Biochem - Metabolism Module

What is the catabolic pathway?

What is the catabolic pathway?

Break down larger molecules into smaller substances

Extract H/e- → Deliver it to the electron transport chain

Controlled by demand

What is the anabolic pathway?

Build larger molecules from smaller substances

• Require ATP

Dinitrophenol (DNP)

• An uncoupler (a molecule that disrupts oxidative phosphorylation)

• Prevent energy being stored as fat in the body (instead releasing as heat)

ATP, ADP and AMP

Free energy required is produced by ATP hydrolysis, which makes them thermodynamically favourable.

• The rate of ATP synthesis = The rate of ATP use

• Cells can’t burn fuel without O2

Energy demand

The rates of catabolic (ATP-demanding) and anabolic (ATP-utilising) pathways are regulated by the energy state within the cell.

• A small change in AMP can significantly affect the whole energy charge.

What are kinases, phosphatases and phosphorylases?

• Kinases → Catalyse a phosphorylation reaction.

• Phosphatases → Catalyse a dephosphorylation reaction.

• Phosphorylases → Catalyse a phosphorolysis reaction.

What are synthases and synthetases?

• Synthases → Catalyse condensation reactions/ synthetic process.

  • Without ATP

  • Reversible

• Synthetases → Catalyse condensation reactions.

  • With ATP

  • Irreversible

What is Coenzyme A?

• Carrier for acyl group

• Great for trapping metabolites within the cell

What are dehydrogenases?

• Catalyse oxidation-reduction reactions

  • Transfer 2 H atoms from organic compounds to electron acceptors.

  • Involved NAD+ or FAD as cofactors.

• NAD+ (Nicotinamide Adenine Dinucleotide)

  • NAD+ is reduced to NADH

  • Loves to oxidise -CH2-COH- to -CH-CO-

• FAD (Flavin Adenine Dinucleotide)

  • Accept 2 H+ and 2 e-

  • Become FADH2

  • Loves to oxidise -CH2-CH2- to -CH=CH-

What is the role of hydrogen/electron carriers?

• H/e- carriers: NAD+ or FAD

  • They are also H/e-strippers.

  • Both are limited supply (once they’re carrying H/e-, they can’t do any more stripping)

Strategy of Fuel Oxidation

Stage 1

• Rip H/e- out of fuels

• Fuels are broken up into 2-carbon pieces (acetate)

Stage 2

• Rip H/e- out of acetate

• Compete for oxidation of C atoms to CO2

Stage 3

• Capturing the energy of H/e- as chemical/potential energy

  • Reaction between H and O liberates lots of energy

• Formation of a proton gradient

• Limited by oxidised NAD+ in resting muscle tissue

How do we make ATP with H+ gradient?

H+ flows under pressure through a channel in the inner mitochondrial membrane.

They come in → Rotate another protein → Interact with subunits of ATP synthase → Generate ATP from ADP and phosphate

The 7 big concepts

1. H/e- carriers are in short supply

2. ADP is in short supply

   • [ATP] = 5 mM.

   • < 3 mM → Cells die

3. ATP is stable

4. Inner mitochondrial membrane is impermeable to H+

5. H+ only flow into the matrix if the ATP is being made

6. H+ pumps don’t work if the H+ gradient is very high

7. No H+ pumping, no H/e- movement down the ETC

Fatty acids and ß-Oxidation

Fatty Acids (FA)

• Nearly all C atoms are fully reduced

• Stored as Triglyceride

• Hydrophobic

• Very energy dense

• Huge store

• Can’t be used by brain

ß-Oxidation

• 2 C atoms are removed in the form of acetyl-CoA at the carbonyl terminal

• FAs trapped in the cytoplasm as Fatty Acyl-CoA

• Transported into mitochondria by H/e- carrier: Carnitine

  Help transport long-chain FAs into mitochondria to oxidise them to produce ATP.

• H/e- ripped out by FAD and NAD+

• FA part loses an acetate chunk

• Cycle repeats

Glucose and Glucose Oxidation

Glucose

• Reasonably reduced

• Stored as Glycogen

• Hydrophilic

• Low store (300g)

• Used by all tissues (esp. by the brain)

• Most readily available fuel (glucose transporters move to the cell surface)

Glucose Oxidation/Glycolysis

• All tissues

• Wholly cytosolic

• No O2

• Very fast but inefficient

• Pyruvate must be transported into mitochondria for full oxidation.

