Gluconeogenesis PDF

BCH 351: PRIMARY METABOLIC PATHWAY I GLUCONEOGENESIS Introduction Glucose occupies a very important position in the metabolism of many organisms including mammals. It not only serves as an excellent energy source, but is also a versatile precursor for many biosyntheti...

BCH 351: PRIMARY METABOLIC PATHWAY I GLUCONEOGENESIS Introduction Glucose occupies a very important position in the metabolism of many organisms including mammals. It not only serves as an excellent energy source, but is also a versatile precursor for many biosynthetic reactions. In mammals, circulating glucose from blood is the sole or major source of energy for certain cells/tissues such as the brain and nervous system, testes, red blood cells, etc. Dietary carbohydrate ingestion is the main source of glucose for the body, however, during long fasts, after vigorous exercise, or sometimes between meals, glycogen stores are depleted. During these times, the liver is able to synthesize glucose from non-carbohydrate sources to maintain blood glucose via the process of gluconeogenesis (new formation of sugar/glucose). Gluconeogenesis is a metabolic process that generates glucose form non-carbohydrate substrates. This process ensures the maintenance of appropriate blood glucose levels when the liver glycogen is almost depleted and no carbohydrates are ingested. In mammals, gluconeogenesis takes place mainly in the liver and, to a lesser extent, in the renal cortex. Reactions of gluconeogenesis Although it shares several steps with the glycolytic pathway, gluconeogenesis and glycolysis are not identical pathways running in opposite directions. In other words, gluconeogenesis is not the reversal of glycolysis even though seven (7) of the ten (10) enzymatic steps of gluconeogenesis are the reversal of the reactions of glycolysis. In glycolysis, there are three (3) irreversible steps that cannot be utilized in gluconeogenesis and therefore need to be bypassed by separate set of enzymes. These reactions include: a. The conversion of glucose to glucose-6-phosphate by hexokinase b. Phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by phosphofructokinase-I c. Conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase. 1 1. Conversion of pyruvate to phosphoenolpyruvate In the first reaction of gluconeogenesis which is the first bypass reaction, pyruvate is converted (via phosphorylation) to phosphoenolpyruvate (PEP). This reaction is not a simple reversal of the pyruvate kinase reaction of glycolysis. Instead, pyruvate is first converted to oxaloacetate by the enzyme pyruvate carboxylase in the mitochondrial matrix in a reaction that requires biotin as a coenzyme. To proceed in the gluconeogenic pathway, the oxaloacetate formed must be transferred back into the cytosol. However, mitochondria lack an efficient transporter for oxaloacetate, therefore, oxaloacetate is reduced to malate by malate dehydrogenase which converts one molecule of NADH to NAD+. Malate is then transported out of the mitochondria and re- oxidized to oxaloacetate, regenerating NADH from NAD+ in the cytosol. In the cytosol, a second enzyme, phosphoenolpyruvate carboxykinase catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate (PEP) using GTP (from TCA cycle) as the phosphate group donor. Note* - The glucogenic precursor determines whether this reaction occurs entirely in the mitochondrion or partially in both the mitochondrion and the cytosol. When pyruvate is the glucogenic precursor, it is first transported from the cytosol into the mitochondrion or generated from alanine via transamination reaction in the mitochondrion. The pyruvate then undergoes the pyruvate carboxylate reaction to yield oxaloacetate which is reduced to malate and transported out of the mitochondrion into the cytosol. In the cytosol, malate is re-oxidized back to oxaloacetate by malate dehydrogenase with a concomitant production of NADH. The oxaloacetate is then simultaneously decarboxylated and phosphorylated to phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase. This reaction requires Mg2+ and GTP and the phosphoryl group donor. *** The oxaloacetate-malate shuttling allows the transport of reducing equivalents (NADH) into