Gierke's disease: causes, symptoms, treatment. Hepatic-hypoglycemic abnormalities

PHOSPHATASES- enzymes that catalyze the cleavage of ester bonds in monoesters of phosphoric acid with the formation of free orthophosphate; belong to the class of hydrolases, a subclass of phosphorus monoester hydrolases (EC 3.1.3).

F. are present in all animals and plant organisms and occupy important place in cellular metabolism; biol. F.'s role is associated with their participation in the metabolism of carbohydrates (see Carbohydrate metabolism), nucleotides (see Nucleic acids) and phospholipids (see Phosphatides), as well as with the formation bone tissue(see Bone). Changes in the activity of certain F. in the blood serve as a valuable diagnostic sign for a number of diseases. A genetically determined violation of the synthesis or enzymatic usefulness of certain enzymes is the cause of a severe hereditary disease (see Hypophosphatasia).

By the nature of their catalytic action, all phosphorus enzymes are phosphomonoesterases, which cleave the ester bond hydrolytically. The systematic name of these enzymes always includes the term “hydrolase” (the name “phosphatase” is a working name derived from the name of the substrate). F. can be considered as phosphotransferases (q.v.), since they are capable of catalyzing the transfer of a phosphate residue to molecules of acceptors other than water, but since water is physiologically the main and most active acceptor, phosphatases are classified as hydrolases (q.v.).

Substrate specificity

Most F. are among the enzymes (see) that have a relatively broad substrate specificity. However, some F. are distinguished by a limited range of converted substrates. These are, first of all, enzymes that act on phosphorus derivatives of sugars, as well as nucleotidases (see), which break down mononucleotides. In many tissues, phosphorus is represented by multiple forms, differing in their catalytic and physical properties(see Isoenzymes). Phosphatases from different biol. sources, differences in substrate specificity and catalytic activity are also observed. Some enzymes show similarities with enzymes belonging to other groups. Thus, there are phosphorylations that can catalyze transphosphorylation reactions (see) or cleave the acid-anhydride pyrophosphate bond (see Pyrophosphatases). For example, glucose-6-phosphatase (D-glucose-b-phosphate phosphohydrolase; EC 3.1.3.9) is very similar in substrate specificity and catalytic properties to phosphotransferases (EC 2.7.1.62 and 2.7.1.79), as well as inorganic pyrophosphatase (EC 3.6 .1.1).

Mechanism of action

For many phosphorus, the three-dimensional structure of their molecules has been established and detailed chemistries have been proposed. mechanisms of catalytic action. It is assumed that several cooperative (combined) participants take part in the process of the catalytic act. various groups, localized on the surface of the enzyme molecule in the active center. One of these enzymes is glucose-6-phosphatase. This enzyme, associated with the microsomal fraction of cells, along with the hydrolysis of glucose-6-phosphate, catalyzes the transfer of a phosphate group from inorganic pyrophosphate (see Phosphorus) to glucose (see), as well as the exchange reaction between glucose and glucose-6-phosphate. Studies of the kinetics of hydrolytic, transferase and exchange reactions (see Kinetics of biological processes) have shown that their mechanism is in the nature of a two-stage transfer, in which a phosphoenzyme, or phosphoryl enzyme, is formed as an intermediate compound. In this case, the transferred phosphate group in the enzyme molecule binds to the histidine residue (see). To exhibit activity, glucose-b-phosphatase requires a divalent metal ion. In accordance with the proposed (with some simplification) reaction mechanism, the metal ion binds to the negatively charged phosphate group of the substrate, and the reactive histidine residue, which has nucleophilic properties, binds to the phosphorus atom, which leads to the formation of a phosphoenzyme. The latter then either undergoes hydrolysis or reacts with the nucleophilic groups of acceptor molecules (eg, with the hydroxyl groups of sugars) to form the final reaction products and release the phosphate-free enzyme.

Not all phosphatase reactions occur with the formation of an intermediate phosphoenzyme, in which the histidine residue is phosphorylated. When the reaction is catalyzed by alkaline phosphatase (EC 3.1.3.1), isolated from mammalian tissues or bacteria, the serine residue in the enzyme molecule undergoes phosphorylation (see). The enzyme is a zinc-containing metalloprotein (see Metalloproteins), in which 2-3 grams of zinc atoms per 1 mole of protein. Zinc or other metal ions are necessary for the catalytic activity of alkaline phosphatase and, possibly, for stabilizing the native structure of the enzyme molecule. Divalent cations Co 2+, Mg 2+, and Mn 2+ activate enzymes isolated from various tissues, while Be 2+ ions and complexing agents (eg, EDTA) are inhibitors of these enzymes. The mechanism of action of alkaline phosphatase is similar to the mechanism postulated for glucose-6-phosphatase, but the phosphorus atom interacts not with the histidine, but with the serine residue of the enzyme molecule.

For other phosphatases, for example, for fructose bisphosphatase (EC 3.1.3.11), there is no data yet on the formation of the phosphoenzyme. It is possible that the enzymatic reaction it catalyzes occurs by a one-step concerted mechanism rather than by two-step transfer.

Determination methods

Most methods for determining F.'s activity are based on measuring the amount of inorganic phosphate (formed as a result of the reaction catalyzed by these enzymes) using various colorimetric methods (see Colorimetry), which are associated with the reduction of phosphomolybdenum acid. Classic way determination of F.'s activity is the Bodansky method using beta-glycerophosphate as a substrate (see Bodansky method). It is often more convenient in practice to measure the amount of phenol released from the aryl phosphomonoester. Thus, to determine the activity of alkaline phosphatase in blood serum, the King-Armstrong method (see King-Armstrong method), the Jenner-Kay method based on the same principle, or their modifications are widely used. The most sensitive method for determining the activity of alkaline phosphatase in blood serum is the Bessey method (see Bessey methods). To determine the activity of acid phosphatase, the Gutman-Gutman method is widely used. These standard methods Determinations of phosphorus activity in blood serum involve the use of monophosphorus esters of phenol, n-nitrophenol, phenolphthalein, or thymolphthalein as substrates. The free phenols (see) formed as a result of the reaction are determined spectrophotometrically (see Spectrophotometry). Methods for measuring phosphatase activity using fluorescent substrates such as beta-naphthyl phosphate and 3-O-methylfluorescein phosphate are highly sensitive (see Fluorochromes). Trace amounts of 32P-labeled pyrophosphate can be determined by precipitation with ammonium molybdate and triethylamine in the presence of an unlabeled carrier. The sensitivity of this radioisotope method is approx. 3 ng.

Acid and alkaline phosphatases

Among phosphorus, the two most widely distributed and studied groups of enzymes are alkaline and acid phosphatases. Possessing broad substrate specificity, these enzymes differ markedly in their properties depending on the source from which they are isolated. Their substrates can be various monoesters of orthophosphoric acid - both aliphatic, for example, glycerol-1- and glycerol-2-phosphates, and aromatic, for example. 4-nitrophenylphosphate; at the same time, these enzymes are inactive towards di- and tri-esters phosphoric acids(cm.). A big difference between acidic and alkaline phosphorus is observed in their action on sulfur-containing ethers. Alkaline phosphatase hydrolyzes S-substituted monoesters of thiophosphoric acid, for example. cpsteamine-S-phosphate; For the action of acid phosphatase, oxygen of the cleaved ester bond is apparently necessary: ​​acid phosphatase hydrolyzes O-substituted monoesters of thiophosphoric acid, for example. O-4-nitrophenyl phosphate.