Protein

• Channel into pyruvate, acetyl-CoA or Krebs cycle

• Need to dispose of amine groups

• Store 5-20 kg

• Last alternative fuel source

• Don’t store protein since all of it has its functions

• Making protein requires lots of energy

Muscle contraction and ATP

• Use ATP

  • Actin and Myosin interaction - Filaments sliding across each other

  • The faster the contraction, the faster ATP use

• Use ATP even at rest

  • Maintain ion gradients

  • Sacroplasmic reticulum and CA2+

• Compared to resting muscle cells, actively contracting muscle tissue has a higher rate of NAD+/NADH turnover

How many types of muscle?

Type 1 - Red + Slow

• Contract slow

• Many mitochondria

• Good blood supply

Type 2b - White + Fast

• Contract rapid

• Few mitochondria

• Poor blood supply

• Packed full of contractile filaments

Pathways of Fuel Oxidation

What happen once ATP is used?

• ↑ rate of ATP generation

• Once ATP is used → Greater availability of ADP

  • ↑ ATP synthase

  • ↑ ETC

  • ↑ H/e- carriers/trippers

  • ↑ Fuel oxidation

  • Proton gradients diffuse faster, ie. H+ flow back into the matrix more quickly.

• ↓ Blood glucose

  • Need to keep at 5 nM for brain

• Glucose homeostasis

What happen to energy in the body during gentle exercise?

• Glucose is used → Cannot be recycled directly

• After several minutes, fatty acids take over

• Glucose stores (as glycogen) are limited

  • Cannot convert FAs into glucose

    • FAs substitute for glucose as a fuel

    • FAs prevent glucose from being wastefully oxidised

• Glucose still gets into the muscles until lactate is reached

• Lactate (produced from pyruvate) goes to the liver for re-synthesis of glucose

  • Gluconeogenesis

• Low insulin and High glucagon → Stimulate

  • Glycogen breakdown in liver.

  • Fat breakdown in white adipose tissue.

What happen to energy in the body during moderate exercise?

• ↑ Pace → ↑ Rate of FA utilisation

  ↳ Soon, FA oxidation enzymes are at their peak

  • FA oxidation cannot maintain ATP production alone (inhibition on glucose oxidation is removed)

• Glucose oxidation occurs

  • Less glucose recycling

  • A faster depletion of liver glycogen

• Mixture of FA oxidation and glucose oxidation

• Further ↑ pace by ↑ glucose oxidation

  ↳ FA oxidation going at full speed

What happen to energy in the body during strenuous exercise?

• Muscle glycogen is now broken down

  ↳ Endogenously stored

• Limits on blood glucose oxidation

  • The supply and transport of blood cannot keep up with the demand

  • FAs are still going as fast as they can

What happen to energy in the body during very strenuous exercise?

• ATP production cannot be met by oxidative phosphorylation

  ↳ Mitochondrial processes are too slow

• Extra glycolysis boost needed

  • Glycolysis is very fast but inefficient

  • ↑ Blood lactate levels

• Glucose must come from muscle glycogen

  • Transport already at max

What happen to energy in the body during sprinting?

• Use Type 2b muscles → Very rapid ATP consumption.

• Don’t use

  • FAS → Poor O2 supply, low mitochondria

  • Blood glucose → Delay in transporter recruitment, poor fuel supply

• Glycolysis to lactate is very fast but creates a problem

  ↳ Lead to lactic acidosis due to lactate accumulation → Muscle fatigue and disrupt cellular processes

• Lots of lactate produce very quickly

  • Poor blood supply takes away

• ATP regeneration is so inefficient

  • Only 2 ATPs per glucose

• Regeneration of H/e- carrier (NAD+)

Why glycogen is important?

• ATP can only be produced by FA oxidation when glycogen is depleted

  • Power output is lower when using only FAs

• Cannot sprint if there’s no glycogen

  • Glucagon quickly provides glucose for energy production.

Creatine Phosphate (CP)

• An instant store of ATP (< 5 sec supply 15 nM)

• Creatine supplements ↑ CP levels

  • Creatine increases the availability of creatine in the muscles, allowing higher levels of phosphocreatine.

Creatine Phosphate + ADP → ATP + Creatine

Fatty Acid Oxidation/ß-Oxidation

Occurs in the ß-carbon atom

Transport of Fatty Acid

• Transported through the bloodstream bound to a protein called albumin (ab).

• The cells produce ATP after taking up fatty acids and undergoing beta-oxidation.

Transport of Fatty Acid: Mitochondria

Trapping of Fatty Acid

• FA trapped by attachment to CoA

  ↳ The CoA will always be attached from now on

  ➙ Activates FA

• Requires lots of energy

  • ATP is not converted into ADP, but AMP

  • Pyrophosphate is hydrolysed, pulling reaction over

• Coenzyme A

  • Carrier of acyl group

  • Great for trapping metabolites within the cell

First and Second Stripping Steps

ß-Oxidation

• FAs trapped in the cytoplasm as Fatty Acyl-CoA

• Transported into mitochondria - Carrier: Carnitine

• H/e- ripped out by FAD and NAD+

• FA part loses an acetate chunk

• Cycle repeats

• Each round of ß-oxidation gives

  • 1 acetyl CoA

  • 1 NADH

  • 1 FADH2

Glycolysis

Glucose Uptake

Early Glycolysis or Investment Phase

• Requires two ATP molecules to prepare glucose for further breakdown.

