Gluconeogenesis

Hello :) In this post we'll be discussing gluconeogenesis. I'll describe the function of gluconeogenesis and the locations of this pathway. I'll also list the irreversible reactions of glycolysis and the obligatory quartet of enzymes that regulate gluconeogenesis. I'll also talk about the regulation of glycolysis and gluconeogenesis.

Gluconeogenesis

Gluconeogenesis is the conversion of non-carbohydrates to glucose or glycogen. It is located only in the liver and the kidney (but to a lesser extent). It occurs in the cytosol, mitochondria and smooth endoplasmic reticulum of these cells. The function of gluconeogenesis is to maintain glucose levels when dietary carbohydrates are in low supply. Gluconeogenesis also maintains the TCA cycle intermediates and clears metabolites from the blood. It is essential for the long term maintenance of glucose homeostasis and commences after twelve hours of no or little glucose being present in the diet. Gluconeogenesis provides a major source of blood glucose in all starving animals.

Irreversible Reactions

You may think that to convert non-carbohydrates to glucose you could simply just reverse glycolysis. However, this cannot be done because some reactions in glycolysis are irreversible, particularly:

• The conversion of glucose to glucose-6-phosphate which is catalysed by hexokinase and involves the conversion of ATP to ADP (the first reaction). 
• The conversion of Fructose-6-Bisphosphate to Fructose-1,6-Bisphosphate which is catalysed by phosphofructokinase and involves the conversion of one ATP to ADP (the third reaction).
• The conversion of phosphoenolpyruvate to pyruvate which is catalysed by pyruvate kinase and involves the conversion of one ADP to ATP (the ninth step).

However, there are some enzymes which can be used to overcome these "irreversible" reactions.

The Obligatory Quartet of Gluconeogenesis

Four new enzymes are needed to overcome the three irreversible reactions of glycolysis. These are:

1. Pyruvate Carboxylase: an allosteric enzyme which is stimulated by AcetylCoA. Starvation and fat mobilisation stimulates the production of AcetylCoA and thus stimulates this enzyme.
2. Phosphoenolpyruvate CarboxyKinase: this is induced by glucocorticoids
3. Fructose-1,6-bisphosphatase: gluconeogenesis is mainly controlled from this enzyme. It is allosterically regulated by Fructose-2,6-bisphosphate.
4. Glucose-6-phosphatase: this is essential for the release of glucose from the liver cell into the hepatic vein.

Without these enzymes gluconeogenesis cannot occur. The liver and kidney are the only organs which express all four enzymes, thus they are the only places where gluconeogensis can occur. 

The Regulation of Gluconeogenesis

It is important that gluconeogenesis and glycolysis do not occur at the same time, thus reciprocal and co-ordinated regulation of gluconeogenesis and glycolysis is needed. A bifunctional protein, phosphofructokinase-2 (PFK2), as well as the cAMP dependent pathway are important in regulation. 

cAMP-dependent pathway:

When glucagon or adrenaline bind to their receptors on the surface of a cell they stimulate adenyl cyclase to produce cAMP. This causes cAMP Dependent Protein Kinase to be stimulated which phosphorylates several different metabolic enzymes. However, if insulin binds to a cell instead of glucagon or adrenaline, cAMP will not stimulate cAMP Dependent Protein Kinase to phosphorylate other enzymes. 

When glucagon causes cAMP Dependent Protein Kinase to be activated it phosphorylates pyruvate kinase which inhibits the final step of glycolysis. Thus, glycolysis is inhibited which prevents the glycolysis and gluconeogenesis from occuring at the same time.

Phosphofructokinase 2-The Bifunctional Enzyme

The concentration of fructose-2,6-bisphosphatase (F26BP) is critical to the direction of glycolysis or gluconeogenesis. The amount of F26BP is closely regulated through the activity of phosphofructokinase-2. This enzyme has a phosphofructokinase subunit, which adds phosphates to molecules, and a fructose-2,6-bisphosphatase (F26 bis Pase) subunit, which removes the phosphate group from fructose-2,6-bisphosphate. This type of enzyme is known as a bifunctional enzyme because it has two subunits which perform different functions. Which function is performed depends on whether or not the enzyme is phosphorylated. If it is not phosphorylated, it uses its phosphofructokinase activity to convert fructose-6-phosphate to F26BP. This stimulates glycolysis and inhibits gluconeogenesis. When the enzyme is phosphorylated, it uses its F26 bis Pase activity to destroy F26BP so that gluconeogenesis is no longer inhibited at fructose-1,6-bisphosphatase which is the major regulatory enzyme of the gluconeogenic obligatory quartet.

The major influence on the phosphorylation state of phosphofructokinase 2 is the presence of the hormone glucagon. As mentioned before, when glucagon binds to the surface of a cell it stimulates the production of cAMP inside the cell. This increases the activity of cAMP dependent protein kinase which phosphorylates the bifunctional enzyme so that the phosphofructokinase activity is inhibited and the F26 bis Pase activity is stimulated. The net result of the presence of glucagon is a drop in the concentration of fructose-2,6-bisphosphate and therefore an increase in the activity of fructose-1,6-phosphatase so gluconeogenesis is stimulated. Thus it is evident why glucagon is released when an animal is starving, it is because it stimulates gluconeogenesis. 

