Biochemistry Gluconeogenesis - Library

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Biochemistry Metabolism of Carbohydrates

Gluconeogenesis

Dr. Vijaya Khader Dr. MC Varadaraj

Paper : 04 Metabolism of carbohydrates

Module : 23 Gluconeogenesis

Principal Investigator

Paper Coordinator

Content Reviewer

Content Writer

Dr. S.K.Khare,Professor IIT Delhi.

Dr. Ramesh Kothari, Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA

Dr. S. P. Singh Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA

Dr. Vikram Raval, Assistant Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA

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Gluconeogenesis

Description of Module

Subject Name Biochemistry

Paper Name 04 Metabolism of carbohydrates

Module Name/Title Gluconeogenesis

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Gluconeogenesis

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

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Gluconeogenesis

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)

The formation of glucose molecules from various carbon skeletons is often necessary since the

vital organs viz. testes, kidney (renal cortex) exclusively utilize glucose for ATP production

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

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Gluconeogenesis

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 amino 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

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Gluconeogenesis

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

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Gluconeogenesis

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

irreversible

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 reactions

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.

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Gluconeogenesis

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

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-

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Gluconeogenesis

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).

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Gluconeogenesis

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.

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Gluconeogenesis

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.

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Gluconeogenesis

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

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Biochemistry Gluconeogenesis - Library