[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.