Comprehensive Overview of Metabolic Processes

Overview of metabolic pathways Glycolysis: Glucose -> pyruvate Gluconeogenesis: pyruvate -> glucose Glycogenolysis: glycogen -> glucose Glycogenesis: glucose -> glycogen Describe Glycolysis. * Glycolysis is the conversion of glucose into pyruvate, it produces ATP and metabolic intermediates * Other dietary saccharides can also be used as inputs into various parts of glycolysis (after processing by other enzymes) * Occurs in the cell membrane * Red-blood cells rely on glycolysis for ATP, and muscle tissues under anaerobic conditions * Preparatory phase: * Primes glucose through the consumption of ATP (2ATP consumed) * Two key control points (irreversible - requires a different enzyme/pathway to be reversed): glucose -> glucose-6-phosphate & F6P -> F1,6BP * Glucose can be transported across the cell membrane, but G6P cannot. “Locks” it in * The conversion into F1,6BP is a committed step - after this step the glycolysis pathway is the only pathway that can be undertaken by intermediates * Final reaction of prep phase converts one of the 3C-P molecules into the other so it’s the same. * Payoff phase: * Main energy generation phase of glycolysis * Only control point is the final conversion into pyruvate. * 8ATP, 2pyruvate, 4NADH produced per glucose Explain redox reactions and the place of NAD+ and NADH in them. * Redox reactions are the transfer of electrons. (oxidation and reduction. Oxidation is loss of electrons) * In the payoff phase, NAD+ is oxidised into NADH through a dehyrogenate * NAD+ conc in the cell is low, NADH must be recycled to provide it for glycolysis Define the Warburg effect. * Metabolic adaptation of cancer cells * Cancer cells rely on high levels of glycolysis for energy * NAD+ is replenished with lactic acid fermentation (reduces pH) * Observed mechanisms: * Increased synthesis of glycolytic enzymes * Decreased gluconeogenesis * Increased import of glucose into cells * Warburg effect can be seen by PET to detect cancer tissues (based upon increased glycolysis rate) Define the location and purpose of glycogen in humans. * Energy storage in (mainly) liver and muscle * Stored in granules in the cytosol * Muscles have enough glycogen for ~1hr exercise * Liver contains enough for 12-24hrs * Glucose is released as glucose-1P by glycogen phosphate. Explain the structure of glycogen, including the central protein, the non-reducing ends, the branches and the linkages. * glycogen consists of a chain/branches of glucose molecules connected by a1-4 links. Branches are connected by a1,6 links * when broken down, glucose broken off the main chain are in the form of glucose-1p Explain the breakdown of glycogen in glycogenolysis, including the roles of the enzymes. * glycogen phosphorylase adds a phosphate group to the released glucose, breaking the bond from its glycogen molecule (glucose-1P made) * the debranching enzyme has two main functions: * a transferase activity to take 3 (of the remaining 4) glucose units and transfer them to the longer branch * a glycosides activity of a1-6 links to release the final glucose left on the branch (glucose released) Define the possible fates of glucose-1P, including how glucose can be released from the liver and kidney cells. * G1P in the muscle must be altered to G6P before it can be used in glycolysis (done through phosphoglucomutase) * in the liver/kidney G6P can then have its phosphate group removed to allow for glucose transport out of the cell * once glucose is out of the cell it can then go into the blood to increase blood glucose concentration. Explain the synthesis of glycogen – glycogenesis – including the precursors, overall process, and primer and enzyme. * The precursor for glycogenesis is ADP-glucose, a sugar nucleotide * Allows the cell to set aside some sugars for incorporation into glycogen and others for glycolysis * Glucose + ATP -> glucose-6P + ADP (glycolysis) Glucose-6p <-> glucose-1P Glucose-1P + UTP -> UDP-glucose + PPi * Glycogenin is an enzyme that adds glucose to itself from UDP-glucose (begins the chain) * Glycogen synthase takes overr and adds glucose onto the glycogen molecule (extends the chain) * Transferase enzyme modifies it to be branched Explain the fate of pyruvate under anaerobic conditions, for example in muscles during a sprint. * Pyruvate has many possible pathways (mainly TCA cycle) * In anaerobic conditions (hypoxia) the TCA cycle stops and pyruvate is shunted to other pathways * One pathways is the conversion into lactate by lactate dehydrogenase (oxidises NADH into NAD+) Explain the importance of the lactate (lactic acid) production for glycolysis in anaerobic conditions. * Lactate dehydrogenase helps to recycle the built-up materials generated during glycolysis in anaerobic conditions * The TCA cycle stops under anaerobic conditions, so NAD+ needs to be recycled as well, the lactate cycle also recycles NAD+ Explain the role of the Cori cycle. * Lactate generated in the muscles during anaerobic conditions are transported to the liver to be converted into glucose during the recovery phase (consumes ATP) * Glucose can then be transported back to the muscles Explain the roles and the costs of gluconeogenesis. * Gluconeogenesis is pyruvate -> glucose (energy consuming reaction) * Its an emportant source of glucose during the fasting state, mainly done by the liver * Enzymes are the same as glycolysis, only 3 are different to ‘bypass’ the ‘irreversible’ steps * Pyruvate is converted into oxaloacetate (pyruvate carboxylase) then into phosphophenol pyruvate (PEP carboxylase) as opposed to the single step from PEP into pyruvate in glycolysis * Pyruvate -> oxaloacetate (done in mito), oxaloacetate -> PEP (in cyto). Oxaloacetate is transported into the cytoplasm by the malate-aspartate shuttle * Loss: 2 pyruvate, 2 GTP, 4 ATP, 2 NADH. Gain: 1 glucose Explain the use of the malate-aspartate shuttle in gluconeogenesis. * In the mitochondria oxaloacetate is converted into malate to allow for transport into the cyto. Once in the cyto, it is then re-converted into oxaloacetate to be used in gluconeogenesis * Oxaloacetate in the cyto can also be converted into aspartate to allow for transport back into the mito * This shuttle replenishes NADH in the cytoplasm to drive the rest of gluconeogenesis. Explain the roles of the pentose phosphate pathway (PPP) and their importance in different conditions. * The PPP is used to generate NADPH, and ribose-5P which is a precursor for nucleotide synthesis. It also generates intermediates for glycolysis and gluconeogenesis * Glucose-6P (from glycolysis) can also be shunted into the PPP pathway to be converted into ribose-5P * In the cytosol, using NADH+ as an electron acceptor * In the oxidative phase: NADPH is reduced to NADP+ during the conversion of glucose-6P to ribose-5P, CO2 is also released * 2x redox reactions, so 2x NADPH produced * Rapidly dividing cells is it mainly used for nucleotide synthesis for RNA/DNA * In non-dividing cells its mainly used for the production of NADPH as a protector of oxidative damage, or for FFA synthesis Explain broadly the nonoxidative phase of PPP. * Replenishes glucose-6P when the cell needs alot of NADP+ and other byproducts for biosynthesis * Ribose-5P is recycled by shuffling the carbons around to generate more glucose-6P Explain the role of Xylulose 5-P in regulation of glycolysis. * Regulates glycolysis, carbon and fat metabolism * In response to using glucose in the cytoplasm, xylulose 5P causes PFKFBP2 to be dephosphorylated, stimulating glycolysis and inhibiting gluconeogenesis Define isozymes and provide and example on isozymes in regulation of glycolysis. * Izozymes are different proteins which have the same enzymatic activity * Can differ in: kinetics, regulation, cell type, subcellular distribution, cofactor use * Eg. hexokinase * Humans have 4 izozymes of hexokinase (the enzyme in the first step of glycolysis) Explain how the different enzymatic rates of Hexokinase I and Hexokinase IV provide important differences in glycolysis in different tissues. * Hexokinase 1/2: (myocytes) * Produced in muscle cells * Both as maximal rate at very low concentrations of glucose * Allosterically inhibited by G^P to maintain steady state * Max rate is much lower than that of kexokinase 4 * Hexokinase 4: (hepatocytes) * Produced in liver cells * Much more responsive to blood glucose changes. Rate increases with increased glucose * Not inhibited by G6P, regulated by a liver specific regulatory protein. Explain how the sub-cellular localisation of hexokinase IV is regulated and its effect on glucose regulation. * GLUT2 rapidly brinds glucose into hepatocytes (where hexokinase 4 is) * GKRP regulates hexokinase by binding to it in response to high levels of F6P and sequesters it in the nucleus * In the nucleus, hexokinase 4 can no longer contribute to glycolysis (this binding is counterbalanced by glucose) Explain the regulation of PFK1 and how this regulates glucose utilisation pathways. * PFK1 catalyses the conversion of F6P into F1,6BP for use in glycolysis * Inhibited by ATP and citrate, and catalysed by ADP and AMP Explain how Glucagon regulates glucose utilisation and storage pathways. * Glucagon response is mediated by F26BP, glucose utilisiation and storage pathways such as glycolysis and gluconeogenesis are allosterically regulated by both glucagon and F26BP * Increased blood glucose = insulin release, decreased blood glucose = glucagon release (both by pancreas) Explain the production of Fructose 2,6-bisphosphate, and how it alters enzymatic activity in glucose utilisation pathways * PFK2 catalyses F26BP formation. * Increased insulin +glucose = increased PFK2 + F26BP = increased glycolysis * F26BP activates PFK1 which activates glycolysis, and inhibits FBPase1 which decreases gluconeogenesis Explain how the different isozymes of pyruvate kinase are regulated, and how this contributes to glucose regulation. * 2 important isozymes: liver and muscle pyruvate kinase. Both regulate glycolysis in the same way * Liver: glucagon activates the isozyme by modifying it, allowing it to inhibit glycolysis * The muscle isozyme is not modified in response to glucagon, but still inhibits glycolysis Explain how pyruvate is central in the regulation of glucose utilisation pathways. * Pyruvate is the final product of glycolysis and the starting step of gluconeogenesis, can be converted into acetyl-Coa to be used in the TCA cycle (irreversible step) Explain how redox potential is balanced in the cell by the regulation of pathways and shuttles (but full details of the shuttles is elsewhere). * Redox is balanced by balancing levels of NADH/NAD+ * Lactic acid fermentation: replenishes NAD+ for anaerobic glycolysis (increased ATP, decrease NADH requirements) * Malate shuttle: moves oxaloacetate from mito to cyto as malate, replenishes NADH in cyto for gluconeogenesis * Glycerol-3P shuttle: dihydroxyacetone phosphate from glycolysis is reduced to glycerol-3P. Passes electrons for ox-phos. Compare the responses of liver and muscle to the stimulus of epinephrine and glucagon * Glucagon and epinephrine bind to different receptors but activate the same intracellular signalling pathway * Activates a g-protein coupled receptor, which activates the enzyme adenylyl cyclase, which activates PKA * Activates glycogenolysis in both. Liver: increase gluconeogenesis, decrease glycolysis. Muscle: increase glycolysis Describe the pathway taken by a TAG from the diet into the adipose tissue and then into the bloodstream for use by the cell. * Fats are first solubilised with bile salts forming micelles * After ingestion, fatty acids and other TAG sub-products are taken up by intestinal mucosa and converted into TCGs * TCGs -> chylomicrons -> (into blood) -> (lipase breaks it into) FFA + glycerol * Allows for FFA to enter cells for storage and usage, FFAs require transporters to be moved as they’re not water soluble * TAGs are stored in adipose cells as lipid droplets * Epinephrine and glucagon stimulate adenylyl cyclase to produce cAMP -> PKA activation -> lipase breaks down TAGs -> FFA and glycerol release * Glycerol can be fed into glycolysis or gluconeogenesis * For glycolysis, it must be phosphorylated (1 ATP used) and dehydrogenated (1 NAD+ used) before entry * Majority of energy in TAGs is stored in the FFA * <12C FFAs = no transporteres needed * >14C FFAs = carnitine shuttle needed to move into mitochondria for metabolism Describe the carnitine shuttle, and the overall process of the beta-oxidation pathway. * The carnitine shuttle is used for transporting FFAs of >14C into the mitochondria * FFA + CoA + ATP <-> Fatty acyl-CoA + AMP + 2Pi * Fatty acyl-CoA is a high energy compound * FA-CoA can then be attached to carnitine -> FA-Carnitine (in the outer mito membrane) * FA-Carnitine can diffuse through the outer membrane and