Alkaline phosphatase (phosphomonoesterase; EC 3.1.3.1) exhibits maximum activity at pH 8.4-9.4 and catalyzes the hydrolysis of almost all phosphomonoesters with the formation of inorganic phosphate and the corresponding alcohol, phenol, sugar, etc. Alkaline phosphatase is found in most tissues and fluids in humans and animals, as well as in plants and microorganisms. In humans, especially high activity of this enzyme is observed in the epithelium small intestine, kidneys, bones, liver, leukocytes, etc. A widely used source of alkaline phosphatase is ossifying cartilage, which indicates the possible role of this enzyme in the processes of bone tissue calcification. The presence of active alkaline phosphatase is characteristic of tissues associated with transport nutrients, it is often present in developing tissues and secretory organs. Alkaline phosphatase is practically absent in muscles, mature connective tissue and erythrocytes, the walls of blood vessels and hyaline cartilage are also poor in this enzyme.

Alkaline phosphatase has an extremely wide isoenzyme spectrum. Using immunochemical and electrophoretic methods, it was shown that there are pronounced physicochemical and catalytic differences between its isoenzymes (see). During electrophoresis in a polyacrylamide gel, alkaline phosphatase obtained from the intestinal mucosa remains near the place where the enzyme solution was added to the gel (start line), and alkaline phosphatase isolated from the liver moves towards the anode along with the fraction of ά1- or α2-globulins (rice.). Electrophoretic separation of serum alkaline phosphatase with an increase in its activity makes it possible to establish the bone or liver origin of the enzyme, the release of which caused the increased activity of alkaline phosphatase in the blood. In normal blood serum, the liver appears to be the main source of alkaline phosphatase. The appearance of the isoenzyme, characteristic of the mucous membrane of the small intestine, is under genetic control: there is evidence that its presence in the blood is typical for people with blood group zero.

Distribution of enzyme activity even in one morphological education inhomogeneous. Thus, the activity of alkaline phosphatase varies in different parts of the intestine; in the renal cortex it is much higher than in the brain. The activity of alkaline phosphatase is influenced by hormonal factors: the activity of the enzyme in the blood decreases after hypophysectomy, castration, and also as a result of the use of corticosteroid drugs. After administration of thyroxine, enzyme activity increases. In humans various factors, causing stress, contribute to an increase in alkaline phosphatase activity in leukocytes.

The activity of alkaline phosphatase in the blood depends to some extent on age and gender. In men, the activity of the enzyme in the blood is 20-30% higher than in women, however, during pregnancy, women experience a significant (2-3 times) increase in the activity of this phosphatase, which can be explained by the growth of the embryo, especially the process of fetal osteogenesis.

The functions of alkaline phosphatase in each tissue have not yet been precisely established. In bone tissue, it appears to be involved in calcification processes. In a cell, alkaline phosphatase is usually associated with the lipoprotein membrane, and in some microorganisms, as shown by histochemical studies. research, it is located between the membrane and the cell wall. The localization of the enzyme on absorbent surfaces indicates its possible role in transmembrane transport.

Mol. weight (mass) of alkaline phosphatase isolated from different sources, varies between 70,000-200,000; the enzyme from the human placenta, obtained in crystalline form, has a mol. weight 125,000. It is believed that its molecule consists of two subunits of equal mol. weight, but not identical to each other. The results of genetic studies indicate the existence of three types of alkaline phosphatase subunits, various combinations of which give six phenotypic variants that differ in electrophoretic mobility and represent the main multiple forms (isoforms) of the enzyme. It is assumed that the difference in the composition of the subunits is due to the presence in the molecules of certain alkaline phosphatases of a carbohydrate part covalently bound to the protein.

Alkaline phosphatase is stable at neutral and alkaline pH values, but is sensitive to acidification. In the pH range of 7.0-8.0 and at a concentration of Zn 2+ ions above 10 -5 M, the enzyme forms an active tetramer that binds 16 Zn 2+ ions. Microbial alkaline phosphatase, isolated from different sources, is capable of forming active hybrids using monomers from different enzymes, which indicates the closeness of the secondary structure of microbial phosphatases, despite differences in composition and immunol. properties of subunits.

The substrate specificity of alkaline phosphatases from different sources is not the same. Thus, an enzyme from bone tissue hydrolyzes a number of phosphorus compounds, including hexose phosphates, glycerophosphates, ethyl phosphate, adenylate and phenyl phosphate. The enzyme from Escherichia coli is capable of hydrolyzing various polyphosphates, including metaphosphates of various chain lengths, as well as phosphoserine, phosphothreonine, pyridoxal phosphate and phosphocholine. A number of alkaline phosphatases from mammalian tissues at pH 8.5 exhibit irophosphatase activity, and an enzyme from the chicken intestinal mucosa hydrolyzes cysteamine S-phosphate and other S-phosphates to form inorganic phosphate and the corresponding thiol. Some alkaline phosphatases also have transferase activity and, in transphosphorylation reactions, can catalyze the transfer of phosphate from the phosphoester to the alcohol group of the acceptor.

Thus, alkaline phosphatase is capable of hydrolyzing compounds containing P - F, P - O - C, P - O - P, P - S and P - N bonds, and the catalyzed reaction involves the transfer of phosphate from a donor type

(where X can be represented by fluorine, oxygen, sulfur, nitrogen, and R can be a hydrogen atom, an alkyl substituent or completely absent) to an acceptor of the type R" - OH (where R" is represented by a hydrogen atom or an alkyl substituent) with the cleavage of the P - X bond Since the enzyme also catalyzes the reverse reaction, acceptor specificity extends to all compounds of the R - CN type. Alkaline phosphatase catalyzes the transfer of only the terminal phosphate; a characteristic feature of the enzyme is that the relative rates of hydrolysis of various substrates are very similar.

Determination of alkaline phosphatase activity in the blood has diagnostic value for liver diseases and skeletal system. Thus, hyperphosphatasemia is noted with hron. liver diseases, sarcoidosis (see), tuberculosis (see), amyloidosis (see) and lymphogranulomatosis (see). With rickets (see), an increase in the activity (sometimes 2-4 times) of alkaline phosphatase was noted in 65% of cases. Paget's disease (see Paget's disease), and osteogenic sarcoma(see), phosphate diabetes (see) are accompanied by a significant increase in the activity of alkaline phosphatase in the blood serum.

Genetically determined low activity of alkaline phosphatase in the blood (hypophosphatasia) causes severe hereditary disease accompanied by skeletal abnormalities due to disruption of ossification processes; The enzyme defect is inherited in an autosomal recessive manner.

Acid phosphatase (phosphomonoesterase; EC 3.1.3.2) is also widespread in nature. It is found in yeast, molds, bacteria, plant and animal tissues and biol. liquids In humans, acid phosphatase activity in the prostate gland is particularly high. Red blood cells also contain a lot of acid phosphatase. An extract from prostate tissue exhibits phosphatase activity in a slightly acidic environment, which is almost 1000 times higher than the phosphatase activity of extracts from the liver or kidneys. Histochem. studies show that the enzyme is contained in Ch. arr. in the glandular epithelium of the prostate gland; large quantities enzyme found in sperm. Available close connection between the synthesis of acid phosphatase in the prostate gland and the content of sex hormones (see). With a low concentration of androgens (see) in the urine, low activity of acid phosphatase in sperm is noted. The same is observed with cryptorchidism (see) and hypogonadism (see).

The pH optimum for acid phosphatase is between pH 4.7 and 6.0 (however, spleen-derived acid phosphatase activity peaks at pH values ​​between 3.0 and 4.8). Substrate spectrum and rates of hydrolysis of various substrates by acid phosphatase and alkaline phosphatase quite different. Thus, acid phosphatase is not capable of hydrolyzing S-substituted monoesters of thiophosphoric acid, while O-substituted monoesters are actively hydrolyzed by it under the same conditions (in the case of alkaline phosphatase, the opposite is observed).

