Abstract
The influence of the insulin secretagogues, glucose and K+, to activate the multifunctional, Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in isolated rat pancreatic islets has been examined. Glucose (28 mM) and K+ (40 mM) were demonstrated to induce a 1.89 +/- 0.19- and 1.75 +/- 0.15-fold increase, respectively, in phosphorylation of a subunit of CaM kinase II immunoprecipitated by an anti-CaM kinase II alpha antibody. In intact islets, glucose and K+ also induced the generation of an autonomous, Ca2+/calmodulin-independent protein kinase II activity characteristic of autophosphorylated enzyme. Maximal activation, 2.9 +/- 0.2- and 3.0 +/- 0.5-fold for glucose and K+, respectively, relative to basal glucose control, was achieved at 2.5-5 min followed by a decline to near basal levels by 20 min. Glucose induced the production of autonomous CaM kinase II activity that, in terms of -fold stimulation, correlated closely with the extent of insulin release over a glucose concentration range of 3-28 mM. This stimulated activity was completely prevented by an inhibitor of glucose metabolism, mannoheptulose. These data demonstrate that the exposure of islets to stimulatory glucose concentrations activates CaM kinase II. The close correlation of enzyme activation with insulin secretion is consistent with the hypothesis that CaM kinase II plays an important role in the regulation of insulin secretion or related beta-cell processes.
Highlights
The influence of the insulin secretagogues, glucose these processes is thought to involve an increased intracellular and K+, to activate the multifunctional, Ca2+/calmodu-ATP:ADP ratio as the result of glucose metabolism and the lin-dependent proteinkinase I1 (CaM kinase 11) in iso- depolarization of the @-cellvia the closing of ATP-sensitive lated rat pancreaticislets has been examined
CaM kinase I1 has long been implicated in the regulation of insulin secretion, but a definitive evaluation of this hypothesis has been hindered by the lack of information concerning the function and regulation of this enzyme in the @cell
Until recently, there have not been methods of sufficient sensitivity to detect the activation of islet CaM kinase I1 in situ in response to insulin secretagogues
Summary
Increase in the proportion of islet CaM kinase I1 in the autonomous form. By 2.5 min, autonomous CaM kinase I1. Mr the resultantgeneration of autonomous kinase activity (Hanson and Schulman, 1992). An immunoprecipitation procedure using polyclonal anti-CaM kinase IIa. Exposed to stimulatory concentrations of the insulin secretagogues,K' (40mM) and glucose (28mM). Control (Con) islets were incubated in basal KRB medium (3 mM glucose, 5 mM K+).CaM kinase I1 was immunoprecipitated and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography as described under "Experimental Procedures." Markers to the left of the gel indicate electrophoretic mobilities of molecular weight markers on the gel.
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