through a transporter into the matrix * FA-Carnitine gets turned into FA-CoA by an enzyme in the inner mito membrane before the matrix Describe the B-oxidation pathway of FFA, and calculate the amount of energy generated using beta-oxidation of a FA. 1. Removal of chain in two-carbon units as acetyl-coa 2. Acetyl-coa enters the TCA cycle for oxidation 3. NADH and FADH2 donate electrons to the mitochondrial respiratory chain and the electrons pass to oxygen, with phosphorylation of ADP->ATP * Carboxyl group on FA is removed first as acetyl-coa, the remaining carbons are attached to a new CoA * For one round of B-ox: +1 FADH2, 1NADH, 1H+, 1 Acetyl-Coa * For one 16C FA, 7 rounds are needed, so 7x(above)+ acetyl-coa * Each FADH2 = 1.5ATP, NADH=2.5ATP * One round ~4ATP molecules List the differences for an unsaturated and an odd-numbered FA. * Unsaturated FAs have a double bond (counted from ‘open’ end) * For a cis-unsaturated FA, B-oxidation is normal until the double bond. Isomerase enzyme turns cis- into trans-FA * For odd-numbered fatty acids the last round of b-oxidation results in a 3C molecule (propionyl-Coa) * Molecule is carboxylated (requires ATP) * Rearranged to form succinyl-coa (which can then enter the TCA cycle Define ketone body and explain when and where and why they are produced. * 3 forms of ketone bodies: acetone, acetoacetate, D-B-hydroxybutyrate * Acetyl-coa in the liver can enter the TCA cycle or get converted into ketone bodies * Acetone gets excreted, the other two can be converted into acetyl-coa * Brain can use them as emergency fuel when glucose isnt available * Ketone production = ketogenesis * In starvation/uncontrolled diabetes the liver produces a large amount of ketone bodies * Oxaloacetate is diverted from the TCA for gluconeogenesis, TCA cycle slows. * Increased fat intake = increased B-oxidation = increased acetyl-coa production = increased ketone bodies = lowering of blood pH = acidosis Define peroxisome * Peroxisome is the major site of b-oxidation in plants * Produces less ATP but some heat * Mainly useful for very long chained FA, branched FAs, animal fat, meat, fish… Explain how beta-oxidation pathway is cross-regulated with FA synthesis and glucose utilisation. * The first enzyme in FA synthesis is activated by insulin and inhibited by glucagon * High levels of FA synthesis intermediates inhibit the carnitine shuttle of FA into the mitochondria * Balances the use of glucose and FA as fuel and balances FA synthesis/degredation Explain how pyruvate is transported into the mitochondria, and explain the role of this process in the Warburg effect * Pyruvate diffuses through the outer mito membrane through large openings, transported through the inner membrane by a passive transporter (mitochondrial pyruvate carrier) * MPC is encoded by two genes, both of which are mutated in 80% of cancers * Contributor of the warburg effect by shifting emphasis to glycolysis rather than TCA/Oxphos Broadly describe the structure of the Pyruvate Dehydrogenase complex * Located in mitochondria, oxidises pyruvate into acetyl-coa * 5 cofactors -> multi-enzyme complex * Uses a 3 step process -> 3 diff enzymes in the complex * Has a cubic core (E2) core with a channel * E1 and E3 are linked on the outside by other enzymes Define the location of the metabolites and enzymes in the TCA cycle * Aconitase is an enzyme used to create isocitrate, eukaryotic cells have two isozymes, one in the mito (TCA cycle) and one in the cytoplasmic aconitase * Contains an active iron sulfur centre which is used to carry out its enzymatic activity * In low iron is undergoes a conformational change to open up a mRNA binding site for its regulatory role * Isocitrate dehydrogenase either reduces NAD+ in the mito or NADPH+ in the mito or cyto Explain how much energy comes out of the TCA cycle * Overall (-2ATP)+6ATP+10NADH+2FADH2 = 30-32ATP List the other biological molecules that can be synthesised from metabolites from the TCA cycle * Citrate can be turend into fatty acids/sterols * Alpha-ketoglutarate -> glutamate -> byproducts * succinyl-CoA -> polyphyrins, heme * Oxaloacetate -> PEP, glucose, amino acids Explain why some metabolites need to be replenished to keep the cycle moving * Oxaloacetate is used for many different purposes besides the TCA cycle, it is also the biggest driving force of the TCA cycle. (very important to replenish it to keep it moving) * Many cycles replenish but mainly by using pyruvate Explain the importance of oxidative phosphorylation in life * OxPhos generates most of the ATP in non-photosynthetic organisms - uses the chemiosmotic theory * Electrons flow through electron carriers (respiratory chain) to a final electron acceptor (oxygen) * Free-energy generated is coupled to the proton transport across the membrane * Oxidative energy gets turned into membrane potential * Flow of protons down the concentration gradient (through channels) provides energy needed for ATP synthesis * The respiratory chain is part of OxPhos Define the electron carriers of the respiratory chain and elsewhere in metabolism and how many electrons they can carry * Electron carriers and the respiratory chain is embedded in the inner mito membrane. +ve potential in the inner-memb space, -ve in the mito matrix * Electrons are passed from the initial reactions via NADH, NADPH, FADH2 * Ubiquinone (Q) can accept one electron (*QH) or two (QH2) -> takes the proton H+ as well * QH2 = ubiquinol, which is a small hydrophobic molecule that can freely diffuse in the lipid bilayer of the inner mito membrane * Heme groups in cytochromes Define the roles of the protein complexes in the respiratory chain * 4 complexes (complex 2 is also a TCA cycle enzyme) * 2 entry points: * Electrons from NADH enter complex 1, bypass complex 2, goes into 3, passed into 4 * Electrons from FAD enter through 2, pass through 3, then 4 * Complex 1: (NADH dehydrogenase) * Electrons pass from NADH to ubiquinone -> ubiquinol * 45 different polypeptides * Electrons pass through FMN-containing flavoproteins and 8 iron-sulfur centres before QH2 * 4H+ are pumped into the intermembrane space * COmplex 2: (succinate dehydrogenase) * Electrons from succinate are passed to FAD (as part of the TCA cycle) * Electrons then pass through Fe-S centres to Q to reduce it to QH2 * Acts as a coordination point between the TCA cycle and OxPhos * Heme group is to protect against leakage of electrons, no H+ being pumped * Complex 3: * Large complex with multiple heme grorups, a cavern, and Cyt C (intermemb side) * Electrons pass through QH2 to Cyt C * Simultaneous pumping of 4H+ into intermembrane * Stage 1: 1 electron passes to Cyt C, stage 2: other electrons passes to 2nd Cyt C. * Complex 4: (cytochrome oxidase) * Takes electrons from Cyt C through a Cu Centre and a heme, to oxygen, reducing it to H2O * For every 2 electrons = 1 H2O * 2 protons pumped into intermembrane space Explain how the movement of electrons through the respiratory chain causes the pumping of H+ * Power generated from redox reactions in the electron chain is converted into power used to pump protons into the intermembrane space (maintanance of membrane potential) * 10 H+ pumped all together by OxPhos Explain the role of the ATP synthase in respiration * Pumping of protons to the intermembrane space = membrane potential = drives ATP synthesis enzyme * ATP synthas is coupled with respiration and allows for the electron transport chain to continue working Define coupled in terms of oxphos and respiration * Electron transport chain itself is the proteins embedded in the mitochondrial membrane, OxPhos is the process of conversion into ATP from electrons from electron transport as well as the pumping of protons into the intermembrane space * If one isnt working, the other wont work. Relief of protons by ATPsynthase allows for the respiration chain to function Explain the overall structure of the F0F1 ATP synthase * The ATPsynthase is separate but also embedded in the inner mito membrane. The C-ring is embedded * The C-ring: has 8-15 subunits in ring, embedded in membrane, proton path is between c and alpha subunits * F1 - 3 beta subunits, 3 alpha subunits, ATP synthesis domain (on the matrix side) * F0 - imbedded in the inner mito membrane, a and b subunits lock the proton channel to the f1 domain Explain how the rotation of the ATP synthase changes the structure of the ATP/ADP binding region, and how this contributes to the formation of ATP * 3 phases of movement of the F1 domain, causes the gamma subunit shaft to rotate and interact with the ab dimers to change their shape and function * b-ADP conformation: binds ADP, Pi * b-ATP conformation: stabilises ADP and Pi as ATP * B-empty: low affinity for ATP = release of ATP * One full rotation catalyses the formation of 3ATPs Define how the movement of the H+ through the ATP synthase drives the rotational movement * Proton enters C-ring half-channel on P side (intermemb) * Displaces ARg219 to adjacent C subunit, rotates, displaces H+ from Asp * Displaced H+ exists on N side (matrix) * C-Ring rotates, Arg219 returns to P-Side half channel Explain how many ATP are produced per NADH, and per FADH2 * C-ring = 8 subunits, 1 rotation = 8H+ = 3ATP * Transport of Pi into matrix = -1H+ * Transport of 3Pi = -3H+ * 3ATP costs 11H+, 3.7protons per ATP * Oxidising one NADH moves 10protons. 1NADH = 2.7ATP * Oxidising one FADH2 move 6protons. 1 FADH2 = 1.6ATP Explain how the malate aspartate shuttle allows respiration to occur after glycolysis in some tissues * Used to move glycolysis electrons from the cytoplasm into the matrix * Oxaloacetate is reduced to malate in the cytoplasm before transport into the matrix * Aspartate is used to replenish oxaloacetate Explain how the Glycerol 3 phosphate shuttle allows respiration to occur after glycolysis in some tissues * Used in skeletal muscle and brain * Electrons are passed to FAD to make FADH2 * Through reduction of DHAP (from glycolysis) * Less ATP produced from this transporter (3 ATP) Explain the regulation of OxPhos * Depends on the availability of ADP as the phosphate acceptor * ATP synthase is inhibited by the absence of oxygen * All pathways of ATP-production are coordinately regulated, all respond to concentrations of ATP, ADP, AMP, NADH Explain the result of uncoupling the respiratory chain, and give an example of its utility * Uncoupling can produce heat instead of ATP * Newborn animals have brown adipose tissue (BAT) which contains uncoupling protein 1 which allows protons to flow back through the matrix side and increase heat production * Energy of electron transport chain is lost as heat Explain the overall symptoms that would be expected from a mutation affecting mitochondrial function * Disease symptoms often affect tissues with high ATP production requirements * Blindness, stroke, deafness, structural defects in heart, anemia * Mutations are inherited Inborn Errors of Metabolism What are inborn errors of metabolism? * A group of genetic disorders (including mitochondrial disease) that cause a block in a metabolic pathway * Mutations alter metabolism results in accumulation of toxic metabolites, or deficiencies in energy production * Monogenic disorders = one gene is mutated * Usually inherited, often very severe and present early but can arise at any time * IEMs are the most common of the rare diseases, treatment is usually symptomatic (highly dependant on genotype) How are monogenic inborn errors of metabolism different to other metabolic diseases? * Mongenic = one gene containing mutations that critically impacts its function * Other metabolic disorders tend to have alot of interplay between other genes, environment, epigenetic influences * Common themes of IEMs is defects in pathways involved in catabolism or storage of carbs/fatty acids/proteins * Some diseases require multiple deficiencies/mutations to completely lose function/be diagnosed How does amino acid catabolism, in particular phenylalanine, contribute to energy production? * AAC counts for 10-15% of energy production, it can contribute to both gluconeogenesis and ketone bodies, generating 6 products all of which enter the TCA cycle * 7 different amino acids generated by AAC can be degraded into acetyl-coa (which feeds into TCA = energy) What are some disorders of phenylalanine catabolism? * The breakdown of pehnylalanine (and tyrosine) yields: 1xacetoacetate (->acetyl-coa), 1x fumerate. Both feed into TCA * Genetic defects in enzymes involved cause IEMs: * Loss of function in enzyme = buildup of previous metabolite. * Phenylketonia (PKU) is a disorder relating to dysfunction in amino acid catabolism * Caused by loss of function mutations in the PAH gene encoding the enzyme phenylalanine hydroxylase (normally converts tyrosine into phenylalanine) * High buildup of phenylalanine results in the use of a secondary pathways being used -> undergoes