• It involves phosphorylation and rearrangement steps to convert glucose into fructose-1,6-bisphosphate.

Second Glycolysis or Return Phase

• Conversion of fructose-1,6-bisphosphate into 2 molecules of glyceraldehyde-3-phosphate (G3P).

• Pyruvate is produced through an enzymatic reaction that converts G3P molecules into ATP and NADH.

What happen completing glycolysis?

• 2 ATP, 2 pyruvate and 2 NADH (need to generate NAD+)

• Fate of pyruvate (Aerobic and Anaerobic)

• Get more ATP from full oxidation of pyruvate

  • Need to transport into mitochondria

  • Oxidise with pyruvate dehydrogenase (PDH)

• Reoxidise NADH quickly → Important

  • Maintain the supply of NAD+

    • Lactate production

    • Alcohol production (in yeast)

  • Keep everything cytosolic

The Krebs Cycle

• Substrate: Acetyl CoA

  ↳ From FA oxidation and/or glucose oxidation

• Everything is in mitochondria

• Strategy

  • Completely oxidise acetate carbons to CO2

  • Produce lots of NADH, FADH2 and even ATP (not directly)

  • Performing the reactions on a carrier molecule

    • Regenerate the carrier

What are the important features of Krebs Cycle?

• 2C atoms come in and 2C atoms release

• Generate:

• 3 NADH, 1 reduced FAD + 1 GTP

  1 ADH → 2.5 ATP in oxidative phosphorylation

  1 FADH2 → 1.5 ATP

  With GTP, ≈ 10 ATP per acetyl CoA

• ATP is not directly generated

• Oxaloacetate is not net consume in the cycle (acts as carrier)

Regulation pathways of the Krebs Cycle

• Mainly by availability of cofactors

  • NAD+, FAD, ADP (more of these → Faster they go)

• Inhibited by a high ‘energy charge’ – ATP : ADP ratio

What happens if there is no proton gradient?

• Will burn all of stored fuel

• No driving force for ATP synthesis

  • No back-pressure to stop H+ pumping

  • No restriction, no H/e- movement down the transport chain to O2

• Instant regeneration of NAD from NADH

  • Massive fuel oxidation rate

  • Massive O2 consumption

• No ATP production → Low ATP synthesis and cell death (<3 mM)

What does uncoupling mean?

ATP synthesis and electron transport chain are disrupted.

Dinitrophenol (DNP)

• An uncoupler

  • Disrupts the normal coupling between electron transport and ATP synthesis in oxidative phosphorylation.

  • Prevent energy from being stored as fat in the body (instead releasing as heat)

• Hydrophobic when protonated

  ↳ Can move freely across membrane

• Weak acid

  Part of molecule can take up or release H+, depending on surrounding pH

• When H+ comes off → Negative charge can be delocalised (e- shared 2+ in a molecules)

  ↳ Still hydrophobic

The mechanism of DNP

• DNP is a protonophore, allowing protons to cross the inner mitochondrial membrane freely.

• Protons leak back into the matrix without passing through ATP synthase, disrupting electron transport and ATP synthesis.

• Dissipation of proton gradient

  • ↓ Rate of ATP synthesis (Prevent ATP production)

  • Proton gradient dissipates

  • ↑ Oxygen consumption.

  • ↑ Rate of ß-oxidation

• Massive weight loss and heat production

• Later used as a weight loss agent

Natural uncoupler - Uncoupling Protein 1 (UCP-1)

• UCP-1 or Thermogenin

• Found only in brown adipose tissue

• Function: Generate heat

  • Esp in small mammals and hibernating animals

• Under hormonal control

  • Noradrenaline binds to ß-receptors (only in white adipose tissue) on the cell surface.

  • Stimulates FA secretion

  • Open proton channel

  ➜ Targeted and controllable

High in neonates, less as we grow up

What are e- transport and H+ pumping?

• The strippers and carriers of H/e-

• Components of the ETC

  • H/e- carriers in the chain

  • Proteins that support them

• Matrixed fuel system

  • Movement of protons out of the matrix

What does Electron Transport Chain (ETC)?

