GLYCOGEN SYNTHESIS AND REGULATION


The body stores glucose as glycogen, lipids as fat (which can then be oxidised to produce energy but NOT be converted back into pyruvate – pyruvate into acetyl CoA is a completely irreversible reaction), and then amino acids which are not stored at all, but are in the blood in a small pool of amino acids which needs to be maintained as having all the 20aa that we need.

What happens to fructose and galactose?

Fructose is metabolised almost completely in the liver (unlike glucose all over the body), and it is directed towards replenishment of liver glycogen and triglyceride synthesis, some of it goes to skeletal muscle where it can be metabolised to carbon dioxide, and in fat cells where it is metabolised primarily to glycerol phosphate for triglyceride synthesis and energy production. About 10% of western diet calories come from fructose. The main thing here is that even when it is converted in the liver to glycogen, it has to first be converted into glucose, therefore it is really important to understand the fate of glucose. The same thing is true of galactose, it is converted into glucose-6-phosphate in the liver.

Therefore, when it comes to monosaccharides, glucose is the form of energy which is of the most importance to understand.

Glucose (relative to fructose and galactose) is quite a stable molecule. It is usually found in its ring structure, which makes not too reactive. However, it is still toxic and a small molecule, so it affects osmolarity: so we don’t want it in the blood – we keep it inside cells. Because it is small it affects osmolarity, so even if we trapped it within the cell by phosphorylating it, when it would increase in amounts after food consumption, it would keep drawing water in which can be catastrophic. So we need another form of energy storage for glucose from food: Glycogen

Outline the important features of the structure of glycogen Indicate the importance of liver glycogen in maintaining constant levels of glucose in the blood stream (glucose homeostasis) Indicate the importance of muscle glycogen as a rapid source of glucose for muscle contraction

Glycogen

can be a single granule (skeletal muscle) or clusters of granules (liver). In white-twitch muscles it is converted primarily into lactate (fast-twitch), and in red muscles it is completely oxidised (slow-twitch).

So glycogen is a glucose polymer which we produce in our livers. We do not consume it in western diets (only in oysters because by the time the meat comes to us after it’s been killed, all the glycogen has been depleted). Glycogen is therefore produced when we have too much glucose in our blood: the hormone responsible for this synthesis and storage is insulin. Glycogen is a polysaccharide. It has a low osmolarity. It is a medium-term fuel storage – all our liver glycogen storages would be depleted after 24h of fasting. Unlike starch, it is not linear but branched, with 1-6alpha glycoside links forming the linear connections, and the alpha1-6glycoside links forming the branches. Two different enzymes are responsible for the formation of these bonds: the 1-4glycoside by glycogen synthase, and the 1-6linkages by branching enzyme.

There are two types of glycogen: liver and muscle glycogen.

Liver glycogen is sensitive to blood glucose concentrations: the function of the liver is to ensure that the levels of glucose in the blood remain ideal for all tissues at all times, and it does this by continuously breaking down and synthesising glycogen. This is done under the control of insulin (storage and synthesis of glycogen) and glucagon (breaking down glycogen). Skeletal muscle has its own glycogen storage, in this way it can have its own personal reserves of energy in times of need for exercise, therefore it is sensitive to its own needs for energy. It can be affected by adrenaline, calcium, AMP (to break down glycogen) and ATP (to synthesise and store glycogen)

Describe the enzyme reactions for the breakdown of glycogen, emphasising the importance of glycogen phosphorylase Briefly indicate the control mechanisms for regulating the activity of these two key enzymes

How is glycogen formed? Glucose-6-phosphate is formed from glucose using hexokinase/glucokinase (the first step in any glucose-involving reaction), which is then transformed into glucose-1-phosphate by phosphoglucomutase. Then UDP glucose is formed: a transferase adds UTP to glucose-1-phosphate forming UDP glucose and PPi. This phosphorylated glucose is now capable of acting as a monomer for the formation of glycogen. A protein primer is first needed: glycogenin (with an OH group to which the glycogen synthase can then add UDP glucose monomers). This is then extended – these are all alpha 1,4 bonds, to form alpha 1,6 bonds, the branching enzyme breaks up the chain at every 10-12 glucose subunits, and then adds them to another glucose on the chain.

