Alex's Metabolism

MCD Metabolism

Alexandra Burke-Smith

1. Introduction to Protein Structure James Pease (j.pease@imperial.ac.uk)

1. Outline the reaction by which amino acids are joined together.

Definition of a protein: any of a group of organic compounds composed of one or more chains of amino acids and forming an essential part of all living organisms. 

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Individual amino acids are joined in condensation reactions (i.e. a molecule of lost) to form peptide chains The polypeptide chain of a protein rarely forms a structure (random coil) as proteins generally have fulfil, and these functions rely upon specificity.

Functionality requires a definite 3D structure or conformation of the polypeptide chain. Proteins possess a degree of flexibility necessary for function, e.g. muscle fibres.

water is disordered functions to

generally

Sketch a trimeric peptide, illustrating the amino -terminus, carboxyl terminus and side chains.      

A trimeric peptic consists of three amino acids joined by peptide bonds An amino acid consists of an amino group, carboxyl group, hydrogen atom and variable R side chain arranged around a central chiral carbon atom, i.e. with four different substituents bound to it This gives rise to optical isomers (enantiomers) of each amino acids each of which is a mirror image of the other. All amino acids found in proteins are of the L form The whole of the amino acid minus the side chains is known as the backbone Substitutions at the R position or side chain, give rise to the 20 different amino acids Glycine (Gly) has no side chain (only an H atom) and is therefore the only non-chiral amino acid

Amino acids with non-polar side chains  Have an even distribution of electrons, so have no polarity and are hydrophobic  Glycine; gly, Alanine; ala, Valine; val, Leucine; leu, Isoleucine; ile), Proline; pro, methionine; met, Phenylalanine; phe Amino acids with polar side chains  Have an uneven distribution of electrons (often due to excess O2- or OH-) therefore are charged and hydrophilic  Often found on surface membranes  Asparagine; Asn, Glutamine; gln, Cysteine; cys, Histidine; his, Serine; ser, Threonine; thr, Tyrosine; thr, Tryptophan; trp  Happy in aqueous solution.  Arginine (Arg) and Lysine (Lys) at physiological pH are always protonated and therefore basic (NH4+)  Glutamic acid (Glu, E) and Aspartic acid (Asp, D) at physiological pH are always negatively charged due to proton donation (COO-) The state of ionisation of an amino acid provides vital biological properties to many proteins and enzymes, and for this reason cells cannot generally tolerate wide changes in pH. The ability to act as proton donors and proton acceptors gives amino acids some buffering capacity to resist some changes in pH.

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MCD Metabolism 3.

Alexandra Burke-Smith

Appreciate the different types of bond that combine to stabilise a particular protein conformation.

Characteristics of a peptide bond  There is no free rotation around the peptide bond.  The C=O and N-H are in the same plane of the molecule.  The other two bonds in the backbone of the polypeptide are able to rotate, therefore only conformations which are allowed by this rotation occur (in which side chains do not interfere with the main chain) What holds a protein together? (pg 78-79 ECB) 1. Covalent bonds (in which two atoms share electrons) are the strongest bonds within protein and exist in the primary structure itself. Covalent bonds can also exist as disulphide bridges. These occur when cysteine side chains within a protein are oxidised resulting in a covalent link between the two amino acids 2. Hydrogen bonds occur when two atoms bearing partial negative charges share a partially positively charged hydrogen (e.g. O and H), the atoms are engaged in a hydrogen bond (H-bond). These can occur either between atoms on different sidechains and the backbone of the protein or between water molecules.

3. Ionic interactions arise from the electrostatic attraction between charged side chains e.g. Glu, Asp, Lys and Arg. They are relatively strong bonds, particularly when the ion pairs are within the protein interior (hydrophobic core) and excluded from water. Within any particular protein molecule, the majority of charged groups are at the surface of the folded protein. There, they can be neutralised by counterions such as salts.

4. Van der Waals Forces are transient, weak electrostatic attractions between two atoms, due to the fluctuating electron cloud surrounding each atom which has a temporary electric dipole. Although relatively weak and transient in nature, because of the sheer number of these interactions within a protein, they can still have a large part to say in the overall conformation of a protein. Provided that the two atoms are quite close, the transient dipole in one atom can induce a complementary dipole in another atom, with weak attractive properties. Alternatively, if the two electron clouds of adjacent atoms are too close, repulsive forces come into play because of the negatively-charged electrons.

5. Hydrophobic Interactions are a major force driving the folding of proteins into their correct conformation.

When proteins fold, they juxtapose hydrophobic side chains by packing them into the interior of the protein.

This creates a hydrophobic core and a hydrophilic surface to the majority of proteins.

4. Understand the concepts of primary structure, secondary structure, tertiary structure & quaternary structure with respect to proteins.        

Proteins fold into the conformation of lowest energy.

This can occur spontaneously Chaperones bind to the partly folded polypeptide chain and ensure that the folding continues along the most energetically favourable pathway.

We can denature the protein into the original flexible polypeptide e.g. urea (breaks hydrogen bonds) and 2mercaptoethanol (breaks disulphide bonds).

Primary structure: linear sequence of amino acids that make up the protein. Standard nomenclature dictates the sequence from amino to carboxyl terminus.

Secondary structure: Defined as local structural motifs within a protein, e.g. a-helices and b-pleated sheets, dictated by the primary structure or amino acid sequence.

Tertiary Structure: Defined as the arrangement of the secondary structure motifs into compact globular structures called domains.

Quaternary structure: Defined as the three dimensional structure of a multimeric protein composed of several subunits (e.g. the four subunits of the globular protein; haemoglobin) 2

MCD Metabolism

Alexandra Burke-Smith

5. Distinguish between an α-helix and a β-pleated sheet and appreciate the bonds that stabilise their formation.

The main chain is highly polar and hydrophilic (due to presence of C=O and N-H bonds), however folding into the interior can be achieved through neutralisation of the polar groups by Hydrogen bonding in the alpha helix or betapleated sheets α-helix  Hydrogen Bonds between the C=O of one residue and the N-H of another residue, 4 amino acids along the helix, stabilise the entire structure  As a result of the L-enantiomer form of amino acids, right hand helices are formed e.g. DNA is a double right hand helix  Side chains project outwards from helix β-pleated sheet  hydrogen bonds between the N-H and C=O groups of two or more b-strands hold the b -pleated sheet sheet together, and point out at right angles to the line of the backbone  Alternate β -strands can run in the same direction to give a parallel β -pleated sheet or in opposite directions to give an antiparallel β -pleated sheet, allowing best alignment of the hydrogen bonds Proline  When proline is joined to a polypeptide chain, the NH group of the amino acid is lost  This prevents the N atom from hydrogen bonding with C=O groups of another residue within the helix, thereby distorting the helical conformation, putting a ‘kink’ into it.

6. Outline how warfarin works with reference to the post translational modification of glutamate.   

Even after synthesis, (post translation), the starting set of 20 amino acids can be modified to create novel amino acids, enhancing the capabilities of the protein e.g. hydroxylation(introduction of OH group), glycosaylation (addition of a saccharide), and carboxylation (introduction of a carboxyl group) Carboxylation of glutamic acid within several proteins of the blood clotting cascade (e.g. factor IX) is critical for their normal function by increasing their calcium binding capabilities The anticoagulant warfarin works by inhibiting the carboxylation reaction, so coagulation factors cannot bind to phospholipid surfaces inside blood vessels, on the vascular endothelium

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2. Energetics and enzymes James Pease (j.pease@imperial.ac.uk)

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Energetically unfavourable reactions can be carried out occur by coupling them to energetically favourable reactions, generally involving the hydrolysis of a high-energy phosphoanhydride such as found in ATP. Enzymes are specific catalysts that increase the speed of biochemical reactions by lowering the energy barriers that impede chemical reactions.

Since enzymes are proteins, they are sensitive to extremes of temperature and pH. Enzymes often require the help of cofactors to catalyse reactions.

1. Explain the concept of free energy and how we can use changes in free energy to predict the outcome of a reaction.

First Law of Thermodynamics: Energy can neither be created nor destroyed. i.e. it is simply converted from one form to another.

Second Law of Thermodynamics: In any isolated system, e.g. a single cell or the universe, the degree of disorder can only increase, i.e. reactions proceed in the direction of increasing entropy.  

However, biological systems are very well ordered, which achieved by investing taking energy from the environment surrounding the cell and investing it in chemical reactions which maintain order.

At the single cell level, this can be seen as increased intracellular order as the surrounding environment becomes more disordered and hotter.

Free Energy  Entropy changes during a chemical reaction are very difficult to measure, so the concept of Free energy was set up, combining both the 2st and 2nd laws of thermodynamics.  (Gibb’s) Free Energy is defined as the amount of energy within a molecule that could perform useful work at a constant temperature.  Changes in G (DG) measure the amount of disorder that results from a particular reaction, including both the change in order within the cell and also upon the change in entropy of the system.  A reaction can only occur spontaneously if DG is negative. Conversely, a reaction cannot occur spontaneously if DG for the reaction is positive.  Therefore the change in free energy can be used to predict the outcome of a reaction.

2. Draw the chemical structure of ATP and explain how it acts as a carrier of free energy and is used to couple energetically unfavourable reactions. 

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Pathways within the cell that synthesise molecules are generally energetically unfavourable e.g. peptide synthesis. They take place because they are coupled to an energetically favourable one, e.g. the hydrolysis of high-energy phosphate bonds such as those found in ATP (ΔG°'= - 31 kJ/mol ΔG°' = standard free energy change at pH 7) Phosphoanhydride bonds have a large negative DG of hydrolysis, and are thus said to be "high energy" bonds.

Providing that the sum of the DG for the overall reaction is still negative, the reaction will proceed. ΔG°' (both reactions coupled)= (-31 + 23) =-8 kJ/mole i.e. energetically favourable 4

MCD Metabolism 3.

Alexandra Burke-Smith

Describe how enzymes act as catalysts of reactions with reference to the reactions catalysed by lysozyme and glucose-6-phosphatase.

