Biochemistry Notes - Lecture 1 - Membranes

Biochemistry notes 26/09/2024- lecture 1 **Membranes and their ability to transport materials** **Learning outcomes:** - Describe and explain the structure and functions of the phospholipids and how they interact with each other and the aqueous compartments to form a bilayer membrane...

Biochemistry notes 26/09/2024- lecture 1 **Membranes and their ability to transport materials** **Learning outcomes:** - Describe and explain the structure and functions of the phospholipids and how they interact with each other and the aqueous compartments to form a bilayer membrane - Explain why the term "fluid mosaic model" is applied to this structure - Discuss the structure and function of cholesterol - Describe and explain the role of diffusion within membranes with respect to lipids and membrane proteins - Compare and contrast different membrane compartments, identifying how they differ in their constituents - Apply the properties of constituents within the membrane to explain their influence on the physical properties of the compartments. **The Lipid bilayer: A fragile barrier** - Interactions between the components of the cell membranes are necessary to maintain its integrity. **The phospholipid bilayer** - Components of the cell membranes: - Phospholipids - Cholesterol - Protein - Glycoprotein - Glycolipids **Phospholipids** **Properties:** - 2 Fatty acid chains - Glycerol - Highly polar or charged group - Amphipathic - They are a major component of cell membranes. - Cis fatty acids are commonly found, trans fatty acids are obtained from your diet. - The phospholipid can either have a saturated tail or unsaturated fatty acid tail. - 4 ringed structure at the centre, this provides stability. - Saturated hydrocarbon tail (single c-c bonds) - Generated in the liver from Acetyl-CoA - Often converted into other physiological products - Dietary intake can cause toxicity - HMG-CoA reductase is the limiting step in this process - This is inhibited if there is no need for extra cholesterol - Acetyl CoA is the starting product for cholesterol synthesis - Low density lipid proteins contain excess cholesterol - Cholesterol is made in the smooth endoplasmic reticulum, in the liver - Hepatocytes (liver cells) have a high composition of smooth ER - Cholesterol moves from hepatocytes bile large intestine reabsorbed into circulation - Cholesterol is lipophilic so it must be bound to a protein or be part of a larger protein. - Fat soluble vitamins, due to their ring structure - Vitamins A and D both have ring/ steroid structures - Lipid soluble hormones - Steroid hormones- Cortisol, aldosterone, testosterone - T3 and T4 - Ligands are able to cross the plasma membrane due to its lipophilicity - Bind to ligand dependent transcription factors, which can only bind to specific DNA sequences when bound to a ligand. - DNA binding induces transcription of specific genes and therefore changes in specific protein expression. - More time is required for generating a protein than activating a protein **Bile Acids/ Salts** - Synthesised from cholesterol - Essential for lipid digestion - Increase solubility - Increase surface area **Cholesterol: Bile acids** Importance in digestion - Cholesterol is used to synthesise bile acids, without bile acids there is no breakdown of fatty acids - Bile acids are amphiphilic so they can enable solubilisation of dietary fats in a process called emulsification - Emulsification is essential to enable the uptake if dietary fats by enabling the formation of mixed micelles, which allow access of triacylglycerols to intestinal lipases for breakdown. - Triglycerides monoglycerides **Functions of bile**  - Bile is dehydrated and stored in the gall bladder at a much smaller volume - The gall bladder then dilutes in response to fats detected In the small intestine through the release of hormones cholecystokinin (chole-sisto-kinin) - Increased solubility in aqueous solutions increased surface area - Increases access for interstitial lipase - Generates mixed micelles - Modified by conjugation - Formation of a link between an amino acid or other organic molecules with a waste or toxic product - Makes the original product: - Less toxic - More hydrophilic - Easier to excrete - Modification is self limiting as cholic Acid turns of an enzyme that breaks down the secondary bile acid - Tertiary bile acids are much less reactive, so not as toxic - The process of refining bile Acid is required to uptake the maximum amount of lipid **Types of bile acids** **Primary-** - Most toxic - Generated in the liver from cholesterol **Secondary-** - Generated by oxidation reaction in the liver **Tertiary-** - Least toxic - Conjugates to glycine or taurine A diagram of a diagram of a plant Description automatically generated with medium confidence Second lecture **Membranes and their ability to transport materials** **The storage and importance in digestion** - Cholesterol is also present in lipoproteins - Lipoproteins are how lipids are transported around the body - They contain; - Hydrophobic core (contains triacylglycerols and cholesterol esters) - Amphipathic coat of: - Apolipoproteins - Phospholipids Cholesterol \- Metabolised fatty acids Acetyl CoA - Acetyl CoA then feeds into the crebs cycle.. or stored if in excess - Fatty acids that are being taken up can then be generated into a cinomicron, with a layer of phospholipids on the outside. Phospholipids are amphipathic as they are both hydrophilic and hydrophobic - They are important in activating the enzymes - Lipids are stored in adipocytes/ or can be metabolised in cells **Functions- Fat transport** Phospholipids on the outside, this enables the body to carry them around - A chylomicron has the lowest density and contains dietary lipid and cholesterol esters. It transports absorbed fats to the liver for processing. - No carbohydrates in lipoproteins - Lipoproteins can be quite large, - Low density lipoprotein is large, full of fats, low density- only when there is more lipid and fat than protein. **Lipoproteins** - Several classes of lipoproteins, named by density. As central triacylglycerols are degraded, the density increases. - VLDL transports lipids generated in the liver to the tissues - IDL and HDL transport remaining lipids from the tissues back to the liver - LDL recycles back to the liver and to tissues - Apolipoproteins act as signals for cellular uptake and metabolism. - Chylonmicron- largest protein, this then gets smaller as it gets degraded - These take the lipids from the GI track to the Liver - The LDL and VLDL take excess lipids along the body to the target muscle - The High Density Lipid Proteins- take them back. - The different densities have different jobs. **Summary** - Cholesterol is a versatile molecule with a rigid sterol ring structure, a tail and a hydroxyl group - Uptake of all lipids from the diet requires emulsification (requires bile acids) and formation of chylomicrons for transport to the liver, for processing. - Bile acids not bound to lipid droplets can be toxic to the liver, as a result their binding to nuclear hormone receptors enables them to increase expression of enzymes that will degrade them through a transcription mechanism. - The liver generated lipoproteins enable transport of lipids around the body to tissues where it can either be stored or used, and then back to the liver. These use the amphipathic nature of some lipids to envelope those without property. - The contents of the outside of lipoproteins dictate where the particles are destines for **Back to Membranes-** **Fitting into the membrane** **Cholesterol interacts with the top 3^rd^ of the phospholipid** it can have 2 effects; - Interactions between phospholipid tails and the steroid ring give "stiffening " effect by reducing the movement of saturated fatty-acyl chains of the phospholipids. - In bilayers where long chain, unsaturated fatty acid tails are prevalent the presence of cholesterol will decrease van der Waals