What is phosphorylation? Phosphorylation is the addition of a phosphate group to a protein. It can only be added to a serine or threonine (because those are the only ones with the OH side chains needed). (Tyrosine Kinases are a special group of protein kinases that phosphorylate tyrosine residues). This changes the proteins structural properties, its stability and dynamics: activates or inactivates it.

Remember: kinases require ATP (that’s where the phosphate group comes from) phosphatases do not require ATP (it is a passive process, they are considered housekeeping enzymes)

It was already mentioned that a glycogen synthase enzyme is needed for the formation of glycogen. Let’s talk about it. Glycogen synthase, like any other enzyme can be either active (a) or inactive (b). This regulation can either be allosteric (in which case it can be quick and non-permanent) or covalent: via phosphorylation (also slower). It is active when un-phosphorylated and inactive when phosphorylated. Since phosphorylation is done by a kinase enzyme, action of kinase therefore inactivates glycogen synthase. (ATP is needed for kinases) Action of a phosphatase (an enzyme which dephosphorylates) would therefore activate glycogen synthase. Insulin promotes glycogen synthase activation through dephosphorylation of the enzyme through activation of phosphatase. Where does insulin come from? Beta cells in the pancreas produce insulin once they get glucose in them: they have GLUT2 receptors, with a low affinity for glucose = if glucose manages to get in, its levels in the blood are too high.

On the other hand, we have glycogen breakdown. Why would we need to break down glycogen? When the levels of glucose in the blood are low, this is detected by the pancreas, and signals in the form of hormones – glucagon, are sent to the liver to break down those glycogen stores we formed earlier and bring back the glucose levels in the blood to the required amounts. How did the pancreas detect this low level of glucose? Well, it has GLUT2 receptors: which have low affinity for glucose, if glucose doesn’t manage to get in the cell, levels are probably low. (when it comes to muscle glycogen breakdown, this happens when stimulated by adrenaline) Firstly we can focus on the actual importance of the branched structure of glycogen: it allows for many glycogen breaking-down enzymes to attach. What are these enzymes called? Glycogen phosphorylase. Unlike glycogen synthase, glycogen phosphorylase is activated by phosphorylation.

How does a phosphorylase enzyme work to break down a molecule? It works like a hydrolase, just uses Pi instead of H2O.

So, when glucagon attaches to the receptor on the muscle and liver cell, it activates adenylate cyclase, which produces cAMP. The activated catalytic subunit of cAMP phosphorylates glycogen phosphorylase kinase, which then phosphorylates glycogen phosphorylase – activating it (and turning it into it’s a-form form b). A kinase also however phosphorylates glycogen synthase, which is inactivated by this process: so this results in glycogen being broken down.

Regulation

Insulin = synthesis High blood glucose – insulin release – activation of phosphatase – dephosphorylation of glycogen synthase (activates it) and of glycogen phosphorylase (inactivates it) – glucose is converted into glycogen Glucagon = breakdown Low blood glucose – glucagon release – activation of kinase – phosphorylations of glycogen synthase (inactivates it) and of glycogen phosphorylase (activates it) – glycogen is broken down into glucose

The liver can also respond to insulin or glucagon itself: when glucose is high, it can bind to glycogen phosphorylase (the enzyme which breaks off glucose from glycogen) and deactivate it – kind of like end-product inhibition, I think. The muscles have calcium calmodulin dependent kinases – which can activate glycogen phosphorylase (which breaks down glycogen into glucose). So if you have muscle contraction, you have calcium release, formation of the calcium-calmodulin complex, and activation of this kinase which will allow glycogen breakdown and sustain movement in muscle for longer times without the need of adrenaline-induced glycogen breakdown – you can therefore exercise without the need to be terrified.

ATP and AMP act as allosteric regulators too. AMP activates glycogen phosphorylase (increases glycogen breakdown). ATP is its allosteric inhibitor.

What is the difference between hexokinase and glucokinase, and what is the significance of this difference?

