A pyruvate and lactate

Pyruvate is formed in the liver from lactate and alanine. Lactate dehydrogenase oxidizes lactate to pyruvate to form NADH. Alanine aminotransferase transfers the amino group from alanine to α-ketoglutarate to form glutamate and pyruvate.

B Glucogenic amino acids

Amino acids that are catabolized to pyruvate or CTC metabolites are potential substrates for gluconeogenesis (pyruvate and CTC metabolites are able to form oxaloacetate and be involved in gluconeogenesis). Such amino acids are called glucogenic. The amino acids alanine and glutamine, which carry amino groups from the muscles to the liver, are especially important glucogenic amino acids in our body.

To Glycerol

Glycerol enters our body with food and is synthesized in the liver and adipose tissue. During starvation, triacylglycerols (TAGs) in adipocytes are broken down into glycerol and fatty acids. Glycerol enters the bloodstream and is transported to the liver. Further, during two enzymatic reactions, it is converted to dihydroxyacetone phosphate, which is a metabolite of glycolysis and gluconeogenesis.

G Fatty acids

Fatty acids with an odd number of atoms are oxidized to form propionyl-CoA. It is converted to methylmalonyl-CoA, which forms succinyl-CoA in another enzymatic reaction. Succinyl-CoA is a metabolite of TCA, therefore it is potentially able to be involved in gluconeogenesis. This is confirmed by studies with carbon isotopes C-14.

2.3 Reactions of gluconeogenesis

A Reaction equations

	Pyruvate + ATP + HCO3 - + H2 O Oxaloacetate + ADP + Fn + 2H+
	Oxaloacetate + GTP Phosphoenolpyruvate + GDP + CO2
	Phosphoenolpyruvate + H2 O 2-Phosphoglycerate
	2-Phosphoglycerate 3-Phosphoglycerate
	3-Phosphoglycerate + ATP 1,3-Bisphosphoglycerate + ADP
	1,3-Bisphosphoglycerate + NADH + H+ Glyceraldehyde-3-phosphate + NAD+ + Fn (× 2)
	Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate

8. Dihydroxyacetone phosphate + Glyceraldehyde-3-phosphate Fructose-1,6-bisphosphate

9. Fructose 1,6-bisphosphate+ H2 O Fructose-6-phosphate + Fn

10. Fructose 6-phosphate Glucose-6-phosphate

11. Glucose-6-phosphate + H2 O Glucose + Fn

32 Chapter 2 Gluconeogenesis

B Energy Barriers and Unique Reactions of Gluconeogenesis

IN glycolysis are irreversible 1st, 3rd and 10th reactions. These reactions only go in one direction and are called energy barriers. In gluconeogenesis, they are bypassed by 4 reactions. The remaining reactions are common for glycolysis and gluconeogenesis, since they are able to go both in the forward and reverse directions, depending on the excess of the product or substrate.

Reaction 1

In the first reaction of gluconeogenesis pyruvate carboxylase catalyzes the carboxylation of pyruvate with the formation of oxaloacetate with the expenditure of 1 molecule of ATP. The reaction proceeds in mitochondria in two phases:

1. Breaking the macroergic bond in the ATP molecule with the formation of ADP. A high-energy carboxyphosphate molecule is formed, which then binds to biotin and is "activated".

2. The active carboxyl group is transferred from carboxybiotin to the pyruvate molecule to form oxaloacetate.

Reaction 2

Gluconeogenesis reactions 33

Hormonal regulation:

Some hormones have a stimulating effect on the expression of the PEPcarboxykinase gene.

The second reaction of gluconeogenesis leads to the formation of a high-energy molecule - phosphoenolpyruvate. During this reaction, oxaloacetate is decarboxylated at the cost of 1 molecule of GTP.

Rice. 7. Transport of oxaloacetate and phosphoenolpyruvate from mitochondria to the cytosol.

This reaction is catalyzed by an enzyme PEP-carboxykinase. In humans, it is found both in mitochondria and in the cytosol. However, in some tissues it is present only in the cytosol, so oxaloacetate must be transported there from the mitochondria. The inner mitochondrial membrane has protein carriers for malate and aspartate, but not for oxaloacetate, so it must be converted to one of these compounds, for which there are transport proteins in the membrane.

