Lecture 7: Gluconeogenesis, Metabolic Regulation, and Glycogen Metabolism (Biochemistry)

Gluconeogenesis

"New formation of sugar" - Occurs in all animals, plants, fungi and microorganisms The important precursors of glucose in animals are three-carbon compounds such as lactate, pyruvate, and glycerol as well as certain amino acids In mammals glucoenogensis mainly takes place in the liver and a to a lesser extent in the renal cortex and in the epithelial cells that line the inside of the small intestine Gluconeogenesis and glycolysis are NOT identical pathways running in opposite directions, although they do share several steps. - 7 steps are the reverse of glycolytic reactions - 3 reactions of glycolysis are essentially irreversible and cannot be used in gluconeogenesis (Step 1, 3 and 10)

Hypothetically, what would be the cost (in ATP) of transforming glucose to pyruvate via glycolysis and then immediately back again to glucose via gluconeogenesis?

-4 ATP Glycolysis = makes 2 net ATP Gluconeogenesis = requires 6 ATP

How can the activity of enzymes be regulated?

1. Change the total levels of the enzyme - Alter rate of transcription/translation of protein - Alter rate of mRNA/protein degradation - Sequester the enzyme within the cell 2. Directly Alter Enzyme Activity (more common) - Alter substrate concentrations - Alter the concentration of allosteric effectors - Covalent modifications of enzymes (add phosphate groups onto enzymes to change how they behave)

We can use other sugars than Glucose in Glycolysis:

1. Glycogen: Cellular stored glycogen - Extract off a glucose and use it in glycolysis 2. Galactose 3. Fructose

Cori Cycle

1. Glycolysis: Muscles ATP produced by glyoclysis for rapid contraction. Hard working muscles produces Lactate 2. Gluconeogensis: Transported through blood to liver where it is converted to glucose - ATP used in synthesis of glucose during recovery

Galactose Metabolism

1. Uses an ATP to add a phosphate group to carbon 1 of galactose - Enzyme: Galactokinase - Same as step 1 - USES an ATP 2. Enzyme converts Galactose 1 phosphate into glucose 1 phosphate 3. Phosphoglucomutase Enzyme can convert glucose 1 to glucose 6 Net 2 ATP

Starting from 2 molecules of Pyruvate how much ATP is needed to make a molecule of Glucose?

6 ATP Step 1: Pyruvate to PEP - Use 1 ATP to oxaloacetate - Use 1 GTP OOA to PEP Step 4: 3PG to 1,3 BPG - Use 1 ATP

How many ATP and how many NADH would be produced from the conversion of 3 molecules of fructose 6-P into pyruvate?

ATP = 9 (3 each molecule) (Invest 1 ATP, Produce 4 ATP (7x2) and (10x2)) NADH = 6 (2 each molecule) (Step 6x2 NADH)

When glycogen phosphorylase is phosphorylated its inactive or active?

Active because PKA phosphorylates things when glucagon is present, meaning blood sugar is low. Glycogen phosphorylase is active when phosphorylated. - PKA does not directly phosphorylate Glycogen phosphorylase though, it phosphoylates a kinase that can then phosphorylate glycogen phosphorylase - Phosphorylase Kinase: enzyme that adds phosphate to glycogen phosphorylase, PKA phosphorylates this kinase activating it.

Three Bypasses

All steps of gluconeogensis are just glycolysis in reverse except for Step (10) 1: Conversion of Pyruvate to Pep 1. Carboxylation: Bicarbonate + Pyruvate to Oxaloacetate (- 1 ATP) 2. Oxaloacetate to PEP (-1 GTP) Step (3) 8: Conversion of Fructose 1,6- bis phosphate to fructose 6-phosphate Step (1) 10: Conversion of Glucose 6-phosphate to Glucose

Biotin and Step 1 of Gluconeogenesis

Biotin is a CO2 carrier - Using biotin to add CO2 onto pyruvate Biotin is a swinging arm cofactor, can swing from one portion of active site and swing to another site, site 2. It picks up CO2 from one site and swings it over to Pyruvate at other active site. CO2 from bicarbonate to Pyruvate

Transcriptional Regulation of Glycolysis and Gluconeogenesis

Controlling through amount of enzyme present - Lots of enzyme promote pathway, little enzyme inhibit pathway. Insulin can also promote the transcription of hexokinase and decreases transcription of G6Pase and PEPCK Insulin is high, high blood glucose, glycolysis promoted. - Activate hexokinase, activate glycolysis.