By electrophoretic separation of acid phosphatase isolated from various tissues, it was established that this enzyme has four components - A, B, C and D. The combination of ABD components is dominant in the kidneys; BD - in the liver, intestines, heart and skeletal muscles; component B predominates in the skin, and D - in the pancreas; component C is present in the placenta and is not found in any organ of the adult body. In general, the BD combination is characteristic of acid phosphatase in most human tissues with the exception of skin, kidneys and pancreas. All 4 electrophoretic components are genetically determined isoforms of acid phosphatase. Characteristic feature acid phosphatase is sensitive to inactivation at the interface; the addition of surfactants (see Detergents) to the enzyme solution protects acid phosphatase from inactivation.

Mol. the weight of acid phosphatase is different for enzymes obtained from different sources, for example, two immunologically different molecular isoenzymes of acid phosphatase from the human prostate gland have a mol. weight 47,000 and 84,000.

Determination of acid phosphatase activity in blood serum is important diagnostic test when prostate cancer is detected (see Prostate, pathology). In patients with prostate cancer without metastases, an increase in acid phosphatase activity in the blood is detected in 25% of cases, and in prostate cancer with tumor metastases to other organs - in 80-90% of cases. The dynamics of the activity of this enzyme in the blood in prostate cancer can serve as a criterion for the effectiveness of the therapy.

The determination of acid phosphatase is also of significant importance in forensic medicine. The high activity of the enzyme in sperm makes it possible to identify suspicious spots with great certainty in cases of chemistry. examination of physical evidence.

Histochemical methods for detecting phosphatases

Alkaline phosphatase is detected in histochemistry using the Gomori method, methods using tetrazolium, azoindoxyl and the azo coupling method. When using the tetrazolium method and the azo coupling method, it is recommended to use cryostat sections treated with acetone, as well as cryostat unfixed sections. Metal salt methods require the use of cryostat sections fixed in formaldehyde or frozen sections after fixation of tissue blocks in formaldehyde or glutaraldehyde. The Gomori method is the most recommended, followed by methods using tetrazolium and azoindoxyl. The tetrazolium method for determining alkaline phosphatase uses 5-bromo-4-chloro-3-indoxyl phosphate, toluidine salt, nitrotetrazolium blue, 0.1 - 0.2 M Tris-HCl buffer or veronal acetate buffer pH 9.2-9, 4. Azose coupling reactions and the tetrazolium method for histochemistry. detection of alkaline phosphatase is more sensitive than the Gomori method, however, the diffusion of the enzyme that occurs when using naphthols and tetrazolium salts may prevent the establishment of its exact localization.

Gomori method using metal salts

Incubation medium:

3% alpha-glycerophosphate solution 10 ml

2 -10% Medinal solution 10 ml

2% chloride solution calcium CaCl 2 (anhydrous) 15 ml

2% solution of magnesium sulfate MgSO 4 10 ml

distilled water 5 ml

Total volume 50 ml

The incubation medium is thoroughly mixed and, if cloudy, filtered. Incubate for 1-60 minutes. at 37° or at room temperature, then drain the incubation medium, wash the sections in running water, transfer to a 1 - 2% solution of cobalt chloride CoCl 2 or another soluble cobalt salt (cobalt acetate or nitrate) for 5 minutes. Then wash in running water for 2-5 minutes. When incubating unfixed sections, it is necessary to carry out postfixation at room temperature in 4% paraformaldehyde solution for 2 - 5 minutes. and rinse in running water for 2 minutes. The sections are treated with ammonium sulfate solutions of increasing concentrations (0.1 - 1%) for 2 minutes. and washed in running water for 10 minutes, after which they are placed in glycerin gel or Apati syrup or (after dehydration) in entellane or a similar medium. Alkaline phosphatase localization sites turn black. Control reactions are carried out without adding substrate to the incubation medium.

Barston's method of simultaneous azo coupling

Incubation medium:

naphthol AS, AS-MX, AS-D, AS-B1 or naphthol phosphate AS-TR 10 - 25 mg dissolved in stable diazonium salt (N, N "-dimethylformamide or dimethyl sulfoxide) 0.5 ml

0.1 - 0.2 M veronal acetate or Tris-HCl buffer, pH 8.2-9.2 50 ml

strong blue B, BB, RR, strong red TR, strong blue VRT (variamin blue, (gol RT), strong blue VB (variamin blue B) or strong violet B 50 mg

The incubation medium is thoroughly mixed and filtered. Instead of the stable diazonium salt, 0.5 ml of freshly prepared hexazotated new fuchsin can be used. In this case, the desired pH value is established by adding caustic soda drop by drop. Incubate for 5 - 60 minutes. at 37° or at room temperature. The incubation medium is drained, the sections are rinsed in distilled water, placed in a 4% formaldehyde solution for several hours at room temperature, then washed in running water, if necessary, the nuclei are stained with strong red or hematoxylin and enclosed in glycerin gel or Apati syrup. Depending on the type of diazonium salt included in the incubation medium, structures with alkaline phosphatase enzymatic activity are colored blue-violet or red.

For histochemical For the detection of acid phosphatase, it is recommended to use cryostat or frozen sections after preliminary fixation in formaldehyde, as well as cryostat sections that have been frozen and dried and covered with celloidin, and cryostat sections that have been frozen and covered with celloidin. The best results are achieved when tissue is fixed with glutaraldehyde or formaldehyde. To identify the enzyme, azo coupling reactions, the Gomori method and indigogenic reactions are used. The method of simultaneous azo coupling with naphthol phosphates and hekeazotized n-rosaniline or new fuchsin is considered universal. The second most commonly used method is the indigogenic method using 5-bromo-4-chloro-3-indoxyl phosphate as a substrate. The Gomori method makes it possible to accurately identify lysosomes (see).

Gomori method with metal salts (modified)

Incubation medium:

0.1 M acetate buffer, pH 5.0 or 6.0 50 ml

0.24% lead nitrate solution 50 ml

3% sodium alpha-glycerophosphate solution or 0.1% sodium cytidine monophosphate solution 10 ml

Total volume 110 ml

The incubation medium is mixed well and left to stand for 15-30 minutes. at incubation temperature, then filtered. Incubation is carried out in cuvettes at 37°C for 10-60 minutes. or at room temperature for up to 2 hours, free-floating sections can be incubated. The incubation medium is drained, the sections are rinsed in two changes of distilled water for 1 minute each. in each and placed in 0.5 - 1% solution yellow ammonium sulfide for 1 - 2 minutes. Rinse again in distilled water and place in glycerin gel or Apati syrup. Structures with acid phosphatase activity appear brown.

Method of simultaneous azo coupling with naphthol ethers AS

Incubation medium:

naphthol phosphate AS-BI or naphthol AS-TR 20 - 25 mg dissolved in N,N"-dimethylformamide - 1 ml

Buffered hexazotated n-rosaniline or new fuchsin (1.5 - 4.5 ml of hexazotated n-rosaniline or 1.25 ml of new fuchsin is dissolved in 45.5 - 48.5 ml of 1.36-2.72% acetate solution sodium CH 3 CONa 3H 2 O or 48.75 ml of 0.1 M seronal acetate buffer, pH about 6.0, adjusted to pH 5.0 - 5.5) - 50 ml

Total volume 51 ml

The incubation medium is thoroughly mixed and filtered. Incubate for 30 - 60 minutes. at 37° or 1-2 hours. at room temperature or several hours (days) in the refrigerator at +4°. The incubation medium is drained, the sections are rinsed in distilled water and placed in a 4% formaldehyde solution for several hours at room temperature. Rinse in running water, if necessary, stain the nuclei with hematoxylin and enclose in glycerin gel or Apati syrup. Structures with acid phosphatase activity are colored red.

