Energetics, Glucose Metabolism and Aerobic Metabolism - Lectures 146-147, 151

Energetics, Glucose Metabolism and Aerobic Metabolism - Lectures 146-147, 151 

1. Appreciate that diabetes is associated with a fundamental metabolic imbalance, centred on defective utilisation of carbohydrates

Cat diabetes:

• usually Type II
• generally severely insulin dependant by the time symptoms show
• can be treated successfully
• treatment can lead to remission
• low carb diet - there is a deficiency in carb metabolism
• can lead to neuropathy

Causes:

• pancreas producing insufficient insulin - Type I
• Failure of cells to respond to insulin - Type II

Results in: 

• inability of cells to absorb glucose
• overly high levels of glucose in blood ---> hyperglycaemia

Dog Diabetes:

• Usually Type I
• auto-immune disease

2. Discuss the regulation of the 3 enzymes catalysing the essentially irreversible reactions of glycolysis in terms of substrate and product levels, other forms of allostery, and hormonal control, highlighting the dynamic nature of liver metabolism

Glycolysis: 

• Glycolysis happens most of the time, in most tissues. 
• Very controlled in Liver and Muscles
• Liver is sensor/metaboliser of body
• has more pathways with more controls
• anaerobic
• fast reactions, so it can provide immediate energy

3 enzymes catalysing irreversible reactions: 
Points of control

1. hexokinase - phosphorylates glucose
2. phosphofructaokinase - PFK
3. pyruvate kinase - phosphorylates PEP

Each has allosteric regulator(s): 

1. hexokinase
• inhibitor = glucose 6-P
2. PFK: key regulator in glycolysis
• inhibitor = ATP, citrate, H⁺
• stimulator = AMP
3. pyruvate kinase
• inhibitor = ATP
• stimulator = Fructose 1,6-BP

• When lacking ATP and glycolysis is not producing, the following reaction occurs: 
•  2ADP <---> ATP + AMP
• The AMP stimulates more  PFK and thus glycolysis

Energy consumed points: 

• blood glucose ---> glucose 6-P
• 1 ATP
• fructose 6-P ---> fructose 1,6-BP
• 1 ATP

Energy generation points: 

• Fructose ---> PEP 
• 2 ATP
• 2 NADH
• PEP ---> pyruvate
• 2 ATP

Net Production: 

• 2 ATP
• 2 NADH
• 2 Pyruvate

Kinase = enzyme that phosphorylates a substrate

Regulatory methods of glycolysis: 
Mass Action: 

• substrate v. product ratio regulation

Allostery: 

• substrate binding at non-active site to change activity
• changes the shape of the substrate, so active site cannot be used
• active site has higher affinity than allosteric site

Hormones: 

• influence throughout cycle
• regulate enzyme or expression of enzyme (translation/transcription or rate of destruction) 
• Glucagon
• inhibits glycolysis in the liver
• acts on cell surface recpetor
• it tells liver to export glucose to the rest of the body where it's needed
• acts on pyruvate kinase by adding phosphate to it which inhibits glycolysis
• indirect effect on PFK2 - changes Fructose 1,6-BP back to Fructose 6-P
• Insulin
• increases glycolysis in liver
• acts on it's own cell surface receptor
• to make glycogen and store it
• make ATP and some fatty acids, TAGs
• activates a protein, PP1, which increases glycolysis

3. Explain the fates of pyruvate, which reflect the need to regenerate NAD⁺ and the oxygen status of the tissue

Pyruvate and NADH: 

• usually converted to AcetylCoA
• used in CAC
• NADH donates electrons to ETC to generate more ATP

When hypoxic (not enough oxygen): 

• ETC cannot accept more electrons
• therefore pyruvate is converted to lactate
• regenerates NAD⁺ so glycolysis can continue
• specific microorganisms anaerobically convert pyruvate into ethanol, regenerating NAD⁺

4. Discuss how the link reaction and citric acid cycle are regulated, reflecting the need for ATP and use of NADH, through allosteric effects on key enzymes

Link Reaction: 

• link between glycolysis and CAC
• irreversible reaction catalyzed by pyruvate dehydrogenase (PDH)
• inhibitors: ATP, NADH, AcetylCoA
• stimulators: pyruvate, Ca²⁺, insulin
• CA²⁺ in muscle due to increased contractions, thus need more ATP made

Citric Acid Cycle (CAC):