transamination with pyruvate to yield phenylpyruvate * Accumulation of phenylalanine and its metabolites in early life impairs normal brain development Why is limiting the intake of the artificial sweetener aspartame important in phenylketonuria? * If PKU is diagnosed under 3yrs after birth, intellectual disability and other longterm symptoms can be prevented with strict dietary control. * Restrict phenylalanine/tyrosine/protein-rich intake to just meet the needs of protein synthesis * Aspartame is a dipeptide of aspartate and a methyl ester of phenylalanine -> breaks down in the gut to become phenylalanine. What is the ‘Guthrie test’ and what has replaced it in modern newborn screening programs? * Guthrie test: * Heel prick and blood drop is placed on filter paper * Filter paper is then placed on agar with bacillus subtilis that requires the presence of phenylalanine to grow * Increased phenylalanine (PKU) will show a ‘halo’ of bacteria * Heel prick tests: * Heel of an infant is pricked and blood is dropped onto a part of paper * Sent to lab and holes are punched out of each blood drop or placed into wells * Multiple tests are ran -> testing for increases in hormones or proteins or other genetic biomarkers * Also uses mass spec to measure metabolites * Nowadays: mainly ESI-MSMs and qPCR are used * ESI-MSMs detection method established in 2002 * Involves dried blood sports extracted with methanol and spiked with internal standards * Direct injection in positive ion mode, MRM to detect metabolites (amino acids and acyl carnitines) Newborn screening (heel prick test) reports a baby with high levels of carnitines, what group of inborn errors of metabolism is this result indicative of? * Indicative of fatty acid metabolism disorders * Most FFA enter the mitochondria via the carnitine shuttle * Fatty acids are the major source of energy in the muscle and the heart * Carnitine shuttle is needed to transport FFA into mito for b-oxidation to occur * In FFA metabolism disorders, it leads to less FFA being oxidised into energy, and thus the accumulation in the cytosol or mito * Fatty acids get shunted into carnitine, producing acylcarnitines which are detected in newborn screens * IEMs in b-oxidation results in a severe decrease in the production of ATP in mitochondria * Deficiencies can be managed with early diagnosis Why do mitochondria have their own ribosomes and how might genetic defects in the mitochondrial ribosome cause mitochondrial disease? * Mitochondria are also essential for thermogenesis, amino acid synthesis, lipid metabolism, ect… * mtDNA encodes for 13 proteins in humans, 2rRNA and 22tRNA. All proteins encoded by mtDNA are found in the OxPhos system * Mito ribosomes are needed to translate the mtDNA into proteins in the mito * ‘Primary’ disorders are due to defects in proteins directly involved in OxPhos themselves, eg: * Monogenic disorders of mtDNA * Differences in heteroplasmy means siblings may not present wit hteh same symptoms, generally adult onset and less severe than mito disorders caused by nDNA * Most common is leigh syndrome. * Disorders of the respiratory chain * Gene encodes subunit of the chain, encoding for assembly factors, encoding translation factors * Disorders associated with mtDNA replication (mtDNA deletions or depletion) * Disorders of mito membranes * ‘secondary’ disorders are defects in other mito pathways which indirectly impact OxPhos. In the context of mitochondrial disease, what is heteroplasmy? * A typical cell may contain both wild and mutant type mtDNA, when a cell replicates and divides it can lead to different ratios of wild and mutant type mtDNA in different cells in the same organism * Same organism, cells have different mtDNA ratios between ‘healthy’ and ‘mutant’ mtDNA Why are chronic high levels of lactate in blood suggestive of mitochondrial disease? * High levels of lactate can indicate: a defect in OxPhos, and an increase in anaerobic glycolysis Describe how respiratory chain enzymology for mitochondrial Complex I deficiency works. * Performed on crude mitochondria isolated from clinically relevant tissue * Spectrophotometric assays that analyse teh change in absorbance of specific compounds * OxPhos indicators are used to selectively analyse a single compound in a crude isolate * Mito treated with digitonin to isolate inner memb fragments -> OxPhos inhibitors added * Eg. Complex 1 deficiency: * Isolate crude metabolite * Add antimycin A and potassium cyanide (inhibits complex 3 and 4) * Add ubiquinone to act as electron acceptor * Add NADH * Monitor absorbance of NADH at spectrophotometer at 340nm * Similar assays for all OxPhos complexes -> monitoring full enzymatic reaction. Assays arent specific to a single gene How are mitochondrial diseases diagnosed and treated? Use leighs disease as an example. * Nowadays whole genome sequencing is done on the patient and in 50% of the time there is a known variant of a known disease gene, in other cases with a novel variant functional tests are performed * Functional tests: * Biochemical experiments that aim to demonstrate a connection between genotype and phenotype * Spectrophotometric assay with OxPhos inhibitors is one method * Understanding of mito diseases relys on research tests done by academic labs outside of clinical accreditation * Leigh syndrome case report: * Infant presented with elevated blood lactate, mild developmental dely * Sequencing identified homozygous missense variant of unknown significance in a subunit of complex1 in OxPhos * Skin fibroblasts were taken from child and mother, quantitative proteomics were done demonstrating a clear reduction in abundance of complex 1 subunits * Treatment of mito disease: * For most there is no cure, only symptom management * Some can be ‘cured’ through diet * Diagnosis needs to be early enough to do so, to avoid developmental and other lifelong problems = ethical dilemma What is mitochondrial donation and what kinds of primary mitochondrial diseases might it be used to prevent transmission of? * An IVF technique combining the mtDNA from a healthy donor with the genomic DNA of a healthy egg * Mothers nDNA is taken from egg and places into healthy donor egg (without nucleus) Mitonuclear Communications Explain the mitochondrial bottleneck phenomenon * Discuss reproductive options for women with deleterious mutated mtDNA Evaluate the importance of stakeholder engagement in development of mitochondrial donation Identify different mitochondrial donation techniques Discuss ethical considerations involved in mitochondrial donation Mitonuclear communication - anterograde signalling Explain how cellular metabolism is maintained and coordinated with mitochondria Analyse the importance of mito-nuclear communication Explain the cells’ bidirectional adaptation to stress with anterograde and retrograde signaling Explain how anterograde signals are activated by upstream sensors to activate nuclear and mt-encoded mitochondrial proteins and fine tuning involved Explain how anterograde signals are activated by upstream sensors to detect changes in metabolic conditions (exercise, Ca2+, cold and nuclear stress) Mitonuclear communication - retrograde signalling Explain what retrograde signals are Explain retrograde energetic, Ca2+and ROS dependent stress responses Mitonuclear feedback and proteostasis Explain the importance of mitonuclear feedback and proteostasis Explain the three different mito-nuclear proteostasis responses ie UPRmt, proteolytic stress and heat shock responses in mammals Compare the UPRmt, proteolytic stress and heat shock responses with the integrated stress response Integrated stress responses introduction Describe the control of the integrated stress response (ISR) Detail how the ISR regulation is dependent upon the eIF2 ternary complex (TC) formation Detail how the eIF2 phosphorylation regulates TC formation Explain how eIF2 phosphorylation transforms eIF2 from an eIF2B substrate into an eIF2B inhibitor Explain how ISRIB works as an inhibitor of ISR and its effect at different concentrations of eIF2-P Detail how the ISR reprograms gene expression at both translational and transcriptional levels Describe the different mechanisms that uORF-mediated translational control can occur Explain how eIF2-P can be reset to counteract ISR activation Broadly explain why the resulting consequences of ISR activation are complex Mitochondrial stress effects Discuss that the ISR phenomenon that can be protective or deleterious to the organism Give general examples of mitochondrial stress and dysfunction and its effects Explain that mitochondrial stress signals can be effected by mitokines triggering non-cell autonomous responses Diabetes Define hyperglycaemia and hypoglycaemia versus normal blood glucose Detail how pancreatic beta cells release insulin in response to elevated blood glucose Evaluate how some drugs such as sulfonylureas exploit pathway events triggered in pancreatic beta cells as a treatment for type 2 diabetes Describe the important biochemical features of insulin leading to why insulin is not suitable for oral administration as a treatment for diabetes Explain how glucose enters cells and how insulin increases glucose uptake by target tissues Describe features of the insulin receptor and discuss its signal transduction mechanisms Detail the function of insulin receptor substrate 1 (IRS1) and other proteins containing PTB and SH2 domains Compare how insulin can have rapid and also slower metabolic effects in its target tissues Explain generalized pathways in insulin signaling to support glucose homeostasis Describe changes in insulin signaling during nutrient excess Investigate whether hyperinsulinemia or insulin resistance is the primary impairment Define insulin resistance and its effect on blood glucose Detail one of the risk factors contributing to insulin resistance Argue why obesity alone is not sufficient change for development of Type 2 diabetes Explain the underlying molecular mechanisms and biochemical changes that cause insulin resistance Describe the 3 main phases in the development of overt Type 2 diabetes and explain the transition from the pre-diabetic state to overt diabetes Explain how both insulin resistance and pancreatic b-cell loss/failure contribute to development of type 2 diabetes Explain the triggers for pancreatic beta cell loss and failure in Type 2 diabetes Elaborate on the similarities and differences between Type 1 and Type 2 diabetes Provide a simple definition of Type 2 diabetes Discuss tests that help diagnose pre-diabetes and diabetes Distinguish changes in pancreatic beta cell numbers in Type 1 and Type 2 diabetes and events that bring about the changes in pancreatic beta cell number Describe metabolic events in diabetes Explain the biochemical basis underlying the acute symptoms of diabetic hyperketonemia Explain why some diabetic patients can present with hypoglycaemia Discuss long term consequences of diabetes Diabetic ketoacidosis Explain why a small change in blood plasma pH can be life threatening Detail how and when ketone bodies are produced in the liver and what ketosis is Explain what happens in uncontrolled diabetes Discuss how ketoacidosis occurs and its effects Compare starvation and diabetes Metabolic techniques, approaches, and applications: Explain the major analytical challenges of metabolomics Explain the steps in a typical metabolomics workflow Describe the advantages and disadvantages of major analytical platforms Explain the basics of mass spectrometry Explain the differences between mass accuracy and mass resolution Describe the requirements for different levels of metabolite ID Explain the options for metabolite data analysis Explain the advantages of metabolomics over other ‘omics Identify the key applications and frontiers of metabolomics Give examples where metabolomics bridges basic and translational research Explain the limitations of steady state metabolomics Explain what metabolic flux is Explain the advantages of stable isotopes over radioactive isotopes Describe the workflow and consideration of a labeling study Explain the typical statistical workflow to analyse metabolomic data Explain the purpose of data scaling and its potential problems Explain what you can infer from a volcano plot Explain the common dimension reduction approaches for multivariate analysis and their differences Explain why spatial information is important Explain the key ionization techniques for mass spectrometry imaging (MSI) Describe a typical MSI workflow Explain strategies that can improve the confidence of metabolite ID in MSI Describe the key emerging technologies in the field of metabolomics Explain the key strengths of anion-exchange chromatography mass spectrometry Explain why spatial multi-omics is an exciting approach Explain the advantages of system automation in metabolomic studies

