Development of an organ bath technique for isolated rat pancreas preparations to assess the effect of 1,5-AG on insulin secretion

Abstract

To investigate substances related to insulin secretion, we reported a convenient experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath. While the method has experimental utility for investigating insulin secretion, optimization of the experimental design is still needed. The level of insulin outflow in the control decreased over time in our previous study. Decreasing serum 1,5-anhydroglucitol (1,5-AG) levels is also known to be shown in patients with worsening glycemic control. There is one in vitro report demonstrated that 1,5-AG induced insulin release. It appears that discussion needs to be deepened further on it. In this study, we investigated the effect of 1,5-AG on insulin secretion through to optimize the condition of endocrine function using the ex vivo organ bath technique. The level of insulin outflow in the control and 1,5-AG groups decreased over time in the organ bath experiment. To analyze the effect of trypsin on reduced insulin secretion, pancreas preparation was treated with soybean trypsin inhibitor (TI). Insulin outflow levels of the TI group were significantly higher than the control group. An enzyme indicator of tissue damage tended to be lower in the TI group. There was no significant enhancement of insulin secretion by 1,5-AG. The present study demonstrated the utility of TI application for the organ bath technique. This finding supported the development of an organ bath technique for the assessment of the effects of novel therapeutics on insulin secretion.

The Effects of Trypsin Inhibitor on Insulin Secretion Using Rat Pancreas in an Organ Bath

“…We identified the marked change that was recorded in insulin outflow, caused by TI treatment, was not observed in amylase outflow. We previously confirmed the variation in amylase outflow with and without 100 μg/ml TI at the experimental end-point (data not shown) (7). Herein, we focused more on insulin outflow than on amylase and lipase outflow.…”

“…LDH is also present in pancreas (20). We reported previously that there was a significant difference in results of LDH activity with 100 μg/ml TI addition, compared to control (7). In the present study, there was no significant difference in LDH activity (data not shown).…”

“…Pancreatic organ bath preparations have been found to be sufficiently sensitive and reproducible as a method to assess the effects of novel therapeutics for diabetes mellitus on insulin secretion. However, in our previous two studies, we observed that the level of insulin outflow decreased over time in the control group without stimulation (6,7). Thus, we presumed that adding a bioactive substance would create a situation where basal insulin secretion would be sustained.…”

“…In our earlier studies, we added soybean-derived trypsin inhibitor (TI) to the isolation buffer with collagenase for isolating rat pancreatic acini (8) and parotid gland acini (9), to prevent cell damage from trypsin and trypsin-like proteases. Hence, we focused on TI as a candidate bioactive substance and studied the effect of a moderately high concentration (100 μg/ml) of TI (7). Addition of TI to the experimental solution rescued the reduction in insulin secretion.…”

The progress of clinical research on the detection of 1,5-anhydroglucitol in diabetes and its complications

“…( 70 ), through a study of 284 T2DM patients, found that salivary 1,5-AG was closely correlated with the C-peptide production index at 0–30 min and the ratio of the area under the C-peptide curve to the area under the glucose curve. However, A Morita ( 71 ) deemed that 1,5-AG had no significant correlation with insulin secretion function. Whether there is a link between the two remains to be verified.…”