• CAC is the final common pathway for the oxidation of fuel molecules
• Serves as a source of building blocks for biosyntheses
• hormones do not have much of an effect on CAC
• 3 enzymes with alloseteric controllers:
• citrate synthase
• inhibitor: ATP
• isocitrate dehydrogenase
• inhibitors: ATP, NADH
• stimulators: ADP
• α-KG dehydrogenase
• inhibitors: ATP, NADH, succinyl-CoA
• ATP is common allosteric inhibitor
• main purpose of CAC is to make ATP
• therefore if ATP exists, CAC is not necessary
• GTP turns into ATP easily and can be considered 'an ATP'

Dehydrogenase - enzymes which take off CO₂ and adds electrons

Net Production for CAC per turn: 
(will turn 2x for every glucose molecules as 2 pyruvates are produced)

• 3 NADH
• 1 FADH₂
• 1 GTP/ATP
• 2 CO₂
• 1 Oxaloacetate

Summary of CAC: 

• 2 carbons enter (acetylCoA) ---> leave as 2CO₂
• in 4 redox reactions, 3 pairs of electrons are transferred to 3NAD⁺ ---> 3NADH and one pair to FAD ---> FADH₂
• Needs oxygen. Requires NAD⁺ and FAD and are regenerated in ETC when NADH and FADH pass elections to O₂ in ETC
• Outcome is synthesis of ATP
• tightly regulated through alloseteric effects on 3 regulated enzymes and control of supply of acetylCoA by regulation of PDH in Link reaction

5. Explain the functioning of the electron transport chain in creating a proton gradient over the inner mitochondrial membrane

• Oxidative phosphorylation: Electrons on NADH and FADH₂ yield energy when transferred to O₂ in ETC - generates ATP
• If no Oxygen is available, all components of ETC will accumulate in reduced form (accumulate electrons)
• If oxygen is added, the electron carriers in cytochrome oxidase will become oxidised before NADH-Q reductase 

ETC - NADH (from CAC)

1. NADH in and 2 electrons taken off, pumps out 4H⁺
2. electrons continue to 2nd enzyme, pumps out 2H⁺
3. electrons continue to 3rd enzyme, accepted by O₂ to form H₂O, pump 4H⁺out

This forms an electrochemical gradiant

Potential energy created

Total: 10H⁺

ETC- for FADH₂ (from CAC):

1. FADH₂ in and 2 electrons taken off
2. electrons continue to 2nd enzyme, pumps out 2H⁺
3. electrons continue to 3rd enzyme, accepted by O₂ to form H₂O, pump 4H⁺out

This forms an electrochemical gradiant

Potential energy created

Total: 6H⁺

ETC for NADH from Glycolysis:

1. NADH in and 2 electrons taken off - different starting point, skips 1st enzyme
2. electrons continue to 2nd enzyme, pumps out 2H⁺
3. electrons continue to 3rd enzyme, accepted by O₂ to form H₂O, pump 4H⁺out

This forms an electrochemical gradiant

Potential energy created

Total: 6H⁺

6. Explain how the proton gradient drives ATP synthesis 

NADH pumps 10H⁺

FADH₂ pumps 6H⁺
Therefore, one cycle of CAC is (3NADH x 10H⁺) + (1FADH₂ x 6H⁺) = 36 H⁺

• A Proton Motive Force (pH gradient and transmembrane electric potential) is generated over inner mitochondrial membrane due to the accumulation of H ions.
• As protons flow back into mitochondrial matrix through ATP synthase, ATP is made
• ADP + Pi ---> ATP
• therefore oxidation and phosphorylation are coupled
• 26 of 30 ATP molecules from glucose are generated in this way

• 3 protons come through, adds Pi to ADP to make ATP 
• reversible pump

Question: 

A single proton moving down the electrochemical gradient into the mitochondrial matrix space liberates approximately 4.6kcal/mole of free energy.

a) How many protons have to flow across the inner mitochondrial membrane to synthesise one molecule of ATP (from ADP + Pi) if the ΔG for ATP synthesis under intracellular conditions is between 11 and 13 kcal/mole?
= 3 (4.6) = 13.8
b) Why is a range given for this value and not a precise number? Under which conditions would the lower value apply? ratio of ATP:ADP available, therefore it is unknown exact levels of ATP present  ΔG = +11        
          +13
+11 - more substrate (ADP), more likely to occur (needs less energy) 
+13 - more product (ATP), less energetically probably

ATP production from 1 glucose molecule:  
lose 4H⁺ for each Phosphate bond (each ATP) 

therefore:

• 2.5 ATP for each matrix NADH (10H⁺)
• 1.5 ATP for each  matrix FADH₂ (6H⁺)
• 2.5 ATP for each cytosolic NADH (6H⁺) 

