Gluconeogenesis PDF

Tymoczko Berg Gatto Stryer Biochemistry: A Short Course Fourth Edition CHAPTER 17 Gluconeogenesis © 2019 Macmillan Learning Gluconeogenesis Glucose is a primary fuel not only for humans but for almost all organisms including animals, plants, fungi, and microorganisms. It is therefore not surprising...

Tymoczko Berg Gatto Stryer Biochemistry: A Short Course Fourth Edition CHAPTER 17 Gluconeogenesis © 2019 Macmillan Learning Gluconeogenesis Glucose is a primary fuel not only for humans but for almost all organisms including animals, plants, fungi, and microorganisms. It is therefore not surprising that organisms have a process whereby glucose can be synthesized from non-carbohydrate precursors. This process is called gluconeogenesis. Learning Objectives When we have finished this section, you should be able to do the following: 1. Recognize the biological function of gluconeogenesis. 2. Describe the reactions, enzymes and intermediates of gluconeogenesis. 3. Recall the mechanisms of regulation of gluconeogenesis. 4. Explain the functional relationship between glycolysis and gluconeogenesis as well as their regulatory relationship. 5. Characterize the mechanism of contribution of the Cori Cycle to Gluconeogenesis In this section, we continue our consideration of glucose metabolism with consideration of gluconeogenesis, the production of glucose from noncarbohydrate precursors. These precursors include: Lactate (lactic acid) amino acids glycerol. While glycolysis and gluconeogenesis share many reactions that lie near equilibrium, the two processes are not mere reversals of each other. Specifically, there are three reactions of glycolysis that are irreversible that must be bypassed for gluconeogenesis. Gluconeogenesis These three reactions of gluconeogenesis will be considered in detail and involve: 1) The synthesis of phosphoenolpyruvate from pyruvate by a two-step reaction with oxaloacetate as an intermediate. 2) The synthesis of fructose-6-phosphate from fructose-1,6-bisphosphate, and finally 3) The production of the final product, glucose, from glucose-6-phosphate. Given the close relationship between glycolysis and gluconeogenesis, emphasis will be placed on the reciprocal regulation of these pathways Biological Function of Gluconeogenesis In animals, the primary function of gluconeogenesis is to assist in maintaining adequate glucose levels in the blood. There are organs and cells in our bodies that depend on glucose as their primary fuel. Red blood cells, for example, use glucose as their sole energy source. The biggest consumer of glucose is the brain, about 120 g per day on average. While glucose is the primary fuel for the brain, this organ can also use ketone bodies when glucose is in short supply (to be covered later). Muscle also uses a significant amount of glucose for its energy needs although it also relies heavily on fatty acids. For these reasons, gluconeogenesis is particularly important during periods of fasting (e.g., when you are sleeping) or starvation. Biological Function of Gluconeogenesis It is important to note that in animals, gluconeogenesis occurs only in liver and kidney, with liver being the largest contributor. Maintaining blood glucose levels is a major function of the liver. As will be shown, when the liver or kidney produces glucose, this molecule passes across the cell membrane into the blood so that other tissues such as brain and muscle can extract it in order to meet their metabolic demands. In plants, most of the glucose produced is used to synthesize starch, cellulose and sucrose. Glycolysis Irreversible Reactions Gluconeogenesis The pyruvate is formed from non-carbohydrate precursors such as lactate and some amino acids, while other precursors such as glycerol and certain amino acids enter the pathway at later stages. The yellow boxes in the figure show where these precursors enter the pathway. Gluconeogenic precursors Lactate: In glycolysis, one of the fates of pyruvate is that it can be reduced to lactate. This reaction, catalyzed by lactate dehydrogenase, is a reversible reaction. Most of the lactate formed in our bodies is produced in active muscle during anaerobic metabolism, which is released into the blood and eventually taken up by liver. There, it is converted back to pyruvate by lactate dehydrogenase and thereby enters the gluconeogenesis pathway. Amino acids: Some amino acids, that are derived from the diet or from protein degradation, can be metabolized to intermediates in the gluconeogenic pathway as shown in previous figure. This will be discussed in more detail in a later section. Glycerol: Triacylglycerols, the form of fat that we store in our bodies, can be broken down into fatty acids and glycerol. While fatty acids are used by various organs in our body for fuel, the glycerol is released from the adipose tissue into the blood and is taken up by the liver. There, glycerol is converted to dihydroxyacetone phosphate in a two-step process: Gluconeogenesis