Gluconeogenesis and other pathway interactions

Glucose-6-phosphate Dehydrogenase (G6PDH) Deficiency

Typically, the enzyme is not lacking or nonfunctional, it simply functions at lower efficiency

◦ This means for most people, they’re asymptomatic So why does this matter then?

◦ G6PDH helps to produce more NADPH for the cell, which in turn is used to help reduce glutathione, which is an important red cell antioxidizing compound

How does a lack of G6PDH do its damage?

If little G6PDH is functioning normally, no NADPH is present No NADPH present, GSH can not be reformed from GSSG, the oxidized form of GSH GSH regenerated into its oxidized form from NADPH

◦ GSSG + NADPH + H+ = 2GSH + NADP+

If too many peroxide compounds are produced, you end up with structures in erythrocytes known as Heinz bodies

◦ Formation of these bodies often leads to the lysis of the cell, causing hemolytic anemia

Its connection to malaria

beneficial if youre in an area where having more oxidative stress means more resistant to malaria

Gluconeogenesis – In a nutshell

Very similar to glycolysis, but uses slightly different enzymes for some reactions ◦ Three reactions require so much free energy to reverse, if they were using their original enzyme, that different enzymes are required: Most of the steps that are difficult to reverse are the ones that attached a phosphate group (steps 1 and 3), and their enzymes do not work as well in reverse The last step, going from PEP to pyruvate, also requires two different enzymes that we’ll get into

Glycolysis and Gluconeogenesis The reactions of these two pathways, and their differences:

Reaction 1: ◦ Glucose-6-phosphotase instead of hexokinase

◦ Reaction 3: ◦ Fructose-1-6-bisphosphatase instead of phosphofructokinase

◦ Reaction 10: ◦ Pyruvate carboxylase instead of pyruvate kinase

Overall energy investment required for gluconeogenesis

4 ATP ◦ 2 GTP (another energy transfer molecule we’ll discuss more in citric acid cycle) ◦ 2 NADH → 2 NAD+

To (re)use these compounds for gluconeogenesis, they need to be converted via different enzymes and bypass the enzymes in the glycolysis system.

Bypass 1

Pyruvate to PEP

Turning pyruvate back into phosphoenolpyruvate ◦ We are now bypassing the pyruvate carboxylase enzyme Does so by two enzymes 1. Pyruvate carboxylase

I. However, because oxaloacetate is a substance found in the citric acid cycle, and therefore the mitochondria, it needs to be converted to malate

2. Phosphoenolpyruvate carboxykinase

Bypasses 2 & 3

Dephosphorylation

The removal of both of the phosphate groups from two intermediaries to get back glucose

◦ Each of these are done by hydrolysis reactions

The first, in bypass 2, is done by Fructose 1-6- bisphosphatase ◦ This transforms F1-6B back into F6P

The next, bypass 3, is done by Glucose-6-phosphatase ◦ This transforms G6P back into glucose

Substrates for Gluconeogenesis

he main point of gluconeogenesis is not always to work back from pyruvate to glucose

◦ If that was the case, why bother converting the glucose in the first place?

Rather, many of the substances that are used in the gluconeogenesis pathway are recovered from other types of molecules in different biochemical pathways.

? 1. Lactate ◦ The most reconverted in gluconeogenesis, happens via the Cori cycle

2. Amino acids ◦ Virtually all can be degraded via glycolysis / gluconeogenesis

Cori Cycle

Recovery of Lactate

The cycle by which lactate is repurposed to produce (more) energy Lactate is produced in the muscles under anaerobic conditions, when ATP synthesis is required quickly

From there, it is shunted to the liver where it is converted back into pyruvate, then glucose, then back into the blood stream

Amino Acids & Glycolysis

Through a combination of glycolysis and the citric acid cycle, almost all amino acids are fully metabolized.

◦ Most of the “simple” ones via glycolysis, the larger and more complex ones via citric acid The only two not metabolized in this fashion: leucine and lysine

◦ What happens to them? Portions of them get transferred to the citric acid cycle via acetoacetate

◦ Remaining portions of them get fashioned into ketone bodies

Reciprocal regulation of these two pathways

Much like all other pathways and regulations in biochemical reactions in the body, homeostasis and balance reigns supreme

◦ The same is true for glycolysis and gluconeogenesis

◦ These two pathways are reciprocally regulated

These two pathways are regulated at multiple control points through the activation or inhibition of enzymes in each of the pathways

How can glycolysis be regulated? At the three most energy intensive steps:

1. Hexokinase:

◦ Decreased hexokinase activity if there are increased levels of G6P

◦ Substrate level control of activity

2. Phosphofructokinase:

I. Can be increased by increased levels of AMP or ADP through their binding to allosteric sites II.

Can be decreased by increased levels of ATP or citrate I. Why citrate? It’s necessary for the citric acid cycle.

