Cytofluorimetric research on the content of glycogen and its fractions in the hepatocytes of patients with liver cirrhosis of different etiologies]

By cytofluorometric method, a study was made of the total glycogen and its two fractions in liver parenchymal cells both in the donors (20 men) and in patients with cirrhosis of different etiology (39 men). The examination was performed on preparations--smears of isolated hepatocytes, obtained from the live functional liver biopsies. The quantitative analysis has shown an increase in the total glycogen content in hepatocytes of patients with cirrhosis by 3 times compared to the norm, and this increase is independent on the etiology of liver cirrhosis. To study the mechanism of the discovered glycogenosis, the activity of key enzymes of glycogenolyses was determined. It was shown that glucose-6-phosphatase and glycogen-phosphorylase activity in the liver with cirrhosis was lower than in the norm. The most considerable changes were shown in hepatocytes of patients with liver cirrhosis in fractional glycogen composition and, even more significant, in the content of a hard soluble fraction. The hard soluble fraction portion was higher in hepatocytes of the patients with liver cirrhosis of alcohol etiology. The quantitative analysis of glycogen fraction contents in liver cells may be the best marker in the differential diagnosis of symptomless elapsing liver cirrhosis.     

Cytofluorimetric study of glycogen fractions of the liver cells of patients with chronic viral and alcoholic hepatitis

A cytofluorometric study was made of total glycogen and two glycogen fractions--the one easily soluble (ES) and the other hard soluble (HS) in isolated liver cells (needle aspiration biopsy) of patients in the norm and with chronic viral hepatitis (CVH) and chronic alcoholic hepatitis (CAH). The amount of LS in hepatocytes of patients with CAH was lower than that in patients with the norm or with CVH. This distinction was shown already at the beginning of chronic disease, and then, in spite of a considerable increase in the total glycogen content in hepatocytes, with progression of the disease did not change. The quantitative analysis of glycogen fraction contents in liver cells may be an additional differential diagnostic marker for the etiological distinction of chronic hepatitis.     

Cytofluorimetric study of the glycogen content in hepatocytes of varying ploidy in adult rats]

A combined method, that allows measuring glycogen and DNA contents in one of the same cell, was applied for quantitative determination of these in mono- and binucleate hepatocytes with different ploidy obtained from adult rats. The mean glycogen content was shown to increase proportionally to the genome number within the changes of the hepatocyte ploidy from 2 to 8c.     

Metabolic heterogeneity of glycogen in hepatocytes of patients with liver cirrhosis

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.     

Hepatic glutathione content in patients with alcoholic and non alcoholic liver diseases

Reduced and oxidized hepatic glutathione was evaluated during alcoholic and non alcoholic liver injury. We studied 35 chronic alcoholics, 20 patients with non alcoholic liver diseases, 15 control subjects. Hepatic glutathione was measured in liver biopsies and correlated with histology and laboratory tests. Alcoholic and non alcoholic patients exhibited a significant decrease of hepatic glutathione compared to control subjects (controls: 4.14 +/- 0.1 mumol/g liver; alcoholics: 2.55 +/- 0.1, p less than 0.001; non alcoholics 2.77 +/- 0.1, p less than 0.001). Oxidized glutathione was significantly higher in the two groups of patients compared to controls (controls: 4.4 +/- 0.2% of total; alcoholics 8.2 +/- 0.3, p less than 0.001; non alcoholics: 8.5 +/- 0.8, p less than 0.001). The decreased hepatic glutathione levels in patients with alcoholic and non alcoholic liver diseases may represent a contributing factor of liver injury and may enhance the risk of toxicity in these patients.     

Cytofluorometric study of the glycogen content in DNA-synthesizing and -nonsynthesizing hepatocytes in the regenerating liver

Using a combined cytochemical method that allows to determine glycogen, DNA and 3H-thymidine label in the same cell, glycogen amounts were measured both in 3H-TdR-marked and non-marked hepatocytes of the regenerating 3H-thymidine. During mitosis, the glycogen amount is reduced if compared with that in cells being in presynthetic phase. It is proposed that the decrease in glycogen content in the regenerating liver may partly depend on energetic expenses of cells that started DNA synthesis and mitotic division. The phenotypic expression of genes responsible for glycogen synthesis and splitting in di-, tetra- and octoploid hepatocytes of the regenerating liver liver was proportional to the corresponding values.     

The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments

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.     

Cytofluorimetric study of the content of glycogen and its fractions in rat liver cells in the course of the 1st week of postnatal ontogeny]

A cytofluorometric study of the total glycogen and its fractions in rat liver cells using the fluorescent PAS reaction was made during 1--7 days of the postnatal development. It was established that glycogen content was small on the first two days of development. The glycogen content increases only on the third day after birth. The glycogen of the rat liver cells during a first week of the postnatal development is different from that detected in adult liver cells in two aspects: in 3 day old hepatocytes soluble and stable glycogen fractions are equal, while in adult rat liver cells the former makes 80--90%; during the first week of the postnatal development, the stable fraction of rat liver cell is more labile, while in the adult rat liver the soluble fraction of glycogen is more labiles.     

