Spatial Structure of Glycogen Molecules in Cells.

Glycogen is a strongly branched polymer of α-D-glucose, with glucose residues in the linear chains linked by 1→4-bonds (~93% of the total number of bonds) and with branching after every 4-8 residues formed by 1→6-glycosidic bonds (~7% of the total number of bonds). It is thought currently that a fully formed glycogen molecule (ß-particle) with the self-glycosylating protein glycogenin in the center has a spherical shape with diameter of ~42 nm and contains ~ 55,000 glucose residues. The glycogen molecule also includes numerous proteins involved in its synthesis and degradation, as well as proteins performing a carcass function. However, the type and force of bonds connecting these proteins to the polysaccharide moiety of glycogen are significantly different. This review presents the available data on the spatial structure of the glycogen molecule and its changes under various physiological and pathological conditions.

[Dynamics of pro- and macroglycogen content in hepatocytes of normal and cirrhotic rat liver at different stages of glycogenesis].

The content and structure of glycogen in hepatocytes of normal and cirrhotic rat liver has been studied at definite time intervals after the administration of glucose to starving animals. In the study, an original cytofluorimetric method for detection and quatification of proglycogen (PG) and macroglycogen (MG) content in isolated hepatocytes was applied. This method is based on using Schiff reagents with different spectral characteristics. It has been determined that the content MG content in the hepatocytes of control rats increases in 10 min after initiation of glycogenesis by 52% (P < 0.01). MG content in the cells of cirrhotic liver increased only after 20 min (43%, P < 0.05) after glucose administration to starving animals. The coefficient of correlation between MG content and the total glycogen content in the hepatocytes at different stages of glycogenesis ranged from 0.90 to 0.99 (P < 0.001) in both groups of rats. Increase in PG content in hepatocytes of control rats appeared within 10-30 and 45-70 min. In the case of cirrhosis PG content increased only 60 min after the start of glycogenesis, but after 120 min it was 1.5 times higher than the control values (P < 0.001). The correlation coefficient between the PG and the total glycogen content in rat liver cells averaged 0.86 (P < 0.001) and 0.77 (P < 0.001) in control and experimental groups, respectively. Thus, the change in total glycogen content in hepatocytes of normal and cirrhotic liver is associated mainly with the level of MG. In normal cells, contribution of PG is most significant in the early glycogenesis (10-30 min), and in the cirrhotic liver--in the later stages.

Heterogeneity of left ventricular cardiomyocytes from rat heart.

Contractile cardiomyocytes in various parts of the heart differ in shape, size, ploidy, and other parameters. However, it is not known whether their population is heterogeneous within each heart chamber. In this paper, dry weight and ploidy of cardiomyocytes were estimated in different parts of rat left ventricle. It was found that the dry weight of cardiomyocytes in medial part of left ventricular anterior wall is higher than in other parts of the ventricle. Cardiomyocyte ploidy is the same in different regions of the left ventricle.

[Analysis of glycogen structure in rat hepatocytes using cytochemical and FRET methods].

Using cytochemical and FRET (Forster, Resonance Energy Transfer) methods, the glycogen structure in rat hepatocytes was investigated during fasting and at different time intervals after per os glucose administration to animals. Hepatocytes on slides were stained with fluorescent PAS-reaction. Staining the slides with ethidium bromide-SO2 (EtBr-SO2) for 40 min revealed a labile glycogen fraction (LE), and the subsequent staining the same samples with auramine-SO2 (Au-SO2) for 50 min showed a stable glycogen fraction (SF) in the cells. The total glycogen content (LF and SF) in the hepatocytes at different stages of refeeding was determined by means of cytofluorimetry, and then efficiency of FRET was measured in the same cells. Registration of FRET in several areas of the cells was carried out on a laser scanning confocal microscope Leica TCS SP5 with application of FRET AB (Acceptor Photobleaching) procedure. In this procedure, auramine served as a donor (D) and ethidium bromide was an acceptor (A). It was shown that the efficiency of FRET varied from 10 to 14 % during refeeding, while the glycogen structure had a marked influence on the value of this parameter. FRET efficiency was shown to correlate with the ratio A/D in the cells of hungry rats and at the early stages after glucose administration to animals, which reflected the degree of filling of the external tiers of glycogen molecules of glucose residues. At later stages, this correlation was either less pronounced or absent. It was found that the FRET efficiency can vary by 3-4 times at the same value of A/D. Since the probability of energy transfer from D to A is proportional to 1/R6, where R is a distance between D and A, such variations of the FRET efficiency indicate that the glycogen molecules possess a labile structure in which the chain of glucose residues can deviate from its axis by a distance of about half their diameter.

