The endocrine cells in the epithelium of the gastrointestinal mucosa of the rat. An electron microscope study.

The authors of this study examine the question of whether the so-called enterochromaffin or argentaffin cells of the gastrointestinal tract should be considered as a single cell type. The systematic application of purely morphologic methods has led to the conclusion that the epithelium of the gastrointestinal mucosa comprises endocrine cells of several types. This conclusion is primarily based on the uneven and characteristic distribution of the various cell types along the intestinal tract, an observation precluding the interpretation that the different types correspond to diverse functional stages of the same cell. A specific endocrine function may be attributed to each of the given cell types recognized so far on account of their appearance and their localization in characteristic areas of the gastrointestinal tract. It is acknowledged, however, that a purely morphological study leaves room for doubt. The first cell type is probably responsible for the formation of 5-hydroxytryptamine. Cells of type II are morphologically comparable to the pancreatic A cells and may, therefore, be called intestinal A cells. Cell type III comprises intestinal D cells since their appearance corresponds to that of pancreatic D cells. Cell type IV might well be responsible for catecholamine production, whereas gastrin is in all probability produced in endocrine cell type V. As yet, the thorough morphological study of the gastrointestinal epithelium does not provide information as to additional distinct cellular sites of production of the several other hormones isolated from different parts of the gut.

Morphological and biochemical studies of B cells of fetal rat endocrine pancreas in organ culture. Evidence for (pro) insulin biosynthesis.

Fetal rat pancreases explanted on the 18th day of gestation and maintained in organ culture for 1-10 days were utilized for this series of studies. Ultrastructurally, at the time of explantation, the majority of fetal B cells was sparsely granulated and characterized by numerous free ribosomes and undeveloped rough endoplasmic reticulum (RER) and Golgi complexes. During the culture period, extensive development of the RER and Golgi complexes preceded an increasing accumulation of beta-granules. This later increase in the number of beta-granules and in the concentration of immunoreactive insulin was paralleled by a reduction of RER and Golgi complex activity. High resolution radioautographic studies of pulse-chase experiment over a 1 hr period demonstrated the shift of silver grains from the elements of the RER, through the Golgi region, and finally to the beta-granules. Incubation with (14)C-labeled leucine demonstrated the incorporation of radioactivity into molecules possessing the immunological and electrophoretic properties of insulin. These studies indicate that de novo synthesis of (pro)insulin occurs also during culture of fetal rat pancreas explanted relatively late in gestation.

Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell.

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.

Gap junction development is correlated with insulin content in the pancreatic B cell.

The development of gap junctions between insulin-containing B cells was quantitatively analyzed in islets of Langerhans isolated from rats treated with the sulfonylurea glibenclamid for 1, 2, or 7 days. Glibenclamid treatment was associated with a marked depletion of the insulin content of B cells and with an increase in the number and size of gap junctions between these cells. A significance correlation was found between these two events.

Insulin release by emiocytosis: demonstration with freeze-etching technique.

The technique of freeze-etching for electron microscopy applied to isolated islets of Langerhans has permitted a successful evaluation of emiocytotic events on the cell surface. The frequency of these events in stimulated cells suggests that emiocytosis represents a significant mechanism for insulin release.

Exocytosis-endocytosis coupling in the pancreatic beta cell.

The stimulation of the release of insulin by glucose is accompanied by an enhanced uptake of cytochemically demonstrable horseradish peroxidase into endocytotic vesicles within the beta cells. An exocytosis-endocytosis coupling might represent a mechanism by which membrane constituents are recycled within the beta cells under conditions of increased secretory activity.

Pronase effect on pancreatic beta cell secretion and morphology.

Pronase at low concentration (4 micrograms per milliliter) produces a reversible increase of glucose-stimulated insulin release in isolated islets of Langerhans. Pronase also affects the ultrastructure of the beta cells by inducing extensive development of tight junctions as well as the accumulation of secretory product within the extracellular spaces.

Insulin release and cyclic AMP accumulation in response to glucose in pancreatic islets of fed and starved rats.

