Natalia N. Bezborodkina

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 the rat regenerating cirrhotic liver after a partial hepatectomy

Rat liver punctate biopsies were used for cytofluorimetric determinations of the content of glycogen and its fractions in hepatocytes, and also for microchemical measurements of the activity of glucose-6-phosphatase, glycogen phosphorylase, and glycogen synthase, in liver tissue with cirrhosis produced by carbon tetrachloride (CCl4) poisoning, during regeneration of the liver after the cessation of poisoning and after a partial resection of the cirrhosed liver. The liver cirrhosis was shown to be characterized by an accumulation of glycogen (predominantly of its metabolically less active fraction) in hepatocytes and by a decrease in the activities of the glycogenolytic enzymes in the liver parenchyma. On the cessation of poisoning, there was a partial or complete return to normal levels of the glycogen metabolism parameters. Some of them returned to normal more quickly if a partial hepatectomy was performed after the cessation of poisoning.

Effects of the 2-ethylthiobenzimidazole hydrobromide (bemithyl) on carbohydrate metabolism in cirrhotic rat liver

The effect of the actoprotector bemithyl (2-ethylthiobenzimidazole hydrobromide) on the content of glycogen and activities of glycogen synthase, glycogen phosphorylase, and glucose-6-phosphatase was studied in the cirrhotic rat liver. The content of glycogen and its fraction was determined by a cytofluorimetric method (Kudryavtseva et al. 1974). It has been shown that in cirrhosis the content of total glycogen in hepatocytes increases about 3 times and the content of its stable fraction increases 7.5 times. The activity of glucose-6-phosphatase fell to a level as low as 25% of normal. Activities of glycogen synthase and glycogen phosphorylase in the cirrhotic liver did not differ from normal. In the cirrhotic liver, bemithyl produced a decrease of the total glycogen content which was associated with a decrease of the glycogen synthase activity and an increase of the glucose-6-phosphatase and glycogen phosphorylase activities. Thus, the results of our studies indicate a favorable effect of bemithyl on the cirrhotic liver.

Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose

Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120min after per os administration of glucose or fructose to fasted rats with CCl4-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity.

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.

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

Using cytochemical and Förster resonance energy transfer (FRET) methods, the structure of glycogen was studied in rat hepatocytes during starvation and in some time intervals after the peroral administration of glucose to the animals. Hepatocytes were stained with a fluorescent variant of PAS reaction on object glasses. The staining of preparations for 40 min with ethidium bromide-SO2 (EtBr-SO2) revealed the labile fraction (LF) of glycogen, while their subsequent staining with auramine-SO2 (Au-SO2) for 50 min revealed the stable fraction (SF) of glycogen in cells. The total glycogen content (LF + SF) in hepatocytes at various stages of rat refeeding was determined using a cytofluorimeter; then, in the same cells, the FRET efficiency was measured. Recording FRET at several sites of cells was performed using a Leica TCS SP5 laser scanning confocal microscope by using the FRET Acceptor Photobleaching (FRET AB) procedure. In this procedure, auramine was used as the donor (D), while ethidium bromide was used as the acceptor (A). The efficiency of FRET in the course of rat refeeding with glucose has been shown to change from 10 to 14%, and the glycogen structure markedly affects the value of this parameter. It is found that, in cells of starved rats and in early terms after the administration of glucose, the FRET efficiency correlates with the A/D ratio, which reflects the degree of filling of external tiers of glycogen molecules with glucose residues. At later terms of refeeding, this correlation is either less pronounced or completely absent. It has been established that, at the same A/D value, the FRET efficiency can change by three to four times. Since the probability of energy transduction from D to A is proportional to 1/R6, where R is the distance between D and A. These fluctuations of the FRET efficiency mean that the glycogen molecules have the labile structure, in which chains of glycoside residues can deviate from its axis at a distance of about a half of their diameter.