Quantitative analysis of glycogen content in hepatocytes of human liver allograft after ischemia and reperfusion.

In order to examine glucose metabolism in liver grafts after cold ischemia and reperfusion, the heterogeneous lobular distribution pattern of glycogen content was studied using histochemical quantitative analysis. In most of the cases, this heterogeneous pattern of glycogen was observed after preservation and reperfusion. However, a 42% reduction of glycogen content, expressed as the ratio between stained surface and total surface of liver biopsies, was observed in biopsies after reperfusion. Moreover, both periportal and centrilobular hepatocytes showed a significant decrease in mean optical density after reperfusion (18% and 25%, respectively). The comparison of our results to early postoperative liver function tests and cold ischemia times showed no significant correlation (p<0.05).     

Ultrastructural localization of Cu, Zn-SOD in hepatocytes of patients with various liver diseases

The ultrastructural localization of copper, zinc-superoxide dismutase (Cu, Zn-SOD) in the liver of patients with acute hepatitis, chronic hepatitis, liver cirrhosis and alcoholic fatty liver was studied by means of the indirect immunoperoxidase technique. In hepatocytes Cu, Zn-SOD was found to be localized in perinuclear cisternae, rough endoplasmic reticulum (rER), vesicles and Golgi apparatus. The Cu, Zn-SOD was also detected around the lipid droplets in hepatocytes as well as on the cytoplasmic membrane in cases of liver cirrhosis. These findings suggest that Cu, Zn-SOD is produced in the rER in hepatocytes and protects the cells from cellular injury caused by superoxide anion radical in various disorders of the liver.     

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.     

Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes. Inverse relationship to glycogen content

Cultured rat hepatocytes were used to characterize the relationship between cellular glycogen content and the basal rate, as well as response to insulin of glycogen synthesis. Depending on the concentration of medium glucose, glycogen-depleted monolayers accumulated glycogen between 24 and 48 h of culture up to the fed in vivo level. Insulin at 100 nM stimulated glycogen deposition 20-fold at 1 mM and 1.5-fold at 50 mM glucose. The rate of further glycogen storage decreased with time and increasing glycogen content. In hepatocytes preincubated with 1-50 mM glucose during 24-48 h, short-term basal and insulin-dependent incorporation of 10 mM [14C]glucose into glycogen was inversely related to the actual cellular glycogen content. This was not due to different intracellular dilution of the label, since the specific radioactivity of UDP-glucose was similar in all groups. 125I-Insulin binding indicated that insulin receptors were also not involved in this phenomenon. An inverse relationship was also found between glycogen content and the stimulation of glycogen synthase I activity by insulin, whereas the basal activity of the enzyme was dissociated from the rate of incorporation of [14C]glucose. Basal net glycogen deposition at 10 mM glucose was also inversely related to cellular glycogen; however, no such relation was evident in the presence of insulin due to the overlapping inhibition of glycogenolysis. These studies suggest that the glycogen-mediated inhibition of the activation of glycogen synthase I is operative in the cultured hepatocyte and leads to an apparent inverse relationship between the actual glycogen content and basal as well as insulin-dependent glycogenesis.     

Effect of cobalt on the liver glycogen content in the streptozotocin-induced diabetic rats

Cobalt decreases blood glucose in diabetic rats but the mechanisms involved are unclear. To determine the contribution of glycogen metabolism to glycemia-lowering effect, glycogen contents of liver and muscle in the streptozotocin-induced diabetic rats were determined. The liver glycogen was depleted in diabetic rats. But when cobalt was administered to the rats, the glycogen returned to the level of healthy rats, concomitantly with the decrease in blood glucose. The cobalt treatment had no effect on the muscle glycogen in the diabetic rats. The tissue-specific responses of glycogen metabolism suggest the involvement of suppressed glucagon signaling due to cobalt treatment.     

Serum alpha-mannosidase in patients with alcoholic liver disease

Sera from 9 persons with either biopsy-proven alcoholic liver disease or a history of chronic, excessive ethanol consumption were analyzed for their content of various hydrolases. Compared to controls, significant elevations in the following enzyme activities were seen in sera from the patient population: acid phosphatase (2.0-fold), beta-glucuronidase (2.1-fold), hexosaminidase (1.4-fold), and alpha-L-fucosidase (2.3-fold). In addition, alpha-mannosidase activity, previously reported to be unchanged in cases of hepatic cirrhosis [Reglero et al., Clinica chim. Acta 130: 155-158], (1980) was found to be significantly increased (p less than 0.001) when assays were performed at acid (pH 4.5) or intermediate (pH 5.5) hydrogen ion concentrations. Fractionation of sera on DEAE-Sephadex columns showed that the increase in alpha-mannosidase activity in the serum of patients with alcoholic liver disease was due to increases in the level of at least one 'acid alpha-mannosidase' and two intermediate pH optimum alpha-mannosidases. The general increase in the activity of a group of glycosidases is consistent with a hypothesis involving decreased clearance of glycoproteins from the blood of persons with hepatic cirrhosis.