Enzymes Definition of an enzyme: A protein that acts as a catalyst to induce chemical changes in other substances, itself remaining apparently unchanged by the process    

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Increase the speed at which a chemical reaction takes place by a factor of at least 1 x 106 yet at the same time, they exhibit quite exquisite specificity due to their conformation.

Enzymes function by lowering the activation energy of a reaction All the molecules within a cell possess energy in the form of rotation and vibrations and also in the form of the bonds holding the various atoms together.

Enzymes bind one or more substrate molecules tightly within a part of the protein known as the active site, and arrange them in such a way that certain bonds are strained, or making/ breaking of bonds by altering the arrangement of electrons within the substrate(s).

This can be oxidation or reduction reactions, but since the cellular environment is generally aqueous, often, when a molecule gains an electron, it also simultaneously gains a proton. This makes the substrate resemble their transition state.

The transition state is the particular conformation of the substrate in which the atoms of the molecule are rearranged both geometrically and electronically so that the reaction can proceed.

This makes them more amenable to reaction with other molecules.

Lysozyme  component of tears and nasal secretions  one of the first lines of defence against bacteria- catalyses the hydrolysis of sugar molecules within bacterial cell walls that are necessary for their structure. With this bond broken, the bacteria lyse and die.  In detail, hydrolyzes alternating polysaccharide copolymers of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) which represent the polysaccharide structure of many bacterial cell walls.  It cleaves at the b(1-4) glycosidic linkage, connecting the C1 carbon of NAM to the C4 carbon of NAG.  Side chains within lysozyme, (non-polar) Glu35 and (polar) Asp52 are essential for this hydrolysis.  Glu35 protonates the oxygen in the glycosidic bond breaking the bond holding the two sugar molecules together.  A water molecule enters and is de-protonated by Glu35.  Asp52 stabilises the positive charge in the transition state.  The hydroxide ion attacks the remaining sugar molecule adding an OH group.  Both Glu35 and Asp52 are in their original state to continue catalysis.  At pH 5.0, Asp52 is ionised (COO-) and Glu35 is unionised (COOH), providing an optimum Ph Glucose-6-phosphotase  G-6-Pase is predominantly a liver enzyme that catalyses the reaction of G-6-P into glucose when blood glucose levels are low.  A deficiency in G-6-Pase is known as Von Gierke’s disease and leads to low blood sugar, slow growth, large livers and short stature  In the presence of water, G-6-P is converted into Glucose and inorganic phosphate, with a GP=-13.8Kj/mole.

Although energetically favourable, this reaction will still not occur at a useful without catalysis by G-6-Pase 4.

Outline the differences between lock and key and induced fit models of substrate-enzyme interactions. 

Lock and Key: the shape of the substrate (key) matches that of the active site (lock) of the enzyme, forming the enzyme-substrate complex. This model explains the specificity of most enzymes for a single substrate.

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Alexandra Burke-Smith

Induced Fit Theory: the substrate induces a change in the conformation of the enzyme which results in the formation of the active site. Upon release of products, the enzyme reverts back to its original conformation.

The induced fit theory is correct, as Proteins generally possess a degree of flexibility necessary for function. e.g. muscle fibres, and side chains in the active site catalyse the reaction

5. Describe graphically, the effects of substrate concentration, temperature and pH on reactions catalysed by enzymes. 

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The effect of substrate concentration: The rate of an enzyme reaction increases as the substrate concentration increases until the maximum value is reached, when all active sites on the enzymes have formed substrate complexes. The rate of reaction is then limited by the rate of the catalytic process and enzyme concentration.

The effect of temperature: Chemical reactions speed up as temperature is increased.In general, catalysis increases at higher temperatures (e.g. fever when ill). However beyond the enzymes optimum temperature, the conformation is denatured so the enzyme becomes inactive, The effect of PH: most enzymes have an optimum pH for their activity, at which the catalytic side chains are in the correct state of ionisation. Wide changes in pH therefore reduce enzyme activity.

6. Illustrate the role of the coenzyme NAD in the reaction catalysed by lactate dehydrogenase.  

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NAD+ (Nicotinamide adenine dinucleotide) is a coenzyme and is a cofactor for many dehydrogenation reactions within the body.

It functions only after binding to a protein, catalysing the reaction below by readily accepting a hydrogen atom and two electrons During intense exercise, skeletal muscles have to function anaerobically, as oxygen is a limiting factor. To do this, the metabolite pyruvate is converted into lactate, which also generates free NAD+ which is needed by the muscle for other reactions.

Lactate then diffuses from the muscle into the blood stream and is picked up by the liver, where the high levels of NAD+ can be used by lactate dehydrogenase to regenerate pyruvate.

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MCD Metabolism

Alexandra Burke-Smith

3. Metabolic pathways and ATP production I James Pease (j.pease@imperial.ac.uk)

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Glycolysis is central to metabolism in mammals, producing 2 moles of ATP for every mole of glucose It relies upon the formation of a high energy compound which is subsequently split to liberate energy.

Under aerobic conditions, pyruvate produced by glycolysis can be dehydrogenated by the actions of a giant multimeric enzyme, PDH, to generate acetyl CoA, a substrate for the TCA cycle in mitochondria and a prelude to oxidative phosphorylation.

1. Sketch a cartoon of and describe the three stages of cellular metabolism that convert food to waste products in higher organisms, illustrating the cellular location of each stage.

Cellular Metabolism  High energy bonds such as those found in ATP are a convenient way for the body to harness the energy that is liberated from food molecules during cellular metabolism  The three principle forms of food molecules used by the cells are: 1) Polysaccharides= simple sugars 2) Proteins= amino acids 3) Fats= fatty acids and glycerol Metabolism Definition of metabolism: the sum total of the chemical reactions that take place in the cells of a living organism resulting in growth, division, energy production, excretion of waste and so on. A highly controlled, highly ordered process in three main stages:   

Digestion: Enzyme mediated, liberates small molecules, takes place in the intestines after food is initially broken down by teeth and saliva.

Cellular Metabolism I/Glycolysis: Oxidation of the small molecules within the cytosol of individual cells, generating ATP and NADH. Cellular Metabolism II/Oxidative Phosphorylation: Oxidation of the small molecules generated by the first stage of cellular metabolism within the mitochondria of individual cells, generating ATP and waste products.

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MCD Metabolism

Alexandra Burke-Smith

2. Outline the metabolism of glucose by the process of glycolysis, listing the key reactions, in particular those reactions that consume ATP and those that generate ATP. Glucose Combustion

Glucose Metabolism - broken down into steps to overcome the high activation energy

Glycolysis is Central to metabolism  Glycolysis - glycos (sugar) and lysis (breaking)  Glycolysis is essentially an anaerobic process, occurring in the cytoplasm of cells and is probably a throwback to the pathways used by prehistoric anaerobic bacteria.  1 6-carbon glucose molecule is broken down into 2 3-carbon pyruvate molecules Glycolysis involves ten reactions, with two main concepts:  Formation of a High Energy Compound- involves the investment of energy in the form of ATP  Splitting of a High Energy Compound- produces useful energy in the form of ATP generation The Ten Reactions 1. Glucose into glucose-6-phosphate involving hexokinase and ATP

All kinases catalyse the transfer of a phosphate group from a donor such as ATP, onto a substrate, which makes the molecule more reactive- and hence easier to split This reaction is irreversible and commits the cell to the subsequent reactions.

It also traps glucose inside the cell by means of the negative charge.

2. Glucose-6-phosphate into fructose-6-phosphate involving phosphoglucose isomerase

The isomerisation shuffles the glucose chair to give fructose

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MCD Metabolism

Alexandra Burke-Smith

The logic behind this reaction is that fructose can be split into equal halves when subsequently cleaved as is more symmetrical 3. Fructose-6-phosphate into fructose-1,6-bisphosphate involving phosphofructokinase and ATP

Here a highly symmetrical, high energy compound is generated, which can be easily split Regulation of phosphofructokinase exquisitely controls the entry of sugars into the glycolysis pathway.

4. fructose-1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate involving aldolase

Opening of the fructose ring to generate two high energy compounds (which require less energy again to split), one of which, (dihydroxyacetone phosphate) subsequently undergoes isomerisation.

5. Isomerisation of dihydroxyacetone phosphate into glyceraldehydes 3-phosphate using triose phosphate isomerise (TPI)

Essentially just re-shuffling of the C=O bond and the hydroxyl group Metabolic Diseases - Deficiency in TPI is extremely rare, but most sufferers die within the first 6 years of their lives.

AT THIS HALF-WAY POINT ONE MOLECULE OF GLUCOSE HAS GIVEN RISE TO TWO MOLECULES OF GLYCERALDEHYDE-3-PHOSPHATE, USING 2 MOLECULES OF ATP.

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MCD Metabolism

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6. 2X glyceraldehydes 3-phosphate into 1,3-bisphosphoglycerate involving glyceraldehyde 3-phosphate dehydrogenase, NAD+ and inorganic phosphate (generating NADH)

NADH is generated here which can be later used to generate yet more ATP within the mitochondria in a process known as oxidative phosphorylation(METABOLISM 5) 7. 1,3-bisphosphoglycerate into 3-phosphoglycerate involving phosphoglycerate kinase and ADP

A phosphate group is transferred to an ADP molecule to give ATP. Resulting molecule is of lower energy 8. 3-phosphoglycerate into 2-phosphoglycerate involving phosphoglycerate mutase

Shuffling of the phosphate group from the 3 to the 2 position 9. Dehydration of 2-phosphoglycerate into phosphoenolpyruvate using enolase

Removal of a water molecule

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MCD Metabolism

Alexandra Burke-Smith

10. Phosphoenolpyruvate into pyruvate involving pyruvate kinase and ADP

Transfer of the high energy phosphate group to ADP, generating one ATP molecule in the process.

