Rastogi K S, Cooper R L, Shi Z Q, Vranic M
Department of Physiology, Faculty of Medicine, University of Toronto, Ontario, Canada.
Endocrine. 1997 Dec;7(3):367-75. doi: 10.1007/BF02801332.
We have shown that the glucagon irresponsiveness to hypoglycemia in diabetic rats is markedly improved by correction of hyperglycemia independent of insulin. In contrast, normalization of glycemia by insulin did not improve this response. To find out whether these glucagon responses reflect changes in islet glucagon, we directly quantified glucagon area and content in each pancreatic islet by using fluorescent immunostaining 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 rats. 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-treated, 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 i.v. insulin injection. The rats were sacrificed 2 h after insulin injection and the pancreas was removed. By imaging with CLSM, we quantified: 1. Percent of glucagon containing A-cell area/islet area, 2. Fluorescence intensity per islet area, which indicated glucagon content in the islet. 3. Fluorescence intensity per glucagon area indicating glucagon concentration in A-cells. In NC, glucagon containing A cell area was 21 +/- 2% of the islet 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 glucagon intensity increased by 235% (O) (p < 0.05), but decreased to 135% 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 increase in glucagon content indicating increased synthesis. In contrast, DI 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 concentration followed the same pattern as its intensity.
我们已经表明，糖尿病大鼠中胰高血糖素对低血糖的无反应性可通过纠正高血糖而显著改善，且与胰岛素无关。相比之下，胰岛素使血糖正常化并未改善这种反应。为了弄清楚这些胰高血糖素反应是否反映胰岛胰高血糖素的变化，我们使用荧光免疫染色和共聚焦激光扫描显微镜（CLSM）结合计算机图像分析，直接对每个胰岛中的胰高血糖素面积和含量进行了定量。对四组大鼠的胰腺进行了分析。1.正常对照组（NC，n = 4），链脲佐菌素（65 mg/kg）诱导的糖尿病大鼠。2.未治疗的糖尿病大鼠（DU，n = 4）。3.用根皮苷治疗的糖尿病大鼠（0.4 g/kg），每日两次，共4天（DP，n = 4）。4.用缓释胰岛素植入物（2 - 3 U/d）治疗5天的糖尿病大鼠（DI，n = 4）。NC组的基础血浆葡萄糖为7.4±0.3 mM，DU组升至14.5±2.2 mM，DP组（5.5±0.5）和DI组（6.7±0.8）使其恢复正常。通过静脉注射胰岛素诱导急性低血糖（H）。胰岛素注射2小时后处死大鼠并取出胰腺。通过CLSM成像，我们进行了以下定量：1.含胰高血糖素的A细胞面积/胰岛面积的百分比；2.每个胰岛面积的荧光强度，其表示胰岛中的胰高血糖素含量；3.每个胰高血糖素面积的荧光强度，表示A细胞中的胰高血糖素浓度。在NC组中，含胰高血糖素的A细胞面积在基础（O）状态下为胰岛面积的21±2%，胰高血糖素强度和浓度分别为11±1 U和36±3.0 U，低血糖（H）状态下无变化。在DU组中，胰高血糖素面积在基础（O）状态下增加183%，在低血糖（H）状态下增加166%，胰岛胰高血糖素强度在基础（O）状态下增加235%（p < 0.05），但在低血糖（H）状态下降至135%。DP组和DI组的胰高血糖素面积与DU组无显著差异。然而，DP组的低血糖使胰岛中的胰高血糖素强度进一步增加至正常对照组的306%（p < 0.05），表明胰高血糖素含量显著增加，提示合成增加。相比之下，DI组与DP组相比，在低血糖（H）状态下胰岛中的胰高血糖素强度降低（DP组为46±3，DI组为22±2；p < 0.05）。胰高血糖素浓度与其强度变化模式相同。
1.糖尿病大鼠胰岛胰高血糖素含量增加与每个胰岛中含胰高血糖素的A细胞面积增加有关。2.根皮苷诱导的不依赖胰岛素的高血糖纠正增加了低血糖状态下每个胰岛的胰高血糖素含量。这可能部分解释了之前观察到的胰高血糖素对低血糖反应的改善。3.胰岛素使血糖正常化降低了低血糖期间每个胰岛的胰高血糖素含量。这可能部分解释了在强化胰岛素治疗的胰岛素依赖型糖尿病（IDDM）中经常观察到的胰高血糖素对低血糖无反应性。