interactions, accompanied by an increase in fluidity. **Hydrophobic molecules in water** - Saturated fatty acids linear, generate a cone shape, this is the original idea of how cells are generated around the DNA - Unsaturated fatty acids are more cylindrical. - This can change the properties of the membrane - Amphipathic molecules enable a hydrophobic core and a hydrophilic outer surface - Saturated fatty acids are more of a cone shape due to their fatty acid tails are more linear. **Formation of a sphere** - Hydrophobic "middle" of the bilayer forces a shape change because it can not interact with the aqueous environment - This is because it is not energetically favourable as a sheet so creates a sphere. - Inner leaflet is usually negatively charges, faces the cytoplasm - The inner leaflet flips inwards, creating a sphere, this movement is energetically favourable **Freedom of movement** - Main ways for phospholipids to be dynamic within the membrane - Lateral diffusion, flexion and rotation are the most frequent occur without enzymatic activity. - Flip- flop happens rarely unless it is enabled by an enzyme/channel as it is un energetically favourable **Lipid density** - Affects membrane properties; - Phospholipid tails saturation can affect movement and the thickness of the membrane - Saturated fatty acid tails extend further down if the same number of carbons as the 30 degree kink has the "diagonal length" effect. - This causes the membrane to become thicker - Unsaturated fatty acid tails have a 30 degree kink, this makes them more condense packed together, this makes it easier to compress the membrane and there is a reduction in the thickness of the membrane. **Diffusion-** Fusion if the two cells both colours will be dispersed- this proves that diffusion happens in the cell. - Protens evenly distributed across - Polarity in a cell- e.g. GI gut epithelium cells Cell's need to have two ends/ polarities. So some cells have mechanisms to restrict diffusion within a cell. - E.g. tight junctions- stop things from moving around, ensure that the cells hold onto each other, this has an impact on the movement of the particles in the membrane. **Differences between cell typer and organelles** - The composition in a liver cell in comparison to a red blood is different - RBD, phospholipid to cholesterol ratio 1-1 meaning that t=is ridged - The ratio changes in relation to a cells function - There is also a change in the intracellular compartments. **Lipid raft formation** - **Lipid raft is a combination of proteins and lipids which cluster together to form a large cluster** - An artificial membrane with only phospholipids and cholesterol - With a greater number of saturated phospholipids, the membrane becomes thicker - With more cholesterol, there are more formations of lipid rafts, cholesterol can concentrate into small aggregates called lipid rafts - Lipid rafts are inhibitory to diffusion - **Lipid Raft:** Dynamic assemblies of proteins and lipids that float freely within bilayer of plasma membrane but can also cluster to form large, ordered platforms. - Cholesterol gathers to stabilise the structure in that location **Properties;** - Acts as an anchor for proteins - Prevent movement of membrane components including proteins around the "fluid mosaic" membrane, therefore reducing movement and diffusion **Used when;** - Require location specific functions e.g. receptors need to be in the same synapses - Often contain specific protein subunits e.g. associate in the membrane to be activated... or inactivated Lipid rafts support structures are close enough to assist when required, they can also separate structures to ensure that signals are prevented **Lipinski's rule of 5** Five key physiochemical parameters for being able to move passively through a lipid membrane: 1. Molecular weight (MW) is less than 500Da 2. The calculated lop P value is above five 3. There are no more than five hydrogen bond donors (e.g. -NH-,-OH) 4. No more than 10 hydrogen bond acceptors (e.g.-O-) 5. Low overall charge - Log P is also known as K ( constant)- - Low overall charge as phospholipids are generally already charged- this is important for drugs, hormones... this allows them to cross the membrane - Lipid soluble hormones oestrogen - Generating an action potential - Muscle contraction- movement of Ca - Glucose transporters in the epithelial cells - Excretion of toxic waste products What principles govern the free movement across the membrane? - Lipinski's 5 rules - E.g.O2 diffuses across the cell membrane passively (without energy or proteins) Why do bacteria not use cholesterol? - Has a cell wall made up of peptidoglycan-constricts movement. An Antibody is a protein designed to detect foreign pathogens - Fab region -- recognises some antigens - FC region is important for attaching itself to the surface to other cells **Membrane components p2-** **Membrane asymmetry-30/09/24** What is Fluorescence? - Electromagnetic radiation in the form of light How do we detect fluorescence? - Fluorescent microscope, UV light... **Membrane Components** Components of a cell membrane? - Cholesterol with either saturated or unsaturated fatty acid chains- can make the with of the membrane longer, provides structural support - Phospholipid structure has hydrophobic and hydrophilic regions, allows certain things to pass through - Glycoproteins/ glycolipids How do the properties of the components influence the properties of the membrane? - Cholesterol changes the fluidity of the membrane, it can interact through the hydroxyl groups with the phospholipid, increasing the packing of the phospholipids if they are saturated due to the **stiff ring sterol structure** - The packing will also increase van der walls forces, the longer the fatty acid chains on the phospholipids, the more tightly the packing will be, the tail lengths will be longer, so more van der walls forces, this will also increase the thickness of the fatty acid. The membrane will be thicker and squashed in more tightly making the cell membrane less fluid. Continued... **Week 2 lecture 1** **Membrane asymmetry** **Protein-membrane association** **Membrane components** - How to insert a protein into the membrane 8 mechanisms for insertion  1. **Single path protein** - Transmembrane protein - Alpha helices only go through the membrane as the parts sticking out of the membrane must be lipophilic, the hydrophilic components will be in the membrane - E.g. cell-cell junctions 2. **Multipass proteins** - there or more distinct hydrophobic alpha helices. - Many forms multi-subunit channels - Passes through the membrane multiple times - The link regions, loop structures are hydrophilic as they stick out, they need to interact with water - There is a changing of amino aids, this allows the protein to interact with the phospholipid, ensuring the protein is secure in the membrane. Via van der walls forces - E.g. Nicotinic acetyl choline receptors 3-**Channel** - More complex as it has a charged or polar group facing inwards and vice versa outside. - Pore in the membrane, leak channels - Channel proteins, multipass proteins that make a circle in the membrane, hydrophilic properties are pointed inwards - E.g. GI track, needs pros 4- **Not membrane spanning but embedded in the membrane;** - Protein is considered to be amphipathic - It does not go all the way into the membrane - Not a transmembrane protein - Comes out of the membrane from the same side it originated from - E.g. Adenal cyclase, May be providing an enzyme on the inner surface or Passing a message from outside to inside 5-**Lipid anchored;** - Long chain fatty acid tethers protein to the membrane through Van der Waals forces between phospholipid tails and the fatty acid chain - E.g. Protein Kinase C - An accessories protein 6- **Glycolipid anchored;** - Fatty acid part enables tethering to the membrane but the carbohydrate part acts as a flexible linker enabling proteins to be near but not immediately next to the membrane - E.g. proteases - Lipid