NET RESULT OF GLYCOLYSIS Substrate Level Phosphorylation  Substrate-level phosphorylation can be defined as the production of ATP by the direct transfer of a highenergy phosphate group from an intermediate substrate in a biochemical pathway to ADP, such as occurs in glycolysis.  This is in contrast to oxidative phosphorylation, where ATP is produced using energy derived from the transfer of electrons in an electron transport system 3.

Distinguish between the aerobic and anaerobic metabolism of glucose with reference to the enzymes involved and the comparative efficiencies of each pathway with respect to ATP generation.

Pyruvate has 3 possible fates 1. Alcoholic fermentation to form ethanol

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MCD Metabolism

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This is characteristic of yeasts and can occur under anaerobic conditions.

2. Generation of Lactate

This is also anaerobic and is characteristic of mammalian muscle during intense activity when oxygen is a limiting factor. This generates NAD+ 3. Generation of Acetyl CoA (See learning objective 5) Regeneration of NAD+ is essential  Both alcoholic fermentation and the generation of lactate allow NAD+ to be regenerated and thus glycolysis to continue, in conditions of oxygen deprivation.  i.e. conditions in which the rate of NADH formation by Glycolysis is greater than its rate of oxidation by the respiratory chain (oxidative phosphorylation)  NAD+, you recall, is needed for the dehydrogenation of glyceraldehyde 3-phosphate, which is the first step in generating ATP for the body (Step 6)

Aerobic vs. Anaerobic Respiration  From the anaerobic metabolism of one molecule of glucose we only generate 2 molecules of pyruvate and 2ATP molecules (net during Glycolysis)  This contrasts poorly to the complete aerobic metabolism of a molecule of glucose which can yield 38 molecules of ATP (including TCA Cycle and oxidative phosphorylation)

4. Describe the reactions catalysed by lactate dehydrogenase and creatine kinase and explain the diagnostic relevance of their appearance in plasma.

Lactate dehydrogenase (LDH) as a Diagnostic Tool  LDH is present in many body tissues, especially the heart, liver, kidney, skeletal muscle, brain blood cells and lungs.  LDH catalyses the inter-conversion of pyruvate and lactate  Elevated levels in plasma can be used to diagnose several disorders, as is a sign of damage to respiring tissues  E.g. stroke, heart attack, liver disease (e.g. hepatitis), muscle injury, muscular dystrophy, pulmonary infarction

Creatine Phosphate  In muscle, the amount of ATP needed during exercise is only enough to sustain contraction for around one second.

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MCD Metabolism  

Alexandra Burke-Smith

a large reservoir of creatine phosphate is on hand to sustain contraction by buffering demands for phosphate (25mM creatine phosphate c.f. 4mM ATP in resting muscle) DG (hydrolysis) = -31 kJ/mole (ATP) & -43.1 kJ/mole (CP)

Creatine kinase as a Diagnostic tool  When a muscle is damaged, creatine kinase leaks into the bloodstream  Either total levels of creatine kinase or the tissue specific isoform can be measured to help to determine which tissue has been damaged  Elevated levels can be used to diagnose myocardial infarction, determine the extent of muscular disease, evaluate cause of chest pain, help discover carriers of muscular dystrophy (Duchenne)  The total creatine kinase test is about 70% accurate whilst isoenzyme testing is about 90% accurate.

5. Outline the oxidative decarboxylation reaction catalysed by pyruvate dehydrogenase, with reference to the product and the five co-enzymes required by this enzyme complex.   

Third fate of pyruvate- aerobic respiration series of reactions occurs in the mitochondria of the cell The acetyl CoA thus formed is committed to entry into the citric acid cycle and can ultimately produce ATP by the process of oxidative phosphorylation (lecture 5).

The Pyruvate Dehydrogenase Complex  This is the committed step for the entry of pyruvate into the TCA cycle and the overall reaction is as follows

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The pyruvate dehydrogenase complex is gigantic (in molecular terms 60 polypeptides) and consists of three individual enzymes and five co-factors: Thiamine pyrophosphate (TPP), lipoamide, FAD, CoA and NAD+.

Reaction intermediates are passed directly from one enzyme to another, with products CO2, NADH, and acetyl CoA. Enzyme 1: pyruvate decarboxylase, with the prosthetic group TPP. Enzyme 2: lipoamide reductase-transacetylase, with the prosthetic group lipoamide.

Enzyme 3: dihydrolipoyl dehydrogenase, with the prosthetic group FAD. Prosthetic groups such as lipoamide are a permanent part of the complex, whereas NAD+ and CoA are cofactors bind reversibly to enzymes

Thiamine pyrophosphate (TPP)  Prosthetic group of pyruvate decarboxylase  Derivative of vitamin B1 (thiamine).  Readily loses a proton and the resulting carbanion attacks of pyruvate to yield hydroxyethyl-TPP.

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MCD Metabolism 

Alexandra Burke-Smith

Deficiency of thiamine (vitamin B1) is the cause of Beri-Beri, whose symptoms include damage to the peripheral nervous system, weakness of the musculature and decreased cardiac output. The brain is particularly vulnerable as it relies heavily on glucose metabolism.

Lipoamide  Prosthetic group of lipoamide reductase-transacetylase.  Functional group (undergoes oxidation and reduction).  The long flexible arm of the molecule allows the dithiol group to swing from one active site to another within the complex.  Arsenite and mercury have a high affinity for neighbouring sulphydryl groups, such as those that occur in reduced lipoamide and will readily inhibit pryuvate dehydrogenase.

Flavine Adenine Dinucleotide (FAD)  Prosthetic group of dihydrolipoyl dehydrogenase.  FAD accepts and donates two electrons and 2 protons to form FADH2

The complex

1. Decarboxylation of pyruvate to give hydroxyethyl TPP. 2. Oxidation & transfer to lipoamide to give acetylipoamide.

3. Transfer of the acetyl group to CoA to give acetyl CoA. 4. Regeneration of oxidised lipoamide.

5. Regeneration of oxidised FAD, generating NADH. 14

MCD Metabolism

Alexandra Burke-Smith

4. Metabolic Pathways and ATP Production II James Pease (j.pease@imperial.ac.uk)

1. Describe the processes by which the fatty acid palmitate and the amino acid alanine are converted into acetylCoA. Pyruvate can be used to make Acetyl CoA. (See METABOLISM 3 notes) These reactions occurs in the mitochondria of the cell, and the acetyl CoA thus formed in commited to entry into the citric acid cycle (Krebs Cycle) Fatty Acid Metabolism also leads to Acetyl CoA Production

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More Acetyl CoA is produced from both types of major food molecules (products of digestion) within the mitochondria of cells than during glycolysis It is the location where most of the cell's oxidation reactions occur and also where the majority of the cell’s ATP is made.

Acetyl CoA Definition of Acetyl CoA: small, water-soluble molecule that carries acetyl groups in cells. Contains an acetyl group linked to coenzyme A by a thioester bond

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The thioester bond (C—S) is a high-energy linkage, so it is readily hydrolysed (energetically favourable as releases a lot of energy when broken), enabling acetyl CoA to donate the acetate (2C) to other molecules.

RNA ancestry suggests it is of primeval origin Molecule required for Krebs cycle and therefore necessary for aerobic respiration to occur

FATTY ACID METABOLISM   

The oxidation/metabolism of fatty acids yields more usable chemical energy (ATP) than carbohydrates (e.g. glucose) can deliver because they are fully reduced, i.e. fully saturated by hydrogen The caloric yield from fatty acids is about double that from carbohydrates, providing more than half of the body’s energy needs including the liver. This need is enhanced by fasting.

However metabolism in the brain is completely dependent on glucose

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Fatty Acids are metabolized in the mitochondria in several stages. Firstly they are converted into an activated fatty acyl CoA species. This involves Acetyl CoA, ATP and Acyl CoA synthetase.

- Hydroxyl (OH) group is lost on the fatty acid - energetically favourable as coupled to ATP hydrolysis (ΔG°'= - 31 kJ/mol)

Palmitic acid can make Acetyl CoA The fatty Acyl CoA is then broken down into Acetyl CoA and an acyl CoA species (2 carbons shorter than the original) involving several enzymes in a sequence of reactions called b-oxidation       

Palmitic acid (16 carbon species) is one of the most commonly found saturated fatty acids. It is first converted to palmitoyl CoA by the reaction shown above.

The -oxidation reactions consecutively remove 2-carbon units from the acyl (e.g. palmitoyl) CoA thereby producing acetyl CoA. On the final cycle (4-carbon fatty acyl CoA intermediate), two acetyl CoA molecules are formed.

From just 7 b-oxidation reactions, the 16-carbon palmitoyl CoA molecule produces 8 molecules of acetyl CoA and a 14-carbon CoA (myristyl-CoA) During each cycle one molecule each of FADH2 and NADH are produced, which are used for ATP generation in oxidative phosphorylation (lecture 5) The overall reaction of -oxidation of palmitoyl CoA is: palmitoyl CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA  8 acetyl CoA + 7 FADH2 + 7 NADH

PROTEIN METABOLISM Degration of Amino Acids  Basis is to remove the amino group (excreted as urea), with the carbon skeleton being fed into the production of glucose (gluconeogenisis) or the Krebs Cycle (Lecture 9)  Degradation of all twenty amino acids gives rise to only seven molecules: pyruvate, acetyl CoA, acetoacetyl CoA, a-ketoglutarate, succinyl CoA, fumarate and oxaloacetate.  Involves transamination reactions

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Transamination: Defined as a reaction in which an amine group is transferred from one amino acid to a keto acid thereby forming a new pair of amino and keto acids.

Amino acid: organic molecule containing both an amino group and a carboxyl group. Α- amino acids (those in which the amino and carboxyl group are linked to the same central carbon) are the building blocks of proteins.

Keto acid: are organic compounds that contain two functional groups, a carboxylic acid group and a ketone group.

The alpha-keto acids are especially important in biology as they are involved in the Krebs citric acid cycle and in glycolysis.