Azoindoxyl method according to Gossrau

Incubation medium: toluidine salt of 5-bromo-4-chloro-3-indoxyl phosphate 1.5 - 3 mg dissolved in 0.075 - 0.15 ml of N,N"-dimethylformamide 0.1 M acetate buffer, pH 5.0 10 ml

Hexazotated new fuchsin 0.25 ml

or strong blue B 5 -10 mg

Total volume ~10 ml

The incubation medium is thoroughly mixed and filtered, attached or free-floating sections are incubated for 15-60 minutes. at 37°. The incubation medium is drained, the sections are rinsed in distilled water and placed in a 4% formaldehyde solution for several hours at room temperature, then rinsed in running water and placed in distilled water, after which they are enclosed in glycerin gel or Apati syrup. Structures with acid phosphatase activity appear bluish-brown.

Bibliography: Dixon M. and Webb E. Enzymes, trans. from English, p. 364, 458, M., 1982; Lilly R. Pathohistological techniques and practical histochemistry, trans. from English, M., 1969; Loida Z., Gossrau R. and Schibler T. Histochemistry of enzymes, trans. from English, M., 1982; Nomenclature of enzymes, trans. from English, ed. A. E. Braunstein, M., 1979; Pierce A. Histochemistry, trans. from English, M., 1962; Enzymes, ed. by P. D. Boyer, v. 7, N.Y.-L., 1972.

P. L. Ivanov (biochemistry), A. G. Ufimtseva (hist.).

Glucose-6-phosphatase is a complex of various proteins located in the endoplasmic reticulum. The catalytic subunit is responsible for the main function. In humans, there are three isoenzymes of this subunit: glucose-6-phosphatase-α, encoded by the G6PC gene; IGRP, encoded by the G6P2 gene; and glucose-6-phosphatase-β, encoded by the G6P3 gene.

The alpha and beta isoenzymes are both functionally phosphohydralases, and have similar active site structure, topology, mechanism of action, and kinetic properties for the hydrolysis of glucose-6-phosphate. In turn, the IGRP isoenzyme has virtually no hydrolase activity and may play a different role in insulin secretion in the pancreas.

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Excerpt characterizing Glucose-6-phosphatase

Suddenly Prince Hippolyte stood up and, stopping everyone with hand signs and asking them to sit down, spoke:
- Ah! aujourd"hui on m"a raconte une anecdote moscovite, charmante: il faut que je vous en regale. Vous m"excusez, vicomte, il faut que je raconte en russe. Autrement on ne sentira pas le sel de l"histoire. [Today I was told a charming Moscow joke; you need to teach them. Sorry, Viscount, I will tell it in Russian, otherwise the whole point of the joke will be lost.]
And Prince Hippolyte began to speak Russian with the accent that the French speak when they have been in Russia for a year. Everyone paused: Prince Hippolyte so animatedly and urgently demanded attention to his story.

Glycogenosis type 1 was first described in 1929 by Gierke. The disease occurs in one case out of two hundred thousand newborns. The pathology affects both boys and girls equally. Next, let's look at how Gierke's disease manifests itself, what it is, and what therapy is used.

General information

Despite its relatively early detection, it was not until 1952 that Cory's enzyme defect was identified. Inheritance of the pathology is autosomal recessive. Gierke's syndrome is a disease in which the cells of the liver and convoluted tubules of the kidneys are filled with glycogen. However, these reserves turn out to be unavailable. This is indicated by hypoglycemia and the absence of an increase in blood glucose concentration in response to glucagon and adrenaline. Gierke's syndrome is a disease accompanied by hyperlipemia and ketosis. These signs are characteristic of the state of the body with a deficiency of carbohydrates. At the same time, low glucose-6-phosphatase activity is observed in the liver, intestinal tissues, and kidneys (or it is completely absent).

Progress of the pathology

How does Gierke's syndrome develop? The disease is caused by defects in the liver enzyme system. It converts glucose-6-phosphate into glucose. With defects, both gluconeogenesis and glycogenolysis are impaired. This, in turn, provokes hypertriglyceridemia and hyperuricemia, lactic acidosis. Glycogen accumulates in the liver.

Gierke's disease: biochemistry

In the enzyme system that transforms glucose-6-phosphate into glucose, in addition to itself, there are at least four more subunits. These include, in particular, the regulatory Ca2(+)-binding protein compound, translocases (transport proteins). The system contains T3, T2, T1, which ensure the transformation of glucose, phosphate and glucose-6-phosphate through the endoplasmic reticulum membrane. There are certain similarities in the types that Gierke's disease has. The clinical picture of glycogenosis Ib and Ia is similar; therefore, to confirm the diagnosis and accurately establish the enzyme defect, the activity of glucose-6-phosphatase is also examined. Difference in clinical manifestations between glycogenosis type Ib and Ia is that with the first, transient or permanent neutropenia is noted. In especially severe cases, agranulocytosis begins to develop. Neutropenia is accompanied by dysfunction of monocytes and neutrophils. In this regard, the likelihood of candidiasis increases and staphylococcal infections. Some patients experience inflammation in the intestines similar to Crohn's disease.

Signs of pathology

First of all, it should be said that Gierke’s disease manifests itself differently in newborns, infants and older children. Symptoms appear as fasting hypoglycemia. However, in most cases the pathology is asymptomatic. This is due to the fact that infants often receive nutrition and the optimal amount of glucose. Gierke's disease (photos of patients can be found in medical reference books) is often diagnosed after birth several months later. In this case, the child exhibits hepatomegaly and an enlarged abdomen. Low-grade fever and shortness of breath without signs of infection may also accompany Gierke's disease. The causes of the latter are lactic acidosis due to insufficient glucose production and hypoglycemia. Over time, the intervals between feedings increase and a long period of time appears. night sleep. At the same time, its duration and severity begin to gradually increase, which, in turn, leads to systemic metabolic disorders.

Consequences

In the absence of treatment, changes in the child's appearance are noted. In particular, muscular and skeletal malnutrition, slowdown in physical development and growth are characteristic. There are also fatty deposits under the skin. The child begins to resemble a patient who has no impairment in the development of social and cognitive skills, unless the brain is damaged during repeated hypoglycemic attacks. If hypoglycemia of fasting persists and the child does not receive the required amount of carbohydrates, the delay in physical development and growth becomes clearly pronounced. In some cases, children with hypoglykenosis type I die due to pulmonary hypertension. If there is a violation, repeated nosebleeds or bleeding are observed after dental or other surgical intervention.

Disorders in platelet adhesion and aggregation are noted. The release of ADP in response to contact with collagen and adrenaline is also impaired. Systemic metabolic disorders provoke thrombocytopathy, which disappears after therapy. Enlarged kidneys are detected by ultrasound and excretory urography. Most patients do not have significant renal impairment. In this case, only an increase in Most severe cases accompanied by tubulopathy with glycosuria, hypokalemia, phosphaturia and aminoaciduria (type In some cases, adolescents have albuminuria. In young people, renal damage is observed severe course with proteinuria, increased blood pressure and decreased creatinine clearance, which is caused by interstitial fibrosis and focal segmental glomerulosclerosis. All these disorders provoke end-stage renal failure. The size of the spleen remains within normal limits.

Liver adenomas

They occur in many patients various reasons. As a rule, they appear between the ages of 10 and 30 years. They can become malignant, and hemorrhages into the adenoma are possible. These formations are presented on scintigrams as areas of reduced isotope accumulation. Ultrasound is used to identify adenomas. In case of suspicion malignancy More informative MRI and CT are used. They allow us to trace the transformation of a clear limited formation small size into a larger one with enough blurred edges. In this case, periodic measurement of serum levels of alpha-fetoprotein (a marker of liver cell cancer) is recommended.