• Contains 4 complexes

  • All embedded in the inner mitochondrial membrane

  • Complex I skip complex II (I and II are distinct entries)

I → III → IV, and II → III → IV

• Each complex consists of many proteins

  • Structural - Maintain the shape of complex

  • Prosthetic group (a subset of cofactor) - Bits that transport H/e-

• Proteins are arranged so that

  • H+ expelling reactions on the outside

  • H+ consuming reactions on the matrix side

• ≈ 10 H+ are pumped out for each NADH

Nicotinamide Adenine Dinucleotide (NAD+) in ETC

• Donates H/e- to complex I (re-deoxides NADH to NAD+)

• NAD+ accepts a H+ and 2 e- = A hydride ion H

• NAD+ likes to rip H/e- off from the -CH-OH group converting them to -C=O groups

• Nicotinamide group derived from nicotic acid (niacin)

Why NADH but not NADH2?

• NADH is the reduced form of NAD+.

• In cellular respiration, it carries 2 high-energy electrons and 1 proton.

Flavin Adenin Nucleotide (FAD) in ETC

• Present inside and stuck in Complex II

• Acceptor and donator of Hs

• Rip H from a saturated hydrocarbon chain

  • 2 H ripped out and being carried

• Built-up ADP

Ubiquinone (UQ or Q pool) in ETC

• Reduced form: UQH2 (transfers Hs to Complex III)

• Electrons move around in Complex I from 1 prosthetic group to another until they reach the Q pool.

• Very hydrophobic

  • Lives in the inner mitochondrial membrane

• Accept all H and e- from Complex II

• Never sees the light

Cytochrome C (Cyt C) and Iron in ETC

• Cyt C picks up e- from Complex III and gives e- to Complex IV.

• Cyt C has a prosthetic group that contains a Fe atom

  • Changes from ferrous (Fe2+) to ferric (Fe3+) as it loses, and vice versa, as it accepts e-

  • Fe does not carry Hs

• Only deal with e-

  Very good at moving e- from 1 place to another

• How are Fe atoms held in place?

  • In mid of porphyrin rings

  • In Iron-Sulphur complexes

What is the proton motive force?

• Local pH is important

• Proton motive force has a charge and [component]

• Energy in the gradient is based on both charge, conc, chemical and electrical gradient

• 2 components come together to make free energy in gradient that much greater

Getting Cytoplasmic NADH to the ETC

What does Glycerol 3-Phosphate Shuttle do?

• Effectively bypassing Complex I

• After glycolysis, dihydroxyacetone phosphate is converted to NAD+ by reacting with NADH → Glycerol 3-Phosphate.

  • Then oxidised by FAD in the mitochondrial membrane.

  • Allow e- to pass through the chain to Q and then through the chain again.

• Losing H+ pumping potential

• Functions:

  • Transfers e- between cytosolic NADH and mitochondria, producing ATP through oxidative phosphorylation.

  • Maintains energy production in tissues where NADH is efficiently produced in the cytosol and transported to the mitochondria.

What does Malate Aspartate Shuttle do?

• Moves e- around to get them across the inner mitochondrial membrane

• Purpose:

  • Take NADH from the cytoplasm and make NADH in the matrix

    ↳ e- transferred into the matrix with no loss of H+ potential

• Function:

  • Transfers reducing equivalents from the cytosol to mitochondria by oxidative phosphorylation, where NADH contributes to ATP production.

  • Allows efficient energy utilization and maintains redox balance.

Organise the four separate routes that feed into UQ (Complex I, Complex II, G3P shuttle and beta-oxidation)

1. From Complex I (NADH dehydrogenase)

   • Transfers electrons from NADH to ubiquinone (UQ) in the ETC.

2. From Complex II (Succinate dehydrogenase)

   • Directly transfers electrons from succinate to UQ in the ETC.

3. From the 1st step of ß-oxidation

4. From the Glycerol 3-P shuttle

   • Generates NADH and FADH2 during the breakdown of fatty acids, which transfer electrons to UQ in the ETC.

• Once in the Q pool, the e- will always go to complex III

The mechanisms involved in the generation and destruction of free radicals

• Free radicals: Very dangerous → Mutations to DNA

• Electron Leakage:

  • Electrons leak and react with molecular oxygen, forming superoxide anion (O2·−).

  • This primarily occurs at Complex I and Complex III.