Transamination of Alanine  Alanine is a 3-carbon amino acid  Undergoes transamination by the action of the enzyme alanine aminotransferase in the liver Alanine a-ketoglutarate pyruvate glutamate

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Pyruvate can enter the TCA cycle glutamate is re-converted to a-ketoglutarate by glutamate dehydrogenase, generating NH4+ which is ultimately converted to urea.

Persistently elevated levels of alanine aminotransferase are a diagnostic for hepatic disorders such as Hepatitis C.

2. Outline the Krebs or TCA (tricarboxylic acid cycle/citric acid cycle) with particular reference to the steps involved in the oxidation of acetyl Co-A and the formation of NADH and FADH2 and the cellular location of these reactions. 

A continuous cycle of eight reactions:

The 8 Reactions 1) Oxoacetate (4 carbon) into citrate (6 carbon), transfering the 2 carbon atoms from acetyl CoA- involving citrate synthase

The high energy thio-ester linkage of the acetyl CoA allows it to be readily donated to oxaloacetate.

Regenerates coenzyme A

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2) Isomerisation of citrate to give isocitrate- involving aconitase

loss of a molecule of water 3) Oxidation of isocitrate to give a-ketoglutarate- involving isocitrate dehydrogenase and NAD+

Loss of a 2 hydrogen atoms, which reduces cofactor carbon dioxide given off as a waste product 4) A-ketglutarate into succinyl-CoA- involving a-ketglutarate dehydrogenase complex, CoA and NAD+. Similar to the reaction catalysed by Pyruvate dehydrogenase PDH (lecture 3).

loses COO-, then attaches coenzyme A 5) CoA is displaced from the succinyl-CoA by a phosphate molecule (which is subsequently transferred to GTP) to form succinate- involving succinyl CoA synthetase and water

- GTP: Guanosine Triphosphate (In bacteria and plants, ATP is formed instead).

- Can act as a phosphoryl donor in protein synthesis or signal transduction processes (e.g. protein-coupled receptors) - Is a high energy molecule that can be readily broken down by hydrolysis.

- Alternatively, its g-phosphate group can be transferred to that of ADP to generate ATP and form GDPinvolving nucleoside diphosphokinase 18

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6) Oxidation of succinate to form fumarate- involving succinate dehydrogenase and FAD

Loss of one hydrogen molecule, which reduces cofactor 7) Addition of a water molecule to fumarate, breaking a double bond to form malatate- involving fumerase

8) THE LAST STEP: Dehydrogenation of malate to give oxaloacetate, the starting point of the cycle- involving malate dehydrogenase and NAD+

OVERVIEW OF THE KREBS CYCLE

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Location    

The Krebs cycle enzymes are soluble proteins located in the mitochondrial matrix space, except for succinate dehydrogenase, which is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane Here, it can communicate directly with components in the respiratory chain during oxidative phosphorylation.

The majority of the energy that derives from the metabolism of food is generated when the reduced coenzymes are re-oxidised by the respiratory chain in the mitochondrial inner membrane in a process known as oxidative phosphorylation (lecture 5).

The Krebs cycle only operates under aerobic conditions, as the NAD+ and FAD needed are only regenerated via the transfer of electrons to O2 during oxidative phosphorylation.

3. Outline the glycerol phosphate shuttle and the malate-aspartate shuttle, in particular stating why these mechanisms are required.

NADH Transportation  NADH produced in Glycolysis needs to enter the mitochondria to be utilised by the process of oxidative phosphorylation and to regenerate NAD+.  Remember, there is only a finite amount of NAD+ and unless it is regenerated, glycolysis will very quickly grind to a halt.  How does NADH, or more accurately, its high-energy electrons, cross from the cytosol into the matrix of the mitochondria ?

1. The Glycerol Phosphate Shuttle – skeletal muscle, brain 2. The Malate-Aspartate Shuttle - liver, kidney and heart The Glycerol Phosphate Shuttle  Electrons from NADH are carried across the mitochondrial membrane instead of NADH itself  A cytosolic glycerol dehydrogenase (G-DH) transfers electrons from NADH to glycerol 3-phosphate, which can diffuse into the mitochondria.  There, a membrane bound form of the same enzyme transfers them to FAD, and dihydroxyacetone phosphate is formed in The Malate-Aspartate Shuttle The net result in terms of NADH is: NADH cytoplasmic + NAD+mitochondrial



+ NAD cytoplasmic + NADH mitochondrial





This system uses two membrane carriers and four enzymes  Hydrogen is transferred from cytoplasmic NADH to oxaloacetate to give malate, a reaction catalysed by cytosolic malate dehydrogenase (MDH).

Malate can be transported into the mitochondria where it is rapidly re-oxidised by NAD+ to give oxaloacetate and NADH (catalysed by mitochondrial MDH).

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Transamination in the Malate-Asparate Shuttle

4. Calculate the theoretical maximum yield of ATP per glucose molecule oxidized by aerobic respiration and compare this to the theoretical maximum yield of ATP per molecule of palmitic acid ATP Production by Glycolysis and the Krebs Cycle is only a Prelude to Oxidative Phosphorylation Re-oxidation of the reduced co-factors NADH and FADH2 by the process of oxidative phosphorylation (lecture 5) yields the following:  Three ATP molecules are formed by the re-oxidation of each NADH molecule.  Two ATP molecules are formed by the re-oxidation of each FADH2 molecule.

Therefore, from Glycolysis and the Krebs Cycle:  Splitting of 1 glucose molecule gives 2 x ATP + 2 x NADH = 8 ATP  Oxidation of 1 X acetyl CoA molecule gives 3 x NADH + 1 x FADH2 + 1x GTP = 12 ATP Glucose Metabolism vs Palmitate Metabolism

Glucose Metabolism Glycolysis (1x Glucose)= 8 x ATP (2 ATP + 2X NADH) PDH (2x Pyruvate)= 6 x ATP Krebs Cycle(2x Acetyl CoA)= 24 x TOTAL= 38 ATP

ATP

Palmitate Metabolism b-oxidation = 35 x ATP Krebs Cycle= 96 x ATP TOTAL= 131 ATP BUT The first step of b-oxidation converts the fatty acid into an acyl CoA species which uses 2 ATP, therefore…

TOTAL= 129 ATP

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5. Give two examples of the use of NADPH in reductive biosynthesis.

Glycolysis and the Krebs Cycle Provide the Starting Point for Many Biosynthetic Reactions

  

The amino acids, nucleotides, lipids, sugars, and other molecules shown here as products, in turn, become the precursors for the many of the macromolecules of the cell.

Each black arrow in this diagram denotes a single enzyme-catalysed reaction.

Red arrows generally represent multi-step pathways.

The Relationship Between Catabolic and Anabolic Pathways in Metabolism  NADPH takes part in anabolic reactions, whereas NADH takes place in catabolic reactions.  Anabolism: Reaction pathways by which large molecules are made from smaller ones, e.g. breakdown of food molecules  Catabolism: General term for the enzyme-catalyzed reactions in a cell by which complex molecules are degraded to simpler ones with release of energy. Intermediates in these catabolic reactions are sometimes called catabolites. E.g. build up on macromolecules that make up a cell  The use of different co-factors for sets of reactions allows electron transport in catabolism to be kept separate to that of anabolism.

NADP+ (Nicotinamide Adenine Dinucleotide Phosphate)  NADP+ is a relative of NAD+, differing only by a phosphate group attached to one of the ribose rings.  Like NAD+, NADP+ can pick up two high energy electrons and in the process, a proton (H+) collectively known as a hydride ion (H-).  The phosphate group of NADP+ does not participate in electron transfer, but gives it a slightly different conformation, meaning that it will bind to different enzymes than NAD+.  The hydride ion is held in a high-energy linkage, allowing it to be easily transferred to other molecules.

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NADPH is a Co-factor in the Biosynthesis of RNA

NADPH is a Co-factor in the Biosynthesis of Cholesterol  NADPH helps to catalyse the final reaction of several, that lead to cholesterol synthesis.  The C=C bond is reduced by the transfer of a hydride ion (two electrons plus a proton from solution, H-).

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5. Mitochondria and oxidative phosphorylation James Pease (j.pease@imperial.ac.uk)

1. Draw a cross sectional representation of a mitochondrion, and label its component parts.    

 

A rod-shaped organelle with two membranes which enclose a fluid matrix Outer membrane (which limits the size of the organelle).

Inner membrane (folds that project inward called cristae).

The reactions of oxidative phosphorylation take place in the inner membrane, in contrast to the Krebs Cycle reactions which occur in the matrix.

Numerous folds within the cristae increase the surface area upon which oxidative phosphorylation can take place.

Distributed within rapidly respiring cells

2. Outline the proposed evolutionary origins of mitochondria. 



believed to be the evolutionary descendants of a prokaryote that established an endosymbiotic relationship with the ancestors of eukaryotic cells and that following this, many of the genes needed for mitochondrial function were moved (translocated) to the nuclear genome.

More recently, the elucidation of the complete genome of Rickettsia prowazekii has revealed that several genes are closely related to those found today in mitochondria. This brings up the question of whether Rickettsias the nearest living descendants of the endosymbionts that became the mitochondria of eukaryotes?

Supporting Evidence for the Theory  Mitochondria can only arise from pre-existing mitochondria and chloroplasts, i.e. like bacteria divide by binary fission to form identical clones  Mitochondria possess their own genome; a single circular molecule of DNA, with no associated histones and it resembles that of prokaryotes.  Mitochondria have their own protein-synthesizing machinery, which again resembles that of prokaryotes not that of eukaryotes.  The first amino acid of their transcripts is always fMet as it is in bacteria and not methionine (Met) that is the first amino acid in eukaryotic proteins).  A number of antibiotics (e.g. streptomycin) that act by blocking protein synthesis in bacteria also block protein synthesis within mitochondria and chloroplasts. They do not interfere with protein synthesis in the cytoplasm of the eukaryotes.

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3. Outline the chemiosmotic theory.

Electron Donation by NADH

The protons are donated readily to the solvent surrounding the enzyme complex whilst the electrons join the transport chain.

