Metabolic signals in the regulation of insulin release

Abstract: In the present thesis, an attempt was made to elucidate the role of some of the glucose-induced intracellular metabolic effector signals in insulin secretion and to develop new applications that will enable further investigations of oscillatory signalling and secretion from pancreatic ß-cells. The sequence of early metabolic and ionic events was examined in clonal pancreatic insulin secreting cells (HIT) in response to glucose stimulation. It was determined that the earliest metabolic changes that occur in response to glucose were in pyridine and flavin nucleotides as well as in the ATP/ADP ratio. These changes were followed sequentially by increases in oxygen consumption, intracellular pH and cytoplasnidc free Ca2+ concentration ([Ca2+]i). The results support the hypothesis that metabolism is the driving force for changes in [Ca2+]i and not the reverse. The time sequence above does not include insulin secretion due to the difficulty in measuring this parameter in a cuvette system. Perifusion methods for studying oscillations in insulin secretion also prevent linking the secretory and metabolic events to each other, due to the fact that the cells are inaccessible in a chamber or column. By making use of multiwell culture plates, we have measured oscillations in secretion and the ATP/ADP ratio and have been able to demonstrate that the patterns of change in the ATP/ADP ratio and insulin release are similar. A comparison of the clonal pancreatic cell lines HIT and INS-1 showed a period of 4 min and 1.5 min, respectively, for oscillations in both secretion and the ATP/ADP ratio. The period of oscillations in [Ca2+]i was 5 min in HIT cells and 1.4 min in INS-1 cells. These data support the role of oscillations in the ATP/ADP ratio in pulsatile insulin release. Measurements of exocytosis can be accomplished by monitoring the release of serotonin from preloaded single ß-cells with carbon fibers using the amperometric technique. We show that preloading INS-1 cells with 3H-serotonin allows the detection of exocytosis from populations of both intact and permeabilized cells. Oscillations in insulin release correlated with oscillations in 3H-serotonin release from INS-1 cells. Insulin and 3H-serotonin were measured from the same cells, validating the use of serotonin as an indicator of insulin release. This technique allows for rapid on-line detemiination of exocytosis from cells loaded with the tritiated label. Permeabilized HIT cells were used to study the effects of long-chain acyl-CoA (LC-CoA) on Ca2+ handling and exocytosis. Acyl-CoA was shown to reduce the Ca2+ set point maintained by saponin permeabilized cells, exhibiting both dose and chain length dependencies. The effect was shown to be mediated by the endoplasmic reticulum (ER) Ca2+-ATPase, using inhibitors of mitochondlial Ca2+ uptake and the specific Ca2+-ATPase inhibitor, thapsigargin. In addition, the acyl- CoA effect was influenced by the ATP/ADP ratio, which is a regulator of the ER Ca2+-ATPase in these cells. This interaction with the ATP/ADP ratio suggested that the CoA moiety of the LC-CoA, which resembles ADP, acted to alleviate the inhibition of the ATPase by ADP, thereby increasing Ca2+ sequestration into the ER. We have shown that LC-CoA can acutely stimulate exocytosis from streptolysin-O permeabilized HIT cells. This effect is also dose and chain length dependent, with longer chain lengths being most effective. The effect is not mediated by classical protein kinase C (cPKC) isoforms and seems not to be dependent on the free Ca2+ concentration. LC-CoA simulates exocytosis in the absence of ATP, suggesting that the effect on insulin release comes after ATP dependent docking of secretory granules to the plasma membrane. cPKC activation has been shown to stimulate glucose-induced insulin release. The translocation of PKC to the plasma membrane has been shown, by some but not others, to be stimulated by glucose. The fact that some investigators have failed to observe such a glucose-induced translocation of CPKC seems to lie in the extraction procedures for assessing translocation of the enzyme. We show that upon cell disruption, cPKC translocation is Ca2+-dependent and reversible. Diacylglycerol and phorbol 12-myristate 13-acetate (TPA,) activators of cPKC, stimulate glucose-induced insulin release with different time courses. The activation of glucose-induced insulin release continued for hours after removal of TPA from the media, suggesting the existence of a long lasting phosphorylation event. A dissociation between the changes in [Ca2+]i and insulin secretion was shown subsequent to stimulation with TPA. Under these conditions, the amplitude of the oscillations in secretion were stimulated two-fold with no major effect on [Ca2+]i. It is well accepted that glucose-induced insulin release is coupled to signals generated by glucose metabolism. In many cases the effects of these signals are not completely understood. The studies presented here were designed to increase our knowledge of how some of these signals affect the insulin secretory process.