Overview of metabolic pathways
Glycolysis: Glucose -> pyruvate
Gluconeogenesis:  pyruvate -> glucose
Glycogenolysis:  glycogen -> glucose
Glycogenesis: glucose -> glycogen

Describe Glycolysis.
* Glycolysis is the conversion of glucose into pyruvate, it produces ATP and metabolic intermediates
   * Other dietary saccharides can also be used as inputs into various parts of glycolysis (after processing by other enzymes)
* Occurs in the cell membrane
* Red-blood cells rely on glycolysis for ATP, and muscle tissues under anaerobic conditions
* Preparatory phase:
   * Primes glucose through the consumption of ATP (2ATP consumed)
   * Two key control points (irreversible - requires a different enzyme/pathway to be reversed): glucose -> glucose-6-phosphate & F6P -> F1,6BP
   * Glucose can be transported across the cell membrane, but G6P cannot. “Locks” it in
   * The conversion into F1,6BP is a committed step - after this step the glycolysis pathway is the only pathway that can be undertaken by intermediates
   * Final reaction of prep phase converts one of the 3C-P molecules into the other so it’s the same. 
*  Payoff phase:
   * Main energy generation phase of glycolysis 
   * Only control point is the final conversion into pyruvate.
   * 8ATP, 2pyruvate, 4NADH produced per glucose

Explain redox reactions and the place of NAD+ and NADH in them.
* Redox reactions are the transfer of electrons. (oxidation and reduction. Oxidation is loss of electrons)
* In the payoff phase, NAD+ is oxidised into NADH through a dehyrogenate
* NAD+ conc in the cell is low, NADH must be recycled to provide it for glycolysis

Define the Warburg effect.
* Metabolic adaptation of cancer cells
* Cancer cells rely on high levels of glycolysis for energy
* NAD+ is replenished with lactic acid fermentation (reduces pH)
* Observed mechanisms:
   * Increased synthesis of glycolytic enzymes
   * Decreased gluconeogenesis
   * Increased import of glucose into cells
* Warburg effect can be seen by PET to detect cancer tissues (based upon increased glycolysis rate)

Define the location and purpose of glycogen in humans.
* Energy storage in (mainly) liver and muscle
   * Stored in granules in the cytosol
   * Muscles have enough glycogen for ~1hr exercise
   * Liver contains enough for 12-24hrs
* Glucose is released as glucose-1P by glycogen phosphate.