• Ubiquinone (Coenzyme Q) Reaction:

  • After electrons pass through the ETC, the ubiquinone (UQH2) reduced form can react with molecular oxygen to produce superoxide anion (O2)→ Escape ETC → Free radicals

    • Things getting out of ETC before getting to O2

      ↳ Problems bc things in ETC are very reactive (environment in chain keep it safe

ATP Synthase and its structure

• Using H+ gradient to make ATP

• Movement of 3 H+ → 1 ATP per 1 rotation

• F0 channel: composed of 12 cylindrical proteins

  • As protons enter →  γ subunit rotates

• Causes ß subunit of F1 to change its conformation in 3 ways:

  • Accepting ADP + Pi

  • Reacting them together to give ATP

  • Releasing ATP

The alternate states of the ß-subunit

• Every time 3 H+ come in → ß-subunit change conformation

• Start at any point and follow the ß-subunit ways

• 3 ß-subunits

The contribution of the proton gradient to processes other than the ATP synthase

• Swapping of ATP/ADP (brings ADP + Pi) takes negative charge outside

  • ATP goes out of into the cytoplasm

  • 3- charges come in, 4- go out

  • Need positive charge to do movement → Use a H+ (proton gradient)

• The import of Pi consumes H+

Counting ATP

Inhibitors and Acceptors in ETC

• Rotenone

  • Inhibit at Complex I

  • Whole chain stops → H+ pumping stops

  • Everything downstream is oxidised (stop consuming O2)

• Cyanize, Azide and CO

  • Inhibit at Complex IV

  • Whole chain stops → H+ pumping stops

  • Everything upstream is reduced

• Alternative receptors, e.g. Methylene blue

  • Accepts e- from Complex IV before cyanine blockage point

  • Allow transport to continue

Starvation and Some rules

Starvation

• Begin at the start of the post-absorptive period

  • When all food digested

  • No substrates coming in from gut

• Reliant on blood and stored fuel

Some Rules

• Need to keep [blood glucose] ≈ 5 mM (> 4 mM)

  • Euglycemia or Normoglycemia (normal [blood glucose])

• Under normal circumstances, brain can only use glucose

  • Cannot use FAs which cannot cross Blood Brain Barrier (BBB)

  • Uses ≈ 120g glucose/day

  • Transported by GLUT-1

• Although we store most of our energy as fat, we cannot convert FA into CHO (carbohydrate/glucose)

  • Acyl CoA can’t be made into Gluconeogenic precursors.

  • Pyruvate (3C) → Acetyl CoA (2C → Glucose is lost) is Irreversible

Glucose requirements during the first few hours and what happens to them

• Parts of the kidney, skin and RBCs have obligatory requirements for glucose

  ↳ Cannot use anything else but glucose

• Other tissues (primarily muscle)

  ↳ Can switch to FAs as an alternate fuel during starvation

General Strategy

• Glucose conservation: Don’t use it unless you must to

• Glucose recycling: Don’t fully oxidise it - Generate from Lactate

• New glucose formation: Make it from other things

First Few Hours

• Tissues are using glucose → ↓ [blood glucose]

• Prevent hypoglycemia, liver releases glucose into bloodstream

  • [blood glucose] stays constant to at least euglycemia at ≈ 4 mM

What happen at first 24 hours?

Glycogen Mobilisation or Glycogenolysis

• Breakdown of glycogen to release glucose.

• Signal: The binding of glucagon to the receptor on liver cell membrane

• Glycogen phosphorylase

  • Cleaves glucose units from the glycogen molecule, and a branching enzyme removes branch points using Phosphates.

  • Produce G-1P

  • Rapidly converted into G 6-P

Phosphorylase Activation by Glucagon

• Glucagon activates glycogen phosphorylase via cAMP and protein kinase A (PKA).

• The PKA activates glycogen phosphorylase, leading to glycogen breakdown and glucose release.

How much ATP is used?

• Not much ATP is consumed

• The amount of ATP being used and amount of cAMP being made are very tiny → Not really affect [ATP]cell

  • cAMP is the 2nd messenger in the pathway

  • Tiny changes in conc are detected by PKA (Protein Kinase A)

• PKA is activated by removing a regulatory inhibitory subunit

Why it’s so complicated?

• Amplification through 2nd messenger and cascade, rather than directed binding

  • Massive response from small signal

    ↳ Each step catalysed by an enzyme

  • More control over the whole process

    ↳ Each enzyme can be further influenced by other factors (e.g. Ca2+ and AMP)

• ≠ in muscle → Adrenaline is the stimulant

Branch points of Glucose

• Debranching enzyme

• At the branch points, a simple hydrolysis is used

• ≈ 10% of the glucose residues are released as glucose (not glucose 1-P)

Does muscle contribute to blood glucose?

• Muscle doesn’t breakdown glycogen much in starvation, bc

  • NO glucagon receptors

  • NO G6Pase (only liver has)

    ↳ Cannot convert G6P to glucose → Cannot release glucose into blood.