anchors - Phosphate group and carbohydrate chain - Glycolipid anchors, on the outside - Carbohydrate anchors outside act as a safety mechanism 7- **Adaptor proteins** - Use a transmembrane protein as an adapter to associate with the inner leaflet of the membrane - E.g. enzyme linked receptors 8- **Accessory proteins** - Similar to adapter proteins but extracellular, enables extracellular matrix proteins to provide additional mechanical strength **Lipid anchors** - Rely on interactions between fatty acid chains and phospholipids tails - Raft formation ensures proximity to transmembrane proteins **Inserting proteins into membranes** - Polypeptide chain needs to form a cylindrical structure, allowing charged/polar amino acid to be hidden from the hydrophobic middle of the bilayer - Uncharged/ non-polar amino acids on the outside of the cylinder enable interaction with fatty acid tails. **Multiple transmembrane regions** - Polar or charged amino acids link transmembrane sections, which interact with aqueous environment - Amino acids have both hydrophobic and hydrophilic regions. **Formation of large transmembrane proteins within membranes** - Alpha helices may be twisted sideways, may be located in other places... - Complex pores can be generated by different structures **Filters** - Charges and polarity in the centre of the pore of the channel make it selective for its ligands. - E.G. for a Na + channel the charge in the channel must be negatively charged, but also the right size, how close the loop domains are to each other. **The components of the membrane and their interactions** **Functions of protein in the membrane** **Structures that represent proteins in the membrane and its functions** structures glycoproteins, e.g. lipophilic hormone receptors G protein coupled receptors, thyroid stimulating receptor, glucagon receptor... Nerve conduction, **Giving membranes strength** When something is ridged the membrane becomes less fluid - Spectrin heterodimers from a mesh using junctional complexes - Junctional complexes are formed of short actin filaments - The cytoskeleton is linked to the membrane through two transmembrane proteins; band 3 and glycophorin - Sectrin tetramers bind to some band 3 proteins via ankyrin molecules, and sto glycoproteins and band 3 via band 4.1. proteins. - The proteins can be in the membrane but they do not make the membrane ridged - Proteins attached to other things int the membrane that make them strong e.g. erythrocytes - Transmembrane proteins still affect the movement of proteins in the transmembrane cortex 01/10/24 2.1 **Membrane Asymmetry** **Controlling membrane movement** - A domain is formed if there is a physical blockage to movement - Lateral diffusion is possible within a domain of the membrane - Tight junctions are an example as they restrict movement within the bilayer. - Associated with large lipid rafts (Combination of proteins and lipids which cluster together to form a large cluster) this keeps it strong, and provides protection for microbes, this reduces the lateral diffusion in the membrane **Limiting diffusion** - The cortical cytoskeleton network restricts diffusion of plasma membrane proteins directly anchored to it - Cytoskeleton filament form mechanical barriers that obstruct the free diffusion, partitioning them into small domains - Proteins diffuse rapidly, but are confined within an individual domain - Transmembrane proteins associated with many other proteins are limited in their diffusion depending on interactions between them and size of its cytoplasmic domain. **Sorting membrane contents** - Formation of domains helps concentrate such signalling complexes, increasing the speed and efficiency of the signalling process - Domains can be permanent, as in spermatozoa, or transient e.g.Anterior head involved in fertilisation, needs digestive enzymes, which are located in the anterior head - In cells, with several structurally and functionally distinct parts covered by a continuous plasma membrane miniature rafts enables domains of the membrane to form that contain specific proteins. **Proteins as receptors** - Transmembrane proteins are needed as they are important in cell signalling - Docking station for signals - Transmit a signal from outside the cell to the inside of the cell - Often multi-component: - Multi-Subunit - Enzyme linked - Enzyme associated - Accessory proteins **Other membrane components** **Glycolipids** - Made of a diglyceride attached to a carbohydrate - Diglyceride acts as the anchor - Immune recognition \]involved in determination of self - Cyanic acid- has a H and a N- enzymes which can break down the cyanic acid residues - Used for recognition, anchoring things, keeping things close to the membrane but not part of it **Glycoproteins** - Usually part of complex, multi subunit receptors - Carbohydrate chains acts to restrict movement or scaffold structures - E.g. Proteoglycans: - Essential for interaction with the extracellular matrix **Functions of glycol-additions** - Support interactions of a receptor with its ligand - Can act as part of ligand recognition site **Membrane Dynamics** **Learning Objectives;** - **Explain the need for vesicular transport and describe the movement of vesicles between compartments within the cell** - **Describe how clathrin, as a coat protein, induces budding** - **Explain the importance of snare complexes and Rab proteins in vesicle fusion and docking with the target compartment** - **Apply principles of fusion and docking with membrane components to explain how organelles/ compartment functions are maintained by vesicle transport** - **Discuss the synthesis of lipids within the ER and how these migrate to the cell surface.** **How everything links together** - Components of the membrane determine the properties of the membrane - Different cells have different functions - Parts of the plasma membrane may have different functions - Organelles have different functions - So... different cell types (or organelles types) must have different components in their membranes **Endocytosis and exocytosis** **Vesicle transport** - Donor membrane or compartment the vesicle ends up at its target membrane and merges with it. **Adress labels** - Generation of vesicles starts of in the ER - The vesicle will only move off if it has the right carbohydrate composite - The lysosome have acid hydrolysis, enzymes with low pH levels (enzymes that work in acidic conditions) - Membrane components act as address labels - Glyco-additions such as mannose enable receptor binding and direct transport to specific compartments **Cellular compartments** - Endosome coming in from the plasma membrane, the contents comes in from the plasma membrane, sends things that need to be degraded to the lysosome - Things come into the early endosome to then be re-recycled - Endocytosis is a way of sampling the environment - Type of coat protein determines destination of the vesicle - COP I Golgi to ER - COP II ER to Golgi - Clathrin Golgi to lysosome Plasma membrane to endosome Golgi to plasma membrane **Lipids and compartment summary** - The contents of the membrane can be regulated by the endocytosis and exocytosis - Membrane components made in the ER contain a low level of cholesterol increasing as the membrane matures through the golgi and to the plasma membrane - Membrane bound proteins generated in the ER can act as cargo receptors and/or are proteins destined for other compartments e.g. lysosome, plasma membrane etc. - Vesicular transport enables secretory proteins and membrane bond and associated proteins to be transported to the plasma membrane while also allowing retrograde transported to the plasma membrane while also allowing retrograde transport into the cell of extracellular environment, membrane recycling and receptor recycling and degradation. - Membrane and ER contain a low level of cholesterol to increase fluidity as the ER has to form vesicles - The membrane becomes more ridged as you move towards the golgi as the