QUANTITATIVE MEASUREMENT OF ISLET GLUCAGON-RESPONSE TO HYPOGLYCEMIA BY CONFOCAL FLUORESCENCE IMAGING IN DIABETIC RATS - EFFECTS OF PHLORHIZIN TREATMENT

We have shown that the glucagon irresponsiveness to hypoglycemia in di abetic rats is markedly improved by correction of hyperglycemia indepe ndent of insulin, In contrast, normalization of glycemia by insulin di d not improve this response. To find out whether these glucagon respon ses reflect changes in islet glucagon, we directly quantified glucagon area and content in each pancreatic islet by using fluorescent immuno staining and computerized image analysis with confocal laser scanning microscopy (CLSM). The pancreases were analyzed in four groups of rats . 1. Normal controls (NC, n = 4), streptozotocin (65 mg/kg) diabetic r ats. 2. Diabetic untreated (DU, n = 4). 3. Diabetic Phlorizin-treated, (0.4 g/kg), twice daily for 4 d (DP, n = 4). 4. Diabetic insulin-trea ted, using sustained release (2-3 U/d) insulin implant for 5 d (DI, n = 4). Basal plasma glucose was 7.4 +/- 0.3 mM in NC, increased to 14.5 +/- 2.2 mM in DU, which was normalized in DP (5.5 +/- 0.5) and DI (6. 7 +/- 0.8). Acute hypoglycemia (H) was induced by iv insulin injection . The rats were sacrificed 2 h after insulin injection and the pancrea s was removed. By imaging with CLSM, we quantified: 1. Percent of gluc agon containing A-cell area/islet area, 2. Fluorescence intensity per islet area, which indicated glucagon content in the islet. 3. Fluoresc ence intensity per glucagon area indicating glucagon concentration in A-cells. In NC, glucagon containing A cell area was 21 +/- 2% of the i slet area, and glucagon intensity and concentration was 11 +/- 1 U and 36 +/- 3.0 U, respectively, in basal (O) state and did not change in (H). In DU, glucagon area increased 183% (O) and 166% (H), and islet g lucagon intensity increased by 235% (O) (p < 0.05), but decreased to 1 35% in H. Glucagon area in DP and DI did not differ significantly from DU. However, hypoglycemia in DP increased glucagon intensity in islet further to 306% of normal control (p < 0.05), suggesting marked incre ase in glucagon content indicating increased synthesis. In contrast, D I compared to DP showed a decrease in glucagon intensity in islet (46 +/- 3, DP to 22 +/- 2 DI; p < 0.05) in (H) state. Glucagon concentrati on followed the same pattern as its intensity. Conclusion: 1. Increase in islet glucagon content in diabetic rats was associated with increa se in glucagon containing A-cell area per islet. 2. Phlorizin-induced insulin independent correction of hyperglycemia increased glucagon con tent per islet in hypoglycemic state. This, in part, probably contribu ted to improved glucagon response to hy poglycemia observed earlier 3. Normalization of glycemia with insulin reduced glucagon content of ea ch islet during hypoglycemia. This may explain, in part, unresponsiven ess of glucagon to hypoglycemia often observed in insulin-dependent di abetes mellitus (IDDM) with intensive insulin therapy.