Marie H. Greider

Regional distribution and concentration of pancreatic polypeptide in the human and canine pancreas.

The regional concentrations of pancreatic polypeptide (PP), insulin, and glucagon and the cellular distribution of PP were studied in 13 human and nine canine pancreases by radioimmunoassay, immunoperoxidase localization, and cell quantitation. PP concentration was highest in both the uncinate process and the head of the human pancreas and in the right lobe of the canine pancreas. In contrast, glucagon and insulin levels were higher in the body and tail of both the human and canine pancreases. Human F-cells, which contain PP, were located primarily at the periphery of the islets, although a few F-cells were scattered throughout the ducts and acini. Canine F-cells were located in ducts, acini, and islets; the relative proportion of canine F-cells in the endocrine and exocrine tissues differed according to location. Cellular quantitation of F-cells in both species correlated significantly with the tissue concentration of PP in all regions studied, validating the use of morphometric techniques to quantitate the regional distribution of PP.

Cellular Localization of Gastrin in the Human Pancreas

Cryostat-cut sections of unfixed and formalin-fixed human pancreata obtained at autopsy were stained by the direct method with fluoresceinated antibodies to human gastrin I. The cytoplasm of a small population of cells in the islets of Langerhans exhibited a bright yellow-green fluorescence. Specificity of the inimunochemical reaction was indicated by a decrease in intensity of stain after preincubation of the fluoresceinated antibody with gastrin and after pretreating the section with nonfluoresceinated antibody-preparation. Immunological cross reactivity with cholecystokinin-pancreozymin was excluded by use of highly specific fluoresceinlabelled antibodies to human gastrin I which exhibit no binding of cholecystokinin-pancreozymin. The presence of immunoreactive gastrin in the human pancreas was also demonstrated by radioimmunoassay measurements of pancreatic tissue extracts. The gastrin-containing cells were selectively stained by the Hellerstrom-Hellman method, indicating that the gastrin cell of the pancreatic islet is the classical delta cell of Bloom (or A1 cell of Hellerstrom and Hellman).

Indomethacin-responsive pancreatic cholera.

Pancreatic cholera, otherwise known as the Verner-Morrison1 syndrome or the syndrome of watery diarrhea, hypokalemia and achlorhydria,2 is a rare clinical entity caused by non-β islet-cell tumors. ...

Uncontrolled diabetes mellitus and hyperglucagonemia associated with an islet cell carcinoma

A 53 year old woman presented with diabetes mellitus, hyperglucagonemia (600 to 1,500 pg/ml), clinical hyperparathyroidism and an abdominal mass diagnosed on biopsy as an islet cell carcinoma. Glucagon content of the tumor was 0.78 mug/g wet weight. Hourly blood samples during a 24 hour period revealed a direct correlation between plasma glucose and glucagon. The oral administration of glucose paradoxically increased whereas the intravenous administration decreased plasma glucagon. Circulating glucagon levels were markedly increased with arginine and epinephrine infusion. Both short- and long-term administration of alpha adrenergic blockade depressed the glucagon response to epinephrine infusion. In contrast, long-term alpha adrenergic blockade increased glucagon secretion despite improved glucose tolerance during a second 24 hour study. Although the patient demonstrated overt clinical and chemical findings of hyperparathyroidism, parathyroid hormone (PTH) was not detected in her plasma. The pattern of tumor growth was consistent with an origin from pancreatic islets. We conclude that (1) the tumor was responsive to physiologic stimuli known to affect glucagon secretion; (2) elevations of plasma glucagon levels with oral and dietary glucose suggest regulation of secretion by intestinal factors; and (3) improvement of glucose tolerance with alpha adrenergic blockade may be related to increased insulin secretion.

The human pancreatic islet cells and their tumors. II. Ulcerogenic and diarrheogenic tumors

A comparative histologic examination of hematoxylin‐eosin stained sections of 34 ulcerogenic tumors of the pancreas (UTP), 5 diarrheogenic tumors of die pancreas (DTP), 13 beta cell tumors, and 4 alpha cell tumors revealed two correlative features: all tumors characterized by a gyriform pattern of growth throughout the lesion were either of alpha or beta cell origin, and all but 1 of the tumors containing glandular formations were of either UTP or DTP type. No differential morphological features were found between the tumors which gave rise to metastases and those which did not. A combination of histochemical, ultrastructural, and biochemical studies easily differentiated UTP‐DTP from alpha‐beta cell tumors in most instances. Histochemical stains of 18 UTP and 4 DTP revealed that except for 1 DTP the tumors did not stain with reagents that differentiated normal islet cells. One of the 4 UTP tested gave a positive reaction with a direct immunofluorescent technique using fluoresceinated antibodies to gastrin. Ultrastructurally, all 22 tumors contained secretory granules; in most tumor cells these granules had a diameter range of 150–200 nm, but in others they were pleomorphic and larger (150–360 nm). In this study no morphological, histochemical, or ultrastructural differences were observed between UTP and DTP neoplasms.