[Comparative morphofunctional analysis of rat hepatocyte cultures isolated from the normal and pathologically changed liver due to experimental toxic hepatitis].

The goal of the study was to examine the state of primary hepatocytes of rats with toxic hepatitis induced by combination of CCl4 and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed actin cytoskeleton, cell-cell and cell-matrix contacts. To investigate morphology and localization of mitochondria the hepatocytes were stained with Rhodamine 123. Glycogen and DNA contents in hepatocytes were determined by fluorescent cytophotometry during the lifetime of the culture. Cells were maintained for 5 days, and there were no changes in ploidy distribution observed. The mean ploidy was not changed too. Thus hepatocytes of different ploidy demonstrated similar survival rate. The glycogen content was 50% higher in experimental group compared to the control. The glycogen content decreased in control and cyrrotic hepatocytes after collagenase isolation. It has been found that the control hepatocytes accumulated glycogen within 3 days. On the contrary, the glycogen levels remained to be low in the pathologic hepatocytes.

[Interrelation between the glycogen content in hepatocytes and their size in normal and cirrhotic rat liver].

The dependence between the size of hepatocytes and glycogen content in them was investigated in normal and cirrhotic liver from fasting rats as well as from rats in 10 and 60 minutes after per os glucose administration. The cytophotometric method used allows glycogen and DNA contents determining in the same cell and its dry mass as well. It has been shown that the dry mass and the glycogen content in hepatocytes from normal and cirrhotic liver are proportional to genes dose. Normal liver hepatocytes of various ploidy classes show distinct correlation between the cell size and the glycogen content. No similar dependence is observed in hepatocytes population from the cirrhotic liver, apparently, because the liver lobule structure is disturbed, and heterogeneity of the hepatocytes microenvironment condition is increased.

[Application of Schiff's reagents with various spectral characteristics to determine the content of labile and stable glycogen fractions in isolated hepatocytes].

The dynamics of the total glycogen accumulation, its labile and stable fraction contents were studied in hepatocytes after administration of 30 % glucose to rats that had been starving for 48 h. In the study, the original method based on the use of Schiffs reagents with various spectral characteristics (auramine-SO2 - Au-SO2 - and ethidium bromide-SO2 - EtBr-SO2) was applied allowing detection and quantitative determination of the labile and stable glycogen fractions in the same hepatocytes to be achieved. Staining of the preparation by Au-SO2 during 40 min revealed the labile glycogen fraction (green fluorescence, lambda(max) = 526 nm). The following staining of the same sample by EtBr-SO2 during 50 min revealed stable glycogen fractions (red fluorescence, lambda(max) = 620 nm) in the cells. The measurement of fluorescence intensity in the corresponding spectrum permits the quantity of each glycogen fractions in isolated liver cells to be determined. It has been shown that as the glycogen accumulates in the hepatocytes its labile fraction content increases. The correlation coefficient (r) between the labile fraction content and the total glycogen content in hepatocytes obtained from the liver of starving rats was 0.95, and in hepatocytes isolated 20 and 30 min after the beginning of glucose refeeding to starving rats it was 0.95 and 0.98, respectively. 90 and 120 min after administration of glucose to starving rats, when the hepatocyte population becomes non-synchronous with respect to synthesis and degradation of glycogen in cells, the dependence of the labile fraction content upon the quantity of the total glycogen becomes less expressed (r= 0.71 and 0.82, respectively). Thus the application of Schiff's reagents with various spectral characteristics enables us to obtain valuable information about glycogen structure in cells and about the processes of its synthesis and degradation in the cell population.

[Morphometry of hepatocyte mitochondrial apparatus in normal and cirrhotic rat liver].

Morphometric electron microscopy study of the hepatocyte mitochondrial apparatus and morphofunctional analysis of the degree of pathological alterations were carried out on the liver of rats with CCL4-cirrhosis (experimental group). Chronic poisoning of rats with CCL4 for 6 months led to a 4.2-fold increase in proportion of connective tissue and to a decrease in the number of hepatocytes in the liver by 21.8 %. Dry mass and ploidy of hepatocytes in the cirrhotis liver rose as compared with norm by 20.6 and 9.3%, respectively. Activities of alanine and aspartate aminotransferases in blood of rats of experimental group exceeded normal ones 2.0 and 1.4 times, respectively. Concentration of total bilirubin in blood of the cirrhotic animals increased 1.7 times, while concentration of total protein decreased by 22%. Concentration of diene conjugates in the liver of rats of experimental group increased 2.1 times as compared with normal one, while the level of malonic dialdehyde - by 34%. Activities of superoxide dismutase and catalase in the cirrhotic liver were lower than in the normal liver were lower than in the normal liver by 16 and 23 %, respectively. Morphometry of the hepatocyte mitochondrial apparatus has shown that in spite of an increase in the voluminous density of mitochondria in hepatocytes of the cirrhotic liver (by 28 %), concentration of internal mitochondrial membranes in the cells was reduced almost 1.5 times, while the total length of internal membrane in a single mitochondrion was reduced about twice as compared with norm. Thus, despite compensation of the partial loss of hepatocytes because of their polyploidization and hypertrophy, the specific synthetic activity of cells in the case of cirrhosis is decreased due to deterioration of the antioxidant system and electron transport chain of the mitochondrial apparatus.

