BIOCHEMISTRY AND HUMAN NUTRITION

Module 3. Metabolism

Lesson 17
GLUCONEOGENESIS

17.1 Introduction

• Gluconeogenesis is especially important in periods of starvation or vigorous exercise.
• During starvation, the formation of glucose via gluconeogenesis particularly uses amino acids from protein breakdown and glycerol from fat breakdown.
• During exercise, the blood glucose levels required for brain and skeletal muscle function are maintained by gluconeogenesis in the liver using lactate produced by the muscle.
• Gluconeogenesis synthesizes glucose from noncarbohydrate precursors, including lactate and pyruvate, citric acid cycle intermediates, the carbon skeletons of most amino acids and glycerol.
• This is extremely important since the brain and erythrocytes rely almost exclusively as their energy source under normal conditions. The store of liver glycogen is sufficient to supply the brain with glucose for only about half a day during fasting.
• The main site of gluconeogenesis is the liver, although it also occurs to a far lesser extent in the kidneys. Very little gluconeogenesis occurs in brain or muscle.

Fig. 17.1 Gluconeogenesis

17.2 The Pathway

In principle, gluconeogenesis appears to be a reversal of glycolysis. Indeed, some of the reactions of glycolysis are reversible and so the two pathways have these steps in common.

• However, three steps in glycolysis are essentially irreversible; those catalyzed by the enzymes hexokinase, phosphofructokinase (PFK) and pyruvate kinase.
• Indeed it is the large negative free-energy change in these reactions that normally drives glycolysis forward towards pyruvate formation.
• Therefore, in gluconeogenesis, these three steps have to be reversed by using other reactions so gluconeogenesis is not a simple reversal of glycolysis.

17.2.1 Precursors for gluconeogenesis

• Glycerol can act as a substrate for glucose synthesis by conversion to dihydroxy-acetone phosphate, an intermediate in gluconeogenesis.
• Lactate, pyruvate, citric acid cycle intermediates and the carbon skeletons of most amino acids to act as precursors for gluconeogenesis, these compounds must first be converted to oxaloacetate. Some of the carbon skeletons of the amino acids give rise to oxaloacetate directly.
• Other precursors feed into the citric acid cycle as intermediates and the cycle then converts these molecules to oxaloacetate.
• Lactate is converted to pyruvate by the lactate dehy-drogenase reaction and some amino acids also give rise to pyruvate.
• Therefore, for these precursors, the first step in the gluconeogenic pathway is the conversion of pyruvate to oxaloacetate.

17.2.2 The steps in gluconeogenesis are as follows

• Pyruvate is converted to oxaloacetate by carboxylation using the enzyme pyruvate carboxylase that is located in the mitochondrial matrix. The other enzymes of the pathway are located in the cytosol.

• This enzymes uses biotin as an activated carrier of CO₂, the reaction occurring in two stages:

• The oxaloacetate is now acted on by phosphenolpyruvate carboxykinase which simultaneously decarboxylates and phosphorylates it to form phosphoenolpyruvate (PEP), releasing CO2 and using GTP in the process:

• In the conversion of PEP to pyruvate in glycolysis synthesizes ATP, it is not surprising that the overall reversal of this step needs the input of a substantial amount of energy, one ATP for the pyruvate carboxylase step and one GTP for the PEP carboxykinase step.
• PEP is converted to fructose 1,6-bisphosphate in a series of steps that are a direct reversal of those in glycolysis, using the enzymes enolase, phosphoglycerate mutase, phosphoglycerate kinase, glyceraldehydes 3-phosphate dehydrogenase, triose phosphate isomerase and aldolase. This sequence of reactions uses one ATP and one NADH for each PEP molecule metabolized.
• Fructose 1,6-bisphosphate is dephosphorylated to form fructose 6-phosphate by the enzyme fructose 1,6-bisphosphatase, in the reaction:

• Fructose 6-phosphate is converted to glucose 6-phosphate by the glycolytic enzyme phosphoglucoisomerase.
• Glucose 6-phosphate is converted to glucose by the enzyme glucose 6-phosphatase. This enzyme is bound to the smooth endoplasmic reticulum and catalyzes the reaction:
  

17.3 Energy used

As would be expected, the synthesis of glucose by gluconeogenesis needs the input of energy. Two pyruvate molecules are required to synthesize one molecule of glucose. Energy is required at the following step

This compares with only two ATPs as the net ATP yield from glycolysis. Thus an extra four ATPs per glucose are required to reverse glycolysis.