• However, some glucose residues in glycogen are released as neat glucose

  • B/c debranching enzyme uses water to hydrolysed the glycosidic linkages, not phosphate

  • ≈ 10% potentially released

• Muscle is selfish with its glucagon, but what if PDH is inhibited, G6P will go to lactate

White Adipose Tissue (WAT) Lipolysis

• WAT lipolysis is the breakdown of stored fat in white adipose tissue.

• Accessing the large reserves of fat in WAT

• Glucagon → ↑ [cAMP] → ↑ activity of PKA

• PKA then phosphorylates Hormone Sensitive Lipase (HSL) (breakdown fat) → Cleaves triglycerides into fatty acids and glycerol.

  • PKA also phosphorylates perilipin (shell surrounding the vacuole)

    ↳ Allow the activated HSL to interact with the fat

• FAs released into the bloodstream

• Glycerol can return to liver → Convert back to glucose (small amount)

What are the effects of FA oxidation?

• FAs will be oxidised to provide the acetyl CoA for the Krebs Cycle

  • But need to avoid oxidation

  • PDH ( pyruvate → AcCoA) is Irreversible

• GLUT-1 is still present in muscle

  • Even though GLUT-4s were endocytosed due to a lack of insulin

    ↳ Muscle can still take up glucose

• Need to preserve glucose

  • Get tissues to stop oxidising glucose

Activating PDH

Glucose-Fatty Acid Cycle

In starvation, PDH needs to be off

• PDH Kinase activity >> PDH phosphate activity

• Acetyl CoA stimulates PDH

• PDH is inactive when phosphorylated

• Prevents wasteful oxidation of pyruvate

• Pyruvate only converted into lactate

When PDH is off

• Pyruvate cannot be oxidised to acetyl CoA

  • Then, there is only 1 fate for pyruvate in the muscle to be converted into lactate by LDH.

• Lactate can be taken up by liver.

  • Remade into glucose by gluconeogenesis

• Called Cori-cycle

  • Muscle glucose → Pyruvate → Lactate → Liver → Glucose (via gluconeogenesis) → Glucose into bloodstream again

• Gluconeogenesis can also happen from glycerol

  • Made by 30g glucose per day

  • Glycerol (from lipolysis) is the only source of de novo gluconeogenesis

    • Lactate-fueled gluconeogenesis is recycling

What is Proteolysis?

• The process of breaking down proteins into smaller peptide fragments or individual amino acids.

• After a few a hrs, [blood glucose] < 5 mM → Insulin secretion stops

  • Important in stimulating lipolysis

• Hypoinsulinemia → Proteolysis

  • Release of amino acids from tissues (mainly muscles)

• Many amino acid ‘carbon skeletons’ are used for gluconeogenesis

  • Need to get amino acids to the liver

  • Need to do sth with an amine group (Ammonia is poisonous)

• Carbon skeletons – the portion of the molecule remaining after the removal of nitrogen

What are Processing Amino Acids and the Fate of Amine Groups?

Processing Amino Acids

• Channel amine groups to 3 amino acids

  ↳ Alanine, Glutamate and Aspartate

• 3 acceptors are all found in the main pathways

  • Pyruvate → Alanine

  • α-ketoglutarate → Glutamate

  • Oxaloacetate → Aspartate

• Result in α-keto acids used in gluconeogenesis

Fate of Amine Groups

• Urea Cycle – Only in liver

  • The body’s way of converting toxic ammonia into urea.

• Ammine groups are channelled into urea.

  • Synthesised from aspartate and glutamate

  • Consume lots of ATP

• Urea is non-toxic

What is Gluconeogenesis?

• A reversal of Glycolysis

  • Amino acids and glycerol are converted into glucose.

• Maintains blood glucose levels during fasting or low carbohydrate intake.

• Except 3 ‘rate limiting ‘ steps bypassed

  • Hexokinase → Glucose trapping step

  • Phosphofructokinase → Rate limiting step

  • Pyruvate Kinase → Final and Energy releasing step

• Completing the pathway only in liver

  • Mainly cytoplasmic → Pyruvate carboxylase

• Substrates:

  • Lactate → Enter as pyruvate at the bottom

  • Glycerol → Enter as dihydroxyacetone phosphate

  • Amino acid carbon skeletons → Enter as various places

Can all amino acid skeleton make glucose?

• Not all AA skeletons can make glucose

• A carbon skeleton can be converted into an intermediate for gluconeogenesis.

• Cost for lots of energy to make proteins (made for reasons)

  • Lots of ATP is required to dispose of the amine groups

• Not all amino acids made into glucose

• Many amino acids from proteolysis are burnt before release from tissue.

• Need extra 90g glucose (breakdown 180g) per day

Lipolysis + ß-Oxidation

• After 2-3 days of starvation, the rate of lipolysis will be at max.