composition of cholesterol is higher there. **Clathrin coats** **Structure of a clathrin coats** - To bend the membrane - Only recruited to receptor associated with plasma membrane - triskelion structure (has three legs that make a cage like structure, gives vesicles a spherical shape rather than a circular shape) - The ability to generate a vesicle needs a fluid membrane **Coat formation** - Cargo receptor binds to the cargo - Adaptor proteins then have a different shape to the cargo receptor, this shape difference is what allows it to attract its caot protein, the coat protein associates to the cargo protein - As more and more coat proteins bind, a lolypop formation takes place, this structure then is released - At the bottom of the structure, the membrane binding and fission protein is twisted and the vesicle is released - The vesicle must then get to its target compartment but with the coat protein it is unable to as it can not fuse with its target compartment as the membrane is unable to fusse - The removal of the coat protein exposes the membrane, this allows it to bind to its target compartment - **Coat assembly and cargo selection:** Proteins on the ER membrane act as receptors to keep cargo in a specific place. Binding leads to a confirmational change that enables adapter proteins to be recruited - **Bud formation:** Adaptor Proteins (such as AP2) bind to clathrin triskelions. Bar-domain containing proteins aid the bending of the membrane to increase access to the adapter proteins to clathrin. - **Vesicle formation:** Continuation of formation of a vesicle and generation of a stalk, which then recruits dynamin and accessory proteins to pinch off the vesicle. - **Uncoating:** After the vesicle is released there is no longer need for the coat proteins and associated adapters, so they are disassembled and recycled. This leaves the vesicle with cargo-bound receptors on the vesicle surface (and snare proteins to direct the vesicle to its destination). **Initiation of Coat formation** - AP2 structure is made up of two different subunits - Binding of µ2 and σ2 to PIP2 is required to initiate cargo receptor binding as it results in a conformational shape change - Change in receptor conformation leads to membrane deformation, making it more accessible for coat proteins. - There are two shape changes needed for this. - Clathrin is made up of multiple alpha helices - BAR domain dimer **has alpha helices on the outie, on the outside there are charged amino acids which repel the phospholipid heads, which push together and arch the membrane.** - Membrane-bending proteins that contain crescent-shaped BAR domains, these cause a shape change to the membrane via electrostatic interactions with the lipid head groups - Multiple BAR domains need to work together inorder to cause a shape change to the membrane. - BAR-domain proteins are thought to help AP2 recruit clathrin by shaping the plasma membrane to allow a clathrin-coated bud to form. - Some proteins also contain amphiphilic helices that cause membrane bending when inserted into the cytoplasmic leaflet - These are essential to shaping the neck of a budding vesicle through stabilization of sharp membrane bends - Clathrin machinery recruits local assembly of actin filaments that introduce tension to help pinch off and propel the forming vesicle away from the membrane. - The binding of the clathrin initiates coat formation - The coat associating with the adaptor allows recruiting of coat proteins to interact with microtubules and dynamin **Vesicle release** - Dynamin recruits a protein complex to initiate vesicle release, pinching of the vesicle. - The inner leaflets of the bilayer merge, ensuring that it is completely sealed, then the vesicle "pinches off" **Dynamin** - Needs GTP in order to allow a shape change - The GTPase domain of dynamic changes shape with GTO hydrolysis. - This then breaks down and changes to GD, which changes the shape causing it to squeeze what itt is surrounding, the fussion process pushes the membranes together. - The GTPase domain of dynamin changes shape with GTP hydrolysis - Shape change likely results in the "squeezing" of the neck of budding vesicles. - Coat proteins are snare proteins, they have the address lablle on - V snares are complementary to the T snares, which are on the target membrane - They must be complementary for the fusions process to happen - V snares also have Rab GTPases - Rab is off when bound to GDP - When bound to GTP Rab is on - When on it ensures that the V-snares come together ensuring winding - Rab GTP is associated with Rab effector which causes a change in shape - The Rab is needed on the vesicle to change the shape on the Rab effector - GDI is a Rab inhibitor, which binds it to the Rab effector - Monomeric GTPase - Each Rab protein is associated with one or more organelle - At least one type of Rab is present on the cytosolic side of each organelles membrane. **Lipid droplets** - E.g. digestion with lipid proteins and micceles - Same story: different cargo - The cargo is cholesterol which is generated through HMG CoA reductase (rate limiting step for the generation of cholesterol ) - When the ER is generating things that are amthypathic e.g. phospholipid bi-layer - Phospholipids in the membrane are generated between the two leaflest of the ER membrane, they are created in the space between the space in the leaflet - Lipid droplets are single layered, vesicles are double layered - When these are generated, the outer layer of the phospholipid bi-layer gets larger, not the inner layer - This causes the outer leaflet to grow, this causes limited integrity, which may cause leakage, - Lipid droplets are released and they go into circulation - They are secreted inside a Golgi particle **LDL uptake** Excess LDL can control cholesterol synthesis in the liver; - Endocytosis of LDL particles using clathrin coated pits - Recycling of LDL receptors to the plasma membrane - Formulation of a vesicle containing cholesterol esters which is integrated into the ER - High cholesterol levels in the ER results in a decrease in the synthesis of HMG-CoA reductase and LDL receptors **Vesicle transport: summary** - Organelle and membrane make-up can be regulated by endocytosis and exocytosis - Donor compartments require specific lipids (e.g.PIP2) to encourage recruitment of adapter proteins to load cargo receptors during bud formation - The recruitment of a coat protein and associated BAR domain containing proteins bends the membrane to generate a vesicle, which is then released with the help of dynamin (GTP binding protein kept on the system by PIP2 too) - Monomeric GTPases such as Rab help to ensure that the vesicle reaches its target, and with its complementary Rab-effector enables docking on the target membrane to enable SNAR proteins to cause membrane fusion. - Endocytosis and exocytosis can be observed in the regulation of lipoprotein and cholesterol levels in the body through LDL receptor feedback system. Products are not amphipathic therefor need to be coated in membrane to enable transport through the aqueous cytosol Linking tings together LDL uptake - Low density lipid receptors bind to the LDL receptor, this causes a shape change which recuits clathrin to the surface causin clathrin pit formation, forming a vesicle - The endospme changes shape releasing the vesicle - Cholesterol works in a negative feedback, acting on a receptor that prevents the transcription og HMG reductase, this controls how much cholesterol is generated - LDL brings things back from the tissues after storage - If you have high cholesterol this process may not work, due to a change in one of these proteins in the pathway - Look at vesicle transport summary **Membrane asymmetry** LO: - Compare and contrast the functions of flippase, floppase and scramblease in maintaining membrane organisation. - Explain how phospholipids are organised within a normal healthy cell and in a cell which is undergoing apoptosis. - Discuss how proteins associate with the plasma membrane and the types of protein structures seen within the plasma membrane. **Structures to remember** - Difference in phospholipids - Cerine only phospholipid with a negative charge - Negatively charged are usually on the inner surface - An addition of carbons on the outer membrane - Incoming vesicles are randomly assorted - Enzymes are required to ensure that membrane phospholipid addition occurs equally on both leaflets.  - When making vesicles and lipid droplets there must be a nice even spread around to maintain integrity - If another phospholipid is added on the outer leaflet one s needed on the inner membrane, but when generating phospholipids they are generated on the outer leaflet **Scramblases** - Scramblases- enzyme that ensures that both leaflets have an even amount of phospholipids - Is a calcium dependent enzyme - In the membrane of the ER - Enables the movement of phospholipids from the when Ca is available inner to the outer leaflet of the plasma membrane - Flipases family proteins- flip phospholipids on the other membrane, these proteins are only located on the plasma membrane **Scramblase action** - Is important in apoptosis - Maintain mitochondrial membrane function - Also present at the cell surface e.g. exo and endo cytosis **Flippase and Floppase** - Flippase and Floppase are part of the flippase family of proteins - They are ATP dependent - If a cell is undergoing Apoptosis then there will be no Flippase Floppase action Week 3 lecture 1 **Membrane asymmetry** **Scramblases** - Calcium dependent enzymes - Important in apoptosis - On all the time in the ER, as that is where calcium is stored, in the ER vesicles are generated all the time, phospholipids are generated on the outer leaflet - Scramblases flips phospholipids from the outer leaflet onto the inner leaflet **Flippase and floppase** - Part of the flippase family need ATP - If a cell is undergoing apoptosis no ATP so no Flippases/ Flopases movement **Flippase action** - Exchanges from extracellular to internal surface - Floppase exchanged in the opposite direction - Both require ATP - Scrambalses not usually turned on unless the Ca levels go up (Ca released from the ER) **Apoptosis** - Ca levels go up as the membrane is dying early sign that the cell is undergoing apoptosis **Experimentally identifying membrane components** **Flow Cytometry?** - Lazers excite fluroflors then detected by detectors - Either use an antibody or dye to detect different cells, as long as their emissions are on different wavelengths that are not detected on the same detector **Q;** What are the differences between flow cytometry and confocal microscopy? - Target for antibody would be different Lipophilic dyes - Dyes that are lipophilic that will embed themselves into the membrane, this can help us see how the membrane changes over time - Use an ER tracker to see how many vesicles are generated - When Apoptosis is occurring we can use dyes to see how that happens **Q:** How does the membrane and its integrity change during apoptosis? - Membrane starts blebbing - The membrane becomes more fluid caused by the loss of cholesterol - ER releases Ca this causes the activation of scrambaslases (not flipases and flopases as they need ATP) this allows phosphor cerine to flip onto the outer membrane. This is what we track when looking at apoptosis - Propidiumiodie goes into your cell and under health conditions it the leaves the cell, if a cell is undergoing apoptosis there will be a greater amount of Propimiumiodide in the cell - Release of Ca happens earlier on than the reduction of ATP so cyrine on the outside= early apoptosis - Dye = late apoptosis Experimentally identifying membrane components - FAcs separation **Q:** Advantages/ disadvantages of using flow cytometry over the confocal microscopy? **Data interpretation** What are the increased benefits **Cell signalling** LO **Signal transduction** - extracellular signal, hormone - cargo and receptor must be complementary to one another - The receptor sends signals internally in a cell - Transcriptional effect this may activate a transcriptional activation protein - This may also cause activation of a metabolic enzyme, this may cause a change of the nucleus - Effector proteins cause a change change cell shape Examples of signalling pathways? - Acetly choline A - Synapse transmission - Glucose pathway/ insulin - Endocrine signalling - Apoptosis signalling **Transmission of signals** **Modes of transport** - Paracrine neighbouring cells- no contact release of something from one cell but affects surrounding cells local mediator - Synaptic transmission nerve... - Endocrine signalling release of signal happens far away from the target cell/ organ the signal must be more robust than any other signal **Cell communication** Generally 4 different ways a message can be received - Cell survival - Cell growth - Cell specialisation - Cell death There are different signals depending on the outcome of the cell Contact dependent - T cell receptors/ b cell receptors if they happen to bump into each other they eill bind, causing a secondary activation Paracrine signalling - Signals released into the extracellular space - This signal will move across this space to the cells that are around it - Has an all or nothing response - Happens during chemotaxis chemical messengers (tetropeptide -- 4 AA) detected by immune cells essential for the body to protect itself Cell communication - Neurotransmitter binds to complementary receptors on near by cells there is a delayed period where you can not generate a action potential the lipid rafts - Endocrine signalling not in a fixed space no regulatory effects so the receptors have different sensitivity to the neurotransmitters Speed of transmission - Extracellular signalling molecule act slowly Lecture 3**Interpreting the signal** Method of interpretation: secondar messengers Phosphorylation - Opportunity for the addition of phosphates - Kinase adds a phosphate group, adds a phosphate to a protein, phosphate is lost from ATP When bond to GDP off - GEF remove GDP and allows GTP to replace it, this causes a conformational shape change it will be on its on form **Transduction-passing the message** LO: Signalling cascade - Sends a signal inside the cell via phosphorylation - It can dimerise inside the nucleus - Can activate the next thing in the nucleus phosphatase removes the phosphate, the protein then moves out and the process happens again How can we ensure that transmission is effective? - Use of lipid rafts to keep components of the receptors together using accessory for ligand binding - Using adaptor proteins for signal transmission Ensuring transmission :scaffolding - Attached to the scaffolding protein - In a inactive from - When the signal molecule binds the receptor is active - Proximity activation, if the one next to the molecule is activated : complex formation :docking sites - Inactive receptor - Proteins that associate with the inactive receptor might have the recognition site Secondary messengers - Small molecules e.g. cyclic AMP activates protein kinases A Signalling cascade Receptor types - Channel linked receptors Na channels, ATP channels... something goes through and activates something - Enzyme linked receptors go through the membrane, process of passing the message along happens inside, passing the message on - G protein-coupled receptor Ion channel receptors - Can be created by multiple different components - Signal molecule binds to the protein that is sat in the membrane - Ions move through the channel, they are not the signalling molecules thou - Only briefly allows the membrane to be permeable to the ion increases the intracellular concentration of the ion - Opening of the structure, sometimes the ligands are on the inside, letting things out - Still must be the right charge for the ligand to enter GABA binds to the beta subunit causing it to open but as there are 2 beta subunits, there must be 2 GABA units **G protein-Coupled