The dose as well as the time kinetics of insulin and adenosine-3', 5' -monophosphate (cyclic AMP) responses to glucose were compared in pancreatic islets of fed and starved rats. There was a preferential impairment of the early phase of glucose-induced insulin release in perifused islets of rats starved for 16 and 48 h. Similarly, the accumulation of 3H cyclic AMP in islets prelabeled with 3H-2-adenine was less in islets of 48 h starved than fed rats, during the first 10-min of stimulation with 26.7 mM glucose in the presence of 0.1 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, whereas at 30 and 60 min 3H cyclic AMP responses to glucose were similar in fed and starved islets. Also, in 10-min incubations with glucose 3.3, 6.7, 10.0, 13.3, and 26.7 mM without and with 0.1 mM and 1.0 mM 3-isobutyl-1-methylxanthine, insulin release correlated strongly with the accumulation of 3H cyclic AMP in the islets of fed as well as starved rats. The thresholds for glucose-induced insulin and 3H cyclic AMP responses were higher and the maximal responses were lower in starved than fed islets. Preincubation of islets of 48-h starved rats with 16.7 mM glucose for 60 min corrected the impaired insulin and 3H cyclic AMP responses to glucose. Starvation-induced impairment of insulin secretory responses to glucose, and their restoration by preincubation with glucose in vitro, may represent acute regulatory effects of glucose on the adenylate cyclase-cyclic AMP system in the pancreatic beta cell.

Evidence for the involvement of Na/Ca exchange in glucose-induced insulin release from rat pancreatic islets.

Glucose-induced inhibition of Ca(++) extrusion from the beta-cell may contribute to the rise in cytosol Ca(++) that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na(+) gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of (45)Ca(++). Insulin release and (45)Ca(++) efflux were then measured during perifusion. In Ca(++)-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited (45)Ca(++) efflux. Initially 1 mM ouabain inhibited (45)Ca(++) efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca(++), 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca(++) was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca(++) removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca(++) efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.

Diabetogenic action of streptozotocin: relationship of dose to metabolic response.

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 two-thirds 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.

The roles of intracellular and extracellular Ca++ in glucose-stimulated biphasic insulin release by rat islets.

Verapamil, an agent known rapidly to block calcium uptake into islets of Langerhans, has been used to study the roles of intra- and extracellular calcium in the two phases of glucose-induced insulin release. Rates of calcium uptake and insulin release during the first phase were measured simultaneously over 5 min in rat islets after maintenance in tissue culture for 2 days. Rates of (45)Ca(++) efflux and insulin release during the first and second phases were also measured simultaneously under perifusion conditions. For this, islets were loaded with (45)Ca(++) during the entire maintenance period to complete isotopic equilibrium. Under static incubation conditions 5 muM Verapamil had no effect upon Ca(++) uptake or insulin release in the presence of 2.8 mM glucose. By contrast, glucose-stimulated calcium influx was totally abolished without there being any significant effect upon first phase insulin release. Thus first phase insulin release is independent of increased uptake of extracellular calcium. The lack of effect of 5 muM Verapamil blockade on first phase insulin release was confirmed, under perifusion conditions, and was in marked contrast to the observed 55% inhibition of second phase release. (45)Ca(++) efflux was inhibited during both phases of the insulin release response. The results show that increased calcium uptake in response to glucose is not involved in the mechanism of first phase insulin release but is required for the full development and maintenance of the second phase release. It seems possible that intracellular calcium is the major regulatory control for first phase insulin release and that intracellular calcium and increased uptake of extracellular calcium contribute almost equally to the second phase of glucose-induced release.

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice.

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.

Hepatic autography in uncontrolled experimental diabetes and its relationships to insulin and glucagon.

Exogenous glucagon is known to increase hepatic lysosomes, but the relationships between endogenous glucagon and insulin levels and hepatic lysosomes have not been examined. To determine if the hormones of the pancreatic islets influence the development of these organelles glycogenosomes, dense bodies, and autophagosomes were morphometrically quantitated in normal rats, in rats with mild streptozotocin diabetes with normal hormone levels, and in rats with severe streptozotocin diabetes with hyperglucagonemia, hypo-insulinemia, and clinical evidence of uncontrolled diabetes and ketoacidosis. In the latter volume density of lysosomes averaged 222.8x10(-4) (SEM +/-19.8x10(-4)), significantly above the control value of 75x10(-4) (SEM +/-7.0x10(-4)) (P<0.0005); glycogenosomes were absent in the diabetics, the increase being largely the result of increased autophagosomes. Insulin treatment corrected the hyperglucagonemia, hypoinsulinemia, and other manifestations of uncontrolled diabetes and reduced the volume density of lysosomes to 37.4x10(-4) (SEM +/-2.0x10(-4)), significantly below both the untreated diabetic rats and the nondiabetic controls (P<0.0025). In mild streptozotocin diabetes, in which hyperglucagonemia, hypoinsulinemia, and other evidence of uncontrolled diabetes were absent, lysosomes averaged 77.6x10(-4) (SEM +/-5.5x10(-4)), not different from the controls. A statistically significant correlation between all measurements of lysosomal volume density and plasma glucagon was observed (r=0.79; P<0.001). It is concluded that uncontrolled streptozotocin diabetes in rats is accompanied by hepatic autophagy which may be related to the increased plasma glucagon level and/or the decreased insulin and which is corrected by insulin therapy.