  • FA released into bloodstream → ↑ [FA] in blood → More FA than is needed

• ß-Oxidation in Liver

  • Rate depends on the demand of ATP by the tissues

    • Generation of CoA by Krebs Cycle needed to keep FA oxidation going

    • Rate of Krebs Cycle strictly regulate by demand for ATP

  • BUT ß-oxidation can occur even if ATP isn't required

    • Other pathways can regenerate CoA from acetyl-CoA

What is the function of Fate of Acetoacetate (Ketone body)?

• Acetoacetate, or a Ketone body, is used as an alternative energy source by various tissues for energy.

• It can be converted

  • Back into acetyl-CoA to generate ATP or

  • Into other ketone bodies like beta-hydroxybutyrate and acetone.

• Split in the mitochondria to acetyl CoA

  • An instant source of fuel for the Krebs Cycle

  • AcCoA inhibits PDH and stimulates PDH kinase

• Reduces brain glucose consumption

Functions of Acetoacetate (Ketone body)

Inefficiency in Ketone Body Metabolism

• Nothing inefficient about the oxidation

• But ketone body

  • Lost in the urine

  • Spontaneously decarboxylate

Have Ketone Bodies Saved Us?

• Make 30g glucose per day from glycerol

• After 2 days of starvation

  • Brain using 120g glucose a day

  • Protein loss > 100g protein/day

• After 3-4 days

  • Ketone bodies are lowering the brain’s need for glucose

  • Protein losses ≈ 75g/day

• By day 5

  • Brain using 30g glucose/day

How is the protein related to tissues' demand performing?

• Proteins are lost from all tissues

  • Inactive muscles slightly preferentially degraded

• Will reach equilibrium

  • The loss of body protein is ultimately what kills us

  • Loss of function

  • Much more prone to infection

Amount of protein breakdown = (amount of glucose needed - 30) x ≈ 2

The importance of Glucagon

• Glucagon promotes glycogen breakdown (glycogenolysis) and stimulates gluconeogenesis, which raises blood glucose.

• Maintains glucose homeostasis during fasting.

Energy Charge and ATP

• ATP is not the most energy-containing molecule in metabolism

• Need to keep at 5 mM

• Instant reserves

  • Compounds that phosphorylate substrates

• Only few seconds supply

  • Still need ↑ catabolic pathway

ATP

• Energy released when any of the terminal phosphates are hydrolysed

• ATP → ADP or ADP → AMP releasing energy

Adenylate Kinase (AK)

• Converts ATP to ADP, helping to buffer energy fluctuations.

• 2ADP ↔ ATP + AMP

• Translate small change in ATP and large change in AMP

• Ratio of [Adenine molecule] = Energy charge

  • Key molecule: AMP

  • ATP:ADP kept high

• Key enzymes very sensitive to [ADP]

Which enzymes are controlled these?

Rate-limiting step (RLS)

• Slowest enzyme-catalysed reaction in a metabolic pathway.

• Control the overall rate of metabolic activity

• Irreversible

  • Need alternative enzymes to go back

  • NOT equilibrium under physiological conditions

• Saturated (bao hoa) with substrate.

  • Low Km or [S] > Km

  • Working at Vmax

Flux generating step

• Contributes significantly to the overall flux or rate of metabolite flow through the pathway.

• Often associated with the rate-limiting step, as it determines the overall speed of the pathway.

• 3 major ways to regulate

  • Make the rate-limiting enzyme go faster/slower

  • Turn the rate-limiting enzyme on/off or make it work the other way

  • ↑ rate of transcription/translation of the RLS or change its rate of degradation

Overall catabolic pathway

Allosteric PFK (Phosphofructokinase)

• Involved in the anabolic pathway of glycolysis.

• Catalyses the breakdown of glucose for energy by converting fructose-6-phosphate to fructose-1,6-bisphosphate.

  F6P + ATP → F1,6BP

• In the catabolic pathway, PFK acts as an allosteric regulator of glycolysis and ATP production.

• The binding of ATP and AMP to PFK ensures efficient energy use.

  • Has binding sites for AMP away from the active site.

    ↳ Biniding AMP changes the way PFK responds to ATP

• Also binds Citrate allosterically

  • Citrate inhibits PFK

  • [Citrate] high → ↑ PFK → ↓ Glycolysis

Hexokinase – Feedback Inhibition

• Involved in the first step of glycolysis, it catalyses glucose phosphorylation to produce glucose-6-phosphate (G6P).

  • G6P levels control the activity of hexokinase through feedback inhibition.

• In feedback inhibition, G6P acts as an allosteric inhibitor of hexokinase.

• ↑ G6P levels = Sufficient glucose or saturated downstream metabolic pathways.