Additional Regulation of Phosphofructokinase (PFK-1) in the Liver

Enzyme (Step 3 glycolysis): Phosphorylates F-6P phosphate group is transferred from ATP to fructose-6-phosphate, producing fructose-1,6-bisphosphate. - First Committed Step of Glycolysis - Where most regulation happens AMP sign of low energy. ATP rises sign of high energy Liver: Reciprocal Regulation Because the liver plays such a critical role in maintaining constant blood glucose it requires additional glycolytic and gluconeogenic regulatory mechanisms. Fructose 2,6 Bisphosphate (F26BP): Potent allosteric activator of PFK-1, while inhibiting fructose 1,6 BP - Fructose 1,6 BP enzyme of step 7 of gng. - Promotes Glycolysis - Is made by an enzyme called PFK-2. PFK-2 is high then we make F26BP, promote glycolysis PFK-2 production: (high=F26BP=glycolysis) (low=gng) If blood sugar is high, insulin is in blood - Insulin promotes a phosphatase, PP1 (removes phosphate) - Insulin activates PFK-2 by dephosphorylation If blood sugar low, Glucagon is in blood. - Glucagon activates Protein Kinase A: deactivates PFK-2 by phosphorylating it to FBPase 2. Phosphorylated PFK-2 acts as FBPase 2: bifunctional - Enzyme: FBPase-2 converts fructose 26BP back to fructose 6 phosphate - Phosphorylated: FBPase-2 activity on (low blood glucose, promotes gng) Energy Regulation in Liver: PFK-1 activated by signs of Low Energy (AMP, ADP) - AMP allosterically activates PFK-1, also simultaneously binds to fuctose 1,6 Bisphosphate and inhibits it. - F16BP enzyme for step 7 in gng. - Energy in liver very low (rare) we want to stop gng and promote glycolysis.

Phosphofructokinase (PFK-1) in Muscle

Enzyme (Step 3 glycolysis): Phosphorylates F-6P phosphate group is transferred from ATP to fructose-6-phosphate, producing fructose-1,6-bisphosphate. - First Committed Step of Glycolysis - Where most regulation happens AMP sign of low energy. ATP rises sign of high energy Muscle (mostly, little bit liver): PFK-1 Inhibited by signs of High Energy (ATP, Citrate) - ATP acts as allosteric inhibitor of PFK-1 enzyme. - Negative Feedback: High ATP signals to cell that there is sufficient energy and glycolysis is not needed. - ATP is both a substrate for reaction, but at high levels becomes allosteric inhibitor - Citrate is an Indicator of high energy, also binds to PFK-1 and allosterically inhibits it PFK-1 activated by signs of Low Energy (AMP, ADP) - AMP, ADP is a positive allosteric regulator - High AMP, ADP signals to the cell that more energy is needed and glycolysis must be engaged. Only in Muscle: ATP also indirectly leads to hexokinase inhibition - Backup pathway - ATP rises very highly, PFK-1 is inhibited, so Fuctose 6-Phosphate (reactant) accumulates. Causing high levels of glucose 6-Phosphate (product of step 1). - High levels of product cause G to be very high inhibiting step 1 Low pH also inhibits PFK-1 in muscle only - Lactic acid accumulation, pH too low. Important for glycolysis to let up a bit to clear out lactic acid to blood to go to liver to do gng

Phosphorylisis (Glycogen Metabolism)

Enzyme: Glycogen Phosphorylase - Helps to catalyze a free phosphate attacking and breaking bond - Phosphorylisis: breaking bond with phosphate attack When you free a glucose from glycogen you use a free inorganic phosphate to break the bond.