Functional differences between rat islets of ventral and dorsal pancreatic origin.

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 an 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 microgram/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.

Perifusion of rat pancreatic tissue in vitro: substrate modification of theophylline-induced biphasic insulin release.

The immunoreactive insulin (IRI) release patterns produced by continuous theophylline stimulation of rat pancreas have been defined, using an in vitro perfusion system. In the presence of glucose, citrate, and pyruvate at concentrations which were nonstimulatory by themselves, continuous stimulation with theophylline produced a biphasic IRI release profile. In the absence of substrate, continuous theophylline stimulation produced only an abrupt and limited primary response. Of the substrates tested, only glucose significantly enhanced this primary response. With increasing theophylline concentrations, whether in the presence or absence of substrate, significant increases were noted in the primary response as estimated by either the maximum rate of IRI release attained or by the total amount of IRI released during this time. Similarly, the secondary responses to theophylline increased with theophylline concentration in the presence of either citrate or pyruvate. With glucose as substrate, however, increasing theophylline concentrations from 2.5 to 5, then 10 mM produced a progressive reduction in both indices of the secondary response, which was inversely related to the primary response. These findings suggest that cyclic AMP not only mediates IRI release in quantitative terms but is also implicated in the qualitative nature of the response pattern. They also indicate a possible metabolic basis for biphasic IRI release, the acute or primary response being dependent upon the basal state of the cell and the availability of endogenous energy sources, the secondary response upon the availability of exogenous substrate.

Diazoxide effects on biphasic insulin release: "adrenergic" suppression and enhancement in the perifused rat pancreas.

An in vitro system for perifusion of rat pancreas has been used to investigate the effects of diazoxide on glucose-induced insulin release. Administration of diazoxide with a stimulating concentration of glucose produced a dose-dependent suppression of insulin release. This effect was partly reversed by phentolamine. In the presence of nonstimulatory concentrations of glucose, diazoxide plus phentolamine, but neither alone, stimulated a biphasic release of insulin similar to that observed with 1-isopropyl norepinephrine. A prior period of perifusion with a low concentration of diazoxide enhanced the primary component of subsequent glucose-stimulated insulin release, an effect inhibited by addition of either phentolamine or propranolol to the diazoxide during this "prestimulation" period. These effects are similar to those observed with epinephrine. By contrast with epinephrine however, increasing the concentration of diazoxide during the period before glucose stimulation enhanced both the primary and secondary components of subsequent glucose-induced insulin release. These data suggest that at least some of the direct effects of diazoxide on the pancreas are mediated through alpha- and beta-adrenergic receptor mechanisms.

Somatostatin- and epinephrine-induced modifications of 45Ca++ fluxes and insulin release in rat pancreatic islets maintained in tissue culture.

The effects of somatostatin and epinephrine have been studied with regard to glucose-induced insulin release and (45)Ca(++) uptake by rat pancreatic islets after 2 days in tissue culture and with regard to (45)Ca(++) efflux from islets loaded with the radio-isotope during the 2 days of culture. (45)Ca(++) uptake, measured simultaneously with insulin release, was linear with time for 5 min. (45)Ca(++) efflux and insulin release were also measured simultaneously from perifused islets. Glucose (16.7 mM) markedly stimulated insulin release and (45)Ca(++) uptake. Somatostatin inhibited the stimulation of insulin release by glucose in a concentration-related manner (1-1,000 ng/ml) but was without effect on the glucose-induced stimulation of (45)Ca(++) uptake. Similarly, under perifusion conditions, both phases of insulin release were inhibited by somatostatin while no effect was observed on the pattern of (45)Ca(++) efflux after glucose.Epinephrine, in contrast to somatostatin, caused a concentration-dependent inhibition of the stimulation of both insulin release and (45)Ca(++) 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 (45)Ca(++) efflux (e.g., with 10 nM epinephrine). The inhibitory effect of epinephrine on (45)Ca(++) uptake and insulin release appeared to be mediated via an alpha-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.