There are two ways to do this (see Fig. 7): 1) oxaloacetate is reduced to malate; 2) oxaloacetate accepts an amino group in the transamination reaction and forms aspartate. The first path requires the participation of NADH. The second has little in the liver: aspartate, which is transferred to the cytosol from mitochondria, is deaminated in the urea cycle to oxaloacetate.

Reactions 3-8

These reactions are catalyzed by glycolysis enzymes, however, they do not proceed in the forward (for glycolysis), but in the opposite direction.

Reaction 9

In the 9th reaction of gluconeogenesis, fructose-1,6-bisphosphate is hydrolyzed to fructose-6-phosphate with the help of an enzyme fructose-1,6-bisphosphatase. Several allosteric regulators of this enzyme are known (listed above).

Reaction 10

Fructose-6-phosphate isomerizes to glucose-6-phosphate. This reaction is catalyzed by the glycolytic enzyme phosphoglucoisomerase.

Reaction 11

The final reaction of gluconeogenesis is the dephosphorylation of glucose in the endoplasmic reticulum, catalyzed by glucose-6-phosphate- zoi. This reaction produces glucose. The phosphoric acid residue and glucose are carried back into the cytosol by the T3 and T2 proteins, respectively. Further, free glucose is carried outside the cell by GLUT2 proteins.

The enzyme of this reaction is found only in the liver, kidneys and small intestine, so these organs are able to export glucose into the blood. The remaining cells (not all) synthesize glucose only for their own needs.

Glycogen stores in the liver are limited and after 12-18 hours of fasting they disappear completely. Many cells need a constant supply of glucose (erythrocytes, neurons, muscle cells under anaerobic conditions). Gluconeogenesis is the metabolic pathway that solves this problem. Gluconeogenesis is the metabolic pathway for the conversion of non-carbohydrate compounds into glucose. Many compounds may be involved in this process. These are lactic acid, and PVC, and amino acids that decompose to pyruvate (alanine, cysteine, glycine, serine, threonine, etc.), and glycerol, and propiononyl-CoA, and Krebs cycle substrates (oxalacetate, etc., Fig. 5.8 ).

Gluconeogenesis is a modification of processes such as glycolysis and the Krebs cycle. Most of the reactions of glycolysis are reversible. The exceptions are three reactions catalyzed by hexokinase, phosphofructokinase-1 and pyruvate kinase, and special enzymes are used to overcome these reactions, which are called the key reactions of gluconeogenesis. These enzymes are concentrated in the liver and renal cortex. Table 5.2. the names of enzymes catalyzing irreversible reactions of glycolysis and corresponding key enzymes of gluconeogenesis are given.

Table 5.2. Key Enzymes of Glycolysis and Glyconeogenesis

When such enzymes work together, there is a problem of the so-called. “empty” substrate cycles. Under the condition that the forward and reverse reactions are catalyzed by different enzymes, the product obtained in the forward reaction becomes the substrate of another enzyme, which catalyzes the reverse reaction, turning the product back into the substrate of the first enzyme. There is a danger of “idle” scrolling of the reaction substrates. The problem is solved by the organization of multilevel regulation, including reciprocal allosteric regulation and covalent modification of the structure of enzymes.

It is generally accepted that the initial stage of gluconeogenesis is the reaction that bypasses the pyruvate kinase reaction of glycolysis. Pyruvate kinase is an object of influence of regulatory systems (Fig. 5.9) that control the rate of glycolysis, therefore, under conditions favorable for gluconeogenesis (starvation, etc.), the activity of this enzyme should be inhibited. This is facilitated by an increase in the amount of alanine, which is an allosteric inhibitor of pyruvate kinase, and an increase in glucagon secretion. The latter stimulates the formation of cAMP in hepatocytes, which activates protein kinase A. Phosphorylation of pyruvate kinase under the influence of protein kinase A causes its transition to an inactive state. Inhibition of pyruvate kinase favors the inclusion of gluconeogenesis.