the cytosol where their concentration is relatively low. This reaction is important because gluconeogenesis cannot proceed without the availability of NADH. 2 When lactate (from erythrocytes and vigorously exercising muscles) is the glucogenic precursor, it is first converted to pyruvate by the lactate dehydrogenase reaction to yield pyruvate and a molecule of NADH. The pyruvate is then transported into the mitochondrion where it is converted to oxaloacetate. The oxaloacetate produced is not however, transported out of the mitochondrion. Rather, it is converted directly to PEP by the mitochondrial isozyme of PEP carboxykinase. The PEP is then transported out of the mitochondrion to continue in the gluconeogenesis pathway. *** Oxaloacetate is not shuttled out (as malate) in this reaction because NADH is already generated in the cytosol during the lactate dehydrogenase reaction and does not need to be shuttled out of the mitochondrion. 3 2. Conversion of fructose 1,6-bisphosphate to fructose 6-phosphate The second step of gluconeogenesis is the bypass of the reaction catalyzed by phosphofructokinase-1 (PFK-1) in the glycolytic pathway. This reaction involves the dephosphorylation of fructose 1,6-bisphosphate to fructose 6-phosphate catalyzed by fructose 1,6-bisphosphatase, a Mg2+-dependent enzyme located in the cytosol, leading to the hydrolysis of the C-1 phosphate of fructose 1,6-bisphosphate, without production of ATP. 3. Conversion of glucose 6-phosphate to glucose The third step of gluconeogenesis is the bypass of the reaction catalyzed by hexokinase in glycolysis. This involves the dephosphorylation of glucose 6-phosphate to glucose catalyzed by glucose 6-phosphatase. Glucose 6-phosphatase is found in the lumen of the endoplasmic reticulum (ER) rather than in the cytoplasm. Thus, for the final step of gluconeogenesis, glucose 6-phosphate must be transported into the ER where the phosphate is cleaved off, and then glucose and phosphate are transported back out. **This enzyme is absent in skeletal muscle and adipose tissue and that is why gluconeogenesis cannot take place in these tissues. 4 Summary of the Reactions of Gluconeogenesis 5 Substrates for gluconeogenesis In addition to pyruvate and lactate, other non-carbohydrate precursors can be used as substrates for gluconeogenesis in animals. These include most of the amino acids, as well as glycerol and all the TCA cycle intermediates. 1. Lactate is produced by exercising skeletal muscle and in cells that lack mitochondria (e.g. erythrocytes) and transported back to the liver where it is converted to pyruvate by lactate dehydrogenase and used for gluconeogenesis. 2. Amino acids generated from dietary protein or from the breakdown of muscle protein during starvation, also undergo transamination or deamination in the mitochondrial matrix to yield pyruvate or intermediates of the tricarboxylic acid (TCA) cycle. 6 3. Glycerol released from lipolysis of adipose tissue triacylglycerol and glycerophospholipids are also utilized for gluconeogenesis. Glycerol enters gluconeogenesis, or glycolysis depending on the cellular energy charge, as dihydroxyacetone phosphate (DHAP). It is first phosphorylated to glycerol 3-phosphate by glycerol kinase, which utilizes one ATP molecule for phosphorylation of glycerol. Glycerol 3-phosphate is then oxidized to dihydroxyacetone phosphate by glycerol 3- phosphate dehydrogenase. In this reaction NAD+ is reduced to NADH. During prolonged fasting, glycerol is the major gluconeogenic precursor, accounting for about 20% of glucose production. Regulation of Gluconeogenesis Glycolysis and gluconeogenesis are reciprocally regulated to prevent wasteful operation of both pathways at the same time. The points of regulation of gluconeogenesis are highlighted below. 1. Conversion of pyruvate to phosphoenolpyruvate Pyruvate carboxylase, the enzyme that catalyzes the first step in gluconeogenesis, is activated by acetyl-CoA and inhibited by ADP which is also an inhibitor of phosphoenolpyruvate carboxykinase. Acetyl-CoA can be seen as a reciprocal regulator of glycolysis and gluconeogenesis since it acts on the enzymes that interconvert pyruvate and phosphoenolpyruvate. It is an activator of pyruvate carboxylase and an inhibitor of 7 pyruvate kinase and the pyruvate dehydrogenase complex. When its levels rises, it signals the availability of substrates for the TCA cycle, allowing more carbon to be shuttled into gluconeogenesis and ultimately stored as glycogen. 