Increased levels of citrate indicate a fully saturated citric acid cycle 3.

Pyruvate kinase: I. Increased by high levels of fructose 1 bisphosphate, as to be expected

II. Decreased by high levels of Acetyl-CoA and ATP

Pathway controls in gluconeogenesis Like you would expect, many of these controls work in reverse of glycolytic activators and inhibitors:

Conversion of pyruvate to PEP is increased by both acetyl-CoA and glucagon, and decreased by insulin

◦ Again, this is to be expected knowing the roles of glucagon and insulin

◦ Conversion of fructose-1-6-bisphosphate to fructose-6-phosphate is inhibited by both

◦ AMP and fructose-2-6-bisphosphate, a potent signalling molecule

◦ Conversion of glucose-6-phosphate to glucose

◦ The exact opposite of glycolysis: high concentrations of G6P allow for hexokinase to return G6P to glucose

Use of other substrates in the glycolytic pathway

Monosaccharides:

◦ Galactose

◦ Exists as a product of lactose breakdown, a sugar found in milk

◦ Broken down by galactokinase and a few other enzymes, requiring ATP.

◦ Fructose

◦ Exists as a breakdown product of sucrose, a sugar found in fruits

◦ Broken down by fructokinase Glycerol:

◦ Exists because of the breakdown of fats: Remember, true fats are a combination of three fatty acids attached to a glycerol

◦ Broken down into dihydroxyacetone (DHAP) a main constituent of the glycolysis pathways

Overview of alternate saccharide substrates (con’t) Disaccharides, a bit easier to understand:

All of the disaccharides we examine have glucose as one of the two monomers, so that part is simple

Three major ones to examine in the context of human metabolism: ◦ Maltose – broken down by maltase

◦ Made of two glucose molecules, so it’s very easy ◦ Lactose (combination of galactose and glucose)

◦ Can be converted directly into galactose via lactase ◦ Sucrose (combination of glucose and fructose)

◦ Fructose enters glycolysis either by being converted directly to fructose-6-phosphate, or by being converted to fructose-1- phosphate

How to build up glycogen?

This is done via the creation of the molecule UDP-Glucose, or uridine diphosphate glucose

◦ Hey, uridine! Nucleic acids, superficially mRNA. It’s one of the five nucleosides.

Why do we need UDP-glucose? Can’t we just attach the glucoses together?

◦ No, we need a substrate that can be primed, which therefore needs a specific phosphate substrate to allow the reaction to proceed.

Glycogen storage signalling

So we want to use stored glucose from glycogen, and we want to do this quickly ◦ Think about situations where we need glucose very quickly... ◦ How does the body signal to do this? Can do this via hormonal and non-hormonal signalling

Hormonal Glycogen storage signalling

Glucagon or epinephrine (depends on the cell type) binds to receptors on the cell surface

◦These are G-protein coupled receptors, so they in turn activate cyclic AMP

◦ Through a few other mechanisms, activate glycogen phosphorylase enzymes, allowing for quick release of glucose

The pentose-phosphate pathway

• Other method to break down glucose

• Multipurpose – Can be used to:

• Produce new reducing agents for energy generation in other alternate pathways (NADPH)

• Produces ribose-5-phosphate for the production of nucleic acids and nucleotides

• Produce energy via glycolysis when only a small amount of pentose phosphates are needed

The pentose-phosphate pathway phases

Works in two phases: oxidative, then reductive

◦ Oxidative: ◦ G6P oxidized to ribulose-5-phosphate, each G6P produces 2 reduced structures: 2 NADPH molecules

◦ Reductive: ◦ Products are reduced to alternate carbon/phosphate structures, depending on what’s needed in the cell at the time

Main steps of pentose phosphate pathway

1. Oxidation

◦ For the total pathway to proceed, three units of glucose-6-phosphate are oxidized to 3 ribulose-5-phosphate molecules

◦ This has the by-product of producing carbon dioxide, therefore removing some carbon from the pathway

◦ Also has the benefit of producing NADPH

2. Reduction

◦ Produces ribose-5-phosphate, necessary for nucleotides

◦ Can also converts these three, 5-carbon sugars into two 6-carbon sugars and one 3-carbon sugar, to be reused in NADPH production or sent to citric acid cycle

Purposes of pentose phosphate pathway

• Nucleotide formation

• NADPH formation

• Energy formation

Part A, Nucleotide synthesis. When the primary need is for nucleotide biosynthesis, the primary product is ribose 5 phosphate.

Part B, N A. D P H synthesis. When the primary need is for reducing power, N A. D P H, fructose phosphates are reconverted to glucose 6 phosphate for re oxidation in the oxidative phase.

Part C, Energy generation. When only moderate quantities of pentose phosphates and N A D P H are needed, the pathway can also be used to supply energy, with the reaction products being oxidized through glycolysis and the citric acid cycle.