22 Gluconeogenesis

Dr.Vikram Raval

GLUCONEOGENESIS

Objectives

1. To understand glucose synthesis from non-carbohydrate intermediates.
2. Energy efficiency of glycolysis and gluconeogenesis
3. Gluconeogenesis pathway
4. Bypass energy inefficient glycolytic reactions

Introduction

• Gluconeogenesis is defined as the biosynthetic pathway for formation of glucose de-novo (i.e. not glucose from glycogen a regular stored form in most animals)

• Gluconeogenesis is a metabolic pathway that is actually responsible for the generating glucose from non-carbohydrate carbon containing substrates such as pyruvate, lactate, glycerol, and glucogenic amino acids

• Gluconeogenesis is a ubiquitous process, observed in all of living kingdom including plants, animals, fungi and bacteria. This process is also referred to as an endogenous glucose production (EGP)

• Erythrocytes and human brain also heavily dependent on glucose formed from gluconeogenesis for energy requirements and utilize large amounts of glucose consumed as well as produced daily via gluconeogenesis

• Gluconeogenesis it is the process that occurs chiefly in liver. While a very limited extent of the reactions occurs in kidney as well as in small intestine, but that requires specific physiological conditions

• However, in addition to glucose, the brain derives its energy from ketone bodies via acetyl-CoA and shunted into the TCA cycle. The glucose requirement of the brain in an adult human being is approx 120 g, which accounts for majority of glucose needed by body (160 g) on day-to-day basis. The amount of glucose in body fluids is about 20 g, and that readily available from glycogen is approx 190 g. These glucose reserves are sufficient to meet day to day glucose requirements

• But under conditions of longer period of starvation, glucose must compulsorily be formed from non-carbohydrate sources

• The preliminary carbon skeletons in gluconeogenesis is mainly from pyruvate, lactate, glycerol, and the amino acids alanine and glutamine

• Gluconeogenesis and glycogenolysis are the two mechanism that help in maintaining blood glucose levels in the body

• In few ruminants, this is a continuous process. While in many other animals, the process mainly occurs during fasting, starvation, low-carbohydrate foods, or intense physical activity. The process is highly endergonic but due to coupling of ATP/GTP hydrolysis it ends up to be exergonic

• For gluconeogenesis from non-carbohydrate precursors of glucose they are first converted into pyruvate or enter the pathway at later stages of glucose metabolic pathways such as oxaloacetate (OAA) and dihydroxyacetone phosphate (DHAP)

• Lactate is primarily formed by skeletal muscles when the rate of glycolysis outnumbers the oxidative metabolism. Conversion of lactate into pyruvate is catalysed by lactate dehydrogenase. During starvation the skeletal muscles breakdown the proteins and thus a mino acids are derived from these dietary proteins

• The reactions constitutes the Cori cycle wherein a pyruvate is synthesised from lactate in muscle tissues and in another reaction of transamination in muscles, alanine is formed from pyruvate. The amino group released is reduced in the form of urea. The reaction are popularly called Alanine cycle. Both of these Cori cycle and alanine cycle reactions allow generation of pyruvate and thereby favour entry into gluconeogenesis.

• The hydrolysis of triacylglycerols in adipocytes yields fatty acids and glycerol. Glycerol acts as a precursor of glucose, but animals are unable to transform fatty acid residues to glucose. Glycerol can either enter glycolysis or glyconeogenesis through dihydroxyacetone phosphate

For your information (FYI): Synthesis of glucose from three and four carbon precursors is essentially a reversal of glycolysis. We all are familiar with the process of glycolysis wherein two molecules of glucose are synthesised from pyruvate by various enzymatic reactions

For your information (FYI): Cori cycle for the formation of pyruvate and further glucose from lactate by active muscle metabolism. The Cori cycle generates glucose at the expense of 6 ~ATP in liver for every 2 ~ATP made available in muscle. Thus a net expense of 4 ~ATP is incurred in cori cycle. Inspire of less efficient on energy, the Cori Cycle allows an organism to withstand energy demands of skeletal muscle between resting and active physical exerts. Glutamate or lactate in muscle is transaminated to alanine, which is released into the bloodstream. In the liver, alanine is taken up and converted into pyruvate for further metabolism

Is Gluconeogenesis a kind of reverse Glycolysis?

