A B S T R A C T The relationship between the dose of intravenously administered streptozotocin (a N-nitroso derivative of glucosamine) and the diabetogenic response has been explored by use of the following indices of diabetogenic action: serum glucose, urine volume, and glycosuria, ketonuria, serum immunoreactive insulin (IRI), and pancreatic IRI content. Diabetogenic activity could be demonstrated between the doses of 25 and 100 mg/kg, all indices used showing some degree of correlation with the dose administered. Ketonuria was only seen with the largest dose, 100 mg/kg. The most striking and precise correlation was that between the dose and the pancreatic IRI content 24 hr after administration of the drug, and it is suggested that this represents a convenient test system either for both related and unrelated beta cytotoxic compounds or for screening for modifying agents or antidiabetic substances of a novel type. Ability to produce graded depletion of pancreatic IRI storage capacity led to an analysis of the relationship between pancreatic IRI content and deranged carbohydrate metabolism. Abnormal glucose tolerance and insulin response were seen when pancreatic IRI was depleted by about one-third, while fasting hyperglycemia and gross glycosuria occurred when the depletion had reached twothirds and three-quarters, respectively. The mild yet persistent anomaly produced by the lowest effective streptozotocin dose, 25 mg/kg, exhibits characteristics resembling the state of chemical diabetes in humans and might thus warrant further study as a possible model. Finally, the loss of the diabetogenic action of streptozotocin by pretreatment with nicotinamide was confirmed and was shown to be a function of the relative doses of nicotinamide and streptozotocin and of the interval between injections.

1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded . Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C-peptide, immunoreactivity was detectable . The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected . However, in the final stage of lysosomal digestion, i .e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable . Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.The pancreatic B-cell shares with other polypeptide-secreting cells the ability to store its secretory products within cytoplasmic granules available for release by exocytosis (1, 2). However, the totality of insulin manufactured is not destined to be secreted, since it has been shown that the B-cell can degrade a significant portion of its own secretory product (3, 4). One commonly assumed mechanism of intracellular degradation ofinsulin by the B-cell is the fusion ofsecretory granules with primary lysosomes (5, 6), a mechanism called granulolysis or crinophagy and initially described in the mammotroph cells ofthe anterior pituitary (7) and pancreatic A-cells (8) . In the B-cell, morphological evidence for crinophagy consists of cytoplasmic organelles (secondary lysosomes) that present a very variable morphology but usually contain dense masses resembling a-secretory granule cores. The lysosomal nature of these structures (also called crinophagic or multigranular bodies) is evidenced by their content in marker lysosomal enzymes such as acid phosphatase or arylsulfatase (9), but whether the content ofthe granule core-like material is related to insulin polypeptides has not been ascertained. For this purpose, we have followed, by electron microscopic autoradiography, the labeling of the multigranular bodies in pulsechase experiment with tritiated leucine, and determined their 222 immunoreactive content using anti-insulin and anti-C-peptide antisera revealed by the protein A-gold method. MATERIALS AND METHODSRat islets of Langerhans in sections of pancreatic tissue or isolated by collagenase digestion (10) were studied throughout . The material examined originated from the tissue store of the laboratory . All blocks were from five untreated normal rats, the pancreas (or isolated islets) of which were fixed in 2 .5% glutaraldehyde alone or with a mixture of 1 .25% glutaraldehyde, I % paraformaldehyde, and 0 .02% trinitrocresol (11), then...

A B S T R A C T Do functional linkages between islet endocrine cells exist? The effect of differences in frequency and distribution of islet endocrine cells on B cell function was examined in islets from the ventral (ventral islets) and dorsal (dorsal islets) areas of the rat pancreas.Dorsal islets contained 10 times as much glucagon as ventral islets, whereas insulin and total protein contents were similar. Basal rates of insulin secretion and proinsulin biosynthesis were similar in the two types of islet, but, under conditions of glucose stimulation, both insulin secretion and proinsulin biosynthesis were significantly greater in the glucagon-rich dorsal islets. Similarly, glucose utilization rates and ATP levels were greater in dorsal islets. In contrast, the rates of processing of newly synthesized proinsulin were similar in ventral and dorsal islets. That the islet glucagon content may have affected B cell function is inferred from two independent findings. Firstly, basal and glucose-stimulated cyclic AMP contents of glucagon-rich dorsal islets were greater than those of ventral islets. Secondly, in the presence of excess exogenous glucagon (1 ug/ml), the differences in glucose-induced insulin secretion and proinsulin biosynthesis rates between the two types of islets were eliminated.These results strongly suggest that changes in the relative proportions of the different islet endocrine cells exert marked effects on islet function. In particular, a greater A cell and glucagon content is associated with higher rates of glucose-induced insulin secretion and biosynthesis.

A B S T R A C T The effects of somatostatin and epinephrine have been studied with regard to glucoseinduced insulin release and 45Ca++ uptake by rat pancreatic islets after 2 days in tissue culture and with regard to 45Ca++ efflux from islets loaded with the radioisotope during the 2 days of culture. 45Ca++ uptake, measured simultaneously with insulin release, was linear with time for 5 min. 45Ca++ efflux and insulin release were also measured simultaneously from perifused islets.Glucose (16.7 mM) markedly stimulated insulin release and 45Ca++ uptake. Somatostatin inhibited the stimulation of insulin release by glucose in a concentration-related manner (1-1,000 nglml) but was without effect on the glucose-induced stimulation of 45Ca++ uptake. Similarly, under perifusion conditions, both phases of insulin release were inhibited by somatostatin while no effect was observed on the pattern of 45Ca++ efflux after glucose.Epinephrine, in contrast to somatostatin, caused a concentration-dependent inhibition of the stimulation of both insulin release and 45Ca++ uptake by glucose. Both phases of insulin release were inhibited by epinephrine and marked inhibition could be observed with no change in the characteristic glucose-evoked pattern of 45Ca++ efflux (e.g., with 10 nM epinephrine). The inhibitory effect of epinephrine on 45Ca++ uptake and insulin release appeared to be mediated via an a-adrenergic mechanism, since is was abolished in the presence of phentolamine.Somatostatin inhibits insulin release without any detectable effect upon the handling of calcium by the islets. In contrast, inhibition of insulin release by epinephrine is accompanied by a partial inhibition of glucose-induced Ca++ uptake.