Regulation of Glycogen Breakdown

Enzyme: Glycogen phosphorylase - Breaks glycogen down to glucose 1 phosphate - glucose 6 phosphate Liver: break down to produce glucose for body - Enzyme active when blood glucose levels are low - Break down to glucose-6 phosphate in liver to use in gng to make free glucose to release into blood Glycogen phosphorylase is active when phosphorylated. - PKA does not directly phosphorylate Glycogen phosphorylase though, it phosphoylates a kinase that can then phosphorylate glycogen phosphorylase - Phosphorylase Kinase: enzyme that adds phosphate to glycogen phosphorylase, PKA phosphorylates this kinase activating it. Muscle: break down glycogen to make energy Hormone involved in breaking down glycogen in muscle, Adrenaline (need muscle to produce ATP quickly) - Adrenaline activates glucose phosphorylase also activates protein kinase A in muscle (PKA). - To do glycolysis to make energy Insulin: makes adrenaline less effective, because insulin dephosphorylates the glycogen phosphorylase of muscle, making it inactive. - Dephosphorylates because of PP1 High Levels of glucose in blood, glucose itself can bind to glycogen phosphorylase aiding in dephosphorylation - Glucose very high we want glycogen phosphorylase to be inactive (dephosphorylated) - by assisting it to reveal the phosphate group making it easier to cleave off.

Pyruvate Kinase Regulation

Enzyme: Step 10 of Glycolysis converts PEP to Pyruvate, donates phosphate to ADP producing ATP Muscles: Energy Regulation High energy levels inhibit Pyruvate Kinase - ATP, Acetyl-CoA, long-chain fatty acids, and alanine all allosterically inhibits Pyruvate Kinase - All are signs of high energy. Feed Forward Activation: - Fructose 1,6 Bisphosphate (product of step 3 glycolysis) acts as allosteric activator of pyruvate kinase - When accumulates binds to Pyruvate Kinase activating it to promote glycolysis. - Step 3 is committed step so if we've gone through it we need to promote later steps. - Calling ahead to step 10 Liver: Blood Glucose Regulation Pyruvate Kinase can be phosphorylated (only in the liver) that inhibiting it. - Glucagon promotes PKA which promotes phosphorylation when blood glucose is low (promoting gng) Pyruvate Kinase can be dephosphorylated and activated - Insulin promotes PP1 which de-phosphorylates when blood glucose is high. (promoting glycolysis)

Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction: Would shortening glycolysis in this way be beneficial to the cell? Why or why not?

GAP directly to 3PG Would not be more efficient to the cell because we would not create 1,3BPG so we would not be able to create net ATP.

2 Paths for Gluconeogenesis

Gluconeogenesis can happen in the mitochondria or in the cytoplasm 2 pathways depend on the availability of lactate or pyruvate and they cystolic requirements of NADH for gluconeogenesis. Pyruvate Comes from Two Main sources: 1. Lactate (Cori Cycle, lactate from muscles) - oxidizing lactate back to pyruvate in cytoplasm produces NADH, which allows gluconeogenesis to occur - pyruvate enters mitochondria to oxaloacetate to PEP in mitochondria 2. Pyruvate (When we're starving we turn our protein into sugar in the liver, beginning from alanine) - No NADH is made (Ala and pyruvate are same oxidation state), not enough NADH in cytoplasm to run step 6 in reverse - Must move oxaloacetate out of mitochondria, but the mitochondrial membrane has no transporter for OAA Malate: OAA must be reduced to malate by mitochondrial malate dehydrogenase (using NADH producing NAD+) - Malate leaves the mitochondria through a specific transporter in the inner membrane - Reoxidize Malate back to OAA, which makes us use an NAD+ creating NADH in the cytoplasm - Final step to make PEP in cytoplasm Mitochondrial pool of NADH is not in equilibrium with the cytoplasm pool of NADH - NADH is not limiting in mitochondria

Can you generate free energy by doing glycolysis followed by gluconeogenesis?