• Glycolysis is process that is anaerobic breakdown of glucose molecules in to pyruvate and further into TCA cycle intermediates yielding abundant energy for bodily processes.

• Generally reactions of glycolysis are reversible under cellular environment except the three reactions which have a large negative ΔG in the forward direction and thus they are essentially irreversibl

These are the reactions catalysed by

1. Conversion of glucose into Glucose-6-Phosphate a reaction catalysed by hexokinase,

2. Conversion of Fructose-6-phosphate to fructose 1-6-bis phosphate catalysed by phosphofructokinase and

3. Formation of pyruvate from phosphoenol pyruvate catalysed by pyruvate kinase

• Since we are discussing glucose generation de-novo for gluconeogenesis pathway it becomes necessary that we bypass this reaction

• Often these 3 reactions are referred to as Bypass reactions, two of which are kind of simple hydrolysis while the third one involves action of two enzymes pyruvate carboxylase and phosphoenol pyruvate carboxy kinase

General reactions of gluconeogenesis

• The process of gluconeogenesis is not very cost effective from energy point of view as oxidation of glucose through two moles pyruvate finally yields a mere two moles of ATP while generation of glucose via gluconeogenesis consumes at least 6 moles of ATP at various stages

• The gluconeogenetic reactions which occur in mitochondria are conversion of pyruvate to oxaloacetate and then further from oxaloacetate to malate

• The gluconeogenetic reactions which occur in mitochondria are conversion of pyruvate to oxaloacetate and then further from oxaloacetate to malate

• From cytosol pyruvate is transported across the outer mitochondrial membrane involves a voltage-dependent porin transporter while transport across the inner mitochondrial membrane is by a pyruvate transporter protein called monocarboxylic acid transporter 1(MCT1) and a hetero-tetramer transport protein complex

• While oxaloacetate after reduction is converted to malate and is transported to cytosol by a malate transporter

• In the cytosol oxidation of malate into oxaloacetate takes place. Oxaloacetate is converted to phosphoenol pyruvate by enzyme phosphoenol pyruvate carboxy kinase and then as an intermediate it enters gluconeogenesis pathway. The reaction consumes energy in the form of GTP that has energy equivalence to ATP

For your information (FYI): The reversal reaction of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) action requires a supply of NADH. This depends upon the initial intermediate or precursor of gluconeogenesis. If lactate is precursor then NADH will be supplied by action of lactate dehydrogenase enzyme, while if precursors are pyruvate or amino acids like alanine then the NADH supply will be catalysed by malate dehydrogenase.

• Next is the reaction wherein one mole of glyceraldehyde-3-phosphate needs to  isomerize into DHAP  and  then   further   upon  a   condensation   reaction   giving   one   mole   of   fructose-1,6 bisphosphate a reaction exactly reverse of aldolase enzyme action. In liver hepatocytes glucose-6-phosphatase enzyme allows free glucose supply to blood

   Higher the Km of liver glucokinase majority of glucose will remain in dephosphorylated form and will be removed from hepatocytes into the blood

BYPASS 1: The Conversion of Pyruvate into Phosphoenol pyruvate via Oxaloacetate

• The above reaction requires activity of two enzymes that is pyruvate carboxylase (PC) and phosphoenol pyruvate carboxy kinase (PEPCK).

Pyruvate Carboxylase Reaction

• This reaction utilizes the energy from ATP, enzyme pyruvate kinase requires biotin as a cofactor in presence of carbon dioxide. The CO2 utilized in the above reaction occurs as bicarbonate (HCO3-) ion

• This is the first reaction of gluconeogenesis process and as the name of the enzyme suggests the substrate pyruvate is carboxylated (addition of CO2) to form oxaloacetate (OAA).

• Pyruvate carboxylase is stringent in its requirement of activator. In absence of activator i.e. acetyl-co-A the enzyme becomes inactive.

• Chief source of acetyl-co-A is beta oxidation of fatty acids in liver and adipocytes. The enzyme mainly functions to generate carbon skeleton from non-carbon intermediates. Pyruvate carboxylase works for formation of pyruvate, lactate and alanine. Additionally it works to drive oxaloacetate and thus TCA cycle.