Receptors** - Have 7 transmembrane domains crosses the membrane 7 times - N terminus is on the outside - C terminus is on the inside - C3 largest loop - Alph subunit shown by the dark green always binds to the GDP or the GTP. this induces its change in shape - GDP binding site in the middle of the protein **Calcium signalling** - Ca dependent enzymes involved in apoptosis - Process that requires a g protein coupled receptor - Swapping out of GTP for GTA - The alpha subunit from phospholipase c beta - Phospholipase c is kept by the membrane and activated by pip 2 - Pip2 have long chain fatty acids that sit to the carbon **GPCRs that activate or inhibit Adenylyl cyclase** - Acts as inhibitors/ chaperones - G protein coupled receptor are associated with G alpha S **Week 6Overview of Glycobiology and carbohydrate metabolism** **Per-reading:** - **Monosaccharides and disaccharides** - **Polysaccharides** - **Glycoconjugates: Proteoglycans, Glycoproteins and glycolipids** - **Carbohydrates as informational molecules: the sugar code** - **Working with carbohydrates** **Carbohydrates and Glycobiology** - Carbohydrates most abundant biomolecules - Photosynthesis converts CO2 and H2O into cellulose and other plant products - Carbohydrate polymerase called glycans serve as structural and protein elements in in cell walls of bacteria, fungi, and plants, and In connective tissues of animals - Other carbohydrate polymers lubricate skeletal joints in participate in cell-cell recognition and adhesion - Some complex carbohydrate polymers covalently attached to proteins/ lipids act as signals that determine the intracellular destination or metabolism. - Aldehydes or Ketones, with at least two hydroxyl groups, or substances that yield such compounds on hydrolysis. - Carbohydrates have the empirical formula CH20n - Some carbohydrates also contain nitrogen, phosphorus or Sulfur - There are three major size classes of carbohydrates: - **Monosaccharides** (simple sugars) - Consists of a single polyhydroxy aldehyde or ketone unit - The most abundant monosaccharide in nature is the six-carbon sugar D-glucose (dextrose) - **Oligosaccharides** - Consists of short chains of monosaccharide units/ residues joined by glycosidic bonds - The most abundant are disaccharides (two or more monosaccharide units) - E.g. sucrose consists of the six-carbon sugars D-glucose and B-fructose - All common monosaccharides and disaccharides have the names ending in "-ose". - In Glycoconjugates oligosaccharides consist of three more units not occur as free entities but are joined by no sugar molecules - **Polysaccharides** - Sugar polymers containing more than 10 monosaccharide units - E.g. cellulose cellulose, a linear chains - E.g. glycogen branched - Both cellulose glycogen consists of recurring units of beta glucose but they differ in type of glycosidic linkage different properties - Carbohydrates - Multiple chiral carbons, configuration of groups around each carbon atom determines how the compound interacts with other biomolecules - This can result in L and D antomers - Monomeric subunits, monosaccharides (the building blocks of large carbohydrate polymers) - Specific sugar the way the units are linked whether the polymer is branched determine its properties and thus its functions - Storage of low molecular weight metabolites in polymeric forms avoid high osmolarity that would result from storing them as individual monomers - The sequence of complex polysaccharides are determined by the intrinsic properties of the biosynthetic enzymes that add each monomeric unti to the growing polymer **Monosaccharides and Disaccharides** - Simple carbohydrates - Can either be Aldehydes or Ketones with two or more hydroxyl groups - Ciral carbon centre give rise to many stereoisomers - Enzymes that act on sugars are strictly stereospecific, this is why Stereoisomerism in sugars are important - **The Two families of Monosaccharides: Aldoses and Ketones** - Monosaccharides are colourless, crystalline solids - Freely soluble in water but insoluble in nonpolar solids - Most have a sweet taste shorter ones are sweeter - Have an unbranched backbone carbon- carbon chains are single bonded - One carbon atom is double-bonded to an oxygen atom to form a carbonyl group; each of the other carbon atoms has a hydroxyl group - If the carbonyl group is at the end of the chain **Aldehyde group** - If the carbonyl group is at any other position the monosaccharide is a **Ketose** - The simplest monosaccharides are the two three- carbon triose: glyceraldehyde, an aldotriose and dihydroxyacetone, A **Ketose** - **Monosaccharides: Asymmetrical centres** - All the monosaccharides except from dihydroxyacetone contain one or more asymmetrical (chiral) carbon atoms have optical active isomeric forms - **Monosaccharides: Cyclic structures** - Aldos **Week 6** **Lecture 1** **Week 6- Lecture 2** Monosaccharides can be categorised by the number of carbons Disaccharides 2 monomeric carbon units Which characteristics distinguish starch and glycogen why is this important? - Starch compact - Composed of teo different units Amylose - Amylopectin - Glycogen has multiple branches branching helps when energy is needed easily accessible **Glycoconjugates** - Carbohydrates/ glycans that are attached to proteins and lipids - There are three types of Glycoconjugates; - **Proteoglycans** - **Glyco** - - They paly an important role in cell signalling **Glycosaminolyglycans** - Increased mobility in joints **ECM** - Gell like substance outside any mammalian cell not present in plants - Combination of amino sugar - Negatively charged attract more water so they can be more aqueous - Different cell types have a different composition of the ECM (composed of different proteins), more fluid of solid - Lectins cell cell communication and recognition - Maintain cell integrity - Some microbes secrete enzymes and degrade the matrix how bacteria enters the cell Proteoglycans - Contain proteins - Composed of sugar and small amounts of proteins - Chains are long linear and negatively charged increased inclusion of water and therefor increased hydration - Keritan sulfate and chondroitin sulfate hair like structure - Part of spinal chord can take up liquid (cushioning effect) and compression - Chronic degenerative diseases cleave glycan chains and the aggrecan looses its function - Proteoglycans contain more carbohydrates than sugars Glycoprotein - Proteins with oligosaccharides attached - Carbon=hydrae chains act to restrict movement - Three specific types N-Linked O-Linked OH group as its functional unit GPI anchored O-GlcNAc - N and O linked are on the cell surface or secreated out - Mucins released in secretion, prescence of glycans negatively charged and start to replell each other Glycolipids - Classification of blood group androgen - Difference in glycan chain - Glycolipids can determine what blood group you belong to - Ways pathogens can invade the immune cells - COV-2 predetermine step attached to glycolipids and then recruits the receptor in order to enter the cell Differences and similarities between glycolipids and phospholipids Phospholipids - Have a hydrophobic and hydrophilic region - Is phosphate bound - Make up the lipid-bi layer Glycolipids - Carbohydrate bound - Within the cell membrane - Secreted out the cell - Located outside the cell membrane - Involved in cell signalling and cell recognition Membrane transporters - Different ways nutrients can enter the membrane diffusion, channel proteins, co-transporters... - Transport of nutrients against the concentration gradient requires ATP Glucose transporters - Facilitate transport of glucose/hexoses across the plasma membrane - Glucose transporters independent of sodium Glute 5 intestine fructose transporter Factors that determine what types of Glutes are expressed : - Tissue specificity - Substrate specificity Sodium-independent facilitated diffusion transport - Glute 2 glucose absorption released into the blood stream - GLUT-1 Opens on the inside of the