[New protective effect of simvastatin in rats with experimental steatohepatitis].

The intragastric introduction of carbon tetrachloride (CCl4) (0.2 ml/kg in 50% oil suspension, twice a week) and ethyl alcohol (5% solution ad libitum as the only available drink) in rats over a period of four weeks results in the development of inflammation, fibrosis, and fatty dystrophy in the liver. Such a fast formation of liver damage is obviously caused by potentiating effect of alcohol in combination with CCl4. Simultaneous injection of simvastatin (1 mg/kg, intragastrically) in rats with ethanol--CCl4 hepatitis decreased fatty dystrophy and produced certain anticytolytic and anticholestatic effects without potentiation of microsomal oxidation system damage by hepatotoxins. In addition, simvastatin shows hypolipidemic activity, which is manifested primarily by a decrease in the general holesterol level in the blood serum.

[Studying the hepatoprotector effect of bemithyl on a model of chronic toxic liver damage].

A prophylactic and therapeutic introduction of the synthetic adaptogen bemithyl (2-ethylthiobensimidasole hydrobromide) produces a hepatoprotector effect in rats with experimental cirrhosis. The drug exhibits an anticytolytic activity, restores liver participation in the pigment exchange, and normalizes the function of the microsomal oxidation system responsible for the metabolism of xenobiotics. The treatment with bemithyl also leads to a certain improvement of a histologic picture of the damaged liver and to a decrease in the degree of fibrosis. The drug is also capable of increasing the activity of the antioxidant system and inhibiting the process of lipid peroxidation of proteins and lipids in the liver.

[The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments]. / Vliianie gepatoprotektora 2-étiltiobenzimidazola gidrobromida (bemitila) na soderzhaniie glikogena v gepatotsitakh tsirroticheski izmenennii pecheni, nakhodiashchikhsia v razlichnykh usloviiakh mikrookruzheniia.

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.

[Dynamics of glycogen content in the normal and cirrhotic liver following glucose administration to starving rats]. / Dinamika soderzhaniia glikogena v normal'noi i tsirroticheskaia izmenennoi pecheni posle vvedeniia gliukozy golodnym krysam.

Using biochemical, cytofluorimetric and television cytophotometric methods, glycogen contents were studied in normal and cirrhotic rat liver at various intervals after glucose administration to fasting animals. The obtained data indicate that after a 48 h fasting glycogen contents in normal and cirrhotic liver are equally poor. A marked rise of glycogen content in cirrhotic liver was observed only 20-30 min after glucose administration to rats. It has been established that at all intervals after glucose administration to rats hepatocytes of the portal lobule zone, both in normal and in cirrhotic liver, accumulate more glycogen than those of the central zone. Again, the intensity of glycogen accumulation in cirrhotically altered liver is significantly lower than in normal liver, due, presumably, to a lower rate of glycogen synthesis in pathologically changed liver.

[Features of the effect of bemethyl on glycogen metabolism in hepatocytes of pathological changed human liver]. / Osobennosti vliianiia bemitila na metabolizm glikogena v gepatotsitakh patologicheski izmenennoi pecheni cheloveka.

Effect of actoprotector bemithyl (2-ethylthiobenzimidazole hydrobromide) on glycogen metabolism in hepatocytes of patients with chronic hepatitis and liver cirrhosis was investigated. Using cytofluorimetric method, the content of glycogen and its fractions in isolated hepatocytes was measured. The treatment with bemithyl resulted in a decrease in glycogen levels in hepatocytes, and in a marked restoration of fractional glycogen composition as compared to the basic therapy. Besides, it was established that the degree of glycogen decrease in cells of patients with chronic hepatitis depended on the increase of glucose-6-phosphatase activity (r = 0.75, P < 0.05), and on the levels of glycogen in hepatocytes prior to bemitil treatment (r = = 0.87, P < 0.01). Positive changes in glycogen metabolism after bemithyl treatment are pronounced in patients with chronic hepatitis. These positive alterations take place simultaneously with the conservation of basic structural disturbances in the liver parenchyma. However, even in this case, the indices of glycogen metabolism do not reach the normal levels.