Fig.5.9. Regulation of pyruvate kinase activity

Fig.5.10. Main substrates and enzymes of gluconeogenesis:

1-lactate dehydrogenase; 2– pyruvate carboxylase; 3-malate dehydrogenase; 4-phosphoenolpyruvate carboxykinase; 5-fructose-1,6-diphosphatase; 6- glucose-6-phosphatase; 7-glycerol kinase; 8-a-glycerol phosphate dehydrogenase

If the conversion of phosphoenolpyruvate to PVA, which is catalyzed by pyruvate kinase, is one chemical reaction, then the reverse conversion of PVA to phosphoenolpyruvate requires several reactions. The first reaction is the carboxylation of pyruvate. The reaction is catalyzed by pyruvate carboxylase and proceeds with the participation of carboxybiotin, the active form of CO 2 in the cell. The product of carboxylation, oxaloacetate, occupies a special place in the metabolism of mitochondria, where this reaction takes place. It is the most important substrate of the Krebs cycle (see below) and its exit from mitochondria is difficult. To overcome the mitochondrial membrane, oxaloacetate is reduced by mitochondrial malate dehydrogenase to easily penetrating malic acid through the membrane. The latter, having left the mitochondria, is oxidized again in the cytosol to oxaloacetate already under the influence of cytosolic malate dehydrogenase. Further conversion of oxaloacetate to FEPVC occurs in the cytosol of the cell. Here, with the help of phosphoenolpyruvate carboxykinase, okaloacetate is decarboxylated with the expenditure of energy released during the hydrolysis of GTP and PEPVC is formed.

After the formation of FEPVC, subsequent reactions represent reversible reactions of glycolysis. For every two 3-PHA formed, one molecule is converted into FDA with the participation of phosphotriose isomerase, and both trioses are condensed into fructose-1,6-diphosphate under the influence of aldolase. A certain amount of FDA is formed by the oxidation of glycerol phosphate, which occurs under the influence of glycerol kinase from glycerol, which enters the liver from adipose tissue. It is the only lipid substrate involved in gluconeogenesis. The conversion of fructose-1,6-diphosphate to fructose-6-phosphate is catalyzed by fructose-1,6-diphosphatase-1 . This is followed by the reverse reaction of glycolysis. The final reaction of gluconeogenesis is catalyzed by the enzyme glucose-6-phosphatase, which catalyzes the hydrolysis of glucose-6-phosphate and the resulting free glucose can exit the cell.

The total reaction for the synthesis of a glucose molecule:

2 PVC + 4 ATP + 2 GTP + 2NADH + 2H + + 6H2O → Glucose + 2NAD + + 4ADP+ 2 GDP + 6 Fn + 6H +

Thus, the synthesis of one glucose molecule “costs” the cell six macroergs. 2 ATP molecules are consumed to activate CO2, 2 GTP molecules are used in the phosphoenolpyruvate carboxykinase reaction, and 2 ATP molecules are used to form 1,3-diphosphoglyceric acid.

Gluconeogenesis is activated in liver cells during fasting, after prolonged exercise, when eating food rich in proteins with a low content of carbohydrates, etc.

The intensity of the process depends on the number of substrates, and activity, and the number of key enzymes of glycolysis and gluconeogenesis.

The main suppliers of substrates for the liver are muscles, erythrocytes, and adipose tissue. The latter has rather limited possibilities, since only glycerol can be used for the synthesis of glucose, and this is only about 6% of the weight of a fat droplet.

Lactate, produced from muscle work under anaerobic conditions or from erythrocytes, is a more significant source of glucose. The most important sources are glycogenic amino acids, which can come from a protein-rich diet or from muscle under fasting conditions.

Rice. 5.11. Corey cycle

In order to continuously supply glucose to cells for which it is the main source of energy, but they cannot oxidize it completely due to the absence of mitochondria (erythrocytes) or due to work in anaerobic conditions, cyclic processes for the exchange of substrates are established between the liver and these cells. One of these is the Cori cycle: lactic acid formed in the muscles (erythrocytes) enters the general circulation, is captured by the liver and used by it as a substrate for gluconeogenesis; the glucose synthesized in this case is released into the bloodstream and metabolized by muscles or red blood cells for energy (Fig. 5.11).