Oxidative Phosphorylation Oxidative Phosphorylation: process in bacteria and mitochondria in which ATP formation is driven by the transfer of electrons from food molecules to molecular oxygen. Involves the intermediate generation of a pH gradient across a membrane and chemiosmotic coupling Substrate-level phosphorylation: the production of ATP by the direct transfer of a high-energy phosphate group from an intermediate substrate in a biochemical pathway to ADP, such as occurs in glycolysis.

Within the mitochondria, the co-enzymes NADH and FADH2 are re-oxidised by molecular oxygen in the reactions: NADH + H+ + ½ O2 NAD+ + H20 (NADH  NAD+ + H+ + 2e-) FADH2 + ½ O2  FAD + H20 (FADH2  FAD +     

Each reaction has a DG of -220 and -167 kJ/mol respectively.

You may recall that DG ATP hydrolysis was -31 kJ/mol.

Both cofactors are effectively reducing molecular oxygen, and re-oxidised releasing energy This energy released is enough to generate several phosphoanhydride bonds, i.e. generate ATP. Part of this energy is recovered by the components of the electron transport chain and used to synthesise ATP. This is not 100% efficient, but most of the energy is recovered in the electron transport chain.

Chemiosmotic Hypothesis of Oxidative Phosphorylation  Put forward by Peter Mitchell in 1961.  Hypothesis: “Electron transport is coupled to ATP synthesis by proton gradients “.  Oxidative phosphorylation proceeds in two steps: 1) The translocation or movement of protons from within the matrix of the mitochondria into the membrane space. This is controlled by the electron transport or respiratory chain.

2) The pumped protons are allowed back into the mitochondrial matrix space through a specific channel, which is coupled to an enzyme which can synthesise ATP known as ATP synthase. It is the chanelling of the protons that generates the energy used to synthesise ATP. 25

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It is the accumulation of H+ that drives the protons back into the matrix, i.e. the proton motive force. This is because the accumulation of H+ in the membrane space lowers the PH and increases the positive charge outside of the matrix, thereby generating a PH gradient and a transmembrane electric potential.

4. Describe the electron transport chain in mitochondria with reference to the functions of coenzyme Q (ubiquinone) and cytochrome c.

The electron transport chain A chain of three protein complexes and two mobile carriers which act as electron carriers: Enzymes NADH Dehydrogenase complex Cytochrome b-c1 complex Cytochrome oxidase complex Mobile Carriers Ubiquinone (a.k.a. coenzyme Q) Cytochrome C. 

These integral membrane proteins accept electrons from the coenzymes and in doing so, translocate a proton (H+) from the aqueous solution into the intermembrane space 26

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Each unit (protein complex) of the chain has a higher affinity for electrons than the previous unit, allowing them to flow in a logical order/ensures Unidirectionality.

The electrons therefore lose energy as passed from one complex/unit to the other, and this energy is used to pump the protons/hydrogen through the complexes into the inter-membrane space The transfer of electrons from one complex to another is energetically favourable and as they progress along the chain, the electrons lose energy.

Ubiquone is a mobile carrier of electrons

  

Aromatic carbon ring structure with a hydrophobic hydrocarbon tail Its hydrophobic tail confines it to the lipid bilayer of the membrane where it is needed rather than moving within the mitochondrial matrix It can pick up either one or two electrons (together with a H+ from solution) and pass them to cytochrome bc1 complex.

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Ubiquone is the entry point for electrons donated by FADH2  This means that it bypasses the first electron carrier.  As one hydrogen is pumped into the inter-membrane space per electron transfer, this means only two hydrogens are translocated therefore only two can be channelled back into the matrix synthesising two ATP molecules instead of three (as with NADH)  The same is true for electrons donated by FADH2 generated by other reactions, e.g. glycerol phosphate shuttle, b-oxidation of fatty acids.

Cytochrome Oxidase  In the final electron transfer step, Cytochrome Oxidase receives 4 electrons from CYTOCHROME C and passes them to oxygen to generate water. Therefore cytochrome c acts as the final mobile carrier of electrons, and oxygen is the final electron acceptor.  4e- + 4H+ + O2  2H2O  In addition, 4 protons are also pumped to the intermembrane space, enhancing the proton gradient, which helps to ensure protons return to the matrix (channelled through ATP synthase)  Molecular oxygen is an ideal terminal electron acceptor as it has a high affinity for electrons, providing a driving force for oxidative phosphorylation.

Redox Reactions Defined as electron transfer reactions involving a reduced substrate (which donates electrons and therefore becomes oxidised) and an oxidised substrate (or oxidant) which accepts electrons and becomes reduced in the process.   

A substrate than can exist in both oxidised and reduced forms is known as a redox couple. e.g. NAD+ / NADH; FAD / FADH2; Fe3+ / Fe2+; ½ 02 / H20 The ability of a redox couple to accept or donate electrons can be determined experimentally and is known as the reduction potential or redox potential.

Standard Redox Potentials (E’0 ): dictates the tendency of a redox couple to donate electrons, and hence allows reducing power comparisons to be made.   

 

Convention dictates that a negative E’0 implies that the redox couple has a tendency to donate electrons and therefore has more reducing power than hydrogen. e.g.

NAD+/NADH E’0 = -0.32 V Similarly, a positive E’0 implies that the redox couple has a tendency to accept electrons and therefore has more oxidising power than hydrogen. e.g.

Fe3+ / Fe2+ E’0 = +0.77 V ½ O2 +2 H+/ H20 E’0 = +0.82 V Along the chain, the carriers have an increasing oxidising power, therefore is more likely to accept the electrons, so the transfer electrons from one complex to another is energetically favourable

of

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5. Describe how ATP synthase is able to generate and utilise ATP respectively, with reference to its structure.   

 



A multimeric enzyme consisting of a membrane bound part (FO ) and a part which projects into the matrix space (F1) The F1 and F0 parts each consists of three different subunits. F0 = a, b and c. F1 = alpha, beta and gamma When hydrogen ions flow through the membrane via a pore from the inter-membrane space into the matrix (along the membrane potential and pH gradient), the disc of c subunits is compelled to rotate, i.e. the enzyme rotates The γ-subunit in the F1 unit is fixed to the disc and therefore rotates with it, but the alpha and beta-subunits in the F1 unit cannot rotate because they are anchored together in a fixed position in the membrane The rotation of the γ-subunit drives structural changes in the catalytic portions of the beta-subunits, which alter their affinities for ATP and ADP. As a consequence, torsional energy/conformational energy flows from the catalytic subunit into the bound ADP and inorganic phosphate to promote the formation of ATP (conformational  chemical energy) There are three conformations of the enzyme: open, loose binding and tight binding, the latter of which is the conformation which forces the ADP and inorganic phosphate into ATP

The Direction of Proton Flow dictates ATP Synthesis v ATP Hydrolysis I.e. depending on the direction of the flow of protons through the ATP synthase, we can either generate ATP or consume it.

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Oxidative Phosphorylation – An Overview

Location of the Krebs Cycle Enzymes Succinate dehydrogenase is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane. There, it can communicate directly with ubiquinone. As such one less proton is pumped to the intermembrane space, c.f. NADH and as a consequence, less ATP is produced. The same is true for electrons donated by other FADH2 species, e.g. glycerol phosphate shuttle, β-oxidation of fatty acids.

th

6 reaction of Krebs Cycle

ATP Consumption – A Matter of Life and Death     

The average human body synthesizes around 70kg of ATP per day.

Each of these ATP molecules has a lifespan of between 1 and 5 minutes.

Consequently, any interruption to the process of oxidative phosphorylation and therefore to ATP synthesis, means that a cell rapidly becomes depleted of ATP and is likely to die.

The most common cause of a failure of oxidative phosphorylation is simply a lack of oxygen e.g. hypoxia (diminished), anoxia (total).

Depending on the cell type and their metabolic requirements, death will be within a few minutes (neurons) or a few hours (muscle).

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6. Explain why carbon monoxide, cyanide, malonate and oliogomycin are poisonous in terms of their effects on specific components of the electron transport chain. 

 



Cyanide: bind with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complex.This blocks the flow of electrons through the respiratory chain and consequently, the production of ATP. Carbon monoxide: binds to the ferrous (Fe2+) form of the haem group, also blocking the flow of electrons.

Malonate: closely resembles succinate and acts as a competitive inhibitor of succinate dehydrogenase. This is the one Krebs Cycle enzyme that resides in the inner mitochondrial membrane and passes its electrons directly to ubiquinone via FAD. It effectively slows down the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumarate (reaction 6 in the Krebs cycle) Oligomycin: an antibiotic produced by Streptomyces that inhibits oxidative phosphorylation by binding within the ‘stalk’ of ATP synthase. In doing so, it blocks the flow of protons through the enzyme. As a result, ATP synthesis is inhibited and a backlog of protons will build up in the intermembrane space. This in turn, will eventually inhibit the flow of electrons through the electron transport chain as the [H+] outside the mitochondrion will build up to saturation point at which no more protons can be pumped out against this proton gradient.

