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.

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.

In vitro incubated rat islet B cells differ in their individual rates of protein synthesis. The number of cells in biosynthetic activity increases with the glucose concentration. Flow cytometric monitoring of the cellular redox states indicated that islet B cells differ in their individual metabolic responsiveness to glucose. A shift from basal to increased NAD(P)H fluorescence occurred for 18% of the cells at 1 mM glucose, for 43% at 5 mM, and for 70% at 20 mM. The functional significance of this metabolic heterogeneity was assessed by comparing protein synthesis in metabolically responsive and unresponsive subpopulations, shortly after their separation by autofluorescenceactivated cell sorting. The glucose-sensitive subpopulation exhibited four-to fivefold higher rates of insulin synthesis during 60-min incubations at 2.5-10 mM glucose. Its higher biosynthetic activity was mainly caused by recruitment of cells into active synthesis and, to a lesser extent, by higher biosynthetic activity per recruited cell. Cells from the glucose-sensitive subpopulation were larger, and presented a threefold higher density of a pale secretory vesicle subtype, which is thought to contain unprocessed proinsulin. It is concluded that intercellular differences in metabolic responsiveness result in functional heterogeneity of the pancreatic B cell population. (J. Clin. Invest. 1992. 89:117-125.)

Similar to other endocrine glands, the endocrine pancreas displays a characteristic topography of its constituent cells. The functional significance of this structural organization was examined by measuring-the secretory activity of the B cell in rat islet cell preparations of different composition. Glucose released 30-fold more insulin from B cells lodged within intact islets as from purified single B cells; structurally coupled B cells and single B cells isolated with A cells or incubated with glucagon responded 4-and .2-fold, respectively, more effectively to glucose than single B cells alone. Glucose homeostasis is thus dependent not only on the number and integrity of the insulin-containing B cells but also on their interactions with neighboring B and non-B cells. This study provides direct support for the concept that the microanatomy of the islet creates the anatomical basis for functional cooperation between islet cells and hence for an appropriate glucose-induced insulin release..The endocrine tissue in adrenal cortex, pituitary, hypothalamus, and pancreas consists of various cell, types that display close contacts with homologous and heterologous cells (1-5). It is so far unknown whether this structural organization is a prerequisite for a normal endocrine response and whether it represents a possible site for endocrine disease.We have examined -the eventual role of the microanatomy of the pancreatic islet in its insulin, response to glucose. The mammalian pancreas is composed ofmore than a thousand islets of Langerhans, each containing at least three different cell types, which are arranged according to a precise topography (6-9). Within each islet, the various endocrine cells are thought to communicate with each other, either extracellularly via their secretory products or intracellularly via cell junctions. (10). The paracrine route was proposed in early reports on the insulinstimulating effect of glucagon (11) and, the inhibitory effect of another islet peptide (12), later identified as somatostatin (13,14). An intercellular. exchange through membrane channels was first suggested by the detection of gap junctions in islets (5) and later documented by electrotonic and metabolic coupling experiments (15-17). Evidence implicating both forms of intercellular communication in islet cell function and in particular in the insulin release process remains, however, largely indirect (10). To examine such eventual participation more directly, we developed a purification procedure for islet cells, which resulted in three different preparations of insulin-containing B cells: the first consisted of25% B and 75% non-B cells, the second consisted of more than 90% single B cells, and the third consisted of 30% single and 65% structurally coupled B cells (18). Comparison of the insulin response of each of these cell suspensions with that of intact islets indicated the dependency of glucose-induced insulin release on an intercellular communication between islet cells. MATERIALS AND METHODSIslets were isolate...

The role of nutrients and hormones in the regulation of glucagon release is investigated in pancreatic A cells purified by autofluorescence-activated cell sorting. Purified A cells lack secretory activity in 1-h incubation at 1.4 mM glucose. Their release mechanism can be activated by arginine, alanine, and glutamine, alone or in combination. Glucose inhibits amino acid-induced glucagon release through a direct insulin-independent action upon pancreatic A cells. Nutrient-induced glucagon release is suppressed by somatostatin and amplified by (Bu)2cAMP or epinephrine. The epinephrine stimulus is inhibited by 10(-11) M somatostatin and abolished by 10(-10) M of this peptide. The effects of somatostatin and epinephrine are associated with parallel changes in cellular cAMP levels, which is not the case for the variations induced by amino acids or glucose. It is confirmed that calcium is an essential requirement for glucagon release. In contrast to its exquisite sensitivity for somatostatin, the glucagon release process is relatively insensitive to insulin during a 1-h exposure. The hormone affects solely epinephrine-induced glucagon release and its inhibitory action is partial and only observed at 10(-7) M. This suppressive effect of insulin is not attributable to variations in glucose handling but appears associated with the stimulatory effect of epinephrine. It is concluded that a nutrient-induced signal interacts with a hormone-inducible cAMP signal to activate the secretory process in pancreatic A cells.