Fig. 5.12. Alanine cycle

Unlike the Cori cycle, the alanine cycle (Fig. 5.12) proceeds under the condition that peripheral tissues consume oxygen and require mitochondria. When eating food rich in proteins or during fasting, there is a rather active exchange between the liver and muscles of alanine and glucose. Alanine from the muscles is transferred to the liver cells, where it is reaminated and PVC is used to synthesize glucose. As needed, glucose enters the muscles and is oxidized to PVC, and then, by transamination, it turns into alanine, which can repeat this cycle again. Energetically, this is a more profitable path than the Corey cycle.

Synthesis of glucose from lactic acid

During physical activity in muscles a large amount of lactic acid is produced, especially if the load is intense, maximum power. Also, lactic acid is continuously formed erythrocytes regardless of the state of the organism. With the blood flow, it enters the hepatocyte and here it turns into pyruvate. Further reactions proceed according to the classical scheme.

The overall reaction of gluconeogenesis from lactic acid is:

Lactate + 4ATP + 2GTP + 2H 2 O → Glucose + 4ADP + 2GDP + 6P n

Synthesis of glucose from amino acids

A number of amino acids are glucogenic, that is, their carbon skeletons are more or less able to be incorporated into glucose. Most of the amino acids are except leucine and lysine, whose carbon atoms never participate in the synthesis of carbohydrates.

As an example of the synthesis of glucose from amino acids, consider the participation in this process of glutamate, aspartate, serine and alanine.

Aspartic acid(after the transamination reaction) and glutamic acid(after deamination) are converted into TCA metabolites, respectively, into oxaloacetate and α-ketoglutarate.

Alanine, transaminating, forms pyruvic acid, which is capable of carboxylation to oxaloacetate. Oxaloacetate, being the first element in the process of gluconeogenesis, is then included in the synthesis of glucose.

Serene in a three-step reaction, under the influence of serine dehydratase, it loses its amino group and turns into pyruvate, which enters gluconeogenesis.

Inclusion of amino acids in glucose synthesis

Synthesis of glucose from glycerol

During physical exertion under the influence of adrenaline or during fasting under the influence of glucagon and cortisol, adipocytes actively breakdown of triacylglycerols(lipolysis). One of the products of this process is alcohol. glycerol that enters the liver. Here it is phosphorylated, oxidized to dihydroxyacetone phosphate, and involved in gluconeogenesis reactions.

16.2.1. Gluconeogenesis is the biosynthesis of glucose from various non-carbohydrate compounds. The biological role of gluconeogenesis is to maintain a constant level of glucose in the blood, which is necessary for the normal energy supply of tissues, which are characterized by a continuous need for carbohydrates. This is especially true of the central nervous system.

The role of gluconeogenesis increases with insufficient intake of carbohydrates from food. So, in the body of a starving person, up to 200 g of glucose per day can be synthesized. Gluconeogenesis reacts faster than other metabolic processes to dietary changes: the introduction of a large amount of proteins and fats with food activates the processes of gluconeogenesis; an excess of carbohydrates, on the contrary, inhibits the formation of glucose.

Intense physical activity is accompanied by a rapid depletion of glucose reserves in the body. In this case, gluconeogenesis is the main way to replenish carbohydrate resources, preventing the development of hypoglycemia. Gluconeogenesis in the body is also closely related to the processes of neutralizing ammonia and maintaining the acid-base balance.

16.2.2. main site of glucose biosynthesis de novo is the liver. Gluconeogenesis also occurs in the cortical layer of the kidneys. It is generally accepted that the contribution of the kidneys to gluconeogenesis under physiological conditions is about 10% of the glucose synthesized in the body; in pathological conditions, this proportion can increase significantly. Insignificant activity of gluconeogenesis enzymes was found in the mucosa of the small intestine.