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6. Lipids and Membranes Professor Miguel Seabra (m.seabra@imperial.ac.uk)

Membranes are essential to life, as they serve as barriers which distinguish the cell and its intracellular organelles from their external environment There are two types of membranes; plasma and intracellular Membrane model: the fluid mosaic of Singer and Nicholson (1972)  Membranes are 2D solutions of oriented proteins and lipids, that form a 5nm thick bilayer.  The lipids and proteins are held non-covalently, and proteins are either integral or peripheral.  Integral proteins span both sides of the bilayer  Membranes are heterogeneous

1. Describe the structure of: fatty acids, triglycerides, phospholipids, cholesterol, sphingomyelin.

Phospholipids:  Main consitituents of the lipid bilayer  Hydrophilic heagroup containing phosphate  Glycerol (3-carbon) backbone  Two hydrophobic fatty acid tails, where any double bonds present kink the chain  Organisation of the bilayer is such so that the hydrophobic tails avoid water, and the hydrophilic head faces an aqueous environment, i.e. the most energetically favourable position  Rigity of the membrane is affected by the “kinkness” and length of the fatty acid chains; short kinked chains are more fluid as they are unable to pack together as densely  In addition to their structural role, phospholipids also have a role is signal transduction CommonPhospholipids:  Phosphatidylcholine  Phosphatidylserine  Phosphatidylethanolamine  Phosphatidylglycerol  Phosphatidylinositol Cholesterol: (see next lecture) Sphyingolipids and glycosiphingolipids Ceramide is the most basic sphingolipid which can then be glycolysed E.g. Sphingomyelin (seen right) Fatty Acids  The simplest lipids  Constituents of more complex lipids  In triglycerides as well as phospholipids  Are linear hydrocarbons of varying chain length with a carboxylic acid attached to it  Often sterified with an alcohol to form a stable ion Triglycerides  Glycerol backbone with three fatty acid chains of varying length  Function is energy storage  Can be saturated of unsaturated, depending on the presence of C=C fatty acid tails  Tristearin: triglyceride formed by a particular saturated fatty-acid certain length.

bonds in the chain of a

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2. Give examples of how the lipid composition can differ for different cellular membranes, and indicate the significance of this 





 





Glycolipids and cholesterol are diferentially present in different types of membranes, e.g. human erythrocyte plasma membrane and E. Coli cell membrane, and this difference is related to their function etc. For example more glycolipids and cholesterol are present in membranes which are exposed to the extra-cellular environment.

Phospholipids are the most common part of membranes, although phosphatidylinositol is not common, but is very important in signal transduction MICRODOMAINS There are not only asymmetries between different types of membrane, but also within a membrane- certain lipids are associated with the inner or outer leaflet. These are known as microdomains. Asymmetry within a bilayer is an active process as the molecules “flip” leaflets.

Extracellular leaflet is composed of more glycolipids, sphingomyelins and phosphaidylcholin. The cytosol exposed leaflet is composed of phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol.

Dead cells present with more phosphatidylserine on the outer (extracellular) leaflet, as it helps macrophages to identify and destroy the cells.

Rafts are specialised microdomains of the plasma membrane enriched in certain lipids (glicolipids and cholesterol) and proteins They are small and transient, and have functional significance, i.e. they are concentrated areas of signal transduction.

3. Explain the process of fat digestion, absorption, mobilisation and storage Digestion and Absorption in the GI tract  Large fat globules are emulsified by bile salts in the duodenum, and broken down into smaller fat droplets  Digestion of fat by the pancreatic enzyme lipase yields free fatty acids and monoglycerides by breaking the bonds between glycerol and fatty acids, which then form micelles Mobilisation and Storage  Fatty acids and monoglycerides leave micelles and enter epithelial cells by diffusion  Chylomicrons containing fatty substances are transported out of the epithelial cells and into lacteals, where they are carried away from the intestine by lymph.  Triacylglycerols are transported through the bloodstream via very low-density lipoproteins  These are then stored as triacyglycerols in special tissue which releases them when energy is needed to generate ATP, i.e. through beta-oxidation At the cellular level Digestion= lipids  fatty acids Beta-oxidation- fatty acids  acetyl-CoA 33

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4. Outline the pathway for synthesis of fatty acids.     

Also called lipogenesis Essentially occurs in the cytoplasm Source is Acetyl-CoA, which is mainly produced in mitochondria, and is the key intermediate between fat and carbohydrate metabolism The mitochondrial membrane is impermeable to acetyl-CoA, thus is transported via citrate (look at Krebs Cycle notes) Acetyl CoA (2C)  Palmitate (16C)

Step 1: Production of Malanoyl CoA (3C)  Acetyl CoA only serves to start the reaction, as very little is available in the cytoplasm  Catalysed by Acetyl CoA carboxylase, the cofactor of which is BIOTIN  Involves ATP hydrolysis Step 2: Activation by Acyl Carrier protein  Exchanges CoA for ACP (acyl carrier protein), using Malanoyl-Coa-ACP transacylase  Cofactor for enzyme is phosphopantetheine Step 3: Elongation by successive adhesion of 2C units  Condensation  Reduction  Dehydration  Reduction  Catalysed by FA Synthase  A phosphopantetheine (ACP) is bound to the FA synthase, but acts as a swinging arm which transports the substrates (Acyl groups) into contact with all of the active sites on the FA synthase  There is a pre-existing Acetyl-CoA which is bound to the enzyme  In each cycle, malanoyl- CoA comes in (releasing 1C as carbon dioxide) to form a 4 carbon compound  This means 7 cycles are required to produce palmitate 34

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1. The cycle begins with the transfer of the acetyl group from acetyl-CoA to the phosphopantetheine swinging arm (site 1 ) 2. The acetyl group is then transferred to a cysteine thiol group on the beta-ketoacyl-ACP synthase (site 2) 3. At the end of the first cycle, the resultant butyryl group is transferred to the same cysteine thiol group, so that another group can begin by transfer of a malanoyl group from malanoyl-CoA to the phosphopantetheine swinging arm

Overall Reaction

Acetyl CoA (C2) + 7 Malonyl-CoA (C3) + 14 NADPH +14 H+ 7 HCO2+ 6 H2O + 8 CoA SH + 14 NADP+

ďƒ

Palmitate (C16) +

Note: the reaction is reversible Further metabolism of Palmitate 1. Esterification to form triacyglycerols 2. Formation of other fatty acids, e.g. unsaturated and longer chains There are some essential fatty acids which cannot be synthesised therefore need to be acquired from diet Regulation of Lipogenesis 1. Feedback inhibition of palmitoyl CoA to: acetyl CoA carboxylase fatty acid synthesis pentose phosphate pathway 2. Acetyl CoA carboxylase regulation by hormones 3. Transcriptional regulation of acetyl CoA carboxylase and FA synthase (activated by insulin and inhibited by glucagon)

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7. Cholesterol Professor Miguel Seabra (m.seabra@imperial.ac.uk)

1.

2.

3.

4.

5.

6.

7.

Explain the physiological functions of cholesterol in membrane stability Outline the synthesis of cholesterol from acetate Outline the synthesis of bile acids and steroid hormones from cholesterol Describe the mechanism of transport of cholesterol around the body and its uptake into cells.

Draw a diagram of low density lipoprotein (LDL) Explain why disturbances in cholesterol homeostasis cause disease Give an example of how a selective enzyme inhibitor can be used as a pharmalogical agent controlling cholesterol metabolism Structure  Derivative of saturated tetracyclic hydrocarbpn  All cyclohexane rings are in the chain conformation, giving cholesterol a planar, rigid structure  The storage form is acylated (addition of fatty acid) at the third cyclohexane ring Function  In membranes: high cholesterol leads to reduced fluidity due to: - reducing phase transitions of lipids - reducing lateral motility of polar lipids (by helping to pack the hydrophobic chains in the core)  Precursor of steroid hormones  Precursor of bile salts  Signalling molecule

Cholesterol Biosynthesis Step 1: Formation of Mevalonate from Acetyl-CoA  

3 enzymes involved: thiolase, HMG-CoA synthase, HMG-CoA reductase HMG-CoA reductase catalyses the regulated step. This is important as allows cholesterol production to be shut down, which has many uses, e.g. pharmacolody - statins

Step 2: Isoprenoid Metabolism- conversion of Mevalonate (C6) to Squalene (C30) Note: this is also important for modification of proteins and signalling intermediates   

2.1 Mevalonate (C6) to Isoprene Units (C5) 2.2 Head-to-tail condensations of isoprene units (C10 then C15) 2.3 Brached pathway at farnesyl pyrophosphate involving squalene synthetase into squalene (C30)

Step 3: Squalene to Cholesterol 

Cyclization reaction whereby squalene is given a basic cyclohexan structure with a hydroxyl group

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Synthesis of Steroid Hormones (diagram in powerpoint)   

All steroid hormones are synthesized from pregnenolone (cholesterol) Pregnenolone is generated by cholesterol desmolase, an enzyme which is only expressed in particular tissues, i.e. specific sites of synthesis The levels of hormone are controlled by the rate of synthesis, and hence the rate of pregenenolone production

There are 5 classes of steroid hormones:     

Progestins Corticosteroids Mineralosteroids Androgens Estrogens

Synthesis of bile salts/acids (diagram in powerpoint)  

 

This pathway represents the major route for elimination of cholesterol via the GI tract, i.e. these are excreted although in small quantities.

They are synthesized in the liver; the regulated step being the conversion of cholesterol into 7-alphahydroxycholesterol involving cytochrome P450 monooxygenase, which then goes through further reactions to form conjugated bile salts, which are secreted in polarised aqueous form into the GI tract.

The bile acids are transported into the enterohepatic circulation (liver  GI tract  liver) The bile acids are also conjugated further into other compounds

Function of bile salts Emulsification of dietary fats Emulsifier: a substance that disperses one ingredient into another in which it would not ordinarily dissolve, e.g. oil and water Fat Absorption in GI tract Cell membranes are not permeable to triesterglycerols, so they need to be broken down into constituents, and then repacked to form plasma lipoproteins 1. Large fat globules are emulsified by bile salts in the duodenum 2. Digestion of fat by the pancreatic enzyme lipase yields free fatty acids and monoglycerides, which then form micelles 3. Fatty acids and monoglycerides leave micelles and enter epithelial cells by diffusion 4. Chylomicrons (large lipoproteins) containining fatty substances are then transported out of the epithelial cells and into lacteals (lymphatic capillary that absorbs dietary fats in the villi of the small intestine), where they are carried away from the intestine by the lymph

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Transport of Lipids within the Body Lipoproteins  



Lipids are packaged as lipoproteins Cholesterylesters and triacylglycerolare (hydrophobic) found in the core and free cholesterol, phospholipids and proteins (hydrophilic) are in the surface layer.