Explain the structure of glycogen, including the central protein, the non-reducing ends, the branches and the linkages.
* glycogen consists of a chain/branches of glucose molecules connected by a1-4 links. Branches are connected by a1,6 links
* when broken down, glucose broken off the main chain are in the form of glucose-1p

Explain the breakdown of glycogen in glycogenolysis, including the roles of the enzymes.
* glycogen phosphorylase adds a phosphate group to the released glucose, breaking the bond from its glycogen molecule (glucose-1P made)
* the debranching enzyme has two main functions:
   * a transferase activity to take 3 (of the remaining 4) glucose units and transfer them to the longer branch
   * a glycosides activity of a1-6 links to release the final glucose left on the branch (glucose released)

Define the possible fates of glucose-1P, including how glucose can be released from the liver and kidney cells.
* G1P in the muscle must be altered to G6P before it can be used in glycolysis (done through phosphoglucomutase)
* in the liver/kidney G6P can then have its phosphate group removed to allow for glucose transport out of the cell
* once glucose is out of the cell it can then go into the blood to increase blood glucose concentration.

Explain the synthesis of glycogen – glycogenesis – including the precursors, overall process, and primer and enzyme.
* The precursor for glycogenesis is ADP-glucose, a sugar nucleotide
   * Allows the cell to set aside some sugars for incorporation into glycogen and others for glycolysis
* Glucose + ATP -> glucose-6P + ADP (glycolysis)
Glucose-6p <-> glucose-1P
Glucose-1P + UTP -> UDP-glucose + PPi
* Glycogenin is an enzyme that adds glucose to itself from UDP-glucose (begins the chain)
* Glycogen synthase takes overr and adds glucose onto the glycogen molecule (extends the chain)
* Transferase enzyme modifies it to be branched

Explain the fate of pyruvate under anaerobic conditions, for example in muscles during a sprint.
   * Pyruvate has many possible pathways (mainly TCA cycle)
   * In anaerobic conditions (hypoxia) the TCA cycle stops and pyruvate is shunted to other pathways
   * One pathways is the conversion into lactate by lactate dehydrogenase (oxidises NADH into NAD+)

Explain the importance of the lactate (lactic acid) production for glycolysis in anaerobic conditions.
   * Lactate dehydrogenase helps to recycle the built-up materials generated during glycolysis in anaerobic conditions 
   * The TCA cycle stops under anaerobic conditions, so NAD+ needs to be recycled as well, the lactate cycle also recycles NAD+

Explain the role of the Cori cycle.
   * Lactate generated in the muscles during anaerobic conditions are transported to the liver to be converted into glucose during the recovery phase (consumes ATP)
   * Glucose can then be transported back to the muscles

Explain the roles and the costs of gluconeogenesis.
   * Gluconeogenesis is pyruvate -> glucose (energy consuming reaction)
   * Its an emportant source of glucose during the fasting state, mainly done by the liver
   * Enzymes are the same as glycolysis, only 3 are different to ‘bypass’ the ‘irreversible’ steps
   * Pyruvate is converted into oxaloacetate (pyruvate carboxylase) then into phosphophenol pyruvate (PEP carboxylase) as opposed to the single step from PEP into pyruvate in glycolysis
   * Pyruvate -> oxaloacetate (done in mito), oxaloacetate -> PEP (in cyto). Oxaloacetate is transported into the cytoplasm by the malate-aspartate shuttle 
   * Loss: 2 pyruvate, 2 GTP, 4 ATP, 2 NADH. Gain: 1 glucose

Explain the use of the malate-aspartate shuttle in gluconeogenesis.
   * In the mitochondria oxaloacetate is converted into malate to allow for transport into the cyto. Once in the cyto, it is then re-converted into oxaloacetate to be used in gluconeogenesis
   * Oxaloacetate in the cyto can also be converted into aspartate to allow for transport back into the mito
   * This shuttle replenishes NADH in the cytoplasm to drive the rest of gluconeogenesis.

Explain the roles of the pentose phosphate pathway (PPP) and their importance in different conditions.
   * The PPP is used to generate NADPH, and ribose-5P which is a precursor for nucleotide synthesis. It also generates intermediates for glycolysis and gluconeogenesis
   * Glucose-6P (from glycolysis) can also be shunted into the PPP pathway to be converted into ribose-5P
   * In the cytosol, using NADH+ as an electron acceptor
   * In the oxidative phase: NADPH is reduced to NADP+ during the conversion of glucose-6P to ribose-5P, CO2 is also released
   * 2x redox reactions, so 2x NADPH produced
   * Rapidly dividing cells is it mainly used for nucleotide synthesis for RNA/DNA
   * In non-dividing cells its mainly used for the production of NADPH as a protector of oxidative damage, or for FFA synthesis

Explain broadly the nonoxidative phase of PPP.
   * Replenishes glucose-6P when the cell needs alot of NADP+ and other byproducts for biosynthesis
   * Ribose-5P is recycled by shuffling the carbons around to generate more glucose-6P

Explain the role of Xylulose 5-P in regulation of glycolysis.
   * Regulates glycolysis, carbon and fat metabolism
   * In response to using glucose in the cytoplasm, xylulose 5P causes PFKFBP2 to be dephosphorylated, stimulating glycolysis and inhibiting gluconeogenesis

Define isozymes and provide and example on isozymes in regulation of glycolysis.
   * Izozymes are different proteins which have the same enzymatic activity
   * Can differ in: kinetics, regulation, cell type, subcellular distribution, cofactor use
   * Eg. hexokinase
   * Humans have 4 izozymes of hexokinase (the enzyme in the first step of glycolysis)

Explain how the different enzymatic rates of Hexokinase I and Hexokinase IV provide important differences in glycolysis in different tissues.
   * Hexokinase 1/2: (myocytes)
   * Produced in muscle cells
   * Both as maximal rate at very low concentrations of glucose
   * Allosterically inhibited by G^P to maintain steady state
   * Max rate is much lower than that of kexokinase 4
   * Hexokinase 4: (hepatocytes)
   * Produced in liver cells
   * Much more responsive to blood glucose changes. Rate increases with increased glucose
   * Not inhibited by G6P, regulated by a liver specific regulatory protein.

Explain how the sub-cellular localisation of hexokinase IV is regulated and its effect on glucose regulation.
   * GLUT2 rapidly brinds glucose into hepatocytes (where hexokinase 4 is)
   * GKRP regulates hexokinase by binding to it in response to high levels of F6P and sequesters it in the nucleus
   * In the nucleus, hexokinase 4 can no longer contribute to glycolysis (this binding is counterbalanced by glucose)

Explain the regulation of PFK1 and how this regulates glucose utilisation pathways.
   * PFK1 catalyses the conversion of F6P into F1,6BP for use in glycolysis
   * Inhibited by ATP and citrate, and catalysed by ADP and AMP

Explain how Glucagon regulates glucose utilisation and storage pathways.
   * Glucagon response is mediated by F26BP, glucose utilisiation and storage pathways such as glycolysis and gluconeogenesis are allosterically regulated by both glucagon and F26BP
   * Increased blood glucose = insulin release, decreased blood glucose = glucagon release (both by pancreas)

Explain the production of Fructose 2,6-bisphosphate, and how it alters enzymatic activity in glucose utilisation pathways
   * PFK2 catalyses F26BP formation.
   * Increased insulin +glucose = increased PFK2 + F26BP = increased glycolysis
   * F26BP activates PFK1 which activates glycolysis, and inhibits FBPase1 which decreases gluconeogenesis

Explain how the different isozymes of pyruvate kinase are regulated, and how this contributes to glucose regulation.
   * 2 important isozymes: liver and muscle pyruvate kinase. Both regulate glycolysis in the same way
   * Liver: glucagon activates the isozyme by modifying it, allowing it to inhibit glycolysis
   * The muscle isozyme is not modified in response to glucagon, but still inhibits glycolysis

Explain how pyruvate is central in the regulation of glucose utilisation pathways.
   * Pyruvate is the final product of glycolysis and the starting step of gluconeogenesis, can be converted into acetyl-Coa to be used in the TCA cycle (irreversible step)

Explain how redox potential is balanced in the cell by the regulation of pathways and shuttles (but full details of the shuttles is elsewhere).
   * Redox is balanced by balancing levels of NADH/NAD+
   * Lactic acid fermentation: replenishes NAD+ for anaerobic glycolysis (increased ATP, decrease NADH requirements)
   * Malate shuttle: moves oxaloacetate from mito to cyto as malate, replenishes NADH in cyto for gluconeogenesis
   * Glycerol-3P shuttle: dihydroxyacetone phosphate from glycolysis is reduced to glycerol-3P. Passes electrons for ox-phos.