cell Substrate specificity determined by the AA residue GLUT-1 can cause GLUT -1 deficiency seen around 2 yrs old, absence of glucose can cause mental defects this occurs during the fasting state when glucose levels are low in the morning Kinetics of glucose transport into erythrocytes Maintains blood glucose homeostasis Contributes to the re-absorption of glucose back into the blood stream Glute -4 - Only insulin dependent action - Largely expressed in skeletal muscle and adipose sites (where glucose is conserved) - Insulin binds to its receptor triggers the fusion of vesicles increasing the expression of Glute 4 on the cell membranes in a fed state - Responsible for the insulin regulated transport of glucose in tissues Week 6 Lecture 3 Transport of glucose into cells Part -2 **Sodium dependent glucose transporters** Co-transporters - Driven by high extracellular sodium - Pump occurs with 2 K PUMPED IN AND 2 Na pumped out - The excess K that is being pumped in is also pumped out - This process created a diffusion gradient allowing movement of glucose from the lumen cells SGLT2 contributes to 90% of the absorption - SGLT inhibitors prescribed for diabetics - Decreases glucose levels - Increased glucose levels can cause abnormal glycation non enzymatic addition of glucose to cells, metabolic problems, various toxic bi products.... - SGLT inhibitors block the reabsorption of Glucose An overview on metabolic... - Glucose can be utilised for the production of other hexoses galactose, manose, sugars, glycan diversity.... Glucose can be converted into glycogen - During fasting glycogen is released into glucose ti increase glucose levels - Gluconeogenesis also occurs in fasting stares - Glucose is largely obtained from the diet - Glycolysis Key Points - Glucose homeostasis within the body is maintained by glucose transporters **Week 8** **Week 8, lecture 1 Metabolism in Health and disease** **Pentose phosphate pathway (reading chapter 14; glycolysis, gluconeogenesis and the Pentose Phosphate pathway)** **Learning Outcomes:** - **Generic overview- Pentose Phosphate Pathway** - **NADPH and its roles** - **Inborn errors of metabolism- Glucose-6-phosphate dehydrogenase deficiency.** **Overview of glucose metabolism** - Glucose trnasporters newly port passive glucose transporters - Sodium and ATP independent transporters - There are around 14 different glucose transporters - Once glucose enters a cell primary production is energy production **Pentose Phosphate pathway** - Glucose -Oxidizing pathway runs parallel to upper glycolysis - Not used for energy production - Occurs in the cytosol - Has two phases: - Oxidative Phase (irreversible) - Nonoxidative phase (reversible) - Anabolic pathway builds - Produces ATP - Caters to specialized metabolic needs: - NADPH - Ribose-5-Phosphate - Dietary pentose sugar metabolism - Synthesis of aromatic amino acids **NADPH vs NADH** - Structurally similar but have different functions - NADH in the electron transport chain, produced in Glycolysis to generate ATP, 1 NADH produces 2.5 ATP... so 1 molecule of Glycolysis produces 5 ATPs - NADPH is used in biosynthetic reactionsthis is seen in fatty acid and cholesterol synthesis, redox homeostasis, response to stress responses. **Oxidative phase: Pentose Phosphate Pathway** - An oxidative phase involves an oxidative reaction, this is catalysed by three different pathways. **NADPH regulates partitioning: Glycolysis versus Pentose Phosphate Pathway** - Occurs in a fed state - Produce NADPH for metabolic needs **Nonoxidative phase regenerates G-6-P from R-5-P** - Interconversion for sugar phosphates - Ribulose 5-phosphare Ribose-phosphate - Silde 10 pathways change depending on the biochemical needs of the cell **Modes of Pentose phosphate operation** - More ribose non-oxidative phase is activated - Gives rise to Ribose 5 phosphate - Oxidative Phase is active - Non Oxidative phase recycles immune cells, macrophages and epithelial cells constantly encounter pathogens this is needed to dampen a cell - Glucose 6-phosphate is recycled both are NADP requirement **Regulation of the Pentose Phosphate Pathway** - Substrate regulated - Activation of the first enzyme required this depends on the relationship between NADP and NADPH - Insulin required for an increased rate of transcription for the first enzyme required **Week 8 lecture 3** **Metabolism in health and disease Inborn errors of metabolism** **Learning Outcomes:** - **Dfkhv** **Recap** - Non-oxidative phase can generate ATP **Roles of NADPH** - Metabolism of drugs - Reductive biosynthesis (fatty acid synthesis) - Reduction of hydrogen peroxides (GSH/GSSG) - Cytochrome P-450 - Phagocytosis - Nitric Oxide synthesis **Inborn errors of metabolism** - Heterogeneous group of disorders that may be inherited or may occur as the result of spontaneous mutations. - GLUT 1 expressed in red blood cells and brain cells, this contributes to neurological development - SGLT1 intestienes glucose is transported into the cells by a sodium ATP dependent channel. **Wernicke-Korsakoff Syndrome** - A neurological/memory disorder that results from Vitamin B1 (thymaine) deficiency (Beriberi) - Associated with alcoholism, malnutrition and thiamine transporter variants - Catalyses conversion of **Glucose -6-phosphate dehydrogenase deficiency** - Inhereted - Is X-linked - Meditaranian, middle eastern, asian and African are more likely to be effected - Can confer resistance to malaria, malaria is common in african and medeterian regions. - A mutation can effect the enzyme - The mutation essect on the enzyme can be classified into 4 different sections: - Depend on the severity of anemia **Why does this cause aneami Biochemical basis of G6PD** - Uptake of glucose is needed for energy uptake and unptake of NAPDH needs - NADPH is important as heam in heamoglobin needs NADP it stops oxygen from taking up electrons ensuring there are no free radicals present. **Glutathiol is needed for this, NADPH keeps glutathyold in its functional state.** - **Increased breakdown of RBC Heamolyctin Anemia** **GPC6 Mostly affects RBC, why?** - Other cells have other mechanism for NADPH production - NADP+ is dependant malatate dehydrogenase (Malic Enzyme) - RBC lack mitochondria different way to generate NADPH so increased hemolysis **Consequences of G6PH deficiency** - Affects portiens, lipids - Proteins disulfide bonds are oxidised and the protein becomes denatured, denatured heamoglobin tend to precipitate causing Heinz Bodies - Heinz heam containing precipitates **AAA** - Contribute to oxidative steess - Beans are oxidants can trigure anemai Heinze - Infection need for NADPH in phagocytosis causes G6PD deficiency **Patients with sickle cell are resistant to malaria** **Patients with G6DP are resistant to malaria** - RBC dying so malaria parasites have no were to go **Summary** - **G6PD AAA exposure and consumption of beans AND INFECTIONS can trigure G6PD** - **Is self limited in most cases and early diagnostics** **Week 11** **Diabetes mellitus** **Pathophysiology Type 1 diabetes** **Pathophysiology** - **Genes, diet obesity = instant resistance** **Type 1 diabetes** - Auto immune attack - Beta cells are gradually destroyed - in young children the immune attack is more aggressive - attack on T cells happens in adults **differentiation between different diabetes** - **Endogenous** - Insulin therapy - Age of onset onset of type 2 in younger children is becoming younger - Progression faster in type 2 - Anthropometry (reflects the background population) favours obesity in type 2 - Measuring C peptide biproduct (is not in any pharmaceutical products) - C-peptide biomarker of endogenous insulin measure in urine of a patient thought to have type 1 diabetes - Can discriminate between type 1 where the c-peptide in indictable and other types of diabetes - C-peptide biproduct of post translation modification so it is purified out of the pharmacological drugs used in diabetes treatment. **Part 2** **Insulin secretion glucose levels in