In a well fed animal, an intermediate of glycolysis, fructose-6-phosphate, is present at high concentrations. Fructose-6-phosphate stimulates the phosphofructokinase activity of phosphofructokinase 2 and inhibits the fructose-2,6-bisphosphatase activity. This results in an increase in the amount of fructose-2,6-bisphosphate present which increases the activity of phosphofructokinase. Thus, glycolysis is stimulated.

The Tricarboxylic Acid Cycle and Oxidative Phosphorylation

Hi, in this post we'll be discussing the TCA cycle and Oxidative Phosphorylation. I'll discuss where the TCA cycle and oxidative phosphylation occur and how much energy they produce. I'll also describe the location and function of the malate/aspartate and glycerol phosphate shuttles as well as how much energy they produce.

The Tricarboxylic Acid (TCA) Cycle

The TCA cycle, which is also known as the Citric Acid cycle or the Krebb's cycle, occurs in the mitochondria of a cell. This cycle is the final metabolic pathway for oxidising carbohydrates, lipids and proteins and plays a major role in gluconeogenesis, transamination, deamination and lipogenesis. The first enzymatic step in the TCA cycle is the conversion of Acetyl CoA to Citrate by the citrate synthetase enzyme. The cycle then continues as shown in the diagram below:

The TCA Cycle

The TCA cycle produces three NADH molecules and one NADH molecule produces 2.5ATP molecules. Thus 7.5 ATPs are produced from NADH. The TCA cycle also releases one FADH2 molecule, which releases 1.5 ATPs, in addition to one GTP molecule which results in the release of 1 ATP molecule through substrate level phosphorylation. Thus, 10 ATP molecules are produced per molecule of AcetylCoA. Since two molecules of Acetyl CoA are produced per molecule of glucose, 20ATP molecules are produced per glucose by the TCA cycle.   

Oxidative Phosphorylation

Oxidative phosphorylation occurs in the mitochondria of a cell and couples respiratory oxidation to ATP generation. It consists of the electron transport (ETC) chain which are located on the inner mitochondrial membrane.

The TCA cycle produces three NADH molecules and one NADH molecule yields 2.5ATP molecules via the electron transport chain. Thus 7.5 ATPs are produced from the production of NADH. The TCA cycle also releases one FADH2 molecule, which yields 1.5 ATPs through the ETC chain, in addition to one GTP molecule which results in the release of 1 ATP molecule through substrate level phosphorylation. Thus, 10 ATP molecules are produced per molecule of AcetylCoA.  

NADH Substrate Shuttles

The membrane of the mitochondria is impermeable to NADH and so cannot deliver its electrons to the electron transport chain. Because of this, NADH is transferred into the mitochondria by the glycerophosphate and malate/aspartate shuttles.

The Glycerol Phosphate Shuttle

In skeletal muscle, brain and other tissues, electrons are delivered from the NADH generated in the cytoplasm from glycolysis to the mitochondrial  electron transport chain by a shuttle that uses FAD as the electron acceptor. This is the glycerol phosphate shuttle. Dihydroxyacetone phosphate (DHAP) is reduced to glycerol-3-phosphate, which is transported into the mitochondrion. There, it is reoxidised to DHAP by an enzyme that uses FAD instead of NAD+ as its electron receptor. Because of this, these electrons bypass the first complex of the ETC, generating only 1.5ATP molecules instead of 2.5.

The Malate-Aspartate Shuttle

In the liver, kidney and heart cells, electrons from NADH produced in the cytoplasm from glycolysis are transferred into the mitochondrion via the malate-aspartate shuttle. NADH reduces oxaloacetate in the cytoplasm to malate which is transported into the mitochondrial matrix where it reduces NAD+ to NADH. Thus, the electrons from the NADH in the cytoplasm pass through the first three complexes of the ETC which generates 2.5 ATP.

ATP Yield

As discussed in a previous post, glycolysis produces 2 ATP molecules per molecule of glucose. During anaerobic respiration, this is all that is produced because the TCA cycle or oxidative phosphorylation aren't used.

During aerobic respiration, glycolysis produces 2ATP molecules per glucose. In addition, Pyruvate Dehydrogenase causes the release of 5ATP molecules per glucose. The TCA cycle produces 20ATP molecules per glucose. If the malate-aspartate shuttle is used, 2.5 ATP molecules are produced per NADH. Since glycolysis releases 2 NADH molecule, 5 ATP molecules are produced per glucose. This results in a total of 32 ATP molecules per glucose during aerobic respiration. If the glycerol phosphate shuttle is used, 30 ATP molecules will be produced. This is because the glycerol phosphate shuttle produces 1.5 ATPs per NADH and thus 3 molecules per glucose.  

That's it for this post, if you have any questions please feel free to ask :)