Compare the responses of liver and muscle to the stimulus of epinephrine and glucagon
   * Glucagon and epinephrine bind to different receptors but activate the same intracellular signalling pathway
   * Activates a g-protein coupled receptor, which activates the enzyme adenylyl cyclase, which activates PKA
   * Activates glycogenolysis in both. Liver: increase gluconeogenesis, decrease glycolysis. Muscle: increase glycolysis

Describe the pathway taken by a TAG from the diet into the adipose tissue and then into the bloodstream for use by the cell.
   * Fats are first solubilised with bile salts forming micelles
   * After ingestion, fatty acids and other TAG sub-products are taken up by intestinal mucosa and converted into TCGs
   * TCGs -> chylomicrons -> (into blood) -> (lipase breaks it into) FFA + glycerol
   * Allows for FFA to enter cells for storage and usage, FFAs require transporters to be moved as they’re not water soluble
   * TAGs are stored in adipose cells as lipid droplets
   * Epinephrine and glucagon stimulate adenylyl cyclase to produce cAMP -> PKA activation -> lipase breaks down TAGs -> FFA and glycerol release
   * Glycerol can be fed into glycolysis or gluconeogenesis
   * For glycolysis, it must be phosphorylated (1 ATP used) and dehydrogenated (1 NAD+ used) before entry
   * Majority of energy in TAGs is stored in the FFA
   * <12C FFAs = no transporteres needed
   * >14C FFAs = carnitine shuttle needed to move into mitochondria for metabolism

Describe the carnitine shuttle, and the overall process of the beta-oxidation pathway.
   * The carnitine shuttle is used for transporting FFAs of >14C into the mitochondria
   * FFA + CoA + ATP <-> Fatty acyl-CoA + AMP + 2Pi
   * Fatty acyl-CoA is a high energy compound
   * FA-CoA can then be attached to carnitine -> FA-Carnitine (in the outer mito membrane)
   * FA-Carnitine can diffuse through the outer membrane and through a transporter into the matrix
   * FA-Carnitine gets turned into FA-CoA by an enzyme in the inner mito membrane before the matrix

Describe the B-oxidation pathway of FFA, and calculate the amount of energy generated using beta-oxidation of a FA.
   1. Removal of chain in two-carbon units as acetyl-coa
   2. Acetyl-coa enters the TCA cycle for oxidation
   3. NADH and FADH2 donate electrons to the mitochondrial respiratory chain and the electrons pass to oxygen, with phosphorylation of ADP->ATP
   * Carboxyl group on FA is removed first as acetyl-coa, the remaining carbons are attached to a new CoA
   * For one round of B-ox: +1 FADH2, 1NADH, 1H+, 1 Acetyl-Coa
   * For one 16C FA, 7 rounds are needed, so 7x(above)+ acetyl-coa
   * Each FADH2 = 1.5ATP, NADH=2.5ATP
   * One round ~4ATP molecules

List the differences for an unsaturated and an odd-numbered FA.
   * Unsaturated FAs have a double bond (counted from ‘open’ end)
   * For a cis-unsaturated FA, B-oxidation is normal until the double bond. Isomerase enzyme turns cis- into trans-FA
   * For odd-numbered fatty acids the last round of b-oxidation results in a 3C molecule (propionyl-Coa)
   * Molecule is carboxylated (requires ATP)
   * Rearranged to form succinyl-coa (which can then enter the TCA cycle

Define ketone body and explain when and where and why they are produced.
   * 3 forms of ketone bodies: acetone, acetoacetate, D-B-hydroxybutyrate
   * Acetyl-coa in the liver can enter the TCA cycle or get converted into ketone bodies
   * Acetone gets excreted, the other two can be converted into acetyl-coa
   * Brain can use them as emergency fuel when glucose isnt available
   * Ketone production = ketogenesis
   * In starvation/uncontrolled diabetes the liver produces a large amount of ketone bodies
   * Oxaloacetate is diverted from the TCA for gluconeogenesis, TCA cycle slows.
   * Increased fat intake = increased B-oxidation = increased acetyl-coa production = increased ketone bodies = lowering of blood pH = acidosis

Define peroxisome
   * Peroxisome is the major site of b-oxidation in plants
   * Produces less ATP but some heat
   * Mainly useful for very long chained FA, branched FAs, animal fat, meat, fish…

Explain how beta-oxidation pathway is cross-regulated with FA synthesis and glucose utilisation.
   * The first enzyme in FA synthesis is activated by insulin and inhibited by glucagon
   * High levels of FA synthesis intermediates inhibit the carnitine shuttle of FA into the mitochondria
   * Balances the use of glucose and FA as fuel and balances FA synthesis/degredation
Explain how pyruvate is transported into the mitochondria, and explain the role of this process in the Warburg effect
   * Pyruvate diffuses through the outer mito membrane through large openings, transported through the inner membrane by a passive transporter (mitochondrial pyruvate carrier)
   * MPC is encoded by two genes, both of which are mutated in 80% of cancers
   * Contributor of the warburg effect by shifting emphasis to glycolysis rather than TCA/Oxphos

Broadly describe the structure of the Pyruvate Dehydrogenase complex
   * Located in mitochondria, oxidises pyruvate into acetyl-coa
   * 5 cofactors -> multi-enzyme complex
   * Uses a 3 step process -> 3 diff enzymes in the complex
   * Has a cubic core (E2) core with a channel
   * E1 and E3 are linked on the outside by other enzymes

Define the location of the metabolites and enzymes in the TCA cycle
   * Aconitase is an enzyme used to create isocitrate, eukaryotic cells have two isozymes, one in the mito (TCA cycle) and one in the cytoplasmic aconitase
   * Contains an active iron sulfur centre which is used to carry out its enzymatic activity
   * In low iron is undergoes a conformational change to open up a mRNA binding site for its regulatory role
   * Isocitrate dehydrogenase either reduces NAD+ in the mito or NADPH+ in the mito or cyto

Explain how much energy comes out of the TCA cycle
   * Overall (-2ATP)+6ATP+10NADH+2FADH2 = 30-32ATP

List the other biological molecules that can be synthesised from metabolites from the TCA cycle
   * Citrate can be turend into fatty acids/sterols
   * Alpha-ketoglutarate -> glutamate -> byproducts
   * succinyl-CoA -> polyphyrins, heme
   * Oxaloacetate -> PEP, glucose, amino acids

Explain why some metabolites need to be replenished to keep the cycle moving
   * Oxaloacetate is used for many different purposes besides the TCA cycle, it is also the biggest driving force of the TCA cycle. (very important to replenish it to keep it moving)
   * Many cycles replenish but mainly by using pyruvate

Explain the importance of oxidative phosphorylation in life
   * OxPhos generates most of the ATP in non-photosynthetic organisms - uses the chemiosmotic theory
   * Electrons flow through electron carriers (respiratory chain) to a final electron acceptor (oxygen)
   * Free-energy generated is coupled to the proton transport across the membrane
   * Oxidative energy gets turned into membrane potential
   * Flow of protons down the concentration gradient (through channels) provides energy needed for ATP synthesis
   * The respiratory chain is part of OxPhos

Define the electron carriers of the respiratory chain and elsewhere in metabolism and how many electrons they can carry
   * Electron carriers and the respiratory chain is embedded in the inner mito membrane. +ve potential in the inner-memb space, -ve in the mito matrix
   * Electrons are passed from the initial reactions via NADH, NADPH, FADH2
   * Ubiquinone (Q) can accept one electron (*QH) or two (QH2) -> takes the proton H+ as well
   * QH2 = ubiquinol, which is a small hydrophobic molecule that can freely diffuse in the lipid bilayer of the inner mito membrane
   * Heme groups in cytochromes