• The pyruvate carboxylase enzyme is a homotetramer with three domains, the biotin carboxylase (BC) domain, the carboxyl transferase (CT) domain, and the biotin carboxyl carrier protein (BCCP) domain.

• The reaction occurs in two simple stages one in which biotin is carboxylated to carboxybiotin in presence of HCO3- and spending energy from ATP. Then through carboxyphosphate intermediate it transfers the CO2 (biotin decarboxylation) to pyruvate forming oxaloacetate and regenerating biotin.

• The carboxybiotin is activated form and has ΔG°´ for its cleavage which is equivalent to -4.7 kcal mol-1 (-20 kJ mol-1). This negative ΔG°´ indicates that carboxybiotin is able to transfer its CO2 without any further energy inputs.

Phosphoenol pyruvate Carboxy kinase Reaction

• Next is the formation of phosphoenol pyruvate from oxaloacetate a reaction catalysed by PEP carboxy kinase (PEPCK) utilizing energy from GTP.

• There is no net fixation of CO2 as at the end of this reaction the CO2 that was initially incorporated by pyruvate carboxylase into pyruvate is subsequently released by phosphoenolpyruvate carboxy kinase.

• For gluconeogenesis to proceed further, the oxaloacetate must be transported to cytoplasm for which no mechanism exists in cell not a free diffusion is possible. Three distinct reactions help with this. They are as follows:

1. Conversion to PEP as indicated above through the action of the PEPCK
2. Transamination to aspartate
3. Reduction to malate

• For the transamination reaction of OAA to aspartate or reduction of OAA to malate, both malate and aspartate levels should be adequate in cytosol that ensures the above two reactions are continuously executed.

BYPASS 2: The Conversion of Fructose 1,6-bisphosphate into Fructose 6-phosphate

• Phosphoenol pyruvate upon formation is immediately metabolized by the glycolytic enzymes in reverse reactions and intracellular conditions favour gluconeogenesis.

• It is a simple hydrolysis reaction catalysed by fructose-1, 6-bisphosphatase that converts fructose 1-6-bisphosphate to fructose-6-phosphate releasing an inorganic phosphate (Pi).

• Similarly to its glycolytic counterpart, fructose-1, 6-bisphosphatase is an allosteric enzyme involved in regulation of gluconeogenesis.

BYPASS 3: The generation of free Glucose from Glucose-6-phosphate

• This final step in gluconeogenesis is the generation of glucose. This does not take place in the cytosol instead, glucose 6-phosphate is transported into the endoplasmic reticulum, where it is hydrolyzed to glucose by glucose 6-phosphatase, a membrane bound enzyme in inner lumen of endoplasmic reticulum

• In majority of tissues gluconeogenesis ends when glucose 6-phosphate is formed from fructose 6-phosphate since it cannot diffuse out of cell like free glucose.

• This in one way helps tissues to capture glucose and maintain homeostasis in tissues of liver and kidney

References

• Exton JH. Regulation of gluconeogenesis by glucocorticoids. Monogr Endocrinol. 1979;12:535-46.
• Nuttall FQ, Ngo A, Gannon MC Regulation of hepatic glucose production and the role of gluconeogenesis in humans: is the rate of gluconeogenesis constant? Diabetes Metab Res Rev. 2008 Sep;24(6):438-58. doi: 10.1002/dmrr.863.
• Veldhorst MAB, Westerterp-Plantenga MS, and Westerterp KR Gluconeogenesis and energy expenditure after a high-protein, carbohydrate-free diet. Am J Clin Nutr September 2009 vol. 90 no. 3 519-526

Books

• Harper’sIllustrated Biochemistry by Robert K.. Murray, Daryl K. Granner, Peter A. Mayes, 26th Edition (2003)
• Lehninger’S Principle of Biochemistry David L. Nelson and Michael M. Cox,5th Edition (2008)
• Instant Notes: Biochemistry, 2nd Edition, B.D. Hames & N. M. Hooper (2005)
• Textbook of Biochemistry, 4th Edition Donald Voet, Judith G. Voet (2011)

Web site

• http://www.wiley.com/legacy/college/boyer/0470003790/animations/gluconeogenesis/gluconeogenesis.htm
• http://www.genome.jp/kegg/pathway/map/map00010.html