Diagnostics: mandatory studies

Patients have their levels measured uric acid, lactate, glucose, fasting liver enzyme activity. In infants and newborns, the concentration of glucose in the blood after 3-4 hours of fasting decreases to 2.2 mmol/liter or more; with a duration of more than four hours, the concentration is almost always less than 1.1 mmol/liter. Hypoglycemia is accompanied by a significant increase in lactate and metabolic acidosis. The serum is usually cloudy or milk-like due to very high concentrations of triglycerides and moderately elevated cholesterol levels. Increased activity of ALT (alanine aminotransferase) and AST (aspartaminotransferase), and hyperuricemia are also observed.

Provocative tests

To differentiate type I from other glycogenoses and accurately determine the enzyme defect in infants and older children, the level of metabolites (free fatty acids, glucose, uric acid, lactate, ketone bodies), hormones (GH (somatotropic hormone), cortisol, adrenaline, glucagon) is measured , insulin) after glucose and on an empty stomach. The research is carried out according to a certain scheme. The child receives glucose (1.75 g/kg) orally. Then blood is drawn every 1-2 hours. Glucose concentration is quickly measured. The last analysis is taken no later than six hours after taking glucose or when its content has decreased to 2.2 mmol/liter. A provocative test with glucagon is also performed.

Special studies

During this procedure, a liver biopsy is performed. Glycogen is also examined: its content is significantly increased, but its structure is within normal limits. Measurements of glucose-6-phosphatase activity in disrupted and intact liver microsomes are carried out. They are destroyed by repeated freezing and thawing of the biopath. Against the background of type Ia glycogenosis, activity is not detected either in destroyed or intact microsomes; in type Ib, in the former it is normal, and in the latter it is significantly reduced or absent.

Gierke's disease: treatment

With glycogenosis type I, metabolic disorders associated with insufficient glucose production appear several hours after eating. With prolonged fasting, the disorders increase significantly. In this regard, treatment of the pathology comes down to increasing the frequency of feeding the child. The goal of therapy is to prevent glucose levels from falling below 4.2 mmol/liter. This is the threshold level at which the secretion of contravascular hormones is stimulated. If the child receives a sufficient amount of glucose in a timely manner, a decrease in liver size is noted. Laboratory indicators at the same time, they approach normal, and growth stabilizes, bleeding disappears.

This is the most severe form of glycogenosis, the immediate severity of which is directly related to the possibility acute manifestations hypoglycemia, acidosis and sometimes hemorrhage.

Symptoms. This glycogenosis manifests itself starting from the first weeks of life. The abdomen increases in volume. After a few hours of fasting, signs of hypoglycemia appear: imperative hunger, pallor, profuse sweat, and less commonly, general malaise and seizures. When examining infant there is some degree of fatness of the face and torso, with rounded cheeks that contrast with the thin limbs. There is significant enlargement of the liver, sometimes to the ridges iliac bones, solid consistency; palpation of the lower edge of the liver is often difficult. An older child may develop xanthomas and gradually experience severe growth retardation.

Laboratory data. The biochemical consequences of glucose-6-phosphatase deficiency are revealed quite easily by studying the glycemic cycle, which reveals poor tolerance to delayed feeding. Indeed, glucose is released only under the influence of amylo-1,6-glucosidase; molecules of glucose-1-phosphate, released under the influence of the phosphorylase system, and metabolites of neoglucogenesis lead to the formation of glucose-6-phosphate. Therefore, 3-4 hours after a meal, a rapid decrease in glucose levels occurs, while lactic acidemia increases. These disorders concern the metabolism of carbohydrates, lipids and uric acid.

Clinically, hypoglycemia is quite well tolerated, probably because the brain uses different substrates. This hypoglycemia is accompanied by peripheral hypoinsulinism, as evidenced by the paradiabetic nature of the hyperglycemic curve during exercise testing, as well as a decrease in the absorption curve of intravenous glucose and an insufficient rise in insulinemia after glucose administration. These changes in glycemia are combined with an increase in the content of lactic and pyruvic acids in the blood. The first of them can increase very significantly, reaching 800-1000 mg/l; this causes a state of chronic acidosis that can suddenly decompensate. In this aspect, delayed feeding and intercurrent infections appear dangerous.

Disorders of fat metabolism are constantly observed in the form milky looking blood serum, a significant increase in blood triglycerides, phospholipids and total cholesterol. Circulating NEFAs are also elevated. These changes in fat metabolism manifest themselves cytologically in the form of fat accumulation in the liver, combined in varying degrees with glycogen accumulation.

An increase in uric acid in the blood is often observed and can exceed 120 mg/l. This explains the possibility of the appearance of urate tophi after a few years, and later of attacks of gout or nephropathy. The mechanism of hyperuricemia is likely controversial. It is mainly associated with a decrease in the renal clearance of uric acid compared with the excretion of organic acids, especially lactic acid. Was also installed increased synthesis uric acid from glucose-6-phosphate.

Other observed anomalies include an increase in the volume of the kidneys, which are usually not palpable due to hepatomegaly, but are clearly visible radiographically. Osteoporosis is detected, in the origin of which the role of chronic hypercortisolism is assumed; possible thrombopathy with an increase in the number of platelets in the blood; Bleeding time may be prolonged, which is associated with dysfunction of the platelets. The consequences of this can be dramatic, in the form of spontaneous or provoked bleeding, sometimes fatal. Detection of thrombopathy is necessary during surgery or liver biopsy. Liver function tests are usually normal except for persistent but mild elevations in serum transaminases.

The study of carbohydrate metabolism has a dual purpose: to determine the child’s individual tolerance to delayed food intake and to indirectly assess the activity of glucose-6-phosphatase.

Assessing tolerance to delayed food intake is of fundamental importance, as it determines the rhythm of eating. Tolerance is assessed by studying the glycemic cycle and glucose levels before each meal.

Functional tests allow you to indirectly determine the deficiency of glucose-6-phosphatase activity, which is more convenient than the direct method of determination enzymatic activity, requiring obtaining a liver fragment via biopsy. Various tests have been proposed: with glucagon (0.1 mg/kg, in total no more than 1 mg, intravenously or intramuscularly); with a galactose load (1 g/kg intravenously). The likelihood of glucose-6-phosphatase deficiency is high if these tests do not result in an increase in glucose levels; the latter even continues to decrease during the test due to the continued fasting required for the test. Given the poor tolerance to hunger, these various tests should be performed only after 3-4 hours of fasting. It is very typical for glycogenesis of this type that the introduced galactose disappears from the blood faster than that of healthy children. With these tests, there is a clear increase in the level of lactic acid, already elevated in the initial state. For this reason, and because of the risk of hypoglycemia, one should be prepared to interrupt the test if the slightest sign intolerance and administer intravenous glucose and sodium bicarbonate.

Evidence for glucose-6-phosphatase deficiency was also obtained by direct definition enzyme in a liver fragment obtained from a puncture biopsy performed with normal hemostasis. Liver biopsy allows for histological examination. Liver cells are larger than normal, light, closely spaced, with clear boundaries, generally creating a picture of “plant” tissue. The nuclei are clearly visible, sometimes vacuolated, and liver cells often contain numerous vacuoles containing fat. Staining with Best's carmine or Schiff's reagent shows, subject to good fixation, the presence large quantity glycogen, which disappears after exposure to amylase.

The amount of glycogen in the liver is increased over 5-7 g per 100 g of liver. The reaction to iodine in this glycogen is normal. Glucose-6-phosphatase activity, measured by the release of inorganic phosphorus from glucose-6-phosphate as a substrate, is absent or very weak.