Glycolysis made 2 net ATP Gluconeogensis requires 6 ATP No, you cannot generate free energy by doing glycolysis followed by gluconeogenesis, because it costs energy in the long run. - So it is very important that a cell regulate these two pathways Reciprocal Regulation: Hormone regulation of pathways. - While glycolysis is happening you do not want to do gluconeogenesis and vice versa. - Insulin promotes glycolysis and inhibits gluconeogenesis Futile Cycling: cycling two pathways just ends in losing ATP and get nothing out of it.

Regulation in 2 Tissue Types

Glycolysis regulated differently in these two tissues: 1. Muscle: Use the energy from glucose to contract and produce motion - Energy Charge Based Regulation (tiny bit liver) - No Gng in muscles really 2. Liver: Maintain blood glucose levels at proper concentration and produce glucose for the rest of the body when needed. Soak up extra glucose to convert to glycogen. - Blood Glucose Regulation (liver specific) Energy Charge Based Regulation: High ATP, glycolysis off Low ATP, glycolysis on Blood Glucose Regulation (Liver): High Blood Glucose, Gng Off, Glycolysis on - (liver needs to use glucose and soak it up to convert to glycogen and fat) Low Blood Glucose, Gng On, Glycolysis off

The liver the most unselfish organ

Helps us regulate blood glucose levels. When blood sugar gets too low The liver is going to make new sugar through gluconeogenesis to fuel our brain and muscles When blood sugar gets too high The liver is going to soak up blood glucose and bring it inside of liver Brain and muscles are very selfish organs

Hexokinase Regulation

Hexokinase: Enzyme (Step 1 Glycolysis, Step 10 gng) - phosphorylation of glucose to form glucose-6-phosphate. Two types of Hexokinase (Isozymes) Catalyze same chemistry, but have different properties 1. Hexokinase I: Predominates in skeletal muscles - lower Km (higher affinity for glucose) - Inhibited by the reaction product (glucose 6 phosphate) while liver glucokinase is not. 2. Hexokinase IV (glucokinase): predominates in liver - Higher Km (lower affinity for glucose) - Cooperative: Sigmoidal showing that the substrate (glucose) can activate the enzyme. (more glucose/ more active) Why would muscle have a higher Km than liver? It's important that muscle have a higher affinity for glucose that it can get first call. If only a little available the muscle needs it to make energy. Energy requirements of liver are so low. Why is it important that hexokinase IV (glucokinase) is highly active at high blood sugar concentrations ? Glucokinase is cooperative so more glucose more active. When blood glucose is very high, we need the liver glycolysis to take place to soak up glucose in blood Why is it that Liver hexokinase IV is not regulated by its product (G-6P) while muscle hexokinase I is? ***

Regulation of Gluconeogenesis

High levels of AcetylCoA are a sign of high energy and means you have lots of pyruvate you can use to make new glucose. AcetylCoA is an allosteric activator of pyruvate carboxylate. (First step of gluconeogensis) *talk more about this later with Acetyl CoA*

Insulin and Glucagon

Hormones produced by the pancreas that help to regulate blood glucose levels. Insulin and Glucagon can bind to cell surface receptors and activate a signal transduction pathway that leads to the activation of various proteins inside of our cells. Insulin: Released in response to an increase in blood sugar. Insulin activates a very important phosphatase: PP1 - Dephosphorylates: phosphatase takes a phosphate off of things. - PP1 has many many substrates. Glucagon: Hormone released in response to low blood sugar. Activates a very common enzyme Protein Kinase A: PKA - phosphorylates lots and lots of things (hundreds of substrates, many of which involved in glycolysis + gng)

High levels of Galactose Can be Toxic

If the conversion of galactose to glucose 1P is impaired, excess galactose can be converted to the toxic byproduct called galactitol.

During strenuous activity, the demand for ATP in muscle tissue is vastly increased. In rabbit leg muscle or turkey flight muscle, ATP is produced almost exclusively anaerobically (without oxygen). Suppose skeletal muscle were devoid of lactate dehydrogenase, could it carry out strenuous physical activity? (i.e could it generate ATP at a high rate by glycolysis?) Explain

If you continue to do glycolysis, without being able to further oxidize pyruvate, we must keep running glycolysis and we cannot do that without lactate dehydrogenase because we would run out of NAD+, which would limit glycolysis.