This allows the lipids to be circulated in the aqueous environment of blood

Separation, Identification and composition  Lipoproteins can be separated by ultracentrifugation or electrophoresis  Ultracentrifugation: by subjecting a sample to a very high speed, the denser molecules will migrate further down the tube, therefore a sample can be separated into its different constituents  Lipoproteins that contain more proteins, and less fat will be denser

Circulation  VLDL: lipids are repackaged into these in the liver. These are similar to chylomicrons, except they consist of more cholesterol.  Cholesterol cannot be metabolism on the outside of the cell, therefore the fatty acids and triglycerides within chylomicrons and VLDL are “used up”, but cholesterol returns to the liver by HDL to be repackaged, secreted with bile salts, or to be used as a precursor for steroid hormones.

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Apoproteins The protein constituent of a lipoprotein that binds to lipids  Triglycerides within chylomicrons and VLDL are used by at the surface of capillary cells  Firstly the particles are slowed down by lipoprotein lipase, which hydrolyses the triglycerides. This involves Apo C-11, which is a classic functional apoprotein  The free fatty acids and glycerol then diffuse into the endothelial cells Trafficking of the LDL receptor i.e. how is cholesterol taken up by cells Endocytosis: the active process whereby cells take up components from the extracellular matrix The hydrolytic enzymes of the lyzosyme break down the endosome, releasing the cholesterol and amino acids (i.e. the apoproteins) ACAT is then used to form storages of cholesterol HMG-CoA regulates cholesterol levels by deciding whether cholesterol is made in the cell or extraceullar cholesterol (storages formed by ACAT) is used

Familial Hypercholesterolaemia Pathogenisis LDL receptors becaome defective, therefore LDL accumulates in the blood High levels of cholesterol is associated with arteriosclerosis, stroke, myocardial infarction etc Hymozygous recessive for FH often have severe heart attacks Control  Dietary restriction: if there is less cholesterol in the blood to begin with, less will be taken up into cells  Increased excretion resins: by stimulating bile acids less cholesterol will be absorbed into the blood and more will be excreted  Feedback effect: inhibition of cholesterol synthase, HMG-CoA reductase inhibitors- if the cell needs more cholesterol, more LDL receptors will present at the cell surface, therefore more cholesterol is removed from the blood

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8. Membrane Trafficking Professor Tony Magee (p.magee@imperial.ac.uk)

1. Explain the terms “endocytosis” and “exocytosis”.

2. Describe the pathway and cellular locations for synthesis, post-translational modification and exocytosis of a secreted protein.

3. Distinguish “constitutive” and “regulated” secretion.

4. Describe the process of receptor-mediated endocytosis and the roles played by endocytic vesicles, early endosomes, late endosomes, and lysosomes.

5. Give a general description of the molecular mechanisms of vesicular transport within cells.

6. Give examples of diseases resulting from defects in the secretory and endocytic pathways.

Intracellular Membrane Organisation  

Intracellular Transport; the trafficking of proteins within a cell There are a large number of membrane bound organelles within a cell, leading to a complex system of membranes which allow proteins to move between them

There are three types of Intracellular Transport: 1. Gated transport: e.g. nuclear import- nuclear pores are specialised protein complexes within the nuclear protein which control the movement of substances in/out of the nucleus by the opening and closing of a “gate” 2. Trans-membrane transport: e.g. import of newly synthesized proteins into the ER 3. Vesicular transport: e.g. inter-organellar transport- protein trafficking between organelles. Vesicular transport is complex; involving secretory/exocytotic and endocrytic pathways.

Intracellular trafficking is involved in many diseases including: - Genetic (> 75 genetic diseases or syndromes) - Infections (bacteria, viruses, toxins) - Cancer

The secretory/exocytic pathway Overview  Synthesised proteins are translocated into the RER  These are then transported by vesicles into the Cis side of the Golgi apparatus  Proteins move through the Golgi complex, undergoing post-translational modifications  The proteins are then transported in vesicles. These vesicles may be secretory (transporting the proteins to the plasma membrane) or storage vesicles 1. Translocation into the RER  The proteins that are transported into the ER (which may be secretory or transmembrane) are synthesized from a common pool of ribosomes which also synthesize the proteins that stay in the cytosol  It is the ER signal peptide on the protein that directs the engaged ribosome to the ER membrane  The mRNA that encodes a protein destined for transport into the ER may remain permanently bound to the ER by NASCENT CHAINS as part of a polyribosome, while the ribosomes involved in the translation of the 40

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mRNA into proteins are recycled (they are released after the protein synthesis and rejoin the common pool in the cytosol) Cytosolic proteins are synthesized freely in the cytosol

Post-transational modifications and quality control  Secretory proteins then fold, form disulphide bridges (using protein disulphide isomerases), may undergo glycolysation and specific proteolytic cleavages as they are transported through the RER  Multimeric proteins are also assembled, before they “bud out” in secretory vesilces for transport to the Golgi apparatus  When proteins are “mis-folded”, they are recognised and degraded in the cytosol- this acts as a quality control  If this degradation process is overwhelmed, misfolded non-functioning proteins will accumulate in the cell and may cause disease, e.g. cystic fibrosis Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)  CFTR is classified as an ABC gene.  Mutations of the CFTR gene affect functioning of the chloride channels in the cell membranes of epithelial cells, which leads to the accumulation of thick mucus (principle of Cystic Fibrosis)  The most common mutation (ΔF508) results from deletion (Δ) of three nucleotides which causes loss of the phenylalanine at the 508th position on the protein  This deletion affects the protein folding, therefore the “quality control” mechanism recognizes it as nonfunctioning and it is degraded in the cytosol Robinow Syndrome  Cell surface tyrosine kinase receptor (ROR2) – responsible for aspects of cartilage and bone growth involved in the early formation of chondrocytes and growth plate development  Mutation causes retention and degradation in ER  Symptoms: Dysmorphic facial appearance Dwarfism and vertebral malformations Congenital heart defects Genital hypoplasia (underdevelopment) 2. Transport of Proteins into the Golgi Apparatus  Occurs after the proteins are folded, processed and packaged in the RER through vesicular and tubular transport  Forward Pathway: Secretory Proteins become concentrated by the membrane of the RER, and bud off in secretory vesicles, where they are transported to the Golgi apparatus and fuse with the cis golgi network membrane, releasing their contents  Return/Retrieval Pathway: Organelles have resident proteins which do not move through the secretory pathway, but remain in the organelle for a specific function.

Occasionally the ER resident proteins “escape” in vesicles. Receptors in the Golgi recognize these ER resident proteins, so bind to them forming returning vesicles- this is a conservative system

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3. Movement through the Golgi apparatus   

At the cis golgi network (facing the ER), vesicles fuse with the membrane and release the proteins into the golgi complex. Oligosaccharides (carbohydrate group) on lysosomal proteins are then phosphorylated.

The proteins then move through the golgi stack, where they are processed and undergo further modification, including the changing of glycan and carbohydrate chains on the proteins.

At the trans golgi network, the proteins are sorted- this involves packaging the proteins into specific vesicles for different destinations e.g. the cell surface

4. Sorting at the trans Golgi network Exocytosis  Conservative Secretory Pathway: this is the unregulated pathway that exists in all cells. It is required for plasma membrane and extracellular proteins to be transported to the cell surface.  Lipids are also constantly synthesized and packaged in secretory vesicles  Regulated secretory pathway: exists in specialised cells e.g. nerve cells and beta cells in the pancreas.

Proteins are stored in the vesicles until signalled to fuse with the plasma membrane by hormones or neurotransmitters.

Lysosomal Enzymes  Lysosome is a major organelle involved with degradation of substances  It contains hydrolytic enzymes which are bound in a membrane in order to protect the rest of the cell  The lysosome is more acidic than cytosol (approx 5- 5.5), as the enzymes only function at this pH. This is another protective mechanism, as if the enzymes are released into the cytosol they will not function and damage the cell  The PH is maintained by proton pumps in the membrane of the lysosome  The hydrolytic enzymes are tagged with a phosphate marker (catalysed by PHOSPHOTRANSFERASE), forming mannose 6-phosphate. This marker allows the enzymes to be recognized by and bind to the M6P receptor in the trans golgi network and then packaged in specific transport vesicles  The vesicle then fuses with a late endosome, and the acidic ph causes the enzymes o dissociate from the M6P receptor (involving the removal of the phosphate), therefore the M6P receptor can be recycled  Lysosomal Storage Disorders: if inappropriate proteins are stored in lysosomes, or the hydrolytic enzymes are not tagged (hence will go through secretory pathway, and the lysosome will lose its degrading function leading to an accumulation of toxic proteins in the cell) e.g. I-cell (inclusions) disease results from mutations in phosphotransferase

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Endocytosis There are three types of endocytosis: 1. Receptor-mediated endocytosis: certain proteins have receptors on membrane, form coated pits, invaginate and form coated vesicles  Recycling: occurs with specific proteins e.g. trasnferrin receptor (which carries iron). The transferring is taken up into an early endosome, and at the lower ph releases the iron. The transferring is then recycled.  Degradation: early endosome will mature into a late endosome before either fusing with hydrolytic enzymes to form a lysosome, or fusing with the golgi apparatus,  Transcytosis: in specialised cells, e.g. polarised epithelial cells, endocytosis can transport proteins across the tight epithelium from the apical to basolateral membrane.

Low Density Lipoprotein Receptor (liver)  

  

LDL proteins contain cholesterol These are recognised by receptors, then cluster to form a CLATHRIN coated pit.

Endocytosis to form vesicles, which are then uncoated and fuse with an early endosome.

The LDL receptors and then returned to the plasma membrane The endosome can then fuse with a lysosome to release the cholesterol Familial Hypercholesterolaemia: mutations block the function in different pathways during the release of cholesterol, including: - synthesis of receptor in RER - transport of receptor within cell - blocked binding of LDL to receptor - stops clustering of LDL into coated pits - blocks the recycling of the LDL receptors, therefore they accumulate in the cell

2. Pinocytosis: effectively grabbing bits of extracellular matrix and the molecules it contains 3. Macropinocytosis/Phagocytosis: takes up larger molecules, e.g. microbes are taken up by macrophages and fuse with lysosomes so they can be destroyed.

