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Q.1-Comment on the statement – ‘Glycolysis and Gluconeogenesis are reciprocally regulated’


Discuss the regulation of Gluconeogenesis. 

Answer- 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. Three mechanisms are responsible for regulating the activity of enzymes (1) changes in the rate of enzyme synthesis, (Induction/Repression) (2) covalent modification by reversible phosphorylation, and (3) allosteric effects.

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 eating, stimulates the expression of phosphofructokinase-1, pyruvate kinase, and the bifunctional enzyme that makes and degrades Fructose-2,6-BP (PFK-2 and Fructose 2,6 bisphosphatase). PFK2 is mainly affected by Insulin to increase the concentration of Fructose-2,6 bisphosphate.

Glucagon, which rises during starvation, inhibits the expression of these enzymes and stimulates instead the production of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and fructose 1,6-bisphosphatase. 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 pyruvate kinase. They also affect the concentration of fructose 2,6-bisphosphate by activating Fructose 2,6 bisphoaphatase and therefore glycolysis and gluconeogenesis are appropriately regulated. Low concentration of fructose 2,6  bisphosphate reduces the activity of PFK-I and thereby reduces the rate of glycolysis, while at the same time since fructose 2,6  bisphosphate  inhibits the activity of Fr 1,6 bis phosphatase,its low concentration overcomes the inhibition and gluconeogensis is stimulated.

3) Allosteric Modification

It is an instantaneous process.The role of various allosteric modifiers can be explained as follows-

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 (CO2 is added in the form of bicarbonate).

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-1). 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 is a positive modifier of PFK-1, and a negative modifier of Fr, 1,6 bisphoaphatase.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-1). 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-1- Reciprocal Regulation of Gluconeogenesis and Glycolysis in the Liver,  The level of fructose 2,6-bisphosphate is high in the fed state and low in starvation. Another important control is the inhibition of pyruvate kinase by phosphorylation during starvation.

Q.2-What do you know about substrate cycle or futile cycle? 

Answer- A pair of reactions such as the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate and its hydrolysis back to fructose 6-phosphate is called a substrate cycle. Both reactions are not simultaneously fully active in most cells, because of reciprocal allosteric controls. However, the results of isotope-labeling studies have shown that some fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate in gluconeogenesis. There also is a limited degree of cycling in other pairs of opposed irreversible reactions. This cycling was regarded as an imperfection in metabolic control, and so substrate cycles have sometimes been called futile cycles

In muscle, both phosphofructokinase and fructose 1,6-bisphosphatase have some activity at all times, so that there is indeed some measure of (wasteful) substrate cycling. This permits the very rapid increase in the rate of glycolysis necessary for muscle contraction. At rest, the rate of phosphofructokinase activity is some tenfold higher than that of fructose 1,6-bisphosphatase; in anticipation of muscle contraction, the activities of both enzymes increase, fructose 1,6-bisphosphatase ten times more than phosphofructokinase, maintaining the same net rate of glycolysis. At the start of muscle contraction, the activity of phosphofructokinase increases further, and that of fructose 1,6-bisphosphatase falls, so increasing the net rate of glycolysis (and hence ATP formation) as much as a thousand fold. Indeed, there are pathological conditions, such as malignant hyperthermia, in which control is lost and both pathways proceed rapidly with the concomitant generation of heat by the rapid, uncontrolled hydrolysis of ATP.

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