Gluconeogensis takes place in

In the Liver Takes place in the mitochondria, cytoplasm and ER.

Lactose Metabolism

Is made up of Glucose + Galactose Lactose is metabolized in the intestine by a secreted enzyme called lactase. - Only glucose and galactose can be taken up by intestine Hypolactasia: Lactose intolerance occurs because most adults lack lactase, the enzyme that degrades lactose. - Northern Europeans have a mutation that prevents the decline of lactase activity after weaning In lactase-deficient individuals gut bacteria metabolize lactose instead of being absorbed, generating CH4 (gas) and H2 and disrupt water balance in the intestine

In step 6 of glycolysis, a phosphate is added on to GAP to form 1,3 BPG. Does this phosphate come from ATP? What is the relevance of the source of this phosphate to the overall net ATP production from glycolysis

No, it is a free inorganic phosphate. Allows us to make net ATP in glycolysis

The conversion of phosphoenol pyruvate (PEP) to pyruvate is a highly thermodynamically favorable step in glycolysis. Why is this? How is it that the conversion of pyruvate back to PEP (in gluconeogenesis) is also able to be highly thermodynamically favorable as well?

PEP is very high energy and Pyruvate is very low energy due to tautomerization Coupled with ADP to ATP producing ATP Pyruvate back to PEP is able to be highly thermodynamically favorable because coupled with ATP to ADP

Step 2 of glycolysis is catalyzed the enzyme Phosphohexose Isomerase. Is this step of glycolysis reversible or irreversible? What is the substrate and product for this reaction and what is the purpose of this step in glycolysis?

Reversible Substrate: Product: Purpose: To create a more symmetrical molecule, fructose, to break into a 3 carbon sugar

Step 10 Gluconeogenesis: Conversion of Glucose 6-phosphate to Glucose

Step 1 of Glycolysis Intermediates: Glucose 6-phosphate + H2O to Glucose + Pi Enzyme: Glucose 6-phosphatase - Take off the phosphate Happens in the ER of our liver cells. When we reverse this step, although very energetically favorable, no ATP is produced

Step 1 Gluconeogenesis: Conversion of Pyruvate to PEP

Step 10 in Glycolysis. 1. Carboxylation: Bicarbonate + Pyruvate to Oxaloacetate - Enzyme: Pyruvate Carboxylase (PC) - Biotin co-factor involved in carrying CO2 (B7) - Add CO2 to Pyruvate, - Happens in mitochondria - Requires 1 ATP (x2) 2. Oxaloacetate to PEP - Enzyme: PEP carboxykinase - Requires 1 GTP (x2) - Happens in mitochondria or the cytoplasm - Acquire phosphate on 2nd carbon. PEP and Pyruvate can move in and out of the mitochondria - Pyruvate entered mitochondria to start gluconeogenesis. - PEP once made will leave the mitochondria PEP is very high energy and Pyruvate is very stable so we have to use multiple ATP to do it.

Given liver metabolism of fructose, why might excess fructose consumption be problematic?

Step 3 of Glycolysis is the committed step and highly thermodynamically favorable and highly regulated. - Fructose 6 phosphate and glucose 6 phosphate can move to other pathways but once you make fructose 1,6 Bisphosphate you are committed. Step 3 is skipped, and this is the step with the most regulation. - Example if the body has too much energy it doesn't want to go through glycolysis. ATP buildup prevents glycolysis Fructose will move through glycolysis even if cell has enough energy. - With high levels of glucose your body has a way to shut off glycolysis by regulating Step 3 (and 1 but mostly 3) - With fructose you bypass regulation, so it will always go through.

