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.
Flow Cytometry/methods , Glycogen/analysis , Hepatocytes/chemistry , Animals , Male , Rats , Rosaniline Dyes
