Search

Isolated rat pancreatic islets were studied to determine the dynamic regulatory effects of glucose stimulation on the expression of messenger RNA (mRNA) and protein levels for inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) isoforms I, II, and III. The relative isoform abundance was: IP3R-III > IP3R-II approximately IP3R-I. Culture of islets with glucose (G; 20 mM) or alpha-ketoisocaproic acid for 30 min increased only IP3R-III mRNA expression above control (5.5 mM glucose). 2-Deoxyglucose was without effect. Islet culture for 2 h with G (20 mM) or alpha-ketoisocaproic acid reduced IP3R-III mRNA expression levels below control, and cycloheximide blocked the response. Culturing islets for 1 day or 7 days with G (11 mM) reduced the expression of IP3R-III mRNA but increased the expression of IP3R-II mRNA in a time-dependent manner. Cytosine arabinoside lowered cultured islet IP3R-II and -III mRNA levels, but glucose effects remained evident. IP3R-II mRNA levels were also significantly higher in islets from hyperglycemic 90% partial pancreatectomized rats, compared with sham animals. Islet IP3R mRNA expression also showed osmotic sensitivity. Islet IP3R-III protein levels increased after 2 h islet culture at 20 mM G, were unchanged after 1 day culture at 11 mM G, and were lower than control after 7 days culture at 11 mM G. In contrast, IP3R-II levels increased after 1 day and 7 days culture at 11 mM G, whereas IP3R-I protein levels remained unchanged. Thus, G stimulation rapidly increases transcription and expression of IP3R-III mRNA and protein levels in rat islets. However, chronic G stimulation up-regulates IP3R-II mRNA in cultured islets and in islets from partial pancreatectomized rats. Metabolic regulation of IP3R-II and III expression may mediate beta-cell IP3-responsive Ca2+ mobilization and insulin secretion.

Adenine nucleotides are thought to serve as second messengers in the control of beta-cell function by glucose, e.g. by regulating the activity of ATP-dependent K+ channels. However, their localization in different intracellular pools may mask the biologically relevant changes and complicate the interpretation of measurements in whole cells. The plasma membrane of mouse islet cells was selectively permeabilized by the alpha-toxin from Staphylococcus aureus to allow diffusion of cytoplasmic nucleotides. After permeabilization of cells from freshly isolated islets, approximately 68% of ATP, 45% of ADP, and 52% of AMP rapidly diffused out of the cells, whereas the insulin content hardly varied. The nondiffusible pool of nucleotides was stable for at least 90 min at 4 C, which suggests that it is contained in cellular organelles. The size of this nondiffusible pool decreased proportionally to insulin stores when these were lowered by stimulating secretion to different degrees during culture before permeabilization. From these results, it can be calculated that nondiffusible nucleotides are mainly contained in insulin secretory granules, with a small proportion in another, probably mitochondrial, compartment. Approximately 80% GTP and 30% GDP were present in the diffusible pool, and their relative proportions in the granular pool were only about 20% that of adenine nucleotides. Incubation of the cells in 20 instead of 2 mM glucose before permeabilization did not affect the nondiffusible pool, which indicates that the increase in the ATP/ADP ratio measured in intact cells occurred in the diffusible pool. Cytoplasmic nucleotide levels could be evaluated by subtracting the nondiffusible pool from the measurements in intact cells. It emerges that glucose induces large changes in the ATP/ADP ratio in the cytoplasmic pool, and that these changes are largely due to a fall in ADP.