1. Quantitative study of insular tissue has revealed that the number of B cells is greatly diminished in Patients with acute juvenile diabetes from the time of clinical onset of the disease. The number of these cells is as a rule less than 10 per cent of normal. Such B cells as are still present show the cytological signs of marked activity. 2. The normal or supranormal insular activity that is usually found in juvenile diabetics in this stage of the disease cannot therefore be due to the presence of a normal insular tissue, but is produced by a small number of hyperactive B cells. 3. On the basis of histological findings (presence of islets of large size, signs of new islet formation), it may be assumed that during the preclinical phase of juvenile diabetes, an extrapancreatic factor has exerted a strong stimulant action on the insular tissue. In the long run this must lead to exhaustion of the islet-forming capacity on the pancreatic parenchyma and to a decrease in the number of the B cells. By the time the disease becomes clinically manifest only the latter stage of this process can be observed and the majority of islets consist of A cells or of atrophic tissue devoid of B cells. 4. Peri- and intra-insular inflaminatory infiltrates have been found in 68 per cent of those patients with juvenile diabetes who died soon after the clinical onset of their disease. In other words, and contrary to the generally held view, this lesion is not uncommon. It is specific for diabetes and has never been observed in the chronic cases 5. In patients with chronic juvenile diabetes, the B cells are completely absent, except in occasional cases. The islets consist of small, atrophic cells. 6. A valid assessment of the functional capacity of insular tissue can only be achieved if as much use as pos sible is made of quantitative technics and of cytological examination

A method is developed for the preparation of single, pure, and viable rat pancreatic A and B cells in numbers sufficient for in vitro analysis. Islet isolation and dissociation techniques have been modified to increase the yield in islet cells per pancreas and per experiment. Islet cells are separated on the basis of their light scatter activity and flavin adenine dinucleotide autofluorescence into single non-B cells, single B cells, and structurally coupled B cells. Islet non-B cells are further purified into single A cells by autofluorescence-activated sorting according to the cellular nicotinamide adenine dinucleotide phosphate content at 20 mM glucose. Apart from offering the advantage of separating cells according to their functional characteristics, this procedure succeeds in the simultaneous isolation of 95-100% pure A and B cells. More than 50% of the cells in the initial islet preparation are recovered as single purified cells which can be maintained in culture. The isolated pancreatic A and B cells have been defined in terms of their cell volume, DNA and hormone content, and ultrastructural characteristics. The availability of pure pancreatic A and B cells is expected to contribute to our understanding of the regulation of glucagon and insulin release.

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Single pancreatic B cells are purified by autofluorescence-activated cell sorting, and their secretory activity is measured after overnight culture. Compared to intact islets, the isolated cells release 2-fold more insulin under basal conditions and 5-fold less during nutrient stimulation. Their secretory activity can be induced by glucose, leucine, or arginine, but only 0.3-1.7% of their hormone content is liberated at 20 mM nutrient concentrations. This poor nutrient-induced insulin release from purified B cells is attributed to their low cAMP levels and is markedly increased after addition of (Bu)2cAMP, of glucagon, or of pancreatic A cells. These results strongly support the concept that the potent in vivo insulin-releasing action of glucose and leucine is not only dependent on their fuel capacity in pancreatic B cells but also on the concurrent cAMP levels in these cells. In isolated islets, endogenously released glucagon apparently determines the cAMP production in B cells and thus participates in the nutrient-induced secretory process. Somatostatin and epinephrine were shown to exert their suppressive effects via the glucagon-dependent messenger system. It is concluded that nutrients and hormones interact with two different messenger systems which amplify each others' stimulatory effect upon insulin release. cAMP might represent the hormone-induced messenger which sets the B cell's sensitivity and secretory capacity for nutrient stimuli such as glucose. The higher insulin secretory response observed after reaggregation of single B cells could not be attributed to an altered activity in the nutrient or hormonal regulatory units, raising the possibility that the aggregated state of the cells is rather responsible for a better organization or cooperation of the secretory effector unit.