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Three mechanisms are responsible for regulating the activity of  key enzymes of gluconeogenesis –

(1) Changes in the rate of enzyme synthesis (Induction/Repression)

(2) Covalent modification by reversible phosphorylation, and

(3) Allosteric effects. 

Gluconeogenesis and glycolysis are coordinated so that within a cell one pathway is relatively inactive while the other is highly active. The amounts and activities of the distinctive enzymes of each pathway are controlled so that both pathways are not highly active at the same time. Changes in the availability of substrates are responsible for most changes in metabolism either directly or indirectly acting via changes in hormone secretion.

1) Induction & Repression of Key Enzymes

The amounts and the activities of essential enzymes are regulated by hormones. The enzymes involved catalyze nonequilibrium (physiologically irreversible) reactions. Hormones affect gene expression primarily by changing the rate of transcription, as well as by regulating the degradation of mRNA.

Insulin, which rises subsequent to hyperglycemia after meals, stimulates the expression of glycolytic enzymes such as phosphofructokinase-1, pyruvate kinase, and the bifunctional enzyme (PFK-2 – Fr-2,6 bisphosphatase) that makes and degrades F-2,6-BP. Thus insulin stimulates glycolysis and inhibits gluconeogenesis.

Glucagon, which rises during starvation, inhibits the expression of glycolytic enzymes and stimulates instead the production of two key gluconeogenic enzymes, phosphoenolpyruvate carboxy kinase and fructose 1,6-bisphosphatase. As a result glycolysis is inhibited and gluconeogenesis is stimulated.

Transcriptional control in eukaryotes is much slower than allosteric control; it takes hours or days in contrast with seconds to minutes.

2) Covalent Modification by Reversible Phosphorylation

It is a rapid process. Glucagon and epinephrine, hormones that are responsive to a decrease in blood glucose, inhibit glycolysis and stimulate gluconeogenesis in the liver by increasing the concentration of cAMP. This in turn activates cAMP-dependent protein kinase, leading to the phosphorylation and inactivation of mainly two enzymes –

1) Pyruvate kinase- Pyruvate kinase exists in two forms, phosphorylated (inactive ) and dephosphorylated (active) forms. Insulin activates this enzyme by causing dephosphorylation  through stimulating phosphatase enzyme, while glucagon inactivates this enzyme by bringing about c AMP mediated phosphorylation (Figure-1).

In the inhibited state of pyruvate kinase, phosphoenol pyruvate is channeled towards glucose production .

 Reaction catalyzed by pyruvate kinase

Figure- 1-Pyruvate kinase catalyzes the conversion of phosphoenol pyruvate to pyruvate. This is a regulatory enzyme and catalyzes the last step of glycolysis.ATP is produced at this step by substrate level phosphorylation. The enzyme is inhibited by excess of ATP and alanine while it is stimulated by Fr, 1,6 bisphosphate the product of PFK-1 catalyzed reaction.

2) Bifunctional enzyme (PFK-2- Fr 2,6 bisphosphatase)– The second enzyme to be affected by phosphorylation cascade is phosphofructokinase-2 that is inactivated but Fr 2,6 bisphosphatase becomes active upon phosphorylation (Figure-2 ). As a result net concentration of fructose 2,6 bisphosphate is lowered , Phosphofructo kinase-1 enzyme gets inhibited but Fr 1,6 bisphosphatase enzyme gets stimulated (See the details below) .


 Reciprocal regulation of glycolysis and gluconeogenesis

Figure-2-The activities of PFK2 and FBPase2 are reciprocally controlled by phosphorylation of a single serine residue. When glucose is scarce, a rise in the blood level of the hormone glucagon triggers a cyclic AMP cascade, leading to the phosphorylation of this bifunctional enzyme by protein kinase A. This covalent modification activates FBPase2 and inhibits PFK2, lowering the level of F-2,6-BP. Thus, glucose metabolism by the liver is curtailed. Conversely, when glucose is abundant, the enzyme loses its attached phosphate group. This covalent modification activates PFK2 and inhibits FBPase2, raising the level of F-2,6-BP and accelerating glycolysis. Thus, when glucose is abundant as during fed state, glycolysis is stimulated and when glucose is limiting as during fasting or starvation glycolysis is inhibited.  