Define the roles of the protein complexes in the respiratory chain
   * 4 complexes (complex 2 is also a TCA cycle enzyme)
   * 2 entry points:
   * Electrons from NADH enter complex 1, bypass complex 2, goes into 3, passed into 4
   * Electrons from FAD enter through 2, pass through 3, then 4
   * Complex 1: (NADH dehydrogenase)
   * Electrons pass from NADH to ubiquinone -> ubiquinol
   * 45 different polypeptides
   * Electrons pass through FMN-containing flavoproteins and 8 iron-sulfur centres before QH2
   * 4H+ are pumped into the intermembrane space
   * COmplex 2: (succinate dehydrogenase)
   * Electrons from succinate are passed to FAD (as part of the TCA cycle)
   * Electrons then pass through Fe-S centres to Q to reduce it to QH2
   * Acts as a coordination point between the TCA cycle and OxPhos
   * Heme group is to protect against leakage of electrons, no H+ being pumped
   * Complex 3:
   * Large complex with multiple heme grorups, a cavern, and Cyt C (intermemb side)
   * Electrons pass through QH2 to Cyt C
   * Simultaneous pumping of 4H+ into intermembrane
   * Stage 1: 1 electron passes to Cyt C, stage 2: other electrons passes to 2nd Cyt C.
   * Complex 4: (cytochrome oxidase)
   * Takes electrons from Cyt C through a Cu Centre and a heme, to oxygen, reducing it to H2O
   * For every 2 electrons = 1 H2O
   * 2 protons pumped into intermembrane space

Explain how the movement of electrons through the respiratory chain causes the pumping of H+
   * Power generated from redox reactions in the electron chain is converted into power used to pump protons into the intermembrane space (maintanance of membrane potential)
   * 10 H+ pumped all together by OxPhos

Explain the role of the ATP synthase in respiration
   * Pumping of protons to the intermembrane space = membrane potential = drives ATP synthesis enzyme
   * ATP synthas is coupled with respiration and allows for the electron transport chain to continue working

Define coupled in terms of oxphos and respiration
   * Electron transport chain itself is the proteins embedded in the mitochondrial membrane, OxPhos is the process of conversion into ATP from electrons from electron transport as well as the pumping of protons into the intermembrane space
   * If one isnt working, the other wont work. Relief of protons by ATPsynthase allows for the respiration chain to function

Explain the overall structure of the F0F1 ATP synthase
   * The ATPsynthase is separate but also embedded in the inner mito membrane. The C-ring is embedded
   * The C-ring: has 8-15 subunits in ring, embedded in membrane, proton path is between c and alpha subunits
   * F1 - 3 beta subunits, 3 alpha subunits, ATP synthesis domain (on the matrix side)
   * F0 - imbedded in the inner mito membrane, a and b subunits lock the proton channel to the f1 domain

Explain how the rotation of the ATP synthase changes the structure of the ATP/ADP binding region, and how this contributes to the formation of ATP
   * 3 phases of movement of the F1 domain, causes the gamma subunit shaft to rotate and interact with the ab dimers to change their shape and function
   * b-ADP conformation: binds ADP, Pi
   * b-ATP conformation: stabilises ADP and Pi as ATP
   * B-empty: low affinity for ATP = release of ATP
   * One full rotation catalyses the formation of 3ATPs

Define how the movement of the H+ through the ATP synthase drives the rotational movement
   * Proton enters C-ring half-channel on P side (intermemb)
   * Displaces ARg219 to adjacent C subunit, rotates, displaces H+ from Asp
   * Displaced H+ exists on N side (matrix)
   * C-Ring rotates, Arg219 returns to P-Side half channel

Explain how many ATP are produced per NADH, and per FADH2
   * C-ring = 8 subunits, 1 rotation = 8H+ = 3ATP
   * Transport of Pi into matrix = -1H+
   * Transport of 3Pi = -3H+
   * 3ATP costs 11H+, 3.7protons per ATP
   * Oxidising one NADH moves 10protons. 1NADH = 2.7ATP
   * Oxidising one FADH2 move 6protons. 1 FADH2 = 1.6ATP

Explain how the malate aspartate shuttle allows respiration to occur after glycolysis in some tissues
   * Used to move glycolysis electrons from the cytoplasm into the matrix
   * Oxaloacetate is reduced to  malate in the cytoplasm before transport into the matrix
   * Aspartate is used to replenish oxaloacetate

Explain how the Glycerol 3 phosphate shuttle allows respiration to occur after glycolysis in some tissues
   * Used in skeletal muscle and brain
   * Electrons are passed to FAD to make FADH2
   * Through reduction of DHAP (from glycolysis)
   * Less ATP produced from this transporter (3 ATP)

Explain the regulation of OxPhos
   * Depends on the availability of ADP as the phosphate acceptor
   * ATP synthase is inhibited by the absence of oxygen
   * All pathways of ATP-production are coordinately regulated, all respond to concentrations of ATP, ADP, AMP, NADH

Explain the result of uncoupling the respiratory chain, and give an example of its utility
   * Uncoupling can produce heat instead of ATP
   * Newborn animals have brown adipose tissue (BAT) which contains uncoupling protein 1 which allows protons to flow back through the matrix side and increase heat production
   * Energy of electron transport chain is lost as heat

Explain the overall symptoms that would be expected from a mutation affecting mitochondrial function
   * Disease symptoms often affect tissues with high ATP production requirements
   * Blindness, stroke, deafness, structural defects in heart, anemia
   * Mutations are inherited

Inborn Errors of Metabolism
What are inborn errors of metabolism?
   * A group of genetic disorders (including mitochondrial disease) that cause a block in a metabolic pathway
   * Mutations alter metabolism results in accumulation of toxic metabolites, or deficiencies in energy production
   * Monogenic disorders = one gene is mutated
   * Usually inherited, often very severe and present early but can arise at any time
   * IEMs are the most common of the rare diseases, treatment is usually symptomatic (highly dependant on genotype)

How are monogenic inborn errors of metabolism different to other metabolic diseases?
   * Mongenic = one gene containing mutations that critically impacts its function
   * Other metabolic disorders tend to have alot of interplay between other genes, environment, epigenetic influences
   * Common themes of IEMs is defects in pathways involved in catabolism or storage of carbs/fatty acids/proteins
   * Some diseases require multiple deficiencies/mutations to completely lose function/be diagnosed

How does amino acid catabolism, in particular phenylalanine, contribute to energy production?
   * AAC counts for 10-15% of energy production, it can contribute to both gluconeogenesis and ketone bodies, generating 6 products all of which enter the TCA cycle
   * 7 different amino acids generated by AAC can be degraded into acetyl-coa (which feeds into TCA = energy)

What are some disorders of phenylalanine catabolism?
   * The breakdown of pehnylalanine (and tyrosine) yields: 1xacetoacetate (->acetyl-coa), 1x fumerate. Both feed into TCA
   * Genetic defects in enzymes involved cause IEMs:
   * Loss of function in enzyme = buildup of previous metabolite.
   * Phenylketonia (PKU) is a disorder relating to dysfunction in amino acid catabolism
   * Caused by loss of function mutations in the PAH gene encoding the enzyme phenylalanine hydroxylase (normally converts tyrosine into phenylalanine)
   * High buildup of phenylalanine results in the use of a secondary pathways being used -> undergoes transamination with pyruvate to yield phenylpyruvate
   * Accumulation of phenylalanine and its metabolites in early life impairs normal brain development

Why is limiting the intake of the artificial sweetener aspartame important in phenylketonuria?
   * If PKU is diagnosed under 3yrs after birth, intellectual disability and other longterm symptoms can be prevented with strict dietary control.
   * Restrict phenylalanine/tyrosine/protein-rich intake to just meet the needs of protein synthesis
   * Aspartame is a dipeptide of aspartate and a methyl ester of phenylalanine -> breaks down in the gut to become phenylalanine.

What is the ‘Guthrie test’ and what has replaced it in modern newborn screening programs?
   * Guthrie test:
   * Heel prick and blood drop is placed on filter paper
   * Filter paper is then placed on agar with bacillus subtilis that requires the presence of phenylalanine to grow
   * Increased phenylalanine (PKU) will show a ‘halo’ of bacteria
   * Heel prick tests:
   * Heel of an infant is pricked and blood is dropped onto a part of paper
   * Sent to lab and holes are punched out of each blood drop or placed into wells
   * Multiple tests are ran -> testing for increases in hormones or proteins or other genetic biomarkers
   * Also uses mass spec to measure metabolites
   * Nowadays: mainly ESI-MSMs and qPCR are used
   * ESI-MSMs detection method established in 2002
   * Involves dried blood sports extracted with methanol and spiked with internal standards
   * Direct injection in positive ion mode, MRM to detect metabolites (amino acids and acyl carnitines)

Newborn screening (heel prick test) reports a baby with high levels of carnitines, what group of inborn errors of metabolism is this result indicative of?
   * Indicative of fatty acid metabolism disorders
   * Most FFA enter the mitochondria via the carnitine shuttle
   * Fatty acids are the major source of energy in the muscle and the heart
   * Carnitine shuttle is needed to transport FFA into mito for b-oxidation to occur
   * In FFA metabolism disorders, it leads to less FFA being oxidised into energy, and thus the accumulation in the cytosol or mito
   * Fatty acids get shunted into carnitine, producing acylcarnitines which are detected in newborn screens
   * IEMs in b-oxidation results in a severe decrease in the production of ATP in mitochondria
   * Deficiencies can be managed with early diagnosis

Why do mitochondria have their own ribosomes and how might genetic defects in the
mitochondrial ribosome cause mitochondrial disease?
   * Mitochondria are also essential for thermogenesis, amino acid synthesis, lipid metabolism, ect…
   * mtDNA encodes for 13 proteins in humans, 2rRNA and 22tRNA. All proteins encoded by mtDNA are found in the OxPhos system
   * Mito ribosomes are needed to translate the mtDNA into proteins in the mito
   * ‘Primary’ disorders are due to defects in proteins directly involved in OxPhos themselves, eg:
   * Monogenic disorders of mtDNA
   * Differences in heteroplasmy means siblings may not present wit hteh same symptoms, generally adult onset and less severe than mito disorders caused by nDNA
   * Most common is leigh syndrome.
   * Disorders of the respiratory chain
   * Gene encodes subunit of the chain, encoding for assembly factors, encoding translation factors
   * Disorders associated with mtDNA replication (mtDNA deletions or depletion)
   * Disorders of mito membranes
   * ‘secondary’ disorders are defects in other mito pathways which indirectly impact OxPhos.