  ↳ Hexokinase is inhibited → Slowing down the phosphorylation of glucose and ↓ the flux of glucose through glycolysis → ↓ Glycolysis

• Prevent unnecessary accumulation of G6P and ensures that glucose is utilized efficiently.

  • Prevent waste of ATP

  • Allow glucose to go back out of the cell

• If G6P is not used, glucose is not trapped

PDH – Covalent Modification

• Inactivated by phosphorylation (totally OFF)

  • Phosphorylation inhibits PDH activity, while dephosphorylation activates it.

• Help control the conversion of pyruvate to acetyl-CoA based on energy demands.

• The total amount of enzyme doesn’t change

  • Phosphorylated : Dephosphorylated ratio

• Reactivation by phosphate

  • Release of phosphate

  • Totally ON

• PDH activity balance between kinase and phosphate

Fuel Selection

• Catabolism vs. Anabolism

  • Glycolysis vs. Gluconeogenesis

  • ß-Oxidation vs. FA synthesis

• When 1 pathway is stimulated, the opposite is inhibited

• When they both occur at the same time → Futile Cycle

  • A net loss of energy without any productive outcome.

  • Regulatory purposes but generally leads to wasteful energy expenditure.

Gluconeogenesis and its pathway

• Getting from 3C to 6C

  • Bypassing hexokinase and PFK → PFK-1

• G6Pase (only in liver) and F1

• NO ATP gained from the loss of phosphate at these steps

• PC (Pyruvate Carboxylase)

  • In the mitochondria.

  • Crucial role in gluconeogenesis, converting pyruvate into oxaloacetate

    ↳ Further converted into glucose.

  • Stimulated by acetyl-CoA (FA oxidation)

  • Acetyl-CoA levels high when ß-Oxidation prominent

  • Inhibition of PDH → Prevenr wasteful oxidation of glucose

• PEPCK (phosphoenolpyruvate carboxykinase)

  • Involved in gluconeogenesis, converting oxaloacetate to phosphoenolpyruvate (PEP).

  • Crucial role in maintaining blood glucose levels during fasting

  • Regulated by hormones such as glucagon and cortisol.

  • Stimulated by ↑ transcription/translation of gene

• These enzymes can exist in tissues other than liver

  • Enables glycerol to be made from pyruvate

Synthesis of PEP from Pyruvate

• Pyruvate is carboxylated to form oxaloacetate by pyruvate carboxylase (PC).

• Oxaloacetate is then converted to PEP by phosphoenolpyruvate carboxykinase (PEPCK).

• Essential for gluconeogenesis, allowing the body to generate glucose from non-carbohydrate sources.

Why 2-deoxy glucose can’t be used in glycolysis

• B/c it lacks a hydroxyl group (OH-) at the C2 position

• Preventing it from being efficiently phosphorylated by hexokinase.

• A key step in glycolysis.

• Acts as a competitive inhibitor of hexokinase and blocks the entry of glucose into the glycolytic pathway.

F26BP affects PFK-1 and FBPase-1

• Reversal of F6P → F16BP

• PFK-2 and FBPase-2 are the same enzyme

  • Swapping from 1 form to another after reversible phosphorylation

• Interconversion catalysed by Protein Kinase A (PKA) and [glucagon] and [insulin]

  • Sensitive to cAMP

Gluconeogenesis & Glycolysis

• During starvation

  • ↑ Glucagon → ↑ [cAMP)

  • ↓ [F2,6]

    • NO stimulus for PFK → NO glycolysis

    • NO inhibition of F1,6BPase → Gluconeogenesis

• F6O → F16BP stimulated by allosteric effector F26BP

  • F26BP made by PFK-2

  • F26BP inhibits F16BPase and stimulates PFK

  • When F26BP is high → Glycolysis is favoured

• Phosphorylation of PFK-2 converts it into F26BPase

  • ↓ [F26BP]

  • PFK is inhibited

  • ↑ F16BPase activity

  • When F26BP is low → Gluconeogenesis is favoured

• Phosphorylation is catalysed by cAMP-dependant protein kinase (or PKA)

  • cAMP stimulates PKA

  • cAMP are high when glucagon bound to receptors on liver cell

• F16BPase is more active when [glucagon] high

  • As in starvation

Anaplerosis

• In the citric acid cycle, anaplerotic reactions refill oxaloacetate after consumption.

• Maintain adequate ATP levels for continuous cellular respiration.

Is Glucose toxic?

• Brain can’t live without it

  • Need to keep [blood glucose] at 4-5 mM

• Very reactive in vivo

  • Glycation: Non-enzymatic glycosylation of protein

  • Destroys protein function

  • Rate is directly proportional to [glucose]