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To determine which of the major isoenzymes of pyruvate kinase pancreatic islet pyruvate kinase most resembled, it was compared to pyruvate kinase from other tissues in kinetic and immunologic studies. The pattern of activation by fructose bisphosphate and the patterns of inhibition by alanine and phenylalanine were most similar to those of the M2 isoenzyme from kidney and were dissimilar to those of the isoenzymes from skeletal muscle (type M1) and liver (type L). The islet pyruvate kinase was inhibited by anti-M1 pyruvate kinase serum (which crossreacts with the M2 isoenzyme), but not by anti-L pyruvate kinase. These results are most consistent with islets possessing predominantly, if not exclusively, the M2 isoenzyme of pyruvate kinase. We previously showed that rat pancreatic islet cytosol contains protein kinases that can catalyze a calcium-activated phosphorylation of an endogenous peptide that has properties, such as subunit molecular weight and isoelectric pH, that are identical to those of the M2 and M, isoenzymes of pyruvate kinase, and that islet cytosol can catalyze phosphorylation of muscle pyruvate kinase. In the present study it was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruvate kinase activity. It is concluded that phosphorylation of the islet pyruvate kinase has no immediate effect on enzyme activity.

We previously showed 5-androstenediol (5-AED) injected sc enhanced survival in mice exposed to whole-body gamma-irradiation, ameliorated radiation-induced neutropenia in mice, dogs, and monkeys, and induced increases in bone marrow GM-CFC in irradiated mice. Radiation-induced decreases in platelets, natural killer cells, red blood cells, and monocytes were also blunted by the steroid. Administration of 5-AED caused functional activation of circulating granulocytes (phagocytic ability), monocytes (oxidative burst), and natural killer cells (surface CD11b expression). To test our hypothesis that 5-AED acts via initiation of a cytokine cascade in hematopoietic tissue, we performed real time quantitative PCR on spleen samples taken from mice and correlated the results with serum ELISA. PCR was performed for mRNAs of GM-CSF, IL-6, IL-10, IL-12, and IFNg. Spleens and serum were taken at 3 time points: 4 h after 5-AED or vehicle (PEG-400) injection, and 4 or 24 h after irradiation (7.5 Gy whole-body gamma) or sham-irradiation. Each group contained 5 mice. Results were analyzed by 2-way ANOVA for PCR results 4 and 24 h after irradiation, and by a t-test 4 h after injection. Radiation alone induced increases in mRNAs for all cytokines at both 4 and 24 h, except for IFNg, which was not elevated at 4 h. 5-AED alone did not induce a significant increase for any cytokine, but the increases bordered on significance for IL-12 at 4 h after irradiation or sham-irradiation (28 h after injection, p = 0.07), and for GM-CSF 24 h after irradiation or sham-irradiation (48 h after injection, p = 0.08). There was a significant positive interaction between radiation and 5-AED for IL-6, 4 h after irradiation (p = 0.006). The interaction effect bordered on significance for IL-12 4 h after irradiation (p = 0.07) and for GM-CSF 24 h after irradiation (p = 0.10). ELISA for serum cytokine protein was measured for G-CSF, IL-6, IL-10, and IL-12. Results were analyzed by the Mann-Whitney test for exact p-values. IL-6 was not detected in serum. G-CSF was detected 4 and 24 h after irradiation or sham-irradiation only in 5-AED-treated mice. IL-10 and IL-12 were detected only 24 h after irradiation, and the values in 5-AED-treated mice were significantly higher than those in vehicle-treated mice (p = 0.02). The results support our hypothesis that the previously observed increases in numbers of circulating innate immune cells and platelets, and functional activation of granulocytes, monocytes, and NK cells, result from a cytokine cascade induced by 5-AED.