Dependency of cyclic AMP-induced insulin release on intra- and extracellular calcium in rat islets of Langerhans.

Calcium and cyclic AMP are important in the stimulation of insulin release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) raises islet cAMP levels and causes insulin release at nonstimulatory glucose concentrations. In isolated rat pancreatic islets maintained for 2 d in tissue culture, the effects of IBMX on insulin release and 45Ca++ fluxes were compared with those of glucose. During perifusion at 1 mM Ca++, 16.7 mM glucose elicited a biphasic insulin release, whereas 1 mM IBMX in the presence of 2.8 mM glucose caused a monophasic release. Decreasing extracellular Ca++ a monophasic release. Decreasing extracellular Ca++ to 0.1 mM during stimulation reduced the glucose effect by 80% but did not alter IBMX-induced release. Both glucose and IBMX stimulated 45Ca++ uptake (5 min). 45Ca++ efflux from islets loaded to isotopic equilibrium (46 h) was increased by both substances. IBMX stimulation of insulin release, of 45Ca++ uptake, and of efflux were not inhibited by blockade of Ca++ uptake with verapamil, whereas glucose-induced changes are known to be inhibited. Because IBMX-induced insulin release remained unaltered at 0.1 mM calcium, it appears that cAMP-stimulated insulin release is controlled by intracellular calcium. This is supported by perifusion experiments at 0 Ca++ when IBMX stimulated net Ca++ efflux. In addition, glucose-stimulated insulin release was potentiated by IBMX. These results suggest that cAMP induced insulin release is mediated by increases in cytosolic Ca++ and that cAMP causes dislocation of Ca++ from intracellular stores.

A morphological basis for intercellular communication between alpha- and beta-cells in the endocrine pancreas.

By degranulating beta-cells in the islets of Langerhans of the rat with sulfonylurea, it has been possible to distinguish unambiguously alpha-cells from beta-cells in freeze-fracture replicas. In such preparations, we found morphologically typical tight and gap junctions occurring between alpha- and beta-cells. The presence of gap junctions offers indirect evidence that these cells are coupled with one another; coupling may influence the secretory behavior of alpha- and beta-cells maintaining glucose homeostasis within tightly constricted limits.

Increased clearance and degradation of [3H]insulin in streptozotocin diabetic rats.

The role of the insulin-receptor compartment in the pharmacokinetics of intravenously injected insulin in rats was studied. Since streptozotocin-diabetes in rats results in increased insulin binding to tissues in vitro, insulin pharmacokinetics in streptozotocin-diabetic rats were compared to controls, using semisynthetic [(3)H]insulin as the tracer. The initial distribution volume for [(3)H]insulin was elevated by 60% in diabetic rats. By contrast, no difference in initial distribution volume for [(14)C]inulin was observed, and the absolute values were lower than those found for [(3)H]insulin. The metabolic clearance rate of [(3)H]insulin was elevated by 44% in diabetic rats. That these differences were the result of increased binding of insulin to a specific receptor compartment in diabetic rats was shown by three additional experiments. The first involved receptor saturation by injection of 10 U native insulin 2 min before the tracer injection, resulting in identical [(3)H]insulin disappearance rates in the two groups of rats. The second consisted of displacing [(3)H]insulin from receptors by injecting 10 U unlabeled insulin 6 min after the tracer injection. Displacement of intact [(3)H]insulin from receptors and subsequent reappearance in the circulation occurred in both control and diabetic animals; however, such displacement was 25% greater in the diabetic rats. Finally, treatment of diabetic rats with insulin for 8 d normalized [(3)H]insulin clearance even though the tracer was injected at a time when the animals were again hyperglycemic and hypoinsulinemic. This suggests that down-regulation of insulin receptors had occurred during insulin therapy. These results confirm that a specific compartment for insulin exists (the insulin-receptor compartment) and that this compartment plays an important role in insulin clearance.