Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets microdissected from ob/ob mice. The islet content of endogenous manganese was 80 mumol/kg dry wt., which is about half as much as found in the exocrine pancreas. The initial uptake was characterized by two components, with approximate Km values of 35 microM and 3.7 microM respectively. After 60 min of incubation with 0.25 mM-Mn2+, the intracellular concentration of manganese corresponded to an almost 25-fold accumulation compared with that of the extracellular medium. When exposed to 20 mM-D-glucose, the islets retained more manganese, owing to suppression of its mobilization. The glucose inhibition of efflux was prompt and reversible, as indicated from direct recordings of manganese in a perifusion medium. D-Glucose was an equally potent inhibitor of efflux in the presence of 15 microM- and 1.28 mM-Ca2+. The inhibitory action disappeared when metabolism was suppressed by adding 0.1 mM-N-ethylmaleimide or by lowering the temperature from 37 degrees C to 2 degrees C. At a concentration of 0.25 mM, Mn2+ abolished the insulin-releasing action of D-glucose, exerting only moderate suppression of its metabolism. The addition of Mn2+ resulted in inhibition of basal insulin release in the presence of 1.28 mM-Ca2+, but not in a Ca2+-deficient medium. The studies indicate that the previously observed phenomenon of glucose inhibition of 45Ca efflux has a counterpart in the suppression of manganese mobilization from the pancreatic islets. With the demonstration of a pronounced glucose inhibition of manganese efflux, it is evident that Mn2+ may represent a useful tool for exploring the mechanism of glucose-induced retention of calcium in the pancreatic beta-cells.

/pdf/manganese-accumulation-in-pancreatic-beta-cells-and-its-4lk07fh65v.pdf

Manganese accumulation in pancreatic beta-cells and its stimulation by glucose.

Cryogenic preservation of isolated islets of Langerhans: Two-step cooling☆

Publisher Summary This chapter discusses the purification of islets and cells from islets. The pancreatic islets that are composed of several hormone-producing cells can be isolated by collagenase digestion of the pancreas. The yield of islets relative to the total number is usually small. Sufficient number of islets for a variety of functional and biochemical studies can be obtained by gradient centrifugations or by purification in organ culture. The isolated pancreatic islets are dispersed by enzymatic digestion or mechanical disruption of the islets in Ca2+free medium. The single cells are functionally intact and attempts are made to separate islet cell subpopulations by elutriation or gradient centrifugations, cell sorting, or other techniques such as free flow electrophoresis, sedimentation at unit gravity, or by immunocytochemical techniques. A major task in islet cell purification is to achieve preparations of highly purified endocrine cells and to separate the individual cell types from each other. Flow cytofluorophotometers, which can analyze and separate cells at a high speed based on either light scattering or fluorescence signals, have been found useful in attempts to separate islet cells. The fluorescence-activated cell sorter also allows analysis and sorting of islet cells based on indirect immunofluorescence. Tissue or cell-specific monoclonal antibodies represent useful reagents to allow sorting of certain cell types based on immunochemical labeling. A number of monoclonal antibodies reactive with specific cell-surface antigens on endocrine islet cells have been described in the chapter.

Purification of islets and cells from islets

Morphological investigations into the structure of frozen-thawed rat islets of Langerhans.

3 – Transport Systems of Islet Cells

A previous study showed that the ability of glucose to stimulate insulin release was retained in islets stored at 8 °C for one week provided that glucose was present in a high concentration in the storage medium. The metabolic properties of islets stored in the cold have now been further explored in an attempt to clarify the protective effect of glucose. During storage in the cold the islet formation of 3H2O from (5-3H) glucose and oxygen consumption were only a few per cent of that of fresh islets whereas the uptake of 86Rb+ was 20–48%. Rewarming the cold-stored islets to 37 °C after one week of cold-storage restored the 86Rb+ uptake, the formation of 3H2O and 14CO2 from labelled glucose and oxygen consumption to 75, 80, 60 and 40% respectively of fresh islet levels. The results emphasize the usefulness of cold-storage for preservation of functionally intact isolated islets.

/pdf/metabolism-of-cold-stored-pancreatic-islets-4jzklysavu.pdf

Metabolism of cold-stored pancreatic islets.

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Metabolism of cold-stored pancreatic islets.