Metformin normalizes the structural changes in glycogen preceding prediabetes in mice overexpressing neuropeptide Y in noradrenergic neurons

Hepatic insulin resistance and increased gluconeogenesis are known therapeutic targets of metformin, but the role of hepatic glycogen in the pathogenesis of diabetes is less clear. Mouse model of neuropeptide Y (NPY) overexpression in noradrenergic neurons (OE‐NPYDβH) with a phenotype of late onset obesity, hepatosteatosis, and prediabetes was used to study early changes in glycogen structure and metabolism preceding prediabetes. Furthermore, the effect of the anti‐hyperglycemic agent, metformin (300 mg/kg/day/4 weeks in drinking water), was assessed on changes in glycogen metabolism, body weight, fat mass, and glucose tolerance. Glycogen structure was characterized by cytofluorometric analysis in isolated hepatocytes and mRNA expression of key enzymes by qPCR. OE‐NPYDβH mice displayed decreased labile glycogen fraction relative to stabile fraction (the intermediate form of glycogen) suggesting enhanced glycogen cycling. This was supported by decreased filling of glucose residues in the 10th outer tier of the glycogen molecule, which suggests accelerated glycogen phosphorylation. Metformin reduced fat mass gain in both genotypes, but glucose tolerance was improved mostly in wild‐type mice. However, metformin inhibited glycogen accumulation and normalized the ratio between glycogen structures in OE‐NPYDβH mice indicating decreased glycogen synthesis. Furthermore, the presence of glucose residues in the 11th tier together with decreased glycogen phosphorylase expression suggested inhibition of glycogen degradation. In conclusion, structural changes in glycogen of OE‐NPYDβH mice point to increased glycogen metabolism, which may predispose them to prediabetes. Metformin treatment normalizes these changes and suppresses both glycogen synthesis and phosphorylation, which may contribute to its preventive effect on the onset of diabetes.

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 (β-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.

Glycogen content in hepatocytes is related with their size in normal rat liver but not in cirrhotic one.

Background & Aims: Hepatocytes differ from one another by the degree of the ploidy, size, position in the liver lobule, and level of the DNA‐synthetic processes. It is believed, that the cell size exerts substantial influence on the metabolism of the hepatocytes and the glycogen content in them. The aim of the present study was to test this hypothesis. Methods: Dry weight of hepatocytes, their ploidy and glycogen content were determined in the normal and the cirrhotic rat liver. Liver cirrhosis in rats was produced by chronic inhalation of CCl4 vapours in the course of 6 months. A combined cytophotometric method was used. Dry weight of the cell, its glycogen and DNA content were successively measured on a mapped preparation. Result: Hepatocytes of each ploidy class in the normal and the cirrhotic rat liver accumulated glycogen at the same rate. In the normal liver, there was a distinct correlation between the size of hepatocytes and glycogen content in them. This correlation was observed in each ploidy class, and was especially pronounced in the class of mononucleate tetraploid hepatocytes. In the cirrhotic liver, there was no correlation between the size of the cells and their glycogen content. Conclusions: The impairment of liver lobular structure probably explains the observed lack of correlation between hepatocyte size and their glycogen content in the cirrhotic liver.

Dynamics of Proglycogen and Macroglycogen in Hepatocytes of Normal and Cirrhotic Rat Liver at Various Stages of Glycogenesis

The content and structure of glycogen in hepatocytes of normal and cirrhotic rat liver were examined at different time intervals after glucose administration to starving animals. We used an original cytofluorimetric method for detection and quantification of proglycogen (PG) and macroglycogen (MG) of isolated hepatocytes. The method is based on the use of reagents of the Schiff type with different spectral characteristics. The content of MG in hepatocytes of control rats was increased by 52% (p < 0.01) as early as after 10 min. The MG content in the cirrhotic liver cells was increased by 43% (p < 0.05) only 20 min after glucose administration to the starving animals. The correlation coefficient between MG content and the total glycogen content at various stages of glycogenesis in rats of both groups was from 0.90 to 0.99 (p < 0.001). Increase in the PG content in hepatocytes of control rats was observed in intervals of 10–30 and 45–75 min. The PG content in cirrhosis was increased only in 60 min after the beginning of glycogenesis, but in 120 min it was 1.5 times higher than the control values (p < 0.001). The correlation coefficients between PG and the total glycogen content in the cells were on average 0.86 (p < 0.001) and 0.77 (p < 0.001) in the control and experimental groups, respectively. Thus, the change in the total glycogen content in hepatocytes of normal and cirrhotic liver are associated mainly with changes in the MG level. The contribution of PG was most significant in normal liver at the beginning of glycogenesis (10–30 min); in cirrhotic liver, at later stages (75–120 min).