Flow. The course of glycogenosis type I is especially severe. In the first years of life, the child is at risk of attacks of hypoglycemia, which can affect psychomotor development, as well as frequent exacerbations of chronic acidosis. Attacks of hypoglycemia and acidosis are easily provoked by infection, surgical interventions, fasting. The need for repeated meals often leads to severe anorexia, which in turn increases the risk of attacks of hypoglycemia and acidosis. In several cases there were hemorrhagic complications, sometimes fatal.

Significant growth retardation gradually becomes evident, while fasting tolerance appears to improve. IN adolescence problems arise due to severe retardation of growth and puberty, persistent hypercholesterolemia and sometimes complications associated with hyperuricemia. With long-term observation, these children are often diagnosed with liver adenomas and sometimes even hepatocarcinomas. Three out of five of our children over 3 years old had several liver adenomas.

GLUT-1 ensures a stable 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 ensures a constant flow of glucose to the cells of the nervous and other tissues;

GLUT-4 is the main transporter 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 integrated 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 muscle 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 - part of the insulin receptor, facing the inside of the cell, stimulates the movement of glucose transporters; 3, 4 - transporters, as part 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 disturbances in the functioning of glucose transporters are known. An inherited defect in these proteins may underlie non-insulin-dependent diabetes mellitus. GLUT-4 dysfunction is possible at the following stages:

transmission of the insulin signal to move this transporter to the membrane;

movement of the transporter in the cytoplasm;

inclusion in the membrane;

unlacing from the membrane, etc.

DISORDERS IN DIGESTION AND ABSORPTION OF CARBOHYDRATES

The pathology of carbohydrate digestion and absorption may be based on two types of reasons:

defects in enzymes involved in the hydrolysis of carbohydrates in the intestine;

impaired absorption of carbohydrate digestion products into the cells of the intestinal mucosa.

In both cases, unsplit disaccharides or monosaccharides appear. These unclaimed carbohydrates enter the distal intestine, changing the osmotic pressure of the intestinal contents. In addition, the carbohydrates remaining in the intestinal lumen are partially subject to enzymatic breakdown by microorganisms with the formation of organic acids and gases. All together leads to an influx of water into the intestines, an increase in the volume of intestinal contents, increased peristalsis, spasms and pain, as well as flatulence.

GLUCOSE METABOLISM IN THE CELL

After absorption in the intestine, monosaccharides enter the portal vein and then mainly to the liver. Since glucose predominates in the composition of the main carbohydrates in food, it can be considered the main product of carbohydrate digestion. Other monosaccharides coming from the intestines during metabolism can be converted into glucose or its metabolic products. Part of the glucose in the liver is deposited in the form of glycogen, and the other part is delivered and used by various tissues and organs through the general bloodstream. With a normal diet, the concentration of glucose in the blood is maintained at a level of -3.3-5.5 mmol/l. And during digestion, its concentration can increase by approximately 8 mmol/l.

Phosphorylation of glucose

The metabolism of glucose in the cells of all tissues begins with the reaction of phosphorylation and conversion to glucose-6-phosphate (using ATP). There are two enzymes that catalyze the phosphorylation of glucose: in the liver and pancreas - the enzyme glucokinase, in all other tissues – hexokinase. Phosphorylation of glucose is an irreversible reaction, since it occurs using a significant amount of energy. The plasma membrane of cells is impermeable to phosphorylated glucose (there are no corresponding transport proteins) and, therefore, it can no longer leave them. In addition, phosphorylation reduces the concentration of free glucose in the cytoplasm. As a result, favorable conditions are created for facilitated diffusion of glucose into cells from the blood.

These enzymes differ in their affinity for glucose.Gexokinase has a high affinity for glucose, i.e. this enzyme, unlike glucokinase, is active at low blood glucose concentrations. As a result, the brain, red blood cells and other tissues can use glucose when its concentration in the blood decreases 4-5 hours after eating and during fasting. The enzyme hexokinase can catalyze the phosphorylation of not only D-glucose, but also other hexoses, although at a lower rate. Hexokinase activity changes depending on the energy needs of the cell. The regulators are the ATP/ADP ratio and the intracellular level of glucose-6-phosphate. With a decrease in energy consumption in the cell, the level of ATP (relative to ADP) and glucose-6-phosphate increases. In this case, hexokinase activity decreases and, consequently, the rate of glucose entering the cell decreases.

Phosphorylation of glucose in hepatocytes during digestion is ensured by the properties glucokinase. The activity of glucokinase, unlike hexokinase, is not inhibited by glucose-6-phosphate. This circumstance ensures an increase in the concentration of glucose in the cell in phosphorylated form, corresponding to its level in the blood. Glucose enters hepatocytes through facilitated diffusion with the participation of the GLUT-2 transporter (independent of insulin). GLUT-2, like glucokinase, has a high affinity for glucose and helps to increase the rate of glucose entry into hepatocytes during digestion, i.e. accelerates its phosphorylation and further use for storage.

Although insulin does not affect glucose transport, it increases the influx of glucose into hepatocytes during digestion indirectly by inducing the synthesis of glucokinase and thereby accelerating glucose phosphorylation.

The preferential consumption of glucose by hepatocytes, due to the properties of glucokinase, prevents an excessive increase in its concentration in the blood during the absorption period. This, in turn, reduces the consequences of undesirable reactions involving glucose, such as protein glycosylation.

Dephosphorylation of glucose-6-phosphate

The conversion of glucose-6-phosphate to glucose is possible in the liver, kidneys and intestinal epithelial cells. The cells of these organs contain the enzyme glucose-6-phosphatase, which catalyzes the removal of the phosphate group by hydrolytic means:

Glucose-6-phosphate +H 2 O → Glucose + H 3 RO 4

The resulting free glucose is able to diffuse from these organs into the blood. There is no glucose-6-phosphatase in other organs and tissues, and therefore dephosphorylation of glucose-6-phosphate is impossible. An example of such irreversible penetration of glucose into a cell is muscle, where glucose-6-phosphate can only be used in the metabolism of that cell.

Metabolism of glucose-6-phosphate

Depending on the physiological state of the body and the type of tissue, glucose-6-phosphate can be used in the cell in various transformations, the main of which are: glycogen synthesis, catabolism with the formation of CO 2 and H 2 O, and pentose synthesis. The breakdown of glucose into final products serves as a source of energy for the body. At the same time, during the metabolism of glucose-6-phosphate, intermediate products are formed that are subsequently used for the synthesis of amino acids, nucleotides, glycerol and fatty acids. Thus, glucose-6-phosphate is not only a substrate for oxidation, but also a building material for the synthesis of new compounds.

GLYCOGEN METABOLISM

Many tissues synthesize glycogen as a reserve form of glucose. The reserve role of glycogen is due to two important properties: it is osmotically inactive and highly branched, due to which glucose quickly attaches to the polymer during biosynthesis and is cleaved off during mobilization. The synthesis and breakdown of glycogen ensures a constant concentration of glucose in the blood and creates a depot for its use by tissues as needed.

Structure and functions of glycogen

Glycogen is a branched polysaccharide in which glucose residues are connected in linear sections by an α-1,4-glycosidic bond. At branch points, the monomers are connected by α-1,6-glycosidic bonds. These bonds are formed with approximately every tenth glucose residue, i.e. Branch points in glycogen occur approximately every ten glucose residues. Thus, in the glycogen molecule there is only one free anomeric OH group and, therefore, only one reducing end.

A. The structure of the glycogen molecule: 1 - glucose residues connected by an α-1,4-glycosidic bond; 2 - glucose residues connected by an α-1,6-glycosidic bond; 3 - non-reducing terminal monomers; 4 - reducing terminal monomer.