[Effect of bemythyl on carbohydrate metabolism in cirrhotic rat liver]. / Issledovanie vliianiia bemitila na uglevodnyi obmen tsirroticheski izmenennoi pecheni krys.

Effect of actoprotector bemitil (2-ethylthiobenzimidazole hydrobromide) on glycogen content and activities of glycogen synthase, glycogen phosphorylase, and glucose-6-phosphatase was studied in cirrhotically altered rat liver. The contents of glycogen and its fraction were determined a cytofluorimetrically (Kudryavtseva et al., 1974). In cirrhosis, the total glycogen content in hepatocytes increases by nearly 3 times, while the amount of a stable fraction of glycogen rises by 7.5 times. Glucose-6-phosphatase activity fell to the level of 25% compare to the norm. Activities of glycogen synthase and glycogen phosphorylase in the cirrhotic liver did not differ from the norm. In cirrhotically altered liver, bemitil produced a decrease in the total glycogen content due to a decrease in glycogen synthase activity in an increase in glucose-6-phosphatase and glycogen phosphorylase activities. The above results suggest a favorable effect of bemitil on cirrhotic liver.

[Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver]. / Vliianie chastichnoi gepatéktomii na urovni glikogena v gepatotsitakh portal'noi i tsentral'noi zon dol'ki tsirroticheski izmenennoi pecheni krys.

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.

Metabolic heterogeneity of glycogen in hepatocytes of patients with liver cirrhosis: the glycogen of the liver lobule zones in cirrhosis.

The concentrations of total glycogen (TG) and its labile (LF) and stable (SF) fractions were determined in hepatocytes of portal and central zones of the normal human liver and in the liver of patients with cirrhosis of viral and alcohol aetiologies. Using PAS reaction, TG, LF and SF were revealed in histological sections of the material obtained by the liver punch biopsies. The concentrations of TG and its fractions were measured by televisional cytophotometry. In liver cirrhosis, the concentrations of TG, LF and SF in both zones of the hepatic lobule have been found to be much higher than in the normal liver. It has been shown that the ratio of the hepatocyte TG concentrations in the portal zone to the central zone both in the normal liver and in viral cirrhosis exceeds 1.0, amounting to 1.264 +/- 0.021 and 1.030 +/- 0.009, respectively. The glycogen fraction composition in the cells of both the liver lobule zones in viral cirrhosis does not differ significantly from the norm. On the contrary, in the liver of patients with alcoholic cirrhosis, the ratio of the TG concentrations in the portal zone to the central zone is reduced to 0.815 +/- 0.016 and is accompanied by qualitative changes of the glycogen composition.

Glycogen-forming function of hepatocytes in cirrhotically altered rat liver after treatment with chorionic gonadotropin.

Using cytofluorimetric and biochemical studies on serial supravital liver punctate biopsies, effects of chorionic gonadotropin (CG) on recovery of hepatocyte glycogen-forming function in the cirrhotically altered rat liver were analyzed. The biopsies were taken first from rats with experimental cirrhosis produced by their 6-month-long poisoning with the hepatotoxic poison CCl4, then from the same animals in 1, 3, and 6 month after cessation of their poisoning, either on treatment with CG or with no treatment. In smears of isolated hepatocytes, the contents of the total glycogen (TG) and of its labile and stable fractions (LF and SF, respectively) were measured. In liver homogenates, activities of glucose-6-phosphatase (G6Pase), glycogen phosphorylase, and glycogen synthetase were determined. It was found that the threefold increased TG content in hepatocytes of cirrhotic liver returned to the normal level in 3 months without treatment, while as soon as in 1 month in the case of the treatment with CG. The CG treatment for 3 months resulted in normalization of the glycogen fraction composition that had been changed in cirrhotic liver, whereas without treatment, the glycogen LF/SF ratio remained changed even after 6 months after cessation of the poisoning with CCl4. Activity of G6Pase was fourfold reduced in cirrhosis; in 3 months after the end of poisoning, under effect of CG, the activity increased to the normal level, but somewhat decreased subsequently. In the animals that were not treated with CG, the decrease in the G6Pase activity after the cessation of the CCl4 poisoning was even more marked than in the CG-treated rats. Activities of two other enzymes of glycogen metabolism did not differ statistically significantly from the norm throughout the entire experiment. The data obtained indicate that the use of CG for rehabilitation of the glycogen-forming function of the cirrhotically altered liver is more efficient than other ways of treatment studied previously, such as partial hepatectomy or a high-carbohydrate diet.

[Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes]. / Vliianie preparata "vilon" na tsirroticheski izmenennuiu pecheni krys. Regeneratsiia pecheni i sostoianie glikogenoobrazovatel'noi funktsii gepatotsitov.

Effects of a dipeptide preparation "Vilon" on rehabilitation of functional activity of hepatocytes and regeneration of the cirrhotically altered rat liver were studied. The liver cirrhosis was produced by poisoning of rats for 4 months with carbon tetrachloride (CCl4). On the end of the poisoning with CCl4, one group of animals was not submitted to any further actions, whereas animals of the other group were injected "Vilon" (1.7 micrograms/kg) daily for 5 days. On smears of isolated hepatocytes, contents of total glycogen (TG), and its labile and stable fractions (LF and SF) were determined in addition to cell ploidy levels and the total protein content. In liver homogenates, activities of glucose-6-phosphatase (G6P), glycogen synthase (GS), and glycogen phosphorylase (GP) were measured. In 2 weeks after the drug application, G6P activity being reduced in cirrhosis 1.2 times, elevated under effect of "Vilon". In non-treated rats the contents of TG and its fractions and of G6P activity remained at the level characteristic of the cirrhotic liver prior to "Vilon" administration. In both groups of rats, GP and GS activities in the cirrhotically altered liver did not differ from their control values throughout the entire experiment. "Vilon" has been shown to exert a weak stimulating effect on regeneration of the cirrotically altered rat liver: in hepatocytes of the second group of rats the total protein content and ploidy levels were higher than those in the first group by 4.7 and 11.5%, respectively.

[Metabolic heterogeneity of glycogen in hepatocytes of patients with liver cirrhosis]. / Metabolicheskaia geterogennost' glikogena v gepatotsitakh bol'nykh tsirrozom pecheni.

Concentrations of the total glycogen (TG) and of its labile and stable fractions (LF and SF, respectively) were determined in hepatocytes of portal and central zones of the normal human liver and in the liver of patients with cirrhosis of viral and alcohol etiology. Using the PAS reaction, TG and its LF and SF were revealed in histological sections of the material obtained by liver punction biopsies. Concentrations of TG and its fractions were measured by television cytophotometry. In liver cirrhosis, concentrations of TG, LF, and SF in both zones of the hepatic lobule were much higher than in the normal liver. The ratio between hepatocyte TG concentration in the portal zone and that in the central zone (P/C ratio), both in norm and in viral cirrhosis, exceeds 1.0 to reach, respectively, 1.26 +/- 0.02 and 1.03 +/- 0.01. The glycogen fraction composition in cells of both liver lobule zones in viral cirrhosis does not significantly differ from that in norm. On the contrary, in the liver of patients with alcoholic cirrhosis, the P/C ratio falls to 0.82 +/- 0.02 to be accompanied by qualitative changes in glycogen composition.

[Glycogen synthesizing function of hepatocytes in rats with liver cirrhosis after treatment with chorionic gonadotropin]. / Sostoianie glikogenobrazovatel'noi funktsii gepatotsitov tsirroticheski izmenennoi pecheni krys posle vozdeistviia khorionicheskim gonadotropinom.

Using rat liver hepatocytes, methods of cytofluorimetry (Kudryavtseva et al., 1974) and biochemistry were applied to comparative studies of the total glycogen content, including its labile (LF) and stable (SF) fractions, and activities of glucose-6-phosphatase, glycogen phosphorylase and glycogensynthetase in these. The liver hepatocytes were examined in norm, and under conditions of CCl4 poisoning of rats, both 6 months after a chronic poisoning, and 1, 3 and 6 months following poisoning cessation. All the experimentally poisoned rats were divided into two conventional groups: rats of one group received, apart from poisoning, a complex treatment with chorionic gonadotropin (CG); the other group rats received, no treatment. The material used for examination was obtained from serial functional biopsies of each experimental animal. It has been shown that under cirrhosis the content of the total glycogen in hepatocytes increased by 3 times, and that of its SF even by 9.7 times. The treatment with CG for 1 month resulted in its reducing to the norm, and 3 to 6 months treatments normalized contents of both the glycogen fractions. In the group of non-treated rats no similar changes were registered. Besides, in the cirrotic rats the activity of glucose-6-phosphatase was shown to increase by 4 times. After CG treatment it was seen to decrease by 3 times. Thus, CG may be regarded as an optimum and more effective agent for restoring abnormalities in cirrotic liver, compared to some other stimulating factors, such as hepatectomy (Kudryavtseva et al., 1996) or rich-carbohydrate diet (Kudryavtseva et al., 1998).