Step 8 Gluconeogenesis : Conversion of Fructose 1,6- bis phosphate to fructose 6-phosphate

Step 3 of glycolysis Fructose 1,6 bisphosphonate + H20 = fructose 6 phosphate + Pi Enzyme: Fructose 1,6 Bisphosphatase - catalyzes the irreversible hydrolysis of the ester bond at carbon 1 - The enzyme is a critical step of regulation Since Step 3 and glycolysis consumed an ATP, do we get an ATP back from Step 8 of gluconeogenesis? No - It is a very favorable step, but not favorable enough to make ATP

Step 3 of gluconeogenesis

Step 7 of glycolysis produced ATP so you need ATP to reverse it

Glycogen Metabolism

Storage and mobilization of stored cellular glucose by extracting a glucose off glycogen and using it for glycolysis. 1. Break Glycosidic Linkage to extract out glucose molecule. - Enzyme 1: Glycogen Phosphorylase - Helps to catalyze a free phosphate attacking and breaking bond. - Phosphorylisis: breaking bond with phosphate attack. When you free a glucose from glycogen you use a free inorganic phosphate to break the bond. 2. Glucose 1-phosphate: phosphorylated glucose, must be converted to glucose 6 phosphate to go through Glycolysis - What mechanism does it remind you of? Step 8 of glycolysis - Phosphoglucomutase: Move phosphate from 1 position of glucose to 6. - Similarly phosphate does not move, new one comes in and old one leaves.

Metabolic Regulation

The cell wants to avoid futile cycling of anabolic and catabolic pathways. The cell can control flux through a metabolic pathway by controlling the activity of specific enzymes within that pathway.

Fructose Metabolism

The vast majority of fructose is metabolized in the liver (99%) - Fructokinase: Enzyme is almost entirely liver specific 1. Phosphorylation of Fructose - Traps fructose inside of cell, makes it higher energy. - Fructose 1 phosphate - Uses 1 ATP 2. Cut fructose in half with phosphate at carbon 1 - Enzyme: Fructose 1 phosphate aldolase - Glyceraldehyde + Dihydroxyacetone phosphate - Glyceraldehyde to glyceraldehyde 3 phosphate (Uses 1 ATP) 3. Now we Have GAP and DHP and can proceed with glycolysis Net ATP: 2 (uses 2 ATP in process) Do not need to know name of enzymes here

Glycogen Synthase

Unlike glycogen phosphorylase, glycogen synthase is inactive when phosphorylated active when dephosphorylated Insulin results in dephosphorylation (PP1) - high insulin, high glucose, make glycogen Ephinephrin/glucagon results in its phosphorylation (PKA) - low glucose, use glycogen

Metabolic Regulation of Glycolysis and Gluconeogenesis

Within a metabolic pathway most reactions operate near equilibrium - Key enzymes operate far from equilibrium. These are sites of regulation that control flow through the pathway Glycolysis: 1. Hexokinase (Step 1) 2. Phosphofructokinase-1: PFK-1 (Step 3) Most Important 3. Pyruvate Kinase (Step 10) Gluconeogenesis: 1. Pyruvate Carboxylase + PEPck (Step 1) 2. Fructose 1,6 bisphosphatase (Step 8) 3. Glucose 6-phosphatase (Step 10)

When you start from glycolysis with glycogen 3 ATP Net are made. Why?

You don't have to use an ATP to phosphorylate your glucose. You are starting with already phosphorylated glucose When you're in need of energy it is better to use glucose from your own glycogen.

Malate

a reduced form of oxaloacetate

10. How many molecules of ATP are produced in the following: a. The conversion of 1 molecule of galactose into pyruvate: b. The conversion of 1 molecule of fructose into pyruvate: c. The conversion of 1 molecule of glucose (released from glycogen) into pyruvate:

a. The conversion of 1 molecule of galactose into pyruvate: 3 b. The conversion of 1 molecule of fructose into pyruvate: 2 c. The conversion of 1 molecule of glucose (released from glycogen) into pyruvate: 2 Conversion of fructose to pyruvate is same net ATP as glucose, but importantly fructose bypasses step 3, so not a lot of regulation there and the cell may overconsume fructose

Where does glycolysis occur?

cytoplasm