blood** - Glucose levels between lean and obese are similar but the insulin levels are higher in obese compared to lean this shows the insulin resistance in obese people. - Endogenous insulin is secreted as hexamers with a Zn ion in the middle - Monomer binds to the insulin receptor this gives the degsins for pharmaceutically degsined insulin - Insulin enters the hepatic portal vein and enters the liver - After entering the liver dilutions and clearance - Operating concentrations in insulin in obese people are less efficient. - To manage type 1 diabetes more insulin is needed when injected as it travels through different organs - B-chain sequencs subsitiutions - Suitable for meal time insulin dosages **Reproduction of basal insulin (NPH) long acting insulin** - Protamine and zinc - Protamine stabilises insulin crystals essential to mix- otherwise there will be a high dose to dose activity - Crystals are absorbs slowly prolong insulin absorption/a action **Fatty acid tail long acting insulin** - Injected as hexamers and absorbed rapidly under the skin - Hexamers are protected by degradation by albumin **Summary** - **In liver high concentration** - **Exogenous injected** **Part 3** **Diabetic ketoacidosis** - Insulin deficiency - Causes glucagon excess and cortisol and catecholamine excess - Leads to a decrease in glucose oxidation...... - Oxaloacetate is consumed in glucogenesis prevents acetyl co A build up causes acidosis (32 mins) **Treatment** - Correct dehydration - Switch off ketogenesis - Prevent life-threatening hypokemia Switch off ketone formation combination of insulin and fluids will drive the K down **Part IV** **Hypoglycaemia** - Rande of blood glucose is wider in diabetes as insulin is used to control glucose peaks **Whippl's triad** **42 mins** **Week 11 Lecture 2** **Type 2 diabetes** **Part 1-Abdominal adiposity and insulin resistance** - A greater amount of insulin is needed - Insulin sensitivity the less insulin sensitive they are - Subcutaneous AT - AT has a visceral layer intrabdominal compartment on the inside - Excess fat is stored in the skin, not abdomen **Visceral adiposity** - Fat distribution changes with male and female- this changes with age - After the menopause females start storing fat in the abdomen **Visceral vs subcutaneous adipose tissue** - fat is being stored in the subcutaneous tissue but with overeating- the will be no more storage of fat available in the fat stores in the skin this ability is maximised. This is the spithola hypothesis and fat is stored in other parts of the body, where fat will not normally get strred e.g. abdominal - cells in the abdomen -- not designed to store fat - this generates reactive oxygen species and hypoxia this causes an influx of macrophages in the abdomen which causes inflammation which can cause insulin resistance and problems with related signalling pathways. - This causa lo lead to oxidative stress no blood supply **Intra-abdominal and glucose metabolism** - Obese group insulin sensitive as impacted with fat in the abdomen - Prescence of obesity and type 2 diabetes are intertwined - As BMI goes up there is a greater risk of diabetes - Also linear relationship between wast size and development of type 2 diabetes as fat is stored in their abdomen **Abdominal obesity and increased risk of cardiovascular disease** - Depending on waste circumfrance, the greater your waist size- insulin resistance increases overall cardiovascular risk **Insulin resistance characterises type 2 diabetes** - Insulin goes up after a meal signal fat cells to not break down as there is no longer a need for fat to breakdown - This is achieved by switching off hormone sensitive lipase **Ketone pathway** - Verry little insulin is needed to prevent the fat breakdown - Insulin never goes down to 0 in healthy people as insulin prevents un necessary fat breakdown, otherwise there would be uncontrolled fat breakdown - Trace ketone natural bi-product of fat breakdown - In type 2 diabetes low amount of insulin to control fat breakdown still able to regulate fat breaks=down - Insulin works better on fat cells fatty acid begins to drop a little bit. Examples **of prandial insulin concentration in insulin resistance** - Insulin resistance will have to generate more insulin to overcome the glucose levels **Primary means by which insulin maintiands blood glucose levels** - Insulin converts glucose into glycogen, reducing the glucose concentration- reduces sugar in the blood - Insulin tells the liver to stop producing glucose how it regulates the blood glucose levels (primary mechanism) - Pancreas produces insulin which goes straight to the liver stops producing glucose **Insulin resistance and insulin secretion in type 2 diabetes** - 0- diagnosis of type 2 diabetes - Before that time- no high sugar but has insulin resistance - The body was able to overcome that insulin resistance by boosting the level of insulin produced -- this kept the sugar levels maintained - When the pancreas can not no longer compensate for insulin resistance- type 2 diabetes develop - Insulin resistance -- first then pancreas decreases in action - many people with type 2 diabetes have high insulin levels and high sugar levels **Genetics of insulin resistance** - genetic risk of type 2 diabetes - greater genetic rist for type 2 than 1 diabetes - type 1 few genes that lead to diagnosis - type2 over 400 genes identified - - insulin acts binds to receptor signalling GLUT4 tells glucose to go tho the cells pathways that lead to GLUT4 signalling in type 2 (left) - Insulin in liver gluconeogenesis, conversion of glucose into other storage forms (right) **Part 3** **Clinical presentation of type 2 diabetes** - Often asymptomatic - Same symptoms as type 1 diabetes - Increased risk of infection - No weight loss key thing that distinguished between type 1 and 2 -- type 1 losing weight, type 2 gaining weight. **What is meant by 'micro -or** - Affect small blood vessels back of eyes, kidneys or blood flow to the nerves - This may lead to complications with the nerves, eyes or kidneys - Other consequences of insulin resistance hight blood pressure, high cholesterol affect the microcirculation ( large blood vessesl) greater risk of stroke and heart attack **Association of glycaemia with macrovascular and microvascular complications of** - Diabetes increased rist of mycroardial infaction and diseases **Insulin resistance and cardiovascular disease** - Increase CD risk also influenced by other factors e.g. smoking, genetics Insulin resistance - Hight blood pressure, fat and inflammation **Multiple risk factor lowering** - Blood pressure, cholesterol and glucose only 50% are able to control them - Sometimes low blood glucose is caused when trying to treat diabetes- this causes a lower life expectancy if too low Treatment of type 2 diabetes - Insulin is used for type 1 not for type 2 as they are insulin resistance - Insulin prevents fat breakdown will lead to weight gain - Glucose lowering treatment for T2DM increased the release of insulin/ increased the efficiency of insulin **Incretin effect** - Different insulin concentrations when glucose is given oraly compared to intravenously - Glucose entering the gut caused a increase in insulin - GLP-1 -- peptide produced by linings in the gut, increases insulin release and slows how quickly food is released, improves insulin sensitivity, creates a 'full' fealing licenced as a weight loss drug - Incretin drugs lose 20% of body weight **Normal renal Glucose handling** - Glucose enters gulmorelus and is filtered out into the tubule but then immediately reabsorbed by a Na channel - Drugs target blocking this channel more glucose is in the urine then - Also reduces cardiovascular risk by around 40% - Increases the drive to use these drugs which improve insulin sensitivity and reduce Cardiovascular risk - When treating type 2 diabetes have to treate the cardiovascular risk use statin drugs to reduce this and drugs that reduce platlet agregation to reduce the overall CD risk