16.2.3. The sequence of reactions of gluconeogenesis is the reversal of the corresponding reactions of glycolysis. Only three reactions of glycolysis are irreversible due to the significant energy shifts that occur during their course:

a) glucose phosphorylation; b) phosphorylation of fructose-6-phosphate; c) conversion of phosphoenolpyruvate to pyruvate.

These energy barriers are bypassed by key enzymes of gluconeogenesis.

The reverse conversion of pyruvate to phosphoenolpyruvate requires the participation of two enzymes. The first one is pyruvate carboxylase - catalyzes the formation of oxaloacetate (Figure 16.4, reaction 1). The coenzyme of pyruvate carboxylase is biotin (vitamin H). The reaction proceeds in mitochondria. Its role is also to replenish the oxaloacetate fund for the Krebs cycle.

All subsequent reactions of gluconeogenesis proceed in cytoplasm . The mitochondrial membrane is impermeable to oxaloacetate, and it is transported into the cytoplasm in the form of other metabolites: malate or aspartate. In the cytoplasm, these compounds are again converted to oxaloacetate. Starring phosphoenolpyruvate carboxykinase from oxaloacetate, phosphoenolpyruvate is formed (Figure 16.4, reaction 2).

Phosphoenolpyruvate, as a result of the reversal of a number of glycolysis reactions, passes into fructose-1,6-diphosphate. The conversion of fructose-1,6-diphosphate to fructose-6-phosphate is catalyzed by fructose diphosphatase (Figure 16.4, reaction 3).

Fructose-6-phosphate isomerizes to glucose-6-phosphate. The final reaction of gluconeogenesis is the hydrolysis of glucose-6-phosphate with the participation of the enzyme glucose-6-phosphatase (Figure 16.4, reaction 4).

Figure 16.4. Bypass reactions of gluconeogenesis .

16.2.4. The main sources of glucose in gluconeogenesis are lactate, amino acids, glycerol, and metabolites of the Krebs cycle.

lactate is the end product of anaerobic glucose oxidation. May be involved in gluconeogenesis after oxidation to pyruvate in the lactate dehydrogenase reaction (see section "Glycolysis", Figure 15.4, reaction 11). During prolonged physical work, the main source of lactate is the skeletal muscles, in the cells of which anaerobic processes predominate. The accumulation of lactic acid in the muscles limits their performance. This is due to the fact that with an increase in the concentration of lactic acid in the tissue, the pH level decreases (lactic acidosis). The change in pH leads to the inhibition of enzymes of the most important metabolic pathways. In the utilization of the resulting lactic acid, an important place belongs to glucose-lactate Cori cycle (Figure 16.5).

Figure 16.5. Corey cycle and glucose-alanine cycle (explanations in the text).

Glucogenic amino acids which include most protein amino acids. The leading place in gluconeogenesis among amino acids belongs to alanine , which can be converted to pyruvate by transamination. During fasting, physical work and other conditions, the body functions glucose-alanine cycle , similar to the Cori cycle for lactate (Figure 16.2). The existence of the alanine-glucose cycle prevents the poisoning of the body, since there are no enzymes in the muscles that utilize ammonia. As a result of training, the power of this cycle increases significantly.

Other amino acids can, like alanine, be converted to pyruvate, as well as to Krebs cycle intermediates (α-ketoglutarate, fumarate, succinyl-CoA). All these metabolites are capable of being converted to oxaloacetate and involved in gluconeogenesis.

Glycerol- a product of lipid hydrolysis in adipose tissue. This process is greatly enhanced during fasting. In the liver, glycerol is converted to dihydroxyacetone phosphate, an intermediate product of glycolysis and can be used in gluconeogenesis.

Fatty acid And acetyl-CoA are not glucose precursors. The oxidation of these compounds provides energy for the process of glucose synthesis.

16.2.5. Energy balance. The pathway for the synthesis of glucose from pyruvate (Figure 16.6) contains three reactions, accompanied by the consumption of energy by ATP or GTP:

a) the formation of oxaloacetate from pyruvate (an ATP molecule is consumed); b) the formation of phosphoenolpyruvate from oxaloacetate (a GTP molecule is consumed); c) reversal of the first substrate phosphorylation - the formation of 1,3-diphosphoglycerate from 3-phosphoglycerate (an ATP molecule is consumed).