2. Interconversion of fructose-6-phosphate and fructose-1,6- bisphosphate. Fructose 1,6-bisphosphatase is inhibited by adenosine monophosphate (AMP) and activated by citrate. Adenosine monophosphate is a product of ATP breakdown, and a rise in its concentration indicates a low energy charge in the cell and signals the need for ATP. This stimulates the glycolytic pathway by activating phosphofructokinase-1. On the other hand, high levels of ATP and citrate indicate high energy charge and the abundance of biosynthetic intermediates. Under this condition (high energy levels), glycolysis is greatly slowed down and gluconeogenesis is accelerated. Thus AMP levels indicate the energy state of the cell, consequently regulating the activity of PFK-1 in response to cellular energy needs. Phosphofructokinase-1 and Fructose 1,6-bisphosphatase are also reciprocally controlled by Fructose 2,6-bisphosphate (F 2,6-BP). This molecule is structurally related to fructose 1,6-bisphosphate, but is not an intermediate in glycolysis or gluconeogenesis. The level of F 2,6-BP is high in fed state and low during starvation. Therefore, at high F 2,6-BP levels, gluconeogenesis in inhibited while the reaction is activated at low F 2,6-BP levels. 8 3. Regulation by the action of Glucagon/Insulin Two hormones, namely glucagon and insulin, are also involved in the regulation of glycolysis and gluconeogenesis. These hormones act intracellularly through fructose 2,6- bisphosphate, an allosteric effector of Phosphofructokinase-1 (PFK-1) and Fructose 1,6- bisphosphatase-1. By binding to the allosteric site on PFK-1, fructose 2,6-bisphosphate reduces the affinity of the enzyme for ATP and citrate (allosteric inhibitors) and at the same time increases the affinity of the enzyme for fructose 6-phosphate (its substrate). Therefore, in the absence of fructose 2,6-bisphosphate, and in the presence of physiological concentrations of ATP, fructose 6-phosphate, and of allosteric effectors AMP and citrate, PFK-1 is practically inactive. Summarily, high concentration of fructose 2,6- bisphosphate activates PFK-1, thus stimulating glycolysis in the liver, while at the same time slowing down gluconeogenesis by inhibiting fructose 1,6-bisphosphatase. During starvation or fasting, glucagon is released into the circulation in response to low blood glucose signaling the liver to reduce glucose consumption for its own needs and to increase de novo synthesis of glucose and its release from glycogen stores. By binding to specific membrane receptors, glucagon stimulates hepatic adenylate cyclase to synthesize 3′,5′-cyclic AMP (cAMP), which activates cAMP-dependent protein kinase or protein kinase A (PKA). The kinase catalyzes the phosphorylation of PFK-2/FBPase-2 increasing phosphatase activity while decreasing kinase activity. This causes a decrease in the levels of fructose 2,6-bisphosphate, that, in turn, inhibits glycolysis and stimulates gluconeogenesis. Therefore, in response to glucagon, hepatic production of glucose increases, enabling the organ to counteract the fall in blood glucose levels. In activating gluconeogenesis, glucagon simultaneously stimulates the release of fatty acids from adipose tissue into the liver, increasing fatty acid oxidation and a consequent increase in acetyl-CoA production. On the other hand, insulin released into circulation after a carbohydrate-rich meal binds to its specific membrane receptors and activates a protein phosphatase (phosphoprotein phosphatase 2A or PP2A) that catalyzes the removal of the phosphate group from PFK- 2/FBPase-2, thus increasing PFK-2 activity and decreasing FBPase-2 activity. At the same 9 time, insulin also stimulates a cAMP phosphodiesterase that hydrolyzes cAMP to AMP. This increases the level of fructose 2,6-bisphosphate, that, in turn, inhibits gluconeogenesis and stimulates glycolysis. In addition, fructose 6-phosphate allosterically inhibits FBPase-2, and activates PFK-2. 10