Abstract
In order to clarify the characteristics of the cell membrane in insulin release, the effect of trypsin treatment on the release of insulin from islets of Langerhans isolated from rat pancreas was studied.
Pancreatic islets were isolated from male Wistar rats by the conventional collagenase method. The islets were pretreated with 1 mg/ml trypsin for 60 min in a vial containing 0.5 ml of Krebs Henseleit bicarbonate buffer (KHBB), 5 mg/ml bovine serum albumin and 3.3 mM glucose under he gas phase of 95% 02-5% CO2.
After rinsing 3 times in 5 ml of KHBB, 5 islets were transferred to an incubation vessel containing various agents and were incubated for 60 min. Insulin released in the second incubation medium and insulin contained in the islets were determined by double antibody radioimmunoassay. The islets were frozen at -80° immediately after incubation, and freeze-dried at -40°, 0.001 mmHg. Islet ATP and glucose were assayed by the enzymatic cycling method.
Results were summarized as follows;
1. Pretreatment with various concentrations of trypsin (0.01 to 10mg/ml) for 5, 15, 30, 60 or 90 min brought about a tendency to suppress 16.7 mM glucose-induced release of insulin from the islets. Significant decrease of insulin release was observed as a result of treatment with 1 mg/ml trypsin for 30 min. For assurance, treatment with 1mg/ml of trypsin for 60 min was adopted in successive xperiments.
2. The insulin content of trypsin-pretreated islets was not modified significantly compared with that of non-treated control groups. Furthermore, treatment with trypsin did not change the glucose or ATP concentration of the islet.
3. Trypsin slightly increased the basal level of insulin release, while high concentrations of glucose significantly reduced insulin response.
4. The decrement of 16.7 mM glucose-induced insulin release by trypsin treatment was restored to a certain degree by addition of adenosine, glucagon or db-cAMP, but not to the level of insulin release by these agents from the trypsin non-pretreated islets.
5. The observations mentioned above suggest that:
i) Trypsin modified a process other than the intra-islet insulin synthesis and glucose metabolism; possibly the B-cell membrance which has been thought to be associated with the site of glucoreceptor and/or final site of insulin release. The latter was affected very slightly by trypsin because there was no difference between the insulin contents of trypsin-treated and nontreated islets.
ii) Intracellular events were necessary to maintain function of islet cells, especially insulin release, while incomplete restoration showed the importance of the cell membrane, possiblly thatof the glucoreceptor in insulin release as well.
Further studies are needed to clarify the exact mechanism of trypsin action and thereby the significance of the B-cell memberance as a glucoreceptor in insulin release.