Overview of Steps in Vesicular Transport 1. Cargo sorting in lumen and vesicle formation in donor membrane 2. Vesicle movement, involving microtubules and actin (in cytoskeleton) 3. Protein mediated vesicle tethering (when they reach the membrane- recognised by the proteins on the surface membrane of the vesicle)/docking 4. Fusion with acceptor membrane

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9. Integration of Metabolism Gabriela da Silva Xavier (g.dasilva-xavier@imperial.ac.uk) 1.

Outline general features of metabolic activity in liver, brain, muscle, adipose tissue and endocrine pancreas

2.

Know the effects of eating and fasting on metabolism

3.

Describe glucose interactions with lipid and amino acid synthesis & breakdown

4.

Know basic details of metabolism in muscle

5.

Know basic details of diabetes as e.g. of dysregulation

Overview The whole range of biochemical processes that occur within an organism. Metabolism consists both of anabolism and catabolism (the buildup and breakdown of substances, respectively). The biochemical reactions are known as metabolic pathways and involve enzymes that transform one substance into another substance, either breaking down a substance or building a new chemical substance.     

Measurable as: O2 uptake, CO2 release, heat production Rate of energy intake: sporadic peaks relating to intake of food, but otherwise 0 Rate of energy output: energy is expanded between intakes of food The body must be able to metabolise different types of food Different tissues also have different requirements

Specialised tissues  Muscle - (40 % of total body weight) - can have periods of very high ATP requirement during vigorous contraction and rely on carbohydrate and fat oxidation - during light contractions ATP consumption is met by oxidative phosphorylation (O2 and blood-borne glucose and fatty acids are used) - during vigorous contraction ATP consumption is faster than ATP supply by oxidative phosphorylation (O2 and blood-borne substrate diffusion is limiting)  muscle store of glycogen is broken down to produce ATP - under anaerobic conditions pyruvate is converted to lactate, which can leave muscle and reach the liver via the blood. Lactate is then utilised by the liver in gluconeogenisis in order to generate a supply of glucose, the accumulation of lactate causes “cramp” - further ATP by interconversion from creatine-phosphate  Brain and nervous tissue - (2 % of total body weight) - uses 20 % of resting metabolic rate as it has a continuous high ATP requirement; cannot utilise fats but relies of glucose and pentose phosphate intermediates - requires continuous supply of glucose for metabolism - too little glucose (hypo-glycaemia) causes faintness and coma - too much glucose (hyper-glycaemia) can cause irreversible damage - cannot metabolise fatty acids - ketone bodies (-hydroxy-butyrate) can partially substitute for glucose (pentose phosphate pathways) - in diabetes, both hypo and hyper-glycaemia occur

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 Adipose tissue - (15 % of total body weight) - long term storage site for fats  -

Heart (1 % of total body weight) 10 % of resting metabolic rate and can oxidise fats and carbohydrate heart must beat constantly designed for completely aerobic metabolism, with a large number of mitochondria can use TCA cycle substrates, e.g. free fatty acids, ketone bodies loss of O2 supply is devastating, leading to cell death and myocardial infarction (energy demand > energy supply)

 -

Liver (2.5 % of total body weight) 20 % of resting metabolic rate; the body’s main carbohydrate (glycogen) store and source of blood glucose. immediate recipient of nutrients absorbed at the intestines wide repertoire of metabolic processes (glycolysis, glucose production and gluconeogenesis, glucose storage) highly metabolically active and can interconvert nutrient types central role in maintaining blood glucose at 4.0-5.5 mM storage organ (glycogen) lipoprotein metabolism (transport of triglycerides & cholesterol)

-

Pathways Glucose See previous metabolism lecture notes for details

Gluconeogenesis       

process for making glucose or glycogen from oxaloacetate (a TCA cycle intermediate) is essentially only a function of liver (activity in kidney<<liver) requires ATP hydrolysis not an exact reversal of glycolysis - different enzymes bypass some irreversible glycolysis steps.

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Other nutrients

Note: The majority of metabolism occurs in the mitochondria

Energy Stores and Consumption What happens during exercise?

Aerobic Contractions increase ATP demand

Anaerobic ATP demand cannot be matched by O2 delivery

Contractions increase glucose transport into cells

Transport cannot keep up with the demand by cells for glucose

Muscle glycolysis increases (adrenalin)

Muscle glycogen breakdown increases

Gluconeogensis increases (adrenalin)

Lactate increases

Fatty acid metabolism increase (adrenalin)

Liver uses lactate to form glucose (recovery)

Metabolic Control Glucose metabolism Negative feedback mechanism - Blood glucose increasesď&#x192; uptake of glucose into muscle and liver cells increase involving hexokinase I (muscle) and IV (liver) and the conversion of glucose into glucose-6-phosphate (G6P)

Muscle Hexokinase I

Liver Hexokinase IV (involving Glucose-6-phosphatase)

HKI allows for a rapid production of G6P for glycolysis

Glucose-6-phosphatase allows the interconversion of glucose and G6P, therefore glucose can be released when needed

HKI has a high glucose affinity, which makes sure rate of glucose uptake is rapid

HKIV has a low glucose affinity, which means glucose is released easily if required by respiring cells

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MCD Metabolism HKI is highly sensitive to G6P inhibition, therefore the conversion takes place more rapidly when the product is removed- when G6P increases, there is increased binding of the enzyme to the product therefore cannot bind to the glucose substrate

Alexandra Burke-Smith HKIV has a low sensitivity to G6P inhibition

Hormonal Control  Insulin- secreted when glucose levels rise: stimulates uptake and use of glucose and storage as glycogen and fat  Glucagon- secreted when glucose levels fall: stimulates production of glucose by gluconeogenesis and breakdown of glycogen and fat (both secreted by the islets of the pancreas) – the balance of these hormones allows regulation of blood glucose levels  Adrenaline (or epinephrine): strong and fast metabolic effects to mobilise glucose for “flight or fight”  Glucocorticoids: steroid hormones which increase synthesis of metabolic enzymes concerned with glucose availability (both secreted by the adrenal glands)

The endocrine pancreas and diabetes Islets of Langerhans  Large blood supply  Balls of cells within the pancreas  Consists of different cell types: - alpha  glucagon - beta  insulin - delta somatostatin - epsilon  pancreatic polypeptides  Majority are beta cells  <10% are alpha cells  <1% are delta and epsilon cells  Blood flow through islets are directional- from core to periphery Insulin        

Production is glucose dependent Glucose is metabolised to produce ATP, which acts as an intracellular signalling molecule An increase in ATP affects the K+ATP channel protein Increase of Ca2+ into cell Increase release of insulin, which binds to insulin receptor The binding of insulin with the insulin receptor increases the protein kinase pathway, which activates the insulin gene This leads to a further increase in insulin production The increase in insulin production leads to an increase in the conversion of G6P into glucose (gluconeogenisis) involving HKIV and glucose-6-phosphatase (ANABOLIC)

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After a meal Blood glucose initially increases, and is then controlled by:     

increased secretion of insulin (and reduced glucagon) from islets increased glucose uptake by liver and conversion to G6P- used for glycogen synthesis and glycolysis (acetylCoA produced is used for fatty acid synthesis) increased glucose uptake and glycogen synthesis in muscle increased triglyceride synthesis in adipose tissue increased usage of metabolic intermediates throughout the body

Glucagon  Hormone secreted by pancreatic a-cells.  Where insulin is the hormone of the fed state, glucagon is the hormone of the fasted state.  Regulation of glucagon secretion has been reported to be regulated by glucose, insulin and zinc.  Regulation of glucagon secretion is dependent on ion channels that allow the flux of sodium, calcium and potassium ions.  A decrease in glucose concentration decreases ATP production, which causes an increase of Ca2+ out of the cell, which leads to an increase in glucagon production  Regulation of glucagon secretion has been reported to be regulated by both the sympathetic and parasympathetic nervous system.  Alpha cells are also regulated by insulin; an increase in insulin secretion inhibits glucagon release. Glucose concentration regulates insulin production, therefore one feedback loop.

Incretins  Glucagon like peptide 1 (GLP-1) is released from the L-cells in the small intestine during food intake. It is encoded by the same gene as glucagon.  GLP-1 increases insulin release.  GLP-1 decreases glucagon release.  Effects are glucose-dependent.  GLP-1 analogues inhibit gastric emptying and promote satiety (feeling full), therefore can be useful in managing obesity Between meals After a meal blood glucose starts to fall and is controlled by:   

increased glucagon secretion (and reduced insulin) from islets glucose production in liver resulting from glycogen breakdown and gluconeogenesis utilisation of fatty acid breakdown as alternative substrate for ATP production (important for preserving glucose for brain) Note: adrenaline has similar effects on liver, but also stimulates skeletal muscle towards glycogen breakdown and glycolysis, and adipose tissue towards fat lipolysis to provide other tissues with alternative substrates to glucose Fasting I.e. where glycogen reserves cannot cover it     

glucagon/insulin ratio increases further adipose tissue begins to hydrolyse triglyceride to provide fatty acids for metabolism TCA cycle intermediates are reduced in amount to provide substrate for gluconeogenesis protein breakdown provides amino acid substrates for gluconeogenesis ketone bodies are produced from fatty acids and amino acids in liver to substitute partially the brain’s requirement for glucose, which increases the PH and can lead to acidosis of the blood.

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Diabetes a disorder of insulin release and signalling    

Type I diabetics cannot make insulin Type II diabetics have reduced responsiveness to insulin, and a disregulation in glucagon release The effect is that metabolism is controlled as if for starvation, regardless of dietary uptake.

Complications include: - hyperglycaemia with progressive tissue damage resulting - increase in plasma fatty acids and lipoproteins with possible cardiovascular complications - increase in ketone bodies with possible acidosis - hypoglycaemia with consequent coma if insulin dosage imperfectly controlled

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