• In fact, the glyceraldehydes 3-phosphate dehydrogenase reaction also consumes NADH, equivalent to two molecules of NADH for each molecule of glucose synthesized.
• Since each cytosolic NADH would normally be used to generate approximately three ATP molecules via the glycerol 3-phosphate shuttle and oxidative phosphorylation, this is equivalent to the input of another six ATPs per glucose synthesized.

17.4 Transport of Oxaloacetate

• Pyruvate carboxylase is a mitochondrial matrix enzyme whereas the other enzymes of gluconeogenesis are located outside the mitochondrion.
• Thus oxaloacetate, produced by pyruvate, needs to exit the mitochondrion. However, the inner mitochondrial membrane is not permeable to this compound.
• Thus oxaloacetate is converted to malate inside the mitochondrion by mitochondrial malate dehydrogenase, the malate is transported through the mitochondrial membrane by a special transport protein and then the malate is converted back to oxaloacetate in the cytoplasm by a cytoplasmic malate dehydrogenase.

 

17.5 Reciprocal Regulation of Glycolysis and Gluconeogenesis

• Glycolysis generates two ATPs net per glucose whereas gluconeogenesis uses four ATPs and two GTPs per glucose.
• Thus, if both glycolysis and gluconeogenesis were allowed to operate simultaneously, converting glucose to pyruvate and back again, the only net result would be the utilization of two ATPs and two GTPs, a so-called futile cycle.
• This is prevented by tight coordinate regulation of glycolysis and gluconeogenesis. Since many of the steps of the two pathways are common, the steps that are distinct in each pathway are the sites of this regulation, in particular the inter conversions between fructose 6-phosphate and fructose 1,6-bisphosphate and between PEP and pyruvate.

17.5.1 Regulation of PFK and fructose 1,6-bisphosphatase

• When the level of AMP is high, this indicates the need for more ATP synthesis. AMP stimulates PFK, increasing the rate of glycolysis, and inhibits fructose 1,6- bisphosphatase, turning off gluconeogenesis. Conversely, when ATP and citrate levels are high, this signals that no more ATP need be made. ATP and citrate inhibit PFK, decreasing the rate of glycolysis, and citrate stimulates fructose 1,6-bisphosphatase, increasing the rate of gluconeogenesis.
• Glycolysis and gluconeogenesis are made responsive to starvation by the level of the regulatory molecule fructose 2,6-bisphosphate (F-2,6-BP).
• F-2,6-BP is synthesized from fructose 6-phosphate and hydrolyzed back to fructose 6-phosphate by a single polypeptide with two enzymatic activities (PFK2 and FBPase2). Since F-2, 6-BP strongly stimulates PFK and inhibits fructose 1,6-bisphosphatase, glycolysis is stimulated and gluconeogenesis is inhibited in the fed animal. Conversely, during starvation, the low level of F-2,6-BP allows gluconeogenesis to predominate.

17.5.2 Regulation of pyruvate kinase, pyruvate carboxylase and PEP carboxykinase

• In liver, pyruvate kinase is inhibited by high levels of ATP and alanine so that glycolysis is inhibited when ATP and biosynthetic intermediates are already plentiful. Acetyl CoA is also abundant under these conditions and activates pyruvate carboxylase, favoring gluconeogenesis. Conversely, when the energy status of the cell is low, the ADP concentration is high and this inhibits both pyruvate carboxylase and PEP carboxykinase, switching off gluconeogenesis. At this time, the ATP level will be low so pyruvate kinase is not inhibited and glycolysis will operate.
• Pyruvate kinase is also stimulated by fructose 1,6-bisphosphate so that its activity rises when needed, as glycolysis speeds up. During starvation, the priority is to conserve blood glucose for the brain and muscle. Thus, under these conditions, pyruvate kinase in the liver is switched off. This occurs because the hormone glucagon is secreted into the bloodstream and activates a cAMP cascade that leads to the phosphorylation and inhibition of this enzyme.

17.6 The Cori Cycle

Under the limiting oxygen conditions experienced during vigorous exercise, the formation of NADH by glycolysis exceeds the ability of the respiratory chain to oxidize it back to NAD⁺. The pyruvate produced by glycolysis in muscle is then converted to lactate by lactate dehydrogenase, a reaction that regenerates. NAD⁺ and so allows glycolysis to continue to produce ATP. However, lactate is a metabolic dead-end in that it cannot be metabolized further until it is converted back to pyruvate. Lactate diffuses out of the muscle and is carried in the bloodstream to the liver. Here it diffuses into liver cells and is converted back to pyruvate by lactate dehydrogenase. The pyruvate is then converted to glucose by gluconeogenesis and the glucose is released back into the bloodstream ready to be taken up by skeletal muscle (and brain). This cycle of reactions is called the Cori Cycle.

Fig. 17.2 Cori cycle