3) Allosteric Modification

It is an instantaneous process.

a) Role of Acetyl co A

In gluconeogenesis, pyruvate carboxylase, which catalyzes the synthesis of oxaloacetate from pyruvate, requires acetyl-CoA as an allosteric activator. The addition of acetyl-CoA results in a change in the tertiary structure of the protein, lowering the Km for bicarbonate. This means that as acetyl-CoA is formed from pyruvate, it automatically ensures the provision of oxaloacetate and, therefore, its further oxidation in the citric acid cycle, by activating pyruvate carboxylase (Figure-3). The activation of pyruvate carboxylase and the reciprocal inhibition of pyruvate dehydrogenase by acetyl-CoA derived from the oxidation of fatty acids explain the action of fatty acid oxidation in sparing the oxidation of pyruvate and in stimulating gluconeogenesis. The reciprocal relationship between these two enzymes alters the metabolic fate of pyruvate as the tissue changes from carbohydrate oxidation (glycolysis) to gluconeogenesis during the transition from the fed to fasting state.

b)  Role of ATP and AMP

The interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate is stringently controlled (Figure-3). Phosphofructokinase (phosphofructokinase-1) occupies a key position in regulating glycolysis and is also subject to feedback control.  AMP stimulates phosphofructokinase, whereas ATP and citrate inhibit it. Fructose 1,6- bisphosphatase, on the other hand, is inhibited by AMP and activated by citrate. A high level of AMP indicates that the energy charge is low and signals the need for ATP generation. Conversely, high levels of ATP and citrate indicate that the energy charge is high and that biosynthetic intermediates are abundant. Under these conditions, glycolysis is nearly switched off and gluconeogenesis is promoted.

The interconversion of phosphoenolpyruvate and pyruvate also is precisely regulated. Pyruvate kinase is controlled by allosteric effectors and by phosphorylation. High levels of ATP and alanine, which signal that the energy charge is high and that building blocks are abundant, inhibit the enzyme in liver. Likewise, ADP inhibits phosphoenolpyruvate carboxykinase. Hence, gluconeogenesis is favored when the cell is rich in biosynthetic precursors and ATP.

c) Role of Fructose 2,6-Bisphosphate

The most potent positive allosteric activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase in liver is fructose 2,6-bisphosphate. It relieves inhibition of phosphofructokinase-1 by ATP and increases the affinity for fructose 6-phosphate. It inhibits fructose 1,6-bisphosphatase by increasing the Km for fructose 1,6-bisphosphate. Its concentration is under both substrate (allosteric) and hormonal control (covalent modification) (Figure-2). Fructose 2,6-bisphosphate is formed by phosphorylation of fructose 6-phosphate by phosphofructokinase-2. The same enzyme protein is also responsible for its breakdown, since it has fructose 2,6-bisphosphatase activity. This bifunctional enzyme is under the allosteric control of fructose 6-phosphate, which stimulates the kinase and inhibits the phosphatase.

Hence, when there is an abundant supply of glucose, the concentration of fructose 2,6-bisphosphate increases, stimulating glycolysis by activating phosphofructokinase-1 and inhibiting fructose 1,6-bisphosphatase. In the fasting state, glucagon stimulates the production of cAMP, activating cAMP-dependent protein kinase, which in turn inactivates phosphofructokinase-2 and activates fructose 2,6-bisphosphatase by phosphorylation. Hence, gluconeogenesis is stimulated by a decrease in the concentration of fructose 2,6-bisphosphate, which inactivates phosphofructokinase-1 and relieves the inhibition of fructose 1,6-bisphosphatase (Figure-2).


 Allosteric regulation

Figure-3- Reciprocal Regulation of Gluconeogenesis and Glycolysis in the Liver. PFK-1 and Fr1.6 bisphophase are reciprocally regulated. The negative modifiers of PFK-1(ATP and citrate act as positive modifiers of Fr1,6 bisphosphatase enzyme. Similarly is the case of positive modifiers of PFK-1 which act as negative modifiers of Fr1,6 bisphosphatase. Another regulation is at the level of Pyruvate kinase and pyruvate carboxylase enzymes. Pyruvate carboxylase is stimulated by Acetyl co A and inhibited by ADP while pyruvate kinase(glycolytic enzyme)is inhibited by ATP and Alanine but stimulated by ADP (low energy state).


Gluconeogenesis is stimulated under conditions of fasting and starvation under the effect of glucagon and catecholamines. The regulation is mainly brought about by induction/repression , covalent and allosteric modifications. The key enzymes of regulation are pyruvate carboxylase , phospho enol pyruvate carboxy kinase and fr1,6 bisphosphatase which are stimulated if there is need for glucose production . These effects are reversed in the fed state in the presence of insulin where the glycolytic enzymes are stimulated to promote glucose utilization and enzymes of gluconeogenesis are inhibited. Thus glycolysis and gluconeogenesis do not occur at the same pace at the same time, they are reciprocally regulated.

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