B. The structure of a separate fragment of the glycogen molecule.

Glycogen is stored in the cytosol of the cell in the form of granules with a diameter of 10-40 nm. Some enzymes involved in glycogen metabolism are also associated with the granules, which facilitates their interaction with the substrate. The branched structure of glycogen determines a large number of terminal monomers, which facilitates the work of enzymes that remove or add monomers during the breakdown or synthesis of glycogen, since these enzymes can simultaneously work on several branches of the molecule. Glycogen is deposited mainly in the liver and skeletal muscles.

After eating a meal rich in carbohydrates, the glycogen reserve in the liver can be approximately 5% of its mass. About 1% of glycogen is stored in muscles, but the mass of muscle tissue is much larger and therefore the total amount of glycogen in muscles is 2 times greater than in the liver. Glycogen can be synthesized in many cells, for example in neurons, macrophages, and adipose tissue cells, but its content in these tissues is insignificant. The body can contain up to 450 g of glycogen.

The breakdown of liver glycogen serves primarily to maintain blood glucose levels. Therefore, the glycogen content in the liver changes depending on the rhythm of nutrition. With prolonged fasting, it decreases to almost zero. Muscle glycogen serves as a reserve of glucose, a source of energy during muscle contraction. Muscle glycogen is not used to maintain blood glucose levels.

Glycogen synthesis (glycogenogenesis)

Glycogen is synthesized during digestion (1-2 hours after eating carbohydrate foods). It should be noted that the synthesis of glycogen from glucose requires energy.

Glucose actively moves from the blood into the tissues and is phosphorylated, turning into glucose-6-phosphate. Then glucose-6-phosphate is converted by phosphoglucomutase into glucose-1-phosphate, from which UDP-glucose is formed under the action of (UDP)-glucopyrophosphorylase and with the participation of (UTP).

But due to the reversibility of the reaction glucose-6-phosphate ↔ glucose-1-phosphate, the synthesis of glycogen from glucose-1-phosphate and its breakdown would also be reversible and therefore uncontrollable. For glycogen synthesis to be thermodynamically irreversible, an additional stage of formation of uridine diphosphate glucose from UTP and glucose-1-phosphate is necessary. The enzyme that catalyzes this reaction is named after the reverse reaction: UDP-glucopyrophosphorylase. However, in the cell the reverse reaction does not occur, because the pyrophosphate formed during the direct reaction is very quickly split by pyrophosphatase into 2 phosphate molecules.

Educated UDP-glucose further used as a donor of glucose residue during glycogen synthesis. This reaction is catalyzed by an enzyme glycogen synthase (glucosyltransferase). Because this reaction does not use ATP, the enzyme is called a synthase rather than a synthetase. The enzyme transfers glucose residue per oligosaccharide, consisting of 6-10 glucose residues and representing primer (seed), attaching glucose molecules with α-1,4-glycosidic bonds. Since the primer is connected at the reducing end to the OH group of the tyrosine residue of the glycogenin protein, glycogen synthase sequentially attaches glucose to the non-reducing end. When the number of monomers in the synthesized polysaccharide reaches 11-12 monosaccharide residues, the branching enzyme (glycosyl-4,6-transferase) transfers a fragment containing 6-8 monomers, then the end of the molecule closer to its middle and attaches it to an α-1,6-glycosidic communication As a result, a highly branched polysaccharide is formed.

Glycogen breakdown (glycogenolysis)

Glycogen breakdown or mobilization occurs in response to an increase in the body's need for glucose. Liver glycogen breaks down mainly in the intervals between meals, in addition, this process in the liver and muscles accelerates during physical work.

First the enzymeglycogen phosphorylase cleaves only α-1,4-glycosidic bonds with the participation of phosphoric acid, sequentially cleaves glucose residues from the non-reducing ends of the glycogen molecule and phosphorylates them to form glucose-1-phosphate. This leads to shortening of branches.

When the number of glucose residues in the glycogen branches reaches 4, the enzyme oligosaccharide transferase cleaves the α-1,4-glycosidic bond and transfers a fragment consisting of 3 monomers to the end of a longer chain.

Enzyme α-1,6-glycosidase hydrolyzes the α-1,6-glycosidic bond at the branch point and cleaves off a glucose molecule. Thus, when glycogen is mobilized, glucose-1-phosphate and a small amount of free glucose are formed. Next, glucose-1-phosphate, with the participation of the enzyme phosphoglucomutase, is converted into glucose-6-phosphate.

Glycogen mobilization in the liver and muscles proceeds equally until the formation of glucose-6-phosphate. In the liver under the influence glucose-6-phosphatase Glucose-6-phosphate is converted into free glucose, which enters the blood. Consequently, the mobilization of glycogen in the liver ensures the maintenance of normal blood glucose levels and the supply of glucose to other tissues. There is no enzyme glucose-6-phosphatase in muscles and glucose-6-phosphate is used by the muscles themselves for energy purposes.

Biological significance of glycogen metabolism in the liver and muscles

Comparison of the processes of synthesis and breakdown of glycogen allows us to draw the following conclusions:

the synthesis and breakdown of glycogen proceed through different metabolic pathways;

the liver stores glucose in the form of glycogen not so much for its own needs, but to maintain a constant concentration of glucose in the blood, and, therefore, ensures the supply of glucose to other tissues. The presence of glucose-6-phosphatase in the liver causes this main function liver in glycogen metabolism;

the function of muscle glycogen is to release glucose-6-phosphate, which is consumed in the muscle itself for oxidation and energy use;

Glycogen synthesis requires 1 mole of ATP and 1 mole of UTP;

the breakdown of glycogen to glucose-6-phosphate does not require energy;

The irreversibility of the processes of synthesis and breakdown of glycogen is ensured by their regulation.

Glycogen metabolism disorders lead to various diseases. They arise from mutations in genes encoding enzymes that are involved in glycogen metabolism. In these diseases, there is an accumulation of glycogen granules in the liver, muscles and other tissues, leading to cell damage.

REGULATION OF GLYCOGEN SYNTHESIS AND METABOLISM

The metabolism of glycogen in the liver and muscles depends on the body's need for glucose as an energy source. In the liver, the deposition and mobilization of glycogen is regulated by the hormones insulin, glucagon and adrenaline.

Insulin and glucagon are antagonist hormones; their synthesis and secretion depend on the concentration of glucose in the blood. Normally, the concentration of glucose in the blood corresponds to 3.3-5.5 mmol/l. The ratio of insulin concentration to glucagon concentration in the blood is called insulin-glucagon index.

When blood glucose levels increase, insulin secretion increases (insulin-glucagon index increases). Insulin promotes the entry of glucose into insulin-dependent tissues and accelerates the use of glucose for glycogen synthesis in the liver and muscles.

When blood glucose levels decrease, insulin secretion decreases (insulin-glucagon index decreases). Glucagon accelerates the mobilization of glycogen in the liver, resulting in an increase in the flow of glucose from the liver into the blood.

Insulin- synthesized and secreted into the blood by β-cells of the islets of Langerhans of the pancreas. β-cells are sensitive to changes in blood glucose and secrete insulin in response to increases in glucose levels after meals. Transport protein(GLUT-2), which ensures the entry of glucose into β-cells, has a low affinity for it. Consequently, this protein transports glucose into the pancreatic cell only after its content in the blood is above the normal level (more than 5.5 mmol/l). In β-cells, glucose is phosphorylated by glucokinase; the rate of glucose phosphorylation by glucokinase in β-cells is directly proportional to its concentration in the blood.

Insulin synthesis is regulated by glucose. Glucose is directly involved in the regulation of insulin gene expression.

