phosphate Pathway  Regulation of Glycolysis & Gluconeogenesis

11 Pentose Phosphate Pathway

G-6-P Dehydrogenase Glucose-6-Phosphate 6-Phosphogluconolactone

+ NADP+ NADPH + H

H2O

H+ 6-Phosphogluconolactonase 6-Phosphogluconate Phosphogluconate Dehydrogenase

NADP+ Ribulose-5-Phosphate + CO NADPH + H+ 2

Pentose Phosphate Pathway

• A secondary pathway of glucose oxidation

• A source of reducing equivalents: NADPH

• Production of 5-carbon sugar phosphates - Ribose 5-phosphate - Xylulose 5-phosphate

• Takes place in the cytosol

Pentose Phosphate Pathway

• NADPH is required for synthesis of fatty acids, cholesterol, and sterols - mammary gland, adipose tissue, liver, adrenal cortex and testis

• NADPH is required for maintenance of the integrity of the red blood cells

• Ribose 5-phosphate is essential for biosynthesis of nucleotide and nuclear acids (ATP, coenzyme A, NAD, NADP, FAD, RNA and DNA)

12 Regulation of Glycolysis & Gluconeogenesis

 Glucose transport  Substrate cycle  Substrate levels can control the rate of metabolic pathways  Coordinates glycolysis and gluconeogenesis  Hormonal regulation  Enzyme activity  Gene expression

Glucose Transporters

Transporter Major tissue site Affinity for Km glucose GLUT1 Brain, Placenta High Low

GLUT2 Liver, Pancreatic β-cells Low High

GLUT3 Brain, Placenta High Low

GLUT4 Skeletal and cardiac High Low muscle, Adipose tissue

GLUT5 Small intestine *Transport fructose

Michaelis Constant Km

• The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity.

• The amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum.

13 Glucose Transporters

 GLUT1 and GLUT3:  Low Km or high affinity for glucose  Active when blood glucose levels are low  Responsible for basal glucose uptake in most tissues

 GLUT2:  High Km or low affinity for glucose  Active when blood glucose levels are high  A key transporter responding to elevated blood glucose

 GLUT4:  Low Km or high affinity for glucose  Transport basal glucose uptake, BUT to a small extent – WHY?  Mainly responsible for insulin-stimulated glucose uptake

 GLUT2 and GLUT4 transport glucose into the cells for glucose utilization through different mechanisms  GLUT2: regulated by glucose levels  GLUT4: regulated by insulin 40

Regulation of Glycolysis & Gluconeogenesis - substrate cycles -

 Glycolysis is regulated at 3 steps:  Non-equilibrium reactions (irreversible) catalyzed by:  Glucokinase  Phosphofructokinase  Pyruvate kinase

 Gluconeogenesis  Phosphoenolpyruvate carboxykinase (PEPCK)  Pyruvate carboxylase  Fructose 1,6-biphosphatase  Glucose 6-phosphatase

Substrate Cycles Regulation of Glycolysis and Gluconeogenesis

Stipanik’s chapter 12, Figure 12-8 42

14 Regulation of Glycolysis and Gluconeogenesis

Nutrients Physiological stimuli

Hormonal Regulation Insulin Glucogan Glucocorticoids Epinerpherine 1 Short-term regulation 2 2 Long-term regulation 1 2 3 Enzyme Activity Gene Expression Allosteric regulation Enzymes involved in Phosphorylation glycolysis

and gluconeogenesis 43

Protein Phosphorylation

 Phosphorylation is the addition of a phosphate (PO4) group to a protein molecule on serine, threonine, and tyrosine residues.

 It is a reversible process and an important regulatory mechanism.

 Enzymes called kinases and phosphatases are involved in this process.

 Kinases - phosphorylation (addition of a phosphate (PO4) group)  Phosphatases – dephosphorylation (removal of a phosphate (PO4) group)

 Phosphorylation results in a conformational change in the structure in enzymes and receptors, causing them to become activated or deactivated.

 Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation.

Hormonal Regulation

INSULIN • In the ______state, insulin levels rise.

• ____ glycolysis and glycogen storage

• ____ gluconeogenesis and glycogenolysis • ______expression of the glucokinase gene and ______expression of the PEPCK gene

• Stimulates glucose transport into ______and ______• Net result: ____ blood glucose levels 45

15 Hormonal Regulation Glucagon, Glucocorticoids and Epinephrine - counterregulatory hormones to insulin action -

• In the ______, levels are increased.

• _____is the main targeting tissue of glucagon

• ____ glycogenolysis and gluconeogenesis

• Glucagon ______expression of PEPCK gene

• Stimulates glucose output from the liver

• Net result: ____ blood glucose levels

What you need to know - glucose transport -

 How different GLUTs (1-4) are distributed in different tissues?  How different GLUTs in different tissues play a different role in regulation of glucose uptake in the fed and fast state?  What characteristics of GLUTs determine their roles in transporting glucose in different tissues?  How GLUTs activity is regulated?

What you need to know - hormonal regulation -

 In which physiological condition or when, do hormone (insulin/glucagon/epinepherine) levels rise or fall?  Which organs/cells produce/release hormones?  What is the role of hormones or why our body secret them in terms of glucose metabolism?  What are the target/responsive tissues/cells of hormones?  What do hormones do on glucose metabolism and how?

16 What you need to know - substrate cycle -

 Which substrates are involved in regulation of glucose metabolism?  Fructose 6-P  Fructose 1-P  Fructose 1,6 bisphosphate (Fructose 1, 6P2)  Fructose 2,6 bisphosphate (Fructose 1, 6P2)  Citrate  ATP/AMP  Where do these substrates come from? Or from which part of pathway reactions are they produced?  How they regulate glycolysis and gluconeogenesis in different physiological conditions?  What are the target enzymes of these substrates? or Which enzymes are regulated by these substrates and how?