Each of these reactions is repeated twice, since 2 pyruvate molecules (C3) are used to form 1 glucose molecule (C6). Therefore, the energy balance for the synthesis of glucose from pyruvate is 6 molecules of nucleoside triphosphates (4 ATP molecules and 2 GTP molecules). When using other precursors, the energy balance of glucose biosynthesis is different.

Figure 16.6. Energy balance of glucose biosynthesis from lactate.

16.2.6. regulation of gluconeogenesis. The rate of gluconeogenesis is determined by the availability of substrates - precursors of glucose. An increase in the blood concentration of any of the precursors of glucose leads to stimulation of gluconeogenesis.

Some metabolites are allosteric effectors of gluconeogenesis enzymes. For example, acetyl-CoA in elevated concentrations allosterically activates pyruvate carboxylase, which catalyzes the first reaction of gluconeogenesis. Adenosine monophosphate, on the contrary, has an inhibitory effect on fructose diphosphatase, and excess glucose inhibits glucose-6-phosphatase.

The pancreatic hormone glucagon, adrenal hormones adrenaline and cortisol increase the rate of glucose biosynthesis in the body by increasing the activity of key enzymes of gluconeogenesis or by increasing the concentration of these enzymes in cells. The pancreatic hormone insulin helps to reduce the rate of gluconeogenesis in the body.

From the blood into tissue cells, glucose enters by the mechanism of facilitated diffusion with the participation of carrier proteins. The exceptions are muscle cells and adipose tissue, where facilitated diffusion is regulated by insulin (pancreatic hormone). In the absence of insulin, the plasma membrane of these cells is impermeable to glucose because it does not contain glucose carrier proteins.

Glucose transporters(GLUT) are found in all tissues.

Types of GLUT	Localization in organs
	Predominantly in the brain, placenta, kidneys, large intestine
	Mainly in the liver, kidneys, β-cells of the islets of Langerhans, enterocytes
	Many tissues including brain, placenta, kidneys
	In muscles (skeletal, cardiac), adipose tissue
(insulin dependent)	Contained in the absence of insulin almost entirely in the cytoplasm
	In the small intestine. Possibly a fructose carrier.

The described 5 types of GLUTs have similar primary structure and domain organization.

GLUT-1 ensures a steady flow of glucose into the brain;

GLUT-2 is found in the cells of organs that secrete glucose into the blood. It is with the participation of GLUT-2 that glucose passes into the blood from enterocytes and the liver. GLUT-2 is involved in the transport of glucose into pancreatic β-cells;

GLUT-3 has a greater affinity for glucose than GLUT-1. It also provides a constant supply of glucose to the cells of the nervous and other tissues;

GLUT-4 is the main carrier of glucose into muscle cells and adipose tissue;

GLUT-5 is found mainly in the cells of the small intestine. Its functions are not well known.

All types of GLUTs can be found both in the plasma membrane and in membrane vesicles in the cytoplasm. However, only GLUT-4, localized in cytoplasmic vesicles, is incorporated into the plasma membrane of muscle and adipose tissue cells with the participation of the pancreatic hormone insulin. Due to the fact that the supply of glucose to the muscles and adipose tissue depends on insulin, these tissues are called insulin-dependent.

The effect of insulin on the movement of glucose transporters from the cytoplasm to the plasma membrane.

1 - binding of insulin to the receptor; 2 - the site of the insulin receptor, facing the inside of the cell, stimulates the movement of glucose transporters; 3, 4 - transporters in the composition of the vesicles containing them move to the plasma membrane of the cell, are included in its composition and transfer glucose into the cell.

Various disorders in the work of glucose transporters are known. An inherited defect in these proteins may underlie non-insulin dependent diabetes mellitus. Violations of GLUT-4 function are possible at the following stages:

transmission of the insulin signal about the movement of this transporter to the membrane;

movement of the transporter in the cytoplasm;

inclusion in the membrane;

lacing off the membrane, etc.