Total ATP for 1 glucose molecule
glycolysis:

• 2 ATP directly
• 2 cystolic NADH = 3 ATP

pyruvate dehydrogenase:

• 2 matrix NADH = 5 ATP

CAC:

• 6 Matrix NADH = 15 ATP
• 2 Matrix FADH₂ = 3 ATP
• 2 GTP direct = 2 ATP

TOTAL: 30 ATP

7. Explain how uncoupling agents play a role in thermogenesis

Uncoupling protein - a mitochondrial inner membrane protein that can dissipate the proton gradient before it can be used to provide the energy for oxidative phosphorylation

• Rate of oxidative phosphorylation regulated by level of ADP
• electrons do not flow through ETC to oxygen unless ADP is simultaneously phosphorylated to ATP 
• Coupling is known as respiratory control

Uncoupling oxidation from phosphorylation:

• H⁺ gradient can be disrupted to generate heat (instead of generating ATP) via uncoupling agents
• e.g. 2,4-dinitrophenol can carry H⁺ over inner mitochondrial membrane and thus dissipate proton motive force
• In non-shivering thermogenesis (hibernating animals, some neonates, and some mammals adapted to cold)
•  The inner mitochondrial membrane of brown adipose tissue contains many copies of a transmembrane protein (thermogenin) 
• This protein transfers protons down the concentration gradient from cytosol to mitochondrial matrix. 
• Heat is thereby generated. 
• This activity is controlled by hormones

Question: 

When the chemical dinitrophenol (DNP) is added to mitochondria, the inner membrane becomes permeable to protons. In contrast, when the chemical valinomycin is added to mitochiondria, the inner membrane becomes permeable to K⁺.

a) How will the electrochemical proton gradient change in response to DNP?

• At high dose it dissipates the proton motive force gradient

b) How will it change in response to valinomycin?

• K⁺ flows into mitochondrial matrix
• partially dissipates charge gradient
• concentration gradient doesn't change

c) Compare the relative effects of these 2 compounds on the ability to produce ATP

• DNP more severe

6. Understand that glycogen deposition and breakdown are reciprocally regulated by hormones and allosteric factors controlling the 2 key enzymes, to satisfy the physiological roles for glycogen in muscle and liver

Glycogen metabolism - Reciprocal cycle - prevent futile cycles

• deposit glycogen from excess glucose - Glycogenesis
• breakdown depends on tissue - Glycogenolysis
• Liver - to buffer blood glucose (very important)
• Muscle - to provide glucose to generate energy during strenuous exercise (more glycogenolysis happens in muscle ~80%)
• only one side can work at a time
• hormone controlled - insulin/glucagon

Glycogenesis: 

• Insulin driven - stores glucose
• dephosphorylated enzyme activates pathway

Glycogenolysis:

• glucagon drives - release glucose
• in phosphorylated state it is active

7. Discuss the regulation of gluconeogenesis in the liver in terms of unique enzymes, reciprocity with glycolysis, the interlink with acetyl CoA and oxaloacetate levels, and the potential gluconeogenic substrates that are available in different animals and under different metabolic conditions (e.g. fasting versus strenuous exercise)

Gluconeogenesis:

• Liver+/-kidney
• Pathway to increase the levels of glucose when the body is deficient (e.g. during a fast)
• Not the simple reversal of glycolysis (need to bypass the essentially irreversible steps with new reactions)
• make glucose from non-carbohydrates 
• Pyruvate is moved into the mitochondria then out again after converted to oxaloacetate
• Acetyl CoA made from fast β-oxidation
• There can be too much Acetyl CoA from CAC, so will activate pyruvate carboxylase to make more oxaloacetate (OAA)
• OAA - most goes back to make glucose if not in CAC - some stays in CAC

Allosteric control = milliseconds

Hormone control = minutes

Levels of gene expression = hours 

Gluconeogenesis - make glucose from non-carbohydrates - metabolic intermediates

• while fasting
• substrates are amino acids, glycerol
• during strenuous exercise
• substrate is lactate
• converting absorbed nutrients into glucose
• substrate - propionate in ruminants - they don't absorb much glucose
• Neonate piglets cannot make glucose - no gluconeogenesis
• must suckle, only have ~1 day supply of glucose at birth

Cori cycle - during anaerobic exercise 

• prevents waste of lactate in urine
• prevents acidosis in muscle 
• shifts energy burden to liver (from muscle) 
• Lactate produced from glycolysis is turned via gluconeogenesis into glucose
• Costly process, as it requires 6 ATP to convert into glucose