Isolating the Elusive Islet

The lack of a technique that allows mass isolation of intact, viable human islets is part of the reason that islet transplantation has not become available to the human diabetic. This report outlines the history of islet isolation and presents two new technical modifications that have been developed in the dog. Many of the current problems in islet isolation are presented, including the difficulty in obtaining enough human pancreatic tissue with minimal warm-ischemia time; inadequate distention of the pancreas to provide sufficient disruption for maximal enzymatic reaction to release intact islets; inefficient chopping methods; the use of collagenase of variable composition; different digestion methods for obtaining isolated islets; and inefficient methods for separating and purifying the islets from the ductal, acinar, and fibrous components. The first new modification involves distention of the dog pancreas through the venous system of the gland rather than the ductal system. This results in improved intralobular disruption, which improved the yield of isolated dog islets by permitting more efficient collagenase digestion. The second new modification eliminates the concept of isolating intact islets: the dog pancreas is digested by trypsin to a single-cell preparation that is partially purified by Ficoll gradients; further purification of the endocrine cells results from selective aggregation using rotational culture. This process produces pseudoislets that contain all the islet cell types and can be kept in culture for up to 4 wk, releasing their hormones in response to appropriate stimuli. These modifications may assist in the struggle to isolate the elusive human islet for safe and effective islet transplantation in the diabetic patient.

Correlative Immunochemical and Light Microscopic Studies of the Gastrin Cell of the Antral Mucosa

The specific gastrin-containing epithelial cell of the hog pyloric antral mucosa was studied and identified by immunochemical techniques using γG-immunoglobulin-containing antibodies to human gastrin I labeled with fluorescein and horseradish peroxidase. Cells demonstrated to contain gastrin in this study did not stain by the alkaline diazonium procedure or by the argyrophilic procedures of Grimelius, Bodian, Sevier-Munger, Hellerstron-Hellman, and Goldner-Volk. These results do not agree with conclusions and suggestions of other investigators who have attempted to identify and segregate cells which might contain gastrin on the basis of positive staining with these procedures. Autofluorescent cells of the antral mucosa were stained by the argyrophilic and diazonium procedures. This study reaffirms the previous evidence from this laboratory which did not permit, on the basis of staining techniques utilized, the classification of gastrin-containing cells as argyrophilic or argentaffinic. This study serves to emphasize the caution which must be urged in the interpretation of the specificity of the hormonal content of a population of cells on the basis of its general morphological characteristics, distribution, or nonspecific tinctorial reactions.

Use of a High Voltage Technique to Determine the Molecular Requirements for Exocytosis in Islet Cells

SUMMARY Pancreatic islet cells were subjected to high voltage discharges, which induced pore formation in the plasma membrane. This technique was used to determine the molecular requirements of intracellular sites involved in the control of insulin release in β-cells. Islets, preloaded with 86Rb+ and then shocked, released 92% of the radioisotope within 1 min as compared with only a 55% loss from nonshocked islets. Exposure of the islets to 14C-urea and 3H-sucrose at 0 to 10 min after exposure to high voltage discharges indicated that 68% of the intracellular space was occupied by sucrose, whereas sucrose was excluded from nonshocked islet cells. The pores in the plasma membrane resealed about 30 min after their initial formation, as was indicated by the cellular exclusion of sucrose. The Ca++ concentrations yielding half-maximal and maximal secretory responses from shocked islets were 0.05 × 10−6 M and 0.35 × 10−6 M, respectively; the presence of 16.7 mM glucose did not alter these values. In intact islets, a variation of extracellular Ca++ (only in the presence of 16.7 mM glucose) generated a dose-response curve yielding half-maximal and maximal secretory responses at 2.0 × 10−3 M and 4.0 × 10−3 M, respectively. The total amount of insulin released from shocked islets was three times that released from nonshocked islets during a 15 min incubation period. The addition of 1 or 5 mM ATP during an initial shock and incubation period did not augment the secretory response to 0.05 × 10−6 M Ca++, but the presence of ATP was necessary, or the islets would not respond to 0.35 × 106 M Ca++ during a subsequent shock and incubation period. The presence of 1.0 mM 3-phosphoglycerate or phosphoenolpyruvate augmented the secretory response to 0.05 × 10−6 MCa++ only in the presence of 1.0 mM ATP. Glucose-6- phosphate or fructose-1,6-diphosphate had no influence on the secretory response to Ca++ in the presence of ATP. An increase in Mg++ from 1.0 to 10 mM reduced the secretory response to 0.35 × 10−6 M Ca++ by 63%. Islets, subjected to the high voltage discharges and allowed 30 min to reseal, exhibited a normal secretory response to 16.7 mM glucose. The results indicate that the high voltage technique induces reversible pore formation in β-cells to introduce ions and solutes into the intracellular environment so that the factors controlling exocytosis can be determined.

Neonatal Pig Pseudo-Islets: A Product of Selective Aggregation

Pseudo-islets have been formed from single cell preparations of neonatal porcine pancreas by selective aggregation using the gyrotational tissue culture method for 7 days. Small, distinct aggregates present on the second day of culture gradually enlarged by the seventh day and at that time were taken off for assessment of secreting capabilities and cell type identification. Provocative stimulation and immunocytochemical examination confirmed viable, well-preserved pseudo-islets which contain all four islet cell types. Neonatal pig pseudo-islet formation will allow us to pursue the suitability and growth potential of this source of islet tissue under appropriate immunoaltered conditions as a xenograft source for future transplantation studies.

Ultrastructural localization of pancreatic polypeptide in the F cell of the dog pancreas.

The F cell of the dog pancreas has been identified as the specific cell type containing pancreatic polypeptide. This localization of pnacreatic polypeptide was accomplished by immunocytochemical staining of ultrathin sections and direct electron microscopic identification. Verification of the specificity of the reaction was obtained by blocking experiments on serial sections of the same cell. It is proposed that the name F cell be used for defining in all species the islet cell that contains pancreatic polypeptide.