Glucagon- produced by α-cells of the pancreas in response to a decrease in blood glucose levels. By chemical nature, glucagon is a peptide.

Insulin and glucagon secretion are also regulated by glucose, which stimulates insulin secretion from β cells and inhibits glucagon secretion from α cells. In addition, insulin itself reduces glucagon secretion.

During intense muscular work and stress, it is secreted into the blood from the adrenal glands. adrenalin. It accelerates the mobilization of glycogen in the liver and muscles, thereby providing cells of various tissues with glucose.

Regulation of glycogen phosphorylase and glycogen synthase activity

The action of these hormones ultimately comes down to changing the rate of reactions catalyzed by key enzymes in the metabolic pathways of glycogen metabolism - glycogen synthase And glycogen phosphorylase, whose activity is regulated allosterically and by phosphorylation/desphorylation.

Glycogen phosphorylase exists in 2 forms:

1) phosphorylated - active (form a); 2) dephosphorylated - inactive (form c).

Phosphorylation occurs by transfer of a phosphate residue from ATP to the hydroxyl group of one of the serine residues of the enzyme. The consequence of this is conformational changes in the enzyme molecule and its activation.

Interconversions of the 2 forms of glycogen phosphorylase are ensured by the action of the enzymes phosphorylase kinase and phosphoprotein phosphatase (an enzyme structurally associated with glycogen molecules). In turn, the activity of phosphorylase kinase and phosphoprotein phosphatase is also regulated by phosphorylation and dephosphorylation.

Phosphorylase kinase activation occursunder by the action of protein kinase A - PKA (cAMP-dependent). cAMP first activates protein kinase A, which phosphorylates phosphorylase kinase, converting it into an active state, which, in turn, phosphorylates glycogen phosphorylase. The synthesis of cAMP is stimulated by adrenaline and glucagon.

Activation of phosphoprotein phosphatase occurs as a result of a phosphorylation reaction catalyzed by a specific protein kinase, which in turn is activated by insulin through a cascade of reactions involving other proteins and enzymes. Insulin-activated protein kinase phosphorylates and thereby activates phosphoprotein phosphatase. Active phosphoprotein phosphatase dephosphorylates and therefore inactivates phosphorylase kinase and glycogen phosphorylase.

Effect of insulin on the activity of glycogen synthase and phosphorylase kinase. FP-phosphatase (GR) is a phosphoprotein phosphatase of glycogen granules. PC (pp90S6) is a protein kinase activated by insulin.

Glycogen synthase activity also changes as a result of phosphorylation and dephosphorylation. However, there are significant differences in the regulation of glycogen phosphorylase and glycogen synthase:

phosphorylation of glycogen synthase catalyzes PK A and causes its inactivation;

dephosphorylation of glycogen synthase by phosphoprotein phosphatase, on the contrary, activates it.

Regulation of glycogen metabolism in the liver

An increase in blood glucose levels stimulates synthesis and secretionβ-cells of the pancreas produce the hormone insulin. Insulin transmits a signal into the cell through a membrane catalytic receptor - tyrosine protein kinase. The interaction of the receptor with the hormone initiates a series of sequential reactions leading to the activation of phosphoprotein phosphatase of glycogen granules. This enzyme dephosphorylates glycogen synthase and glycogen phosphorylase, causing glycogen synthase to be activated and glycogen phosphorylase to become inactive.

Thus, glycogen synthesis is accelerated in the liver and its breakdown is inhibited.

During fasting, a decrease in blood glucose levels is a signal for the synthesis and secretion of glucagon by the α-cells of the pancreas. The hormone transmits a signal to cells through the adenylate cyclase system. This leads to the activation of protein kinase A, which phosphorylates glycogen synthase and phosphorylase kinase. As a result of phosphorylation, glycogen synthase is inactivated and glycogen synthesis is inhibited, and phosphorylase kinase becomes active and phosphorylates glycogen phosphorylase, which becomes active. Active glycogen phosphorylase accelerates the mobilization of glycogen in the liver.

1 - glucagon and adrenaline interact with specific membrane receptors. The hormone-receptor complex affects the conformation of the G protein, causing its dissociation into protomers and the replacement of GDP with GTP in the α-subunit;

2 - the α-subunit associated with GTP activates adenylate cyclase, which catalyzes the synthesis of cAMP from ATP;

3 - in the presence of cAMP, protein kinase A reversibly dissociates, releasing subunits C with catalytic activity;

4 - protein kinase A phosphorylates and activates phosphorylase kinase;

5 - phosphorylase kinase phosphorylates glycogen phosphorylase, converting it into an active form;

6 - protein kinase A also phosphorylates glycogen synthase, rendering it inactive;

7 - as a result of inhibition of glycogen synthase and activation of glycogen phosphorylase, glycogen is included in the breakdown process;

8 - phosphodiesterase catalyzes the breakdown of cAMP and thereby interrupts the action of the hormonal signal. The α-subunit-GTP complex then disintegrates.

During intense physical work and stress, the concentration of a increases in the blood. adrenaline. There are two types of membrane adrenaline receptors in the liver. The effect of adrenaline in the liver is due to phosphorylation and activation glycogen phosphorylase. Adrenaline has a mechanism of action similar to glucagon. But it is also possible to include another effector signal transduction system in the liver cell.

Regulation of the synthesis and breakdown of glycogen in the liver by adrenaline and Ca 2+ .

FIF 2 - phosphatidylinositol bisphosphate; IP 3 - inositol 1,4,5-triphosphate; DAG - diacylglycerol; ER - endoplasmic reticulum; PS - phosphodithylserine.

1 - the interaction of adrenaline with the α 1 receptor transforms the signal through the activation of the G protein to phospholipase C, transferring it to an active state;

2 - phospholipase C hydrolyzes PIF 2 into IF 3 and DAG;

3 - IF 3 activates the mobilization of Ca 2+ from the ER;

4 - Ca 2+, DAG and phosphodithylserine activate protein kinase C. Protein kinase C phosphorylates glycogen synthase, rendering it inactive;

5 - complex 4Ca 2+ - calmodulin activates phosphorylase kinase and calmodulin-dependent protein kinases;

6 - phosphorylase kinase phosphorylates glycogen phosphorylase and thereby activates it;

7 - active forms of three enzymes (calmodulin-dependent protein kinase, phosphorylase kinase and protein kinase C) phosphorylate glycogen synthase in various centers, transferring it to an inactive state.

Which cell signal transmission system will be used depends on the type of receptors with which adrenaline interacts. Thus, the interaction of adrenaline with β 2 receptors of liver cells activates the adenylate cyclase system. The interaction of adrenaline with α 1 receptors “switches on” the inositol phosphate mechanism of transmembrane hormonal signal transmission. The result of the action of both systems is the phosphorylation of key enzymes and the switching of processes from glycogen synthesis to its breakdown. It should be noted that the type of receptor that is most involved in the cell's response to adrenaline depends on its concentration in the blood.

During digestion the influence of insulin predominates, since the insulin-glucagon index in this case increases. In general, insulin has the opposite effect on glycogen metabolism than glucagon. Insulin reduces blood glucose concentrations during digestion by affecting liver metabolism in the following ways:

reduces the level of cAMP in cells and thereby activating protein kinase B. Protein kinase B, in turn, phosphorylates and activates cAMP phosphodiesterase, an enzyme that hydrolyzes cAMP to form AMP;

activates phosphoprotein phosphatase of glycogen granules, which dephosphorylates glycogen synthase and thus activates it. In addition, phosphoprotein phosphatase dephosphorylates and therefore inactivates phosphorylase kinase and glycogen phosphorylase;

induces the synthesis of glucokinase, thereby accelerating the phosphorylation of glucose in the cell.