Hexokinase and glucokinase are both kinases: which means they phosphorylate by using ATP. This is the first step in any glucose-including reaction, as it is the addition of the first phosphate to glucose, converting it into glucose-6-phosphate. But we have different types of cells in the body, which all have needs for glucose to produce energy, but have different relative importance for survival. For example, we do not want to have muscle competing with the brain for glucose, or the liver competing with the brain. Therefore, there are two different types of kinases, each with different affinities for glucose, different Km values. Glucokinase has a high Km for glucose (it will be half saturated with glucose at a high glucose concentration = it has a low affinity for the molecule), this means that even if glucose gets in a tissue which has glucokinase as its metabolic enzyme, it will not metabolize glucose unless its concentrations are high: so less glucose will keep entering the cell, allowing more of it to remain in the circulation and be available for tissues that really need it and are important (such as the brain). Such a tissue is the liver – it contains glucokinase, letting glucose remain available for body tissues until its concentrations become high. Hexokinase has a low Km for glucose (low concentrations of glucose are required for it to be half saturated = it has a high affinity for glucose), this means that it will work at any glucose concentration. Such a tissue is muscle: it can readily use glucose. Here we start wondering how we ensure that muscle doesn’t compete with the brain for glucose: it doesn’t. We achieve this by controlling the glucose actually entering the muscle cell. By having insulin dependent GLUT4 transporters on its membrane, glucose won’t actually enter the muscle cell unless insulin is present, which would only happen if glucose levels are high enough (the brain is getting enough therefore). (GLUT 4 is also found in adipose tissue, striated: skeletal and cardiac tissue). GLUT 3 is the glucose transporter of the brain, which has a high affinity for glucose, allowing its transport even at low concentrations. (also the glucose transporter of placenta, another organ for which energy is really important) GLUT1 is the foetal tissue type, also expressed in endothelial cells of barrier tissues like the blood brain barrier. Responsible for the low level of basal glucose uptake required for respiration in all cells. Its presence is actually regulated by the levels of glucose available. Its expression is upregulated by many tumours. GLUT2 is a bidirectional glucose transporter in renal tubular cells, liver, pancreatic beta cells, basolateral small intestine membrane. (allows glucose, galactose and fructose to be transported) – high-frequency, low affinity Why is bidirectionality important? In liver cells: allows them to uptake glucose for glycolysis and glycogenesis, and release glucose for gluconeogenesis. In pancreatic beta cells: free flow of glucose so the intracellular environment can accurately gauge the serum glucose levels. If you have bidirectional flow with low affinity, then they can enter only in times of high concentration and leave only in times of low concentration – so they can allow these cells to “measure” the concentrations in the blood.

Enzymes and ATP There are two enzymes responsible for enzyme regulation: kinases and phosphatases. Kinases phosphorylate a protein (this either activates or deactivates it, depending on the protein). This process always requires ATP. Phosphatases dephosphorylate a protein (also either activates or deactivates the protein depending on its type). This is a passive process, doesn’t require ATP. Many enzymatic reactions run in many steps and in both directions. Regulation of these enzymes (especially the first and last enzyme) can drive the reaction in one direction or the other. Therefore, often the enzymes which work in either direction for the same step react in opposite ways to the same event: phosphorylation. If you activate a kinase which phosphorylates enzymes and it phosphorylates both the forward reaction enzyme and the backward reaction enzyme, and one of these proteins gets activated by this event and the other gets inactivated, you now have control of this pathway, and can drive it in the direction that you want. (To drive it in the opposite direction, you activate the phosphatase, which will remove the phosphate groups and therefore activate the other enzyme and deactivate the other).

EG. This is what happens in smooth muscle contraction. You have myosin light chain Kinase and myosin light chain Phosphatase. Rho kinase is a phosphorylator: it adds phosphate groups to myosin light chain phosphatase, which deactivates it. (another) protein kinase also phosphorylates myosin light chain kinase, which activates it and results in an increase in phosphorylation of the myosin light chain. (myosin light chain kinase is activated by a calcium-calmodulin-dependent protein kinase).

Liver vs muscle enzymes

The muscles do not have glucose-6-phosphatase – so they are unable to cause the conversion of glucose-6-phosphate into glucose to enter the bloodstream – their glucose is trapped inside their cells. This is why muscles cannot contribute to increasing the levels of blood glucose, and they keep their glucose for themselves.

#metabolism