Question: What changes in the level of ribonuclease activity (RNase activity) can be observed in the regenerating liver of healthy and alloxan diabetic rats following partial hepatectomy? What coincidental alterations of the ratio A-cells to B-cells do occur in the islets of Langerhans?
Materials and methods: 120 male Wistar rats of averagely 180 g body weight were fed a standard diet ("Rehbrücke") with drinking water ad libitum. After deprivation of food for 15 hours the animals were sacrificed by heart puncture under ether anesthesia. The caudal part of the body was rinsed free from blood by ice-cooled physiological saline via the aorta. After removal of the livers each 2 g of liver tissue were homogenized in 0.25 M saccharose or 70% ethyl alcohol. For the study of impairment of ribonuclease activity in the liver following different lesions the animals were divided into 3 test groups of 35 animals each: group 1: alloxan diabetes; group 2: liver regeneration after partial hepatectomy; group 3: alloxan diabetes in combination with liver regeneration after partial hepatectomy. From each group always 4 to 6 animals were sacrificed at the following time intervals: 2nd day and 4th day as well as after 3, 4 and 6 weeks. In 8 animals the enzymatic activity was not influenced. RNA content and dry weight of the livers of all test animals were determined. In liver homogenates with saccharose the ribonuclease activity was measured according to the method of FIERS (1961) using highly purified yeast RNA for substrate. This technique is especially suitable for raw tissue homogenates. After 30 min incubation at 37 degrees C and precipitation of the high-molecular polynucleotides as well as of protein by means of methylglycol and barium perchlorate the not precipitable low-molecular oligonucleotides which were produced by ribonuclease action, were spectrophotometrically measured in the supernatant at 260 nm. The deltaE-value was calculated for the employed quantity of RNA related to mg of protein brought into action. Demonstration and determination of the ribonuclease inhibitor was abandoned. Two different RNA substrate concentrations were used; in the higher concentration the activity was measured at pH 5.5 to 6 and 7.5, in the lower concentrations at pH 7. Protein measurement was performed after the method of LOWREY (LOWREY et al. 1951) as modified by GLASER and KLEINE (1962); blood sugar was determined in tail vein blood by means of o-toluidine technique. In the liver homogenate with ethyl alcohol the RNA extraction was modified according to the data given by OGUR and ROSEN (1950); RNA determination was done colorimetrically using orcin hydrochloric acid. For dry weight determination the same homogenate was used. Alloxan diabetes was induced by injecting the rats 175 mg/kg b.w. of alloxan after fasting for 24 hours. The 5% alloxan solution in citrate buffer (pH = 4) was prepared immediately before injection. For further experimentation only those animals with blood sugar values of greater than 300 mg/100 ml were employed...