María C. Semino

Sequential Morphological Changes in Pancreatic Islets of Spontaneously Diabetic Rats

This study describes the sequential morphological changes in pancreaticislets from 1-, 6-, and 18-month-old male eSS rats, as compared to aged-matched control animals. Spontaneous diabetes mellitus was confirmed in 6- and 18-month-old eSS rats after an oral glucose tolerance test. Lightmicroscopic immunocytochemical and morphometric techniques were used to study islet-cell populations. The pancreas was normal, and the morphometric methods did not reveal significant changes in islets from 1-month-old rats. However, 6-month-old eSS animals showed disruption of islet architecture and fibrosis in the stroma. The volume density (Vvi) of endocrine tissue and the Vvi and percentage of B cells were increased, whereas the Vvi of exocrinetissue and the Vvi and percentage of A cells were diminished. Eighteen month-old eSS rats also exhibited conspicuous islet lesions. Nevertheless, the Vvi of endocrine tissue and the Vvi and percentage of B cells were diminished, while the Vvi of exocrine tissue and the Vvi and percentage of D cells were increased. Our results provide further quantitative evidence for the sequentialmor phological events occurring in the pancreatic islets of a useful animalmodel of diabetes mellitus.

Quantitative morphological changes in endocrine pancreas of rats with spontaneous diabetes mellitus

SummaryThe present study describes the cytopathology of the pancreatic islets in 18-old male eSS rats with spontaneous diabetes mellitus as compared to aged-matched normal animals. Lightmicroscopic immunocytochemical and morphometric techniques were used to study islet-cell populations, while quantitative methods were employed specifically for the analysis of B-cell ultrastructure. The diabetic rats showed disruption of the islet structure and fibrosis in the stroma. The volume density (Vvi) of endocrine tissue and the Vvi and percentage of B cells were diminished, whereas the Vvi of exocrine tissue and the Vvi and percentage of D cells were increased. The number of medium and large islets as well as their mean volume (μm3) decreased in these animals. Pancreatic B cells from eSS rats showed an increase in the Vvi of endoplasmic reticulum, immature secretory granules and lysosomes. Conversely, the Vvi of total secretory granules and microtubules appeared diminished. The current observations contribute to our understanding of this useful animal model of diabetes mellitus, in the attempt to clarify the pathogenesis of the disease.

Early Changes in the Rat Pancreatic B Cell Size Induced by Glucose

The perimeter, cell area and volume density (Vvi) of B cells and exocytotic images present in these cells were measured in rat pancreas perfused with 3.3 or 16.6 mM glucose. Four minutes after the beginning of 16.6-mM glucose perfusion and coincident with the appearance at the apex of the first phase of insulin secretion, all these parameters underwent a significant increase. The changes observed in the perimeter, the cell area and the Vvi of B cells suggest an increase in their surface area. An imbalance in the rate of endocytosis:exocytosis processes with a relative predominance of the latter would increase the length of the plasma membrane and could be responsible, at least partly, for the changes in the B cell size.

Sequential determination of calcium distribution in B cells at the various phases of glucose-induced insulin secretion

SummaryLocalization and quantification of calcium pyroantimonate precipitates within the B cells, and determination of insulin secretion were performed in rat pancreas perfused with 3.3 and 16.6 mmol/l glucose. Observations were carried out during the peak, the refractory period, and at 10 and 20 min in the second phase of glucose secretion after the start of a glucose challenge. Specific calcium pyroantimonate precipitates, assessed by EGTA cross-incubation, appeared attached to plasma membrane, Golgi complex, mitochondria, cytoplasmic matrix and secretory granules. The total number of cellular calcium pyroantimonate precipitates increased with perfusion time, being significantly higher at every time-point with the higher concentration of glucose (16.6 mmol/l) than with the 3.3 mmol/l glucose concentration. Calcium pyroantimonate precipitates showed a progressive increment both in plasma membranes and mitochondria. In the cytoplasmic matrix, B granules and Golgi complex, a sharp increase in the number of precipitates was detected at the refractory period, followed by a continuous decrease until the end of the experiment. These results show that the number of calcium pyroantimonate precipitates, localized in different organelles, changes according to the functional state of B cells. They stress the importance of intracellular readily exchangeable pools as regulators of calcium availability for insulin stimulus-secretion coupling.

Ultracytochemical Nuclear Calcium Distribution in Pancreatic B Cells: Its Relation to Glucose-Stimulated Insulin Secretion

Calcium distribution in pancreatic B cells was studied, with the aid of the pyroantimonate technique, at different times of glucose-induced secretory activity in the perfused rat pancreas. Specificity of the pyroantimonate precipitates for calcium was assessed by EGTA cross-incubation. Quantitative studies for these calcium pyroantimonate precipitates were performed by a morphometric technique. Pyroantimonate precipitates within the B cell show both time and glucose dependence. At any time-point studied, in the nucleus as well as in other organelles, they were more numerous when glucose was increased in the medium from 3.3 to 16.6 mmol/l. The total number of nuclear calcium pyroantimonate precipitates rose sharply at the time corresponding to the refractory period, falling after that to almost the number found at the prestimulatory period. Otherwise, glucose significantly modifies the temporal distribution of precipitates bound to euchromatin, heterochromatin and perichromatin. These changes in nuclear calcium pyroantimonate precipitates during different periods of B cell secretory activity may indicate an active role of the cation in some nuclear regulatory function during glucose-induced release of insulin.

Stereological-Ultrastructural Study of Pancreatic B Cells in Metabolic Alkalosis

The secretion of insulin in response to glucose and the changes in the B cell at the ultrastructural level were studied in rat pancreas perfused at pH 7.4 and 7.8 with different concentrations of glucose. Raising the extracellular pH from 7.4 to 7.8 significantly inhibits glucose-induced insulin secretion. Coincidentally, morphometric studies showed significant evidences of low secretory activity in B cells from pancreas submitted to high glucose stimulation under alkalosis, namely lower number of emiocytotic figures and microtubules as well as a decrease in the volume density of the granular endoplasmic reticulum and the Golgi complex. On the other hand, a significant increment in the number of images of granulolysis was also demonstrated. These secretory and ultrastructural results confirm the inhibitory effect of pH 7.8 upon B cell secretory activity induced by glucose. Moreover, they lend further support to the role of intracellular hormone degradation as a regulator of B cell insulin content.