Overview The following video highlights the unique reactions of gluconeogenesis: https://www.youtube.com/watch?v=5oDjsuH-Rms&t=168s As we watch recall the forces of free energy and the requirement for a net negative ΔG for reactions to proceed. Conversion of Pyruvate to Phosphoenolpyruvate Conversion of Pyruvate to Phosphoenolpyruvate Begins with the Formation of Oxaloacetate. In glycolysis, the conversion of phosphoenolpyruvate to pyruvate is catalyzed by pyruvate kinase and generates ATP as a second product. It is a strongly exergonic reaction, and thus not reversible. For gluconeogenesis to proceed, another mechanism involving different enzyme(s) has to be found in order to convert pyruvate to phosphoenolpyruvate. The process starts in the mitochondria with the carboxylation of pyruvate to form a four-carbon molecule called oxaloacetate. Note that the reaction involves the hydrolysis of ATP which provides the energy to drive this reaction forward. Conversion of Pyruvate to Phosphoenolpyruvate Figure: The conversion of pyruvate to oxaloacetate involves a carboxylation reaction and the breakdown of ATP. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, p. 316. Permission: Courtesy of MacMillan Learning. Pyruvate carboxylase requires a covalently bound prosthetic group called biotin (derivative of vitamin B7) that carries the CO2 in a manner that facilitates its reactivity with pyruvate. Pyruvate carboxylase also requires that acetyl CoA is bound to the enzyme in order for it to catalyze carboxylation. In the absence of bound acetyl CoA, carboxylation does not occur. Because of this, acetyl CoA is referred to as an obligate allosteric activator. Borrowed from: https://en.wikipedia.org/wiki/Citric_acid_cycle#/media/File:Citric_acid_cycle_with_aconitate_2.svg Oxaloacetate that is formed now has to be transported out of the mitochondria and into the cytosol. There’s one small problem - there is no transporter for oxaloacetate so it is stuck in the mitochondria. There is a way around that problem as shown in the figure. Oxaloacetate is converted to malate (by malate dehydrogenase), which is able to leave the mitochondria. Figure : How oxaloacetate gets out of the mitochondria. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, Fig.17.4, p. 318. Permission: Courtesy of MacMillan Learning. Once in the cytosol, malate is converted back to oxaloacetate by the same enzyme. From Oxaloacetate to Phosphenolpyruvate Once the oxaloacetate is in the cytosol, the final step in the conversion of pyruvate to phosphoenolpyruvate can occur since the enzyme that catalyzes this, phosphoenolpyruvate carboxykinase, is found in this cellular compartment. Figure: Formation of phosphoenolpyruvate from oxaloacetate. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, p. 318. Permission: Courtesy of MacMillan Learning. Once phosphoenolpyruvate is formed, it is metabolized by the enzymes of glycolysis to fructose 1,6-bisP. This is possible because the reactions are all close to equilibrium at intracellular conditions, and therefore are reversible. Conversion of Fructose 1,6-Bisphosphate to Fructose 6-P In glycolysis, the conversion of fructose 6-P to fructose 1,6-bisP involves ATP cleavage and is an irreversible reaction. So the reverse conversion required for gluconeogenesis clearly needs a different mechanism and enzyme in order to proceed. The enzyme used is fructose 1,6-bisphosphatase, a hydrolase which cleaves off the phosphate group using water from carbon 1 on fructose. Figure: Fructose 1,6-bisP is converted to fructose 6-P by a hydrolysis reaction. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, p. 318. Permission: Courtesy of MacMillan Learning. Final Step: The Generation of Free Glucose The fructose 6-P formed in the reaction above is readily converted to glucose 6-P since the reaction catalyzed by phosphoglucose isomerase, which we encountered in glycolysis, is reversible. Free glucose is then produced by the hydrolytic removal of the phosphate group from glucose 6-P by glucose 6-phosphatase, which resides in the lumen (i.e., inside) of the endoplasmic reticulum, thus requiring that the glucose 6-P produced in the cytosol be shuttled into the ER. The products of the reaction, Pi and glucose, are subsequently transported back out to the cytosol which allows glucose to be transported out of the cell into the blood. Figure: The hydrolysis of glucose-6-P to form free glucose occurs in the lumen of the ER. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, Fig.17.5, p. 319.Permission: Courtesy of MacMillan Learning. Generation of Glucose from Glucose 6-phosphate in lumen of the endoplasmic reticulum Notably - Glucose 6-phosphatase (G6Pase) is only present in liver and kidney Six High-Transfer-Potential Phosphoryl Groups are Spent in Synthesizing Glucose from Pyruvate The synthesis of glucose from pyruvate is energetically unfavourable except for the fact that it is coupled with reactions that release energy, such as the hydrolysis of ATP and GTP. The overall stoichiometry of gluconeogenesis is: Figure: Overall stoichiometry of gluconeogenesis where two pyruvate are converted to one glucose. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, p. 319. Permission: Courtesy of MacMillan Learning. Note the large negative free energy change in the gluconeogenesis pathway (-38 kj/mol). In contrast, the simple reversal of glycolysis has an overall free energy change of +22 kcal/mol, which is not possible. Thus, gluconeogenesis is a good example of a metabolic process that is powered forward by the coupling of reactions which makes this process energetically favorable. Gluconeogenesis and Glycolysis Are Reciprocally Regulated Because these two pathways essentially go in opposite directions i.e., glucose to pyruvate vs. pyruvate to glucose, it would not make sense that these pathways would be highly active at the same time. Instead, it would make lots of sense to control these pathways in a coordinated and reciprocal way, so that while one is activated, the other is inhibited. This is in fact what happens. A fundamental principle is that when glucose is abundant, glycolysis is favoured, while when glucose is scarce, gluconeogenesis is prominent. How this occurs is through the control of key regulatory enzymes in each pathway. The Energy Charge in the Cell Determines Which Pathway Is More Active The major point of regulation of gluconeogenesis is the step where fructose 1,6-bisP is converted to fructose 6-P catalyzed by fructose 1,6bisphosphatase. Interestingly but not surprisingly, the major point of regulation of glycolysis is the conversion of fructose 6-P to fructose 1,6-bisP catalyzed by phosphofructokinase. Figure: The two key interconversions that are reciprocally regulated by allosteric modifiers. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, Fig.17.6, p. 321. Permission: Courtesy of MacMillan Learning Both of these enzymes are allosterically regulated in liver by biomolecules that reflect the energy charge in the cell Note the reciprocal effects of fructose-2,6-bisP, AMP, and citrate on the two key enzymes. AMP, which reflects a low energy charge, activates phosphofructokinase and thus stimulates glycolysis and ATP production, while at the same time it inhibits fructose 1,6bisphosphatase, thereby slowing down gluconeogenesis. Thus, under a low energy state of the cell, the net flux through this step would highly favour glycolysis. Fructose 2,6-bisP has a similar effect, and interestingly the concentration of this biomolecule increases in the fed state, when one would want to metabolize the glucose that is available. Citrate has the opposite effect. Citrate is an intermediate of the citric acid cycle which is the major pathway for oxidizing fuels when oxygen is available. Thus, citrate reflects a high-energy state in the cell. Not surprisingly, citrate inhibits phosphofructokinase but stimulates fructose 1,6-bisphosphatase, and thus signals for a net flux in the direction of gluconeogenesis. The interconversion of pyruvate and phosphoenolpyruvate is another point of reciprocal regulation. Pyruvate kinase is inhibited by biomolecules that reflect a high-energy charge, such as ATP and alanine. Conversely, high levels of ADP which occur in a low energy state of the cell, inhibit the conversion of pyruvate to phosphoenolpyruvate, which leads to a net flux favouring glycolysis and ATP production. Important Generalization! An important general point about allosteric regulators is worth making here. The effect of an allosteric regulator, whether an activator or an inhibitor, is highly dependent on its concentration. The higher its concentration, the greater its effect, and vice versa. The effect is rarely all or nothing (there are rare exceptions), but rather acts like a dimmer switch, that gradually changes the amount of light coming from a fixture and not like an on/off switch. This allows for a very fine-tuned control of enzyme activity to match the needs of the cell. Balance Between Glycolysis and Gluconeogenesis in Liver Is Sensitive to the Blood Glucose Concentration Rates of Glycolysis and Gluconeogenesis are adjusted to maintain blood sugar levels. How does blood sugar levels influence the relative activities of these two pathways? It turns out – through production and degradation of fructose 2,6-bisP fructose 2,6-bisP activates phosphofructokinase, thus activating glycolysis fructose 2,6-bisP inhibits fructose 1,6-bisphosphatase – thus slowing gluconeogenesis OK. … so how do blood sugar levels affect production of fructose 2,6-bisP? Making and Breaking Fructose 2,6-bisphosphate Phosphofructokinase-2 is encoded by a different family of genes from PFK-1 (used in glycolysis). It’s only function is production of the allosteric regulator fructose 2,6P, Figure: Fructose 2,6-bisP is synthesized from fructose 6-P by phosphofructokinase-2, and degraded by fructose 2,6-bisphosphatase-2. Source: Nelson, D. &. Cox, M. (2008). Lehninger Principles of Biochemistry (5th ed.). New York, NY: W. H. Freeman and Company, Fig.15.17a. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Interesting Protein(s)! The relative activities of these two enzymes determine the concentration of fructose 2,6-bisP in the cell and thus the activities of glycolysis and gluconeogenesis. As scientists began researching these two enzymes in order to understand how they were regulated, they found something very surprising: both PFK-2 and FBPase-2 were present in the same protein! Two enzymes in one! Upon determining the structure of this bifunctional enzyme, it became evident that it has two distinct domains or parts to it: one that has the PFK-2 kinase activity, the other which has the phosphatase domain. Figure : The bifunctional domain structure of PFK-2/F 2,6-bisphosphatase-2. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, Fig.17.7, p. 322. Permission: Courtesy of MacMillan Learning. Wait, But How Does Blood Sugar Levels Regulate These Two Enzyme Activities? Further research found that there was a single serine residue in the regulatory domain that became phosphorylated when glucose was scarce, such as when we are asleep and not eating. This occurred as a result of a rise in the hormone glucagon which is secreted by the pancreas as blood sugar levels fall. Glucagon recognition by the cell stimulates a cAMP signal cascade that leads to the phosphorylation of the single serine residue. Phosphorylation of the serine residue activates the phosphatase activity domain but at the same inhibits the kinase domain activity. Wait, But How Does Blood Sugar Levels Regulate These Two Enzyme Activities? Increased phosphatase and lowered kinase activity results in the lowering of fructose 2,6-bisP in the cell. Recall that that this results in an increased flux towards glucose synthesis, precisely what is needed during an overnight fast in order to maintain blood glucose levels. Conversely, after a meal, glucagon levels fall, insulin levels increase, which leads to less phosphorylation of the serine in the bifunctional enzyme, which activates the kinase activity and inhibits the phosphatase activity. This increases the production of fructose 2,6-bisP, which shifts the net flux to that favouring glycolysis. This is a beautiful, elegant example of how metabolism is regulated at the molecular level in response to our eating patterns in order to keep our blood sugar at proper levels. Figure: Control of the synthesis and degradation of fructose 2,6-bisP. Source: Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, Fig.17.8, p. 322. Permission: Courtesy of MacMillan Learning. Clinical Insight: Insulin Fails to Inhibit Gluconeogenesis in Type 2 Diabetes Normally, insulin inhibits gluconeogenesis. This makes sense, since the pancreas releases insulin following a meal to help the tissues take up glucose. After a meal, gluconeogenesis is obviously not required, so insulin shuts it down. However, people with type 2 diabetes have insulin resistance, a condition where the body doesn’t response properly to insulin. As a result, the liver of people with untreated type 2 diabetes continuously produce glucose even when glucose levels are sufficient, leading to hyperglycemia. The large amounts of sugar increase the osmolarity of blood, which draws water out of tissues, leading to excessive thirst and frequent urination. While the exact cause of insulin resistance in many cases is not known, it is strongly associated with obesity. Drugs used to treat type 2 diabetes target different aspects of this disease, including inhibiting liver gluconeogenesis, increasing insulin sensitivity, and stimulating the ability of the pancreas to release insulin. The Cori Cycle Lactate Formed by Muscle Can Be Converted to Glucose in the Liver via the Cori Cycle. Recall that during strenuous activity when oxygen may become limiting, through glycolysis, muscle produces a significant amount of lactate from pyruvate which is released into the blood. Much of this lactate is taken up by the liver, converted back to pyruvate by the enzyme lactate dehydrogenase, and then converted to glucose by the gluconeogenesis pathway. In this way, the liver helps to replenish the glucose levels in the blood, which provides the contracting muscle with the glucose it needs to generate ATP via glycolysis. These combined reactions in the muscle and liver constitute the Cori Cycle The Cori Cycle Lactate Formed by Muscle Can Be Converted to Glucose in the Liver via the Cori Cycle. Discovery was awarded the Nobel Prize in Physiology or Medicine in 1947 https://www.nobelprize.org/p rizes/medicine/1947/corigt/facts/ Figure: The Cori Cycle. Lactate produced in muscle is taken to the liver via the blood, where it is converted back to glucose which provides further fuel to the muscle. Tymoczko, J., Berg, J. & Stryer, L. (2015). Biochemistry: A Short Course (3rd ed.). New York, NY: W. H. Freeman and Company, Fig.17.11, p. 324. Permission: Courtesy of MacMillan Learning.