In the context of mitochondrial disease, what is heteroplasmy?
   * A typical cell may contain both wild and mutant type mtDNA, when a cell replicates and divides it can lead to different ratios of wild and mutant type mtDNA in different cells in the same organism
   * Same organism, cells have different mtDNA ratios between ‘healthy’ and ‘mutant’ mtDNA

Why are chronic high levels of lactate in blood suggestive of mitochondrial disease?
   * High levels of lactate can indicate: a defect in OxPhos, and an increase in anaerobic glycolysis

Describe how respiratory chain enzymology for mitochondrial Complex I deficiency works.
   * Performed on crude mitochondria isolated from clinically relevant tissue
   * Spectrophotometric assays that analyse teh change in absorbance of specific compounds
   * OxPhos indicators are used to selectively analyse a single compound in a crude isolate
   * Mito treated with digitonin to isolate inner memb fragments -> OxPhos inhibitors added
   * Eg. Complex 1 deficiency:
   * Isolate crude metabolite
   * Add antimycin A and potassium cyanide (inhibits complex 3 and 4)
   * Add ubiquinone to act as electron acceptor
   * Add NADH
   * Monitor absorbance of NADH at spectrophotometer at 340nm
   * Similar assays for all OxPhos complexes -> monitoring full enzymatic reaction. Assays arent specific to a single gene

How are mitochondrial diseases diagnosed and treated? Use leighs disease as an example.
   * Nowadays whole genome sequencing is done on the patient and in 50% of the time there is a known variant of a known disease gene, in other cases with a novel variant functional tests are performed
   * Functional tests:
   * Biochemical experiments that aim to demonstrate a connection between genotype and phenotype
   * Spectrophotometric assay with OxPhos inhibitors is one method
   * Understanding of mito diseases relys on research tests done by academic labs outside of clinical accreditation
   * Leigh syndrome case report:
   * Infant presented with elevated blood lactate, mild developmental dely
   * Sequencing identified homozygous missense variant of unknown significance in a subunit of complex1 in OxPhos
   * Skin fibroblasts were taken from child and mother, quantitative proteomics were done demonstrating a clear reduction in abundance of complex 1 subunits
   * Treatment of mito disease:
   * For most there is no cure, only symptom management
   * Some can be ‘cured’ through diet
   * Diagnosis needs to be early enough to do so, to avoid developmental and other lifelong problems = ethical dilemma

What is mitochondrial donation and what kinds of primary mitochondrial diseases might it be used to prevent transmission of?
   * An IVF technique combining the mtDNA from a healthy donor with the genomic DNA of a healthy egg
   * Mothers nDNA is taken from egg and places into healthy donor egg (without nucleus)

Mitonuclear Communications
Explain the mitochondrial bottleneck phenomenon
   *

Discuss reproductive options for women with deleterious mutated mtDNA

Evaluate the importance of stakeholder engagement in development of mitochondrial donation

Identify different mitochondrial donation techniques

Discuss ethical considerations involved in mitochondrial donation

Mitonuclear communication - anterograde signalling

Explain how cellular metabolism is maintained and coordinated with mitochondria

Analyse the importance of mito-nuclear communication

Explain the cells’ bidirectional adaptation to stress with anterograde and retrograde signaling

Explain how anterograde signals are activated by upstream sensors to activate nuclear and mt-encoded mitochondrial proteins and fine tuning involved

Explain how anterograde signals are activated by upstream sensors to detect changes in metabolic conditions (exercise, Ca2+, cold and nuclear stress)

Mitonuclear communication - retrograde signalling

Explain what retrograde signals are

Explain retrograde energetic, Ca2+and ROS dependent stress responses

Mitonuclear feedback and proteostasis

Explain the importance of mitonuclear feedback and proteostasis

Explain the three different mito-nuclear proteostasis responses ie UPRmt, proteolytic stress and heat shock responses in mammals

Compare the UPRmt, proteolytic stress and heat shock responses with the integrated stress response

Integrated stress responses introduction

Describe the control of the integrated stress response (ISR)

Detail how the ISR regulation is dependent upon the eIF2 ternary complex (TC) formation

Detail how the eIF2 phosphorylation regulates TC formation

Explain how eIF2 phosphorylation transforms eIF2 from an eIF2B substrate into an eIF2B inhibitor

Explain how ISRIB works as an inhibitor of ISR and its effect at different concentrations of eIF2-P

Detail how the ISR reprograms gene expression at both
translational and transcriptional levels

Describe the different mechanisms that uORF-mediated translational control can occur

Explain how eIF2-P can be reset to counteract ISR activation

Broadly explain why the resulting consequences of ISR activation are complex
 
Mitochondrial stress effects

Discuss that the ISR phenomenon that can be protective or deleterious to the organism

Give general examples of mitochondrial stress and dysfunction and its effects

Explain that mitochondrial stress signals can be effected by mitokines triggering non-cell autonomous responses

Diabetes
Define hyperglycaemia and hypoglycaemia versus normal blood glucose

Detail how pancreatic beta cells release insulin in response to elevated blood glucose

Evaluate how some drugs such as sulfonylureas exploit pathway events triggered in pancreatic beta cells as a treatment for type 2 diabetes

Describe the important biochemical features of insulin leading to why insulin is not suitable for oral administration as a treatment for diabetes

Explain how glucose enters cells and how insulin increases glucose uptake by target tissues

Describe features of the insulin receptor and discuss its signal transduction mechanisms

Detail the function of insulin receptor substrate 1 (IRS1) and other proteins containing PTB and SH2 domains

Compare how insulin can have rapid and also slower metabolic effects in its target tissues
 
Explain generalized pathways in insulin signaling to support glucose homeostasis

Describe changes in insulin signaling during nutrient excess

Investigate whether hyperinsulinemia or insulin resistance is the primary impairment

Define insulin resistance and its effect on blood glucose

Detail one of the risk factors contributing to insulin resistance

Argue why obesity alone is not sufficient change for development of Type 2 diabetes

Explain the underlying molecular mechanisms and biochemical changes that cause insulin resistance

Describe the 3 main phases in the development of overt Type 2 diabetes and explain the transition from the pre-diabetic state to overt diabetes

Explain how both insulin resistance and pancreatic b-cell loss/failure contribute to development of type 2 diabetes

Explain the triggers for pancreatic beta cell loss and failure in Type 2 diabetes

Elaborate on the similarities and differences between Type 1 and Type 2 diabetes

Provide a simple definition of Type 2 diabetes

Discuss tests that help diagnose pre-diabetes and diabetes

Distinguish changes in pancreatic beta cell numbers in Type 1 and Type 2 diabetes and events that bring about the changes in pancreatic beta cell number

Describe metabolic events in diabetes

Explain the biochemical basis underlying the acute symptoms of diabetic hyperketonemia

Explain why some diabetic patients can present with hypoglycaemia

Discuss long term consequences of diabetes
 
Diabetic ketoacidosis

Explain why a small change in blood plasma pH can be life threatening

Detail how and when ketone bodies are produced in the liver and what ketosis is

Explain what happens in uncontrolled diabetes

Discuss how ketoacidosis occurs and its effects

Compare starvation and diabetes

Metabolic techniques, approaches, and applications:
Explain the major analytical challenges of metabolomics

Explain the steps in a typical metabolomics workflow

Describe the advantages and disadvantages of major analytical platforms

Explain the basics of mass spectrometry

Explain the differences between mass accuracy and mass resolution

Describe the requirements for different levels of metabolite ID

Explain the options for metabolite data analysis

Explain the advantages of metabolomics over other ‘omics

Identify the key applications and frontiers of metabolomics

Give examples where metabolomics bridges basic and translational research

Explain the limitations of steady state metabolomics

Explain what metabolic flux is

Explain the advantages of stable isotopes over radioactive isotopes

Describe the workflow and consideration of a labeling study

Explain the typical statistical workflow to analyse metabolomic data

Explain the purpose of data scaling and its potential problems

Explain what you can infer from a volcano plot

Explain the common dimension reduction approaches for multivariate analysis and their differences

Explain why spatial information is important

Explain the key ionization techniques for mass spectrometry imaging (MSI)

Describe a typical MSI workflow

Explain strategies that can improve the confidence of metabolite ID in MSI

Describe the key emerging technologies in the field of metabolomics

Explain the key strengths of anion-exchange chromatography mass spectrometry

Explain why spatial multi-omics is an exciting approach

Explain the advantages of system automation in metabolomic studies

Transcript for:Comprehensive Overview of Metabolic Processes

Transcript for:
Comprehensive Overview of Metabolic Processes